The ability of insulin to stimulate glucose uptake into skeletal muscle accounts for most of the postprandial clearance of glucose from the circulation. At the cellular level, this clearance of blood glucose is mediated via a specific glucose transporter isoform whose expression is limited to muscle and fat, the two tissues that respond to insulin by dramatically increasing their rate of glucose uptake. This glucose transporter isoform, called GLUT 4, exerts its function by being recruited or translocated from an intracellular locus to the cell surface after cellular insulin exposure. Evidence suggests that the targeting and intracellular movement of the GLUT 4 protein are a result of a program of gene expression that is restricted to fat and muscle and that causes the synthesis and maintenance of a unique organelle or a unique response of an existing organelle. The overall aim of this proposal is to identify these additional proteins in the organelle that are the products of the putative """"""""insulin response"""""""" gene family in muscle. We expect that doing so will provide new insight as to how communication occurs between cell surface insulin receptors and the transporter-containing vesicles. Our specific goals are as follows: 1. We will purify abundant proteins from transporter-rich vesicles derived from muscle with the idea that one or more of these might be an amino acid transporter. 2. We will develop a set of criteria by which the """"""""insulin response"""""""" gene family can be identified. These criteria include tissue specificity of expression and down regulation in a form of diabetes and after denervation. 3. We will employ in situ hybridization to gain a more detailed picture of glucose transporter expression during development and, in the adult rodent, a picture of expression as a function of fiber type. 4. We will also employ a cloning strategy to identify relatively rare """"""""insulin response"""""""" genes by combining the preparation of a subtractive cDNA library with a differential screening protocol. This strategy will identify genes whose expression is regulated like GLUT 4, namely those restricted to fat and muscle and appearing 10-15 days postpartum. The genetic locus (loci) of type II diabetes mellitus is unknown, but it does not appear to result from abnormal expression of the two cellular proteins definitely implicated in insulin's actions, namely the insulin receptor and the glucose transporter. This application seeks to identify novel genes involved in insulin's cellular actions. It seems likely that altered expression of one or more of these putative """"""""insulin response"""""""" genes will be found in some or all patients with type II diabetes.
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