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Insulin-stimulated muscle glucose uptake is a key process in glycemic control. This process depends on the redistribution of glucose transporters to the surface membrane, a process which involves regulatory proteins such as TBC1D1 and TBC1D4. Accordingly, a TBC1D4 loss-of-function mutation in human skeletal muscle is associated with increased risk of type 2 diabetes, and observations from carriers of a TBC1D1 variant associate this protein to a severe obesity phenotype. Here, we identified interactors of the endogenous TBC1D4 in human skeletal muscle by an unbiased proteomics approach. We detected 76 proteins as candidate TBC1D4 interactors. The binding of 12 of these interactors were regulated by insulin, including proteins known to be involved in glucose metabolism (e.g. 14-3-3 proteins and ACTN4). TBC1D1 also co-precipitated with TBC1D4 and vice versa in both human and mouse skeletal muscle. This interaction was not regulated by insulin nor exercise in young, healthy, lean individuals. Similarly, the exercise- and insulin-regulated phosphorylation of the TBC1D1-TBC1D4 complex was intact. In contrast, we observed an altered interaction as well as compromised insulin-stimulated phospho-regulation of the TBC1D1-TBC1D4 complex in muscle of obese individuals with type 2 diabetes. Altogether, we provide a repository of TBC1D4 interactors in human and mouse skeletal muscle, which serve as potential regulators of TBC1D4 function and, thus, insulin-stimulated glucose uptake in human skeletal muscle.

Exercise profoundly influences glycemic control by enhancing muscle insulin sensitivity, thus promoting glucometabolic health. While prior glycogen breakdown so far has been deemed integral for muscle insulin sensitivity to be potentiated by exercise, the mechanisms underlying this phenomenon remain enigmatic. We have combined original data from 13 of our studies that investigated insulin action in skeletal muscle either under rested conditions or following a bout of one-legged knee extensor exercise in healthy young male individuals (n = 106). Insulin-stimulated glucose uptake was potentiated and occurred substantially faster in the prior contracted muscles. In this otherwise homogenous group of individuals, a remarkable biological diversity in the glucometabolic responses to insulin is apparent both in skeletal muscle and at the whole-body level. In contrast to the prevailing concept, our analyses reveal that insulin-stimulated muscle glucose uptake and the potentiation thereof by exercise are not associated with muscle glycogen synthase activity, muscle glycogen content, or degree of glycogen utilization during the preceding exercise bout. Our data further suggest that the phenomenon of improved insulin sensitivity in prior contracted muscle is not regulated in a homeostatic feedback manner from glycogen. Instead, we put forward the idea that this phenomenon is regulated by cellular allostatic mechanisms that elevate the muscle glycogen storage set point and enhance insulin sensitivity to promote the uptake of glucose toward faster glycogen resynthesis without development of glucose overload/toxicity or feedback inhibition.

Protein phosphorylation dynamically integrates environmental and cellular information to control biological processes. Identifying functional phosphorylation amongst the thousands of phosphosites regulated by a perturbation at a global scale is a major challenge. Here we introduce 'personalized phosphoproteomics', a combination of experimental and computational analyses to link signaling with biological function by utilizing human phenotypic variance. We measure individual subject phosphoproteome responses to interventions with corresponding phenotypes measured in parallel. Applying this approach to investigate how exercise potentiates insulin signaling in human skeletal muscle, we identify both known and previously unidentified phosphosites on proteins involved in glucose metabolism. This includes a cooperative relationship between mTOR and AMPK whereby the former directly phosphorylates the latter on S377, for which we find a role in metabolic regulation. These results establish personalized phosphoproteomics as a general approach for investigating the signal transduction underlying complex biology.

Skeletal muscle conveys several of the health-promoting effects of exercise; yet the underlying mechanisms are not fully elucidated. Studying skeletal muscle is challenging due to its different fiber types and the presence of non-muscle cells. This can be circumvented by isolation of single muscle fibers. Here, we develop a workflow enabling proteomics analysis of pools of isolated muscle fibers from freeze-dried human muscle biopsies. We identify more than 4000 proteins in slow- and fast-twitch muscle fibers. Exercise training alters expression of 237 and 172 proteins in slow- and fast-twitch muscle fibers, respectively. Interestingly, expression levels of secreted proteins and proteins involved in transcription, mitochondrial metabolism, Ca2+ signaling, and fat and glucose metabolism adapts to training in a fiber type-specific manner. Our data provide a resource to elucidate molecular mechanisms underlying muscle function and health, and our workflow allows fiber type-specific proteomic analyses of snap-frozen non-embedded human muscle biopsies.

Background: Being born with low birth weight (LBW) is a risk factor for muscle insulin resistance and type 2 diabetes (T2D), which may be mediated by epigenetic mechanisms programmed by the intrauterine environment. Epigenetic mechanisms exert their prime effects in developing cells. We hypothesized that muscle insulin resistance in LBW subjects may be due to early differential epigenomic and transcriptomic alterations in their immature muscle progenitor cells. Results: Muscle progenitor cells were obtained from 23 healthy young adult men born at term with LBW, and 15 BMI-matched normal birth weight (NBW) controls. The cells were subsequently cultured and differentiated into myotubes. DNA and RNA were harvested before and after differentiation for genome-wide DNA methylation and RNA expression measurements. After correcting for multiple comparisons (q ≤ 0.05), 56 CpG sites were found to be significantly, differentially methylated in myoblasts from LBW compared with NBW men, of which the top five gene-annotated CpG sites (SKI, ARMCX3, NR5A2, NEUROG, ESRRG) previously have been associated to regulation of cholesterol, fatty acid and glucose metabolism and muscle development or hypertrophy. LBW men displayed markedly decreased myotube gene expression levels of the AMPK-repressing tyrosine kinase gene FYN and the histone deacetylase gene HDAC7. Silencing of FYN and HDAC7 was associated with impaired myotube formation, which for HDAC7 reduced muscle glucose uptake. Conclusions: The data provides evidence of impaired muscle development predisposing LBW individuals to T2D is linked to and potentially caused by distinct DNA methylation and transcriptional changes including down regulation of HDAC7 and FYN in their immature myoblast stem cells.

Women with polycystic ovary syndrome (PCOS) have been shown to be less insulin sensitive compared with control (CON) women, independent of BMI. Training is associated with molecular adaptations in skeletal muscle, improving glucose uptake and metabolism in both healthy individuals and patients type 2 diabetes. In the current study, lean hyperandrogenic women with PCOS (n = 9) and healthy CON women (n = 9) completed 14 weeks of controlled and supervised exercise training. In CON, the training intervention increased whole body insulin action by 26% and insulin-stimulated leg glucose uptake by 53%, together with increased insulin-stimulated leg blood flow and a more oxidative muscle fiber type distribution. In PCOS, no such changes were found, despite similar training intensity and improvements in VO2max. In skeletal muscle of CON but not PCOS, training increased GLUT4 and HKII mRNA and protein expressions. These data suggest that the impaired increase in whole-body insulin action in women with PCOS with training is caused by an impaired ability to upregulate key glucose-handling proteins for insulin-stimulated glucose uptake in skeletal muscle and insulin-stimulated leg blood flow. Still, other important benefits of exercise training appeared in women with PCOS, including an improvement of the hyperandrogenic state.

Objective: Exercise is a cornerstone in the management of skeletal muscle insulin-resistance. A well-established benefit of a single bout of exercise is increased insulin sensitivity for hours post-exercise in the previously exercised musculature. Although rodent studies suggest that the insulin-sensitization phenomenon involves enhanced insulin-stimulated GLUT4 cell surface translocation and might involve intramuscular redistribution of GLUT4, the conservation to humans is unknown.Methods: Healthy young males underwent an insulin-sensitizing one-legged kicking exercise bout for 1 hour followed by fatigue bouts to exhaustion. Muscle biopsies were obtained 4h post-exercise before and after a 2h hyperinsulinemic-euglycemic clamp.Results: A detailed microscopy-based analysis of GLUT4 distribution muscle specimen in 7 different myocellular compartments revealed that prior exercise increased GLUT4 localization in insulin-responsive storage vesicles and T-tubuli. Furthermore, insulin-stimulated GLUT4 localization was augmented at the sarcolemma and in the endosomal compartments.Conclusion: An intracellular redistribution of GLUT4 post-exercise is proposed as a molecular mechanism contributing to the insulin-sensitizing effect of prior exercise in human skeletal muscle.

A single bout of exercise enhances insulin action in the exercised muscle. However, not all human studies find that this translates into increased whole-body insulin action, suggesting that insulin action in rested muscle or other organs may be decreased by exercise. To investigate this, eight healthy men underwent a euglycemic hyperinsulinemic clamp on two separate days: One day with prior one-legged knee-extensor exercise to local exhaustion (∼2.5 hours) and another day without exercise. Whole-body glucose disposal was ∼18% lower on the exercise day as compared to the resting day due to decreased (-37%) insulin-stimulated glucose uptake in the non-exercised muscle. Insulin signaling at the level of Akt2 was impaired in the non-exercised muscle on the exercise day suggesting that decreased insulin action in non-exercised muscle may reduce GLUT4 translocation in response to insulin.Thus, the effect of a single bout of exercise on whole-body insulin action depends on the balance between local effects increasing and systemic effects decreasing insulin action. Physiologically, this mechanism may serve to direct glucose into the muscles in need of glycogen replenishment. For insulin-treated patients this complex relationship may explain the difficulties in predicting the adequate insulin dose for maintaining glucose homeostasis following physical activity.

Prolonged intervention studies investigating molecular metabolism are necessary for a deeper understanding of dietary effects on health. Here we provide mechanistic information about metabolic adaptation to fat-rich diets. Healthy, slightly overweight men ingested saturated or polyunsaturated fat-rich diets for 6 weeks during weight maintenance. Hyperinsulinemic clamps combined with leg balance technique revealed unchanged peripheral insulin sensitivity, independent of fatty acid type. Both diets increased fat oxidation potential in muscle. Hepatic insulin clearance increased, while glucose production, de novo lipogenesis, and plasma triacylglycerol decreased. High fat intake changed the plasma proteome in the immune-supporting direction and the gut microbiome displayed changes at taxonomical and functional level with polyunsaturated fatty acid (PUFA). In mice, eucaloric feeding of human PUFA and saturated fatty acid diets lowered hepatic triacylglycerol content compared with low-fat-fed control mice, and induced adaptations in the liver supportive of decreased gluconeogenesis and lipogenesis. Intake of fat-rich diets thus induces extensive metabolic adaptations enabling disposition of dietary fat without metabolic complications.

Glucose phosphorylation by hexokinase (HK) is a rate-limiting step in glucose metabolism. Regulation of HK includes feedback inhibition by its product glucose-6-phosphate (G6P) and mitochondria binding. HK affinity for G6P is difficult to measure because its natural product (G6P) inhibits enzyme activity. HK phosphorylates several hexoses and we have taken advantage of the fact that 2-deoxyglucose (2-DG)-6-phosphate does not inhibit HK activity. By this, we have developed a new method for rapid radiochemical analysis of HK activity with 2-DG as substrate, which allows control of the concentrations of G6P to investigate HK affinity for inhibition by G6P. We verified that 2-DG serves as a substrate for the HK reaction with linear time and concentration dependency as well as expected maximal velocity and KM. This is the first simple assay that evaluates feedback inhibition of HK by its product G6P and provides a unique technique for future research evaluating the regulation of glucose phosphorylation under various physiological conditions.

Objective: A single bout of exercise followed by intake of carbohydrates leads to glycogen supercompensation in prior exercised muscle. Our objective was to illuminate molecular mechanisms underlying this phenomenon in skeletal muscle of man. Methods: We studied the temporal regulation of glycogen supercompensation in human skeletal muscle during a 5 day recovery period following a single bout of exercise. Nine healthy men depleted (day 1), normalized (day 2) and supercompensated (day 5) muscle glycogen in one leg while the contralateral leg served as a resting control. Euglycemic hyperinsulinemic clamps in combination with leg balance technique allowed for investigating insulin-stimulated leg glucose uptake under these 3 experimental conditions. Cellular signaling in muscle biopsies was investigated by global proteomic analyses and immunoblotting. We strengthened the validity of proposed molecular effectors by follow-up studies in muscle of transgenic mice. Results: Sustained activation of glycogen synthase (GS) and AMPK in combination with elevated expression of proteins determining glucose uptake capacity were evident in the prior exercised muscle. We hypothesize that these alterations offset the otherwise tight feedback inhibition of glycogen synthesis and glucose uptake by glycogen. In line with key roles of AMPK and GS seen in the human experiments we observed abrogated ability for glycogen supercompensation in muscle with inducible AMPK deletion and in muscle carrying a G6P-insensitive form of GS in muscle. Conclusion: Our study demonstrates that both AMPK and GS are key regulators of glycogen supercompensation following a single bout of glycogen-depleting exercise in skeletal muscle of both man and mouse.

Exercise stimulates the release of molecules into the circulation, supporting the concept that inter-tissue signaling proteins are important mediators of adaptations to exercise. Recognizing that many circulating proteins are packaged in extracellular vesicles (EVs), we employed quantitative proteomic techniques to characterize the exercise-induced secretion of EV-contained proteins. Following a 1-hr bout of cycling exercise in healthy humans, we observed an increase in the circulation of over 300 proteins, with a notable enrichment of several classes of proteins that compose exosomes and small vesicles. Pulse-chase and intravital imaging experiments suggested EVs liberated by exercise have a propensity to localize in the liver and can transfer their protein cargo. Moreover, by employing arteriovenous balance studies across the contracting human limb, we identified several novel candidate myokines, released into circulation independently of classical secretion. These data identify a new paradigm by which tissue crosstalk during exercise can exert systemic biological effects.

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. AMPK's 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.

Hypoglycemia is the leading limiting factor in glycemic management of insulin-treated diabetes. Skeletal muscle is the predominant site of insulin-mediated glucose disposal and our study was designed to test to what extent insulin induced hypoglycemia affects glucose uptake in skeletal muscle and whether hypoglycemia counter-regulation modulates insulin and catecholamine signaling and glycogen synthase activity in skeletal muscle.Nine healthy volunteers were examined on three randomized study days in a crossover design: i) hyperinsulinemic hypoglycemia (bolus insulin), ii) hyperinsulinemic euglycemia (bolus insulin and glucose infusion) and iii) saline control with skeletal muscle biopsies taken just before, 30 min and 75 min after insulin/saline injection.During hypoglycemia glucose levels reached a nadir of ∼2.0mmol/l and epinephrine rose to ∼900pg/ml.Insulin stimulated glucose disposal and glucose clearance in skeletal muscle were impaired whereas insulin signaling to glucose transport was unaffected by hypoglycemia. Insulin-stimulated glycogen synthase activity was completely ablated during hyperinsulinemic hypoglycemia and catecholamine signaling via PKA as well as phosphorylation of inhibiting sites on glycogen synthase all increased.

Perifer insulin resistens, som er forbundet med nedsat insulin-stimuleret glukoseoptagelse, anses som en af de væsentligste defekter i sygdomsforløbet af type 2 diabetes (T2D). Dette tilskrives primært nedsat glykogensyntese i skeletmuskulaturen, og en række studier har fundet lavere insulinstimuleret aktivering af glykogen syntase (GS). Et enkeltstående fysisk arbejde øger aktiviteten af GS i perioden efter arbejdets ophør. Det er dog ikke blevet undersøgt, om et enkelt arbejde forbedrer en nedsat insulin-stimuleret regulering af GS i T2D patienter. Når et enkelt akut arbejde til udmattelse følges af indtag af kulhydrater, så kan muskelcellen øge glykogensyntesen i flere dage efter arbejdets ophør og glykogenniveauet i musklen øges over den basale tilstand. Dette fænomen kaldes ”glykogen superkompensering” og er forbeholdt til den tidligere arbejdende muskulatur, indikerende at et enkelstående arbejde kan potensere faktorer i musklen, som øger kapaciteten for at lagre glukose. Selvom fænomenet har været kendt i flere årtier, så er de molekylære mekanismer, der ligger til grund herfor, ikke opklaret. I studie I blev der fundet at et enkeltstående arbejde øger GS-aktiviteten i samme omfang i T2D patienter sammenlignet med overvægtige kontrolpersoner. I perioden efter arbejdet blev der observeret nedsat GS-aktivitet og affinitet overfor substratet UDP-glukose i sammenspil med øget fosforylering af GS på de hæmmende sites 2+2a. Dette fund indikerer et kompromitteret udbytte af et enkelt arbejde på glykogen syntase i T2D patienter men bevirkede ikke at glukosemetabolismen skiftede fra oxidation til lagring. I Studie II blev der fundet højere aktivitet af GS og AMPK samt øget protein ekspression af HK II i den tidligere arbejdende muskel under et 5 dages et-bens glykogen superkompenseringsforløb i mennesker. Vores forsøg i transgene mus indikerede, at AMPK er afgørende for at øge glykogensyntesen efter et enkelt arbejde ved at øge niveauet af G6P, og den

AIMS/HYPOTHESIS: Insulin and exercise stimulate skeletal muscle glycogen synthase (GS) activity by dephosphorylation and changes in kinetic properties. The aim of this study was to investigate the effects of insulin, exercise and post-exercise insulin stimulation on GS phosphorylation, activity and substrate affinity in obesity and type 2 diabetes.METHODS: Obese men with type 2 diabetes (n = 13) and weight-matched controls (n = 14) underwent euglycaemic-hyperinsulinaemic clamps in the rested state and 3 h after 60 min of cycling (70% maximal pulmonary oxygen uptake [[Formula: see text]]). Biopsies from vastus lateralis muscle were obtained before and after clamps, and before and immediately after exercise.RESULTS: Insulin-stimulated glucose uptake was lower in diabetic patients vs obese controls with or without prior exercise. Post exercise, glucose partitioning shifted away from oxidation and towards storage in both groups. Insulin and, more potently, exercise increased GS activity (fractional velocity [FV]) and substrate affinity in both groups. Both stimuli caused dephosphorylation of GS at sites 3a + 3b, with exercise additionally decreasing phosphorylation at sites 2 + 2a. In both groups, changes in GS activity, substrate affinity and dephosphorylation at sites 3a + 3b by exercise were sustained 3 h post exercise and further enhanced by insulin. Post exercise, reduced GS activity and substrate affinity as well as increased phosphorylation at sites 2 + 2a were found in diabetic patients vs obese controls.CONCLUSIONS/INTERPRETATION: Exercise-induced activation of muscle GS in obesity and type 2 diabetes involves dephosphorylation of GS at sites 3a + 3b and 2 + 2a and enhanced substrate affinity, which is likely to facilitate glucose partitioning towards storage. Lower GS activity and increased phosphorylation at sites 2 + 2a in type 2 diabetes in the recovery period imply an impaired response to exercise.

Subjects with a low birth weight (LBW) display increased risk of developing type 2 diabetes (T2D). We hypothesized that this is associated with defects in muscle adaptations following acute and regular physical activity, evident by impairments in the exercise-induced activation of AMPK signaling. We investigated 21 LBW and 21 normal birth weight (NBW) subjects during 1 hour of acute exercise performed at the same relative workload before and after 12 weeks of exercise training. Multiple skeletal muscle biopsies were obtained before and after exercise. Protein levels and phosphorylation status were determined by Western blotting. AMPK activities were measured using activity assays. Protein levels of AMPK isoforms a1 and ¿1 were significantly increased while ¿3 levels decreased with training independent of group. The LBW group had higher exercise-induced AMPK Thr(172) phosphorylation before training and higher exercise-induced ACC2 Ser(221) phosphorylation both before and after training compared with NBW. Despite exercise being performed at the same relative intensity (65% of VO2peak), the acute exercise response on AMPK Thr172, ACC2 Ser(221), AMPK a2ß2¿1 and a2ß2¿3- activities, GS activity, adenine nucleotides as well as Hexokinase II mRNA levels were all reduced after exercise training. Increased exercise-induced muscle AMPK activation and ACC2 Ser(221) phosphorylation in LBW subjects may indicate a more sensitive AMPK system in this population. Long term exercise training may reduce the need for AMPK to control energy turnover during exercise. Thus, the remaining ¿3-associated AMPK activation by acute exercise after exercise training might be sufficient to maintain cellular energy balance.