【作者】 王振基；

【导师】 申竹芳；

【作者基本信息】 中国协和医科大学 ， 药理学， 2006， 博士

【摘要】 糖尿病是目前对人类健康威胁最大的常见疾病之一,其中90%以上是以胰岛素抵抗为主要病理特征的2型糖尿病。胰岛素抵抗不仅是2型糖尿病的病理基础,还与心血管疾病的发生密切相关。胰岛素抵抗常常伴随葡萄糖耐量异常、肥胖、脂质代谢紊乱、高血压,称为胰岛素抵抗综合征。贝特类PPARα激动剂具有良好的调脂作用,并能降低体重,改善炎症状态和血管机能。噻唑烷二酮类(TZDs)PPARγ激动剂具有胰岛素增敏作用,不但促进脂肪细胞分化、降低血糖和血胰岛素水平,还能降低血压、改善血管内皮功能和动脉粥样硬化,减轻心血管危险因子如TNFα等的损害,保护胰岛β细胞功能,在糖尿病和心血管疾病的治疗和预防方面取得了良好效果。但是部分病人出现的水肿和体重增加等副作用限制了TZDs的使用。GCP-02是本所合成的新型α-烷氧基苯丙酸衍生物,体外反向激活实验证实为PPARα/γ双激动剂,期望它能够保持PPARα和PPARγ两类激动剂的调脂和胰岛素增敏作用,同时减少和减轻副作用。本研究采用胰岛素抵抗的肥胖MSG小鼠模型,以PPARγ激动剂罗格列酮为对照研究了GCP-02对胰岛素耐量、葡萄糖耐量、糖异生和血糖、血脂、血胰岛素水平等的影响,测定了肝脏、骨骼肌、心肌的脂质和糖原含量及心肌抗氧化能力,测定了肝脏与葡萄糖代谢相关基因的mRNA表达。为进一步探讨其作用机制,采用4种体外培养的细胞研究了GCP-02对葡萄糖转运、葡萄糖消耗、细胞活力和前脂肪细胞分化的影响,分别采用培养的人脐静脉内皮细胞和大鼠离体主动脉模型观察丁GCP-02对内皮细胞功能的影响。结果1.GCP-02能够降低MSG小鼠非禁食血糖和空腹血糖,提高胰岛素敏感性,改善胰岛素耐量和葡萄糖耐量,抑制以左旋丙氨酸为底物的糖异生。2.GCP-02降低MSG小鼠体重,降低血胆固醇(Cho1)、甘油三酯(TG)和游离脂肪酸(FFA)水平。3.GCP-02降低MSG小鼠肝脏TG、FFA和糖原含量。4.GCP-02对血糖、胰岛素耐量、葡萄糖耐量、糖异生和肝脂、肝糖原的作用均强于阳性对照药罗格列酮。5.GCP-02降低骨骼肌糖原含量,降低心肌TG含量,提高心糞OD活性。6.GCP-02上调MSG小鼠肝脏IRS-2的mRNA表达。7.GCP-02促进HepG2细胞的葡萄糖消耗,促进HIRc-B细胞的葡萄糖消耗,促进3T3-L1前脂肪细胞的成熟分化,但不影响WB-F344细胞葡萄糖消耗。8.与HUVEC培养24小时,GCP-02对NO和ET-1合成分泌均无显著影响。9.GCP-02能降低去甲肾上腺素所致离体大鼠胸主动脉的收缩程度,其血管松弛作用与PGs和NO无关。结论1.GCP-02增加胰岛素抵抗肥胖MSG小鼠的胰岛素敏感性、抑制糖异生、降低血糖,表明有PPARγ激动作用;有效降低血Cho1、TG和FFA,说明有PPARα激动作用。2.PPARα/γ双激动剂GCP-02的胰岛素增敏和对血糖、血脂的调节作用均强于PPARγ激动剂罗格列酮。3.GCP-02的作用可能与上调肝脏IRS-2的mRNA表达、促进葡萄糖消耗、促进脂肪细胞的分化有关。4.GCP-02抑制去甲肾上腺素性离体大鼠胸主动脉收缩,其血管松弛作用与NO和PGs无关。更多还原

【Abstract】 Background and Objective Diabetes mellitus has become one of the main threats to human health in the 21 st century.The diabetes epidemic relates particularly to type 2 diabetes mellitus（T2DM）,which accounts for 90%of cases globally.T2DM pathophysiologically arises from two defects:impaired insulin action and impairedβ-cell function/insulin secretion.Impaired insulin action,or insulin resistance,is not only the pathophysiologic basis for T2DM,but also associated closely with cardiovascular diseases.Insulin resistance often exsits together with obesity, dyslipidemia and hypertension,that are called insulin resistance syndrome.Fibrates PPARαagonists are hypolipidemic drugs by promoting fatty acid oxidation and can reduce lipid accumulation and body weight,but are not effective in blood glucose control.Thiazolidinediones（TZDs） PPARγagonists are a new class of anti-diabetic drugs with insulin-sensitizing properties.In addition to lowering blood glucose,TZDs may also benefit cardiovascular parameters,such as lipids,blood pressure, inflammatory biomarkers,endothelial function,and fibrinolytic status.However,side effects such as edema and weight gain restrict the use of TZDs in some patients.Then it’s of interest to develop substances with combined PPARα/γeffects that could maintain the beneficial effects on insulin resistance and its associated atherogenic dyslipidemia while reducing the side effects.GCP-02,anα-ethoxy-phenyl-propionic acid derivative synthesized by the synthesis division of Institute of Materia Medica, Chinese Academy of Medical Sciences,has been identified by in vitro transactivation studies as a potent,selective dual activator of both PPARαand PPARγ.Here we report the primary study on the anti-diabetic effects and its mechanism of action of this compound.Methods The research includes three parts.1.Monosodium glutamate-induced insulin resistant obese mice（MSG mice） were used to examine the action of GCP-02 in vivo.MSG mice were divided into four groups:MSG mice control group（Con）, rosiglitazone group(7μmol·kg^-1,RSG),high dose of GCP-02 group(7μmol·kg^-1, GCP-02-H) and low dose of GCP-02 group(3.5μmol·kg^-1,GCP-02-L).Agents were given orally once daily for 19 consecutive days,during which insulin tolerance test, oral glucose tolerance test and gluconeogenesis test were performed and in each test serum cholesterol（Chol）,triglyceride（TG） and free fatty acids（FFA） were monitored. On day 19,mice were decapitated.Body length and body weight were measured. Serum was collected for the determination of insulin,alanine aminotransferase （ALT）,and aspartate aminotransferase（AST）.Intraperitoneal adipose,heart,liver were weighed.Liver,soleus muscle and myocardium were assayed for glycogen, Chol,TG and FFA contents.Myocardium superoxide dismutase（SOD） and malonaldehyde（MDA） were determined.The mRNA expressions of insulin receptor （IR）,insulin receptor substrate 1 and -2（IRS-1,IRS-2）,glucose transportor 1 and -2 （GLUT-1,GLUT-2）,PPARαand PPARγ,in liver were analysed by reverse-transcription PCR.To further investigate the mechanism of action of GCP-02, 2.HepG2,HIRc-B and WB-F344 cells were used to test the effects of GCP-02 on glucose transportation,consumption and cell viability.3T3-L1 cells were used to test the effect of GCP-02 on adipocyte differentiation.3.Human umbilical vein endothelial cells（HUVEC） were cultured to test the effects of GCP-02 on NO and ET-1 secretion.The effect of GCP-02 on artery contractility was performed in rat aortic rings.Results 1.In insulin resistant and obese MSG mice,GCP-02 improved insulin sensitivity,insulin tolerance and glucose tolerance,suppressed gluconeogenesis,the effects were more potent than RSG.2.GCP-02 prodeced more potent,rapid and stable effects than RSG in lowering serum Chol,TG and FFA.3.GCP-02 reduced body weight of MSG mice.4.GCP-02 significantly decreased glycogen,TG and FFA contents of liver,slightly decreased glycogen content of soleus muscle,reduced TG content of myocardium and increased myocardial SOD activity.5.The expression of IRS-2 mRNA in the liver was down-regulated in MSG mice and was up-regulated by GCP-02.6.GCP-02 promoted glucose consumption in both HepG2 and HIRc-B cells. 7.GCP-02 elicited similar promoting effect as RSG on 3T3-L1 cell differentiation.8. GCP-02 didn’t affect NO and ET-1 production significantly in HUVEC after 24 hours of treatment.9.GCP-02 caused dose-dependent reduction of norepinephrine-induced aortic contraction by mechanisms independent of NO and PGs.Conclusion 1.The effects on insulin sensitivity,insulin tolerance,glucose tolerance,blood glucose and gluconeogenesis indicate the activation of PPARγby GCP-02;the effects on serum Chol,TG,FFA and body weight indicate the activation of PPARαby GCP-02.2.PPARα/γdual agonist GCP-02 is more potent than PPARγagonist rosiglitazone in insulin sensitizing and in blood glucose,blood lipids controlling.3.The mechanism of GCP-02 action correlates possibly to the up-regulation of mRNA expression of IRS-2 in liver and the promotion of glucose consumption and pre-adipocyte differentiation.4.GCP-02 reduced norepinephrineinduced rat aortic contractilion by mechanisms independent of NO and PGs.更多还原