脂肪组织ClC-3容积调节性氯通道在胰岛素抵抗中的作用与机制研究

The enhanced apoptosis in preadipocyte of type 2 diabetes patients has been shown in the recent work. Our preliminary study have shown that ClC-3 volume-regulated chloride channel is associated with the insulin resistance by using ClC-3 KO mice subjected to type 2 diabetes mellitus (T2DM). The ClC-3 KO mice have indicated the significant reduction in HOMA-IR and serum glucose levels, but no significant change in serum insulin levels.These data suggested that ClC-3 should be involved in the development of insulin resistence, but not be interpreted by the modulation of insulin secretion.We further found that there is an increase in the ClC-3 protein expression in adipose tissue in T2DM rat models induced by high-sucrose-high-fat diet combined with a low dose of STZ,and which has a positive correlation with HOMA-IR levels,but such correlation could not been found in liver and skeletal muscle. In 3T3-L1 preadipocyts, we have shown that palmitate, the main component of saturated free fatty acids(FFA), induced an increase in ClC-3 protein expression, in parallel with promoting preadipocyte apoptosis in a concentration- dependent manner, ClC-3 siRNA silencing significantly reversed the above both change induced by palmitate.These preliminary data suggested that ClC-3 chloride channel may play a role in free fatty acid-related preadipocyte apoptosis. Our research group has been focused on the role of ClC-3 volume-regulated chloride channel for two decades. We have shown that ClC-3 channels are important mediator contributing to cellular volume regulation, proliferation and apoptosis and thereby accounting for many pathological processes in response to volume stress,such as hpertension,stroke and atherosclerosis. Enlarged adipocytes is taken as an independent predictor for T2DM, and which is mainly due to reduced differentiation ability of preadipocytes or the enhanced apoptosis of preadipocytes. Based on our preliminary data and the suitable molecular tools including ClC-3 KO mice, we will investigate the role and the molecular mechanism of ClC-3 chloride channel during obesity-related insulin resistance. The main objectives of this project are: 1) to investigate the role and molecular mechanisms that ClC-3 volume-regulated chloride channel in FFA-induced preadipocyte apoptosis;2) to examine whether ClC-3 volume-regulated chloride channel is therefore involved in adipose tissue dysfuntion and IR developmemt by using several in vivo T2DM models(including KKAy and/or db/db mouse model, ClC-3 KO T2DM mouse model); and the related signaling pathway,such as preadipocyte apoptosis, proliferation and differentiation ability,inflammation and insulin signal in adipose tissue. The novelty of this proposal is to identify ClC-3 volume-regulated chloride channel as a novel molecular mechanism for adipose tissue derived insulin resistance, and will help in the search of new drug target for improving insulin sensitivity.

以前脂肪细胞凋亡为中心的脂肪组织更新障碍是胰岛素抵抗的重要起始环节。我们前期研究在ClC-3基因敲除2型糖尿病小鼠等整体模型上发现，脂肪组织ClC-3容积调节性氯通道与胰岛素抵抗发生相关；体外实验发现ClC-3参与饱和游离脂肪酸诱导前脂肪细胞凋亡。本项目拟在已有工作基础上，应用ClC-3 siRNA和ClC-3 cDNA全长转染技术, 阐明ClC-3容积调节性氯通道在饱和游离脂肪酸诱导前脂肪细胞凋亡中的作用和机制；结合不同机制2型糖尿病模型 （包括ClC-3基因敲除和自发性肥胖2型糖尿病小鼠等），明确ClC-3容积调节性氯通道能否通过调控前脂肪细胞凋亡参与胰岛素抵抗发生，并揭示其与胰岛素抵抗过程中脂肪组织增殖与分化、炎症和胰岛素信号通路变化的内在联系。本研究将从ClC-3容积调节性氯通道这一新角度，揭示胰岛素抵抗发生新机制，并为寻找改善胰岛素抵抗的新药靶提供实验室依据和新思路。

本项目已全部按原计划研究内容按时完成，并达到预期研究目标。先前研究提示ClC-3 是编码容积调控性氯通道的一个候选基因，本项目应用了ClC-3全基因敲除小鼠和特异的siRNA 和腺病毒介导全长cDNA过表达等基因干预手段，并建立了两种不同机制的2型糖尿病（T2DM）动物模型和收集了人类大网膜脂肪标本进行研究。我们的研究结果表明ClC-3容积调节性Cl-通道可通过调节饱和游离脂肪酸诱导前脂肪细胞凋亡，进而参与调控T2DM小鼠血糖、血脂和慢性、低度脂肪炎症。在ClC-3全基因敲除小鼠上建立高糖高脂+小剂量STZ诱导的T2DM模型，发现ClC-3全基因敲除可改善T2DM小鼠的胰岛素抵抗并降低其空腹血糖水平、改善血糖和血脂代谢紊乱，并揭示ClC-3全基因敲除可通过降低前脂肪细胞内质网应激信号通路抑制T2DM诱导的腹部内脏脂肪组织的前脂肪细胞凋亡。另外，在野生型和ClC-3全基因敲除小鼠建立的高脂饮食诱导肥胖和T2DM模型上，发现ClC-3全基因敲除可显著降低高脂饮食诱导的脂肪细胞体积增大、降低内脏脂肪重量和小鼠体重，并显著抑制高脂饮食诱导的肥胖、慢性低度脂肪炎症以及改善糖耐量减低，ClC-3全基因敲除并不影响T2DM小鼠的肝脏体积和形态学变化。进而，在收集的人类大网膜脂肪标本上，明确了ClC-3 mRNA和蛋白质表达与内脏脂肪细胞体积、体质量指数（BMI）、血FFA水平成显著正相关。本项目已取得的研究结果揭示了脂肪组织ClC-3容积调节性Cl-通道通过调控内脏前脂肪细胞凋亡、脂肪组织脂肪细胞体积和脂肪炎症从而影响胰岛素抵抗发生发展，表明ClC-3可成为改善胰岛素抵抗的新靶点，并可能成为干预肥胖及其相关疾病的新靶点和新策略。

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