Characterization of the role of AMP-activated protein kinase in the regulation of glucose-activated gene expression using constitutively active and dominant negative forms of the kinase

In the liver, glucose induces the expression of a number of genes involved in glucose and lipid metabolism e.g., those encoding L-type pyruvate kinase and fatty acid synthase. Recent evidence has indicated a role for the AMP- activated protein kinase (AMPK) in the inhibition of glucose-activated gene expression in hepatocytes. It remains unclear, however, whether AMPK is in volved in the glucose induction of these genes. In order to study further t he role of AMPK in regulating gene expression, we have generated two mutant forms of AMPK. One of these (alpha 1(312)) acts as a constitutively active kinase, while the other (alpha 1DN) acts as a dominant negative inhibitor of endogenous AMPK. We have used adenovirus-mediated gene transfer to expre ss these mutants in primary rat hepatocytes in culture in order to determin e their effect on AMPK activity and the transcription of glucose activated genes. Expression of alpha 1(312) increased AMPK activity in hepatocytes an d blocked completely the induction of a number of glucose-activated genes i n response to 25 mM glucose. This effect is similar to that observed follow ing activation of AMPK by 5-amino-imidazolecarboxamide riboside. Expression of alpha 1DN markedly inhibited both basal and stimulated activity of endo genous AMPK but had no effect on the transcription of glucose-activated gen es. Our results suggest that AMPK is involved in the inhibition of glucose- activated gene expression but not in the induction pathway. This study demo nstrates that the two mutants we have described will provide valuable tools for studying the wider physiological role of AMPK.