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

Metabolomics and lipidomics: Expanding the molecular landscape of exercise biology

Dynamic changes in circulating and tissue metabolites and lipids occur in response to exercise-induced cellular and whole-body energy demands to maintain metabolic homeostasis. The metabolome and lipidome in a given biological system provides a molecular snapshot of these rapid and complex metabolic perturbations. The application of metabolomics and lipidomics to map the metabolic responses to an acute bout of aerobic/endurance or resistance exercise has dramatically expanded over the past decade thanks to major analytical advancements, with most exercise-related studies to date focused on analyzing human biofluids and tissues. Experimental and analytical con-siderations, as well as complementary studies using animal model systems, are warranted to help overcome challenges associated with large human interindividual variability and decipher the breadth of molecular mechanisms underlying the metabolic health-promoting effects of exercise. In this review, we provide a guide for exercise researchers regarding analytical techniques and experimental workflows commonly used in metabolomics and lipidomics. Furthermore, we discuss advancements in human and mammalian exercise research utilizing metabolomic and lipidomic approaches in the last decade, as well as highlight key technical considerations and remaining knowledge gaps to continue expanding the molecular landscape of exercise biology

Metabolomics reveals mouse plasma metabolite responses to acute exercise and effects of disrupting AMPK-glycogen interactions

Introduction: The AMP-activated protein kinase (AMPK) is a master regulator of energy homeostasis that becomes activated by exercise and binds glycogen, an important energy store required to meet exercise-induced energy demands. Disruption of AMPK-glycogen interactions in mice reduces exercise capacity and impairs whole-body metabolism. However, the mechanisms underlying these phenotypic effects at rest and following exercise are unknown. Furthermore, the plasma metabolite responses to an acute exercise challenge in mice remain largely uncharacterized. Methods : Plasma samples were collected from wild type (WT) and AMPK double knock-in (DKI) mice with disrupted AMPK-glycogen binding at rest and following 30-min submaximal treadmill running. An untargeted metabolomics approach was utilized to determine the breadth of plasma metabolite changes occurring in response to acute exercise and the effects of disrupting AMPK-glycogen binding. Results: Relative to WT mice, DKI mice had reduced maximal running speed (p \u3c 0.0001) concomitant with increased body mass (p \u3c 0.01) and adiposity (p \u3c 0.001). A total of 83 plasma metabolites were identified/annotated, with 17 metabolites significantly different (p \u3c 0.05; FDR \u3c 0.1) in exercised (↑ 6; ↓ 11) versus rested mice, including amino acids, acylcarnitines and steroid hormones. Pantothenic acid was reduced in DKI mice versus WT. 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 plasma showed greater decreases in amino acids following exercise versus WT. Conclusion : This is the first study to map mouse plasma metabolomic changes following a bout of acute exercise in WT mice and the effects of disrupting AMPK-glycogen interactions in DKI mice. Untargeted metabolomics revealed alterations in 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. Reduced pantothenic acid levels may contribute to differences in fuel utilization in DKI mice

Metabolomics and Lipidomics: Expanding the Molecular Landscape of Exercise Biology

Dynamic changes in circulating and tissue metabolites and lipids occur in response to exercise-induced cellular and whole-body energy demands to maintain metabolic homeostasis. The metabolome and lipidome in a given biological system provides a molecular snapshot of these rapid and complex metabolic perturbations. The application of metabolomics and lipidomics to map the metabolic responses to an acute bout of aerobic/endurance or resistance exercise has dramatically expanded over the past decade thanks to major analytical advancements, with most exercise-related studies to date focused on analyzing human biofluids and tissues. Experimental and analytical considerations, as well as complementary studies using animal model systems, are warranted to help overcome challenges associated with large human interindividual variability and decipher the breadth of molecular mechanisms underlying the metabolic health-promoting effects of exercise. In this review, we provide a guide for exercise researchers regarding analytical techniques and experimental workflows commonly used in metabolomics and lipidomics. Furthermore, we discuss advancements in human and mammalian exercise research utilizing metabolomic and lipidomic approaches in the last decade, as well as highlight key technical considerations and remaining knowledge gaps to continue expanding the molecular landscape of exercise biology

Higher strength gain after hypoxic vs normoxic resistance training despite no changes in muscle thickness and fractional protein synthetic rate.

Acute hypoxia has previously been suggested to potentiate resistance training-induced hypertrophy by activating satellite cell-dependent myogenesis rather than an improvement in protein balance in human. Here, we tested this hypothesis after a 4-week hypoxic vs normoxic resistance training protocol. For that purpose, 19 physically active male subjects were recruited to perform 6 sets of 10 repetitions of a one-leg knee extension exercise at 80% 1-RM 3 times/week for 4 weeks in normoxia (FiO : 0.21; n = 9) or in hypoxia (FiO : 0.135, n = 10). Blood and skeletal muscle samples were taken before and after the training period. Muscle fractional protein synthetic rate was measured over the whole period by deuterium incorporation into the protein pool and muscle thickness by ultrasound. At the end of the training protocol, the strength gain was higher in the hypoxic vs the normoxic group despite no changes in muscle thickness and in the fractional protein synthetic rate. Only early myogenesis, as assessed by higher MyoD and Myf5 mRNA levels, appeared to be enhanced by hypoxia compared to normoxia. No effects were found on myosin heavy chain expression, markers of oxidative metabolism and lactate transport in the skeletal muscle. Though the present study failed to unravel clearly the mechanisms by which hypoxic resistance training is particularly potent to increase muscle strength, it is important message to keep in mind that this training strategy could be effective for all athletes looking at developing and optimizing their maximal muscle strength