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.

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.

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.

Human muscle fibre type-specific regulation of AMPK and downstream targets by exercise.

AMP-activated protein kinase (AMPK) is a regulator of energy homeostasis during exercise. Studies suggest muscle fibre type-specific AMPK expression. However, fibre type-specific regulation of AMPK and downstream targets during exercise has not been demonstrated. We hypothesized that AMPK subunits are expressed in a fibre type-dependent manner and that fibre type-specific activation of AMPK and downstream targets is dependent on exercise intensity. Pools of type I and II fibres were prepared from biopsies of vastus lateralis muscle from healthy men before and after two exercise trials: (1) continuous cycling (CON) for 30 min at 69 ± 1% peak rate of O2 consumption (V̇O2 peak ) or (2) interval cycling (INT) for 30 min with 6 × 1.5 min high-intensity bouts peaking at 95 ± 2% V̇O2 peak . In type I vs. II fibres a higher ß1 AMPK (+215%) and lower γ3 AMPK expression (-71%) was found. α1 , α2 , ß2 and γ1 AMPK expression was similar between fibre types. In type I vs. II fibres phosphoregulation after CON was similar (AMPK(Thr172) , ACC(Ser221) , TBC1D1(Ser231) and GS(2+2a) ) or lower (TBC1D4(Ser704) ). Following INT, phosphoregulation in type I vs. II fibres was lower (AMPK(Thr172) , TBC1D1(Ser231) , TBC1D4(Ser704) and ACC(Ser221) ) or higher (GS(2+2a) ). Exercise-induced glycogen degradation in type I vs. II fibres was similar (CON) or lower (INT). In conclusion, a differentiated response to exercise of metabolic signalling/effector proteins in human type I and II fibres was evident during interval exercise. This could be important for exercise type-specific adaptations, i.e. insulin sensitivity and mitochondrial density, and highlights the potential for new discoveries when investigating fibre type-specific signalling.

Epinephrine-stimulated glycogen breakdown activates glycogen synthase and increases insulin-stimulated glucose uptake in epitrochlearis muscles.

Epinephrine increases glycogen synthase (GS) phosphorylation and decreases GS activity but also stimulates glycogen breakdown, and low glycogen content normally activates GS. To test the hypothesis that glycogen content directly regulates GS phosphorylation, glycogen breakdown was stimulated in condition with decreased GS activation. Saline or epinephrine (0.02 mg/100 g rat) was injected subcutaneously in Wistar rats (∼130 g) with low (24-h-fasted), normal (normal diet), and high glycogen content (fasted-refed), and epitrochlearis muscles were removed after 3 h and incubated ex vivo, eliminating epinephrine action. Epinephrine injection reduced glycogen content in epitrochlearis muscles with high (120.7 ± 17.8 vs. 204.6 ± 14.5 mmol/kg, P < 0.01) and normal glycogen (89.5 ± 7.6 vs. 152 ± 8.1 mmol/kg, P < 0.01), but not significantly in muscles with low glycogen (90.0 ± 5.0 vs. 102.8 ± 7.8 mmol/kg, P = 0.17). In saline-injected rats, GS phosphorylation at sites 2+2a, 3a+3b, and 1b was higher and GS activity lower in muscles with high compared with low glycogen. GS sites 2+2a and 3a+3b phosphorylation decreased and GS activity increased in muscles where epinephrine decreased glycogen content; these parameters were unchanged in epitrochlearis from fasted rats where epinephrine injection did not decrease glycogen content. Incubation with insulin decreased GS site 3a+3b phosphorylation independently of glycogen content. Insulin-stimulated glucose uptake was increased in muscles where epinephrine injection decreased glycogen content. In conclusion, epinephrine stimulates glycogenolysis in epitrochlearis muscles with normal and high, but not low, glycogen content. Epinephrine-stimulated glycogenolysis decreased GS phosphorylation and increased GS activity. These data for the first time document direct regulation of GS phosphorylation by glycogen content.

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

We investigated the phosphorylation signatures of two Rab-GTPase activating proteins TBC1D1 and TBC1D4 in human skeletal muscle in response to physical exercise and physiological insulin levels induced by a carbohydrate rich meal using a paired experimental design. Eight healthy male volunteers exercised in the fasted or fed state and muscle biopsies were taken before and immediately after exercise. We identified TBC1D1/4 phospho-sites that (1) did not respond to exercise or postprandial increase in insulin (TBC1D4: S666), (2) responded to insulin only (TBC1D4: S318), (3) responded to exercise only (TBC1D1: S237, S660, S700; TBC1D4: S588, S751), and (4) responded to both insulin and exercise (TBC1D1: T596; TBC1D4: S341, T642, S704). In the insulin-stimulated leg, Akt phosphorylation of both T308 and S473 correlated significantly with multiple sites on both TBC1D1 (T596) and TBC1D4 (S318, S341, S704). Interestingly, in the exercised leg in the fasted state TBC1D1 phosphorylation (S237, T596) correlated significantly with the activity of the α2/ß2/γ3 AMPK trimer, whereas TBC1D4 phosphorylation (S341, S704) correlated with the activity of the α2/ß2/γ1 AMPK trimer. Our data show differential phosphorylation of TBC1D1 and TBC1D4 in response to physiological stimuli in human skeletal muscle and support the idea that Akt and AMPK are upstream kinases. TBC1D1 phosphorylation signatures were comparable between in vitro contracted mouse skeletal muscle and exercised human muscle, and we show that AMPK regulated phosphorylation of these sites in mouse muscle. Contraction and exercise elicited a different phosphorylation pattern of TBC1D4 in mouse compared with human muscle, and although different circumstances in our experimental setup may contribute to this difference, the observation exemplifies that transferring findings between species is problematic.

Akt2 influences glycogen synthase activity in human skeletal muscle through regulation of NH2-terminal (sites 2 + 2a) phosphorylation.

Type 2 diabetes is characterized by reduced muscle glycogen synthesis. The key enzyme in this process, glycogen synthase (GS), is activated via proximal insulin signaling, but the exact molecular events remain unknown. Previously, we demonstrated that phosphorylation of Thr³°8 on Akt (p-Akt-Thr³°8), Akt2 activity, and GS activity in muscle were positively associated with insulin sensitivity. Here, in the same study population, we determined the influence of several upstream elements in the canonical PI3K signaling on muscle GS activation. One-hundred eighty-one nondiabetic twins were examined with the euglycemic hyperinsulinemic clamp combined with excision of muscle biopsies. Insulin signaling was evaluated at the levels of the insulin receptor, IRS-1-associated PI3K (IRS-1-PI3K), Akt, and GS employing activity assays and phosphospecific Western blotting. The insulin-stimulated GS activity was positively associated with p-Akt-Thr³°8 (P = 0.01) and Akt2 activity (P = 0.04) but not p-Akt-Ser47³ or IRS-1-PI3K activity. Furthermore, p-Akt-Thr³°8 and Akt2 activity were negatively associated with NH2-terminal GS phosphorylation (P = 0.001 for both), which in turn was negatively associated with insulin-stimulated GS activity (P < 0.001). We found no association between COOH-terminal GS phosphorylation and Akt or GS activity. Employing whole body Akt2-knockout mice, we validated the necessity for Akt2 in insulin-mediated GS activation. However, since insulin did not affect NH2-terminal phosphorylation in mice, we could not use this model to validate the observed association between GS NH2-terminal phosphorylation and Akt activity in humans. In conclusion, our study suggests that although COOH-terminal dephosphorylation is likely necessary for GS activation, Akt2-dependent NH2-terminal dephosphorylation may be the site for "fine-tuning" insulin-mediated GS activation in humans.

Effect of birth weight and 12 weeks of exercise training on exercise-induced AMPK signaling in human skeletal muscle.

Subjects with a low birth weight (LBW) display increased risk of developing type 2 diabetes (T2D). We hypothesized that this is associated with defects in muscle adaptations following acute and regular physical activity, evident by impairments in the exercise-induced activation of AMPK signaling. We investigated 21 LBW and 21 normal birth weight (NBW) subjects during 1 h of acute exercise performed at the same relative workload before and after 12 wk of exercise training. Multiple skeletal muscle biopsies were obtained before and after exercise. Protein levels and phosphorylation status were determined by Western blotting. AMPK activities were measured using activity assays. Protein levels of AMPKα1 and -γ1 were significantly increased, whereas AMPKγ3 levels decreased with training independently of group. The LBW group had higher exercise-induced AMPK Thr(172) phosphorylation before training and higher exercise-induced ACC2 Ser(221) phosphorylation both before and after training compared with NBW. Despite exercise being performed at the same relative intensity (65% of Vo2peak), the acute exercise response on AMPK Thr(172), ACC2 Ser(221), AMPKα2ß2γ1, and AMPKα2ß2γ3 activities, GS activity, and adenine nucleotides as well as hexokinase II mRNA levels were all reduced after exercise training. Increased exercise-induced muscle AMPK activation and ACC2 Ser(221) phosphorylation in LBW subjects may indicate a more sensitive AMPK system in this population. Long-term exercise training may reduce the need for AMPK to control energy turnover during exercise. Thus, the remaining γ3-associated AMPK activation by acute exercise after exercise training might be sufficient to maintain cellular energy balance.

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.

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.

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.

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.

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

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

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.

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-769662 activates AMPK beta1-containing complexes but induces glucose uptake through a PI3-kinase-dependent pathway in mouse skeletal muscle.

5'-AMP-activated protein kinase (AMPK) regulates several aspects of metabolism. Recently, A-769662 was shown to activate AMPK in skeletal muscle. However, no biological effects of AMPK activation by A-769662 in this tissue have been reported. We hypothesized that A-769662 would increase glucose uptake in skeletal muscle. We studied incubated soleus and extensor digitorum longus (EDL) muscles from 129S6/sv and C57BL/6 mice. Glucose uptake increased only in soleus from 129S6/sv when concentrations of A-769662 were 500 microM (approximately 15%, P < 0.05) and 1 mM (approximately 60%, P < 0.01). AMPK beta1- but not beta2-containing complexes were dose dependently activated by A-769662 in muscles from both genotypes (approximately 100% at 200 microM and 300-600% at 1 mM). The discrepancy between the A-769662-induced AMPK activation pattern and stimulation of glucose uptake suggested that these effects were unrelated. A-769662 increased phosphorylation of Akt in both muscles from both genotypes, with phosphorylation of T308 being significantly higher in soleus than in EDL in 129S6/sv mice (P < 0.01). In soleus from 129S6/sv mice, insulin receptor substrate 1-associated phosphatidylinositol 3 (PI3)-kinase activity was markedly increased with A-769662, and Akt phosphorylation and glucose uptake were inhibited by wortmannin while phosphorylation of acetyl-CoA carboxylase (S227) was unaffected. Thus, A-769662 activates beta1-containing AMPK complexes in skeletal muscle but induces glucose uptake through a PI3-kinase-dependent pathway. Although development of A-769662 has constituted a step forward in the search for AMPK activators targeting specific AMPK trimers, our data suggest that in intact muscle, A-769662 has off-target effects. This may limit use of A-769662 to study the role of AMPK in skeletal muscle metabolism.