Functional heterogeneity of CD11c-positive adipose tissue macrophages in diet-induced obese mice.

Obesity represents a state of chronic, low grade inflammation and is associated with infiltration of increased numbers of adipose tissue macrophages (ATMs). Diet-induced obesity leads to an increase in non-inflammatory M1-like ATMs displaying the CD11c surface marker. We assessed the function of CD11c-positive ATMs when insulin resistant high fat diet (HFD) mice become insulin-sensitive after switching from HFD to normal chow (NC). HFD mice rapidly become insulin-sensitive in all major insulin-target tissues, including muscle, liver, and adipose tissue, after the diet switch. In adipose tissue the CD11c-positive macrophages remain constant in number despite the presence of insulin sensitivity, but these macrophages now assume a new phenotype in which they no longer exhibit increased inflammatory pathway markers. Adipose tissue markers of apoptosis and necrosis were elevated on HFD and remain high after the HFD --> NC diet switch. Furthermore, ATM accumulation preceded detectable adipocyte necrosis at the early phase of HFD. Together, these results indicate that 1) CD11c-positive M1-like ATMs can exhibit phenotypic plasticity and that the polarization of these cells between inflammatory and non-inflammatory states is well correlated to the presence of absence of insulin resistance, and 2) adipocyte necrosis and apoptosis can be dissociated from ATM accumulation.

Glucocorticoids and thiazolidinediones interfere with adipocyte-mediated macrophage chemotaxis and recruitment.

The link between intra-abdominal adiposity and type II diabetes has been known for decades, and adipose tissue macrophage (ATM)-associated inflammation has recently been linked to insulin resistance. However, the mechanisms associated with ATM recruitment remain ill defined. Herein, we describe in vitro chemotaxis studies, in which adipocyte conditioned medium was used to stimulate macrophage migration. We demonstrate that tumor necrosis factor alpha and free fatty acids, key inflammatory stimuli involved in obesity-associated autocrine/paracrine inflammatory signaling, stimulate adipocyte expression and secretion of macrophage chemoattractants. Pharmacological studies showed that peroxisome proliferator-activated receptor gamma agonists and glucocorticoids potently inhibit adipocyte- induced recruitment of macrophages. This latter effect was mediated by the glucocorticoid receptor, which led to decreased chemokine secretion and expression. In vivo results were quite comparable; treatment of high fat diet-fed mice with dexamethasone prevented ATM accumulation in epididymal fat. This decrease in ATM was most pronounced for the proinflammatory F4/80(+), CD11b(+), CD11c(+) M-1-like ATM subset. Overall, our results elucidate a beneficial function of peroxisome proliferator-activated receptor gamma activation and glucocorticoid receptor/glucocorticoids in adipose tissue and indicate that pharmacologic prevention of ATM accumulation could be beneficial.

Impaired oxidative metabolism and inflammation are associated with insulin resistance in ERalpha-deficient mice.

Impaired estrogen action is associated with the metabolic syndrome in humans. We sought to determine whether impaired estrogen action in female C57Bl6 mice, produced by whole body Esr1 ablation, could recapitulate aspects of this syndrome, including inflammation, insulin resistance, and obesity. Indeed, we found that global knockout (KO) of the estrogen receptor (ER)alpha leads to reduced oxygen uptake and caloric expenditure compared with wild-type (WT) mice. In addition, fasting insulin, leptin, and PAI-1 levels were markedly elevated, whereas adiponectin levels were reduced in normal chow-fed KO. Furthermore, ERalpha-KO mice exhibited impaired glucose tolerance and marked skeletal muscle insulin resistance that was accompanied by the accumulation of bioactive lipid intermediates, inflammation, and diminished PPARalpha, PPARdelta, and UCP2 transcript levels. Although the relative glucose intolerance and insulin resistance phenotype in KO mice became more severe with high-fat feeding, WT mice were refractory to these dietary-induced effects, and this protection coincided with a marked increase in circulating adiponectin and heat shock protein 72 levels in muscle, liver, and fat. These data indicate that ERalpha is critical for the maintenance of whole body insulin action and protection against tissue inflammation during both normal chow and high-fat feeding.

Macrophage PPAR gamma is required for normal skeletal muscle and hepatic insulin sensitivity and full antidiabetic effects of thiazolidinediones.

PPAR gamma is required for fat cell development and is the molecular target of antidiabetic thiazolidinediones (TZDs), which exert insulin-sensitizing effects in adipose tissue, skeletal muscle, and liver. Unexpectedly, we found that inactivation of PPAR gamma in macrophages results in the development of significant glucose intolerance plus skeletal muscle and hepatic insulin resistance in lean mice fed a normal diet. This phenotype was associated with increased expression of inflammatory markers and impaired insulin signaling in adipose tissue, muscle, and liver. PPAR gamma-deficient macrophages secreted elevated levels of factors that impair insulin responsiveness in muscle cells in a manner that was enhanced by exposure to FFAs. Consistent with this, the relative degree of insulin resistance became more severe in mice lacking macrophage PPAR gamma following high-fat feeding, and these mice were only partially responsive to TZD treatment. These findings reveal an essential role of PPAR gamma in macrophages for the maintenance of whole-body insulin action and in mediating the antidiabetic actions of TZDs.

Adenovirus-mediated chronic "hyper-resistinemia" leads to in vivo insulin resistance in normal rats.

We investigated the chronic in vivo effect of resistin on insulin sensitivity and glucose metabolism by overexpressing resistin protein in male Wistar rats using intravenous administration of an adenovirus encoding mouse resistin. After 7 days of elevated resistin levels at a supraphysiological concentration, the animals displayed glucose intolerance and hyperinsulinemia during glucose tolerance tests, and insulin tolerance tests demonstrated an impaired glucose-lowering effect of insulin. The glucose clamp studies were performed at submaximal (4 mU/kg/min) and maximal (25 mU/kg/min) insulin infusion rates and demonstrated the presence of insulin resistance induced by elevated resistin levels. Indeed, the insulin-stimulated glucose infusion rate was decreased by 12-31%; suppression of hepatic glucose output was attenuated by 28-55%; and insulin suppression of circulating FFA levels was inhibited by 7%. Insulin receptor substrate-1 and -2 phosphorylation and Akt activation were impaired in muscle and adipose tissue. Interestingly, activation of AMP-activated protein kinase in skeletal muscle, liver, and adipose tissue was also significantly downregulated. Together, these results indicate that chronic "hyper-resistinemia" leads to whole-body insulin resistance involving impaired insulin signaling in skeletal muscle, liver, and adipose tissue, resulting in glucose intolerance, hyperinsulinemia, and hypertriglyceridemia. Thus elevated resistin levels in normal rats fed a regular chow diet produce many of the features of human syndrome X.

Lentiviral short hairpin ribonucleic acid-mediated knockdown of GLUT4 in 3T3-L1 adipocytes.

Adipose tissue is an important insulin target organ, and 3T3-L1 cells are a model cell line for adipocytes. In this study, we have used lentivirus-mediated short hairpin RNA (shRNA) for functional gene knockdown in 3T3-L1 adipocytes to assess the molecular mechanisms of insulin signaling. We chose to target GLUT4 to validate this approach. We showed that lentiviruses efficiently delivered transgenes and small interfering RNA (siRNA) into fully differentiated 3T3-L1 adipocytes. We established a strategy for identifying efficient siRNA sequences for gene knockdown by transfecting 293 cells with the target gene fluorescent fusion protein plasmid along with a plasmid that expresses shRNA. Using these methods, we identified highly efficient siGLUT4 sequences. We demonstrated that lentivirus-mediated shRNA against GLUT4 reduced endogenous GLUT4 expression to almost undetectable levels in 3T3-L1 adipocytes. Interestingly, insulin-stimulated glucose uptake was only reduced by 50-60%, suggesting that another glucose transporter mediates part of this effect. When siGLUT1 was introduced into GLUT4-deficient adipocytes, insulin-stimulated glucose uptake was essentially abolished, indicating that both GLUT4 and GLUT1 contribute to insulin-stimulated glucose transport in 3T3-L1 adipocytes. We also found that GLUT4 knockdown led to impaired insulin-responsive aminopeptidase protein expression that was dependent on whether GLUT4 was knocked down in the differentiating or differentiated stage. We further found that GLUT4 expression was not required for adipogenic differentiation but was necessary for full lipogenic capacity of differentiated adipocytes. These studies indicate that lentiviral shRNA constructs provide an excellent approach to deliver functional siRNAs into 3T3-L1 adipocytes for studying insulin signaling and adipocyte biology.

Adenovirus-mediated adiponectin expression augments skeletal muscle insulin sensitivity in male Wistar rats.

In this study, we investigated the chronic in vivo effect of adiponectin on insulin sensitivity and glucose metabolism by overexpressing the adiponectin protein in male Wistar rats using intravenous administration of an adenovirus (Adv-Adipo). Virally infected liver secreted adiponectin as high and low molecular weight complexes. After 7 days of physiological or supraphysiological hyperadiponectinemia, the animals displayed enhanced insulin sensitivity during the glucose tolerance and insulin tolerance tests. Glucose clamp studies performed at submaximal and maximal insulin infusion rates (4 and 25 mU x kg(-1) x min(-1), respectively) also demonstrated increased insulin sensitivity in Adv-Adipo animals, with the insulin-stimulated glucose disposal rate being increased by 20-67%. In contrast, insulin's effect on the suppression of hepatic glucose output and plasma free fatty acid levels was not enhanced in Adv-Adipo rats compared with controls, suggesting that high levels of adiponectin expression in the liver may lead to a local desensitization. Consistent with the clamp data, the activation of AMP-activated protein kinase was significantly enhanced in skeletal muscle (by 50%) but not in liver. One interesting finding was that in male Wistar rats, both AdipoR1 and AdipoR2 expression levels were higher in skeletal muscle than in liver, as it is the case in humans. These results indicate that chronic adiponectin treatment enhances insulin sensitivity and could serve as a therapy for human insulin resistance.

Regulation of chemokine and chemokine receptor expression by PPARγ in adipocytes and macrophages.

BACKGROUND: PPARγ plays a key role in adipocyte biology, and Rosiglitazone (Rosi), a thiazolidinedione (TZD)/PPARγ agonist, is a potent insulin-sensitizing agent. Recent evidences demonstrate that adipose tissue inflammation links obesity with insulin resistance and that the insulin-sensitizing effects of TZDs result, in part, from their anti-inflammatory properties. However the underlying mechanisms are unclear. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, we establish a link between free fatty acids (FFAs) and PPARγ in the context of obesity-associated inflammation. We show that treatment of adipocytes with FFAs, in particular Arachidonic Acid (ARA), downregulates PPARγ protein and mRNA levels. Furthermore, we demonstrate that the downregulation of PPARγ by ARA requires the activation the of Endoplamsic Reticulum (ER) stress by the TLR4 pathway. Knockdown of adipocyte PPARγ resulted in upregulation of MCP1 gene expression and secretion, leading to enhanced macrophage chemotaxis. Rosi inhibited these effects. In a high fat feeding mouse model, we show that Rosi treatment decreases recruitment of proinflammatory macrophages to epididymal fat. This correlates with decreased chemokine and decreased chemokine receptor expression in adipocytes and macrophages, respectively. CONCLUSIONS AND SIGNIFICANCE: In summary, we describe a novel link between FAs, the TLR4/ER stress pathway and PPARγ, and adipocyte-driven recruitment of macrophages. We thus both describe an additional potential mechanism for the anti-inflammatory and insulin-sensitizing actions of TZDs and an additional detrimental property associated with the activation of the TLR4 pathway by FA.

Suppression of PPAR-gamma attenuates insulin-stimulated glucose uptake by affecting both GLUT1 and GLUT4 in 3T3-L1 adipocytes.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) plays a critical role in regulating insulin sensitivity and glucose homeostasis. In this study, we identified highly efficient small interfering RNA (siRNA) sequences and used lentiviral short hairpin RNA and electroporation of siRNAs to deplete PPAR-gamma from 3T3-L1 adipocytes to elucidate its role in adipogenesis and insulin signaling. We show that PPAR-gamma knockdown prevented adipocyte differentiation but was not required for maintenance of the adipocyte differentiation state after the cells had undergone adipogenesis. We further demonstrate that PPAR-gamma suppression reduced insulin-stimulated glucose uptake without affecting the early insulin signaling steps in the adipocytes. Using dual siRNA strategies, we show that this effect of PPAR-gamma deletion was mediated by both GLUT4 and GLUT1. Interestingly, PPAR-gamma-depleted cells displayed enhanced inflammatory responses to TNF-alpha stimulation, consistent with a chronic anti-inflammatory effect of endogenous PPAR-gamma. In summary, 1) PPAR-gamma is essential for the process of adipocyte differentiation but is less necessary for maintenance of the differentiated state, 2) PPAR-gamma supports normal insulin-stimulated glucose transport, and 3) endogenous PPAR-gamma may play a role in suppression of the inflammatory pathway in 3T3-L1 cells.

A subpopulation of macrophages infiltrates hypertrophic adipose tissue and is activated by free fatty acids via Toll-like receptors 2 and 4 and JNK-dependent pathways.

Obesity and type 2 diabetes are characterized by decreased insulin sensitivity, elevated concentrations of free fatty acids (FFAs), and increased macrophage infiltration in adipose tissue (AT). Here, we show that FFAs can cause activation of RAW264.7 cells primarily via the JNK signaling cascade and that TLR2 and TLR4 are upstream of JNK and help transduce FFA proinflammatory signals. We also demonstrate that F4/80(+)CD11b(+)CD11c(+) bone marrow-derived dendritic cells (BMDCs) have heightened proinflammatory activity compared with F4/80(+)CD11b(+)CD11c(-) bone marrow-derived macrophages and that the proinflammatory activity and JNK phosphorylation of BMDCs, but not bone marrow-derived macrophages, was further increased by FFA treatment. F4/80(+)CD11b(+)CD11c(+) cells were found in AT, and the proportion and number of these cells in AT is increased in ob/ob mice and by feeding wild type mice a high fat diet for 1 and 12 weeks. AT F4/80(+)CD11b(+)CD11c(+) cells express increased inflammatory markers compared with F4/80(+)CD11b(+)CD11c(-) cells, and FFA treatment increased inflammatory responses in these cells. In addition, we found that CD11c expression is increased in skeletal muscle of high fat diet-fed mice and that conditioned medium from FFA-treated wild type BMDCs, but not TLR2/4 DKO BMDCs, can induce insulin resistance in L6 myotubes. Together our results show that FFAs can activate CD11c(+) myeloid proinflammatory cells via TLR2/4 and JNK signaling pathways, thereby promoting inflammation and subsequent cellular insulin resistance.

Topiramate treatment causes skeletal muscle insulin sensitization and increased Acrp30 secretion in high-fat-fed male Wistar rats.

We show that Topiramate (TPM) treatment normalizes whole body insulin sensitivity in high-fat diet (HFD)-fed male Wistar rats. Thus drug treatment markedly lowered glucose and insulin levels during glucose tolerance tests and caused increased insulin sensitization in adipose and muscle tissues as assessed by euglycemic clamp studies. The insulin-stimulated glucose disposal rate increased twofold (indicating enhanced muscle insulin sensitivity), and suppression of circulating FFAs increased by 200 to 300%, consistent with increased adipose tissue insulin sensitivity. There were no effects of TPM on hepatic insulin sensitivity in these TPM-treated HFD-fed rats. In addition, TPM administration resulted in a three- to fourfold increase in circulating levels of total and high-molecular-weight (HMW) adiponectin (Acrp30). Western blot analysis revealed normal AMPK (Thr(172)) phosphorylation in liver with a twofold increased phospho-AMPK in skeletal muscle in TPM-treated rats. In conclusion, 1) TPM treatment prevents overall insulin resistance in HFD male Wistar rats; 2) drug treatment improved insulin sensitivity in skeletal muscle and adipose tissue associated with enhanced AMPK phosphorylation; and 3) the tissue "specific" effects are associated with increased serum levels of adiponectin, particularly the HMW component.

Tankyrase-1 overexpression reduces genotoxin-induced cell death by inhibiting PARP1.

Poly(ADP-ribose) polymerases or PARPs are a family of NAD(+)-dependent enzymes that modify themselves and other substrate proteins with ADP-ribose polymers. The founding member PARP 1 is localized predominantly in the nucleus and is activated by binding to DNA lesions. Excessive PARP 1 activation following genotoxin treatment causes NAD(+) depletion and cell death, whereas pharmacological PARP 1 inhibition protects cells from genotoxicity. This study investigates whether cellular viability and NAD(+) metabolism are regulated by tankyrase-1, a PARP member localized predominantly in the cytosol. Using a tetracycline-sensitive promoter to regulate tankyrase-1 expression in Madin-Darby canine kidney (MDCK) cells, we found that a 40-fold induction of tankyrase-1 (from 1,500 to 60,000 copies per cell) lowers steady-state NAD(+) levels but does not affect basal cellular viability. Moreover, the induction confers protection against the oxidative agent H(2)O(2) and the alkylating agent MNNG, genotoxins that kill cells by activating PARP 1. The cytoprotective effect of tankyrase-1 is not due to enhanced scavenging of oxidants or altered expression of Mcl-1, an anti-apoptotic molecule previously shown to be down-regulated by tankyrase-1 in CHO cells. Instead, tankyrase-1 appears to protect cells by preventing genotoxins from activating PARP 1-mediated reactions such as PARP 1 automodification and NAD(+) consumption. Our findings therefore indicate a cytoprotective function of tankyrase-1 mediated through altered NAD(+) homeostasis and inhibition of PARP 1 function.

JNK and tumor necrosis factor-alpha mediate free fatty acid-induced insulin resistance in 3T3-L1 adipocytes.

Lipid infusion and high fat feeding are established causes of systemic and adipose tissue insulin resistance. In this study, we treated 3T3-L1 adipocytes with a mixture of free fatty acids (FFAs) to investigate the molecular mechanisms underlying fat-induced insulin resistance. FFA treatment impaired insulin receptor-mediated signal transduction and decreased insulin-stimulated GLUT4 translocation and glucose transport. FFAs activated the stress/inflammatory kinases c-Jun N-terminal kinase (JNK) and IKKbeta, and the suppressor of cytokine signaling protein 3, increased secretion of the inflammatory cytokine tumor necrosis factor (TNF)-alpha, and decreased secretion of adiponectin into the medium. RNA interference-mediated down-regulation of JNK blocked JNK activation and prevented most of the FFA-induced defects in insulin action. Blockade of TNF-alpha signaling with neutralizing antibodies to TNF-alpha or its receptors or with a dominant negative TNF-alpha peptide had a partial effect to inhibit FFA-induced cellular insulin resistance. We found that JNK activation by FFAs was not inhibited by blocking TNF-alpha signaling, whereas the FFA-induced increase in TNF-alpha secretion was inhibited by RNA interference-mediated JNK knockdown. Together, these results indicate that 1) JNK can be activated by FFAs through TNF-alpha-independent mechanisms, 2) activated JNK is a major contributor to FFA-induced cellular insulin resistance, and 3) TNF-alpha is an autocrine/paracrine downstream effector of activated JNK that can also mediate insulin resistance.