Metabolic effects of mitochondrial uncoupling in murine skeletal muscle: Essential role of AMP-activated protein kinase in metabolic improvements of UCP1-transgenic mice?

Background and aim: Altering the efficiency of mitochondrial respiration may represent an attractive target for the treatment of obesity as well as related disorders. Transgenic (UCP1-TG) mice with ectopic expression of UCP1 in skeletal muscle (SM) show a phenotype of increased energy expenditure and improved glucose tolerance counteracting most of the detrimental effects of high fat diet. We could show an increased AMPK phosphorylation in SM of UCP1-TG mice, likely due to the energy depletion through uncoupling by UCP1. However, the full molecular mechanisms leading to the ameliorated metabolic phenotype of UCP1-TG mice are still not known. The aim of this study was to investigate the potential role of skeletal muscle AMPK activation in the healthy metabolic phenotype of UCP1-TG mice.

Material and Methods: We generated double transgenic (DTG) mice, by crossing of UCP1-TG mice with DN-AMPK mice overexpressing a dominant negative alpha2 subunit of AMPK in SM which results in decreased AMPK activity. For basal characterization we investigated body composition, energy metabolism, glucose homeostasis and physical activity under standard chow diet in young male and female wild-type (WT) and TG mice (DN-AMPK, UCP1-TG, DTG). At 12 weeks of age mice were killed for analysis of substrate metabolism in SM (AMPK activity, ex vivo glucose uptake).

Results: Skeletal muscle AMPK activity was assayed in vitro by incorporation of 32P into a synthetic SAMS peptide after immunoprecipitation of AMPKalpha; subunits. As expected, AMPKalpha2 activity was increased by 50±20% (p 0.01) in SM of UCP1-TG mice, whereas in DN-AMPK and DTG mice AMPKalpha2 activity was impaired by 90±9% (p 0.001). AMPKalpha1 activity was not affected in any genotype. Biometric analysis showed decreased body weight, lean, and fat mass for both UCP1-TG and DTG compared to WT and DN-AMPK mice. DTG mice showed slightly reduced body weight and significant decreased body length (p 0.001) compared to UCP1-TG mice, but no differences in body composition. Energy intake and weight-specific total energy expenditure were increased, both in UCP1-TG and DTG mice. Moreover, weight-specific resting energy expenditure was increased in DTG mice only (p 0.001). In addition, voluntary physical activity during night was decreased in DTG mice (p 0.05). Basal glucose uptake in EDL muscle ex vivo was significantly increased in UCP1-TG (p 0.01), but not in DTG mice compared to WT. However, maximum insulin stimulated glucose uptake was similar in all genotypes. No gender differences were observed.

Conclusion: Collectively, the first basal characterization shows that the reduction of AMPKalpha2 activity in SM slightly affects biometrics, energy metabolism and physical activity of young DTG compared to UCP1-TG mice. In vivo metabolic challenges (dietary intervention, exercise endurance) and further ex vivo/in vitro pathway analysis will follow to elucidate the role and significance of AMPK on energy and substrate metabolism in UCP1-TG mice in more detail.