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

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1 598 Diabetes Volume 66, March 2017 Rasmus Kjøbsted, 1,2 Nanna Munk-Hansen, 1 Jesper B. Birk, 1 Marc Foretz, 3,4,5 Benoit Viollet, 3,4,5 Marie Björnholm, 6 Juleen R. Zierath, 2,6 Jonas T. Treebak, 2 and Jørgen F.P. Wojtaszewski 1 Enhanced Muscle Insulin Sensitivity After Contraction/Exercise Is Mediated by AMPK Diabetes 2017;66: DOI: /db METABOLISM Earlier studies have demonstrated that muscle insulin sensitivity to stimulate glucose uptake is enhanced several hours after an acute bout ofexercise.usingaicar, 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 priorinsitucontractionofm.extensordigitorumlongus (EDL) and treadmill exercise increased muscle and whole-body insulin sensitivity in wild-type (WT) mice, respectively. These effects were not found in AMPKa1a2 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 Thr 649 and Ser 711.Suchfindings 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. Skeletal muscle from human, sheep, dog, and rodents demonstrates increased insulin-stimulated glucose uptake in the period after a single bout of exercise (1 10). This phenomenon is observed in both healthy and insulin-resistant muscle (11 13) and has been suggested to involve an increased abundance of GLUT4 at the plasma membrane (14). Moreover, changes in muscle insulin sensitivity occurs independent of changes in protein synthesis (15), indicating involvement of posttranslational mechanisms. Interestingly, studies of human and rodent muscle suggest that prior exercise does not improve the ability of insulin to stimulate components of the proximal insulin signaling cascade, including the insulin receptor, insulin receptor substrate 1, PI3K, and Akt (5,15 18). This supports the notion that improved insulin sensitivity after exercise is not caused by enhanced delivery of insulin to the muscle and indicates an important role for more distal intramyocellular signaling events. AMPK is a heterotrimeric complex containing catalytic a and regulatory b and g subunits, of which several isoforms exist (a1, a2, b1, b2, g1, g2, and g3) (19). In human and mouse skeletal muscle, three (a2b2g1, a2b2g3, and a1b2g1) and five (a2b2g1, a2b2g3, a2b1g1, a1b2g1, and a1b1g1) heterotrimeric combinations have been found, respectively (20,21). Interestingly, mouse skeletal muscle contains two b1-associated complexes that are not found in human skeletal muscle. Furthermore, in mouse extensor digitorum longus (EDL) and human vastus lateralis muscle, the a2b2g3 complex represents ;20% of all AMPK heterotrimer complexes, whereas in mouse soleus (SOL) muscle, it comprises,2% (20,21). AMPK is considered an important sensor of cellular energy balance, and in skeletal muscle, AMPK is activated during conditions of cellular stress, such as muscle contraction and hypoxia (22). When activated, AMPK stimulates ATP-generating processes (e.g., glucose 1 Section of Molecular Physiology, Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen, Copenhagen, Denmark 2 Section of Integrative Physiology, Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark 3 INSERM, U1016, Institut Cochin, Paris, France 4 CNRS, UMR8104, Paris, France 5 Université Paris Descartes, Sorbonne Paris Cité, Paris, France 6 Integrative Physiology, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden Corresponding author: Jørgen F.P. Wojtaszewski, jwojtaszewski@nexs.ku.dk. Received 25 April 2016 and accepted 24 October by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at

2 diabetes.diabetesjournals.org Kjøbsted and Associates 599 uptake and lipid oxidation) while inhibiting ATP-consuming processes (e.g., protein and lipid synthesis) in an attempt to restore cellular energy homeostasis (22,23). TBC1D4 is phosphorylated by multiple kinases (including Akt) during insulin stimulation (24,25). This modification has been suggested to be important for insulin-stimulated glucose uptake (26). AMPK is also upstream of TBC1D4, and both contraction- and AICAR induced AMPK activation increase phosphorylation of TBC1D4 (25). Within recent years, TBC1D4 has emerged as a likely candidate for mediating the insulin-sensitizing effect of prior exercise on skeletal muscle glucose uptake. In support of this, phosphorylation of TBC1D4 is elevated in prior exercised human and rat muscle, concomitant with enhanced insulin sensitivity (11,17,18,27 29). Prior AICAR stimulation increases insulin sensitivity to stimulate glucose transport in rat muscle (15), and we have recently provided evidence that this is mediated by AMPK in muscle of mice (30). We also reported a positive association between insulin-stimulated glucose uptake and phosphorylation of regulatory sites on TBC1D4 (30). This suggests a mechanism by which AICAR, through AMPK, potentiates a subsequent effect of a submaximal concentration of insulin on TBC1D4, leading to improved insulin-stimulated glucose uptake. During AICAR stimulation, cells maintain energy and fuel homeostasis. In contrast, the myocyte is subjected to energy and fuel disturbances during exercise/contraction, which likely contributes to AMPK activation. Furthermore, while AMPK regulates muscle glucose uptake, fatty acid uptake, gene activation, and mitochondrial protein content in response to AICAR treatment (31 34), activation of AMPK is not necessary for inducing such effects in response to exercise/contraction (31 36). Hence, little evidence exists to support the assumption that AICAR- and exercise/ contraction-induced biological responses are equally dependent on AMPK activation. Since the first proposal of an insulin-sensitizing effect of prior exercise by Bergström and Hultman (37) and the subsequent proof of this in rat and human skeletal muscle (1,2), an ongoing search for molecular interactions between exercise and insulin signaling has occurred. To further study this, we established an experimental protocol in which mouse muscle displays enhanced insulin sensitivity to stimulate glucose uptake after in situ contraction. We used this model to provide genetic evidence for the hypothesis that AMPK acts as a molecular transducer between exercise and insulin signaling and, thus, is necessary for the ability of prior contraction/exercise to increase muscle insulin sensitivity. RESEARCH DESIGN AND METHODS Animals All experiments were approved by the Danish Animal Experiments Inspectorate ( and ), as well as the regional ethics committee of Northern Stockholm, and complied with the EU convention for protection of vertebra animals used for scientific purposes (Council of Europe, Treaty 123/170, Strasbourg, France, 1985/1998). Animals used in this study were AMPKa1a2 muscle-specific double-knockout (mdko) and whole-body AMPKg3 KO female mice with corresponding wild-type (WT) littermates as controls (35,36,38). Animals ( weeks [means 6 SD]) were maintained on a 12:12 light-dark cycle (6:00 A.M. to 6:00 P.M.) with unlimited access to standard rodent chow and water. Glucose Uptake During In Situ Contraction of EDL and SOL Muscle For all experiments, fed mice were anesthetized by an intraperitoneal injection of pentobarbital (10 mg/100 g body weight) before both common peroneal or tibial nerves were exposed. Subsequently, an electrode was placed on a single common peroneal or tibial nerve followed by in situ contraction of EDL or SOL muscle, respectively. The contralateral leg served as a rested control. The contraction protocol consisted of 0.5-s trains (100 Hz, 0.1 ms, 2 5 V) repeated every 1.5 s for 10 min. To determine glucose uptake during in situ contraction, tail blood was collected at time points 0, 5, and 10 min. After the first blood sample, a bolus of [ 3 H]2-deoxyglucose (12.3 MBq/kg body weight) dissolved in isotonic saline was injected retroorbitally. After the last blood sample, EDL or SOL muscles were rapidly dissected and frozen in liquid nitrogen. Uptake of [ 3 H]2- deoxyglucose into muscle was assessed based on accumulated [ 3 H]2-deoxyglucose-6-phosphate and tracer-specific activity in plasma as previously described (39). Muscle Insulin Sensitivity After In Situ Contraction For measurements of insulin sensitivity after in situ contraction, electrodes were connected to either the common peroneal nerve (EDL) or the tibial nerve (SOL) of both legs of the anesthetized animals. One-half of the animals served as sham-operated controls. Immediately after in situ contraction of EDL or SOL, muscles were dissected and suspended at low tension (;1 mn) in incubation chambers (model 610/820M; Danish Myo Technology, Aarhus, Denmark) containing Krebs-Ringer buffer (KRB) (117 mmol/l NaCl, 4.7 mmol/l KCl, 2.5 mmol/l CaCl 2, 1.2 mmol/l KH 2 PO 4, 1.2 mmol/l MgSO 4, 0.5 mmol/l NaHCO 3 [ph 7.4]) supplemented with 0.1% BSA, 5 mmol/l mannitol, and 5 mmol/l D-glucose. During the entire incubation period, the buffer was oxygenated with 95% O 2 and 5% CO 2 and maintained at 30 C. SOL and EDL muscles were allowed to recover for 2 and 3 h, respectively. These time points were selected based on measurements demonstrating reversal of muscle glucose uptake after in situ contraction. During recovery, the incubation medium was replaced once every 30 min to maintain an adequate glucose concentration. Subsequently, basal, submaximal (100 mu/ml/694.5 pmol/l), and maximal (10,000 mu/ml/69,450 pmol/l; only EDL) insulin-stimulated 2-deoxyglucose uptake was measured during the last 10 min of a 30-min stimulation period by adding 1 mmol/l [ 3 H]2-deoxyglucose (0.028 MBq/mL), 7 mmol/l [ 14 C]mannitol ( MBq/mL), and 2 mmol/l pyruvate to a glucosefree incubation medium. 2-Deoxyglucose uptake was

3 600 AMPK and Muscle Insulin Sensitivity Diabetes Volume 66, March 2017 assessed by the accumulation of [ 3 H]2-deoxyglucose into musclewiththeuseof[ 14 C]mannitol as an extracellular marker (30). Radioactivity was measured on 200 ml lysate by liquid scintillation counting (Ultima Gold and Tri-Carb 2910 TR; PerkinElmer) and related to the specific activityof the incubation media. Postexercise Insulin Tolerance Test and In Vivo Muscle Glucose Uptake All mice were acclimatized to treadmill running on five consecutive days. The acclimatization consisted of a 2-min warm up ( m/min) followed by 5 min of running at 10.2 m/min and 0 incline. Two days after the acclimatization, mice were subjected to a graded maximal running test, as previously described (36). For insulin tolerance tests (ITTs),micewerefastedinsinglecagesfor2h(;8:00 10:00 A.M.) before performing a single bout of treadmill exercise (30 min, 15 incline, and 55% of maximal running speed).restingcontrolmicewereleftinthecage.after exercise, mice were returned to their individual cage without access to food for 1 h, after which they were administered witheither0.3units(2.09nmol)or0.4units(2.78nmol)of insulin per kilogram body weight intraperitoneally, respectively. Throughout the ITT, blood was collected from the tail vein at 0, 20, 40, 60, 90, and 120 min, and blood glucose concentration was determined using a glucometer (ContourXT;Bayer,Leverkusen,Germany).Areaoverthe curve values were calculatedfromtimepoints 0 40 min, since changes in blood glucose concentrations at later time points may largely reflect the ability to counteract hypoglycemia rather than peripheral glucose disposal. Three to four weeks after the last ITT, in vivo muscle glucose uptake during the first40minofanitt(0.3units/kginsulin)wasmeasuredin the same mice 1 h after treadmill exercise. All mice received an intraperitoneal injection of insulin dissolved in isotonic saline (10 ml/g body weight) containing 0.1 mmol/l [ 3 H]2-deoxyglucose (1.78 MBq) 1 h after rest and exercise. Immediately before, as well as 20 and 40 min into the ITT, blood glucose concentration was measured from the tail vein and blood samples were obtained for determination of radioactivity. After the last blood sample, mice were euthanized by cervical dislocation and tissues were rapidly dissected and frozen in liquid nitrogen. Uptake of [ 3 H]2-deoxyglucose into muscle was assessed as previously described (39). Ex Vivo Contraction of Incubated Skeletal Muscle Whole-body AMPKg3 KO mice were anesthetized and EDL muscles were isolated and preincubated in KRB (40 min) before they were stimulated to contract (10 min), as previously described (40). Muscle Processing Muscles were homogenized in 400 ml ice-cold homogenization buffer (30) and rotated end-over-end for 1 h at 4 C. Part of the homogenate was centrifuged at 16,000g for 20 min at 4 C, after which lysate (supernatant) was collected and frozen in liquid nitrogen for later analyses. Total protein abundance inmusclelysateandhomogenatewasdeterminedby the bicinchoninic acid method (Thermo Fisher Scientific, Waltham, MA). Glycogen Synthase Activity Muscle glycogen synthase (GS) activity was measured in 75 mgmusclehomogenateusing96-wellmicrotitreplatesas previously described (11,41). Samples were assayed in triplicate in the presence of 0.02 and 8.0 mmol/l glucose-6- phosphate and presented as percent glucose-6-phosphate independent activity (GS 0.02 * 100/GS 8.0 ; %I-form) and total GS activity (GS 8.0 ;total),respectively. AMPK Activity Heterotrimer-specific AMPK activity in mouse skeletal muscle was determined as previously described (30). AMPK activity was measured on 300 mg of muscle lysate protein using AMPK-g3, -a2, and -a1 antibodies for three consecutive immunoprecipitations. Glycogen Content Muscle glycogen content was measured on 200 mgofmuscle protein homogenate after acid hydrolysis, as previously described (36). SDS-PAGE and Western Blot Analyses Muscle lysates and homogenates were boiled in Laemmli buffer for 10 min before being subjected to SDS-PAGE and immunoblotting as previously described (30). Quantification of protein phosphorylation has not been related to protein abundance since abundance of all measured proteins did not change in response to any specified intervention. Small differences in total abundance were observed for some proteins between genotypes; however, this did not affect phosphorylation dynamics or interpretation of data. Antibodies Antibodies against phospho-ampk-thr 172, phospho-acca/ b-ser 79/212, Akt2, phospho-akt-ser 473, phospho-akt-thr 308, phospho-tbc1d1-thr 590, phospho-tbc1d4-(ser 318,Ser 588, Thr 642 ), phospho-erk1/2-thr 202 /Tyr 204, and hexokinase II (HKII) were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against phospho-tbc1d1-ser 231 and AS160 (TBC1D4) were from Millipore (Temecula, CA), and antibodies against AMPKa2andGLUT4werepurchased from Santa Cruz Biotechnology (Dallas, TX) and Thermo Fisher Scientific, respectively. ACC protein was determined using horseradish peroxidase conjugated streptavidin from Dako (Glostrup, Denmark). TBC1D1, pyruvate dehydrogenase (PDH), AMPKa1,andGSprotein,aswellasphosphorylation of TBC1D4-Ser 711, GS site 2+2a and site 3a+3b, were determined using specific antibodies as previously described (11,36,41). Antibodies used for AMPK activity measurements were against AMPKa2 (Santa Cruz Biotechnology), AMPKg3 (provided by D.G. Hardie, University of Dundee, Scotland, U.K.), and AMPKa1 (purchased from GenScript Jiangning, Nanjing, China). Statistics Statistical analyses were performed using SigmaPlot (version 13.0; Systat, Erkrath, Germany). Two-way ANOVA with or

4 diabetes.diabetesjournals.org Kjøbsted and Associates 601 without repeated measures and paired/unpaired Student t tests were used to assess statistical differences within and between genotypes, where appropriate. A three-way ANOVA was used to assess differences in total muscle protein abundance between genotypes. The Student-Newman-Keuls test wasusedforposthoctesting,andallmaineffectshavebeen indicated by lines. Correlation analyses were performed by calculating Pearson product moment correlation coefficient. Data are expressed as the means 6 SEM unless stated otherwise. Differences were considered statistically significant at P, RESULTS In Situ Contraction Increases Glucose Uptake and AMPK Signaling in EDL and SOL Muscle During in situ contraction, glucose uptake in EDL and SOL muscle increased similarly in AMPK WT and mdko mice (Fig. 1A). Furthermore, in situ contraction decreased muscle glycogen content (Fig. 1B) and increased Erk1/2 Thr 202 / Tyr 204 phosphorylation (Fig. 1C) toanextentthatdidnot differ between genotypes. This suggests that the electrical stimulation protocol induced similar changes in WT and mdko muscle. In situ contraction markedly increased phosphorylation of AMPK Thr 172 (Fig. 1D) and downstream targets ACC Ser 212 (Fig. 1E), TBC1D1 Ser 231 (Fig. 1F), and TBC1D4 Ser 711 (Fig. 1G) in EDL and SOL muscle from WT mice, whereas only minor, if any, changes were seen in EDL and SOL muscle from mdko mice. Contraction did not alter total protein abundance of Erk1/2, AMPKa1, AMPKa2, ACC, TBC1D1, and TBC1D4 in either EDL or SOL muscle (Fig. 1H). As expected, EDL and SOL muscle from AMPK mdko mice showed a substantial loss of AMPKa1 and AMPKa2 protein abundance. Identical to previous observations (36), ACC and TBC1D1 protein abundance was decreased in AMPK mdko skeletal muscle compared with WT littermates. Intriguingly, protein abundance of Erk1/2 was increased (;35%, P, 0.01) whereas TBC1D4 protein abundance was decreased (;20%, P, 0.05) in SOL muscle from mdko mice compared with WT mice (Fig. 1H). Prior In Situ Contraction Increases Insulin Sensitivity in EDL Muscle via an AMPK-Dependent Mechanism To test whether the effect of muscle contraction on insulin sensitivity is dependent on AMPK, we measured submaximal insulin-stimulated glucose uptake ex vivo after in situ contraction. Three hours after in situ contraction, basal glucose uptake was not significantly different between prior contracted and rested EDL muscle (Fig. 2A). However, prior in situ contraction increased submaximal insulin-stimulated glucose uptake in isolated EDL muscle from WT mice but failed to do so in EDL muscle from AMPK mdko mice (Fig. 2A). Maximal insulin-stimulated glucose uptake was similar between prior contracted and rested EDL muscle in both genotypes (Fig. 2A). The incremental increase in submaximal insulin-stimulated glucose uptake (delta insulin: submaximal insulin-stimulated glucose uptake minus basal glucose uptake) was significantly higher after prior in situ contraction in WT mice only (Fig. 2B). Interestingly, prior in situ contraction did not increase submaximal insulin-stimulated glucose uptake ex vivo in WT SOL muscle (Fig. 2C and D). On the basis of these results, we performed the subsequent in situ experiments in EDL muscle from WT and mdko mice with SOL muscle from WT mice as a negative control. Prior Exercise Enhances Whole-Body Insulin Sensitivity and Insulin-Stimulated Muscle Glucose Uptake in WT Mice but Not in AMPK mdko Mice To evaluate the involvement of skeletal muscle AMPK in regulating whole-body insulin sensitivity after an acute exercise bout in vivo, we performed intraperitoneal ITTs on AMPKWTandmdKOmiceatrestand1hafterasingle bout of acute treadmill exercise. Prior exercise enhanced the blood glucose lowering response to a submaximal concentration of insulin (0.3 units/kg body weight) (Fig. 2E) and improved insulin tolerance by ;250% in WT mice (Fig. 2G). In contrast, prior exercise did not induce a greater insulin response to lower blood glucose concentrations in AMPK mdko mice (Fig. 2F and G). Furthermore, insulin-stimulated glucose uptake during the first 40 min of an ITT (0.3 units/kg body weight) was significantly improved 1 h after exercise in m. tibialis anterior from WT mice but not inmusclefromampkmdkomice(fig.2e and F). Total Protein Abundance in EDL and SOL Muscle Is Not Affected by Prior Muscle Contraction Prior contraction and submaximal insulin did not alter total protein abundance of Akt2, HKII, GS, ACC, PDH, TBC1D1, TBC1D4, and GLUT4 in EDL muscle within either genotype (Fig. 3A). Total protein abundance of ACC, TBC1D1, HKII, and PDH was significantly lower (;20 25%; n = 12 13, P, ) in EDL muscle from AMPK mdko mice compared with WT mice. In contrast, Akt2 muscle protein level was ;33% higher (n =12 13, P, 0.001) in EDL muscle from mdko mice compared with WT mice. No differences in GS, TBC1D4, and GLUT4 muscle protein level were observed between genotypes. Like in EDL muscle, prior contraction and submaximal insulin did not alter total protein abundance of Akt2, TBC1D1, TBC1D4, ACC, and AMPKa2 in WT SOL muscle (Fig. 3B) Proximal Insulin Signaling Is Not Enhanced by Prior Muscle Contraction Previousstudiesofhuman,sheep,rat,andmouseinvestigating muscle insulin sensitivity in the postexercise state suggest that the increased ability of insulin to stimulate glucose uptake occurs independent of enhanced proximal insulinsignaling(ir, IRS1, PI3K, andakt)(5 7,15 18). In accordance, in the current study, prior contraction also did not affect submaximal insulin-induced phosphorylation of Akt Thr 308 and Ser 473 in EDL (Fig. 4A and B) or SOL muscle (Fig. 4C and D). Prior Muscle Contraction Increases Site-Specific Phosphorylation of TBC1D4 in Response to Insulin TBC1D4 is phosphorylated by Akt and AMPK (24 26). TBC1D4 has been suggested to regulate muscle insulin

5 602 AMPK and Muscle Insulin Sensitivity Diabetes Volume 66, March 2017 Figure 1 In situ contraction promotes muscle glucose uptake in WT and AMPK mdko mice. 2-Deoxyglucose uptake (A), glycogen content (B), perk1/2 Thr 202 /Tyr 204 (C), pampk Thr 172 (D), pacc Ser 212 (E), ptbc1d1 Ser 231 (F), and ptbc1d4 Ser 711 (G) in EDL and SOL muscle from WT (white bars) and AMPK mdko (black bars) mice immediately after 10 min of in situ contraction of the lower hind limb. EDL and SOL muscle were stimulated to contract through the common peroneal and tibial nerve, respectively. Data were analyzed by twoway repeated-measures ANOVA within each muscle type. A, B, C, D (only SOL), and F: ***P < 0.001, **P < 0.01, and *P < 0.05 main effect of contraction. D (only EDL), E, F, and G: Treatment 3 genotype interaction (P < 0.05), ###P < and ##P < 0.01 effect of genotype within treatment; ***P < 0.001, **P < 0.01, and *P < 0.05 effect of contraction within genotype. Representative Western blot images (H). Quantification of protein phosphorylation has not been related to protein abundance (see RESULTS). Values are means 6 SEM. For all SOL data, n =5 6 per group. For all EDL data, n =3 4 per group except muscle glycogen in mdko, which has n = 10. sensitivity, as insulin-stimulated phosphorylation of TBC1D4 is enhanced during conditions in which muscle displays increased insulin sensitivity after exercise and AICAR treatment (27 30). In the current study, prior contraction of EDL muscle increased the effect of submaximal insulin stimulation on TBC1D4 Thr 649 and Ser 711 phosphorylation compared with rested control muscle from WT mice (Fig. 5A and B). Interestingly, this effect was dependent on AMPK since insulin-induced phosphorylation of TBC1D4 was similar between rested and prior contracted EDL

6 diabetes.diabetesjournals.org Kjøbsted and Associates 603 Figure 2 Improvements in muscle and whole-body insulin sensitivity after contraction and exercise are impaired in AMPK mdko mice. Glucose uptake (A and C) and delta glucose uptake (submaximal insulin minus basal) (B and D) in EDL and SOL muscle from AMPK WT and mdko mice incubated without or with insulin 2 h (SOL) and 3 h (EDL) after prior in situ contraction of the lower hind limb. Blood glucose concentration (% Basal) as well as insulin-stimulated muscle glucose uptake (E and F) from AMPK WT and mdko mice during an ITT (0.3 or 0.4 units/kg) after rest or 1 h after exercise. TA, m. tibialis anterior. Absolute blood glucose concentrations at time point 0 during the 0.3 units/kg ITT in WT mice (rest: mmol/l, prior exercise: mmol/l; P = 0.36) and in mdko mice (rest: mmol/l, prior exercise: mmol/l; P < 0.01). Area over the curve calculations (G) were extracted from the 0.3 units/kg ITT in E and F and related to

7 604 AMPK and Muscle Insulin Sensitivity Diabetes Volume 66, March 2017 Figure 3 Insulin and prior contraction do not affect total protein expression. Protein abundance for Akt2, HKII, GS, ACC, AMPKa2, PDH, TBC1D1, TBC1D4, and GLUT4 in EDL muscle from AMPK WT and mdko mice (A) as well as protein abundance for Akt2, TBC1D1, TBC1D4, ACC, and AMPKa2 in SOL muscle (B). Data were analyzed by a three-way (A) and a two-way repeated-measures (B) ANOVA. No significant differences were found within each genotype (A) and SOL muscle (B) in response to prior contraction or submaximal insulin stimulation. For EDL data, n =10 12 per group. For SOL data, n =10 11 per group. muscle from AMPK mdko mice. Submaximal insulin-stimulated phosphorylation of TBC1D4 Ser 324 and Ser 595 was unaffected by prior muscle contraction of EDL muscle (Fig. 5C and D), suggesting a highly selective interaction between these stimuli. Correlation analyses revealed that delta insulin for glucose uptake and delta insulin for phosphorylation of TBC1D4 Thr 649 and Ser 711 in EDL muscle were positively correlated in WT mice (r = , P, ) (Fig. 5E G). Interestingly, prior contraction did not affect insulin-stimulated phosphorylation of TBC1D4 Thr 649 and Ser 711 in WT SOL muscle (Fig. 5H and I) inparallelwith unchanged insulin sensitivity. AMPKg3-Associated Activity Is Increased in EDL Muscle 3 Hours After In Situ Contraction As muscle contraction acutely increases AMPK activity, we investigated whether this effect was maintained in EDL muscle recovered for 3 h ex vivo. In muscle from AMPK mdko mice, phosphorylation of AMPK Thr 172 and downstream target ACC Ser 212 was reduced by ;80 90% compared with WT littermates (Fig. 6A and B). Phosphorylation of AMPK Thr 172 and ACC Ser 212 had returned to resting levels 3 h after contraction. Submaximal insulin did not affect phosphorylation of AMPK Thr 172 but induced a minor increase in ACC Ser 212 phosphorylation in EDL muscle from AMPK mdko mice (Fig. 6A and B). When measuring AMPK heterotrimer complex activity 3 h into recovery, we found that AMPKg3-associated activity was increased in prior contracted WT EDL muscle (P, 0.01) (Fig. 6C), whereas no significant differences for the remaining AMPKa2- and AMPKa1-associated activities were found between rested and prior contracted muscle (Fig. 6D and E). Phosphorylation of AMPK Thr 172 and ACC Ser 212 was similar between prior rested and contracted WT SOL muscle (Fig. 6F and G). Also, no significant differences in AMPK activity were observed between prior contracted and rested WT SOL muscle (Fig. 6H). Taken together, this demonstrates that AMPKg3- associated activity is increased concomitant with enhanced muscle insulin sensitivity. To elucidate a possible role of AMPKg3 to enhance muscle insulin sensitivity after contraction, we investigated phosphorylation of TBC1D4 Ser 711 in EDL muscle from AMPKg3 KO mice. Interestingly, ex vivo contraction increased phosphorylation of TBC1D4 Ser 711 in WT muscle but not in muscle from AMPKg3KOmice(Fig.6I). individual rest groups. Data were analyzed by a two-way ANOVA (A, C, E, and F) and a Student t test (B, D, and G) within each genotype (A, B, C, D, and G) and insulin concentration (E and F). A: WT: treatment 3 insulin interaction (P < 0.05), ***P < vs. basal group (0 mu/ml) within genotype; ###P < effect of prior contraction within group; P < vs. submaximal group (100 mu/ml) within genotype. mdko: ***P < vs. basal group (0 mu/ml); P < vs. submaximal group (100 mu/ml). B: Data are extracted from the raw data in A. #P < 0.05 vs. rest within genotype. C: ***P < main effect of insulin. E: 0.3 units/kg; group 3 time interaction (P < 0.05), ***P < 0.001, **P < 0.01, and *P < 0.05 effect of group within time; ##P < 0.01 effect of prior exercise. G: #P < 0.05 vs. rest within genotype. Values are means 6 SEM. For all SOL data, n = 9 11 per group. For all EDL data, n = per group. For ITTs, n = 3 4 (0.4 units/kg) and n = 6 8 (0.3 units/kg). For in vivo insulin-stimulated muscle glucose uptake, n = 5 8.

8 diabetes.diabetesjournals.org Kjøbsted and Associates 605 Figure 4 Prior contraction does not affect regulation of Akt by insulin. Phosphorylation of Akt Thr 308 (A and C) and Ser 473 (B and D) in EDL muscle from AMPK WT and mdko mice as well as WT SOL muscle incubated with or without submaximal insulin 2 h (SOL) and 3 h (EDL) after prior in situ contraction of the lower hind limb. Data were analyzed by two-way repeated-measures ANOVA within each genotype (EDL) and muscle (SOL). A D: ***P < main effect of insulin. Quantification of protein phosphorylation has not been related to protein abundance (see RESULTS). Values are means 6 SEM. For all WT SOL data, n = 11 per group. For all EDL data, n =11 13 per group. Phosphorylation of TBC1D1 Does Not Parallel Changes in Muscle Insulin Sensitivity Phosphorylation of TBC1D1 has been proposed to regulate muscle glucose uptake in response to insulin and contraction (42,43). In the current study, submaximal insulin stimulation increased phosphorylation of TBC1D1 Thr 590 in EDL muscle of AMPK mdko and WT mice, with no significant differences between rested and prior contracted muscle (Fig. 7A). Furthermore, phosphorylation of TBC1D1 Ser 231 had returned to resting levels 3 h after contraction and did not respond to submaximal insulin stimulation (Fig. 7B). Also, phosphorylation of TBC1D1 Ser 231 was reduced in EDL muscle from AMPK mdko mice compared with WT mice (Fig. 7B). Phosphorylation of TBC1D1 Thr 590 and Ser 231 in WT SOL muscle (Fig. 7C and D) was similar to findings in WT EDL muscle, indicating that TBC1D1 is not involved in regulating muscle insulin sensitivity after contraction. Increased GS Activity Does Not Seem to Be Necessary for Enhanced Muscle Insulin Sensitivity After Contraction To determine whether GS was secondarily affecting 2-deoxyglucose uptake in skeletal muscle, we measured GS phosphorylationandactivity.inwtmice,basalgsactivity (%I-form) was similar between prior rested and contracted EDL muscle (Fig. 8A). In contrast, GS activity was significantly higher in previously contracted EDL muscle compared with rested muscle in AMPK mdko mice. Submaximal insulin stimulation significantly increased GS activity in both rested and prior contracted EDL muscle independent of genotype (Fig. 8A). Total GS activity was similar between genotypes and did not change in response to prior contraction or submaximal insulin stimulation (Fig. 8B). GS activity increases by dephosphorylation(44).in the current study, phosphorylation at COOH-terminal GS residues (3a+3b) decreased similarly in rested and prior contracted EDL muscle during submaximal insulin stimulation in both genotypes (Fig. 8C). No significant differences in phosphorylation at N-terminal GS residues (2+2a) were observed in response to prior contraction or submaximal insulin stimulation (Fig. 8D). Notably, EDL muscle from AMPK mdko mice had an ;40% reduction in GS site 2+2a phosphorylation in line with the notion that AMPK is a kinase for GS site 2 (45,46). This may explain the higher GS activity observed in EDL muscle of the AMPK mdko mouse (Fig. 8A). Decreased Muscle Glycogen Content Is Not Sufficient to Enhance Muscle Insulin Sensitivity After Contraction In skeletal muscle, insulin-stimulated glucose uptake is suggested to be regulated by glycogen content per se (47). Three hours after in situ contraction, glycogen content was lower in previously contracted EDL muscle compared with rested muscle (Fig. 8E). Interestingly, glycogen content was significantly lower in prior contracted EDL muscle from

9 606 AMPK and Muscle Insulin Sensitivity Diabetes Volume 66, March 2017 Figure 5 Prior contraction increases site-specific phosphorylation of TBC1D4 by insulin. Phosphorylation of TBC1D4 Thr 649 (A and H), Ser 711 (B and I), Ser 324 (C), and Ser 595 (D) in EDL muscle from AMPK WT and mdko mice as well as WT SOL muscle incubated with or without submaximal insulin 2 h (SOL) and 3 h (EDL) after prior in situ contraction of the lower hind limb. Data were analyzed by two-way repeated-measures ANOVA within each genotype (EDL) and muscle (SOL). A and B: WT: treatment 3 insulin interaction (P < 0.05), ***P < effect of insulin within treatment; ###P < and ##P < 0.01 effect of treatment within insulin. mdko: ***P < main effect of insulin. C, D, H, and I: ***P < main effect of insulin. Values are means 6 SEM. Pearson correlations between delta insulin (submaximal insulin minus basal) on glucose uptake and phosphorylation of TBC1D4 Thr 649 (E), glucose uptake and phosphorylation of TBC1D4 Ser 711 (F), and phosphorylation of TBC1D4 Thr 649 and Ser 711 (G). Rest, open symbols; prior contraction, closed symbols. For all WT SOL data, n =11per group. For all EDL data, n =11 13 per group. r values and significance level are indicated in the respective panel. AU, arbitrary units.

10 diabetes.diabetesjournals.org Kjøbsted and Associates 607 Figure 6 AMPKg3-associated activity is elevated in WT EDL muscle in the postcontraction period. Phosphorylation of AMPK Thr 172 (A and F) and ACC Ser 212 (B and G) in EDL muscle from AMPK WT and mdko mice as well as WT SOL muscle incubated with or without submaximal insulin 2 h (SOL) and 3 h (EDL) after prior in situ contraction of the lower hind limb. AMPKg3-associated (C), AMPKa2bg1- associated (D), and AMPKa1-associated (E) activity in EDL muscle from AMPK WT and mdko mice as well as WT SOL muscle (H) 2 h (SOL) and 3 h (EDL) after prior in situ contraction. Ex vivo contraction-induced phosphorylation of TBC1D4 Ser 711 (I) in EDL muscle from AMPK WT and AMPKg3 KO mice. Data were analyzed by two-way repeated-measures ANOVA (A, B, F, G, and I) and paired Student t tests (C E and H). B: ***P < main effect of insulin. C and I: ##P < 0.01 and #P < 0.05 vs. rest within genotype. Quantification of protein phosphorylation has not been related to protein abundance (see RESULTS). Values are means 6 SEM. For WT SOL data, n =10 11 per group in F and G and n =7 8 inh. For EDL data, n =12 13 per group in A and B and n =7 10 per group in C E and I.

11 608 AMPK and Muscle Insulin Sensitivity Diabetes Volume 66, March 2017 Figure 7 Prior contraction does not affect regulation of TBC1D1 by insulin. Phosphorylation of TBC1D1 Thr 590 (A and C) and Ser 231 (B and D) in EDL muscle from AMPK WT and mdko mice as well as WT SOL muscle incubated with or without submaximal insulin 2 h (SOL) and 3 h (EDL) after prior in situ contraction of the lower hind limb. Data were analyzed by two-way repeated-measures ANOVA within each genotype (EDL) and muscle (SOL). A and C: ***P < main effect of insulin. Quantification of protein phosphorylation has not been related to protein abundance (see RESULTS). Values are means 6 SEM. For all WT SOL data, n = 11 per group. For all EDL data, n =12 13 per group. AMPK mdko mice compared with EDL muscle from WT littermates, suggesting that any potential influence of glycogen per se is not a factor explaining the loss of contractioninduced insulin sensitization in muscle from AMPK mdko mice. Notably, glycogen content was similar between prior contracted and rested WT SOL muscle in parallel with normal insulin sensitivity (Fig. 8F). DISCUSSION The current study represents a further contribution to the search of molecular transducers involved in the insulinsensitizing effect of exercise. Here, we provide evidence to support that AMPK is necessary for increasing insulin sensitivity to stimulate glucose uptake in EDL muscle after in situ contraction, as well as enhancing whole-body insulin sensitivity and insulin-stimulated muscle glucose uptake after a single bout of acute exercise. We establish a causal link between a contraction-regulated signal and the subsequent improvement in muscle insulin sensitivity. On the basis of our findings, we propose that contraction-induced activation of AMPK potentiates the ability of insulin to increase phosphorylation of TBC1D4 leading to enhanced muscle glucose uptake. Theoretically, synthesis of new proteins involved in muscle glucose uptake may mediate improvements in skeletal muscle insulin sensitivity after contraction. However, we found that greater insulin-stimulated glucose uptake after contraction occurred without an increase in the abundance of multiple proteins involved in insulin-mediated signaling, as also supported by findings from others (12,15) and our previous observations in man (5,11,28). We found that the HKII protein level was decreased in EDL muscle from AMPK mdko mice compared with WT mice. However, since maximal insulin-stimulated glucose uptake was similar between genotypes, this indicates that lower HKII protein abundance inedlmusclefromampkmdkomiceisnotratelimiting for the ability of insulin to stimulate glucose uptake after contraction. Similar to previous findings in humans and rodents (4 6,17), the increase in skeletal muscle insulin sensitivity after contraction was not associated with enhanced proximal insulin signaling measured at the level of phosphorylated Akt Thr 308 and Ser 473. This further supports the notion that the intracellular mechanism responsible for increasing muscle insulin sensitivity after contraction is located downstream of Akt or involves insulin-regulated parallel pathways converging with elements regulating glucose transport. Improved insulin sensitivity after exercise is associated with enhanced translocation of GLUT4 to the cell surface membrane in skeletal muscle (14). Contraction-induced phosphorylation of TBC1D1, as well as insulin-induced phosphorylation of TBC1D4 regulates glucose uptake and GLUT4 translocation in skeletal muscle (26,41,42,48), indicating a role of these proteins in enhancing skeletal muscle insulin sensitivity after exercise/contraction. Since phosphorylation

12 diabetes.diabetesjournals.org Kjøbsted and Associates 609 Figure 8 Glycogen synthase activity does not drive improvements in muscle insulin sensitivity after prior contraction. Glycogen synthase (GS) activity expressed as %I-form (A) and total (B) as well as phosphorylation of GS site 3a+3b (C) and 2+2a (D) in EDL muscle from AMPK WT and mdko mice incubated with or without submaximal insulin 3 h after prior in situ contraction of the lower hind limb. Glycogen content (E and F) in EDL muscle from AMPK WT and mdko mice as well as WT SOL muscle 2 h (SOL) and 3 h (EDL) into recovery from prior in situ contraction. Data were analyzed by two-way repeated-measures ANOVA within each genotype (A D) and between genotypes (E) as well as a paired Student t test (F). A and C: ***P < main effect of insulin within genotype. E: Treatment 3 genotype interaction (P < 0.05), ###P < and ##P < 0.01 effect of treatment within genotype; P < 0.05 effect of genotype within treatment. Values are means 6 SEM. For WT SOL data, n = 8 per group. For EDL data, n =12 13 per group in A D and n = 10 per group in E. of TBC1D1 was similar between genotypes (WT vs. mdko) and muscle type (EDL vs. SOL), this indicates that TBC1D1 is not involved in the insulin-sensitizing effect of prior contraction. This is supported by several other studies in humans and rats (11,17,18,49). In contrast to TBC1D1, we observed an increased effect of a submaximal dose of insulin to stimulate phosphorylation of TBC1D4 Thr 649 and Ser 711 in prior contracted WT EDL muscle but not in EDL muscle from AMPK mdko mice. Furthermore, insulin-stimulated phosphorylation of TBC1D4 was not enhanced in prior contracted WT SOL muscle. We hypothesize that the potentiating effect of AMPK activation by prior contraction on insulin-stimulated phosphorylation of TBC1D4 Ser 711 induces a subsequent increase in TBC1D4 Thr 649 phosphorylation, which may facilitate the enhanced effect of insulin on glucose uptake. These observations are supported by positive and significant correlations between TBC1D4 Thr 649 and Ser 711

13 610 AMPK and Muscle Insulin Sensitivity Diabetes Volume 66, March 2017 phosphorylation, as well as between glucose uptake and phosphorylation of TBC1D4 Thr 649 /Ser 711,ofwhichThr 649 has previously been reported to be important for muscle glucose uptake in response to insulin (50). Moreover, these observations are fully in line with our previous findings in prior AICAR-stimulated EDL muscle (30). In the current study, contraction-induced phosphorylation of TBC1D4 Ser 711 is dependent on AMPKa1a2 and more specifically on AMPKg3. This suggests that the increase in muscle insulin sensitivity after prior contraction may be mediated through increased AMPKg3 activity during and/or after contraction. This idea is supported by observations in the AMPKg3-scarce SOL muscle (21) in which prior in situ contraction failed to enhance insulin sensitivity, AMPKg3 activity, and phosphorylation of TBC1D4 Ser 711. It is also in line with our previous findings showing that the increased effect of insulin on muscle glucose uptake after prior AICAR stimulation is dependent on AMPKg3 (30). Studies in humans and rats have not found evidence to support increased AMPK activity at time points of enhanced muscle insulin sensitivity after exercise/contraction (11,18,29). This may be related to measures of AMPK phosphorylation or surrogate measures of this (e.g., pacc and ptbc1d1) that potentially conceal differential regulation among the AMPK heterotrimers (51,52), as also evident in the current study. The amount of muscle glycogen consumed during an exercise bout may play a role in regulating postexercise insulin sensitivity (53). However, we found that the electrical stimulation protocol decreased glycogen content to similar levels in muscle from both genotypes, indicating that glycogen depletion per se does not mediate changes in muscle insulin sensitivity as also previously suggested (54). Skeletal muscle glycogen content and insulin-stimulated glucose uptake display an inverse relationship (47,55). Thus, glycogen levels at the time of insulin stimulation (rather than immediately after contraction) may be of importance for muscle insulin sensitivity. However, immediately before insulin stimulation, muscle glycogen content was lower in prior contracted mdko muscle compared with WT muscle, indicating that this is not the reason for the lost ability of prior contraction to enhance muscle insulin sensitivity in AMPK mdko mice. Since glycogen content was lower in prior contracted muscle compared with rested muscle in WT mice, we cannot rule out a functional role of decreased glycogen content for mediating the insulinsensitizing effect of prior contraction. This may be supported by observations in prior contracted WT SOL muscle in which glycogen content had returned to resting levels concomitant with normal insulin sensitivity. In fact, it may be hypothesized that reduced glycogen levels signal via AMPK to enhance muscle insulin sensitivity after exercise/contraction. At present we do not possess solid evidence to support this idea, and studies using AICAR (15,30) indicate at least that it is possible to bypass this association. In human and rodent skeletal muscle displaying increased insulin sensitivity after exercise, GS activity is higher in prior exercised muscle compared with nonexercised muscle (1,5,9,11). Thus, higher GS activity may be needed for the prior exercised muscle to handle the increased amount of glucose taken up during insulin stimulation. Because ;30% of 2-deoxyglucose taken up by muscle is incorporated into glycogen during insulin stimulation (56, and J.R. Hingst and J.F.P.W., unpublished observations in mouse muscle), our findings on 2-deoxyglucose uptake may be influenced by possible dysregulation of GS activity in skeletal muscle of AMPK mdko mice. However, we found that in vitro GS activity and phosphorylation were regulated similarly in muscle of AMPK WT and mdko mice in response to insulin. In fact, GS activity was elevated in prior rested and prior exercised muscle from AMPK mdko mice compared with WT mice. This suggests that elevated GS activity is not a primary driver for improvements in muscle insulin sensitivity at the level of glucose uptake. In conclusion, we provide evidence to support that prior contraction increases insulin sensitivity in EDL muscle to stimulate glucose uptake by an AMPK-dependent mechanism. Since the relative distribution of AMPK heterotrimeric complexes in human vastus lateralis greatly resembles that of mouse EDL muscle (20,21), our findings may be of high relevance for glucose metabolism in human skeletal muscle as well. Furthermore, we recently found intact regulation of the AMPK signaling network in skeletal muscle of patients with type 2 diabetes (51), and several findings of enhanced insulin-stimulated phosphorylation of TBC1D4 in prior exercised human muscle (11,28) support the notion of an AMPK-TBC1D4 signaling axis regulating muscle insulin sensitivity. Altogether, we provide basic insight to a physiological role of AMPK in skeletal muscle, strengthening the idea of AMPK being a relevant target for physiological and pharmacological interventions in the prevention and treatment of muscle insulin resistance in various conditions. Acknowledgments. The authors thank Betina Bolmgren, Irene Bech Nielsen, and Jeppe Kjærgaard Larsen (Department of Nutrition, Exercise and Sports, Faculty of Science, University of Copenhagen) for their skilled technical assistance. Funding. This study was supported by grants from the Danish Council for Independent Research, Medical Sciences; the research programme (2016) Physical activity and nutrition for improvement of health funded by the University of Copenhagen; the Lundbeck Foundation; the Novo Nordisk Foundation; and the Novo Nordisk Foundation Center for Basic Metabolic Research. The Novo Nordisk Foundation Center for Basic Metabolic Research is an independent research center at the University of Copenhagen that is partially funded by an unrestricted donation from the Novo Nordisk Foundation ( Duality of Interest. No potential conflicts of interest relevant to this article were reported. Author Contributions. R.K. designed and performed the experiments, analyzed the data, and wrote the manuscript. N.M.-H. performed the experiments and analyzed the data. J.B.B. performed biochemical assays and analyzed the data. M.F., B.V., M.B., and J.R.Z. provided founder mice for the study cohort. J.T.T. and J.F.P.W. designed the experiments and wrote the manuscript. All authors

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