| Abstrakt: |
The glucose-6-phosphatase catalytic subunit (G6Pase) catalyzes the final step in gluconeogenesis and glycogenolysis and, as such, plays a key role in hepatic glucose production. G6Pase gene expression is markedly stimulated by the transcriptional co-activator PGC-1 alpha. We have previously shown that PGC-1 stimulates mouse G6Pase fusion gene expression, at least in part, through hepatocyte nuclear factor-4 alpha (HNF-4 alpha) which binds an element located between -76 and -64 in the mouse promoter. Surprisingly we show here that PGC-1 alpha fails to stimulate human G6Pase fusion gene expression even though the sequence of this HNF-4 alpha binding site is perfectly conserved in the human promoter. Experiments were therefore designed to elucidate the molecular basis for this differential regulation by PGC-1 alpha. As a first step, a minimal promoter region was defined that was sufficient to mediate the action of PGC-1 alpha on mouse G6Pase fusion gene expression. This region, which was narrowed to between -85 and +1, only differs at 7 bp between the mouse and human promoters. A mutational analysis was then performed to identify the critical sequence variants responsible for the differential regulation by PGC-1 alpha. This led to the identification of 3 bp, located between -48 and - 45, which, when switched to the human sequence in the context of the mouse promoter, resulted in the complete loss of PGC-1 alpha stimulated fusion gene expression. Importantly the inverse experiment, in which these 3 bp were switched to the mouse sequence in the context of the human promoter, resulted in the generation of a G6Pase fusion gene that was now induced by PGC-1. This critical 3 bp region is located immediately adjacent to a consensus nuclear hormone receptor half site (TGACCT) that is perfectly conserved between the mouse and human G6Pase promoters. Mutation of this element was found to also abolish the PGC-1 alpha response leading to the hypothesis that PGC-1 alpha acts through a bridging mechanism that requires simultaneous contacts with both HNF-4 alpha and the factor bound to the nuclear hormone receptor half site. Further experiments identified the latter as the peroxisome proliferator activated receptor (PPAR) and revealed that the critical 3 bp region influences the affinity of PPAR binding to the nuclear hormone receptor half site. These observations suggest that PGC-1 alpha may be more important in the control of mouse than human hepatic glucose production. [ABSTRACT FROM AUTHOR] |
