【作者】 冯晓丽；

【导师】 祝之明；

【作者基本信息】 第三军医大学 ， 内科学， 2009， 博士

【摘要】 背景和目的:代谢综合征(metabolic syndrome,MS)是以中心性肥胖为核心,合并血压、血糖、甘油三酯升高和/或高密度脂蛋白胆固醇(HDL C)降低,可显著增加2型糖尿病和心血管病的危险性。一类抗高血压药物,血管紧张素Ⅱ受体阻断剂(ARBs)常用于治疗合并有糖尿病或糖尿病肾病的高血压患者。几个临床试验的亚组分析发现这类药物改善了患者的胰岛素抵抗、降低了高血压患者中新发糖尿病的发生率。在动物试验也证实ARBs改善肥胖或高血压大鼠的胰岛素抵抗。由于2型糖尿病目前在全球普遍流行,ARBs的这些药理作用值得进一步关注,以便制定防治该病的更好策略。但是,ARBs对葡萄糖稳态的作用还没有完全阐明。人们广泛认为,ARBs潜在的抗糖尿病作用主要与它们能阻断肾素血管紧张素系统(RAS),纠正血管紧张素Ⅱ(AngⅡ)对代谢的副作用有关。AngⅡ通过对胰岛素信号通路、组织血流、氧化应急、交感神经活性和脂肪生成的作用损害葡萄糖代谢。ARBs与AngⅡ受体结合,竞争地阻断AngⅡ的作用从而发挥增加胰岛素敏感性、降血糖的作用。但是,也有一些研究认为ARBs对胰岛素敏感性的作用相当小或没有作用。这说明ARBs可能存在独立于RAS阻断以外的抗糖尿病作用。在众多的ARBs中,替米沙坦(Tel)在结构上不同与临床上用的其他ARBs。替米沙坦结构类似于胰岛素増敏剂比格咧酮,能部分激活过氧化物酶体增值激活受体γ(PPARγ),从而影响炭水化合物和脂代谢。然而,PPARγ主要分布于脂肪组织,肌肉组织才是外周进行葡萄糖处理的主要组织,而在肌肉组织中主要分布的PPARs为δ型。PPARδ在代谢综合征的作用最近才开始被阐明,已有的研究表明PPARδ在调节脂质代谢、改善胰岛素敏感性以及抗动脉粥样硬化中发挥重要的作用,有望成为防治MS、2型糖尿病的新型药物靶点。因此我们推论替米沙坦在肌肉组织中通过激活PPARδ发挥其增加胰岛素敏感性,改善胰岛素抵抗的作用。为了明确替米沙坦与PPARδ在肌肉组织是否存在相互作用及与葡萄糖代谢的关系,本研究分别于在体和离体水平对上述推论进行探讨,以期为高危人群中药物预防糖尿病发生提供理论依据。本研究分为三个部分:1.用替米沙坦急性刺激C2C12和原代培养的C57BL/6J和PPARδ基因敲除小鼠的骨骼肌细胞,观察替米沙坦对不同细胞葡萄糖摄取的不同影响。2.从分子水平观察替米沙坦对骨骼肌组织及细胞PPARδ、PPARγ、RAS组份及其它相关蛋白表达的影响,初步探讨替米沙坦影响骨骼肌葡萄糖代谢的分子机制。3.用替米沙坦长期喂养C57BL/6J和PPARδ基因敲除小鼠,观察替米沙坦对这两种小鼠模型体重、肌纤维组成及葡萄糖耐量等表型的不同影响。材料方法:整个研究分在体和离体实验两部分。离体研究以C2C12细胞和原代培养的C57BL/6J(C57)和PPARδ基因敲除小鼠的骨骼肌细胞为研究对象。在体试验以C57和PPARδ基因敲除小鼠,以及自发性高血压大鼠(SHR)作为模型。1.小鼠骨骼肌系C2C12细胞诱导成熟后分别用胰岛素,胰岛素+AngII,胰岛素+AngII+替米沙坦刺激,然后用3H 2脱氧葡萄糖检测细胞对2脱氧葡萄糖(2 DG)的摄取率。2.采用原代骨骼肌细胞培养技术,培养C57和PPARδ基因敲除小鼠骨骼肌细胞。细胞融合后换用无血清培养基,在有或无Compound C存在的情况下加用不同浓度的替米沙坦刺激24小时,再用3H 2脱氧葡萄糖测其对2 DG摄取率。3.提取原代培养骨骼肌细胞及小鼠模型腓肠肌的总RNA和蛋白,用半定量RT PCR技术检测PPARγ和PPARδmRNA表达;用Western blot技术分析PPARγ、PPARδ、RAS组份(ACE、AngⅡ、Renin和AT1R)以及其它相关蛋白(AMPKα1、p AMPKα、TroponinⅠss)的表达。SHR大鼠肌肉组织PPARγ、PPARδmRNA和蛋白表达也被分析。4.用替米沙坦干预C57BL/6J和PPARδ基因敲除小鼠共24周,观测喂养前10天的进食量、饮水量;每2周观测一次体重;第23 24周时进行运动耐量、氧耗量测试、腹腔内注射葡萄糖耐量实验和胰岛素耐量实验;第24周过夜空腹麻醉后处死,取血测胰岛素、甘油三酯和总胆固醇;取双侧腓肠肌,一侧冰冻切片后做ATP染色,观察骨骼肌纤维成分组成,另一侧70℃冻存,备提蛋白和RNA。结果:1.胰岛素刺激成熟C2C12细胞的2 DG摄取,AngⅡ抑制胰岛素的该作用;替米沙坦的存在则逆转了AngⅡ对胰岛素刺激的2 DG摄取的抑制作用。2.原代培养的C57小鼠骨骼肌细胞在胰岛素刺激下2 DG摄入增加,替米沙坦剂量依赖性增加C57骨骼肌细胞2 DG摄入,该作用在AMPK抑制剂Compound C存在时受抑制;胰岛素仍能刺激原代培养的PPARδ基因敲除小鼠骨骼肌细胞的2 DG摄入,但替米沙坦对此骨骼肌无增加2 DG摄入的作用。3.替米沙坦急性干预显著增加原代培养的C57骨骼肌细胞PPARδmRNA和PPARδ、AMPK、p AMPK蛋白表达,对PPARγmRNA和PPARγ、RAS组份蛋白表达无影响;替米沙坦长期干预增加C57骨骼肌组织PPARδmRNA和PPARδ、p AMPK蛋白表达,对PPARγmRNA和PPARγ、RAS组份蛋白表达无影响;替米沙坦长期或急性干预都不影响PPARδ基因敲除小鼠骨骼肌细胞和组织的PPARγmRNA和PPARγ、AMPK、p AMPK、RAS组份蛋白表达。替米沙坦长期干预促进SHR肌肉组织PPARδmRNA和蛋白表达,也促进肌肉组织PPARγmRNA和蛋白表达。4.替米沙坦喂养的小鼠与普食喂养的小鼠摄食量、饮水量无区别。C57小鼠替米沙坦喂养组较普通饮食喂养组的小鼠体重增加缓慢,12周以后体重明显减轻。实验23 24周时,血总胆固醇下降,运动耐量增加,运动后氧耗量增加,腓肠肌中Ⅰ型纤维肌肉成分增多,腹腔内注射葡萄糖耐量曲线下面积减少,胰岛素耐量实验中胰岛素注射后血糖下降更明显,曲线下面积明显减少。PPARδ基因敲除小鼠替米沙坦喂养组和普通喂养组之间以上观察指标间无显著差异。结论:1.替米沙坦对骨骼肌细胞的葡萄糖摄取有直接促进作用,该作用通过PPARδ/AMPK途径发挥作用。当RAS活性增强时,替米沙坦还通过抑制AngⅡ作用改善胰岛素刺激下的骨骼肌细胞葡萄糖摄取。2.替米沙坦通过激活PPARδ/AMPK途径,抑制小鼠体重增加,增加腓肠肌中Ⅰ型纤维含量和运动后氧耗量,提高运动耐量,改善葡萄糖耐量,增加胰岛素敏感性。3.在普食或正常细胞培养状态下,替米沙坦不影响肌肉组织和细胞RAS组份表达和活性。更多还原

【Abstract】 Background and Purpose: The metabolic syndrome, which describes the presence of visceral obesity, insulin resistance or glucose intolerance, atherogenic dyslipidemia, hyperuricemia, and hypertension in a patient, increases the risk for type 2 diabetes and cardiovascular disease. A class of anti hypertensive drugs, angiotensin II type 1 receptor blockers (ARBs), has been reported to have favorable effects on insulin sensitivity. Particularly, sub analysis of several recent clinical trials has revealed that these drugs reduce the incidence of new onset diabetes. Additionally, blockade of the AT1R has been shown to improve insulin sensitivity in animal models of insulin resistance. However, the mechanisms underlying the insulin sensitizing and antidiabetic effects of the ARBs have not been defined. They may include enhancing blood flow through the microcirculation of skeletal muscles and, thereby, promotion insulin and glucose delivery to the insulin sensitive tissues. As a result, insulin signaling is facilitated at the cellular level and insulin secretion by beta cells is improved. Some ARBs, such as, telmisartan (Tel), have been found to effectively activate the peroxisome proliferator activated receptorγ(PPARγ) , a well known target for insulin sensitizing.PPARs are ligand - inducible transcription factors which isoforms display tissue specific expression and gene regulatory profiles. PPARγis a key regulator of adipocyte differentiation and adipose insulin sensitivity, whereas it is barely expressed in skeletal muscle. By contrast, PPARδ(also referred to as PPARβ) is expressed in a wide variety of tissues, with high levels in skeletal muscle. Studies in transgenic mice show that targeted expression of activated PPARδincreases the predominance of oxidative typeⅠmuscle fibers, enhances whole body insulin sensitivity, and increases exercise endurance capacity . The PPARδspecific agonist GW501516 improves insulin sensitivity in skeletal muscle in several animal models. Thus, activation of PPARδmay offer an efficacious strategy for the improving glucose homeostasis. However, this particular PPARδagonist is not available for clinical use due its undesired side effects. It is important to know whether ARBs, such as Tel affect PPARδexpression and/or function. Given the importance of skeletal muscle insulin resistance in the development of type 2 diabetes, we hypothesized that Tel has direct effects on glucose metabolism in skeletal muscle through an action on PPARδ.In the present study, we first confirmed whether Tel directly improved glucose intake in primary cultured myotubes of mice throught PPARδ. Then we detected the glucose metabolism related key molecules in skeletal muscle cell and tissue of mice to find out whether PPARδwere regulated by Tel. Finally, we examined the glucose metabolism related struction and function changes of skeletal muscle in C57BL/6J and PPARδknockout mice fed with or without Tel.Methods:The present study includes in vivo and in vitro experiments. In vitro models were primary cultured myotubes from C57BL/6J (C57)and PPARδknockout mice, as well as myotubes differentiated from control C2C12 myoblasts. In vivo models include wild type mice and PPARδknockout mice fed with or without telmisartan, and spontaneous hypertensive rats (SHR) were also observed.1. The differentiated C2C12 myotubes were divided into groups as follow: Control, Insulin, Insulin+angtensionⅡ(AngⅡ)and Insulin+ AngⅡ+Tel groups. After treatment, the 2 deoxyglucose (2 DG) uptake was assayed.2. The skeletal muscle cells from C57 and PPARδknockout mice were cultured in DMEM using the primary culture technique. After confluent, cells were given DMEMs without FBS. Telmisartan was added to media in present or absence of Compound C. After 24 h, 2 Deoxyglucose uptakes assay in cells were carried out.3. Protein expression of glucose metabolism related molecules including PPARδ, PPARγ, AMPKα1, p AMPKα, TroponinⅠss as well as RAS components (included ACE、AngⅡ、Renin and AT1R) in skeletal muscle cells and tissue from C57 and PPARδknockout mice, were detected by immunoblotting and mRNA expression of PPARδand PPARγwere detected by RT PCR. Protein and mRNA expression of PPARδand PPARγin SHR were also analyized.4. Mice were fed a normal diet or normal diet plus telmisartan. Daily food intake per mouse was recorded during the ?rst 10 days after the start of telmisartan administration. Body weights were measured every two weeks throughout the experimental period. During 23 24 weeks, exercise endurance, oxygen consumption, intraperitoneal glucose tolerance test (IPGTT) as well as insulin tolerance test (ITT) were analyzed in mice. After sacrificed, metabolic parameters were determined and fiber typing of gastronomies muscle was observed using the metachromatic dye ATPase method.Major Research Results:1. The 2 DG uptake in differentiated C2C12 myotubes increased by insulin and inhibited by AngⅡprincubation. Telmisartan inverted the inhibition effect of AngⅡon the insulin stimulated 2 DOG uptake .2. Insulin and telmisartan significantly increased 2 DG uptake in cultured myotubes from C57 mice compared with control myotubes. Administration of compound C, an AMPK inhibitor, significantly reduced telmisartan’s action in cultured C57 myotubes. In the cultured PPARδdeficient myotubes, insulin increased 2 DG uptake, but telmisartan no effect on glucose uptake.3. Telmisartan significantly increased expression of PPARδmRNA and protein expression in cultured myotubes in skeletal muscle tussue from C57 mice, and no effect on PPARγexpressions in skeletal muscle cell and tissue from either C57 or PPARδdeficient mice. Protein expression of AMPKα1and p AMPKαin myotubes , p AMPKαand TroponinⅠss in skeletal muscle tussue of C57 was significantly increased by telmisartan stimulation. Telmisartan did not influence RAS components of primary cultured myotubes and skeletal muscle in either wild type or PPARδdeficient mice.4. 24 week telmisartan treatment reduced weight gain in C57 mice, but not in PPARδdeficient mice. Food and water intake was not affected in either group. Serum insulin and triglyceride levels did not differ between mouse strains with or without telmisartan treatment, but cholesterol lower in telmisartan treatment group of C57 micel. Compared with control mice, C57 mice treated with telmisartan had higher exercise endurance, post exercise oxygen consumption andⅠskeletal muscle fiber composition, lower AUC of IPGTT and ITT, as well as lower blood glucose level after insulin injection in ITT. However, PPARδdeficient mice treated with telmisartan displayed no phenotypic change relative to untreated PPARδdeficient mice. Conclusion1. Telmisartan directly increases glucose uptake in skeletal muscle cell through PPARδ/AMPK path. When RAS actitivity enhancement, telmisartan improves the insulin stimulated 2 DOG uptake by inhibition effect of AngⅡon skeletal muscle cell.2. Through activating PPARδ/AMPK path, telmisartan reduces weight gain, increasesⅠskeletal muscle fiber composition in gastronomies muscle and post exercise oxygen consumption, enhances exercise endurance, improves glucose tolerance and increases insulin sensitivity in mice.3. Telmisartan has no effect on RAS components in myotubes and skeletal muscle tissue of mice in normal condition.更多还原