【作者】 崔巍；

【导师】 焦凯；

【作者基本信息】 第四军医大学 ， 内科学， 2010， 硕士

【摘要】 研究背景糖尿病,是一种由于胰岛素对糖代谢的调节障碍所致的代谢性疾病,以血糖水平持续升高为特征,最终会导致机体各器官的损害。由于发病率的不断升高,目前已成为世界性的健康问题。肥胖是导致糖尿病的危险因素之一,研究显示,肥胖与胰岛素抵抗和2型糖尿病(type 2 diabetes mellitus,T2DM)密切相关。有研究者认为,脂代谢紊乱是糖尿病的原发病理生理变化,即脂毒性学说,并提出糖尿病其实是糖脂病的观点。肥胖所致的内脏脂肪沉积和血游离脂肪酸(free fatty acid,FFA)水平的升高是导致脂毒性的关键因素。FFA通常被认为通过其进入细胞后的一系列代谢反应及产生脂质衍生物调节胰岛素的分泌。2003年,Itoh等研究者提出G蛋白偶联受体40(G protein coupled receptor 40,GPR40)作为中、长链游离脂肪酸的受体在胰岛β细胞高表达,并指出其参与了FFA介导的胰岛素分泌并且可能与肥胖介导的T2DM有关,使GPR40成为治疗T2DM可能的药物靶点。研究显示,FFA能够通过激活胰岛β细胞的GPR40增加葡萄糖刺激的胰岛素分泌(glucose-stimulated insulin secretion,GSIS)[1, 2]。通过siRNA下调β细胞GPR40的表达会导致FFA介导的细胞内Ca2+应答及胰岛素分泌的显著抑制,提示胰岛β细胞GPR40通过介导β细胞Ca2+信号反应参与了FFA刺激的胰岛素分泌[3、4]。表明GPR40可能对肥胖状态下葡萄糖刺激的胰岛素分泌存在调控作用。随后的研究对于GPR40是否参与了FFA水平长期升高导致的胰岛素抵抗(insulin resistance,IR)及T2DM的发生进行了进一步探讨,然而结果却不尽相同。Steneberg[5]等认为GPR40参与了FFA慢性作用对胰岛素分泌的损害作用,提出GPR40拮抗剂可以作为有效的治疗策略预防和治疗肥胖介导的T2DM。而Naqasumi[6]则认为GPR40对改善IR状态下的GSIS发挥了作用。因此,对于GPR40与肥胖和胰岛素抵抗的关系还需进一步深入研究。已知肥胖时内脏脂肪沉积增加,但GPR40在胰岛β细胞表达水平的变化与内脏脂肪含量的关系,及其表达是否对正常大鼠GSIS也存在调控作用目前还未见报道,本研究应用免疫组织化学法结合激光扫描共聚焦显微镜(laser scanning confocal microscope,LSCM)技术研究GPR40在不同体重组SD大鼠胰岛β细胞的表达水平,分析GPR40表达与内脏脂肪含量及胰岛素1相分泌的关系。目的观察不同体重SD大鼠胰岛β细胞G蛋白偶联受体40(GPR40)表达水平与内脏脂肪含量及胰岛素1相分泌之间的关系。方法1、标准饮食的SD大鼠按体重分为3组(100g组、200g组、300g组);2、行静脉糖耐量实验;3、放射免疫法检测血胰岛素水平;4、采用胶原酶原位灌注法分离胰岛;5、留取内脏脂肪及肝脏称重;6、应用免疫组织化学法结合激光扫描共聚焦显微镜技术对胰岛β细胞表达的GPR40进行定位,并行半定量分析。结果1、随着大鼠体重的增加,内脏脂肪含量、肝脏重量和体脂比明显增加。2、3组大鼠空腹血糖、静脉糖耐量及胰岛素1相分泌之间无显著差异。3、300g组大鼠胰岛β细胞GPR40表达较100g及200g大鼠明显增加。结论1、大鼠胰岛β细胞GPR40表达的变化可能与内脏脂肪含量及年龄因素有关。2、GPR40表达的增多对正常大鼠葡萄糖刺激的胰岛素分泌并无明显影响。更多还原

【Abstract】 IntroductionDiabetes, a diease in which glucose metabolism is regulated improperly by insulin, is a serious worldwide health issue now, because of its increasing high incidence. Diabetes is charicaterized by high blood sugar in longterm, it can impair the functions of mang organs of our body. In recent years, obesity has drawn a lot of attention because it has benn thought as one of the risk factors of type 2 diabetes (T2DM). The reaserchers have showed that obesity is typically associated with insulin resistance (IR) and T2DM. They put forward the view that the disorder of lipid metabolism is the foundation of T2DM, it is called“lipotoxicity”, so T2DM can be also named as“diabetes mllipitus”. The deposition of visceral fat and high level of free fatty acid (FFA) in plasma induced by obesity are the critical factors which can cause lipotoxicity. Until recently, it was considered that fatty acids must enter the beta cells in order to elicit insulin secretion. In 2003, Itoh proposed that GPR40 is activated by long-chain FFA, and its expression is high inβcells. They indicated that GPR40 may take a part in FFA induced insulin secretion, and may have links with T2DM. So GPR40 may become a new durg target against T2DM. Reaserches showed that FFA can inhance glucose stimulated insulin secretion (GSIS) by activate GPR40 inβcells. GPR40 RNAi impaired the ability of FFA to increase both intracellular calcium ([Ca2+]i) and insulin secretion, indicate that GPR40 make a role in FFA stimulated insulin secretion by induce the signals of Ca2+ inβcells. So GPR40 may regulate GSIS in the condition of obesity. After that, many reaserchers studied that whether GPR40 really take a part in insulin resistance and T2DM which are caused by the high level of FFA, but the results are quite different. Steneberg thought that GPR40 participate the impairment in insulin secretion which is caused by chronic effects of FFA, and propose that GPR40 antagonists may present a useful therapeutic strategy for the prevetion and treatment of obesity-associated T2DM. but Naqasumi thought that GPR40 can improve GSIS in IR. In a word, the relationship between GPR40, obesity and IR need further study.Visceral fat will increase in the condition of obesity, but so far there are few reaserches show the relationship between visceral fat and the level of GPR40 expression inβcells, whether its expression regulate GSIS in normal rats. Thus, our study use immunohistochemical and laser-scanning confocal microscopy to investigate the expression of GPR40 in beta cells, analyze the relationship between visceral fat、first phase of insulin secretion and GPR40 expressing in beta cells in rats. Objective To investigate the relationship between visceral fat、first phase of insulin secretion and G-protein coupled receptor 40 expressing in beta cells in SD rats with different weight. Methods1、18 male SD rats were divided into three groups averagely by body weight(100g、200g、300g).2、Intravenous glucose tolerance test (IVGTT) was measured.3、Islets of rats were isolated by the digestion of pancreas with collagenase V.4、Immunohistochemical and laser-scanning confocal microscopy were used to observe the expression of GPR40 in beta cells.Results1、Visceral fat、liver weight and body fat proportion are increased with rats body weight gain in each group(P < 0.05).2、The expression of GPR40 in beta cells in 300g group was significantly increased as compared with 100g and 200g group(P < 0.05).3、IVGTT has no obvious change among these groups.ConclusionsThe expression of GPR40 in pancreatic beta cells may relate to visceral fat and age, and the increased expression of GPR40 in beta cells did not show any effect on GSIS.更多还原