Carbenoxolone treatment attenuates symptoms of metabolic syndrome and atherogenesis in obese, hyperlipidemic mice

Glucocorticoids, which are well established to regulate body fat mass distribution, adipocyte lipolysis, hepatic gluconeogenesis, and hepatocyte VLDL secretion, are speculated to play a role in the pathology of metabolic syndrome. Recent focus has been on the activity of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1), which is capable of regenerating, and thus amplifying, glucocorticoids in key metabolic tissues such as liver and adipose tissue. To determine the effects of global 11beta-HSD1 inhibition on metabolic syndrome risk factors, we subcutaneously injected "Western"-type diet-fed hyperlipidemic mice displaying moderate or severe obesity [LDL receptor (LDLR)-deficient (LDLR(-/-)) mice and mice derived from heterozygous agouti (A(y)/a) and homozygous LDLR(-/-) breeding pairs (A(y)/a;LDLR(-/-) mice)] with the nonselective 11beta-HSD inhibitor carbenoxolone for 4 wk. Body composition throughout the study, end-point fasting plasma, and extent of hepatic steatosis and atherosclerosis were assessed. This route of treatment led to detection of high levels of carbenoxolone in liver and fat and resulted in decreased weight gain due to reduced body fat mass in both mouse models. However, only A(y)/a;LDLR(-/-) mice showed an effect of 11beta-HSD1 inhibition on fasting insulin and plasma lipids, coincident with a reduction in VLDL due to mildly increased VLDL clearance and dramatically decreased hepatic triglyceride production. A(y)/a;LDLR(-/-) mice also showed a greater effect of the drug on reducing atherosclerotic lesion formation. These findings indicate that subcutaneous injection of an 11beta-HSD1 inhibitor allows for the targeting of the enzyme in not only liver, but also adipose tissue, and attenuates many metabolic syndrome risk factors, with more pronounced effects in cases of severe obesity and hyperlipidemia.     

Carbenoxolone alters the morphology of adipose tissues and downregulates genes involved in adipogenesis, glucose transport and lipid metabolism in high-fat diet-fed mice

Glucocorticoid (GC) excess promotes adipose tissue accumulation, and 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) plays an important role in the local amplification of GC. Therefore, in this study, we investigated the effects of carbenoxolone (CBX), an 11β-HSD1 inhibitor, on morphological changes in visceral fat, and the expression of genes involved in adipogenesis and lipid metabolism in high-fat (HF) diet-fed mice. Mice were fed a HF diet from 5 weeks of age. At 10 weeks of age, the mice received an intraperitoneal injection of CBX or vehicle every day for 2 weeks. CBX decreased body weight and visceral fat mass, and improved insulin sensitivity in HF-fed mice. This was accompanied by reduced adipocyte size and a decrease in large-sized adipocytes in visceral fat. The expression of adipogenesis (PPARγ and C/EBPα), glucose transport (GLUT4) and lipid metabolism (LPL, ATGL, and HSL)-related genes were suppressed in CBX mice. CBX treatment induced beneficial morphological changes in visceral fat and decreased the expression of adipogenesis, glucose transport and lipid metabolism-related genes. These findings reveal a potential mechanism underling the effects of CBX on reduced fat accumulation and improved insulin sensitivity.     

Metabolic effects of sub-chronic ablation of the incretin receptors by daily administration of (Pro3)GIP and exendin(9-39)amide in obese diabetic (ob/ob) mice

Effects of chemical ablation of the GIP and GLP-1 receptors on metabolic aspects of obesity-diabetes were investigated using the stable receptor antagonists (Pro3)GIP and exendin(9-39)amide. Ob/ob mice received a daily i.p. injection of saline vehicle, (Pro3)GIP, exendin(9-39)amide or a combination of both peptides over a 14-day period. Non-fasting plasma glucose levels were significantly (p<0.05) lower in (Pro3)GIP-treated mice compared to control mice after just 9 days of treatment. (Pro3)GIP-treated mice also displayed significantly lower plasma glucose concentrations in response to feeding and intraperitoneal administration of either glucose or insulin (p<0.05 to p<0.001). The (Pro3)GIP-treated group also exhibited significantly (p<0.05) reduced pancreatic insulin content. Acute administration of exendin(9-39)amide immediately prior to re-feeding completely annulled the beneficial effects of sub-chronic (Pro3)GIP treatment, but non-fasting concentrations of active GLP-1 were unchanged. Combined sub-chronic administration of (Pro3GIP) with exendin(9-39)amide revealed no beneficial effects. Similarly, daily administration of exendin(9-39)amide alone had no significant effects on any of the metabolic parameters measured. These studies highlight an important role for GIP in obesity-related forms of diabetes, suggesting the possible involvement of GLP-1 in the beneficial actions of GIP receptor antagonism.     

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.     

Diabetic pregnancy in rats leads to impaired glucose metabolism in offspring involving tissue-specific dysregulation of 11beta-hydroxysteroid dehydrogenase type 1 expression

Population-based studies have shown that the offspring of diabetic mothers have an increased risk of developing obesity, insulin resistance, type 2 diabetes and hypertension in later life. To investigate mechanism for the high incidence of metabolic diseases in the offspring of diabetic mothers, we focused on the tissue-specific glucocorticoid regulation by 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) and studied offspring born to streptozotocin-induced diabetic rats. The body weights of newborn rats from diabetic mothers were heavier than those from control mothers. Offspring born to diabetic mothers demonstrated insulin resistance and mild glucose intolerance after glucose loading at 10 weeks and showed significantly increased 11beta-HSD1 mRNA and enzyme activity in adipose tissue at 12 weeks of age without obvious obesity. Hepatic 11beta-HSD1 mRNA was also elevated. We propose that the 11beta-HSD1 in adipose tissue and liver may play a key role in the development of metabolic syndrome in the offspring of diabetic mothers. Tissue-specific glucocorticoid dysregulation provides a candidate mechanism for the high incidence of metabolic diseases in the offspring of diabetic mothers. Therefore early analyses before apparent obesity are needed to elucidate the molecular mechanisms that may be programmed during the fetal period.     

丁酸通过增强肝线粒体功能缓解高脂诱导的小鼠肥胖

丁酸可以预防高脂日粮诱导的小鼠肥胖和胰岛素抵抗,但是否有治疗作用尚不清楚。本研究证明,高脂日粮诱导小鼠肥胖模型后,用80 mg/mL丁酸钠水溶液灌胃能够缓解肥胖。表观指标检测发现,丁酸钠显著降低肥胖小鼠的肝重（1.24 g ± 0.03 g 至1.08 g ± 0.04 g）、体重（32.46 g ± 0.50 g至28.35 g ± 0.58 g）和附睾脂重（1.33 g ± 0.13 g至0.81 g ± 0.08 g）及其与体重的比（4.06% ± 0.37%至2.83% ± 0.22%）。葡萄糖耐受实验和血液激素含量检测表明,丁酸钠部分缓解由高脂引起的葡萄糖不耐受,并显著降低血液中瘦素（3.71 ng/mL ± 0.62 ng/mL至1.50 ng/mL ± 0.26 ng/mL）和胰岛素（2.39 ng/mL ± 0.30 ng/mL至1.25 ng/mL ± 0.09 ng/mL）的水平。肝中脂质和糖原的生化检测表明,丁酸钠对肝中的甘油三酯、胆固醇和糖原的含量没有显著影响。通过RT-PCR实验发现,丁酸钠显著上调线粒体β氧化和解耦联相关的关键基因以及线粒体自身编码的8个基因的mRNA水平的表达,Western印迹检测表明,丁酸钠显著升高肝葡萄糖转运蛋白GLUT2和调控线粒体功能的关键蛋白PGC-1α的表达。上述结果提示,丁酸钠可能通过增强肝线粒体功能缓解食源性小鼠肥胖。     

Intermittent hypoxia exacerbates metabolic effects of diet-induced obesity

Obesity causes insulin resistance (IR) and nonalcoholic fatty liver disease (NAFLD), but the relative contribution of sleep apnea is debatable. The main aim of this study is to evaluate the effects of chronic intermittent hypoxia (CIH), a hallmark of sleep apnea, on IR and NAFLD in lean mice and mice with diet-induced obesity (DIO). Mice (C57BL/6J), 6-8 weeks of age were fed a high fat (n = 18) or regular (n = 16) diet for 12 weeks and then exposed to CIH or control conditions (room air) for 4 weeks. At the end of the exposure, fasting (5 h) blood glucose, insulin, homeostasis model assessment (HOMA) index, liver enzymes, and intraperitoneal glucose tolerance test (1 g/kg) were measured. In DIO mice, body weight remained stable during CIH and did not differ from control conditions. Lean mice under CIH were significantly lighter than control mice by day 28 (P = 0.002). Compared to lean mice, DIO mice had higher fasting levels of blood glucose, plasma insulin, the HOMA index, and had glucose intolerance and hepatic steatosis at baseline. In lean mice, CIH slightly increased HOMA index (from 1.79 ± 0.13 in control to 2.41 ± 0.26 in CIH; P = 0.05), whereas glucose tolerance was not affected. In contrast, in DIO mice, CIH doubled HOMA index (from 10.1 ± 2.1 in control to 22.5 ± 3.6 in CIH; P < 0.01), and induced severe glucose intolerance. In DIO mice, CIH induced NAFLD, inflammation, and oxidative stress, which was not observed in lean mice. In conclusion, CIH exacerbates IR and induces steatohepatitis in DIO mice, suggesting that CIH may account for metabolic dysfunction in obesity.     

Hypoglycemic and hypolipidemic effects of processed Aloe vera gel in a mouse model of non-insulin-dependent diabetes mellitus

The effects of processed Aloe vera gel (PAG) on the course of established diet-induced non-insulin-dependent diabetes mellitus (NIDDM) were studied in C57BL/6J mice. NIDDM was induced in C57BL/6J mice by feeding them a high-fat diet. Mice exhibiting diet-induced obesity (DIO) with blood glucose levels above 180 mg/dl were selected to examine the antidiabetic effects of PAG. Oral administration of PAG for 8 weeks reduced circulating blood glucose concentrations to a normal level in these DIO mice. In addition, the administration of PAG significantly decreased plasma insulin. The antidiabetic effects of PAG were also confirmed by intraperitoneal glucose tolerance testing. PAG appeared to lower blood glucose levels by decreasing insulin resistance. The administration of PAG also lowered triacylglyceride levels in liver and plasma. Histological examinations of periepididymal fat pad showed that PAG reduced the average size of adipocytes. These results demonstrate that the oral administration of PAG prevents the progression of NIDDM-related symptoms in high-fat diet-fed mice, and suggest that PAG could be useful for treating NIDDM.     

Mice lacking NKT cells but with a complete complement of CD8+ T-cells are not protected against the metabolic abnormalities of diet-induced obesity

The contribution of natural killer T (NKT) cells to the pathogenesis of metabolic abnormalities of obesity is controversial. While the combined genetic deletion of NKT and CD8(+) T-cells improves glucose tolerance and reduces inflammation, interpretation of these data have been complicated by the recent observation that the deletion of CD8(+) T-cells alone reduces obesity-induced inflammation and metabolic dysregulation, leaving the issue of the metabolic effects of NKT cell depletion unresolved. To address this question, CD1d null mice (CD1d(-/-)), which lack NKT cells but have a full complement of CD8(+) T-cells, and littermate wild type controls (WT) on a pure C57BL/6J background were exposed to a high fat diet, and glucose intolerance, insulin resistance, dyslipidemia, inflammation, and obesity were assessed. Food intake (15.5±4.3 vs 15.3±1.8 kcal/mouse/day), weight gain (21.8±1.8 vs 22.8±1.4 g) and fat mass (18.6±1.9 vs 19.5±2.1 g) were similar in CD1d(-/-) and WT, respectively. As would be expected from these data, metabolic rate (3.0±0.1 vs 2.9±0.2 ml O(2)/g/h) and activity (21.6±4.3 vs 18.5±2.6 beam breaks/min) were unchanged by NKT cell depletion. Furthermore, the degree of insulin resistance, glucose intolerance, liver steatosis, and adipose and liver inflammatory marker expression (TNFα, IL-6, IL-10, IFN-γ, MCP-1, MIP1α) induced by high fat feeding in CD1d(-/-) were not different from WT. We conclude that deletion of NKT cells, in the absence of alterations in the CD8(+) T-cell population, is insufficient to protect against the development of the metabolic abnormalities of diet-induced obesity.     

Increased energy expenditure, dietary fat wasting, and resistance to diet-induced obesity in mice lacking renin

An overactive renin-angiotensin system is associated with obesity and the metabolic syndrome. However, the mechanisms behind it are unclear. Cleaving angiotensinogen to angiotensin I by renin is a rate-limiting step of angiotensin II production, but renin is suggested to have angiotensin-independent effects. We generated mice lacking renin (Ren1c) using embryonic stem cells from C57BL/6 mice, a strain prone to diet-induced obesity. Ren1c^−/− mice are lean, insulin sensitive, and resistant to diet-induced obesity without changes in food intake and physical activity. The lean phenotype is likely due to a higher metabolic rate and gastrointestinal loss of dietary fat. Most of the metabolic changes in Ren1c^−/− mice were reversed by angiotensin II administration. These results support a role for angiotensin II in the pathogenesis of diet-induced obesity and insulin resistance.     

Magainin-AM2 improves glucose homeostasis and beta cell function in high-fat fed mice

Background

Magainin-AM2, a previously described amphibian host-defense peptide, stimulates insulin- and glucagon-like peptide-1-release in vitro. This study investigated anti-diabetic effects of the peptide in mice with diet-induced obesity and glucose intolerance.

Methods

Male National Institute of Health Swiss mice were maintained on a high-fat diet for 12-weeks prior to the daily treatment with magainin-AM2. Various indices of glucose tolerance were monitored together with insulin secretory responsiveness of islets at conclusion of study.

Results

Following twice daily treatment with magainin-AM2 for 15 days, no significant difference in body weight and food intake was observed compared with saline-treated high fat control animals. However, non-fasting blood glucose was significantly (P < 0.05) decreased while plasma insulin concentrations were significantly (P < 0.05) increased. Oral and intraperitoneal glucose tolerance and insulin secretion following glucose administration via both routes were significantly (P < 0.05) enhanced. The peptide significantly (P < 0.001) improved insulin sensitivity as well as the beta cell responses of islets isolated from treated mice to a range of insulin secretagogues. Oxygen consumption, CO₂ production, respiratory exchange ratio and energy expenditure were not significantly altered by sub-chronic administration of magainin-AM2 but a significant (P < 0.05) reduction in fat deposition was observed.

Conclusion

These results indicate that magainin-AM2 improves glucose tolerance, insulin sensitivity and islet beta cells secretory responsiveness in mice with obesity-diabetes.

General significance

The activity of magainin-AM2 suggests the possibility of exploiting this peptide for treatment of type 2 diabetes.     

Hypoglycemic effects of brassinosteroid in diet-induced obese mice

The prevalence of obesity is increasing globally, and obesity is a major risk factor for metabolic diseases such as type 2 diabetes. Previously, we reported that oral administration of homobrassinolide (HB) to healthy rats triggered a selective anabolic response that was associated with lower blood glucose. Therefore, the aim of this study was to evaluate the effects of HB administration on glucose metabolism, insulin sensitivity, body composition, and gluconeogenic gene expression profiles in liver of C57BL/6J high-fat diet-induced obese mice. Acute oral administration of 50-300 mg/kg HB to obese mice resulted in a dose-dependent decrease in fasting blood glucose within 3 h of treatment. Daily chronic administration of HB (50 mg/kg for 8 wk) ameliorated hyperglycemia and improved oral glucose tolerance associated with obesity without significantly affecting body weight or body composition. These changes were accompanied by lower expression of two key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase), and increased phosphorylation of AMP-activated protein kinase in the liver and muscle tissue. In vitro, HB treatment (1-15 μM) inhibited cyclic AMP-stimulated but not dexamethasone-stimulated upregulation of PEPCK and G-6-Pase mRNA levels in H4IIE rat hepatoma cells. Among a series of brassinosteroid analogs related to HB, only homocastasterone decreased glucose production in cell culture significantly. These results indicate the antidiabetic effects of brassinosteroids and begin to elucidate their putative cellular targets both in vitro and in vivo.     

Inactivation of MARK4, an AMP-activated Protein Kinase (AMPK)-related Kinase,Leads to Insulin Hypersensitivity and Resistance to Diet-induced Obesity

MARK4, also known as Par-1d/MarkL1, is a member of the AMP-activated protein kinase (AMPK)-related family of kinases, which are implicated in the regulation of dynamic biological functions, including glucose and energy homeostasis. However, the physiological function of MARK4 in mammals remains elusive. Here, we investigated a role for MARK4 in regulating energy homeostasis by generating mice with targeted inactivation of the mark4 gene. We show that MARK4 deficiency in mice caused hyperphagia, hyperactivity, and hypermetabolism, leading to protection from diet-induced obesity and its related metabolic complications through up-regulation of brown fat activity. Consequently, MARK4 deficiency mitigated insulin resistance associated with diet-induced obesity by dramatically enhancing insulin-stimulated AKT phosphorylation in major metabolic tissues. Ablation of MARK4 also significantly improved glucose homeostasis by up-regulating the activity and expression of AMPK kinase in key metabolic tissues. Taken together, these data identify a key role of MARK4 in energy metabolism, implicating the kinase as a novel drug target for the treatment of obesity and type 2 diabetes.     

Beta-arrestin-1 protein represses diet-induced obesity

Diet-related obesity is a major metabolic disorder. Excessive fat mass is associated with type 2 diabetes, hepatic steatosis, and arteriosclerosis. Dysregulation of lipid metabolism and adipose tissue function contributes to diet-induced obesity. Here, we report that β-arrestin-1 knock-out mice are susceptible to diet-induced obesity. Knock-out of the gene encoding β-arrestin-1 caused increased fat mass accumulation and decreased whole-body insulin sensitivity in mice fed a high-fat diet. In β-arrestin-1 knock-out mice, we observed disrupted food intake and energy expenditure and increased macrophage infiltration in white adipose tissue. At the molecular level, β-arrestin-1 deficiency affected the expression of many lipid metabolic genes and inflammatory genes in adipose tissue. Consistently, transgenic overexpression of β-arrestin-1 repressed diet-induced obesity and improved glucose tolerance and systemic insulin sensitivity. Thus, our findings reveal that β-arrestin-1 plays a role in metabolism regulation.     

Correction of diet-induced hyperglycemia, hyperinsulinemia, and skeletal muscle insulin resistance by moderate hyperleptinemia

Human obesity and high fat feeding in rats are associated with the development of insulin resistance and perturbed carbohydrate and lipid metabolism. It has been proposed that these metabolic abnormalities may be reversible by interventions that increase plasma leptin. Up to now, studies in nongenetic animal models of obesity and in human obesity have concentrated on multiple injection therapy with mixed results. Our study sought to determine whether a sustained, moderate increase in plasma leptin, achieved by administration of a recombinant adenovirus containing the leptin cDNA (AdCMV-leptin) would be effective in reversing the metabolic abnormalities of the obese phenotype. Wistar rats fed a high-fat diet (HF) were heavier (P < 0.05), had increased fat mass and intramuscular triglycerides (mTG), and had elevated plasma glucose, insulin, triglyceride, and free fatty acids compared with standard chow-fed (SC) control animals (all P < 0.01). HF rats also had impaired glucose tolerance and were markedly insulin resistant, as demonstrated by a 40% reduction in insulin-stimulated muscle glucose uptake (P < 0.001). Increasing plasma leptin levels to 29.0 +/- 1.5 ng/ml (from 7.0 +/- 1.4 ng/ml, P < 0.001) for a period of 6 days decreased adipose mass by 40% and normalized plasma glucose and insulin levels. In addition, insulin-stimulated skeletal muscle glucose uptake was normalized in hyperleptinemic rats, an effect that correlated closely with a 60% (P < 0.001) decrease in mTG. Importantly, HF rats that received a control adenovirus containing the beta-galactosidase cDNA and were calorically matched to AdCMV-leptin-treated animals remained hyperglycemic, hyperinsulinemic, insulin resistant, and maintained elevated mTG. We conclude that a gene-therapeutic intervention that elevates plasma leptin moderately for a sustained period reverses diet-induced hyperglycemia, hyperinsulinemia, and skeletal muscle insulin resistance, and that these improvements are tightly linked to leptin-induced reductions in mTG.     

肥胖大鼠模型的建立及其脂代谢相关分子机制研究

目的建立饮食诱导的肥胖(diet-induced obesity,DIO)大鼠模型并初步探讨其发病的分子机制。方法用脂肪含量30%的高脂饲料饲喂雄性SD大鼠25周,观察大鼠体重、Lee’s指数、肝组织病理改变,检测大鼠空腹血糖及空腹血清胰岛素水平,并通过real-time PCR,检测成模大鼠肝脏中乙酰辅酶A羧化酶(ACC)、脂肪酸合酶(FAS)、激素敏感酯酶(HSL)以及固醇调节元件结合蛋白-1c(SREBP-1c)的表达变化。结果高脂饲料饲喂6周后,DIO组大鼠体重、Lee’s指数均显著增加;25周后肝脏脂肪异常蓄积,出现中重度脂肪肝,空腹血糖及胰岛素水平显著升高,出现明显的胰岛素抵抗。肝脏中ACC、FAS和HSL表达显著增加,SREBP-1c表达水平达到正常组的2.56倍,两组间差异极其显著。结论成功建立了DIO大鼠模型,通过检测脂代谢相关基因的表达水平,初步阐释了营养性肥胖的发生与脂代谢变化之间的关系,SREBP-1c,ACC,FAS和HSL参与了DIO的形成,从而初步揭示了脂代谢变化与营养性肥胖的发生的关系。