2042 Diabetes Volume 64, June 2015 Rasmus Kjøbsted,1,2 Jonas T. Treebak,1,2 Joachim Fentz,1 Louise Lantier,3,4,5 Benoit Viollet,3,4,5 Jesper B. Birk,1 Peter Schjerling,6 Marie Björnholm,7 Juleen R. Zierath,2,7 and Jørgen F.P. Wojtaszewski1 Prior AICAR Stimulation Increases Insulin Sensitivity in Mouse Skeletal Muscle in an AMPK-Dependent Manner Diabetes 2015;64:2042–2055 | DOI: 10.2337/db14-1402 An acute bout of exercise increases glucose uptake in rodents (1–6) and may persist for up to 48 h after exercise, skeletal muscle by an insulin-independent mechanism. depending on carbohydrate availability (7–9). Improved mus- In the period after exercise, insulin sensitivity to in- cle insulin sensitivity postexercise is mediated by one or creased glucose uptake is enhanced. The molecular several local contraction-induced mechanisms (10) involving mechanisms underpinning this phenomenon are poorly both enhanced transport and intracellular processing of glu- understood but appear to involve an increased cell cose. This period is characterized by increased GLUT4 protein surface abundance of GLUT4. While increased proximal abundance at the plasma membraneandenhancedglycogen insulin signaling does not seem to mediate this effect, synthase activity (11,12). These changes occur independent elevated phosphorylation of TBC1D4, a downstream target of both insulin (Akt) and exercise (AMPK) signal- of global protein synthesis (13), including both total GLUT4 ing, appears to play a role. The main purpose of this and glycogen synthase protein content (4,11), and are inde- study was to determine whether AMPK activation pendent of changes in proximal insulin signaling, including increases skeletal muscle insulin sensitivity. We found Akt activation (3,4,13–17). that prior AICAR stimulation of wild-type mouse muscle AMPK is a heterotrimeric complex consisting of catalytic increases insulin sensitivity to stimulate glucose uptake. (a1/a2) and regulatory subunits (b1/b2andg1/g2/g3). Of However, this was not observed in mice with reduced or the 12 heterotrimeric combinations, only 3 and 5 combina- SIGNAL TRANSDUCTION ablated AMPK activity in skeletal muscle. Furthermore, tions have been found in the skeletal muscle of human and prior AICAR stimulation enhanced insulin-stimulated mouse, respectively (18,19). AMPK is activated in response 649 711 phosphorylation of TBC1D4 at Thr and Ser in to various stimuli that increase cellular energy stress (e.g., wild-type muscle only. These phosphorylation events metformin, hypoxia, hyperosmolarity, muscle contraction, were positively correlated with glucose uptake. Our and exercise) (20). With energy stress, intracellular concen- results provide evidence to support that AMPK activa- trations of AMP and ADP accumulate. This activates AMPK tion is sufficient to increase skeletal muscle insulin sen- allosterically and decreases the ability of upstream phospha- sitivity. Moreover, TBC1D4 phosphorylation may tases to dephosphorylate Thr172, which further increases facilitate the effect of prior AMPK activation to enhance glucose uptake in response to insulin. AMPK phosphorylation and activity (21). Like exercise, AICAR increases AMPK activity in skeletal muscle (22), which partly mimics the metabolic changes observed dur- The effect of insulin on skeletal muscle glucose uptake is ing muscle contraction (23). increased in the period after a single bout of exercise. This TBC1D4 is involved in insulin-stimulated glucose phenomenon is observed in muscle from both humans and transport in skeletal muscle (24) and is regulated via 1Section of Molecular Physiology, August Krogh Centre, Department of Nutrition, 7Integrative Physiology, Department of Molecular Medicine and Surgery, Karolinska Exercise and Sports, University of Copenhagen, Copenhagen, Denmark Institutet, Stockholm, Sweden 2 The Novo Nordisk Foundation Center for Basic Metabolic Research, Section of Corresponding author: Jørgen F.P. Wojtaszewski, [email protected]. Integrative Physiology, University of Copenhagen, Copenhagen, Denmark Received 11 September 2014 and accepted 20 December 2014. 3INSERM, U1016, Institut Cochin, Paris, France 4CNRS, UMR8104, Paris, France R.K. and J.T.T. share first authorship. 5Université Paris Descartes, Sorbonne Paris Cité, Paris, France © 2015 by the American Diabetes Association. Readers may use this article as 6Institute of Sports Medicine, Department of Orthopedic Surgery, Bispebjerg long as the work is properly cited, the use is educational and not for profit, and Hospital and Center for Healthy Aging, Faculty of Health Sciences, University of the work is not altered. Copenhagen, Copenhagen, Denmark See accompanying article, p. 1901. diabetes.diabetesjournals.org Kjøbsted and Associates 2043 phosphorylation at multiple sites by Akt (25), thereby and extensor digitorum longus (EDL) muscles were increasing translocation of GLUT4 to the plasma mem- dissected and suspended in incubation chambers (Multi brane. AMPK also targets TBC1D4; however, this does not Wire Myograph System; DMT, Aarhus, Denmark) con- seem to directly affect glucose uptake (26). As insulin taining Krebs-Ringer buffer (KRB) (117 mmol/L NaCl, (Akt) and exercise/AICAR (AMPK) signaling pathways 4.7 mmol/L KCl, 2.5 mmol/L CaCl2, 1.2 mmol/L converge on TBC1D4, this may explain how exercise KH2PO4, 1.2 mmol/L MgSO4, 0.5 mmol/L NaHCO3, modulates insulin action to regulate glucose transport in pH 7.4) supplemented with 0.1% BSA, 8 mmol/L man- skeletal muscle. Supporting this concept, TBC1D4 phos- nitol, and 2 mmol/L pyruvate. During the entire incuba- phorylation is elevated in skeletal muscle several hours tion period, the buffer was oxygenated with 95% O2 and after an acute bout of exercise in both rodents and 5% CO2, and maintained at 30°C. After 10 min of pre- humans, concomitant with increased insulin sensitivity incubation, muscles were incubated for 50 min in the to stimulate glucose uptake in the postexercise period absence or presence of 1 mmol/L AICAR (Toronto Re- (15,16,27–30). search Chemicals, Toronto, Ontario, Canada) in 100% Prior AICAR stimulation increases skeletal muscle human serum from healthy overnight-fasted men. The insulin sensitivity (13). However, because AICAR exerts use of serum is necessary to elicit an effect of AICAR on multiple AMPK-independent effects (31), the direct re- muscle insulin sensitivity (13). Soleus and EDL muscles lationship between AMPK and muscle insulin sensitivity were allowed to recover in the absence of AICAR in has not been established. Thus, the primary purpose of modified KRB supplemented with 5 mmol/L glucose, 5 the current study was to determine whether AMPK di- mmol/L mannitol, and 0.1% BSA for 4 h (soleus muscle) rectly regulates skeletal muscle insulin sensitivity on or 6 h (EDL muscle). During recovery, the medium was glucose uptake. We established an ex vivo protocol using replaced once every hour to maintain an adequate glu- mouse muscle to study insulin sensitivity after prior cose concentration. Subsequently, paired muscles from AICAR stimulation and tested the hypothesis that each animal were incubated for 30 min in KRB in the AMPK is necessary for the effect of AICAR to enhance absence or presence of a submaximal concentration insulin sensitivity. Furthermore, we evaluated TBC1D4 (100 mU/mL) of insulin (Actrapid; Novo Nordisk, Bagsvaerd, phosphorylation status because this protein is a conver- Denmark). The uptake of 2-deoxyglucose was measured gence point for insulin- and exercise-mediated signaling during the last 10 min of the 30-min period by adding events. 1 mmol/L [3H]2-deoxyglucose (0.056 MBq/mL) and 7 mmol/L [14C]mannitol (0.0167 MBq/mL) to the incu- RESEARCH DESIGN AND METHODS bation medium. After incubation, muscles were harvested, fi Animals/Humans washed in ice-cold KRB, quickly dried on lter paper, and All experiments were approved by the Danish Animal frozen in liquid nitrogen. Experimental Inspectorate and the regional animal ethics committee of Northern Stockholm and complied with Muscle Processing the European Union Convention for the Protection of Muscles were homogenized in 400 mLofice-cold Vertebrate Animals Used for Scientific Purposes (Council buffer (10% glycerol, 20 mmol/L sodium pyrophos- of Europe 123, Strasbourg, France, 1985). Except for phate, 1% NP-40, 2 mmol/L phenylmethylsulfonyl fl the wild-type (WT) mice (C57BL/6J; Taconic, Ejby, uoride [PMSF], 150 mmol/L sodium chloride, 50 Denmark) used in Figs. 1, 3E, and 8, the animals used mmol/L HEPES, 20 mmol/L b-glycerophosphate, 10 fi mmol/L sodium fluoride, 1 mmol/L EDTA, 1 mmol/L in this study were muscle-speci c kinase-dead a2-AMPK fi EGTA, 10 mg/mL aprotinin, 3 mmol/L benzamidine, (AMPK KD) (32), muscle-speci c a2- and a1-AMPK double-knockout (AMPK mdKO) (33), and g -AMPK KO 10 mg/mL leupeptin, and 2 mmol/L sodium orthova- 3 3 mice (34) with corresponding WT littermates used as nadate, pH 7.5) for 2 30 s at 30 Hz using steel beads controls. All mice in this study were female (mean weight and a TissueLyzer II (QIAGEN, Hilden, Germany). 24.3 6 0.2 g) and were maintained on a 12:12 light-dark Homogenates were rotated end over end for 1 h before cycle (6:00 A.M. to 6:00 P.M.) with unlimited access to centrifugation at 16,000g for 20 min. The supernatant standard rodent chow and water. Serum was obtained (lysate) was collected, frozen in liquid nitrogen, and 2 from healthy young men in accordance with a protocol stored at 80°C for later analyses. approved by the Ethics Committee of Copenhagen (pro- tocol #H-3–2012–140) and complied with the ethical Glucose Uptake Measurements 3 guidelines of the Declaration of Helsinki II. Informed con- Glucose uptake was assessed by the accumulation of [ H] 14 sent was obtained from all participating subjects before 2-deoxyglucose in muscle with the use of [ C]mannitol they entered the study. (PerkinElmer, Waltham, MA) as an extracellular marker. Radioactivity was measured in 250 mL of lysate by liquid Muscle Incubations scintillation counting (Ultima Gold and Tri-Carb 2910 TR; Fed animals were anesthetized by intraperitoneal injec- PerkinElmer) and was related to the specific activity of the tion of pentobarbital (10 mg/100 g body wt) before soleus incubation buffer.