Keywords: macrophage, O-GlcNAcylation, inflammation, LPS, metabolic syndrome

O-GlcNAcylation corresponds to the addition of N-Acetylglucosamine (GlcNAc) on serine and threonine residues of proteins. This reversible post-translational modification regulates the activity of cytosolic and nuclear proteins according to glucose availability and its metabolism through the hexosamine biosynthesis pathway (HBP). Only two enzymes, OGT and OGA, catalyse the addition and removal of GlcNAc from proteins, respectively. This modification has been involved in several pathologies including diabetes, cancer and neurological diseases. Several lines of evidence indicate that O-GlcNAcylation also participates in inflammatory processes, but contrasting results have been obtained, with both pro-and anti-inflammatory effects depending on the cell type and experimental setting. In addition, little is known concerning the role of O-GlcNAcylation in macrophage functions.

In this work, we demonstrate that LPS (lipopolysaccharide) induces an increase in O-GlcNAcylation of intracellular proteins in different macrophage models from rodent and human origin. We observed that LPS stimulation has no effect on the expression or the activity of the O-GlcNAc cycling enzyme OGT and OGA, but significantly increases the activity of glutamine fructose amido-transferase (GFAT), the rate limiting enzyme of the HBP. Stimulation of protein O-GlcNAcylation by LPS is mediated by a rapid and marked induction of the expression of the GFAT2 isoform. We identified FoxO1 binding sites on the GFAT2 promoter, and we showed that pharmacological inhibition or genetic ablation of the transcription factor FoxO1 impairs the effect of LPS on GFAT2 expression in macrophages. This indicates that induction GFAT2 occurs through a FoxO1 dependent mechanism.

To evaluate the role of LPS-induced O-GlcNAcylation in macrophages, we also developed a mice model with targeted OGT deletion in the myeloid lineage. We observed that deletion of OGT in macrophage exacerbates the inflammatory response triggered by LPS, characterized by increased expression of Nitric oxide synthase 2 (NOS2) and elevated secretion of pro-inflammatory cytokines. This result suggests that O-GlcNAcylation may restrain excessive inflammation upon LPS stimulation.

Altogether, our results suggest that upon LPS stimulation, induction of GAFT2, which permits an increase in protein O-GlcNAcylation, may be part of a regulatory feed-back mechanism self-limiting inflammatory processes in macrophages.

Aknowlegement

I would like to begin by thanking the doctors and professors DUGAIL, YAZIDI, BENOMAR, LAUZIER and ISSAD, thank you for having done me the honor of judging this work. I sincerely thank professor EL-Yazidi and doctor BENOMAR for having taken so much interest in this work by agreeing to be the rapportors for this thesis, despite their busy schedules. I would also like to thank doctor DUGAIL and doctor LAUZIER for having kindly accepted to be examiners of this work.

I would like to express my deepest gratitude to Tarik ISSAD who welcomed me into his laboratory during these four years. Tarik, I sincerely thank you for having supervised me in this thesis work and for having transmitted to me your scientific knowledge and your experience in the field of research and especially for having supported me and trusting me during these years. I will not forget that without you I would not be here today. Finally thank you for your careful rereading of my manuscript.

I warmly thank the whole team "Signaling of insulin and glucose, and glucotoxicity", in particular Lea BAUDOIN who supervised me with patience and teaching throughout my master training. Thanks also to Abdelouhab Bouaboud, Patrick Pagesy without you, I could not reach to where I am today with very nice work atmosphere in the labratory. Abdelouhab, I sincerely thank you for giving me your support and your great work that you did it for me. I will never forget your help. I would also like to express my thanks to the alumni and members of team phagocytes directed by doctor Florence Niedergang for the sympathy and help they have shown me during these four years, especially Floriane Herit. I of course do not forget to thank all the people of the third floor of the Mechain building for the first part of my Phd and the people of 4 th floor of faculty building and all the friendly and kind people that I had the chance to meet and with whom I sympathized. A very big thank you also to Dr. George bismuth for his collaboration and help during my Phd. Thank to all of you, my life in Cochin would be less interesting without you.

Resumé

La O-GlcNAcylation correspond à l'ajout de N-acétylglucosamine (GlcNAc) sur les résidus sérine et thréonine des protéines cytosoliques, nucleaires et mitochondriales. Cette modification post-traductionnelle réversible régule l'activité des protéines en fonction de la disponibilité du glucose et de son métabolisme par la voie de biosynthèse de l'hexosamine (HBP). Seules deux enzymes, l'OGT et l'OGA, catalysent l'addition et l'élimination de GlcNAc des protéines, respectivement. Cette modification a été impliquée dans plusieurs pathologies dont le diabète, le cancer et les maladies neurologiques. Plusieurs travaux indiquent que la O-GlcNAcylation participe également aux processus inflammatoires, mais des résultats contrastés ont été obtenus, avec des effets à la fois pro-et anti-inflammatoires selon le type cellulaire et le cadre expérimental. De plus, on sait peu de choses sur le rôle de la O-GlcNAcylation dans les fonctions des macrophages.

Dans ce travail, nous démontrons que le LPS (lipopolysaccharide) induit une augmentation de la O-GlcNAcylation des protéines intracellulaires dans différents modèles de macrophages murins et humains. Nous avons observé que la stimulation par le LPS n'a aucun effet sur l'expression ou l'activité des enzymes OGT et OGA, mais augmente de manière significative l'activité de la glutamine fructose amido-transférase (GFAT), enzyme limitante de la voie HBP. La stimulation de la O-GlcNAcylation par le LPS est médiée par une induction rapide et marquée de l'expression de l'isoforme GFAT2. Nous avons identifié des sites de liaison FoxO1 sur le promoteur GFAT2, et nous avons montré que l'inhibition pharmacologique ou l'ablation génétique du facteur de transcription FoxO1 inhibe l'effet du LPS sur l'expression de GFAT2 dans les macrophages. Ceci indique que l'induction de la GFAT2 se produit via un mécanisme dépendant de FoxO1.

Pour évaluer le rôle de la O-GlcNAcylation induite par le LPS dans les macrophages, nous avons également développé un modèle de souris présentant une invalidation de l'OGT dans la lignée myéloïde. Nous avons observé que la suppression de l'OGT dans les macrophages exacerbe la réponse inflammatoire déclenchée par le LPS, avec en particulier une expression accrue de l'oxyde nitrique synthase 2 (NOS2) et une sécrétion plus élevée de cytokines pro-inflammatoires. Ce résultat suggère que la O-GlcNAcylation permet de limiter l'inflammation excessive lors de la stimulation du LPS. Dans l'ensemble, nos résultats suggèrent que l'induction de la GAFT2 permet une augmentation de la O-GlcNAcylation, ce qui pourrait faire partie d'un mécanisme de régulation visant à limiter l'inflammatoires lors de la stimulation des macrophages par le LPS.

Liste of figures

Regulation of glucose homeostasis

Maintaining blood glucose within physiological limits around 0.8 to 1 g/L in fasting conditions and around 1.2 to 1.4 g/L in the postprandial state is necessary for the proper functioning of the organism. Some tissues are totally or partially dependent on glucose for their metabolism. For example, the glucose is the only fuel for red blood cells (which do not possess mitochondria) and renal medulla cells, which are under low oxygen tension and express low levels of oxidative enzymes. The brain gets also most of its energy from glucose, but ketone bodies can a major alternative source. Other cells of the organism preferentially use fatty acids such as oxidative muscles. However, in the case of an extreme emergency (e.g., fighting) glucose is a rapidly usable fuel by all tissues. Therefore, glucose metabolism is a highly regulated process.

Several tissues are involved in managing glucose homeostasis in the body, such as skeletal muscle, adipose tissue and the liver. Glucose uptake is a limiting step for the use and the storage of glucose in muscle and adipose tissue. In mammals, the blood glucose levels are maintained in a relative narrow range despite wide daily variations between fasted state and fed states. This is accomplished by a series of hormonal and physiologic responses during each state.

Fed state

Insulin is a polypeptide hormone with high anabolic power. It is stored in the pancreatic β cells, and will be secreted by exocytosis following an increase in the concentration of glucose in the blood circulation [START_REF] Biddinger | FROM MICE TO MEN: Insights into the Insulin Resistance Syndromes[END_REF]. Upon the arrival of glucose (Figure 1), containing food to the gut, the incretin glucagon-like peptide (GLP)-1 will be secreted from enteroendocrine cells in the gut which promotes insulin release from the endocrine pancreas in a glucose-dependent mechanism. Insulin stimulates the uptake of glucose in its target tissues (adipose tissues, skeletal muscles) by inducing the translocation of the glucose transporter 4 (GLUT4) from an intracellular compartment to the plasma membrane. Once glucose has entered the cell, it is rapidly phosphorylated to glucose 6phosphate (G6P) by a hexokinase. This phosphorylation of glucose traps it inside the cell. In presence of insulin, glucose is stored in a macromolecular form. For example, in adipose tissue, glucose is stored in the form of triglycerides via activation of the enzymes involved in lipid synthesis (lipogenesis). In muscle, glucose is stored as glycogen by activation of glycogen synthase. In the liver, insulin also promotes the storage of glucose in the form of glycogen. These reserves will provide the energy necessary to maintain the functioning of the body in the event of prolonged fasting. Thus insulin in the postprandial state promotes the storage of energy in these tissues. In addition to the storage, glucose is also used to produce energy after its metabolism through glycolysis (see below). Moreover, insulin not only induces glucose uptake by its targets organs, but also prevents hepatic production of glucose (by inhibition of glycogenolysis and gluconeogenesis which occurred during fasting conditions). The net effect is a drop in blood sugar. As a consequence, in a healthy individual, blood sugar levels only moderately rise after a meal.

Figure 1Schematic representation of glucose homeostasis during fed and fasted states Blood glucose concentrations must be kept stable at around one gram per liter for the proper functioning of the body. During the fasted state, low blood glucose leads to diminished circulating insulin and high glucagon levels. This results in increased hepatic glucoe production through glycogenolysis, and then gluconeogenesis and lower muscular and adipose tissue glucose uptake.

In the fed state, after a meal, glucose induces insulin secretion from the pancreas. Insulin promotes the uptake, use and storage of excess glucose (in the liver and muscle, glucose is stored as glycogen while it is stored as lipids in adipose tissue). Insulin also ihibits hepatic glucose production.

Fasting state

During fasting (Figure 1), blood glucose concentrations and insulin are low. Under these conditions the glucagon secreted by the pancreas stimulates the hepatic production of glucose via different mechanisms. The first mechanism is glycogenolysis, which is the process by which glycogen in the liver is transformed into glucose. When the glycogen stores are exhausted a second mechanism intervenes called the gluconeogenesis. Gluconeogenesis allows the synthesis of glucose from precursors such as pyruvate, lactate, glycerol and glucoforming amino acids. When the fasting continues, adipose tissues start to break-down triglycerides and secrete free fatty acids (FFA) and glycerol (lipolysis). Glycerol can serve as a precursor to produce glucose by gluconeogenesis in the liver. FFA can be used by oxidative skeletal muscles and heart. FFA can also be used by the liver to produce ketones bodies through ketogenesis. Indeed, ketogenesis supplies the energy under the circumstances of low glucose levels and prolonged fasting to certain organs, particularly brain, heart and skeletal muscles.

Glucose transporters

Glucose entry into the cells is facilitated by glucose transporter proteins called (GLUTs). Different transporter isoforms have varying affinities for glucose and the expression of these isoforms has some tissue specificity. Indeed, there are ubiquitous isoforms (GLUT 1 and 3), present in all tissues, and tissue specific isoforms (GLUT 2 and 4):

 GLUT 1 is the predominant facilitative glucose transporter and it is widely distributed in different tissues. GLUT1 is highly conserved with 98% identity in the amino acid sequence between humans and the rat (De [START_REF] Vos | Human and Rat Beta Cells Differ in Glucose Transporter but Not in Glucokinase Gene Expression[END_REF].

 GLUT 2 is mainly expressed in the hepatocytes and the β cells of the islets of Langerhans,  GLUT 3 is mainly expressed in neurons,  GLUT 4 is highly expressed in muscle cells and adipocytes. GLUT4 is the insulino-sensitive glucose transporter which facilitates the metabolism or the storage of glucose in these cells in response to insulin.

 GLUT 5 is mainly expressed in the enterocytes and spermatozoa.

GLUT1 is insulin-independent whereas GLUT4 is insulin-dependent. In the absence of insulin GLUT4 transporters are stored in intracellular vesicles. In the presence of insulin, there is an increase in the glucose transporters at the membrane of adipocytes and muscle cells.

In the liver, after a meal, glucose enters through the transporter GLUT2 which is present constitutively at the membrane of hepatocytes. Due to its high Km, GLUT2 can let glucose enter according to blood glucose concentration. This transporter will also allow the release of glucose by the liver during periods of fasting [START_REF] Ogawa | Functional Localization of Glucose Transporter 2 in Rat Liver[END_REF][START_REF] Hubbard | Crystal Structure of the Activated Insulin Receptor Tyrosine Kinase in Complex with Peptide Substrate and ATP Analog[END_REF].

GLUT2 is also present on the surface of pancreatic β cells where it allows the diffusion of glucose according to its blood concentrations, which confers it a role of "sensor" of the concentration of glucose and makes it possible to secrete insulin in response to hyperglycemia.

Glycolysis pathway

Glycolysis is an almost universal pathway and corresponds to the first steps in the breakdown of glucose to extract energy for cellular metabolism. Glycolysis includes 10 enzymatic reactions which take place in the cytoplasm, resulting in conversion of glucose into pyruvate together with synthesis nicotinamide adenine dinucleotide (NADH) and adenosine triphosphate (ATP).

In aerobic conditions, the pyruvate enters into mitochondria where it is converted into Acetyl CoA and NADH by pyruvate dehydrogenase (PDH). Acetyl CoA then goes into the TCA (Krebs cycle) where it undergoes a series of redox reactions taking place in the mitochondria. These reactions provide NADH and FADH2 that are then used by the electron transport chain in the mitochondria to generate energy in the form of ATP (adenosine triphosphate). In the absence of sufficient oxygen, the pyruvate is reduced by the NADH to produce lactate. (See schema below).

Insulin signaling pathway

Insulin exerts its physiological effects on insulin-sensitive tissues by activating intracellular signaling cascades which will be described briefly below.

The insulin receptor (IR) is present on the cell plasma membrane. It consists of two extracellular α subunits which bind to the hormone and two β subunits which comprises an intracellular domain that possesses a tyrosine kinase activity [START_REF] Hubbard | Crystal Structure of the Activated Insulin Receptor Tyrosine Kinase in Complex with Peptide Substrate and ATP Analog[END_REF]. The binding of insulin to its receptor induces a change in conformation of the latter which in turn induces its auto-phosphorylation. This allows activation of the receptor tyrosine kinase and the recruitment and phosphorylation of many substrates (IRS1-4, Shc). The best characterized substrates belong to the family of IRS (Insulin receptor substrates) [START_REF] Voliovitch | Tyrosine Phosphrylation of Insulin Receptor Substrate-1 in Vivo Depends upon the Presence of Its Pleckstrin Homology Region[END_REF].

The IRS1 and IRS2 isoforms are most abundant in mammals and contain many sites which can be phosphorylated on tyrosines. Male knockout (KO) mice for IRS1 have increased blood glucose levels [START_REF] Araki | Alternative Pathway of Insulin Signalling in Mice with Targeted Disruption of the IRS-1 Gene[END_REF] and KO mice for IRS2 develop type 2 diabetes [START_REF] Withers | Disruption of IRS-2 Causes Type 2 Diabetes in Mice[END_REF]. The IRSs are phosphorylated on tyrosines by the insulin receptor tyrosine kinase.

From the activation of the IRS two main pathways are activated. These are the MAPK (mitogen-activated protein kinase) pathway which has effects on cell differentiation and growth, and the PI3K (phosphatidylinositide-3-kinase) pathway which, in addition to a role in cell growth, differentiation and apoptosis, is the main pathway responsible for the metabolic effects of insulin. PI3K consists of a p85 regulatory subunit and a p110 catalytic subunit [START_REF] Rei | Phosphatidylinositol 3-Kinase Signals Activate a Selective Subset of Rac/Rho-Dependent Effector Pathways[END_REF]. The phosphorylation of IRS proteins on tyrosines leads to the recruitment of PI3K to the plasma membrane and to its activation. The activated PI3K phosphorylates phosphatidylinositol 4, 5-bisphosphate (PIP2), into phosphatidylinositol [START_REF] Fardini | O-GlcNAcylation: A New Cancer Hallmark?[END_REF]4,[START_REF] Ma | Hyper-O-GlcNAcylation Is Anti-apoptotic and Maintains Constitutive NF-kappaB Activity in Pancreatic Cancer Cells[END_REF] -triphosphate (PIP3), a second messenger that allows the recruitment of the protein Akt (also called PKB) to the plasma membrane. Once at the membrane, Akt is phosphorylated and activated by PDK1 (PI3,4,5P3-Dependent Protein Kinase) and mTORC2 (mammalian target of rapamycin complex 2). Activated Akt phosphorylates substrates and thereby controls many metabolic pathways. The PI3K also allows the translocation of GLUT4 to the membrane and thus increase the entry of glucose into muscle cells and adipocytes (Figure 3). Binding of insulin to its receptor induces its phosphorylation on tyrosine residues. This in turn leads to phosphorylation of the insulin receptor substrates (IRS1-4, Shc). Through the activation of Grb2 / SOS, the IRSs and Shc activate the MAP Kinase pathway (MAPK) which is mainly responsible for cell proliferation in response to insulin. IRSs also induce the activation of phosphatidylinositol-3-kinase (PI3K) which catalyses the formation of Phosphatidylinositol [START_REF] Fardini | O-GlcNAcylation: A New Cancer Hallmark?[END_REF]4,[START_REF] Ma | Hyper-O-GlcNAcylation Is Anti-apoptotic and Maintains Constitutive NF-kappaB Activity in Pancreatic Cancer Cells[END_REF] -triphosphate (PIP3). PIP3 recruits phosphoinositide dependent kinase 1 (PDK1) and PKB / Akt. PDK and mTORC2 then phosphorylates PKB / Akt which will then be mainly responsible for the metabolic effects of insulin (From Jung and Choi 2014).

Diabetes mellitus

Diabetes mellitus commonly known as diabetes is a group of metabolic disorders characterized by a disturbance of glucose metabolism which lead to a high blood glucose level over a prolonged period of time. Its cardinal symptoms include polyuria (excessive production of urine), polydipsia (increased thirst), and polyphagia (increased hunger). If left untreated, diabetes can cause to development of many complications related to hyperglycaemia like microvascular complications (retinopathy, nephropathy and neuropathy)

and macrovascular damage that promotes the development of cardiovascular complications such as atherosclerosis. Indeed, the higher the amount of glucose in the blood (hyperglycemia) for a long period of time, the more the risks are likely to be numerous and severe.

The clinical diagnosis is based on one of any of the following observations according to WHO (World health organization):

 Two abnormal fasting blood glucose tests performed in separate days with values equal to or greater than 1.26 g / l.

 Glycated hemoglobin HbA1 C ≥6.5% (which gives the average level of blood sugar over the past 2 to 3 months).

 A blood sugar level greater than or equal to 2 g / l (11.1 mmol / l) 2 hours after an oral load of 75 g of glucose.

 Only one measure of blood glucose randomly (without previous fasting) and a level equal to or greater than 2 g /l associated with cardinal symptoms (polyuria, polydipsia and polyphagia). America (26 Million) and Africa (16 million). 312.000 persons were estimated to have died of diabetes in 2017 and about 73% of the deaths due to diabetes were people under the age of 60.

In addition, the AFR (the IDF -Africa Region), which includes 49 diverse sub-Saharan countries and territories, estimated 19.4 million adults aged of 20-79 years were living with diabetes in 2019, representing a regional prevalence of 3.9%.

Two forms of diabetes are mostly known. Type 1 diabetes is an autoimmune disease due to the destruction of pancreatic β cells, which will lead to a total absence of insulin production. Type 1 diabetes accounts for 10% of the forms of diabetes and generally occurs in young people.

Type 2 diabetes represents approximately 85% of the forms of diabetes. It is a metabolic disease due to the development of insulin resistance as well as to the dysfunction of pancreatic β cells. It is a multifactorial disease partly linked to the environment, which develops in older subjects, between 40 and 50 years on average. However, with the increase in juvenile forms of obesity, type 2 diabetes is found in increasingly young people.

Indeed, type 2 diabetes mellitus is one of the complications of the metabolic syndrome (also known as X syndrome). People with metabolic syndrome are 2-fold more likely to develop cardiovascular disease and 5-fold more likely to develop type 2 diabetes than the general population [START_REF] Alexander | NCEP-Defined Metabolic Syndrome, Diabetes, and Prevalence of Coronary Heart Disease Among NHANES III Participants Age 50 Years and Older[END_REF]).

The metabolic syndrome

In recent decades, the lifestyle has changed considerably. This resulted in a decrease in physical expenditure, associated with a modification of the diet, with a generalization of the western type diet rich in saturated fatty acid and refined sugars. This has led to a very large increase in the prevalence of various metabolic diseases such as obesity, diabetes and atherosclerosis. These diseases are major threats to human health in the 21st century (Eckel et al. 2010).

According to the international diabetes federation (IFD), a patient with metabolic syndrome is defined as the person with central obesity (waist circumference ≥94 cm for a male; ≥80 cm for a female) as obligatory criteria with at least 2 of the following abnormalities Blood pressure ≥ 130/85 mm Hg Fasting blood glucose ≥ 1, 10 g/l Serum High density lipoprotein (HDL) ≤0,4 g/l for a male; ≤0,5 g/l for a female Serum Triglycerides (TG) ≥ 1, 5 g/l

While the pathogenesis of the metabolic syndrome and each of its components is complex and not well understood, central obesity and insulin resistance are recognized as important causative factors [START_REF] Nakamura | Contribution of Visceral Fat Accumulation to the Development of Coronary Artery Disease in Non-Obese Men[END_REF][START_REF] Bonora | Prevalence of Insulin Resistance in Metabolic Disorders: The Bruneck Study[END_REF][START_REF] Rutter | Impact of Glucose Intolerance and Insulin Resistance on Cardiac Structure and Function: Sex-Related Differences in the Framingham Heart Study[END_REF].

Central obesity

According to WHO, obesity is a chronic disease which corresponds to an excessive accumulation of fat in different parts of the body to the extent that it is associated with an adverse effect on health. Excessive intake of energy-dense foods, physical inactivity and genetic susceptibility are known as causative factors of obesity.

The World Health Organization classifies the obesity according to the body mass index BMI (which is a person's weight in kilograms (kg) divided by his or her height in meters squared).

Classification of obesity

Grade 1 overweight (commonly called overweight) -BMI between 25-29.9 kg/m 2 .

Grade 2 overweight (commonly called obesity) -BMI between 30-39.9 kg/m 2 .

Grade 3 overweight (commonly called severe or morbid obesity) -BMI equal to or greater than 40 kg/m 2 .

According to WHO, in 2016, 39% of adults aged 18 years and over (39% of men and 40% of women) were overweight. Overall, about 13% of the world's adult population (11% of men and 15% of women) were obese in 2016. The worldwide prevalence of obesity nearly tripled between 1975 and 2016. In the United States, where the epidemic is particularly large, more than a third of adults are obese [START_REF] Ogden | Trends in Obesity Prevalence Among Children and Adolescents in the United States, 1988-1994 Through 2013-2014[END_REF]) and worryingly the prevalence of this disease has increased very rapidly in children and adolescents. It now reaches 17% of young subjects [START_REF] Ogden | Trends in Obesity Prevalence Among Children and Adolescents in the United States, 1988-1994 Through 2013-2014[END_REF]. This pathology is considered to be the leading cause of preventable deaths [START_REF] Jia | Trends in Quality-Adjusted Life-Years Lost Contributed by Smoking and Obesity[END_REF] and is associated with a very significant cost for society [START_REF] Finkelstein | Annual Medical Spending Attributable To Obesity: Payer-And Service-Specific Estimates: Amid Calls for Health Reform, Real Cost Savings Are More Likely to Be Achieved through Reducing Obesity and Related Risk Factors[END_REF][START_REF] Misra | Obesity and the Metabolic Syndrome in Developing Countries[END_REF].

Although overall obesity confers a significant threat to the health of individuals, the distribution of body fat is also of great importance in determining its adverse effects [START_REF] Bhaskaran | Body-Mass Index and Risk of 22 Specific Cancers: A Population-Based Cohort Study of 5•24 Million UK Adults[END_REF][START_REF] Flegal | Association of All-Cause Mortality With Overweight and Obesity Using Standard Body Mass Index Categories: A Systematic Review and Meta-Analysis[END_REF]). An excess of body fat in the abdomen, measured simply by waist circumference, is more indicative for the metabolic syndrome profile than BMI.

Obesity is an established risk factor for type 2 diabetes mellitus. Anthropometric measures of overall and central obesity serve as predictors of type 2 diabetes. For example, in a survey study of 43,581 women, was achieved by Carey VJ et al, the relative risk of type 2 DM was 10 folds higher in women with a BMI of 30 kg/m 2 than in women with a BMI 20 kg/m 2 . Moreover, the relative risk of type 2 diabetes is 5 fold higher in women with a waist circumference of 92 cm than women with a waist circumference of 67 cm. These results strongly suggest that body fat distribution is a powerful independent predictor for metabolic syndrome and particularly for type 2 diabetes mellitus [START_REF] Vincent | Body Fat Distribution and Risk of Non-Lnsulin-Dependent Diabetes Mellitus in Women[END_REF]. It has been well demonstrated that central obesity can be considered as a significant added risk factor for the development of cardiovascular diseases in addition to other risk factors such as hypertension, diabetes and current smoking [START_REF] Yusuf | Effect of Potentially Modifiable Risk Factors Associated with Myocardial Infarction in 52 Countries (the INTERHEART Study): Case-Control Study[END_REF]).

Insulin resistance

Insulin resistance is defined as a state of decreased responsiveness of target tissues (liver, skeletal muscle and adipose tissue) to normal circulating levels of insulin.

Insulin resistance in liver manifests itself primarily as an increased production of glucose through gluconeogenesis. In adipocytes, insulin resistance leads to the inhibition of lipogenesis and to the stimulation of lipolysis, which in turn results in an increase in circulating lipids, whereas in skeletal muscles manifests itself by a reduction in insulinstimulated glycogen synthesis

To maintain normal glucose levels, insulin secretion at first increases to overcome insulin resistance (hyperinsulinism). Indeed, during the first phases of insulin resistance, there is a compensatory effect with an expansion of the mass of pancreatic β cells (hyperplasia and hypertrophy) in the islets of Langerhans [START_REF] Butler | '-Cell Deficit and Increased -Cell Apoptosis in Humans With Type 2 Diabetes[END_REF]. The production of over-increasing amounts of insulin weakens and may eventually result in a decrease in the mass of β cells in the islets of Langerhans [START_REF] Kahn | Mechanisms Linking Obesity to Insulin Resistance and Type 2 Diabetes[END_REF].

This leads to progressive accumulation of glucose and free fatty acids in the circulation. Excess of glucose has deleterious effect on different tissues in the body, a phenomenon called glucotoxicity. Moreover, pathological accumulation of fatty acids also has deleterious effect a phenomenon called lipotoxixity. Both glucotoxicity and lipotoxicity play important roles in the pathogenesis of type 2 diabetes mellitus through different mechanisms (Figure 4). The mechanisms that lead to further impairment of insulin sensitivity and pancreatic β cells will be detailed below.

Glucotoxicity

Although glucose is necessary for cell survival, it has harmful effects when it is present in a high concentration. Hyperglycemia is characterized by blood glucose levels above 5.5mM or 100 mg/dl. Chronic hyperglycemia has deleterious effects on insulin target tissues (liver, adipose tissues and muscles) by disrupting insulin signaling, thereby decreasing insulin sensitivity of these tissues. On the other hand, chronic hyperglycemia also has deleterious effects on the pancreatic β cells resulting in decreased secretion of insulin by the pancreas.

Thus, chronic hyperglycemia worsens hyperglycemia creating a vicious cycle of glucotoxicity.

Glucotoxicity is related to the toxic effect of increased intracellular glucose metabolites. Body cells have the ability to downregulate the expression of glucose transporters in response to chronic hyperglycemia to reduce glucose uptake as a protective mechanism. It has been reported that endothelial cells are not capable of adapting the expression of glucose transporter GLUT1 expression and activity when medium glucose concentration are highly elevated whereas vascular smooth muscle exhibited a reduction of about 50% under the same condition. This results in increased intracellular glucose concentrations in endothelial cells [START_REF] Kaiser | Differential Regulation of Glucose Transport and Transporters by Glucose in Vascular Endothelial and Smooth Muscle Cells[END_REF]). This explains the increased incidence of predominant vascular complications in patients with diabetes mellitus.

Several mechanisms have been involved in the deleterious effects of glucose on cells (Michael Brownlee. 2001) that will be briefly described below.

The formation of advanced glycation end products (AGEs)

Hyperglycemia contributes to the production of advanced glycation end products or AGE which irreversibly modify proteins or lipids by glycation [START_REF] Basta | Advanced Glycation End Products and Vascular Inflammation: Implications for Accelerated Atherosclerosis in Diabetes[END_REF]). The formation of AGEs is non-enzymatic. The first step consists in adding a reducing sugar (glucose or fructose) on the amino residues, mainly lysine and the N-terminal amine function of intra-or extracellular proteins, leading to the formation a Schiff base. This step is dependent on the glucose concentration and is quite unstable. Following this step, the Schiff bases undergo a molecular rearrangement, called Amadori rearrangement, which is reversible by hydrolysis.

The amount of Amadori products formed is proportional to the blood sugar concentration.

This rearrangement is followed by a more complex reaction, called the Maillard reaction. It leads to the formation of AGEs. Unlike previous reactions, this last step is independent of blood sugar and is irreversible.

AGEs have harmful effects through several mechanisms. First, the formation of AGEs on intracellular proteins alters their functions. In addition, at the extracellular level, plasma proteins modified by AGEs can bind to their receptors, RAGEs, on the cell surface. Among other things, this activates NFκB (nuclear factor κB) causing alterations in gene expression, and may also contribute to the production of nitric oxide (NO) and pro-inflammatory cytokines [START_REF] Muñoz | Nutritionally Mediated Oxidative Stress and Inflammation[END_REF]. AGEs can also activate NADPH oxidase which will lead to the production of superoxide and thus contribute to oxidative stress (M. [START_REF] Zhang | Glycated Proteins Stimulate Reactive Oxygen Species Production in Cardiac Myocytes: Involvement of Nox2 (Gp91phox)-Containing NADPH Oxidase[END_REF].

Furthermore, the precursors of AGEs can diffuse out of cells and the extracellular matrix is also the target of glycations. This causes cellular dysfunctions by disrupting the signaling between cells and the extracellular matrix. Hemoglobin can be changed by AGEs to form glycated hemoglobin. The percentage of glycated hemoglobin is commonly used as a marker to measure the progression of diabetes as well as the effectiveness of treatments. Numerous studies have shown beneficial effects of decreases in AGEs in diabetic patients, whether by inhibitors (Aminoguanidine) or dietary treatment by reducing food intake rich in AGE (lipids and proteins). Thus, in diabetics with dietary AGEs restriction, it is possible to decrease both blood concentrations of AGEs and the activity of MAPK, NFκB and VCAM1 [START_REF] Goldin | Advanced Glycation End Products: Sparking the Development of Diabetic Vascular Injury[END_REF][START_REF] Vlassara | Inflammatory Mediators Are Induced by Dietary Glycotoxins, a Major Risk Factor for Diabetic Angiopathy[END_REF].

Figure 5: Different possible pathways of formation of advanced glycation end products (AGE)

The initial interaction between an aldehyde group of glucose and a free amine group on proteins creates a Schiff base, which spontaneously rearranges into an Amadori product. Thereafter, the reactions which are less and less reversible, continue (reactions not shown), leading to the definitive formation of AGEs. In addition, a variety of highly reactive carbonyl intermediates, such as 3-deoxy-glucosone, glyoxal and methyl-glyoxal, can be formed by auto-oxidation of glucose, Schiff base or Amadori products. These carbonyl intermediates can react with free amine groups to form final AGE products such as CML, CEL, GOLD and MOLD. Abbreviations:

CML for N-e-carboxy-methyl-lysine, CEL for N-e-carboxy-ethyl-lysine, GOLD for glyoxal-lysine dimer and MOLD for methyl-glyoxal-lysine dimer (image from [START_REF] Basta | Advanced Glycation End Products and Vascular Inflammation: Implications for Accelerated Atherosclerosis in Diabetes[END_REF]).

Activation of the different PKC isoforms

Hyperglycemia also leads to increased glucose flux in the glycolysis pathway. This results in a greater production of dihydroxyacetone phosphate from fructose 6-P and glyceraldehyde 3-P. The dihydroxyacetone phosphate is then reduced into glycerol-3phosphate, which allows the synthesis of diacylglycerol (DAG). DAG is an activator of a family of serine-threonine kinases, the protein kinase C (PKC) α, β, and δ, [START_REF] Sheetz | Molecular Understanding of Hyperglycemia's Adverse Effects for Diabetic Complications[END_REF]. The activation of PKC by hyperglycemia has harmful effects on the vascular level and has been implicated in the development of microvascular complications associated with diabetes (M. [START_REF] Brownlee | The Pathobiology of Diabetic Complications: A Unifying Mechanism[END_REF]. Hyperglycemia can also activate PKC indirectly via activation of AGEs receptors (RAGEs) or via an induction of the polyol pathway (Figure 6).

Increased flux of glucose through the polyol pathway

The amount of glucose entering the polyol pathway is negligible under physiological conditions [START_REF] Morrison | Glucose Utilization by the Polyol Pathway in Human Erythrocytes[END_REF]. However, the flux through this pathway during hyperglycemia varies from 33% of total glucose used in the rabbit lens to 11% in human erythrocytes (Yabe-Nishimura. 1998). This is due to the fact that the aldose reductase, which catalyzes the first reaction of this pathway, has a very low affinity for glucose and is therefore only able to metabolize glucose in the presence of a high glucose concentration. Aldose reductase makes it possible to reduce glucose to sorbitol which, in the second reaction of the pathway, is oxidized to fructose by sorbitol dehydrogenase. Several mechanisms are implicated in the deleterious effects associated with the activation of this pathway. One of them includes sorbitol-induced osmotic stress via decreased Na+,K (+)-adenosine triphosphatase activity in a PKC-activation dependent mechanism [START_REF] Xia | Characterization of the Mechanism for the Chronic Activation of Diacylglycerol-Protein Kinase C Pathway in Diabetes and Hypergalactosemia[END_REF]). It has also been proposed that the reduction of glucose to sorbitol by NADPH consumes NADPH.

NADPH is an important cofactor for the regeneration of the reduced form of glutathione (GSH). Depletion of NADPH induced by the polyol pathway could thus contribute to generation of oxidative stress (H.-M. [START_REF] Cheng | The Effect of High Glucose and Oxidative Stress on Lens Metabolism, Aldose Reductase, and Senile Cataractogenesis[END_REF]. Furthermore, the second reaction of the polyol pathway produces NADH, a substrate for NADH oxidase which leads to the production of superoxide anions and thus contributes to oxidative stress [START_REF] Tang | Aldose Reductase, Oxidative Stress, and Diabetic Mellitus[END_REF]. NADH can also inhibit glyceraldehyde-3-phosphate dehydrogenase (GAPDH) resulting in inhibition of glycolysis which in turn leads to decrease of glucose uptake further and thus worsening of glycemia (Figure 7). During the second reaction of the polyol pathway, sorbitol dehydrogenase (SDH) will oxidize sorbitol to fructose using NAD + as a cofactor, which will produce NADH. NADH is also involved in oxidative stress (M. Brownlee 2005).

Increased glucose flux in the hexosamine biosynthetic pathway

Another mechanism that may contribute to the deleterious effects of hyperglycemia is the entry of glucose into the hexosamine biosynthetic pathway. This pathway is significantly increased in the event of hyperglycemia. The end product of this pathway is Uridine diphosphate N-acetylglucosamine or UDP-GlcNAc. UDP-GlcNAc serves as a substrate of O-GlcNAc transferase (OGT) enzyme to modify proteins reversibly by the addition of Nacetylglucosamine (GlcNAc) motif on the serine or threonine residues of cytoplasmic and nuclear proteins.

Augmentation of the glucose flux through this pathway has different effect on proteins stability, localization and activity and thus on the cell function. The role of O-GlcNAc in different tissues in normal and pathological hyperglycemic conditions will be described in details in the following chapter (O-GlcNAcylation and metabolic diseases).

Production of Reactive Oxygen Species (ROS)

As previously mentioned, the intracellular oxidation of glucose generates electron donors, NADH and FADH2 which are used to yield ATP via the electron transport chain. In case of hyperglycemia, a significant amount of glucose will be oxidized, which in turn generates high levels of NADH and FADH2. These excessive levels of electron donors lead to an increase in the proton gradient (H +) across the mitochondrial membrane which then exceeds a critical threshold. This results in blockage of electron transfer within complex III, which induces production of superoxide by coenzyme Q [START_REF] Korshunov | High Protonic Potential Actuates a Mechanism of Production of Reactive Oxygen Species in Mitochondria[END_REF][START_REF] Nishikawa | Normalizing Mitochondrial Superoxide Production Blocks Three Pathways of Hyperglycaemic Damage[END_REF]. A high concentration of superoxide anion has been observed in diabetic patients and the level of superoxide anion is well correlated with the degree of hyperglycemia.

Oxidative stress is viewed as an imbalance between the production of reactive oxygen species (ROS) and their elimination by protective anti-oxidant mechanisms, which can lead to multiple of complications. Oxidative stress is a known pathway in the pathogenesis of diabetic complications [START_REF] Giacco | Oxidative Stress and Diabetic Complications[END_REF]. It has been shown that exposure of isolated rat islet to oxidative stress inhibits the promoter activity and mRNA expression of the insulin gene, thereby, decreasing insulin gene expression [START_REF] Kaneto | Activation of the Hexosamine Pathway Leads to Deterioration of Pancreatic β-Cell Function through the Induction of Oxidative Stress[END_REF].

Figure 8: Production of superoxide by the electron transport chain within the mitochondria

An increase in electron donors (NADH and FADH2) induced by hyperglycemia, generates a high potential of mitochondrial membrane (∆uH +) by pumping protons through the inner mitochondrial membrane.

This prevents the transport of electrons to complex III, allowing an overproduction of superoxide by coenzyme Q, which reduces O2 to superoxide (image is adapted from [START_REF] Brownlee | Biochemistry and Molecular Cell Biology of Diabetic Complications[END_REF]).

In addition to its direct effect, oxidative stress can activate a variety of transcription factors, which lead to the differential expression of genes involved in inflammatory pathways.

The inflammation triggered by oxidative stress is the cause of many chronic diseases such as type 2 diabetes mellitus. Increased secretion of tumour necrosis factor alpha (TNFα) has been observed to be linked to obesity-related insulin resistance, a risk factor for the development of type 2 diabetes (X. [START_REF] Du | Inhibition of GAPDH Activity by Poly(ADP-Ribose) Polymerase Activates Three Major Pathways of Hyperglycemic Damage in Endothelial Cells[END_REF].

Brownlee proposed a unifying theory for the deleterious effects of glucose. The various mechanisms exposed above could be the consequence of the same dysregulation induced by hyperglycemia: the overproduction of the superoxide anion in the mitochondria.

Indeed, his group has shown that the excessive production of the superoxide anion associated with hyperglycemia leads to inhibition of the activity of GAPDH (C. [START_REF] Du | Smac, a Mitochondrial Protein That Promotes Cytochrome c-Dependent Caspase Activation by Eliminating IAP Inhibition[END_REF]. This inhibition then induces an accumulation of all of the glycolysis intermediates located upstream of the GAPDH. Indeed, following the inhibition of GAPDH, the intracellular glucose level increases, leading to a significant increase in the flux in the polyol pathway. The level of fructose-6 phosphate is also increased resulting in an increase in the flux in the hexosamine biosynthetic pathway and thus in O-GlcNAcylation. Finally, the inhibition of GAPDH increases the level of glyceraldehyde-3 phosphate which will activate both the AGEs and the PKCs pathways. Indeed, glyceraldehyde-3 phosphate allows the formation of methylglyoxal, a precursor of AGEs. Glyceraldehyde-3 phosphate also allows the formation of DAG, the activator of PKCs.

Thus an excessive glucose metabolism leads to the production of ROS, which could be at the origin of all the deleterious effects of chronic hyperglycemia (Michael Brownlee 2001;

M. Brownlee 2005).

Figure 9: Hyperglycemia induced ROS activates glucotixic pathways

The unifying mechanism by which the mitochondrial overproduction ofhydrogen peroxide, induced by hyperglycemia, activates four major pathways that lead to damage associated with hyperglycemia. The superoxide produced in excess leads to DNA damage which results in activation of poly-ADP-ribose polymerase (PARP). Activated PARP inhibits GAPDH activity through poly (ADP-ribosyl)ation, which increases the concentration of the set of glycolysis metabolites located upstream of GAPDH (X. [START_REF] Du | Inhibition of GAPDH Activity by Poly(ADP-Ribose) Polymerase Activates Three Major Pathways of Hyperglycemic Damage in Endothelial Cells[END_REF]. This leads to an increase in the transformation of glyceraldehyde-3-phosphate into methylglyoxal, the main intracellular precursor of AGEs, a higher formation of DAG, an activator of PKC, from DHAP (dihydroxyacetone phosphate), an increase in flow of fructose-6-phosphate which will be converted into UDP-GlcNAc, the substrate used to modify proteins by O-GlcNAc, and to an increase in the entry of glucose into the polyol pathway which consumes NADPH and depletes GSH.

Lipotoxicity

Lipotoxicity is defined by William I. Sivitz as the diabetogenic effect of increased circulating free fatty acids or increased cellular fat content. This condition causes functional impairments in several metabolic pathways, in adipose tissue, liver, heart, pancreas and muscle [START_REF] Sivitz | Lipotoxicity and Glucotoxicity in Type 2 Diabetes: Effects on Development and Progression[END_REF]. Elevated plasma level of saturated fatty acids is associated with increased incidence of type 2 diabetes [START_REF] Mozaffarian | Saturated Fatty Acids and Type 2 Diabetes: More Evidence to Re-Invent Dietary Guidelines[END_REF]. G. Boden et al showed the infusion of lipids in humans for longer than 2 hours reduces insulin-mediated muscle glucose uptake and increases hepatic glucose output (G. [START_REF] Boden | Effects of Acute Changes of Plasma Free Fatty Acids on Intramyocellular Fat Content and Insulin Resistance in Healthy Subjects[END_REF].

Several mechanistic explanations for lipotoxicity have emerged.

Fatty acids-glucose cycle

For many years, the Randle glucose-fatty acid cycle has been invoked to explain insulin resistance in skeletal muscle of patients with type 2 diabetes or obesity. The principal site for regulation of glucose oxidation is the mitochondrial pyruvate dehydrogenase (PDH) complex. Accordingly, fatty acid and glucose oxidation can be thought of as competitive in that excess fat metabolism impairs the oxidation of glucose. The main feature of the lipotoxicity model was that increased fat oxidation in muscle leads to intra-mitochondrial accumulation of Acetyl-CoA and NADH which are both allosteric inhibitors of pyruvate dehydrogenase (PDH). Increased intra-mitochondrial Acetyl-CoA leads to the production of citrate by its reaction with oxaloacetate. Citrate itself is an inhibitor phosphofructokinase. The inhibition of phosphofructokinase blocks glycolysis: These road blocks placed in the glycolytic pathway would lead to increased glucose 6-phosphate concentration, inhibiting hexokinase and resulting in reduced glucose uptake and oxidation. This homeostatic mechanism became known as the glucose fatty acid-glucose cycle or the Randle cycle [START_REF] Randle | Mechanisms Decreasing Glucose Oxidation in Diabetes and Starvation: Role of Lipid Fuels and Hormones[END_REF]Randle et al. 1963) (Figure 10).

Figure 10: The glucose-fatty acid cycle (the Randle cycle)

proposed by Randle, Garland, Hales and Newsholme first described in 1963 An increase in fatty acid concentration results in an elevation of the intra-mitochondrial acetyl CoA/CoA and NADH/NAD+ ratios, with subsequent inactivation of pyruvate dehydrogenase. This in turn causes citrate concentrations to increase, leading to inhibition of phosphofructokinase. Subsequent increases in intracellular glucose-6-phosphate concentration would inhibit hexokinase II activity, which would result in an increase in intracellular glucose concentration and a decrease in muscle glucose uptake. HK, hexokinase II; PFK, phosphofructokinase; PDH, pyruvate dehydrogenase.

Fatty acids impair glucose transporter 4 (GLUT4)

A second alternative mechanism to Randle cycle mechanism has been proposed for fatty-acid induced insulin resistance in skeletal and cardiac muscles. In these tissues glucose uptake is controlled mainly by the translocation of glucose transporter GLUT4 and not by hexokinase. The vesicular traffic of GLUT4 from intracellular stores to the plasma membrane is stimulated by insulin, muscle contraction, and energy stress. Hexokinase inhibition by accumulation of its product glucose 6-phosphate, initially proposed by Randle and his team.

probably has little impact on the overall glucose uptake under basal conditions. It has been suggested that hexokinase is not responsible for inhibition of glucose uptake at least in skeletal muscles [START_REF] Sakamoto | Emerging Role for AS160/TBC1D4 and TBC1D1 in the Regulation of GLUT4 Traffic[END_REF]J. C. Hou and Pessin 2008). In vivo human study demonstrated that lipid infusion induced elevation of serum fatty acids was observed to be associated with a significant reduction of skeletal muscle glycogen synthesis and glucose oxidation. Moreover, they revealed that reduction of muscle glycogen synthesis by elevated plasma free fatty acids was preceded by a fall of muscle glucose-6-phosphate concentrations.

These results indicate that the control of glucose metabolism by fatty acids is exerted mainly at the level of glucose uptake and not at the level of PDH as reported by Randle. However, these findings have been observed only in muscles not in other tissues [START_REF] Roden | Mechanism of Free Fatty Acid-Induced Insulin Resistance in Humans[END_REF].

Free fatty acids may interfere directly with glucose uptake by impairment GLUT4 gene expression in muscle and fat [START_REF] Boden | Role of Fatty Acids in the Pathogenesis of Insulin Resistance and NIDDM[END_REF] or indirectly by disruption of the insulinsignaling cascade responsible for GLUT4 translocation and the regulation of anabolic metabolism. It has been reported in several studies that high fat diet increases skeletal myocytes contents of lipid metabolites. The increased intracellular lipid content (diacylglycerol and long chain fatty acyl CoA) activates protein kinase C θ (PKCθ) in high fat-fed rats [START_REF] Schmitz-Peiffer | Alterations in the Expression and Cellular Localization of Protein Kinase C Isozymes e and 0 Are Associated With Insulin Resistance in Skeletal Muscle of the High-Fat[END_REF]. This in turn leads to the activation of a serine/threonine kinase cascade which is responsible for the phosphorylation of serine/threonine sites on insulin receptor substrates. Serine-phosphorylated forms of these proteins fail to associate with or to activate PI3-kinase. PI3 kinase activation is an important step for triggering the movement of intracellular GLUT4 molecules to the cell surface [START_REF] Dresner | Effects of Free Fatty Acids on Glucose Transport and IRS-1-Associated Phosphatidylinositol 3-Kinase Activity[END_REF]).

Accordingly, it has been shown that elevations in plasma fatty acid concentrations, after lipid infusion for 5h, was associated with impaired IRS-1-associated phosphatidylinositol 3-kinase (PI 3-kinase) activity in human muscle biopsy samples. The same team also showed that a similar lipid infusion protocol in rats resulted in a reduction of insulin-stimulated IRS-1 tyrosine phosphorylation, which was associated with activation of protein kinase C [START_REF] Griffin | Free Fatty Acid-Induced Insulin Resistance Is Associated With Activation of Protein Kinase C and Alterations in the Insulin Signaling Cascade[END_REF]) (Figure 11). As a consequence, GLUT4 translocation and other events downstream of insulin receptor signaling are diminished.

Fatty acids induce insulin resistance through inflammation

Chronic inflammation is a hallmark of obesity and is linked to the development of insulin resistance and numerous other diseases. The activation of toll-like receptor 4 (TLR4) by long chain saturated fatty acids (lcSFAs) is an important process involved in obesityassociated inflammation. TLR4 is a member of the family transmembrane receptors for the recognition of molecular patterns of pathogens and danger signals associated with tissue damage. TLR4 is the best characterized of all pattern recognition receptor (PRRs). TLR4 stimulation results in the activation of pro-inflammatory signaling pathways, including the activation of mitogen-activated protein kinases (MAPKs), nuclear factor kappa B (NF-kB) and interferon regulatory factors (IRFs) [START_REF] Hoebe | Upregulation of Costimulatory Molecules Induced by Lipopolysaccharide and Double-Stranded RNA Occurs by Trif-Dependent and Trif-Independent Pathways[END_REF][START_REF] Horng | The Adaptor Molecule TIRAP Provides Signalling Specificity for Toll-like Receptors[END_REF][START_REF] Wang | Association of Specific Dietary Fats With Total and Cause-Specific Mortality[END_REF][START_REF] Yamamoto | Cutting Edge: A Novel Toll/IL-1 Receptor Domain-Containing Adapter That Preferentially Activates the IFN-β Promoter in the Toll-Like Receptor Signaling[END_REF]. These pathways in turn lead to the expression and the secretion of inflammatory cytokines. These pathways will be described in the next chapter).

Studies in animals fed high-fat diets have revealed that TLR4 deletion selectively protects high fat-induced obesity and improves peripheral insulin sensitivity [START_REF] Poggi | C3H/HeJ Mice Carrying a Toll-like Receptor 4 Mutation Are Protected against the Development of Insulin Resistance in White Adipose Tissue in Response to a High-Fat Diet[END_REF][START_REF] Davis | Tlr-4 Deficiency Selectively Protects Against Obesity Induced by Diets High in Saturated Fat[END_REF]. Other studies using TLR2 or TLR4 deficient mice are also observed protected from high saturated fat diet-induced inflammation and insulin resistance (J. Y. [START_REF] Lee | Saturated Fatty Acids, but Not Unsaturated Fatty Acids, Induce the Expression of Cyclooxygenase-2 Mediated through Toll-like Receptor 4[END_REF][START_REF] Glass | Inflammation and Lipid Signaling in the Etiology of Insulin Resistance[END_REF]. These results suggest a link between increased circulating saturated fatty acids and TLRs activation. Thus, TLR4 emerged as a candidate intermediary linking obesity and dyslipidaemia to enhanced inflammatory cytokine production. However, the mechanism by which free fatty acids activate TLR4 is not clearly understood. As described below contradictory results have been reported concerning the direct effect of free fatty acids on TLR4.

Fatty acids are TLR4 ligands

The earliest evidence that linked TLR4 to lipid-induced inflammation predates its identification as the receptor for liposaccharide (LPS) [START_REF] Liao | Genetic Control of Inflammatory Gene Induction and NF-Kappa B-like Transcription Factor Activation in Response to an Atherogenic Diet in Mice[END_REF]. Later, in vitro and in vivo studies [START_REF] Caesar | Crosstalk between Gut Microbiota and Dietary Lipids Aggravates WAT Inflammation through TLR Signaling[END_REF]S. Huang et al. 2012; J. Y. [START_REF] Lee | Saturated Fatty Acids, but Not Unsaturated Fatty Acids, Induce the Expression of Cyclooxygenase-2 Mediated through Toll-like Receptor 4[END_REF][START_REF] Nagareddy | Adipose Tissue Macrophages Promote Myelopoiesis and Monocytosis in Obesity[END_REF] have confirmed these earlier findings, and the activation of TLR4 by lipids has become an important paradigm for understanding how obesity initiates inflammation (Gökhan S. Indeed, HFD increases serum level of saturated fatty acids and also LPS which is the wall component of the gut grame negative bacteria (microbiote).

The human intestine contains from 1.10 13 to 1.10 14 bacterial cells, which are thus 10 times more numerous than human cells and express 150 times more genes than the human genome [START_REF] Gill | Metagenomic Analysis of the Human Distal Gut Microbiome[END_REF]. The gut microbiota is a source of LPS and plays a role in obesity and insulin resistance [START_REF] Cani | Metabolic Endotoxemia Initiates Obesity and Insulin Resistance[END_REF]2012). In patients with metabolic disorders, there is an increased passage in the blood of LPS from the intestinal microbiota [START_REF] Ghoshal | Chylomicrons Promote Intestinal Absorption of Lipopolysaccharides[END_REF]).

This could be due to changes in the microbiota which leads to disruption of tight intestinal junctions, resulting in an increase in intestinal permeability [START_REF] Poltorak | Defective LPS Signaling in C3H/HeJ and C57BL/10ScCr Mice: Mutations in Tlr4 Gene[END_REF][START_REF] Leber | The Influence of Probiotic Supplementation on Gut Permeability in Patients with Metabolic Syndrome: An Open Label, Randomized Pilot Study[END_REF].

Thus zonulin, which is a marker of intestinal permeability, is increased in obese and diabetic patients [START_REF] Moreno-Navarrete | Circulating Zonulin, a Marker of Intestinal Permeability, Is Increased in Association with Obesity-Associated Insulin Resistance[END_REF]. In healthy subjects, after a meal rich in fats, there is a rapid increase in blood LPS concentrations, [START_REF] Erridge | A High-Fat Meal Induces Low-Grade Endotoxemia: Evidence of a Novel Mechanism of Postprandial Inflammation[END_REF]). This increase in LPS levels may at least partially explain the activation of TLR4 in patients with metabolic syndrome.

"Germ-free" mice lacking a microbiota are thus protected from metabolic alterations induced by diets rich in lipids [START_REF] Backhed | The Gut Microbiota as an Environmental Factor That Regulates Fat Storage[END_REF].

LPS, the classical ligand of TLR4 [START_REF] Poltorak | Defective LPS Signaling in C3H/HeJ and C57BL/10ScCr Mice: Mutations in Tlr4 Gene[END_REF], contains Lipid A moiety which possesses most of its biological activities. Lipid A is acylated with hydroxy saturated fatty acids. The 3-hydroxyl groups of these saturated fatty acids are further 3-acylated by saturated fatty acids (Raetz et al. 2002) (Figure 12). Interestingly, the replacement of Lipid A saturated fatty acids by monounsaturated or polyunsaturated fatty acids stops the pro-inflammatory activity of the LPS (Hoshino et al. 1999). Myeloid differentiation factor 2 MD-2, is a co-receptor that physically interacts with the extracellular domain of TLR4. MD-2 is principally responsible for LPS binding to TLR4 [START_REF] Park | The Structural Basis of Lipopolysaccharide Recognition by the TLR4-MD-2 Complex[END_REF]). In the inactive state, TLR4 exists as a hetero-dimer with MD-2. The binding of LPS to TLR4/MD-2 creates a dimerization interface that facilitates the formation of the active complex consisting of a ''dimer of dimers'' (TLR4 2 /MD-2 2 ), arranged symetrically in an M-like shape. Park et al. studied ligand specificity and receptor activation mechanism of the TLR4-MD-2-LPS complex. They performed X-ray crystallographic and amino acid sequence analyses of leucine rich repeat (LRR) domains of TLR4 which provided an important clue about structural features of the ligands and their interactions with TLR4.

The authors revealed that LPS interacts with a receptor hydrophobic lipid pocket and directly bridges the two components of the heterodimers. Interestingly, they observed that acyl chains of Lipid A occupy the hydrophobic lipid-binding pocket. This suggests that saturated fatty acids play an important role for TLR4 ligand recognition and receptor activation [START_REF] Park | The Structural Basis of Lipopolysaccharide Recognition by the TLR4-MD-2 Complex[END_REF]).

In addition, Lee and his team demonstrated that saturated fatty acids alone, particularly lauric acid and palmitic acid, are able to stimulate TLR4 receptor (J. Y. [START_REF] Lee | Saturated Fatty Acids, but Not Unsaturated Fatty Acids, Induce the Expression of Cyclooxygenase-2 Mediated through Toll-like Receptor 4[END_REF]). This has been confirmed later by another team [START_REF] Wong | Fatty Acids Modulate Toll-like Receptor 4 Activation through Regulation of Receptor Dimerization and Recruitment into Lipid Rafts in a Reactive Oxygen Species-Dependent Manner[END_REF]). Accordingly, it was reported that long-chain saturated fatty acids (lcSFAs) are TLR4 agonists [START_REF] Osborn | The Cellular and Signaling Networks Linking the Immune System and Metabolism in Disease[END_REF].

However, the question of these saturated fatty acids as ligands of TLR4 remains debated.

Fatty acids are not a direct TLR4 ligands

Although all previous arguments supported the assumption that TLR4 is a receptor of FFA, another set of experiments and studies emerged to prove that TLR4 is not the receptor of saturated fatty acids. Erridge and Samani first hypothesized an alternative mechanism that links dietary fat intake with TLR4-associated pathologies. These authors suggested LPS and lipopeptide contamination of the fatty-acid-free bovine serum albumin (BSA). Indeed, in cell culture experiments, fatty acids are poorly soluble in the culture media and thus usually studied complexed to BSA. In this study, they reconstituted SFAs in ethanol, and diluted it directly either into warmed culture medium or complexed with BSA. They revealed that, in the absence of BSA, dietary saturated fatty acids (palmitate and lauric acids) alone were not able to elicit TLR4 activation [START_REF] Erridge | Saturated Fatty Acids Do Not Directly Stimulate Toll-Like Receptor Signaling[END_REF]. However, Huang et al. defended the initial proposition. In this study they revealed that saturated fatty acids sodium palmitate or laureate without BSA induced phosphorylation of inhibitor of nuclear factor-kB, c-Jun Nterminal kinase (JNK), p44/42 mitogen-activated-kinase (ERK), and nuclear factor-kB subunit p65, and TLR2 and TLR4 target gene expression in macrophages (S. [START_REF] Huang | Saturated Fatty Acids Activate TLR-Mediated Proinflammatory Signaling Pathways[END_REF]. However, recent study by Lancaster et al. using atomically detailed molecular dynamics (simulations of palmitate leading to TLR4 2 /MD-2 2 complex, they revealed that palmitate is unable to induce the dimerization and endocytosis of TLR4, events that are fundamental for the activation of TLR4 signaling. Furthermore, LPS treatment of macrophage, induced as expected the activation of JNK (MKK4/7 phosphorylation) as well as NF-kB (phosphorylation of p65 and degradation of IkBα). Surprisingly, they observed that palmitate induced JNK and MKK4/7 phosphorylation, but was unable to activate NF-kB.

Moreover, they reported that, in TLR4 deficient macrophages, palmitate lost its ability to activate JNK and to accumulate intracellular fatty acids metabolites in particular ceramides.

The effect of fatty acids was restored when TLR4 deficient macrophage were primed with agonists of alternate TLRs (TLR2 or TLR3). Collectively, these data revealed that TLR4 is not the receptor of SFA but is essential for SFAs biological effect (Figure 13). Furthermore, it has been previously shown that TLR4 is an upstream signaling component required for saturated fatty acid-induced ceramide biosynthesis. In this study, the authors showed that TLR4 through activation the inhibitory kappa B kinase (IKKβ) increases the expression of genes driving ceramides biosynthesis such as the single tube protein system 2 (STP2) and CerS1, -2, and -6. IKKβ inhibition with sodium salicylate (NS) in vivo, blocked high fatinduced accumulation of ceramides in soleus, liver, and hypothalamus and improved glucose uptake. Furthermore, they indicated that increased-ceramides production was essential for TLR4-dependent inhibition of Akt phosphorylation leading to insulin resistance [START_REF] Holland | Lipid-Induced Insulin Resistance Mediated by the Proinflammatory Receptor TLR4 Requires Saturated Fatty Acid-Induced Ceramide Biosynthesis in Mice[END_REF]. These results establish that TLR4 signaling plays a critical role in linking fatty acids to insulin resistance through modulation of ceramides biosynthesis on one hands, and via JNK activation (we will describe it in the next paragraph) on the other hand. LPS and free fatty acids (FFAs) are increased, due to high fat diet (HFD) consumption. Serum FFAs are also increased due to insulin resistance induced-lipolysis in adipocytes. In addition to TLR4 mediated LPS induced NFkB and JNK activation, TLR4 is also essential for FFAs to activate JNK and to induce AKT phosphorylation inhibition and thus inhibition of GLUT4 membrance traslocation. This occurs via TLR4mediated upregulation of ceramides biosynthesis genes STP2 (single tube protein system 2) and CerS through an IKKB-dependent mechanism.

Fatty acids induce oxidative stress

Saturated fatty acids also induce oxidative stress (ROS) and inflammation. ROS can mediate indirectly the effect of lipid on insulin-resistance. Anderson et al. have revealed that feeding mice or humans a HFD induced a significant elevation of H2O2 production by mitochondria isolated from the skeletal muscle [START_REF] Anderson | Mitochondrial H2O2 Emission and Cellular Redox State Link Excess Fat Intake to Insulin Resistance in Both Rodents and Humans[END_REF]). Additionally, an adipogenic diet and the accumulation of adipose tissue can trigger ROS accumulation in mitochondria isolated from different tissues such as adipose tissues [START_REF] Curtis | Downregulation of Adipose Glutathione S-Transferase A4 Leads to Increased Protein Carbonylation, Oxidative Stress, and Mitochondrial Dysfunction[END_REF], liver [START_REF] Raffaella | Alterations in Hepatic Mitochondrial Compartment in a Model of Obesity and Insulin Resistance[END_REF], and kidney [START_REF] Ruggiero | High-Fat Diet Induces an Initial Adaptation of Mitochondrial Bioenergetics in the Kidney despite Evident Oxidative Stress and Mitochondrial ROS Production[END_REF]. Moreover, mice with an adiposespecific knockout of carnitine palmitoyltransferase 2, the enzyme that catalyses an obligate step in mitochondrial long-chain fatty acid oxydation, are protected from high fat induced oxidative stress and inflammation in visceral white adipose tissue (WAT) ) (J. J. [START_REF] Lee | Efficacy of Thigh Volume Ratios Assessed via Stereovision Body Imaging as a Predictor of Visceral Adipose Tissue Measured by Magnetic Resonance Imaging: Regional Body Volume Ratios and Visceral Adipose Tissue[END_REF]. In another hand, it has been reported that antioxidant enzyme activities such as superoxide dismutase (SOD), catalase, glutathione peroxidase, and glutathione reductase were significantly reduced in mice liver fed a HFD [START_REF] Valenzuela | Molecular Adaptations Underlying the Beneficial Effects of Hydroxytyrosol in the Pathogenic Alterations Induced by a High-Fat Diet in Mouse Liver: PPAR-α and Nrf2 Activation, and NF-ΚB down-Regulation[END_REF].

The mechanisms underlying the elevation of oxidative stress by increasing circulating lipids are not fully understood. The metabolism of fatty acids in mitochondria is performed via β-oxidation which is a multi-step process through which fatty acids are broken down yielding Acetyl-CoA, NADH and FADH2. The Acetyl-CoA then enters the mitochondrial Krebs cycle. The electron transport chain to produce ATP uses NADH and FADH2 produced by both fatty acid β-oxidation and the Krebs cycle. Therefore, the high consumption of fatrich diets should promote mitochondrial β-oxidation of FFAs and subsequently lead to an excess electron flux and accumulation of ROS.

NADPH oxidase is a mitochondrial multi-component enzyme composed of membrane-bound components (gp91 PHOX and p22 PHOX ). NADPH uses NAD(P) H as its substrate and catalyzes the one-electron reduction of molecular oxygen to superoxide. NAD(P)H oxidase activity has been widely recognized as an important source of superoxide in phagocytes (neutrophils and monocytes) [START_REF] Pithon-Curi | Glutamine Plays a Role in Superoxide Production and the Expression of P47phox, P22phox and Gp91phox in Rat Neutrophils[END_REF]. Accumulated mitochondrial H2O2 diffuses across membranes and promotes the activation of Ser/Thr protein kinases such as PKCθ and the member of mitogen-activated protein kinase (MAPK) JNK. Activation of PKC θ and JNK leads to phosphorylation of IRS-1/2 which in turn inhibits IRS-1/2 association with the IR. This promotes IRS-1/2 degradation and suppress the recruitment and activation of PI3K (review; Tiganis 2011). It has been suggested that fat accumulation-induced ROS plays a pathogenic mechanism of obesity associated metabolic syndrome in mice and human. In this study, the authors revealed that fat accumulation induced NADPH oxidase mRNA expression and activity and increased ROS production in adipose tissues of obese patients and in cultured 3T3-L1 adipocytes treated with FFA. They observed that, in adipose tissue of obese mice, the production of ROS increased selectively accompanied by augmented expression of NADPH oxidase and decreased expression of antioxidative enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase [START_REF] Furukawa | Increased Oxidative Stress in Obesity and Its Impact on Metabolic Syndrome[END_REF]).

In addition, a study showed that lipids accumulation in skeletal muscles and liver is associated with mitochondrial dysfunction due to mitochondrial DNA damage. In this study, the authors reported that HFD fed mice exhibit activated phosphorylation of c-Jun N-terminal kinases (JNK) in both liver and skeletal muscle. JNK activation plays an important role in the conditions of both increased oxidative and endoplasmic reticulum stress [START_REF] Yuzefovych | Mitochondrial DNA Damage and Dysfunction, and Oxidative Stress Are Associated with Endoplasmic Reticulum Stress, Protein Degradation and Apoptosis in High Fat Diet-Induced Insulin Resistance Mice[END_REF]. Accordingly, palmitate treatment of macrophages induces JNK activation and MKK4/7 phosphorylation but not NF kappa B signaling pathway [START_REF] Lancaster | Evidence That TLR4 Is Not a Receptor for Saturated Fatty Acids but Mediates Lipid-Induced Inflammation by Reprogramming Macrophage Metabolism[END_REF]).

Moreover, it has been shown that inhibition of JNK activity and JNK depletion with siRNA reduced the level of reactive oxygen species (ROS). In this study, they revealed that mitochondrial superoxide stimulates JNK through upstream kinases, primarily apoptosis signaling regulated kinase 1 (ASK1), a kinase responsive to mitochondrial ROS. Activated JNK translocates to mitochondria where it is responsible for amplification of mitochondrial ROS production through the inhibition of respiratory Complex I, contributing to decreased respiratory function independent on NADPH oxidase activity [START_REF] Chambers | Mitochondrial C-Jun N-Terminal Kinase (JNK) Signaling Initiates Physiological Changes Resulting in Amplification of Reactive Oxygen Species Generation[END_REF]. Furthermore, JNK signaling pathway is also implicated in insulin resistance by direct phosphorylation of serine residues of IRS-1, thus inhibiting its tyrosine phosphorylation by insulin [START_REF] Aguirre | The C-Jun NH 2 -Terminal Kinase Promotes Insulin Resistance during Association with Insulin Receptor Substrate-1 and Phosphorylation of Ser 307[END_REF]. Therefore, saturated fatty acids may also participate in insulin resistance through a vicious circle involving increased ROS production and JNK activation (Figure 14). (apoptosis signaling regulated kinase 1), which amplifiyes ROS production via impairement of mitochondrial respiratory complex 1, creating a vicious circle. JNK itself can impair insulin-induced tyrosine phosphorylation of IRS-1 through promoting its serine phosphorylation. This leads to insulin resistance through inhibition of AKT, resulting in impairement of GLUT4 membrane translocation and thus reduced glucose uptake.

Further, it has been reported that HFD induced-ROS can trigger nuclear factor NF-κB, which in turn leads to the expression of pro-inflammatory agents such as TNF-α, inducible nitric oxide synthase (NOS2), and interferon-γ (IFN-γ) [START_REF] Vial | Effects of a High-Fat Diet on Energy Metabolism and ROS Production in Rat Liver[END_REF][START_REF] Ruggiero | High-Fat Diet Induces an Initial Adaptation of Mitochondrial Bioenergetics in the Kidney despite Evident Oxidative Stress and Mitochondrial ROS Production[END_REF][START_REF] Weisberg | Dietary Curcumin Significantly Improves Obesity-Associated Inflammation and Diabetes in Mouse Models of Diabesity[END_REF]. Overall, these data imply that high-fat diet induced-Oxidative stress can stimulate inflammatory transcription factors such as NF-kB and JNK and thereby activate inflammatory pathways in infiltrating immune cells including monocytes, T cells, mast cells, and macrophages. This in turn results in the impairment of insulin sensitivity and pancreatic beta cells function and thus in the development of insulin resistance and type 2 diabetes mellitus. They are professional phagocytes capable of recognizing, phagocytizing and degrading pathogens as well as apoptotic or necrotic cells. They are the first line of defense against pathogens. Macrophages are derived from monocytes, which are produced by the myeloid lineage, and are recruited from the blood stream in tissues where they differentiate into macrophages in response to injury or infection. Macrophages are found ubiquitously within the organism. Resident macrophages in tissues have a role of "sensor" of the microenvironment [START_REF] Pollard | Trophic Macrophages in Development and Disease[END_REF]. Like the majority of completely differentiated cells, macrophages do not divide anymore. They play a key role in the link between innate and adaptive immunity. Indeed, these cells allow the establishment of the adaptive immune response by secreting pro-inflammatory cytokines. Macrophages also have a role in presenting microbial peptides associated with the class II histocompatibility complex (CMH II) to lymphocytes (i.e. a role of antigen presenting cell). Macrophages like other member of innate immune cells express pattern recognition receptor (PRRs) that detect various microbial components, the so-called pathogen-associated molecular patterns (PAMPs) [START_REF] Newton | Signaling in Innate Immunity and Inflammation[END_REF][START_REF] Mogensen | Pathogen Recognition and Inflammatory Signaling in Innate Immune Defenses[END_REF]. PRRs also recognize damage associated molecular patterns (DAMPs), which are molecules released by necrotic cells and damaged tissues. Mammalian cells express five families of PRRs, including toll-like receptors (TLRs), RIG-I-like receptors, NOD-like receptors (NLRs), C-type lectin-like receptors and cytosolic DNA sensors. However, they share many similarities in their downstream signal transduction pathways. The Toll like receptors TLR4 and TLR2 are strongly expressed on the surface of macrophages. To be active, the TLRs must be in the form of dimers. Most of them form homodimers although certain TLRs such as TLR2 form heterodimers (in particular with TLR1 and TLR6).

Macrophage signaling pathways

Structurally, all TLRs are type I integral membrane proteins consisting of an ectodomain comprising of leucine-rich repeats (LRRs) and a cytoplasmic domain containing a Toll/IL-1R homology (TIR) domain, which is required for signaling [START_REF] Medzhitov | A Human Homologue of the Drosophila Toll Protein Signals Activation of Adaptive Immunity[END_REF][START_REF] Medvedev | Inhibition of Lipopolysaccharide-Induced Signal Transduction in Endotoxin-Tolerized Mouse Macrophages: Dysregulation of Cytokine, Chemokine, and Toll-Like Receptor 2 and 4 Gene Expression[END_REF][START_REF] Kawai | TLR Signaling[END_REF][START_REF] Kumar | Toll-like Receptors and Innate Immunity[END_REF]. Whereas TLR2 heterodimer (TLR2/1 or TLR2/6), TLR4 and TLR5 are located on the outer membrane of cells, TLR3, TLR7, TLR8, and TLR9 are located on endosomes.

Upon stimulation by ligands, TLRs can interact via TIR domain with adaptors molecules such as MyD88 (myeloid differentiation factor 88) or TRAM (Toll-like receptor adaptor molecule) [START_REF] Brown | Mammalian Target of Rapamycin Complex 2 (MTORC2) Negatively Regulates Toll-like Receptor 4-Mediated Inflammatory Response via FoxO1[END_REF]. All TLRs, with the exception of TLR3, utilize MyD88 which in turn, provides the necessary framework to recruit and activate downstream kinases and transcription factors that regulate the host inflammatory response.

The best studied among TLRs is TLR4 which is activated in response to LPS, an endotoxin present on the surface of gram-negative bacteria. When stimulated, TLR4 activates several signaling pathways that are either dependent or independent on MyD88.

MYD88 dependent signaling pathway:

Upon the activation of TLR4, MyD88 is recruited through TIR-TIR interactions to the TLR4 receptor. MyD88 links the IL1 receptor associated kinase 4 IRAK-4 to TLRs via interaction of death domain of each molecule resulting in its phosphorylation and activation. MYD88-IRAK4 plays crucial roles as adaptor molecules in signal transduction of the TLR superfamily. IRAK-4 in turn phosphorylates and activates IRAK-1 (Figure 15). IRAK-1 consists of an N-terminal death domain, which is involved in the binding of MyD88 and a central serine/ threonine kinase domain. The C-terminal region death domain of IRAK-1 contains three potential TRAF6-binding sites. Thus, IRAK1 phosphorylation and activation, results in the recruitment of TRAF6 (TNF associated factor 6) to TLRs where it is activated by IRAK-1. TRAF6/IRAK-1 complex then dissociates from the receptor to activate downstream signaling pathway.

TRAF6 mediates the activation of IKK (inhibitor kappa B kinase) and it has also been shown to activate MAPKK6 (mitogen-activated protein kinase kinase 6) pathway.

IKK activation by TRAF6 requires two intermediary factors, TRAF6-regulated IKK activator 1 (TRIKA1) and TRIKA2. TRIKA1 is a dimeric ubiquitin-conjugating enzyme complex composed of Ubc13 and Uev1A. TRAF6 together with Ubc13 and Uev1A catalyze the formation of a Lys 63 (K63)-linked polyubiquitin to activate IKK [START_REF] Deng | Activation of the IB Kinase Complex by TRAF6 Requires a Dimeric Ubiquitin-Conjugating Enzyme Complex and a Unique Polyubiquitin Chain[END_REF].

In addition, TRIKA2, which is a protein kinase complex, composed of TAK1 (Transforming growth factor-β (TGF-β)-activated kinase 1) and its binding partners TAB1 and TAB2 (TAK-Binding protein), phosphorylates and activates IKK and MKK6. MKK6 activates JNK (Jun amino-terminal kinase) (C. [START_REF] Wang | TAK1 Is a Ubiquitin-Dependent Kinase of MKK and IKK[END_REF].

Activation IKK-NF-κB signaling pathways (see the paragraph below), is responsible for the expression and secretion of mediators of inflammation such as IL1β, IL6, TNFα and MCP1.

JNK activation is also involved in the regulation of inflammatory genes via stimulation transcription factor activator proteins-1 (AP-1).

MYD88 independent signaling pathways:

The activation of TLR recruits and activates adapter proteins such as TRIF (Toll / IL1 receptor domain containing adaptor protein-inducing interferon β) and TRAM (TRIF-related adaptor molecule). This in turn leads to activation of the transcription factor IRF3 (interferon regulatory factor 3), and thereby induces interferon β (IFNβ). IFNβ activates the transcription factor signal transducer 1 (STAT1), leading to the induction of several IFN-inducible genes from [START_REF] Takeda | TLR Signaling Pathways[END_REF].

NFKB signaling pathway

The transcription factor NFκB is found in all cell types and is involved in many cellular functions. This family comprises five proteins (p65 (also called RelA), RelB, c-Rel, p105 / p50 (NFκB1) and p100 / 52 (NFκB2)) which combine to form homo or heterodimeric complexes.

The NF-κB proteins are normally sequestered in the cytoplasm by a family of inhibitory proteins, the IκB family members. To date, the best studied and most important IκB This phosphorylation leads to the ubiquitination of IκB and its degradation by the proteasome, resulting in rapid and transient translocation of canonical NF-κB members in the nucleus, predominantly, the p50/RelA and p50/c-Rel dimers (Sen and Baltimore, 1986.;[START_REF] Oeckinghaus | The NF-B Family of Transcription Factors and Its Regulation[END_REF].

A well-recognized function of NF-κB is the regulation of inflammatory responses. Indeed, NF-κB is known as a key transcription factor of M1 macrophages and is required for induction of a large number of pro-inflammatory genes, including those encoding NOS2, TNF-α, IL-1β, IL-6, IL-12p40 and cyclooxygenase-2 (Review: Zen. 2014). In addition, NF-κB regulates also the activation, differentiation and effector function of inflammatory T cells [START_REF] Tak | NF-ΚB: A Key Role in Inflammatory Diseases[END_REF][START_REF] Lawrence | The Nuclear Factor NF-B Pathway in Inflammation[END_REF]. Recent evidence reported that NF-κB has a role in regulating the activation of the multi-protein complex inflammasome that trigger the maturation of pro-inflammatory cytokines such as interleukin-1β to engage innate immune defenses [START_REF] Sutterwala | Mechanism of NLRP3 Inflammasome Activation: Mechanism of NLRP3 Inflammasome Activation[END_REF]. Not surprisingly, deregulated NF-κB activation is a hallmark of chronic inflammatory diseases. The complex of TRAF6, TAK1, TAB1, and TAB2 further forms a larger complex with Ubc13 and Uev1A, which induces the activation of IKK complex (IKKα, IKKβ, and NEMO/IKKγ). TRAF6 activates also MAP kinases, such as JNK. The result of IKK and MAPKs activation is the activation of the transcription factors NF-kB and AP-1, respectively.

The NLRP3 inflammasome

Inflammasomes are molecular complexes involved in innate immunity. Several inflammasomes have been discovered, the most studied in the context of metabolic diseases being the NLRP3 inflammasome. NLP3 is a complex made up of three proteins: the protein NLRP3 (Nucleotidebinding domain Leucinerich Repeat protein), the adapter protein ASC (Apoptosisassociated Specklike protein containing a CARD) and caspase 1. The recognition of bacterial components or danger signals by the NOD-like receptor (NLR) allows the activation of the inflammasome. Once activated, the inflammasome cleaves pro-caspase 1 and thus leads to the production of active caspase 1 which will contribute to the maturation and secretion of IL1β. The NLRP3 inflammasome also allows the maturation and secretion of IL18.

The Infammasome has been shown to be involved in metabolic syndrome. The NLRP3 receptor appears to play an important role in the development of inflammation, especially in adipose tissue, and can lead to insulin resistance. All of the inflammasome proteins, as well as mature IL1β, have been shown to have increased expression in obese mice [START_REF] Vandanmagsar | The NLRP3 Inflammasome Instigates Obesity-Induced Inflammation and Insulin Resistance[END_REF]. IL1β impairs insulin signaling in insulin targets tissues in vitro and in vivo partially through a tumor necrosis factor alpha TNF α-dependent pathway (H. [START_REF] Wen | Fatty Acid-Induced NLRP3-ASC Inflammasome Activation Interferes with Insulin Signaling[END_REF], thus it plays a role in insulin resistance. Several mechanisms have been suggested that link TNFα to insulin resistance such as phosphorylation of serine residues on IRS-1 via PKCξ dependent mechanism. TNFα also increases intracellular ceramides levels (G. S. [START_REF] Hotamisligil | Mechanisms of TNF-Alpha-Induced Insulin Resistance[END_REF]. Moreover, analysis of obese mice with genetic absence of TNF signaling: (i)signignicantly improves insulin receptor signaling capacity and consequently insulin sensitivity; (ii) prevents brown adipose tissue atrophy and β3-adrenoreceptor deficiency and improves thermo-adaptive responses, (iii) increases muscular GLUT4 protein levels compared to wild type mice; and (iv) lowers hyperlipidemia and hyperleptinemia (G. [START_REF] Hotamisligil | Molecular Mechanisms of Insulin Resistance and the Role of the Adipocyte[END_REF].

The NLRP3 inflammasome can also be activated by saturated fatty acids as well as ceramides, which can play the role of second signal, necessary for the activation of the inflammasome, after stimulation by LPS [START_REF] Lukens | Inflammasome Activation in Obesity-Related Inflammatory Diseases and Autoimmunity[END_REF][START_REF] Vandanmagsar | The NLRP3 Inflammasome Instigates Obesity-Induced Inflammation and Insulin Resistance[END_REF]. Mice deleted for NLRP3 or for caspase 1 have adipocytes which exhibit greater adipogenesis as well as better insulin sensitivity, which is correlated with a decrease in the inflammation of the adipose tissue [START_REF] Stienstra | The Inflammasome-Mediated Caspase-1 Activation Controls Adipocyte Differentiation and Insulin Sensitivity[END_REF]). After weight loss in obese and diabetic patients, there is a decrease in the expression of NLRP3 and IL1β in adipose tissue. These beneficial effects could be due to the fact that the loss of NLRP3 induces a shift of macrophages from an M1 phenotype to an M2 phenotype (Nan Wang, Hongwei Liang, and Ke Zen 2014).

In addition, inflammasome-activated IL1β is implicated in type 2 diabetes [START_REF] Maedler | Glucose-Induced β Cell Production of IL-1β Contributes to Glucotoxicity in Human Pancreatic Islets[END_REF]. Indeed, this interleukin contributes to the deleterious effects of high concentrations of glucose by causing in particular the death of pancreatic β cells. Clinical trials in type 2 diabeteic patients have shown that by blocking the effects of IL1β using IL1RA, it is possible to improve insulin secretion by β cells and blood sugar levels [START_REF] Larsen | Interleukin-1-Receptor Antagonist in Type 2 Diabetes Mellitus[END_REF][START_REF] Donath | Type 2 Diabetes as an Inflammatory Disease[END_REF][START_REF] Donath | Inflammation in Obesity and Diabetes: Islet Dysfunction and Therapeutic Opportunity[END_REF]).

PI3K/Akt signaling pathway in macrophage

The family of phosphatidylinositol [START_REF] Fardini | O-GlcNAcylation: A New Cancer Hallmark?[END_REF]4,[START_REF] Ma | Hyper-O-GlcNAcylation Is Anti-apoptotic and Maintains Constitutive NF-kappaB Activity in Pancreatic Cancer Cells[END_REF]-trisphosphate kinases (PI3K) enzymes is largely responsible for the phosphorylation of phosphatidylinositol lipids in response to cellular stimuli. PI3K is a heterodimeric enzyme that consists of a regulatory (p85) and a catalytic (p110) subunit [START_REF] Cantley | The Phosphoinositide 3-Kinase Pathway[END_REF]. The exact molecular mechanism by which TLRs stimulate PI3K/Akt in macrophage is not fully understood. Several studies showed that TLR2, TLR3, TLR5, TIRAP and MyD88, can directly bind the p85 subunit of PI3K after tyrosine phosphorylation in overexpression studies, many of which were performed in nonhematopoietic cells [START_REF] Sarkar | Novel Roles of TLR3 Tyrosine Phosphorylation and PI3 Kinase in Double-Stranded RNA Signaling[END_REF][START_REF] Laird | TLR4/MyD88/PI3K Interactions Regulate TLR4 Signaling[END_REF][START_REF] Rhee | Role of MyD88 in Phosphatidylinositol 3-Kinase Activation by Flagellin/Toll-like Receptor 5 Engagement in Colonic Epithelial Cells[END_REF].

The PI3 kinase/Akt signaling pathway plays a critical role in macrophage proliferation, survival and migration. Early studies have reported that PI3-kinase plays a negative role in the process of macrophage activation. It has been reported that the inhibition of PI3K with Wortmannin or LY294002 in LPS-stimulated macrophage increases the amount of inducible nitric oxide synthase (NOS2). Therefore, it has been suggested that this enzyme might suppress the action of pro-inflammatory cytokines (Marı á J. et al. 1999). Moreover, activation of the PI3K/Akt pathway may play a critical role in the restriction of proinflammatory responses in LPS-stimulated macrophages, confirming its role as a gate-keeper that prevents excessive innate immune responses [START_REF] Fukao | PI3K and Negative Regulation of TLR Signaling[END_REF]. Indeed, several mechanisms have been shown to be implicated downstream of PI3K in the inhibition of TLR responses in macrophages.

Furthermore, Akt exerts a negative feedback via inhibition of the transcription factor forkhead box 1 (FoxO1) (Figure 16). FoxO1 is a member of the forkhead family of transcription factors which is composed of four members FoxO1, FoxO3, FoxO4 and FoxO6.

FoxO1 in particular plays a key role in glucose metabolism by promoting the expression of gluconeogenic enzymes in liver. Its activity is regulated by the PI3K/Akt pathway. In the absence of PI3K/Akt activation, Foxo1 is found in the nucleus bound to Foxo1 consensus DNA sequences. After activation of the PI3K pathway, active Akt phosphorylates Foxo1 on three amino acids (Thr24/Ser256/Ser319) resulting in the export of FoxO1 from the nucleus to the cytoplasm, causing the termination of Foxo1-dependent transcription (Review:

Eijkelenboom and Burgering 2013). Interestingly, nuclear export of Foxo1 has been implicated in terminating the transcription of several genes downstream of LPS-induced activation in macrophages via PI3K, Akt1. A study revealed that impairment of Akt phosphorylation by deletion of mTORC2 in dendritic cells led to hyperinflammatory response due to elevated FoxO1 nuclear levels [START_REF] Brown | Mammalian Target of Rapamycin Complex 2 (MTORC2) Negatively Regulates Toll-like Receptor 4-Mediated Inflammatory Response via FoxO1[END_REF]. Furthermore, in another study, it has been documented that the gene coding for the pro-inflammatory cytokine IL1β is a potential target of FoxO1 in cultured macrophage. The authors showed that FoxO1 synergistically interacts with NFκB p50 subunit to enhance the activity of the IL1β promoter luciferase reporter gene upon LPS stimulation (Su et al. 2009a). Moreover, Fan et al demonstrated that FoxO1 acts as a key molecular adaptor integrating inflammatory responses in LPS-stimulated macrophages. In this study, they revealed, using chromatin immunoprecipitation, that FoxO1 is bound to TLR4 gene and regulates its transcription in unstimulated macrophage. This regulation has been shown to be terminated upon activation of the PI3K-Akt pathway and subsequent FoxO1 phosphorylation (Fan et al. 2010). These data demonstrated the role of PI3/Akt via FoxO1 as a self-limiting mechanism of inflammation and the implication of this pathway in context of insulin resistance associated with a hyperinflammatory phenotype. Upon TLR4 activation, PI3K/AKT signalling pathway plays as a TLR4 self-limiting step via phosphorylation of the transcription factor FoxO1. Phosphorylated FoxO1 translocates from nucleus into cytoplasm where it is degraded. FoxO1 Nuclear translocation attenuates NFkB pro-inflammatory effect and thus terminates inflammatory process.

Macrophage phenotypes and their metabolic characteristics

Macrophages phenotypes M1/M2

Macrophages, which are related to myelomonocytic cell populations, are dynamic and heterogeneous cells. This is due to different mechanisms governing their differentiation, tissue distribution, and responsiveness to stimuli [START_REF] Gordon | Alternative Activation of Macrophages[END_REF][START_REF] Mosser | The Many Faces of Macrophage Activation[END_REF]. The heterogeneity of undifferentiated circulating monocytes may affect their polarization once they arrive in tissues [START_REF] Gordon | Monocyte and Macrophage Heterogeneity[END_REF][START_REF] Strauss-Ayali | Monocyte Subpopulations and Their Differentiation Patterns during Infection[END_REF]. Polarized macrophages have been broadly classified into two groups: M1 and M2 macrophages.

-Classically activated macrophages in response to T helper type 1 derived lymphokine stimuli (Th-1) such as tumor necrosis factor α (TNFα) and interferon γ (IFNγ) [START_REF] Nathan | IDENTIFICATION OF INTERFERON-3[END_REF] or to patterns conserved on the surface of pathogens (PAMPs) such as LPS (lipopolysacharride), polarize towards a proinflammatory type and are then called macrophages of type M1 (also called proinflammatory phenotype). These macrophages have a large tumoricidal and bactericidal capacity. They strongly express NOS2 (nitric oxide synthase) and therefore produce a large amount of nitric oxide •NO, necessary for the degradation of phagocytized particles. They also produce pro-inflammatory type cytokines such as IL6, TNFα, IL1β, IL12 and IL23.

-On the contrary, M2 macrophages have been characterized when Siamon Gordon team revealed that IL-4 (a Th-2 lymphocyte product) induces alternative activation of macrophage distinct from that induced by LPS and IFNγ. IL4 enhances the expression of the mannose receptor MMRs (CD206) and of the major histocompatibility class II antigen (MHC-II) in these macrophages but it inhibits pro-inflammatory cytokine production on their cell surface [START_REF] Thornberry | A Novel Heterodimeric Cysteine Protease Is Required for Interleukin-l FJ Processing in Monocytes[END_REF]. They appear to have a key role in tissue remodeling after the inflammatory process. They produce antiinflammatory cytokines like IL10 and TGFβ. M2 macrophages strongly express arginase 1 which inhibits the production of NO, by limiting the levels of available arginine [START_REF] Rath | Metabolism via Arginase or Nitric Oxide Synthase: Two Competing Arginine Pathways in Macrophages[END_REF]. This anti-inflammatory phenotype seems tightly linked to the activation of PPARγ (peroxisomal proliferator activator receptor γ).

Moreover, M1 and M2 macrophages differ in terms of receptors, cytokine and chemokine expression, and effector functions. Recently, macrophage activation has been shown to be plastic, rapid, and fully reversible, suggesting that macrophage populations are dynamic and may first take part in inflammation and then participate in its resolution [START_REF] Porcheray | Macrophage Activation Switching: An Asset for the Resolution of Inflammation[END_REF]. However, there are many intermediate types which neither correspond to the M1 nor to the M2 type [START_REF] Murray | Macrophage Activation and Polarization: Nomenclature and Experimental Guidelines[END_REF]. In addition, this classification established in vitro is not very representative of macrophages in vivo which often have mixed profiles and express both pro-inflammatory and anti-inflammatory markers [START_REF] Morris | The Answer Is 17 Years, What Is the Question: Understanding Time Lags in Translational Research[END_REF]. In addition, the same tissue can contain macrophages of type M1 and type M2 at the same time (review: Martinez and Gordon 2014).

.

Metabolic characteristics of M1 and M2 macrophage

To achieve their different functions (detecting and killing pathogens, clearing apoptotic cells and subcellular debris, triggering inflammation and initiating wound-healing programs) macrophages possess the capacity to acquire pro-inflammatory (M1 phenotype) and anti-inflammatory (M2 phenotype) programs in response to extrinsic signals. Macrophage activation/polarization is tightly linked to and controlled by their metabolic reprogramming (metabolic shifts) which gives rise to different phenotypes in response to changes resulting from changes in the microenvironment such as hypoxia, cytokines, pathogen-derived TLRligands, and lipid mediators [START_REF] Mills | Reprogramming Mitochondrial Metabolism in Macrophages as an Anti-Inflammatory Signal[END_REF].

How polarizing signals coordinate metabolic and functional reprogramming, and the potential implications for control of macrophage activation is described below.

Glycolysis dependent metabolism

As with most hematopoietic cells, glucose is the main energy source for monocytes and macrophages (Calder, Dimitriadis and Newsholme 2007) Classically activated, or M1 macrophages (Figure 18), in response to different stimuli such as hypoxia or bacterial products such as LPS, switch their metabolism from oxidative phosphorylation to glycolysis and lactate production (reminiscent of the Warburg effect in cancer cells) [START_REF] Ganeshan | Metabolic Regulation of Immune Responses[END_REF]. Enhanced glucose flux through glycolysis not only produces ATP as energy to sustain their high secretory and phagocytic functions but also feeds the pentose phosphate pathway (PPP) which leads to a significant production of ribulose-5-phosphate (R5P) and NADPH. Using a functional screening for kinases involved in M1 macrophage, Haschemi and his colleagues demonstrated that LPS downregulates CARKL (carbohydrate kinase like protein), a kinase that catalyses the production of sedoheptulose-7-phosphate (S7P), an intermediate of the non-oxidative activity of the pentose phosphate pathway (PPP). Constitutive expression of CARKL in primary macrophage repressed LPS induced pro-inflammatory cytokine production suggesting a critical role in M1 macrophage polarization. The authors revealed that CARKL down-regulation results in refocusing cellular metabolism in to oxidative activity of PPP which is caracterized by high redox state [START_REF] Haschemi | The Sedoheptulose Kinase CARKL Directs Macrophage Polarization through Control of Glucose Metabolism[END_REF] . Furthermore, M1 macrophages are characterized by upreregulation of the mitochondrial enzyme NADPH oxidase which catalyzes the production of reactive oxygene species. [START_REF] Tannahill | Succinate Is an Inflammatory Signal That Induces IL-1β through HIF-1α[END_REF].

Increased ROS levels have been reported to stabilize the transcription factor hypoxiainducible factor-1α (HIF-1α) in LPS-stimulated macrophage during normoxia. However, the mechanism by which ROS stabize HIF1α is unexplained [START_REF] Chandel | Reactive Oxygen Species Generated at Mitochondrial Complex III Stabilize Hypoxia-Inducible Factor-1α during Hypoxia: A MECHANISM OF O 2 SENSING[END_REF][START_REF] Sumbayev | LPS-Induced Toll-like Receptor 4 Signalling Triggers Cross-Talk of Apoptosis Signal-Regulating Kinase 1 (ASK1) and HIF-1α Protein[END_REF]). On the other hand, Tannahill et al revealed, through the use of metabolomics analysis, that despite mitochondrial respiration in M1 macrophage, TCA cycle intermediates fumarate, malate and succinate accumulated. Succinate, which is a glutamine metabolite, has also been shown to stabilize HIF1α through the impairment of the activity of prolyl hydroxylases (PHD), resulting in IL1β production [START_REF] Selak | Succinate Links TCA Cycle Dysfunction to Oncogenesis by Inhibiting HIF-α Prolyl Hydroxylase[END_REF]. In addition, succinate also stabilizes HIF1α indirectly via mediating ROS production. It has been shown that increased mitochondrial oxidation of succinate by succinate dehydrogenase (SDH) and elevation of mitochondrial membrane potential combine to drive mitochondrial reactive oxygen species (ROS) production [START_REF] Mills | Reprogramming Mitochondrial Metabolism in Macrophages as an Anti-Inflammatory Signal[END_REF]. Consequently, HIF-1α then accumulates in the nucleus, where it binds to short DNA sequences called hypoxia response elements (HREs) near oxygen-sensitive genes. This in turn results in induction of the expression of hundreds of genes, which regulate angiogenesis, metabolism, growth and survival [START_REF] Esser-Von Bieren | Antibodies Trap Tissue Migrating Helminth Larvae and Prevent Tissue Damage by Driving IL-4Rα-Independent Alternative Differentiation of Macrophages[END_REF]). Moreover, it has been revealed that in human monocytes derived macrophage, using a luciferase reporter gene, GLUT1 promoter activity is directly regulated by HIF1α [START_REF] Burke | Hypoxia-Induced Gene Expression in Human Macrophages[END_REF]. Moreover, Tannahill et al demonstrated that upon LPS treatment, using ChIP-PCR analysis, HIF1α binding on IL1β promoter was increased. And finally, blocking of glycolysis by 2-deoxyglucose inhibited the secretion of IL1β whereas TNFα secretion was not affected in myeloid derived macrophage [START_REF] Tannahill | Succinate Is an Inflammatory Signal That Induces IL-1β through HIF-1α[END_REF]). Furthermore, it has been reported that using genetic and pharmacological approaches, HIF1α rather than NF-kB is the main mediator for pro-inflammatory response of macrophage during hypoxia [START_REF] Fang | Hypoxia-Inducible Factors 1 and 2 Are Important Transcriptional Effectors in Primary Macrophages Experiencing Hypoxia[END_REF]). On the other hand, macrophages infected with bacteria and mice subjected to hypoxia showed a defective HIF-1α expression upon deletion of the IKKβ-encoding gene [START_REF] Rius | NF-ΚB Links Innate Immunity to the Hypoxic Response through Transcriptional Regulation of HIF-1α[END_REF]. This suggests a cross talk between hypoxia and innate immune response. In addition, the M1 marker NOS2 (inducible nitric oxide synthase 2) has been identified among the transcriptional targets of HIF-1α under hypoxia [START_REF] Peyssonnaux | HIF-1α Expression Regulates the Bactericidal Capacity of Phagocytes[END_REF]. NOS2 mediates nitric oxide •NO synthesis that is an important mediator for macrophage bactericidal activity (•NO production and function that will be described later in this chapter).

β oxidation dependent metabolism

Conversely to M1 macrophage, M2 macrophages differentiated with the antiinflammatory cytokine interleukin-4 (IL-4) and interleukin 13 (IL-13) induce a transcriptional program associated with mitochondrial biogenesis and oxidative mitochondrial metabolism [START_REF] Vats | Oxidative Metabolism and PGC-1β Attenuate Macrophage-Mediated Inflammation[END_REF]. Pharmacological targeting of oxidative phosphorylation reprogramming perturbs the acquisition of anti-inflammatory phenotypes and/or wound-healing functions of macrophage (Odegaard et al. 2007a). Moreover, M2 type macrophages have an oxidative type of glucose metabolism (Krebs cycle). Thus, the induction of oxidative metabolism in macrophages of the M1 type helps to polarize them into macrophages of the M2 type [START_REF] Vats | Oxidative Metabolism and PGC-1β Attenuate Macrophage-Mediated Inflammation[END_REF][START_REF] O'neill | Metabolic Reprograming in Macrophage Polarization[END_REF].

Peroxisome proliferator activated receptors (PPARs) are lipid activated transcription factors of the nuclear hormone receptor superfamily. Endogenous ligands that regulate the PPAR family remain poorly characterized and include free native and modified fatty acids. In mammals, there are three PPAR isoforms (PPARα,γ, δ ) expressed in a large variety of cells and playing pleiotropic functions in fatty acid metabolism including transport, synthesis, storage, mobilisation and oxidation [START_REF] Chawla | Control of Macrophage Activation and Function by PPARs[END_REF].

Binding of IL-4 or IL-13 to their receptors, IL-4Rα/IL-2R γ and IL-4Rα/IL-13Rα1, respectively, initiates a cytoplasmic signaling cascade leading to tyrosine phosphorylation of signal transducers and activators of transcription-6 (STAT6). Phosphorylated STAT6 dimerizes and translocates to the nucleus, where it induces the expression of numerous target genes including markers Arg1(arginase 1), Chi3l3 (Chitinase 3-like-3), Mrc1(C-type mannose receptor 1), Mgl1(The macrophage galactose-type lectin-1), and Retnla (resistin-like alpha) and regulators PPARγ, PPARδ, and PPARγ coactivator 1β (PGC-1β) of alternatively activated macrophages (Odegaard et al. 2007a;2008). PPARγ in turn mediates the expression of the main M2 phenotype markers such as fatty acid translocase CD36 which regulates the uptake of fatty acids and their subsequent metabolism serves elevated mitochondrial oxidative phosphorylation (OXPHOS) (S. C.-C. [START_REF] Huang | Cell-Intrinsic Lysosomal Lipolysis Is Essential for Alternative Activation of Macrophages[END_REF]. Furthermore, PPARγ also blocks the expression of numerous pro-inflammatory mediators induced by LPS and IFN-γ such as IL-1β and NOS2. PPARγ KO mice deficient in M2 macrophages developed spontaneous chronic inflammation in lung [START_REF] Malur | Deletion of PPARγ in Alveolar Macrophages Is Associated with a Th-1 Pulmonary Inflammatory Response[END_REF]). Interestingly, these mice were more susceptible to obesity and insulin resistance. The authors suggested that homeostatic functions performed by M2 macrophages might allow animals to more efficiently store and oxidize incoming lipids, thereby maintaining insulin sensitivity and glucose tolerance by attenuating inflammation (Odegaard et al. 2007b).

Congruent with this notion, deletion of PPARγ in macrophages morbidly impairs their ability to induce oxidative metabolism, as evidenced by reduced rates of β-oxidation of fatty acids and the blunted mitochondrial biogenic response. Consequently, PPARγ null macrophages are unable to perform this metabolic shift, which is required for full expression of the alternative phenotype, both in vitro and in vivo (Figure 19).

L-arginine metabolism

Amino acid catabolism also represents a key mechanism whereby M1 and M2-type macrophages exert their anti-microbial and immune-regulatory roles.

In addition to a switch between glycolytic versus oxidative phosphorylation dependent fuel supply, there is also an established difference between M1 and M2 macrophages is the way by which they metabolize L-arginine (Figure 20). L-arginine is a non-essential amino acid. The average arginine ingestion with a Western style diet is around 4-5 g/day and the normal plasma arginine level is 100-200μM. Besides dietary intake, arginine is derived from cellular protein break down or endogenous de novo arginine production.

L-arginine elevated catabolism has been linked to immune-regulation and antimicrobial immunity (review [START_REF] Rath | Metabolism via Arginase or Nitric Oxide Synthase: Two Competing Arginine Pathways in Macrophages[END_REF].

As mentioned previously, M2 macrophages are characterized by the expression of the enzyme arginase1. L-arginine is used as a substrate for Arginase 1 (Arg1) to produce ornithine and urea. Ornithine itself can further feed into the downstream pathways of polyamine and proline synthesis, which are important for cellular proliferation and tissue repair. It has been reported that increased L-arginine metabolism is an immune escape strategy adopted by pathogens resulting in disease progression [START_REF] Butcher | Toxoplasma Gondii Rhoptry Kinase ROP16 Activates STAT3 and STAT6 Resulting in Cytokine Inhibition and Arginase-1-Dependent Growth Control[END_REF][START_REF] Osorio | Progressive Visceral Leishmaniasis Is Driven by Dominant Parasite-Induced STAT6 Activation and STAT6-Dependent Host Arginase 1 Expression[END_REF]. As an example, active induction of Arg1 gene transcription in infected macrophages through modulation of the STAT6 signaling pathway has been demonstrated for both Toxplasma gondii and Leishmania donovanii [START_REF] Ong | Toxoplasma Rhoptry Protein 16 (ROP16) Subverts Host Function by Direct Tyrosine Phosphorylation of STAT6[END_REF][START_REF] Butcher | Toxoplasma Gondii Rhoptry Kinase ROP16 Activates STAT3 and STAT6 Resulting in Cytokine Inhibition and Arginase-1-Dependent Growth Control[END_REF][START_REF] Osorio | Progressive Visceral Leishmaniasis Is Driven by Dominant Parasite-Induced STAT6 Activation and STAT6-Dependent Host Arginase 1 Expression[END_REF]). The discovery that T cells are particularly sensitive to local arginine concentrations has further confirmed the important immunoregulatory role of Arg1, able therefore to inhibit both innate (•NO production) and adaptive immune responses to parasites [START_REF] Modolell | Local Suppression of T Cell Responses by Arginase-Induced L-Arginine Depletion in Nonhealing Leishmaniasis[END_REF][START_REF] Munder | L -Arginine Deprivation Impairs Leishmania Major -Specific T-Cell Responses: Immunity to Infection[END_REF]). In addition to its immune-regulatory role, increased arginine metabolism has been shown to directly promote intracellular growth of several Leishmania species via accumulation of polyamines [START_REF] Iniesta | Arginase I Induction during Leishmania Major Infection Mediates the Development of Disease[END_REF].

M1 macrophages (after stimulation with LPS or/and IFNγ) express the enzyme nitric oxide synthase, which metabolizes arginine into nitric oxide (•NO) and citrulline. •NO can be metabolized to further downstream reactive nitrogen species, while citrulline might be reused for efficient •NO synthesis via the citrulline-•NO cycle. Nitric oxide is the most potent bactericides secreted by macrophages. •NO plays a critical role in macrophage as described below.

Figure 20: schematic presentation of L-arginine metabolism in macrophage polarization

Role of nitric oxide (•NO) in the inflammation

•NO is one of the factors allowing pathogens to be eliminated by macrophages through its bactericidal effect. In macrophages, as mentioned, •NO is produced by the inducible nitric oxide synthase or iNOS enzyme also called NOS2. This enzyme was discovered in macrophages [START_REF] Xie | Cloning and Characterization of Inducible Nitric Oxide Synthase from Mouse Macrophages[END_REF]) and is also expressed in muscle, liver and adipocytes.

In the basal state, this enzyme is practically not expressed. However, many stimuli induce the expression of this enzyme. For example, the amount of NOS2 is greatly increased in response to pro-inflammatory cytokines, while anti-inflammatory cytokines inhibit the synthesis of NOS2. LPS, via TLR4 and NFκB, very strongly stimulates the expression of NOS2. In order to form •NO, NOS2 has to dimerize. When NOS2 is activated the •NO levels can be up to 1000 times higher than those observed in basal condition. •NO is a gas involved in many functions of the body. It thus participates in vasodilation and scarring, and plays a key role in the non-specific immune response to infection. Indeed, •NO allows the elimination of bacteria as well as the degradation of phagocytosed particles by macrophages.

In the presence of O2, •NO establishes a covalent bond with the cysteine residues of proteins and thus form S-nitrosothiol, resulting in a reversible post-translational modification called Snitrosylation (or S-nitrosation). This post-translational modification is found in mammals as well as in plants and bacteria. It is involved in many cellular functions such as protein localization, protein-protein interactions and the regulation of enzyme activities. Snitrosylation does not appear to be mediated by an enzyme. On the other hand, the reverse process, de-nitrosylation, can be non-enzymatic as well as being mediated by various enzymes such as S-Nitrosoglutathione reductase (GSNOR) (J. S.-H. [START_REF] Liu | Expression of Inducible Nitric Oxide Synthase and Nitrotyrosine in Multiple Sclerosis Lesions[END_REF]) and thioredoxin (Trx) [START_REF] Benhar | Regulated Protein Denitrosylation by Cytosolic and Mitochondrial Thioredoxins[END_REF][START_REF] Sengupta | Thioredoxin Catalyzes the Denitrosation of Low-Molecular Mass and Protein S-Nitrosothiols †[END_REF]. Since •NO is not very stable, it is difficult to measure it directly. Thus, the variations in •NO are evaluated by measuring the levels of the stable products of •NO: nitrite (NO2) and nitrate (NO3). These products are increased in both type 2 diabetes and obese patients.

However, this can be corrected by weight loss and is positively correlated with improved insulin sensitivity [START_REF] Lin | Nitric Oxide Production Is Paradoxically Decreased after Weight Reduction Surgery in Morbid Obesity Patients[END_REF]). In addition, by measuring NO2 and NO3, it has been shown indirectly that in diabetic rats the plasma and urinary levels of NO are high compared to control rats [START_REF] Bank | Role of EDRF (Nitric Oxide) in Diabetic Renal Hyperfiltration[END_REF]. The secretion of •NO by macrophages in response to LPS is significantly increased in the event of hyperglycemia. The mechanism inducing the increase in NOS2 in response to glucose remains poorly understood. However, it could be an effect of the activation of PKC, p38 and NFκB by high glucose concentrations [START_REF] Hua | High Glucose Increases Nitric Oxide Generation in Lipopolysaccharide-Activated Macrophages by Enhancing Activity of Protein Kinase C-α/δ and NF-ΚB[END_REF]).

An increase in the production of •NO, in response to fatty diets or to pharmacological donors, leads to S-nitrosylation of the insulin receptor, IRS1 and Akt in the muscle of mice, which induces insulin resistance [START_REF] Carvalho-Filho | S-Nitrosation of the Insulin Receptor, Insulin Receptor Substrate 1, and Protein Kinase B/Akt: A Novel Mechanism of Insulin Resistance[END_REF]. Indeed, the S-nitrosylation of the proteins of the insulin signaling pathway (notably Akt) leads to their degradation or to the inhibition of their function [START_REF] Charbonneau | Inducible Nitric Oxide Synthase Induction Underlies Lipid-Induced Hepatic Insulin Resistance in Mice: Potential Role of Tyrosine Nitration of Insulin Signaling Proteins[END_REF]. Thus, IRS1 is ubiquitinated and degraded by the proteasome in the presence of •NO, which leads to a decrease in the protein level of IRS1 [START_REF] Sugita | Inducible Nitric-Oxide Synthase and NO Donor Induce Insulin Receptor Substrate-1 Degradation in Skeletal Muscle Cells[END_REF]).

An increase in S-nitrosylation of the insulin receptor and of Akt has been observed in the adipocytes of wild-type mice submitted to a high fat diet or of ob/ob mice, as well as in obese patients. The increase in S-nitrosylation correlates with BMI and seems to be linked both to the increase in expression of NOS2 and to the decrease in protein levels of thioredoxine reductase [START_REF] Ovadia | Increased Adipocyte S -Nitrosylation Targets Anti-Lipolytic Action of Insulin: RELEVANCE TO ADIPOSE TISSUE DYSFUNCTION IN OBESITY[END_REF]. Using 3 different approaches in aged-mice (KO mice for NOS2, mice treated with an inhibitor of NOS2 or mice whose level of expression of NOS2 was decreased by exercise) it has been shown that these mice were protected from insulin resistance by reducing aging induced-protein S-nitrosylation [START_REF] Ropelle | Targeted Disruption of Inducible Nitric Oxide Synthase Protects Against Aging, S-Nitrosation, and Insulin Resistance in Muscle of Male Mice[END_REF]).

•NO is responsible for two opposite effects on inflammation. •NO induces the activation of NFκB at early times of activation of murine macrophages by LPS, whereas in the longer term, •NO inhibits NFκB [START_REF] Connelly | Biphasic Regulation of NF-ΚB Activity Underlies the Pro-and Anti-Inflammatory Actions of Nitric Oxide[END_REF]. S-Nitrosylation of the p65 subunit of NFκB induces a decrease in its DNA binding activity, thus making it possible to attenuate inflammation [START_REF] Kelleher | NOS2 Regulation of NF-ΚB by S -Nitrosylation of P65[END_REF]). Experiments carried out on KO mice for NOS2 have shown that NOS2 also inhibits the beneficial effects of PPARγ activation on the insulin response, in particular by decreasing the expression of adiponectin [START_REF] Dallaire | Obese Mice Lacking Inducible Nitric Oxide Synthase Are Sensitized to the Metabolic Actions of Peroxisome Proliferator-Activated Receptor-Agonism[END_REF].

Another team has demonstrated that, using a macrophage and adipocyte co-culture model, pro-inflammatory macrophages recruited from adipose tissue secrete •NO, which in turn induces S-Nitrosylation of PPARγ in adipocytes. S-nitrosylation of PPARγ on Cys168 prevents its binding to the promoter of its target genes and also leads to the degradation of PPARγ by the proteasome [START_REF] Yin | Pro-Inflammatory Macrophages Suppress PPARγ Activity in Adipocytes via S-Nitrosylation[END_REF].

The protein S-nitrosylation, which is increased in the event of overproduction of •NO, as well as the decrease in denitrosylation, thus constitute molecular mechanisms linking inflammation and insulin resistance.

Macrophage response during metabolic syndrome

Several studies have demonstrated monocyte deregulation associated with metabolic diseases. Obese mice have a greatly increased number of circulating pro-inflammatory (CD16 +) monocytes [START_REF] Tsou | Critical Roles for CCR2 and MCP-3 in Monocyte Mobilization from Bone Marrow and Recruitment to Inflammatory Sites[END_REF]. In obese and diabetic patients, there is also an increase in the number of pro-inflammatory monocytes, which is quickly corrected by weight loss after surgery (Poitou et al. 2011). The increase in the number of monocytes could be due to the fact that obesity stimulates myelopoiesis and thus increases the production of monocytes in the bone marrow. This is due to the overproduction of IL1β in obese individuals, through activation of TLR4 pathway and the NLRP3 inflammasome. IL1β binding to its receptor on the surface of myeloid progenitors stimulates the production of neutrophils and monocytes [START_REF] Nagareddy | Adipose Tissue Macrophages Promote Myelopoiesis and Monocytosis in Obesity[END_REF]. Furthermore, in obese patients, the expression of the chemokine receptor CCR2 on the surface of the monocytes is greatly increased, which play a critical role in the establishment of an inflammatory response resulting in a greater chemotactic activity and increase in the migration of monocytes. This increase in the migration capacity of monocytes is also correlated with insulin resistance [START_REF] Krinninger | Peripheral Monocytes of Obese Women Display Increased Chemokine Receptor Expression and Migration Capacity[END_REF].

This increased number of pro-inflammatory monocytes having a high migration capacity allows in turn an increased recruitment of monocytes in different tissues and these convert into pro-inflammatory macrophages. The recruitment of macrophages in these tissues and its consequences will be discussed below.

Macrophages in adipose tissue

Adipose tissue has long been considered a simple place to store nutrients. However, it is a complex and active tissue capable, for example, of secreting many molecules such as adipokines. In addition to adipocytes, many different cells make up adipose tissue, such as pre-adipocytes, endothelial cells and immune cells.

Macrophages represent 5 to 10% of adipose tissue cells in thin subjects, but can represent up to 50% of all cells in hypertrophic adipose tissue of obese mice and human [START_REF] Weisberg | Obesity Is Associated with Macrophage Accumulation in Adipose Tissue[END_REF][START_REF] Ferrante | The Immune Cells in Adipose Tissue[END_REF]. The proportion of macrophages in adipose tissue is thus positively correlated with BMI (Vieira-Potter 2014). Macrophages of adipose tissue are the main source of cytokines in this tissue [START_REF] Weisberg | Obesity Is Associated with Macrophage Accumulation in Adipose Tissue[END_REF].

Thus, the infiltration of macrophages into adipose tissue seems to play a major role in the inflammation associated with metabolic diseases and has therefore been widely studied [START_REF] Boutens | Adipose Tissue Macrophages: Going off Track during Obesity[END_REF][START_REF] Ferrante | The Immune Cells in Adipose Tissue[END_REF]).

Macrophages in adipose tissue of lean subjects

In thin subjects the adipose tissue secretes a large amount of IL4, which maintains an anti-inflammatory phenotype of type M2 macrophages [START_REF] Wu | Eosinophils Sustain Adipose Alternatively Activated Macrophages Associated with Glucose Homeostasis[END_REF]. "Thin" adipose tissue also produces a large amount of adiponectin, which helps in maintaining M2 polarization and has a positive role in insulin sensitivity [START_REF] Castoldi | The Macrophage Switch in Obesity Development[END_REF]. In mice, global or macrophage specific PPARδ KO inhibits the polarization of the macrophages towards an antiinflammatory type M2. This leads to inflammation and insulin resistance and contributes to glucose intolerance (K. [START_REF] Kang | Adipocyte-Derived Th2 Cytokines and Myeloid PPARδ Regulate Macrophage Polarization and Insulin Sensitivity[END_REF]. M2 Macrophages represent the major source of IL10 in adipose tissue, since they produce 100 times IL10 more than adipocytes. Several studies suggest that this cytokine has beneficial effects on the insulin response of adipocytes.

The expression of this cytokine improves glucose uptake in response to insulin in adipocytes of the 3T3-L1 line [START_REF] Lumeng | Obesity Induces a Phenotypic Switch in Adipose Tissue Macrophage Polarization[END_REF]. The overexpression of IL10 by adenoviral strategy improves the insulin sensitivity of mice placed both on a standard diet and on a diet rich in fat. Thus, IL10 produced by macrophages of adipose tissue, in addition to its anti-inflammatory role, seems to improve the signaling of insulin in adipocytes [START_REF] Fujisaka | Regulatory Mechanisms for Adipose Tissue M1 and M2 Macrophages in Diet-Induced Obese Mice[END_REF]).

Macrophage in adipose tissue of obese patients

In a schematic view, macrophages of the adipose tissue of obese patients are of the pro-inflammatory type whereas those of the "healthy" adipose tissue are rather of the antiinflammatory type. The macrophages of the adipose tissue of obese patients indeed exhibit a strong activation of pro-inflammatory pathways, which leads to a strong secretion of cytokines such as TNF-α and IL1β [START_REF] Olefsky | Macrophages, Inflammation, and Insulin Resistance[END_REF]. The pro-inflammatory macrophages of adipose tissue very strongly express the CD11c differentiation marker, which is a pro-inflammatory marker. The number of CD11c + macrophages in adipose tissue is also negatively correlated with insulin sensitivity [START_REF] Fujisaka | Regulatory Mechanisms for Adipose Tissue M1 and M2 Macrophages in Diet-Induced Obese Mice[END_REF]. In mice, a conditional KO of CD11c positive cell helps to reduce the inflammation of adipose tissue and improve glucose tolerance [START_REF] Patsouris | Ablation of CD11c-Positive Cells Normalizes Insulin Sensitivity in Obese Insulin Resistant Animals[END_REF]. Furthermore, in the event of an excessive nutritional intake, the adipocytes become hypertrophic, and present local hypoxia, which promotes proinflammatory signaling pathways in the adipocytes with, in particular, the activation of the NFκB pathway [START_REF] Hosogai | Adipose Tissue Hypoxia in Obesity and Its Impact on Adipocytokine Dysregulation[END_REF][START_REF] Sell | Adaptive Immunity in Obesity and Insulin Resistance[END_REF]. Adipocytes also secrete chemokines such as MCP1 (chemotactic protein-1 monocytes), leukotriene B4 and RANTES [START_REF] Meshkani | Tissue Resident Macrophages: Key Players in the Pathogenesis of Type 2 Diabetes and Its Complications[END_REF]. This secretion of chemokines will set up a chemo-attractor gradient which attracts the monocytes into the adipose tissue, where they will then differentiate into macrophages. The macrophages recruited in adipose tissue will in turn produce chemokines which will help attract monocytes and result in recruitment of additional macrophages.

MCP1 (also called CCL2) is a chemokine which binds to the surface receptor CCR2 (for C-C chemokine receptor 2) of monocytes and thus contributes to their recruitment in the tissues where they then become macrophages. MCP1 is produced in large quantities by hypertrophic adipocytes (Figure 15). KO mice for CCR2 have low levels of circulating inflammatory monocytes and are protected from adipose tissue inflammation associated with obesity [START_REF] Obstfeld | C-C Chemokine Receptor 2 (CCR2) Regulates the Hepatic Recruitment of Myeloid Cells That Promote Obesity-Induced Hepatic Steatosis[END_REF][START_REF] Weisberg | CCR2 Modulates Inflammatory and Metabolic Effects of High-Fat Feeding[END_REF]. Conversely, transgenic mice overexpressing MCP1 in adipocytes have an increased number of macrophages in adipose tissue, associated with the development of insulin resistance [START_REF] Kamei | Overexpression of Monocyte Chemoattractant Protein-1 in Adipose Tissues Causes Macrophage Recruitment and Insulin Resistance[END_REF]). In addition, preclinical studies seem to indicate that CCR2 antagonists block the migration of monocytes and thus improve obesity and its consequence (Y. S. [START_REF] Kang | Novel C-C Chemokine Receptor 2 Antagonists in Metabolic Disease: A Review of Recent Developments[END_REF][START_REF] Tamura | C-C Chemokine Receptor 2 Inhibitor Improves Diet-Induced Development of Insulin Resistance and Hepatic Steatosis in Mice[END_REF].

Other chemokines are overexpressed in mice on a fatty diet such as CCL3, CCL6, CCL7, CCL8, and CCL9, and could therefore also participate in the excessive recruitment of monocytes in adipose tissue [START_REF] Jiao | Obesity-Related Upregulation of Monocyte Chemotactic Factors in Adipocytes: Involvement of Nuclear Factor-B and c-Jun NH2-Terminal Kinase Pathways[END_REF].

Furthermore, a recent study has shown that, unlike the majority of macrophages of other tissues which are non-proliferative cells, macrophages of adipose tissue have the possibility of dividing in response to their activation [START_REF] Haase | Local Proliferation of Macrophages in Adipose Tissue during Obesity-Induced Inflammation[END_REF]). Dividing macrophages are found mainly in crown like structures, which are microscopic foci of dying adipocytes surrounded by macrophages mostly derived from blood monocytes), and their proliferation rate is increased both in mice on a fatty diet and in obese patients. In addition, this study seems to show that MCP1 is involved in this process.

Macrophages in liver

Resident liver macrophages are called Kupffer cells. They represent about 15% of all liver cells and 80 to 90% of tissue resident macrophages [START_REF] Bouwens | Quantitation, Tissue Distribution and Proliferation Kinetics of Kupffer Cells in Normal Rat Liver[END_REF]). The proinflammatory pathways are strongly activated in these cells in obese individuals, although the number of Kupffer's cells does not seem to have changed [START_REF] Cai | Local and Systemic Insulin Resistance Resulting from Hepatic Activation of IKK-β and NF-ΚB[END_REF]. The depletion of Kupffer cells in the liver of mice placed on a fatty diet reduces the amount of hepatic TNFα, which prevents the development of steatosis while improving insulin signaling (W. [START_REF] Huang | Depletion of Liver Kupffer Cells Prevents the Development of Diet-Induced Hepatic Steatosis and Insulin Resistance[END_REF]). Kupffer's cells can also change polarization from an inflammatory type to an antiinflammatory type. Thus, in response to IL4, the Kupffer cells move towards an alternative phenotype via the activation of PPARδ [START_REF] Odegaard | Alternative M2 Activation of Kupffer Cells by PPARδ Ameliorates Obesity-Induced Insulin Resistance[END_REF]. The removal of PPARδ also leads to the development of steatosis and insulin resistance (K. [START_REF] Kang | Adipocyte-Derived Th2 Cytokines and Myeloid PPARδ Regulate Macrophage Polarization and Insulin Sensitivity[END_REF].

Physiologically, Kupffer's cells appear to secrete IL10, which has beneficial effects on the metabolism of hepatocytes [START_REF] Clementi | Loss of Kupffer Cells in Diet-Induced Obesity Is Associated with Increased Hepatic Steatosis, STAT3 Signaling, and Further Decreases in Insulin Signaling[END_REF]).

In addition to the Kupffer cells which reside physiologically in the liver, in the event of metabolic diseases, there is a recruitment of macrophages in this organ via the secretion of MCP1 which leads to recruitment of the monocytes from the circulating blood [START_REF] Obstfeld | C-C Chemokine Receptor 2 (CCR2) Regulates the Hepatic Recruitment of Myeloid Cells That Promote Obesity-Induced Hepatic Steatosis[END_REF]. TNF α and IL-6 generated by recruited macrophages contribute to the dysfunction of hepatocytes [START_REF] Baffy | Kupffer Cells in Non-Alcoholic Fatty Liver Disease: The Emerging View[END_REF]. Finally, depletion studies of all liver macrophages by different approaches have demonstrated that macrophages are key players in hepatic insulin resistance [START_REF] Lanthier | Kupffer Cell Activation Is a Causal Factor for Hepatic Insulin Resistance[END_REF][START_REF] Neyrinck | Critical Role of Kupffer Cells in the Management of Diet-Induced Diabetes and Obesity[END_REF].

A human study also found that an increase in the number of Kupffer cells was the first observable difference in liver biopsies from steatosis patients compared to biopsies of healthy livers. In addition, the multiplication of these macrophages precedes the recruitment of other inflammatory cells such as T and B lymphocytes. This study suggests the involvement of the activation of Kupffer cells in the initiation of fatty liver disease [START_REF] Gadd | The Portal Inflammatory Infiltrate and Ductular Reaction in Human Nonalcoholic Fatty Liver Disease[END_REF]).

Macrophages in pancreas

Surprisingly, relatively few studies have looked at the role of macrophages in the pancreas. Under physiological conditions, macrophages are mainly located in the exocrine pancreas. They are however constitutively present inside the islets of Langerhans of the pancreas where they contribute to the development of the endocrine pancreas as well as to the maintenance of the homeostasis of the islets [START_REF] Banaei-Bouchareb | Insulin Cell Mass Is Altered in Csf1op/Csf1op Macrophage-Deficient Mice[END_REF]Carrero, Ferris, and Unanue 2016). Macrophages are the only myeloid cells of the endocrine pancreas and represent a very small cell population, i.e. 1 to 3% of all the cells of the islets [START_REF] Calderon | The Pancreas Anatomy Conditions the Origin and Properties of Resident Macrophages[END_REF]. However, several studies have shown the crucial contribution of islet macrophages in patients with type 2 diabetes, involving significant infiltration of macrophages in the islets of Langerhans in these subjects [START_REF] Ehses | Increased Number of Islet-Associated Macrophages in Type 2 Diabetes[END_REF][START_REF] Rhodes | Type 2 Diabetes-a Matter of b-Cell Life and Death?[END_REF]. This recruitment is enabled by the secretion of chemokines by pancreatic β cells, which has been shown to be increased both in response to glucose and to saturated fatty acids. Indeed, the infusion of palmitate in mice induces the secretion of chemokines such as MCP1 by the islets, and leads to the recruitment of macrophages in a manner dependent on TLR4 and MyD88. There are thus twice as many macrophages expressing TLR4 in the islets of diabetic patients as in healthy islets. While stimulation of the TLR4 pathway by LPS does not induce apoptosis of β cells, activation of this pathway in macrophages induces apoptosis of β cells. This can be done both when the macrophages and the β cells are in contact, as well as when these two cell types are at a distance [START_REF] Cucak | Macrophage Contact Dependent and Independent TLR4 Mechanisms Induce β-Cell Dysfunction and Apoptosis in a Mouse Model of Type 2 Diabetes'. Edited by Christina Bursill[END_REF]). In addition, the macrophages recruited from the islets of diabetic mice are of pro-inflammatory M1 phenotype, whereas under normal conditions the macrophages present are of M2 phenotype (K. [START_REF] Eguchi | Saturated Fatty Acid and TLR Signaling Link β Cell Dysfunction and Islet Inflammation[END_REF].

The macrophages recruited being of the M1 type [START_REF] Cucak | Macrophage Contact Dependent and Independent TLR4 Mechanisms Induce β-Cell Dysfunction and Apoptosis in a Mouse Model of Type 2 Diabetes'. Edited by Christina Bursill[END_REF], they produce cytokines, including TNF-α and IL1β. However, this latter cytokine is known to have two opposite effects on pancreatic β cells. In response to physiological and transient stimulation,

IL1β improves the secretion of insulin by β cells [START_REF] Dror | Postprandial Macrophage-Derived IL-1β Stimulates Insulin, and Both Synergistically Promote Glucose Disposal and Inflammation[END_REF]). On the other hand, when the expression of IL1β is chronically elevated, it induces the death of the pancreatic β cells [START_REF] Donath | Cytokine Production by Islets in Health and Diabetes: Cellular Origin, Regulation and Function[END_REF]). In addition, in response to stimulation by IL1β, pancreatic β cells produce more IL1β. The strong response of β cells to IL1β could be due to the very strong expression of the IL1β type 1 receptor by these cells. There is then a vicious cycle in which the IL1β self-induces its production by the β cell [START_REF] Böni-Schnetzler | Increased Interleukin (IL)-1β Messenger Ribonucleic Acid Expression in β-Cells of Individuals with Type 2 Diabetes and Regulation of IL-1β in Human Islets by Glucose and Autostimulation[END_REF]). In the islets of diabetic subjects, there is also deposits of amyloid plaques over the long term (after one year of diabetes in mice), which also contributes to the M1 polarization of macrophages and causes them produce IL1β. By blocking the IL1β receptor in human pancreatic β cells with IL1RA (Interleukin 1 receptor antagonist), or by neutralizing IL1β with blocking antibodies, it is possible to inhibit the production of cytokines and chemokines induced by stimulation with fatty acids [START_REF] Böni-Schnetzler | Free Fatty Acids Induce a Proinflammatory Response in Islets via the Abundantly Expressed Interleukin-1 Receptor I[END_REF].

All these studies demonstrate the role of macrophage recruitment in different tissues on inflammation associated with metabolic diseases and its deleterious effects on blood sugar and tissue sensitivity to insulin. Recent clinical trials have also shown that inhibiting the recruitment of macrophages, by inhibiting the action of MCP-1 thanks to molecules capable of binding to it and thus preventing its function (by using a chemokine CCL2/monocyte chemo-attractant protein-1 inhibitor called Emapticap pegol), or by blocking its receptor, has beneficial effects on blood sugar, which are largely due to an improvement in insulin secretion [START_REF] Menne | C-C Motif-Ligand 2 Inhibition with Emapticap Pegol (NOX-E36) in Type 2 Diabetic Patients with Albuminuria[END_REF][START_REF] Di Prospero | CCR2 Antagonism in Patients with Type 2 Diabetes Mellitus: A Randomized, Placebo-Controlled Study[END_REF]).

Effect of hyperglycemia on macrophages

The result of pro-inflammatory effects of macrophages on the pancreatic insulin secreting β cells and on insulin sensitivity in insulin targets tissues (liver and adipose tissues) leads to worsening of glycaemia. Hyperglycemia itself influences macrophage behavior resulting in a vicious circle that worsens the inflammation and glycaemia, which ends, with type 2 diabetes mellitus.

At present, the role of non-immunological stimuli such as glucose on the activation of macrophages is still poorly understood (Martinez and Gordon 2014b).

Numerous studies carried out on cell lines have shown that monocytes or macrophages exposed to high concentrations of glucose for a prolonged period will develop a phenotype of the pro-inflammatory type. Thus, exposure of THP1 cells, a human monocyte cell line, to 15 mM of glucose for 72 hours, increases the expression of the pro-inflammatory cytokines IL1β and TNFα genes as well as that of the chemokine MCP1, in a NFκB signaling dependent manner [START_REF] Shanmugam | High Glucose-Induced Expression of Proinflammatory Cytokine and Chemokine Genes in Monocytic Cells[END_REF]). In the same cell type, high glucose concentrations also induce an increase in the expression of TLR2 and TLR4, which is at least partly mediated by PKC [START_REF] Dasu | High Glucose Induces Toll-Like Receptor Expression in Human Monocytes: Mechanism of Activation[END_REF]). In the murine macrophage line RAW264.7, high glucose concentrations do not seem to induce the production of inflammatory mediators, but potentiate the effects of stimulation by LPS, in particular by increasing the protein level of NOS2 and the production of •NO, as well as the secretion of IL1β. The mechanism of action of glucose on the potentiation of the effects of LPS seems to be dependent both on a transcriptional increase in activity NFκB and on an activation of PKC [START_REF] Hua | High Glucose Increases Nitric Oxide Generation in Lipopolysaccharide-Activated Macrophages by Enhancing Activity of Protein Kinase C-α/δ and NF-ΚB[END_REF].

However, in this same study, the levels of TNFα and IL6 secreted in response to LPS are decreased in response to glucose, suggesting complex effects of glucose on inflammation.

In vivo, chronic hyperglycemia also affects the phenotype of macrophages. Thus, two weeks after the development of diabetes induced by streptozotocin or alloxan, the peritoneal macrophages of the diabetic mice have a more marked pro-inflammatory profile than those of control mice [START_REF] Ptak | Macrophage Function in Alloxan Diabetic Mice: Expression of Adhesion Molecules, Generation of Monokines and Oxygen and NO Radicals[END_REF]Y. Wen et al. 2006). In addition, prolonged hyperglycemia in diabetic mice seems to prevent the action of IL4 to polarize macrophages towards an alternative type M2 [START_REF] Parathath | Diabetes Adversely Affects Macrophages During Atherosclerotic Plaque Regression in Mice[END_REF]. Another study supports pro-inflammatory effects.

Thus, murine peritoneal macrophages have mRNA levels of TNF-α, IL-1β, IL-6, and IL-12 increased in response to high glucose concentrations [START_REF] Pan | Inhibition of High Glucose-Induced Inflammatory Response and Macrophage Infiltration by a Novel Curcumin Derivative Prevents Renal Injury in Diabetic Rats: C66 Prevents HG-Induced Inflammatory Responses[END_REF]). In addition, patients with type 2 diabetes have significantly fewer monocytes expressing IL10 than control subjects [START_REF] Satoh | Unbalanced M1/M2 Phenotype of Peripheral Blood Monocytes in Obese Diabetic Patients: Effect of Pioglitazone[END_REF]. Finally, in hyperglycemic patients, an increase in the number of CD11c + macrophages expressing NOS2 is observed associated with a decrease in macrophages of the M2 type expressing arginase 1 and IL10. This seems to be due to a modification of the polarity of the macrophages which are more of a pro-inflammatory type in these patients [START_REF] Torres-Castro | Human Monocytes and Macrophages Undergo M1-Type Inflammatory Polarization in Response to High Levels of Glucose[END_REF]).

Short term versus long term macrophage exposure to hyperglycemia appears to alter macrophage behavior. Thus two weeks after the development of diabetes induced by streptozotocin or alloxan, the peritoneal macrophages of mice have a more marked proinflammatory profile than those of control mice [START_REF] Ptak | Macrophage Function in Alloxan Diabetic Mice: Expression of Adhesion Molecules, Generation of Monokines and Oxygen and NO Radicals[END_REF]Y. Wen et al. 2006).

However, after 4 months of diabetes and the establishment of chronic hyperglycemia, a decrease in the production of TNFα and IL6 is observed, although the secretion of •NO in response to LPS remains increased (C. [START_REF] Sun | The Phenotype and Functional Alterations of Macrophages in Mice with Hyperglycemia for Long Term[END_REF]. In a recent study in which BMDM were cultured in high glucose concentration either for short or long period, the authors observed that although BMDM in both conditions were of M1 phenotype, their proinflammatory response is different. They showed that long term high glucose exposed BMDM expressed IL1β and TNFα at levels similar to that short term exposed BMDM, but the expression of IL12 and •NO are diminished. Moreover, the phagocytic and bactericidal activities of macrophage are decreased in long-term cultured macrophage in high glucose and also in peritoneal macrophage issued from diabetic mice.

Taking all together, hyperglycaemia appears to have complex effect on the immune response and the clearance of pathogens by macrophages in diabetic subjects. A lack of glucose homeostasis can be an important key to macrophage deregulation in a hyperglycaemic environment under a variety of stimuli. This may increase the susceptibility to infections and elevated risk of developing complications in diabetic patients. Failures in inflammation resolution also contribute to the high rates of morbidity and mortality in diabetic subjects.

Chapter 3: O-N-acetylglucosaminylation and its regulation 3.1 Discovery of O-N-acetylglucosaminylation

To date over 450 types of post-translational modifications of proteins have been discovered (including acetylation, phosphorylation, methylation, ubiquitination, and glycosylation). These modifications can modulate protein activity, function, localization and stability [START_REF] Marshall | Discovery of a Metabolic Pathway Mediating Glucose-Induced Desensitization of the Glucose Transport System. Role of Hexosamine Biosynthesis in the Induction of Insulin Resistance[END_REF]. Phosphorylation is probably one of the most studied post-translational modifications. However, O-N-acetylglucosaminylation (O-GlcNAcylation), which refers to the addition of β-D-N-acetylglucosamine (GlcNAc) units linked to serine or threonine residues of intracellular proteins, has been gaining increasing attention in recent years.

Prior to its discovery, it was believed that proteins were glycosylated in the endoplasmic reticulum and Golgi apparatus, and then either transported outside of the cell or confined in intracellular compartments after processing.

In 1984, Torres and Hart discovered this new type of glycosylation. The authors were able to detect the O-GlcNAc modification sites on cell-surface proteins of lymphocytes specifically on the inner side of the plasma membrane. Other teams showed that Nacetylglucosamine is added to serine residues and probably also to threonine residues of proteins (Hanover et al. 1987, Holt et al. 1987). Subsequent studies have confirmed this observation by demonstrating that nuclear pore-associated proteins (Holt et al. 1992;Forbes et al.1992) and chromatin proteins (Kelly and Hart. 1989) are modified by O-GlcNAcylation.

The authors then showed that O-GlcNAc is ubiquitous and abundant on nuclear and cytosolic proteins of virtually all eukaryotes, including protozoans (Ortega- Barria et al. 1990;Dieckmann-Schuppert etal. 1993, 1994;Stanley et al. 1995) and fungi (Machida and Jigami, 1994). Two decades later, some mitochondrial proteins were also found to be O-GlcNAcylated in cardiac myocytes in response to high glucose exposure (Yong Hu et al.

2008).

Since its discovery, a large number of proteins with different biological functions have been shown to be modified by O-GlcNAc, such as nuclear pore-associated proteins, RNA polymerase II (Kelly et al. 1993), proteasome proteins, and transcription factors. To date, more than 4000 proteins have been identified as modified by O-GlcNAc (Junfeng Ma and Gerald W Hart 2014). This modification regulates virtually all important cellular processes, such as metabolism, cell cycle and apoptosis [START_REF] Hart | Cycling of O-Linked β-N-Acetylglucosamine on Nucleocytoplasmic Proteins[END_REF][START_REF] Slawson | O-GlcNAc Cycling: How a Single Sugar Post-Translational Modification Is Changing the Way We Think about Signaling Networks[END_REF].

General characteristics of O-GlcNAc glycocylation

O-GlcNAc glycocylation or O-GlcNAcylation is a reversible post-translational modification analogous to phosphorylation. Only one pair of enzymes is responsible for this modification. O-GlcNAc transferase (OGT) which uses uridine diphosphate Nacetylglucosamine or UDP-GlcNAc (the end product of the hexsamine biosynthetic pathway) as a substrate to carry out the addition of the N-acetylglucosamine (GlcNAc) motif on serine or threonine residues of its target proteins. The removal of this GlcNAc is catalyzed by O -GlcNAcase (OGA) (Figure 22).

The characteristics of these two enzymes will be described in more detail in the section (enzymes of O-GlcNAcylation). This glycosylation is considered to be atypical due to the characteristics described below:

(1) Its simple structure A single saccharide (N-acetylglucosamine or GlcNAc) is attached to the hydroxyl groups of serine or threonine residues of proteins. This monosaccharide is not lengthened into a more complex oligosaccharide (structure often having developed ramifications), as is the case with extracellular glycoproteins. Therefore, the apparent "shift" phenomenon in gel electrophoresis is generally not observed for proteins modified by O-GlcNAc since the GlcNAc motif, small and without charge, does not change the migration of proteins on a polyacrylamide gel (SDS-PAGE).

(

UDP GlcNAc is an integrator of the nutritional state

Uridine diphosphate N-acetylglucosamine or UDP-GlcNAc is the most abundant sugar nucleotide in mammalian cells [START_REF] Oikari | Borate-Aided Anion Exchange High-Performance Liquid Chromatography of Uridine Diphosphate-Sugars in Brain, Heart, Adipose and Liver Tissues[END_REF]. UDP-GlcNAc is the obligate substrate of OGT and is therefore necessary for O-GlcNAcylation. UDP-GlcNAc also serves as a GlcNAc donor for the de novo synthesis in the endoplasmic reticulum and in the Golgi apparatus, of secreted and membrane glycoproteins, as well as membrane glycolipids (Ghosh et al. 1960, Johnston et al. 1966).

UDP-GlcNAc synthesis is at the crossroad of several metabolic pathways: metabolism of glucose, amino acids (glutamine), fatty acids (Acetyl-CoA) and nucleotides (Uridine) (Wang et al. 1998). UDP-GlcNAc is synthesized through the hexosamine biosynthetic pathway (HBP) and can therefore be considered as an integrator of the nutritional environment of the cell (Figure 23).

The Hexosamines Biosynthetic Pathway (HBP)

Once the glucose enters inside the cell through a glucose transporter dependent mechanism, a small portion of glucose (about 2 to 3%) is directed to the hexosamine biosynthetic pathway [START_REF] Marshall | Discovery of a Metabolic Pathway Mediating Glucose-Induced Desensitization of the Glucose Transport System. Role of Hexosamine Biosynthesis in the Induction of Insulin Resistance[END_REF], which leads to the formation of UDP-GlcNAc. However, this percentage was estimated from a single study in rat adipocyte cultures. A very recent study demonstrated, in isolated murine heart perfused with glucose, that only 0,003 to 0,006% of glucose is directed in the hexosamine biosynthetic pathway [START_REF] Olson | First Characterization of Glucose Flux through the Hexosamine Biosynthesis Pathway (HBP) in Ex Vivo Mouse Heart[END_REF].

Indeed, after the phosphorylation of glucose by hexokinase (HK) to form glucose 6phosphate, the latter is then converted to fructose 6-phosphate (F6P) by glucose 6-phosphate isomerase (GP1). These first two steps are common for HBP and glycolysis and then both pathways diverge.

A fraction of this F6P is metabolized by glutamine fructose-6-phosphate amidotransferase (GFAT), the rate limiting enzyme of the hexosamine biosynthetic pathway, which uses glutamine to convert fructose-6-phosphate to glucosamine-6 phosphate [START_REF] Wells | A Role for N -Acetylglucosamine as a Nutrient Sensor and Mediator of Insulin Resistance[END_REF]. The glucosamine 6-phosphate then undergoes several successive enzymatic reactions. This results at the end in the formation of UDP-GlcNAc (Figure 24).

Glutamine-fructose amidotransferase (GFAT)

The GFAT consists of two domains : a glutaminase domain that catalyzes the hydrolysis of glutamine to glutamate and ammonia, and a synthase domain that allows the amination and isomerization of fructose 6-phosphate to glucosamine 6-phosphate [START_REF] Massière | The Mechanism of Glutamine-Dependent Amidotransferases[END_REF].

The gene coding for GFAT is located on chromosome 2p in humans and on chromosome 6 in mice (Jianxin [START_REF] Zhou | Human Glutamine: Fructose-6-Phosphate Amidotransferase: Characterization of MRNA and Chromosomal Assignment to 2p13[END_REF]. GFAT is expressed ubiquitously in whole body tissues.

GFAT isoforms

In 1999, a gene coding for a protein having about 75% homology with GFAT at the level of the amino acid sequence (75.6% and 74.7% homology to GFAT in humans and mice, respectively) was cloned (Oki et al. 1999). The product of this gene was called GFAT2 on the basis of sequence homology to GFAT and the first discovered form of GFAT was renamed GFAT1. The gene encoding human GFAT2 (called GFPT2) has been located on chromosome 5q in humans and that encoding mouse GFAT2 (Gfpt2) on chromosome 11 in mice. At the amino acid level, murine GFAT2 has 97.2% homology with human GFAT2. By Northern Blot, it has been shown that GFAT2 is expressed in the central nervous system, and mainly in the spinal cord. Until 2004, it was not known whether GFAT2 had an enzymatic activity similar to that of GFAT1. Hu and his team then showed for the first time that GFAT2 has GFAT activity (Hu et al. 2004). This has been then confirmed in retina of HFD fed mice where it was observed that increased GFAT2 expression enhanced protein O-GlcNAcylation [START_REF] Dai | Consumption of a High Fat Diet Promotes Protein O-GlcNAcylation in Mouse Retina via NR4A1-Dependent GFAT2 Expression[END_REF].

In

GFAT Structure

GFAT consists of two structurally and functionally distinct domains. The N-terminal 27 kDa glutaminase domain catalyzes the hydrolysis of glutamine to glutamate and ammonia.

The C-terminal 40 kDa isomerase domain utilizes ammonia for the conversion of fructose 6 phosphate (Fru6P) to glucosamine 6 phosphateGlcN6P. The GFAT1 of Escherichia coli has been previously characterized, based on structural and biochemical analysis. It has been revealed that Glucosamine 6-phosphate synthase GlmS is a functional dimer that catalyses the rate limiting step of HBP, converting fru6P into GlcN6P, whereas glutaminase and isomerase domains are linked by a solvent-inaccessible hydrophobic channel which is responsible for ammonia transfer between the two domains [START_REF] Teplyakov | Involvement of the C Terminus in Intramolecular Nitrogen Channeling in Glucosamine 6-Phosphate Synthase: Evidence from a 1.6 å Crystal Structure of the Isomerase Domain[END_REF]). The three-dimensional structure of the human GFAT1 isomerase domain, which is the active domain in human, was described and characterized for the first time in 2009 [START_REF] Nakaishi | Structural Analysis of Human Glutamine:Fructose-6-Phosphate Amidotransferase, a Key Regulator in Type 2 Diabetes[END_REF]) (Figure 25). The GFAT2 structure has not been determined yet.

Regulation of GFAT1

The relative expression of GFAT1 and GFAT2 varies among tissues allowing differential regulation of the hexosamine biosynthetic pathway in specialized tissues.

GFAT is the rate limiting enzyme that determines the synthesis of UDP-GlcNAc, which is essential for eukaryotic cells. The activity and expression of this enzyme have been described as being finely regulated [START_REF] Nerlich | Expression of Glutamine:Fructose-6-Phosphate Amidotransferase in Human Tissues[END_REF]). GFAT1 regulation was extensively studied as its activity was linked to insulin desensitization.

Regulation of GFAT1 activity

It has been shown that GFAT1 is phosphorylated by cAMP-dependent protein kinase (PKA) on Ser205. This phosphorylation decreases its activity [START_REF] Chang | Phosphorylation of Human Glutamine:Fructose-6-Phosphate Amidotransferase by CAMP-Dependent Protein Kinase at Serine 205 Blocks the Enzyme Activity[END_REF][START_REF] Zhou | Regulation of Glutamine:Fructose-6-Phosphate Amidotransferase by CAMP-Dependent Protein Kinase[END_REF]). More recently, Ser243 was reported as another a phosphorylation site on GFAT1. The activity of GFAT is also regulated by the intracellular contents of its downstream products. For example, UDP-GlcNAc has been shown to exert a negative feedback on the activity of GFAT. A study revealed that UDP-GlcNAc can inhibit a purified GFAT from rat liver allosterically (via binding at a site on the enzyme distinct from the active site) (Kornfeld 1967). It is not known if this regulation includes both isoforms of GFAT (GFAT1 and GFAT2).

Regulation of GFAT1 expression

Several studies demonstrated that GFPT1 transcription is up-regulated during enhanced demand for nutrients especially in proliferating cells such as cancer cells. Increased In addition, Wang et al showed that the spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the Gfpt1 gene (Z. V. [START_REF] Wang | Spliced X-Box Binding Protein 1 Couples the Unfolded Protein Response to Hexosamine Biosynthetic Pathway[END_REF]). It has been indicated that nutrient deprivation promotes Xbp1 nuclear accumulation and Gfpt1 transcriptional upregluation via a mTORC2 dependent mechanism. They suggested that reducing glutamine metabolite levels stimulates mTORC2 which in turn promotes α ketoglutarate production through glutaminolysis. α ketoglutarate was found to be essential to stimulate Xbp1 protein expression and its nuclear translocation and thereby GFAT1 mRNA up-regulation. [START_REF] Moloughney | MTORC2 Responds to Glutamine Catabolite Levels to Modulate the Hexosamine Biosynthesis Enzyme GFAT1[END_REF]). However, the mechanism of Xbp1 regulation by α ketoglutarate was not explained.

On the other hand, the protein and gene expression of GFAT have also been shown to be regulated by saturated fatty acids such as palmitate and stearate [START_REF] Weigert | Palmitate-Induced Activation of the Hexosamine Pathway in Human Myotubes: Increased Expression of Glutamine:Fructose-6-Phosphate Aminotransferase[END_REF])

Regulation of GFAT2

Very few studies explored the regulation of GFAT2 expression and enzymatic activity regulation

GFAT2 activity regulation

Like GFAT1, GFAT2 has been found also phosphorylated on serine 205 by cAMPdependent protein kinase (PKA). On the contrary to GFAT1, GFAT2 activity was increased by approximately 2.2-fold by this modification (Hu et al. 2004).

Transcriptional regulation

Yamazaki et al, after the discovery of GFAT2 gene by his team, performed sequence analysis of mouse Gfat2 gene promoter. They revealed several putative transcription factor binding sites: Sp1, a CCAAT box, AP-1 and AP-2, but no TATA box. Reporter assays using deletion and mutant variants in NIH3T3 fibroblast cells revealed that two binding sites for Sp1 play an important role in the basal promoter activity [START_REF] Yamazaki | Cloning and Characterization of Mouse Glutamine:Fructose-6-Phosphate Amidotransferase 2 Gene Promoterq[END_REF]. A recent study reported that high fat diet induced GFAT2 mRNA expression in mice retina via NR4A1 (a lipotoxicity sensor nuclear receptor subfamily 4 group A member 1) dependent mechanism.

In this study, they observed that mice fed with a HFD for 4 weeks exhibited elevated protein O-GlcNAcylation and increased of GFAT2 but not of GFAT1 mRNA in retina. This was associated with increased NR4A1 expression. Moreover, they confirmed these findings in retinal Müller cells by showing that treatment with palmitate or a ceramide analog Cer6 promoted Gfat2 up-regulation. Finally, the transfection of NR4A1 was sufficient to increase GFAT2 mRNA expression selectively [START_REF] Dai | Consumption of a High Fat Diet Promotes Protein O-GlcNAcylation in Mouse Retina via NR4A1-Dependent GFAT2 Expression[END_REF]. Indeed, the lipotoxicity sensor nuclear receptor subfamily 4 group A member 1 (NR4A1, also known as NGF1B, TR3, and Nur77) has been shown to mediate early genes induced by multiple stressors. NR4A1 play an important role in maintaining cellular homeostasis and is also involved in metabolic, cardiovascular and neurological functions and diseases (Safe et al. 2016). Furthermore, Zitzler et al detected that GFAT2 gene expression is significantly induced in neuronal cells in response to oxidative stress (H2O2). They suggested that GFAT2 plays a protective role in CNS by detoxification of peroxide [START_REF] Zitzler | High-Throughput Functional Genomics Identifies Genes That Ameliorate Toxicity Due to Oxidative Stress in Neuronal HT-22 Cells: GFPT2 Protects Cells Against Peroxide[END_REF]).

O-GlcNAc cycling enzymes

While the phosphorylation is regulated by a myriad of kinases and phosphatases (approximately 1000 and 150 respectively), the O-GlcNAcylation is controlled by only two enzymes, the OGT that O-GlcNAcylates proteins and the OGA that removes the O-GlcNAc motif from proteins.

O-GlcNAc transferase (OGT)

Structure of OGT

OGT protein was first isolated in 1992 from rat liver by using a combination of conventional and affinity chromatographic techniques (Haltiwanger S, Blomberg, and Harts.1992). The gene coding for OGT was isolated later from rat, Caenorhabditis elegans, and human genomes [START_REF] Lubas | O -Linked GlcNAc Transferase Is a Conserved Nucleocytoplasmic Protein Containing Tetratricopeptide Repeats[END_REF][START_REF] Kreppel | Dynamic Glycosylation of Nuclear and Cytosolic Proteins: CLONING AND CHARACTERIZATION OF A UNIQUE O-GlcNAc TRANSFERASE WITH MULTIPLE TETRATRICOPEPTIDE REPEATS[END_REF] and was found to be highly conserved among these organisms, sharing over 65% amino acid identity. The ogt gene resides on the X chromosome.

The importance of OGT has been demonstrated by experiments to invalidate of ogt gene in embryonic cells. The authors showed that the removal of one or two exons from the OGT results in death of embryonic cells. This suggests an essential role of the OGT in the survival and development of the embryo [START_REF] Shafi | The O-GlcNAc Transferase Gene Resides on the X Chromosome and Is Essential for Embryonic Stem Cell Viability and Mouse Ontogeny[END_REF].

Hanover et al found that ogt gene encodes a protein consisting of 2 distinct functional regions which are described below [START_REF] Hanover | Mitochondrial and Nucleocytoplasmic Isoforms of O-Linked GlcNAc Transferase Encoded by a Single Mammalian Gene[END_REF]:

The N-terminal domain comprises a series of tetratricopeptide repeats (TPRs), which are helix-turn-helix motifs that are commonly involved in mediating protein-protein interactions (Zeytuni N and Zarivach R 2012;Blatch GL and L¨assle M 1999). The number of TPRs varies according to the isoform of OGT. Three predominant human OGT isoforms are generated by alternative splicing (Figure 26).

The nucleocytoplasm OGT (ncOGT) isoform is the longest OGT isoform which comprises 13.5 complete TPR repeats that gives a protein with an apparent molecular weight of about 116 kDa on SDS gel. ncOGT, as its name indicates, is localized in the nucleus and the cytoplasm [START_REF] Hanover | Mitochondrial and Nucleocytoplasmic Isoforms of O-Linked GlcNAc Transferase Encoded by a Single Mammalian Gene[END_REF].

The mitochodrial OGT (mOGT) is the second longest isoform with 9 complete TPRs producing a ~ 103 kDa protein with an alternatively spliced N-terminal mitochondrialtargeting sequence (MTS) [START_REF] Love | Mitochondrial and Nucleocytoplasmic Targeting of O-Linked GlcNAc Transferase[END_REF]; X. L. [START_REF] Du | Hyperglycemia Inhibits Endothelial Nitric Oxide Synthase Activity by Posttranslational Modification at the Akt Site[END_REF].

The shortest OGT isoform or sOGT has only 2.5 TPRs. sOGT is a protein of ~ 75 kDa [START_REF] Bond | A Little Sugar Goes a Long Way: The Cell Biology of O-GlcNAc[END_REF]. This isoform is localized also in the nucleus and in the cytoplasm (B. D. [START_REF] Lazarus | Recombinant O-GlcNAc Transferase Isoforms: Identification of O-GlcNAcase, Yes Tyrosine Kinase, and Tau as Isoform-Specific Substrates[END_REF] and is suggested to have an anti-apoptotic function [START_REF] Fletcher | Functional Cloning of SPIN-2, a Nuclear Anti-Apoptotic Protein with Roles in Cell Cycle Progression[END_REF].

The C terminal domain contains the catalytic domain CD which catalyzes the glycosylation of cytoplasmic and nuclear protein. It is identical for the 3 isoforms of the enzyme. Based on GalNac-transferase similarities, the catalytic domain of the OGT has been divided into 2 subdomains. The CD I catalytic domain that would be responsible for the activity and binding UDP-GlcNAc and the CD II that would be a "lectin-like" region [START_REF] Roos | Structure of O-Linked GlcNAc Transferase: Mediator of Glycan-Dependent Signaling[END_REF].

Notably, a region containing amino acids from 958 to 1001, adjacent to catalytic domain II (CD II) has been identified as "PIP-binding region" which interacts with the phospholipid PI [START_REF] Fardini | O-GlcNAcylation: A New Cancer Hallmark?[END_REF]4,[START_REF] Ma | Hyper-O-GlcNAcylation Is Anti-apoptotic and Maintains Constitutive NF-kappaB Activity in Pancreatic Cancer Cells[END_REF] P3. In the absence of homology with known sequences of "PIP-binding region" present in other proteins, the authors have named this new motif PPO (PIPbinding activity of OGT) domain (Xiaoyong Yang et al. 2008).

Figure 26: The 3 OGT isoforms

The OGT is made up of three domains: in N-terminal a domain made up of repetitions from 2.5 to 13.5 TPRs which is followed by a binding domain called "linker" and finally by a catalytic domain.

There are 3 isoforms of OGT:

-The nucleo-cytoplasmic form or ncOGT has 13.5 TPR and is the longest form of OGT. It is found both in the nucleus and in the cytoplasm.

-The mitochondrial form or mOGT has in the N-terminal a mitochondrial targeting sequence (MTS for mitochondrial targeting sequence) and comprises 9 TPRs.

-The short form or sOGT has only 2.5 TPR and is found in the same location as ncOGT. Image adapted from [START_REF] Bond | A Little Sugar Goes a Long Way: The Cell Biology of O-GlcNAc[END_REF] Michael

OGT Regulation

Regulation of OGT by post-translational modification: OGT activity has been shown to be regulated by post-translational modifications also. Indeed, the OGT may be phosphorylated at many sites. In response to insulin, the OGT can be recruited by phosphatidylinositol 3,4,5-trisphosphate to the plasma membrane through its PIP3 binding site. OGT will then be tyrosine phosphorylated by the insulin receptor which increases its activity [START_REF] Whelan | Regulation of the O -Linked β-N -Acetylglucosamine Transferase by Insulin Signaling[END_REF][START_REF] Yang | Phosphoinositide Signalling Links O-GlcNAc Transferase to Insulin Resistance[END_REF]. It was also shown that neuronal cell depolarisation induces activation of OGT by a Calcium/calmodulin-dependent protein kinase type IV (CAMKIV) dependent -phosphorylation (M. [START_REF] Song | O-GlcNAc Transferase Is Activated by CaMKIV-Dependent Phosphorylation under Potassium Chloride-Induced Depolarization in NG-108-15 Cells[END_REF]. OGT is also able to GlcNAcylate itself at the 9th TPR as well as in its catalytic domain [START_REF] Lubas | Functional Expression of O -Linked GlcNAc Transferase: DOMAIN STRUCTURE AND SUBSTRATE SPECIFICITY[END_REF]. Multiple O-GlcNAc sites have been identified on sOGT and ncOGT, both of which locate in the nuclear and cytoplasm in cell. Moreover, O-GlcNAcylation of Ser389 in ncOGT (1036 aa) affects its nuclear translocation in Hela cells. Using LC MS/MS analysis, 6O-GlcNAcylation sites have been detected in the tetratricopeptide repeat (TPR) domain of sOGT. However, it has been revealed that O-GlcNAcylation does not affect sOGT activity but dose affect sOGT interaction with proteins (L. Liu et al. 2019).

Regulation of OGT protein stability:

A study suggested that the heat shock protein Hsp90 is involved in the regulation of OGT protein stability, as they observed that OGT interacts with the tetratricopeptide repeat binding site of Hsp90 in endothelial cells and inhibition of Hsp90 destabilizes OGT and enhances its degradation by the proteasome (F. [START_REF] Zhang | Hsp90 Regulates O -Linked β-N -Acetylglucosamine Transferase: A Novel Mechanism of Modulation of Protein O -Linked β-N -Acetylglucosamine Modification in Endothelial Cells[END_REF]. However, the precise mechanism underlying the regulation of OGT stability by Hsp90 remains unclear. Moreover, in a mice model of relative hypoxia (pups placed in 75% O2 chamber for 5 days and then transferred to room air for 5 days) or in endothelial cells cultured with hypoxia-mimetic agents, OGT protein levels were reduced (H. [START_REF] Liu | Proteasomal Degradation of O-GlcNAc Transferase Elevates Hypoxia-Induced Vascular Endothelial Inflammatory Response †[END_REF]). The authors found that hypoxia doest not reduce ogt gene expression rather than it induces OGT protein degradation by stimulation of 26S proteasome activity. Mechanistically, activated 26S proteasome works in coordination with E3 ubiquitin ligase β-TrCP1 resulting in proteasomal degradation of OGT (H. [START_REF] Liu | Proteasomal Degradation of O-GlcNAc Transferase Elevates Hypoxia-Induced Vascular Endothelial Inflammatory Response †[END_REF]). OGT stability is also regulated by phosphorylation. Recently, the Checkpoint kinase 1 (Chk1) (a kinase instrumental for orchestrating DNA replication, DNA damage checkpoints, the spindle assembly checkpoint and cytokinesis) has been found to interact with and phosphorylate OGT at serine 20. The authors observed that this phosphorylation is required for inducing OGT stability (Z. Li et al. 2017).

Regulation of OGT at the post-transcriptional level: OGT has been found to be also regulated at the post-tanscriptional level. Several miRNAs have been shown to interact with 3'-UTRs of OGT mRNAs and lead to the suppression of OGT expression. MiR-423-5p significantly regulated the expression of OGT. It has been documented that MiR-423-5p can induce apoptosis of cardiomyocytes through binding to OGT mRNA and decreasing OGT protein level. [START_REF] Luo | MicroRNA-423-5p Targets O-GlcNAc Transferase to Induce Apoptosis in Cardiomyocytes[END_REF]. OGT has also been described as a potential target of miR-15b which behave as a negative regulator of its expression (R. Liu et al. 2017).

Transcriptional regulation of OGT: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor which activates the cellular antioxidant response to fight the harmful effects of extrinsic and intrinsic insults, such as toxic substances (drugs, pollutants) and oxidative stress. A recent study showed, by chromatin immune-precipitation, that Nrf2 binds to ogt gene promoter in myeloid derived macrophage. Moreover, they observed that promoting Nrf2 nuclear accumulation through inhibition of its ubiquitination regulates positively the transcription of OGT (X. Li et al. 2017).

Drugs inhibiting of OGT activity

Several compounds have been designed and employed to inhibit OGT activity in the last few years. Nevertheless, lacks of specificity, off-target effects and/or limited cell permeability are still obstacles to select the ideal molecule.

The crystallography of OGT published at the beginning of the decade (M. B. [START_REF] Lazarus | Structure of Human O-GlcNAc Transferase and Its Complex with a Peptide Substrate[END_REF][START_REF] Schimpl | O-GlcNAc Transferase Invokes Nucleotide Sugar Pyrophosphate Participation in Catalysis[END_REF]) should allow progressing in the synthesis of molecules capable of specifically inhibiting OGT with high affinity. The common OGT inhibitors are described below: UDP: as mentioned previously UDP-GlcNAc is the OGT donor substrate for glycosyltransferase reaction. The transfer of GlcNAc on the peptide substrate yielding UDP as a product of reaction product UDP. UDP is considered as the most potent OGT feed-back inhibitor, as it has been shown to inhibit OGT in vitro with an IC 50 of 1.8 μM [START_REF] Dorfmueller | Substrate and Product Analogues as Human O-GlcNAc Transferase Inhibitors[END_REF]. Unfortunately, it is unsuitable for cell biology studies because it is not cell permeable and it is a substrate for a wide range of other enzymes.

Alloxan or (2,4,5,6-tetraoxypyrimidine), an uracil analogue, is a cell permeable compound. It is known as a diabetogenic drug as it selectively destroys pancreatic β cells. It was first known to have OGT inhibitory activity in 1993 (H. [START_REF] Zhang | Alloxan Cytotoxicity Is Highly Potentiated by Plasma Membrane-and Lysosomal-Associated Iron ? A Study on a Model System of Cultured J-774 Cells[END_REF]. The OGT-inhibitory effect of alloxan was then confirmed in isolated in β cells [START_REF] Konrad | Alloxan Is an Inhibitor of the Enzyme O-Linked N-Acetylglucosamine Transferaseq[END_REF]. It is less potent than UDP (IC 50 =18 μM). Nevertheless, it is not specific and is quite toxic since it can generate reactive oxygen species. BADGP (benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside) is another compound that has been used to inhibit OGT. However, this inhibitor modulates the activity of all glycolsyl-transferases. For example it has been shown to reduce secreted and membrane glycoproteins such as mucin in the epithelial colon cell line HT-29 (Hennebicq-Reig et al.

1998).

Ac 4 -5S-GlcNAc is a newer inhibitor that seems to have more interesting activity. It exhibits an OGT inhibitory activity after its conversion into UDP-5S-GlcNAc in the cell. The UDP-5S-GlcNAc mimics the structure of the donor substrate UDP-GlcNAc and thereby it strongly inhibits OGT activity. This inhibitor has high specificity and potency for OGT inhibition with an IC 50 of 5 μM. Many studies have recently used it [START_REF] Ma | Hyper-O-GlcNAcylation Is Anti-apoptotic and Maintains Constitutive NF-kappaB Activity in Pancreatic Cancer Cells[END_REF][START_REF] Perez-Cervera | Insulin signaling controls the expression of O-GlcNAc transferase and its interaction with lipid microdomains[END_REF]Donovan et al. 2014).

The second group of OGT inhibitors have been discovered by high-throughput screening studies. These inhibitors have been selected for their potent donor displacement activity.

For example, ST045849,

3-(2-adamantanylethyl)-2-[(4-chlorophenyl)

azamethylene]-4-oxo-1,3-thiazaperhydroine-6-carboxylic acid, has an IC 50 of 53 μM. This inhibitor has been used to study the role of O-GlcNAcylation on the development of pancreatic cells [START_REF] Filhoulaud | The Hexosamine Biosynthesis Pathway Is Essential for Pancreatic Beta Cell Development[END_REF] and in several other studies [START_REF] Itkonen | Inhibition of O-GlcNAc Transferase Activity Reprograms Prostate Cancer Cell Metabolism[END_REF].

OSMI-1 is also a small cell membrane permeable molecule. Its IC50 for OGT is 2.7

O-GlcNAcase (OGA)

The enzyme that hydrolyzes the GlcNAc group of proteins is the β-Nacetylglucosaminidase, also called O-GlcNAcase or OGA. It is a hexosaminidase located both in the cytoplasm and in the cell nucleus. This enzyme was first characterized in 1994 by Hart and his team as an OGA, but prior to this study, it was already known as hexosaminidase C (Braidman et al. 1974). The optimal pH for the OGA is between 5.5 and 7 while hexasominidases A and B have maximum activity at a more acidic pH between 3.5 and 5.5 (Dong and Hart 1994;[START_REF] Gao | Dynamic O -Glycosylation of Nuclear and Cytosolic Proteins: Cloning and characterization of a neutral cytosolic β -N-Acetylglucosaminidase from human brain[END_REF]).

The gene coding for OGA

Gao and his colleagues cloned the gene encoding O-GlcNAcase in 2001 [START_REF] Gao | Dynamic O -Glycosylation of Nuclear and Cytosolic Proteins: Cloning and characterization of a neutral cytosolic β -N-Acetylglucosaminidase from human brain[END_REF]. The authors discovered that the sequences of the tryptic digest peptides from purified O-GlcNAcase correspond to a known protein (MGEA5) which was originally identified as a hyaluronidase having a similar sequence with the hyaluronidase of C. elegans. Initially, this enzyme was called MGEA5 (for Meningioma expressed antigen 5) because of its association with human meningiomas [START_REF] Heckel | Novel Immunogenic Antigen Homologous to Hyaluronidase in Meningioma[END_REF]). The gene encoding MGEA5 is located on chromosome 10 in position q24.1-q24.3, a locus associated with many neurodegenerative diseases including Alzheimer's disease. Furthermore, a single nucleotide polymorphism (SNP) of the MGEA5 gene is correlated with sensitivity to type 2 diabetes in a Mexican-American population [START_REF] Lehman | A Single Nucleotide Polymorphism in MGEA5 Encoding O-GlcNAc-Selective N-Acetyl--D Glucosaminidase Is Associated With Type 2 Diabetes in Mexican Americans[END_REF].

Structure of OGA

OGA exists in 2 isoforms resulting from alternative splicing [START_REF] Comtesse | Identification of a Nuclear Variant of MGEA5, a Cytoplasmic Hyaluronidase and a β-N-Acetylglucosaminidase[END_REF].

-The long form of OGA (OGA-L) has an expected molecular weight of 102 kDa, but migrates with an apparent molecular weight of 130 kD in SDS-PAGE. It is the isoform with the highest activity and is found preferentially in the cytoplasm (E. J. Kim et al. 2006).

-The other isoform is a short isoform (OGA-S) of 75 kDa that does not have the last third of the C-terminal domain of the complete protein, and these does not have a HAT domain. This isoform is localized mainly in the endoplasmic reticulum and lipid droplets (Ruan HB. et al.2013;Hanover JA. et al. 2012) (Figure 30). The 3-D structure of human OGA has been revealed with an unusual obligate dimer and intertwined helical bundle domains that leads to residues from both domains contributing to formation of the substrate-binding site (Figure 31) [START_REF] Roth | Structural and Functional Insight into Human O-GlcNAcase[END_REF].

OGA bi-functional enzyme activity

Bioinformatic analysis predicts that O-GlcNAcase has two catalytic domains, a Nterminal region homologous to hyaluronidase and a C-terminal region homologous to acetyltransferase (aa 772-898) [START_REF] Comtesse | Identification of a Nuclear Variant of MGEA5, a Cytoplasmic Hyaluronidase and a β-N-Acetylglucosaminidase[END_REF][START_REF] Schultz | Prediction of Structure and Functional Residues for O -GlcNAcase, a Divergent Homologue of Acetyltransferases[END_REF].

An intrinsic histone acetyltransferase (HAT) activity of this protein was subsequently demonstrated (Clifford [START_REF] Toleman | Characterization of the Histone Acetyltransferase (HAT) Domain of a Bifunctional Protein with Activable O -GlcNAcase and HAT Activities[END_REF] The demonstration of this HAT activity seems important since it has been demonstrated that OGT can repress transcription by interacting with the co-repressor mSin3A and histone deacetylase 1 (HDAC 1) [START_REF] Yang | Recruitment of O-GlcNAc Transferase to Promoters by Corepressor MSin3A: Coupling Protein O-GlcNAcylation to Transcriptional Repression[END_REF]. OGT and NCOAT could participate in the chromatin remodeling process associated with transcriptional regulation. Whisenhunt et al. have also shown that NCOAT and OGT are very strongly associated. This complex of OGT and NCOAT, called the GlcNAczyme complex, occupies the promoter repressed with histone deacetylase HDAC1 within the transcriptional co-repression complex. When transcription is stimulated, this GlcNAczyme complex as well as HDAC1 are released from the promoter. The authors also showed that the activity of O-GlcNAcase is important for the transition from a repressed state of transcription to an active state, since the overexpression of an NCOAT variant from the Goto-Kakizaki rat which has no O-GlcNAcase activity blocks the release of the GlcNAczyme complex from promoters upon stimulation of transcription [START_REF] Whisenhunt | Disrupting the Enzyme Complex Regulating O-GlcNAcylation Blocks Signaling and Development[END_REF]. The discovery of the GlcNAczyme complex suggests a central role in the regulation of gene expression.

Post-translational modification of O-GlcNAcase

It has been predicted by bioinformatic analysis that O-GlcNAcase may be subject to

Cleavage of O-GlcNAcase by caspase-3

It (Kim et al. 2006). They did not find an explanation for this reassociation, but speculated that the reassociation process between the two fragments of O-GlcNAcase after cleavage by caspase-3 could modify its specificity towards its substrates during apoptosis.

Drugs modulating the activity of OGA

PUGNAc (O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenyl

carbamate) is certainly the most widely used OGA inhibitor (Haltiwanger, Grove, and Philipsberg 1998) (Figure 32). The use of PUGNAC leads to an increase in O-GlcNAcylation. However, this inhibitor also acts on other hexosaminidases and is therefore not totally specific for OGA (E. J. Kim et al. 2006).

Several specific OGA inhibitors have been developed after the characterization of OGA crystalized structure.

GlcNAcstatin has a 100,000-fold higher selectivity for OGA than for other hexosaminidases and is thus a highly effective and selective drug for OGA inhibition [START_REF] Dorfmueller | GlcNAcstatin: A Picomolar, Selective O -GlcNAcase Inhibitor That Modulates Intracellular O -GlcNAcylation Levels[END_REF]).

-

NButGT (1,2-dideoxy-2'-propyl-α-D-glucopyranoso-[2,1-d] -Δ 2'-thiazoline) allows
a more optimal occupancy of the active site of OGA than PUGNAc and is thus more specific for this hexosaminidase [START_REF] Macauley | O -GlcNAcase Uses Substrate-Assisted Catalysis: KINETIC ANALYSIS AND DEVELOPMENT OF HIGHLY SELECTIVE MECHANISM-INSPIRED INHIBITORS[END_REF].

Thiamet G (3aR, 5R, 6S, 7R, 7aR) -2-(ethylamino) -3a, 6,7,7a-tetrahydro-5-(hydroxymethyl) -5H-pyrano [3,2-d] thiazole-6.7-diol) appears to be now the most commonly used compound to inhibit OGA [START_REF] Yuzwa | A Potent Mechanism-Inspired O-GlcNAcase Inhibitor That Blocks Phosphorylation of Tau in Vivo[END_REF]. It is as specific to the OGA as the NButGT but has the advantage of being more stable.

The general functions of O-GlcNAc

Direct regulation of protein function

The dynamic addition of O-GlcNAc has been implicated in the modulation of protein function through various mechanisms that we will briefly describe below.

Regulation of protein phosphorylation

O-GlcNAcylated proteins are often also modified by phosphorylation. These posttranslational modifications occur both on serine or threonine residues. These two modifications can be mutually exclusive, suggesting the existence of a possible competition

Modification of protein stability

It has been reported that O-GlcNAc can modify the stability of proteins by two different mechanisms, on the one hand by decreasing the degradation of certain proteins by the proteasome, and on the other hand by modifying the activity of the proteasome itself. ERβ O-GlcNAcylation has been shown to extend its half-life, suggesting that O-GlcNAc may prevent its degradation by changing its protein phosphorylation status. The authors showed that a mutation in an ER-β (S16A) O-GlcNAcylation site leads to a longer half-life compared to non-mutated ER. In contrast, another S16E mutant that mimics ER-β phosphorylation has a shorter half-life (X. [START_REF] Cheng | Alternative O -Glycosylation/ O -Phosphorylation of Serine-16 in Murine Estrogen Receptor β: POST-TRANSLATIONAL REGULATION OF TURNOVER AND TRANSACTIVATION ACTIVITY[END_REF]. This suggests that O-GlcNAcylation of S16 impairs its phosphorylation resulting in higher stability.

On the other hand, Zhang and his colleagues have shown that OGT inhibits the activity of the 26S proteasome, leading to a decrease in the breakdown of the transcription factor Sp1. The O-GlcNAc state of Sp1 does not affect its degradation, suggesting that inhibition by OGT is due to O-GlcNAc of the proteasome itself. These authors showed that Rpt2 ATPase, associated with the 19S proteasome subunit, is modified and inhibited by O-GlcNAc. This decrease in ATPase activity may be responsible for the inhibition of proteasome 26S activity induced by OGT, since degradation by proteasome 26S is energydependent. This result demonstrates the involvement of O-GlcANc in the modulation of protein degradation by the proteasome. This suggests that O-GlcNAc could function as a detector of the nutritional status and regulate degradation by the proteasome depending on the availability of nutrients (F. [START_REF] Zhang | O-GlcNAc Modification Is an Endogenous Inhibitor of the Proteasome[END_REF].

Change in protein localization

It

Regulation of protein interactions

The signal transducer and activator of transcription 5 (STAT5) has been shown to be modified by O-GlcNAc and threonine 92 has been identified as an O-GlcNAc site by mass spectrometry. Co-immunoprecipitation experiments have shown that O-GlcNAc is necessary for the interaction of Stat5a with the co-activator CBP (CREB-Binding Protein), since the mutation on threonine 92 inhibits the interaction between Stat5a and CBP [START_REF] Gewinner | The Coactivator of Transcription CREB-Binding Protein Interacts Preferentially with the Glycosylated Form of Stat5[END_REF]). It has also been shown that the O-GlcNAcylation of NFκB p65 decreases the interaction of p65 NFκB with its inhibitor IκB.

Targeting of OGT to its substrates

More and more data support the idea that OGT interacts with different protein partners that address the enzyme to specific locations within cells or increase its specificity towards certain substrates. This could explain how "only one" OGT modifies a myriad of proteins and why no consensus sequence of recognition by OGT was identified. For example, TRAK1 (for trafficking protein, kinesin binding 1) interacts with OGT via its TPR domain. Coimmunoprecipitation experiments have shown the presence of an RNA polymerase II / TRAK1 / OGT complex, suggesting that the interaction with TRAK1 is important for the targeting of OGT at transcription sites, thus enabling it to exercise its regulatory activity on transcription [START_REF] Iyer | Identification and Cloning of a Novel Family of Coiled-Coil Domain Proteins That Interact with O -GlcNAc Transferase[END_REF]. OGT can also be addressed to promoters through its interaction with mSin3A and with a histone deacetylase (HDAC1) to suppress the transcription of NOS2 (Xiaoyong [START_REF] Yang | Recruitment of O-GlcNAc Transferase to Promoters by Corepressor MSin3A: Coupling Protein O-GlcNAcylation to Transcriptional Repression[END_REF]. As already mentioned, numerous protein complexes have been described to date, such as the OGT-phosphatase complex (PP1), the GlcNAczyme complex including OGT and O-GlcNAcase and a more sophisticated complex comprising 4 enzymatic activities (kinase / phosphatase / OGT / O-GlcNAcase) [START_REF] Slawson | A Mitotic GlcNAcylation/Phosphorylation Signaling Complex Alters the Posttranslational State of the Cytoskeletal Protein Vimentin[END_REF]).

Riu et al have found that very little (if any) of bacterial proteins are modified by OGT when one of the three isoforms of human OGT is overexpressed in Escherichia coli.

However, some bacterial proteins appear to be modified by O-GlcNAc when ncOGT and the Sp1 protein are co-expressed in E. coli. The demonstration that the purified Sp1 protein can also help ncOGT in vitro to glycosylate bacterial proteins in a time-dependent manner, led the authors to conclude that Sp1 could help OGT (specifically for the ncOGT isoform) to recognize some of its substrates. Under these conditions, ncOGT can recognize bacterial proteins as target substrates and induce their O-GlcNAcylation [START_REF] Riu | Sp1 Modulates NcOGT Activity to Alter Target Recognition and Enhanced Thermotolerance in E. Coli[END_REF].

It has also been observed by two-hybrid screening that MYPT1 (myosin phosphatase targeting subunit 1) and CARM1 (coactivator-associated arginine methyltransferase 1)

interact separately with OGT and that MYPT1 and CARM1 change the specificity of OGT towards certain substrates (Cheung et al. 2008).

Furthermore, Cheung et al discovered that OGT interacts with MAP kinase p38 during cellular stress induced by glucose depletion in Neuro-2a cells (neuroblastoma cells). p38

MAP kinase directs OGT to neurofilament H (NF-H), which is a substrate for OGT, leading to an increase in O-GlcNAc on NF-H and an increase in its solubility. It should be noted that p38 interacts with the C-terminal of the OGT and not with the TPR part, which is the part most often involved in the interaction with its partners (Cheung and Hart 2008). Our laboratory revealed that the transcription factor FoxO1 is O-GlcNAcylated in hepatocytes and in β cells, resulting in an increase in its transcriptional activity (Kuo et al. 2008;Fardini et al. 2014).

O-GlcNAcylation involvement in transcriptional regulation

On other hand, O-GlcNAc modification can also inactivates gene transcription. Sp1 is the first transcription factor which was found to be O-GlcNAcylated (Jackson, S. P. and Tjian, R.

1988). It has been reported that O-GlcNAcylation of the activation domain of the transcription

factor Sp1 inhibits its TATA-binding protein-associated factor II 110 which results in inhibition of transcription activity in vitro and in vivo (X. [START_REF] Yang | O-Linkage of N-Acetylglucosamine to Sp1 Activation Domain Inhibits Its Transcriptional Capability[END_REF]).

In addition, Gerald Hart's team revealed that the COOH-terminal domain (CTD) of RNA polymerase II is O-GlcNAcylated. The CTD domain of RNA polymerase II is extensively phosphorylated and is essential for cell viability. However, they observed that only the unphosphorylated form of RNA polymerase II has detectable O-GlcNAc motifs, which suggests that glycosylation and phosphorylation of the CTD may be mutually exclusive events (William G. Kelly et al. 1992).

Xiaoyong [START_REF] Yang | Recruitment of O-GlcNAc Transferase to Promoters by Corepressor MSin3A: Coupling Protein O-GlcNAcylation to Transcriptional Repression[END_REF] revealed that OGT interacts with a histone deacetylase complex (HDACs) by binding with the corepressor mSin3A. The authors suggested that mSin3A targets OGT to promoter to inactivate transcription factors and RNA polymerase II by O-GlcNAc modification, which acts in concert with histone deacetylation to promote gene silencing in an efficient and specific manner.

OGT can also act as an epigenetic regulator through its interaction with Ten-eleven translocation (TET) enzymes such as TET1, TET2 and TET3 (Q. [START_REF] Chen | TET2 Promotes Histone O-GlcNAcylation during Gene Transcription[END_REF]) revealed that, using mass spectrometry analysis, that OGT is found as a functional a partner of TET2 and TET3 enzymes. TET2 and TET3 are TET proteins which oxidize 5-methylcytosine and contribute to active DNA demethylation, and thus regulate gene transcription. The authors showed that TET2 directly interacts with OGT, which is important for the chromatin association of OGT in vivo. Although this specific interaction does not regulate the enzymatic activity of TET2, it facilitates OGT-dependent histone O-GlcNAcylation. Furthermore, it has been reported that TET 1, 2, 3 are also O-GlcNAcylated. Interestingly, O-GlcNAcylation of TET proteins reduces TET phosphorylation site number and occupancy, resulting in modulation in their enzymatic activity [START_REF] Bauer | Phosphorylation of TET Proteins Is Regulated via O -GlcNAcylation by the O -Linked N -Acetylglucosamine Transferase (OGT)[END_REF]. These data demonstrate that O-GlcNAc can act as the link between the nutritional status of the cell and epigenetic regulation.

O-GlcNAcylation in metabolic syndrome

Given the role of nutritional "sensor" of O-GlcNAcylation, it was early suspected that it could be involved in the development of chronic metabolic diseases. Various observations in cultured cells, animal models and in human patients supported this notion. It is now well documented that hyperglycemia associated with metabolic syndrome leads to an abnormally high O-GlcNAcylation rate, which contributes to glucotoxicity [START_REF] Issad | O-GlcNAc Modification, Insulin Signaling and Diabetic Complications[END_REF]. It has been observed that the concentration of UDP-N-acetyl hexosamines (UDP-HexNAc) in muscle is higher in ob/ob mice than in control mice, and the activity of GFAT is also increased in muscle in ob/ob mice (Buse et al.1997). In 1996, Yki-Jarvinen et al reported that the activity of GFAT is increased by 46% in the muscle in patients with NIDDM (noninsulin dependent diabetes mellitus) (Yki-Jarvinen et al. 1996). Since then, numerous studies both in vitro and in vivo (in rodents) have been carried out to validate the fact that the flux of glucose in the HBP pathway could represent a mechanism by which hyperglycemia leads to insulin resistance.

Heart et al have shown that glucosamine, which provides the UDP-GlcNAc substrate to OGT, decreases insulin-induced translocation of GLUT4 to plasma membrane, and this was associated with a decrease in AKT activity in cultured 3T3-L1 adipocytes (Heart et al., 2000).

It should be noted that it is necessary to take certain precautions to interpret the results of (Park et al., 2005;[START_REF] Wells | Dynamic O-GlcNAcylation of Nuclear and Cytosolic Proteins[END_REF] or skeletal muscles (Arias et al., 2004) with PUGNAc induces insulin resistance in both cell types. Moreover, our team has shown that in hepatocytes, treatment with glucosamine and PUGNAc induced O-GlcNAcylation of FoxO1. O-GlcNAcylation of FoxO1 increases its transcriptional activity of gluconeogenic enzyme glucose 6-phosphatase. This would promote hepatic glucose production and could lead to hyperglycemia (Kuo et al. 2008).

Lessons from transgenic animal models

To avoid potential non-specific effects of O-GlcNAc by using glucosamine or pharmacological agents, several groups have devoted their efforts to develop transgenic animal models that overexpress one of the key enzymes of the O-GlcNacylation pathway.

This work provides more direct evidence of the involvement of HBP and O-GlcNacylation in the development of insulin resistance. The overexpression of GFAT in the skeletal muscles and adipose tissue of mice leads to a decrease in the uptake of glucose by the muscle (Cooksey et al., 1999;Hebert et al., 1996). In mice that overexpress GFAT in the liver develop obesity and glucose intolerance and insulin resistance with age (Veerababu et al. 2000). Transgenic mice overexpressing OGT under the control of the GLUT4 promoter are also resistant to insulin and are hyperleptinemics (McClain et al. 2002). Moreover, transgenic mice overexpressing GFAT specifically in β cells under the control of the RIP (Rat Insulin Promoter develop a progressive loss of glucose-induced insulin secretion and an overt diabetes (Cooksey et al. 2006). All of these data support the idea that an increase in O-GlcNAc linked to overexpression of GFAT or OGT leads to insulin resistance. This results in increase in its level of O-GlcNAcylation and induces its nuclear translocation (Andrali et al. 2008). Similarly, Pdx-1 has many sites that are O-GlcNAcylated in response to high concentrations of glucose [START_REF] Gao | The Transcription Factor PDX-1 Is Post-Translationally Modified by O-Linked N-Acetylglucosamine and This Modification Is Correlated with Its DNA Binding Activity and Insulin Secretion in Min6 β-Cells[END_REF]. The O-GlcNAcylation of these transcription factors promotes their binding to the promoter of the insulin gene and thus increases its expression.

O-GlcNAcylation and insulin secretion

However, it seems that an excessive and prolonged level of O-GlcNAcylation in the β cells, in the case of chronic hyperglycemia, can participate in the deleterious effects of diabetes.

Indeed, a prolonged over-activation of the HBP pathway has deleterious consequences on pancreatic β cells. Thus, it has been observed an increase in the level of OGT and protein O-GlcNAcylation in the pancreas of various animal models of diabetes such as the rat Goto-Kakizaki (Akimoto et al. 2007), or diabetes induced by streptozotocin (Akimoto et al. 2000).

On the other hand, numerous studies have established that excessive O-GlcNAcylation leads to a deterioration of the function and to apoptosis of the pancreatic β cells (Liu et al., 2000).

One of the mechanisms involved is an increase in oxidative stress in these cells, induced by an increase in the flow of glucose through the HBP pathway, which can lead to a loss of function as well as the induction of cell death [START_REF] Kaneto | Activation of the Hexosamine Pathway Leads to Deterioration of Pancreatic β-Cell Function through the Induction of Oxidative Stress[END_REF]. Moreover, our laboratory also Furthermore, increased O-GlcNAcylation was also observed to induce β cells apoptosis (D'Alessandris et al. 2004;[START_REF] Kang | Adipocyte-Derived Th2 Cytokines and Myeloid PPARδ Regulate Macrophage Polarization and Insulin Sensitivity[END_REF]. These authors suggested that O-GlcNAcylation induced apoptosis by inhibiting of various steps in PI3K/Akt signaling pathway. Consistent with this, the overexpression of OGT in the liver by adenovirus in mice leads to insulin resistance, which is manifested by increased hepatic glucose production and decreased glycogen synthesis. This insulin resistance indeed depends on the PPO domain of OGT, since overexpression of OGT mutant in which two lysine residues essential for association with PIP3 are replaced by alanine, does not make the mice resistant to insulin (Xiaoyong Yang et al. 2008). and the cyclin kinase inhibitor p27 (O'Donnell and al., 2004).

O-GlcNAcylation and insulin signaling

Therefore, it has been proposed that the establishment of tissue specific and / or an inducible OGT KO is necessary to study the impact of the deletion of this enzyme on tissue function.

OGT knock out in the central nervous system

Since OGT is considered a nutritional sensor, several studies have also focused on the potential role of OGT deletion in central regulator of food intake. The orexigenic neurons expressing the agouti-related protein or AgRP neurons are neurons that are strongly activated during the fasting period to promote feeding via inhibition of anorexigenic neurons [START_REF] Takahashi | Fasting Induces a Large, Leptin-Dependent Increase in the Intrinsic Action Potential Frequency of Orexigenic Arcuate Nucleus Neuropeptide Y/Agouti-Related Protein Neurons[END_REF]. Ruan and his colleagues observed that OGT expression as well as O-GlcNAcylation levels are increased in the AgRP neurons during fasting [START_REF] Ruan | O-GlcNAc Transferase Enables AgRP Neurons to Suppress Browning of White Fat[END_REF]. They developed selective OGT knockout in these neurons. This knock out inhibits the activity of these neurons to suppress the browning of white adipose tissue. These mice were then protected from obesity induced by fatty diets. This study suggests that in these neurons OGT has a regulatory role on body weight by inducing a brown phenotype in adipose tissue [START_REF] Ruan | O-GlcNAc Transferase Enables AgRP Neurons to Suppress Browning of White Fat[END_REF]. Another group studied the effect of the deletion of OGT in neurons expressing αCaMKII [START_REF] Lagerlof | The Nutrient Sensor OGT in PVN Neurons Regulates Feeding[END_REF]). These neurons are mainly located in the paraventricular nucleus or PVN. In this nucleus, the authors observed that fasting decreases the levels of O-GlcNAc, while food intake activates the αCaMKII neurons of the PV. The loss of OGT specifically in these neurons l completely blocks their activation in response to food intake. OGT KO mice showed rapid weight gain compared to control mice. This weight gain is explained by a very large increase in the mass of adipose tissue, due to the development of hyperphagia in these mice. This suggests that in PVN, OGT has a role in regulating satiety (Lagerlöf et al., 2016). Therefore, deletion of OGT in the brain has different consequences depending on the type of neurons targeted.

OGT knock out in adipose tissues

Increased flux through HBP has been shown to induce insulin resistance in transgenic mice models. However, the mechanism by which OGT promotes obesity and insulin resistance not In contrast to WAT, OGT in brown adipose tissues may promote weight loss by increasing thermogenic function. A study showed that deletion of OGT in brown adipose tissues led to severe cold intolerance. The authors revealed that deletion of OGT induced proteosomal degradation of mitochondrial PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1-α) which is essential for enhancing oxidative phosphorylation, mitochondrial biogenesis and oxygen consumption via transcriptional stimulation of uncoupling protein 1(UCP1) protein [START_REF] Ohashi | Pivotal Role of O -GlcNAc Modification in Cold-Induced Thermogenesis by Brown Adipose Tissue Through Mitochondrial Biogenesis[END_REF]). However, OGT KO in BAT had no effect on body weight.

OGT knock out in skeletal muscle

Mice with conditional OGT invalidation in skeletal muscle exhibited an increase in insulin sensitivity and in glucose uptake. Interestingly, high-fat diet failed to induce insulin resistance in these mice. The authors found that IL15 mRNA in skeletal muscles and its protein level in blood circulation were increased in KO OGT mice. Increased IL15 expression was due to decreased its gene repressor EZH2 (histone methyltransferase enhancer of zeste homolog 2) which it is stabilized by its O-GlcNAcylation [START_REF] Shi | Skeletal Muscle O-GlcNAc Transferase Is Important for Muscle Energy Homeostasis and Whole-Body Insulin Sensitivity[END_REF]). IL-15 has been shown previously shown to promote regulatory T cell function and protects against diabetes development (Jinxing Xia et al., 2009). These data suggest OGT in skeletal muscles promotes insulin resistance, in agreement with results obtained with OGT transgenic mice models.

OGT knock out in liver

Metabolic liver diseases such as non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) characterized by fibrosis and alterations in organ architecture and function. This is due to chronic hepatocyte turnover, recruitment of immune cells and activation of hepatic stellate cells. Chronic cell death contributes to the development of liver fibrosis, cirrhosis and cancer. It has become evident that, besides apoptosis, necroptosis is a highly relevant form of programmed cell death in the liver [START_REF] Schwabe | Apoptosis and Necroptosis in the Liver: A Matter of Life and Death[END_REF]. It has been reported that mice in which OGT is deleted in liver exhibited hepatomegaly and ballooning degeneration at an early age. The authors observed that OGT-deficient hepatocytes underwent excessive necroptosis. Indeed, these cells exhibited elevated protein expression levels of key mediators of necroptosis such as receptor-interacting protein kinase 3 (RIPK3)

and mixed lineage kinase domain-like (MLKL). These two proteins have been previously

shown to form the necrosome (Gautheron J et al., 2018). Phosphorylated RIPK3 recruits and activates MLKL, which translocate to the cell membrane and causes membrane rupture (Linkermann A and Green DR., 2014). Interestingly, in this study they found that RIPK3 is O-GlcNAcylated. Glycosylation of RIPK3 by OGT is associated with reduced RIPK3 protein stability (Zhang et al.2019). These data suggest that OGT plays an important role in hepatic protection against liver fibrosis and subsequent cirrhosis by negative regulation of the necroptosis pathway.

OGT knock out in pancreas

Mice deficient in OGT specifically in pancreatic β cells developed severe diabetes due to βcell failure. They observed that insulin content was lower in β islet of these mice. This was associated with down regulation of insulin genes (ins1, ins2, Pdx-1, and NueroD1) mRNA levels. The authors observed that these events were preceded by increased endoplasmic reticulum ER stress induced-apoptosis. In addition, critical pro-survival proteins Akt1/2 and Pdx-1 were down-regulated in these mice [START_REF] Alejandro | Disruption of O-Linked N-Acetylglucosamine Signaling Induces ER Stress and β Cell Failure[END_REF]. These findings identify

O-GlcNAcylation and inflammation

O-GlcNAcylation was discovered on the surface of lymphocytes, more precisely on the internal side of the plasma membrane, more than 30 years ago (Carmen-Rosa Torres and Gerald W. [START_REF] Torres | Topography and Polypeptide Distribution of Terminal N-Acetylglucosamine Residues on the Surfaces of Intact Lymphocytes[END_REF]. It was quickly shown that O-GlcNAcylation is modulated during activation of these cells (Kearse and Hart 1991). This suggested a potential role for O-GlcNAcylation in the activation of the immune response.

Despite these observations, studying the role of O-GlcNAcylation in inflammatory process only recently started to bring the attention. Several studies have been conducted on the role of O-GlcNAcylation in pro-inflammatory diseases and have given conflicting results.

In an in vitro model of acute pancreatitis, using rat acinar pancreatic cells treated with caerulin, a drug that induces strong pancreatic inflammation [START_REF] Zaninovic | Cerulein Upregulates ICAM-1 in Pancreatic Acinar Cells, Which Mediates Neutrophil Adhesion to These Cells[END_REF], the levels of O-GlcNAcylation and OGT are increased compared to those of control cells (Zhang et al. 2015). A decrease in O-GlcNAcylation using OGT siRNA helps reduce inflammation.

Conversely, an increase in O-GlcNAcylation levels by treatments with PUGNAc contributes to amplifying the inflammation in this cellular model. These observations suggest a proinflammatory role for this post-translational modification in acute pancreatitis (Zhang et al. 2015). Likewise, in a mouse model of inflammatory colitis (induced by sodium dextran sulfate (DSS)), heterozygous OGA KO, had an increase in O-GlcNAcylation levels (notably in the colon). This was associated with an increase in intestinal inflammation (Y. R. Yang et al. 2015). O-GlcNAcylation therefore seems to play a pro-inflammatory role in this disease.

Conversely, in the context of inflammation linked to hypoxia (H. [START_REF] Liu | Proteasomal Degradation of O-GlcNAc Transferase Elevates Hypoxia-Induced Vascular Endothelial Inflammatory Response †[END_REF], O-GlcNAcylation has protective anti-inflammatory effects. Thus, in endothelial cells placed in hypoxia, or in murine models of hypoxia, hypoxia induces inflammation which is associated with a decrease in OGT levels. An increase in OGT levels (by adenoviral strategy), or the use of glucosamine, reduces the inflammatory response induced by hypoxia. These results suggest an anti-inflammatory effect of O-GlcNAcylation in hypoxic endothelium. This has also been observed in models of arterial lesions in rats (Xing et al. 2011a).

Although O-GlcNAcylation seems to have opposite effects on inflammation according to the diseases studied, a mechanism often proposed by these authors is a regulation of the NFκB pathway by O-GlcNAcylation.

O-GlcNAcylation and NFκB signaling pathway

Several groups have studied the potential role of O-GlcNAcylation on the modulation of the NFκB signaling pathway. Initial studies were performed on mesangial cells treated with glucosamine or high glucose concentrations (L. R. [START_REF] James | Flux Through the Hexosamine Pathway Is a Determinant of Nuclear Factor B-Dependent Promoter Activation[END_REF]. By studying the modification of DNA mobility on electrophoresis gel, the authors observed an increase in the binding of nuclear proteins on NFκB consensus sequences. This was associated with enhanced vascular cell adhesion molecule-1 (Vcam-1) promoter activity in glomerulus cells isolated from male Sprague-Dawley rats. This observation suggested that O-GlcNAcylation of NFκB could be an integral part of inflammatory processes. However, in this first study, neither the O-GlcNAcylation sites of the NFκB p65 subunit, nor the mechanisms by which O-GlcNAcylation allows the activation of NFκB, were identified (L. R. [START_REF] James | Flux Through the Hexosamine Pathway Is a Determinant of Nuclear Factor B-Dependent Promoter Activation[END_REF].

Since then, several mechanisms that modulate the activation of the NFκB signaling pathway through O-GlcNAcylation have been demonstrated, but with diverging results. All these mechanisms could explain the over-activation of NFκB associated with hyperglycemia, and thus at least partially explain the chronic inflammation associated with hyperglycemia that characterizes diabetes and metabolic diseases. However, O-GlcNAcylation also has been shown to negatively regulate NFκB signaling.

O-GlcNAcylation activates

O-GlcNAcylation negatively regulates

O-GlcNAcylation and oxidative stress

Several evidences suggest that O-GlcNAcylation could play an important role in the fight against oxidative stress via regulation of different anti-oxidant mediators.

For example, the transcription factor FoxO1, in addition to its role in metabolic regulation, it has also been shown to be involved in oxidative stress. Interestingly, our laboratory revealed that O-GlcNAcylation of FoxO1 increased its transcriptional activity in liver (Kuo et al. 2008) and in pancreas (Fardini et al. 2014). Housely et al observed that O-GlcNAcylation of FoxO1

activates the transcription of genes MnSOD and catalase that are involved in the detoxification of redox upon exposure to high glucose (25mM) [START_REF] Housley | O -GlcNAc Regulates FoxO Activation in Response to Glucose[END_REF].

Moreover, one recent study demonstrated that hydrogen peroxide treatment induced the O-GlcNAcylation of the sirtuin deacetylase SIRT1 on Ser549. Indeed, SIRT1, a class III protein deacetylase, is a crucial cellular survival protein, which is also has anti-oxidant protective role. Interestingly, SIRT1 O-GlcNAcylation potentiated its deacetylase activity in vitro and in vivo. This has been shown to result in enhanced cellular stress resistance, at least in part by promoting the deacetylation of the tumor suppressor protein p53 (C. Han et al.

2017).

Furthermore, Nrf1 (Nuclear factor erythroid-derived 2-related factor-1) belongs to the Cap-N-Collar (CNC) family of transcription factors that includes, Nrf2, Nrf3 and p45NFE2. Nrf1 is essential for the maintenance of cellular homeostasis. It has been documented that Nrf1stimulates antioxidant genes via cis-active sequences known as the anti-oxidant response element such as (Myhrstad et al., 2000). [START_REF] Han | Nuclear Factor-Erythroid-2 Related Transcription Factor-1 (Nrf1) Is Regulated by O-GlcNAc Transferase[END_REF] revealed that Nrf1 is O-GlcNAcylated. O-GlcNAcylation catalysed by OGT was essential for Nrf1 stabilization and consequent up-regulation its activity.

In addition, it has been shown that O-GlcNAcylation mediates hyperglycemia and oxidative stress induced activation of the anti-oxidant enzyme glutathione peroxidase 1 (GPX1) in rat vascular smooth muscle cells (VSMCs). The anti-oxidant glutathione peroxidase (GPX) is a selenium-containing enzyme that converts H2O2 to H2O by oxidizing glutathione to its disulphide form, resulting in cell protection from oxidative stress. The authors showed that GPX is O-GlcNAcylated at its C-terminal. O-GlcNAcylation of GPX1 was revealed to be essential for its activation by promoting its binding to tyrosine kinases c-Abl or Arg which are responsible for its phosphorylation and activation (Won Ho Yang et al.

2010).

However, other work indicates that the OGT could on the contrary increase oxidative stress through negatively controls the Nrf2-Keap1 signaling pathway.

The main function of Keap1 (Kelch-like erythroid cell-derived protein with CNC homology [ECH]-associated protein 1)-ARE (antioxidant response element) signaling pathway is to activate the cellular antioxidant response by inducing the transcription of a wide array of genes that are able to fight the harmful effects of extrinsic and intrinsic insults, such as toxic substances (drugs and polluants) and oxidative stress. In unstressed cells, NRF2

associates with a complex which comprises the cytoplasmic KEAP1 adaptor protein and CUL3 E3 ubiquitin ligase, which ubiquitinates NRF2, targeting it for proteasome-mediated destruction [START_REF] Jaramillo | The Emerging Role of the Nrf2-Keap1 Signaling Pathway in Cancer[END_REF]. Oxidative stress or electrophiles can cause a conformational change in the Keap1-Cul3-E3 ubiquitin ligase by acting on specific cysteine residues in Keap1. These changes disrupt Nrf2-Keap1 binding [START_REF] Jaramillo | The Emerging Role of the Nrf2-Keap1 Signaling Pathway in Cancer[END_REF][START_REF] Menegon | The Dual Roles of NRF2 in Cancer[END_REF]. Free NRF2 then migrates into the nucleus, where it binds to promoters containing antioxidant response elements (AREs) and up-regulates the transcription of a variety of genes involved in cytoprotection and metabolism (Itoh et al. 1997;Nguyenet al. 2000;Motohashi et al. 2004;Hirotsu et al. 2012).

Several biochemical and functional connections between O-GlcNAcylation and NRF2 have been reported. These reports hint at an inverse correlation between global O-GlcNAcylation and NRF2 signaling. Consistent with this notion, a recent report found that sustained elevated Recently, Chen et al revealed that KEAP1, the primary negative regulator of NRF2, is a direct substrate of OGT. Moreover, they showed that O-GlcNAcylation of KEAP1 at serine 104 is required for the efficient ubiquitination and degradation of NRF2. In addition, they found that KEAP1 physically interacts with OGT and is thereby O-GlcNAcylated under unstressed conditions. Furthermore, transcriptional modification associated with pharmacological manipulation of O-GlcNAc by the OGT inhibitor 5SGlcNAc or by the OGA inhibitor Thiamet G was mediated in part by NRF2 transcriptional activity (P. [START_REF] Chen | Glycosylation of KEAP 1 Links Nutrient Sensing to Redox Stress Signaling[END_REF].

Therefore, a complex relationship exists between these pathways with both protective and deleterious effect of O-GlcNAcylation on oxidative stress.

O-GlcNAcylation in immune cells

O-GlcNAcylation in T cells

The role of O-GlcNAcylation in immune cells has been extensively studied in T.cell. O-GlcNAcylation was shown to be rapidly modulated during activation of these cells (Kearse and Hart 1991). Lund and his collegues performed a global analysis of O-GlcNAcylated proteins in human T cells upon activation of T cell receptor TCR, using a glycoproteomics approach [START_REF] Lund | Global Analysis of O -GlcNAc Glycoproteins in Activated Human T Cells[END_REF]. They revealed more than 200 O-GlcNAc proteins in human T cells. O-GlcNAcylation plays an important role in T-cell activation via its regulation of major inflammatory molecules. As mentioned previously, c-Rel and p65 subunits of NF-κB are found to be O-GlcNAcylated in T cells, and this results in their activation and in the expression of pro-inflammatory molecules (Ramakrishnan et al. 2013;[START_REF] Ma | Hyper-O -GlcNAcylation Activates Nuclear Factor κ-Light-Chain-Enhancer of Activated B Cells (NF-ΚB) Signaling through Interplay with Phosphorylation and Acetylation[END_REF].

In addition, the transcription factor NFAT, which regulates several genes critical for thymocyte development, T-cell differentiation and self-tolerance, has also been shown to be O-GlcNAcylated shortly after T/ B-cell activation, which correlated well with its translocation to the nucleus (Ramakrishnan et al. 2013). Moreover, the transcription factors c-JUN and JUNB were also observed to display higher levels of O-GlcNAcylation in activated T cells. [START_REF] Woo | Mapping and Quantification of Over 2000 O-Linked Glycopeptides in Activated Human T Cells with Isotope-Targeted Glycoproteomics (Isotag)[END_REF]. These data confirm that O-GlcNAcylation plays an important role in T cell activation.

Consistently, Swamy et al demonstrated that loss of O-GlcNAcylation blocked T cell selfrenewal, clonal expansion as well as malignant transformation of T cells (Swamy et al. 2016).

O-GlcNAcylation may protect T cells from apoptotic cell death. It has been shown that O-GlcNAcylation of DNA Fragmentation Factor 45 (DFF45) confers resistance to its cleavage by caspases during DNA damage-induced apoptosis in T cells and protect them from death [START_REF] Johnson | Implications of the O-GlcNAc Modification in the Regulation of Nuclear Apoptosis in T Cells[END_REF].

O-GlcNAcylation in macrophage

Monocytes and macrophages are strongly implicated in inflammation associated with chronic hyperglycemia. Chronic hyperglycemia may affect macrophage functions through different mechanisms. O-GlcNAcylation is one of the mechanisms known to mediate high glucose effect. Several studies revealed a role for O-GlcNAcylation in the modulation of inflammatory response in macrophage with contradictory results.

O-GlcNAc promotes macrophage pro-inflammatory phenotype

In the human THP1 monocyte line, high glucose concentrations and PUGNAc increase the secretion of MIP1 α and β (Macrophage Inflammatory Proteins) via an OGT-dependent epigenetic mechanism [START_REF] Chikanishi | Glucose-Induced Expression of MIP-1 Genes Requires O-GlcNAc Transferase in Monocytes[END_REF]). These studies are therefore in favor of a proinflammatory role of O-GlcNAcylation in macrophages.

Recently, it has been identified that O-GlcNAcylation exerts a pro-inflammatory effect in macrophage by antagonizing the activation of the transcription factor STAT3. The signal transducer and activator of transcription 3 (STAT3) is a key mediator of inflammation by induction of the anti-inflammatory cytokine IL10 [START_REF] Takeda | Enhanced Th1 Activity and Development of Chronic Enterocolitis in Mice Devoid of Stat3 in Macrophages and Neutrophils[END_REF]). Li el al revealed that O-GlcNAcylation of STAT3 at threonine 717 negatively regulates its phosphorylation and and thereby induces STAT3-dependent gene expression in LPS-stimulated myeloid-lineage derived macrophage resulting in exacerbation of inflammatory process (X. Li et al. 2017).

O-GlcNAcylation promotes anti-inflammatory phenotype

In contrast to the previous observations, it has also documented that O-GlcNAcylation can exert anti-inflammatory effects in macrophage. Ryu et al showed, in RAW264.7 and peritoneal macrophages, that LPS increases the levels of O-GlcNAcylation (Ryu and Do 2011). In this study the authors suggest that this increase in O-GlcNAcylation is related to an increase in the activity of the OGT. In addition, in this study, it was found that LPS increases O-GlcNAcylation levels of p65, and participate in the secretion of IL1β and NO in response to LPS (Ryu and Do 2011).

Moreover, a study revealed a neuroprotective effect O-GlcNAcylation. They observed that Thiamet G inhibits the nuclear translocation of NFκB resulting in an inhibition of production of pro-inflammatory cytokines and induces a M2 type polarization of microglial cells [START_REF] He | Thiamet G Mediates Neuroprotection in Experimental Stroke by Modulating Microglia/Macrophage Polarization and Inhibiting NF-ΚB P65 Signaling[END_REF]. In addition, another study by Hwang and his team showed that overexpression of OGT in microglial Bv2 cells will unexpectedly decrease the transcriptional activity of NFκB both in the absence and in the presence of glucosamine. This overexpression inhibits the increase in NOS2 expression in response to LPS (S.-Y. Hwang et al. 2013b).

Xinghui Li et al very recently demonstrated that LPS stimulation of macrophage decreased

protein O-GlcNAcylation. They then showed that in a septic shock mice model, either by intraperitoneal LPS injection or by caecal ligation, the deletion of OGT in macrophage induced a higher pro-inflammatory profile than wild type mice (X. Li et al. 2019). Another study was published in parallel was also in same line for the anti-inflammatory role of O-GlcNAcylation in macrophage. They revealed that mitochondrial antiviral-signaling protein (MAVS), a key mediator of interferon signaling, is also O-GlcNAcylated in macrophages.

MAVS O-GlcNAcylation was found to be critical host defense against virus (N. Song et al.

2019).

In conclusion, although the involvement of hyperglycemia in the inflammation associated with the metabolic syndrome is now well known, the role of O-GlcNAcylation on the modulation of inflammation remains poorly studied and seems to be a complex phenomenon. In addition, few studies concern the functions and regulation of this posttranslational modification in immune cells. Among them, very few have been particularly focused in macrophages. Moreover, the molecular mechanism by which immune cell activation modulates protein O-GlcNAcylation remains elusive.

Thesis Project

Although the implication of inflammation and O-GlcNAcylation in the glucotoxicity phenomenon is now well known, the relationships between inflammatory processes and O-GlcNAcylation of proteins remain elusive. In addition, very few studies have studied the involvement of O-GlcNAcylation in immune cells. In particular, the role of O-GlcNAcylation of proteins in the macrophage is poorly studied and contradictory results have been obtained.

Several studies indicate that O-GlcNAcylation may have a pro-inflammatory role in LPSstimulated macrophages whereas other groups showed an anti-inflammatory effect.

Moreover, little is known concerning the mechanisms that regulate protein O-GlcNAcylation level in these cells. Thus, the aim of this thesis work was to assess the involvement of the O-GlcNAcylation pathway in the macrophage.

Our main objectives were:

I. To investigate the effect of activation of the TLR4 pathway by LPS on the levels of O-GlcNAcylation in macrophages.

II. To elucidate the mechanisms by which TLR4 activation regulates protein O-GlcNAcylation in macrophages.

III.

To evaluate the impact of OGT deletion on the pro-inflammatory effects of LPS.

IV. To study In vivo consequences of ogt invalidation in macrophages on inflammation associated with High fat/ High sucrose (HF/HS) diet 5 Results

The stimulation of the TLR4 pathway by LPS induces an increase in protein O-GlcNAcylation levels

We first wanted to determine the effect of activation of the TLR4 pathway by LPS on O-GlcNAcylation levels in macrophages. In order to study the effects of LPS on O-GlcNAcylation in living cells, we have developed a BRET biosensor from a FRET biosensor previously described by the team of L.K. Mahal [START_REF] Carrillo | Targeted in vivo O-GlcNAc sensors reveal discrete compartment-specific dynamics during signal transduction[END_REF], allowing us to follow variations in O-GlcNAcylation levels. To do this, we replaced the CFP (Cyan fluorescent protein) of the FRET biosensor with an Rluc8 (Figure 35). These biosensors permit detection proteins O-GlcNAcylation activity in different cell compartments: nucleus, cytosol and plasma membrane.

We transfected RAW264.7 cells, using electroporation, with different biosensors. 18 hours after transfection, we treated the cells with LPS (100ng / ml) Thiamet G (an OGA inhibitor, 10µM), or both for 24 hours. We then observed that in the three compartments studied, LPS induces a significant increase in the BRET signal, at a level similar to that observed with Thiamet G. These results suggest that stimulation by LPS leads to a general increase in O-GlcNAcylation levels in macrophages and that O-GlcNAcylation could therefore have a role in the TLR4 signaling pathway.

Interestingly, we observed that when cells are treated with both LPS and Thiamet G, the effects on O-GlcNAcylation are additive, suggesting that the effects of LPS and Thiamet G are mediated by different mechanisms. These results indicate that LPS induces an increase in O-GlcNAcylation activity in all 3 compartments (Figure 36).

By western bloting using anti-O-GlcNAc anti-bodies, we confirmed that LPS induced O-GlcNAcylation in RAW264.7 macrophages. These observation was also obtained in primary macrophages from human (HMDM) and mouse (BMDM and peritoneal).

Investigation of the molecular mechanism by which LPS induces an increase in protein O-GlcNAcylation

Activation of TLR4 by LPS stimulates GFAT2 expression in macrophage

We further explored the mechanism involved in LPS-induced O-GlcNAcylation in RAW264.7 macrophages. For that, we treated RAW264.7 cells with or without LPS for 24 hrs. We then lysed the cells and precipitated the glycosylated proteins on wheat germ lectin (WGL) coupled with agarose beads. As expected, we observed an increase in the general retention of O-GlcNAcylated proteins on WGL, detected using an anti-O-GlcNAc (RL2)

antibody (Figure 37.A) confirming our previous observations. Moreover, we wanted to know if the increase in O-GlcNAcylation levels associated with LPS stimulation could be due to a modification of the expression of O-GlcNAcylation / de-GlcNAcylation enzymes. Cell lysates were analysed by western blot, as shown in Figure (37.B) and (37.C). We did not observe any changes in the expression of neither OGT, the enzyme which O-GlcNAcylates proteins, or OGA, the enzyme which allows the deglycosylation of proteins, neither at protein level nor messenger RNA. This suggests that LPS-induced O-GlcNAcylation was not mediated by regulation of O-GlcNAc cycling enzymes.

We then looked at the effect of LPS on the expression of GFAT, the limiting enzyme in the hexosamine pathway. In the basal state we observed that only the GFAT1 isoform was expressed in RAW264.7 cells. LPS stimulation increases modestly the protein and mRNA expression. In contrast, although the GFAT2 protein was almost undetectable under basal conditions, LPS induces markedly its expression. The increase in GFAT2 protein levels by LPS was associated to a large induction of mRNA levels (by a factor of 10) of this enzyme.

Figure 37: LPS up-regulates proteins O-GlcNAcylation and HBP rate limiting enzymes expression

A) RAW264.7 cells were cultured for 24 hours in the absence or presence of LPS. After cells lysis, the proteins were precipitated on wheat germ lectin (WGL) fixed on agarose beads. The proteins precipitated on WGL were subjected to a western blot and revealed with an anti-O-GlcNAc antibody.

B) The total cell lysate (TCL) was also analyzed by western blot is with anti OGT, OGA, GFAT1, GFAT2, NOS2 antibodies. On the right side, protein quantification by densitometry using image J relative to the expression of housekeeping protein (clathrin) for 8 independent experiments.

C) The mRNAs were extracted and their expression levels were analyzed by RT-qPCR. The results are expressed relative to the level of expression obtained in the untreated cells and are the means ± SEM of 6 independent experiments. Statistical analyzes were performed using a Student t test (*: p< 0.05; **: p <0.01, ***: p< 0.001).

Gfpt2 is an early TLR4 inducible gene

In order to further characterize the expression profile of both GFAT isoform, a timecourse experiment was performed. We observed that the mRNA expression of GFAT1 displayed a gradual increase over time. In contrast, GFAT2 was markedly induced after 1-3 hours post-LPS treatment and stabilize at law level until the end of stimulus (Figure 38).

These data indicate that Gfpt2 is an early inducible gene in macrophage. Distinct transcriptional profile suggests a different regulatory mechanism for each isoform in macrophage. We were interested particularly to investigate the molecular mechanism that is implicated in the transcriptional regulation of Gfpt2 since its expression behaves like other TLR4-induced genes.

LPS induces GFAT2 expression and enhances protein O-GlcNAcylation in primary macrophage

We then evaluated the effect of TLR4 stimulation on protein O-GlcNAcylation and GFAT2 protein expression in primary macrophage. For this purpose, we used macrophages differentiated from mice bone marrow cells (BMDM) and human primary macrophages which were isolated from blood of healthy donors (HMDM). As observed in RAW264.7 cells, LPS treatment for 24h enhanced protein O-GlcNAcylation, and this was associated with induction of GFAT2 mRNA and protein expression in BMDM (Fig. 39.A), and in HMDM (Fig. 40.B).

Figure 39: LPS effect on O-GlcNAcylation and O-GlcNAc regulatory enzymes in BMDM

A) BMDM generated from C57Blck6J male mice were left untreated or stimulated with the 100ng/ml of LPS for 24h. After cell lysis, proteins were submitted to western blot and hybridized OGT, GFAT1, GFAT2, NOS2 antibodies on (left). Using image J software, densitometric analysis was performed.

Results are the mean of 8 independent experiments corrected by clathrin. The mRNAs were extracted and their expression levels analyzed by RT-qPCR (right down) for 9 experiments.

LPS stimulates GFAT enzymatic activity in macrophage

Furthermore, to determine whether increased in GFAT expression resulted in increase in its enzymatic activity, we investigated the effect of TLR4 stimulation by LPS on GFAT activity by spectrophotometric assay using the protocol that has been previously described [START_REF] Ye | A Simple and Sensitive Method for Glutamine:Fructose-6-Phosphate Amidotransferase Assay[END_REF]) (Figure 41).

We observed that LPS treatment for 24h significantly increased GFAT activity in RAW264.7 cells; bone marrow derived macrophage and in human monocyte derived macrophage (figure 42 A, B and C respectively). This suggests that LPS induced GFAT1/2 protein and mRNA expression leads to increase GFAT activity. GFAT being the rate limiting step of the HBP, an increase in its activity results in increase the flux through this pathway.

This may explain the increased in protein O-GlcNAcylation observed upon LPS stimulation.

LPS induces GFAT2 expression in vivo

After the demonstration of the effect of LPS on protein O-GlcNAcylation and GFAT2

induction in vitro, we wanted to confirm this effect in vivo. Therefore, we injected mice with 0.6 g per kg of body weight of LPS intra-peritoneally for 6h. Then we harvested peritoneal macrophage for analysis by western blot. As expected, we observed that LPS induced proinflammatory cytokines (IL1β and NOS2) protein expression, but also GFAT2 expression.

This was associated with an increased O-GlcNAc profile in these cells with no change in OGT expression (Figure 43).

Figure 43: LPS effect on proteins O-GlcNAcylation in vivo

C57Blck6J male mice (5 mice in each group) were injected intra-peritoneally either with physiological serum or 0.6 g/kg of LPS for 6h. Peritoneal macrophages were collected and incubated for 2 h then washed to eliminate non adherent cells. Adherent macrophages were lysed for protein analysed by western blot. Densitometric analysis (right side) for indicated proteins was corrected relatively to clathrin expression. Statistical analyzes were performed using a Student t test (*: p< 0.05; **: p <0.01, ***: p< 0.001).

Molecular mechanism by which LPS induces GFAT2 in macrophage

We then wanted to investigate the molecular mechanism that mediates LPS in induction GFAT1/2 expression in macrophage.

HIF1α has no effect on the expression of HBP enzymes

Manzari and his team have shown that in macrophages, the expression of GFAT1 was increased in response to hypoxia (or in response to factors mimicking hypoxic stimuli such as picolinic acid and desferrioxamine). They demonstrated that this increase was due to the activation of the GFPT1 promoter through a Hypoxia Responsive Element (HRE) sequence.

They suggested that the transcriptional activation of the GFPT1 gene is due at least in part to the Hypoxia Inducible Factor 1 α (HIF1α) (Manzari et al. 2007). Therefore, we studied the effect of HIF1α deletion on LPS induced expression of both GFAT isoforms. For this purpose, we used mice in which HIF1α deleted specifically in macrophages (LysM cre lox/lox HIF1α), kindly provided by Dr. Carole Peyssonnaux. We observed that the deletion of HIF1α

has no effect on LPS induced GFAT1 and GFAT2 mRNA and protein expression (Figure 44). This result suggests that other mechanisms could be involved to explain LPS induced expression of GFAT1 and GFAT2.

Inhibition of NFκB activity partially attenuates LPS induced GFAT2 expression

We were particularly interested in the molecular mechanism that regulates GFAT2 expression, since its transcription behaves as an early TLR4 inducible gene. We hypothesized that GFPT2 may be a target gene of the transcription factor NFκB. To test this hypothesis, we used BAY 11-7082, an inhibitor of IKK which blocks TLR4 induced phosphorylation and degradation of IκB inhibitor and thus prevents NFκB nuclear translocation. RAW264.7 were incubated either with this inhibitor or DMSO for 30 min, then treated or not with LPS for further 24 h. We observed that the inhibition of NFκB activity partially decreased LPS-induced GFAT2 transcription (Figure 45). This suggests although NFkB may participate in the regulation of GFPT2, other mechanisms may be involved.

FoxO1 is essential for LPS-induced GFAT2 expression and O-GlcNAc stimulation in macrophage 5.3.3.1 The promoter of GFAT2 contains FoxO1 functional binding sites

The transcription factor Forkhead box 1 (FoxO1) has been shown to paly a critical role in linking glucose metabolism to inflammation in skeletal muscle [START_REF] Lundell | Regulation of Glucose Uptake and Inflammation Markers by FOXO1 and FOXO3 in Skeletal Muscle[END_REF].

Moreover, FoxO1 has been also reported to regulate the expression of several TLR4-inducible genes in macrophages (Su et al. 2009b;Fan et al. 2010). We hypothesized that FoxO1 may be involved in the regulation of GFAT2 expression in macrophages.

We analysed mouse and human GFPT2 putative promoters and we revealed canonical FoxO1 recognition sites within the 500bp and 800pb regions upstream of the transcription start site of the mouse and human genes, respectively. We inserted these upstream sequences in a luciferase reporter gene plasmid. Transfection in HEK293 cells with this plasmid revealed that the activities of both human and mouse reporter genes were markedly increased by co-transfection with a constitutively active form of FOXO1, FOXO1-TM (Figure 46.A).

The mouse 500bp promoter contains two FoxO1 recognition sites (referred to as Site1 .

Invalidation of FoxO1 blocks LPS induced GFAT2 expression

To evaluate the potential involvement of FoxO1 in the regulation of GFAT2 expression, we used a small molecule inhibitor of FoxO1 activity, AS1842858. We incubated cells in the absence or presence AS1842858 for 72h. Cells were then treated with or without LPS for further 24h before cell lysis. We observed, both in RAW264.7 cells (Figure.

OGT deletion promotes LPS-induced pro-inflammatory response

In order to study the role of LPS-induced O-GlcNAcylation, we have developed in our laboratory a mouse model (LyzM-cre mouse lox/lox OGT or OGT-KO mac ) with a targeted deletion of OGT in the macrophage.

OGT deletion promotes macrophage M1 polarization

In a preliminary experiment, we evaluated the role of OGT in the polarization of Bmacrophages; we used macrophages derived from bone marrow of wild type or KO-OGT mac mice. Macrophages were cultured for 48 hours in the presence or absence of IFNγ to induce their polarization into M1 phenotype. We then performed surface labeling with a mixture of antibodies including: anti-CD45 (LCA, leukocyte common antigen), anti-CD11b, anti-SiglecF (sialic acid-binding Ig superfamily receptor that is highly expressed on eosinophils), anti-F4 /80 (marker for macrophage), anti-CD11c (expressed highly in M1 pro-inflammatory macrophage) and anti-CD206 (expressed mainly in M2 anti-inflammatory phenotype). After elimination of non-macrophage cells, we sorted cells mainly in two populations either, F4/80-CD11c for pro-inflammatory M1 macrophage and F4/80-CD206 for anti-inflammatroy M2 macrophage.

We observed that in basal conditions, the frequency of F4/80-CD11c expressing macrophages (59%) is significantly higher in the OGT KO macrophage population than in the WT macrophage population (46%). Moreover, the frequency of F4/80-CD206 expressing macrophages is higher in the population of WT macrophages (51%) than in the population of KO macrophages (38%). These results suggest that OGT in macrophage prevents M1 polarization in in basal condition (Figure 48).

Upon IFNγ treatment, we observed that the frequency of F4/80-CD11c macrophages is higher in the population of OGT-KO macrophages in comparison with the population of WT macrophages (74% vs 65%). Conversely, the frequency of F4/80-CD206 macrophages is lower in the OGT-KO macrophage population than in the wild type population (24% vs 32%).

These data indicate that deletion of OGT promotes M1 polarization both in absence and presence of IFNγ, suggesting that O-GlcNAcylation favors M2 phenotype.

Ogt deletion exacerbates pro-inflammatory effect of LPS in macrophage in vitro and in vivo

we performed ex vivo experiments, using peritoneal macrophages of these mice. We incubated these cells in the absence or presence of LPS for 24 h. Before cell lysis, we collected the supernatant to measure the secretion of pro-inflammatory cytokines by multiplex assay and ELISA kit.

As expected, in western blot using an anti-O-GlcNAc antibody, we observed that the levels of O-GlcNAcylation are totally inhibited in the macrophages of KO-OGT mac mice, both in the presence or absence of LPS. The effect of LPS on the expression of NOS2, the enzyme responsible for the production of nitric oxide during the activation of macrophages, was also increased in KO-OGT mac macrophage cells compared to control cells. Moreover, the expression of GFAT, and particularly GFAT2, was highly up-regulated in KO mice compared to wild type (Figure 49.A).

Analyses of inflammatory cytokines in the supernatant enabled us to observe that the effect of LPS on the production of pro-inflammatory cytokines IL1β and IFNγ was greatly increased in OGT cells in KO macrophage compared to WT control cells ( Figure 49.B).

Moreover, we performed a time-course experiment to study specifically the secretion of IL1β in the supernatant. We found that the secretion of IL1β is significantly higher in the supernantant of KO-OGT maccrophage at all time points compared to wild type cells (Figure 49.C). Finally, to confirm the effect of O-GlcNAc in inflammatory process in vivo, we injected wild type and KO-OGT mac mice either with 0.6 g/kg of LPS or physiological serum.

We collected the serum of these mice to measure circulating cytokines by MSD multiplex kits. We found that, similar to ex vivo results, pro-inflammatory cytokines IL1β and IFNγ in the serum was higher in KO-OGT mac mice than wild type after LPS injection (figre 49.D).

Moreover, in the serum, TNFα was also higher in KO-OGT mac mice in comparison to wild type. These data indicate that OGT has a negative effect on pro-inflammatory response of macrophage upon TLR4 activation.

Invalidation of ogt in macrophage impairs glucose tolerance in mice fed high fat/ high sucrose diet

In order to further characterize the effect of OGT invalidation in the macrophage during low grade inflammation associated with metabolic syndrome, we submitted wild-type and OGT-KO mac mice to a HF / HS diet for 10 weeks. We monitored the body weight and the food intake of the animals weekly.

We observed that OGT-KO mac mice fed HF/HS diet exhibited a higher tendency to gain weight compared to wild-type mice fed with the same diet, although the difference was not statistically significant. There was no difference in food intake between OGT-KO mac and wild-type mice suggesting that deletion of OGT in macrophage has no effect on the appetite (Figure 50.A). To determine whether the deletion of OGT in macrophage may affect glucose hemostasis, we performed a glucose tolerance test (OGTT) after 10 weeks of diet. Blood sugar was measured at [START_REF] Swamy | Glucose and glutamine fuel protein O-GlcNAcylation to control T cell self-renewal and malignancy[END_REF][START_REF] Xing | O-GlcNAc modification of NFkappaB p65 inhibits TNF-alpha-induced inflammatory mediator expression in rat aortic smooth muscle cells[END_REF][START_REF] Issad | Isoproterenol inhibits insulin-stimulated tyrosine phosphorylation of the insulin receptor without increasing its serine/threonine phosphorylation[END_REF][START_REF] Sayeski | The murine glutamine:fructose-6-phosphate amidotransferase-encoding cDNA sequence[END_REF]90 and 120 min after oral glucose load of 20 mg/ml. We observed that fasting blood glycaemia was normal in both groups. After the glucose challenge, we found that the group OGT-KO mac mice displayed a significant glucose intolerance compared to wild type mice (Figure 50.B). This indicates that OGT deletion promotes glucose intolerance in response to HF/HS diet. These results suggest that increased inflammatory profile of OGT-KO macrophage may favor glucose intolerance when mice are fed a HF/HS diet.

Discussion

The aim of our study was to investigate in macrophages a potential cross talk between the inflammatory signaling pathway and protein O-GlcNAcylation, one of the mechanisms implicated in glucotoxicity. This was achieved at first by studying the effect of TLR4 activation by LPS on general protein O-GlcNAcylation level in different macrophage models.

Then, the work was followed by evaluation the impact of invalidation of the enzyme OGT on the inflammatory processes induced by LPS in macrophage.

We were able to demonstrate that: (iii) O-GlcNAcylation plays a negative feed-back role in inflammatory process induced by LPS.

Activation of TLR4 by LPS stimulates protein O-GlcNAcylation in macrophages

At the beginning of our project, the effects of TLR4 activation by LPS on O-GlcNAcylation in macrophages were poorly studied, with contradictory results. Whereas some studies showed an increase in O-GlcNAcylation levels in macrophages of the RAW264.7 cell line after stimulation with LPS (Ryu and Do 2011;J.-S. Hwang et al. 2017).

Other studies showed the converse. In our work, we showed, using a BRET-based method, that LPS induces an increase in O-GlcNAcylation in RAW264.7 macrophages. We confirmed this result by western-blotting with anti-O-GlcNAc antibody, and then extended this observation to macrophages from different origins (mice bone marrow-derived and peritoneal macrophages, as well as in human monocyte-derived macrophages). Furthermore, we showed that peritoneal macrophages stimulated with LPS in vivo exhibited an increase in protein O-GlcNAcylation. All these data with different approaches strongly establish that TLR4 stimulation by LPS enhanced intracellular level of protein O-GlcNAcylation. This suggests an important role of O-GlcNAc in TLR4 biological effect in macrophages.

As mentioned previously, whereas similar observations were made by other groups, some groups found that LPS decreases O-GlcNAcylation in macrophages. The reasons for these discrepancies are unknown at the present time. We can speculate that some differences in the experimental procedure may be responsible for these discrepancies. As mentioned in the introduction, O-GlcNAcylation is a dynamic, rapidly reversible modification due to the interplay between O-GlcNAc cycling enzymes, OGT and OGA. During cell lysis and sample preparation, loss of O-GlcNAcylation may occur (Hart et al. 2011). To avoid this potential artefact, we always add the inhibitor PugNAc to the extraction buffer, in order to inhibit the de-GlcNAcylation of O-GlcNAcylated proteins. The other groups did not mention this precaution. Moreover, LPS induced protein O-GlcNAcylation was also clearly detected in intact living cells using a totally independent methodology (BRET biosensors), which does not involve extraction and manipulation of glycosylated protein. This strongly support our results obtained by classical biochemical technique in different macrophages models.

LPS-induced GFAT expression and activity may mediate increase O-GlcNAcylation in macrophages

After having demonstrated that LPS stimulates an increase in protein O-GlcNAcylation in macrophages, we wanted to know if this increase could be mediated by regulation of the two O-GlcNAc cycling enzymes, OGT and OGA. Indeed, an increase in protein O-GlcNAcylation could be due to an increase in OGT activity, a decrease in OGA activity or both. We did not observe any significant change in OGT expression of after macrophage stimulation with LPS. We also did not observe any differences in the activity of OGT in cells treated with LPS, unlike Ryu and his team who suggested an increase in its activity following its denitrosylation in response to LPS. Experiments performed in our laboratory by Léa Baudoin evaluated the implication of NOS2 on protein O-GlcNAcylation using NOS2 KO mice. LPS-induced protein O-GlcNAcylation was similar in macrophages from NOS2 KO mice and in their wild-type counterpart. This suggests that a potential regulation of OGT nitrosilation by NOS2 does not play a significant role in LPS-induced O-GlcNAcylation in macrophages.

Moreover, we did not observe any change in the expression nor in the activity of OGA in response to LPS in macrophages. This is consistent with the fact that LPS still strongly induced O-GlcNAcylation in cells in which OGA activity is inhibited by Thiamet G.

Therefore, changes in protein O-GlcNAcylation upon LPS stimulation do not appear to be due to a regulation of O-GlcNAcylation cycling enzymes. In contrast, we observed, in different cell models (RAW264.7, BMDM and HMDM), that the stimulation by LPS leads to a significant increase in the activity of GFAT, the rate limiting enzyme of HBP. This increased activity of the rate limiting enzyme of HBP is likely to result in an increase in the synthesis of UDP-GlcNAc, the substrate of OGT. An increase in the concentration of UDP-GlcNAc will then allow an increase in the level of O-GlcNAcylation. OGT has been shown to display three distinct Km for UDP-GlcNAc, suggesting that the concentration of UDP-GlcNAc may directly affect the specificity of OGT action [START_REF] Kreppel | Regulation of a Cytosolic and Nuclear O -GlcNAc Transferase: ROLE OF THE TETRATRICOPEPTIDE REPEATS[END_REF].

This increase in GFAT activity appears to result from an increase in its expression.

Very little data are available concerning the expression of GFAT in macrophages. GFAT exists in two isoforms, encoded by different genes. The expression of GFAT in macrophage was first studied by Manzari et al, they indicated that GFAT1 was constitutively expressed in ANA1 cell macrophages, and they mentioned (as data not shown) that LPS had no effect on its expression. GFAT2 was not mentioned in this study (Manzari et al. 2007).

Consistently with the work of Manzari et al, we observed that GFAT1 is constitutively expressed in macrophages. LPS treatment modestly increased the expression of GFAT1. In contrast, we found that whereas the expression of GFAT2 is undetectable in the basal state in macrophages, LPS very strongly induces its expression (both at mRNA and protein levels). GFAT2 has been suggested as the GFAT isoform preferentially expressed in the central nervous system [START_REF] Dehaven | A Novel Variant of Glutamine: Fructose-6-Phosphate Amidotransferase-1 (GFAT1) MRNA Is Selectively Expressed in Striated Muscle[END_REF]). Here we were able to show for the first time that GFAT2 can be expressed in macrophage as an isoform inducible by TLR4 activation.

Thus, the increase in GFAT activity upon LPS stimulation is likely due to the major induction of GFAT2 expression observed in these conditions. Therefore, while GFAT1 may mainly control basal flux through the HBP pathway, GFAT2 induction may mediate LPS stimulated protein O-GlcNAcylation in macrophage. Nevertheless, it should be mentioned that glucose transporter GLUT1 has also been shown to be upregulated upon LPS stimulation. This is what we also found it in our experiments in RAW264.7 and mouse primary macrophages (data not shown). Therefore, an increase in glucose uptake may also play a role in increasing O-GlcNAcylation, particularly if the flux through the rate limiting step catalyzed by GFAT is also increased.

FoxO1 mediates LPS induced GFAT2 expression in macrophage

We demonstrated that GFAT1 and GFAT2 exhibit distinct expression behaviour upon LPS stimulation. Whereas GFAT1 expression gradually (and moderatly) increased overtime, GFAT2 displays maximum expression level 1-3 hours post-LPS stimulation. These data indicate that GFPT2 is an early LPS inducible gene in macrophage. Distinct transcriptional profile suggests a different LPS regulatory mechanism for each isoform in macrophage.

It has been suggested that Gfpt1 gene promoter can be activated by Hypoxia Inducible Factor 1 α (HIF1α) during hypoxia (Manzari et al. 2007). On the other hand, in normoxia, HIF1α expression itself is up-regulated by LPS in macrophage [START_REF] Tannahill | Succinate Is an Inflammatory Signal That Induces IL-1β through HIF-1α[END_REF]. In an attempt to determine whether HIF1α could mediate regulation of GFAT1/2 expression upon LPS stimulation, we studied macrophages from mice with targeted deletion of HIF1α. We showed that the deletion of HIF1α had no effect on GFAT1/2 expression in BMDM and in peritoneal macrophages. Thus, whereas HIF1α may regulate GFAT1 expression under hypoxic conditions, this transcription factor does not appear to mediate LPS effects on GFAT1 and GFAT2 expression.

We then investigated a potential regulation of nuclear factor NFκB on GFAT2 expression in our system. We observed that pharmacological inhibition of IKK complex by which results in inhibition of NFκB nuclear translocation, leads to a partial attenuation of LPS-induced GFAT2 expression. These data suggested that nuclear factor NFκB activity may participate in the induction of GFAT2 expression upon LPS stimulation.

Previous studies have revealed a role for FoxO1 in the expression of LPS-inducible genes. Su et al have previously revealed that FoxO1 binds selectively to the promoter of IL1β

and acts synergistically with p50/NFκB upon LPS stimulation to induce IL1β expression in macrophages. We analyzed the putative promoter of human and mouse GFPT2 gene and we detected two potential binding sites for the transcription factor FoxO1 in the 500bp or 800pb regions upstream of the transcription start site. We validated these two sequences as FoxO1 binding sites in macrophage in chromatin immunoprecipitation experiments. Moreover, we showed, in HEK293 cells transfected with a Gfpt2 promoter-reporter gene that FoxO1 induced the luciferase activity of this reporter gene whereas it had no effect on this reporter gene when these sites were mutated. These results confirm that FoxO1 may regulate GFAT2 transcription. Furthermore, we showed that pharmacological inhibition of FoxO1 (AS1842858) in RAW264.7 cells and HMDM blocked LPS-induced GFAT2 expression.

Moreover, we showed that genetic ablation of FoxO1 totally impaired LPS-induced GFAT2 expression and protein O-GlcNAcylation. Collectively these data suggest that FoxO1 is necessary for LPS-induced protein O-GlcNAcylation in macrophages via regulation of GFAT2 expression.

However, the molecular mechanism that mediates FoxO1 activation during LPS stimulation remains to be elucidated.

FoxO1 regulation is principally achieved by post-translational modifications that regulate its nuclear localisation and/or its transcriptional activity. Whereas phosphorylation of FoxO1 by Akt inhibits its transcriptional activity, several studies revealed that FoxO1 can be acetylated by the histone acetyltransferases CBP (cAMP response element binding protein CREB binding protein) and p300, whereas NAD-dependent deacetylase SIRT1 mediates its deacetylation. However, contradictory results emerged for the regulation of FoxO1 transcriptional activity by acetylation. It has been shown that CBP mediates acetylation of FoxO1 at Lys-242, Lys-245 and Lys-262, which augments its transactivation. However, others showed that the acetylation of FoxO1 attenuates its transcriptional activity [START_REF] Daitoku | Silent Information Regulator 2 Potentiates Foxo1-Mediated Transcription through Its Deacetylase Activity[END_REF]).

As mentioned in the introduction, LPS stimulation induces oxidative stress in macrophages. It has been indicated that oxidative stress stimulates FoxO1 transcriptional activity via promoting its acetylation in pancreatic β cells [START_REF] Kitamura | FoxO1 Protects against Pancreatic β Cell Failure through NeuroD and MafA Induction[END_REF].

Moreover, FoxO1 regulation also occurs through its binding to 14-3-3 protein, which retains it in the cytosol and thereby impairs its transcriptional activity. Oxidative stress can also stimulate FoxO1 activity by disruption of FoxO1-14-3-3 interaction through JNKmediated phosphorylation of 14.3.3 (Weng et al. 2016). Therefore, it is possible that LPSinduced ROS production stimulates FoxO1 activity in macrophages. FoxO1 is known to stimulate the expression of genes involved in ROS detoxification (MnSOD2, Catalase, Peroxiredoxins 3 and 5). ROS-induced FoxO1 activation could constitute a regulatory loop to provide cells with protection against oxidative stress [START_REF] Akasaki | FoxO Transcription Factors Support Oxidative Stress Resistance in Human Chondrocytes: FoxO Function in Human Chondrocytes[END_REF][START_REF] Kitamura | FoxO1 Protects against Pancreatic β Cell Failure through NeuroD and MafA Induction[END_REF]. Interestingly, GFPT2 gene has been identified in a high-throughput functional genomics screening aimed at revealing human genes that protect from oxidative stress induced by H2O2 in HT-22 neuronal cells. In this screen, among 5000 human cDNAs individually transfected into HT-22 cells, GFPT2 was found to have a protective effect against H2O2-induced toxicity, together with 5 other well-known genes involved in anti-oxidative response (Catalase, glutathione peroxidase-1, peroxiredoxin-1, peroxiredoxin-5, and nuclear factor erythroid-derived 2-like 2) [START_REF] Zitzler | High-Throughput Functional Genomics Identifies Genes That Ameliorate Toxicity Due to Oxidative Stress in Neuronal HT-22 Cells: GFPT2 Protects Cells Against Peroxide[END_REF]. It is therefore tempting to speculate that FoxO1-dependent GFAT2 induction may participate in a mechanism that protects cells against oxidative stress upon LPS stimulation.

O-GlcNAcylation attenuates LPS-induced pro-inflammatory response in macrophages

The role of O-GlcNAcylation in inflammatory processes in macrophage is also To evaluate the role of O-GlcNAc on inflammatory processes, we studied the effect of invalidation of ogt in macrophage by developing OGT KO macrophage mice in our laboratory. We studied the effect of ogt deletion in macrophages upon LPS treatment in vitro and in vivo. In an attempt to study the role of macrophage OGT during metabolic syndrome, we also challenged these mice with a high fat/high sucrose diet.

Unexpectedly, we observed that the deletion of ogt in macrophages induced pro-inflammatory M1 polarization in unstimulated cells as well as in IFNγ stimulated macrophages, when compared to wild-type macrophages. In agreement with this finding, we observed that upon LPS stimulation, OGT KO mac expressed higher level of nitric oxide synthase NOS2.

Moreover, we also showed that LPS-induced secretion of pro-inflammatory cytokines (IL1β, TNFα and IFNγ) was exacerbated in OGT-KO macrophages in comparison to wild-type macrophages.

These results suggest that the presence of OGT in macrophage acts in a self-limiting pathway of inflammatory processes induced by LPS. Our present observation is in agreement with several studies reporting that O-GlcNAcylation inhibits NFκB transcription activity.

Xing et al have shown that PugNAc and glucosamine treatment inhibited TNFα induced-NFκB activity through O-GlcNAcylation of its p65 subunit in rat aortic smooth muscle cells (Xing et al. 2011b). Moreover, Hwang et al revealed that O-GlcNAcylation inhibited LPSinduced NOS2 expression in RAW264.7 via regulation of the transcription corepressor complex mSin3A-HDACs. They suggested that OGT acts as transcriptional repressor by directing the transcription corepressor complex mSin3A-HDACs to NOS2 promoter upon LPS stimulation (S.-Y. Hwang et al. 2013b). Therefore, it would be important to verify in our system the transcriptional activity of NFκB and/or the nuclear translocation of p65 in OGT -KO macrophages as well as to measure nitric oxide production by these cells.

Another possible mechanism that could explain this hyperinflammatory phenotype is the fact that O-GlcNAc deficiency could promote reactive oxygen species ROS production. As previously mentioned in the introduction, O-GlcNAcylation has been suggested as a protective pathway against oxidative stress via regulation of the transcriptional activities of FoxO1 (Kuo et al. 2008;[START_REF] Housley | O -GlcNAc Regulates FoxO Activation in Response to Glucose[END_REF]Fardini et al. 2014) and Nrf1 (J. W. [START_REF] Han | Nuclear Factor-Erythroid-2 Related Transcription Factor-1 (Nrf1) Is Regulated by O-GlcNAc Transferase[END_REF]). These factors are known to control the expression of genes implicated in the detoxification of ROS. Moreover, oxidative stress stimulates the O-GlcNAcylation of glutathione peroxidase 1 (GPX1), which stimulates its enzymatic activity for ROS detoxification (Won Ho [START_REF] Yang | O-GlcNAcylation Regulates Hyperglycemia-Induced GPX1 Activation[END_REF]). Furthermore, it has been shown that inhibition of ROS by anti-oxidants (N-acetylcysteine or α tocopherol) in LPS-stimulated neutrophils abolished the production of pro-inflammatory cytokines (TNF-α, macrophage inflammatory protein-2, and IL-1β), as well as IL-1R-associated kinase-1 (IRAK-1) and IRAK4 [START_REF] Asehnoune | Involvement of Reactive Oxygen Species in Toll-Like Receptor 4-Dependent Activation of NF-ΚB[END_REF]). These authors suggested that proximal events in TLR4 signaling, at IRAK-1 and IRAK-4 activation steps, are oxidant-dependent. Therefore, ROS can modulate NF-κB-dependent transcription through their involvement in early TLR4-mediated cellular responses [START_REF] Asehnoune | Involvement of Reactive Oxygen Species in Toll-Like Receptor 4-Dependent Activation of NF-ΚB[END_REF]). Because of the protective effect of O-GlcNAc against oxidative stress, it is possible that OGT deletion in macrophages leads to a defect in the detoxification mechanism and to increase ROS production. This in turn will exacerbate inflammatory process triggered by LPS. Therefore, it would be interesting to measure ROS production by macrophage of KO OGT mac treated with LPS. It would also be interesting to evaluate the effect of inhibition of ROS with ROS scavengers (N-acetylcysteine or αtocopherol) on increased cytokines secretion in KO-OGT LPS-stimulated macrophages.

At the end of my thesis, a study was published on the role of OGT in macrophage by Li et al. They have also observed that OGT has an anti-inflammatory effect in macrophages via inhibition of necroptosis signaling (an inflammatory form of cell death). In this study they demonstrated that the member of the receptor-interacting protein family of serine/threonine kinases (RIPK3) is O-GlcNAcylated at T467. RIPK3 activation has been shown to form a necrosome complex with RIPK1 and this complex is required to induce necroptosis, leading to enhanced immune cell activation and exacerbated inflammation [START_REF] Galluzzi | Necroptosis: Mechanisms and Relevance to Disease[END_REF]). Li et al showed that OGT mediated O-GlcNAcylation of RIPK3 blocks its interaction with RIPK1 and that deletion of OGT in BMDM leads to exacerbated inflammatory phenotype with increased secretion of pro-inflammatory cytokines (X. Li et al. 2019). Surprisingly, they found an increase in RIPK3 O-GlcNAcylation upon LPS-stimulation, in a cellular context where they describe a general decrease in protein O-GlcNAcylation induced by LPS. Despite this discrepancy with our results concerning the effect of LPS on global O-GlcNAcylation, the final consequences of OGT deletion in their study is in line our observation, i.e. an exacerbated inflammatory phenotype of OGK-KO macrophages.

After evaluation of the impact of OGT deletion in macrophage upon acute TLR4 stimulation by LPS, we wanted to study the effect of OGT deletion in macrophage during a situation known to induce chronic low-grade inflammation by the intake of a high-fat/high-sucrose diet. Indeed, LPS is produced physiologically continuously in the intestine by the death of gram negative bacteria. It can then reach the blood circulation and thus create inflammation in different tissues. The increase in circulating LPS levels appears to be caused at least in part by an increase in intestinal permeability, which appears to be attributed to the change in the composition of the microbiota induced by fatty diets [START_REF] Cani | Metabolic Endotoxemia Initiates Obesity and Insulin Resistance[END_REF]). In a pilot experiment, we fed wild-type and OGT KO mice with a HF/HS diet for 10 weeks. We observed that OGT deletion in macrophages promoted weight gain without affecting food intake, suggesting that OGT activity in macrophages may somehow affect body weight regulation but have no effect on appetite. Interestingly, KO-OGT mac mice exhibited glucose intolerance compared to wildtype mice. Although these data are very preliminary and need to be confirmed and completed, it is tempting to speculate that the pro-inflammatory phenotype of OGT KO mice may favour low-grade inflammation upon HF/HS diet, and as a consequence, increased glucose intolerance in these animals.

Conclusions and perspectives

Our results indicate that stimulation of TLR4 by LPS increases protein O-GlcNAcylation in macrophage via the induction of the expression of GFAT2, the rate limiting enzyme of HBP, through a FoxO1 dependent mechanism. Our work shows also that deletion of OGT enhances the pro-inflammatory effect of LPS. Together, our results suggest that LPSinduced O-GlcNAcylation may constitute a self-protecting mechanism for the macrophage, permitting to avoid exacerbated inflammation upon acute TLR4 stimulation.

O-GlcNAcylation is known to have protective effect against oxidative stress through various mechanisms, and LPS is known to induce oxidative stress in macrophages. One of our hypotheses is that GFAT2, by inducing a marked increase in protein O-GlcNAcylation, may participate in ROS detoxification and attenuation of the inflammation induced by TLR4 activation.

To test this hypothesis, it will be interesting to evaluate the effect of pharmacologically induced oxidative stress on GFAT2 expression and protein O-GlcNAcylation in macrophages. This can be achieved by treatment of macrophages with hydrogen peroxide H2O2. Conversely, it will be interesting to study the effect of ROS scavenging by pretreatment with NAC (N-acetylcysteine) or α tocopherol on LPS induced GFAT2 expression and on protein O-GlcNAcylation.

On the other hand, it would be important to measure ROS production in cultured OGT KO macrophage, to determine whether lack of O-GlcNAcylation exacerbates LPS-induced ROS production. It will also be important to compare the activities of enzymes implicated in the regulation of oxidative stress (Sirt1, GPX1, Catalase, Superoxyde dismutase) in macrophages from wild-type and OGT-KO mice, in order to determine whether lack of OGT results in decreased anti-oxidant defenses in these cells. It will also be interesting to determine whether the inflammatory profile of OGT-KO macrophages can be corrected by incubation with ROS scavengers.

Finally, although we could only perform a pilot experiment, our preliminary study in vivo suggested that deletion of OGT in macrophage can affect the metabolic profile of mice submitted to HF/HS diet for 10 weeks. This intolerance could be due to an exacerbation of the inflammatory phenotype induced by the diet. It will now be important to fully characterize the pro-inflammatory and metabolic profile of WT and OGT-KO mac mice subjected to HF/HS diet for longer periods (4-5months).

In these mice, the consequence of OGT deletion on the metabolic profile (body weight, fat mass, glucose and insulin tolerance, insulin signaling in different tissues) and on the inflammatory profile (circulating cytokines and chemokines, recruitment of macrophages in different tissues) should be evaluated. This will permit to determine whether OGT deletion in macrophages induces a metabolic phenotype and whether this phenotype is linked to exacerbated inflammation in insulin target tissues.

Potential clinical application of this thesis work

The Recently, some clinical studies have suggested that the level of O-GlcNAcylation and the expression of its regulatory enzymes in circulating blood cells may serve as biomarkers of glycemic control in type 2 diabetic patients (Wang et al. 2009b;Park, Saudek, and Hart 2010).

During these studies, they detected an increased expression of OGA in diabetic patients without change in the expression of the enzyme OGT. Although several studies have suggested that the increase in OGA expression in diabetic patients reflects hyperglycemia in these patients, one recent study that was carried out by our laboratory in collaboration with the diabetology department of Cochin hospital indicated a link between OGA and inflammation in diabetic patients. In this study, the expression of OGA mRNA and its enzymatic activity were significantly higher in leukocytes from diabetic patients compared to that of blood leukocytes from healthy donors. Unexpectedly, OGA activity was not correlated with markers of hyperglycemia (glycemia, fructosamine, HbA1c) but was rather positively correlated with inflammatory markers such as TNFα and TxNIP in these patients [START_REF] Filhoulaud | O-GlcNacylation Links TxNIP to Inflammasome Activation in Pancreatic beta Cells[END_REF]). However, it is known that in situations where O-GlcNAcylation is increased, OGA expression is also increased as an adaptive regulatory mechanism permitting to avoid excessive protein O-GlcNAcylation. In the present thesis, we revealed that inflammation is associated with increased O-GlcNAcylation due to increase GFAT activity, notably through increased GFAT2 expression. Therefore, the correlation between OGA and inflammation markers observed by Pagesy et al. may indirectly reflect the increase in O-GlcNAc associated with inflammation, rather than a direct link between OGA and inflammation.

These observations lead us to propose that detection of GFAT2 in monocytes derived from the blood of obese or prediabetes patients may serve as a sensitive early biomarker reflecting metabolic endotoxemia induced inflammatory processes in these patients. This can be performed in clinical practice by measuring the activity of GFAT (and/or GFAT2 mRNA expression) in the blood cell-derived monocytes. Early detection of metabolic disturbances may permit intervention to prevent the development of type 2 diabetes mellitus.

On the other hand, O-GlcNAcylation has also been linked to autoimmunity through activation of T and B cells (Review: Y. Li et al. 2019). The activation of these cells is recognized to contribute in the development of various chronic autoimmune diseases, such as Type 1 diabetes. This autoimmune disease, in genetically predisposed childrens with the DR4-DQ8 haplotype, is triggered by chronic autoimmune reaction, which is mediated by autoantibodies. These autoantibodies are directed against pancreatic beta cells antigens such as GAD (glutamic acid decarboxylase), IA-2 (insulinoma associated antigen), IA (insulin) and Zn8T (zinc 8 transporter) and is ended with total destruction of beta cells mass and overt diabetes. Detection of one or more of these autoantibodies is the earliest appearing markers of autoimmunity directed towards the pancreatic islet beta cells during the first asymptomatic normoglycemic phase. However, it has been reported that up to 60% of persons who are positive for a single autoantibody may revert to seronegativity [START_REF] Knip | Prediction of Type 1 Diabetes in the General Population[END_REF].

Furthermore, although B and T cells activity is known as the key mediator of beta cell destruction, the role of the innate immune system in type 1 diabetes is also well characterized.

Introduction

O-GlcNAcylation is a post-translational modification that regulates the activity of cytosolic, nuclear and mitochondrial proteins. This modification is controlled by only two enzymes: OGT, that adds N-Acetylglucosamine (GlcNAc) on serine or threonine residues, and OGA, that removes it. O-GlcNAcylation, which regulates a wide array of biological processes [START_REF] Hart | Cycling of O-linked beta-N-acetylglucosamine on nucleocytoplasmic proteins[END_REF], depends on the availability of glucose and its metabolism through the hexosamine biosynthesis pathway (Suppl. participate in several important human pathologies [START_REF] Issad | O-GlcNAc modification, insulin signaling and diabetic complications[END_REF][START_REF] Fardini | O-GlcNAcylation: A New Cancer Hallmark?[END_REF][4]. Moreover, a number of studies indicated a link between O-GlcNAcylation and inflammatory conditions, including diabetes, auto-immune diseases and cancer [START_REF] Ma | Hyper-O-GlcNAcylation Is Anti-apoptotic and Maintains Constitutive NF-kappaB Activity in Pancreatic Cancer Cells[END_REF][START_REF] Yang | Elevated O-GlcNAcylation promotes colonic inflammation and tumorigenesis by modulating NF-kappaB signaling[END_REF][START_REF] Liu | MicroRNA-15b Suppresses Th17 Differentiation and Is Associated with Pathogenesis of Multiple Sclerosis by Targeting O-GlcNAc Transferase[END_REF][8][START_REF] Pagesy | Increased OGA expression and activity in leukocytes from patients with diabetes: correlation with inflammation markers[END_REF][10][START_REF] Szymura | NF-kappaB upregulates glutamine-fructose-6-phosphate transaminase 2 to promote migration in nonsmall cell lung cancer[END_REF]. Moreover, several lines of evidence have suggested a role for O-GlcNAcylation in immune cell signalling. Indeed, O-GlcNAcylation was first discovered at the surface of lymphocytes [START_REF] Torres | Topography and polypeptide distribution of terminal Nacetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc[END_REF] and dynamic changes upon lymphocyte activation were described at the beginning of the nineties [START_REF] Kearse | Topology of O-linked N-acetylglucosamine in murine lymphocytes[END_REF]. More recently, several studies indicated that protein O-GlcNAcylation plays major roles in the immune system. In T cells, TCR (T-cell receptor) activation results in global elevation of protein O-GlcNAcylation [START_REF] Lund | Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells[END_REF][START_REF] Swamy | Glucose and glutamine fuel protein O-GlcNAcylation to control T cell self-renewal and malignancy[END_REF], increased O-GlcNAcylation of several transcription factors [START_REF] Ramakrishnan | Activation of the transcriptional function of the NF-kappaB protein c-Rel by O-GlcNAc glycosylation[END_REF][START_REF] Juang | Phosphorylation and O-linked glycosylation of Elf-1 leads to its translocation to the nucleus and binding to the promoter of the TCR zeta-chain[END_REF][START_REF] Liu | The lineage stability and suppressive program of regulatory T cells require protein O-GlcNAcylation[END_REF][START_REF] Golks | Requirement for O-linked Nacetylglucosaminyltransferase in lymphocytes activation[END_REF] and accumulation of OGT at the immunological synapse [START_REF] Lund | Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells[END_REF]. Moreover, in B lymphocytes, activation of the Bcell receptor (BCR) induces O-GlcNAcylation of several signalling molecules involved in BCR signalling [START_REF] Wu | Temporal regulation of Lsp1 O-GlcNAcylation and phosphorylation during apoptosis of activated B cells[END_REF][START_REF] Wu | O-GlcNAcylation is required for B cell homeostasis and antibody responses[END_REF]. Overall, these studies strongly support a major role in O-GlcNAcylation in adaptive immunity. However, the molecular mechanisms by which immune cell activation modulates protein O-GlcNAcylation remain elusive.

In macrophages, contradictory results have been obtained concerning the effect of LPS on protein O-GlcNAcylation and its role in inflammatory processes. Whereas some studies indicated that LPS induced a general increase in protein O-GlcNAcylation [START_REF] Ryu | Denitrosylation of S-nitrosylated OGT is triggered in LPSstimulated innate immune response[END_REF][START_REF] Hwang | Lipopolysaccharide (LPS)-stimulated iNOS Induction Is Increased by Glucosamine under Normal Glucose Conditions but Is Inhibited by Glucosamine under High Glucose Conditions in Macrophage Cells[END_REF], others found that LPS decreases protein O-GlcNAcylation in macrophages [START_REF] Hwang | O-GlcNAc transferase inhibits LPSmediated expression of inducible nitric oxide synthase through an increased interaction with mSin3A in RAW264.7 cells[END_REF][START_REF] Li | O-GlcNAc Transferase Suppresses Inflammation and Necroptosis by Targeting Receptor-Interacting Serine/Threonine-Protein Kinase 3[END_REF]. Moreover, whereas several lines of evidence indicate that increased O-GlcNAcylation of signaling proteins in the NFκB pathway potentiates inflammatory processes [START_REF] Yang | NFkappaB activation is associated with its O-GlcNAcylation state under hyperglycemic conditions[END_REF][START_REF] Allison | Modification of RelA by O-linked N-acetylglucosamine links glucose metabolism to NF-kappaB acetylation and transcription[END_REF][START_REF] Pathak | O-GlcNAcylation of TAB1 modulates TAK1-mediated cytokine release[END_REF][START_REF] Kawauchi | Loss of p53 enhances catalytic activity of IKKbeta through O-linked beta-N-acetyl glucosamine modification[END_REF], other studies indicate that increased O-GlcNAcylation may inhibit pro-inflammatory signals [START_REF] Hwang | O-GlcNAc transferase inhibits LPSmediated expression of inducible nitric oxide synthase through an increased interaction with mSin3A in RAW264.7 cells[END_REF][START_REF] Xing | O-GlcNAc modification of NFkappaB p65 inhibits TNF-alpha-induced inflammatory mediator expression in rat aortic smooth muscle cells[END_REF]. More recently, O-GlcNAcylation of the signalling adaptor MAVS in macrophages has been involved in antiviral signalling response [START_REF] Li | O-GlcNAc Transferase Links Glucose Metabolism to MAVS-Mediated Antiviral Innate Immunity[END_REF], whereas increased RIPK3 O-GlcNAcylation was shown to regulate necroptosis signalling upon LPS stimulation [START_REF] Li | O-GlcNAc Transferase Suppresses Inflammation and Necroptosis by Targeting Receptor-Interacting Serine/Threonine-Protein Kinase 3[END_REF]. Thus, whereas numerous evidences suggest that modulation of O-GlcNAcylation signalling constitutes an important facet of innate immune response, the mechanisms by which macrophage activation regulates O-GlcNAc, and its consequences on inflammatory processes, remain to be firmly established.

In the present work, using different macrophage cell models, we clearly demonstrated that TLR4 activation markedly stimulates protein O-GlcNAcylation in macrophages through increased expression and activity of the rate-limiting enzyme of the hexosamine biosynthetic pathway, glutamine fructose-6-phosphate amidotransferase (GFAT). More specifically, we showed that whereas resting macrophages express mainly the GFAT1 isoform, upon LPS stimulation, a marked increase in GFAT2 expression was observed, revealing GFAT2 as a new TLR4-inducible gene in macrophages that promotes a general increase in protein O-GlcNAcylation. In macrophages with conditional OGT deletion, lack of LPS-induced O-GlcNAyclation was associated with an increased in the production of pro-inflammatory molecules, suggesting that GFAT2 induction may be part of a regulatory loop that may limit inflammation and/or permit its resolution after TLR4 activation. 1.

Materiel and Methods

Chemicals and antibodies

cDNA constructs

The cDNA coding for nuclear, cytosolic and plasma membrane FRET O-GlcNAc biosensors [START_REF] Carrillo | Targeted in vivo O-GlcNAc sensors reveal discrete compartment-specific dynamics during signal transduction[END_REF] were generously provided by Prof. L.K. Mahal (University of Texas, Austin, USA). BRET biosensors were developed based on these FRET constructs by replacing the CFP by a Rluc8 sequence [START_REF] Groussaud | Hijacking of the O-GlcNAcZYME complex by the HTLV-1 Tax oncoprotein facilitates viral transcription[END_REF].

Human (-801>>>0) and mouse GFAT2 (-501>>>0) putative promoter sequences were amplified by PCR and cloned in a firefly luciferase plasmid (pGL4-20, Promega). FoxO1 binding sites on GFAT2 promoter were identified using Regulatory Sequence Analysis Tools (RSAT) web server (http://rsat.ulb.ac.be/rsat/). Mutagenesis of Foxo1 binding sites on the mouse GFAT2 reporter gene was performed using mutagenesis kit (QuikChangeII XL-Agilent Technologies).

Constitutively active FOXO1-TM (mutated on the 3 Akt phoshorylation sites) has been described previously [START_REF] Kuo | O-glycosylation of FoxO1 increases its transcriptional activity towards the glucose 6-phosphatase gene[END_REF].

Cell culture and transfection RAW264.7 murine macrophage cells were maintained in media constituted of RPMI 1640-glutamax medium supplemented with 10% fetal calf serum (FCS), 50µM βmercaptoethanol, 1 mM sodium pyruvate, 10 mM HEPES and 2 mM L-Glutamine (GIBCO).

Plasmid transfections were performed by cell electroporation using the Electrobuffer kit (Cell projects Ltd). For each transfection, cells grown to sub-confluence in a 100 mm plate were transfected with 15 µg of plasmid DNA. Cells were electroporated at 250 V, 900 μF in 0.4 cm cuvettes (Biorad) using the Gene Pulser X electroporation system (Bio-Rad Laboratories). After electroporation, cells were immediately re-suspended in culture medium, For peritoneal macrophages collection, after mice sacrifice, the peritoneum was infused with 10mL of cold RPMI. Cells were seeded in 6 well-plates at 10 6 cells/ml in complete media and allowed to adhere overnight. Non-adherent cells were then washed away and macrophages treated with LPS.

In some experiments, mice were injected intraperitoneally with LPS (0.6 mg/kg). 6h after injection, mice were sacrificed and peritoneal cells were harvested as described above and plated in a 6 well-plate at 37 °C with 5% CO2. After 2 h, non-adherent cells were washed away, and adherent cells were lysed for analysis by western blot.

To evaluate the effect of LPS on cytokine production in vivo in OGT-KO mice, LPS was injected intraperitoneally, mice were sacrificed 6 h after injection and blood was collected by cardiac puncture. Blood was centrifuged at 1500 rpm for 10 min at 4°C and the serum was collected and frozen at -80°C for subsequent determination of cytokine concentrations using MSD kit.

Preparation of Human monocytes derived macrophages

Human primary macrophages were isolated from blood of healthy donors (Etablissement Français du Sang, Ile-de-France, Site Trinité, Agreement number INSERM-EFS:18/EFS/030) by density gradient sedimentation on Ficoll (GE Healthcare), followed by negative selection on magnetic beads (Stem cells, Cat n°19059) and adhesion on plastic at 37°C during 2 h. Cells were then cultured in the presence of complete culture medium (RPMI 1640 supplemented with 10 % FCS (Eurobio), 100 mg/ml streptomycin/penicillin and 2 mM L-glutamine (Gibco) containing 10 ng/ml rhM-CSF (R&D systems) during 4-5 days [START_REF] Mazzolini | Inhibition of phagocytosis in HIV-1-infected macrophages relies on Nef-dependent alteration of focal delivery of recycling compartments[END_REF].

RNA extraction, reverse transcription and qPCR

Macrophages cultured in 6-well plates were lysed in Trizol reagent (Life Technologies). RNA was isolated and reverse transcribed [START_REF] Strobel | Effect of thiazolidinediones on expression of UCP2 and adipocyte markers in human PAZ6 adipocytes[END_REF]. Levels of the cDNA of interest were measured by qPCR (LightCycler FastStart DNA Master SYBR Green 1 kit; Roche Diagnostics). The absence of genomic DNA contamination was verified by treating RNA samples in parallel without reverse transcriptase and controlling for the absence of amplification by qPCR. Gene expression was normalized over cyclophilin and HPRT (Hypoxanthine-guanine phosphoribosyltransferase) RNA levels. The sequences of the primers used for qPCR are given in Supplementary Table 2.

OGA enzymatic assay.

Control and LPS-treated RAW264.7 cells were lysed in extraction buffer containing 50mM Tris, 137 mM NaCl, 1% triton, 10% glycerol, phosphatases (50 mM NaF, 10 mM disodium β-glycerophosphate, 1 mM Na 3 VO 4 ) and proteases (AEBSF, leupeptine, antipaine aprotinine and pepstatine, 1µg/ml each) inhibitors. OGA activity was measured using 4methylumbellifery-N-acetylβ-D-glucosamine (MU-GlcNAc, Sigma), which is converted into fluorescent 4-methylumbelliferon upon hydrolysis by OGA and other hexosaminidases [START_REF] Kim | Enzymatic characterization of O-GlcNAcase isoforms using a fluorogenic GlcNAc substrate[END_REF].

4-methylumbelliferon fluorescence was measured at 448 nm after excitation at 362 nm.

Fluorescent measurements were performed after 30 min and 60 min of incubation at 37 °C, to ensure that the determination was performed during the linear phase of the reaction. To specifically determine OGA activity versus other hexosaminidase present in the lysate, all reactions were performed both in absence and presence of 100 µM Thiamet G (a specific OGA inhibitor). The difference between the fluorescent signals obtained in absence (activity of OGA plus other hexosaminidases) and presence of Thiamet G (activity of the other hexosaminidases) reflected the amount of 4-methylumbelliferon produced by OGA [START_REF] Pagesy | Increased OGA expression and activity in leukocytes from patients with diabetes: correlation with inflammation markers[END_REF].

OGT enzymatic assay.

Control and LPS-treated RAW264.7 cells were lysed in extraction buffer containing 50mM Tris, 137 mM NaCl, 1% triton, 10% glycerol, phosphatases (50 mM NaF, 10 mM di-sodium β-glycerophosphate, 1 mM Na 3 VO 4 ) and proteases (AEBSF, leupeptine, antipaine aprotinine and pepstatine, 1µg/ml each) inhibitors. Immunoprecipitation of OGT was performed as described below. 400 μg of proteins were incubated with 1.5 μg of anti-OGT antibody (Sigma-Aldrich) for 2h at 4°C. Precipitation was performed by incubating 25µL equilibrated protein G-sepharose beads (GE Healthcare) for 30 min at 4°C. After 3 washes, the precipitated proteins were submitted to an additional wash in OGT assay buffer containing 50 mM Tris-HCl and 12.5 mM MgCl 2 , pH7.5. OGT assay was then performed on protein-G sepharose bound OGT [START_REF] Groussaud | Hijacking of the O-GlcNAcZYME complex by the HTLV-1 Tax oncoprotein facilitates viral transcription[END_REF] using the bioluminescent UDP-Glo TM glycosyltransferase assay (Promega) exactly as described in the manufacturer instructions [START_REF] Rodriguez | Enhanced transfer of a photocross-linking N-acetylglucosamine (GlcNAc) analog by an O-GlcNAc transferase mutant with converted substrate specificity[END_REF].

GFAT enzymatic assay.

Glutamine fructose-6-phosphate amidotransferase enzymatic activity was measured as described previously [START_REF] Marshall | Discovery of a metabolic pathway mediating glucose-induced desensitization of the glucose transport system. Role of hexosamine biosynthesis in the induction of insulin resistance[END_REF]. Control and LPS-treated RAW264.7 cells, human monocytederived macrophages or mouse bone-marrow-derived macrophages were lysed in extraction buffer containing 50mM Tris, 137 mM NaCl, 1% triton, 10% glycerol, phosphatases (50 mM NaF, 10 mM di-sodium β-glycerophosphate, 1 mM Na 3 VO 4 ) and proteases (AEBSF, leupeptine, antipaine aprotinine and pepstatine, 1µg/ml each) inhibitors. 15-30 µg of proteins were incubated in a buffer containing 10 mM fructose 6-phosphate, 6 mM glutamine, 0.3 mM 3-acetylpyridine adenine dinucleotide (APAD), 50 mM KC1, 100 mM KH 2 PO 4 (pH 7.5), 1 mM dithiothreitol and 30 unit/ml of glutamate dehydrogenase. After incubation for 30 min at 37°C, the change in absorbance due to reduction of APAD to APADH was monitored spectrophotometrically at 365 nm using TECAN™ infinite M1000 Pro Microplate Reader.

Western blotting

Cells were lysed with buffer containing 50mM Tris-HCl (pH 8), 137mM NaCl, 10%

(v/v) glycerol, 1% (v/v) triton, 50mM NaF, 10mM di-sodium β-glycerophosphate, 1mM Na 3 VO 4 , protease inhibitors (1µg/ml pepstatin, antipain, leupeptin, aprotinin and AEBSF), supplemented with 10µM PUGNAc in order to preserve the GlcNAcylation state of proteins during the extraction procedure. Proteins were then analysed by SDS-PAGE followed by western-blotting as described previously [START_REF] Liu | Fibroblast growth factor-2 has opposite effects on human breast cancer MCF-7 cell growth depending on the activation level of the mitogen-activated protein kinase pathway[END_REF].

In some experiments, O-GlcNAcylated proteins were precipitated on wheat germ lectin as described previously [START_REF] Issad | Isoproterenol inhibits insulin-stimulated tyrosine phosphorylation of the insulin receptor without increasing its serine/threonine phosphorylation[END_REF][START_REF] Fardini | O-GlcNAcylation of FoxO1 in pancreatic beta cells promotes Akt inhibition through an IGFBP1-mediated autocrine mechanism[END_REF]. Briefly, cell lysates (400 µg of proteins) were incubated for 2 hours at 4° C on a rotating wheel with 20 μl of agarose beads coupled to wheat germ lectin (which binds the N-Acetyl-Glucosamine pattern). At the end of the incubation, the beads were pelleted by centrifugation at 3000 g for 2 min. The supernatant was removed and the pellet was washed 3 times in extracting buffer. Twenty μL of Laemmli sample buffer were added to the beads, and the samples were boiled at 95°C for 5 minutes and then submitted to westernblotting as described previously.

Statistical analysis

Statistical analyses were performed using PRISM software. Comparison between groups were performed using Student's t test, or ANOVA followed by Dunnett's or Tukey's post-test for multiple comparison analysis.

Results

LPS stimulation markedly increased protein O-GlcNAcylation in macrophages

Sub-cellular relocalisation of OGT in different cell compartments has been observed upon stimulation of membrane receptors [START_REF] Yang | Phosphoinositide signalling links O-GlcNAc transferase to insulin resistance[END_REF][START_REF] Perez-Cervera | Insulin signaling controls the expression of O-GlcNAc transferase and its interaction with lipid microdomains[END_REF], resulting in compartment specific changes in O-GlcNAcylation activity. In order to evaluate whether TLR4 stimulation affects protein O-GlcNAcylation in different cellular compartments in macrophages, we used BRET-based O-GlcNAc biosensors [START_REF] Groussaud | Hijacking of the O-GlcNAcZYME complex by the HTLV-1 Tax oncoprotein facilitates viral transcription[END_REF]. These biosensors are composed of Rluc8 luciferase fused to a lectin domain (GafD), a known OGT substrate peptide derived from casein kinase II, followed by the Venus variant of the yellow fluorescent protein (Fig. 1A). Upon O-GlcNAcylation, the casein kinase peptide binds to the lectin, resulting into a conformational change detected as an increased BRET signal. These biosensors were fused to addressing sequences for targeting to the internal face of the plasma membrane (using Lyn myristoylation/palmitoylation sequence), the cytosol (using the HIV-1 Rev protein nuclear exclusion sequence) or the nucleus (using the SV40 nuclear localisation sequence) [START_REF] Carrillo | Targeted in vivo O-GlcNAc sensors reveal discrete compartment-specific dynamics during signal transduction[END_REF].

RAW264.7 cells transfected with these biosensors were incubated for 24h in presence of LPS, Thiamet G (an inhibitor of OGA) or both. We observed that LPS treatment increased BRET signal with all three biosensors (Fig. 1A), indicating that TLR4 stimulation promotes a general rather than compartment-specific increase in O-GlcNAcylation in RAW264.7 macrophages. Interestingly, the effects of LPS and Thiamet G were additive, suggesting that the effect of LPS on O-GlcNAcylation is independent of OGA activity.

LPS-induced O-GlcNAcylation of proteins, both in absence and presence of Thiamet G, was further demonstrated by western-blotting using anti-O-GlcNAc antibody in RAW 264.7 cells, but also in mouse bone marrow-derived and peritoneal primary macrophages, as well as in human monocyte-derived macrophages (Fig. 1B). Therefore, increased O-GlcNAcylation upon LPS stimulation also appears to occur in primary macrophages from different origins and species, suggesting a general mechanism elicited by TLR4 activation.

GFAT2 is a TLR4-inducible gene in macrophages

We further explored, using RAW264.7 macrophages, the mechanism involved in this LPSinduced O-GlcNAcylation. As shown in Figure 2, LPS-induced increase in protein O-GlcNAcylation (Fig. 2A) was not associated with any detectable change in OGT and OGA mRNA or protein expression (Fig. 2B andC), suggesting that it was not mediated by regulation of the expression level of O-GlcNAc cycling enzymes. GFAT, the enzyme that catalyzes the rate-limiting step of the hexosamine biosynthesis pathway (Suppl Fig. 1), exists as two isoforms (GFAT1 and GFAT2), encoded by two separate genes (also denominated GFPT1 and GFPT2, respectively) that are differentially expressed in a cell-type specific manner. We observed that in the basal state, only GFAT1 protein was highly expressed in macrophages, whereas GFAT2 protein expression was barely detectable (Fig. 2B). GFAT1

protein expression was moderately increased (1.3 fold) by LPS treatment, whereas LPS induced a major increase (7 fold) in GFAT2 protein expression (Fig. 2B). In agreement with these results, Figure 2C shows that LPS increased the expression of GFAT1 mRNA by 2 to 3fold, and markedly induced (by 10 to 15-fold) the expression of GFAT2 mRNA, suggesting a regulation of GFAT1 and 2 at the transcriptional level. Enzymatic assays indicated that whereas LPS treatment had no detectable effect on OGT or OGA activities, it significantly increased the activity of GFAT in macrophages (Fig. 2D). These results suggest that LPSinduced increase in the expression of GFAT, and more specifically GFAT2, translates into an increase in the activity of the rate limiting step of the hexosamine biosynthesis pathway.

Time-course experiments indicated that a major induction of GFAT2 mRNA could be detected 1h after LPS stimulation, whereas GFAT1 mRNA expression was barely modified at early time points (Suppl. Fig. S2), revealing that GFAT2 is an early TLR4-target gene.

Increased GFAT2 protein and mRNA expression was also observed in human monocytederived macrophages (Fig. 3A), mouse bone marrow-derived macrophages (Fig. 3B), as well as well as peritoneal macrophages (Fig. 3C). In agreement with these results, GFAT enzymatic activity was also increased upon LPS stimulation in human and mouse primary macrophages (Suppl. Fig. S3). Moreover, intraperitoneal injection of LPS in mice also induced a modest increase in GFAT1 and a marked increase in GFAT2 protein expression in peritoneal cells, indicating that this response was also operative in vivo (Suppl. Fig. S4).

Altogether, our results suggest that LPS induces an increase in protein O-GlcNAcylation through stimulation of the expression of the rate-limiting enzyme of the hexosamine biosynthesis pathway.

Impaired O-GlcNAcylation promotes pro-inflammatory response in macrophages

To evaluate the role of LPS-induced O-GlcNAcylation in macrophages, we isolated peritoneal macrophages from mice with conditional deletion of OGT in the myeloid lineage.

As expected, OGT expression and LPS-induced O-GlcNAcylation were markedly impaired in OGT-KO cells (Fig. 4A). We noticed that basal GFAT1 and both basal and LPS-induced GFAT2 expression were increased in OGT-KO macrophages, suggesting a compensatory response to the low level of O-GlcNAcylation in these cells. Interestingly, OGT deletion resulted in an increased in LPS-induced NOS2 expression. Moreover, we observed an increased production of IFNγ and IL1β in the culture medium of OGT-KO macrophages.

These results suggest that increased O-GlcNAcylation upon LPS stimulation may have a counter-regulatory effect that restrains excessive cytokines production by macrophages (Fig. 4B). In agreement with this notion, significant increases in pro-inflammatory cytokines IFN γ, IL1β and TNFα were also observed in vivo in the serum of OGT-KO mice intraperitoneally injected with LPS (Fig. 4C).

These results suggest that increased O-GlcNAcylation upon TLR4 activation participates in the counter-regulation of proinflammatory signalling in macrophages. Induction of GFAT2 expression may therefore constitute a new mechanism to limit excessive inflammation upon LPS stimulation.

GFAT2 expression is dependent on FoxO1 transcription factor in macrophages

The transcription factor FoxO1 have been shown previously to display dual regulatory effects on inflammatory signals in myeloid cells, with either pro- [START_REF] Su | FoxO1 links insulin resistance to proinflammatory cytokine IL-1beta production in macrophages[END_REF][START_REF] Fan | FoxO1 regulates Tlr4 inflammatory pathway signalling in macrophages[END_REF][START_REF] Becker | FOXO-dependent regulation of innate immune homeostasis[END_REF][START_REF] Seiler | FOXO transcription factors regulate innate immune mechanisms in respiratory epithelial cells[END_REF] or anti-inflammatory [START_REF] Baumgartl | Myeloid lineage cell-restricted insulin resistance protects apolipoproteinEdeficient mice against atherosclerosis[END_REF][START_REF] Senokuchi | Forkhead transcription factors (FoxOs) promote apoptosis of insulin-resistant macrophages during cholesterol-induced endoplasmic reticulum stress[END_REF][START_REF] Hwang | FOXO3 deficiency leads to increased susceptibility to cigarette smoke-induced inflammation, airspace enlargement, and chronic obstructive pulmonary disease[END_REF][START_REF] Tsuchiya | Expanded granulocyte/monocyte compartment in myeloid-specific triple FoxO knockout increases oxidative stress and accelerates atherosclerosis in mice[END_REF] effects, depending on the pathophysiological context. Analysis of mouse and human GFAT2 putative promoters revealed canonical FoxO1 recognition sites within the 500bp and 800pb regions upstream of the transcription start site of the mouse and human genes, respectively (Suppl. Fig. S5). We inserted these upstream sequences in a luciferase reporter gene plasmid. Transfection in HEK293 cells with this plasmid revealed that the activities of both human and mouse reporter genes were markedly increased by co-transfection with a constitutively active form of FOXO1, FOXO1-TM (Fig. 5A).

The mouse 500bp promoter contains two FoxO1 recognition sites (referred to as Site1 and Site 2 on Fig. 5B and Suppl Fig. S5). To further demonstrate the contribution of these sites, we mutated either one or both FoxO1 binding sites and measured the activity of the mutated promoters in the luciferase assay.

As shown in Fig. 5B, FOXO1-TM effect was markedly reduced by mutation of Site 1 and totally abolished when both Site 1 and Site 2 were mutated. This suggests that both sites might be important for regulation of GFAT2 expression by FoxO1. Binding of Foxo1 to Site 1

and Site 2 was further demonstrated on the endogenous GFAT2 promoter in RAW264.7 cells, in chromatin immunoprecipitation experiments using a FoxO1-specific antibody (Fig. 5C).

These results suggested that FoxO1 may be involved in the regulation of GFAT2 expression in macrophages. To evaluate the potential involvement of FoxO1 in the regulation of GFAT2 expression, we used a small molecule specific inhibitor of FoxO1 activity, the AS1842856 drug [START_REF] Roux | FOXO1 transcription factor plays a key role in T cell-HIV-1 interaction[END_REF]. We observed, both in RAW264.7 cells (Fig. 6A) and human monocytederived macrophages (Fig. 6B), that inhibition of FoxO1 by this compound markedly impaired the effect of LPS on induction of GFAT2 mRNA and protein expression.

To further demonstrate the role of Foxo1 in LPS-induced GFAT2 expression, we also evaluated the effect of LPS on peritoneal macrophages from mice with conditional Foxo1 deletion in the myeloid lineage. We observed that LPS-induced GFAT2 protein and mRNA expression was markedly impaired in Foxo1 KO macrophages (Fig. 6C). Inhibition of GFAT2 expression was associated with inhibition of LPS effect on protein O-GlcNAcylation. These results demonstrate that Foxo1 is necessary for induction of GFAT2 by LPS and support the notion that increased GFAT2 mediates LPS-induced increase in protein O-GlcNAcylation in macrophages.

Discussion

Several lines of evidence have suggested a role for O-GlcNAcylation in the regulation of inflammatory processes in macrophages [START_REF] Baudoin | O-GlcNAcylation and Inflammation: A Vast Territory to Explore[END_REF]. However, contradictory results have been obtained concerning the effect of TLR4 activation on protein O-GLNAcylation. Indeed, depending on the experimental setting, both increases [START_REF] Ryu | Denitrosylation of S-nitrosylated OGT is triggered in LPSstimulated innate immune response[END_REF][START_REF] Hwang | Lipopolysaccharide (LPS)-stimulated iNOS Induction Is Increased by Glucosamine under Normal Glucose Conditions but Is Inhibited by Glucosamine under High Glucose Conditions in Macrophage Cells[END_REF] and decreases [START_REF] Hwang | O-GlcNAc transferase inhibits LPSmediated expression of inducible nitric oxide synthase through an increased interaction with mSin3A in RAW264.7 cells[END_REF][START_REF] Li | O-GlcNAc Transferase Suppresses Inflammation and Necroptosis by Targeting Receptor-Interacting Serine/Threonine-Protein Kinase 3[END_REF] in the general O-GlcNAcylation profile were observed upon LPS stimulation.

In the present work, we showed, using two different methodological approaches (BRETbased assay (Fig. 1A) and western-blotting on crude cell lysate as well as WGL-bound fraction (Fig. 1B Given that O-GlcNAc is a very dynamic and labile modification, that can be rapidly hydrolyzed upon cellular damage or during protein isolation [START_REF] Hart | Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease[END_REF], it is quite possible that loss of O-GlcNAc might occur during the sample preparation. In contrast, we always included PUGNAc at a concentration of 10µM in our extraction buffer, in order to preserve O-GlcNAcylation state of proteins obtained in cells after LPS treatment. In addition, the confirmation of our western-blotting results by an independent technique based on the use of a BRET biosensor, which monitors O-GlcNAcylation changes in intact living cells without any processing of cellular proteins, strongly argues in in favour of an LPS-induced general increase in O-GlcNAcylation in macrophages.

Interestingly, the effect of LPS on protein O-GlcNAcylation were additive to those of a maximally inhibitory concentration of the OGA inhibitor, Thiamet G. This suggested that LPS-induced O-GlcNAcylation was not mediated by regulation of OGA activity. In agreement with this notion, using a fluorogenic substrate, we observed that OGA activity was similar in cell extracts from control and LPS stimulated RAW264.7 macrophages (Fig. 2 D).

Moreover, using a luminescent assay [START_REF] Groussaud | Hijacking of the O-GlcNAcZYME complex by the HTLV-1 Tax oncoprotein facilitates viral transcription[END_REF][START_REF] Rodriguez | Enhanced transfer of a photocross-linking N-acetylglucosamine (GlcNAc) analog by an O-GlcNAc transferase mutant with converted substrate specificity[END_REF], we found that LPS treatment had no detectable effect on OGT activity in cell extracts from RAW264.7 cells (Fig. 2D). Therefore, LPSinduced O-GlcNAcylation does not appear to be mediated by regulation of O-GlcNAc cycling enzymes.

One of the most important finding of our study is that LPS treatment resulted in an increased expression and activity of GFAT (Fig. 2). GFAT is the enzyme that catalyses the rate limiting step of the hexosamine biosynthesis pathway, which eventually leads to the production of UDP-GlcNAc, the substrate used by OGT for protein O-GlcNAcylation. GFAT exists in two isoforms, GFAT1 and GFAT2, encoded by two different genes [START_REF] Sayeski | The murine glutamine:fructose-6-phosphate amidotransferase-encoding cDNA sequence[END_REF][START_REF] Oki | cDNA cloning and mapping of a novel subtype of glutamine:fructose-6-phosphate amidotransferase (GFAT2) in human and mouse[END_REF]. Although little data are available concerning differential roles of these enzymes, these two isoforms present different tissue distribution, with GFAT1 mRNA being predominantly expressed in pancreas, placenta and testis, whereas GFAT2 mRNA were found throughout the central nervous system [START_REF] Oki | cDNA cloning and mapping of a novel subtype of glutamine:fructose-6-phosphate amidotransferase (GFAT2) in human and mouse[END_REF]. Whereas some differences in the regulation of their catalytic activities by cAMP-induced phosphorylation has been described [START_REF] Hu | Phosphorylation of mouse glutaminefructose-6-phosphate amidotransferase 2 (GFAT2) by cAMP-dependent protein kinase increases the enzyme activity[END_REF], very little is known about differential regulation of GFAT1 versus GFAT2 expression in different cell types. Most surprisingly, to our knowledge, only one study evaluated in macrophages the expression of GFAT in a mouse macrophage cell line (ANA-1) [START_REF] Manzari | Induction of macrophage glutamine: fructose-6-phosphate amidotransferase expression by hypoxia and by picolinic acid[END_REF]. These authors reported that GFAT1 was constitutively expressed in these cells and they indicated (as data not shown) that no effect of LPS or IFNγ on its expression was observed. However, GFAT2 expression was not evaluated in this study. Interestingly, we found that GFAT1 mRNA and protein were indeed expressed at significant levels in resting macrophages, whereas GFAT2 expression was barely detectable.

LPS stimulated both GFAT1 and GFAT2 expression, although the stimulatory effect was much higher for GFAT2, which turned-out to be an early TLR4-inducible gene in macrophages (Suppl. Although the exact mechanism by which TLR4 stimulates GFAT2 expression remains elusive, we provide strong evidence for a role of FoxO1 in this process. Indeed, using gene reporter as well as chromatin immunoprecipitation assays, we demonstrated the presence of FoxO1 binding sites on GFAT2 putative promoter. Moreover, pharmacological inhibition or genetic deletion of FoxO1 in macrophages markedly impaired LPS-induced GFAT2 expression, confirming the involvement of FoxO1 in this regulation.

Previous studies have shown that O-GlcNAcylation can either promote inflammation or reduces it, according to the cellular context and type of insult. Thus, whereas O-GlcNAcylation has pro-inflammatory effects in situations of chronic hyperglycaemia, it appears to be protective in acute stress conditions, such as ischemia-reperfusion injury in the heart [START_REF] Baudoin | O-GlcNAcylation and Inflammation: A Vast Territory to Explore[END_REF]. We observed that impaired O-GlcNAcylation in OGT-KO macrophages resulted in marked increase in NOS2 expression and pro-inflammatory cytokines production, suggesting that the O-GlcNAc tone may exerts a break on inflammatory processes. Therefore, the rapid induction of GFAT2 expression may constitute a protective mechanism to limit exacerbated inflammation upon LPS stimulation.

In summary, we have shown that LPS stimulation promotes a general increase in protein O-GlcNAcylation in macrophages. This effect is at least in part mediated by increased expression and activity of the rate-limiting enzyme of the hexosamine biosynthesis pathway, GFAT, with the GFAT2 isoform being the most responsive to TLR4 activation. Indeed, while GFAT1 may control the activity of the HBP in the basal state, our work revealed that GFAT2

is a new TLR4-inducible gene in macrophages, permitting a rapid adaptive response to environmental changes.

Figure 1 :

 1 Figure 1: Schematic representation of glucose homeostasis during fed and fasted states . Error! Bookmark not defined.

Figure 2 :

 2 Figure 2: Schematic representation of glucose metabolism through glycolysis and Krebs cycle. .........................................................................................................................................................

Figure 3 :

 3 Figure 3: Insulin signaling pathway ......................................................................................

Figure 4 :

 4 Figure 4: pathophysiology of metabolic syndrome and type 2 diabetes mellitus .....................

Figure 5 :

 5 Figure 5: Different possible pathways of formation of advanced glycation end products (AGE) .........................................................................................................................................................

Figure 6 :

 6 Figure 6: Deleterious consequences of PKC activation ..........................................................

Figure 7 :

 7 Figure 7: Increased glucose flux in the polyol pathway .........................................................

Figure 8 :

 8 Figure 8: Production of superoxide by the electron transport chain within the mitochondria ..

Figure 9 :

 9 Figure 9: Hyperglycemia induced ROS activates glucotixic pathways ...................................

Figure 10 :

 10 Figure 10: The glucose-fatty acid cycle (the Randle cycle) ...................................................

Figure 11 :

 11 Figure 11: Schematic representation of FFAs impair GLUT4 in skeletal muscles ..................

Figure 12 :

 12 Figure 12: The structure of Lipid A component of LPS .........................................................

Figure 13 :

 13 Figure 13: Schematic representation of FFA induced insulin resistance through TLR4 ..........

Figure 14 :

 14 Figure 14: Model proposed for the mechanism of FFAs induced-insulin resistance through ROS..................................................................................................................................................

Figure 15 :

 15 Figure 15: TLR-mediated MyD88-dependent signaling pathway ...........................................

Figure 16 : 54 Figure 17 :

 165417 Figure 16: PI3K/Akt signaling pathway as a negative feed-back of TLR4 signaling pathway.54 Figure 17: Schematic representation of macrophage polarization ..........................................

Figure 18 :

 18 Figure 18: Schematic model for metabolic characteristic of M1 phenotype............................

Figure 19 :

 19 Figure 19 : Schematic representation of metabolic reprogramming of M2 macrophage ..........

Figure 20 :

 20 Figure 20: schematic presentation of L-arginine metabolism in macrophage polarization ......

Figure 21 :

 21 Figure 21: Macrophages of adipose tissue in physiological and pathological situations..........

Figure 22 :

 22 Figure 22: A single pair of enzymes regulates O-GlcNAc, OGT and OGA. ...........................

Figure 23 :

 23 Figure 23: UDP-GlcNAc sources ..........................................................................................

Figure 24 :

 24 Figure 24: Hexosamine biosynthetic pathway and O-GlcNAcylation ....................................

Figure 25 :

 25 Figure 25: human GFAT1 structure ......................................................................................

Figure 26 :

 26 Figure 26: The 3 OGT isoforms ............................................................................................

Figure 27 :

 27 Figure 27: Structure of human OGT complexed to UDP .......................................................

Figure 28 :

 28 Figure 28: Overall crystalized structures of the OGT-UDP-HCF-1 complex ..........................

Figure 29 :

 29 Figure 29: Biochemical structure of OGT inhibitors ..............................................................

Figure 30 :

 30 Figure 30: OGA enzyme isoforms ........................................................................................

Figure 31 :

 31 Figure 31: Crystalized human OGA-L structure ....................................................................

Figure 32 :

 32 Figure 32: OGA inhibitors ....................................................................................................

Figure 33 :

 33 Figure 33: Schematic representation of the dynamic interplay between O-GlcNAcylation and phosphorylation. ...............................................................................................................................

Figure 34 :

 34 Figure 34: Schematic representation of O-GlcNAc effect on insulin signaling pathway .......

Figure 35 :

 35 Figure 35: The BRET biosensors used to monitor of O-GlcNAcylation levels .....................

Figure 36 :

 36 Figure 36: Monitoring of LPS-induced O-GlcNAcylation using a BRET Biosensor in RAW264.7 Cells .............................................................................................................................

Figure 37 :

 37 Figure 37: LPS up-regulates proteins O-GlcNAcylation and HBP rate limiting enzymes expression .......................................................................................................................................

Figure 38 :

 38 Figure 38: Time-course experiment for GFAT2 expression .................................................

Figure 39 :Figure 41 :

 3941 Figure 39: LPS effect on O-GlcNAcylation and O-GlcNAc regulatory enzymes in BMDM Figure 40: LPS effect on O-GlcNAcylation and O-GlcNAc regulatory enzymes in HMDM Figure 41: Principle of spectrophotometric assay of GFAT activity .....................................

Figure 42 :

 42 Figure 42: Measurement of GFAT enzyme activity in LPS-stimulated macrophages ...........

Figure 43 :

 43 Figure 43: LPS effect on proteins O-GlcNAcylation in vivo ................................................

Figure 44 :

 44 Figure 44: HIF1α KO effect on HBP enzymes ....................................................................

Figure 45 :

 45 Figure 45: Effect of NFκB inhibitor BAY11-7082 on GFAT2 expression ...........................

Figure 46 :

 46 Figure 46: FoxO1 binds directly to GFAT2 promoter and enhances its transcription ...........

Figure 47 :

 47 Figure 47: FoxO1 is essential for LPS induced-GFAT2 expression in macrophage. .............

Figure 48 :

 48 Figure 48: Effect of OGT deletion on macrophage polarization ...........................................

Figure 49 :

 49 Figure 49: ogt deficient macrophage exhibited exacerbated LPS-pro-inflammatory response in vitro and in vivo ..............................................................................................................................

Figure 50 :

 50 Figure 50: Effect OGT deletion in macrophage on HF/HS diet induced-inflammation ........

Figure 2 :

 2 Figure 2: Schematic representation of glucose metabolism through glycolysis and Krebs cycle .

Figure 3 :

 3 Figure 3: Insulin signaling pathway

Figure 4 :

 4 Figure 4: pathophysiology of metabolic syndrome and type 2 diabetes mellitus Genetic predisposition and environmental factors contribute in the pathogenesis of type 2 diabetes. Glucotoxicity and lipotoxicity may induce insulin resistance and reduce pancreatic B-cell function by several different mechanisms. Defective insulin secretion and insulin resistance leads to a vicious circle involved in the progression from impaired glucose tolerance to type 2 diabetes. Source Br. J Diabetes vasc Disease 2011 Sherbourne Gibbs, Ltd.

Figure 6 :

 6 Figure 6: Deleterious consequences of PKC activation Hyperglycemia induces the synthesis of diacylglycerol (DAG), which activates PKC, mainly the β and δ isoforms. PKC activation has pathogenic consequences by affecting the expression of numerous genes, such as eNOS (endothelial nitric oxide synthetase), ET-1 (endothelin-1), VEGF (vascular endothelial growth factor),

Figure 7 :

 7 Figure 7: Increased glucose flux in the polyol pathway In conditions of hyperglycemia the aldose reductase reduces the glucose to sorbitol. This reaction requires the use of NADPH as a cofactor which in turn causes a decrease in the intracellular concentration of NADPH. This leads to the appearance of oxidative stress via a decrease in the production of reduced glutathione (GSH), a powerful antioxidant, from the oxidized form of glutathione (GSSG), this reaction requiring NADPH.

Figure 11 :

 11 Figure 11: Schematic representation of FFAs impair GLUT4 in skeletal muscles Another mechanism has been proposed for fatty-acid induced insulin resistance in skeletal muscle. An increase in delivery of fatty acids to muscle or a decrease in intracellular metabolism of fatty acids leads to an increase in intracellular fatty acid metabolites such as diacylglycerol, fatty acyl CoA, and ceramides. These metabolites activate a serine/threonine kinase cascade (possibly initiated by protein kinase Cθ) leading to phosphorylation of serine/ threonine sites on insulin receptor substrates (IRS-1 and IRS-2), which in turn reduces the ability of the insulin receptor substrates to activate PI 3-kinase resulting in inhibition Akt phosphorylation.

Hotamisligil 2017 ;

 2017 [START_REF] Mcnelis | Macrophages, Immunity, and Metabolic Disease[END_REF].

Figure 12 :

 12 Figure 12: The structure of Lipid A component of LPS In the lipid A of the most studied organism Escherichia coli illustrated here, there are six fatty acids in total. The four hydroxy fatty acids are C 14 in chain length. The hydroxyl groups of the two (R)-3-hydroxy fatty

Figure 13 :

 13 Figure 13: Schematic representation of FFA induced insulin resistance through TLR4

Figure 14 :

 14 Figure 14: Model proposed for the mechanism of FFAs induced-insulin resistance through ROS Increased serum free fatty acids promotes the production of reactive oxygene species (ROS) via increased mitochondrial β oxidation. ROS in turn stimulates cytoplasmic JNK activation, through ASK1

Chapter 2 :

 2 Macrophage and metabolic syndrome 2.2 Macrophage and its principal signaling pathwaysMacrophages were first described by the Russian biologist Elie Metchnikoff in 1883.

  Toll like receptor, are trans-membrane receptors for the recognition of molecular patterns of pathogens and danger signals associated with tissue damage. They enable the activation of innate immunity by activating numerous signaling pathways. They were named after their homology to the Toll gene in Drosophila. Hoffman and his team were the first to demonstrate the implication of TLRs in immunity in 1996. There are 11 members of the TLR family in humans and 13 in mice. The majority of TLRs are localized in the cell membrane such as TLR1, TLR2, TLR4, TLR5, TLR6 and TLR11 whereas TLR3, TLR7 and TLR9 are localized in intracellular endosomes. TLRs allow the recognition of patterns conserved on the surface of pathogens (PAMPs), as well as danger signals produced for example in the event of tissue damage (DAMPs).

  family member is IκBα. When cells are stimulated by pro-inflammatory cytokines, growth factors or LPS, IκB is phosphorylated by the kinase of IκB, IKK. IKK is composed of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit named NF-κB essential modulator (NEMO) or IKKγ (S.-C. Sun and Ley 2008).

Figure 15 : 1 (

 151 Figure 15: TLR-mediated MyD88-dependent signaling pathway Schematic representation of main inflammatory signaling pathways localized downstream of the TLR4 receptor in macrophage. Upon TLR4 stimulation, MyD88 binds to the cytoplasmic portion of TLR4 through interaction between individual TIR domains. IRAK-4 (Interleukin 1 Receptor Associated Kinase 6), IRAK-1 (Interleukin 1 Receptor Associated Kinase 1), and TRAF6 (TNF associated factor 6) are then recruited to the receptor, which induces association of IRAK-1 and MyD88 via the death domains. IRAK-4 then phosphorylates IRAK-1. Phosphorylated IRAK-1, together with TRAF6, dissociates from the receptor and then TRAF6 interacts with TAK1 (Transforming growth factor-β (TGF-β)-activated kinase 1) and TAB1 (TAK-Binding protein 1).

Figure 16 :

 16 Figure 16: PI3K/Akt signaling pathway as a negative feed-back of TLR4 signaling pathway.

Figure 17 :

 17 Figure 17: Schematic representation of macrophage polarization In culture, M1 macrophages (inflammatory, pathogen clearing) are induced by IFNγ or LPS and are characterized by production of NOS2 (nitric oxide synthase) and pro-inflammatory cytokines such as TNFα (tumor necrosis factor α), IL-1β (interleukin beta), IL6 (interleukin 6), ROS (reactive oxygen specis), IFNγ (interferon gamma), •NO (nitric oxide) and IL12 (interleukin 12). M2 macrophages (resolving, apoptotic cell and damaged molecule clearing) are induced by IL-4 and IL13 and are characterized by the synthesis of Arg1 (arginase 1) and the anti-inflammatory cytokine IL-10, CCL5 (chemokine ligand 5) CCL18 (chemokine ligand 18), EGF (epidermal growth factor), CD206 (mannose receptor), Fizz1(Resistin-like molecule 1) and Ym1 (chitinase 3-like 3) synthesis.

Figure 18 :

 18 Figure 18: Schematic model for metabolic characteristic of M1 phenotype Classically or M1 activated macrophages by LPS and IFNγ promote glycolysis-pentose phosphate pathway (PPP) axis of metabolism. Anaerobic glycolytic metabolism results in increased lactate production and increased levels of intermediates of the Krebs cycle such as succinate. LPS reduces CARKL (carbohydrates kinase like protein) that catalyses S7P (sedoheptulose phosphate) production and favors oxidative activity of PPP, resulting in an overproduction of R5P (ribulose-5-phosphate) and NADPH. The transcription factor HIF1α is stabilized by succinate and reactive oxygene species (ROS). HIF1α can drive the production of proinflammatory cytokines, mainly IL1β. The key functional consequences of M1 phenotype are bacterial killing, mostly through the production of ROS by mitochondria, •NO production through metabolism of L-arginine by NOS2, and inflammation, which occurs via cytokine production. G6P, glucose-6-phosphate; F6P, fructose-6phosphate, NO, nitric oxide. (Image adapted from Galvan-Pena et al. 2014).

Figure 19 :

 19 Figure 19 : Schematic representation of metabolic reprogramming of M2 macrophage Signal transducer and activator of transcription 6 (STAT6) signaling pathway is activated by Interleukins 4 and 13 leading to stimulation of PPARγ co-activator 1β PGC1β. This results in turn in stimulation of PPARγ and PPARδ. These transcription factors induce the transcription of M2 macrophage anti-inflammatory genes Fizz1 (Resistine like molecule 1), Ym1 (chitinase 3-like 3), Arg1 (Arginase 1) and CD36: (Fatty acid translocase). Arg1 mediates metabolism of L-arginine into urea, L-ornithine and polyamines which have wound repairing action. PPARγ promotes β-oxidation of fatty acids and mitochondrial respiration .

Figure 21 :

 21 Figure 21: Macrophages of adipose tissue in physiological and pathological situations "Thin" adipose tissue is made up of small fat cells and M2-type macrophages that secrete IL-10, which maintains an anti-inflammatory environment. Excess nutrient intake leads to the development of a proinflammatory environment in the fat tissue of obese subjects. The fat cells become hypertrophic and may become necrotic, and this is associated with a significant increase in the infiltration of macrophages. The proinflammatory environment leads to a polarization of the macrophages towards an M1 phenotype which accumulate around ring structures made up of necrotic adipocytes. Both adipocytes and macrophages secrete inflammatory cytokines, which in turn exacerbates the inflammation. Fat tissue inflammation is involved in both insulin resistance associated with obesity and in type 2 diabetes.

Figure 22 :

 22 Figure 22: A single pair of enzymes regulates O-GlcNAc, OGT and OGA.

  ) Its dynamic aspect Unlike to the so-called classical glycosylations which are characterized by their stable appearance, O-GlcNAc is a dynamic post-translational modification. Kearse and Hart illustrated the dynamism of this modification by demonstrating a rapid and a transient change in the level as well as the localization of O-GlcNAc after activation of murine T lymphocytes by mitogenic agents. The observation of a significant decrease in O-GlcNAc one hour after activation and a re-increase in O-GlcNAc of certain proteins in a few hours after activation of lymphocytes suggests the idea of a "turnover of O-GlcNAc" (Kearse and Hart 1991). In 1992, Chou et al. have also demonstrated that human cytokeratins 8 and 18 are O-GlcNAcetylated at different sites by O-GlcNAc and that this O-GlcNAc is a dynamic process since the half-lifeof the saccharide part is shorter than that of the protein itself[START_REF] Chou | Characterization and Dynamics of O-Linked Glycosylation of Human Cytokeratin 8 and 18[END_REF].

Figure 23 :

 23 Figure 23: UDP-GlcNAc sourcesBeing at the crossroads of several metabolic pathways, UDP-GlcNAc can play the role of an integrator of the nutritional environment. From(Issad et al. 2010).

Figure 24 :

 24 Figure 24: Hexosamine biosynthetic pathway and O-GlcNAcylation

  rate limiting enzyme of the HBP pathway is L-glutamine: D-fructose-6phosphate-amidotransferase (GFAT). It carries out the first reaction of the HBP pathway. It consists of 681 amino acids and has an approximate molecular weight of 77 kD. The human form was cloned for the first time in 1992[START_REF] Mcknight | Molecular Cloning, CDNA Sequence, and Bacterial Expression of Human Glutamine:Fructose-6-Phosphate Amido transferase[END_REF]). The murine form was cloned in 1994 and has 91% homology with the human sequence[START_REF] Sayeski | The Murine Glutamine:Fructose-6-Phosphate Amidotransferase-Encoding CDNA Sequence[END_REF].

  2001, DeHaven. et al revealed a novel variant of GFAT1 mRNA (GFAT1Alt [Muscle selective variant of GFAT1]). This variant is expressed selectively in striated muscles whereas it was not detected in brain and other tissues (DeHaven et al. 2001).

Figure 25 :

 25 Figure 25: human GFAT1 structure A) Ribbon diagram of the structure of the human GFAT1 isomerase domain B) A model of full length of GFAT1 structure designed by superimposition of GFAT1 structures: In green, isomerase active sites of human GFAT1. In yellow, the glutaminase domain was built on the basis of the crystal structure of the full-length E. coli GlmS (PDB ID 1JXA) (Y. Nakaishi et al.2009).

  This residue was shown to be phosphorylated by AMP-activated protein kinase (AMPK) and calcium / calmodulin-dependent kinase II (CaMKII) dependent mechanisms in vitro. This phosphorylation is associated with an increase in GFAT1 enzymatic activity by 1.4 fold[START_REF] Li | Identification of a Novel Serine Phosphorylation Site in Human Glutamine:Fructose-6-Phosphate Amidotransferase Isoform 1 †[END_REF]. Furthermore, this phosphorylation on Ser243 has been shown to be induced by glucose deprivation through AMPK (S.[START_REF] Eguchi | AMP-Activated Protein Kinase Phosphorylates Glutamine : Fructose-6-Phosphate Amidotransferase 1 at Ser243 to Modulate Its Enzymatic Activity[END_REF]) supporting the notion that GFAT1 phosphorylation on Ser243 promotes the HBP flux to maintain the production of UDP-GlcNAc when nutrients are limiting. However, this serine 243 has been suggested later to be the target of mammalian Target of Rapamycin Complex 2 (mTORC2) rather than by AMPK. Indeed, these authors observed that blocking glycolysis by 2-deoxyglucose combined with glutamine deprivation leads to reduce AMPK activation whereas GFAT1 Ser243 phosphorylation and mTORC2 activation remained elevated[START_REF] Moloughney | MTORC2 Modulates the Amplitude and Duration of GFAT1 Ser-243 Phosphorylation to Maintain Flux through the Hexosamine Pathway during Starvation[END_REF]).

  GFAT1 and thus flux through HBP leads to increased macromolecular synthesis such as glycoproteins, which are essential for cellular mitosis.Paterson and Kudlow (1995) have revealed that the epidermal growth factor EGF, in a breast cancer cell line (MDA468), stimulated the accumulation of GFAT mRNA by 4-fold. Interestingly, the authors reported that cell exposure to high glucose concentrations blocked this effect of EGF. Moreover, it has been indicated that angiotensin 2 increases GFAT1 mRNA and protein expression levels in mesangial cells. The authors indicated that angiotensin 2 through angiotensin receptor 1 (AT1) leads to transactivation of epidermal growth factor EGF receptor in a calcium dependant manner resulting in the activation of Gfpt gene transcription (Leighton R.[START_REF] James | Angiotensin II Activates the GFAT Promoter in Mesangial Cells[END_REF]).

  B. Lazarus et al have revealed the crystalized structure of human OGT in 2011(M. B. Lazarus et al. 2011) (Figure 27).

Figure 27 :

 27 Figure 27: Structure of human OGT complexed to UDP

Figure 28 :

 28 Figure 28: Overall crystalized structures of the OGT-UDP-HCF-1 complexThe 26-residue peptide containing a proteolytic repeat revealed a HCF-1 threonine-rich region implicated in binding to the TPR domain of OGT. OGT catalytic domain in red and TPR domain in grey along with the HCF-111-26 peptide in cyan and UDP in yellow are shown. From(Lazarus MB et al. 2013).

  μM. OSMI-1 has OGT inhibitory activity without qualitatively altering cell surface N-or Olinked glycans. Its low toxicity made it used successfully to inhibit OGT in vivo in animal model (Ortiz-Meoz et al. 2015). L01 is a novel inhibitor with low toxicity in vitro and in zebrahfish. It has been discovered by a structure-based virtual screening analysis. Molecular dynamics and sitedirected mutagenesis indicated a new mechanism of action in which L01 can interact with Asn557 near the UDP binding pocket of OGT. The IC 50 of L01 for inhibition of OGT is of 21.8 μM (Y. Liu et al. 2017).

Figure 29 :

 29 Figure 29: Biochemical structure of OGT inhibitors

Figure 30 :

 30 Figure 30: OGA enzyme isoforms The long OGA isoform (OGA-L) consists of a catalytic domain with hyaluronidase activity at the Nterminal and a domain with histone acetyltransferase (HAT) activity at the C-terminal level. The short form of OGA (OGA-S) only contains the N-terminal domain with O-GlcNAcase activity, the HAT domain being replaced by a tail of 15 amino acids (aa). Based on Bond and Hanover (2015).

Figure 31 :

 31 Figure 31: Crystalized human OGA-L structure A) Ribon diagram of human Hoga-L dimer, colored by chain. The position of N-terminal catalytic domain or the active site (-1 sugar binding site) is indicated in blue-green. B) Binding of the C-terminal peptide (shown in bluegreen) of the helical bundle fragment to the hOGA peptide-binding groove.

  in addition to hyaluronidase activity. The authors demonstrated that the C-terminal region of O-GlcNAcase (a.a 583-917) was capable of exerting its HAT activity on histones. However, the histone acetyltransferase activity of OGA requires post-translational modifications or the presence of accessory proteins. Indeed, the recombinant O-GlcNAcase derived from bacterial preparations does not have HAT activity, but have a HAT activity comparable to that derived from mammals if it is pre-incubated with cellular lysates of mammals. The authors also identified some essential sites for the HAT activity of O-GlcNAcase. These are residues D853, D884 and Y891. The authors showed that these two catalytic domains function independently, since mutations on these residues essential for HAT activity do not affect O-GlcNAcase activity. Conversely, an inhibitor of O-GlcNAcase activity (streptozotocin) does not affect HAT activity. O-GlcNAcase was thus renamed NCOAT for "Nuclear and Cytoplasmic O-GlcNAcase and Acetyltransferase" which takes into account that it is an enzyme having two different biological activities. The same team confirmed that the active site of O-GlcNAcase is located in the Nterminal part of NCOAT (aa 63-283) by demonstrating that the enzyme mutated in essential residues (D175 and D177) has no O-GlcNAcase activity (C Toleman, Paterson, and Kudlow 2006). Wells et al. could not detect O-GlcNAcase activity from the short isoform (v-O-GlcNAcase). Elsewhere, it has been shown that the mutant containing only the O-GlcNAcase domain (a.a. 1-413) has a lower enzymatic activity (about 5 times) compared to the whole protein. These results suggest that the C-terminal part is important for the catalytic function of O-GlcNAcase[START_REF] Butkinaree | Characterization of β-N -Acetylglucosaminidase Cleavage by Caspase-3 during Apoptosis[END_REF][START_REF] Wells | Dynamic O-GlcNAcylation of Nuclear and Cytosolic Proteins[END_REF].

  phosphorylation by kinases CKII (casein kinase II) and PKC (Protein Kinase C). O-GlcNAc on O-GlcNAcase has been highlighted (B. D. Lazarus, Love, and Hanover 2006), but the effect of this post-translational modification remains unknown. In addition, it has been shown that post-translational modifications of the O-GlcNAcase are necessary for HAT activity (Clifford Toleman et al. 2004).

  has been shown that caspase-3 can cleave O-GlcNAcase in vitro and that this cleavage has no effect on its activity, neither for the activity of O-GlcNAcase nor for HAT activity (Clifford[START_REF] Toleman | Characterization of the Histone Acetyltransferase (HAT) Domain of a Bifunctional Protein with Activable O -GlcNAcase and HAT Activities[END_REF][START_REF] Wells | Dynamic O-GlcNAcylation of Nuclear and Cytosolic Proteins[END_REF]. This cleavage by caspase-3 could be involved in the regulation of this enzyme.Butkinaree et al have shown that caspase-3 can cleave O-GlcNAcase in vivo into two fragments during apoptosis. They succeeded in identifying a "non-classical" cleavage sequence of caspase-3, SVVD (a.a 410-413), between the N-terminal domain and the C-terminal domain. Consequently, an N-terminal part containing the O-GlcNAcase activity and a C-terminal part possessing histone acetyltransferase activity are thus obtained at the end of this cleavage. In agreement with the previous results, cleavage by caspase-3 did not change its activity since the two parties rejoined after the cleavage

Figure 32 :

 32 Figure 32: OGA inhibitors

  between phosphorylation and O-GlcNAcylation in the regulation of proteins. This reciprocal relationship between O-GlcNAcylation and phosphorylation has been called the "Yin-Yang" phenomenon. By modifying the availability of the Ser/Thr sites accessible to phosphorylation, O-GlcNAcylation can modify the phosphorylation of the protein and therefore regulate its activity.Thus, it has been shown that the alternance between O-GlcNAcylation and the phosphorylation of the estrogen receptor ER-β on serine 16 could regulate its stability as well as its activity (see next paragraph)[START_REF] Cheng | Alternative O -Glycosylation/ O -Phosphorylation of Serine-16 in Murine Estrogen Receptor β: POST-TRANSLATIONAL REGULATION OF TURNOVER AND TRANSACTIVATION ACTIVITY[END_REF]. It has also been reported that c-Myc is modified by O-GlcNAc on threonine 58, a residue known to be phosphorylated. The phosphorylated form of c-Myc on threonine 58 mainly accumulates in the cytoplasm. The alternance between O-GlcNAcylation and phosphorylation on threonine 58 could regulate different functions of c-Myc(Kamemura et al., 2002).In addition, O-GlcNAcylation and phosphorylation not only take place on the same residues: these two modifications can occur on adjacent residues or on residues spaced by few amino acids, allowing much more sophisticated regulation as is the case for IRS1 which can be O-GlcNAcylated on Ser1036 and phosphorylated on Thr 1045 at the same time(Ball et al., 2006). It is known that the phosphorylation of p53 on threonine 155 promotes the degradation of p53 by the ubiquitin-proteasome pathway. p53 O-GlcNAcylation on serine 149 prevents its degradation by decreasing the phosphorylation of Thr 155[START_REF] Whisenhunt | Disrupting the Enzyme Complex Regulating O-GlcNAcylation Blocks Signaling and Development[END_REF]). Alzheimer's disease (AD) is characterized by abnormal function of Tau proteins. A common feature of these pathologies is hyperphosphorylation of Tau, the main component of fibrillar assemblies such as Paired Helical Filaments (PHFs). O-GlcNAcylation is another important post-translational modification involved in regulation of tau pathophysiology. Recently, it has been reported that an increase in tau O-GlcNAcylation reduces tau phosphorylation levels, leading to inhibition of tau aggregation properties. In contrast, decreased O-GlcNAcylation could be involved in the raise of tau phosphorylation associated with AD ([START_REF] Bourré | Direct Crosstalk Between O-GlcNAcylation and Phosphorylation of Tau Protein Investigated by NMR Spectroscopy[END_REF]).

Figure 33 :

 33 Figure 33: Schematic representation of the dynamic interplay between O-GlcNAcylation and phosphorylation.

  has been shown that O-GlcNAc can modify the subcellular localization of certain proteins. Andrali and his colleagues have demonstrated that glucose in high concentrations induces a translocation of NeuroD1 from the cytoplasm to the nucleus in pancreatic β cells. In the presence of low concentrations of glucose, this nuclear translocation can be mimicked by PUGNAc, an inhibitor which prevents the de-glycosylation of proteins. In addition, inhibition of the expression of OGT by siRNAs blocks the nuclear translocation of NeuroD1 induced by glucose in high concentrations, confirming that the NeuroD1 nuclear translocation is regulated by O-GlcNAc[START_REF] Andrali | Glucose Mediates the Translocation of NeuroD1 by O -Linked Glycosylation[END_REF]. Nuclear translocation induced by O-GlcNAc has also been reported for other proteins such as CRTC2 (CREB regulated transcription coactivator 2)[START_REF] Dentin | Hepatic Glucose Sensing via the CREB Coactivator CRTC2[END_REF]) and p65 NFκB (W. H.Yang et al. 2008).

O-

  GlcNAc has been shown to regulate transcription via direct O-GlcNAcylation of transcription factors, thereby modifying their transcriptional activity. For example, the increase in nuclear translocation of NeuroD1 induced by O-GlcNAc leads to stimulation of the expression of the insulin gene in MIN6 pancreatic β cells(Andrali, Qian, and Özcan 2007).Furthermore, it has been reported that the O-GlcNAc level of PDX-1 is correlated with its ability to bind DNA and with the secretion of insulin in β cells[START_REF] Gao | The Transcription Factor PDX-1 Is Post-Translationally Modified by O-Linked N-Acetylglucosamine and This Modification Is Correlated with Its DNA Binding Activity and Insulin Secretion in Min6 β-Cells[END_REF]. Yang et al have demonstrated that glucose in high concentrations induces an increase in O-GlcNAc on p65 NFκB, inducing its transcriptional activity. Indeed, this O-GlcNAcylation decreases the interaction of NFκB p65 with its inhibitor IkB, allowing the nuclear translocation of NFκB p65. Moreover, O-GlcNAcylation increases its affinity for DNA and therefore increases its transcriptional activity. The authors also identified threonine 352 as an important O-GlcNAc site for NFκB p65. The substitution of threonine 352 by alanine (T352A) results in a loss of O-GlcNAc-induced transcriptional activation. O-GlcNAcinducing treatments no longer have any effect on DNA affinity on nuclear translocation of this T352A mutant (W. H. Yang et al. 2008).

  experiments using glucosamine to increase O-GlcNAc, since Hresko et al have demonstrated that induction to insulin resistance by glucosamine in 3T3-L1 adipocytes is the consequence of a depletion of cellular ATP (Hresko et al., 1998). However, subsequent results obtained by controlling the intracellular concentration of ATP have demonstrated the involvement of the HBP pathway in the development of glucosamine-induced insulin resistance (Buse 2006; review: Darley-Usmar, Ball, et Chatham 2012). PUGNAc, an O-GlcNAcase inhibitor, increases O-GlcNAc levels in cells. It has been used to demonstrate the involvement of O-GlcNAcylation pathway in insulin resistance. Incubation of adipocytes

  An important physiological role for O-GlcNAcylation in the regulation of insulin secretion by endocrine cells of the pancreas has been suggested. By electron microscopy, OGT was observed located near the insulin secretion granules. Pharmacological inhibition of OGA increases the secretion of insulin by MIN6 cells, a murine β-pancreatic cell line. This is due to an increase in cellular insulin content, linked to the increased transcription of the Ins1 and Ins2 genes(Durning et al., 2016). Conversely, the pharmacological inhibition of GFAT by azaserine in cultured islets decreases the secretion of insulin in response to glucose(Zraika et al., 2002).Two major transcription factors involved in the regulation of insulin gene expression in β cells, Pdx-1 (pancreatic and duodenal homeobox 1) and NeuroD1 (neurogenic differentiation 1), areO-GlcNAcylated (Babu et al., 2008). NeuroD1 is O-GlcNAcylated in a glucose dependent manner. Thus in the basal state, NeuroD1 is mainly cytoplasmic and interacts with the OGA which keeps it deglycosylated. In hyperglycemia, NeuroD1 interacts with OGT.

  showed that the transcription factor FoxO1 downstream of PI3K/Akt signaling pathway is O-GlcNAcylated in INS1 832/13 β cells treated with high glucose concentrations. O-GlcNAcylation of FoxO1 in β cells leads to increase its transcriptional activity, resulting in increased mRNA expression and secretion of IGFBP1. IGFBP1 over secretion exerts an autocrine effect by inhibition of the activity of the insulin like growth factor 1 receptor (IGF1R)/phosphatidyl inositol 3 kinase (PI3K)/Akt pathway in β cells. This may be one of the mechanisms by which O-GlcNAcylation mediates high glucose induced β cell failure(Fardini et al. 2014).

  By studying the mechanisms leading to insulin resistance induced by O-GlcNAc, researchers have discovered that several proteins in the insulin signaling cascade, namely IRS-1, PI 3-kinase, and AKT, are modified by O-GlcNAc. Despite the fact that the mechanisms involved are far from being clear and that these results are quite variable depending on the experiences, it is recognized that O-GlcNAc on these proteins attenuates the action of insulin in different tissues. Yang et al have demonstrated a mechanism by which OGT can glycosylate these proteins. The authors have shown that OGT can interact with the phospholipid PI[START_REF] Fardini | O-GlcNAcylation: A New Cancer Hallmark?[END_REF]4,[START_REF] Ma | Hyper-O-GlcNAcylation Is Anti-apoptotic and Maintains Constitutive NF-kappaB Activity in Pancreatic Cancer Cells[END_REF] P3 (PIP3) via its PPO domain (PIP-binding domain of OGT) in vitro, and that the production of PIP3 by PI3-kinase induced by insulin recruits OGT to the plasma membrane. This recruitment to the membrane allows the OGT to glycosylate certain members of the insulin signaling, such as IRβ, IRS1 and PKB/AKT, which leads to decreased in insulin-induced phosphorylation of these proteins and decreased biological action of insulin.

Figure 34 :

 34 Figure 34: Schematic representation of O-GlcNAc effect on insulin signaling pathway

  well characterized. Li et al revealed that OGT in adipocyte links dietary fat sensing to hyperphagia via regulation of N-arachidonyl ethanolamine (AEA) production, which acts as an endogenous ligand of appetite-inducing cannabinoid receptor 1 (CB1). They revealed that adipocyte OGT KO (OGT-FKO) mice were protected from HFD induced hyperphagia compared to wild-type mice (M.-D.Li et al. 2018). Moreover, another recent study by Yang et al reported that OGT prevents visceral fat lipolysis and promotes central obesity. The authors showed that OGT inhibits lipolysis via O-GlcNAcylation of lipid droplet-associated perilipin 1 (PLIN1). PLIN1, a lipid droplet coating protein, is known to regulate lipid metabolism in adipocytes by serving as a physical barrier as well as a recruitment site for lipases to the lipid droplet. Phosphorylation of PLIN1 is crucial step to initiate lipolysis. Interestingly, they found that PLIN1 is O-GlcNAcylated at serine 492 and serine 517 sites which are located adjacent to the sites phosphorylated during fasting. They observed that an inducible deletion of OGT in visceral fat in mice resulted in a rapid weight loss by specifically promoting lipolysis in visceral fat (Y.[START_REF] Yang | O-GlcNAc Transferase Inhibits Visceral Fat Lipolysis and Promotes Diet-Induced Obesity[END_REF]. Conversely, in the same study, overexpression of OGT in mice visceral adipose tissue inhibited lipolysis and promoted diet induced-obesity (Y.[START_REF] Yang | O-GlcNAc Transferase Inhibits Visceral Fat Lipolysis and Promotes Diet-Induced Obesity[END_REF]. These results indicate that the nutrient sensor activity of OGT in white adipose tissues promotes obesity, which may explain the high fat induced-insulin resistance.

  OGT as a regulator of β-cell mass and function, and provide a direct link between O-GlcNAcylation and β-cell survival by regulation of ER stress responses and modulation of PI3K/Akt signaling pathway. Therefore, whereas chronic increase in O-GlcNAcylation may have deleterious effect on β cells, OGT and O-GlcNAc appear necessary for proper development of β cell function.

  NFκB signaling pathwayO-GlcNAcylation can activate NFκB signaling pathway and thus promote proinflammatory responses at different levels. The p65 subunit of the transcription factor NFκB is the most studied as a target of O-GlcNAc. In rat vascular smooth muscle, O-GlcNAcylation of NF-κB p65 on T352 inhibits the interaction between p65 NF-κB and IκB, thus increasing the translocation of NF-κB into the nucleus and the transcription of VCAM-1 under hyperglycemic conditions (W. H.Yang et al. 2008). These results suggest that O-GlcNAcylation of NF-κB p65 on T352 may contribute to an increase in NF-κB activity during the progression of diabetes (W. H.Yang et al. 2008).Moreover, p65 has also been shown to be O-GlcNAcylated on T305. Interestingly, this potential O-GlcNAcylation site seems to increase the transcriptional activity of NFκB by promoting its acetylation by p300 on Lys310 (K310)(Allison et al. 2012).Similarly, Ma and Vosseller have confirmed that O-GlcNAcylation of p65 on T305 and also that of S319 increase CBP/p300-dependent acetylation of p65 on K310, facilitating NF-κB transcriptional activation. In addition to enhancing O-GlcNAcylation, by overexpression of OGT, increases the expression of p300, IKKα and IKK and elevates IKK induced p65 phosphorylation on S536, leading to NF-κB activation. However, O-GlcNAcylation of p65 at T305 is likely impaired by the phosphorylation of p65 at T308[START_REF] Ma | Hyper-O -GlcNAcylation Activates Nuclear Factor κ-Light-Chain-Enhancer of Activated B Cells (NF-ΚB) Signaling through Interplay with Phosphorylation and Acetylation[END_REF]. Furthermore, Kim et al investigated the role of N-acetyl glucosamine on inflammatory process associated with arthritis by using the OGA inhibitor Thiamet G (H. B.[START_REF] Kim | O-Linked N-Acetylglucosamine Glycosylation of P65 Aggravated the Inflammation in Both Fibroblast-like Synoviocytes Stimulated by Tumor Necrosis Factor-α and Mice with Collagen Induced Arthritis[END_REF]. They revealed that Thiamet G increased TNFα induced pro-inflammatory gene expression in vitro in synovial cells and in vivo in mice with collageninduced arthritis (CIA). The authors suggested that O-GlcNAcylation of p65 enhanced inflammatory processes in TNF-α-stimulated Fibroblast-like synoviocytes (FLS) via increased nuclear translocation of p65, its DNA binding affinity, and enhanced its transcriptional activity. Finally, using siRNA, the authors also highlighted that this mechanism is dependent on OGT.Furthermore, in in vivo study, hyperglycemia has been found to augment O-GlcNAcylation of NF-κB, particularly the p65 subunit. This was associated with an increase in the production of pro-inflammatory cytokines TNF-α and IL-6 in rat placenta (Dela[START_REF] Justina | Increased O-Linked N-Acetylglucosamine Modification of NF-ΚB and Augmented Cytokine Production in the Placentas from Hyperglycemic Rats[END_REF]).In addition, the inhibitor β kinase (IKK) of NF-κB has also been found as a target of O-GlcNAcylation at serine 733. Interestingly, IKKβ O-GlcNAcylation at serine 733, in the C terminal domain, competes and impairs the inactivating phosphorylation at the same site. This results in activation of catalytic activity of IKKβ and phosphorylation of IKK complex and increases in the phosphorylation and therefore degradation of IκB by the proteasome. This in turn ultimately results in activation of p65 and its translocation in the nucleus(Kawauchi et al. 2009). TAK1 (TGFß activated kinase 1) is a serine threonine kinase that activates the MAPK pathway as well as the NFκB pathway in response to pro-inflammatory cytokines TNFα and IL1β (Sato et al. 2005). TAK1 activity depends on the phosphorylation and ubiquitination of its binding partner TAB1 (transforming growth factor-β-activated kinase 1 binding protein). Pathak et al revealed that TAB1 is O-GlcNAcylated on Ser395 in response to high glucose concentrations and IL1β. The O-GlcNAcylation of TAB1 allows the activation of TAK1, which in turn induces the activation of NFκB by promoting the phosphorylation of IκB, resulting in the expression of pro-inflammatory cytokines (Pathak et al. 2012). Nucleotide-binding oligomerization domain-containing protein 2 receptor (Nod2) is an intracellular receptor for innate immunity, which recognizes the bacterial wall and then activates the inflammatory response in particular by activating the inflammatory response pathway via NFkB. A study has shown that the Nod2 receptor is O-GlcNAcylated. This O-GlcNAcylation increases its half-life, thereby enhancing inflammation and inducing an increase in the transcriptional activity of NFκB (C.-W. Hou et al. 2015). The c-Rel subunit has also been shown to be O-GlcNAcylated on Ser350, resulting in increases in its transcriptional activity. This mechanism has been observed in T cells after activation of the TCR (T-cell receptor). c-Rel O-GlcNAcylation leads to increased expression of IL-2 and IFNγ (Ramakrishnan et al. 2013).

  NFκB signaling pathwayAlthough increasing O-GlcNAc levels are usually accompanied by NF-κB activation in diabetes and insulin resistance, O-GlcNAcylation inducing treatments can also have antiinflammatory and pro-survival effects. Zou and his team demonstrated that, in a traumahemorrhage rat model, the administration of glucosamine or PUGNAc during resuscitation improves cardiac function and decreases circulating levels of pro-inflammatory cytokines such as IL6 and TNFα[START_REF] Zou | The protective effects of pugnac on cardiac function after trauma-haemorrhage are mediated via increased protein O-GlcNAc levels[END_REF]. Later, the same team revealed that the protective effect of glucosamine-induced O-GlcNAcylation was mediated by attenuation of traumahaemorage induced IκB-α phosphorylation, NF-κB DNA binding activity and proinflammatory cytokines (TNFα and IL6) mRNA expression. Moreover, they showed that glucosamine decreased LPS stimulated-IkB phosphorylation in cultured primary cardiomyocytes and RAW264.7 cells[START_REF] Zou | Glucosamine Improves Cardiac Function Following Trauma-Hemorrhage by Increased Protein O -GlcNAcylation and Attenuation of NF-ΚB Signaling[END_REF].Moreover, in rat aortic smooth muscle cells, GlcN and PUGNAc treatment protected against TNF-α induced inflammatory stress by enhancing O-GlcNAcylation of p65 at S536 and by inhibiting TNF-α induced phosphorylation of NF-κB p65, thus inhibiting NF-κB signaling(Xing et al. 2011a). It has also been shown that O-GlcNAc could prevent colon carcinogenesis. In this study, they generated colon specific OGT-transgenic mice in which they evaluated the effect of in vivo administration 1,2 dimethylhydrazine/ dextran sodium sulfate induced colon carcinogenesis model. They observed that NF-κB p65 phosphorylation and IL-1β mRNA expression are significantly lower in Ogt-transgenic than in wild-type mice. Moreover, by histochemical staining, they demonstrated shedding of colon epithelial cells in wild type mice a few days after dextran sodium sulfate treatment, whereas they remained essentially intact in OGT-transgenic mice. This suggests that O-GlcNAcylation could prevent acute colitis by reducing acute inflammation and carcinogenesis (Yoshimasa[START_REF] Hirata | Augmented O-GlcNAcylation Alleviates Inflammation-Mediated Colon Carcinogenesis via Suppression of Acute Inflammation[END_REF]. Another study indicated that, in BV2 mouse microglial cells, GlcN alleviates the O-GlcNAcylation of both nuclear and cytosolic forms of c-Rel and inhibits the binding of c-Rel to the NF-κB site in the NOS2 promoter upon stimulation by LPS. This effect was mediated by O-GlcNAc induced inhibition of OGT-c-Rel interaction through unknown mechanism. (S.-Y.Hwang et al. 2013a).

  level of O-GlcNAcylation in SH-SY5Y neuroblastoma cells using the OGA inhibitor Thiamet-G or glucosamine (GlcN) reduced cellular respiration and ROS generation. The authors showed that cells with elevated O-GlcNAc levels had elongated mitochondria and increased mitochondrial membrane potential, and RNA-sequencing analysis indicated transcriptome reprogramming and down-regulation of the NRF2-mediated antioxidant response. Conversely, genetic ablation of OGT in the murine liver caused also increased ROS, but increased NRF2 levels and activity (E. P.[START_REF] Tan | Sustained O-GlcNAcylation Reprograms Mitochondrial Function to Regulate Energy Metabolism[END_REF] N. S. Tan and Wahli 2014).

Figure 35 :Figure 36 :

 3536 Figure 35: The BRET biosensors used to monitor of O-GlcNAcylation levels A) Structure of BRET biosensors: The BRET biosensor consists of a luciferase and a yellow fluorescent protein (Venus) separated by a lectin (GafD) and a peptide carrying an O-GlcNAcylation site derived from casein kinase 2. When the peptide is O-GlcNAcylated, it binds to the lectin. This induces a change in conformation of the biosensor and allows energy transfer from the Rluc to the Venus, resulting in a BRET signal. The biosensor has been fused to different targeting sequences allowing its nuclear, cytosolic or plasma membrane localization. B) Fluorescence microscopy performed in the laboratory in RAW 264.7 macrophages transfected with the nuclear, cytoplasmic and plasma membrane BRET biosensors. Left image: Venus fluorescence of the O-GlcNAc BRET biosensor. Middle image: DAPI staining of nuclei. Right image: Merge image showing localization of the BRET biosensor in different cellular compartments

Figure 38 :

 38 Figure 38: Time-course experiment for GFAT2 expression RAW264.7 cells were cultured in absence or presence of LPS (100ng/mL) for 1, 3, 6, and 24h. RNAs were extracted and GFAT2 expression was analysed by RT-qPCR. Results are the mean±SEM of 3 to 4 experiments. Statistical analysis was performed using Student's t test (*, **: p<0.05 and p<0.01, respectively).

Figure 40 :

 40 Figure 40: LPS effect on O-GlcNAcylation and O-GlcNAc regulatory enzymes in HMDM B) HMDM derived from blood of healthy donor were incubated in presence or absence of LPS 100ng/ml for 24h. After cell lysis, proteins and m RNA expression were analysed either by western blot which has been hybridized with the indicated antibodies or by RT-Qpcr respectively for 8 independent experiments. Statistical analyzes were performed using a Student t test (*: p< 0.05; **: p <0.01, ***: p< 0.001).

Figure 41 :

 41 Figure 41: Principle of spectrophotometric assay of GFAT activity GFAT (Glutamine Fructose-6-phosphate Amido-Transferase) catalyze the reaction of fructose-6phosphate (F6P) with glutamine which produces glucosamine-6-phosphate and glutamate. The assay here involves the enzyme Glutamate Dehydrogenase (GDH) which uses this glutamate as well as 3-acetylpyridine adenine dinucleotide (APAD), a chemical analogue of NAD, to form (among others) the molecule detectable by the spectrophotometer at 365 nm: APADH.

Figure 42 :

 42 Figure 42: Measurement of GFAT enzyme activity in LPS-stimulated macrophages A) RAW cells treated with LPS and lysed after 24 hours. (N = 6) B) BMDM primary cells treated with LPS and lysed after 24 h. (N = 5) C) Human HMDM cells treated with LPS and lysed after 24 h. (N = 3) A), B) and C). The results are represented in the form of optical density per microgram of proteins. (*, **, ****: respectively p≤0.05, p≤0.01, p≤0.0001)

Figure 44 :

 44 Figure 44: HIF1α KO effect on HBP enzymes Peritoneal macrophage from HIF1α treated or not with LPS for 24 h. Cells were then lysed and the proteins analyzed by western blot with antibodies directed against GFAT1, GFAT2, OGT and Clathrin respectively. mRNA were extracted and analysed by RTqPCR. (n=2).

Figure 45 :

 45 Figure 45: Effect of NFκB inhibitor BAY11-7082 on GFAT2 expression RAW264.7 cells were incubated with DMSO or BAY11-7082 (17µM) for 30 min followed by LPS treatment for 24h. Aftert cell lysis, RNA were extracted and analyzd by RTqPCR. Statistical analyzes were performed using an ANOVA followed by a Tukey post-test on 3 independent experiments. (*, **: p <0.05, p <0.01, respectively).

and

  Site 2 on (Figure 46.B). To further demonstrate the contribution of these sites, we mutated either one or both FoxO1 binding sites and measured the activity of the mutated promoters in the luciferase assay. As shown in Figure 46.B, FOXO1-TM effect was markedly reduced by mutation of Site 1 and totally abolished when both Site 1 and Site 2 were mutated. This suggests that both sites might be important for regulation of GFAT2 expression by FoxO1. Binding of Foxo1 to Site 1 and Site 2 was further demonstrated on the endogenous Gfpt2 promoter in RAW264.7 cells, in chromatin immunoprecipitation experiments using a Foxo1-specific antibody (Fig. 46.C).

Figure 46 :

 46 Figure 46: FoxO1 binds directly to GFPT2 promoter and enhances its transcription (A) HEK 293-T cells were co-transfected with a firefly luciferase reporter gene comprising the 500bp or 800pb regions upstream of the transcription start site of the mouse and human genes, respectively, a Renilla luciferase expression plasmid, and either pcDNA3 or the cDNA coding for the constitutively active FOXO1-TM. Results are expressed as fold-activation of the promoter by FOXO1 and are the mean ± SEM of 5 independent experiments. Statistical analysis was performed using Student's t test (***: p<0.001). (B) Mutation of FoxO1 binding sites on the mouse GFAT2 promoter inhibits FoxO1 induced activation of the GFAT2 promoter reporter gene. Results are expressed as fold-activation of the wild-type promoter by FOXO1 and are the mean ± SEM of at least 4 independent experiments. Statistical analysis was performed using ANOVA followed by Tukey's test (**, ****: p<0.01, p<0.0001, respectively; NS: nonsignificant). (C) Binding of Foxo1 on Site 1 and Site 2 of the endogenous mouse promoter was shown by chromatin immunoprecipitation of Foxo1 in RAW264.7 cells. Chromatin was immunoprecipitated with either control IgG or anti-FoxO1 antibody and recovered DNA was amplified by qPCR. Results are the mean ± SEM of 5 independent experiments, each performed in biological duplicate. Statistical analysis was performed using Student's t test (**, p<0.01).

  47.A)and HMDM (Figure47.B), that inhibition of FoxO1 by this compound totally impaired the effect of LPS on induction of GFAT2 mRNA and protein expression. Finally, to further confirm the role of FoxO1 in LPS-induced GFAT2 expression, we evaluated the effect of LPS on peritoneal macrophages from mice with targeted deletion of FoxO1 in the myeloid lineage (LysM-Cre Foxo1). After cell treatment with LPS for 24 h, protein lysis and RNA extraction were performed. We observed that LPS-induced GFAT2 protein and mRNA expression was markedly impaired in Foxo1 KO macrophages, whereas GFAT1 and OGT expression was not significantly affected (Figure47.C). Furthermore, inhibition of GFAT2 expression was associated with inhibition of LPS effect on protein O-GlcNAcylation. These data suggest that FoxO1 is critical for TLR4 induced GFAT2 expression and subsequent enhanced protein O-GlcNAcylation in macrophages.

Figure 47 :

 47 Figure 47: FoxO1 is essential for LPS induced-GFAT2 expression in macrophage. RAW264.7 cells (A) or HMDM (B) were incubated in the absence or presence of 500 nM of AS1842858 for 72h. Cells were then treated with or without LPS for 24h before lysis. Proteins were submitted to western blot and revealed with the indicated antibodies. mRNA were extracted and analysed by RTqPCR. (N = 5 for each cell type). (C) Peritoneal macrophages from LysM cre lox/lox Foxo1 mice were either stimulated with LPS or left untreated for 24h. Cells were then lysed and the proteins analyzed by western blot with antibodies directed against O-GlcNAc, FoxO1, GFAT2 and OGT. mRNA were extracted and analysed by RTqPCR. Statistical analyzes were performed using an ANOVA followed by a Tukey post-test on 8 independent experiments. (*, **,***: p <0.05, p <0.01, p< 0.001 respectively).

Figure 49 :

 49 Figure 49: ogt deficient macrophage exhibited exacerbated LPS-pro-inflammatory response in vitro and in vivo A) Peritoneal macrophages, from wild type and LysM cre lox/lox ogt (KO-OGT mac ) male mice, were cultured in the presence or absence of 100ng/ml of LPS for 24h before lysis. Proteins were submitted to western blot and then hybridized with anti-(O-GlcNAc, OGT, GFAT1, GFAT2, NOS2). (n=8) B) IL1β, TNFα and IFNγ concentrations in the supernantant of peritoneal macrophage after 24h of LPS treatment were measured by MSD multiplex kit. (n=6). C) Secreted IL1β concentration in the supernatant of peritoneal macrophages (WT and KO-OGT mac ) was measured by ELISA after stimulation with LPS for 3,6 and 24h. (n=3). D) wild type mice and KO-OGT mac male mice (9 mice per group) were injected in vivo intraperitoneally either with physiological serum or 0.06 g/kg of LPS. After 6h, serum was collected. IL1β, TNFα and IFNγ concentration in pg/ml were measured by MSD multiplex kit. Statistical analyzes were performed using an ANOVA followed by a Tukey post-test on 8 independent experiments. (*, **,***: p <0.05, p <0.01, p< 0.001 respectively).

Figure 50 :

 50 Figure 50: Effect OGT deletion in macrophage on HF/HS diet induced-inflammation

  (i) TLR4 stimulation by LPS increased protein O-GlcNAcylation in macrophage (ii) This enhanced O-GlcNAcylation was at least in part mediated by FoxO1 dependent GFAT2 gene experession.

  Hwang et al tested the effects of LPS on O-GlcNAcylation in RAW264.7 cells and they described a decrease in O-GlcNAcylation levels induced by LPS (S.-Y. Hwang et al. 2013a). Moreover, a very recent study also demonstrated that TLR4 stimulation by LPS attenuated O-GlcNAcylation in myeloid macrophages (X. Li et al. 2019).

  debated. Several key molecules in TLR4-NFκB signaling pathway have been shown to be O-GlcNAcylated such as IKKβ, NF-κB p65, c-Rel, and TAB1. Their O-GlcNAcylation enhances their activities and promotes the transcription of NF-kB target genes (Özcan, Andrali, and Cantrell 2010; Pathak et al. 2012; Y. R. Yang et al. 2015). Moreover, Zanotto et al (2015) demonstrated that increased cellular O-GlcNAcylation favour macrophage M1 polarization. In this study they showed that glucosamine and ThiametG enhanced LPSinduced bone marrow derived macrophage M1 polarization and IL1β secretion. On the other hand, other studies demonstrated that O-GlcNAcylation exerts an anti-inflammatory effect (N. Song et al. 2019; X. Li et al. 2019).

  identification of individuals at high risk for developing type 2 diabetes and the early diagnosis of diabetes are useful for prevention and early treatment of the disease, as well as for reducing the already increased risk of cardiovascular disease (Zinman et al. 2010). It has been established that adequate lifestyle and pharmacological interventions delay the development of diabetes in people at high risk, such as patients with metabolic syndrome (Diabetes Prevention Program Research Group 2009).

  The activation and pancreatic infiltration of monocytes promotes the synthesis and release of pro-inflammatory mediators such as IL1β, TNF α and IFNγ after the stimulation of TLRs by local production of chemokines and cytokines, and danger signals from dying β cells. This results in the amplification and maintaining of local inflammation (insulitis) which induces progressive and prolonged β-cell loss with symptomatic dysglycemia. Therefore, we suggest blood test that measures monocytes GFAT activity (and/or GFAT2 mRNA expression) for patients with positive autoantibodies. Increased GFAT activity could serve as a marker that predicts the active β cell destruction as the result of symptomatic T1D-associated autoimmunity. Early prediction should guide new strategies for the prevention of type 1 diabetes.

  Fig S1). This modification has been involved in the modulation of various signalling pathways, and perturbations of O-GlcNAcylation

  and 2A)) that LPS induced a major increase in global protein O-GlcNAcylation in RAW264.7 macrophages. The use of plasma membrane-, cytosol-or nucleus-targeted BRET O-GlcNAc biosensors indicated that the LPS-induced increased in O-GlcNAcylation was not restricted to a specific cell compartment. Increased protein O-GlcNAcylation upon LPS treatment was also confirmed in primary mice and human macrophages, indicating that our observation was not a cell line specific effect. The reason underlying the discrepancies between different laboratories are unknown at the present time. However, it is possible technical differences in the extraction procedure may affect the detection of O-GlcNAc by western-blotting. For instance, whereas investigators generally add proteases and phosphatases inhibitors in their cell lysis buffer, they do not mention the use of any hexosaminidase inhibitor to prevent loss of O-GlcNAc during the extraction procedure.

Fig 3 )

 3 . Thus, LPS-induced increase in O-GlcNAcylation is likely to be at least in part mediated by the induction of the expression of the GFAT2 isoform in macrophages.

Figure 1 :

 1 Figure 1: LPS induces O-GlcNAcylation in macrophages (A) RAW264.7 cells were transfected with plasma membrane, cytosol or nucleustargeted BRET biosensors. 18h after transfection, medium was changed and cells were cultured for an additional 24h in presence of LPS (100ng/mL), Thiamet G (TG, 10µM) or both. BRET signal was then measured every 45 seconds during 15 min. In each experiment, the mean of 20 repeated BRET measurements in a given experimental condition was taken as the BRET value obtained in this experimental condition. Left panels show the signals obtained during typical BRET experiments with each biosensor (white circles, control; grey squares, LPS; grey circle, Thiamet G; black squares, LPS+ Thiamet G). Right panels correspond to the increase BRET above basal induced by LPS, Thiamet G or LPS+Thiamet G (delta BRET expressed in milliBRET Units) and are the mean±SEM of 8 independent BRET experiments. Statistical analysis was performed using ANOVA followed by Dunnett's posttest (*, **: p<0.05, p<0.01, respectively, when compared to the control condition).(B) RAW264.7 cells, bone marrow-derived and peritoneal primary mouse macrophages, and human monocyte-derived macrophages were cultured during 24h in absence or presence of LPS (100ng/mL), Thiamet G (10µM) or both. Proteins were extracted and analysed by western-blotting using an anti-O-GlcNAc antibody (RL2). Membranes were then re-probed with anti-clathrin antibody to control for protein loading in each well. Each blot is representative of at least 4 experiments.

Figure 2 :

 2 Figure 2: Effect of LPS on OGT, OGA and GFAT expression and activity in RAW264.7 cells. RAW264.7 cells were cultured during 24h in absence or presence of LPS (100ng/mL) and lysed for protein and RNA extraction. (A) O-GlcNAcylated proteins were precipitated on wheat-germ lectin (WGL) agarose beads and submitted to western-blotting using anti-O-GlcNAc antibody. Left panel, a typical western blot is shown. Right panel, the effect of LPS on global protein O-GlcNAcylation was quantified by densitometric analysis of the blots (B) Protein expression level of OGA, OGT, GFAT1, GFAT2, and NOS2 in total cell lysates. Left panel: a typical western blot is shown. Right panel, densitometric analysis of the blots. (C) mRNA expression levels of OGA, OGT, GFAT1, GFAT2 and NOS2 were evaluated by RT-qPCR. (D) OGT, OGA, and GFAT enzymatic activities in cell lysates from control and LPStreated cells. Results are expressed as LPS-induced fold-effect and are the mean ± SEM of 4

Figure 3 :

 3 Figure 3: Effect of LPS on GFAT2 expression in human and mouse primary macrophages Human monocyte-derived macrophages (A), mouse bone marrow-derived (B) and peritoneal (C) macrophages were cultured during 24h in absence or presence of LPS (100ng/mL) and lysed for protein and RNA extraction. The effect of LPS on global protein O-GlcNAcylation level and on the expression of OGT, OGA, GFAT1 and GFAT2 was evaluated by densitometric analysis of the western-blots. mRNA expression levels of GFAT2, GFAT1 and OGT were evaluated by RT-qPCR. Results are expressed as LPS-induced foldeffect and are the mean ± SEM of at least 4 independent experiments. Statistical analysis was performed using Student's t test (*, **, *** p<0.05, p<0.01, p<0.001, respectively; NS: nonsignificant).

Figure 4 :

 4 Figure 4: Targeted deletion of OGT in macrophages increases the proinflammatory effect of LPS (A, B) Peritoneal macrophages from control or OGT-KO mice were cultured during 24h in absence or presence of LPS (100ng/mL). The culture medium was collected for measurement of cytokine production in the medium, and cell were lysed for protein analysis. (A) Typical western blot showing protein O-GlcNAcylation and expression of OGT, GFAT1, GFAT2 and NOS2 in control and OGT-KO macrophages. (B) The concentration of IL1β, TNFα and IFNγ secreted in the culture medium by control and OGT-KO macrophages were determined using MSD kit. (C) Control and OGT-KO mice were intraperitoneally injected with 0.6 mg/kg of LPS. 6h after injection, mice were sacrificed and blood was collected. The concentration of IL1β, TNFα and IFNγ in the serum were determined using MSD kit. Results are expressed are the mean ± SEM of 4 to 9 independent experiments. Statistical analysis was performed using ANOVA followed by Tukey's post-test (*, **, ***: p<0.05, p<0.01, p<0.001, respectively).

Figure 5 :

 5 Figure 5: FoxO1 stimulates the activity of a GFAT2 promoter reporter gene.

  Moreover, the distribution of the diabetic patients in different regions is reported as following: Western Pacific (159 million), Southeast Asia (82 million), North America and Caribbean (46 million), Middle East and North Africa (39 million), South and Central

According to the International Diabetes Federation (IDF) in 2017, diabetes mellitus is recognized as the third highest risk factor for worldwide premature mortality. 425 million people have diabetes in the world and more than 58 million people in the European Union region; by 2045 this will rise to 66.7 million. There were 3.276.400 cases of diabetes in France in 2017.

  LysM-Cre transgenic mice (LysMCre-OGT knock-out (KO) mice, thereafter referred to as OGT-KO mice). All mice were housed in the Institut Cochin animal facility. All experiments were performed in accordance with accepted standards of animal care, as established in the Institut National de la Santé et de la Recherche Médicale (INSERM) and the Centre National de la Recherche Scientifique (CNRS) guidelines and were approved by the national ethical committee (APAFIS N°A751402).

	Preparation of bone-marrow-derived and peritoneal macrophages from mice.
	Mice were sacrificed by CO 2 asphyxiation. Bone marrow-derived macrophages
	(BMDM) were prepared from bone marrow cells flushed from femurs and tibias. Cells were
	seeded in sterile Petri dishes at the concentration of 10 6 /ml in RPMI medium supplemented
	with 10% FCS, 100μg/ml gentamycin, 10mM HEPES, 1mM sodium pyruvate, 50μM β-
	mercaptoethanol and 20ng/mL mouse MCSF (Miltenyi Biotec). Cells were differentiated
	during 6 days, then washed to eliminate non-adherent cells, and macrophages were detached in

cold PBS -/-and seeded in 6 well-plates at 10 6 cells/ml in complete media. Cells were treated the day after.

Acknowledgments

We are very grateful to Prof. L.K. Mahal for the cDNA coding for the FRET O-GlcNAc biosensors. We also thank Laura Francese for her help in some of the BMDM experiments. This work was performed within the Département Hospitalo-Universitaire AUToimmune and HORmonal diseases.

Funding

This work was supported by the SFD (Société Francophone du Diabète) and the FRM (Fondation pour la Recherche Médicale). Léa Baudoin and Hasanain Al-Mukh hold PhD.

grants from the CORDDIM-Région Ile-de-France. José-Luis Sanchez Salgado was a recipient of a post-doctoral grant from CONACYT (Consejo Nacional de Ciencia y Tecnologia, Mexico).

Figure 48: Effect of OGT deletion on macrophage polarization

Representative image of the effect of treatment for 48 h with or without 20 ng/ml IFNγ on the percentage of CD11c + and CD206 + cells in the populations of OGT-KO and WT macrophages.

Article

Article available on bioarchive https://www.biorxiv.org/content/10.1101/2020.03.22.002303v1

Abstract

O-GlcNAc glycosylation is a reversible post-translational modification that regulates the activity of intracellular proteins according to glucose availability and its metabolism through the hexosamine biosynthesis pathway (HBP). This modification has been involved in the regulation of various immune cell types, including macrophages. However, little is known concerning the mechanisms that regulate protein O-GlcNAcylation level in these cells. In the present work, we demonstrate that LPS treatment induces a marked increase in protein O-GlcNAcylation in RAW264.7 cells, bone-marrow-derived and peritoneal mouse macrophages, as well as human monocyte-derived macrophages. Targeted deletion of OGT in macrophages resulted in an increased effect of LPS on NOS2 expression and cytokine production, suggesting that O-GlcNAcylation may restrain inflammatory processes induced by TLR4 activation. The effect of LPS on protein O-GlcNAcylation in macrophages was associated with an increased expression and activity of glutamine fructose 6-phosphate amido-transferase (GFAT), the enzyme that catalyzes the rate-limiting step of the HBP. More specifically, we observed that LPS rapidly and potently stimulated GFAT2 isoform mRNA and protein expression. Genetic or pharmacological inhibition of FoxO1 impaired LPS effect on GFAT2 expression, suggesting a FoxO1-dependent mechanism. We conclude that GFAT2 should be considered as a new TLR4-inducible gene involved in regulation of protein O-GlcNAcylation, that permits to limit exacerbated inflammation upon macrophage activation. transferred into 96-well White Optiplates previoulsy coated with polylysine (Perkin Elmer) and then cultured for 18 hours at 37°C and 5% CO2 before treatments.

HEK 293-T cells were cultured in DMEM and transfected with Fugene as described previously [START_REF] Kuo | O-glycosylation of FoxO1 increases its transcriptional activity towards the glucose 6-phosphatase gene[END_REF].

Chromatin immunoprecipitation (ChIP):

RAW264.7 cells (1x10 7 cells) were prepared for ChIP assay using HighCell# ChIP kit protein A (Diagenode). After cross-linking with formaldehyde, DNA was sonicated into 200-300bp fragments, and protein-DNA complexes were immunoprecipitated using either FoxO1-ChIP grade antibody (Abcam, ab39670) or control rabbit IgG. Protein-DNA crosslinks were reversed by heating, and precipitated DNA was quantified by real-time PCR using primers (Table 1) that amplify either Foxo1 binding site 1 or 2 present in the 500bp upstream of the transcription start site of the mouse GFAT2 gene.

BRET experiments

Transfected cells were treated with LPS (100ng/mL) and/or Thiamet G (10µM) for 24h. BRET experiments were then performed exactly as described previously [START_REF] Lacasa | Interaction of the insulin receptor with the receptorlike protein tyrosine phosphatases PTPalpha and PTPepsilon in living cells[END_REF][START_REF] Blanquart | Characterization of IRA/IRB hybrid insulin receptors using bioluminescence resonance energy transfer[END_REF] using the Infinite F200 Pro microplate analyser (Tecan). Briefly, cells were pre-incubated for 5 min in PBS in the presence of 5µM coelenterazine. Each measurement corresponded to the signal emitted by the whole population of cells present in a well. BRET signal was expressed in milliBRET Unit (mBU). The BRET unit has been defined previously as the ratio 530 nm/485 nm obtained in cells expressing both luciferase and YFP, corrected by the ratio 530 nm/485 nm obtained under the same experimental conditions in cells expressing only luciferase [START_REF] Issad | A homogenous assay to monitor the activity of the insulin receptor using Bioluminescence Resonance Energy Transfer[END_REF][START_REF] Nouaille | Interaction with Grb14 results in site-specific regulation of tyrosine phosphorylation of the insulin receptor[END_REF].

Duality of Interest

The authors declare to have no conflict of interest related to this work.

Biophysical

Research Communications 408 (1): 52-57. https://doi.org /10.1016/j.bbrc.2011.03.115.