| AMP-activated protein kinase (AMPK) activity has a strong influence on the regulation of whole body glucose metabolism, and may also play a role in the development of type-2 diabetes. In the liver, AMPK activation suppresses glucose production by inhibiting the transcriptional activity of genes involved in glucose synthesis, such as PEPCK. AMPK is a heterotrimer composed of a catalytic alpha subunit, an AMP binding gamma subunit and a structural beta subunit; with either two or three possible isoforms of each subunit. In the liver, AMPK complex containing the alpha2 catalytic subunit is essential in regulation of hepatic glucose production. While AMPK is primarily considered a cytoplasmic protein kinase, complexes containing alpha2 subunit are also present in the nucleus. Hepatocyte nuclear factor 4alpha (HNF4alpha), a downstream target of AMPKalpha2, is a liver-enriched transcription factor that plays a key role in the regulation of gluconeogenic PEPCK gene transcription. It binds to a well characterized response element in the PEPCK promoter, and acts in collaboration with co-activator PGC-1alpha to induce PEPCK gene expression. We hypothesized that AMPKalpha2 carries out an important function in the nucleus, as a component of the transcriptional machinery, to regulate PEPCK gene expression through direct modulation of specific transcription factors and co-activators, such as HNF4alpha and PGC-1alpha.;My thesis examines the mechanism of how nuclear AMPKalpha2 functions to repress PEPCK gene transcription, and specifically tests the hypothesis that it accomplishes this by directly altering HNF4alpha activity. I found that AMPKalpha2 is present in the nucleus and that the nuclear content increases after its activation in hepatocytes using Immunofluorescence and Western blot analysis. I also observed that, AMPKalpha2 is bound to the same region of the endogenous PEPCK gene promoter as HNF4alpha using the Chromatin Immunoprecipitation technique. Using an in vivo protein-protein interaction assay, I demonstrated that AMPKalpha2 physically interacts with HNF4alpha in the nucleus and mapped the interaction domains of the two proteins. Finally, my findings show that AMPKalpha2 negatively regulates HNF4alpha-induced PEPCK gene promoter activity by inhibiting recruitment of co-activator PGC-1alpha.;Taken together, my findings demonstrate that AMPKalpha functions in the nucleus of the Hepatocyte as a novel co-repressor of HNF4alpha and negatively regulates hepatic gluconeogenic PEPCK gene transcription. |
