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.

Pharmacological activation of PDC flux reverses lipid-induced inhibition of insulin action in muscle during recovery from exercise.

Insulin resistance is a risk factor for type 2 diabetes and exercise can improve insulin sensitivity. However, following exercise high circulating fatty acid (FA) levels might counteract this. We hypothesized that such inhibition would be reduced by forcibly increasing carbohydrate oxidation through pharmacological activation of the pyruvate dehydrogenase complex (PDC). Insulin-stimulated glucose uptake was examined with a cross-over design in healthy young men (n = 8) in a previously exercised and a rested leg during a hyperinsulinemiceuglycemic clamp five hours after one-legged exercise with: 1) infusion of saline, 2) infusion of intralipid imitating circulating FA levels during recovery from whole-body exercise, and 3) infusion of intralipid + oral PDC-activator, dichloroacetate (DCA). Intralipid infusion reduced insulin-stimulated glucose uptake by 19% in the previously exercised leg, which was not observed in the contralateral rested leg. Interestingly, this effect of intralipid in the exercised leg was abolished by DCA, which increased muscle PDC activity (130%) and flux (acetylcarnitine 130%) and decreased inhibitory phosphorylation of PDC on Ser293 (∼40%) and Ser300 (∼80%). Novel insight is provided into the regulatory interaction between glucose and lipid metabolism during exercise recovery. Coupling exercise and PDC flux activation upregulated the capacity for both glucose transport (exercise) and oxidation (DCA), which seems necessary to fully stimulate insulin-stimulated glucose uptake during recovery.

Systemic proteome adaptions to 7-day complete caloric restriction in humans.

Surviving long periods without food has shaped human evolution. In ancient and modern societies, prolonged fasting was/is practiced by billions of people globally for religious purposes, used to treat diseases such as epilepsy, and recently gained popularity as weight loss intervention, but we still have a very limited understanding of the systemic adaptions in humans to extreme caloric restriction of different durations. Here we show that a 7-day water-only fast leads to an average weight loss of 5.7 kg (±0.8 kg) among 12 volunteers (5 women, 7 men). We demonstrate nine distinct proteomic response profiles, with systemic changes evident only after 3 days of complete calorie restriction based on in-depth characterization of the temporal trajectories of ~3,000 plasma proteins measured before, daily during, and after fasting. The multi-organ response to complete caloric restriction shows distinct effects of fasting duration and weight loss and is remarkably conserved across volunteers with >1,000 significantly responding proteins. The fasting signature is strongly enriched for extracellular matrix proteins from various body sites, demonstrating profound non-metabolic adaptions, including extreme changes in the brain-specific extracellular matrix protein tenascin-R. Using proteogenomic approaches, we estimate the health consequences for 212 proteins that change during fasting across ~500 outcomes and identified putative beneficial (SWAP70 and rheumatoid arthritis or HYOU1 and heart disease), as well as adverse effects. Our results advance our understanding of prolonged fasting in humans beyond a merely energy-centric adaptions towards a systemic response that can inform targeted therapeutic modulation.

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.

Acute Exercise Increases GDF15 and Unfolded Protein Response/Integrated Stress Response in Muscle in Type 2 diabetes.

CONTEXT: Regular exercise is a key prevention strategy for obesity and type 2 diabetes (T2D). Exerkines secreted in response to exercise or recovery may contribute to improved systemic metabolism. Conversely, an impaired exerkine response to exercise and recovery may contribute to cardiometabolic diseases. OBJECTIVE: We investigated if the exercise-induced regulation of the exerkine, growth/differentiation factor 15 (GDF15) and its putative upstream regulators of the unfolded protein response (UPR)/integrated stress response (ISR) is impaired in skeletal muscle in patients with T2D compared with weight-matched glucose-tolerant men. METHODS: Thirteen male patients with T2D and 14 age- and weight-matched overweight/obese glucose-tolerant men exercised at 70% of VO2max for 1-h. Blood and skeletal muscle biopsies were sampled before, immediately after, and 3-h into recovery. Serum and muscle transcript levels of GDF15 and key markers of UPR/ISR were determined. Additionally, protein/phosphorylation levels of key regulators in UPR/ISR were investigated. RESULTS: Acute exercise increased muscle gene expression and serum GDF15 levels in both groups. In recovery, muscle expression of GDF15 decreased toward baseline, whereas serum GDF15 remained elevated. In both groups, acute exercise increased the expression of UPR/ISR markers, including ATF4, CHOP, EIF2K3 (encoding PERK) and PPP1R15A (encoding GADD34), of which only CHOP remained elevated 3-h into recovery. Downstream molecules of the UPR/ISR including XBP1-U, XBP1-S, and EDEM1 were increased with exercise and 3-h into recovery in both groups. The phosphorylation levels of eIF2α-Ser51, a common marker of UPR and ISR, increased immediately after exercise in controls, but decreased 3-h into recovery in both groups. CONCLUSION: In conclusion, exercise-induced regulation of GDF15 and key markers of UPR/ISR are not compromised in patients with type 2 diabetes compared with weight-matched controls.

AMPKγ3 Controls Muscle Glucose Uptake in Recovery From Exercise to Recapture Energy Stores.

Exercise increases muscle glucose uptake independently of insulin signaling and represents a cornerstone for the prevention of metabolic disorders. Pharmacological activation of the exercise-responsive AMPK in skeletal muscle has been proven successful as a therapeutic approach to treat metabolic disorders by improving glucose homeostasis through the regulation of muscle glucose uptake. However, conflicting observations cloud the proposed role of AMPK as a necessary regulator of muscle glucose uptake during exercise. We show that glucose uptake increases in human skeletal muscle in the absence of AMPK activation during exercise and that exercise-stimulated AMPKγ3 activity strongly correlates to muscle glucose uptake in the postexercise period. In AMPKγ3-deficient mice, muscle glucose uptake is normally regulated during exercise and contractions but impaired in the recovery period from these stimuli. Impaired glucose uptake in recovery from exercise and contractions is associated with a lower glucose extraction, which can be explained by a diminished permeability to glucose and abundance of GLUT4 at the muscle plasma membrane. As a result, AMPKγ3 deficiency impairs muscle glycogen resynthesis following exercise. These results identify a physiological function of the AMPKγ3 complex in human and rodent skeletal muscle that regulates glucose uptake in recovery from exercise to recapture muscle energy stores. ARTICLE HIGHLIGHTS: Exercise-induced activation of AMPK in skeletal muscle has been proposed to regulate muscle glucose uptake in recovery from exercise. This study investigated whether the muscle-specific AMPKγ3-associated heterotrimeric complex was involved in regulating muscle glucose metabolism in recovery from exercise. The findings support that exercise-induced activation of the AMPKγ3 complex in human and mouse skeletal muscle enhances glucose uptake in recovery from exercise via increased translocation of GLUT4 to the plasma membrane. This work uncovers the physiological role of the AMPKγ3 complex in regulating muscle glucose uptake that favors replenishment of the muscle cellular energy stores.

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.

Microtubule-mediated GLUT4 trafficking is disrupted in insulin-resistant skeletal muscle.

Microtubules serve as tracks for long-range intracellular trafficking of glucose transporter 4 (GLUT4), but the role of this process in skeletal muscle and insulin resistance is unclear. Here, we used fixed and live-cell imaging to study microtubule-based GLUT4 trafficking in human and mouse muscle fibers and L6 rat muscle cells. We found GLUT4 localized on the microtubules in mouse and human muscle fibers. Pharmacological microtubule disruption using Nocodazole (Noco) prevented long-range GLUT4 trafficking and depleted GLUT4-enriched structures at microtubule nucleation sites in a fully reversible manner. Using a perifused muscle-on-a-chip system to enable real-time glucose uptake measurements in isolated mouse skeletal muscle fibers, we observed that Noco maximally disrupted the microtubule network after 5 min without affecting insulin-stimulated glucose uptake. In contrast, a 2-hr Noco treatment markedly decreased insulin responsiveness of glucose uptake. Insulin resistance in mouse muscle fibers induced either in vitro by C2 ceramides or in vivo by diet-induced obesity, impaired microtubule-based GLUT4 trafficking. Transient knockdown of the microtubule motor protein kinesin-1 protein KIF5B in L6 muscle cells reduced insulin-stimulated GLUT4 translocation while pharmacological kinesin-1 inhibition in incubated mouse muscles strongly impaired insulin-stimulated glucose uptake. Thus, in adult skeletal muscle fibers, the microtubule network is essential for intramyocellular GLUT4 movement, likely functioning to maintain an insulin-responsive cell surface recruitable GLUT4 pool via kinesin-1-mediated trafficking.

TBC1D4-S711 Controls Skeletal Muscle Insulin Sensitization After Exercise and Contraction.

The ability of insulin to stimulate glucose uptake in skeletal muscle is important for whole-body glycemic control. Insulin-stimulated skeletal muscle glucose uptake is improved in the period after a single bout of exercise, and accumulating evidence suggests that phosphorylation of TBC1D4 by the protein kinase AMPK is the primary mechanism responsible for this phenomenon. To investigate this, we generated a TBC1D4 knock-in mouse model with a serine-to-alanine point mutation at residue 711 that is phosphorylated in response to both insulin and AMPK activation. Female TBC1D4-S711A mice exhibited normal growth and eating behavior as well as intact whole-body glycemic control on chow and high-fat diets. Moreover, muscle contraction increased glucose uptake, glycogen utilization, and AMPK activity similarly in wild-type and TBC1D4-S711A mice. In contrast, improvements in whole-body and muscle insulin sensitivity after exercise and contractions were only evident in wild-type mice and occurred concomitantly with enhanced phosphorylation of TBC1D4-S711. These results provide genetic evidence to support that TBC1D4-S711 serves as a major point of convergence for AMPK- and insulin-induced signaling that mediates the insulin-sensitizing effect of exercise and contractions on skeletal muscle glucose uptake.

Ketone Body Infusion Abrogates Growth Hormone-Induced Lipolysis and Insulin Resistance.

CONTEXT: Exogenous ketone body administration lowers circulating glucose levels but the underlying mechanisms are uncertain. OBJECTIVE: We tested the hypothesis that administration of the ketone body ß-hydroxybutyrate (ßOHB) acutely increases insulin sensitivity via feedback suppression of circulating free fatty acid (FFA) levels. METHODS: In a randomized, single-blinded crossover design, 8 healthy men were studied twice with a growth hormone (GH) infusion to induce lipolysis in combination with infusion of either ßOHB or saline. Each study day comprised a basal period and a hyperinsulinemic-euglycemic clamp combined with a glucose tracer and adipose tissue and skeletal muscle biopsies. RESULTS: ßOHB administration profoundly suppressed FFA levels concomitantly with a significant increase in glucose disposal and energy expenditure. This was accompanied by a many-fold increase in skeletal muscle content of both ßOHB and its derivative acetoacetate. CONCLUSION: Our data unravel an insulin-sensitizing effect of ßOHB, which we suggest is mediated by concomitant suppression of lipolysis.

Greater Phosphorylation of AMPK and Multiple AMPK Substrates in the Skeletal Muscle of 24-Month-Old Calorie Restricted Compared to Ad-Libitum Fed Male Rats.

AMP-activated protein kinase (AMPK), a highly conserved, heterotrimeric serine/threonine kinase with critical sensory and regulatory functions, is proposed to induce antiaging actions of caloric restriction (CR). Although earlier studies assessed CR's effects on AMPK in rodent skeletal muscle, the scope of these studies was narrow with a limited focus on older animals. This study's purpose was to fill important knowledge gaps related to CR's influence on AMPK in skeletal muscle of older animals. Therefore, using epitrochlearis muscles from 24-month-old ad-libitum fed (AL) and CR (consuming 65% of AL intake for 8 weeks), male Fischer-344 × Brown Norway F1 rats, we determined: (a) AMPK Thr172 phosphorylation (a key regulatory site) by immunoblot; (b) AMPKα1 and AMPKα2 activity (representing the 2 catalytic α-subunits of AMPK), and AMPKγ3 activity (representing AMPK complexes that include the skeletal muscle-selective regulatory γ3 subunit) using enzymatic assays; (c) phosphorylation of multiple protein substrates that are linked to CR-related effects (acetyl-CoA carboxylase [ACC], that regulates lipid oxidation; Beclin-1 and ULK1 that are autophagy regulatory proteins; Raptor, mTORC1 complex protein that regulates autophagy; TBC1D1 and TBC1D4 that regulate glucose uptake) by immunoblot; and (d) ATP and AMP concentrations (key AMPK regulators) by mass spectrometry. The results revealed significant CR-associated increases in the phosphorylation of AMPKThr172 and 4 AMPK substrates (ACC, Beclin-1, TBC1D1, and TBC1D4), without significant diet-related differences in ATP or AMP concentration or AMPKα1-, AMPKα2-, or AMPKγ3-associated activity. The enhanced phosphorylation of multiple AMPK substrates provides novel mechanistic insights linking AMPK to functionally important consequences of CR.

Insulin Sensitization Following a Single Exercise Bout Is Uncoupled to Glycogen in Human Skeletal Muscle: A Meta-analysis of 13 Single-Center Human Studies.

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.

Illumination of the Endogenous Insulin-Regulated TBC1D4 Interactome in Human Skeletal Muscle.

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 that involves regulatory proteins such as TBC1D1 and TBC1D4. Accordingly, a TBC1D4 loss-of-function mutation in human skeletal muscle is associated with an 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 protein in human skeletal muscle by an unbiased proteomics approach. We detected 76 proteins as candidate TBC1D4 interactors. The binding of 12 of these interactors was regulated by insulin, including proteins known to be involved in glucose metabolism (e.g., 14-3-3 proteins and α-actinin-4 [ACTN4]). TBC1D1 also coprecipitated with TBC1D4 and vice versa in both human and mouse skeletal muscle. This interaction was not regulated by insulin or 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 phosphoregulation 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 that serve as potential regulators of TBC1D4 function and, thus, insulin-stimulated glucose uptake in human skeletal muscle.

Factors mediating exercise-induced organ crosstalk.

Exercise activates a plethora of metabolic and signalling pathways in skeletal muscle and other organs causing numerous systemic beneficial metabolic effects. Thus, regular exercise may ameliorate and prevent the development of several chronic metabolic diseases. Skeletal muscle is recognized as an important endocrine organ regulating systemic adaptations to exercise. Skeletal muscle may mediate crosstalk with other organs through the release of exercise-induced cytokines, peptides and proteins, termed myokines, into the circulation. Importantly, other tissues such as the liver and adipose tissue may also release cytokines and peptides in response to exercise. Hence, exercise-released molecules are collectively called exerkines. Moreover, extracellular vesicles (EVs), in the form of exosomes or microvesicles, may carry some of the signals involved in tissue crosstalk. This review focuses on the role of factors potentially mediating crosstalk between muscle and other tissues in response to exercise.

Personalized phosphoproteomics identifies functional signaling.

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.

Functional sympatholysis in mouse skeletal muscle involves sarcoplasmic reticulum swelling in arterial smooth muscle cells.

The vasoconstrictive effect of sympathetic activity is attenuated in contracting skeletal muscle (functional sympatholysis), allowing increased blood supply to the working muscle but the underlying mechanisms are incompletely understood. The purpose of this study was to examine α-adrenergic receptor responsiveness in isolated artery segments from non-exercised and exercised mice, using wire myography. Isometric tension recordings performed on femoral artery segments from exercised mice showed decreased α-adrenergic receptor responsiveness compared to non-exercised mice (logEC50 -5.2 ± 0.04 M vs. -5.7 ± 0.08 M, respectively). In contrast, mesenteric artery segments from exercised mice displayed similar α-adrenergic receptor responses compared to non-exercised mice. Responses to the vasoconstrictor serotonin (5-HT) and vasodilator isoprenaline, were similar in femoral artery segments from non-exercised and exercised mice. To study sarcoplasmic reticulum (SR) function, we examined arterial contractions induced by caffeine, which depletes SR Ca2+ and thapsigargin, which inhibits SR Ca2+ -ATPase (SERCA) and SR Ca2+ uptake. Arterial contractions to both caffeine and thapsigargin were increased in femoral artery segment from exercised compared to non-exercised mice. Furthermore, 3D electron microscopy imaging of the arterial wall showed SR volume/length ratio increased 157% in smooth muscle cells of the femoral artery from the exercised mice, whereas there was no difference in SR volume/length ratio in mesenteric artery segments. These results show that in arteries surrounding exercising muscle, the α-adrenergic receptor constrictions are blunted, which can be attributed to swollen smooth muscle cell SR's, likely due to increased Ca2+ content that is possibly reducing free intracellular Ca2+ available for contraction. Overall, this study uncovers a previously unknown mechanism underlying functional sympatholysis.

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice.

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been reported that skeletal muscle may increase the extraction of glucose from the blood by up to 50-fold during exercise compared to resting conditions. The increase in muscle glucose uptake during exercise and insulin stimulation is dependent on the translocation of glucose transporter 4 (GLUT4) from intracellular compartments to the muscle cell surface membrane, as well as phosphorylation of glucose to glucose-6-phosphate by hexokinase II. Isolation and incubation of mouse muscles such as m. soleus and m. extensor digitorum longus (EDL) is an appropriate ex vivo model to study the effects of insulin and electrically-induced contraction (a model for exercise) on glucose uptake in mature skeletal muscle. Thus, the ex vivo model permits evaluation of muscle insulin sensitivity and makes it possible to match muscle force production during contraction ensuring uniform recruitment of muscle fibers during measurements of muscle glucose uptake. Moreover, the described model is suitable for pharmacological compound testing that may have an impact on muscle insulin sensitivity or may be of help when trying to delineate the regulatory complexity of skeletal muscle glucose uptake. Here we describe and provide a detailed protocol on how to measure insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle preparations from mice using radiolabeled [3H]2-deoxy-D-glucose and [14C]mannitol as an extracellular marker. This allows accurate assessment of glucose uptake in mature skeletal muscle in the absence of confounding factors that may interfere in the intact animal model. In addition, we provide information on metabolic viability of incubated mouse skeletal muscle suggesting that the method applied possesses some caveats under certain conditions when studying muscle energy metabolism.

Effect of exercise training on skeletal muscle protein expression in relation to insulin sensitivity: Per-protocol analysis of a randomized controlled trial (GO-ACTIWE).

Exercise training improves peripheral insulin sensitivity and leads to molecular adaptations in the skeletal muscle. We investigated changes in the expression of key muscle proteins in the glucose metabolic pathway following active commuting by bike or leisure-time exercise at two different intensities. In addition, potential associations between insulin sensitivity and muscle protein expression were examined. This per-protocol analysis included 72 out of 130 physically inactive, healthy women and men (20-45 years) with overweight/obesity (BMI: 25-35 kg/m2 ) who completed 6 months of no intervention (CON, n = 12), active commuting by bike (BIKE, n = 14), or leisure-time exercise of moderate (MOD, n = 28) or vigorous (VIG, n = 18) intensity. Exercise was prescribed 5 days/week with a weekly exercise energy expenditure of 1,600 kcal for women and 2,100 kcal for men. Insulin sensitivity was determined by a hyperinsulinemic euglycemic clamp and skeletal muscle biopsies were obtained from m. vastus lateralis and analyzed for protein expression at baseline and after 3 and 6 months of intervention. We found an increased expression of pyruvate dehydrogenase (PDH) in the exercise groups compared with the control group following 6 months of training. No differential effects were observed on the protein expression following moderate versus vigorous intensity exercise. In addition, we found a positive association between insulin sensitivity and the expression of glucose transporter type 4 as well as PDH. The positive association and the increase in expression of PDH after exercise training points toward a role for PDH in the training-induced enhancement of insulin sensitivity.

Direct small molecule ADaM-site AMPK activators reveal an AMPKγ3-independent mechanism for blood glucose lowering.

OBJECTIVE: Skeletal muscle is an attractive target for blood glucose-lowering pharmacological interventions. Oral dosing of small molecule direct pan-activators of AMPK that bind to the allosteric drug and metabolite (ADaM) site, lowers blood glucose through effects in skeletal muscle. The molecular mechanisms responsible for this effect are not described in detail. This study aimed to illuminate the mechanisms by which ADaM-site activators of AMPK increase glucose uptake in skeletal muscle. Further, we investigated the consequence of co-stimulating muscles with two types of AMPK activators i.e., ADaM-site binding small molecules and the prodrug AICAR. METHODS: The effect of the ADaM-site binding small molecules (PF739 and 991), AICAR or co-stimulation with PF739 or 991 and AICAR on muscle glucose uptake was investigated ex vivo in m. extensor digitorum longus (EDL) excised from muscle-specific AMPKα1α2 as well as whole-body AMPKγ3-deficient mouse models. In vitro complex-specific AMPK activity was measured by immunoprecipitation and molecular signaling was assessed by western blotting in muscle lysate. To investigate the transferability of these studies, we treated diet-induced obese mice in vivo with PF739 and measured complex-specific AMPK activation in skeletal muscle. RESULTS: Incubation of skeletal muscle with PF739 or 991 increased skeletal muscle glucose uptake in a dose-dependent manner. Co-incubating PF739 or 991 with a maximal dose of AICAR increased glucose uptake to a greater extent than any of the treatments alone. Neither PF739 nor 991 increased AMPKα2ß2γ3 activity to the same extent as AICAR, while co-incubation led to potentiated effects on AMPKα2ß2γ3 activation. In muscle from AMPKγ3 KO mice, AICAR-stimulated glucose uptake was ablated. In contrast, the effect of PF739 or 991 on glucose uptake was not different between WT and AMPKγ3 KO muscles. In vivo PF739 treatment lowered blood glucose levels and increased muscle AMPKγ1-complex activity 2-fold, while AMPKα2ß2γ3 activity was not affected. CONCLUSIONS: ADaM-site binding AMPK activators increase glucose uptake independently of AMPKγ3. Co-incubation with PF739 or 991 and AICAR potentiates the effects on muscle glucose uptake and AMPK activation. In vivo, PF739 lowers blood glucose and selectively activates muscle AMPKγ1-complexes. Collectively, this suggests that pharmacological activation of AMPKγ1-containing complexes in skeletal muscle can increase glucose uptake and can lead to blood glucose lowering.