【作者】 施劲松；

【导师】 袁文俊；

【作者基本信息】 第二军医大学 ， 生理学， 2010， 博士

【摘要】 研究背景随着人类生活水平和生存环境的改变,内分泌与代谢疾病的发病率逐年上升。超重、肥胖和糖尿病是其中最常见的疾病,对人类健康有极大危害。摄食行为和代谢调节是机体维持能量平衡的重要机制,当摄入远远大于消耗时,将会导致体内能量平衡失调,引起脂肪堆积,导致超重、肥胖以及与肥胖相关联的各种并发症如糖尿病。深入研究机体摄食行为和代谢的调节机制无疑会为肥胖和糖尿病的防治提供新的思路。生长激素释放肽(ghrelin)是1999年Kojima在寻找生长激素促分泌物受体(GHS-R)的内源性配体时发现的。近年来研究发现其是一个重要的食欲和体重调节激素,可通过促进食欲,增加摄食量,以及影响物质和能量代谢的各个方面,导致体重增加、脂肪积累和肥胖。动物实验亦显示直接给予外源性ghrelin可以明显增加大鼠或小鼠的摄食量并导致肥胖。肥胖抑制素(obestatin)是Zhang等在大鼠身上发现的含23个氨基酸的多肽,它由ghrelin前体原产生,ghrelin基因编码产物经翻译后剪切和修饰,其乙酰化产物为ghrelin,其C末端酰胺化产物为obestatin。外源性ghrelin可增加食物的摄入及体重,降低能量消耗,而外源性obestatin则明显抑制其摄食量及体重的增加,且相同剂量的obestatin可以完全拮抗ghrelin促进小鼠摄食和增加体重的作用；以上各种研究提示来源相同的obestatin和ghrelin可能是调控机体食欲和体重的一对功能完全相反的调节因子。胰岛素是体内降低血糖,同时促进糖原、脂肪、蛋白质合成的激素,与肥胖和糖尿病的发生发展密切相关。Ghrelin可以升高血糖水平,而对胰岛素分泌的调节具有葡萄糖浓度依赖性,抑制低浓度葡萄糖刺激的胰岛素分泌,而促进高浓度葡萄糖刺激的胰岛素分泌。Obestatin对胰岛素分泌的影响是obestatin研究领域的新进展,我们课题组观察到obestatin能够抑制葡萄糖刺激引起的胰岛素分泌。目前关于obestatin和胰岛素之间的关系还没有太多研究。因此,本课题拟从以下几个方面探讨obestatin和胰岛素的相互作用：1、葡萄糖和胰岛素对obestatin分泌的影响。2、Obestatin、ghrelin血浆浓度和其基因表达在1型糖尿病大鼠中的变化。3、Obestatin对INS-1细胞在基础状态和高糖刺激状态下胰岛素分泌的影响,并研究其信号通路机制。4、Obestatin对INS-1细胞通道电流的影响。第一部分葡萄糖和胰岛素对大鼠血浆obestatin的影响目的探讨葡萄糖和胰岛素对obestatin血浆浓度的影响。方法SD大鼠禁食4小时,分为3组：A,对照组,灌输生理盐水；B,高血糖钳制组,20%的葡萄糖溶液灌输,调节速度使血糖浓度维持在16.7 mM附近；C,高胰岛素-正常血糖钳制组,按每千克体重7.5mU/min的速度灌输胰岛素,同时灌注20%葡萄糖溶液,调节灌输速度使大鼠血糖维持在对照组水平。实验开始后每15分钟收集5μl血样测血糖；并且每60分钟收集150μl血样,用ELISA法检测血浆obestatin和胰岛素。结果对照组的血浆obestatin浓度在实验过程中缓慢下降,但高血糖钳制组和高胰岛素-正常血糖钳制组血浆obestatin浓度下降更为明显。120分钟时,对照组血浆obestatin浓度为1.188±0.242μg/L,高血糖钳制组和和高胰岛素-正常血糖钳制组分别为0.860±0.180μg/L和0.829±0.203μg/L,均低于对照组,差异有统计学意义(p<0.05,n=6),两个处理组间差异无统计学意义。180分钟时,对照组血浆obestatin浓度为1.049±0.269μg/L,高血糖钳制组和高胰岛素-正常血糖钳制组的血浆obestatin浓度分别为0.398±0.149μg/L和0.369±0.132μg/L,均低于对照组,差异有统计学意义(p<0.01,n=6),两个处理组间差异无统计学意义。结论高浓度胰岛素抑制血浆obestatin浓度,而高浓度的血糖对血浆obestatin浓度可能没有影响。第二部分1型糖尿病大鼠血浆ghrelin、obestatin浓度及其基因表达的变化目的观察1型糖尿病大鼠血浆ghrelin、obestatin浓度及其基因表达的变化方法采用腹腔注射链脲菌素(streptozotocin, STZ)的方法建立大鼠1型糖尿病模型。每周测定大鼠血糖、体重,每2周收集血样,ELISA法测血浆ghrelin、obestatin浓度,取胃组织,定量PCR检测ghrelin/obestatin mRNA表达。结果(1)糖尿病大鼠血浆obestatin浓度在造模后2-6周与对照组差异无统计学意义,8周和10周则高于对照组(p<0.05,n=5)；(2)糖尿病大鼠血浆ghrelin浓度在造模后2周与对照组差异无统计学意义,4周开始高于对照组(p<0.05,n=5)；(3)糖尿病大鼠血浆ghrelin/obestatin比值呈现先升后降的趋势；(4)10周后胃组织ghrelin/obestatin mRNA表达下降(p<0.01,n=5)；(5)10周时糖尿病组大鼠体重变化与ghrelin水平和ghrelin/obestatin比值均呈负相关(p<0.05,n=5)。结论1型糖尿病大鼠血浆ghrelin、obestatin浓度升高,胃组织ghrelin/obestatin mRNA表达下降。第三部分Obestatin对高糖刺激胰岛素分泌的影响及其机制研究目的观察obestatin对高糖刺激引起胰岛素分泌的作用及其机制方法以培养的INS-1细胞为模型,观察胰岛素基础分泌、高糖刺激胰岛素分泌、obestatin对高糖刺激胰岛素分泌的抑制作用,以及各种信号通路阻断剂对obestatin作用的影响。结果(1)高浓度的葡萄糖能够刺激INS-1细胞分泌胰岛素；(2)10 nM和100 nM的obestatin能够抑制INS-1细胞高糖刺激胰岛素分泌(p<0.01,n=6)和INS-1细胞的基础激胰岛素分泌(p<0.05,n=6)；(3)PTX和chelerythrine(PKC抑制剂)能够抑制obestatin的作用(p<0.01,n=6)。Xestospongin C(IP3受体阻断剂),U73122(PLC抑制剂)和neomycin(PLC/PLD抑制剂)对obestatin的作用没有明显影响。结论Obestatin对INS-1细胞葡萄糖刺激胰岛素分泌的抑制作用的部分机制可能是通过PTX敏感G蛋白和细胞膜PKC介导的。第四部分Obestatin对INS-1细胞通道电流的影响目的观察Obestatin对INS-1细胞通道电流的影响方法以培养的INS-1细胞为模型,利用穿孔全细胞膜片钳技术研究obestatin对细胞膜电位、动作电位、ATP敏感钾电流(ATP-sensitive potassium currents, IKATP)和电压敏感钙电流(Voltage-dependent calcium currents, ICaV)的影响。结果(1) Obestatin对静息膜电位无明显作用,但能够降低葡萄糖刺激引起的动作电位的幅度,10 nM和100 nM obestatin作用5 min后,INS-1细胞动作电位幅度分别为给予obestatin之前的69.4±9.6%和39.8±7.0%,与对照(93.1±3.6%)相比,差异有统计学意义(p<0.01,n=5)；(2)Obestatin对IKATP无明显作用,但能够剂量依赖性地抑制ICaV,10 nM和100 nM obestatin作用5 min后,Cav通道电流分别为给药前的60±9%和45±6%,与对照组(97±8%)相比,差异有统计学意义(p<0.01,n=5)。结论Obestatin抑制细胞膜Cav通道电流。结合第三部分的实验结果,推测obestatin对葡萄糖刺激胰岛素分泌的抑制作用的部分机制可能是结合PTX敏感G蛋白偶联受体、激活的G蛋白在细胞膜PKC的协助下抑制细胞膜电压依赖钙通道(Voltage-dependent calcium channels, VDCC)。更多还原

【Abstract】 With great changes in human lifestyle in recent years, the incidence of endocrine and metabolic diseases is rising yearly. Overweight, obesity and diabetes are very big threats to human health. Regulation of feeding behavior and metabolism is an important mechanism to maintain energy balance. When the intake is far more than the consumption, the energy balance is broken, which leads to fat accumulation and may result in overweight, obesity and complications of obesity such as diabetes. Investigation on the regulation mechanisms of feeding behavior and metabolism would benefit the prevention and cure of obesity and diabetes.Ghrelin was initially discovered by Kojima et al. in 1999 as an endogenous ligand for GHSR (growth hormone secretagogue receptor), but subsequent considerable and unequivocal evidence shows that it plays critical roles in the short-and long-term regulation of appetite and body weight. Ghrelin affects appetite and food intake as well as a diverse array of processes involved in energy expenditure and fuel utilization, all of which promote weight gain and fat accumulation. Exogenous ghrelin administration causes hyperphagia and obesity in rodents.Zhang and coworkers reported that ghrelin gene also encodes another 23-amino acid peptide, obestatin. The biological activity of obestatin depends on the amidation at its carboxyl terminus. Obestatin, though derived from the same peptide precursor, suppressed food intake, inhibited jejunal contraction, decreased body-weight gain, and antagonized the actions of ghrelin when both peptides are co-administered. These facts may suggest that the intricate balance of ghrelin and obestatin is important in the regulation of energy homeostasis and body weight control.Insulin can lower blood glucose concentration and promote the synthesis of glycogen, fat and protein. Insulin is closely related to fat and diabetes. Ghrelin can raise blood glucose level and inhibit insulin release. A recent prominent discovery in the obestatin field came from the effect of obestatin on insulin secretion. It is found that administration of exogenous obestatin suppresses insulin secretion in glucose-stimulated insulin secretion.To the best of our knowledge, there is little knowledge about the relationship between obestatin and insulin. Therefore, we attempted to investigate the relationship between obestatin and insulin as follows.1. The effect of glucose and insulin on plasma obestatin level was investigated using hyperglycemic clamp and hyperinsulinemic-euglycemic clamp.2. Diabetic rats were induced by a single intraperitoneal injection of 60 mg/Kg streptozotocin. Plasma obestatin and ghrelin levels and expressions of ghrelin/obestatin mRNA were investigated.3. INS-1 cells were cultured and used to investigate the effect of obestatin on normal and glucose stimulated insulin secretion. The mechanism was explored using blockers.4. The effects of obestatin on membrane potential, action potential, ATP-sensitive potassium currents and voltage-dependent calcium currents of INS-1 cells were investigated using perforated whole cell patch-clamp technique.Part 1 The effect of glucose and insulin on plasma obestatin levelObjectiveTo investigated the effect of glucose and insulin on plasma obestatin level.MethodsSD rats (not fasted, but 4 h after removal of food from cages) were assigned to 3 treatment groups:(A) saline-infused control. (B) hyperglycemic group infused with 20% dextrose for 3 h to goal blood glucose 16.7 mM. (C) hyperinsulinemic-euglycemic clamp (7.5 mU insulin/min per kg body weight, and variable rate 20% dextrose) to reach insulin concentrations similar to group B and glucose similar to group A. After baseline samples (150μl) were obtained, samples (5μl each) were obtained from the catheter in the jugular vein every 15 min. And samples (150μl each) were obtained every 60 min after treatment administration to measure obestatin and insulin using ELISA.ResultsIn control group, plasma obestatin level decreased slightly, but decreased promptly in hyperglycemic group and hyperinsulinemic-euglycemic group. At 120 minutes, plasma obestatin level were 0.860±0.180μg/L and 0.829±0.203μg/L respectively in hyperglycemic group and hyperinsulinemic-euglycemic group, compared with control group (1.188±0.242μg/L), the differences were significant (p<0.05, n=6).At 180 minutes, plasma obestatin level were 0.398±0.149μg/L and 0.369±0.132μg/L respectively in hyperglycemic group and hyperinsulinemic-euglycemic group, compared with control group (1.049±0.269μg/L), the differences were significant (p<0.01, n=6). The differences of plasma obestatin level between hyperglycemic group and hyperinsulinemic-euglycemic group were not statistically significant.ConclusionHigh concentration of plasma insulin reduced plasma obestatin levels, but high concentration of plasma glucose may has no effect on plasma obestatin levels.Part 2 Plasma obestatin and ghrelin levels and expression of ghrelin/obestatin mRNA in type 1 diabetic ratsObjectiveTo investigated plasma obestatin and ghrelin levels and expression of ghrelin/obestatin mRNA in type 1 diabetic rats.MethodsType 1 diabetic rats were induced by a single intraperitoneal injection of 60 mg/Kg streptozotocin (STZ). Plasma glucose levels and weights were measured weekly. Blood samples were collected to measure obestatin and insulin using ELISA, gastric tissues were collected to investigate the expression of ghrelin/obestatin mRNA by Real-time PCR.Results(1) Plasma obestatin levels remained unchanged at 2-6 weeks, but increased at 8 and 10 weeks, compared with control group, the differences were significant (p<0.05, n=5). (2) Plasma ghrelin levels remained unchanged at 2 weeks, and then increased at 4-10 weeks, compared with control group, the differences were significant (p<0.05, n=5). (3) The ghrelin/obestatin ratio first rose and then decreased. (4) The expression of ghrelin/obestatin mRNA in gastric tissues decreased at 10 weeks, compared with control group, the differences were significant (p<0.01, n=5). (5) The weight changes of diabetic rats were negatively correlated with plasma ghrelin levels (r=-0.833, p<0.05, n=5) and ghrelin/obestatin ratio (r=-0.824, p<0.05, n=5).ConclusionPlasma obestatin and ghrelin levels rose and expression of ghrelin/obestatin mRNA decreased in type 1 diabetic rats.Part 3 The effect of obestatin on glucose stimulated insulin secretion and its mechanismsObjectiveTo investigate the effect of obestatin on normal and glucose stimulated insulin secretion, and discuss the mechanisms.MethodsINS-1 cells was cultured and used to investigate the effect of obestatin on normal and glucose stimulated insulin secretion, and the mechanism was explored using blockers.Results(1) High concentration of glucose stimulated the secretion of insulin in INS-1 cells. (2) 10 nM and 100 nM obestatin suppressed the secretion of insulin stimulated by high concentration of glucose (p<0.01, n=6) and basal insulin secretion (p<0.05, n=6). (3) PTX and chelerythrine (PKC inhibitor) antagonized the effects of obestatin (p<0.01, n=6). Xestospongin C (IP3 receptor blocker), U73122 (PLC inhibitor) and neomycin (PLC/PLD inhibitor) had no significant effect.ConclusionOne mechanism by which obestatin decreased insulin secretion stimulated by glucose is by PTX-sensitive G proteins and PKC.Part 4 The effects of obestatin on membrane ion channels of INS-1 cellsObjectiveTo investigate the effects of obestatin on membrane ion channels of INS-1 cellsMethodsCultured INS-1 cells were used for perforated whole cell patch-clamp recording. We investigate the effects of obestatin on membrane potential, action potential, ATP-sensitive potassium currents (IKAP) and voltage-dependent calcium currents (ICaV).Results(1) Obestatin did not affect the membrane potential, but suppressed the amplitudes of action potential stimulated by glucose. Treatment with 10 and 100 nM obestatin for 5min, the amplitudes of action potential were 69.4±9.6%and 39.8±7.0% of the value before treatment, compared with the control group (93.1±3.6%), the differences were significant (p<0.01, n=5). (2) Obestatin had no significant effect on IKAP, but dose dependently inhibited Cav currents. After treatment with 10 and 100 nM obestatin for 5min, Cav currents were 60±9% and 45±6% of the value before treatment, compared with the control group (97±8%), the differences were significant (p<0.01, n=5).ConclusionObestatin suppressed Cav currents of INS-1 cells. Considering the results in part 3, we speculate one mechanism by which obestatin decreased insulin secretion stimulated by glucose is by activating PTX-sensitive G proteins, and then with help of membrane PKC, activated G protein suppressed the voltage-dependent calcium channels (VDCC).更多还原