Search

Chronic inflammation is an important etiology underlying obesity-related disorders such as insulin resistance and type 2 diabetes, and recent findings indicate that the macrophage can be the initiating cell type responsible for this chronic inflammatory state. The mammalian silent information regulator 2 homolog SIRT1 modulates several physiological processes important for life span, and a potential role of SIRT1 in the regulation of insulin sensitivity has been shown. However, with respect to inflammation, the role of SIRT1 in regulating the proinflammatory pathway within macrophages is poorly understood. Here, we show that knockdown of SIRT1 in the mouse macropbage RAW264.7 cell line and in intraperitoneal macrophages broadly activates the JNK and IKK inflammatory pathways and increases LPS-stimulated TNF[alpha] secretion. Moreover, gene expression profiles reveal that SIRT1 knockdown leads to an increase in inflammatory gene expression. We also demonstrate that SIRT1 activators inhibit LPS-stimulated inflammatory pathways, as well as secretion of TNF[alpha], in a SIRT1-dependent manner in RAW264.7 cells and in primary intraperitoneal macrophages. Treatment of Zucker fatty rats with a SIRT1 activator leads to greatly improved glucose tolerance, reduced hyperinsulinemia, and enhanced systemic insulin sensitivity during glucose clamp studies. These in vivo insulin-sensitizing effects were accompanied by a reduction in tissue inflammation markers and a decrease in the adipose tissue macrophage proinflammatory state, fully consistent with the in vitro effects of SIRT1 in macrophages. In conclusion, these results define a novel role for SIRT1 as an important regulator of macrophage inflammatory responses in the context of insulin resistance and raise the possibility that targeting of SIRT1 might be a useful strategy for treating the inflammatory component of metabolic diseases. macrophage; insulin resistance doi:10.1152/ajpendo.00417.2009.

Glucagon-like peptide-1 (GLP-1) is a polypeptide hormone secreted from enteroendocrine L cells and potentiates glucose-dependent insulin secretion in pancreatic [beta] cells. Recently the GLP-1 receptor (GLP-1 R) has been a focus for new anti-diabetic therapy with the introduction of GLP-1 analogues and DPP-IV inhibitors, and this has stimulated additional interest in the mechanisms of GLP-1 signaling. Here we identify a mechanism for GLP-1 action, showing that the scaffold protein [beta]-arrestin-1 mediates the effects of GLP-1 to stimulate cAMP production and insulin secretion in [beta] cells. Using a coimmunoprecipitation technique, we also found a physical association between the GLP-1 R and [beta]-arrestin-1 in cultured INS-1 pancreatic [beta] cells. [beta]-Arrestin-1 knockdown broadly attenuated GLpol signaling, causing decreased ERK and CREB activation and IRS-2 expression as well as reduced cAMP levels and impaired insulin secretion. However, [beta]-arrestin-1 knockdown did not affect GLP-1 R surface expression and ligand-induced GLP-1 R internalization/desensitization. Taken together, these studies indicate that [beta]-arrestin-1 plays a role in GLP-1 signaling leading to insulin secretion, defining a previously undescribed mechanism for GLP-1 action. CREB | IRS-2

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-LI 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. small interfering RNA; lentivirus; peroxisome proliferator activated-receptor-[gamma] doi:10.1152/ajpendo.00695.2006

Objective The aim of this study was to search for novel markers of visceral adiposity. Methods Visceral (omental) and subcutaneous adipose tissues were obtained from 43 Japanese men. Microarray analysis using total RNA from visceral and subcutaneous adipose tissues obtained from five men with abdominal obesity and five nonobese men was first conducted. Then the expression pattern of candidate genes identified in the human study in mouse models of adiposity was examined. Results Among 30,500 genes evaluated, the mRNA expression of CCDC3 (encoding coiled-coil domain-containing protein 3) was upregulated in omental adipose tissues from abdominally obese subjects (3.07-fold) but not in subcutaneous adipose tissues (0.89-fold). Similar expression patterns were found in two distinct mouse models of obesity. In the analysis of all 43 men, CCDC3 mRNA levels in omental, but not in subcutaneous adipose tissue, were positively correlated with waist circumference and body mass index. CCDC3 was predicted to be a secretory protein, which was confirmed by western blotting, as overexpressed CCDC3 was secreted into the culture media. Conclusions The expression of CCDC3 is specifically increased in visceral adipose tissues in abdominally obese subjects. These results suggest that CCDC3 is a potential biomarker for estimating visceral adiposity. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator-activated receptor-γ (PPAR-γ) 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-γ from 3T3-L1 adipocytes to elucidate its role in adipogenesis and insulin signaling. We show that PPAR-γ 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-γ 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-γ deletion was mediated by both GLUT4 and GLUT1. Interestingly, PPAR-γ-depleted cells displayed enhanced inflammatory responses to TNF-α stimulation, consistent with a chronic anti-inflammatory effect of endogenous PPAR-γ. In summary, 1) PPAR-γ is essential for the process of adipocyte differentiation but is less necessary for maintenance of the differentiated state, 2) PPAR-γ supports normal insulin-stimulated glucose transport, and 3) endogenous PPAR-γ may play a role in suppression of the inflammatory pathway in 3T3-L1 cells. [ABSTRACT FROM AUTHOR]

Many studies have suggested that Akt/protein kinase B, in particular Akt2/PKBβ, is required for insulin-stimulated glucose transporter GLUT4 translocation as well as glucose uptake in muscle and fat cells. Furthermore, insulin-stimulated Akt activation is impaired in adipose tissue from insulin resistant or type 2 diabetic animals and humans. However the downstream signaling events following Akt activation are relatively unknown. In this study, we identify an Akt consensus phosphorylation motif (RXRXXpS/T) in the actin based motor protein Myosin 5a, and show that insulin stimulation leads to the phosphorylation of Myosin 5a at serine 1650. Akt mediated phosphorylation of Myosin 5a enhances its ability to interact with actin filaments (F-actin), and siRNA-mediated inhibition of Myosin 5a attenuates insulin-stimulated GLUT4 translocation and glucose uptake in 3T3-LI adipocytes. Furthermore, siRNA-mediated knockdown of Akt2, but not Akt1, or overexpression of dominant negative Akt, abolish insulin-stimulated phosphorylation of Myosin 5a, Myosin 5a binding to F-actin and insulin-stimulated GLUT4 translocatoin. These data indicate that Myosin 5a is a newly identified insulin-stimulated substrate of Akt2, and that phosphorylated Myosin 5a facilitates anterograde movement of GLUT4 vesicles along F-actin. In addition, taken together with our previous findings, showing the connection between PKCλ and the microtubule-based motor KIF3 (MCB vol. 23, p4892), it is suggested that PKCλ and Akt2 sequentially cooperate to translocate GLUT4 vesicles from the perinuclear pool to the cell surface through the PKCλ/KIF3/microtubule and subsequent Akt2/Myosin 5a/Factin mechanisms. [ABSTRACT FROM AUTHOR]

Tumor necrosis factor-α (TNFα) is a proinflammatory cytokine secreted from macrophages and adipocytes. It is well known that chronic TNFα exposure can lead to insulin resistance both in vitro and in vivo and that elevated blood levels of TNFα are observed in obese and/or diabetic individuals. TNFα has many acute biologic effects, mediated by a complex intracellular signaling pathway. In these studies we have identified new G-protein signaling components to this pathway in 3T3-L1 adipocytes. We found that β-arrestin-1 is associated with TRAF2 (TNF receptor-associated factor 2), an adaptor protein of TNF receptors, and that TNFα acutely stimulates tyrosine phosphorylation of Gαq/11 with an increase in Gαq/11 activity. Small interfering RNA-mediated knockdown of β-arrestin-1 inhibits TNFα-induced tyrosine phosphorylation of Gαq/11 by interruption of Src kinase activation. TNFα stimulates lipolysis in 3T3-L1 adipocytes, and β-arrestin-1 knockdown blocks the effects of TNFα to stimulate ERK activation and glycerol release. TNFα also led to activation of JNK with increased expression of the proinflammatory gene, monocyte chemoattractant protein-1 and matrix metalloproteinase 3, and β-arrestin-1 knockdown inhibited both of these effects. Taken together these results reveal novel elements of TNFα action; i) the trimeric G-protein component Gαq/11 and the adapter protein β-arrestin-1 can function as signaling molecules in the TNFα action cascade; 2) β-arrestin-1 can couple TNFα stimulation to ERK activation and lipolysis; 3) β-arrestin-1 and Gαq/11 can mediate TNFα-induced phosphatidylinositol 3-kinase activation and inflammatory gene expression. [ABSTRACT FROM AUTHOR]

Nonalcoholic fatty liver disease is the most frequent liver disease. Ezetimibe, an inhibitor of intestinal cholesterol absorption, has been reported to ameliorate hepatic steatosis in human and animal models. To explore how ezetimibe reduces hepatic steatosis, we investigated the effects of ezetimibe on the expression of lipogenic enzymes and intestinal lipid metabolism in mice fed a high-fat or a high-fructose diet. CBA/JN mice were fed a high-fat diet or a high-fructose diet for 8 wk with or without ezetimibe. High-fat diet induced hepatic steatosis accompanied by hyperinsulinemia. Treatment with ezetimibe reduced hepatic steatosis, insulin levels, and glucose production from pyruvate in mice fed the high-fat diet, suggesting a reduction of insulin resistance in the liver. In the intestinal analysis, ezetimibe reduced the expression of fatty acid transfer protein-4 and apoB-48 in mice fed the high-fat diet. However, treatment with ezetimibe did not prevent hepatic steatosis, hyperinsulinemia, and intestinal apoB-48 expression in mice fed the high-fructose diet. Ezetimibe decreased liver X receptor-α binding to the sterol regulatory element-binding protein-1c promoter but not expression of carbohydrate response element-binding protein and fatty acid synthase in mice fed the high-fructose diet, suggesting that ezetimibe did not reduce hepatic lipogenesis induced by the high-fructose diet. Elevation of hepatic and intestinal lipogenesis in mice fed a high-fructose diet may partly explain the differences in the effect of ezetimibe.

MicroRNAs (miRNAs) are important posttranscriptional regulators of various biological pathways. In this study, we focused on the role of miRNAs during mitochondrial biogenesis in skeletal muscle. The expression of miR-494 was markedly decreased in murine myoblast C₂C₁₂ cells during myogenic differentiation, accompanied by an increase in mtDNA. Furthermore, the expression of predicted target genes for miR-494, including mitochondrial transcription factor A (mtTFA) and Forkhead box j3 (Foxj3), was posttranscriptionally increased during myogenic differentiation. Knockdown of miR-494 resulted in increased mitochondrial content and upregulation of mtTFA and Foxj3 at the protein level. A 3'-untranslated region reporter assay revealed that miR-494 knockdown directly upregulated the luciferase activity of mtTFA and Foxj3. All of these observations were reversed by overexpression of miR-494. Furthermore, the miR-494 content significantly decreased after endurance exercise in C57BL/6J mice, accompanied by an increase in expression of mtTFA and Foxj3 proteins. These results suggest that miR-494 regulates mitochondrial biogenesis by downregulating mtTFA and Foxj3 during myocyte differentiation and skeletal muscle adaptation to physical exercise.

Abnormal secretion of adipocytokines promotes atherosclerosis, diabetes and insulin resistance, and is mainly induced by adipocyte hypertrophy. Recently, the circulating adipocytokine concentrations were reported to change in the postprandial period, as the levels of TNFα, IL-6 IL-8 and MCP-1 increased after a meal, whereas that of adiponectin decreased. These data suggest that prandial modulation of cytokines may be involved in the pathogenesis of atherosclerosis in type 2 diabetes. However, the regulatory mechanism of such change is still unclear. In the present study, we identified this mechanism with a special focus on the functions of protein kinase C (PKC) and of the transcription factor AP-2β, both of which are associated with the pathophysiology of adipocytokine regulation. PKCµ was highly phosphorylated in the re-feeding condition compared to the fasting condition in mouse adipose tissue, while other PKC isoforms remained unchanged. Furthermore, overexpression of PKCµ in 3T3-L1 adipocytes, but not other PKC isoforms, positively regulated the mRNA expression and promoter activity of MCP-1 and IL-6, and negatively regulated those of adiponectin. AP-2β had similar effects on the expression and promoter activity of these adipocytokines. Interestingly, overexpression of PKCµ enhanced the stimulatory and inhibitory effects of AP-2β on the expression of these adipocytokines. Finally, PKCµ could not activate a mutant MCP-1 promoter lacking the AP-2β binding domain. Our results suggest that postprandial activation of PKCµ plays a role in disordered postprandial adipocytokine expression through AP-2β.

Tumor necrosis factor-alpha (TNFalpha) is a proinflammatory cytokine secreted from macrophages and adipocytes. It is well known that chronic TNFalpha exposure can lead to insulin resistance both in vitro and in vivo and that elevated blood levels of TNFalpha are observed in obese and/or diabetic individuals. TNFalpha has many acute biologic effects, mediated by a complex intracellular signaling pathway. In these studies we have identified new G-protein signaling components to this pathway in 3T3-L1 adipocytes. We found that beta-arrestin-1 is associated with TRAF2 (TNF receptor-associated factor 2), an adaptor protein of TNF receptors, and that TNFalpha acutely stimulates tyrosine phosphorylation of G alpha(q/11) with an increase in G alpha(q/11) activity. Small interfering RNA-mediated knockdown of beta-arrestin-1 inhibits TNFalpha-induced tyrosine phosphorylation of G alpha(q/11) by interruption of Src kinase activation. TNFalpha stimulates lipolysis in 3T3-L1 adipocytes, and beta-arrestin-1 knockdown blocks the effects of TNFalpha to stimulate ERK activation and glycerol release. TNFalpha also led to activation of JNK with increased expression of the proinflammatory gene, monocyte chemoattractant protein-1 and matrix metalloproteinase 3, and beta-arrestin-1 knockdown inhibited both of these effects. Taken together these results reveal novel elements of TNFalpha action; 1) the trimeric G-protein component G alpha(q/11) and the adapter protein beta-arrestin-1 can function as signaling molecules in the TNFalpha action cascade; 2) beta-arrestin-1 can couple TNFalpha stimulation to ERK activation and lipolysis; 3) beta-arrestin-1 and G alpha(q/11) can mediate TNFalpha-induced phosphatidylinositol 3-kinase activation and inflammatory gene expression.

During differentiation, expression of protein phosphatase-2Calpha (PP2Calpha) is increased in 3T3-L1 adipocytes. To elucidate the role of PP2Calpha in insulin signaling, we overexpressed wild-type (WT) PP2Calpha by adenovirus-mediated gene transfer in 3T3-L1 adipocytes. Overexpression of PP2Calpha-WT enhanced the insulin sensitivity of glucose uptake without any changes in the early steps of insulin signaling. Infection with adenovirus 5 expressing PP2Calpha-WT increased phosphatidylinositol 3-kinase (PI3K) activities in the immunoprecipitate using antibody against the p85 or p110 subunit under both basal and insulin-stimulated conditions, followed by activation of downstream steps in the PI3K pathway, such as phosphorylation of Akt, glycogen synthase kinase-3, and atypical protein kinase C. In contrast, overexpression of the phosphatase-defective mutant PP2Calpha(R174G) did not produce such effects. Furthermore, overexpression of PP2Calpha-WT (but not PP2Calpha(R174G)) decreased the (32)P-labeled phosphorylation state as well as the gel mobility shift of the p85 subunit, suggesting that dephosphorylation of the p85 subunit by PP2Calpha activation might stimulate PI3K catalytic activity. Moreover, knockdown of PP2Calpha by transfection of small interfering RNA led to a significant decrease in Akt phosphorylation. In addition, microinjection of anti-PP2Calpha antibody or PP2Calpha small interfering RNA led to decreased insulin-stimulated GLUT4 translocation. In conclusion, PP2Calpha is a new positive regulator of insulin sensitivity that acts through a direct activation of PI3K in 3T3-L1 adipocytes.