Fat-specific Protein 27 (FSP27) Interacts with Adipose Triglyceride Lipase (ATGL) to Regulate Lipolysis and Insulin Sensitivity in Human Adipocytes

In adipocytes, lipolysis is a highly regulated process involving hormonal signals, lipid droplet-associated proteins, and lipases. The discovery of new lipid droplet-associated proteins added complexity to the current model of lipolysis. In this study, we used cultured human adipocytes to demonstrate that fat-specific protein 27 (FSP27), an abundantly expressed protein in adipocytes, regulates both basal and stimulated lipolysis by interacting with adipose triglyceride lipase (ATGL, also called desnutrin or PNPLA2). We identified a core domain of FSP27, amino acids 120–220, that interacts with ATGL to inhibit its lipolytic function and promote triglyceride storage. We also defined the role of FSP27 in free fatty acid-induced insulin resistance in adipocytes. FSP27 depletion in human adipocytes increased lipolysis and inhibited insulin signaling by decreasing AKT phosphorylation. However, reducing lipolysis by either depletion of ATGL or expression of exogenous full-length FSP27 or amino acids 120–220 protected human adipocytes against the adverse effects of free fatty acids on insulin signaling. In embryonic fibroblasts derived from ATGL KO mice, exogenous free fatty acids did not affect insulin sensitivity. Our results demonstrate a crucial role for FSP27-ATGL interactions in regulating lipolysis, triglyceride accumulation, and insulin signaling in human adipocytes.     

Mice Lacking C1q Are Protected from High Fat Diet-induced Hepatic Insulin Resistance and Impaired Glucose Homeostasis

Complement activation is implicated in the development of obesity and insulin resistance, and loss of signaling by the anaphylatoxin C3a prevents obesity-induced insulin resistance in mice. Here we have identified C1q in the classical pathway as required for activation of complement in response to high fat diets. After 8 weeks of high fat diet, wild-type mice became obese and developed glucose intolerance. This was associated with increased apoptotic cell death and accumulation of complement activation products (C3b/iC3b/C3c) in liver and adipose tissue. Previous studies have shown that high fat diet-induced apoptosis is dependent on Bid; here we report that Bid-mediated apoptosis was required for complement activation in adipose and liver. Although C1qa deficiency had no effect on high fat diet-induced apoptosis, accumulation of complement activation products and the metabolic complications of high fat diet-induced obesity were dependent on C1q. When wild-type mice were fed a high fat diet for only 3 days, hepatic insulin resistance was associated with the accumulation of C3b/iC3b/C3c in the liver. Mice deficient in C3a receptor were protected against this early high fat diet-induced hepatic insulin resistance, whereas mice deficient in the negative complement regulator CD55/DAF were more sensitive to the high fat diet. C1qa^−/− mice were also protected from high fat diet-induced hepatic insulin resistance and complement activation. Evidence of complement activation was also detected in adipose tissue of obese women compared with lean women. Together, these studies reveal an important role for C1q in the classical pathway of complement activation in the development of high fat diet-induced insulin resistance.     

Selective Insulin Resistance in Adipocytes

Aside from glucose metabolism, insulin regulates a variety of pathways in peripheral tissues. Under insulin-resistant conditions, it is well known that insulin-stimulated glucose uptake is impaired, and many studies attribute this to a defect in Akt signaling. Here we make use of several insulin resistance models, including insulin-resistant 3T3-L1 adipocytes and fat explants prepared from high fat-fed C57BL/6J and ob/ob mice, to comprehensively distinguish defective from unaffected aspects of insulin signaling and its downstream consequences in adipocytes. Defective regulation of glucose uptake was observed in all models of insulin resistance, whereas other major actions of insulin such as protein synthesis and anti-lipolysis were normal. This defect corresponded to a reduction in the maximum response to insulin. The pattern of change observed for phosphorylation in the Akt pathway was inconsistent with a simple defect at the level of Akt. The only Akt substrate that showed consistently reduced phosphorylation was the RabGAP AS160 that regulates GLUT4 translocation. We conclude that insulin resistance in adipose tissue is highly selective for glucose metabolism and likely involves a defect in one of the components regulating GLUT4 translocation to the cell surface in response to insulin.     

Indomethacin Treatment Prevents High Fat Diet-induced Obesity and Insulin Resistance but Not Glucose Intolerance in C57BL/6J Mice

Chronic low grade inflammation is closely linked to obesity-associated insulin resistance. To examine how administration of the anti-inflammatory compound indomethacin, a general cyclooxygenase inhibitor, affected obesity development and insulin sensitivity, we fed obesity-prone male C57BL/6J mice a high fat/high sucrose (HF/HS) diet or a regular diet supplemented or not with indomethacin (±INDO) for 7 weeks. Development of obesity, insulin resistance, and glucose intolerance was monitored, and the effect of indomethacin on glucose-stimulated insulin secretion (GSIS) was measured in vivo and in vitro using MIN6 β-cells. We found that supplementation with indomethacin prevented HF/HS-induced obesity and diet-induced changes in systemic insulin sensitivity. Thus, HF/HS+INDO-fed mice remained insulin-sensitive. However, mice fed HF/HS+INDO exhibited pronounced glucose intolerance. Hepatic glucose output was significantly increased. Indomethacin had no effect on adipose tissue mass, glucose tolerance, or GSIS when included in a regular diet. Indomethacin administration to obese mice did not reduce adipose tissue mass, and the compensatory increase in GSIS observed in obese mice was not affected by treatment with indomethacin. We demonstrate that indomethacin did not inhibit GSIS per se, but activation of GPR40 in the presence of indomethacin inhibited glucose-dependent insulin secretion in MIN6 cells. We conclude that constitutive high hepatic glucose output combined with impaired GSIS in response to activation of GPR40-dependent signaling in the HF/HS+INDO-fed mice contributed to the impaired glucose clearance during a glucose challenge and that the resulting lower levels of plasma insulin prevented the obesogenic action of the HF/HS diet.     

Pathway-selective Insulin Resistance and Metabolic Disease: The Importance of Nutrient Flux

Hepatic glucose and lipid metabolism are altered in metabolic disease (e.g. obesity, metabolic syndrome, and Type 2 diabetes). Insulin-dependent regulation of glucose metabolism is impaired. In contrast, lipogenesis, hypertriglyceridemia, and hepatic steatosis are increased. Because insulin promotes lipogenesis and liver fat accumulation, to explain the elevation in plasma and tissue lipids, investigators have suggested the presence of pathway-selective insulin resistance. In this model, insulin signaling to glucose metabolism is impaired, but insulin signaling to lipid metabolism is intact. We discuss the evidence for the differential regulation of hepatic lipid and glucose metabolism. We suggest that the primary phenotypic driver is altered substrate delivery to the liver, as well as the repartitioning of hepatic nutrient handling. Specific alterations in insulin signaling serve to amplify the alterations in hepatic substrate metabolism. Thus, hyperinsulinemia and its resultant increased signaling may facilitate lipogenesis, but are not the major drivers of the phenotype of pathway-selective insulin resistance.     

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 associates with insulin receptor substrate-1 and enhances insulin actions and adipogenesis

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is a unique enzyme that associates with the pSer/Thr-Pro motif and catalyzes cis-trans isomerization. We identified Pin1 in the immunoprecipitates of overexpressed IRS-1 with myc and FLAG tags in mouse livers and confirmed the association between IRS-1 and Pin1 by not only overexpression experiments but also endogenously in the mouse liver. The analysis using deletion- and point-mutated Pin1 and IRS-1 constructs revealed the WW domain located in the N terminus of Pin1 and Ser-434 in the SAIN (Shc and IRS-1 NPXY binding) domain of IRS-1 to be involved in their association. Subsequently, we investigated the role of Pin1 in IRS-1 mediation of insulin signaling. The overexpression of Pin1 in HepG2 cells markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events: phosphatidylinositol 3-kinase binding with IRS-1 and Akt phosphorylation. In contrast, the treatment of HepG2 cells with Pin1 siRNA or the Pin1 inhibitor Juglone suppressed these events. In good agreement with these in vitro data, Pin1 knock-out mice exhibited impaired insulin signaling with glucose intolerance, whereas adenoviral gene transfer of Pin1 into the ob/ob mouse liver mostly normalized insulin signaling and restored glucose tolerance. In addition, it was also demonstrated that Pin1 plays a critical role in adipose differentiation, making Pin1 knock-out mice resistant to diet-induced obesity. Importantly, Pin1 expression was shown to be up-regulated in accordance with nutrient conditions such as food intake or a high-fat diet. Taken together, these observations indicate that Pin1 binds to IRS-1 and thereby markedly enhances insulin action, essential for adipogenesis.     

Insulin Resistance Induces Posttranslational Hepatic Sortilin 1 Degradation in Mice

Insulin promotes hepatic apolipoprotein B100 (apoB100) degradation, whereas insulin resistance is a major cause of hepatic apoB100/triglyceride overproduction in type 2 diabetes. The cellular trafficking receptor sortilin 1 (Sort1) was recently identified to transport apoB100 to the lysosome for degradation in the liver and thus regulate plasma cholesterol and triglyceride levels. Genetic variation of SORT1 was strongly associated with cardiovascular disease risk in humans. The major goal of this study is to investigate the effect and molecular mechanism of insulin regulation of Sort1. Results showed that insulin induced Sort1 protein, but not mRNA, in AML12 cells. Treatment of PI3K or AKT inhibitors decreased Sort1 protein, whereas expression of constitutively active AKT induced Sort1 protein in AML12 cells. Consistently, hepatic Sort1 was down-regulated in diabetic mice, which was partially restored after the administration of the insulin sensitizer metformin. LC-MS/MS analysis further revealed that serine phosphorylation of Sort1 protein was required for insulin induction of Sort1 in a casein kinase 2-dependent manner and that inhibition of PI3K signaling or prevention of Sort1 phosphorylation accelerated proteasome-dependent Sort1 degradation. Administration of a PI3K inhibitor to mice decreased hepatic Sort1 protein and increased plasma cholesterol and triglyceride levels. Adenovirus-mediated overexpression of Sort1 in the liver prevented PI3K inhibitor-induced Sort1 down-regulation and decreased plasma triglyceride but had no effect on plasma cholesterol in mice. This study identified Sort1 as a novel target of insulin signaling and suggests that Sort1 may play a role in altered hepatic apoB100 metabolism in insulin-resistant conditions.     

Porcine Adiponectin Receptor 1 Transgene Resists High-fat/Sucrose Diet-Induced Weight Gain,Hepatosteatosis and Insulin Resistance in Mice

Adiponectin and its receptors have been demonstrated to play important roles in regulating glucose and lipid metabolism in mice. Obesity, type II diabetes and cardiovascular disease are highly correlated with down-regulated adiponectin signaling. In this study, we generated mice overexpressing the porcine Adipor1 transgene (pAdipor1) to study its beneficial effects in metabolic syndromes as expressed in diet-induced obesity, hepatosteatosis and insulin resistance. Wild-type (WT) and pAdipor1 transgenic mice were fed ad libitum with a standard chow diet (Chow) or a high-fat/sucrose diet (HFSD) for 24 weeks, beginning at 6 to 7 weeks of age. There were 12 mice per genetic/diet/sex group. When challenged with HFSD to induce obesity, the pAdipor1 transgenic mice resisted development of weight gain, hepatosteatosis and insulin resistance. These mice had lowered plasma adiponectin, triglyceride and glycerol concentrations compared to WT mice. Moreover, we found that (indicated by mRNA levels) fatty acid oxidation was enhanced in skeletal muscle and adipose tissue, and liver lipogenesis was inhibited. The pAdipor1 transgene also restored HFSD-reduced phosphoenolpyruvate carboxykinase 1 (Pck1) and glucose transporter 4 mRNA in the adipose tissues, implying that the increased Pck1 may promote glyceroneogenesis to reduce glucose intolerance and thus activate the flux of glyceride-glycerol to resist diet-induced weight gain in the adipose tissues. Taken together, we demonstrated that pAdipor1 can prevent diet-induced weight gain and insulin resistance. Our findings may provide potential therapeutic strategies for treating metabolic syndromes and obesity, such as treatment with an ADIPOR1 agonist or activation of Adipor1 downstream targets.     

Deficiency of a Glycogen Synthase-associated Protein,Epm2aip1, Causes Decreased Glycogen Synthesis and Hepatic Insulin Resistance

Glycogen synthesis is a major component of the insulin response, and defective glycogen synthesis is a major portion of insulin resistance. Insulin regulates glycogen synthase (GS) through incompletely defined pathways that activate the enzyme through dephosphorylation and, more potently, allosteric activation. We identify Epm2aip1 as a GS-associated protein. We show that the absence of Epm2aip1 in mice impairs allosteric activation of GS by glucose 6-phosphate, decreases hepatic glycogen synthesis, increases liver fat, causes hepatic insulin resistance, and protects against age-related obesity. Our work identifies a novel GS-associated GS activity-modulating component of insulin resistance.     

Adipocyte-specific inactivation of Acyl-CoA synthetase fatty acid transport protein 4 (Fatp4) in mice causes adipose hypertrophy and alterations in metabolism of complex lipids under high fat diet

Fatp4 exhibits acyl-CoA synthetase activity and is thereby able to catalyze the activation of fatty acids for further metabolism. However, its actual function in most tissues remains unresolved, and its role in cellular fatty acid uptake is still controversial. To characterize Fatp4 functions in adipocytes in vivo, we generated a mouse line with adipocyte-specific inactivation of the Fatp4 gene (Fatp4(A-/-)). Under standard conditions mutant mice showed no phenotypical aberrance. Uptake of radiolabeled palmitic and lignoceric acid into adipose tissue of Fatp4(A-/-) mice was unchanged. When exposed to a diet enriched in long chain fatty acids, Fatp4(A-/-) mice gained more body weight compared with control mice, although they were not consuming more food. Pronounced obesity was accompanied by a thicker layer of subcutaneous fat and greater adipocyte circumference, although expression of genes involved in de novo lipogenesis was not changed. However, the increase in total fat mass was contrasted by a significant decrease in various phospholipids, sphingomyelin, and cholesteryl esters in adipocytes. Livers of Fatp4-deficient animals under a high fat diet exhibited a higher degree of fatty degeneration. Nonetheless, no evidence for changes in insulin sensitivity and adipose inflammation was found. In summary, the results of this study confirm that Fatp4 is not crucial for fatty acid uptake into adipocytes. Instead, under the condition of a diet enriched in long chain fatty acids, adipocyte-specific Fatp4 deficiency results in adipose hypertrophy and profound alterations in the metabolism of complex lipids.     

Short term high fat diet challenge promotes alternative macrophage polarization in adipose tissue via natural killer T cells and interleukin-4

Inflammation in adipose tissue plays an important role in the pathogenesis of obesity-associated complications. However, the detailed cellular events underlying the inflammatory changes at the onset of obesity have not been characterized. Here we show that an acute HFD challenge is unexpectedly associated with elevated alternative (M2) macrophage polarization in adipose tissue mediated by Natural Killer T (NKT) cells. Upon 4d HFD feeding, NKT cells are activated, promote M2 macrophage polarization and induce arginase 1 expression via interleukin (IL)-4 in adipose tissue, not in the liver. In NKT-deficient CD1d(-/-) mice, M2 macrophage polarization in adipose tissue is reduced while systemic glucose homeostasis and insulin tolerance are impaired upon 4d HFD challenge. Thus, our study demonstrate, for the first time to our knowledge, that acute HFD feeding is associated with remarkably pronounced and dynamic immune responses in adipose tissue, and adipose-resident NKT cells may link acute HFD feeding with inflammation.     

Fish Oil and Fenofibrate Prevented Phosphorylation-dependent Hepatic Sortilin 1 Degradation in Western Diet-fed Mice

Obesity and diabetes are associated with hepatic triglyceride overproduction and hypertriglyceridemia. Recent studies have found that the cellular trafficking receptor sortilin 1 (Sort1) inhibits hepatic apolipoprotein B secretion and reduces plasma lipid levels in mice, and its hepatic expression was negatively associated with plasma lipids in humans. This study investigated the regulation of hepatic Sort1 under diabetic conditions and by lipid-lowering fish oil and fenofibrate. Results showed that hepatic Sort1 protein, but not mRNA, was markedly lower in Western diet-fed mice. Knockdown of hepatic Sort1 increased plasma triglyceride in mice. Feeding mice a fish oil-enriched diet completely restored hepatic Sort1 levels in Western diet-fed mice. Fenofibrate also restored hepatic Sort1 protein levels in Western diet-fed wild type mice, but not in peroxisome proliferator-activated receptor α (PPARα) knock-out mice. PPARα ligands did not induce Sort1 in hepatocytes in vitro. Instead, fish oil and fenofibrate reduced circulating and hepatic fatty acids in mice, and n-3 polyunsaturated fatty acids prevented palmitate inhibition of Sort1 protein in HepG2 cells. LC/MS/MS analysis revealed that Sort1 phosphorylation at serine 793 was increased in obese mice and in palmitate-treated HepG2 cells. Mutations that abolished phosphorylation at Ser-793 increased Sort1 stability and prevented palmitate inhibition of Sort1 ubiquitination and degradation in HepG2 cells. In summary, therapeutic strategies that prevent posttranslational hepatic Sort1 down-regulation in obesity and diabetes may be beneficial in improving dyslipidemia.     

小鼠胰岛素抵抗哮喘模型的建立与评估

目的:探讨小鼠胰岛素抵抗哮喘模型的建立方法,并进行评估。方法:C57BL/6J小鼠随机分为4组:正常对照组(HC)、哮喘组(NIRA)均给予普通饲料喂养;胰岛素抵抗组IRNA)、胰岛素抵抗+哮喘组(IRA)均给予高脂饲料(D12492)喂养。每周称重,第6-14周,每周检测各组小鼠空腹血糖(FPG)、空腹血清胰岛素(FINS)水平,计算稳态模型胰岛素抵抗评价指数(HOMA-IR)评估胰岛素抵抗程度;小鼠胰岛素抵抗模型建立成功后在其基础上诱导哮喘模型,NIRA组和IRA组小鼠给予卵清蛋白(OVA)致敏、激发;HC组和IRNA组小鼠给予生理盐水作为对照,末次激发24后,制作肺病理切片,计数肺泡灌洗液(BALF)中白细胞总数及分类,检测血清和BALF中相关炎性因子的水平,比较各组小鼠胰岛素抵抗指数与哮喘评价指标,评估模型。结果:(1)第9周末,IRNA组、IRA组小鼠的HOMA-IR值均2.5,表明胰岛素抵抗小鼠模型建立成功;(2)肺组织病理切片中,HC组、IRNA组小鼠肺组织无炎症改变,NIRA组、IRA组炎细胞浸润明显,尤以IRA组更甚。(3)与HC组比较,NIRA组(P0.01)、IRA组(P0.01)BALF中白细胞总数、嗜酸性粒细胞比例明显增高;(4)血清中抗OVA特异性Ig E(P0.01)和Ig G1(P0.05)水平显著升高;(5)血清和BALF中IL-4(P0.01)、IL-17(P0.05)的水平明显升高,且IRA组(P0.05)明显高于NIRA组;IFN-γ(P0.05)的水平明显降低,且IRA组(P0.05)明显低于NIRA组。结论:用高脂饲料喂养C57BL/6J小鼠9周,可建立稳定的胰岛素抵抗模型,从第10周开始用OVA致敏、激发诱发哮喘,可成功建立稳定的胰岛素抵抗哮喘小鼠模型,为进一步研究胰岛素抵抗与哮喘相关机制奠定基础。     

Gr-1+ CD11b+ myeloid-derived suppressor cells suppress inflammation and promote insulin sensitivity in obesity

Activation of immune cells, including macrophages and CD8(+) T cells, contributes significantly to the advancement of obesity and its associated medical complications, such as atherosclerosis, insulin resistance, and type 2 diabetes. However, how the activation of these immune cells is regulated in vivo remains largely unexplored. Here we show that a group of immature myeloid cells with cell surface markers of Gr-1(+) CD11b(+) are highly enriched in peripheral tissues (i.e. liver and adipose tissues) during obesity. Down-regulation of these cells in obese animals significantly increases inflammation and impairs insulin sensitivity and glucose tolerance, whereas elevation of these cells via adoptive transfer has the opposite effects. Mechanistically, we show that under obese conditions, the Gr-1(+) cells suppress proliferation and induce apoptosis of CD8(+) T cells and are capable of skewing differentiation of macrophages into insulin-sensitizing, alternatively activated M2 macrophages. Taken together, our study demonstrates that immature myeloid cells provide a checks-and-balances platform to counter proinflammatory immune cells in the liver and adipose tissue during obesity to prevent overt immune responses.     

罗格列酮联合胰岛素对糖尿病大鼠氧化应激及血脂水平的影响

摘要 目的：探讨罗格列酮（RSG）联合胰岛素（INS）对糖尿病大鼠的氧化应激和血脂水平的影响。方法：30只健康雄性Wistar大鼠,随机分为3组：健康对照组、DM模型组和RSG+INS组,每组10只。采用链脲佐菌素（STZ）造模法进行糖尿病大鼠模型诱导。此后以RSG （3 mg/kg/day）灌胃,INS （2.25 U/kg/day）皮下注射,均连续给药8周,对RSG+INS组大鼠进行干预治疗。每2周定期记录各组大鼠的体重和血糖值1次;药物干预治疗8周后,取各组大鼠血清,对血清总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白（LDL）、高密度脂蛋白（HDL）、糖化血红蛋白（HbA1c）和丙二醛（MDA）的含量以及超氧化物歧化酶（SOD）和谷胱甘肽（GSH）的活性进行检测;应用苏木素碱性复红苦昧酸（HBFP）染色分析心肌形态变化。结果：RSG联合INS组治疗糖尿病大鼠,导致血糖发生时间依赖性下降（均P<0.05）;药物干预处理8周后,RSG+INS组大鼠的血清TC、TG、LDL和HbA1c水平均显著低于DM模型组,而HDL显著高于DM模型组（均P<0.05）;与DM模型组相比,RSG+INS组大鼠的血清MDA含量显著下降,而SOD和GSH活性则显著升高（均P<0.05）;与DM模型组相比,RSG+INS组心肌缺血/氧程度减轻。结论：RSG联合INS可有效对抗糖尿病大鼠体内的氧化应激损伤,改善糖尿病大鼠的脂代谢异常情况,并对心肌组织有一定的保护作用。