Whole Body Deletion of AMP-activated Protein Kinase β2 Reduces Muscle AMPK Activity and Exercise Capacity

Tim Naim,Nicolas L. Dzamko,René Koopman,Jonathan D. Schertzer,Bryce J.W. van Denderen,Matthew J. Watt,Gordon S. Lynch,Hayley M. O'Neill,Bruce E. Kemp,Gregory R. Steinberg,Jane Honeyman,Duncan J. Campbell,Sebastian B. Jørgensen,John W. Scott,Sandra Galic,Zhi-Ping Chen,Kimberly Hewitt,Frank Koentgen

doi:10.1074/jbc.m110.102434

Abstract

AMP-activated protein kinase (AMPK) β subunits (β1 and β2) provide scaffolds for binding α and γ subunits and contain a carbohydrate-binding module important for regulating enzyme activity. We generated C57Bl/6 mice with germline deletion of AMPK β2 (β2 KO) and examined AMPK expression and activity, exercise capacity, metabolic control during muscle contractions, aminoimidazole carboxamide ribonucleotide (AICAR) sensitivity, and susceptibility to obesity-induced insulin resistance. We find that β2 KO mice are viable and breed normally. β2 KO mice had a reduction in skeletal muscle AMPK α1 and α2 expression despite up-regulation of the β1 isoform. Heart AMPK α2 expression was also reduced but this did not affect resting AMPK α1 or α2 activities. AMPK α1 and α2 activities were not changed in liver, fat, or hypothalamus. AICAR-stimulated glucose uptake but not fatty acid oxidation was impaired in β2 KO mice. During treadmill running β2 KO mice had reduced maximal and endurance exercise capacity, which was associated with lower muscle and heart AMPK activity and reduced levels of muscle and liver glycogen. Reductions in exercise capacity of β2 KO mice were not due to lower muscle mitochondrial content or defects in contraction-stimulated glucose uptake or fatty acid oxidation. When challenged with a high-fat diet β2 KO mice gained more weight and were more susceptible to the development of hyperinsulinemia and glucose intolerance. In summary these data show that deletion of AMPK β2 reduces AMPK activity in skeletal muscle resulting in impaired exercise capacity and the worsening of diet-induced obesity and glucose intolerance.

Highlights

The AMP-activated protein kinase (AMPK)5 is an evolutionary conserved serine/threonine protein kinase that functions as a metabolic regulatory enzyme at both the intracellular and whole body level [1, 2]
Glycogen—Because oxidative phosphorylation can be limiting for exercise and AMPK ␣2 null mice have reduced mitochondrial content [36] and AMPK ␣2-kinase dead (KD) mice have reduced mitochondrial complex activity [46] we examined protein and mRNA expression of key mitochondrial enzymes
We found that there was no difference in total Akt expression irrespective of genotype or diet and the high-fat diet (HFD) suppressed insulin-stimulated Akt phosphorylation there was no difference between wild type (WT) and ␤2 KO mice (Fig. 7, D and E)

Summary

EXPERIMENTAL PROCEDURES

Generation of AMPK ␤2 KO Mice—␤2 KO mice were generated on a pure C57Bl/6 background by Ozgene Pty. Glucose Uptake Assays—Extensor digitorum longus (EDL) muscles were dissected from anesthetized mice (6 mg of pentobarbital 100 gϪ1 body weight) and transferred to incubation flasks containing 2 ml of essential buffer (Krebs-Henseleit buffer, pH 7.4, with 2.0 mM pyruvate, 8 mM mannitol, and 0.1% BSA), gassed with 95% O2 ϩ 5% CO2 and maintained at 30 °C as described [29, 30]. Muscle lysates were generated as described below, and radioactivity was measured by liquid scintillation counting (Tri-Carb 2000, Packard Instrument Co.). Rates of fatty acid oxidation were determined by collecting 14CO2 produced during the intervention in benzethonium hydroxide and measuring acid soluble metabolites as described [32, 33] Radioactivity in these samples was determined by liquid scintillation counting (Tri-Carb 2000, Packard Instrument Co).

RESULTS

Glucose Uptake but Not Palmitate

We found that AICAR increased

Findings

DISCUSSION