Glucose Transporter Regulation in Obesity and Diabetes

描述（由申请人提供）：在肥胖症和2型糖尿病中，Glut 4葡萄糖转运蛋白表达在脂肪组织中特异性下调。我们最近确定，脂肪特异性下调Glut 4导致分泌一种新的因子，改变肌肉和肝脏中的胰岛素作用。通过对adipse-GLUT 4基因敲除和adipse-GLUT 4基因过表达小鼠脂肪组织的DNA阵列分析，我们鉴定了在胰岛素抵抗啮齿类动物和人类血清中升高的蛋白质，视黄醇结合蛋白4（RBP 4）。我们证明了RBP 4在正常小鼠中引起胰岛素抵抗。降低由于饮食诱导的肥胖而产生胰岛素抵抗的小鼠的RBP 4水平可以改善其胰岛素敏感性。该资助的总体目标是确定1）RBP 4引起胰岛素抵抗的生理、细胞和分子机制，以及2）RBP 4保留增加是否是胰岛素抵抗状态下血清升高的重要机制及其潜在机制。目的1是确定RBP 4影响胰岛素作用的生理机制。我们将使用体内技术来确定哪些组织和生理过程是肥胖和糖尿病小鼠中由升高的RBP 4引起的胰岛素抵抗以及降低RBP 4水平的改善作用的基础。我们将确定RBP 4基因敲除小鼠是否在高脂饮食中受到胰岛素抵抗的保护，以及导致RBP 4排泄的合成类维生素A的治疗作用的机制。目的2是确定RBP 4升高引起胰岛素抵抗的机制是类维生素A依赖性还是非依赖性。为了研究类维生素A依赖性机制，我们将测量血清和组织中视黄醇和类维生素A的水平;研究肥胖小鼠中的视黄酸受体信号;测试饮食中视黄醇的影响。（维生素A）过量和缺乏对胰岛素敏感性的影响;并对注射RBP 4的小鼠的胰岛素靶组织和脂肪组织进行DNA阵列研究，Glut 4基因敲除小鼠，以确定基因表达模式是否与我们将建立的视黄醇过量基因集重叠。为了研究不依赖于视黄醇的机制，我们将研究不结合视黄醇的突变体RBP 4对胰岛素敏感性的影响，缺乏甲状腺素运载蛋白（TTR）的小鼠的代谢表型，以及参与类维生素A代谢的酶的变化。目的3是确定RBP 4保留增加是否是胰岛素抵抗状态下血清中RBP 4升高的重要机制，以及这是否是RBP 4和/或TTR的翻译后修饰的结果。使用质谱法，我们将鉴定和定量胰岛素抵抗小鼠模型血清中的TTR蛋白变体，并确定这些变体是否可能导致RBP 4滞留。由于已知RBP 4的C末端蛋白水解调节其对TTR的亲和力，我们将确定胰岛素抵抗是否与RBP 4蛋白水解减少相关，从而导致血清中RBP 4保留增强。使用纯化的RBP 4和TTR，我们将确定TTR的哪些翻译后修饰改变其对RBP 4的结合亲和力。这些研究将阐明一种新的机制，潜在的组织间通讯，在2型糖尿病的发病机制中起着重要作用。

DESCRIPTION (provided by applicant): In obesity and type 2 diabetes, Glut4 glucose transporter expression is down-regulated specifically in adipose tissue. We recently determined that adipose-specific down-regulation of Glut4 results in secretion of a novel factor that alters insulin action in muscle and liver. Using DNA array analyses of adipose tissue from adipose-Glut4 knockout and adippse-GLUT4 overexpressing mice, we identified such a protein that is elevated in serum of insulin-resistant rodents and humans, retinol binding protein 4 (RBP4). We demonstrated that RBP4 causes insulin resistance in normal mice. Lowering RBP4 levels in mice that are insulin-resistant due to diet-induced obesity improves their insulin sensitivity. The overall goal of this grant is to determine 1) the physiologic, cellular and molecular mechanisms by which RBP4 causes insulin resistance and 2) whether increased retention of RBP4 is an important mechanism for its serum elevation in insulin resistant states and the mechanisms underlying this. Aim 1 is to determine the physiological mechanisms for the effects of RBP4 on insulin action. We will use in vivo techniques to determine which tissue(s) and physiologic processes underlie the insulin resistance resulting from elevated RBP4 and the ameliorative effect of lowering RBP4 levels in obese and diabetic mice. We will determine whether RBP4 knockout mice are protected from insulin resistance on a high fat diet and the mechanism for the therapeutic effects of a synthetic retinoid that results in RBP4 excretion. Aim 2 is to determine whether the mechanisms by which elevation of RBP4 causes insulin resistance are retinoid-dependent or retinoid-independent. To investigate retinoid-dependent mechanisms, we will measure serum and tissue levels of retinol and retinoids; investigate retinoic acid receptor signaling in obese mice; test the effects of dietary retinol (vitamin A) excess and deficiency on insulin sensitivity; and perform DNA array studies on insulin target tissues of RBP4-injected mice and adipose-Glut4 knockout mice to determine whether the gene expression patterns overlap with a gene set we will establish for retinol excess. To investigate retinol independent mechanisms we will investigate the effects of a mutant RBP4 that does not bind retinol on insulin sensitivity, the metabolic phenotype of mice lacking transthyretin (TTR), and changes in enzymes involved in retinoid metabolism. Aim 3 is to determine whether increased retention of RBP4 is an important mechanism for RBP4 elevation in serum in insulin-resistant states and whether this results from post-translational modifications of RBP4 and/or TTR. Using Mass Spectrometry, we will identify and quantitate TTR protein variants in serum of insulin resistant mouse models and determine whether these are likely to cause RBP4 retention. Since C terminus proteolysis of RBP4 is known to regulate its affinity for TTR, we will determine whether insulin resistance is associated with reduced proteolysis of RBP4 leading to enhanced RBP4 retention in serum. Using purified RBP4 and TTR we will determine which post-translational modifications of TTR alter its binding affinity for RBP4. These studies will elucidate a novel mechanism underlying the inter-tissue communication that plays an important role in the pathogenesis of type 2 diabetes.