Acetyl-CoA carboxylase (ACC), a rate-limiting enzyme in fatty acid metabolism, is an important determinant of the rates of fatty acid synthesis, oxidation and elongation. ACC activity is tightly regulated via allosteric mechanisms, covalent phosphorylation, and changes in gene expression by a number of hormones and nutrients. Based on new information, it is clear that its overall roles and regulation are much more complex than previously realized. This complexity has arisen because of the discovery that there is more than one isozymic form of ACC expressed in several species, subject to unique and tissue-specific regulation and because of the recognition of the regulation of ACC by a unique protein kinase, designated the 5'-AMP-activated protein kinase (AMPK).
Three specific aims are proposed to contribute to further understanding of this important enzyme. First, we propose to clone full- length cDNAs for newly discovered ACC isozymes (a Mr 280,000 rat isoform of ACC (ACC 280) and for its human homolog of Mr 275,000). These cDNAs will be used to determine whether ACC isozymic heterogeneity is accountable for by more than one ACC gene,whether there are multiple forms of ACC 280 mRNA. and to map and analyze the 5'-UTR region of the genes of these alternative ACC isozymes. Second, in order to understand the roles and regulation of these new ACC isozymes,. we will study the impact of the expression of ACC 280 on fatty acid metabolism in cultured muscle and hepatoma cells, the influence of the physical association of different ACC isozymes on enzyme catalysis, the rapid regulation of ACC 280 activity in intact cells by hormones via enzyme phosphorylation and mechanisms underlying the variable expression of ACC 280 polypeptide at pre- and post-translational levels. Third, in order to further understand the regulation of ACC by AMPK, we propose to produce and characterize polyclonal antibodies to AMPK, to study the phosphorylation state of AMPK in intact cells coincident with regulation of its activity and to define a potential role for AMPK in the regulation of ACC 280 in muscle. We also propose to identify/characterize other protein kinases active on AMPK, as part of a regulatory kinase cascade system. Taken together, these studies will contribute new and significant information, not only regarding ACC, but of important changes in fatty acid metabolism seen in human health/disease, including diabetes mellitus, hyperlipoproteinemia, the process of cell/tissue differentiation, the biology of human cancer and muscle metabolism during exertion/ischemia.
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