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Objective: Up-regulated expression of transcription-factor E2F1 in human visceral adipose tissue (VAT) characterizes a dysmetabolic obesity sub-phenotype. An E2F1-miRNA network has been described in multiple cancers. Here we investigated whether elevated VAT-E2F1 in obesity is associated with VAT-miRNA alterations similar to, or distinct from, those described in cancer. Furthermore, we assessed if E2F1-associated miRNA changes may contribute to the link between high- VAT-E2F1 and a dysmetabolic obesity phenotype. Methods: We assembled a cohort of patients with obesity and high-VAT-E2F1, matched by age, sex, ±BMI to patients with low-VAT-E2F1, with and without obesity (8 patients/groupX3 groups). We performed Nanostring©-based miRNA profiling of VAT samples from all 24 patients. Candidate E2F1-related miRNAs were validated by qPCR in an independent cohort of patients with extreme obesity, with or without type-2-diabetes (T2DM) (n = 20). Bioinformatic tools and manipulation of E2F1 expression in cells were used to establish the plausibility of the functional VAT-E2F1-miRNA network in obesity. Results: Among n = 798 identified miRNAs, 17 were differentially expressed in relation to E2F1 and not to obesity itself. No evidence for the cancer-related E2F1-miRNA network was identified in human VAT in obesity. In HEK293-cells, overexpression/downregulation of E2F1 correspondingly altered the expression of miRNA-206 and miRNA-210-5p, two miRNAs with reported metabolic functions consistent with those of E2F1. In VAT from both cohorts, the expression of both miRNA-206 and 210-5p intercorrelated, and correlated with the expression of E2F1. In cohort 1 we did not detect significant associations with biochemical parameters. In cohort 2 of patients with extreme obesity, all those with high VAT-E2F1 showed a diabetes-complicated obesity phenotype and higher expression of miRNA-206 and miRNA-210-5p, which also correlated with fasting glucose levels (both miRNAs) and fasting insulin (miRNA-210-5p). Conclusions: Whilst the previously described cancer-related E2F1-miRNA network does not appear to operate in VAT in obesity, miRNAs-206 and 210-5p may link high-E2F1 expression in VAT with diabetes-complicated extreme obesity phenotype. [ABSTRACT FROM AUTHOR]

Am J Physiol Endocrinol Metab 298: E1-E7, 2010. First published November 3, 2009; doi: 10.1152/ajpendo.00562.2009.--Autophagy is a major degradative pathway(s) by which intracellular components are delivered into the lysosomes. It is largely implicated in determining cell death and survival because it eliminates unnecessary, damaged, and/or potentially harmful cellular products and organelles and is an important source for nutrients and energy production under conditions of external nutrient deficiency. As such, autophagy has been suggested to contribute to the regulation of carbohydrate and protein metabolism during fasting. Recently, three papers implicated a role for autophagy in cellular lipid metabolism as well. This Perspectives article presents these novel findings in the context of prior studies on the role of autophagy and lysosomes in metabolic and energy regulation, discusses their points of agreement and opposing propositions, and outlines key outstanding questions. lipolysis; liquid droplet biogenesis and breakdown; liquid droplet-associated proteins; lysosomal proteolysis

Intraabdominal (IA) fat functionally differs from subcutaneous (SC) adipose tissue, likely contributing to its stronger association with obesity-induced morbidity and to differential response to medications. Drug-induced partial lipodystrophy, like in response to antiretroviral agents, is an extreme manifestation of the different response of different fat depots, with loss of SC but not IA. Investigating depot-specific adipocyte differences is limited by the low accessibility to IA fat and by the heterogenous cell population comprising adipose tissue. Here, we aimed at utilizing immortalized preadipocyte cell lines from IA (epididymal) or SC (inguinal) fat to investigate whether they differentially respond to the HIV protease inhibitor nelfinavir. Preadipocytes were readily amenable to adipogenesis, as evidenced by lipid accumulation, expression of adipose-specific genes, measurable lipolysis, and insulin responsiveness. Leptin secretion was higher by the SC line, consistent with known differences between IA and SC fat. As previously reported, nelfinavir inhibited adipogenesis downstream of C/EBP[beta], but similarly in both cell lines. In contrast, nelfinavir's capacity to diminish insulin signaling, decrease leptin secretion, enhance basal lipolysis, and decrease expression of the lipid droplet-associated protein perilipin occurred more robustly and/or at lower nelfinavir concentrations in the SC line. This was despite similar intracellular concentrations of nelfinavir (23.8 [+ or -] 5.6 and 33.6 [+ or -] 12.2 [micro]g/mg protein for inguinal and epididymal adipocytes, respectively, P = 0.46). The cell lines recapitulated depot-differential effects of nelfinavir observed in differentiated primary preadipocytes and with whole tissue explants. Thus, we report the use of fat depot-specific adipocyte cell lines for unraveling depot-differential responses to a drug causing partial lipodystrophy. nelfinavir; lipolysis; perilipin; intra-abdominal adipocytes; subcutaneous adipocytes

Increased oxidative stress is believed to be one of the mechanisms responsible for hyperglycemia-induced tissue damage and diabetic complications. In these studies, we undertook to characterize glucose uptake and oxidative stress in adipocytes of type 2 diabetic animals and to determine whether these promote the activation of PKC-[delta]. The adipocytes used were isolated either from C57Bl/6J mice that were raised on a high-fat diet (HF) and developed obesity and insulin resistance or from control animals. Basal glucose uptake significantly increased (8-fold) in HF adipocytes, and this was accompanied with upregulation of GLUT1 expression levels. Insulin-induced glucose uptake was inhibited in HF adipocytes and GLUT4 content reduced by 20% in these adipocytes. Reactive oxygen species (ROS) increased twofold in HF adipocytes compared with control adipocytes and were largely reduced with decreased glucose concentrations. At zero glucose, ROS levels were reduced to the normal levels seen in control adipocytes. The activity of PKC-[delta] increased twofold in HF adipocytes compared with control adipocytes and was further activated by [H.sub.2][O.sub.2]. Moreover, PKC-[delta] activity was inhibited in HF adipocytes either by glucose deprivation or by treatment with the antioxidant N-acetyl-L-cysteine. In summary, we propose that increased glucose intake in HF adipocytes increases oxidative stress, which in turn promotes the activation of PKC-[delta]. These consequential events may be responsible, at least in part, for development of HF diet-induced insulin resistance in the fat tissue. oxidative stress; protein kinase C-[delta]; insulin resistance; glucose metabolism; C57B1/6J mice

Obesity is a heterogenous condition that affects the life and health of patients to different degrees and in different ways. Yet, most approaches to treat obesity are not currently prescribed, at least in a systematic manner, based on individual obesity sub-phenotypes or specifically-predicted health risks. Adipose tissue is one of the most evidently affected tissues in obesity. The degree of adipose tissue changes – "adiposopathy", or as we propose to relate to herein as Obesity-related Adipose tissue Disease (OrAD), correspond, at least cross-sectionally, to the extent of obesity-related complications inflicted on an individual patient. This potentially provides an opportunity to better personalize anti-obesity management by utilizing the information that can be retrieved by assessing OrAD. This review article will summarize current knowledge on histopathological OrAD features which, beyond cross-sectional analyses, had been shown to predict future obesity-related endpoints and/or the response to specific anti-obesity interventions. In particular, the review explores adipocyte cell size, adipose tissue inflammation, and fibrosis. Rather than highly-specialized methods, we emphasize standard pathology laboratory approaches to assess OrAD, which are readily-available in most clinical settings. We then discuss how OrAD assessment can be streamlined in the obesity/weight-management clinic. We propose that current studies provide sufficient evidence to inspire concerted efforts to better explore the possibility of predicting obesity related clinical endpoints and response to interventions by histological OrAD assessment, in the quest to improve precision medicine in obesity. [ABSTRACT FROM AUTHOR]

The stimulation of glucose transport, lipogenesis and glycogen synthase activity were analyzed in 3T3-L1 adipocyte cell lines to determine the effects of oxidant stress on adipocyte insulin resistance. Prolonged exposure of 3T3-L1 adipocyte cell lines to glucose oxidase increased the uptake of basal 2-deoxyglucose and enhanced basal lipoxygenase activity. However, basal lipoxygenase activity was significantly reduced by insulin stimulation in a dose-dependent manner.

A study using the L6 line of skeletal muscle cells was conducted to determine the effect of the iron chelators deferoxamine and bipyridyl on glucose transport and the expression of the GLUT-1 transmembrane glycoprotein in cultured muscle cells. The results show that L6 muscle cells responded to the reduction of iron availability due to iron chelators by enhancing GLUT-1 expression, which increased glucose transport. Furthermore, muscle cells adapted to reduced iron availability without losing their normal sensitivity to insulin.

Inhibition of oxidative phosphorylation and the electron transport chain as well as hypoxic conditions led to elevated glucose uptake by L6 muscle cells and 3T3-L1 fat cells to twicethe normal levels while quickly depleting intracellular adenosine triphosphate stores. It is postulated that the depletion of energy stores activate glucose transport by synthesis and/or translocation of carrier proteins. This increasesthe availability of substrates for anaerobic energy generation.

Elevated expression of E2F1 in adipocyte fraction of human visceral adipose tissue (hVAT) associates with a poor cardiometabolic profile. We hypothesized that beyond directly activating autophagy and MAP3K5 (ASK)-MAP kinase signaling, E2F1 governs a distinct transcriptome that contributes to adipose tissue and metabolic dysfunction in obesity. We performed RNA sequencing of hVAT samples from age-, sex-, and BMI-matched patients, all obese, whose visceral E2F1 protein expression was either high (E2F1high) or low (E2F1low). Tumor necrosis factor superfamily (TNFSF) members, including TRAIL (TNFSF10), TL1A (TNFSF15), and their receptors, were enriched in E2F1high While TRAIL was equally expressed in adipocytes and stromal vascular fraction (SVF), TL1A was mainly expressed in SVF, and TRAIL-induced TL1A was attributed to CD4+ and CD8+ subclasses of hVAT T cells. In human adipocytes, TL1A enhanced basal and impaired insulin-inhibitable lipolysis and altered adipokine secretion, and in human macrophages it induced foam cell biogenesis and M1 polarization. Two independent human cohorts confirmed associations between TL1A and TRAIL expression in hVAT and higher leptin and IL6 serum concentrations, diabetes status, and hVAT-macrophage lipid content. Jointly, we propose an intra-adipose tissue E2F1-associated TNFSF paracrine loop engaging lymphocytes, macrophages, and adipocytes, ultimately contributing to adipose tissue dysfunction in obesity. [ABSTRACT FROM AUTHOR]

Adipose tissue inflammation and dysfunction are considered central in the pathogenesis of obesity-related dysmetabolism, but their role in the rapid metabolic recovery upon obesity reversal is less well defined. We hypothesized that changes in adipose tissue endocrine and paracrine mechanisms may support the rapid improvement of obesityinduced impairment in cellular lipid handling. C57Bl-6J mice were fed ad libitum either normal chow (NC) or high-fat diet (HFF) for 10 weeks. A dietary obesity reversal group was fed HFF for 8 weeks and then switched to NC for 2 weeks (HFF≤NC). Whole-body glucose homeostasis rapidly nearly normalized in the HFF≤NC mice (fasting glucose and insulin fully normalized, glucose and insulin tolerance tests reversed 82% to the NC group levels). During 2 weeks of the dietary reversal, the liver was significantly cleared from ectopic fat, and functionally, glucose production from pyruvate, alanine or fructose was normalized. In contrast, adipose tissue inflammation (macrophage infiltration and polarization) largely remained as in HFF, though obesity-induced adipose tissue macrophage lipid accumulation decreased by ~50%, and adipose tissue MAP kinase hyperactivation was reversed. Ex vivo, mild changes in adipose tissue adipocytokine secretion profile were noted. These corresponded to partial or full reversal of the excess cellular lipid droplet accumulation induced by HFF adipose tissue conditioned media in hepatoma or macrophage cells, respectively. We propose that early after initiating reversal of nutritional obesity, rapid metabolic normalization largely precedes resolution of adipose tissue inflammation. Nevertheless, we demonstrate a hitherto unrecognized contribution of adipose tissue to the rapid improvement in lipid handling by the liver and by macrophages. [ABSTRACT FROM AUTHOR]

Background: Visceral adipose tissue foam cells are increased in human obesity, and were implicated in adipose dysfunction and increased cardio-metabolic risk. In the circulation, non-classical monocytes (NCM) are elevated in obesity and associate with atherosclerosis and type 2 diabetes. We hypothesized that circulating NCM correlate and/or are functionally linked to visceral adipose tissue foam cells in obesity, potentially providing an approach to estimate visceral adipose tissue status in the non-surgical obese patient. Methods: We preformed ex-vivo functional studies utilizing sorted monocyte subclasses from healthy donors. Moreover, we assessed circulating blood monocyte subclasses and visceral fat adipose tissue macrophage (ATM) lipid content by flow-cytometry in paired blood and omental-fat samples collected from patients (n = 65) undergoing elective abdominal surgery. Results: Ex-vivo, NCM and NCM-derived macrophages exhibited lower lipid accumulation capacity compared to classical or intermediate monocytes/-derived macrophages. Moreover, of the three subclasses, NCM exhibited the lowest migration towards adipose tissue conditioned-media. In a cohort of n = 65, increased %NCM associated with higher BMI (r = 0.250,p<0.05) and ATM lipid content (r = 0.303,p<0.05). Among patients with BMI≥25Kg/m2, linear regression models adjusted for age, sex or BMI revealed that NCM independently associate with ATM lipid content, particularly in men. Conclusions: Collectively, although circulating blood NCM are unlikely direct functional precursor cells for adipose tissue foam cells, their increased percentage in the circulation may clinically reflect higher lipid content in visceral ATMs. [ABSTRACT FROM AUTHOR]

Background: Phosphatidylinositol-3,4,5-trisphosphate (PIP3) is a major lipid second messenger in insulin-mediated signalling towards the metabolic actions of this hormone in muscle and fat. Purpose: Assessing the intracellular transport of exogenous PIP3attached to a polymeric carrier in an attempt to overcome cellular insulin resistance. Methods: Artificial chromatic bio-mimetic membrane vesicles composed of dimyristoylphosphatidylcholine and polydiacetylene were applied to screen the polymeric carriers. PIP3cellular localization and bio-activity was assessed by fluorescent and live-cell microscopy in L6 muscle cells and in 3T3-L1 adipocytes. Results and discussion: We demonstrate that a specific-branched polyethylenimine (PEI-25, 25 kDa) carrier forms complexes with PIP3that interact with the bio-mimetic membrane vesicles in a manner predictive of their interaction with cells: In L6 muscle cells, PEI-25/fluorescent-PIP3complexes are retarded at the cell perimeter. PEI-25/PIP3complexes retain their bio-activity, engaging signalling steps downstream of PIP3, even in muscle cells rendered insulin resistant by exposure to high glucose/high insulin. Conclusions: Inducing insulin actions by intracellular PIP3delivery (PEI-25/PIP3complexes) in some forms of insulin-resistant cells provides the first proof-of-principle for the potential therapeutic use of PIP3in a “second-messenger agonist” approach. In addition, utilization of an artificial bio-mimetic membrane platform to screen for highly efficient PIP3delivery predicts biological function in cells. [ABSTRACT FROM PUBLISHER]

Objective: To examine if, as in obesity, pregnancies complicated by gestational diabetes mellitus (GDM) exhibit increased macrophage in filtration and activated MAP-kinases in omental adipose tissue. Methods: Paired omental (OM) and abdominal subcutaneous (SC) fat samples were collected from 11 GDM and 20 normal pregnancies during cesarean delivery. Tissues were stained to detect macrophages, and analyzed to assess MAP-kinases. Results: OM had higher macrophage counts than SC in GDM (6.10 ± 2.20 versus 2.53± 1.45, p = 0.04), but not in normal pregnancies (p = 0.346). GDM pregnancies had more macrophages than normal pregnancies in OM (6.10± 2.20 versus 1.29± 0.55, p = 0.01), while only a trend was observed in SC fat (p = 0.08). Significant correlation (R = 0.619, p = 0.005) was observed between OM-macrophage in filtration and insulin resistance. Using multivariate analysis, only obesity independently associated with GDM. Expression of total p38MAP-kinase was higher in OM versus SC in both normal and GDM pregnancies, without significant differences between these groups. However, expression of activated p-p38MAP-kinase, and its upstream kinase MKK4, was comparable between fat depots. Conclusion: GDM pregnancies demonstrate increased macrophage in filtration to OM fat, correlating with higher insulin resistance. As in non-pregnant-patients obesity and OM macrophage in filtration may be on the same causal pathway, leading to GDM. Yet, this occurs without activation of p38MAP-kinase signaling. [ABSTRACT FROM AUTHOR]

The inflammasome has been recently implicated in obesity-associated dys-metabolism. However, of its products, the specific role of IL-1β was clinically demonstrated to mediate only the pancreatic beta-cell demise, and in mice mainly the intra-hepatic manifestations of obesity. Yet, it remains largely unknown if IL-1beta;, a cytokine believed to mainly function locally, could regulate dysfunctional inter-organ crosstalk in obesity. Here we show that High-fat-fed (HFF) mice exhibited a preferential increase of IL-1β in portal compared to systemic blood. Moreover, portally-drained mesenteric fat transplantation from IL-1βKO donors resulted in lower pyruvate-glucose flux compared to mice receiving wild-type (WT) transplant. These results raised a putative endocrine function for visceral fat-derived IL-1β in regulating hepatic gluconeogenic flux. IL-1βKO mice on HFF exhibited only a minor or no increase in adipose expression of pro-inflammatory genes (including macrophage M1 markers), Mac2-positive crown-like structures and CD11β-F4/80-double-positive macrophages, all of which were markedly increased in WT-HFF mice. Further consistent with autocrine/paracrine functions of IL-1β within adipose tissue, adipose tissue macrophage lipid content was increased in WT-HFF mice, but significantly less in IL-1βKO mice. Ex-vivo, adipose explants co-cultured with primary hepatocytes from WT or IL-1-receptor (IL-1RI)-KO mice suggested only a minor direct effect of adipose-derived IL-1β on hepatocyte insulin resistance. Importantly, although IL- 1βKOs gained weight similarly to WT-HFF, they had larger fat depots with similar degree of adipocyte hypertrophy. Furthermore, adipogenesis genes and markers (pparg, cepba, fabp4, glut4) that were decreased by HFF in WT, were paradoxically elevated in IL-1βKO-HFF mice. These local alterations in adipose tissue inflammation and expansion correlated with a lower liver size, less hepatic steatosis, and preserved insulin sensitivity. Collectively, we demonstrate that by promoting adipose inflammation and limiting fat tissue expandability, IL-1β supports ectopic fat accumulation in hepatocytes and adipose-tissue macrophages, contributing to impaired fat-liver crosstalk in nutritional obesity. [ABSTRACT FROM AUTHOR]

Insulin responsiveness of adipocytes is acquired during normal adipogenesis, and is essential for maintaining whole-body insulin sensitivity. Differentiated adipocytes exposed to oxidative stress become insulin resistant, exhibiting decreased expression of genes like the insulin-responsive glucose transporter GLUT4. Here we assessed the effect of oxidative stress on DNA binding capacity of C/EBP isoforms known to participate in adipocyte differentiation, and determine the relevance for GLUT4 gene regulation. By electrophoretic mobility shift assay, nuclear proteins from oxidized adipocytes exhibited decreased binding of C/EBPα-containing dimers to a DNA oligonucleotide harboring the C/EBP binding sequence from the murine GLUT4 promoter. C/EBPδ-containing dimers were increased, while C/EBPβ-dimers were unchanged. These alterations were mirrored by a 50% decrease and a 2-fold increase in the protein content of C/EBPα and C/EBPδ, respectively. In oxidized cells, GLUT4 protein and mRNA levels were decreased, and a GLUT4 promoter segment containing the C/EBP binding site partially mediated oxidative stress-induced repression of a reported gene. The antioxidant lipoic acid protected against oxidation-induced decrease in GLUT4 and C/EBPα mRNA, but did not prevent the increase in C/EBPδ mRNA. We propose that oxidative stress induces adipocyte insulin resistance partially by affecting the expression of C/EBPα and δ, resulting in altered C/EBP-dimer composition potentially occupying the GLUT4 promoter. [ABSTRACT FROM AUTHOR]

Increased oxidative stress is believed to be one of the mechanisms responsible for hyperglycemia-induced tissue damage and diabetic complications. In these studies, we undertook to characterize glucose uptake and oxidative stress in adipocytes of type 2 diabetic animals and to determine whether these promote the activation of PKC-δ. The adipocytes used were isolated either from C57B1/6J mice that were raised on a high-fat diet (HF) and developed obesity and insulin resistance or from control animals. Basal glucose uptake significantly increased (8-fold) in HF adipocytes, and this was accompanied with upregulation of GLUT1 expression levels. Insulin-induced glucose uptake was inhibited in HF adipocytes and GLUT4 content reduced by 20% in these adipocytes. Reactive oxygen species (ROS) increased twofold in HF adipocytes compared with control adipocytes and were largely reduced with decreased glucose concentrations. At zero glucose, ROS levels were reduced to the normal levels seen in control adipocytes. The activity of PKC-δ increased twofold in HF adipocytes compared with control adipocytes and was further activated by H[sub 2]O[sub 2]. Moreover, PKC-δ activity was inhibited in HF adipocytes either by glucose deprivation or by treatment with the antioxidant N-acetyl-L-cysteine. In summary, we propose that increased glucose intake in HF adipocytes increases oxidative stress, which in turn promotes the activation of PKC-5. These consequential events may be responsible, at least in part, for development of HF dietinduced insulin resistance in the fat tissue. [ABSTRACT FROM AUTHOR]

HIV protease inhibitors (HPIs) are potent antiretroviral agents clinically used in the management of HIV infection. Recently, HPI therapy has been linked to the development of a metabolic syndrome in which adipocyte insulin resistance appears to play a major role. In this study, we assessed the effect of nelfinavir on glucose uptake and lipolysis in differentiated 3T3-L1 adipocytes. An 18-h exposure to nelfinavir resulted in an impaired insulin-stimulated glucose uptake and activation of basal lipolysis. Impaired insulin stimulation of glucose up take occurred at nelfinavir concentrations >10 micromol/l (EC(50) = 20 micromol/l) and could be attributed to impaired GLUT4 translocation. Basal glycerol and free fatty acid (FFA) release were significantly enhanced with as low as 5 micromol/l nelfinavir, displaying fivefold stimulation of FFA release at 10 micromol/l. Yet, the antilipolytic action of insulin was preserved at this concentration. Potential underlying mechanisms for these metabolic effects included both impaired insulin stimulation of protein kinase B Ser 473 phosphorylation with preserved insulin receptor substrate tyrosine phosphorylation and decreased expression of the lipolysis regulator perilipin. Troglitazone pre- and cotreatment with nelfinavir partly protected the cells from the increase in basal lipolysis, but it had no effect on the impairment in insulin-stimulated glucose uptake induced by this HPI. This study demonstrates that nelfinavir induces insulin resistance and activates basal lipolysis in differentiated 3T3-L1 adipocytes, providing potential cellular mechanisms that may contribute to altered adipocyte metabolism in treated HIV patients. [ABSTRACT FROM AUTHOR]

The protein kinase dependent on adenosine 3′: 5′-monophosphate (cAMP) in homogenates of rat intestinal mucosa was found to be remarkably unstable during its purification due (at least in part) to proteolysis. Since soybean trypsin inhibitor was found to protect the kinase from inactivation, an attempt was made to use this inhibitor, coupled to Sepharose, in order to extract and remove the proteolytic enzyme(s) responsible for the instability at an early stage of the purification. Indeed, this column excluded the undissociated form of cAMP-dependent protein kinase (R2C2) in a considerably more stable form, illustrating the use of immobilized proteolytic inhibitors for stabilizing proteins in crude extracts containing potent proteases. But unexpectedly the same column retarded a protein kinase which was not activated by cAMP. The latter enzyme was identified as the free catalytic subunit (C) of cAMP-dependent protein kinase since it was found to be inhibited by the Walsh-Krebs specific protein inhibitor. On the basis of these observations, a method was developed for the purification of C from rabbit muscle which is based on (a) passage of the crude extract on the column and (b) biospecific dissociation of R2C2 by cAMP and rechromatography of the extract on the same column. This procedure yields > 300-fold purification of C in the last step (with a recovery of 34% and a rapid resolution between R2C2 and C. The same column can also be used for the resolution of type I and type II cAMP-dependent protein kinases, and for a > 40-fold purification of the type II form of the enzyme, in one step. The theoretical and practical implications of these results with regard to the use and abuse of biorecognition in chromatography are discussed. [ABSTRACT FROM AUTHOR]

Protein S-nitrosylation is a reversible protein modification implicated in both physiological and pathophysiological regula- tion of protein function. In obesity, skeletal muscle insulin resistance is associated with increased S-nitrosylation of insu- lin-signaling proteins. However, whether adipose tissue is simi- larly affected in obesity and, if so, what are the causes and func- tional consequences of increased S-nitrosylation in this tissue are unknown. Total protein S-nitrosylation was increased in intra-abdominal adipose tissue of obese humans and in high fat-fed or leptin-deficient ob/ob mice. Both the insulin receptor β-subunit and Akt were S-nitrosylated, correlating with body weight. Elevated protein and mRNA expression of inducible NO synthase and decreased protein levels of thioredoxin reductase were associated with increased adipose tissue S-nitrosylation. Cultured differentiated pre-adipocyte cell lines exposed to the NO donors S-nitrosoglutathione (GSNO) or S-nitroso-N- acetylpenicillamine exhibited diminished insulin-stimulated phosphorylation of Akt but not of GSK3 nor of insulin-stimu- lated glucose uptake. Yet the anti-lipolytic action of insulin was markedly impaired in both cultured adipocytes and in mice injected with GSNO prior to administration of insulin. In cells, impaired ability of insulin to diminish phosphorylated PKA sub- strates in response to isoproterenol suggested impaired insulin- induced activation of PDE3B. Consistently, increased S-nitrosy- lation of PDE3B was detected in adipose tissue of high fat-fed obese mice. Site-directed mutagenesis revealed that Cys-768 and Cys-1040, two putative sites for S-nitrosylation adjacent to the substrate-binding site of PDE3B, accounted for ~ 50% of its GSNO-induced S-nitrosylation. Collectively, PDE3B and the anti-lipolytic action of insulin may constitute novel targets for increased S-nitrosylation of adipose tissue in obesity. [ABSTRACT FROM AUTHOR]