Impact of Modulated Exocyst Activity on Glut4 Trafficking in Metabolic Tissues

摘要 2型糖尿病（Type-2 Diabetes Mellitus，T2 DM）是一种以高血糖、胰岛素抵抗、高脂血症、高脂血症为特征的代谢紊乱性疾病， 缺乏和胰岛素抵抗。胰岛素抵抗细胞在胰岛素诱导的胞吐中表现出缺陷， 葡萄糖转运蛋白GLUT 4，影响葡萄糖摄取。外囊，一个高度保守的八蛋白运输 在培养的脂肪细胞中鉴定了复合物作为响应胰岛素的GLUT 4运输的关键调节剂。 然而，目前尚不清楚外囊组织胞吐的分子机制， 脂肪细胞中的这种葡萄糖转运蛋白在其他胰岛素反应组织中是保守的，如骨骼肌， 肌肉.骨骼肌细胞负责绝大多数胰岛素诱导的葡萄糖摄取，因此， 研究这些细胞中葡萄糖转运蛋白的运输将是更好地理解 葡萄糖稳态Sec 10是外囊复合体的中心亚基，其过表达可导致 总体上增加的外囊活性，而其敲除导致几种其他的蛋白质降解， 亚单位。我们最近已经产生了一种新的转基因小鼠来分析外生囊调节的细胞运输 体内：第一个具有条件性Sec 10等位基因的小鼠品系。这种独特的模型使我们能够研究 组织特异性Sec 10失活的后果，通过促进脂肪和肌肉特异性 条件性Sec 10敲除动物。我们假设，在体内调节外囊复合物， 在胰岛素应答细胞和组织中独立调节GLUT 4胞吐和葡萄糖摄取，影响 糖尿病表型为了验证这一假设，我们提出了以下具体目标：（1）确定是否 胰岛素诱导的GLUT 4胞吐的胞吐调节机制在骨骼肌中是保守的 细胞(2)使用脂肪细胞和骨骼肌特异性Sec 10检测体内葡萄糖稳态缺陷 敲除小鼠品系。(3)确定小鼠中T2 DM相关的代谢缺陷可以通过以下方式改善： 用一种新的组织特异性体内基因递送系统增加GLUT 4胞吐作用。预期 这个项目的结果是第一个在体内的证据，外囊的作用，调节胰岛素诱导的葡萄糖 摄取，并验证这种调节机制是否也在肌肉组织中保守。这些 研究还将表明，如果胞吐和其他直接控制GLUT 4胞吐的分子是潜在的 胰岛素抵抗和糖尿病的治疗靶点。

Abstract Type-2 diabetes mellitus (T2DM) is a metabolic disorder characterized by hyperglycemia, relative insulin deficiency and insulin resistance. Insulin resistant cells show defects in insulin-induced exocytosis of the glucose transporter GLUT4, affecting glucose uptake. The exocyst, a highly conserved eight protein trafficking complex was identified in cultured adipocytes as a key regulator of GLUT4 trafficking in response to insulin. However, it is not known if the molecular mechanisms through which the exocyst orchestrates exocytosis of this glucose transporter in adipocytes are conserved in other, insulin-responsive tissues, such as the skeletal muscle. Skeletal muscle cells are responsible for the vast majority of insulin-induced glucose uptake, therefore investigating glucose transporter trafficking in these cells will be key to better understanding the mechanism of glucose homeostasis. Sec10 is a central subunit of the exocyst complex, and its overexpression can lead to overall increased exocyst activity, while its knockdown leads to protein degradation of several of the other subunits. We have recently generated a new transgenic mouse to analyze exocyst-regulated cellular trafficking in vivo: the first mouse strain with a conditional Sec10 allele. This unique model enables us to investigate the consequences of tissue-specific Sec10 inactivation by facilitating generation of adipose and muscle-specific conditional Sec10 knockout animals. We hypothesize that in vivo modulation of the exocyst complex can independently regulate GLUT4 exocytosis and glucose uptake in insulin-responsive cells and tissues, affecting the diabetic phenotype. To test this hypothesis, we propose the following Specific Aims: (1) Determine if the exocyst mediated regulatory mechanism of insulin-induced GLUT4 exocytosis is conserved in skeletal muscle cells. (2) Test for defects in glucose homeostasis in vivo using adipocyte and skeletal muscle-specific Sec10 knockout mouse strains. (3) Determine T2DM-associated metabolic defects in mice can be ameliorated by increasing GLUT4 exocytosis with a novel tissue-specific in vivo gene delivery system. The anticipated outcome of this project is the first in vivo evidence of the exocyst's role in regulating insulin-induced glucose uptake, and verification whether this regulatory mechanism is conserved in muscle tissues as well. These studies will also show if the exocyst, and other molecules that directly control GLUT4 exocytosis, are potential therapeutic targets for insulin resistance and diabetes.