Recent studies have demonstrated that the addition of glucose to the medium of cultured rat hepatocytes results in the selective induction of the mRNA form malic enzyme (ME) and other mRNA species. Several selected questions raised by these observations will be investigated. (1) What is the intracellular source of the carbohydrate signal which stimulates the formation of malic enzyme mRNA? Since current findings strongly suggest a product distal to pyruvate metabolism is the proximate stimulus, the precise mitochondrial pathway involved in pyruvate metabolism which generates the signal for ME induction will be defined. The content of metabolites which are potential mediators of the carbohydrate signal will be assayed in the intact cell and in nuclei where measurements will be performed both after aqueous and nonaqueous isolation techniques. Mechanistic relationships will be inferred by modulating the concentrations of glucose, insulin, glucagon, and known activators of mitochondrial pyruvate dehydrogenase such as dichloroacetic acid. These factors are known to alter intracellular carbohydrate metabolism with the consequent induction or suppression of malic enzyme activity. (2) Is the induction of other carbohydrate-responsive mRNA sequences triggered by the same intracellular processes responsible for this stimulation of malic enzyme mRNA? Cultures will be maintained under varying carbohydrate and hormonal conditions which alter the content of ME mRNA. ME mRNA will be quantitated by immunoprecipitation of reticulocyte lysate translated product directed by RNA extracted from hepatocyte cultures. A wide range of other mRNA sequences will be quantitated from two-dimensional gel electrophoresis of the reticulocyte lysate translated products. (3) What is the mechanism of specific mRNA induction by glucose? A cDNA probe to an mRNA with response characteristics similar to ME mRNA will be utilized for hybridization studies to determine whether the glucose effect is mediated by changes in the rate of synthesis or degradation of this mRNA. The studies in this proposal should clarify the mechanisms by which carbohydrate regulates specific gene expression. Moreover, they should help to define the potential abnormalities which occur in states of carbohydrate dysmetabolism such as diabetes mellitus.
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