Mara Gallí

Frédéric Van Gool

Oberdan Leo
email: oleo@ulb.ac.

Sirtuins and inflammation: friends or foes?

Abstract

Lysine acetylation/deacetylation has been recognized as an important posttranslational modification regulating numerous cellular processes. Sirtuins represent novel players in these complex regulatory circuits. These NAD-dependent lysine-deacetylases have attracted much interest based on their role in the regulation of lifespan in lower organisms, and their capacity to interfere with cell growth, proliferation and survival in response to stress. Their absolute requirement for NAD suggests that these enzymes may represent an important molecular link between metabolism and several human disorders such as diabetes and cancer. More recently, the identification of several transcription factors known to play a role in the immune system as sirtuin substrates has suggested that this family of enzymes may also play an important role in the regulation of inflammation, a pathological situation with clear links to metabolism and ageing in humans. We review herein the possible links between nuclear sirtuins and the regulation of an immune response, and discuss the possible strategies that may lead to the development of novel therapeutic approaches to treat inflammation by targeting sirtuin activity.

Introduction

The activity, stability, and intracellular location of virtually all proteins are regulated by posttranslational modifications (PTMs). This very diverse set of modifications include phosphorylations, acetylations, sumoylations, ubiquitinations, ADP-ribosylations, nitrations and many others [START_REF] Deribe | Post-translational modifications in signal integration[END_REF]. PTMs are highly dynamic processes and most of them result from the activity of antagonizing enzymes (e.g. kinases vs. phosphatases, acetylases vs. deacetylases, etc). Numerous studies have highlighted the important role of PTMs in signaling pathways, allowing a crosstalk between the cell and its environment and endowing the cell with a certain flexibility towards change [START_REF] Deribe | Post-translational modifications in signal integration[END_REF]. In this context, nicotinamide adenine dinucleotide (NAD) has gained a renewed interest as an important substrate for a series of enzymes catalyzing a set of post-translational modifications, such as deacetylation or ADP-ribosylation. In contrast to its role in energy metabolism, the involvement of NAD in these regulatory processes is based on its ability to act as an ADP-ribose donor, thus requiring NAD resynthesis to avoid depletion of the intracellular NAD pool [START_REF] Grahnert | NAD+: a modulator of immune functions[END_REF]. The central role of NAD in both energy metabolism and protein modification has been shown to have important physiological consequences, in particular in the control of cell metabolism, cell death and longevity [START_REF] Imai | Nicotinamide phosphoribosyltransferase (Nampt): a link between NAD biology, metabolism, and diseases[END_REF].

NAD has been linked to lifespan extension through the activity of a family of deacetylases whose activity is strictly dependent on its availability [START_REF] Imai | Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase[END_REF]. NAD-dependent deacetylases, or sirtuins, have indeed been shown to promote longevity in several model organisms [START_REF] Lin | Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae[END_REF], [START_REF] Tissenbaum | Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans[END_REF] and in mammalian cell cultures [START_REF] Ho | SIRT1 markedly extends replicative lifespan if the NAD+ salvage pathway is enhanced[END_REF], bringing NAD to the forefront of the pharmaceutical struggle against aging [START_REF] Imai | A possibility of nutriceuticals as an anti-aging intervention: activation of sirtuins by promoting mammalian NAD biosynthesis[END_REF]. This family comprises seven members (SIRT1 to SIRT7) that localize to the nucleus (SIRT1,6 and 7), the cytoplasm (SIRT2) and the mitochondria (SIRT3,4 and5) even though some of them can shuttle between these compartments. To date, SIRT4 is the only member that has been shown to lack a deacetylase activity, catalyzing instead an ADP-A c c e p t e d M a n u s c r i p t 4 ribosylation reaction [START_REF] Haigis | SIRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in pancreatic beta cells[END_REF]. The deacetylase activity of the other members of the family consists in the removal of the lysine-linked acetyl group of a target protein [START_REF] Sauve | The biochemistry of sirtuins[END_REF] (Figure 1). During sirtuin-mediated deacetylation, an NAD molecule is hydrolyzed, releasing a metabolite resulting from the condensation of the acetyl group with the ADP-ribose moiety of NAD (OAADPR) [START_REF] Sauve | The biochemistry of sirtuins[END_REF]. The other moiety of NAD, nicotinamide, is also released and acts as an endproduct inhibitor of the deacetylation reaction [START_REF] Sauve | The biochemistry of sirtuins[END_REF]. Although sirtuins share many substrates with classical histone deacetylases (HDACs), their unique NAD-dependency links the activity of these enzymes to the intracellular concentration of available NAD, leading several authors to consider sirtuins as "metabolic sensors" [START_REF] Yu | Protein deacetylation by SIRT1: an emerging key post-translational modification in metabolic regulation[END_REF]. Accordingly, sirtuins have been shown to regulate the cellular response to nutrient availability, intracellular glucose metabolism and cell survival in response to several forms of stress [START_REF] Yu | Protein deacetylation by SIRT1: an emerging key post-translational modification in metabolic regulation[END_REF], [START_REF] Bao | Protein deacetylation by sirtuins: delineating a post-translational regulatory program responsive to nutrient and redox stressors[END_REF]. The discovery that SIRT1 activity can regulate the circadian machinery [START_REF] Nakahata | The NAD+dependent deacetylase SIRT1 modulates CLOCK-mediated chromatin remodeling and circadian control[END_REF] that potently impacts metabolism, further argues for this sirtuin's pivotal role in cellular metabolism, as discussed in several recently published reviews [START_REF] Yu | Protein deacetylation by SIRT1: an emerging key post-translational modification in metabolic regulation[END_REF], [START_REF] Bao | Protein deacetylation by sirtuins: delineating a post-translational regulatory program responsive to nutrient and redox stressors[END_REF].

(Insert Figure 1)

An additional and potentially important role for sirtuins in regulating an immune response has been recently uncovered. Several experimental observations had previously established a potential link between NAD metabolism and inflammation. In particular, expression of Nampt, the enzyme catalyzing the first and limiting reaction allowing NAD biosysnthesis from nicotinamide, has been found overexpressed in several cell lines and tissues during an inflammatory response [START_REF] Luk | Pre-B cell colony-enhancing factor (PBEF)/visfatin: a novel mediator of innate immunity[END_REF]. Although high rates of NAD biosynthesis may represent a biological response to meet an increased metabolic rate, this observation is also compatible with an important role for NAD-dependent enzymes in controlling immune reactions. A series of recent observations concur with this hypothesis, and clearly demonstrate a direct role for

SIRT1

SIRT1 negatively regulates NFkB activity.

The NFkB transcription factor plays a pivotal role in immune function [START_REF] Hayden | Shared principles in NF-kappaB signaling[END_REF]. The observation that several lysines of NFkB p65 subunit can be acetylated [START_REF] Hayden | Shared principles in NF-kappaB signaling[END_REF] has highlighted the potential regulatory role of lysine acetylation on NFkB function. Among these, K310 acetylation confers superior transcriptional activity, while representing a substrate for SIRT1 [START_REF] Yeung | Modulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase[END_REF]. SIRT1 acts therefore as a negative regulator of NFkB activity through the deacetylation of the p65 lysine 310. This NFkB-SIRT1 negative loop has been described in several experimental models, confirming its biological relevance. Kwon and coworkers have recently confirmed and expanded these results, by showing that this pathway is targeted and exploited by the HIV virus. The authors wished to understand the mechanism by which the HIV transcriptional activator Tat can positively regulate NFkB activity and induce IL-2 expression in infected lymphocytes [START_REF] Kwon | Human immunodeficiency virus type 1 Tat protein inhibits the SIRT1 deacetylase and induces T cell hyperactivation[END_REF]. In their work, they elegantly demonstrate that Tat inhibits SIRT1 enzymatic activity by binding to its acetyl-lysine-binding domain. Functional SIRT1 A c c e p t e d M a n u s c r i p t 6 inhibition leads to NFkB hyperacetylation, which in turn causes an increase in IL-2 production and T cell activation, a condition that renders T cells permissive to the virus. This SIRT1-NFkB pathway has subsequently been shown to be active in several tissues and cell types, in which a reduction in SIRT1 protein levels and/or activity is associated with an NFkB-dependent sustained inflammatory response. A decrease in SIRT1 activity has for example been reported in the lungs of smokers and of patients suffering from chronic obstructive pulmonary disease (COPD) [START_REF] Nakamaru | A protein deacetylase SIRT1 is a negative regulator of metalloproteinase-9[END_REF], correlating with increased inflammatory gene expression and p65 K310 acetylation. An oxidative modification of the SIRT1 protein is thought to lead to increased SIRT1 decay in cells exposed to cigarette smoke [START_REF] Rajendrasozhan | SIRT1, an antiinflammatory and antiaging protein, is decreased in lungs of patients with chronic obstructive pulmonary disease[END_REF], possibly explaining the failure to restore adequate SIRT1 activity in this model by increasing the intracellular NAD pool. [START_REF] Caito | PARP-1 inhibition does not restore oxidant-mediated reduction in SIRT1 activity[END_REF]. In organs such as the liver or in adipose tissue that play a major role in metabolic homeostasis, SIRT1 activity can be affected by diet. In most studies, high fat diet (HFD), in which approximately 40 to 60% calories are provided from fat, is accompanied by a decrease in SIRT1 activity [START_REF] Yang | Macrophage alpha1 AMP-activated protein kinase (alpha1AMPK) antagonizes fatty acid-induced inflammation through SIRT1[END_REF], [START_REF] Yoshizaki | SIRT1 exerts anti-inflammatory effects and improves insulin sensitivity in adipocytes[END_REF], while caloric restriction (CR), that consists in a moderate caloric limitation (to approximately 60% of a standard regimen), tends to activate SIRT1 [START_REF] Guarente | Calorie restriction--the SIR2 connection[END_REF]. Thus SIRT1 +/-mice develop hepatic steatosis under a HFD, with a steatosisassociated increase in pro-inflammatory cytokine production and macrophage infiltration [START_REF] Xu | Lack of SIRT1 (Mammalian Sirtuin 1) activity leads to liver steatosis in the SIRT1+/-mice: a role of lipid mobilization and inflammation[END_REF]. The same inflammatory activation is evident in SIRT1-depleted adipocytes, together with an increase in insulin resistance [START_REF] Yoshizaki | SIRT1 exerts anti-inflammatory effects and improves insulin sensitivity in adipocytes[END_REF]. The putative role for NFkB in this model is supported by the observation of an NFkB hyperacetylation status [START_REF] Yoshizaki | SIRT1 exerts anti-inflammatory effects and improves insulin sensitivity in adipocytes[END_REF] and by the finding that NFkB knockdown reverses these metabolic changes [START_REF] Yoshizaki | SIRT1 exerts anti-inflammatory effects and improves insulin sensitivity in adipocytes[END_REF]. Importantly, free fatty acids have been shown to lead to a similar reduction in SIRT1 activity, by downregulating AMPactivated protein kinase (AMPK) [START_REF] Yang | Macrophage alpha1 AMP-activated protein kinase (alpha1AMPK) antagonizes fatty acid-induced inflammation through SIRT1[END_REF]. This study, conducted by Yang and coworkers, shows that, like SIRT1, AMPK is a metabolic sensor that can reduce NFkB activation and that the anti-inflammatory properties of AMPK are strictly dependent on its ability to induce SIRT1-A c c e p t e d M a n u s c r i p t dependent p65 deacetylation [START_REF] Yang | Macrophage alpha1 AMP-activated protein kinase (alpha1AMPK) antagonizes fatty acid-induced inflammation through SIRT1[END_REF], further supporting the physiological relevance of an SIRT1-NFkB anti-inflammatory axis. Interestingly a recent report proposes that the inflammatory character of fatty acids depends on their nature: n-6 unsaturated fatty acids being pro-inflammatory while the n-3 subset has anti-inflammatory properties possibly linked to their ability to induce SIRT1 expression [START_REF] Rahman | The fat-1 transgene in mice increases antioxidant potential, reduces pro-inflammatory cytokine levels, and enhances PPARgamma and SIRT-1 expression on a calorie restricted diet[END_REF]. Besides from its dietary origin, hepatic steatosis can also be caused by excessive alcoholic absorption. Ethanol-derived metabolites have also been shown to decrease SIRT1 levels in macrophages [START_REF] Shen | Role of SIRT1 in regulation of LPS-or two ethanol metabolites-induced TNF-alpha production in cultured macrophage cell lines[END_REF]. NFkB hyperacetylation originates from this SIRT1 depletion and causes a pro-inflammatory cytokine release [START_REF] Shen | Role of SIRT1 in regulation of LPS-or two ethanol metabolites-induced TNF-alpha production in cultured macrophage cell lines[END_REF] that can reduce insulin sensitivity in co-cultured adipocytes [START_REF] Yoshizaki | SIRT1 inhibits inflammatory pathways in macrophages and modulates insulin sensitivity[END_REF]. It must be noted that inflammatory cytokines can be released by macrophages but also by non-immune cells such as hepatocytes and adipocytes. This raises the question of whether the inflammatory state observed in the liver and in the adipose tissue is of immune or hepatic/adipose origin. A similar question has been elegantly addressed by Li's team, who focused on SIRT1's role in maintaining liver homeostasis. In two successive papers, Li's team has knocked out SIRT1 expression in hepatocytes [START_REF] Purushotham | Hepatocyte-specific deletion of SIRT1 alters fatty acid metabolism and results in hepatic steatosis and inflammation[END_REF] and in macrophages [START_REF] Schug | Myeloid deletion of SIRT1 induces inflammatory signaling in response to environmental stress[END_REF], and has assessed how this would affect liver steatosis following a HFD. Purushotham's liver specific SIRT1-KO mice (LKO) have impaired lipid homeostasis and are more prone to developing liver steatosis when fed a HFD, as compared to wt littermates [START_REF] Purushotham | Hepatocyte-specific deletion of SIRT1 alters fatty acid metabolism and results in hepatic steatosis and inflammation[END_REF] . Steatosis is accompanied by liver inflammation, with an increase in TNF and IL-1 synthesis and in total macrophage markers such as F4.80. However, it must be noted that Guarente's team has observed an opposite outcome in a similar model of liver-specific SIRT1 deletion, with SIRT1 absence proving to have a beneficial effect on several metabolic parameters following HFD, even if no effect on NFkB acetylation or liver inflammation was reported [START_REF] Chen | Tissue-specific regulation of SIRT1 by calorie restriction[END_REF]. The increase in F4.80 expression in Purushotham's study suggests an important macrophage infiltration and this prompted the authors to disrupt SIRT1 in macrophages and assess the consequences of HFD feeding [START_REF] Schug | Myeloid deletion of SIRT1 induces inflammatory signaling in response to environmental stress[END_REF].

A c c e p t e d M a n u s c r i p t

Macrophage-specific SIRT1 deletion (Mac-SIRT1 KO) has remarkable effects on several organs and on the entire organism. HFD reduces insulin sensitivity in Mac-SIRT1 KO mice while exacerbating inflammatory cytokine expression, both systemically and locally. SIRT1 deficiency clearly correlates with NFkB hyperacetylation and NFkB knockdown blunts the increase in cytokine production observed in macrophages from SIRT1 KO mice. It is therefore tempting to conclude that, as previously stated, lack of SIRT1 activity results in hyperactive NFkB status and contributes to the development of inflammation in these models.

In line with the previous conclusion, several studies have shown that systemic SIRT1 activation decreases inflammation by reducing NFkB activity [START_REF] Pfluger | Sirt1 protects against high-fat diet-induced metabolic damage[END_REF], [START_REF] Escande | Deleted in breast cancer-1 regulates SIRT1 activity and contributes to high-fat diet-induced liver steatosis in mice[END_REF]. SIRT1 in vivo overexpression has been achieved by Pfluger and colleagues, who generated a transgenic mouse carrying an extra copy of the SIRT1 gene under its own promoter [START_REF] Pfluger | Sirt1 protects against high-fat diet-induced metabolic damage[END_REF]. Alternatively, SIRT1 overactivation can result from the removal of endogenous SIRT1 inhibitors, such as deleted in breast cancer-1 (DBC1) [START_REF] Escande | Deleted in breast cancer-1 regulates SIRT1 activity and contributes to high-fat diet-induced liver steatosis in mice[END_REF], a nuclear protein recently shown to bind and negatively regulate SIRT1 activity [START_REF] Zhao | Negative regulation of the deacetylase SIRT1 by DBC1[END_REF], [START_REF] Kim | DBC1 is a negative regulator of SIRT1[END_REF]. Focusing on the liver, both of these studies showed that SIRT1 activation confers protection against HFD-induced steatosis and the associated inflammation. The authors also conclude that SIRT1 anti-inflammatory effects can be explained by the inhibition of NFkB, since they observe an impaired p65 activation following TNF stimulation [START_REF] Pfluger | Sirt1 protects against high-fat diet-induced metabolic damage[END_REF], [START_REF] Escande | Deleted in breast cancer-1 regulates SIRT1 activity and contributes to high-fat diet-induced liver steatosis in mice[END_REF].

Although numerous reports have illustrated the negative regulatory influence of SIRT1 on NF-kB, the underlying molecular mechanism has not been completely elucidated. Recently, however, two potential mechanisms by which SIRT1 can negatively affect NFkB activity have been uncovered. In a search for proteins able to modulate RelA transcriptional activity, Huang and co-workers have identified Brd4 as a bromodomain-containing protein able to specifically bind and co-activate K310-acetylated RelA [START_REF] Huang | Brd4 coactivates transcriptional activation of NF-kappaB via specific binding to acetylated RelA[END_REF]. Brd4 allows the recruitment of A c c e p t e d M a n u s c r i p t the CDK9 kinase at the site of transcription, an event associated with the phosphorylation of the RNA pol II [START_REF] Huang | Brd4 coactivates transcriptional activation of NF-kappaB via specific binding to acetylated RelA[END_REF] (Figure 2). Notably, not all NF-kB-dependent genes are similarly regulated by Brd4, suggesting that the acetylation state of RelA K310 offers a new and subtle mechanism for modulating NFkB activity. In a separate study, the same group has recently demonstrated a role for the acetylation of the lysine 310 of RelA in modulating protein stability [START_REF] Yang | Functional interplay between acetylation and methylation of the RelA subunit of NF-kappaB[END_REF]. Acetylation of this SIRT1 substrate impairs the Set9-mediated methylation of proximal lysine residues (314 and 315), a posttranslational modification that targets chromatin-associated RelA for ubiquitin-mediated degradation. Acetylated RelA is therefore protected from degradation, a novel mechanism that explains the negative influence of SIRT1 on NFkB function [START_REF] Yang | Functional interplay between acetylation and methylation of the RelA subunit of NF-kappaB[END_REF].

SIRT1 inhibits AP-1 by deacetylating c-Jun and c-Fos

c-Jun and c-Fos are the main components of the dimeric transcription factor AP-1. In a recent study, Gao and coworkers showed that c-Jun transcriptional activity can be downregulated by SIRT1 in vitro [START_REF] Gao | Inhibition of transcriptional activity of c-JUN by SIRT1[END_REF]. A second study rapidly followed, showing that c-Jun is actively deacetylated by SIRT1 in vivo [START_REF] Zhang | The type III histone deacetylase Sirt1 is essential for maintenance of T cell tolerance in mice[END_REF], while a third team reported that both the subunits c-Jun and c-Fos are targeted by SIRT1 [START_REF] Zhang | SIRT1 suppresses activator protein-1 transcriptional activity and cyclooxygenase-2 expression in macrophages[END_REF]. These events play a major role in regulating the function of several immune cells. Indeed, in macrophages, SIRT1 activation was reported to decrease the transcription of COX-2, a typical AP-1 dependent pro-inflammatory gene [START_REF] Zhang | SIRT1 suppresses activator protein-1 transcriptional activity and cyclooxygenase-2 expression in macrophages[END_REF].

In T lymphocytes, AP-1 deacetylation by SIRT1 underlies T cell anergy and permits peripheral tolerance, preventing uncontrolled T cell proliferation and cytokine production [START_REF] Zhang | The type III histone deacetylase Sirt1 is essential for maintenance of T cell tolerance in mice[END_REF]. As a consequence, SIRT1 -/-mice display an increased T cell responsiveness in vitro and A c c e p t e d M a n u s c r i p t 10 are predisposed to develop autoimmune disorders, a conclusion further confirmed by an independent study [START_REF] Sequeira | sirt1-null mice develop an autoimmune-like condition[END_REF].

SIRT1-dependent deacetylation of FoxP3 leads to its degradation

Tregs are CD4 + T cells endowed with suppressive capacities, whose most specific and widely accepted marker is the transcription factor FoxP3. High and stable levels of FoxP3 expression have been proposed to be required for adequate Treg function [START_REF] Zhou | Plasticity of CD4(+) FoxP3(+) T cells[END_REF]. The acetylated form of FoxP3 displays improved repressive functions and is a poor substrate for polyubiquitinmediated degradation [START_REF] Van Loosdregt | Regulation of Treg functionality by acetylation-mediated Foxp3 protein stabilization[END_REF]. SIRT1 has been shown to bind and decrease FoxP3 protein levels, possibly through its deacetylation. Accordingly, nicotinamide mediated SIRT1 inhibition increases both FoxP3 + protein levels and the relative proportion of FoxP3+ cells, resulting in an overall increased Treg suppressive function [START_REF] Van Loosdregt | Regulation of Treg functionality by acetylation-mediated Foxp3 protein stabilization[END_REF]. In line with these results, Yang and coworkers reported that resveratrol, a natural SIRT1 activator [START_REF] Howitz | Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan[END_REF], depletes CD4 + CD25 + Tregs in tumor-bearing mice [START_REF] Yang | Resveratrol induces the suppression of tumorderived CD4+CD25+ regulatory T cells[END_REF]. As previously discussed, SIRT1 appears to limit the abundance and activity of a well described transcription factor affecting T cell functions.

Notably however, and in marked contrast to the previously described observations, the antagonizing role of SIRT1 on the function and development of Tregs appears to promote, rather than inhibit, an inflammatory response.

A c c e p t e d M a n u s c r i p t 11

SIRT1 role in granulopoiesis

A recent report reveals that SIRT1 is not only implicated in immune cell activation but might also play an important role in their development. Indeed, Nampt-mediated NAD synthesis has been shown to trigger neutrophil differentiation from bone-marrow precursors in a SIRT1dependent manner [START_REF] Skokowa | NAMPT is essential for the G-CSF-induced myeloid differentiation via a NAD(+)-sirtuin-1-dependent pathway[END_REF]. This pathway is affected in individuals suffering from congenital neutropenia, whose treatment consists in daily injections of granulocyte colony stimulating factor (G-CSF). Skokowa and colleagues have shown that G-CSF induces both Nampt and SIRT1 expression, leading to an increased expression of the transcription factors C-EBP and . C-EBP activity increases expression of both G-CSF itself and of its receptor, thus leading to a positive autoregulatory loop. This study demonstrates that administration of nicotinamide, the major NAD precursor in vivo, leads to increased NAD synthesis, C-EBP and  expression and consequently granulopoiesis. Since C-EBPis known to be regulated by acetylation [START_REF] Cesena | CCAAT/enhancer-binding protein (C/EBP) beta is acetylated at multiple lysines: acetylation of C/EBPbeta at lysine 39 modulates its ability to activate transcription[END_REF], it is tempting to assume that it could be added to the growing list of transcription factors representing novel SIRT1 substrates.

SIRT6

Similarly to SIRT1, SIRT6 appears to be mainly localized in the nucleus, predominantly associated to chromatin [START_REF] Mostoslavsky | Genomic instability and aging-like phenotype in the absence of mammalian SIRT6[END_REF]. In 2006 the description of SIRT6 knockout mice drew much attention due to the severe premature aging observed in these mice [START_REF] Mostoslavsky | Genomic instability and aging-like phenotype in the absence of mammalian SIRT6[END_REF]. These mice appear normal at birth but undergo an accelerated degenerative process and die at approximately three weeks of age. They show impaired IGF1 and glucose serum levels, a decrease in bone mineral density and, at the immune level, a dramatic and systemic increase in lymphocyte A c c e p t e d M a n u s c r i p t apoptosis. Using these same mice, Kawahara and colleagues later demonstrated that lymphopenia could be rescued by p65 heterozygosity, arguing again for a sirtuin-dependent regulation of NFkB-driven gene expression [START_REF] Kawahara | SIRT6 links histone H3 lysine 9 deacetylation to NF-kappaB-dependent gene expression and organismal life span[END_REF].

SIRT6 inhibits NFkB dependent transcription by affecting chromatin structure.

Kawahara and colleagues focused on the possible link between SIRT6 and NFkB to explain the accelerated aging phenotype of SIRT6 -/-mice. These authors were able to demonstrate that although SIRT6 directly binds to p65, it actually interferes with its transcriptional activity by deacetylating H3 lysine 9 (H3K9) on the promoter of selected NFkB target genes rather than directly modulating p65 activity [START_REF] Kawahara | SIRT6 links histone H3 lysine 9 deacetylation to NF-kappaB-dependent gene expression and organismal life span[END_REF]. Thus, while SIRT1 inhibits NFkB activity by direct posttranslational modification of p65, SIRT6 acts by decreasing promoter accessibility to p65 (Figure 2). Notably, a compensatory role for SIRT6 has been recently demonstrated in SIRT1deficient macrophages [START_REF] Schug | Myeloid deletion of SIRT1 induces inflammatory signaling in response to environmental stress[END_REF]. In these cells, SIRT6 was found to be strongly associated with selected NFkB promoters following TNF stimulation, thus attenuating the increased NFkB activity caused by the loss of SIRT1. Accordingly, siRNA-mediated inhibition of SIRT6 protein expression further increased the expression of selected NF-kB genes (such as IL-1β in SIRT1 KO mice). Even though the nature of this compensatory mechanism was not uncovered, these observations confirm that both SIRT1 and SIRT6, although acting at distinct levels, represent negative regulators of NFkB activity (Figure 2).

A potential role for SIRT6 in post-transcriptional regulation of pro-inflammatory cytokines.

In contrast to the anti-inflammatory role generally attributed to sirtuins, we and others have recently reported a possible positive regulatory role for SIRT6 in the induction of proinflammatory cytokine expression, in both innate and adaptive immune cells [START_REF] Van Gool | Intracellular NAD levels regulate tumor necrosis factor protein synthesis in a sirtuin-dependent manner[END_REF], [START_REF] Bruzzone | Catastrophic NAD+ depletion in activated T lymphocytes through Nampt inhibition reduces demyelination and disability in EAE[END_REF]. In A c c e p t e d M a n u s c r i p t 13 these studies, reduction of intracellular NAD levels, obtained by inhibiting the enzymatic activity of Nampt, led to reduced secretion of selected cytokines (such as TNFIL-6 and/or IFN) by immune cells, while leaving other pro-inflammatory mediators (such as CCL5/Rantes) unaffected [START_REF] Van Gool | Intracellular NAD levels regulate tumor necrosis factor protein synthesis in a sirtuin-dependent manner[END_REF], [START_REF] Bruzzone | Catastrophic NAD+ depletion in activated T lymphocytes through Nampt inhibition reduces demyelination and disability in EAE[END_REF]. Surprisingly, several sirtuin inhibitors could reproduce these effects, suggesting therefore that the NAD-sirtuin axis could also represent a positive regulatory loop required for adequate cytokine secretion. Using different approaches, both studies led to the identification of SIRT6 as the sirtuin member able to positively regulate TNF and IFN synthesis. Notably, , over-expression of an enzymatically active (but not a catalytically inactive) form of SIRT6 led to supraoptimal production of TNF by upregulating the translational efficiency of TNF mRNA [START_REF] Van Gool | Intracellular NAD levels regulate tumor necrosis factor protein synthesis in a sirtuin-dependent manner[END_REF]. In agreement with this observation, dowregulation of intracellular NAD levels and sirtuin inhibitors reduced TNF protein synthesis with no significant effect on TNF mRNA accumulation in response to microbial stimulation. Collectively these data suggest a complex regulatory role for SIRT6 in controlling an inflammatory response, since this sirtuin member could inhibit selected NFkB target genes at the transcriptional level, while increasing the translation of other cytokine mRNAs by a yet to be defined mechanism (Figure 2). Note however that TNF gene transcription does not appear to be affected by siRNA-mediated SIRT6 downregulation [START_REF] Schug | Myeloid deletion of SIRT1 induces inflammatory signaling in response to environmental stress[END_REF],

suggesting that SIRT6 may in fact specifically promote TNF translation, while inhibiting IL-1 gene transcription in the same cells. Collectively, these observations suggest that sirtuins, and in particular SIRT6, may affect the cytokine profile of activated immune cells in a subtle fashion, by acting at distinct steps of the synthetic process.

(Insert Figure 2)

A c c e p t e d M a n u s c r i p t 14

SIRT7

Finally, a function in neutrophil development has also been proposed for the nuclear sirtuin, SIRT7. Vakhrusheva and coworkers have generated a SIRT7 knockout mouse and have shown that SIRT7 disruption predisposes the mice to heart hypertrophy together with increasing cardiac inflammation [START_REF] Vakhrusheva | Sirt7 increases stress resistance of cardiomyocytes and prevents apoptosis and inflammatory cardiomyopathy in mice[END_REF]. These authors observed an increased infiltration of immune cells (especially granulocytes) in SIRT7 -/-hearts, which correlated with constitutively higher levels of in situ pro-and anti-inflammatory cytokine production, in particular in aged mice. SIRT7 has been shown to display a nucleolar localization and to affect RNA polymerase-I transcription [START_REF] Ford | Mammalian Sir2 homolog SIRT7 is an activator of RNA polymerase I transcription[END_REF]. Although this sirtuin member appears to be highly expressed by cells of the immune system (see data available at http://biogps.gnf.org), further work will be required to evaluate whether SIRT7 is a "bona fide" immune regulator, or whether it affects an inflammatory response indirectly by controlling immune cell survival and/or ribosomal RNA synthesis capacities.

Pharmacological perspectives

Modulation of sirtuin activity through NAD metabolism.

Due to their highly dynamic nature and sensitivity to small molecule modulators, enzymes mediating post-translational modifications represent ideal targets for pharmacological intervention. Sirtuins appear as particularly suitable for in vivo modulation. Their strict NADdependency connects these enzymes to other enzymatic pathways [START_REF] Pillai | Poly(ADP-ribose) polymerase-1-dependent cardiac myocyte cell death during heart failure is mediated by NAD+ depletion and reduced Sir2alpha deacetylase activity[END_REF], offering numerous possibilities for affecting their enzymatic activity. Several observations indicate, for example, A c c e p t e d M a n u s c r i p t that increased intracellular NAD levels often lead to increased enzymatic activity of selected sirtuin members [START_REF] Yang | Nutrient-sensitive mitochondrial NAD+ levels dictate cell survival[END_REF]. This is particularly true for SIRT1, whose activity has been clearly

shown to be positively regulated by protocols leading to increased intracellular NAD levels.

Forced expression of Nampt in fibroblasts has been shown to increase intracellular NAD levels, causing an enhanced transcriptional repression by SIRT1. [START_REF] Revollo | The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells[END_REF]. A similar direct relationship between Nampt, intracellular NAD levels and SIRT1 activity has been recently confirmed in several cell types including skeletal myoblasts [START_REF] Fulco | Glucose restriction inhibits skeletal myoblast differentiation by activating SIRT1 through AMPK-mediated regulation of Nampt[END_REF], vascular smooth muscle cells [START_REF] Ho | SIRT1 markedly extends replicative lifespan if the NAD+ salvage pathway is enhanced[END_REF], [START_REF] Van Der Veer | Pre-B-cell colonyenhancing factor regulates NAD+-dependent protein deacetylase activity and promotes vascular smooth muscle cell maturation[END_REF], and chondrocytes [START_REF] Dvir-Ginzberg | Regulation of cartilage-specific gene expression in human chondrocytes by SirT1 and nicotinamide phosphoribosyltransferase[END_REF]. Of interest, the nuclear form of Nicotinamide Mononucleotide Adenylyl Transferase (NMNAT-1), the enzyme catalyzing the last step in NAD biosynthesis from nicotinamide in mammals, has been found to bind to SIRT1 and to be recruited to selected gene promoters [START_REF] Zhang | Enzymes in the NAD+ salvage pathway regulate SIRT1 activity at target gene promoters[END_REF]. This close association strongly suggests a mechanism whereby neo-synthesized NAD is made directly available to SIRT1, thus providing a link between cell metabolism and the regulation of gene transcription.

The possibility to affect sirtuin activity through NAD metabolism has recently led S. Imai to propose the use of "nutriceuticals" as a novel type of sirtuin activators [START_REF] Imai | A possibility of nutriceuticals as an anti-aging intervention: activation of sirtuins by promoting mammalian NAD biosynthesis[END_REF]. Although administration of NAD could represent the most straightforward approach to increase intracellular NAD concentrations, the widespread expression of NAD-degrading ectoenzymes represent a likely mechanism affecting exogenously administered NAD bioavailability before it could reach the initially targeted cells. Moreover, systemic administration of NAD could also affect lymphocyte numbers through the process of NAD-Induced Cell Death (NICD) [START_REF] Adriouch | NAD+ released during inflammation participates in T cell homeostasis by inducing ART2-mediated death of naive T cells in vivo[END_REF]. As previously discussed, exogenous nicotinamide, a vitamin B3 component representing the major NAD precursor in mammalian cells and displaying low toxicity in vivo [START_REF] Skokowa | NAMPT is essential for the G-CSF-induced myeloid differentiation via a NAD(+)-sirtuin-1-dependent pathway[END_REF], may represent an ideal alternative to affect intracellular NAD levels. Accordingly, SIRT1 was successfully activated in humans following oral administration of nicotinamide [START_REF] Skokowa | NAMPT is essential for the G-CSF-induced myeloid differentiation via a NAD(+)-sirtuin-1-dependent pathway[END_REF],

A c c e p t e d M a n u s c r i p t

suggesting that this strategy could prove useful in defined clinical settings. Notably however, nicotinamide can both activate and inhibit sirtuins (by respectively promoting NAD synthesis and acting as an end-product inhibitor of the deacetylation reaction), and it may therefore be difficult to predict which of these activities will prevail in vivo. Administration of nicotinamide mononucleotide (NMN), the product of the Nampt catalyzed reaction, may represent a useful and clinically relevant alternative to modulate sirtuin activity in vivo [START_REF] Imai | The NAD World: a new systemic regulatory network for metabolism and aging--Sirt1, systemic NAD biosynthesis, and their importance[END_REF].

NMN can be viewed as a rather "neutral" molecule, lacking the inhibitory properties of nicotinamide and being unable to activate NAD-consuming reactions directly. Even though NMN could be potentially degraded by the ectoenzyme CD38 [START_REF] Sauve | The reaction mechanism for CD38. A single intermediate is responsible for cyclization, hydrolysis, and base-exchange chemistries[END_REF], it has been shown to effectively increase intracellular NAD levels in vitro, while displaying minimal toxicity when administered to animals [START_REF] Revollo | Nampt/PBEF/Visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme[END_REF] Further in vivo studies are warranted to better define its pharmacokinetic properties, tissue selectivity and overall effect on sirtuin activity.

Inhibition of NAD-consuming enzymes represents an alternative approach to modulate sirtuin activity in vivo. PARP1 (the major intracellular NAD-consuming enzyme) and SIRT1, for example, are two long known rivals that share part of their substrates and whose activity depends upon NAD availability, although with distinct sensitivities [START_REF] Zhang | Are poly(ADP-ribosyl)ation by PARP-1 and deacetylation by Sir2 linked?[END_REF]. PARP1 overactivation inhibits SIRT1 activity by depleting NAD and increasing nicotinamide levels [START_REF] Pillai | Poly(ADP-ribose) polymerase-1-dependent cardiac myocyte cell death during heart failure is mediated by NAD+ depletion and reduced Sir2alpha deacetylase activity[END_REF], and PARP inhibitors have been shown to contribute to maintain high intracellular NAD levels [START_REF] Caito | PARP-1 inhibition does not restore oxidant-mediated reduction in SIRT1 activity[END_REF] resulting in increased SIRT1 activity [START_REF] Caito | PARP-1 inhibition does not restore oxidant-mediated reduction in SIRT1 activity[END_REF], [START_REF] Hwang | Cigarette smoke-induced autophagy is regulated by SIRT1-PARP-1-dependent mechanism: implication in pathogenesis of COPD[END_REF]. Notably, SIRT1 has also been shown to directly interact with PARP1 and inactivate it in a deacetylase-dependent manner [START_REF] Rajamohan | SIRT1 promotes cell survival under stress by deacetylation-dependent deactivation of poly(ADPribose) polymerase 1[END_REF], further illustrating the competing activities of these two NAD-consuming enzymes. In any event, these preliminary observations offer new pharmacological opportunities to activate sirtuins by both promoting NAD biosynthesis and by switching off competing NADconsuming enzymes.

A c c e p t e d M a n u s c r i p t

Small molecule modulators of sirtuin activity

The potential capacity of sirtuins to promote longevity and to increase cell resistance to stress, has led to the blossoming of numerous research activities aiming at identifying sirtuinactivating drugs. This topic has been recently reviewed (see as an example [START_REF] Alcain | Chapter 10 Small Molecule Modulators of Sirtuin Activity[END_REF]) and will not be further discussed herein. It is noteworthy however that although numerous sirtuinactivating compounds have been described, their mode of action, remains often controversial.

Resveratrol, the leading and most used sirtuin-activating drug [START_REF] Howitz | Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan[END_REF] has been shown to affect several non-sirtuin related enzymatic activities in the cell [START_REF] Mader | Identification of a novel proapoptotic function of resveratrol in fat cells: SIRT1-independent sensitization to TRAILinduced apoptosis[END_REF], complicating the interpretation of most in vivo effects of this compound. Moreover, its sirtuin-enhancing capacities, originally established with an in vitro assay using an artificial substrate, have also been recently questioned [START_REF] Pacholec | SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1[END_REF]. Nonetheless, screening of several chemical libraries has led to the identification of several sirtuin-activator candidates, some of which display the expected functional properties of a bona fide sirtuin activity promoting agent. In particular, Nayagam and colleagues [START_REF] Nayagam | SIRT1 modulating compounds from high-throughput screening as anti-inflammatory and insulin-sensitizing agents[END_REF] have recently demonstrated the in vitro anti-inflammatory properties of SIRT1-activating compounds. In vivo administration of a sirtuin activator, has also been shown to decrease inflammation while concomitantly improving insulin sensitivity in naturally obese rats [START_REF] Yoshizaki | SIRT1 inhibits inflammatory pathways in macrophages and modulates insulin sensitivity[END_REF]. Finally, J. Smith and colleagues [START_REF] Smith | Small molecule activators of SIRT1 replicate signaling pathways triggered by calorie restriction in vivo[END_REF] have recently demonstrated the ability of two structurally distinct SIRT1 activators to recapitulate many of the signaling pathways generally controlled by SIRT1. In particular, these compounds have been shown to inhibit NFkB transcriptional activity and TNF secretion in vitro, further confirming the potential therapeutic relevance of sirtuin activators in an inflammatory setting.

Sirtuin inhibitors have also been extensively studied, mostly in the setting of cancer therapy, as shown by [START_REF] Heltweg | Antitumor activity of a small-molecule inhibitor of human silent information regulator 2 enzymes[END_REF]. It is noteworthy however that nicotinamide, the prototypic sirtuin inhibitor, displays anti-inflammatory properties both in vitro and in vivo [START_REF] Van Gool | Intracellular NAD levels regulate tumor necrosis factor protein synthesis in a sirtuin-dependent manner[END_REF], [START_REF] Ungerstedt | Nicotinamide is a potent inhibitor of proinflammatory cytokines[END_REF]. Although nicotinamide may represent a general inhibitor for all NAD-consuming reactions, we and others have been able to confirm the potential beneficial role of sirtuin inhibitors as antiinflammatory compounds both in vitro and in vivo [START_REF] Van Gool | Intracellular NAD levels regulate tumor necrosis factor protein synthesis in a sirtuin-dependent manner[END_REF], [START_REF] Bruzzone | Catastrophic NAD+ depletion in activated T lymphocytes through Nampt inhibition reduces demyelination and disability in EAE[END_REF], although these observations need to be further extended to more clinically relevant settings.

Conclusions

Based on the brief review of the literature presented herein, it is tempting to conclude that sirtuins do represent clinically relevant targets for the development of novel antiinflammatory compounds. Several complicating factors that may limit such development need however to be considered. Although numerous reports concur to identify SIRT1 as a negative regulator of NFkB activity, it is worth noting that SIRT1 does not merely switch off NFkB activity, but rather plays a subtle role in selectively modulating the expression of NFkBdependent genes. This is particularly well illustrated by the finding that SIRT1 KO mice do not display the extremely inflammatory phenotype of mice expressing a constitutively active form of the p65 NFkB subunit [START_REF] Dong | Constitutively active NF-kappaB triggers systemic TNFalpha-dependent inflammation and localized TNFalpha-independent inflammatory disease[END_REF], and by the finding that the acetylation status of the lysine 310 of p65 affects the expression of some, but not all not all NFkB target genes [START_REF] Huang | Brd4 coactivates transcriptional activation of NF-kappaB via specific binding to acetylated RelA[END_REF]. As a consequence, the possibility that the K310 deacetylated forms of p65 could be redirected to other target gene promoters cannot be presently excluded. As an example, in cells expressing the murine p65 mutant S276A, that is unable to recruit histone acetylases, the induction of TNF and IL-6 is lost but the activation of the COX-2 gene is preserved [START_REF] Dong | Repression of gene expression by unphosphorylated NF-kappaB p65 through epigenetic mechanisms[END_REF], arguing in favor of different roles for the Lys310-acetylated and Lys310-deacetylated forms of p65.

Further work is therefore required to precisely identify the target genes that may be preferentially inhibited by sirtuin activators in vivo. SIRT1 appears to negatively affect observations performed using purified cell subpopulations. The potential opposing roles of SIRT1 and SIRT6 on selected target genes is also a potential confounding factor illustrating the complex regulatory role of this enzyme family on the immune response. As previously discussed, although both SIRT1 and SIRT6 appear to negatively regulate the transcriptional activity of several NFkB genes, SIRT6 has also been shown to positively control the translational efficiency of TNF mRNA. During an inflammatory reaction, TNF is synthesized very early and several studies suggest that this cytokine orchestrates the release of other cytokines, potentially explaining the high beneficial impact of TNF blocking reagents in the treatment of several inflammatory disorders [START_REF] Dong | Constitutively active NF-kappaB triggers systemic TNFalpha-dependent inflammation and localized TNFalpha-independent inflammatory disease[END_REF]. Notably, mice expressing a constitutively active p65 form die a few weeks after birth from excessive inflammation, unless TNF signaling is disrupted [START_REF] Dong | Constitutively active NF-kappaB triggers systemic TNFalpha-dependent inflammation and localized TNFalpha-independent inflammatory disease[END_REF], We therefore believe that understanding the respective role of SIRT1 and SIRT6 in controlling TNF production is warranted before a sirtuin-based anti-inflammatory therapy can be envisioned.

Low grade, chronic inflammation is associated with aging and linked to pathological conditions, such as type 2 diabetes and cancer. Noteworthy, sirtuin activity has been shown to glucose homeostasis [START_REF] Revollo | Nampt/PBEF/Visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme[END_REF] and cellular differentiation [START_REF] Van Loosdregt | Regulation of Treg functionality by acetylation-mediated Foxp3 protein stabilization[END_REF], [START_REF] Dvir-Ginzberg | Regulation of cartilage-specific gene expression in human chondrocytes by SirT1 and nicotinamide phosphoribosyltransferase[END_REF]. The work reviewed herein suggests that some of the pathological processes linked to metabolic disorders or aging may in fact be functionally linked to the immunomodulatory function of this family of metabolic sensors.

In conclusion, depending on the family member examined and on the experimental conditions, sirtuins display either pro-or anti-inflammatory properties. Although these activities can be modified by specific activators/inhibitors, a better understanding of the role of each member of the sirtuin family and the development of drugs able to specifically activate/inhibit individual sirtuins isoforms is warranted before clinically relevant applications of such compounds could be envisioned.

A c c e p t e d M a n u s c r i p t 5 sirtuins

 5 in controlling the expression of several inflammatory mediators. Although a conclusive role for sirtuins as pro or anti-inflammatory regulators is still a matter of debate, several recent reports have clearly established that acetylation and sirtuin dependent deacetylation of several transcription factors with well established immunoregulatory functions, play an important role in immune cells, warranting a brief review of the literature on this new emerging field.

A c c e p t e d M a n u

  s c r i p t several transcription factors expressed by immune cells. In particular, the capacity of SIRT1 to negatively regulate the activity of both NFkB and FoxP3 appears as intriguing. When expressed by T cells, NFkB and FoxP3 regulate an immune response in opposite fashion, NFkB being considered as a positive regulator of T cell responses while FoxP3-expressing cells represent regulatory cells endowed with suppressive activity toward multiple immune effectors. This observation suggests that sirtuin inhibitors may act in a cell context dependent fashion, and although SIRT1 KO animals display an autoimmune-prone phenotype (in keeping with an overall anti-inflammatory role for SIRT1 in vivo), caution should be taken in generalizing the in vivo properties of sirtuin modulating compounds based on in vitro

A c c e p t e d M a n u s c r i p t 20 play

 20 a role in related processes such as cellular and organismal lifespan extension[START_REF] Tissenbaum | Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis elegans[END_REF],[START_REF] Ho | SIRT1 markedly extends replicative lifespan if the NAD+ salvage pathway is enhanced[END_REF],

Figure 1 :

 1 Figure 1: Schematic representation of nicotinamide recycling to NAD and of the reaction

Figure 2 :

 2 Figure 2: Putative model of how two NAD-dependent deacetylases, SIRT1 and SIRT6, can

Figure

  Figure


Ph.D Adriana Georgescu
email: adriana.georgescu@icbp.ro

Doina Popov

Anamaria Constantin

Miruna Nemecz

Nicoleta Alexandru

Daniel Cochior

Aura Tudor

Dysfunction of human subcutaneous FAT arterioles in obesity associated or not with Type 2 diabetes

Keywords: arterioles, obesity, type 2 diabetes, insulin signaling, endothelial dysfunction

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Obesity, insulin resistance, and endothelial dysfunction closely coexist throughout the natural history of type 2 diabetes. Insulin resistance contributes to endothelial dysfunction through a complex cascade of events that are not fully understood. Also, obesity, known as a well established risk factor, generates adverse health effects, including cardiovascular disease, diabetes and cancer [START_REF] De Ferranti | The Perfect Storm: Obesity, Adipocyte Dysfunction, and Metabolic Consequences[END_REF]. On the other hand, obesity, particularly "central or abdominal" obesity, usually involving increased visceral fat, leads to an imbalanced production of several metabolic products, hormones and cytokines (adipocytokines), which favor decreased insulin sensitivity in liver and skeletal muscle, and impair endothelial function through direct and/or indirect mechanisms [START_REF] Caballero | Endothelial dysfunction in obesity and insulin resistence: a road to diabetes and heart disease[END_REF]. In humans, the subcutaneous adipose tissue has a greater volume and is more strongly associated with insulin resistance than the visceral one [START_REF] Garg | Regional adiposity and insulin resistance[END_REF]. Type 2 diabetes mellitus significantly alters the adipose tissue distribution with less influence on the subcutaneous one and with more influence on the visceral and intermuscular ones, both fat depots known to exacerbate insulin resistance [START_REF] Gallagher | Adipose tissue distribution is different in type 2 diabetes[END_REF]. The interest in studying the subcutaneous adipose tissue arterioles comes not only from the need to understand the interplay between the adipocytes and vasculature within this fat depot, but also from the therapeutic potential of adipose tissue-derived stroma cells in physiological and pathological wound healing [START_REF] Ebrahimian | Cell therapy based on adipose tissuederived stromal cells promotes physiological and pathological wound healing[END_REF].

Considering the rather disparate studies on the subcutaneous adipose tissue arterioles, our objective was to understand the structural-functional correlates and cellular mechanisms by which obesity and obesity aggravated by type 2 diabetes induce local effects in this particular microvascular bed, with adverse consequences on vascular wall-adipocyte crosstalk.

Methods

Reagents

Noradrenaline, KCl, acetylcholine, insulin, Nε-Nitro-L-Arginine Methyl Ester and sodium orthovanadate were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The primary antibodies to IRS-1, IRS-2, PI3-kinase, AKT, p-AKT, eNOS, p-eNOS were purchased from Santa Cruz (USA), rabbit polyclonal to β-actin was from Abcam (http://www.abcam.com, USA) and the secondary antibody, goat anti-rabbit IgG coupled with peroxidase (HRP) was from Sigma (St. Louis, MO, USA). All others reagents used were of analytical grade.

Study groups and samples

In collaboration with "CFR 2" University Hospital Bucharest, the study was performed on 60 middle-aged subjects (44.32 ± 4.83 years old): 35 men and 25 women. The subjects were divided into three equal groups: healthy as control subjects (C group), obese patients, and obese patients with type 2 diabetes. At hospital, patients evaluation consisted of physical, electrocardiographic and echocardiographic examination, establishment of body mass index (BMI), measurement of waist circumference (WC) and of systolic and diastolic blood pressure; the blood was collected by venipuncture and subsequently used for the assay of glucose and cholesterol concentrations. Small pieces of subcutaneous adipose tissue were removed during surgery from the periumbilical area, at 1.5-2.0 cm deep from the skin surface. These specimens (otherwise discarded) were subsequently transferred to the lab, where the arterioles (internal diameter 220 ± 40 µm) were dissected out, and used in physiological and biochemical assays. Exclusion criteria for the three experimetal groups were considered: the current tobacco use, established cardiovascular disease, medications for hypertension, and hyperlipidaemia. All the subjects had blood pressure of ≤140/90mmHg, fasting total cholesterol ≤200mg/dl, and no family history of cardiovascular disease. The controls were healthy volunteers, all in good physical health and did not take any medication at the time the experiments were performed. None had a family history of diabetes or obesity among first-degree relatives. In addition, they had BMI, WC and glucose concentrations in normal range. For obese patients, we included subjects with evidence of increased values of BMI and WC, but with normal baseline blood tests and without another medication. Obese type 2 diabetic patients were maintained in a good metabolic control with standard oral anti-diabetic agents (see Table 1). They discontinued all drug treatment 3 days before the study. The small pieces of subcutaneous adipose tissue were removed during the esthetic surgery for C group and during the necessary surgery for obese patients and obese patients with type 2 diabetes. The experiments associated with this study have been carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association, and have been approved by the Ethic Committee of the Institute of Cellular Biology and Pathology "N. Simionescu", Bucharest, Romania. Also, each patient or subject taken in consideration in our study gave their consent after full explanation of the purpose, nature and risk of all procedures used.

Electron microscopy

Tissue processing for electron microscopy was performed essentialy as in Popov et al., [START_REF] Popov | Beneficial effects of Larginine supplementation in experimental hyperlipemia -hyperglycemia in the hamster[END_REF]. Briefly, the dissected arterioles were fixed in a mixture made of 2.5% paraformaldehyde and 1.5% glutaraldehyde in 0.1 M sodium cacodylate -HCl buffer, supplemented with 2.5 mmol/l CaCl 2 , pH 7.4, postfixed in 1% OsO 4 , dehydrated in graded concentrations of ethanol, and embedded in Epon 812. The thick sections stained with 0.25% toluidine blue were examined by light microscopy, and used to locate the position were thin sections are to be cut. The thin sections were stained with 7.6% uranyl acetate (in distilled water) and 0.4% lead citrate (in 0.1 N NaOH), and were finally examined by electron microscopy (FEI Tecnai F20 field emission gun TEM, Philips Electron Optics, Eindhoven, The Netherlands).

The isometric myograph technique

One mm long segments of subcutaneous arterioles were dissected out from peri-umbilical fat, and the small amount of connective tissue surrounding their thin wall was removed. Two stainless steel wires (φ= 40 µm) were threaded through the arteriole lumen and the preparation was mounted in a small vessel myograph (Model 410A; J.P. Trading, Aarhus, Denmark) as described by Mulvany and Halpern [START_REF] Mulvany | Contractile properties of small arteriolar resistance vessels in spontaneously hypertensive and normotensive rats[END_REF]. The myograph chamber was filled with HEPES salt solution maintained at 37°C and continuously gassed with O 2 [START_REF] Chulia | Comparative study of elgodipine and nisoldipine on the contractile response of various isolated blood vessels[END_REF][START_REF] Thurston | Changes in endothelial actin cytoskeleton at leakage sites in the rat mesenteric vasculature[END_REF]. Following an equilibration period of 20 min, the arterioles were set to a normalized internal circumference at which they gave the maximum of the isometric response. The mean internal diameter of arterioles used in this study was 220± 40 µm. Then, for each vessel a routine "run-up" procedure consisting of consecutive contractions in 5 x 10 -6 M noradrenaline (NA), 125 x 10 -3 M K + , and 5 x 10 -6 M NA in 125 x 10 -3 K + was performed. To measure arteriolar wall contractility, 10 -8 -10 -4 M NA was cumulatively added (at 2 min intervals) in the organ bath of myograph, and the active wall tension recorded; the contractile force of arteries was expressed in mN mm -1 . To measure arteriolar wall vasodilation, NA contracted vessels (as above) were exposed to 10 -8 -10 -4 M acetylcholine (ACh) cumulatively added at 2 min intervals in the organ bath of myograph; relaxation induced by ACh was expressed as percentage of NA-induced contraction.

In other experiments, the effect of insulin on the vascular reactivity of subcutaneous fat arterioles was investigated. To this purpose, arterioles were first precontracted in NA (10 -8 -10 - 4 M), and then exposed to various concentrations of insulin (0.4 IU/ml -6.4 IU/ml) in the presence or absence of eNOS inhibitor L-NAME (10 -4 M). The force developed by vascular wall was measured after 15 minutes of incubation, and relaxation was expressed as percentage of NAinduced contraction. Time control experiments were not performed, since the aim of study was to compare the responses of wiremounted arterioles from the three equal groups subjected to identical protocols. It should be noted, however, that for subcutaneous arterioles dissected out from peri-umbilical fat, agonist concentration-response relations were repeatable for many hours.

The measurement of endothelial NO production

We measured NO concentration at the luminal side of longitudinally opened arterioles using the NO sensor technique (amiNO-700; Innovative Instruments, Inc., Tampa, FL, USA) as previously described [START_REF] Georgescu | Nebivolol induces a hyperpolarizing effect on smooth muscle cells in the mouse renal artery by activation of the β 2 -adrenoceptors[END_REF]. With the isolated arteriole bathed in 2 ml HEPES salt solution [START_REF] Tschudi | Direct in situ measurement of nitric oxide in mesenteric resistance arteries[END_REF], NO sensor was placed perpendicularly to the luminal arteriole side (upwards), in the close vicinity of endothelial cells layer, in order to minimize diffusion path of NO, and to obtain a high signal. The continuous monitoring of current (in pA) or of NO production (in nmoles/l) by intact endothelium of the arterioles was also performed in presence of 3x10 -5 M ACh or 6.4 IU/ml insulin.

The immunoblotting technique

Lysates of isolated arterioles explanted from human peri-umbilical fat were subjected to SDS-PAGE and transfered on nitrocellulose membranes, as previously described [START_REF] Georgescu | Protective effects of nebivolol and reversal of endothelial dysfunction in diabetes associated with hypertension[END_REF]. The latter were washed in 0.05% Tween-20 in PBS, blocked in 5% BSA in PBS-Tween-20, and incubated with the first antibody (chosen function of the molecules studied, see Reagents) at 4°C. After 17 hrs, membranes were washed again, and incubated for 1 h with the secondary antibody, goat antirabbit IgG coupled with HRP. The bound antibody was detected by ECL technique. The density of immunoreactive bands was measured using a microcomputer imaging system (Scion Image). The densitometric evaluation of protein expression of studied molecules in obese patients with and without type 2 diabetes was compared to C group (considered as 100%) and was calculated as percentage (%).

Data analysis

All values were expressed as mean ± SE of the mean. One-way analysis of variance (ANOVA) was employed to quantify the results. The data were considered significant when P < 0.05.

Results

Clinical characterization of the study subjects

The clinical parameters of middle-age subjects in healthy (C), obese and obese type 2 diabetic groups are presented in Table 1. In C group, plasma glucose concentration was in the range 70 -105 mg/dl, cholesterol concentration was between 120 -190 mg/dl, BMI < 25 kg/m 2 and WC was less than 100 cm in men and less than 87.5 cm in women. In the obese group, plasma glucose concentration exceeded 105 mg/dl, cholesterol concentration was ~ 10 % augmented (versus C group), BMI was over 31 kg/m 2 and WC over 100 cm in men and over 87.5 cm in women. The obese patients with type 2 diabetes displayed ~ 2.4 fold augmented glucose concentration and ~ 15 % increase in cholesterol concentration (versus C group), BMI was in the range 37-42.6 kg/m 2 and WC ~ 122 cm. All middle-age subjects in this study had normal systolic and diastolic blood pressure (Table 1).

Morphological examination of the human subcutaneous adipose tissue arterioles

We questioned whether obesity and obesity associated with type 2 diabetes influence the morphology of subcutaneous adipose tissue arterioles. Light microscopy examination of cross sections of arteriolar wall showed that at obese patients with or without type 2 diabetes the media layer was thickened and the lumen diameter was reduced, compared to arteries in C group (Figure 1A). In these samples the endothelial cells (ECs) appeared as secretor, containing abundant rough endoplasmic reticulum, and an expanded subendothelial extracellular matrix (Figure 1B,b). The two-three layers of smooth muscle cells (SMCs) displayed abundant collagen deposits that separated nearby SMCs (Figure 1B,c). In contradistinction, the subcutaneous arterioles in C group showed a rather normal appearance, with a thin basal lamina separating endothelium from the SMCs layer (Figure 1B,a). These data suggest that, in the human subcutaneous adipose tissue arterioles, obesity associated or not with type 2 diabetes, activates the endothelium, and enhances collagen acumulation within vascular wall.

The vascular reactivity of human subcutaneous adipose tissue arterioles

The isometric myograph technique was used to assess the effect of obesity and obesity associated with type 2 diabetes on arterioles contractile and relaxant properties, in an attempt to correlate their vascular reactivity with the morphological structure. To this purpose, the small amount of connective tissue surrounding their thin wall was removed, and the genuine vascular reactivity of wall was measured, without contribution of anti-contractile effect of perivascular adipose tissue. The results showed that in response to 3x10 -5 M NA the subcutaneous arterioles explanted from lean subjects (C group) developed a maximal contractile force of 0.044 ± 0.015 mN/ mm; at an identical NA concentration, arterioles in obese group contracted to 0.11 ± 0.02 mN/mm, while in obesity associated with type 2 diabetes the force of contractile response was even higher, measuring 0.24 ± 0.027 mN/mm (Figure 2). Analysis of the concentration-dependent curves of contractile response to NA (10 -8 -10 -4 M) showed that half-maximal NA concentrations (EC50) were significantly different between the three groups, i.e. 5.15 ± 0.02 in C group, 6.45 ± 0.02 in the obese group, and 5.99 ± 0.03 in the obese-diabetics group (Figure 2). These data suggest that obesity without or with type 2 diabetes is associated with an increased sensitivity (-log EC50) of the contractile response of subcutaneous arterioles. In another set of experiments, NA-contracted arterioles were exposed to 10 -8 -10 -4 M ACh. The results showed that arterioles in group C relaxed to ACh (EC50: 6.30 ± 0.05), and the maximal vasodilatation was equivalent to 87.19 ± 4.03 % of NA -induced contraction (at 3 x10 -5 M ACh) (Figure 3a). Pre-contracted arterioles in the obese groups without or with type 2 diabetes relaxed very little (2.72 ± 1.56 % and 1.99 ± 0.95 %, respectively) in the presence of ACh (Figure 3a). Taken together, these data suggest that obesity associated or not with type 2 diabetes alters the vascular reactivity of subcutaneous adipose tissue arterioles by enhancing the sensitivity and adrenergic hyperresponsiveness, and by desensitization to ACh induced endothelium-dependent vasodilation.

The effect of insulin on vascular reactivity of human subcutaneous adipose tissue arterioles

As metabolic syndrome is associated with elevated levels of insulin, we tested the effect of insulin (0.4 IU/ml -6.4 IU/ml) on vascular reactivity of subcutaneous adipose fat arterioles explanted from the peri-umbilicat fat of obese and obese with type 2 diabetes patients (versus arterioles from lean, C group). The results showed that arterioles in group C relaxed to insulin (EC50: 0.72 ± 0.07), and the maximal vasodilatation measured 21.2 ± 1.7 % of NA -induced contraction (at 6.4 IU/ml insulin) (Figure 3b). Pre-contracted arterioles in the obese groups without or with type 2 diabetes had a significantly diminished relaxation (1.4 ± 0.6 % and 1.2 ± 0.4 %, respectively) in the presence of insulin (Figure 3b). To investigate whether NO contributes to insulin-induced vasorelaxation, we supplemented the organ bath of myograph with 10 -4 M L-NAME and the responses of wire-mounted arterioles were recorded. In C group vascular relaxation was significantly diminished (P<0.05), measuring only 6.32 ± 1.3 % of NAinduced contraction (Figure 3c). These data suggest that in physiological condition the isolated subcutaneous adipose tissue arterioles respond to insulin in a process NO-dependent, while in obesity and obesity associated with type 2 diabetes, the arterioles respond very little to insulin.

The concentration of NO released by the endothelial cells of human subcutaneous fat arterioles

These experiments examined if the suppression of vessels relaxation, induced by obesity, can be related to a reduced NO production by ECs layer. To this purpose, the endothelial NO production was continuously recorded at the lumen of human subcutaneous fat arterioles exposed to 3x10 -5 M ACh or 6.4 IU/ml insulin, applied for 10 min. The results in Table 2 showed that in presence of ACh, the arterioles explanted from obese and obese-diabetic patients produced ~5.7 and ~11.6 fold less NO, respectively, compared to C group. In presence of insulin, the arterioles explanted from obese and obese-diabetic patients produced ~8.3 and ~13 fold less NO, respectively, compared to C group (Table 2). These results demonstrate that obesity diminishes the endothelial NO production of subcutaneous adipose tissue arterioles, and that type 2 diabetes associated with obesity induces even more severe NO diminishment.

The protein expression of eNOS, and of insulin signaling intermediates in human subcutaneous adipose tissue arterioles

To examine whether the diminished endothelial NO production and the abolished vasodilatation (endothelium-dependent and insulin-induced) observed in obesity and obesity associated with A c c e p t e d M a n u s c r i p t type 2 diabetes is related to a defect in eNOS expression, immunoblotting experiments were performed. The densitometry evaluation of both phosphorylated and non-phosphorylated eNOS show that in obesity these were ~2.51 and ~5.20 fold reduced, respectively compared to C group. Moreover, association between obesity and type 2 diabetes produced the most attenuated p-eNOS and eNOS protein expression, that was diminished by ~13.55 and ~17.48 fold, respectively (Figure 4A,a and 4B).

To have a deeper insight into insulin effects on human adipose tissue arterioles, the protein expression of signaling molecules in insulin pathway was investigated (Figure 4A,b). Densitometry of the bands assessed that, in the subcutaneous arterioles of obese patients both insulin receptor substrates were reduced, i.e. IRS-1 by ~2.61 fold, and IRS-2 by ~3.35 fold (compared to proteins expression in C group) (Figure 4B). Obesity impeded also the expression of downstream molecules in insulin pathway, such as PI3-kinase (by ~1.43 fold), activated AKT (by ~2.14 fold) and AKT (by ~1.44 fold). Furthermore, obesity joined by type 2 diabetes reduced protein expression of all examined molecules (Figure 4A, b and4B). Thus, IRS-1 was reduced by ~3.64 fold, IRS-2 by ~4.73 fold, PI3-kinase by ~4.30 fold, and both phosphorylated and nonphosphorylated AKT by ~2 fold. These data suggest that in the subcutaneous adipose tissue arterioles obesity diminishes the protein expression of insulin signaling molecules, a process more severely impeded when obesity is joined by type 2 diabetes.

Disscusion

This study reports novel findings on the effect of obesity and obesity associated with type 2 diabetes on arterioles isolated from human peri-umbilical subcutaneous adipose tissue. Firstly, we emphasize the structural-functional correlates in endothelial vascular dysfunction; increased contractility and drastically reduced endothelium-dependent vasorelaxation. Secondly, we provide evidence on the diminishment of insulin-induced vasorelaxation associated with obesity, and the molecular defects in its signaling pathway. Thirdly, we show that type 2 diabetes associated with obesity induces even more severe alterations, impeding NO release within arteriolar lumen, and lowering the expression of insulin signaling molecules, such as IRS-1, IRS-2, PI3-kinase, AKT and eNOS.

This study was conducted on subcutaneous fat arterioles explanted from middle-aged pacients, allowing comparisons between groups in the absence of significant comorbidities. As expected, the middle-aged obese and obese-diabetics used as source for subcutaneous fat arterioles had higher BMI and WC compared to C group. In our experiments, the small amount of fat and connective tissue surrounding the thin wall of fat arterioles was removed allowing the direct measurement of endothelium and SMCs layer response. In a different vascular bed (skeletal muscle) it was reported that the fat depot localised around arterioles origin may be involved in local TNF-α signaling, resulting in impaired tissue perfusion and insulin resistance [START_REF] Yudkin | Vasocrine' signaling from perivascular fat: a mechanism linking insulin resistance to vascular disease[END_REF][START_REF] Yudkin | Inflammation, obesity, and the metabolic syndrome[END_REF].

Here we provide morphological and functional evidence on the effect of obesity and obesity joined with type 2 diabetes in human peri-umbilical subcutaneous fat arterioles. The ultrastructural examination of arterioles showed that obesity and obesity aggravated by type 2 diabetes induced similar structural effects, i.e. activate endothelium, thickened the media layer, and apparently reduced the arterioles lumen diameter; the enhanced extracellular matrix acumulation within vascular wall may account for media layer thickening (Figure 1). These structural modifications suggest the installment of a remodeling process, and a possible restriction of blood perfusion of subcutaneous fat in obesity. In another subcutaneous region, such as the gluteal fat, the hypertrophic remodeling of small arteries wall was found to be associated with type 1 diabetes [START_REF] Rizzoni | Structural alterations in subcutaneous small arteries of normotensive and hypertensive patients with non-insulin-dependent diabetus mellitus[END_REF]. Then, it is generaly admitted that impaired perfusion may underline much of tissue and organ dysfunction associated with hypertension, obesity and diabetes mellitus [START_REF] Levy | Impaired tissue perfusion a pathology common to hypertension, obesity, and diabetes mellitus[END_REF].

At the functional level, our data show that obesity associated or not with type 2 diabetes produced an enhanced contractile response to NA of human peri-umbilical subcutaneous fat arterioles, and the suppression of endothelium-dependent vasodilation to ACh (Figure 2). We correlate the enhanced vasoconstriction, the thicker media layer, and the drastic diminishment of relaxation to: (i) extracellular matrix acumulation within vascular wall, (ii) decreased eNOS protein expression, and (iii) reduced NO production (Figure 4 and Table 2). Recent data underline obesity-enhanced vasoconstriction of human gluteal subcutaneous small arteries, and the fact that in obesity are lost both the anticontractile property of perivascular fat and the increase in NO bioavailability caused by adiponectin secreted by nearby adipocytes [START_REF] Greenstein | Local inflammation and hypoxia abolish the protective anticontractile properties of perivascular fat in obese patients[END_REF]. Then, in the subcutaneous microvascular endothelial cells of type 2 diabetic patients endothelin -1 mRNA levels were found to be significantly higher [START_REF] Gogg | Increased MAPK activation and impaired insulin signaling in subcutaneous microvascular endothelial cells in Type 2 diabetes: The role of Endothelin-1[END_REF]. As for the vasorelaxation, previous reports show that in type 1 diabetes, the vasodilatation of subcutaneous gluteal small arteries induced by exposure to ACh or BK was reduced, and ~50% diminished in presence of eNOS inhibitors [START_REF] Rizzoni | Structural alterations in subcutaneous small arteries of normotensive and hypertensive patients with non-insulin-dependent diabetus mellitus[END_REF]. It was reported that NO contributes to reduced BK-mediated dilation of subcutaneous fat microvessels especially in male subjects [START_REF] Sato | Effect of gender on endothelium-dependent dilation to bradykinin in human adipose microvessels[END_REF]. Recently, the reports assess that in diabetes the abnormalities of microvascular blood flow are evident, and become more marked with the development of microvascular complications [START_REF] Brooks | Detection and characterisation of microcirculatory abnormalities in the skin of diabetic patients with microvascular complications[END_REF]. Although it is known that insulin stimulates the production and the release of NO [START_REF] Potenza | Vascular actions of insulin with implications for endothelial dysfunction[END_REF], we show here that this applies also to subcutaneous adipose tissue arterioles in physiological condition; these display a robust protein expression of phosphorylated and total eNOS (Figure 3b and Figure 4). However, in subcutaneous adipose tissue arterioles, obesity significantly reduced insulin-induced vasorelaxation by diminishment of eNOS protein expression and NO production, and all these processes were aggravated in obesity joined by type 2 diabetes (Figure 4 and Table 2). An explanation for these results came from the studies which showed that, in obesity, the enhanced production of circulating tumor necrosis factor (TNF) may reduce insulin-mediated effects on the microvascular function by impairing the balance between endothelial-derived vasodilator and vasoconstrictor substances. Also, there are evidences which demonstrate that TNF down-regulates the expression of eNOS and up-regulates ET-1 expression in the human endothelial cells [START_REF] Jonk | Microvascular dysfunction in obesity: A potential mechanism in the pathogenesis of obesity-associated insulin resistance and hypertension[END_REF]. Examination of insulin signaling proteins in arterioles of lean subjects show the strong expression of insulin receptor substrates IRS-1 and IRS-2, as well as of the downstream molecules PI3-kinase, and AKT (serine phosphorylated AKT included) (Figure 4). In accordance with our data, it was established that: (i) the metabolic action of insulin to stimulate the glucose uptake in skeletal muscle and adipose tissue is mediated through the stimulation of insulin receptor, insulin receptor substrate IRS-1, PI3-kinase, phosphoinositide-dependent kinase 1 (PDK-1), and AKT [START_REF] Kim | Reciprocal relationships between insulin resistance and endothelial dysfunction: molecular and pathophysiological mechanisms[END_REF], (ii) insulin increased directly eNOS activity leading to augmented NO production [START_REF] Kim | Reciprocal relationships between insulin resistance and endothelial dysfunction: molecular and pathophysiological mechanisms[END_REF][START_REF] Potenza | Insulin resistance in spontaneously hypertensive rats is associated with endothelial dysfunction characterized by imbalance between NO and ET-1 production[END_REF]. Beside its vasodilator action, insulin has also vasoconstrictor effects. These vasoconstrictor effects are mainly mediated by vasoconstrictor peptide endothelin-1 (ET-1) [START_REF] Kim | Reciprocal relationships between insulin resistance and endothelial dysfunction: molecular and pathophysiological mechanisms[END_REF]. ET-1 is produced in the vascular endothelium through stimulation of intracellular MAP-kinase signaling pathway and extracellular signal-regulated kinase-1/2 (ERK1/2) [START_REF] Eringa | Vasoconstrictor effects of insulin in skeletal muscle arterioles are mediated by ERK1/2 activation in endothelium[END_REF]. Thus, insulin has opposing vasodilator and vasoconstrictor effects, the net effect being dependent on the balance between these two, and its imbalance causes the development of microvascular dysfunction. In addition, our results revealed that obesity associated or not with type 2 diabetes disrupts the molecules involved in signaling pathway mediated by insulin (IRS-1, IRS-2, PI3-kinase, p-AKT, AKT, p-eNOS, e-NOS), impairs endothelial NO production, and generates insulin resistance condition. In insulin signaling pathway, eNOS is activated through AKT-mediated phosphorylation of serine residue 1179 (p-eNOS serine 1179). The mechanisms regulating eNOS activity and endothelial NO production are complex and sometimes overlapping. For example eNOS contains multiple phosphorylation sites in addition to serine 1179, and increasing evidence has implicated the participation of a variety of kinase and phosphatase activities in the regulation of eNOS activity. The phosphorylation events also participate in controlling the interactions between eNOS and other regulatory proteins [START_REF] Bauer | Compensatory phosphorylation and protein-protein interactions revealed by loss of function and gain of function mutants of the multiple serine phosphorylation sites in endothelial nitric oxide synthase[END_REF]. Therefore, we report here that obesity and obesity associated with type 2 diabetes impede the protein expression of molecules associated with insulin signaling, and also blunt arterioles vasodilation.

Conclusion

The results of this study indicate that obesity associated or not with type 2 diabetes alters human peri-umbilical adipose tissue arterioles in terms of structure, function and biochemsitry, including diminished eNOS expression and reduced levels of IRS-1, IRS-2, PI3-kinase and AKT in insulin-signaling pathway.

Limitations of the study

This study was carried out on specimens to be discarded from peri-umbilical subcutaneous adipose tissue; although we know the clinical data of the patients (Table 1), they underwent surgery for multiple reasons, and were likely taking a variety of medications. Therefore, one can not exclude the interference of the latter in the results presented. However, in the three groups studied the intact arterioles originate allways from the same body area (i.e. peri-umbilical) and from the deep compartment of the subcutaneous fat (i.e. at 1.5-2.0 cm from the skin surface).

Clinical

  Science Immediate Publication. Published on 28 Oct 2010 as manuscript CS20100355 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100355 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 28 Oct 2010 as manuscript CS20100355 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100355 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 28 Oct 2010 as manuscript CS20100355 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100355 A c c e p t e d M a n u s c r i p t

  Clinical Science Immediate Publication. Published on 28 Oct 2010 as manuscript CS20100355 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100355 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 28 Oct 2010 as manuscript CS20100355 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100355

Clinical

  Science Immediate Publication. Published on 28 Oct 2010 as manuscript CS20100355 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100355 A c c e p t e d M a n u s c r i p t

Clinical

  Science Immediate Publication. Published on 28 Oct 2010 as manuscript CS20100355 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100355 A c c e p t e d M a n u s c r i p t

A c c e

  p t e d M a n u s c r i p t Clinical Science Immediate Publication. Published on 28 Oct 2010 as manuscript CS20100355 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100355 A c c e p t e d M a n u s c r i p t

Clinical

  Figure 1A.

Figure 2

 2 Figure 2.

  Clinical Science Immediate Publication. Published on 28 Oct 2010 as manuscript CS20100355 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/CS20100355 A c c e p t e d M a n u s c r i p t

Figure legends Figure 1 .

 legends1 Figure 4B.

Figure 2 .

 2 Figure 2. Dose-dependent contractile response to NA (10 -8 -10 -4 M) of human peri-umbilical subcutaneous adipose tissue arterioles.

Figure 3 .

 3 Figure 3. The vasorelaxation of human peri-umbilical subcutaneous adipose tissue arterioles. (a) dose-dependent vasorelaxation to ACh (10 -8 -10 -4 M); (b) dose-dependent vasorelaxation to insulin (0.4 IU/ml -6.4 IU/ml); (c) the vasorelaxant effect of insulin (0.4 IU/ml -6.4 IU/ml) in the presence and absence of L-NAME (10 -4 M).

Figure 4 .

 4 Figure 4. A. Representative Western blot of the arteries explanted from white adipose tissue of obese patients with and without type 2 diabetes compared to C group. The expression of IRS-1, IRS-2, PI3-kinase, AKT, p-AKT, eNOS, p-eNOS; the presence of βactin on the same transfers was used to prove equal loading of the samples; B. The diagram of densitometric measurements: n=10 individual experiments.

Table 1 .

 1 Clinical characteristics of the study subjects Data are means ± SE. P values represent the statistical significance of comparisons. Symbols indicate which post hoc comparisons were significant: * differs from others; †all three groups differ.

		Clinical Science Immediate Publication. Published on 28 Oct 2010 as manuscript CS20100355
	Clinical	Control group	Obese group	Obese + type 2 diabetes	P
	characteristics	(n=20)	(n=20)	(n=20)
	BM I (kg/m 2 )	23.5 ± 1.9	35.7 ± 4.8	39.8 ± 2.8	<0.0001 †
	WC (cm)	81.25 ± 1.7	110.5 ± 1.9	122.25 ± 2.5	<0.002 †
	Glucose	92.8 ± 2.6	98.95±6.9	217.03 ± 9.86	< 0.001*
	(mg/dl)
	Cholesterol	151 ± 10.2	165.45 ± 9.04 173.6 ± 13.7	0.12
	(mg/dl)
	Systolic blood
	pressure	127 ± 2.5	129 ± 1.9	131 ± 2.3	0.18
	(mm Hg)
	Diastolic blood
	pressure	78 ± 1.2	79 ± 1.6	80 ± 1.8	0. 21
	(mm Hg)

Table 2 .

 2 The NO concentration (nmol/l) delivered at the luminal side of human subcutaneous fat arterioles explanted from lean subjects (control group), obese patients, and obese-type 2 diabetic patients.

		Clinical Science Immediate Publication. Published on 28 Oct 2010 as manuscript CS20100355
	NO (nM)	Control	Obese group	Obese + type 2	P
		group (n=10)	(n=10)	diabetes
				(n=10)
	in the presence of	34.68 ± 1.98	6.11 ± 1.54	2.98 ± 0.7	< 0.0005*
	3x10 -5 M ACh
	in the presence of	21.06 ± 1.89	2.55 ± 0.6	1.62 ± 0.5	< 0.0001*
	6.4 IU/ml insulin
	Data are means ± SE. P values represent the statistical significance of comparisons. * differs
	from others.

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Acknowledgements

We appreciate the dedicated work of Marcela Toader and Marilena Isachi (biochemistry), Marilena Misici, Ana Manole, Elena Florea (electron microscopy). This work was supported by CNCSIS -UEFISCSU, project number 1159, PNII -IDEI code 1043/2008, by CNMP project number 42138, PNII -Parteneriat code 3334 /2008 and by European Social Fund -"Cristofor I. Simionescu" Postdoctoral Fellowship Programme (ID POSDRU/89/1.5/S/55216), Sectoral Operational Programme Human Resources Development 2007 -2013.

Author contribution

All authors of this manuscript had the same contribution to the scientific results.