Christian Pehmøller

AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity

AMP‐activated protein kinase (AMPK) is a sensor of cellular energy status that plays a central role in skeletal muscle metabolism. We used skeletal muscle‐specific AMPKα1α2 double‐knockout (mdKO) mice to provide direct genetic evidence of the physiological importance of AMPK in regulating muscle exercise capacity, mitochondrial function, and contraction‐stimulated glucose uptake. Exercise performance was significantly reduced in the mdKO mice, with a reduction in maximal force production and fatigue resistance. An increase in the proportion of myofibers with centralized nuclei was noted, as well as an elevated expression of interleukin 6 (IL‐6) mRNA, possibly consistent with mild skeletal muscle injury. Notably, we found that AMPKα1 and AMPKα2 isoforms are dispensable for contraction‐induced skeletal muscle glucose transport, except for male soleus muscle. However, the lack of skeletal muscle AMPK diminished maximal ADP‐stimulated mitochondrial respiration, showing an impairment at complex I. This effect was not accompanied by changes in mitochondrial number, indicating that AMPK regulates muscle metabolic adaptation through the regulation of muscle mitochondrial oxidative capacity and mitochondrial substrate utilization but not baseline mitochondrial muscle content. Together, these results demonstrate that skeletal muscle AMPK has an unexpected role in the regulation of mitochondrial oxidative phosphorylation that contributes to the energy demands of the exercising muscle.—Lantier, L., Fentz, J., Mounier, R., Leclerc, J., Treebak, J. T., Pehmøller, C., Sanz, N., Sakakibara, I., Saint‐Amand, E., Rimbaud, S., Maire, P., Marette, A., Ventura‐Clapier, R., Ferry, A., Wojtaszewski, J. F. P., Foretz, M., Viollet, B. AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity. FASEB J. 28, 3211–3224 (2014). www.fasebj.org

Akt and Rac1 signaling are jointly required for insulin-stimulated glucose uptake in skeletal muscle and downregulated in insulin resistance

Skeletal muscle plays a major role in regulating whole body glucose metabolism. Akt and Rac1 are important regulators of insulin-stimulated glucose uptake in skeletal muscle. However the relative role of each pathway and how they interact are not understood. Here we delineate how Akt and Rac1 pathways signal to increase glucose transport independently of each other and are simultaneously downregulated in insulin resistant muscle. Pharmacological inhibition of Rac1 and Akt signaling was used to determine the contribution of each pathway to insulin-stimulated glucose uptake in mouse muscles. The actin filament-depolymerizing agent LatrunculinB was combined with pharmacological inhibition of Rac1 or Akt, to examine whether either pathway mediates its effect via the actin cytoskeleton. Akt and Rac1 signaling were investigated under each condition, as well as upon Akt2 knockout and in ob/ob mice, to uncover whether Akt and Rac1 signaling are independent and whether they are affected by genetically-induced insulin resistance. While individual inhibition of Rac1 or Akt partially decreased insulin-stimulated glucose transport by ~40% and ~60%, respectively, their simultaneous inhibition completely blocked insulin-stimulated glucose transport. LatrunculinB plus Akt inhibition blocked insulin-stimulated glucose uptake, while LatrunculinB had no additive effect on Rac1 inhibition. In muscles from severely insulin-resistant ob/ob mice, Rac1 and Akt signaling were severely dysregulated and the increment in response to insulin reduced by 100% and 90%, respectively. These findings suggest that Rac1 and Akt regulate insulin-stimulated glucose uptake via distinct parallel pathways, and that insulin-induced Rac1 and Akt signaling are both dysfunctional in insulin resistant muscle. There may thus be multiple treatment targets for improving insulin sensitivity in muscle.

Exercise‐induced TBC1D1 Ser237 phosphorylation and 14‐3‐3 protein binding capacity in human skeletal muscle

TBC1D1 is a Rab‐GTPase activating protein involved in regulation of GLUT4 translocation in skeletal muscle. We here evaluated exercise‐induced regulation of TBC1D1 Ser237 phosphorylation and 14‐3‐3 protein binding capacity in human skeletal muscle. In separate experiments healthy men performed all‐out cycle exercise lasting either 30 s, 2 min or 20 min. After all exercise protocols, TBC1D1 Ser237 phosphorylation increased (∼70–230%, P < 0.005), with the greatest response observed after 20 min of cycling. Interestingly, capacity of TBC1D1 to bind 14‐3‐3 protein showed a similar pattern of regulation, increasing 60–250% (P < 0.001). Furthermore, recombinant 5′AMP‐activated protein kinase (AMPK) induced both Ser237 phosphorylation and 14‐3‐3 binding properties on human TBC1D1 when evaluated in vitro. To further characterize the role of AMPK as an upstream kinase regulating TBC1D1, extensor digitorum longus muscle (EDL) from whole body α1 or α2 AMPK knock‐out and wild‐type mice were stimulated to contract in vitro. In wild‐type and α1 knock‐out mice, contractions resulted in a similar ∼100% increase (P < 0.001) in Ser237 phosphorylation. Interestingly, muscle of α2 knock‐out mice were characterized by reduced protein content of TBC1D1 (∼50%, P < 0.001) as well as in basal and contraction‐stimulated (∼60%, P < 0.001) Ser237 phosphorylation, even after correction for the reduced TBC1D1 protein content. This study shows that TBC1D1 is Ser237 phosphorylated and 14‐3‐3 protein binding capacity is increased in response to exercise in human skeletal muscle. Furthermore, we show that the catalytic α2 AMPK subunit is the main (but probably not the only) donor of AMPK activity regulating TBC1D1 Ser237 phosphorylation in mouse EDL muscle.

Acute exercise and physiological insulin induce distinct phosphorylation signatures on TBC1D1 and TBC1D4 proteins in human skeletal muscle.

Phosphorylation signature patterns on TBC1D1 and TBC1D4 proteins in the insulin–glucose pathway were investigated in human skeletal muscle in response to physiological insulin and exercise. In response to postprandial increase in insulin, Akt phosphorylation of T308 and S473 correlated significantly with sites on TBC1D1 (T596) and TBC1D4 (S318, S341, S704). Exercise induced phosphorylation of TBC1D1 (S237, T596) that correlated significantly with activity of the α2/β2/γ3 AMPK trimer, whereas TBC1D4 phosphorylation (S341, S704) with exercise correlated with activity of the α2/β2/γ1 AMPK trimer. TBC1D1 phosphorylation signatures with exercise/muscle contraction were comparable between human and mouse skeletal muscle, and AMPK regulated phosphorylation of these sites in mouse muscle, whereas contraction and exercise elicited different TBC1D4 phosphorylation patterns in mouse compared with human muscle. Our results show differential phosphorylation of TBC1D1 and TBC1D4 in response to physiological stimuli in human skeletal muscle and indicate that Akt and AMPK may be upstream kinases.

LKB1 Regulates Lipid Oxidation During Exercise Independently of AMPK

Lipid metabolism is important for health and insulin action, yet the fundamental process of regulating lipid metabolism during muscle contraction is incompletely understood. Here, we show that liver kinase B1 (LKB1) muscle-specific knockout (LKB1 MKO) mice display decreased fatty acid (FA) oxidation during treadmill exercise. LKB1 MKO mice also show decreased muscle SIK3 activity, increased histone deacetylase 4 expression, decreased NAD+ concentration and SIRT1 activity, and decreased expression of genes involved in FA oxidation. In AMP-activated protein kinase (AMPK)α2 KO mice, substrate use was similar to that in WT mice, which excluded that decreased FA oxidation in LKB1 MKO mice was due to decreased AMPKα2 activity. Additionally, LKB1 MKO muscle demonstrated decreased FA oxidation in vitro. A markedly decreased phosphorylation of TBC1D1, a proposed regulator of FA transport, and a low CoA content could contribute to the low FA oxidation in LKB1 MKO. LKB1 deficiency did not reduce muscle glucose uptake or oxidation during exercise in vivo, excluding a general impairment of substrate use during exercise in LKB1 MKO mice. Our findings demonstrate that LKB1 is a novel molecular regulator of major importance for FA oxidation but not glucose uptake in muscle during exercise.

Exercise Alleviates Lipid-Induced Insulin Resistance in Human Skeletal Muscle–Signaling Interaction at the Level of TBC1 Domain Family Member 4

Excess lipid availability causes insulin resistance. We examined the effect of acute exercise on lipid-induced insulin resistance and TBC1 domain family member 1/4 (TBCD1/4)-related signaling in skeletal muscle. In eight healthy young male subjects, 1 h of one-legged knee-extensor exercise was followed by 7 h of saline or intralipid infusion. During the last 2 h, a hyperinsulinemic-euglycemic clamp was performed. Femoral catheterization and analysis of biopsy specimens enabled measurements of leg substrate balance and muscle signaling. Each subject underwent two experimental trials, differing only by saline or intralipid infusion. Glucose infusion rate and leg glucose uptake was decreased by intralipid. Insulin-stimulated glucose uptake was higher in the prior exercised leg in the saline and the lipid trials. In the lipid trial, prior exercise normalized insulin-stimulated glucose uptake to the level observed in the resting control leg in the saline trial. Insulin increased phosphorylation of TBC1D1/4. Whereas prior exercise enhanced TBC1D4 phosphorylation on all investigated sites compared with the rested leg, intralipid impaired TBC1D4 S341 phosphorylation compared with the control trial. Intralipid enhanced pyruvate dehydrogenase (PDH) phosphorylation and lactate release. Prior exercise led to higher PDH phosphorylation and activation of glycogen synthase compared with resting control. In conclusion, lipid-induced insulin resistance in skeletal muscle was associated with impaired TBC1D4 S341 and elevated PDH phosphorylation. The prophylactic effect of exercise on lipid-induced insulin resistance may involve augmented TBC1D4 signaling and glycogen synthase activation.

AMPK in skeletal muscle function and metabolism

Skeletal muscle possesses a remarkable ability to adapt to various physiologic conditions. AMPK is a sensor of intracellular energy status that maintains energy stores by fine‐tuning anabolic and catabolic pathways. AMPKs role as an energy sensor is particularly critical in tissues displaying highly changeable energy turnover. Due to the drastic changes in energy demand that occur between the resting and exercising state, skeletal muscle is one such tissue. Here, we review the complex regulation of AMPK in skeletal muscle and its consequences on metabolism (e.g., substrate uptake, oxidation, and storage as well as mitochondrial function of skeletal muscle fibers). We focus on the role of AMPK in skeletal muscle during exercise and in exercise recovery. We also address adaptations to exercise training, including skeletal muscle plasticity, highlighting novel concepts and future perspectives that need to be investigated. Furthermore, we discuss the possible role of AMPK as a therapeutic target as well as different AMPK activators and their potential for future drug development.— Kjøbsted, R., Hingst, J. R., Fentz, J., Foretz, M., Sanz, M.‐N., Pehmøller, C., Shum, M., Marette, A., Mounier, R., Treebak, J. T., Wojtaszewski, J. F. P., Viollet, B., Lantier, L. AMPK in skeletal muscle function and metabolism. FASEB J. 32, 1741–1777 (2018). www.fasebj.org

AMPK and insulin action--responses to ageing and high fat diet.

The 5′-AMP-activated protein kinase (AMPK) is considered “a metabolic master-switch” in skeletal muscle reducing ATP- consuming processes whilst stimulating ATP regeneration. Within recent years, AMPK has also been proposed as a potential target to attenuate insulin resistance, although the exact role of AMPK is not well understood. Here we hypothesized that mice lacking α2AMPK activity in muscle would be more susceptible to develop insulin resistance associated with ageing alone or in combination with high fat diet. Young (∼4 month) or old (∼18 month) wild type and muscle specific α2AMPK kinase-dead mice on chow diet as well as old mice on 17 weeks of high fat diet were studied for whole body glucose homeostasis (OGTT, ITT and HOMA-IR), insulin signaling and insulin-stimulated glucose uptake in muscle. We demonstrate that high fat diet in old mice results in impaired glucose homeostasis and insulin stimulated glucose uptake in both the soleus and extensor digitorum longus muscle, coinciding with reduced insulin signaling at the level of Akt (pSer473 and pThr308), TBC1D1 (pThr590) and TBC1D4 (pThr642). In contrast to our hypothesis, the impact of ageing and high fat diet on insulin action was not worsened in mice lacking functional α2AMPK in muscle. It is concluded that α2AMPK deficiency in mouse skeletal muscle does not cause muscle insulin resistance in young and old mice and does not exacerbate obesity-induced insulin resistance in old mice suggesting that decreased α2AMPK activity does not increase susceptibility for insulin resistance in skeletal muscle.

Enhanced voluntary wheel running in GPRC6A receptor knockout mice

GPRC6A is an amino acid-sensing receptor highly expressed in the brain and in skeletal muscle. Although recent evidence suggests that genetically engineered GPRC6A receptor knockout (KO) mice are susceptible to develop subtle endocrine and metabolic disturbances, the underlying disruptions in energy metabolism are largely unexplored. Based on GPRC6As expression pattern and ligand preferences, we hypothesize that the receptor may impact energy metabolism via regulating physical activity levels. Thus, in the present study, we exposed GPRC6A receptor KO mice and their wild-type (WT) littermates to voluntary wheel running and forced treadmill exercise. Moreover, we assessed energy expenditure in the basal state, and evaluated the effects of wheel running on food intake, body composition, and a range of exercise-induced central and peripheral biomarkers. We found that adaptation to voluntary wheel running is affected by GPRC6A, as ablation of the receptor significantly enhances wheel running in KO relative to WT mice. Both genotypes responded to voluntary exercise by increasing food intake and improving body composition to a similar degree. In conclusion, these data demonstrate that the GPRC6A receptor is involved in regulating exercise behaviour. Future studies are highly warranted to delineate the underlying molecular details and to assess if these findings hold any translational value.