Chemical genetic screen identifies Gapex-5/GAPVD1 and STBD1 as novel AMPK substrates

AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis, acting as a sensor of energy and nutrient status. As such, AMPK is considered a promising drug target for treatment of medical conditions particularly associated with metabolic dysfunctions. To better understand the downstream effectors and physiological consequences of AMPK activation, we have employed a chemical genetic screen in mouse primary hepatocytes in an attempt to identify novel AMPK targets. Treatment of hepatocytes with a potent and specific AMPK activator 991 resulted in identification of 65 proteins phosphorylated upon AMPK activation, which are involved in a variety of cellular processes such as lipid/glycogen metabolism, vesicle trafficking, and cytoskeleton organisation. Further characterisation and validation using mass spectrometry followed by immunoblotting analysis with phosphorylation site-specific antibodies identified AMPK-dependent phosphorylation of Gapex-5 (also known as GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1)) on Ser902 in hepatocytes and starch-binding domain 1 (STBD1) on Ser175 in multiple cells/tissues. As new promising roles of AMPK as a key metabolic regulator continue to emerge, the substrates we identified could provide new mechanistic and therapeutic insights into AMPK-activating drugs in the liver

Contraction-stimulated glucose transport in muscle is controlled by AMPK and mechanical stress but not sarcoplasmatic reticulum Ca²⁺ release

Understanding how muscle contraction orchestrates insulin-independent muscle glucose transport may enable development of hyperglycemia-treating drugs. The prevailing concept implicates Ca2+ as a key feed forward regulator of glucose transport with secondary fine-tuning by metabolic feedback signals through proteins such as AMPK. Here, we demonstrate in incubated mouse muscle that Ca2+ release is neither sufficient nor strictly necessary to increase glucose transport. Rather, the glucose transport response is associated with metabolic feedback signals through AMPK, and mechanical stress-activated signals. Furthermore, artificial stimulation of AMPK combined with passive stretch of muscle is additive and sufficient to elicit the full contraction glucose transport response. These results suggest that ATP-turnover and mechanical stress feedback are sufficient to fully increase glucose transport during muscle contraction, and call for a major reconsideration of the established Ca2+ centric paradigm

Periodized low protein-high carbohydrate diet confers potent, but transient, metabolic improvements

Objective: Chronic ad libitum low protein-high carbohydrate diet (LPHC) increases health- and life-span in mice. A periodized (p) LPHC regimen would be a more practical long-term human lifestyle intervention, but the metabolic benefits of pLPHC are not known. Also, the interactions between LPHC diet and exercise training have not been investigated. Presently, we aimed to provide proof-of-concept data in mice of the efficacy of pLPHC and to explore the potential interactions with concurrent exercise training. Methods: A detailed phenotypic and molecular characterization of mice undergoing different durations of 14 d LPHC (5 E% protein)/14 d control diet cycles for up to 4 months with or without concurrent access to activity wheels allowing voluntary exercise training. Results: pLPHC conferred metabolic benefits similar to chronic LPHC, including increased FGF21 and adaptive thermogenesis, obesity-protection despite increased total energy intake and improved insulin sensitivity. The improved insulin sensitivity showed large fluctuations between diet periods and was lost within 14 days of switching back to control diet. Parallel exercise training improved weight maintenance but impaired the FGF21 response to pLPHC whereas repeated pLPHC cycles progressively augmented this response. Both the FGF21 suppression by exercise and potentiation by repeated cycles correlated tightly with Nupr1 mRNA in liver, suggesting dependence on liver integrated stress response. Conclusion: These results suggest that pLPHC may be a viable strategy to promote human health but also highlight the transient nature of the benefits and that the interaction with other lifestyle-interventions such as exercise training warrants consideration. Keywords: Dietary restriction, FGF21, Periodized diet, Glucose metabolism, Obesity, Exercise, Integrated stress respons

Regulation of autophagy in human skeletal muscle: effects of exercise, exercise training and insulin stimulation

KEY POINTS: Regulation of autophagy in human muscle in many aspects differs from the majority of previous reports based on studies in cell systems and rodent muscle. An acute bout of exercise and insulin stimulation reduce human muscle autophagosome content. An acute bout of exercise regulates autophagy by a local contraction‐induced mechanism. Exercise training increases the capacity for formation of autophagosomes in human muscle. AMPK activation during exercise seems insufficient to regulate autophagosome content in muscle, while mTORC1 signalling via ULK1 probably mediates the autophagy‐inhibiting effect of insulin. ABSTRACT: Studies in rodent muscle suggest that autophagy is regulated by acute exercise, exercise training and insulin stimulation. However, little is known about the regulation of autophagy in human skeletal muscle. Here we investigate the autophagic response to acute one‐legged exercise, one‐legged exercise training and subsequent insulin stimulation in exercised and non‐exercised human muscle. Acute one‐legged exercise decreased (P<0.01) lipidation of microtubule‐associated protein 1A/1B‐light chain 3 (LC3) (∼50%) and the LC3‐II/LC3‐I ratio (∼60%) indicating that content of autophagosomes decreases with exercise in human muscle. The decrease in LC3‐II/LC3‐I ratio did not correlate with activation of 5′AMP activated protein kinase (AMPK) trimer complexes in human muscle. Consistently, pharmacological AMPK activation with 5‐aminoimidazole‐4‐carboxamide riboside (AICAR) in mouse muscle did not affect the LC3‐II/LC3‐I ratio. Four hours after exercise, insulin further reduced (P<0.01) the LC3‐II/LC3‐I ratio (∼80%) in muscle of the exercised and non‐exercised leg in humans. This coincided with increased Ser‐757 phosphorylation of Unc51 like kinase 1 (ULK1), which is suggested as a mammalian target of rapamycin complex 1 (mTORC1) target. Accordingly, inhibition of mTOR signalling in mouse muscle prevented the ability of insulin to reduce the LC3‐II/LC3‐I ratio. In response to 3 weeks of one‐legged exercise training, the LC3‐II/LC3‐I ratio decreased (P<0.05) in both trained and untrained muscle and this change was largely driven by an increase in LC3‐I content. Taken together, acute exercise and insulin stimulation reduce muscle autophagosome content, while exercise training may increase the capacity for formation of autophagosomes in muscle. Moreover, AMPK activation during exercise may not be sufficient to regulate autophagy in muscle, while mTORC1 signalling via ULK1 probably mediates the autophagy‐inhibiting effect of insulin