Dietary copper (Cu) deficiency leads to a number of physiological changes, including accelerated rates of lipid synthesis. The response to Cu-deficiency is an enigma because none of the lipogenic pathway's enzymes are cuproproteins. The enhanced lipogenic rate reflects a higher level of activity of fatty acid synthase (FAS), and an induction of FAS gene transcription and a corresponding 3-fold rise in FAS mRNA. The goal of this application is to elucidate the molecular events which operate to modulate hepatic gene transcription in response to a dietary Cu-deficiency. The applicants hypothesize that the hepatocyte contains a transcription factor(s) which is regulated by changes in dietary Cu-status in a manner that alters the trans-factor's interaction with a specific response element, and thereby modifies the rate of transcription for genes encoding lipogenic proteins. Because trans-factor activity may be modulated by a number of potential mechanisms, the primary objective of the proposal is one that focuses on identifying the affected Cu-responsive sequence and its associated trans- factor for the FAS gene, and subsequently utilize the specific target to elucidate the cellular mechanisms which govern trans-factor function.
The specific aims are: (1) identify potential cellular determinants of FAS transcription that respond to dietary Cu-status by characterizing the temporal response of hepatic FAS gene expression to Cu- depletion/repletion; and correlating the pattern of response with hepatic changes in Cu, GSH, lipid hydro peroxides, Cu,Zn-SOD, and mRNA levels for oxidative stress genes; (2) functionally localize the Cu- dependent cis-acting element(s) in the 5'flanking region of the FAS gene; and (3) identify the Cu-responsive sequence, and characterize the impact of dietary Cu depletion/repletion on trans-factor interactions with the Cu-responsive sequence. The approach employs intact animals and isolated hepatocytes and will focuses on methods which identify in vivo responses, e.g. mRNA and nuclear run-on assays, transfection analyses, DNase I hypersensitivity assays, and in vivo foot printing. The studies are directed towards providing a mechanistic understanding of how dietary Cu deficiency enhances hepatic lipid synthesis and elevates blood lipids.
