Metabolomic responses to acute exercise and AMPK-glycogen binding disruption in mice

Background: Exercise is widely accepted as a potent intervention to promote whole-body metabolic health and help prevent and/or treat metabolic diseases. Exercise represents a major challenge to energy homeostasis, both at the whole-body and cellular level. Numerous molecular metabolic responses to acute exercise are activated to preserve energy homeostasis. Central to maintaining cellular energy balance is the AMP-activated protein kinase (AMPK), a heterotrimeric enzyme that senses cellular energy levels by competitively binding to adenosine mono-, di- and triphosphate (AMP, ADP and ATP, respectively). In response to energy stress, AMPK becomes activated and switches on energy-producing catabolic processes while simultaneously switching off energy-consuming anabolic processes. Through its regulatory β subunit, AMPK also binds glycogen – an important energy reserve primarily stored in liver and skeletal muscle. Although growing evidence from AMPK double knock-in (DKI) mice has highlighted physiological consequences of disrupting AMPK-glycogen binding in exercise and metabolic control, the underlying molecular pathways and mechanisms remain unclear. Metabolomics is the unbiased collection and study of small molecules (< 1500 daltons) involved in metabolic reactions to capture molecular snapshots of metabolic pathways, for example associated with given stimuli (e.g., exercise) or genotype. Therefore, metabolomic analysis of biofluids and tissues represents a promising approach to better understand the molecular metabolic responses to acute exercise and the physiological effects of disrupting AMPK-glycogen binding in vivo.

Methods: Plasma, gastrocnemius muscle and liver samples were collected from age-matched male WT and DKI mice with disrupted AMPK-glycogen binding at rest and immediately following 30-min submaximal treadmill running. An untargeted mass spectrometry-based metabolomic approach was utilised to determine changes in plasma and/or tissue metabolites occurring in response to acute exercise and the disruption of AMPK-glycogen interactions in DKI mice. Complementary whole-body mouse phenotyping and real-time metabolic phenotyping assays using the Seahorse XFe24 Analyzer and Oroboros O2k high-resolution respirometer were performed to compare energy metabolism and substrate utilisation profiles in mouse embryonic fibroblast (MEF) cells and skeletal muscle from WT and DKI mice.

Results/Discussion: Relative to WT mice, DKI mice had reduced maximal running speed, concomitant with increased total body mass and adiposity. In plasma, a total of 83 metabolites were identified/annotated, with 17 metabolites significantly different in exercised versus rested mice. These included amino acids, acylcarnitines and steroid hormones. Distinct plasma metabolite profiles were observed between the rest and exercise conditions and between WT and DKI mice at rest, while metabolite profiles of both genotypes converged following exercise. These differences in metabolite profiles were primarily explained by exercise-associated increases in acylcarnitines and steroid hormones as well as decreases in amino acids and derivatives following exercise. DKI mice showed greater decreases in plasma amino acid levels following exercise versus WT. In liver and skeletal muscle, 150 and 92 metabolites were identified/annotated, respectively. Similar to the plasma metabolite responses observed across genotypes and conditions, significant overall metabolite profile shifts were observed between WT and DKI mice at rest, as well as significant metabolite profile differences between the rested and exercised conditions. Differential muscle metabolite responses to acute exercise were also observed between genotypes. Markers of mitochondrial respiration in permeabilised gastrocnemius fibres were not affected by AMPK DKI mutation, although there were reduced total ATP rate and relative contribution of glycolysis in DKI versus WT MEF cells.

Conclusion: The plasma metabolomic analyses performed in Study 1 represent the first study to map mouse plasma metabolomic changes following acute exercise in WT mice and the effects of disrupting AMPK-glycogen interactions using DKI mice. Untargeted metabolomics uncovered alterations in plasma, skeletal muscle and liver metabolite profiles between rested and exercised mice in both genotypes, and between genotypes at rest. This study has uncovered known and previously unreported plasma metabolite responses to acute exercise in WT mice, as well as greater decreases in amino acids following exercise in DKI plasma. These mouse tissue metabolomic datasets, combined with cell and tissue respirometry data complement previous whole-body, tissue and molecular characterisation of WT and DKI mice, revealing potential metabolic pathways and novel molecular biomarkers underlying exercise’s metabolic health benefits and the physiological effects of disrupting AMPK-glycogen binding in mice.