Despite the potential involvement of altered gene expression in the pathogenesis, little is known regarding the normal regulation of expression of human skeletal muscle enzymes which determine rates of glucose metabolism. The overall goal of the proposed studies is to establish an in vivo/in vitro system to determine how plasma insulin, glycemia, acute exercise, and training regulate skeletal muscle hexokinase II (HKII) and glycogen synthase (GS) catalytic activities, protein content, and mRNA abundance. We will use this system to determine whether changes in enzyme activity are due to changes in enzyme transcription or are due to regulation of intrinsic enzyme activity. This system will allow us to define the relationships between enzyme HKII and GS activity and in vivo glucose uptake/glycolysis and glycogen synthesis, respectively. We will use the glucose clamp technique in combination with percutaneous biopsies of the vastus lateralis muscle and the leg arteriovenous balance technique. HKII and GS catalytic activities, protein content (immunoblots), and mRNA (RNAse protection assays) will be measured in the biopsy specimens. Specifically, we propose: 1. To determine the normal time course and dose-response characteristics for the effects of insulin on HKII, HKI, and GS activity, protein content, and mRNA in skeletal muscle. We will focus on how the activities of these enzymes correspond to in vivo rates of glycolysis and glycogen storage and will test the hypotheses that: a. hyperinsulinemia increases skeletal muscle HKII and GS (but not HKI) activities, protein levels, and mRNA content in a time and dose-dependent manner in healthy nondiabetic subjects, and b. HKII activity is positively correlated with leg muscle glucose uptake in normal volunteers. 2. To determine the normal time course and dose- response characteristics for the effects of glucose on HKII, HKI, and GS activity, mRNA and protein content. We will provide mechanistic data for the concept of glucose toxicity by testing the hypothesis that hyperglycemia suppresses GS and HKII mRNA and protein content in a time and dose-dependent manner in healthy nondiabetic volunteers. 3. To determine how acute exercise and physical training alter GS, HKI, and HKII activities, protein content, and mRNA levels in skeletal muscle. We will test the hypotheses that: a. In untrained nondiabetic volunteers, an acute exercise bout increases skeletal muscle HKII (but not HKI) and GS activity and mRNA; b. Acute exercise increases the proportion of HKII activity associated with mitochondrial fractions of skeletal muscle; c. Endurance-trained athletes (long distance runners) have increased GS and HKII mRNA, protein levels, and activities in their leg muscle which is correlated to increased leg muscle glucose uptake. The studies in this proposal will focus especially on the hitherto unknown regulation of HKII in human skeletal muscle. These studies will define the normal role of HKII in the regulation of glucose metabolism in skeletal muscle and provide the ground work for determining whether decreased HK activity in muscle of NIDDM subjects leads to the well-known decrease in muscle glucose uptake in this disease.
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