【作者】 高春林；

【导师】 郭锡熔；

【作者基本信息】 南京医科大学 ， 儿童营养， 2010， 博士

【摘要】 肥胖已成为全球性的公共卫生问题,儿童肥胖的现状不容乐观。美国儿童肥胖的发生率在过去三十年内已增加近3倍,中国儿童、青少年超重和肥胖的发生率已达12%。肥胖不仅影响儿童的心理发育,而且还导致肥胖相关代谢综合症(包括2型糖尿病)的出现。近年来,儿童2型糖尿病的低龄化趋势日趋明显,而2型糖尿病及代谢综合症发生的关键环节是胰岛素抵抗,但其发病机制目前尚未完全阐明,因此进一步系统地对胰岛素抵抗发生发展的机理进行研究非常必要。高血糖、高游离脂肪酸是胰岛素抵抗2型糖尿病最主要、最常见的生化表现。早在上世纪90年代就提出了胰岛素抵抗的葡萄糖毒性(glucose toxicity)和脂质毒性(lipid toxicity)学说,胰岛β细胞在高浓度葡萄糖或游离脂肪酸作用下会出现功能衰竭,致葡萄糖刺激的胰岛素分泌(glucose stimulated insulin secretion,GSIS)功能丧失。近年来越来越多研究则表明,高糖、高游离脂肪酸致胰岛素敏感性降低的机制还不仅仅涉及其对胰岛β细胞的毒性作用,而且还涉及其对骨骼肌细胞、肝细胞、血管内皮细胞等的作用。已知脂肪组织也是胰岛素作用的重要靶组织之一,由于近年来发现脂肪组织已不单纯是能量储存的场所,在胰岛素抵抗发生过程中能通过增加游离脂肪酸释放、分泌大量炎性因子如肿瘤坏死因子(TNF-α)等,加重全身胰岛素的抵抗,因此脂肪细胞胰岛素抵抗已被认为是全身胰岛素抵抗的一部分。脂肪组织中线粒体细胞器十分丰富,近年来胰岛素抵抗的线粒体机制备受关注,但目前有关高糖和/或高游离脂肪酸作用下脂肪细胞线粒体功能变化及与脂肪细胞胰岛素敏感性之间的关系尚无系统论证。解耦联蛋白4(UCP4)是位于线粒体内膜上、与能量代谢密切相关的分子。前期研究中,本研究小组应用基因芯片(cDNA array)技术筛选肥胖相关差异表达基因时发现,UCP4基因在饮食诱导性肥胖大鼠脂肪组织中表达显著上调;UCP4基因过表达于3T3-L1脂肪前体细胞,不但抑制脂肪细胞分化、抑制其脂质积聚,而且还促进前体脂肪细胞增殖、抑制脂肪细胞凋亡,在成熟脂肪细胞中则下调GLUT1、GLUT4而抑制脂肪细胞对基础葡萄糖摄取、胰岛素刺激后葡萄糖摄取。UCP4对脂肪细胞胰岛素敏感性的下调作用是否涉及线粒体机制尚需深入研究。为此,本研究拟评价高糖、高游离脂肪酸、UCP4对脂肪细胞胰岛素敏感性的影响,并分析线粒体功能障碍机制在其中的作用。第一部分,以高糖、高游离脂肪酸干预3T3-L1脂肪细胞,观察此过程中脂肪细胞胰岛素敏感性的变化,分析高糖、高游离脂肪酸对线粒体功能的影响及与胰岛素敏感性变化之间的关系;第二部分,通过构建UCP4真核表达载体,转染3T3-L1脂肪前体细胞,观察UCP4在脂肪细胞中过表达对脂肪细胞胰岛素敏感性的影响,并初步评价线粒体功能障碍机制在其中可能的作用。第一部分:高糖、高游离脂肪酸对3T3-L1脂肪细胞胰岛素敏感性的影响及线粒体机制分析目的:探讨高糖、高游离脂肪酸对3T3-L1脂肪细胞胰岛素敏感性的影响,分析高糖、高游离脂肪酸对线粒体功能的影响及与胰岛素敏感性变化之间的关系。方法:3T3-L1前体脂肪细胞经MDI方案诱导分化成熟后,分别以高游离脂肪酸(含混合游离脂肪酸1mmol/L)、高糖(葡萄糖25 mmol/L)、高糖+高游离脂肪酸干预48hr,低糖(5 mmol/L )为对照,2-Deoxy-[~3H]glucose摄取实验验证脂肪细胞的胰岛素敏感性,透射电镜观察线粒体超微结构,荧光定量Real Time PCR、Western Blotting检测线粒体的生物合成情况,荧光分子探针检测脂肪细胞内活性氧、线粒体膜电位、线粒体内钙等。结果:①高游离脂肪酸、高糖、高糖+高游离脂肪酸干预各组,胰岛素刺激后的葡萄糖摄取量均显著降低;②高糖、高游离脂肪酸单独或联合干预后,脂肪细胞线粒体体积变小,线粒体变致密,线粒体基质浓缩,线粒体嵴断裂、减少、排列紊乱;③线粒体融合相关蛋白mfn1在高游离脂肪酸组、高糖+高游离脂肪酸组表达显著下调,而介导线粒体分裂的蛋白Drp1在高游离脂肪酸组显著增加,线粒体倾向于分裂而融合减少;④各干预组线粒体生物合成的关键因子过氧化物酶体增殖物激活受体γ辅激活子1α(PGC-1α)、过氧化物酶体增殖物激活受体γ辅激活子1β(PGC-1β)、核呼吸因子(NRF-1)、线粒体转录因子A(mtTFA)均表达下调;⑤各干预组脂肪细胞内ROS显著增加、线粒体膜电位显著降低,但线粒体DNA拷贝数、脂肪细胞内ATP浓度无显著变化;⑥高糖、高糖+高游离脂肪酸干预可显著降低脂肪细胞线粒体内的钙含量,但高游离脂肪酸干预对脂肪细胞线粒体内钙含量无显著影响。结论:高糖、高游离脂肪酸可降低脂肪细胞胰岛素的敏感性,同时引发线粒体功能障碍,线粒体功能障碍可能是脂肪细胞胰岛素抵抗的机制之一。第二部分:UCP4过表达对3T3-L1脂肪细胞胰岛素敏感性的影响及线粒体机制分析目的:探讨UCP4基因过表达对3T3-L1脂肪细胞胰岛素敏感性的影响,分析UCP4基因过表达对线粒体功能的影响及与胰岛素敏感性变化之间的关系。方法:构建UCP4真核表达载体,稳定转染体外培养的3T3-L1前体脂肪细胞株,Real Time PCR、Western Blotting验证UCP4稳定转染后,以含空载质粒的3T3-L1脂肪细胞为对照,MDI方案诱导稳定转染UCP4-pcDNATM3.1/myc-His B载体质粒的3T3-L1脂肪细胞分化成熟;2-Deoxy-[~3H]glucose摄取实验验证UCP4过表达对脂肪细胞胰岛素敏感性的影响,透射电镜观察其线粒体超微结构,采用荧光定量Real Time PCR、Western Blotting等技术检测其线粒体的生物合成情况,荧光分子探针检测脂肪细胞内活性氧、线粒体膜电位、线粒体内钙。结果:①UCP4基因过表达可显著降低脂肪细胞的胰岛素敏感性;②透射电镜显示UCP4过表达脂肪细胞的线粒体体积大小不一,线粒体变致密,线粒体基质浓缩,线粒体嵴模糊,断裂;③UCP4过表达可致脂肪细胞中线粒体融合蛋白mfn1表达下调,线粒体分裂蛋白Drp1表达降低;④UCP4过表达脂肪细胞中,PGC-1α、NRF-1表达下调而NRF-2表达显著上调,SIRT1(沉默信息调节因子1)蛋白表达量在无明显差异,雌激素受体相关受体β(ERRβ)蛋白表达显著降低,mtTFA mRNA表达也显著降低;⑤UCP4过表达可显著降低脂肪细胞线粒体DNA拷贝数、脂肪细胞内ATP浓度、线粒体膜电位、线粒体内的钙含量,显著增加脂肪细胞内的ROS。结论:UCP4过表达不但降低脂肪细胞胰岛素的敏感性,同时引发线粒体功能障碍,线粒体功能障碍可能是脂肪细胞胰岛素抵抗的机制之一。更多还原

【Abstract】 Obesity has been a global public health problem, and the situation in children and adolescents is not optimistic. The obesity incidence rate of US childhood has increased nearly 3 times during the past three decades, and 12% of the Chinese children and adolescents are overweight and obese. Obesity has been showed to affect the children’s mental growth, and would lead to develop the obesity related metabolism syndrome( type 2 diabetes included). Recently, the lower age tendency of childhood type 2 diabetes is becoming obvious. The key link of type 2 diabetes and metabolism syndrome is insulin resistance, but the mechanism of which is not fully clarified untill now, so a systematic study for the pathogenesis of insulin resistance is needed.Hyperglycaemia and high level of free fatty acids (FFAs) in blood circulation are the most important and common sceneries. The concept of glucose toxicity and lipid toxicity was raised early in 1990s, which meant that high level of glucose or FFAs would lead toβ-Cell failure and loss of glucose-stimulated insulin secretion (GSIS). There were growing body of evidences demonstrated that not onlyβ-Cell, but other insulin targeted tissues, such as skeletal muscle, hepatic cell and vascular endothelial cell et al, were involved in the development of high glucose and high FFAs caused insulin resistance.As one of the important periphery insulin targeted tissues, the adipose tissue was not merely as an energy storage place, once it became insulin resistance, it would turn to release FFAs and secrete inflammatory factors (TNF-α, for example), the both conditions would exacerbate systematic insulin resistance, today it has been conceived as an important player of systematic insulin resistance. The adipose tissue contained plentiful of mitochondria, on the other hand, the mitochondria mechanism has been implicated closely in insulin resistance, but there is little message for the effect of high level of glucose and FFAs on mitochondrial function and adipocyte insulin sensitivity.UCP4, located in the inner mitochondrial membrane, was closely related to energy metabolism. We previously showed that UCP4 was significently upregulated in omental adipose tissue in diet-induced obese rats using suppression subtractive hybridization (SSH). Ectopic expression of UCP4 can promote proliferation and inhibit differentiation and lipid accumulation as well as apoptosis in 3T3-L1 preadipocytes. The insulin-stimulated glucose uptake and the expression of glucose transporters GLUT1 and GLUT4 in differentiated 3T3-L1 adipocytes were downregulated upon UCP4 overexpression. Whether mitochondrial mechanism is involved in the down regulated insulin sensitivity of UCP4 in adipocytes deserves futhur investigation.The aim of this study was to determine the effect of high glucose、high FFAs and UCP4 on adipocytes insulin sensitivity, and to detemine whether mitochondrial dysfunction plays a role. In the first part, the differentiated 3T3-L1 adipocytes were incubated with high glucose or/and high FFAs, adipocytes insulin sensitivity was observed, the mitochondrial function was analyzed after the treatment, the relationship of mitochondrial function and insulin sensitivity was investigated; In the second part, we examined the effect of UCP4 on adipocytes insulin sensitivity and mitochondrial function in vitro by establishing a stable preadipocyte cell line overexpressing UCP4, we preliminary assessed the possible role of mitochondrial dysfunction in the process. Part I: The mitochondrial mechanism analysis of high level of glucose、high FFAs impacting the insulin sensitivity of 3T3-L1 adipocytesObjective: To investigate the relationship and the effects of high glucose and high FFAs on insulin sensitivity and mitochondrial function in differentiated 3T3-L1 adipocytes.Methods: Differentiated 3T3-L1 adipocytes were treated with high glucose (25 mM) or high FFAs (1 mM) or both for 48 hours, 5 mmol/L glucose as control. Insulin sentivity was determined by insulin-stimulated 2-Deoxy-D-[3H] glucose uptake in differentiated 3T3-L1 adipocytes, mitochondria ultramicrostructure was displayed by electromicrograph morphometry, mitochondrial biogenesis related proteins were examined by western blot, and the mitochondrial DNA copynumber and mRNA expression of PGC-1αwas detected by Realtime PCR. The ATP content of the adipocytes was measured with ATP lite-glo, a luciferase-based luminescence assay kit. Mitochondrial membrane potential and intramitochondrial calcium as well as intracellular ROS was detemined by fluorescent molecular probes and FACS.Results: We found:①High glucose,high FFAs,or high glucose+high FFAs reduced insulin-stimulated glucose uptake in differentiated 3T3-L1 adipocytes;②The treatment of high glucose, high FFAs, or high glucose+high FFAs induced smaller and more compact mitochondria, the mitochondrial matrix condensed, cristae collapsed and confused arranage;③Levels of the mitofusion protein mfn1 decreased in high FFAs group and high glucose+high FFAs group, and levels of the mitofission protein Drp1 increased in high FFAs group;④Levels of the mitochondrial biogenesis key factors PGC-1α、PGC-1β、NRF-1、mtTFA were downregulated in the treated groups ;⑤Intracellular ROS was significently increased while mitochondrial membrane potential was decreased in the treated groups, but there was no difference was detected when it came to mtDNA copy number and intracellular ATP content;⑥High glucose and high glucose+high FFAs treatment induced significent decreased intramitochondrial calcium, but high FFAs had no effect.Conclusion:High glucose and high FFAs could down regulate insulin sensitivity and cause mitochondrial dysfunction of adipocytes, mitochondrial dysfunction mightbe one of the mechanisms of adipocytes insulin resistance.Part II: The mitochondrial mechanism analysis of UCP4 overexpression affecting the insulin sensitivity of 3T3-L1 adipocytesObjective: To explore the effect of UCP4 on adipocytes insulin sensitivity, and to analyze the relationship of UCP4 overexpression induced mitochondrial function changes and insulin sensitivity in differentiated 3T3-L1 adipocytes.Methods: UCP4 eukaryotic expression vector was constructed and was stably transfected into 3T3-L1 preadipocytes. UCP4 expression was confirmed by Real Time PCR and Western-Blot analysis. The transfected cells with an empty expression vector (pcDNA3.1Myc/His B)(as control) or an UCP4 expression vector were induced to differentiated adipocytes; Insulin sentivity was determined by insulin-stimulated 2-Deoxy-D-[3H] glucose uptake in differentiated 3T3-L1 adipocytes, mitochondria ultramicrostructure was displayed by electromicrograph morphometry, mitochondrial biogenesis related proteins were examined by western blot, and the mitochondrial DNA copynumber and mRNA expression of PGC-1αwas detected by Realtime PCR. The ATP content of the adipocytes was measured with ATP lite-glo, a luciferase-based luminescence assay kit. Mitochondrial membrane potential and intramitochondrial calcium as well as intracellular ROS was detemined by FACS. Results:①Adipocytes insulin sensitivity was down regulated upon UCP4 overexpression;②Transmission electron microscopy (TEM) showed that adipocytes overexpressing UCP4 displayed different size and condensed mitochondria with collapsed and unclear cristae;③UCP4 overexpression impaired mitochondrial fusion and fission, as indicated by decreased mitofusin mfn1, and mitofission DRP1;④The adipocytes overexpressing UCP4 also showed decreased mRNA expression of key factors in mitochondrial biogenesis, including PGC-1αand mtTFA. NRF-1 and ERRβlevels were downregulated, while NRF-2 levels were upregulated and no change for SIRT1;⑤The adipocytes overexpressing UCP4 also showed decreased mitochondrial copy number (mtDNA) and intracellular ATP content, higher production of intracellular ROS and diminished levels of intramitochondrial calcium and mitochondrial membrane potential.Conclusion: UCP4 overexpression induced decreased insulin sensitivity and mitochondrial dysfunction in adipocytes, and mitochondrial dysfunction might be one of the mechanisms of adipocytes insulin resistance.更多还原