Benoit Viollet
email: <benoit.viollet@inserm.fr

Fabrizio

Keywords: Anti-Obesity Agents, pharmacology, therapeutic use, Caloric Restriction, Cyclic AMP-Dependent Protein Kinases, antagonists & inhibitors, metabolism, Endothelium, Vascular, drug effects, physiology, Enzyme Inhibitors, pharmacology, therapeutic use, Exercise, physiology, Humans, Hypoglycemic Agents, pharmacology, therapeutic use, Hypolipidemic Agents, pharmacology, therapeutic use, Myocardial Ischemia, drug therapy AMP-activated protein kinase, energy balance, diabetes, obesity, therapeutic strategy

AMP-activated protein kinase and metabolic control.

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AMP-activated protein kinase and metabolic control

Introduction

Obesity (defined as a body-mass index (BMI) of >30 kg.m ) and the metabolic syndrome are related conditions that can be 2 considered as precursors of T2D and increases the risk of developing this disease by >20-fold ( ). Although these [START_REF] Willett | Guidelines for healthy weight[END_REF] conditions clearly have a genetic component, as indicated by the high prevalence in certain ethnic group, the rapid increase in the prevalence of these conditions in populations throughout the world suggests the contribution of environmental factors. A widely accepted explanation for the increasing prevalence of these conditions lays on the frequent consumption of processed foods with high energy and low fibers content and the reduction in physical exercise due to sedentary lifestyle in modern urban environment. Thus, obesity arises due to an imbalance between energy intake and energy expenditure where caloric excess accumulates preferentially as lipids in adipose tissue but also into muscle and liver. Disruption of energy balance has led to an increased prevalence of T2D and related comorbidities such as coronary heart disease, heart failure, hypertension and renal failure (

). [START_REF] Wing | Behavioral science research in diabetes: lifestyle changes related to obesity, eating behavior, and physical activity[END_REF] Type 2 diabetes (T2D) has a high prevalence worldwide and its treatment produces considerable costs for the health budgets.

Prevention and management of T2D has become a major public health challenge around the world. Diabetes is defined by a fasting plasma glucose higher than 7 mM ( ). T2D is characterized by altered lipid and glucose metabolism (fasting or [START_REF] Alberti | New diagnostic criteria and classification of diabetes--again?[END_REF] postprandial hyperglycaemia, dyslipidemia) as a consequence of combined insulin resistance in skeletal muscle, liver and adipose tissue and relative defects of insulin secretion by -cells that may arise due to an imbalance between energy intake and expenditure ( β

Saltiel and

). Insulin is the primary anabolic hormone that stimulates uptake and storage of fuel substrates, while inhibiting substrate [START_REF] Saltiel | Insulin signalling and the regulation of glucose and lipid metabolism[END_REF] production in peripheral tissues (

). It lowers blood glucose levels by facilitating glucose uptake, mainly into skeletal [START_REF] Kahn | Mechanisms linking obesity to insulin resistance and type 2 diabetes[END_REF] muscle and fat tissue, and by inhibiting endogenous glucose production in the liver. Insulin resistance occurs when a normal dose of insulin is unable to elicit its metabolic responses. Peripheral insulin resistance is associated with lipid partitioning in specific compartments, i.e. muscle and liver, more than with obesity ( ; ). In the natural history of per se [START_REF] Defronzo | Skeletal muscle insulin resistance is the primary defect in type 2 diabetes[END_REF]Tripathy 2009 Unger 1995 type 2 diabetes, pancreatic -cells initially compensate for insulin resistance by increasing insulin secretion, but with time, progressive β β

-cell failure leads to insulin deficiency, and hyperglycaemia ensues ( ). [START_REF] Fonseca | Defining and characterizing the progression of type 2 diabetes[END_REF] Lifestyle intervention is now recognized as the first-line strategy for the management of T2D and remains important for optimization of metabolic control. This is supported by observational studies and clinical trials comparing the respective effects of diet, drugs or exercise, in persons at high risk for type 2 diabetes ( ; ;

). The Diabetes [START_REF] Knowler | Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin[END_REF][START_REF] Pan | Effects of diet and exercise in preventing NIDDM in people with impaired glucose tolerance. The Da Qing IGT and Diabetes Study[END_REF][START_REF] Tuomilehto | Prevention of type 2 diabetes mellitus by changes in lifestyle among subjects with impaired glucose tolerance[END_REF] Prevention Program (DPP) Research Group conducted a large, randomized clinical trial involving adults in the United States who were at high risk for the development of this disease (

). In this study, the lifestyle intervention was particularly effective [START_REF] Knowler | Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin[END_REF] more than an oral hypoglycaemic drug) to prevent the onset of diabetes. In clinical practice, when lifestyle modification fails to achieve or sustain adequate glycaemic control, insulin or oral anti-diabetic agents are typically used to manage the disease (

). [START_REF] Nathan | Medical management of hyperglycemia in type 2 diabetes: a consensus algorithm for the initiation and adjustment of therapy: a consensus statement of the American Diabetes Association and the European Association for the Study of Diabetes[END_REF] Treatment options with oral agents are quite diverse, including metformin, thiazolidinediones (TZDs), -glucosidase inhibitors, α sulphonylureas, DPP-4 inhibitors, and GLP-1 analogs. The currently available classes of oral agents differ in mechanism and duration of action, and the degree to which they lower blood glucose and their side-effect profile (including hypoglycaemia, weight gain, dema, fractures, lactic acidosis, and gastrointestinal intolerance). Because it is recognized that T2D is a progressive disease worsening with time, all available drugs can be used alone or in varied associations.

There is a pressing need to develop new therapeutic strategies to prevent and treat T2D. Exciting recent developments have shown that AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, acts as an integrator of regulatory signals monitoring systemic and cellular energy balance, thus providing the emerging concept, as first suggested by Winder and Hardie in (

), that AMPK is an attractive therapeutic target for intervention in many conditions of disordered energy 1999 [START_REF] Winder | AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes[END_REF] balance including T2D and insulin resistance.

Rational for a pharmacological management of T2D by targeting AMPK

Physical activity is an important determinant to prevent and control T2D. Current guidelines recommend practical, regular and moderate regimens of physical activity. The multiple metabolic adaptations that occur in response to physical activity can improve glycaemic control for individuals with T2D or delay the onset of the disease. Indeed, it is now recognized that beneficial effects of physical activity are still maintained in insulin resistant populations. This suggests that some metabolic actions of exercise (as increase in muscular glucose uptake) are dependent of specific intracellular pathways that bypass signalling altered by insulin resistance. In consequence, any drug inducing favourable changes similar to those of physical exercise on whole body metabolism are attractive candidates for treatment and prevention of obesity, metabolic syndrome and T2D. Interestingly, it is now well established that muscle contraction is a prototypical

AMPK activator (

). Thus, it is expected that part of the effect of physical activity in preventing the development of [START_REF] Hayashi | Evidence for 5 AMP-activated protein kinase mediation of the effect of muscle contraction on glucose transport ′[END_REF] metabolic disorders related to a sedentary lifestyle is due to activation of AMPK. Indeed, it has been documented that pharmacological AMPK activation may recapitulate some of the exercise-induced short-term adaptations and is likely to mediate beneficial effects of exercise on insulin sensitivity and glucose transport in skeletal muscle ( ; ). In addition, [START_REF] Bergeron | Effect of AMPK activation on muscle glucose metabolism in conscious rats[END_REF][START_REF] Fisher | Activation of AMP kinase enhances sensitivity of muscle glucose transport to insulin[END_REF] pharmacological AMPK activation resulted in long-term adaptation similar to those induced by endurance exercise training with the induction of genes linked to oxidative metabolism and enhanced running endurance (

). [START_REF] Narkar | AMPK and PPARdelta agonists are exercise mimetics[END_REF] In the Diabetes Prevention Program (DPP) the incidence of diabetes was reduced by 58 with a low-calorie, low-fat diet, as compared % with placebo after 3 years of follow-up (

). The beneficial effect of calorie restriction in reducing T2D incidence was [START_REF] Knowler | Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin[END_REF] confirmed by other clinical studies ( ; ). In overweight and obese humans, calorie restriction [START_REF] Pan | Effects of diet and exercise in preventing NIDDM in people with impaired glucose tolerance. The Da Qing IGT and Diabetes Study[END_REF][START_REF] Tuomilehto | Prevention of type 2 diabetes mellitus by changes in lifestyle among subjects with impaired glucose tolerance[END_REF] improves glucose tolerance, lipid profile and insulin action and reduces mortality associated with T2D ( ; Hammer et al. 2008 Jazet et al. ; ;

). In order to produce a metabolic profile similar to those of calorie restriction in 2008 [START_REF] Larson-Meyer | Effect of calorie restriction with or without exercise on insulin sensitivity, beta-cell function, fat cell size, and ectopic lipid in overweight subjects[END_REF][START_REF] Weiss | Improvements in glucose tolerance and insulin action induced by increasing energy expenditure or decreasing energy intake: a randomized controlled trial[END_REF] diabetic patients, there is an increased interest in developing pharmacological agents acting as calorie-restriction mimetics. Such agents

" "
could provide the beneficial metabolic, hormonal and physiological effects of calorie restriction without altering dietary intake or experiencing any potential adverse consequences of excessive restriction. To this purpose, phytochemicals mimicking the effects of calorie restriction (polyphenols) were recently identified as potent activators for AMPK and ( ; ; in vitro in vivo [START_REF] Baur | Resveratrol improves health and survival of mice on a high-calorie diet[END_REF][START_REF] Collins | Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, suppresses hepatic gluconeogenesis through 5 -AMP-activated protein kinase ′[END_REF] ). [START_REF] Zang | Polyphenols stimulate AMP-activated protein kinase, lower lipids, and inhibit accelerated atherosclerosis in diabetic LDL receptor-deficient mice[END_REF] Additionally, it is now recognized that a dysfunction in AMPK signalling pathway might have sustained, deleterious effects at the systemic levels and might contribute to the events that lead to the metabolic syndrome. It is interesting to note that there is a strong correlation between low activation state of AMPK with metabolic disorders associated with insulin resistance, obesity and sedentary activities ( ; ; ;

). Recent studies showed that AMPK is likely to be Lee et al. 2005aLee et al. 2005b[START_REF] Luo | AMPK, the metabolic syndrome and cancer[END_REF][START_REF] Martin | Diet-induced obesity alters AMP kinase activity in hypothalamus and skeletal muscle[END_REF] under both endocrine and autocrine control in rodents. Thus, in addition to exercise and starvation, AMPK is activated by the fat-cell-derived hormones adiponectin and leptin ( ; ;

) and interleukin-6 [START_REF] Minokoshi | Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase[END_REF][START_REF] Tomas | Enhanced muscle fat oxidation and glucose transport by ACRP30 globular domain: acetyl-CoA carboxylase inhibition and AMP-activated protein kinase activation[END_REF][START_REF] Yamauchi | Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase[END_REF] (IL-6) (

). Conversely, AMPK activity is suppressed in muscle and liver by sustained hyperglycaemia, in liver by [START_REF] Kelly | AMPK activity is diminished in tissues of IL-6 knockout mice: the effect of exercise[END_REF] re-feeding after starvation (

) and by increases in the plasma concentration of others adipocyte-derived hormones, resistin [START_REF] Assifi | AMP-activated protein kinase and coordination of hepatic fatty acid metabolism of starved/carbohydrate-refed rats[END_REF] ) and tumor necrosis factor-(TNF-) ( ). In addition to its role in the periphery, AMPK also [START_REF] Banerjee | Regulation of fasted blood glucose by resistin[END_REF] α α [START_REF] Steinberg | Tumor necrosis factor alpha-induced skeletal muscle insulin resistance involves suppression of AMP-kinase signaling[END_REF] regulates energy intake and body weight by mediating opposing effects of anorexigenic and orexigenic signals in the hypothalamus ( ; ; ;

). In addition, many therapies that are useful in treating [START_REF] Andersson | AMP-activated protein kinase plays a role in the control of food intake[END_REF][START_REF] Kim | Anti-obesity effects of alpha-lipoic acid mediated by suppression of hypothalamic AMP-activated protein kinase[END_REF][START_REF] Kola | Cannabinoids and ghrelin have both central and peripheral metabolic and cardiac effects via AMP-activated protein kinase[END_REF][START_REF] Minokoshi | AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus[END_REF] the metabolic syndrome and associated disorders in humans, including TZDs ( ; ), metformin [START_REF] Fraser | The Anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways[END_REF][START_REF] Saha | Pioglitazone treatment activates AMP-activated protein kinase in rat liver and adipose tissue in vivo[END_REF] Zhou et al.

), calorie deprivation and exercise, have been shown to activate AMPK system. Lastly, the development of transgenic and knockout 2001 (KO) mouse models (see below) have made possible to better understand the physiological role of AMPK and confirm that disruption of AMPK pathway in various tissues induces various phenotypes mimicking the metabolic syndrome observed in humans.

Taken together the physiological functions of AMPK and the suspected role of AMPK in metabolic disorders, activation of AMPK pathway appears as a promising tool to prevent and/or to treat metabolic disorders.

Structure and regulation of AMPK

AMPK is a major regulator of cellular and whole-body energy homeostasis that coordinates metabolic pathways in order to balance nutrient supply with energy demand. Activation of AMPK switches off ATP-consuming anabolic pathways and switches on ATP-producing catabolic pathways (

). This would typically occur when AMPK is activated as a result of energy [START_REF] Viollet | Physiological role of AMP-activated protein kinase (AMPK): insights from knockout mouse models[END_REF] deprivation linked to alterations of the intracellular AMP/ATP ratio (e.g., hypoxia, glucose deprivation, muscle contraction), changes in calcium concentration as well as the action of various adipocytokines. AMPK is composed of three different subunits , and appearing α β γ in several isoforms with different action properties ( ). The -subunit contains the catalytic site whereas regulatory -and Figure 1 α β γ

-subunits are important to maintain the stability of the heterotrimeric complex. The subunit contains a central region that allows AMPK β complex to bind glycogen. The subunit contains four tandem repeats known as cystathionine -synthase (CBS) motifs that bind together γ β two molecules of AMP or ATP in a mutually exclusive manner. Binding of AMP (on subunit) activates AMPK via a complex γ mechanism involving direct allosteric activation and phosphorylation of subunit on Thr172 by upstream kinases as the protein kinase α LKB1 (a tumour suppressor whose germline mutations in humans are the cause of Peutz-Jeghers syndrome), the CaMKKβ (calmodulin-dependent protein kinase kinase ) and TAK1 (mammalian transforming growth factor -activated kinase) ( ).

β β Figure 2
Although it was originally proposed that AMP binding promoted AMPK phosphorylation by upstream kinases, recent work suggested entirely inhibition of dephosphorylation of Thr 172 to be critical ( ; ) ( ). [START_REF] Sanders | Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade[END_REF][START_REF] Suter | Dissecting the role of 5 -AMP for allosteric stimulation, activation, and deactivation of AMP-activated protein kinase ′[END_REF] Figure 2 Beneficial metabolic effects of targeting AMPK pathway

Mimicking the beneficial effects of physical exercise

It has been confirmed in large scale epidemiological and interventional studies that regular physical activity is of great benefit for the metabolic control of subjects with metabolic syndrome or impaired glucose tolerance or T2D ( ; ; [START_REF] Knowler | Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin[END_REF][START_REF] Pan | Effects of diet and exercise in preventing NIDDM in people with impaired glucose tolerance. The Da Qing IGT and Diabetes Study[END_REF] ). Although appropriate diet and exercise regimes should therefore be the first choice of treatment and prevention of [START_REF] Tuomilehto | Prevention of type 2 diabetes mellitus by changes in lifestyle among subjects with impaired glucose tolerance[END_REF] type 2 diabetes, in some patients such management is not appropriate for other medical reasons, or when compliance is difficult because of social factors or poor motivation. In these cases, drugs that act on the signalling pathways involved in physical activity are attractive candidates for treatment and prevention. It is now clearly demonstrated that AMPK is activated by physical training in an intensity-dependent manner both in humans and in rodents (

). AMPK activation during muscle contraction is a Steinberg and Kemp 2009 physiological adaptation in front of increased energy demand and ATP turnover. It has been demonstrated that AMPK activation may recapitulate some of the exercise-induced adaptations and is likely to mediate beneficial effects of exercise not only on insulin sensitivity and glucose transport in skeletal muscle ( ) but also for additional metabolic benefits coming from AMPK activation by [START_REF] Fisher | Activation of AMP kinase enhances sensitivity of muscle glucose transport to insulin[END_REF] exercise in liver and in adipose tissue (

). Conversely, it has also been demonstrated that disruption of muscular AMPK [START_REF] Park | Coordinate regulation of malonyl-CoA decarboxylase, sn-glycerol-3-phosphate acyltransferase, and acetyl-CoA carboxylase by AMP-activated protein kinase in rat tissues in response to exercise[END_REF] signalling can be a key factor in the pathophysiology of metabolic disorders. Indeed, reduction of muscular AMPK activity exacerbates the development of insulin resistance and glucose intolerance during high-fat feeding, disturbs muscle energy balance during exercise (as indicated by a reduced muscular ATP content during muscle contraction) and abolishes mitochondrial biogenesis ( ; [START_REF] Fujii | Ablation of AMP-activated Protein Kinase alpha 2 Activity Exacerbates Insulin Resistance Induced by High-fat Feeding of Mice { }[END_REF] ). [START_REF] Jorgensen | Effects of alpha-AMPK knockout on exercise-induced gene activation in mouse skeletal muscle[END_REF][START_REF] Zong | AMP kinase is required for mitochondrial biogenesis in skeletal muscle in response to chronic energy deprivation[END_REF] As a proof of concept, studies with AMPK activators in animal models of T2D have provided promising results. The first evidence came from treatment with the pharmacological compound AICAR (5-amino-imidazole-4-carboxamide-1 D-ribofuranoside, in vivo β metabolized to ZMP which is an analog of AMP) of various animal models of insulin resistance, causing improvement in most, if not all, of the metabolic disturbances of these animals ( ; ; ; ; Bergeron et al. 2001a[START_REF] Buhl | Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying features of the insulin resistance syndrome[END_REF][START_REF] Iglesias | AICAR administration causes an apparent enhancement of muscle and liver insulin action in insulin-resistant high-fat-fed rats[END_REF]Pold et al. 2005 Song et al. ). In addition, long-term AICAR administration prevents the development of hyperglycaemia in Zucker diabetic fatty (ZDF) rats, 2002 improves peripheral insulin sensitivity in skeletal muscle and delays -cell dysfunction associated with type 2 diabetes ( ).

β [START_REF] Pold | Long-term AICAR administration and exercise prevents diabetes in ZDF rats[END_REF] AICAR increases muscle glucose uptake concomitantly with glucose transporter 4 (GLUT4) translocation to the plasma membrane in insulin-resistant animal models and in humans ( ; ;

). Interestingly, [START_REF] Koistinen | 5-amino-imidazole carboxamide riboside increases glucose transport and cell-surface GLUT4 content in skeletal muscle from subjects with type 2 diabetes[END_REF][START_REF] Kurth-Kraczek | 5 AMP-activated protein kinase activation causes GLUT4 translocation in skeletal muscle ′[END_REF][START_REF] Merrill | AICA riboside increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle[END_REF] AMPK-induced glucose transport occurs through a mechanism distinct from that utilized by the classical insulin signalling pathway because it is not blocked by inhibitors of phosphatidylinositol 3-kinase, and also because the effects of insulin and AMPK activators are additive (

). This metabolic improvement can be also explained partly by increased expression of specific muscle [START_REF] Hayashi | Evidence for 5 AMP-activated protein kinase mediation of the effect of muscle contraction on glucose transport ′[END_REF] proteins mimicking some of the effects of exercise training following chronic pharmacological activation of AMPK . Thus, AICAR in vivo or chronic intake of the creatine analogue -guanadinopropionic acid ( -GPA, which competitively inhibits creatine uptake and lowers ). It has been proposed that the development of skeletal muscle insulin resistance may be partly linked to 1999 [START_REF] Michael | Restoration of insulin-sensitive glucose transporter (GLUT4) gene expression in muscle cells by the transcriptional coactivator PGC-1[END_REF] decreased mitochondrial density (

). Interestingly, chronic activation of AMPK with AICAR or -GPA increases [START_REF] Petersen | Mitochondrial dysfunction in the elderly: possible role in insulin resistance[END_REF] β mitochondrial content and expression of mitochondrial proteins, leading to a mitochondrial biogenesis ( ; Bergeron et al. 2001b Winder et ; ). All of these data argue for AMPK as a key factor for the metabolic adaptation of skeletal muscle to physical al. 2000[START_REF] Zong | AMP kinase is required for mitochondrial biogenesis in skeletal muscle in response to chronic energy deprivation[END_REF] exercise. Supporting this, the effects of chronic activation of AMPK mimicking physical activity on gene expression and mitochondrial biogenesis are abolished in AMPK 2 knock-out (KO) and mAMPK-KD (transgenic mice overexpressing a kinase-dead AMPK 2 mutant biogenesis after chronic activation of AMPK is partly explained by increased expression of nuclear respiratory factor-1 and -2 (which are critical regulators of genes encoding electron chain complexes) ( ). Another critical factor for mitochondrial Bergeron et al. 2001b biogenesis is the inducible coactivator of nuclear receptors, PGC-1 . Regulation of PGC-1 by AMPK is complex. First, it has been α α demonstrated that AMPK directly phosphorylates and activates PGC-1 ( ). In addition, activated PGC-1 in turn α [START_REF] Jager | AMP-activated protein kinase (AMPK) action in skeletal muscle via direct phosphorylation of PGC-1alpha[END_REF] α increased the expression of PGC-1 and of mitochondrial oxidative genes (cytochrome c, uncoupling protein 1). Interestingly, PGC-1 α α activity and expression are reduced in type 2 diabetes in humans ( ). Thus, AMPK activators could be used in order to [START_REF] Mootha | PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes[END_REF] reverse this defect. Additionally, activation of AMPK in response to physical exercise has been also observed in extra-muscular tissues such as liver and adipose tissue (

) and might accounted for additional metabolic benefits. Physical training increases [START_REF] Park | Coordinate regulation of malonyl-CoA decarboxylase, sn-glycerol-3-phosphate acyltransferase, and acetyl-CoA carboxylase by AMP-activated protein kinase in rat tissues in response to exercise[END_REF] circulating adiponectin and mRNA expression of its receptors in muscle, which may mediate the improvement of insulin resistance and the metabolic syndrome in response to exercise by activation of AMPK.

Lastly, increase in blood supply is critical for physiological adaptation during physical activity. Vasodilatation is a vital mechanism of systemic blood flow regulation that occurs during periods of increased energy demand. Thus, because AMPK plays a central role in the adaptation to metabolic stress, it is tempting to speculate that AMPK could be involved in the regulation of metabolic vasomotion. It is well known that moderate-intensity exercise increases nitric oxide synthase (NOS) activity (

). Interestingly, it has been [START_REF] Roberts | Acute exercise increases nitric oxide synthase activity in skeletal muscle[END_REF] recently reported that mAMPK-KD mice are unable to increase total NOS activity during moderate-intensity exercise and may cause an ). This indicates how changes in tissue metabolism can direct blood flow according to demand. [START_REF] Griffee | Skeletal muscle AMP-activated protein kinase is essential for the metabolic response to exercise in vivo[END_REF] In addition, the lower skeletal muscle capillarization in mAMPK-KD mice might also contribute to the reduced blood flow during exercise (

). Nitric oxide (NO) plays a fundamental role in vascular homeostasis and it has been suggested that impaired [START_REF] Zwetsloot | AMPK regulates basal skeletal muscle capillarization and VEGF expression, but is not necessary for the angiogenic response to exercise[END_REF] efflux from contracting mAMPK-KD mice suppressed exercise-induced vascular relaxation (

). Furthermore, it has [START_REF] Griffee | Skeletal muscle AMP-activated protein kinase is essential for the metabolic response to exercise in vivo[END_REF] suggested that AMPK activation is in part regulated by endogenous NO in a positive feedback mechanism, such that increase NO activates AMPK, which further augments NOS activity and NO production ( ; ). Accordingly, the exercise-induced [START_REF] Lira | Nitric oxide increases GLUT4 expression and regulates AMPK signaling in skeletal muscle[END_REF][START_REF] Zhang | Identification of nitric oxide as an endogenous activator of the AMP-activated protein kinase in vascular endothelial cells[END_REF] increase in AMPK signalling was ablated in skeletal muscle of eNOS KO mice (Lee-Young et al.). Therefore, AMPK-eNOS interaction might play an important role in the adaptation processes during exercise in order to maintain cellular energy levels by amending vascular function.

Mimicking the beneficial effects of calorie/dietary restriction

Excessive calorie intake increases the risk of developing chronic disease such as obesity, metabolic syndrome, T2D, systemic low grade inflammation, cardiovascular event and premature mortality. Conversely, calorie restriction improves glucose tolerance and insulin action and reduces mortality linked to type 2 diabetes and cardiovascular diseases ( ; ; [START_REF] Hammer | Prolonged caloric restriction in obese patients with type 2 diabetes mellitus decreases myocardial triglyceride content and improves myocardial function[END_REF]Jazet et al. 2008 Larson-Meyer ; ). Because it is difficult to maintain long-term calorie restriction in modern society, there has been an et al. 2006[START_REF] Weiss | Improvements in glucose tolerance and insulin action induced by increasing energy expenditure or decreasing energy intake: a randomized controlled trial[END_REF] increased interest in developing pharmacological agents that act as calorie-restriction mimetics. Among them, plant-derived polyphenolic ) and coordinates lipid mobilization and utilization [START_REF] Bordone | The stimulatory effect of globular adiponectin on insulin-stimulated glucose uptake and fatty acid oxidation is impaired in skeletal muscle from obese subjects[END_REF][START_REF] Moynihan | Increased dosage of mammalian Sir2 in pancreatic beta cells enhances glucose-stimulated insulin secretion in mice[END_REF] ). The knowledge of SIRT1 action at the molecular level has been more delineated by using chronic treatments with [START_REF] Picard | Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma[END_REF] resveratrol and it has been suggested that SIRT1 promotes LKB1-dependent AMPK stimulation through the direct deacetylation and activation of LKB1 ( ; ). Thus, polyphenols as resveratrol are now recognized as compounds with potential [START_REF] Hou | SIRT1 regulates hepatocyte lipid metabolism through activating AMP-activated protein kinase[END_REF][START_REF] Lan | SIRT1 modulation of the acetylation status, cytosolic localization and activity of LKB1; possible role in AMP-activated protein kinase activation[END_REF] great interest to improve and/or delay or prevent metabolic disorders linked to western life style by activating the complementary metabolic stress sensors SIRT1 and AMPK (

). Accordingly, it has been recently established that AMPK acts as the prime [START_REF] Canto | AMPK regulates energy expenditure by modulating NAD metabolism and SIRT1 activity +[END_REF] initial sensor for fasting-induced adaptations in skeletal muscle and that SIRT1 downstream signalling was blunted in the absence of

AMPK (

). In addition, recent studies demonstrated that resveratrol failed to increase the metabolic rate, insulin [START_REF] Canto | Interdependence of AMPK and SIRT1 for metabolic adaptation to fasting and exercise in skeletal muscle[END_REF] sensitivity, glucose tolerance, mitochondrial biogenesis and physical endurance in the absence of either AMPK 1 or AMPK 2 ( α α Um et al. ). 2010

Mimicking the beneficial effects of hypoglycaemic agents

AMPK action in liver

T2D is the result of an imbalance between glucose production and glucose uptake by peripheral tissues. Elevated hepatic glucose production is a major cause of fasting hyperglycaemia in diabetic subjects (

). From various effectors, AMPK [START_REF] Saltiel | Insulin signalling and the regulation of glucose and lipid metabolism[END_REF] signalling is a key factor that control hepatic glucose production. Indeed, systemic infusion of AICAR in normal and insulin-resistant obese rats leads to the inhibition of hepatic glucose production (

). Additionally, short-term hepatic expression of a ). This effect is achieved at least to al. 2006[START_REF] Foretz | Short-term overexpression of a constitutively active form of AMP-activated protein kinase in the liver leads to mild hypoglycemia and fatty liver[END_REF][START_REF] Lochhead | 5-aminoimidazole-4-carboxamide riboside mimics the effects of insulin on the expression of the 2 key gluconeogenic genes PEPCK and glucose-6-phosphatase[END_REF][START_REF] Viana | Role of hepatic AMPK activation in glucose metabolism and dexamethasone-induced regulation of AMPK expression[END_REF] a large extent via the regulation of a transcriptional coactivator, transducer of regulated CREB activity 2 (TORC2) ( ) [START_REF] Koo | The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism[END_REF] which is known to mediate CREB-dependent transcription of PGC1 and its subsequent gluconeogenic targets PEPCK and G6Pase genes. ). Control of hepatic glucose production by activated AMPK is also demonstrated in resistin KO Koo et al. 2005 mice and in adiponectin treated rodents ( ; ) suggesting that hepatic AMPK is specifically a [START_REF] Banerjee | Regulation of fasted blood glucose by resistin[END_REF][START_REF] Yamauchi | Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase[END_REF] target of both adipocytokines, the former acting as an AMPK inhibitor, the latter as an activator. This was also demonstrated by lack of systemic adiponectin infusion effect on hepatic glucose production in liver-specific AMPK 2 KO mice (

).

α [START_REF] Andreelli | Liver adenosine monophosphate-activated kinase-alpha2 catalytic subunit is a key target for the control of hepatic glucose production by adiponectin and leptin but not insulin[END_REF]

AMPK action in skeletal muscle

After a meal or during the euglycaemic hyperinsulinemic clamp, both situations with high circulating levels of insulin, skeletal muscle is the main site for glucose disposal in the body. This is sustained by the insulin-dependent translocation of glucose transporter GLUT4 from intracellular vesicles to the cell surface, which is impaired in T2D patients. ). In contrast, chronic exposure of IL-6 (as observed in obesity) [START_REF] Glund | Interleukin-6 directly increases glucose metabolism in resting human skeletal muscle[END_REF] promotes insulin resistance both and ( ). The dual effect of IL-6 on insulin sensitivity probably in vitro in vivo [START_REF] Nieto-Vazquez | Dual Role of Interleukin-6 in Regulating Insulin Sensitivity in Murine Skeletal Muscle[END_REF] explains some conflicting results recently discussed in more details elsewhere ( ). Importantly, it has been also [START_REF] Nieto-Vazquez | Dual Role of Interleukin-6 in Regulating Insulin Sensitivity in Murine Skeletal Muscle[END_REF] suggested that AICAR, in addition to activating AMPK, suppresses chronic IL-6 release by an AMPK independent mechanism in insulin-resistant models (

). This strongly suggested that AMPK activators can act at a multi-tissular level in order to [START_REF] Glund | Role of adenosine 5 -monophosphate-activated protein kinase in interleukin-6 release from isolated mouse skeletal muscle ′[END_REF] restore metabolic inter-organs cooperation.

Interestingly, available hypoglycaemic drugs as metformin and TZDs have been reported to activate AMPK ( ; Fryer et al. 2002 Zhou ). Even if it was postulated that blood glucose lowering effects of metformin are mediated by AMPK activation from studies of et al. 2001 mice that are deficient in the upstream AMPK kinase, LKB1, in the liver (

) recent studies have shown that LKB1 [START_REF] Shaw | The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin[END_REF] phosphorylates and activates at least 12 AMPK-related kinases. These data raised the question whether the glucose-lowering function of LKB1 is mediated by AMPK-related kinases rather than AMPK itself.

Because circulating levels of adiponectin are decreased in individuals with obesity and insulin resistance, adiponectin replacement in humans may be a promising approach. It has been demonstrated that full-length adiponectin activates AMPK in the liver, while globular adiponectin did so both in muscle and the liver (

). Blocking AMPK activation by the use of a dominant negative [START_REF] Yamauchi | Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase[END_REF] mutant inhibited the action of full length adiponectin on glucose hepatic production (

). In addition, lack of action of [START_REF] Yamauchi | Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase[END_REF] adiponectin on hepatic glucose production when AMPK 2 catalytic subunit is missing, strongly supports the concept that adiponectin α effect is strictly dependent on AMPK (

). Awaiting adiponectin analogs development, alternative ways to restore [START_REF] Andreelli | Liver adenosine monophosphate-activated kinase-alpha2 catalytic subunit is a key target for the control of hepatic glucose production by adiponectin and leptin but not insulin[END_REF] adiponectin effects have been suggested recently. Improved metabolic disorders following TZDs administration are in part mediated through adiponectin-dependent activation of AMPK since activation of AMPK by rosiglitazone treatment is diminished in adiponectin KO

mice (

). TZDs can markedly enhance the expression and secretion of adiponectin and through the [START_REF] Nawrocki | Mice lacking adiponectin show decreased hepatic insulin sensitivity and reduced responsiveness to peroxisome proliferator-activated receptor gamma agonists[END_REF] in vitro in vivo activation of its promoter and also antagonize the suppressive effect of TNF-on the production of adiponectin ( ).

α [START_REF] Maeda | PPARgamma ligands increase expression and plasma concentrations of adiponectin, an adipose-derived protein[END_REF] Interestingly, in human adipose tissue, AICAR has been shown to increase the expression of adiponectin ( ; [START_REF] Lihn | AICAR stimulates adiponectin and inhibits cytokines in adipose tissue[END_REF][START_REF] Sell | Cytokine secretion by human adipocytes is differentially regulated by adiponectin, AICAR, and troglitazone[END_REF] ) while no change in serum adiponectin concentration or adipocyte adiponectin content was found in type 2 diabetic patients treated with metformin (

). [START_REF] Phillips | Modulation of circulating and adipose tissue adiponectin levels by antidiabetic therapy[END_REF]

AMPK action in -cells

β β-cell failure is a strong determinant in the pathogenesis of type 2 diabetes. This defect inexorably aggravates with time as demonstrated in prospective clinical studies (1995). According to the glucolipotoxicity hypothesis ( ) chronic high [START_REF] Prentki | Malonyl-CoA signaling, lipid partitioning, and glucolipotoxicity: role in beta-cell adaptation and failure in the etiology of diabetes[END_REF] glucose dramatically influences -cell metabolism. Indeed, it has been observed in high glucose condition an increase of cytosolic fatty β acyl-CoA partitioning toward potentially toxic cellular products (e.g., diacylglycerol, ceramide and lipid peroxides) leading to impaired insulin secretory response to glucose and ultimately apoptosis (

). Indeed, decrease in -cell mass is likely to play a role [START_REF] Donath | Mechanisms of beta-cell death in type 2 diabetes[END_REF] β in the pathogenesis of human type 2 diabetes ( ) as it does in rodent models of the disease ( ; [START_REF] Butler | Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes[END_REF]Kaiser et al. 2003 Rhodes ). 2005

Pathways regulating -cell turnover are also implicated in -cell insulin secretory function. In consequence, decrease in -cell mass is ). In contrast, the role of AMPK in the control of -cell death survival remains controversial ( ; ; 2002 β Kefas et al. 2003aKefas et al. 2003b ; ;

). [START_REF] Kim | AICAR potentiates ROS production induced by chronic high glucose: roles of AMPK in pancreatic beta-cell apoptosis[END_REF][START_REF] Riboulet-Chavey | Inhibition of AMP-activated protein kinase protects pancreatic beta-cells from cytokine-mediated apoptosis and CD8 T-cell-induced + cytotoxicity[END_REF][START_REF] Richards | Over-expression of AMP-activated protein kinase impairs pancreatic beta -cell function in vivo { }[END_REF] Beyond a potential role of AMPK for long-term regulation of -cell function and survival, AMPK may also regulate acutely insulin glucose-stimulated insulin secretion ( ). This could be considered as a deleterious effect of AMPK activation. But it is [START_REF] Sun | Ablation of AMP-activated protein kinase alpha1 and alpha2 from mouse pancreatic beta cells and RIP2.Cre neurons suppresses insulin release in vivo[END_REF] hypothesized that pharmacological activation of AMPK and its subsequent decrease in insulin secretion, could be appropriate in insulin resistant conditions characterized by high insulin levels. Indeed, it has been suggested that reduction of the pathological hyperinsulinemia is potentially a mechanism to protect -cell mass. Consistent with this assumption, systemic AICAR infusion in prediabetic Zucker fatty β rats prevented the development of hyperglycaemia and preserved -cell mass (

).

β [START_REF] Pold | Long-term AICAR administration and exercise prevents diabetes in ZDF rats[END_REF] Taken together, these data suggest that AMPK is an emergent factor that could protect by different ways -cell function and -cell

β β
mass from the deleterious effects of glucolipotoxicity.

Mimicking the beneficial effects of hypolipidemic agents

Dyslipidemia of both insulin resistance and type 2 diabetes is a recognized risk factor for cardiovascular disease. Diabetic dyslipidemia is a cluster of potentially atherogenic lipid and lipoprotein abnormalities that are metabolically interrelated. Activated AMPK inhibits cholesterol and fatty acid synthesis. Thus, AMPK suppresses expression of lipogenesis-associated genes such as fatty acid synthase, pyruvate kinase and acetyl CoA carboxylase (ACC) ( ; ; ; [START_REF] Foretz | Short-term overexpression of a constitutively active form of AMP-activated protein kinase in the liver leads to mild hypoglycemia and fatty liver[END_REF][START_REF] Foretz | AMP-activated protein kinase inhibits the glucose-activated expression of fatty acid synthase gene in rat hepatocytes[END_REF][START_REF] Leclerc | The 5 -AMP-activated protein kinase inhibits the transcriptional stimulation by glucose in liver cells, acting through the glucose response complex ′[END_REF] ), and 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase ). These data emphasize the critical role for AMPK in the control of hepatic lipid deposition via [START_REF] Andreelli | Liver adenosine monophosphate-activated kinase-alpha2 catalytic subunit is a key target for the control of hepatic glucose production by adiponectin and leptin but not insulin[END_REF] decreased lipogenesis and increased lipid oxidation, thus improving lipid profile in type 2 diabetes.

It is well documented that changes in adipose tissue mass are frequently associated with alterations in insulin sensitivity (Eckel et al. ; ). AMPK evidenced recently as a regulator of fat mass. Indeed, activation of AMPK in white adipocytes is 2005 [START_REF] Katsuki | Increased visceral fat and serum levels of triglyceride are associated with insulin resistance in Japanese metabolically obese, normal weight subjects with normal glucose tolerance[END_REF] concomitant with a decreased expression of genes coding lipogenic enzyme (

) and leads to a decreased lipogenic flux and [START_REF] Orci | Rapid transformation of white adipocytes into fat-oxidizing machines[END_REF] a decreased triglyceride synthesis ( ; ). In white adipocytes, AMPK activation using AICAR or [START_REF] Daval | Anti-lipolytic action of AMP-activated protein kinase in rodent adipocytes[END_REF][START_REF] Sullivan | Inhibition of lipolysis and lipogenesis in isolated rat adipocytes with AICAR, a cell-permeable activator of AMP-activated protein kinase[END_REF] overexpression of AMPK-CA has been shown to inhibit -adrenergic-induced lipolysis ( ; ).

β [START_REF] Corton | 5-aminoimidazole-4-carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells?[END_REF][START_REF] Sullivan | Inhibition of lipolysis and lipogenesis in isolated rat adipocytes with AICAR, a cell-permeable activator of AMP-activated protein kinase[END_REF] Hormone sensitive lipase (HSL), one of the key protein responsible for the lipolytic activity, is activated by PKA phosphorylation at serines 563, 659 and 660 ( ). AMPK reduces this activation through phosphorylation at serine 565 ( Anthonsen et al. 1998 Garton et al. ; ). This effect has been demonstrated both in white adipocytes and in skeletal muscle in both resting and 1989 Garton and Yeaman 1990 contracting conditions ( ; ;

). Thus, inhibition of HSL by AMPK represents a [START_REF] Muoio | AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target[END_REF][START_REF] Smith | AMP kinase activation with AICAR further increases fatty acid oxidation and blunts triacylglycerol hydrolysis in contracting rat soleus muscle[END_REF][START_REF] Watt | Regulation of HSL serine phosphorylation in skeletal muscle and adipose tissue[END_REF] mechanism to limit this recycling and ensure that the rate at which fatty acids are released by lipolysis does not exceed the rate at which they could be disposed of by export or by internal oxidation.

Beyond its hypolipidemic properties, AMPK system can be also a regulator of ectopic lipids metabolism. Depot of lipids in tissue is a hallmark defect in metabolic syndrome in humans. According to this lipotoxicity hypothesis, insulin resistance develops when excess lipids are deposited in insulin-sensitive cell types. The balance between lipids oxidation and lipids storage in cells is mainly regulated by malonyl-CoA, generated by ACC. Malonyl-CoA is known to inhibit transport of fatty acids into mitochondria via allosteric regulation of carnitine palmitoyltransferase-1, thereby preventing them from being metabolized. Activated AMPK inhibits malonyl-CoA synthesis and shifts the balance towards mitochondrial fatty acid oxidation and away from fat storage. Several studies have shown that activation of AMPK with AICAR, -lipoic acid, leptin, adiponectin and IL-6 enhances muscle fatty acid oxidation ( ; [START_REF] Carey | Interleukin-6 increases insulin-stimulated glucose disposal in humans and glucose uptake and fatty acid oxidation in vitro via AMP-activated protein kinase[END_REF]Lee et al. 2005b ; ; ;

α β
). Chronic leptin treatment increases skeletal muscle fatty acid [START_REF] Merrill | AICA riboside increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle[END_REF][START_REF] Minokoshi | Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase[END_REF][START_REF] Yamauchi | Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase[END_REF] oxidation in an AMPK-dependent manner by increasing AMP/ATP ratio in oxidative muscle fibers and by increasing AMPK 2 nuclear ). Interestingly, recent data have shown that resistin lowers [START_REF] Barnes | The 5 -AMP-activated protein kinase gamma3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle ′[END_REF] AMPK signalling in muscle cells and that this reduction is associated with suppressed fatty acid oxidation (

).

Palanivel and Sweeney 2005

Non-alcoholic fatty liver disease is a serious consequence of obesity increasing the risk of liver cancer or cirrhosis. The origin of this disease is unknown and probably multifactorial. Nevertheless, because insulin resistance is recognized as an associate and/or promoting mediator of the disease, management of insulin resistance becomes an important challenge. For this specific point and because AMPK is a key factor in lipids partitioning (balance between synthesis and oxidation), management of non-alcoholic fatty liver disease by activators of AMPK represents a new therapeutic strategy. Adiponectin treatment restores insulin sensitivity and decreases hepatic steatosis of obese mice (

). This effect is linked to an activation of AMPK in the liver that decreases fatty acid biosynthesis and increases [START_REF] Xu | The fat-derived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver diseases in mice[END_REF] mitochondrial fatty acid oxidation (

). Reduction of liver steatosis when AMPK is activated has been also confirmed [START_REF] Yamauchi | The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity[END_REF] by a decrease in liver triglyceride content in lean and obese rodents during AICAR infusion ( ; ) and Bergeron et al. 2001a[START_REF] Cool | Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome[END_REF] after treatment with small-molecule AMPK activators (

). The synthesis of triglycerides is regulated by both the supply of [START_REF] Cool | Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome[END_REF] glycerol-3-phosphate (from carbohydrate metabolism) and of fatty acyl-coenzyme A. The first step of triglycerides synthesis is catalyzed by glycerol-3-phosphate acyl-transferase (GPAT). AICAR or exercise induced AMPK activation reduces hepatic GPAT activity and triglycerides esterification ( ; ). Fasting, that increases hepatic AMPK inhibits GPAT activity [START_REF] Muoio | AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target[END_REF]Park et al. 2002 Witters et ). In the same way, AMPK activation by resveratrol protects against lipid accumulation in the liver of diabetic mice ( al. 1994 Zang et al.

) in association with increased mitochondrial number ( ) and SIRT1-dependent deacetylation of peroxisome 2006 [START_REF] Baur | Resveratrol improves health and survival of mice on a high-calorie diet[END_REF] proliferator-activated receptor coactivator (PGC)-1 , a master regulator of mitochondrial biogenesis ( ; α Baur et al. 2006 Rodgers and ). The efficacy of metformin as a treatment for fatty liver disease has been confirmed in obese, mice, which develop Puigserver 2007 ob/ob hyperinsulinemia, insulin resistance and fatty livers ( ). [START_REF] Lin | Metformin reverses fatty liver disease in obese, leptin-deficient mice[END_REF] The discovery of new strategies of management of hepatic steatosis in humans is of considerable interest. AMPK activation could be one of them as suggested by recent clinical studies in type 2 diabetic patients. Indeed, it has been demonstrated that AICAR infusion results in significant decline in circulating plasma non-esterified fatty acids (NEFA) levels, suggesting stimulation of hepatic fatty acid oxidation and/or a reduction in whole body lipolytic rate (

). Management of hepatic steatosis by targeting AMPK is also [START_REF] Boon | Intravenous AICAR administration reduces hepatic glucose output and inhibits whole body lipolysis in type 2 diabetic patients[END_REF] suggested by recent successes in treating this disorder with diet, exercise, and TZDs all known as AMPK activators ( ; [START_REF] Carey | Effect of rosiglitazone on insulin sensitivity and body composition in type 2 diabetic patients corrected[END_REF] ). Other studies are needed to analyze the beneficial effect of AMPK activation for the Neuschwander-Tetri and Caldwell 2003 management of fatty liver diseases in humans.

Mimicking the beneficial effects of an anti-obesity drug

Weight reduction is best achieved by behavioural change to reduce energy intake and by increasing physical activity to enhance energy expenditure. Therefore, the AMPK system may be an important pharmacological target to reduce fatty acid storage in adipocytes and to treat obesity. By inducing fatty acid oxidation within the adipocyte, activation of AMPK would reduce fat cell size and also prevent fatty acids from being exported to peripheral tissues and cause deleterious effects. Direct evidence linking AMPK activation to diminished adiposity was first obtained by chronic administration of AICAR to lean and obese rats, an effect attributable, at least in part, to an increase in energy expenditure ( ; ). Furthermore, the anti-obesity hormone leptin increases fatty-acid [START_REF] Buhl | Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying features of the insulin resistance syndrome[END_REF][START_REF] Winder | Activation of AMP-activated protein kinase increases mitochondrial enzymes in skeletal muscle[END_REF] oxidation in skeletal muscle by activating AMPK (this process involves an increase in the AMP/ATP ratio) ( ), [START_REF] Minokoshi | Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase[END_REF] depletes body fat stores by activating AMPK activity and by increasing uncoupling mitochondrial protein (UCP)-1 and UCP-2 expression ( ). 3-adrenoceptor ( 3-AR) agonists were also found to have remarkable anti-obesity and anti-diabetic effects in rodents [START_REF] Orci | Rapid transformation of white adipocytes into fat-oxidizing machines[END_REF] β β and these compounds were found to stimulate AMPK in fat cells (

). In addition, overexpression of UCP-1 in [START_REF] Moule | The activation of p38 MAPK by the beta-adrenergic agonist isoproterenol in rat epididymal fat cells[END_REF] adipocytes leads to an increase in the AMP/ATP ratio and activation of AMPK, inactivation of ACC and a decreased lipogenesis (

). Additionally, a strong mitochondrial biogenesis in response to increased UCP-1 expression in adipocytes has been Matejkova et al. 2004 demonstrated ( ;), features that could enhance the fatty acid oxidation capacity of adipocytes in [START_REF] Orci | Rapid transformation of white adipocytes into fat-oxidizing machines[END_REF][START_REF] Rossmeisl | Expression of the uncoupling protein 1 from the aP2 gene promoter stimulates mitochondrial biogenesis in unilocular adipocytes in vivo[END_REF] response to AMPK activation. During chronic AICAR treatment, activated AMPK increases UCP-3 expression in muscle independently of changes in mitochondrial biogenesis ( ; ). This effect can also explain changes in energy expenditure [START_REF] Stoppani | AMP-activated protein kinase activates transcription of the UCP3 and HKII genes in rat skeletal muscle[END_REF][START_REF] Zhou | UCP-3 expression in skeletal muscle: effects of exercise, hypoxia, and AMP-activated protein kinase[END_REF] during AMPK activation.

Benefits of targeting AMPK pathway for metabolic complications AMPK and ischemic heart

Type 2 diabetes is recognized as an important risk factor for cardiovascular diseases and mortality. In ischemic heart, balance between glucose and lipids is altered. In this situation, activation of AMPK is considered as a metabolic adaptation to rescue energy supply. Indeed, AMPK stimulates glycolysis and sustains energy supply during ischemic stress. Convincing evidence suggests that the more AMPK is activated in ischemic myocardial tissue, the more the size of infarcted tissue is reduced. Because the size of myocardial infarcted tissue is one of the variables that determine the risk of sudden death and the risk of cardiac insufficiency in humans, reduction of the volume of ischemic tissue is an important therapeutic challenge. Thus, promotion of glucose oxidation or inhibition of fatty acid oxidation in ischemic/reperfused hearts could be a promising novel therapeutic approach during myocardial ischemia. Such a mechanism has been demonstrated during the phenomenon called ischemic preconditioning. This phenomenon (consisting in repeated brief episodes of myocardial ischemia) (

) induces endogenous protective mechanisms in the heart which becomes more resistant to [START_REF] Murry | Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium[END_REF] subsequent ischemic episodes. The molecular mechanism of this protective effect is based on AMPK activation in a PKC-dependent manner and promotion of glucose utilization in myocardial cells (

). Attractively, adiponectin protects the heart from [START_REF] Nishino | Ischemic preconditioning activates AMPK in a PKC-dependent manner and induces GLUT4 up-regulation in the late phase of cardioprotection[END_REF] ischemia by activating AMPK and increasing the energy supply to heart cells ( ). For example, high blood levels of [START_REF] Shibata | Adiponectin protects against myocardial ischemia-reperfusion injury through AMPK-and COX-2-dependent mechanisms[END_REF] adiponectin are associated with a lower risk of heart attack, and vice versa (

). Additionally, adiponectin levels rapidly [START_REF] Pischon | Plasma adiponectin levels and risk of myocardial infarction in men[END_REF] decline after the onset of acute myocardial infarction. Similarly, in mice, deletion of adiponectin induces increased heart damage after reperfusion that was associated with diminished AMPK signalling in the myocardium (

). In addition, it has also been [START_REF] Shibata | Adiponectin protects against myocardial ischemia-reperfusion injury through AMPK-and COX-2-dependent mechanisms[END_REF] reported that adiponectin attenuated cardiac hypertrophy through activation of AMPK signalling pathway ( ; Liao et al. 2005 Shibata et al. ). These findings clearly show that adiponectin has a cardioprotective role during ischemia through AMPK-dependent 2004a in vivo mechanisms.

Since AMPK regulates the balance between glucose and fatty acid metabolism at the cellular level, the metabolic response of the heart to global ischemia was studied in AMPK 2 / mice. These hearts displayed a more rapid onset of ischemic contracture, which was α -associated with a decrease in ATP content, in lactate production, in glycogen content and in the phosphorylation state of ACC (

). Importance of metabolic adaptation via AMPK activation during ischemia was also documented in another [START_REF] Zarrinpashneh | Role of the alpha2 isoform of AMP-activated protein kinase in the metabolic response of the heart to no-flow ischemia[END_REF] transgenic mouse model overexpressing a dominant negative form of AMPK 2 in the heart (

). These studies indicate α et al. 2004 that the 2 isoform of AMPK is required for the metabolic response of the heart to ischemia suggesting that AMPK is cardioprotective.

Russell

α Thus, AMPK activators could be of particular interest for the management of myocardial ischemia. Nevertheless, inappropriate activation of AMPK can have deleterious consequences in the heart. Indeed, in humans, a variety of mutations in the 2 subunit ( ) have been γ

Figure 1 shown to produce a glycogen storage cardiomyopathy characterized by ventricular pre-excitation, conduction defects and cardiac hypertrophy ( ). This argues for a restrictive use of AMPK activators during the acute phase of heart ischemia [START_REF] Dyck | AMPK alterations in cardiac physiology and pathology: enemy or ally?[END_REF] and not for a chronic activation of cardiac AMPK. Thus, the balance between benefits and deleterious cardiac effects of AMPK activation has to be studied in more details.

AMPK and endothelial dysfunction

Endothelial cell dysfunction, as manifested by impaired vascular relaxation or an increase in circulating vascular cell adhesion molecules, is present in patients with type 2 diabetes, and it is thought to be one component of the inflammatory process that initiates atherogenesis (

). Based on studies using genetically modified mice, the production of NO via eNOS is crucial in the Van [START_REF] Van Gaal | Mechanisms linking obesity with cardiovascular disease[END_REF] regulation of vascular tone ( ; ). The activity of eNOS is largely determined by posttranslational [START_REF] Lau | nNOS and eNOS modulate cGMP formation and vascular response in contracting fast-twitch skeletal muscle[END_REF][START_REF] Maxwell | Limb blood flow during exercise is dependent on nitric oxide[END_REF] modifications such as multisite phosphorylation and protein interactions. Interestingly, AMPK enhances eNOS activity by direct phosphorylation of Ser1177 ( ; ), Ser633 ( ) and by promoting its association with heat [START_REF] Chen | AMPK signaling in contracting human skeletal muscle: acetyl-CoA carboxylase and NO synthase phosphorylation[END_REF][START_REF] Chen | AMP-activated protein kinase phosphorylation of endothelial NO synthase[END_REF][START_REF] Chen | AMP-activated protein kinase functionally phosphorylates endothelial nitric oxide synthase Ser633[END_REF] shock protein 90 ( ) leading to endothelial NO production. In this respect, metformin has been proposed to improve [START_REF] Davis | Activation of the AMP-activated kinase by antidiabetes drug metformin stimulates nitric oxide synthesis in vivo by promoting the association of heat shock protein 90 and endothelial nitric oxide synthase[END_REF] endothelium function in diabetes by favouring phosphorylation of eNOS by AMPK activation (

). Metformin was also [START_REF] Davis | Activation of the AMP-activated kinase by antidiabetes drug metformin stimulates nitric oxide synthesis in vivo by promoting the association of heat shock protein 90 and endothelial nitric oxide synthase[END_REF] shown to relax endothelium-denuded rat aortic rings pre-contracted with phenylephrine, showing that AMPK can induce vasorelaxation in an endothelium-and NOS-independent manner ( ). Accordingly, AMPK activation in response to hypoxia [START_REF] Majithiya | Metformin reduces blood pressure and restores endothelial function in aorta of streptozotocin-induced diabetic rats[END_REF] or metabolic challenge can induce vasorelaxation of big vessels ( ; ), thereby favouring blood flow. [START_REF] Evans | Does AMP-activated protein kinase couple inhibition of mitochondrial oxidative phosphorylation by hypoxia to calcium signaling in O2-sensing cells?[END_REF][START_REF] Rubin | Metabolic activation of AMP kinase in vascular smooth muscle[END_REF] Interestingly, AMPK-dependent adiponectin vascular effects have been demonstrated for angiogenic repair in an ischemic hind limbs model (

). Similarly, -lipoic acid improves vascular dysfunction by normalizing triglyceride and lipid peroxide levels Shibata et al. 2004b α and NO synthesis in endothelial cells from obese rat by activating AMPK (

). Attractively, adiponectin exhibits potent Lee et al. 2005a anti-atherosclerotic effects and suppresses endothelial cell proliferation via AMPK activation ( ; ). [START_REF] Kubota | Disruption of adiponectin causes insulin resistance and neointimal formation[END_REF][START_REF] Yamauchi | Globular adiponectin protected ob/ob mice from diabetes and ApoE-deficient mice from atherosclerosis[END_REF] Beyond the vascular effects of AMPK activation, it has been recently demonstrated that AMPK can regulate blood pressure. Thus, long-term administration of AICAR reduces systolic blood pressure in an insulin-resistant animal model ( ). In this [START_REF] Buhl | Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying features of the insulin resistance syndrome[END_REF] process, a potential role for AMPK could be the regulation of ion channels or sodium cotransporters including ENaC and the Na-K-2Cl cotransporter ( ; ). These data provide additional support to the hypothesis that AMPK activation [START_REF] Carattino | Epithelial sodium channel inhibition by AMP-activated protein kinase in oocytes and polarized renal epithelial cells[END_REF][START_REF] Fraser | The Anti-diabetic drugs rosiglitazone and metformin stimulate AMP-activated protein kinase through distinct signaling pathways[END_REF] might be a potential future pharmacological strategy for treating the cardiovascular risk factors linked to the metabolic syndrome.

Conclusion

Lifestyle modifications are recognized as an important preventive and therapeutic intervention for impaired glucose tolerance, insulin resistance and type 2 diabetic patients. AMPK activators are potential new therapeutic agents for the treatment of type 2 diabetes by mimicking the beneficial effects of physical activity and of calorie restriction. Accordingly, AMPK-activating agents could also be used as regulators of hyperglycaemia, obesity, lipids disorders, lipotoxicity and cardiovascular risk by targeting specific cellular pathways (Figure ). Resveratrol, metformin, TZDs, adiponectin and leptin are now considered as AMPK activators. However, many other effects of 3 AMPK activation should be carefully evaluated and many questions are not resolved: are new AMPK activators tissue specific? What are the consequences of a long term pharmacological AMPK activation? Additional studies are required to address these critical points.

Acknowledgements:

This work was supported by the European Commission integrated project (LSHM-CT-2004-005272/exgenesis), Agence Nationale de la Recherche (ANR-06-PHYSIO-026), Association Fran aise contre les Myopathies (AFM), Association pour l Etude des Diab tes et des The major upstream kinase is a complex between the tumour suppressor kinase LKB1 and two accessory subunits, STRAD and MO25 which appears to be constitutively active. The CaMKK could also phosphorylate Thr-172 and activate AMPK following a rise in cytosolic Ca . A

β 2 +
third potential upstream kinase is TAK1 but its physiological significance is uncertain. Thr-172 phosphorylation is removed by PP2C phosphatase. Physiological, hormonal and pharmacological stimulatory effectors of AMPK complex are listed.

  increases muscle expression of glucose transporter GLUT4 and hexokinase II, an effectBergeron et al. 2001b partly mediated by the transcriptional coactivator peroxisome proliferator-activated receptor-coactivator-1 (PGC-1 ) (

]

  [START_REF] Holmes | AMP kinase is not required for the GLUT4 response to exercise and denervation in skeletal muscle[END_REF][START_REF] Jorgensen | Effects of alpha-AMPK knockout on exercise-induced gene activation in mouse skeletal muscle[END_REF][START_REF] Zong | AMP kinase is required for mitochondrial biogenesis in skeletal muscle in response to chronic energy deprivation[END_REF]

  impairment in muscle blood flow ( ). This finding is supported by the close association between AMPK and nNOS Lee-Young et al. 2009 μ phosphorylation following moderate-intensity exercise ( ; ) and reduced expression of nNOS in Chen et al. 2000 Stephens et al. 2002 μ mAMPK-KD mice (

α

  AMPK activation causes TORC2 phosphorylation and sequesters the coactivator in the cytoplasm, thus blunting the expression of the gluconeogenic program (

α

  [START_REF] Minokoshi | Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase[END_REF] adipokine, has also been shown to increase glucose transport in both lean and obese skeletal muscle, although the effect was less significant in obese skeletal muscle (). It has also recently been recognized that IL-6 (also called myokine( Bruce et al. 2005 " " Febbraio and )) is released acutely from the skeletal muscle during prolonged exercise, activates AMPK and improves peripheral glucose Pedersen 2005 uptake and insulin sensitivity at the whole body level (

  an intrinsic secretory defect. Because AMPK is important for the balance of intracellular energy homeostasis it was interesting to analyze to what extend AMPK regulates -cell function/survival. AICAR dose dependently improves -cell function β βprobably by reducing apoptosis induced by prolonged hyperglycaemia ( ). In addition to AICAR, -cell AMPK[START_REF] Nyblom | AMP-activated protein kinase agonist dose dependently improves function and reduces apoptosis in glucotoxic beta-cells without changing triglyceride levels[END_REF] β activation (by metformin, TZDs or adenovirus-mediated over-expression of AMPK 1-CA) favours fatty acid -oxidation and prevents et al.2003[START_REF] Eto | Genetic manipulations of fatty acid metabolism in beta-cells are associated with dysregulated insulin secretion[END_REF] Higa et al. 1999 Lupi et al.

ββ

  secretion. Thus, AMPK activity is rapidly decreased when glucose levels increased over the physiological range suggesting that AMPK could be one of the regulator of insulin secretion through its capacity to sense intracellular energy ( ; da Silva Xavier et al. 2003 Leclerc et ). Interestingly, activation of AMPK by AICAR, berberine, metformin and TZDs or by overexpression of AMPK 1-CA markedly al. 2004 α reduced glucose-stimulated insulin secretion in -cell lines and in rodent and human islets ( ; ; Eto et al. 2002 Leclerc et al. 2004 Wang et ; ). Similarly, activation of AMPK selectively in -cells in AMPK 1-CA transgenic mice decreased al. 2007 Zhou et al. 2008 β α

α

  translocation and PPAR transcription ( ). Studies in transgenic animals support these observations since expression of α Suzuki et al. 2007 the activating AMPK 3 R225Q mutation in muscle increased fatty acid oxidation and protected against excessive triglyceride γ accumulation and insulin resistance in skeletal muscle (

Figure 1

 1 Figure 1 Domain organization of the catalytic and regulatory and subunits of AMPK α β γ Each AMPK molecule is comprised of a catalytic ( 1 and 2) and regulatory ( 1 and 2) and ( 1, 2 and 3) subunits. The catalytic α α α β β β γ γ γ γ α subunit is phosphorylated at threonine 172 by upstream kinases (LKB1, CaMKK and TAK1), leading to enzyme activation. The subunit β β contains a glycogen-binding domain. The subunit contains 4 nucleotide-binding modules (CBS domains) capable of cooperative binding to γ 2 molecules of either ATP or AMP. Mutations in the human 2 subunit gene (PRKAG2) causing cardiac hypertrophy associated with γ abnormal glycogen accumulation and conduction system disease are shown.

Figure 2

 2 Figure 2Regulation of AMPK by upstream kinases and phosphatases The major upstream kinase is a complex between the tumour suppressor kinase LKB1 and two accessory subunits, STRAD and MO25 which

Figure 3

 3 Figure 3 AMPK, a potential therapeutic target in metabolic disease AMPK pathway has become the focus of a great deal of attention as a novel therapeutic target in metabolic disease because it has been demonstrated that physiological and pharmacological activation of AMPK results in remodelling different metabolic pathways. AMPK has several important metabolic effects, mimicking the beneficial effects of exercise, including modulation of lipid metabolism, enhanced muscle glucose uptake, increased mitochondrial biogenesis, improvement in insulin sensitivity and reduction in blood glucose. Activation of AMPK by pharmacological agents presents a unique challenge to prevent and treat the metabolic abnormalities associated with the metabolic syndrome.

  Benoit Viollet 1 * , Fabrizio Andreelli 1 2

	1	Institut Cochin	INSERM : U1016 , CNRS : UMR8104 , Universit Paris V -Paris Descartes é	, Paris,FR
	2	Service d endocrinologie diab tologie

  Bergeron et al. 2001a constitutively active form of the 2 catalytic subunit (AMPK 2-CA) leads to mild hypoglycaemia in normal mice ( ;

		α	α		Foretz et al. 2005
	Viana et al. 2006	) and abolishes hyperglycaemia in diabetic	ob/ob	and streptozotocin-induced diabetic mice (	; Foretz et al. 2005 Viana et
	) by inhibition of gluconeogenesis (	;	;

  As described above, it has been clearly demonstrated that muscular AMPK activation, either by exercise or by AICAR, stimulates muscle glucose uptake. Interestingly, even if AMPK and insulin acts through phosphorylation of downstream target of Akt (Akt substrate of 160kDa, AS160) (), AMPK-dependent and[START_REF] Dreyer | Resistance exercise increases human skeletal muscle AS160/TBC1D4 phosphorylation in association with enhanced leg glucose uptake during post-exercise recovery[END_REF] insulin-dependent GLUT4 translocation are distinct pathways (). Additionally, exercise-induced muscularAMPK Treebak et al. 2007 activation and AS160 phosphorylation are both reduced in obese non-diabetic and obese type 2 diabetes subjects ( ) but[START_REF] Musi | AMP-activated protein kinase (AMPK) is activated in muscle of subjects with type 2 diabetes during exercise[END_REF] maintained in lean type 2 diabetes patients () suggesting that dysregulation of muscular AMPK is more dependent ofBruce et al. 2005 obesity than of hyperglycaemia. Discovery of muscular AMPK activators in order to mimic regular physical activity metabolic effects is an important challenge. It was first demonstrated that some adipokines stimulate glucose transport in skeletal muscle in an AMPK-dependent manner. Indeed, leptin is known to stimulate glucose uptake in peripheral tissue (

			; Kamohara et al. 1997 Minokoshi et
	al. 1999	) by stimulating AMPK 2 phosphorylation and activation in skeletal muscle (	). Adiponectin, another

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