The regulation of glucose entry into fat and muscle cells is one of the primary acute effects of insulin essential for the maintenance of whole body glucose homeostasis. This regulation is perturbed in insulin resistance syndromes, including type 2 diabetes, and therefore a better understanding of how insulin controls glucose flux into these cells will ultimately have a significant impact on human health and disease. Insulin regulates glucose flux into fat and muscle cells by controlling the amount of the Glut4 glucose transporter in the plasma membrane. Efforts of numerous labs over the past decade have established that this control is by a surprisingly complex, multi-step process. Recent applications of advanced microscopy methods and molecular cell biological approaches (including gene silencing strategies) have led to the formulation of a detailed model for Glut4 trafficking. In unstimulated cells it is important to limit the amount of Glut4 in the plasma membrane, and thereby restrict glucose flux into fat and muscle, which preserves blood glucose for use by the brain. Limiting Glut4 in the plasma membrane is accomplished by two distinct intracellular transport cycles. One involves traffic of Glut4 between endosomes and a specialized peri-nuclear compartment. The second involves the targeting of Glut4 from endosomes to Glut4-specialized vesicles (GSVs) that inefficiently dock and fuse with the plasma membrane in the absence of insulin-stimulation. Insulin action intersects Glut4 trafficking at a couple of steps that alter Glut4 transport through these pathways. Full understanding of insulin control of glucose transport requires an understanding of each step of this multi-step process. Here I propose to address some of the key outstanding questions in the field. Throughout the work I propose the use of quantitative microscopy methods and molecular cell biological approaches that we have used in our past studies, many of which I have had a hand in introducing to the study of Glut4.
In Aim1 we will analyze insulin control of pre- fusion steps of GSV association with the PM, with a specific emphasis on regulation by rab10. We will also create an adipose-specific rab10 knockout mouse to probe the physiologic role of adipose rab10 in the control of whole body glucose homeostasis.
In Aim2 we will further characterize the role of IRAP in Glut4 sorting to GSVs, and we will use a membrane-tethered yeast 2-hybrid screen to identify proteins that bind to IRAP and Glut4 cytoplasmic sequences. In secondary analyses we will determine the roles of these proteins in the control of Glut4 and IRAP trafficking.
In aim3 we will use SILAC mass spectrometry approaches to identify proteins of the peri-nuclear compartment, and in secondary analyses determine their contributions to the control of Glut4 trafficking. The results of this ambitious and comprehensive study will significantly advance our understanding of the molecular mechanisms controlling the trafficking of Glut4 between the plasma membrane and endomembrane compartments of adipocytes.
Insulin controls blood glucose levels, in part, by controlling the transport of glucose from the blood into muscle and fat cells. This regulation is defective in type 2 diabetes, contributing to elevated blood glucose of that disease. Insulin regulation of glucose transport is by a complex mechanism that controls the amount of a glucose transporter (known as Glut4) on the surface of cells. The details of this mechanism are not understood. In this project I propose studies to better understand how insulin regulates the amount of Glut4 on the surface of adipocytes. The long term objective of this work is to identify the proteins required for normal regulation of Glut4, use this information to identify what is defective in type 2 diabetes and thereby identify potential targets for the development of pharmacologic agents that mimics insulin's effects on glucose transport as a treatment for type 2 diabetes.
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