The long term goal of this research is to determine how insulin and diabetes affect regulation of glycogen metabolism and mechanical permormance of cardiac muscle. The regulation of cardiac glycogen metabolism will be studied at the molecular level as well as the perfused rat hearts. Studies will utilize hearts from normal, alloxan diabetic and BB/W spontaneously diabetic rats. Specific hypotheses to be tested are: 1) The phosphorylation state of the glycogen synthase molecule is altered by the diabetic state making it a poorer substrate for synthase phosphatase, 2) Glycogen synthase phosphatase is an inducible enzyme controlled by insulin, 3) Glycogen synthase phosphatase activity is hormonally controlled by the phosphorylation states of Inhibitor-1 and Inhibitor-2 as well as changes in the activity state of synthase kinase 3, 4) Epinephrine hypersensitivity of phosphorylase activation in diabetic hart is due to elevated intracellular free calcium, and 5) Long term diabetes results in a mechanical weakness in cardiac muscle.
The specific aims directed towards testing these hypotheses are: 1) Purification of glycogen synthase and phosphorylase from diabetic rat heart, characterize the normal and diabetic synthases, and test the ability of purified normal and diabetic synthase and phosphorylase to serve as substrates for their phosphatases, 2) Raise monoclonal and polyclonal antibodies to rat heart synthase and phosphorylase to be used in characterizations, affinity purifications and removal of the respective enzymes from tissue fractions, 3) Purify synthase phosphatase from normal rat heart, characterize it, and prepare antibodies to that purified phosphatase, 4) Isolate and characterize Inhibitors-1 and -2 to determine if and how they are involved in regulation of synthase phosphatase by insulin and diabetes, 5) Investigate the acute action of insulin on synthase phosphatase, synthase kinase and their modulators, 6) Determine if Ca-ATPase is affected by diabetes, and 7) Determine the effects of long term diabetes in the BB/W diabetic rat heart. Results of these studies should lead to a better understanding of how insulin and diabetes affect the regulation of cardiac glycogen metabolism and could serve as a basis of improved treatment of the diabetic population.
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