The cytoplasmic enzyme glycerophosphate dehydrogenase (GPD) plays a pivotal role in lipid synthesis and fuel metabolism in a number of tissues. Our overall objective is to delineate the molecular processes which activate and modulate the expression of the specific gene encoding GPD in skeletal muscle. Towards this goal we have already demonstrated the responsiveness of chimeric GPD genes in myogenic cell lines, which represents the first successful regulation of appropriate GPD expression in vitro. We have now identified by deletional analysis a 1 kb intragenic region downstream from the GPD promoter which confers differentiation-dependent expression on an attached reporter gene in mouse C2-skeletal muscle cells. This DNA segment will be further dissected to localize sequences that specifically activate GPD late in the myogenic program. Our recent studies suggest that GPD regulation involves a novel myoblast silencer plus a muscle-specific enhancer, distinct from characterized muscle enhancers. To elucidate the mechanism of GPD activation, we will use in vitro binding and footprinting assays and site-directed mutagenesis of GPD regulatory sequences to define nuclear factor binding sites, their differentiation-dependent usage, and the profile of nuclear proteins that interact with them. Similarly, the DNA sequences and trans-acting factors which mediate inhibition by two catabolic agents, cyclic AMP-linked lipolytic hormones and tumor necrosis factor (the active agent of cachexia), will be characterized. Novel DNA- binding regulatory factors that control GPD expression will be isolated from cDNA expression libraries. We describe a new strategy, using chimeric c-fos/GPD constructs, to delineate the molecular basis for insulin stimulation of GPD transcription and GPD mRNA stability. Finally, we will determine the pattern of GPD expression in different muscle fibers and how disease-related states that affect insulin responsiveness in skeletal muscle (denervation, high fat feeding, genetic obesity) affect GPD expression in vivo. These experiments should yield novel insights into the mechanisms by which metabolic gene expression is regulated in skeletal muscle during development and in response to hormonal controls.
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