去乙酰化酶1介导2型糖尿病胰岛素抵抗的病理机制及其与运动的关系

<正>研究~([1])表明,体育运动调节骨骼肌能量稳态,促进骨骼肌线粒体生物合成,缓解骨骼肌线粒体功能紊乱和激活胰岛素相关信号通路,进而有效地改善2型糖尿病(T2DM)骨骼肌胰岛素敏感性。去乙酰化酶(SIRT)1是SIRT的第三家族Sirtuin家族成员之一,在运动训练引起的线粒体能量代谢适应性反应中起重要作用。研究~([2])发现,T2DM患者骨骼肌中SIRT1活性与胰岛素敏感性及线粒体功能呈高度正相关。大量研究致力     

胰岛素对高脂诱导胰岛素抵抗L6细胞固醇调节元件结合蛋白-1c表达的影响

目的 探讨胰岛素对高脂诱导胰岛素抵抗的骨骼肌细胞脂代谢及相关基因、蛋白表达的影响和机制.方法 L6成肌细胞诱导分化后行棕榈酸干预建立胰岛素抵抗模型,胰岛素干预后,分别检测3组脂代谢相关基因和蛋白表达的变化.结果 与对照组比较,高脂组诱导胰岛素抵抗后,固醇调节元件结合蛋白(SREBP)-1c mRNA和蛋白水平明显升高,IRS-1 mRNA和蛋白水平明显降低.胰岛素干预后,SREBP-1c表达下降,IRS-1表达升高.3组中肿瘤坏死因子(TNF)-α、stat3 mRNA表达无明显变化.结论 高脂诱导骨骼肌胰岛素抵抗下,胰岛素干预可能通过影响脂质代谢通路中关键的转录因子改善胰岛素抵抗.     

糖尿病性骨骼肌病变发病机制的研究进展

葡萄糖是细胞生长的重要调节因子,骨骼肌是全身利用葡萄糖的重要组织之一[1].研究表明葡萄糖灌注后约75%的糖被骨骼肌摄取,骨骼肌既是胰岛素作用的重要靶组织,也是胰岛素抵抗的主要部位[2].     

SIRT1与糖尿病相关肿瘤的关系

糖尿病不仅增加肿瘤的患病风险,而且促进肿瘤的复发和转移.沉默信息调节因子1(SIRT1)是机体的能量感受器,具有调节糖、脂代谢,抑制慢性非特异性炎性反应,改善胰岛素抵抗等作用,其表达及活性异常参与了糖尿病及其并发症的发生.同时近期研究发现SIRT1也与肿瘤的发生、发展密切相关.通过去乙酰化调节机制SIRT1可调控大量转录因子及组蛋白的活性,从而参与染色质沉默、细胞凋亡、自噬途径及DNA损伤修复的调控,是联系细胞能量代谢与染色质结构的关键蛋白,因而可能是联系糖尿病及糖尿病相关肿瘤的重要中介.积极干预糖尿病患者中SIRT1信号途径异常,可能具有降低肿瘤风险的作用.     

糖代谢与骨骼肌的关系

骨骼肌是体内胰岛素刺激下摄取葡萄糖的主要组织,在糖代谢平衡中发挥着重要的作用.病理状态下的骨骼肌这种代谢调节能力下降.肌糖原及循环中葡萄糖是维持骨骼肌细胞正常代谢及功能的主要物质.糖代谢紊乱尤其是高血糖对骨骼肌的代谢、结构及功能等都有明显影响,包括高血糖导致骨骼肌胰岛素抵抗、对肌糖原代谢的影响、肌萎缩以及血管异常等,肌组织病变反过来又影响代谢的控制,使病情加重.     

自噬与糖尿病骨骼肌病变

骨骼肌占人类体成分的40％～ 50％,是摄取和利用葡萄糖的重要组织,70％的葡萄糖通过胰岛素依赖的方式被骨骼肌摄取;同时骨骼肌也是2型糖尿病患者发生胰岛素抵抗的重要靶组织.现有研究发现,2型糖尿病患者骨骼肌内细胞器发生重构、外膜细胞退化及凋亡、肌膜下线粒体减少、肌细胞内脂质沉积、慢肌纤维与快肌纤维比值降低等,在临床上表现为胰岛素抵抗、骨骼肌萎缩等糖尿病骨骼肌肌病特征.糖尿病骨骼肌病变的发病机制至今不甚明确,自噬可能是主要原因之一.     

2型糖尿病患者及其一级亲属和非糖尿病者骨骼肌的胰岛素信号传导

2型糖尿病与骨骼肌和其他组织的葡萄糖利用能力下降有关。胰岛素抵抗可能与遗传因素和糖尿病本身的代谢异常如高血糖或非脂化脂肪酸(NEFA)升高有关。遗传因素和继发的胰岛素抵抗代谢异常可能是由于调节葡萄糖代谢的胰岛素受体和受体后信号传导的改变引起。胰岛素与其受体结合后,受体自身磷酸化,受体激酶活化,一些细胞间的底物磷酸化,其中在骨骼肌细胞中胰岛素受体底物-1(IRS-1)最重要。酪氨酸磷酸化的IRS与脂肪激酶磷脂酚肌醇3’(PI3’)激     

运动对胰岛素刺激的老年雌性鼠骨骼肌细胞葡萄糖转运因子4转位的影响

我们应用老年小鼠模型制备完整的骨骼肌束，研究运动对胰岛素刺激的骨骼肌细胞葡萄糖转运因子4（Glucose transporter 4，GLUT4）转位的影响，探讨运动改善骨骼肌细胞对胰岛素敏感性和减少骨骼肌细胞胰岛素抵抗（IR）的机制。     

骨骼肌胰岛素抵抗的分子机制

骨骼肌是体内葡萄糖处理的主要靶位 ,葡萄糖在骨骼肌中的转运受损是导致 2型糖尿病患者外周胰岛素抵抗的重要原因 ,这种缺陷可归结为骨骼肌组织中的胰岛素信号转导异常。胰岛素信号转导通路上任一位点发生障碍均可成为骨骼肌胰岛素抵抗的原因。表达在骨骼肌组织中的解偶联蛋白也是参与骨骼肌糖、脂代谢和能量平衡调节的重要因素     

糖尿病性骨骼肌病变的研究进展

骨骼肌是葡萄糖摄取和利用的重要组织,也是胰岛素发挥作用的靶组织.骨骼肌病变可加重胰岛素抵抗(IR),进一步影响机体的糖代谢.糖尿病性骨骼肌病变常因其临床表现不典型而被忽视,其发病机制复杂,与血管性因素、神经性因素、代谢性因素、氧化应激、细胞凋亡、慢性炎性反应等均有关.因此,明确其临床特点及发病机制是近年来研究糖尿病性骨骼肌病变的重点.     

Phosphoinositide 3-kinase as a novel functional target for the regulation of the insulin signaling pathway by SIRT1

The protein deacetylase SIRT1, and its activator resveratrol, exert beneficial effects on glucose metabolism. Different SIRT1 targets have been identified, including PTP1B, AMPK, FOXO, PGC-1α and IRS2. The latter may underscore a tight link between SIRT1 and insulin signaling components. However, whether SIRT1 has a direct effect on insulin resistance and whether resveratrol acts directly or indirectly in this context is still a matter of controversy and this question has not been addressed in muscle cells. Here, we show that SIRT1 protein expression is decreased in muscle biopsies and primary myotubes derived from type 2 diabetic patients, suggesting a contribution of diminished SIRT1 in the determination of muscle insulin resistance. To investigate the functional impact of SIRT1 on the insulin pathway, the activation of insulin downstream effector PKB was evaluated after SIRT1 inactivation by RNAi, SIRT1 overexpression, or resveratrol treatments. In muscle cells and HEK293 cells, downregulation of SIRT1 reduced, while overexpression increased, insulin-induced PKB activatory phosphorylation. Further molecular characterisation revealed that SIRT1 interacts in an insulin-independent manner with the PI3K adapter subunit p85. We then investigated whether resveratrol may improve insulin signaling in muscle cells via SIRT1, or alternative targets. Incubation of muscle cells with resveratrol reverted the insulin-resistant state induced by prolonged TNFα or insulin treatment. Resveratrol-dependent improvement of insulin-resistance occurred through inhibition of serine phosphorylation of IRS1/2, implicating resveratrol as a serine kinase inhibitor. Finally, a functional interaction between PI3K and SIRT1 was demonstrated in C. elegans, where constitutively active PI3K - mimicking increased IIS signaling - lead to shortened lifespan, while removal of sir-2.1 abolished PI3K-induced lifespan shortening. Our data identify SIRT1 as a positive modulator of insulin signaling in muscle cells through PI3K, and this mechanism appears to be conserved from C. elegans through humans.     

The loss of Sirt1 in mouse pancreatic beta cells impairs insulin secretion by disrupting glucose sensing

Aims/hypothesis

Sirtuin 1 (SIRT1) has emerged as a key metabolic regulator of glucose homeostasis and insulin secretion. Enhanced SIRT1 activity has been shown to be protective against diabetes, although the mechanisms remain largely unknown. The aim of this study was to determine how SIRT1 regulates insulin secretion in the pancreatic beta cell.

Methods

Pancreatic beta cell-specific Sirt1 deletion was induced by tamoxifen injection in 9-week-old Pdx1CreER:floxSirt1 mice (Sirt1BKO). Controls were injected with vehicle. Mice were assessed metabolically via glucose challenge, insulin tolerance tests and physical variables. In parallel, Sirt1 short interfering RNA-treated MIN6 cells (SIRT1KD) and isolated Sirt1BKO islets were used to investigate the effect of SIRT1 inactivation on insulin secretion and gene expression.

Results

OGTTs showed impaired glucose disposal in Sirt1BKO mice due to insufficient insulin secretion. Isolated Sirt1BKO islets and SIRT1KD MIN6 cells also exhibited impaired glucose-stimulated insulin secretion. Subsequent analyses revealed impaired α-ketoisocaproic acid-induced insulin secretion and attenuated glucose-induced Ca²⁺ influx, but normal insulin granule exocytosis in Sirt1BKO beta cells. Microarray studies revealed a large cluster of mitochondria-related genes, the expression of which was dysregulated in SIRT1KD MIN6 cells. Upon further analysis, we demonstrated an explicit defect in mitochondrial function: the inability to couple nutrient metabolism to mitochondrial membrane hyperpolarisation and reduced oxygen consumption rates.

Conclusions/interpretation

Taken together, these findings indicate that in beta cells the deacetylase SIRT1 regulates the expression of specific mitochondria-related genes that control metabolic coupling, and that a decrease in beta cell Sirt1 expression impairs glucose sensing and insulin secretion.     

Effects of resveratrol on NO secretion stimulated by insulin and its dependence on SIRT1 in high glucose cultured endothelial cells

To investigate the effects of resveratrol on the secretion of NO induced by insulin in high glucose cultured primary human umbilical vein endothelial cells (HUVEC). HUVEC were treated with 1 μmol/l resveratrol for 24 h before cultured in high glucose medium for 48 h, then all cells were stimulated by 100 nmol/l insulin for 30 min. Method based on nitric acid reductase was used to analyze the NO contents in the supernatant. Cells were collected to analyze the expression of eNOS, endothelin-1, E-selectin, and SIRT1. In order to investigate the dependence of resveratrol on SIRT1, the effects of resveratrol on cells treated by SIRT1 siRNA were also examined. Compared with control cells, high glucose decreased the secretion of NO induced by insulin. Resveratrol treatment increased the expression of SIRT1 and the secretion of NO. After interfering the expression of SIRT1 using SIRT1 siRNA, the effects of resveratrol on the NO secretion induced by insulin was impaired. Resveratrol also counteracted other pro-atherosclerotic effects of high glucose, including the up-regulating roles of high glucose on the expression of endothelin-1 mRNA and E-selectin mRNA, and the down-regulating roles of high glucose on the expression of eNOS mRNA and the basal NO secretion without the stimulating of insulin. Resveratrol can improve the NO stimulating function of insulin in high glucose cultured HUVEC in SIRT1-dependent manner. Thus, our results imply that resveratrol may have the preventive roles of atherosclerosis in diabetic patients.     

Sirtuin 3 regulates mouse pancreatic beta cell function and is suppressed in pancreatic islets isolated from human type 2 diabetic patients

Aims/hypothesis

Sirtuin (SIRT)3 is a mitochondrial protein deacetylase that regulates reactive oxygen species (ROS) production and exerts anti-inflammatory effects. As chronic inflammation and mitochondrial dysfunction are key factors mediating pancreatic beta cell impairment in type 2 diabetes, we investigated the role of SIRT3 in the maintenance of beta cell function and mass in type 2 diabetes.

Methods

We analysed changes in SIRT3 expression in experimental models of type 2 diabetes and in human islets isolated from type 2 diabetic patients. We also determined the effects of SIRT3 knockdown on beta cell function and mass in INS1 cells.

Results

SIRT3 expression was markedly decreased in islets isolated from type 2 diabetes patients, as well as in mouse islets or INS1 cells incubated with IL1β and TNFα. SIRT3 knockdown in INS1 cells resulted in lowered insulin secretion, increased beta cell apoptosis and reduced expression of key beta cell genes. SIRT3 knockdown also blocked the protective effects of nicotinamide mononucleotide on pro-inflammatory cytokines in beta cells. The deleterious effects of SIRT3 knockdown were mediated by increased levels of cellular ROS and IL1β.

Conclusions/interpretation

Decreased beta cell SIRT3 levels could be a key step in the onset of beta cell dysfunction, occurring via abnormal elevation of ROS levels and amplification of beta cell IL1β synthesis. Strategies to increase the activity or levels of SIRT3 could generate attractive therapies for type 2 diabetes.     

Loss of sirtuin 4 leads to elevated glucose- and leucine-stimulated insulin levels and accelerated age-induced insulin resistance in multiple murine genetic backgrounds

Several inherited metabolic disorders are associated with an accumulation of reactive acyl-CoA metabolites that can non-enzymatically react with lysine residues to modify proteins. While the role of acetylation is well-studied, the pathophysiological relevance of more recently discovered acyl modifications, including those found in inherited metabolic disorders, warrants further investigation. We recently showed that sirtuin 4 (SIRT4) removes glutaryl, 3-hydroxy-3-methylglutaryl, 3-methylglutaryl, and 3-methylglutaconyl modifications from lysine residues. Thus, we used SIRT4 knockout mice, which can accumulate these novel post-translational modifications, as a model to investigate their physiological relevance. Since SIRT4 is localized to mitochondria and previous reports have shown SIRT4 influences metabolism, we thoroughly characterized glucose and lipid metabolism in male and female SIRT4KO mice across different genetic backgrounds. While only minor perturbations in overall lipid metabolism were observed, we found SIRT4KO mice consistently had elevated glucose- and leucine-stimulated insulin levels in vivo and developed accelerated age-induced insulin resistance. Importantly, elevated leucine-stimulated insulin levels in SIRT4KO mice were dependent upon genetic background since SIRT4KO mice on a C57BL/6NJ genetic background had elevated leucine-stimulated insulin levels but not SIRT4KO mice on the C57BL/6J background. Taken together, the data suggest that accumulation of acyl modifications on proteins in inherited metabolic disorders may contribute to the overall metabolic dysfunction seen in these patients.     

Inhibition of silent information regulator 1 induces glucose metabolism disorders of hepatocytes and enhances hepatitis C virus replication

Background and objective

Glucose metabolism disorders including insulin resistance (IR) and type 2 diabetes are frequent and important cofactors of chronic hepatitis C (CHC). Silent information regulator 1 (SIRT1) plays a key role in the regulation of hepatic glucose metabolism. We investigated the possible effect of HCV replication on glucose metabolism of hepatocytes and expression of SIRT1 using Huh-7.5 cells harboring the HCV replicon.

Methods

The level of reactive oxygen species (ROS) and value of NAD⁺/NADH and ATP/ADP were detected. Glucose uptake by hepatocytes and glucose production were measured. The activity and expression levels of SIRT1 and expression of its downstream glucose-metabolism genes were measured.

Results

In replicon cells, the level of ROS increased and the value of nicotinamide adenine dinucleotide (NAD⁺)/NADH decreased, then the activity and expression level of mRNA and protein of SIRT1 decreased. Inhibition of SIRT1 not only increased insulin receptor substrate-1 phosphorylation and decreased Akt phosphorylation, inhibited cell surface expression of glucose transporter 2 and suppressed cellular glucose uptake, but it also decreased phosphorylation of forkhead box O1, then upregulated phosphoenolpyruvate carboxykinase and glucose 6-phosphatase genes and downregulated the glucokinase gene, thus promoting glucose production. Interferon treatment restored the aforementioned changes. SIRT1 activator improved glucose metabolism disorders by an increase in glucose uptake and a decrease in glucose production, and it inhibited HCV replication.

Conclusions

HCV replication decreasing the NAD⁺/NADH ratio may downregulate the activity and expression of SIRT1, then change the expression profile of glucose metabolism-related genes, thereby causing glucose metabolism disorders of hepatocytes and promoting HCV replication. Treatment with SIRT1 activator improves glucose metabolic disorders and inhibits HCV replication, suggesting that restoration of SIRT1 activity may be a promising new therapeutic approach for CHC patients with IR.     

Skeletal muscle-specific overexpression of SIRT1 does not enhance whole-body energy expenditure or insulin sensitivity in young mice

Aims/hypothesis

The NAD⁺-dependent protein deacetylase sirtuin (SIRT)1 is thought to be a key regulator of skeletal muscle metabolism. However, its precise role in the regulation of insulin sensitivity is unclear. Accordingly, we sought to determine the effect of skeletal muscle-specific overexpression of SIRT1 on skeletal muscle insulin sensitivity and whole-body energy metabolism.

Methods

At 10 weeks of age, mice with muscle-specific overexpression of SIRT1 and their wild-type littermates were fed a standard diet with free access to chow or an energy-restricted (60% of standard) diet for 20 days. Energy expenditure and body composition were measured by indirect calorimetry and magnetic resonance imaging, respectively. Skeletal muscle insulin-stimulated glucose uptake was measured ex vivo in soleus and extensor digitorum longus muscles using a 2-deoxyglucose uptake technique with a physiological insulin concentration of 360 pmol/l (60 μU/ml).

Results

Sirt1 mRNA and SIRT1 protein levels were increased by approximately 100- and 150-fold, respectively, in skeletal muscle of mice with SIRT1 overexpression compared with wild-type mice. Despite this large-scale overexpression of SIRT1, body composition, whole-body energy expenditure, substrate oxidation and voluntary activity were comparable between genotypes. Similarly, skeletal muscle basal and insulin-stimulated glucose uptake were unaltered with SIRT1 overexpression. Finally, while 20 days of energy restriction enhanced insulin-stimulated glucose uptake in skeletal muscles of wild-type mice, no additional effect of SIRT1 overexpression was observed.

Conclusions/interpretation

These results demonstrate that upregulation of SIRT1 activity in skeletal muscle does not affect whole-body energy expenditure or enhance skeletal muscle insulin sensitivity in young mice on a standard diet with free access to chow or in young mice on energy-restricted diets.     

Downregulation of miR-181a upregulates sirtuin-1 (SIRT1) and improves hepatic insulin sensitivity

Aims/hypothesis  

Sirtuin-1 (SIRT1) is a potential therapeutic target to combat insulin resistance and type 2 diabetes. This study aims to identify a microRNA (miRNA) targeting SIRT1 to regulate hepatic insulin sensitivity.