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.

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.

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.

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.

Skeletal muscle adaptations to exercise are not influenced by metformin treatment in humans: secondary analyses of 2 randomized, clinical trials.

Metformin and exercise both improve glycemic control, but in vitro studies have indicated that an interaction between metformin and exercise occurs in skeletal muscle, suggesting a blunting effect of metformin on exercise training adaptations. Two studies (a double-blind, parallel-group, randomized clinical trial conducted in 29 glucose-intolerant individuals and a double-blind, cross-over trial conducted in 15 healthy lean males) were included in this paper. In both studies, the effect of acute exercise ± metformin treatment on different skeletal muscle variables, previously suggested to be involved in a pharmaco-physiological interaction between metformin and exercise, was assessed. Furthermore, in the parallel-group trial, the effect of 12 weeks of exercise training was assessed. Skeletal muscle biopsies were obtained before and after acute exercise and 12 weeks of exercise training, and mitochondrial respiration, oxidative stress and AMPK activation was determined. Metformin did not significantly affect the effects of acute exercise or exercise training on mitochondrial respiration, oxidative stress or AMPK activation, indicating that the response to acute exercise and exercise training adaptations in skeletal muscle is not affected by metformin treatment. Further studies are needed to investigate whether an interaction between metformin and exercise is present in other tissues, e.g., the gut. Trial registration: ClinicalTrials.gov (NCT03316690 and NCT02951260). Novelty: Metformin does not affect exercise-induced alterations in mitochondrial respiratory capacity in human skeletal muscle. Metformin does not affect exercise-induced alterations in systemic levels of oxidative stress nor emission of reactive oxygen species from human skeletal muscle. Metformin does not affect exercise-induced AMPK activation in human skeletal muscle.

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.

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.

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.

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.

Inducible deletion of skeletal muscle AMPKα reveals that AMPK is required for nucleotide balance but dispensable for muscle glucose uptake and fat oxidation during exercise.

OBJECTIVE: Evidence for AMP-activated protein kinase (AMPK)-mediated regulation of skeletal muscle metabolism during exercise is mainly based on transgenic mouse models with chronic (lifelong) disruption of AMPK function. Findings based on such models are potentially biased by secondary effects related to a chronic lack of AMPK function. To study the direct effect(s) of AMPK on muscle metabolism during exercise, we generated a new mouse model with inducible muscle-specific deletion of AMPKα catalytic subunits in adult mice. METHODS: Tamoxifen-inducible and muscle-specific AMPKα1/α2 double KO mice (AMPKα imdKO) were generated by using the Cre/loxP system, with the Cre under the control of the human skeletal muscle actin (HSA) promoter. RESULTS: During treadmill running at the same relative exercise intensity, AMPKα imdKO mice showed greater depletion of muscle ATP, which was associated with accumulation of the deamination product IMP. Muscle-specific deletion of AMPKα in adult mice promptly reduced maximal running speed and muscle glycogen content and was associated with reduced expression of UGP2, a key component of the glycogen synthesis pathway. Muscle mitochondrial respiration, whole-body substrate utilization, and muscle glucose uptake and fatty acid (FA) oxidation during muscle contractile activity remained unaffected by muscle-specific deletion of AMPKα subunits in adult mice. CONCLUSIONS: Inducible deletion of AMPKα subunits in adult mice reveals that AMPK is required for maintaining muscle ATP levels and nucleotide balance during exercise but is dispensable for regulating muscle glucose uptake, FA oxidation, and substrate utilization during exercise.

A Single Bout of One-Legged Exercise to Local Exhaustion Decreases Insulin Action in Nonexercised Muscle Leading to Decreased Whole-Body Insulin Action.

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 2 separate days: one day with prior one-legged knee-extensor exercise to local exhaustion (∼2.5 h) and another day without exercise. Whole-body glucose disposal was ∼18% lower on the exercise day as compared with the resting day due to decreased (∼37%) insulin-stimulated glucose uptake in the nonexercised muscle. Insulin signaling at the level of Akt2 was impaired in the nonexercised muscle on the exercise day, suggesting that decreased insulin action in nonexercised 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.

Metformin does not compromise energy status in human skeletal muscle at rest or during acute exercise: A randomised, crossover trial.

5´AMP-activated protein kinase (AMPK) is a mediator of a healthy metabolic phenotype in skeletal muscle. Metformin may exacerbate the energy disturbances observed during exercise leading to enhanced AMPK activation, and these disturbances may provoke early muscular fatigue. We studied acute (1 day) and short-term (4 days) effects of metformin treatment on AMPK and its downstream signaling network, in healthy human skeletal muscle and adipose tissue at rest and during exercise, by applying a randomized blinded crossover study design in 10 lean men. Muscle and fat biopsies were obtained before and after the treatment period at rest and after a single bout of exercise. Metformin treat ment elicited peak plasma and muscle metformin concentrations of 31 µM and 11 µM, respectively. Neither of the treatments affected AMPK activity in skeletal muscle and adipose at rest or during exercise. In contrast, whole-body stress during exercise was elevated as indicated by increased plasma lactate and adrenaline concentrations as well as increased heart rate and rate of perceived exertion. Also whole-body insulin sensitivity was enhanced by 4 days metformin treatment, that is reduced fasting plasma insulin and HOMA-IR. In conclusion, acute and short-term metformin treatment does not affect energy homeostasis and AMPK activation at rest or during exercise in skeletal muscle and adipose tissue of healthy subjects. However, metformin treatment is accompanied by slightly enhanced perceived exertion and whole-body stress which may provoke a lesser desire for physical activity in the metformin-treated patients.

AMPK and TBC1D1 Regulate Muscle Glucose Uptake After, but Not During, Exercise and Contraction.

Exercise increases glucose uptake in skeletal muscle independently of insulin signaling. This makes exercise an effective stimulus to increase glucose uptake in insulin-resistant skeletal muscle. AMPK has been suggested to regulate muscle glucose uptake during exercise/contraction, but findings from studies of various AMPK transgenic animals have not reached consensus on this matter. Comparing methods used in these studies reveals a hitherto unappreciated difference between those studies reporting a role of AMPK and those that do not. This led us to test the hypothesis that AMPK and downstream target TBC1D1 are involved in regulating muscle glucose uptake in the immediate period after exercise/contraction but not during exercise/contraction. Here we demonstrate that glucose uptake during exercise/contraction was not compromised in AMPK-deficient skeletal muscle, whereas reversal of glucose uptake toward resting levels after exercise/contraction was markedly faster in AMPK-deficient muscle compared with wild-type muscle. Moreover, muscle glucose uptake after contraction was positively associated with phosphorylation of TBC1D1, and skeletal muscle from TBC1D1-deficient mice displayed impaired glucose uptake after contraction. These findings reconcile previous observed discrepancies and redefine the role of AMPK activation during exercise/contraction as being important for maintaining glucose permeability in skeletal muscle in the period after, but not during, exercise/contraction.

ADAMTS9 Regulates Skeletal Muscle Insulin Sensitivity Through Extracellular Matrix Alterations.

The ADAMTS9 rs4607103 C allele is one of the few gene variants proposed to increase the risk of type 2 diabetes through an impairment of insulin sensitivity. We show that the variant is associated with increased expression of the secreted ADAMTS9 and decreased insulin sensitivity and signaling in human skeletal muscle. In line with this, mice lacking Adamts9 selectively in skeletal muscle have improved insulin sensitivity. The molecular link between ADAMTS9 and insulin signaling was characterized further in a model where ADAMTS9 was overexpressed in skeletal muscle. This selective overexpression resulted in decreased insulin signaling presumably mediated through alterations of the integrin ß1 signaling pathway and disruption of the intracellular cytoskeletal organization. Furthermore, this led to impaired mitochondrial function in mouse muscle-an observation found to be of translational character because humans carrying the ADAMTS9 risk allele have decreased expression of mitochondrial markers. Finally, we found that the link between ADAMTS9 overexpression and impaired insulin signaling could be due to accumulation of harmful lipid intermediates. Our findings contribute to the understanding of the molecular mechanisms underlying insulin resistance and type 2 diabetes and point to inhibition of ADAMTS9 as a potential novel mode of treating insulin resistance.

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.

Exercise-induced molecular mechanisms promoting glycogen supercompensation in human skeletal muscle.

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.

Identifying the Heterotrimeric Complex Stoichiometry of AMPK in Skeletal Muscle by Immunoprecipitation.

The 5'-AMP-activated protein kinase is a complicated enzyme consisting of three different subunits, each of which is expressed as two or three isoforms. This gives the possibility of 12 different heterotrimeric complexes, which could have diverse functions within the cell. To map out which of these complexes are present and to what extent in skeletal muscle, we have used the immunoprecipitation technique and analyzed both the precipitates and the remaining supernatants for coprecipitation of complex partners. We have fine-tuned this method to give the best results on lysates from the skeletal muscle, liver, and heart muscle from mouse to man.

Kinase Activity Determination of Specific AMPK Complexes/Heterotrimers in the Skeletal Muscle.

Measuring the kinase activity of the 5'-AMP-activated protein kinase (AMPK) is an essential part of understanding the regulation of this metabolic master switch. The AMPK heterotrimer can exist in 12 different constellations with potentially diverse activation patterns. It is therefore important to be able to measure heterotrimer-specific activity to discriminate between these patterns. In this chapter we describe how to measure the AMPK activity of specific heterotrimeric complexes by consecutive immunoprecipitations and how the assay can be performed in a medium throughput fashion using 96-well plates.

Enhanced Muscle Insulin Sensitivity After Contraction/Exercise Is Mediated by AMPK.

Earlier studies have demonstrated that muscle insulin sensitivity to stimulate glucose uptake is enhanced several hours after an acute bout of exercise. Using AICAR, we recently demonstrated that prior activation of AMPK is sufficient to increase insulin sensitivity in mouse skeletal muscle. Here we aimed to determine whether activation of AMPK is also a prerequisite for the ability of muscle contraction to increase insulin sensitivity. We found that prior in situ contraction of m. extensor digitorum longus (EDL) and treadmill exercise increased muscle and whole-body insulin sensitivity in wild-type (WT) mice, respectively. These effects were not found in AMPKα1α2 muscle-specific knockout mice. Prior in situ contraction did not increase insulin sensitivity in m. soleus from either genotype. Improvement in muscle insulin sensitivity was not associated with enhanced glycogen synthase activity or proximal insulin signaling. However, in WT EDL muscle, prior in situ contraction enhanced insulin-stimulated phosphorylation of TBC1D4 Thr649 and Ser711 Such findings are also evident in prior exercised and insulin-sensitized human skeletal muscle. Collectively, our data suggest that the AMPK-TBC1D4 signaling axis is likely mediating the improved muscle insulin sensitivity after contraction/exercise and illuminates an important and physiologically relevant role of AMPK in skeletal muscle.

Intact Regulation of the AMPK Signaling Network in Response to Exercise and Insulin in Skeletal Muscle of Male Patients With Type 2 Diabetes: Illumination of AMPK Activation in Recovery From Exercise.

Current evidence on exercise-mediated AMPK regulation in skeletal muscle of patients with type 2 diabetes (T2D) is inconclusive. This may relate to inadequate segregation of trimeric complexes in the investigation of AMPK activity. We examined the regulation of AMPK and downstream targets ACC-ß, TBC1D1, and TBC1D4 in muscle biopsy specimens obtained from 13 overweight/obese patients with T2D and 14 weight-matched male control subjects before, immediately after, and 3 h after exercise. Exercise increased AMPK α2ß2γ3 activity and phosphorylation of ACCß Ser(221), TBC1D1 Ser(237)/Thr(596), and TBC1D4 Ser(704) Conversely, exercise decreased AMPK α1ß2γ1 activity and TBC1D4 Ser(318)/Thr(642) phosphorylation. Interestingly, compared with preexercise, 3 h into exercise recovery, AMPK α2ß2γ1 and α1ß2γ1 activity were increased concomitant with increased TBC1D4 Ser(318)/Ser(341)/Ser(704) phosphorylation. No differences in these responses were observed between patients with T2D and control subjects. Subjects were also studied by euglycemic-hyperinsulinemic clamps performed at rest and 3 h after exercise. We found no evidence for insulin to regulate AMPK activity. Thus, AMPK signaling is not compromised in muscle of patients with T2D during exercise and insulin stimulation. Our results reveal a hitherto unrecognized activation of specific AMPK complexes in exercise recovery. We hypothesize that the differential regulation of AMPK complexes plays an important role for muscle metabolism and adaptations to exercise.