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

Abstract

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 involved in the glucose induction of these genes. In order to study further the role of AMPK in regulating gene expression, we have generated two mutant forms of AMPK. One of these (␣1 312 ) acts as a constitutively active kinase, while the other (␣1DN) acts as a dominant negative inhibitor of endogenous AMPK. We have used adenovirus-mediated gene transfer to express these mutants in primary rat hepatocytes in culture in order to determine their effect on AMPK activity and the transcription of glucose-activated genes. Expression of ␣1 312 increased AMPK activity in hepatocytes and blocked completely the induction of a number of glucose-activated genes in response to 25 mM glucose. This effect is similar to that observed following activation of AMPK by 5-amino-imidazolecarboxamide riboside. Expression of ␣1DN markedly inhibited both basal and stimulated activity of endogenous AMPK but had no effect on the transcription of glucose-activated genes. Our results suggest that AMPK is involved in the inhibition of glucoseactivated gene expression but not in the induction pathway. This study demonstrates that the two mutants we have described will provide valuable tools for studying the wider physiological role of AMPK.

Role of AMP‐activated protein kinase in the metabolic syndrome and in heart disease

“…AMPK inhibits fatty acid synthesis both acutely by phosphorylating and inactivating the ACC1 isoform of acetyl-CoA carboxylase [47], and in the longer term by switching off expression of lipogenic genes including ACC1 and fatty acid synthase [48]. It stimulates fatty acid oxidation by phosphorylating and inactivating the ACC2 isoform of acetyl-CoA carboxylase, lowering malo- nyl-CoA and thus relieving inhibition by the latter of fatty acid uptake into mitochondria [49].…”

Role for AMP-activated protein kinase in glucose-stimulated insulin secretion and preproinsulin gene expression

“…Plasmid pcDNA3.hGH [28] was a gift from Professor R. D. Burgoyne, University of Liverpool, Liverpool, U.K. Plasmids encoding c-Myc-tagged forms of dominant-negative-acting AMPKα1 (AMPK DN) with an Asp-157-to-Ala mutation (D"&(A ; pcDNA3.AMPK DN) [26] and truncated, constitutively active AMPKα1$"# (residues 1-312 ; AMPK CA) with a Thr-172-to-Asp mutation (T"(#D ; pcDNA3.AMPK CA) [29] were kind gifts from Dr D. Carling, Imperial College School of Medicine, University of London, London, U.K. Plasmid pINS-Luc FF contained nucleotides k260 to j60 of the human insulin promoter fused upstream of a minimal herpes simplex thymidine kinase promoter and humanized firefly luciferase cDNA [30]. The expression plasmid for Renilla reniformis luciferase (pRL.CMV) was from Promega.…”

“…An intriguing finding made during these studies is that inhibition of AMPK activity with a dominant-negative form of the enzyme, expected to inhibit both AMPKα1 and α2 complexes [29], leads to the activation of insulin secretion by mechanisms that are at least partly distinct from those which mediate the effects of glucose (Figures 4, 5 and 7). Thus expression of AMPK DN had no apparent effect on the stimulation of ATP synthesis by glucose ( Figures 4B, 4D and 4F), nor on intracellular Ca# +…”

Naturally occurring R225W mutation of the gene encoding AMP-activated protein kinase (AMPK)γ3 results in increased oxidative capacity and glucose uptake in human primary myotubes

“…AMPK may also be regulated through the glycogenbinding domain on the β subunit by the content and structure of intracellular glycogen [9]. When stimulated, AMPK acts to restore cellular energy balance by stimulating ATP-producing pathways (glucose uptake, fatty acid oxidation and mitochondrial biogenesis) [10][11][12][13] and inhibiting ATP-consuming pathways (fatty acid synthesis, glycogen synthesis and protein synthesis) [14][15][16][17].…”