转甲状腺素蛋白在运动改善肥胖小鼠胰岛素抵抗中的作用研究

Studies have shown that the prevention of type 2 diabetes should mainly focus on insulin resistance (IR) interference. It has become a consencus that exercise can improve insulin sensitivity involving AMPK though the upstream molecule regulating the AMPK activity is far from clarified. Our previous studies have shown that Ttr mRNA levels in liver, as well as TTR protein levels in serum were significantly decreased in the exercised rats compared with the sedentary ones, which were independent on exercise-induced weight loss.Ttr expression and secretion were increased in mice maintained on high fat diet comared with those on regular chow. However,TTR protein was significantly decreased in skeletal muscle of the exercised rats, accompanied by an increase in AMPK activity, which plays a key role in the metabolism of glucose and fatty acid. Moreover, exogenous TTR pure protein significantly inhibited the AMPK pathway activity in C2C12 cells in a dose- and time-dependent manner.Based on our previous findings, we assumed that decreased Ttr expression in the liver induced by exercise, together with the decreased serum TTR levels would result in a lower TTR endocytosis in skeletal muscle, contributing to an increased AMPK alpha phosphorylation and higher utilization of glucose and fatty acid and improvement of mitochondrial number and function. The aims of the present study are to further clarify the mechanism of whole body insulin sensitivity improvement due to exercise and try to find a new molecular target for type 2 diabetes prevention. For these aims, in vitro we would like to adopt the C2C12 cells and primary muscle cell from newborn Sparague-Dawley rats and in vivo to use obese and insulin resistant mice models induced by high fat diet, as well as different exercised-mice models (mice of treamill exercise and one blos of exercise ), using the methods of hyperinsulinemic-euglycemic clamp for insulin sensitivity assessment and recombinant adenovirus expression vector for over-expression or knock-down Ttr gene expression in the liver, to see whether the decreased TTR in liver and serum could play a key role in the improvement of insulin sensitivity and AMPK activity in the muscle induced by exercise.

胰岛素抵抗（IR）是2型糖尿病发生的基本环节，运动改善IR的机制主要涉及AMPK活性增加，但调节分子不详。我们前期研究发现高脂饮食诱导的肥胖小鼠（DIO）肝脏转甲状腺素蛋白（TTR）表达分泌增加，而运动显著降低肝脏TTR表达和循环TTR水平，且此变化独立于运动导致的体重下降作用；运动能降低骨骼肌TTR蛋白摄取，伴随糖脂代谢关键信号通路-AMPK活性增加；而体外证实TTR蛋白剂量依赖性抑制骨骼肌细胞C2C12的AMPK活性，由此提出运动可能通过降低肝脏TTR表达分泌、减少骨骼肌TTR蛋白摄取继而增加AMPK活性改善局部糖脂代谢和线粒体功能而改善IR。本研究拟利用DIO小鼠跑台模型，借助原代细胞分离、重组腺病毒表达载体构建、蛋白标记及激光共聚焦等方法，观察慢性运动训练诱导的TTR下降在运动改善IR、调节骨骼肌能量代谢中的介导作用，旨在进一步阐明运动改善IR的机制，为2型糖尿病的防治寻找新的靶点

胰岛素抵抗是肥胖相关疾病如2型糖尿病、代谢综合征的主要病生理机制。运动能够改善胰岛素抵抗，从而降低2型糖尿病等患病率，但运动改善胰岛素抵抗的分子机制远未阐明，而对运动改善胰岛素抵抗关键分子的寻找和研究也是防治胰岛素抵抗相关疾病的新视角。我们最初在探索急性运动如何抑制大鼠食欲的研究中发现了下丘脑转甲状腺素蛋白（transthyretin，TTR）表达增高与运动相关食欲抑制密切相关。肝脏和脉络丛是循环和脑脊液中TTR蛋白的主要来源，运动主要引起肝脏TTR表达、合成和分泌入循环下降。随后我们发现，在高脂饮食喂养肥胖胰岛素抵抗小鼠（DIO）和代谢综合征人群中循环TTR蛋白水平明显高于普通饮食喂养小鼠和正常人群，提示循环中TTR蛋白增高可能与胰岛素抵抗有关。DIO小鼠肝脏TtrmRNA表达增加，而运动则降低肝脏TtrmRNA水平，且其向循环中释放减少，肝脏TTR表达变化极可能与运动适应性改变及运动改善胰岛素抵抗有关。由此，我们建立了尾静脉注射Ttr腺病毒（Ttr-AAV）转基因小鼠，发现尾静脉Ttr-AAV可以引起肝脏TtrmRNA表达增高，循环中TTR水平增高，并引起普通饮食喂养小鼠胰岛素抵抗和葡萄糖耐量异常。.更重要的是我们发现骨骼肌组织尽管不能表达TtrmRNA，但骨骼肌组织以及C2C12细胞中均有TTR蛋白存在。跑台运动小鼠骨骼肌中TTR蛋白量减少。进一步通过配体杂交、质谱分析发现，C2C12细胞能够时间依赖性的摄取生物素标记的TTR蛋白，而介导C2C12细胞摄取TTR蛋白的膜受体系Grp78。运动能够减少肝脏TTR蛋白合成和分泌，而肝脏过表达TtrmRNA不仅引起循环中TTR蛋白水平增高，同时骨骼肌TTR蛋白的“内在化”增加，并抵消运动对高脂饮食肥胖小鼠的胰岛素抵抗改善作用。骨骼肌TTR蛋白“内在化”增加主要抑制骨骼肌能量代谢的枢纽和运动介导胰岛素抵抗改善的关键分子“AMPK”通路活性下降，从而引起骨骼肌葡萄糖利用减少。.本研究基于TTR蛋白建立的肝脏-骨骼肌“对话”很可能是运动适应性的一种改变，也参与运动调节机体胰岛素抵抗改善的过程。但TTR蛋白进入骨骼肌细胞后在何处定位、其参与机体的运动适应性以及在运动调节机体胰岛素作用中的机制有待更多的基础研究。相关研究不仅有助于阐明运动适应性、运动改善胰岛素抵抗机制，也为拟运动效应剂的开发提供思路。