The effects of disrupting AMPK-glycogen binding in mice on glucose homeostasis, glycogen dynamics and exercise metabolism

Background: The AMP-activated protein kinase (AMPK) is a central regulator of cellular energy balance and metabolism. Many of the health benefits of exercise (e.g., improved insulin sensitivity, increased glucose disposal and fat oxidation) are linked to AMPK. AMPK possesses a regulatory β subunit that binds glycogen, the primary storage form of glucose in liver and skeletal muscle. Based on in vitro and in vivo findings indicating an inverse relationship between glycogen availability and AMPK activity, AMPK’s glycogen-binding capacity has been proposed to serve roles in cellular energy sensing. However, in vivo models to test the physiological effects of disrupting AMPK-glycogen binding are limited. Previously, murine models with isoform-specific knock-in mutations to disrupt AMPK-glycogen binding in the β1 or β2 subunit isoform demonstrated increased adiposity, glucose intolerance, decreased maximal exercise capacity, and reductions in AMPK protein content and activity in liver and skeletal muscle. However, the consequences of genetic mutations resulting in whole-body disruption of AMPK-glycogen binding and potential compensatory and/or synergistic roles of the β subunits isoforms’ glycogen binding capacity remains unknown. Therefore, AMPK β double knock-in (DKI) mice were generated with mutations in tryptophan residues critical for AMPK-glycogen binding in both the β1 (W100A) and β2 (W98A) subunit isoforms, providing the first in vivo model to directly assess the consequences of genetic disruption of whole-body AMPK-glycogen binding in vivo. The two experimental chapters described in this thesis aimed to elucidate the phenotypic effects of disrupting AMPK-glycogen interactions via AMPK DKI mutation on 1) whole-body substrate utilisation, glucose homeostasis, and tissue glycogen dynamics; and 2) exercise capacity and whole-body substrate utilisation and tissue metabolism during exercise. Methods: For Study 1, body composition, metabolic caging, glucose and insulin tolerance, serum hormone and lipid profiles, and fed and fasted tissue glycogen and signalling responses were analysed in chow-fed male AMPK DKI and age-matched wild type (WT) mice. For Study 2, maximal treadmill running speed and endurance capacity were determined. Substrate utilisation during submaximal exercise was calculated using indirect calorimetry. Liver and skeletal muscle glycogen content and skeletal muscle AMPK content and signalling were assessed in WT and DKI mice at rest and following maximally exercise. Results: AMPK DKI mice displayed increased whole-body fat mass, hyperinsulinemia, and whole-body glucose intolerance associated with inactivity and reduced rates of fat oxidation in the fed and fasted states relative to WT mice. DKI mice had reduced liver glycogen content in the fed state and demonstrated increased utilisation and little repletion of skeletal muscle glycogen in response to fasting and refeeding, respectively, despite similar glycogen synthase phosphorylation and content of glycogen associated proteins relative to WT. Liver, skeletal muscle, and adipose tissue from DKI mice displayed reductions in AMPK protein content but no associated impairments in AMPK or acetyl-CoA carboxylase (ACC) phosphorylation in liver or skeletal muscle in response to fasting and refeeding versus WT. DKI mice had reduced maximal running speed, but similar endurance capacity, compared to WT. During submaximal running, DKI mice displayed increased respiratory exchange ratio and increased rates of carbohydrate oxidation. During maximal treadmill running, DKI mice utilised similar absolute levels of liver and skeletal muscle glycogen compared to WT, despite reduced running time. DKI mice displayed no impairments in AMPK or ACC phosphorylation in skeletal muscle following maximal exercise. Conclusions: Collectively, these findings determine the phenotypic effects of the AMPK DKI mutation utilised to disrupt whole-body glycogen binding capacity in vivo in the regulation of whole-body metabolism, liver and skeletal muscle AMPK content, and tissue glycogen utilisation and synthesis. Specifically, the DKI mutation results in reduced maximal running capacity, and high rates of skeletal muscle glycogen utilisation during maximal running that were associated with reduced skeletal muscle AMPK content. This thesis addresses existing knowledge gaps regarding the physiological consequences of disruption of whole-body AMPK-glycogen binding, suggesting that AMPK-glycogen binding may serve physiological roles in cellular, glycogen-storing tissues, and whole-body energy homeostasis. Furthermore, these studies highlight potential unappreciated roles for AMPK in regulating tissue glycogen dynamics and further expand AMPK’s known roles in exercise and metabolism. Collectively, the findings in this thesis have broad implications for whole-body, tissue and cellular energy metabolism and homeostasis