应激影响肉鸡骨骼肌葡萄糖代谢的机制研究

Insulin stimulates glucose uptake by target tissues through specific glucose transporters (GLUTs). In mammals, skeletal muscle is the most important organ for the disposal of glucose. Compared with mammals, glucose metabolism in chickens is characterized by hyperglycemia and insulin resistance. Although the homologous gene of GLUT-4, the predominant insulin-responsive GLUT in mammals, has not been found in chickens, GLUT-1, -3 and -8 are expressed, though to a very small extent, in skeletal muscles. Glucose transport across the plasma membrane in skeletal muscles was increased by insulin treatment of growing chicks, suggesting that an alternative mechanism of insulin-responsive glucose transport is present in birds. Thus, the regulatory mechanisms for glycemia in chickens appear to differ from those in mammals..As the final effectors of the hypothalamic-pituitary-adrenal axis, glucocorticoids (GC) participate in the control of whole body homeostasis and stress responses. In mammals, GC play a key role in the pathogenesis of insulin resistance, and the action of GC on GLUTs via different signaling pathways has been described. In chickens, exogenous GC administration also induces insulin resistance. In concert with insulin, GC suppressed muscle development and altered energy deposition in broilers. Our recent work showed that GC suppressed insulin-stimulated glucose uptake in chicken muscles, suggesting that GC are likely to be metabolically involved in the glucose transport mechanism. Whether impaired GLUT regulation underlies GC-induced insulin resistance in glucose transport activity remains unclear..Broiler chickens have a high metabolic rate, blood glucose concentrations, growth rate, and capacity for fat deposition, making their muscles a potentially interesting model of insulin resistance. In the present study, the insulin-sensitive GLUT (GLUTx) will be identified first, followed by an analysis of insulin sinaling pathways in chicken muscles. And then the effect of GC on GLUTx and insulin signaling will be assessed. The accomplishment of this study may not only be helpful to guide the commercial poultry production, but also throw some new light on the pathogenesis of diabetes and obesity in humans.

应激抑制肉鸡骨骼肌的生长发育，导致能量分配发生改变，而葡萄糖代谢障碍是其重要原因之一。家禽的葡萄糖代谢机制与哺乳动物存在很大不同，许多控制的关键点目前都处于研究的空白。本项目利用蛋白质双向电泳、质谱分析、免疫组化、细胞转染、western blot和信号通路的特异性阻断等关键技术，体内和体外试验相结合，首先根据目标蛋白的表达数量和在细胞内膜、细胞质膜上的移位变化，在肉鸡骨骼肌内确定与胰岛素响应的葡萄糖转运载体蛋白（GLUTx）；然后，以此为靶点，研究胰岛素通过MAPK ERK2途径激活PKB/Akt进而调控肉鸡肌肉葡萄糖代谢的可能性；最后，探讨糖皮质激素与GLUTx和胰岛素信号通路的关系，揭示应激影响肉鸡骨骼肌葡萄糖代谢的机制。本项目所得结果，不仅可以为了解家禽应激反应的机理和合理地指导生产实践提供科学依据，而且对于人类糖尿病和肥胖症的研究也具有重大参考价值。

为揭示应激导致肉鸡骨骼肌胰岛素耐受、引起葡萄糖摄取障碍的机制，本项目在糖皮质激素（地塞米松）和胰岛素单独或联合处理情况下，研究了肌肉组织（活体）和鸡胚源成肌细胞（离体）的葡萄糖转运载体（GLUTs）表达、胰岛素MAPK/ERK信号通路活性和与胰岛素响应的GLUT的移位机制。结果发现：（1）在活体条件下，胸肌和腿肌GLUTs的转录对应激的反应性不同；在应激条件下，活体GLUTs的基因表达与葡萄糖摄取速率的变化并不同步。在基础条件下，糖皮质激素对肉鸡骨骼肌葡萄糖摄取的抑制作用与GLUT-1和-3的基因表达下降有关；在胰岛素存在条件下，糖皮质激素对肉鸡骨骼肌葡萄糖摄取的抑制作用与GLUT-1的基因表达下降有关。在不同浓度梯度的胰岛素作用条件下，肉鸡骨骼肌GLUT-1的mRNA、蛋白表达与胰岛素受体（IR）变化一致。（2）在正常条件下，胰岛素处理上调鸡胚源成肌细胞ERK（Tyr204位点）及其下游分子p70S6K（Thr389位点）、mTOR（Ser2448位点）和Akt（Ser473）的磷酸化水平；加入MEK抑制剂U0126后，这些蛋白的磷酸化水平均下降。使用Akt抑制剂（鱼藤素）能显著降低胰岛素诱导的Akt（Ser473）、mTOR（Ser2448位点）和p70S6K（Thr389位点）磷酸化，但不影响ERK（Tyr204位点）；使用mTOR抑制剂（雷帕霉素）能显著降低胰岛素诱导的mTOR（Ser2448位点）和p70S6K（Thr389位点）磷酸化，但不影响Akt（Ser473）和ERK（Tyr204位点）。在应激条件下，糖皮质激素（地塞米松）下调胰岛素诱导的ERK（Tyr204位点）和Akt（Ser473）磷酸化。（3）分离肉鸡骨骼肌细胞质膜和细胞内膜，根据3H-松弛素B结合试验，非特异性确定在家禽骨骼肌内存在与胰岛素响应的GLUT。利用双向电泳（2-DE）和质谱技术分析发现，与对照组相比，注射胰岛素后，肉鸡肌肉细胞内膜和细胞质膜上有多个蛋白重复点分别减少和增加，膜联蛋白（Annexin）A6是其中之一（这一结果尚需进一步证实）。以上结果表明，在肉鸡骨骼肌内，胰岛素通过ERK/Akt（不依赖于mTOR和p70S6K）途径调控葡萄糖摄取。应激（糖皮质激素）抑制ERK（Tyr204位点）和Akt（Ser473）磷酸化活性，下调GLUT-1表达，引发胰岛素耐受。