pH-Gated Succinate Secretion Regulates Muscle Remodeling in Response to Exercise.

In response to skeletal muscle contraction during exercise, paracrine factors coordinate tissue remodeling, which underlies this healthy adaptation. Here we describe a pH-sensing metabolite signal that initiates muscle remodeling upon exercise. In mice and humans, exercising skeletal muscle releases the mitochondrial metabolite succinate into the local interstitium and circulation. Selective secretion of succinate is facilitated by its transient protonation, which occurs upon muscle cell acidification. In the protonated monocarboxylic form, succinate is rendered a transport substrate for monocarboxylate transporter 1, which facilitates pH-gated release. Upon secretion, succinate signals via its cognate receptor SUCNR1 in non-myofibrillar cells in muscle tissue to control muscle-remodeling transcriptional programs. This succinate-SUCNR1 signaling is required for paracrine regulation of muscle innervation, muscle matrix remodeling, and muscle strength in response to exercise training. In sum, we define a bioenergetic sensor in muscle that utilizes intracellular pH and succinate to coordinate tissue adaptation to exercise.

The Rho guanine dissociation inhibitor α inhibits skeletal muscle Rac1 activity and insulin action.

The molecular events governing skeletal muscle glucose uptake have pharmacological potential for managing insulin resistance in conditions such as obesity, diabetes, and cancer. With no current pharmacological treatments to target skeletal muscle insulin sensitivity, there is an unmet need to identify the molecular mechanisms that control insulin sensitivity in skeletal muscle. Here, the Rho guanine dissociation inhibitor α (RhoGDIα) is identified as a point of control in the regulation of insulin sensitivity. In skeletal muscle cells, RhoGDIα interacted with, and thereby inhibited, the Rho GTPase Rac1. In response to insulin, RhoGDIα was phosphorylated at S101 and Rac1 dissociated from RhoGDIα to facilitate skeletal muscle GLUT4 translocation. Accordingly, siRNA-mediated RhoGDIα depletion increased Rac1 activity and elevated GLUT4 translocation. Consistent with RhoGDIα's inhibitory effect, rAAV-mediated RhoGDIα overexpression in mouse muscle decreased insulin-stimulated glucose uptake and was detrimental to whole-body glucose tolerance. Aligning with RhoGDIα's negative role in insulin sensitivity, RhoGDIα protein content was elevated in skeletal muscle from insulin-resistant patients with type 2 diabetes. These data identify RhoGDIα as a clinically relevant controller of skeletal muscle insulin sensitivity and whole-body glucose homeostasis, mechanistically by modulating Rac1 activity.

Metabolic effect of adrenaline infusion in people with type 1 diabetes and healthy individuals.

AIMS/HYPOTHESIS: As a result of early loss of the glucagon response, adrenaline is the primary counter-regulatory hormone in type 1 diabetes. Diminished adrenaline responses to hypoglycaemia due to counter-regulatory failure are common in type 1 diabetes, and are probably induced by exposure to recurrent hypoglycaemia, however, the metabolic effects of adrenaline have received less research attention, and also there is conflicting evidence regarding adrenaline sensitivity in type 1 diabetes. Thus, we aimed to investigate the metabolic response to adrenaline and explore whether it is modified by prior exposure to hypoglycaemia. METHODS: Eighteen participants with type 1 diabetes and nine healthy participants underwent a three-step ascending adrenaline infusion during a hyperinsulinaemic-euglycaemic clamp. Continuous glucose monitoring data obtained during the week before the study day were used to assess the extent of hypoglycaemia exposure. RESULTS: While glucose responses during the clamp were similar between people with type 1 diabetes and healthy participants, plasma concentrations of NEFAs and glycerol only increased in the group with type 1 diabetes (p<0.001). Metabolomics revealed an increase in the most common NEFAs (p<0.01). Other metabolic responses were generally similar between participants with type 1 diabetes and healthy participants. Exposure to hypoglycaemia was negatively associated with the NEFA response; however, this was not statistically significant. CONCLUSIONS/INTERPRETATION: In conclusion, individuals with type 1 diabetes respond with increased lipolysis to adrenaline compared with healthy participants by mobilising the abundant NEFAs in plasma, whereas other metabolic responses were similar. This may suggest that the metabolic sensitivity to adrenaline is altered in a pathway-specific manner in type 1 diabetes. TRIAL REGISTRATION: ClinicalTrials.gov NCT05095259.

Exercise-induced increase in muscle insulin sensitivity in men is amplified when assessed using a meal test.

AIMS/HYPOTHESIS: Exercise has a profound effect on insulin sensitivity in skeletal muscle. The euglycaemic-hyperinsulinaemic clamp (EHC) is the gold standard for assessment of insulin sensitivity but it does not reflect the hyperglycaemia that occurs after eating a meal. In previous EHC investigations, it has been shown that the interstitial glucose concentration in muscle is decreased to a larger extent in previously exercised muscle than in rested muscle. This suggests that previously exercised muscle may increase its glucose uptake more than rested muscle if glucose supply is increased by hyperglycaemia. Therefore, we hypothesised that the exercise-induced increase in muscle insulin sensitivity would appear greater after eating a meal than previously observed with the EHC. METHODS: Ten recreationally active men performed dynamic one-legged knee extensor exercise for 1 h. Following this, both femoral veins and one femoral artery were cannulated. Subsequently, 4 h after exercise, a solid meal followed by two liquid meals were ingested over 1 h and glucose uptake in the two legs was measured for 3 h. Muscle biopsies from both legs were obtained before the meal test and 90 min after the meal test was initiated. Data obtained in previous studies using the EHC (n=106 participants from 13 EHC studies) were used for comparison with the meal-test data obtained in this study. RESULTS: Plasma glucose and insulin peaked 45 min after initiation of the meal test. Following the meal test, leg glucose uptake and glucose clearance increased twice as much in the exercised leg than in the rested leg; this difference is twice as big as that observed in previous investigations using EHCs. Glucose uptake in the rested leg plateaued after 15 min, alongside elevated muscle glucose 6-phosphate levels, suggestive of compromised muscle glucose metabolism. In contrast, glucose uptake in the exercised leg plateaued 45 min after initiation of the meal test and there were no signs of compromised glucose metabolism. Phosphorylation of the TBC1 domain family member 4 (TBC1D4; p-TBC1D4Ser704) and glycogen synthase activity were greater in the exercised leg compared with the rested leg. Muscle interstitial glucose concentration increased with ingestion of meals, although it was 16% lower in the exercised leg than in the rested leg. CONCLUSIONS/INTERPRETATION: Hyperglycaemia after meal ingestion results in larger differences in muscle glucose uptake between rested and exercised muscle than previously observed during EHCs. These findings indicate that the ability of exercise to increase insulin-stimulated muscle glucose uptake is even greater when evaluated with a meal test than has previously been shown with EHCs.

Physical activity attenuates postprandial hyperglycaemia in homozygous TBC1D4 loss-of-function mutation carriers.

AIMS/HYPOTHESIS: The common muscle-specific TBC1D4 p.Arg684Ter loss-of-function variant defines a subtype of non-autoimmune diabetes in Arctic populations. Homozygous carriers are characterised by elevated postprandial glucose and insulin levels. Because 3.8% of the Greenlandic population are homozygous carriers, it is important to explore possibilities for precision medicine. We aimed to investigate whether physical activity attenuates the effect of this variant on 2 h plasma glucose levels after an oral glucose load. METHODS: In a Greenlandic population cohort (n = 2655), 2 h plasma glucose levels were obtained after an OGTT, physical activity was estimated as physical activity energy expenditure and TBC1D4 genotype was determined. We performed TBC1D4-physical activity interaction analysis, applying a linear mixed model to correct for genetic admixture and relatedness. RESULTS: Physical activity was inversely associated with 2 h plasma glucose levels (ß[main effect of physical activity] -0.0033 [mmol/l] / [kJ kg-1 day-1], p = 6.5 × 10-5), and significantly more so among homozygous carriers of the TBC1D4 risk variant compared with heterozygous carriers and non-carriers (ß[interaction] -0.015 [mmol/l] / [kJ kg-1 day-1], p = 0.0085). The estimated effect size suggests that 1 h of vigorous physical activity per day (compared with resting) reduces 2 h plasma glucose levels by an additional ~0.7 mmol/l in homozygous carriers of the risk variant. CONCLUSIONS/INTERPRETATION: Physical activity improves glucose homeostasis particularly in homozygous TBC1D4 risk variant carriers via a skeletal muscle TBC1 domain family member 4-independent pathway. This provides a rationale to implement physical activity as lifestyle precision medicine in Arctic populations. DATA REPOSITORY: The Greenlandic Cardio-Metabochip data for the Inuit Health in Transition study has been deposited at the European Genome-phenome Archive ( https://www.ebi.ac.uk/ega/dacs/EGAC00001000736 ) under accession EGAD00010001428.

AXIN1 knockout does not alter AMPK/mTORC1 regulation and glucose metabolism in mouse skeletal muscle.

KEY POINTS: Tamoxifen-inducible skeletal muscle-specific AXIN1 knockout (AXIN1 imKO) in mouse does not affect whole-body energy substrate metabolism. AXIN1 imKO does not affect AICAR or insulin-stimulated glucose uptake in adult skeletal muscle. AXIN1 imKO does not affect adult skeletal muscle AMPK or mTORC1 signalling during AICAR/insulin/amino acid incubation, contraction and exercise. During exercise, α2/ß2/γ3AMPK and AMP/ATP ratio show greater increases in AXIN1 imKO than wild-type in gastrocnemius muscle. ABSTRACT: AXIN1 is a scaffold protein known to interact with >20 proteins in signal transduction pathways regulating cellular development and function. Recently, AXIN1 was proposed to assemble a protein complex essential to catabolic-anabolic transition by coordinating AMPK activation and inactivation of mTORC1 and to regulate glucose uptake-stimulation by both AMPK and insulin. To investigate whether AXIN1 is permissive for adult skeletal muscle function, a phenotypic in vivo and ex vivo characterization of tamoxifen-inducible skeletal muscle-specific AXIN1 knockout (AXIN1 imKO) mice was conducted. AXIN1 imKO did not influence AMPK/mTORC1 signalling or glucose uptake stimulation at rest or in response to different exercise/contraction protocols, pharmacological AMPK activation, insulin or amino acids stimulation. The only genotypic difference observed was in exercising gastrocnemius muscle, where AXIN1 imKO displayed elevated α2/ß2/γ3 AMPK activity and AMP/ATP ratio compared to wild-type mice. Our work shows that AXIN1 imKO generally does not affect skeletal muscle AMPK/mTORC1 signalling and glucose metabolism, probably due to functional redundancy of its homologue AXIN2.

Quantification of exercise-regulated ubiquitin signaling in human skeletal muscle identifies protein modification cross talk via NEDDylation.

The maintenance of muscle function is extremely important for whole body health and exercise is essential to this process. The ubiquitin-proteasome system (UPS) is required for muscle adaptation following exercise but little is known about acute posttranslational regulation and proteome remodeling during and after high-intensity exercise. Here, we used quantitative proteomics to study ubiquitin signaling dynamics in human skeletal muscle biopsies from healthy males before, during, and after a single bout of high-intensity exercise. Exercise resulted in a marked depletion of protein ubiquitylation in the vastus lateralis muscle consistent with proteasome activation. This was also associated with acute posttranslational modification of protein abundance. Integration of these data with phosphoproteomics identified co-regulated proximal modifications suggesting a cross talk between phosphorylation and ubiquitylation. We also identified additional protein modification cross talk and showed acute activation of protein NEDDylation. In vitro experiments revealed that cAMP-dependent activation of the proteasome requires NEDD8, an ubiquitin-like protein involved in protein NEDDylation, to maintain cellular protein ubiquitylation levels. Our data reveal the complexity of ubiquitin signaling and proteome remodeling in muscle during and after high-intensity exercise. We propose a model whereby exercise and the resulting cAMP signaling activate both the proteasome and ubiquitylation via NEDDylation to rapidly remove potentially damaged proteins.

Colchicine treatment impairs skeletal muscle mitochondrial function and insulin sensitivity in an age-specific manner.

The aim of the study was to investigate the impact of autophagy inhibition on skeletal muscle mitochondrial function and glucose homeostasis in young and aged mice. The transcriptional co-activator PGC-1α regulates muscle oxidative phenotype which has been shown to be linked with basal autophagic capacity. Therefore, young and aged inducible muscle-specific PGC-1α knockout (iMKO) mice and littermate lox/lox controls were used in three separate experiments performed after either saline or colchicine injections on two consecutive days: (1) Euthanization in the basal state obtaining skeletal muscle for mitochondrial respirometry, (2) whole body glucose tolerance test, and (3) in vivo insulin-stimulated 2-deoxyglucose (2-DG) uptake into skeletal muscle. Muscle PGC-1α was not required for maintaining basal autophagy flux, regardless of age. Colchicine-induced inhibition of autophagy was associated with impairments of skeletal muscle mitochondrial function, including reduced ADP sensitivity and altered mitochondrial redox balance in both young and aged mice. Colchicine treatment reduced the glucose tolerance in aged, but not young mice, and similarly in iMKO and lox/lox mice. Colchicine reduced insulin-stimulated 2-DG uptake in soleus muscle in aged mice, independently of PGC-1α, and without affecting insulin-regulated phosphorylation of proximal or distal mediators of insulin signaling. In conclusion, the results indicate that autophagy regulates the mitochondrial ADP sensitivity and redox balance as well as whole body glucose tolerance and skeletal muscle insulin sensitivity in aged mice, with no additional effects of inducible PGC-1α deletion.

The insulin-sensitizing effect of a single exercise bout is similar in type I and type II human muscle fibres.

KEY POINTS: Rodent studies suggest muscle fibre type-specific insulin response in the recovery from exercise.  The current study investigates muscle fibre type-specific insulin action in the recovery from exercise in healthy subjects.  In type I and type II muscle fibres, key proteins in glucose metabolism are similarly regulated by insulin during recovery from exercise.  Our findings imply that both type I and type II muscle fibres contribute to the phenomenon of increased insulin sensitivity in the recovery from a single bout of exercise in humans. ABSTRACT: Human skeletal muscle consists of slow-twitch (type I) and fast-twitch (type II) muscle fibres. Muscle insulin action, regulating glucose uptake and metabolism, is improved following a single exercise bout. Rodent studies suggest that this phenomenon is confined to specific muscle fibre types. Whether this phenomenon is also confined to specific fibre types in humans has not been described. To investigate this, nine healthy men underwent a euglycaemic hyperinsulinaemic clamp (EHC) in the recovery from a single bout of one-legged knee-extensor exercise. Pools of type I and type II fibres were prepared from muscle biopsies taken in the rested and prior exercised leg before and after the EHC. AMPK γ3 and TBC1D4 - two key proteins regulating muscle insulin action following exercise - were higher expressed in type II than type I fibres. However, phosphor-regulation of TBC1D4 was similar between fibre types when related to the total amount of TBC1D4 protein. The activating dephosphorylation of glycogen synthase was also similar in the two fibre types. Thus, insulin-induced regulation of key proteins important for transport and intracellular flux of glucose towards glycogen storage in the recovery from exercise, does not differ between fibre types. In conclusion, the insulin-sensitizing effect of a single bout of exercise includes both type I and type II fibres in human skeletal muscle. This may be an important observation for future pharmacological strategies targeting muscle insulin sensitivity in humans.

Insulin-induced membrane permeability to glucose in human muscles at rest and following exercise.

KEY POINTS: Increased insulin action is an important component of the health benefits of exercise, but its regulation is complex and not fully elucidated. Previous studies of insulin-stimulated GLUT4 translocation to the skeletal muscle membrane found insufficient increases to explain the increases in glucose uptake. By determination of leg glucose uptake and interstitial muscle glucose concentration, insulin-induced muscle membrane permeability to glucose was calculated 4 h after one-legged knee-extensor exercise during a submaximal euglycaemic-hyperinsulinaemic clamp. It was found that during submaximal insulin stimulation, muscle membrane permeability to glucose in humans increases twice as much in previously exercised vs. rested muscle and outstrips the supply of glucose, which then becomes limiting for glucose uptake. This methodology can now be employed to determine muscle membrane permeability to glucose in people with diabetes, who have reduced insulin action, and in principle can also be used to determine membrane permeability to other substrates or metabolites. ABSTRACT: Increased insulin action is an important component of the health benefits of exercise, but the regulation of insulin action in vivo is complex and not fully elucidated. Previously determined increases in skeletal muscle insulin-stimulated GLUT4 translocation are inconsistent and mostly cannot explain the increases in insulin action in humans. Here we used leg glucose uptake (LGU) and interstitial muscle glucose concentration to calculate insulin-induced muscle membrane permeability to glucose, a variable not previously possible to quantify in humans. Muscle membrane permeability to glucose, measured 4 h after one-legged knee-extensor exercise, increased ∼17-fold during a submaximal euglycaemic-hyperinsulinaemic clamp in rested muscle (R) and ∼36-fold in exercised muscle (EX). Femoral arterial infusion of NG -monomethyl l-arginine acetate or ATP decreased and increased, respectively, leg blood flow (LBF) in both legs but did not affect membrane glucose permeability. Decreasing LBF reduced interstitial glucose concentrations to ∼2 mM in the exercised but only to ∼3.5 mM in non-exercised muscle and abrogated the augmented effect of insulin on LGU in the EX leg. Increasing LBF by ATP infusion increased LGU in both legs with uptake higher in the EX leg. We conclude that it is possible to measure functional muscle membrane permeability to glucose in humans and it increases twice as much in exercised vs. rested muscle during submaximal insulin stimulation. We also show that muscle perfusion is an important regulator of muscle glucose uptake when membrane permeability to glucose is high and we show that the capillary wall can be a significant barrier for glucose transport.

Fatty acid type-specific regulation of SIRT1 does not affect insulin sensitivity in human skeletal muscle.

The nicotinamide adenine dinucleotide-dependent deacetylase, sirtuin (SIRT)1, in skeletal muscle is reduced in insulin-resistant states. However, whether this is an initial mechanism responsible for mediating insulin resistance in human skeletal muscle remains to be investigated. Also, SIRT1 acts as a mitochondrial gene transcriptional regulator and is induced by a short-term, high-fat diet (HFD) in human skeletal muscle. Whether saturated or unsaturated fatty acids (FAs) in the diet are important for this is unknown. We subjected 17 healthy, young men to a eucaloric control (Con) diet and 1 of 2 hypercaloric [+75% energy (E%)] HFDs for 3 d enriched in either saturated (Sat) FA (79 E% fat; Sat) or unsaturated FA (78 E% fat; Unsat). After Sat, SIRT1 protein content and activity in skeletal muscle increased ( P < 0.05; ∼40%) while remaining unchanged after Unsat. Whole-body insulin sensitivity and insulin-stimulated leg glucose uptake were reduced ( P < 0.01; ∼20%) to a similar extent compared to Con after both HFDs. We demonstrate a novel FA type-dependent regulation of SIRT1 protein in human skeletal muscle. Moreover, regulation of SIRT1 does not seem to be an initiating factor responsible for mediating insulin resistance in human skeletal muscle.-Fritzen, A. M., Lundsgaard, A.-M., Jeppesen, J. F., Sjøberg, K. A., Høeg, L. D., Deleuran, H. H., Wojtaszewski, J. F. P., Richter, E. A., Kiens, B. Fatty acid type-specific regulation of SIRT1 does not affect insulin sensitivity in human skeletal muscle.

Exercise training reduces the insulin-sensitizing effect of a single bout of exercise in human skeletal muscle.

KEY POINTS: A single bout of exercise is capable of increasing insulin sensitivity in human skeletal muscle. Whether this ability is affected by training status is not clear. Studies in mice suggest that the AMPK-TBC1D4 signalling axis is important for the increased insulin-stimulated glucose uptake after a single bout of exercise. The present study is the first longitudinal intervention study to show that, although exercise training increases insulin-stimulated glucose uptake in skeletal muscle at rest, it diminishes the ability of a single bout of exercise to enhance muscle insulin-stimulated glucose uptake. The present study provides novel data indicating that AMPK in human skeletal muscle is important for the insulin-sensitizing effect of a single bout of exercise. ABSTRACT: Not only chronic exercise training, but also a single bout of exercise, increases insulin-stimulated glucose uptake in skeletal muscle. However, it is not well described how adaptations to exercise training affect the ability of a single bout of exercise to increase insulin sensitivity. Rodent studies suggest that the insulin-sensitizing effect of a single bout of exercise is AMPK-dependent (presumably via the α2 ß2 γ3 AMPK complex). Whether this is also the case in humans is unknown. Previous studies have shown that exercise training decreases the expression of the α2 ß2 γ3 AMPK complex and diminishes the activation of this complex during exercise. Thus, we hypothesized that exercise training diminishes the ability of a single bout of exercise to enhance muscle insulin sensitivity. We investigated nine healthy male subjects who performed one-legged knee-extensor exercise at the same relative intensity before and after 12 weeks of exercise training. Training increased V̇O2peak and expression of mitochondrial proteins in muscle, whereas the expression of AMPKγ3 was decreased. Training also increased whole body and muscle insulin sensitivity. Interestingly, insulin-stimulated glucose uptake in the acutely exercised leg was not enhanced further by training. Thus, the increase in insulin-stimulated glucose uptake following a single bout of one-legged exercise was lower in the trained vs. untrained state. This was associated with reduced signalling via confirmed α2 ß2 γ3 AMPK downstream targets (ACC and TBC1D4). These results suggest that the insulin-sensitizing effect of a single bout of exercise is also AMPK-dependent in human skeletal muscle.

Effect of bariatric surgery on plasma GDF15 in humans.

Bariatric surgery results in marked body weight loss and improves type 2 diabetes in most patients with obesity. The growth differentiation factor 15 (GDF15) has recently emerged as a novel satiety factor. To begin to understand whether GDF15 is involved in mediating the effects of bariatric surgery on body weight and glycemia in humans, we measured plasma GDF15 in patients with obesity ( n = 25) and in patients with obesity and diabetes ( n = 22) before and after Roux-en-Y gastric bypass (RYGB) surgery. GDF15 was increased 1 wk after RYGB compared with before surgery (689 ± 45 vs. 487 ± 28 pg/ml, P < 0.001) and GDF15 remained elevated at 3 mo (554 ± 37 pg/ml, P < 0.05), at 1 yr (566 ± 37 pg/ml, P < 0.05), and at 2.5-4 yr (630 ± 50 pg/ml, P < 0.001) after RYGB surgery. Both age and insulin sensitivity correlated with GDF15 before the surgery ( r = 0.46, P < 0.0001 and r = 0.34, P < 0.001, respectively). These correlations disappeared at 2.5-4 yr following the surgery. Conversely, weight loss magnitude correlated with GDF15, measured 2.5-4 yr postsurgery ( r = 0.21, P < 0.0055). In summary, circulating GDF15 increases and correlates with body weight loss following RYGB surgery.

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. 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.

Multiplexed Temporal Quantification of the Exercise-regulated Plasma Peptidome.

Exercise is extremely beneficial to whole body health reducing the risk of a number of chronic human diseases. Some of these physiological benefits appear to be mediated via the secretion of peptide/protein hormones into the blood stream. The plasma peptidome contains the entire complement of low molecular weight endogenous peptides derived from secretion, protease activity and PTMs, and is a rich source of hormones. In the current study we have quantified the effects of intense exercise on the plasma peptidome to identify novel exercise regulated secretory factors in humans. We developed an optimized 2D-LC-MS/MS method and used multiple fragmentation methods including HCD and EThcD to analyze endogenous peptides. This resulted in quantification of 5,548 unique peptides during a time course of exercise and recovery. The plasma peptidome underwent dynamic and large changes during exercise on a time-scale of minutes with many rapidly reversible following exercise cessation. Among acutely regulated peptides, many were known hormones including insulin, glucagon, ghrelin, bradykinin, cholecystokinin and secretogranins validating the method. Prediction of bioactive peptides regulated with exercise identified C-terminal peptides from Transgelins, which were increased in plasma during exercise. In vitro experiments using synthetic peptides identified a role for transgelin peptides on the regulation of cell-cycle, extracellular matrix remodeling and cell migration. We investigated the effects of exercise on the regulation of PTMs and proteolytic processing by building a site-specific network of protease/substrate activity. Collectively, our deep peptidomic analysis of plasma revealed that exercise rapidly modulates the circulation of hundreds of bioactive peptides through a network of proteases and PTMs. These findings illustrate that peptidomics is an ideal method for quantifying changes in circulating factors on a global scale in response to physiological perturbations such as exercise. This will likely be a key method for pinpointing exercise regulated factors that generate health benefits.

Serum Is Not Necessary for Prior Pharmacological Activation of AMPK to Increase Insulin Sensitivity of Mouse Skeletal Muscle.

Exercise, contraction, and pharmacological activation of AMP-activated protein kinase (AMPK) by 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) have all been shown to increase muscle insulin sensitivity for glucose uptake. Intriguingly, improvements in insulin sensitivity following contraction of isolated rat and mouse skeletal muscle and prior AICAR stimulation of isolated rat skeletal muscle seem to depend on an unknown factor present in serum. One study recently questioned this requirement of a serum factor by showing serum-independency with muscle from old rats. Whether a serum factor is necessary for prior AICAR stimulation to increase insulin sensitivity of mouse skeletal muscle is not known. Therefore, we investigated the necessity of serum for this effect of AICAR in mouse skeletal muscle. We found that the ability of prior AICAR stimulation to improve insulin sensitivity of mouse skeletal muscle did not depend on the presence of serum during AICAR stimulation. Although prior AICAR stimulation did not enhance proximal insulin signaling, insulin-stimulated phosphorylation of Tre-2/BUB2/CDC16- domain family member 4 (TBC1D4) Ser711 was greater in prior AICAR-stimulated muscle compared to all other groups. These results imply that the presence of a serum factor is not necessary for prior AMPK activation by AICAR to enhance insulin sensitivity of mouse skeletal muscle.

Abnormal epigenetic changes during differentiation of human skeletal muscle stem cells from obese subjects.

BACKGROUND: Human skeletal muscle stem cells are important for muscle regeneration. However, the combined genome-wide DNA methylation and expression changes taking place during adult myogenesis have not been described in detail and novel myogenic factors may be discovered. Additionally, obesity is associated with low relative muscle mass and diminished metabolism. Epigenetic alterations taking place during myogenesis might contribute to these defects. METHODS: We used Infinium HumanMethylation450 BeadChip Kit (Illumina) and HumanHT-12 Expression BeadChip (Illumina) to analyze genome-wide DNA methylation and transcription before versus after differentiation of primary human myoblasts from 14 non-obese and 14 obese individuals. Functional follow-up experiments were performed using siRNA mediated gene silencing in primary human myoblasts and a transgenic mouse model. RESULTS: We observed genome-wide changes in DNA methylation and expression patterns during differentiation of primary human muscle stem cells (myoblasts). We identified epigenetic and transcriptional changes of myogenic transcription factors (MYOD1, MYOG, MYF5, MYF6, PAX7, MEF2A, MEF2C, and MEF2D), cell cycle regulators, metabolic enzymes and genes previously not linked to myogenesis, including IL32, metallothioneins, and pregnancy-specific beta-1-glycoproteins. Functional studies demonstrated IL-32 as a novel target that regulates human myogenesis, insulin sensitivity and ATP levels in muscle cells. Furthermore, IL32 transgenic mice had reduced insulin response and muscle weight. Remarkably, approximately 3.7 times more methylation changes (147,161 versus 39,572) were observed during differentiation of myoblasts from obese versus non-obese subjects. In accordance, DNMT1 expression increased during myogenesis only in obese subjects. Interestingly, numerous genes implicated in metabolic diseases and epigenetic regulation showed differential methylation and expression during differentiation only in obese subjects. CONCLUSIONS: Our study identifies IL-32 as a novel myogenic regulator, provides a comprehensive map of the dynamic epigenome during differentiation of human muscle stem cells and reveals abnormal epigenetic changes in obesity.

Intact initiation of autophagy and mitochondrial fission by acute exercise in skeletal muscle of patients with Type 2 diabetes.

Type 2 diabetes (T2D) is characterized by insulin resistance, mitochondrial dysregulation and, in some studies, exercise resistance in skeletal muscle. Regulation of autophagy and mitochondrial dynamics during exercise and recovery is important for skeletal muscle homoeostasis, and these responses may be altered in T2D. We examined the effect of acute exercise on markers of autophagy and mitochondrial fusion and fission in skeletal muscle biopsies from patients with T2D (n=13) and weight-matched controls (n=14) before, immediately after and 3 h after an acute bout of exercise. Although mRNA levels of most markers of autophagy [PIK3C, MAP1LC3B, sequestosome 1 (SQSTM1), BCL-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), BNIP3-like (BNIP3L)] and mitochondrial dynamics [optic atrophy 1 (OPA1), fission protein 1 (FIS1)] remained unchanged, some either increased during and after exercise (GABARAPL1), decreased in the recovery period [BECN1, autophagy-related (ATG) 7, DNM1L] or both [mitofusin (MFN) 2, mitochondrial E3 ubiquitin ligase 1 (MUL1)]. Protein levels of ATG7, p62/SQSTM1, forkhead box O3A (FOXO3A) and MFN2 (only controls) as well as dynamin-related protein 1 (DRP1) Ser616 phosphorylation increased in response to exercise and/or recovery, whereas microtubule-associated protein 1 light chain 3B (LC3B)-II content was reduced immediately after exercise. Exercise increased the activating Ser555 phosphorylation and reduced the inhibitory Ser757 phosphorylation of Unc-51-like kinase-1 (ULK1). The LC3B-II content and phosphorylation of ULK1 and DRP1 returned towards pre-exercise levels in the recovery period. Insulin sensitivity was reduced in T2D, but with no differences in the autophagic response to exercise. Our results demonstrate that initiation of autophagy and mitochondrial fission is activated by exercise in human skeletal muscle, and that these responses are intact in T2D. The exercise-induced decrease in LC3B-II could be due to increased autophagic turnover.

AMPKα is critical for enhancing skeletal muscle fatty acid utilization during in vivo exercise in mice.

The importance of AMPK in regulation of fatty acid (FA) oxidation in skeletal muscle with contraction/exercise is unresolved. Using a mouse model lacking both AMPKα1 and -α2 in skeletal muscle specifically (mdKO), we hypothesized that FA utilization would be impaired in skeletal muscle. AMPKα mdKO mice displayed normal respiratory exchange ratio (RER) when fed chow or a high-fat diet, or with prolonged fasting. However, in vivo treadmill exercise at the same relative intensity induced a higher RER in AMPKα mdKO mice compared to wild-type (WT = 0.81 ± 0.01 (sem); mdKO = 0.87 ± 0.02 (sem); P < 0.01), indicating a decreased utilization of FA. Further, ex vivo contraction-induced FA oxidation was impaired in AMPKα mdKO muscle, suggesting that the increased RER during exercise originated from decreased skeletal muscle FA oxidation. A decreased muscle protein expression of CD36 (cluster of differentiation 36) and FABPpm (plasma membrane fatty acid binding protein) (by ∼17-40%), together with fully abolished TBC1D1 (tre-2/USP6, BUB2, cdc16 domain family member 1) Ser(237) phosphorylation during contraction/exercise in AMPKα mdKO mice, may impair FA transport capacity and FA transport protein translocation to sarcolemma, respectively. AMPKα is thus required for normal FA metabolism during exercise and muscle contraction.

Dysregulation of muscle glycogen synthase in recovery from exercise in type 2 diabetes.

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 [VO2max]). 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.