高脂饮食老年大鼠骨骼肌脂肪酸转运与IR相关机制及药物干预的研究
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摘要
流行病学资料表明,2型糖尿病患病率随年龄增加而上升。胰岛素抵抗(insulin resistance,IR)和胰岛β细胞功能受损是2型糖尿病的主要发病机制。IR是由于肝脏、脂肪和肌肉等组织对胰岛素生物效应的反应性降低而产生的一系列临床表现。大量的研究已经表明IR是大多数2型糖尿病患者始动因素。随着年龄的增长,IR的发生呈增高趋势,对于IR发病率随增龄而增加的机制目前还不清楚。
遗传因素和环境因素均可诱导IR的发生。研究发现,老年内脏脂肪增加使2型糖尿病和心血管发病率增加,其中饮食是重要促发因素之一。长期高脂饮食的人群IR和2型糖尿病发病率明显高于正常人群,动物实验也证实,高脂环境可以损害骨骼肌、肝脏和脂肪在内的多种胰岛素靶器官的胰岛素敏感性。目前认为高脂环境是诱发IR的独立危险因素之一。
骨骼肌是机体葡萄糖、脂质摄取和利用的重要器官。从人和动物的研究中有充分的证据表明在肌肉中脂肪酸代谢异常与胰岛素敏感性有关,骨骼肌细胞内脂质含量增加可引起骨骼肌胰岛素抵抗。脂肪酸的摄入增加和/或氧化减少会导致肌内甘油三酯的积累。骨骼肌细胞膜脂肪酸转运蛋白(fatty acid translocase, FAT/CD36)表达增加被认为有易于长链脂肪酸的转运,是肌肉清除血中脂肪酸增多的原因,与胰岛素抵抗相关疾病联系紧密。研究发现,老年脂肪酸氧化能力下降,可能是导致年龄相关的肌内脂肪堆积原因之一。肉碱棕榈酰转移酶Ⅰβ(carnitine palmitoyltransferaseⅠβ, CPTⅠβ)是骨骼肌脂肪酸进入线粒体进行β氧化的限速酶,是控制能量代谢的关键因素,CPTⅠβ功能缺陷会导致长链酯酰CoA在胞浆过多积聚。骨骼肌为什么随年龄的增长而脂肪堆积增加,目前还不清楚,是否与随年龄的增加这些组织中脂肪酸转运的关键环节发生障碍有关,目前国内外还未见报道。
胰岛素刺激组织摄取利用葡萄糖是通过胰岛素受体-胰岛素受体底物1-磷脂酰激醇-3激酶-蛋白激酶B(PKB)等一系列信号传导过程完成的,最终使葡萄糖转运蛋白4(GLUT4)转移到细胞膜,大量GLUT4的膜转移可加速葡萄糖摄入到细胞内。胰岛素信号转导通路中任一步骤的异常都可能最终阻碍骨骼肌转运葡萄糖,从而引起IR。在青年大鼠有研究发现骨骼肌内脂质代谢中间产物的堆积干扰了胰岛素信号转导的某些环节,但增龄引起的肌内脂肪积聚是否与胰岛素刺激的葡萄糖摄入的关键环节异常有关,目前报道较少。
噻唑烷二酮类药物罗格列酮是人工合成的过氧化物酶体增殖物激活受体γ(peroxisome proliferators activated receptorγ,PPARγ)高亲和力配体,是一类由配体激活的新型降糖药物。罗格列酮可以通过多种途径增加胰岛素敏感性,改善IR。有研究发现PPARγ与肌肉组织中脂肪酸转运蛋白的表达密切相关,但具体机制还有待进一步阐明。
本课题通过观察老年大鼠和给予高脂饮食喂养后骨骼肌脂肪酸代谢情况,研究骨骼肌中脂肪酸转运关键酶FAT/CD36和CPTⅠβ的表达及胰岛素信号转导通路中PKB和GLUT4的表达,探索老年大鼠骨骼肌脂肪酸转运有关的代谢异常与IR形成的机制;通过对高脂喂养老年大鼠罗格列酮药物干预,探讨罗格列酮改善IR的新机制,为改善老年IR状态、降低糖尿病的发生和延缓衰老提供一个新的研究方向。本实验内容主要包括以下四部分:
第一部分高脂饮食对老年大鼠骨骼肌脂肪酸代谢和胰岛素敏感性的影响及罗格列酮的干预研究
目的:探讨老年和高脂饮食及罗格列酮干预对大鼠胰岛素敏感性和骨骼肌内脂质的影响,并对骨骼肌内脂质与胰岛素抵抗进行相关性分析。方法:22-24月龄老年雄性Wistar大鼠40只随机分为2组:老年对照(OC)组16只、高脂(HF)组24只, 4-5月龄青年大鼠16只,为青年对照组(YC)。对照组给予基础饲料,热量组成:碳水化合物65.5%,脂肪10.3%,蛋白质24.2%,总热量为348kcal/100g;HF组给予高脂饲料,其热量组成:碳水化合物20.1%,脂肪59.8 %,蛋白质20.1%,总热量为501kcal/100g;给予老年两组大鼠每日等热量投喂饲料,每周称体重1次,喂养八周。于喂养第四周末心脏采血测空腹血糖(FBG),胰岛素(INS),血清甘油三酯(TG),血清总胆固醇(TC),游离脂肪酸(FFA)的含量。喂养四周末,每组均随机选8只行口服葡萄糖耐量实验(OGTT)评价大鼠对葡萄糖负荷的耐受情况,行高胰岛素-正葡萄糖钳夹试验判断胰岛素抵抗情况,判断造模成功后高脂组随机分为2组:高脂组和罗格列酮干预组(RSG),每组8只,除继续给予高脂饲料外RSG组给予罗格列酮3mg/kg灌胃,高脂组给予等量生理盐水灌胃,继续喂养四周。实验第八周末,行OGTT实验和高胰岛素-正葡萄糖钳夹试验评价各组胰岛素抵抗情况,实验前抽取血样用于测定血清各项指标;实验结束后颈动脉放血处死动物,立即取出股四头肌和脂肪组织放入液氮中冷冻,然后保存于-70℃低温冰箱。骨骼肌甘油三酯(TGm)经氯仿/甲醇抽提后用全自动生化分析仪测定,骨骼肌长链酯酰辅酶A(LCACoA)用荧光分光光度计测定。
结果:1各组一般项目的比较:与青年组相比,老年组大鼠体重增加,高脂组和罗格列酮干预组体重与老年组相比变化不明显,差异无统计学意义(p>0.05)。与青年组相比,老年组空腹胰岛素、空腹血糖和游离脂肪酸在喂养第八周均升高;与老年组比较,高脂组空腹胰岛素、空腹血糖、游离脂肪酸、总胆固醇和甘油三酯明显升高;罗格列酮干预组空腹胰岛素、空腹血糖、游离脂肪酸、总胆固醇和甘油三酯与高脂组比较均下降,差异均有统计学意义(p<0.05或p<0.01)。2在第四周和第八周,与青年组相比,老年组TGm和LCACoA含量明显升高(p<0.05);与老年组相比,高脂组TGm和LCACoA含量进一步明显升高,且随喂养时间的延长,高脂组喂养第八周TGm和LCACoA含量明显高于第四周(p<0.05或p<0.01);罗格列酮干预组TGm和LCACoA含量与高脂组相比明显下降(p<0.05或p<0.01)。3胰岛素抵抗的评价:在喂养第四周和第八周,与青年组相比,老年组钳夹实验的葡萄糖输注率降低(p<0.01);与老年组相比,高脂组葡萄糖输注率明显下降;随喂养时间延长,第八周时高脂组内葡萄糖输注率与第四周相比明显下降(p<0.01);罗格列酮干预组葡萄糖输注率与高脂组相比明显升高(p<0.01)。4 OGTT实验结果:与青年组比较,无论第四周或第八周老年组大鼠60′、120′血糖和葡萄糖曲线下面积(AUC)均升高(p<0.05或p<0.01)。与老年组比较,高脂喂养第四周120′血糖和AUC升高,高脂喂养第八周各时间点血糖和AUC均升高,差异有统计学意义(p<0.01);高脂喂养第八周0′、60′、120′血糖和AUC明显高于第四周,罗格列酮干预组各时间点血糖和AUC均下降,差异有统计学意义(p<0.05或p<0.01)。5相关结果表明:葡萄糖输注率与空腹胰岛素、游离脂肪酸和骨骼肌甘油三酯和长链脂酰辅酶A明显负相关(p<0.05或p<0.01)。
结论:1老年大鼠骨骼肌甘油三酯和长链脂酰辅酶A含量明显高于青年大鼠,葡萄糖输注率和口服糖耐量在老年组明显减低,表明老年大鼠更易发生骨骼肌脂肪堆积和胰岛素抵抗。2高脂饮食可诱导老年大鼠骨骼肌甘油三酯和长链脂酰辅酶A含量进一步升高,葡萄糖输注率和口服糖耐量进一步降低,说明高脂饮食可加重老年骨骼肌脂肪酸代谢异常及胰岛素抵抗。3相关分析表明葡萄糖输注率和骨骼肌脂质明显负相关,说明脂代谢紊乱参与了年龄和饮食诱导的胰岛素抵抗的发生。4罗格列酮可以降低高脂诱导老年胰岛素抵抗大鼠骨骼肌甘油三酯和长链脂酰辅酶A含量,使OGTT各点血糖和AUC下降及增加葡萄糖输注率,改善胰岛素抵抗。
第二部分高脂饮食和罗格列酮干预对老年大鼠骨骼肌FAT/CD36和CPTⅠβ表达的影响
目的:探讨是否随年龄增长及高脂的环境因素影响了骨骼肌脂肪酸转运的关键酶FAT/CD36和CPTⅠβ的表达及与骨骼肌脂质的相关性研究,观察罗格列酮干预对FAT/CD36和CPTⅠβ表达的影响。
方法:动物分组及标本取得同第一部分。骨骼肌FAT/CD36mRNA表达采用实时荧光定量RT-PCR分析技术,CPTⅠβmRNA表达采用半定量RT-PCR分析技术,采用TRIZOL提取组织总RNA,鉴定RNA完整性,测定其在260nm和280nm的吸光度值(OD)。以完整性较好且OD260/OD280在1.8～2.0的RNA进行RT-PCR。Western-blot法分析FAT/CD36蛋白表达。
结果:1与青年组比较,老年组骨骼肌FAT/CD36蛋白表达升高(P<0.05);与老年组相比,高脂组FAT/CD36蛋白表达明显升高,罗格列酮干预组FAT/CD36蛋白表达下降,差异有统计学意义(P<0.05)。2与青年组比较,老年对照组骨骼肌FAT/CD36mRNA表达升高(P<0.01);与老年组相比,高脂组FAT/CD36mRNA表达进一步明显升高,罗格列酮干预组FAT/CD36mRNA表达降低,差异均有统计学意义(P<0.01)。3老年组骨骼肌CPTⅠβmRNA表达与青年组比较差异无统计学意义(P>0.05);与老年组比较,高脂喂养组CPTⅠβmRNA表达下降;与高脂组比较,罗格列酮干预组CPTⅠβmRNA表达升高,差异有统计学意义(P<0.01)。4骨骼肌内长链酯酰辅酶A与FAT/CD36mRNA表达呈正相关,与CPTⅠβmRNA表达负相关(P<0.01)。
结论:1与青年组相比,老年组大鼠骨骼肌细胞FAT/CD36mRNA和蛋白表达升高可能是老年组易发生脂质沉积的原因。2高脂饮食可使骨骼肌FAT/CD36mRNA和蛋白表达明显增加和CPTⅠβmRNA表达下降,可能导致饮食诱导骨骼肌内脂质沉积原因之一。3骨骼肌内长链酯酰辅酶A与FAT/CD36mRNA表达呈正相关,与CPTⅠβmRNA表达负相关,说明老年高脂大鼠骨骼肌脂质堆积与脂肪酸转运关键酶的表达异常有关。4罗格列酮干预使骨骼肌FAT/CD36mRNA和蛋白表达降低和CPTⅠβmRNA表达升高,可能相应减少骨骼肌脂质堆积,改善胰岛素抵抗。
第三部分老年高脂饮食大鼠骨骼肌脂肪酸含量与PKB、GLUT4表达的相关性研究及罗格列酮干预影响
目的:探讨老年和高脂环境因素对骨骼肌PKB和GLUT4mRNA和蛋白表达影响及罗格列酮干预的影响,并对骨骼肌内脂质与PKB和GLUT4表达的量进行了相关分析,以了解骨骼肌内脂质对胰岛素信号转导的影响。
方法:动物分组及标本取得同第一部分。骨骼肌PKB和GLUT4mRNA表达采用实时荧光定量RT-PCR分析技术,PKB和GLUT4蛋白表达用Western-blot分析。
结果:1与青年组相比,老年组PKBmRNA和蛋白表达降低;与老年组相比,高脂组PKBmRNA和蛋白表达进一步降低;罗格列酮干预组PKBmRNA和蛋白表达与高脂组相比明显升高,以上组间差异均有统计学意义(P<0.05)。2与青年组相比,老年组GLUT4mRNA和蛋白表达降低;与老年组相比,高脂组GLUT4mRNA和蛋白表达进一步降低;罗格列酮干预组GLUT4mRNA和蛋白表达与高脂组相比明显升高,以上组间差异均有统计学意义(P<0.05或P<0.01)。3 PKBmRNA表达的量与葡萄糖输注率正相关,GLUT4mRNA表达的量与葡萄糖输注率正相关,与骨骼肌LCACoA负相关(P均<0.01)。
结论:1与青年组相比,老年组大鼠骨骼肌细胞PKBmRNA和蛋白表达下降, GLUT4mRNA和蛋白表达也下降,可能是老年组易出现胰岛素信号转导异常,胰岛素抵抗的发生增加。2高脂饮食可诱导PKBmRNA和蛋白表达及GLUT4mRNA和蛋白表达进一步下降,说明高脂环境因素可通过影响胰岛素信号转导的关键酶而促进胰岛素抵抗的形成。3相关分析表明,PKBmRNA表达的量与葡萄糖输注率明显正相关,GLUT4mRNA表达的量与骨骼肌内LCACoA负相关,和葡萄糖输注率正相关,说明骨骼肌内脂质通过影响胰岛素信号转导通路的关键酶参与胰岛素抵抗的形成。4罗格列酮干预可使高脂喂养老年大鼠骨骼肌PKBmRNA和蛋白表达及GLUT4mRNA和蛋白表达升高,可能是改善老年胰岛素抵抗的机制之一。
第四部分高脂饮食和罗格列酮干预对老年大鼠脂肪和骨骼肌PPARγ表达的影响
目的:通过观察高脂喂养老年大鼠脂肪和骨骼肌组织PPARγ的表达情况,探讨高脂饮食老年大鼠骨骼肌脂质代谢紊乱和罗格列酮改善胰岛素抵抗的机制。
方法:动物分组及标本取得同第一部分。脂肪和骨骼肌组织PPARγmRNA表达采用实时荧光定量RT-PCR技术,脂肪和骨骼肌组织PPAR蛋白表达用Western-blot分析。
结果:1与青年组相比,老年组脂肪组织PPARγmRNA和蛋白表达降低(P<0.05);与老年组相比,高脂组脂肪组织PPARγmRNA和蛋白表达明显降低(P<0.05);罗格列酮干预组脂肪组织PPARγmRNA和蛋白表达与高脂组相比明显升高,各组间差异均有统计学意义(P<0.01)。2与青年组相比,老年组骨骼肌组织PPARγmRNA和蛋白表达差异无显著性意义(P>0.05);与老年组相比,高脂组骨骼肌PPARγmRNA和蛋白表达明显降低(P<0.05或P<0.01);罗格列酮干预组骨骼肌PPARγmRNA和蛋白表达与高脂组相比明显升高,差异有统计学意义(P<0.01)。
结论:1与青年组相比,老年组大鼠脂肪组织PPARγmRNA和蛋白表达下降可能是老年脂肪酸代谢紊乱胰岛素抵抗发生增加原因之一。2高脂饮食可诱导老年大鼠脂肪组织和骨骼肌组织PPARγmRNA和蛋白表达下降,可能参与饮食诱导的骨骼肌脂肪酸代谢异常和胰岛素抵抗的形成。3罗格列酮干预可使高脂喂养老年大鼠骨骼肌和脂肪组织PPARγmRNA和蛋白表达升高,可能是改善老年胰岛素抵抗的机制之一。
A significant increase in type 2 diabetes mellitus with increasing age can be demonstrated from all epidemiological studies. Insulin resistance and dysfunction of beta cell of islet are the main pathogenesis in the type 2 diabetes mellitus. Insulin resistance brings a varied of clinical manifestation because of the lower reactivity to the action of insulin on muscle, liver and fat. Insulin resistance is also the initiating agent for majority of type 2 diabetics. The incidence of insulin resistance is increasing with increasing age on a global scale, but the precise mechanism is not fully elucidated yet.
Genetic and environmental factors play an important role in insulin resistance state, studies demonstrated that the incidence of type 2 diabetics and cadiovascular diseases is increasing for the increased internal fat, diets is one of precipitating factor. The people with high-fat diet habits have higher incidence of type 2 diabetics and insulin resistance than those with normal diet habits, animal experiments also proved that high-fat exposure on impaired insulin sensitivity in several insulin target organs, including skeletal muscles, liver, adipocytes etc, so high-fat diet is a single risk factor leading to insulin resistance.
The skeletal muscle plays a primary role in plasma glucose and lipid regulation. The lipid milieu of skeletal muscle has been associated with skeletal muscle insulin resistance in animals and humans. Several studies suggest that a reduced capacity to oxidize fat or/and an increased capacity to uptake fat may contribute to fat accumulation. Fatty acid translocase (FAT/CD36) is important membrane protein to mediate the uptake of long-chain fatty acid in skeletal muscle, the expression of FAT/CD36 affect fatty acid transport and secondary lipid deposition and insulin resistance.
Aging is associated with reduced fat oxidation.The age-related reduction in fat oxidation, therefore, may promote the lipid accumulation in skeletal muscle. Carnitine palmitoyl transferase 1β(CPTⅠβ) is the rate-limiting enzyme of intracellular fatty acid transport in the mitochondrial outer membrane. The activity of CPTⅠβdecides if long-chain fatty acid can be tansfered into mitochondria to participate in beta-oxidation, so the express and activity of CPTⅠβis the key factor to the accumulation of long-chain fatty acid in kytoplasm. It is not clear why the lipid deposition of skeletal muscle increase with age, whether it is associated with the impaired fatty acid transport, now there are few studies in the field.
In recently published statements , PI3-K/PKB pathway played an important role in insulin-signaling transduction. The effects of insulin on glucose uptake are mediated via the insulin receptor - insulin receptor substrate-1-phosphatidylinositol-3-kinase (PI3-K)-protein kinase B(PKB), and results in the translocation of intracellular glucose transporter 4 (GLUT4) to the cell surface. It is the increased amount of GLUT4 on the cell plasma membrane that results in an increased rate of glucose transport into the cell. According the present study,impairment of the insulin-signaling pathway at any step may lead to the insulin resistant. Recent evidence has demonstrated that lipid metabolic intermediate impaired the insulin-signaling pathway at some link from experiments results of the young rats. But, there was few investigate the relationship between the lipid deposition of skeletal muscle and abnormal insulin-signaling transduction in elderly people.
Thiazolidinediones (TZDs) are a new class of insulin-sensitizing agents for the oral treatment of type 2 diabetes. TZDs are agonists of the peroxisome proliferator-activated receptorγ(PPARγ). PPARγ, a nuclear hormone receptor, is predominantly expressed in adipocytes but also, although at lower levels, in skeletal muscle and heart. TZDs have been proven to influence intramuscular long-chain FA uptake by changing the mRNA expression levels of several key players in the protein-mediated FA uptake process, but the precise mechanism need to further study.
In the present study, we explore the possible mechanisms about high incidence of insulin resistance in aged rats through investigating the fatty acid metabolism of skeletal muscle including the key enzyme expression at protein and mRNA levels. Given a high fat diets and treatment with roglitazone in the aged rats to learn about the reactive potency to intervention factors, providing a new study direction to improve insulin resistance and delay senility. The paper contains four parts below:
Part one: The effects of high fat diets on fatty acid metabolism of skeletal muscle and insulin sensitivity in the aged rats and the study of rosiglitazone treatment.
Objective: To investigate the effects of high fat diets and rosiglitazone treatment on fatty acid metabolism of skeletal muscle and insulin sensitivity in the aged rats and to analyze the correlation between insulin resistance and lipid of skeletal muscle.
Methods: Male Wistar rats aged 22-24 months were divided into old control (OC) group (n=16), high-fat diet (HF) group (n=24), Wistar rats aged 4-5 months were selected as young control (YC) group (n=16), the rats in control group were fed with a regular low fatty acids diet containing 10.3% fat, 24.2% protein, and 65.5% carbohydrate as percentage of total calories. The rats in high-fat diet group were fed regular diets mixed with 30% lard, containing 59.8% fat, 20.1% protein and 20.1% carbohydrate as a percentage of total calories. The rats in every group were fed equal calories every day. The body weights were determined weekly for 8 weeks. The blood sample was collected by cardiac puncture after rats were anesthetized with diethyl ether for the biochemical analysis, insulin resistance was evaluated by glucose infusion rate (GIR) of hyperinsulinemic euglycemic clamp technique and oral glucose tolerance test was done at the end of 4 week(eight rats in each group). After 4 weeks, rats were randomly divided into high fat diets group and high-fat + rosiglitazone group (RSG), the rats were given rosiglitazone (GlaxoSmithKline, UK) 3 mg per kg body weight through intragastric administration once a day for 4 weeks in RSG group(eight rats in each group). At the end of 8 week, the rats were killed with phenobarbital sodium after hyperinsulinemic euglycemic clamp test and OGTT, and skeletal muscles and fat tissues were taken out freeze-clamped with copper clamps precooled in liquid N2 and were stored in -70℃refrigerator. Muscle triglyceride(TGm) was measured by automatic biochemistry analyzer after extracting by methanol and chloroform, muscle long-chain fatty acyl coenzyme A(LCACoA)was measured by fluorospectro- photometer.
Results:1 The body weight in OC group rats were higher than that in YC group rats. Compared with OC group, the body weight in HF group and RSG group didn’t increase significantly(p>0.05).Compared with YC group, the fasting insulin(FINS), fasting blood glucose(FBG) and free fatty acid(FFA) were higher in the OC group at the end of 8 week; Compared with OC group, FINS, FBG, FFA, total cholesterol(TC) and triglyceride(TG) were higher in the HF group(p<0.05 or p<0.01). FINS, FBG, FFA, TC and TG were lower in the RSG group than those in the HFgroup(p<0.05 or p<0.01). 2 At the end of 4 week and 8 week, compared with YC group, TGm and LCACoA were higher in OC group(p<0.05); TGm and LCACoA were significantly higher in HF group than those in OC group(p<0.05 or p<0.01); TGm and LCACoA were higher at 8 week than those at 4 week in HF group(p<0.01); TGm and LCACoA were lower in the RSG group than those in the HF group(p<0.05 or p<0.01). 3 Compared with YC group at the end of 4 week and 8 week, GIR was lower in OC group, GIR was more lower in HF group than that in OC group(p<0.01); Compared with HF group at 4 week, GIR was significantly lower in HF group at 8 week(p<0.01). GIR were higher in the RSG group than that in HF group(p<0.01)4 The results of oral glucose tolerance test. At the end of 4 week and 8 week, the glucose level of the rats in OC group was much higher than that of rats in YC group at the time of 0′and 60′, 120′after glucose loading and area under curve(AUC) significantly increased (p<0.05 or p<0.01). The glucose level at every time point and AUC of the rats in HF group increased compared with those of rats in OC group (P<0.01). The glucose level in RSG group decreased at every time point and AUC (p<0.05 or p<0.01). 5 GIR has a negative correlation with INS, FFA, TGm and LCACoA (p<0.05 or p<0.01).
Conclusions: TGm and LCACoA were higher and GIR was lower in OC group than those in YC group. It demonstrated that insulin resistance and lipid deposition of skeletal muscle always exist in aged rats. High fat diets in aged rats induced a significant increase in plasma lipid and intramusculary lipid paralleled impaired GIR, the results showed that insulin resistance and lipid metabolism were more serious. GIR has a negative correlation with INS, FFA, TGm and LCACoA. It suggested that lipid disorder is associated with insulin resistance. TGm and LCACoA decreased by rosiglitazone treatment and GIR and OGTT improved in aged rats induced by high fat diets, that maybe one of the mechanisms of rosiglitazone to improve insulin rasistance. Part two: Effects of high-fat diets and rosiglitazone treatment on expression of FAT/CD36 and CPTⅠβin aged rat skeletal muscles.
Objective: To observe the effects of aging, high-fat diets and rosiglitazone treatment on the expression at protein and mRNA level of FAT/CD36 and mRNA level of CPTⅠβand to analyze the correlation with lipid in muscle tissue.
Methods: Animal grouping and the samples acquirement were the same as those in part one. The expression at protein level of FAT/CD36 in skeletal muscle was measured by Western-blot method and the expression at mRNA level of FAT/CD36 in skeletal muscle was measured by real time polymerase chain reaction (PCR) method; the expression of CPTⅠβmRNA was measured by semiquantitative PCR method. TRIZOL Reagent was used to extract RNA from the tissues, integrity of RNA was evaluated by formaldehyde degeneration caraphoresis and optical density (OD) was got at 260nm and 280nm. RT-PCR was carried out with better integrity and ratio of OD 260nm and 280nm in 1.8～2.0 of RNA.
Results: 1 Compared with YC group, the expression of FAT/CD36 mRNA and FAT/CD36 protein in skeletal muscle were higher in the OC group(p<0.05 or p<0.01); Compared with OC group, the expression of FAT/CD36 mRNA and FAT/CD36 protein were even higher in HF group rats(p<0.05 or p<0.01); the expression of FAT/CD36 mRNA and FAT/CD36 protein were lower in RSG group rats than those of rats in HF group(p<0.05 or p<0.01). 2 There were no significant difference of the expression of CPTⅠβmRNA between the YC group and OC group rats(P>0.05). Compared with OC group, the expression of CPTⅠβmRNA was lower in HF group rats(p<0.01). In response to the rosiglitazone treatment , the expression of CPTⅠβmRNA was significantly higher compared with HF group. 3 LCACoA has a positive correlation with the expression of FAT/CD36mRNA and has a negative correlation with the expression of CPTⅠβmRNA.
Conclusions: 1 It is possible reason for aged rats to produce more lipid deposition in skeletal muscles than young rats for the higher expression of FAT/CD36 mRNA and protein. 2 The abnormal expression of FAT/CD36 and CPTⅠβof skeletal muscle in the aged rats induced by high fat diets may be one of the mechanisms of lipid deposition. 3 Correlation analyses suggested that lipid accumulation in muscles was associated with the abnormal expression of key enzyme to fatty acid transport. The expression of CPTⅠβmRNA increased and the expression of FAT/CD36mRNA and protein decreased in high-fat diets rats treated with rosiglitazone maybe associate with reduced lipid deposition and improve insulin resistance.
Part three: Effects of high-fat diets on expression of GLUT4 and PKB in aged rat skeletal muscles and rosiglitazone treatment.
Objective: To observe the effects of aging and high-fat diets on the expression at protein and mRNA level of PKB and GLUT4 and to analyze the correlation with lipid in muscle tissue.
Methods: Animal grouping and the samples acquirement were the same as those in the part one. The expression at protein and mRNA level of PKB and GLUT4 in skeletal muscle was measured by Western blot method and real time PCR method respectively.
Results: 1 Compared with YC group, the expression of PKBmRNA and PKB protein was lower in the OC group(P<0.05); Compared with OC group, the expression of PKBmRNA and protein was significantly lower in HF group rats (P<0.05); the expression of PKBmRNA and protein was higher in RSG group than that in HF group(p<0.05). 2 Compared with YC group, the expression of GLUT4mRNA and protein was lower in OC group (P<0.05 or P<0.01); Compared with OC group, and the expression of GLUT4mRNA and protein were significantly lower in HF group (P<0.05 or P<0.01); the expression of GLUT4mRNA and protein was higher in RSG group than that in HF group(p<0.01).3 The expression of PKBmRNA and GLUT4mRNA have possitive correlation with GIR and the expression of GLUT4mRNA has a negative correlation with LCACoA(P <0.01).
Conclusions: 1 It is possible reason for the high incidence of insulin resistance in the aged rats because the impaired insulin-signaling transduction accounting for the abnormal expression of PKB and GLUT4. 2That insulin signal transduction impaired because of the abnormal expression of PKB and GLUT4 induced by high fat diets may be one of the mechnisms of insulin resistance. 3 Correlation analyses suggested that lipid accumulation associated with skeletal muscle insulin resistance through the effect on the key enzyme of insulin signal transduction. 4 The expression of PKB and GLUT4 increased in high-fat diets rats treated with rosiglitazone maybe one of the mechanisms to improve insulin resistance.
Part four: Effects of high-fat diets and rosiglitazone treatment on the expression of PPARγin fat and skeletal muscle tissues in aged rat Objective: To observe the effects of aging and high-fat diets on the expression at protein and mRNA level of PPARγin fat and skeletal muscle tissues and rosiglitazone interference study.
Methods: Animal grouping and the samples acquirement were the same as part one. The expression at protein and mRNA level of PPARγwas measured by Western-blot method and real time PCR method respectively.
Results: 1 Compared with YC group, the expression of PPARγmRNA and PPARγprotein in fat tissue were lower in the old control group rats(P<0.05); Compared with OC group, the expression of PPARγmRNA and PPARγprotein in fat tissue were even lower in HF group (P<0.05); the expression of PPARγwas higher in RSG group than that of rats in HF group(p<0.01). 2 There were no significant differerce of the PPARγmRNA and PPARγprotein in sheletal muscle between YC group and OC group (p>0.05). Compared with OC group, the expression of PPARγmRNA and protein in skeletal muscle were lower in HF group (P<0.05 or P<0.01); the expression of PPARγmRNA and protein in skeletal muscle were higher in RSG group than those of rats in HF group (P<0.01).
Conclusions: 1 It is possible for lipid metabolic disorder in aged rats because the lower expression of PPARγin fat tisssues than that of in young rats.2 That the lower expression of PPARγin fat and skeletal muscle tissues induced by high-fat diets maybe assosiated with abnormal lipid metabolism and insulin resistance. 3 Rosiglitazone treatment maybe affect adipocyte differentiation and improve insulin resistance through increasing the expression of PPARγin fat and skeletal muscle tissues.
遗传因素和环境因素均可诱导IR的发生。研究发现,老年内脏脂肪增加使2型糖尿病和心血管发病率增加,其中饮食是重要促发因素之一。长期高脂饮食的人群IR和2型糖尿病发病率明显高于正常人群,动物实验也证实,高脂环境可以损害骨骼肌、肝脏和脂肪在内的多种胰岛素靶器官的胰岛素敏感性。目前认为高脂环境是诱发IR的独立危险因素之一。
骨骼肌是机体葡萄糖、脂质摄取和利用的重要器官。从人和动物的研究中有充分的证据表明在肌肉中脂肪酸代谢异常与胰岛素敏感性有关,骨骼肌细胞内脂质含量增加可引起骨骼肌胰岛素抵抗。脂肪酸的摄入增加和/或氧化减少会导致肌内甘油三酯的积累。骨骼肌细胞膜脂肪酸转运蛋白(fatty acid translocase, FAT/CD36)表达增加被认为有易于长链脂肪酸的转运,是肌肉清除血中脂肪酸增多的原因,与胰岛素抵抗相关疾病联系紧密。研究发现,老年脂肪酸氧化能力下降,可能是导致年龄相关的肌内脂肪堆积原因之一。肉碱棕榈酰转移酶Ⅰβ(carnitine palmitoyltransferaseⅠβ, CPTⅠβ)是骨骼肌脂肪酸进入线粒体进行β氧化的限速酶,是控制能量代谢的关键因素,CPTⅠβ功能缺陷会导致长链酯酰CoA在胞浆过多积聚。骨骼肌为什么随年龄的增长而脂肪堆积增加,目前还不清楚,是否与随年龄的增加这些组织中脂肪酸转运的关键环节发生障碍有关,目前国内外还未见报道。
胰岛素刺激组织摄取利用葡萄糖是通过胰岛素受体-胰岛素受体底物1-磷脂酰激醇-3激酶-蛋白激酶B(PKB)等一系列信号传导过程完成的,最终使葡萄糖转运蛋白4(GLUT4)转移到细胞膜,大量GLUT4的膜转移可加速葡萄糖摄入到细胞内。胰岛素信号转导通路中任一步骤的异常都可能最终阻碍骨骼肌转运葡萄糖,从而引起IR。在青年大鼠有研究发现骨骼肌内脂质代谢中间产物的堆积干扰了胰岛素信号转导的某些环节,但增龄引起的肌内脂肪积聚是否与胰岛素刺激的葡萄糖摄入的关键环节异常有关,目前报道较少。
噻唑烷二酮类药物罗格列酮是人工合成的过氧化物酶体增殖物激活受体γ(peroxisome proliferators activated receptorγ,PPARγ)高亲和力配体,是一类由配体激活的新型降糖药物。罗格列酮可以通过多种途径增加胰岛素敏感性,改善IR。有研究发现PPARγ与肌肉组织中脂肪酸转运蛋白的表达密切相关,但具体机制还有待进一步阐明。
本课题通过观察老年大鼠和给予高脂饮食喂养后骨骼肌脂肪酸代谢情况,研究骨骼肌中脂肪酸转运关键酶FAT/CD36和CPTⅠβ的表达及胰岛素信号转导通路中PKB和GLUT4的表达,探索老年大鼠骨骼肌脂肪酸转运有关的代谢异常与IR形成的机制;通过对高脂喂养老年大鼠罗格列酮药物干预,探讨罗格列酮改善IR的新机制,为改善老年IR状态、降低糖尿病的发生和延缓衰老提供一个新的研究方向。本实验内容主要包括以下四部分:
第一部分高脂饮食对老年大鼠骨骼肌脂肪酸代谢和胰岛素敏感性的影响及罗格列酮的干预研究
目的:探讨老年和高脂饮食及罗格列酮干预对大鼠胰岛素敏感性和骨骼肌内脂质的影响,并对骨骼肌内脂质与胰岛素抵抗进行相关性分析。方法:22-24月龄老年雄性Wistar大鼠40只随机分为2组:老年对照(OC)组16只、高脂(HF)组24只, 4-5月龄青年大鼠16只,为青年对照组(YC)。对照组给予基础饲料,热量组成:碳水化合物65.5%,脂肪10.3%,蛋白质24.2%,总热量为348kcal/100g;HF组给予高脂饲料,其热量组成:碳水化合物20.1%,脂肪59.8 %,蛋白质20.1%,总热量为501kcal/100g;给予老年两组大鼠每日等热量投喂饲料,每周称体重1次,喂养八周。于喂养第四周末心脏采血测空腹血糖(FBG),胰岛素(INS),血清甘油三酯(TG),血清总胆固醇(TC),游离脂肪酸(FFA)的含量。喂养四周末,每组均随机选8只行口服葡萄糖耐量实验(OGTT)评价大鼠对葡萄糖负荷的耐受情况,行高胰岛素-正葡萄糖钳夹试验判断胰岛素抵抗情况,判断造模成功后高脂组随机分为2组:高脂组和罗格列酮干预组(RSG),每组8只,除继续给予高脂饲料外RSG组给予罗格列酮3mg/kg灌胃,高脂组给予等量生理盐水灌胃,继续喂养四周。实验第八周末,行OGTT实验和高胰岛素-正葡萄糖钳夹试验评价各组胰岛素抵抗情况,实验前抽取血样用于测定血清各项指标;实验结束后颈动脉放血处死动物,立即取出股四头肌和脂肪组织放入液氮中冷冻,然后保存于-70℃低温冰箱。骨骼肌甘油三酯(TGm)经氯仿/甲醇抽提后用全自动生化分析仪测定,骨骼肌长链酯酰辅酶A(LCACoA)用荧光分光光度计测定。
结果:1各组一般项目的比较:与青年组相比,老年组大鼠体重增加,高脂组和罗格列酮干预组体重与老年组相比变化不明显,差异无统计学意义(p>0.05)。与青年组相比,老年组空腹胰岛素、空腹血糖和游离脂肪酸在喂养第八周均升高;与老年组比较,高脂组空腹胰岛素、空腹血糖、游离脂肪酸、总胆固醇和甘油三酯明显升高;罗格列酮干预组空腹胰岛素、空腹血糖、游离脂肪酸、总胆固醇和甘油三酯与高脂组比较均下降,差异均有统计学意义(p<0.05或p<0.01)。2在第四周和第八周,与青年组相比,老年组TGm和LCACoA含量明显升高(p<0.05);与老年组相比,高脂组TGm和LCACoA含量进一步明显升高,且随喂养时间的延长,高脂组喂养第八周TGm和LCACoA含量明显高于第四周(p<0.05或p<0.01);罗格列酮干预组TGm和LCACoA含量与高脂组相比明显下降(p<0.05或p<0.01)。3胰岛素抵抗的评价:在喂养第四周和第八周,与青年组相比,老年组钳夹实验的葡萄糖输注率降低(p<0.01);与老年组相比,高脂组葡萄糖输注率明显下降;随喂养时间延长,第八周时高脂组内葡萄糖输注率与第四周相比明显下降(p<0.01);罗格列酮干预组葡萄糖输注率与高脂组相比明显升高(p<0.01)。4 OGTT实验结果:与青年组比较,无论第四周或第八周老年组大鼠60′、120′血糖和葡萄糖曲线下面积(AUC)均升高(p<0.05或p<0.01)。与老年组比较,高脂喂养第四周120′血糖和AUC升高,高脂喂养第八周各时间点血糖和AUC均升高,差异有统计学意义(p<0.01);高脂喂养第八周0′、60′、120′血糖和AUC明显高于第四周,罗格列酮干预组各时间点血糖和AUC均下降,差异有统计学意义(p<0.05或p<0.01)。5相关结果表明:葡萄糖输注率与空腹胰岛素、游离脂肪酸和骨骼肌甘油三酯和长链脂酰辅酶A明显负相关(p<0.05或p<0.01)。
结论:1老年大鼠骨骼肌甘油三酯和长链脂酰辅酶A含量明显高于青年大鼠,葡萄糖输注率和口服糖耐量在老年组明显减低,表明老年大鼠更易发生骨骼肌脂肪堆积和胰岛素抵抗。2高脂饮食可诱导老年大鼠骨骼肌甘油三酯和长链脂酰辅酶A含量进一步升高,葡萄糖输注率和口服糖耐量进一步降低,说明高脂饮食可加重老年骨骼肌脂肪酸代谢异常及胰岛素抵抗。3相关分析表明葡萄糖输注率和骨骼肌脂质明显负相关,说明脂代谢紊乱参与了年龄和饮食诱导的胰岛素抵抗的发生。4罗格列酮可以降低高脂诱导老年胰岛素抵抗大鼠骨骼肌甘油三酯和长链脂酰辅酶A含量,使OGTT各点血糖和AUC下降及增加葡萄糖输注率,改善胰岛素抵抗。
第二部分高脂饮食和罗格列酮干预对老年大鼠骨骼肌FAT/CD36和CPTⅠβ表达的影响
目的:探讨是否随年龄增长及高脂的环境因素影响了骨骼肌脂肪酸转运的关键酶FAT/CD36和CPTⅠβ的表达及与骨骼肌脂质的相关性研究,观察罗格列酮干预对FAT/CD36和CPTⅠβ表达的影响。
方法:动物分组及标本取得同第一部分。骨骼肌FAT/CD36mRNA表达采用实时荧光定量RT-PCR分析技术,CPTⅠβmRNA表达采用半定量RT-PCR分析技术,采用TRIZOL提取组织总RNA,鉴定RNA完整性,测定其在260nm和280nm的吸光度值(OD)。以完整性较好且OD260/OD280在1.8～2.0的RNA进行RT-PCR。Western-blot法分析FAT/CD36蛋白表达。
结果:1与青年组比较,老年组骨骼肌FAT/CD36蛋白表达升高(P<0.05);与老年组相比,高脂组FAT/CD36蛋白表达明显升高,罗格列酮干预组FAT/CD36蛋白表达下降,差异有统计学意义(P<0.05)。2与青年组比较,老年对照组骨骼肌FAT/CD36mRNA表达升高(P<0.01);与老年组相比,高脂组FAT/CD36mRNA表达进一步明显升高,罗格列酮干预组FAT/CD36mRNA表达降低,差异均有统计学意义(P<0.01)。3老年组骨骼肌CPTⅠβmRNA表达与青年组比较差异无统计学意义(P>0.05);与老年组比较,高脂喂养组CPTⅠβmRNA表达下降;与高脂组比较,罗格列酮干预组CPTⅠβmRNA表达升高,差异有统计学意义(P<0.01)。4骨骼肌内长链酯酰辅酶A与FAT/CD36mRNA表达呈正相关,与CPTⅠβmRNA表达负相关(P<0.01)。
结论:1与青年组相比,老年组大鼠骨骼肌细胞FAT/CD36mRNA和蛋白表达升高可能是老年组易发生脂质沉积的原因。2高脂饮食可使骨骼肌FAT/CD36mRNA和蛋白表达明显增加和CPTⅠβmRNA表达下降,可能导致饮食诱导骨骼肌内脂质沉积原因之一。3骨骼肌内长链酯酰辅酶A与FAT/CD36mRNA表达呈正相关,与CPTⅠβmRNA表达负相关,说明老年高脂大鼠骨骼肌脂质堆积与脂肪酸转运关键酶的表达异常有关。4罗格列酮干预使骨骼肌FAT/CD36mRNA和蛋白表达降低和CPTⅠβmRNA表达升高,可能相应减少骨骼肌脂质堆积,改善胰岛素抵抗。
第三部分老年高脂饮食大鼠骨骼肌脂肪酸含量与PKB、GLUT4表达的相关性研究及罗格列酮干预影响
目的:探讨老年和高脂环境因素对骨骼肌PKB和GLUT4mRNA和蛋白表达影响及罗格列酮干预的影响,并对骨骼肌内脂质与PKB和GLUT4表达的量进行了相关分析,以了解骨骼肌内脂质对胰岛素信号转导的影响。
方法:动物分组及标本取得同第一部分。骨骼肌PKB和GLUT4mRNA表达采用实时荧光定量RT-PCR分析技术,PKB和GLUT4蛋白表达用Western-blot分析。
结果:1与青年组相比,老年组PKBmRNA和蛋白表达降低;与老年组相比,高脂组PKBmRNA和蛋白表达进一步降低;罗格列酮干预组PKBmRNA和蛋白表达与高脂组相比明显升高,以上组间差异均有统计学意义(P<0.05)。2与青年组相比,老年组GLUT4mRNA和蛋白表达降低;与老年组相比,高脂组GLUT4mRNA和蛋白表达进一步降低;罗格列酮干预组GLUT4mRNA和蛋白表达与高脂组相比明显升高,以上组间差异均有统计学意义(P<0.05或P<0.01)。3 PKBmRNA表达的量与葡萄糖输注率正相关,GLUT4mRNA表达的量与葡萄糖输注率正相关,与骨骼肌LCACoA负相关(P均<0.01)。
结论:1与青年组相比,老年组大鼠骨骼肌细胞PKBmRNA和蛋白表达下降, GLUT4mRNA和蛋白表达也下降,可能是老年组易出现胰岛素信号转导异常,胰岛素抵抗的发生增加。2高脂饮食可诱导PKBmRNA和蛋白表达及GLUT4mRNA和蛋白表达进一步下降,说明高脂环境因素可通过影响胰岛素信号转导的关键酶而促进胰岛素抵抗的形成。3相关分析表明,PKBmRNA表达的量与葡萄糖输注率明显正相关,GLUT4mRNA表达的量与骨骼肌内LCACoA负相关,和葡萄糖输注率正相关,说明骨骼肌内脂质通过影响胰岛素信号转导通路的关键酶参与胰岛素抵抗的形成。4罗格列酮干预可使高脂喂养老年大鼠骨骼肌PKBmRNA和蛋白表达及GLUT4mRNA和蛋白表达升高,可能是改善老年胰岛素抵抗的机制之一。
第四部分高脂饮食和罗格列酮干预对老年大鼠脂肪和骨骼肌PPARγ表达的影响
目的:通过观察高脂喂养老年大鼠脂肪和骨骼肌组织PPARγ的表达情况,探讨高脂饮食老年大鼠骨骼肌脂质代谢紊乱和罗格列酮改善胰岛素抵抗的机制。
方法:动物分组及标本取得同第一部分。脂肪和骨骼肌组织PPARγmRNA表达采用实时荧光定量RT-PCR技术,脂肪和骨骼肌组织PPAR蛋白表达用Western-blot分析。
结果:1与青年组相比,老年组脂肪组织PPARγmRNA和蛋白表达降低(P<0.05);与老年组相比,高脂组脂肪组织PPARγmRNA和蛋白表达明显降低(P<0.05);罗格列酮干预组脂肪组织PPARγmRNA和蛋白表达与高脂组相比明显升高,各组间差异均有统计学意义(P<0.01)。2与青年组相比,老年组骨骼肌组织PPARγmRNA和蛋白表达差异无显著性意义(P>0.05);与老年组相比,高脂组骨骼肌PPARγmRNA和蛋白表达明显降低(P<0.05或P<0.01);罗格列酮干预组骨骼肌PPARγmRNA和蛋白表达与高脂组相比明显升高,差异有统计学意义(P<0.01)。
结论:1与青年组相比,老年组大鼠脂肪组织PPARγmRNA和蛋白表达下降可能是老年脂肪酸代谢紊乱胰岛素抵抗发生增加原因之一。2高脂饮食可诱导老年大鼠脂肪组织和骨骼肌组织PPARγmRNA和蛋白表达下降,可能参与饮食诱导的骨骼肌脂肪酸代谢异常和胰岛素抵抗的形成。3罗格列酮干预可使高脂喂养老年大鼠骨骼肌和脂肪组织PPARγmRNA和蛋白表达升高,可能是改善老年胰岛素抵抗的机制之一。
A significant increase in type 2 diabetes mellitus with increasing age can be demonstrated from all epidemiological studies. Insulin resistance and dysfunction of beta cell of islet are the main pathogenesis in the type 2 diabetes mellitus. Insulin resistance brings a varied of clinical manifestation because of the lower reactivity to the action of insulin on muscle, liver and fat. Insulin resistance is also the initiating agent for majority of type 2 diabetics. The incidence of insulin resistance is increasing with increasing age on a global scale, but the precise mechanism is not fully elucidated yet.
Genetic and environmental factors play an important role in insulin resistance state, studies demonstrated that the incidence of type 2 diabetics and cadiovascular diseases is increasing for the increased internal fat, diets is one of precipitating factor. The people with high-fat diet habits have higher incidence of type 2 diabetics and insulin resistance than those with normal diet habits, animal experiments also proved that high-fat exposure on impaired insulin sensitivity in several insulin target organs, including skeletal muscles, liver, adipocytes etc, so high-fat diet is a single risk factor leading to insulin resistance.
The skeletal muscle plays a primary role in plasma glucose and lipid regulation. The lipid milieu of skeletal muscle has been associated with skeletal muscle insulin resistance in animals and humans. Several studies suggest that a reduced capacity to oxidize fat or/and an increased capacity to uptake fat may contribute to fat accumulation. Fatty acid translocase (FAT/CD36) is important membrane protein to mediate the uptake of long-chain fatty acid in skeletal muscle, the expression of FAT/CD36 affect fatty acid transport and secondary lipid deposition and insulin resistance.
Aging is associated with reduced fat oxidation.The age-related reduction in fat oxidation, therefore, may promote the lipid accumulation in skeletal muscle. Carnitine palmitoyl transferase 1β(CPTⅠβ) is the rate-limiting enzyme of intracellular fatty acid transport in the mitochondrial outer membrane. The activity of CPTⅠβdecides if long-chain fatty acid can be tansfered into mitochondria to participate in beta-oxidation, so the express and activity of CPTⅠβis the key factor to the accumulation of long-chain fatty acid in kytoplasm. It is not clear why the lipid deposition of skeletal muscle increase with age, whether it is associated with the impaired fatty acid transport, now there are few studies in the field.
In recently published statements , PI3-K/PKB pathway played an important role in insulin-signaling transduction. The effects of insulin on glucose uptake are mediated via the insulin receptor - insulin receptor substrate-1-phosphatidylinositol-3-kinase (PI3-K)-protein kinase B(PKB), and results in the translocation of intracellular glucose transporter 4 (GLUT4) to the cell surface. It is the increased amount of GLUT4 on the cell plasma membrane that results in an increased rate of glucose transport into the cell. According the present study,impairment of the insulin-signaling pathway at any step may lead to the insulin resistant. Recent evidence has demonstrated that lipid metabolic intermediate impaired the insulin-signaling pathway at some link from experiments results of the young rats. But, there was few investigate the relationship between the lipid deposition of skeletal muscle and abnormal insulin-signaling transduction in elderly people.
Thiazolidinediones (TZDs) are a new class of insulin-sensitizing agents for the oral treatment of type 2 diabetes. TZDs are agonists of the peroxisome proliferator-activated receptorγ(PPARγ). PPARγ, a nuclear hormone receptor, is predominantly expressed in adipocytes but also, although at lower levels, in skeletal muscle and heart. TZDs have been proven to influence intramuscular long-chain FA uptake by changing the mRNA expression levels of several key players in the protein-mediated FA uptake process, but the precise mechanism need to further study.
In the present study, we explore the possible mechanisms about high incidence of insulin resistance in aged rats through investigating the fatty acid metabolism of skeletal muscle including the key enzyme expression at protein and mRNA levels. Given a high fat diets and treatment with roglitazone in the aged rats to learn about the reactive potency to intervention factors, providing a new study direction to improve insulin resistance and delay senility. The paper contains four parts below:
Part one: The effects of high fat diets on fatty acid metabolism of skeletal muscle and insulin sensitivity in the aged rats and the study of rosiglitazone treatment.
Objective: To investigate the effects of high fat diets and rosiglitazone treatment on fatty acid metabolism of skeletal muscle and insulin sensitivity in the aged rats and to analyze the correlation between insulin resistance and lipid of skeletal muscle.
Methods: Male Wistar rats aged 22-24 months were divided into old control (OC) group (n=16), high-fat diet (HF) group (n=24), Wistar rats aged 4-5 months were selected as young control (YC) group (n=16), the rats in control group were fed with a regular low fatty acids diet containing 10.3% fat, 24.2% protein, and 65.5% carbohydrate as percentage of total calories. The rats in high-fat diet group were fed regular diets mixed with 30% lard, containing 59.8% fat, 20.1% protein and 20.1% carbohydrate as a percentage of total calories. The rats in every group were fed equal calories every day. The body weights were determined weekly for 8 weeks. The blood sample was collected by cardiac puncture after rats were anesthetized with diethyl ether for the biochemical analysis, insulin resistance was evaluated by glucose infusion rate (GIR) of hyperinsulinemic euglycemic clamp technique and oral glucose tolerance test was done at the end of 4 week(eight rats in each group). After 4 weeks, rats were randomly divided into high fat diets group and high-fat + rosiglitazone group (RSG), the rats were given rosiglitazone (GlaxoSmithKline, UK) 3 mg per kg body weight through intragastric administration once a day for 4 weeks in RSG group(eight rats in each group). At the end of 8 week, the rats were killed with phenobarbital sodium after hyperinsulinemic euglycemic clamp test and OGTT, and skeletal muscles and fat tissues were taken out freeze-clamped with copper clamps precooled in liquid N2 and were stored in -70℃refrigerator. Muscle triglyceride(TGm) was measured by automatic biochemistry analyzer after extracting by methanol and chloroform, muscle long-chain fatty acyl coenzyme A(LCACoA)was measured by fluorospectro- photometer.
Results:1 The body weight in OC group rats were higher than that in YC group rats. Compared with OC group, the body weight in HF group and RSG group didn’t increase significantly(p>0.05).Compared with YC group, the fasting insulin(FINS), fasting blood glucose(FBG) and free fatty acid(FFA) were higher in the OC group at the end of 8 week; Compared with OC group, FINS, FBG, FFA, total cholesterol(TC) and triglyceride(TG) were higher in the HF group(p<0.05 or p<0.01). FINS, FBG, FFA, TC and TG were lower in the RSG group than those in the HFgroup(p<0.05 or p<0.01). 2 At the end of 4 week and 8 week, compared with YC group, TGm and LCACoA were higher in OC group(p<0.05); TGm and LCACoA were significantly higher in HF group than those in OC group(p<0.05 or p<0.01); TGm and LCACoA were higher at 8 week than those at 4 week in HF group(p<0.01); TGm and LCACoA were lower in the RSG group than those in the HF group(p<0.05 or p<0.01). 3 Compared with YC group at the end of 4 week and 8 week, GIR was lower in OC group, GIR was more lower in HF group than that in OC group(p<0.01); Compared with HF group at 4 week, GIR was significantly lower in HF group at 8 week(p<0.01). GIR were higher in the RSG group than that in HF group(p<0.01)4 The results of oral glucose tolerance test. At the end of 4 week and 8 week, the glucose level of the rats in OC group was much higher than that of rats in YC group at the time of 0′and 60′, 120′after glucose loading and area under curve(AUC) significantly increased (p<0.05 or p<0.01). The glucose level at every time point and AUC of the rats in HF group increased compared with those of rats in OC group (P<0.01). The glucose level in RSG group decreased at every time point and AUC (p<0.05 or p<0.01). 5 GIR has a negative correlation with INS, FFA, TGm and LCACoA (p<0.05 or p<0.01).
Conclusions: TGm and LCACoA were higher and GIR was lower in OC group than those in YC group. It demonstrated that insulin resistance and lipid deposition of skeletal muscle always exist in aged rats. High fat diets in aged rats induced a significant increase in plasma lipid and intramusculary lipid paralleled impaired GIR, the results showed that insulin resistance and lipid metabolism were more serious. GIR has a negative correlation with INS, FFA, TGm and LCACoA. It suggested that lipid disorder is associated with insulin resistance. TGm and LCACoA decreased by rosiglitazone treatment and GIR and OGTT improved in aged rats induced by high fat diets, that maybe one of the mechanisms of rosiglitazone to improve insulin rasistance. Part two: Effects of high-fat diets and rosiglitazone treatment on expression of FAT/CD36 and CPTⅠβin aged rat skeletal muscles.
Objective: To observe the effects of aging, high-fat diets and rosiglitazone treatment on the expression at protein and mRNA level of FAT/CD36 and mRNA level of CPTⅠβand to analyze the correlation with lipid in muscle tissue.
Methods: Animal grouping and the samples acquirement were the same as those in part one. The expression at protein level of FAT/CD36 in skeletal muscle was measured by Western-blot method and the expression at mRNA level of FAT/CD36 in skeletal muscle was measured by real time polymerase chain reaction (PCR) method; the expression of CPTⅠβmRNA was measured by semiquantitative PCR method. TRIZOL Reagent was used to extract RNA from the tissues, integrity of RNA was evaluated by formaldehyde degeneration caraphoresis and optical density (OD) was got at 260nm and 280nm. RT-PCR was carried out with better integrity and ratio of OD 260nm and 280nm in 1.8～2.0 of RNA.
Results: 1 Compared with YC group, the expression of FAT/CD36 mRNA and FAT/CD36 protein in skeletal muscle were higher in the OC group(p<0.05 or p<0.01); Compared with OC group, the expression of FAT/CD36 mRNA and FAT/CD36 protein were even higher in HF group rats(p<0.05 or p<0.01); the expression of FAT/CD36 mRNA and FAT/CD36 protein were lower in RSG group rats than those of rats in HF group(p<0.05 or p<0.01). 2 There were no significant difference of the expression of CPTⅠβmRNA between the YC group and OC group rats(P>0.05). Compared with OC group, the expression of CPTⅠβmRNA was lower in HF group rats(p<0.01). In response to the rosiglitazone treatment , the expression of CPTⅠβmRNA was significantly higher compared with HF group. 3 LCACoA has a positive correlation with the expression of FAT/CD36mRNA and has a negative correlation with the expression of CPTⅠβmRNA.
Conclusions: 1 It is possible reason for aged rats to produce more lipid deposition in skeletal muscles than young rats for the higher expression of FAT/CD36 mRNA and protein. 2 The abnormal expression of FAT/CD36 and CPTⅠβof skeletal muscle in the aged rats induced by high fat diets may be one of the mechanisms of lipid deposition. 3 Correlation analyses suggested that lipid accumulation in muscles was associated with the abnormal expression of key enzyme to fatty acid transport. The expression of CPTⅠβmRNA increased and the expression of FAT/CD36mRNA and protein decreased in high-fat diets rats treated with rosiglitazone maybe associate with reduced lipid deposition and improve insulin resistance.
Part three: Effects of high-fat diets on expression of GLUT4 and PKB in aged rat skeletal muscles and rosiglitazone treatment.
Objective: To observe the effects of aging and high-fat diets on the expression at protein and mRNA level of PKB and GLUT4 and to analyze the correlation with lipid in muscle tissue.
Methods: Animal grouping and the samples acquirement were the same as those in the part one. The expression at protein and mRNA level of PKB and GLUT4 in skeletal muscle was measured by Western blot method and real time PCR method respectively.
Results: 1 Compared with YC group, the expression of PKBmRNA and PKB protein was lower in the OC group(P<0.05); Compared with OC group, the expression of PKBmRNA and protein was significantly lower in HF group rats (P<0.05); the expression of PKBmRNA and protein was higher in RSG group than that in HF group(p<0.05). 2 Compared with YC group, the expression of GLUT4mRNA and protein was lower in OC group (P<0.05 or P<0.01); Compared with OC group, and the expression of GLUT4mRNA and protein were significantly lower in HF group (P<0.05 or P<0.01); the expression of GLUT4mRNA and protein was higher in RSG group than that in HF group(p<0.01).3 The expression of PKBmRNA and GLUT4mRNA have possitive correlation with GIR and the expression of GLUT4mRNA has a negative correlation with LCACoA(P <0.01).
Conclusions: 1 It is possible reason for the high incidence of insulin resistance in the aged rats because the impaired insulin-signaling transduction accounting for the abnormal expression of PKB and GLUT4. 2That insulin signal transduction impaired because of the abnormal expression of PKB and GLUT4 induced by high fat diets may be one of the mechnisms of insulin resistance. 3 Correlation analyses suggested that lipid accumulation associated with skeletal muscle insulin resistance through the effect on the key enzyme of insulin signal transduction. 4 The expression of PKB and GLUT4 increased in high-fat diets rats treated with rosiglitazone maybe one of the mechanisms to improve insulin resistance.
Part four: Effects of high-fat diets and rosiglitazone treatment on the expression of PPARγin fat and skeletal muscle tissues in aged rat Objective: To observe the effects of aging and high-fat diets on the expression at protein and mRNA level of PPARγin fat and skeletal muscle tissues and rosiglitazone interference study.
Methods: Animal grouping and the samples acquirement were the same as part one. The expression at protein and mRNA level of PPARγwas measured by Western-blot method and real time PCR method respectively.
Results: 1 Compared with YC group, the expression of PPARγmRNA and PPARγprotein in fat tissue were lower in the old control group rats(P<0.05); Compared with OC group, the expression of PPARγmRNA and PPARγprotein in fat tissue were even lower in HF group (P<0.05); the expression of PPARγwas higher in RSG group than that of rats in HF group(p<0.01). 2 There were no significant differerce of the PPARγmRNA and PPARγprotein in sheletal muscle between YC group and OC group (p>0.05). Compared with OC group, the expression of PPARγmRNA and protein in skeletal muscle were lower in HF group (P<0.05 or P<0.01); the expression of PPARγmRNA and protein in skeletal muscle were higher in RSG group than those of rats in HF group (P<0.01).
Conclusions: 1 It is possible for lipid metabolic disorder in aged rats because the lower expression of PPARγin fat tisssues than that of in young rats.2 That the lower expression of PPARγin fat and skeletal muscle tissues induced by high-fat diets maybe assosiated with abnormal lipid metabolism and insulin resistance. 3 Rosiglitazone treatment maybe affect adipocyte differentiation and improve insulin resistance through increasing the expression of PPARγin fat and skeletal muscle tissues.
引文
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