The ultrastructural and biochemical studies in this project will increase our knowledge of the mechanisms that regulate the aggregation, internalization and intracellular processing of the insulin-receptor complex and will determine whether those processes, especially nuclear translocation of insulin and insulin's effects on macromolecular uptake into and efflux from the nucleus, are involved in insulin's regulation of cellular metabolism and growth.
The specific aims for the next five years are: 1) To use immunoelectron microscopic techniques to document the organization, distribution, lateral mobility, internalization routes and intracellular processing of insulin receptors on liver and skeletal muscle. These tissues, in addition to the adipocyte that has already been studied, are the primary insulin-sensitive tissues. The three tissues differ in their responses to insulin. Tissue-specific differences in insulin-receptor complex processing, which may be observed, should be valuable in understanding the relationship between hormone-receptor complex processing and insulin action. 2) To use high resolution ultrastructural techniques and cells expressing either normal or mutated human insulin receptors to determine which domains of the insulin receptor are responsible for the normal organization, distribution, lateral mobility, internalization and intracellular processing of the insulin-receptor complex. These studies will provide ultrastructural analysis to complement biochemical data concerning the relationships between the molecular structure of the insulin receptor and the aggregation, internalization and appropriate intracellular targeting of the insulin-receptor complex leading to normal insulin action. Similar studies will be performed using cells expressing normal or mutated human IGF I receptors. 3) To use ultrastructural and biochemical techniques to characterize the intracellular route responsible for the translocation and nuclear accumulation of insulin. By determining this intracellular pathway, we should learn more about the mechanism by which insulin regulates gene transcription and cell growth. In some of these studies agents that affect the nuclear accumulation of insulin will be used to accentuate key components of the intracellular translocation itinerary. 4) To determine whether or not insulin's effects on cell proliferation or gene expression correlate with the nuclear accumulation of insulin and/or insulin's effects on macromolecular uptake into and efflux from the nucleus. Our hypothesis, that the nuclear accumulation of insulin and its effects on macromolecular translocation are physiologically significant and possibly related to cell growth and/or gene expression, will be tested by evaluating these processes in cell types, including those expressing mutated human insulin receptors, with different growth-related responses to insulin. Ultrastructural, cellular and molecular techniques, including in situ electron microscopic hybridization, Northern blot analysis, etc., will be used for these studies. We will determine whether insulin accumulation and insulin-stimulated macromolecular nuclear uptake is cell-cycle dependent. The information gained from studies in this project will provide insights into normal mechanisms of insulin action and potential causes of insulin-resistance and diabetes mellitus. These insights may then provide possible alternatives for intervention or therapy.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Metabolism Study Section (MET)
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University of Pennsylvania
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Smith, R M; Harada, S; Smith, J A et al. (1998) Insulin-induced protein tyrosine phosphorylation cascade and signalling molecules are localized in a caveolin-enriched cell membrane domain. Cell Signal 10:355-62
Harada, S; Smith, R M; Smith, J A et al. (1996) Insulin-induced egr-1 and c-fos expression in 32D cells requires insulin receptor, Shc, and mitogen-activated protein kinase, but not insulin receptor substrate-1 and phosphatidylinositol 3-kinase activation. J Biol Chem 271:30222-6
Harada, S; Smith, R M; Hu, D Q et al. (1996) Dexamethasone inhibits insulin binding to insulin-degrading enzyme and cytosolic insulin-binding protein p82. Biochem Biophys Res Commun 218:154-8
Lee, Y H; Harada, S; Smith, R M et al. (1996) The expression of and insulin binding to cellular thyroid hormone binding protein, but not insulin degrading enzyme, is increased during 3T3-L1 adipocytes differentiation. Biochem Biophys Res Commun 222:839-43
Smith, R M; Zhang, S; White, M F et al. (1996) The role of receptor kinase activity and the NPEY960 motif in insulin-accelerated receptor-mediated insulin internalization. J Recept Signal Transduct Res 16:339-55
Shah, N; Zhang, S; Harada, S et al. (1995) Electron microscopic visualization of insulin translocation into the cytoplasm and nuclei of intact H35 hepatoma cells using covalently linked Nanogold-insulin. Endocrinology 136:2825-35
Harada, S; Smith, R M; Smith, J A et al. (1995) Insulin-induced egr-1 expression in Chinese hamster ovary cells is insulin receptor and insulin receptor substrate-1 phosphorylation-independent. Evidence of an alternative signal transduction pathway. J Biol Chem 270:26632-8
Harada, S; Smith, R M; Smith, J A et al. (1995) Demonstration of specific insulin binding to cytosolic proteins in H35 hepatoma cells, rat liver and skeletal muscle. Biochem J 306 ( Pt 1):21-8
Shi, C Z; Dhir, R N; Kesavan, P et al. (1995) Mouse embryonic stem cells express receptors of the insulin family of growth factors. Mol Reprod Dev 42:173-9
Bao, S; Smith, R M; Jarett, L et al. (1995) The effects of brefeldin A on the glucose transport system in rat adipocytes. Implications regarding the intracellular locus of insulin-sensitive Glut4. J Biol Chem 270:30199-204

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