Skeletal muscle is a site of insulin resistance after denervation and in pathological states characterized by hyperinsulinemia, hyperglycemia and/or persistent elevations of plasma free fatty acid (FFA). This proposal will examine two hypotheses (1) that primary alterations of muscle fuel metabolism in these situations affect the diacylglycerol-protein kinase C (DAG-PKC) signalling system and (2) that these alterations of DAG-PKC signalling, if sustained, contribute to insulin resistance. Based on preliminary data with an incubated soleus muscle preparation, we are proposing a model in which increases in DAG in these conditions (1) occur in a specific pool, (2) are predominantly due to DAG synthesis de novo and (3) are associated initially with an increase in PKC activity, and ultimately with increases or decreases in DAG-PKC signalling that result in insulin resistance. The proposed studies will both test this paradigm and explore the biological role of the DAG-PKC signalling system in insulin action. Using incubated and perfused muscle preparations, we will carry out studies with the following aims: 1. To determine the mechanism for the increase in DAG synthesis in insulin+glucose-stimulated and denervated soleus muscles. 2. To determine the relationship between changes in DAG mass and synthesis and PKC activity. 3. To characterize the temporal relations between changes in DAG-PKC signalling and the development of insulin resistance at specific sites. 4. To examine the relationships between alterations in DAG-PKC and the expression of the glucose transporter genes, glut-4 and glut-1, the early response genes, c-fos and jun-B and myogenin and myoD in denervated muscle. 5. To compare DAG-PKC signalling in denervated muscle with that of muscle in other insulin resistant states, and after exercise, an activity that increases insulin sensitivity and 6. To describe how we would eventually proceed to explore mechanisms by which altered DAG-PKC signalling can affect insulin action. These studies should provide novel information about the linkage between fuel-metabolism, signal transduction, and gene expression in skeletal muscle. They should also yield new insights into the role of DAG-PKC signalling in the pathogenesis of insulin resistance and in the expression of specific genes in adult muscle.
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