【作者】 闫文华；

【导师】 潘长玉； 窦京涛；

【作者基本信息】 中国人民解放军军医进修学院 ， 内科学， 2009， 博士

【摘要】 随着代谢综合征和2型糖尿病发病的日益流行,人们对抗高血压药物代谢效应的关注逐渐增多。血管紧张素Ⅱ1型受体拮抗剂(ARB)是一类作用于肾素血管紧张素系统的抗高血压药物,临床研究提示ARB可显著减少原发性高血压患者新发糖尿病的发生率,并可增强胰岛素敏感性,但其改善胰岛素敏感性的机理目前仍不明确。研究目的:探讨血管紧张素Ⅱ1型受体拮抗剂—坎地沙坦对高脂饲养大鼠胰岛素敏感性的影响及其可能分子机制。研究方法:1、以Wistar雄性大鼠为研究对象,通过高脂饲养的方法建立肥胖胰岛素抵抗大鼠模型,并用坎地沙坦酯(8 mg/kg.d)对高脂饲养的Wistar大鼠进行干预4周,用高胰岛素—正常葡萄糖钳夹实验分别比较普通饲料组(NC组),高脂饲料组(HF组)和高脂+坎地沙坦组(HF+C组)的胰岛素敏感性,收集血检测各组动物血糖、血脂、胰岛素及其他生化指标,处死后分离脏器并称重。2、分别用免疫组化法和Western blot法检测各组中肝脏和脂肪组织PPARγ的表达水平和骨骼肌组织葡萄糖转运蛋白4(GLUT4)的表达水平。3、以RT-PCR和免疫印迹比较各组脂肪组织内两面神激酶(JAK2)和蛋白酪氨酸磷酸酶-1B(PTP-1B)的mRNA和蛋白表达水平。研究结果:1、8周龄时两组大鼠体重差异无统计学意义。16周龄时,HF组大鼠体重、肾周和附睾脂肪、心脏重量、肝脏重量均明显高于NC组和HF+C组。HF+C组总胆固醇(TC)、空腹胰岛素(FINS)水平明显低于HF组,但胰岛素敏感指数(ISI)及血管紧张素Ⅱ(AngⅡ)明显高于HF组,HF+C组稳态葡萄糖输注率(SSGIR)也较HF组明显增高。2、观察肝脏、脂肪PPARγ蛋白表达情况,光镜下所见HF组于肝组织汇管区和中央静脉区的肝细胞核内仅有少量棕黄色颗粒表达,位于脂肪细胞周边的PPARγ核阳性表达量也很低。HF+C组PPARγ在肝脏和脂肪的积分吸光度值(IOD)较HF组增高。经免疫组化及蛋白印迹证实:HF+C组大鼠骨骼肌GLUT4表达水平显著高于HF组和NC组。3、RT-PCR检测发现,JAK mRNA和PTP-1B mRNA在NC组大鼠的脂肪组织中表达水平较低,而在HF组中表达明显增强。坎地沙坦可分别下调高脂饮食诱导的JAK mRNA和PTP-1B mRNA的高表达(0.87±0.16 vs 1.51±0.09,0.91±0.13 vs 1.89±0.14,P＜0.05)。三组脂肪组织中JAK2和PTP-1B蛋白表达趋势与RT-PCR检测结果基本一致:与NC组相比,HF组JAK2、PTP-1B的表达显著增高,分别为(1.01±0.18 vs 0.37±0.11,1.57±0.24 vs 0.98±0.15,P＜0.05);HF+C组JAK2、PTP-1B表达又较NC组显著降低。综上所述,我们得出如下结论:1、大鼠经高脂饲料饲养8周后产生肥胖及高脂血症,钳夹实验证实葡萄糖输注率明显下降,并在空腹血糖水平保持正常的情况下出现了高胰岛素血症,成功复制了瓜大鼠模型。2、坎地沙坦干预后,大鼠体重下降,肾周及附睾脂肪含量减低,血清胰岛素水平降低,葡萄糖输入率增高,提示坎地沙坦能够显著改善高脂饮食大鼠的胰岛素抵抗。3、HF组大鼠的脂肪细胞肥大,经坎地沙坦治疗后脂肪细胞体积变小而总的脂肪细胞数量未发生变化。坎地沙坦能够促进大鼠肝脏、脂肪组织的PPARγ表达。4、HF组大鼠骨骼肌GLUT4蛋白表达较NC组及HF+C组显著下降,提示胰岛素抵抗的发生可能和外周组织GLUT4表达降低有关。坎地沙坦可部分逆转这种改变,从而增加机体对胰岛素的敏感性。5、HF组大鼠脂肪组织JAK2和PTP-1B表达增高,而坎地沙坦能够降低其表达,AngⅡ→JAK2→Iκb/NF-κ3→PKAc→PTP-1B信号通路的激活是引起胰岛素抵抗的可能分子机制。更多还原

【Abstract】 Growing concern about the increasing prevalence of the metabolic syndrome and type 2 diabetes has generated substantial interest in the metabolic effects of antihypertensive drugs.AngiotensinⅡtype 1 receptor blocker(ARB)controls the blood pressure by blockade of the renin angiotensin system.The clinical studies reported a remarkably consistent reduction in the incidence of newly-onset type 2 diabetes in hypertensive patients treated with ARB and increase in insulin sensitivity.However,the precise mechanism of improving the insulin sensitivity remains to be determined.Objectives:To investigate the effects of AngiotensinⅡtype 1 receptor blocker -Candesartan on insulin sensitivity of high-fat diet induced obesity of rats and its possible molecular mechanisms.Methods:1.An obese rat model was induced by high fat feed and rats were orally administered with candesartan(8 mg/kg.d)for 4 weeks.Euglycemic hyperinsulinemic clamp technique were performed to estimate the insulin sensitivity among normal chow group(NC group),high fat diet group(HF group), and high-fat diet with daily candesartan treatment group(HF+C group).Blood samples were collected to test the blood glucose,lipid profile,insulin and other biochemical parameters.Viscera were taken out and weighed after rats were sacrificed from each group.2.The protein expressions of peroxisome proliferator activated receptor gamma(PPARγ)in liver and adipose tissue were respectively determined by immunohistochemistry and western blot.Activation of Glucose Transporter(GLUT4)in skeletal muscles were detected by immunohistochemical SP method,GLUT4 expressions were assayed by western blotting.3.The gene and protein expressions of JAK2 and PTP-1B in adipose tissue were confirmed by both RT-PCR and western blotting. Results:1.There were no significant difference in body weight among 8-week-old rats.The body weight,liver weight,heart weight,epididymal and peri-renal fat weight of HF group were significantly higher than those of NC group and HF+C group(all P＜0.01)when rats were 16 weeks old.Although TC and FINS of HF+C group were lower than those of HF group,ISI,AngⅡand Glucose infusion rate(GIR)of HF+C group were much higher than those of HF group.2.Further study revealed that expression of PPARγin liver and visceral adipose tissue.Under light microscope,positive staining was recognized by brown color.There were less stained nuclei of hepatocytes around portal and central vein areas and less stained peripheral nuclei of adipocytes in HF group than in HF+C group.The integral optical density(IOD)of PPARγin HF+C group were stronger than that in HF group.GLUT4 expression of rat skeletal muscle in HF+C group being identified by immunohistochemistry and western blot analysis were much higher than that in HF and NC group.3.The expressions of JAK mRNA and PTP-1B mRNA in adipose tissue in NC group were at relatively lower level,while in HF group the expressions markedly increased.The mRNA expressions of JAK2 and PTP-1B in HF+C group were significantly lower than those in HF group respectively(0.87±0.16 vs 1.51±0.09,0.91±0.13 vs 1.89±0.14,P＜0.05).The protein expressions of JAK2 and PTP-1B basically conformed with the gene expression:Although the expressions of JAK and PTP-1B in NC group were lower than those in HF group(0.57±0.09 vs 1.01±0.18,0.98±0.15 vs 1.57±0.24,P＜0.05),they were still higher than those in HF+C group(1.01±0.18 vs 0.57±0.09,1.57±0.24 vs 0.43±0.08,P＜0.05).The conclusions of this study were as follows:1.Obesity and hyperlipemia were induced in rats by high-fat diet for 8 weeks. Euglycemic-hyperinsulinemic clamp experiment testified decreased-GIR and hyperinsulinemia with normal FBG.The insulin resistant rat model was successfully duplicated with high fat feeding.2.Candesartan intervention could lower the body weight,epididymal and peri-renal fat weight,decrease the serum insulin and increase the GIR.Thus Candesartan might markedly improve insulin resistance in rats induced by high-fat diet.3.Hypertrophic fat cells displayed in rats of HF group.After candesartan treatment,enlarged adipocytes became smaller without changing the number of adipocytes due to the highly expression of PPARγ.Candesartan could promote the expression of PPARγin liver and adipose tissues.4.GLUT4 protein expressions in skeletal muscles in HF group were much lower than that in NC and HF+C group,which suggested that insulin resistance was associated with decreased GLUT4 expressions in peripheral tissues. Candesartan could partially reverse these effects and thus increase the sensitivity of body for the insulin.5.The gene and protein expressions of JAK2 and PTP-1B were higher in adipose tissue of rats of HF group.Candesartan could decrease their expression. The activation of AngⅡ→JAK2→Iκb/NF-κB→PKAc→PTP-1B pathway was the possible molecular mechanism leading to insulin resistance.更多还原