【作者】 吴勉云；

【导师】 王西明；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 糖尿病分为1型糖尿病(type 1 diabetes mellitus, T1DM)和2型糖尿病(type 2 diabetes mellitus, T2DM),其中2型糖尿病约占90%左右。目前认为2型糖尿病的发病主要是在遗传基础上综合多种环境因素的结果。其中高糖高脂饮食是一种重要的环境因素。胰岛素抵抗(insulin resistance, IR)是2型糖尿病发病的重要基础,贯穿于其发生﹑发展的整个过程。所谓胰岛素抵抗是指一定量的胰岛素生物学反应低于正常水平的一种状态。如何预防2型糖尿病即胰岛素抵抗的发生,已成为摆在人们面前的一个重要课题。无论是流行病学调查还是实验室研究表明,食物中不同类型脂肪酸对胰岛素抵抗发生产生不同影响,饱和脂肪酸过多可引发胰岛素抵抗,而补充多不饱和脂肪酸可改善缓解胰岛素抵抗。花生四烯酸(20: 4, n-6)的化学结构为5,8,11,14-二十碳四烯酸,是哺乳动物体内含量最多,分布最广的一种高不饱和脂肪酸。在健康人群中,肝脏、肌肉和脂肪组织担任着维持血糖平衡的作用,而T2DM患者的这三种组织均存在胰岛素抵抗。新近研究结果表明肝脏是胰岛素作用的一个重要靶器官,肝脏中胰岛素信号转导途径的缺陷,会直接关系到全身胰岛素抵抗的发生和T2DM的发病。因此对肝脏胰岛素抵抗发生机理和预防的研究尤为重要,有利于我们进一步深入研究T2DM的发病机理,并探寻早期预防方法。本课题体内外研究高脂诱导胰岛素抵抗机理及花生四烯酸对胰岛素抵抗的预防作用。HepG2细胞源于人的肝胚胎瘤细胞,保留了肝细胞的许多生物学特性。体外研究以HepG2细胞为研究对象,将饱和脂肪酸软脂酸(palmitate, PA)孵育HepG2细胞,诱导HepG2细胞胰岛素抵抗,加入PKC抑制剂,分析PKC在HepG2细胞形成胰岛素抵抗过程中的作用;将软脂酸和花生四烯酸共孵育HepG2细胞,分析花生四烯酸对HepG2细胞形成胰岛素抵抗的预防作用。体内研究以Wistar大鼠为研究对象,高脂饮食诱导形成大鼠胰岛素抵抗模型,分析花生四烯酸对高脂饮食诱导大鼠全身胰岛素抵抗的预防作用及对肝糖输出和肝脏胰岛素信号传递的影响。第一部分PKC在软脂酸诱导HepG2细胞胰岛素抵抗中的作用目的从蛋白激酶C (PKC)信号通路角度,探讨游离肪肪酸(FFA)引起肝脏胰岛素抵抗的可能机制。方法培养HepG2细胞,同时设立对照组(control组)、软脂酸组(PA组)、高胰岛素组(HI组)。PA组、HI组分别用250μM软脂酸(PA)、5×10-7 M胰岛素处理24小时。然后control组、PA组再根据胰岛素刺激前加(+)与不加(-)PKC抑制剂CC (chelerythrine chloride)5μM预处理1小时,随机分为两亚组:control(-)、control(+)、PA(-)、PA(+)。葡萄糖氧化酶法测定胰岛素刺激后12小时葡萄糖消耗量,蒽酮法测定胰岛素刺激后3小时点细胞内糖原含量, Western-blotting技术检测15分钟点胞内P-Ser473 PKB、P-Ser21/9 GSK-3α/β水平。结果PA(-)组与HI组比较葡萄糖消耗量无统计学差异(P>0.05)。葡萄糖消耗量、细胞内糖原含量、P-Ser473 PKB、P-Ser21 GSK-3α、P-Ser9 GSK-3β水平均显示, PA(-)组与control(-)比较显著降低(P<0.05),control(+)与control(-)组比较略有升高但无显著性差异;PA(+)与PA(-)组比较显著升高(P<0.05)。结论软脂酸(250μM)体外成功诱导了HepG2细胞产生胰岛素抵抗,PKC信号通路在FFA引起肝脏胰岛素抵抗中起着重要的作用。第二部分花生四烯酸对软脂酸诱导HepG2细胞胰岛素抵抗的预防作用目的探讨花生四烯酸(AA)对软脂酸(PA)引起HepG2细胞胰岛素抵抗的预防作用及其可能机制。方法培养HepG2细胞,设立对照组(control组)、软脂酸组(PA组)、软脂酸+花生四烯酸组(PA+AA组)、高胰岛素组(HI组)。PA组、PA+AA组、HI组分别用250μM PA、250μM PA加20μM AA,5×10-7 M胰岛素孵育24h。然后葡萄糖氧化酶法测定各组胰岛素刺激后12h点葡萄糖消耗量,蒽酮法测定胰岛素刺激后3h点细胞内糖原含量, Western-blotting技术检测15min点胞内P-Ser473 PKB、P-Ser21/9 GSK-3α/β水平。结果葡萄糖消耗量PA组与HI组比较差异无统计学意义(P>0.05)。葡萄糖消耗量、细胞内糖原含量、P-Ser473 PKB、P-Ser21GSK-3α、P-Ser9 GSK-3β水平均显示,PA组与control组比较显著降低(P<0.05),PA+AA组与PA组比较显著升高(P<0.05)。结论AA(20μM)能显著预防PA(250μM)引起的HepG2细胞胰岛素抵抗,能在PKB、GSK-3水平上显著维持250μM软脂酸孵育HepG2细胞的胰岛素信号传递。第三部分:花生四烯酸对高脂饮食诱导大鼠全身胰岛素抵抗的预防作用目的观察花生四烯酸对大鼠全身胰岛素抵抗及肝糖输出紊乱的预防作用。方法将230~250g 24只雄性Wistar大鼠随机分为3组:正常组(8例),高脂组(8例),高脂+花生四烯酸组即预防组(8例),饲养12周。正常组:喂以基础饲料;高脂组:喂以高脂饲料;预防组:喂以高脂饲料的同时,每天按3mg.kg-1.d-1剂量灌注花生四烯酸。饲养至第12周实验终点时,进行口服葡萄糖耐受实验,基础状态下称量大鼠体重并处死,分离血清,测定血清中胰岛素、血糖、甘油三酯、胆固醇水平,取肝脏,测定肝糖原含量、磷酸烯醇式丙酮酸羧基酶(PEPCK)酶活性及PEPCK、葡萄糖-6-磷酸酶(G-6-Pase)及糖原合酶(GS)mRNA水平,HE染色观察各组大鼠肝细胞胞浆脂滴分布。结果高脂组与正常对照组比较,基础状态下血清甘油三酯(P<0.05)、胰岛素水平(P<0.05)、肝糖原含量(P<0.01)、葡萄糖-胰岛素指数显著升高(P<0.01),但基础状态下体重无显著性差异。预防组和高脂组比较,肝糖原含量(P<0.05)、甘油三酯水平(P<0.05)、葡萄糖-胰岛素指数(P<0.01)显著降低,但与正常对照组比较,肝糖原含量(P<0.01)、葡萄糖-胰岛素指数(P<0.01)显著升高。高脂组与正常对照组比较,G-6-Pase mRNA水平显著升高(P<0.01),PEPCK酶活性及其mRNA水平略有升高,但无显著性差异。预防组和高脂组比较,PEPCK酶活性及其mRNA水平(P<0.05)、G-6-Pase mRNA水平显著降低(P<0.01),但3组大鼠GS mRNA没有显著性差异。HE染色观察正常组肝细胞胞浆没有脂滴,高脂组有大量大小不一的脂滴,预防组有少量大小不一的脂滴。结论花生四烯酸能显著预防高脂饮食诱导大鼠全身胰岛素抵抗及肝糖输出紊乱,但不彻底。第四部分:花生四烯酸对高脂饮食大鼠肝脏胰岛素信号传递影响的研究目的观察花生四烯酸对高脂饮食大鼠肝脏胰岛素信号传递影响的研究。方法将230~250g 24只雄性Wistar大鼠随机分为3组:正常组(8例),高脂组(8例),高脂+花生四烯酸组即预防组(8例),饲养12周。正常组:喂以基础饲料;高脂组:喂以高脂饲料;预防组:喂以高脂饲料的同时,每天按3mg.kg-1.d-1剂量灌注花生四烯酸。饲养至第12周实验终点时,每组大鼠基础状态下腹腔注射胰岛素(6U/kg), 15分钟后取肝脏,Western blotting技术分析P-Ser473 PKB、P-Ser21/9 GSK-3α/β水平。结果高脂组与正常组比较,P-Ser473 PKB及P-Ser21/9 GSK-3α/β水平显著降低(P<0.01),预防组与高脂组比较,P-Ser473 PKB(P<0.05)、P-Ser21 GSK-3α(P<0.01)及P-Ser/9 GSK-3β(P<0.05)水平显著增加,但预防组与正常组比较,P-Ser473 PKB(P<0.05)及P-Ser21GSK-3α水平(P<0.01)显著降低,P-Ser9 GSK-3β水平没有显著性差异。结论花生四烯酸(3mg.kg-1.d-1)能在PKB、GSK-3水平上显著预防高脂饮食大鼠肝脏胰岛素信号传递障碍,但不能完全彻底地预防。更多还原

【Abstract】 It is well-known that insulin resistance is an important characteristic of Type 2 diabetes. Different free fatty acid play different role in the occurrence of insulin resistance. It is reported that saturated-fatty acid can induce insulin resistance, but unsaturated-fatty acid can improve insulin resistance. However, the mechanism is not fully understood. And liver is one of the most important insulin target tissues, the onset of hepatic insulin resistance typically precedes the appearance of peripheral insulin resistance in human. The 1st part of the present study demonstrates role of PKC in insulin resistance induced by palmitate in HepG2 cells. The 2nd part of the present study demonstrates preventing effect of arachidonic acid, a kind of unsaturated-fatty acid on HepG2 cell.Previous researches reported that only AA had no therapeutic effect in genetically diabetic animal models. AA decrease in blood is the earlier event than hyperglycemia and hyperinsulinemia during the insulin resistance development induced by diet. plasma AA concentrations in human and animal models with typeⅡdiabete are lower than in normal subjects. Thus we hypothesized that optimal AA dose administration could prevent high-fat diet induced insulin resistance by preventing the AA decrease on membranes, maintaining the integrity of membranes and cell normal signal transduction and by other mechanism. To test this hypothesis, the effect of AA on the preventing the insulin resistance was determined in Wistar rats. The 3rd part of the present study demonstrates oral AA administration prevents early whole-body insulin resistance induced by high-fat diet significantly. And the study also demonstrates oral AA administration affects hepatic glycogen storage and relative HGO. The 4th part of the present study demonstrates preventive effect of arachidonic acid on hepatic insulin signaling in high-fat-diet fed rats.Part 1 Role of PKC in insulin resistance induced by palmitate in HepG2 cellsObjective To study on the mechanism of insulin resistance (IR) for HepG2 cells induced by high concentrations of palmitate (PA) through protein kinase C (PKC) signaling pathway analysis. Methods The model of hepatic insulin resistance was established induced by PA.HepG2 cells were randomly didided into control group、PA group (250μM PA) and HI group (5×10-7 M insulin) and treated for 24 hours. Then both of control group and PA group were redivded into subgroups: control(-)、control(+)、PA(-)、PA(+). Only control(+) and PA(+) groups were pretreated by 5μM chelerythrine chloride (CC) of PKC inhibitor for lh before insulin stimulation. Insulin-stimulated glucose consumption was measured using the glucose oxidase method at 12-hour timepoint,cell glycogen was measured with anthrone method at 3-hour timepoint and protein levels of phosphate-protein kinase B (P-Ser473 PKB) and phosphate-glycogen synthase kinase (P-Ser21/9 GSK-3α/β) at 15-min timepoint were determined in total cell lysates by Western-blotting. Results There was no significant difference in glucose consumption between PA(-) group and HI group(P>0.05). All levels of glucose consumption、glycogen conten、P-Ser473 PKB、P-Ser21 GSK-3αand P-Ser9 GSK-3βin PA(-) group were reduced significantly(P<0.05)compared with control(-) group, and were increased significantly(P<0.05)in PA(+) group compared with PA(-) group. Conclusions The model of hepatic insulin resistance was established successfully. Overaction of PKC may play an important role in inducing insulin resistance in HepG2 cells.Part 2 Preventive effect of arachidonic acid on HepG2 cell insulin resistance induced by palmitateObjective To study the effect of arachidonic acid (AA) on preventing HepG2 cell insulin resistance induced by palmitate (PA) and the possible mechanism. Methods The model of hepatic insulin resistance was established induced by PA.HepG2 cells were randomly divided into control group、PA group(250μM PA)、PA+AA group (250μM PA plus 20μM AA) and HI group (5×10-7 M insulin) and cultured for 24 hours. Then insulin-stimulated glucose consumption was measured using the glucose oxidase method at 12 hours, cell glycogen was measured with anthrone method at 3 hours and protein levels of phosphate-protein kinase B(P-Ser473 PKB) and phosphate-glycogen synthase kinase (P-Ser21/9 GSK-3α/β) at 15 minutes were determined in total cell lysates by Western-blotting. Results There was no significant difference in glucose consumption between PA group and HI group. All levels of glucose consumption、glycogen conten、P-Ser473 PKB、P-Ser21 GSK-3αand P-Ser9 GSK-3βin PA group were reduced significantly(P<0.05)compared with control group, and were increased significantly (P<0.05) in PA+AA group compared with PA group. Conclusions AA can prevent HepG2 cell insulin resistance induced by palmitate significantly, keeping insulin-stimulated P-Ser473 PKB and P-Ser21/9 GSK-3α/βlevels. Part 3 Preventive effect of arachidonic acid on early insulin resistance induced by high-fat dietObjective To investigate whole-body metabolic disorder and hepatic glucose output(HGO) disturbance in rats with insulin resistance induced by short-term high-fat diet, and the effect of arachidonic acid(AA). Methods Twenty-four normal male Wistar rats (230-250g) were randomly divided into 3 groups according to their weight and fed for 12 weeks: control group, n=8, to be fed with standard chow diet; high-fat (HF) group, n=8, to be fed with high-fat diet; HF+AA group, n=8, to be fed with high-fat diet and administered orally 3mg.kg-1.d-1AA. By the end of experiment, an oral glucose tolerance test (OGTT) was carried out. At the end of the treatment period, every group was measured in weight and decapitated after an overnight fast. Blood samples were collected and plasma was prepared for the determination of plasma blood glucose (BG), triglyceride (TG), cholesterin (CH) and insulin. Livers were separated and frozen for futher analysis of glycogen content, PEPCK activity, PEPCK, GS and G-6-Pase mRNA levels. HE staining was used to observe lipid deposit in hepatic cells. Results Early insulin resistance was successfully induced in HF rats with hyperinsulinemia (P<0.05), higher plasma TG (P<0.05), higher fasting liver glycogen content (P<0.01) and higher glucose-insulin index (P<0.01) during OGTT. The treatment significantly decreased glucose-insulin index (P<0.01), blood TG (P<0.05) and glycogen content (P<0.05) in liver. Significantly decreased mRNA levels and activity of PEPCK (P<0.05) and lowered G-6-Pase mRNA levels (P<0.01) in liver were also observed. But there were significant differences in glucose-insulin index (P<0.01) during OGTT, and glycogen content (P<0.01) between HF+AA group and control group. There was no significant differences in GS mRNA levels among the three group. No lipid deposit was observed in hepatic cells of control group, but a large quantity of lipid deposit was observed in HF group, and a small quantity was observed in HF+AA group. Conclusions AA can significantly prevent whole-body insulin resistance induced by high-fat diet, as well as accompanied HGO disturbance in overnight fasting state, but not thoroughly.Part 4 Preventive effect of arachidonic acid on hepatic insulin signaling in high-fat-diet fed ratsObjective To investigate P-Ser473 PKB、P-Ser21/9 GSK-3α/βlevels in rats with insulin resistance induced by short-term high-fat diet, and the effect of arachidonic acid(AA). Methods Twenty-four normal male Wistar rats (230-250g) were randomly divided into 3 groups according to their weight and fed for 12 weeks: control group, n=8, to be fed with standard chow diet; high-fat (HF) group, n=8, to be fed with high-fat diet; HF+AA group, n=8, to be fed with high-fat diet and administered orally 3mg.kg-1.d-1AA. By the end of experiment, every group was decapitate 15 minutes afte intraperitoneal injection of insulin (6U/kg). Livers were separated and frozen for futher analysis of P-Ser473 PKB、P-Ser21/9 GSK-3α/βlevels. Results P-Ser473 PKB and P-Ser21/9 GSK-3α/βlevels were significantly decreased in HF rats compared with control rats(P<0.01). The treatment (3mg.kg-1.d-1AA) significantly increased P-Ser473 PKB(P<0.05)、P-Ser21 GSK-3α(P<0.01)and P-Ser9 GSK-3β(P<0.05) levels. But there were significant differences in P-Ser473 PKB(P<0.05)and P-Ser21 GSK-3α(P<0.01) between HF+AA group and control group. Conclusions AA can significantly improve hepatic insulin signaling in high-fat-diet fed rats through increasing P-Ser473 PKB、P-Ser21/9 GSK-3α/βlevels, but not thoroughly.更多还原