Type 2 diabetes is an ever growing epidemic of the developed world and estimates have been made that from 3-5% of Americans are afflicted with this disorder. At the cellular level, type 2 diabetes can be described as failure of insulin to stimulate glucose uptake into fat and muscle, or simply, insulin resistance. While there are obviously some differences in the way these two tissues metabolize nutrients such as glucose, the mechanism of their insulin-responsive glucose transport appears identical and entails the movement of transport proteins of the Glut4 isoform from an intracellular storage site to the cell surface. Adipocyte-specific ablation of Glut4 leads to insulin resistance in skeletal muscle and consequently, in the animal. Thus, the overall goal of this proposal is to use cultured and primary adipocytes to gain a further understanding of the mechanism by which insulin causes the movement of intracellular Glut4 storage vesicles (GSVs) to the cell surface. Knowing this will further our fundamental understanding of vesicular traffic, and it will also provide clues as to the cellular processes that underlie insulin resistance and type 2 diabetes. Glut4 trafficking in adipocytes involves several intracellular compartments in addition to GSVs.
As aim 1 of the proposal, these compartments will be isolated and their protein contents identified. These identifications will help define the mechanism of compartment formation and protein trafficking. Proteins from three families of so-called tetraspan vesicle proteins (TVPs) are known to be associated with various membrane compartments in adipocytes. These TVPs are: 1. cellugyrin, 2. pantophysin and 3. Scamps 1-3 (for secretory component-associated membrane proteins). The functions of these proteins are presumed to be in vesicular traffic but are mechanistically unknown.
In aim 2, it is proposed to characterize the distribution of the adipocyte TVPs in relation to the Glut4 compartments and also, the insulin receptor, a separate marker for intracellular vesicle trafficking. Again, characterization will, along with aim 3, help define mechanism. We have recently generated novel adipocyte cell lines that express the so-called """"""""tet off"""""""" regulatory system for gene expression.
In aim three of the proposal, we will use these cell lines to conditionally modify the expression of genes noted in aims 1 and 2 (and others), with antisense or siRNA for example. This will allow the unambiguous confirmation for the involvement of some genes suspected to be necessary for insulin-regulated Glut4 trafficking. More importantly, it will allow the formation of novel blockades in trafficking that will help identify the gene products necessary for the formation and targeting of GSV components and other proteins.
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