【作者】 张杰；

【导师】 朱德增；

【作者基本信息】 第二军医大学 ， 中西医结合临床， 2010， 博士

【摘要】 胰岛素抵抗是指正常剂量的胰岛素产生低于生物学效应的一种状态,是滋生多种代谢性疾病的共同危险因素,且往往存在于疾病的早期阶段。因此,对胰岛素抵抗进行干预和治疗,对防治各种代谢性疾病特别是糖尿病有重要意义。肥胖(主要是腹型肥胖)是胰岛素抵抗发生、发展的重要危险因素。肥胖可以通过内分泌、脂肪细胞因子、炎性反应和细胞内在信号通路导致胰岛素抵抗。目前已经认识到由脂肪分解产生的游离脂肪酸(FFA)和脂肪细胞因子常常参与肥胖介导的胰岛素抵抗发生,这些因子通过不同的机制影响糖、脂肪代谢,各因子之间也存在着复杂的联系。其中,肿瘤坏死因子-α(TNF-α)与胰岛素抵抗的发生、发展密切相关,TNF-α可以通过抑制脂蛋白酯酶,促进脂肪分解,使血浆FFA浓度升高。FFA水平增高既可增加胰岛素抵抗,又可引起高胰岛素血症。肝脏是胰岛素作用的主要靶器官,在胰岛素代谢效应方面,主要促进肝糖原合成和抑制肝糖输出。肝脏出现胰岛素抵抗时,肝糖原合成减少糖异生增加,从而导致血糖升高。胰岛素信号级联中的胰岛素受体底物-2(IRS-2)分支信号与肝细胞胰岛素敏感性密切相关。IRS-2／磷脂酰肌醇3激酶(PI-3K)信号通路是胰岛素在肝脏发挥生理效应的主要信号转导途径。IRS-2基因表达的降低、蛋白量的减少及磷酸化障碍都会影响其下游PI-3K／蛋白激酶B(Akt)信号的有效传递而导致胰岛素抵抗。目前发现诸多因素均可通过影响IRS-2信号转导而导致肝胰岛素抵抗。蛋白酪氨酸磷酸酶-1B (PTP-1B)是蛋白酪氨酸磷酸酶(PTP)家族中的主要成员之一,可与PTP家族其他成员及蛋白酪氨酸激酶联合作用调节细胞内底物酪氨酸磷酸化水平,使胰岛素受体(InsR)、胰岛素受体底物(IRS)等信号分子的酪氨酸去磷酸化而失活,阻断胰岛素信号级联反应的下传,是影响胰岛素信号转导的关键的负调控因子。我们设想能否通过抑制肝脏PTP-1B表达而增加IRS-2的表达及酪氨酸磷酸化,从而达到改善胰岛素抵抗的目的。本实验以中医“酸入肝”、“酸胜甘”为理论指导,通过动物实验,利用分子生物学手段,以肝脏为靶器官,对酸性活性成分熊果酸改善胰岛素抵抗的作用及机理进行深入研究,希望能够进一步印证传统中医“酸胜甘”理论对防治胰岛素抵抗与糖尿病的指导作用,并为糖尿病的防治和药物研发提供实验依据。第一部分高脂饮食诱导胰岛素抵抗大鼠模型目的：建立高脂饮食诱导的肥胖胰岛素抵抗大鼠模型。方法：8周龄雄性Wistar大鼠60只,体重180～200g,随机分为正常饮食组(12只)和高脂饮食组(48只)。正常饮食组饲以基础饲料(脂肪10%,碳水化合物67%,蛋白23%),高脂饮食组饲以高脂饲料：基础饲料(78.9g)基础上加猪油10g,胆固醇1g,蛋黄粉10g,胆盐0.1g(脂肪46%,碳水化合物40%,蛋白14%)。实验前后称取体重、检测空腹血糖(FBG)、血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)水平；酶联免疫吸附检测法(ELISA)测定空腹血清胰岛素(FINS);通过葡萄糖耐量试验、胰岛素耐量试验判断对糖代谢的调节功能；用胰岛素敏感指数(ISI)和胰岛素抵抗指数(HOMA-IR)来评价胰岛素敏感性。结果：与正常饮食组相比,高脂饮食组大鼠血浆FBG轻度升高,而体重、血脂(TC、TG、LDL-C)、FINS水平都明显升高；出现糖耐量受损和胰岛素耐量异常,呈胰岛素敏感性下降状态。结论：9周高脂饮食饲养成功建立肥胖胰岛素抵抗大鼠模型,该模型与人类肥胖所致的胰岛素抵抗表现特征相似,是研究肥胖胰岛素抵抗的经济实用的实验动物模型。第二部分熊果酸对胰岛素抵抗大鼠糖、脂代谢及FFA、TNF-α的影响目的：观察熊果酸对胰岛素抵抗大鼠糖、脂代谢及血清中FFA、TNF-α的影响。方法：60只大鼠随机分为5组,即正常对照组(正常饲料+PBS溶液灌胃),模型对照组(高脂饮食+PBS溶液灌胃),二甲双胍组(高脂饮食+二甲双胍200mg/kg·d灌胃)和熊果酸高、低剂量组(高脂饮食+熊果酸300mg/kg·d、150mg/kg·d灌胃),每组12只。分4周、8周两个时间点观察各组FBG、FINS、血脂、体重、肝重、腹腔脂肪重量,葡萄糖耐量、胰岛素耐量、肝糖原含量及血清FFA、TNF-α变化；计算肝脏指数(LI)、腹脂指数(AI)、胰岛素敏感指数(ISI)和胰岛素抵抗指数(HOMA-IR);观察各组肝组织(HE染色)病理变化。结果：熊果酸和二甲双胍治疗能够不同程度降低胰岛素抵抗大鼠血清FINS、TC、TG、LDL-C和FFA、TNF-α水平；降低体重、肝脏指数(LI)和腹脂指数(AI),减少腹腔脂肪堆积：改善葡萄糖耐量、胰岛素耐量,增加肝糖原含量,升高胰岛素敏感性指数(ISI)和降低胰岛素抵抗指数(HOMA-IR)。与模型对照组比较差异有统计学意义(P<0.05或P<0.01)。熊果酸高、低剂量组间无明显差异(P>0.05)。结论：熊果酸能够降低体重,调节糖、脂代谢,降低血清中FFA、TNF-α含量,改善胰岛素抵抗。第三部分熊果酸对胰岛素抵抗大鼠肝脏PTP-1B、IRS-2表达的影响目的：观察熊果酸对胰岛素抵抗大鼠肝脏中PTP-1B、IRS-2表达变化的影响,探讨PTP-1B、IRS-2在胰岛素抵抗发生中的作用。方法：60只大鼠随机分为正常对照组,模型对照组,二甲双胍组和熊果酸高、低剂量组,每组12只(方法同第二部分)。采用实时荧光定量PCR法观察各组大鼠肝组织内PTP-1B、IRS-2mRNA的表达情况,并用相对定量法计算表达量。采用蛋白免疫印迹法(Western Blot)检测各组肝组织内PTP-1B、IRS-2蛋白的表达及IRS-2酪氨酸磷酸化情况。结果：肝组织实时荧光定量PCR显示模型对照组的PTP-1BmRNA表达均明显高于正常对照组,IRS-2mRNA表达均明显低于正常对照组,而经过高、低剂量熊果酸和二甲双胍干预后,PTP-1BmRNA的表达减少,IRS-2mRNA表达增加,与模型对照组比较差异有统计学意义(P<0.05或P<0.01)。蛋白印迹结果显示PTP-1B蛋白表达减少,IRS-2蛋白增加,IRS-2酪氨酸磷酸化增强。结论：胰岛素抵抗大鼠肝脏PTP-1B表达升高,IRS-2的表达下降,熊果酸和二甲双胍均可抑制肝内PTP-1B表达,从而促进IRS-2表达增多及酪氨酸磷酸化增强,可能是熊果酸改善胰岛素抵抗的分子机制之一。更多还原

【Abstract】 Insulin resistance (IR) is a state in which a given concentration of insulin produces a less-than-expected biological effect. It is a common risk factor for a variety of metabolic diseases and it often exists in the early stage of these diseases. So the treatment of insulin resistanceit is very important for the prevention and treatment of metabolic diseases, espec-ially diabetes.Obesity (mainly abdominal obesity) was the important risk factor resulting in insulin resistance. Obesity can lead to insulin resistance through endocrine, adipocytokines, infla-ammatory response and intrinsic cell signaling pathways. It is now recognized that free fatty acids (FFA) decomposed by the fat and adipocytokines are often involved in insulin resistance mediated by obesity. These factors can affect the sugar, fat metabolism through different mechanisms, and there are complex linkages among the various factors. Among them, free fatty acid (FFA) and tumor necrosis factor-a (TNF-a) are closely related to insu-lin resistance. TNF-a can promote fat decomposition and FFA concentration. By inhibiting lipoprotein lipase. Increased FFA levels can increase the insulin resistance and can also cause hyperinsulinemia. The liver is the main target organ for insulin. In the metabolic effects of insulin, the main function of liver is in the promotion of liver glycogen synthesis and inhibiting glycogen output. When the liver insulin resistance occurs, the liver glycogen synthesis shoud be reduced and gluconeogenesis shoud be increased, resulting in hypergly-cemia. The insulin receptor substrate-2 (IRS-2) branch signal of Insulin signaling cascade is closely related to insulin sensitivity of liver cells. IRS-2/PI3-K signal pathway is the major signal transduction pathways of insulin playing a physiological effect in the liver. Reduction of insulin receptor substrate-2 (IRS-2) expressions in gene and protein and phosphorylation disorder will affect its the effective signa transmission of its downstream PI3K/Akt pathway, which can cause insulin resistance. At present, a number of factors can be found to lead to liver insulin resistance by affecting the IRS-2 signal transduction. Protein tyrosine phosphatase-1B (PTP-1B) is a major member of the protein tyrosine phosphatase (PTP) family. It could make the tyrosine residues of InsR, IRS and other signaling proteins to be dephosphorylated, and block insulin signaling cascade downstream. PTP-1B is a negative regulator of insulin signaling. So it is supposed that whether we can achieve the purpose of improving insulin resistance by inhibiting PTP-1B expression so as to increase the expression and phosphorylation of IRS-2. This study was carried out under the guidance of Chinese medicine theory of "sour restrains sweet" and "sour enters liver". This study attempts to conduct in-depth study about ursolic acid on the role and mechanism of insulin resistance through animal experiments, and molecular biology tools. The liver will be as a target organ in the study. Hopes to prove the guiding role of traditional Chinese medicine theory of "sour restrains sweet" to the prevention and treatment of insulin resistance and diabetes and provide experimental basis for diabetes prevention and drug discovery.Part I Rats of Insulin Resistance Model Induced by High-Fat-FedObjective:To establish insulin resistance obesity rats model induced by high-fat-fedMethods:60 eight-week-old male Wistar rats, body weight 180-200g, were randomly divided into two groups:normal diet group(n=12) and high-fat diet group(n=48). Normal diet group rats were fed with basic feed(fat10%, carbohydrate67%, protein23%)and high-fat diet group rats were fed with high-fat feed(fat46%, carbohydrate40%, protein14%): basic diet(78.9g) plus lard 10g, cholesterol 1g, egg yolk powder 10g, bile salt 0.1g. Before and after the experiment, the body weight and fasting blood glucose, TC, TG, HDL-C and LDL-C levels were tested; serum insulin levels determined by enzyme-linked immuno-sorbent assay (ELISA); the regulatory function of glucose metabolism determined by the glucose tolerance test and insulin tolerance test; insulin sensitivity evaluated by the insulin sensitivity index (ISI) and insulin resistance index (HOMA-IR).Results:Compared with the normal diet group rats, the fasting blood glucose of high-fat diet group rats is not obvious or mildly elevated, while the body weight, blood lipids (TC, TG, LDL-C), fasting plasma insulin levels were significantly higher. The high-fat diet group rats appear impaired glucose tolerance and insulin tolerance and decline in insulin sensitivity.Conclusion:Insulin resistance obesity rats model can be successfully established by 9-week high-fat diet feeding. The model’s characteristics is similar to human insulin resistance caused by obesity and we think it is a economic and practical animal model for the study of obesity and insulin resistance.PartⅡEffects of ursolic acid on glucose, lipid metabolism and FFA, TNF-a levels in insulin resistance ratsObjective:To observe the effects of ursolic acid on glucose, lipid metabolism and free fatty acid (FFA), tumor necrosis factor-a (TNF-a) levels in insulin resistance rats.Methods:60 rats were randomly divided into five groups:normal group (normal diet+ intragastric administration with PBS solution), model group (high-fat diet+intragastric administration with PBS solution), metformin group (high-fat diet+intragastric administration with metformin,200mg/kg·d), ursolic acid high-dose group(high-fat diet+ ursolic intragastric administration with ursolic acid,300mg/kg·d), and ursolic acid low-dose group (high-fat diet+ursolic intragastric administration with ursolic acid, 150mg/kg-d). Each group is 12. At point 4 weeks and point 8 weeks after administration of each group, to observe the changes of fasting blood glucose (FBG), fasting serum insulin (FINS), blood lipids, body weight, liver weight, abdominal adipose weight, glucose tolerance, insulin tolerance, liver glycogen content and serum free fatty acids (FFA), tumor necrosis factor-a (TNF-a); to calculate the liver index(LI), abdominal adipose index(AI), insulin sensitivity index (ISI) and insulin resistance index (HOMA-IR); to observe the liver tissue pathological changes by light microscopy (HE staining).Results:Ursolic acid and metformin treatment can reduce serum FINS, TC, TG, LDL-C and FFA, TNF-a levels in insulin resistance rats, also they can lower body weight, liver index and abdominal adipose index; reduce intra-abdominal fat accumulation; improve glucose tolerance, insulin tolerance; add liver glycogen content; increase insulin sensitivity index (ISI) and lower insulin resistance index (HOMA-IR). Compared with the model control group, There were significant difference between treatment group and model control group (P<0.05 or P<0.01). There were no significant difference between ursolic acid high-dose group and low-dose group.Conclusion:Ursolic acid can reduce body weight, regulate glucose and lipid metabolism, reduce the levels of FFA, TNF-a in insulin resistance rats and improve insulin resistance.PartⅢEffects of ursolic acid on expressions of PTP-1B and IRS-2 in the liver of insulin resistance rats.Objective:To observe the effects of ursolic acid on expressions of PTP-1B and IRS-2 in the liver of insulin resistance rats and explore the role of PTP-1B and IRS-2 in the process of insulin resistance.Methods:60 rats were randomly divided into five groups:normal group, model group, metformin group, ursolic acid high-dose group, and ursolic acid low-dose group. Each group is 12(administration same to PartⅡ). The mRNA expressions of PTP-1B and IRS-2 in liver were detected by real-time fluorescent quantitative RT-PCR. Relative mRNA quantification methods was used for real-time PCR data analysis. The protein expressions of PTP-1B, IRS-2 and IRS-2 tyrosine phosphorylation in liver were detected by Western blot.Results:The content of liver PTP-1BmRNA expression of model control group detected by real-time fluorescent quantitative RT-PCR is higher than that of the normal control group, while IRS-2mRNA is lower than that of the normal control group. After the therapy of ursolic acid (high-dose and low-dose) and metformin, PTP-1BmRNA levels were decreased and IRS-2mRNA levels were increased. There were significant difference between treatment group and model control group (P<0.05 or P<0.01). Similar protein results were detected in Western blot assay. Western blot results showed that PTP-1B protein expression was decreased; IRS-2 protein expression and tyrosine phosphorylation was increased.Conclusion:PTP-1B expression was increased and IRS-2 expression was decreased in liver of insulin resistance rats. Ursolic acid and metformin can inhibit PTP-1B expression in the liver, thereby contributing to increased IRS-2 expression and IRS-2 tyrosine phosphorylation. That may be one of the molecular mechanisms of ursolic acid improving insulin resistance.更多还原