Prior AICAR stimulation increases insulin sensitivity in mouse skeletal muscle in an. AMPK-dependent manner. Jørgen F.P.

Transcription

1 Page 1 of 38 Prior AICAR stimulation increases insulin sensitivity in mouse skeletal muscle in an AMPK-dependent manner Rasmus Kjøbsted a,b,, Jonas T. Treebak a,b,, Joachim Fentz a, Louise Lantier c,d,e, Benoit Viollet c,d,e, Jesper B. Birk a, Peter Schjerling f, Marie Björnholm g, Juleen R. Zierath b,g, Jørgen F.P. Wojtaszewski a a Section of Molecular Physiology, the August Krogh Centre, Department of Nutrition, Exercise and Sports, University of Copenhagen, DK-21 Copenhagen, Denmark b The Novo Nordisk Foundation Center for Basic Metabolic Research, Section of Integrative Physiology, University of Copenhagen, Copenhagen, Denmark c INSERM, U116, Institut Cochin, Paris, France d CNRS, UMR814, Paris, France e Université Paris Descartes, Sorbonne Paris Cité, Paris, France. f Institute of Sports Medicine, Department of Orthopedic Surgery, Bispebjerg Hospital and Center for Healthy Aging, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. g Integrative Physiology, Department of Molecular Medicine and Surgery, Karolinska Institutet, SE Stockholm, Sweden Indicates shared first-authorship Short running title: AMPK and insulin sensitivity in skeletal muscle Key words: AICAR, exercise, glucose uptake, TBC1D4, AS16 Figures / Tables: 8 / Word count: 433 Corresponding author: Jørgen F.P. Wojtaszewski, PhD Publish Ahead of Print, published online December 31, 214 1

2 Page 2 of 38 The August Krogh Centre Department of Nutrition, Exercise and Sports Section of Molecular Physiology University of Copenhagen Universitetsparken 13 DK-21, Copenhagen, Denmark Phone: jwojtaszewski@nexs.ku.dk Abstract Acute exercise increases glucose uptake in skeletal muscle by an insulin-independent mechanism. In the period after exercise insulin sensitivity to increase glucose uptake is enhanced. The molecular mechanisms underpinning this phenomenon are poorly understood, but appear to involve an increased cell surface abundance of GLUT4. While increased proximal insulin signaling does not seem to mediate this effect, elevated phosphorylation of TBC1D4, a downstream target of both insulin (Akt) and exercise (AMPK) signaling, appears to play a role. The main purpose of this study was to determine whether AMPK activation increases skeletal muscle insulin sensitivity. We found that prior AICAR stimulation of wildtype mouse muscle increases insulin sensitivity to stimulate glucose uptake. However, this was not observed in mice with reduced or ablated AMPK activity in skeletal muscle. Furthermore, prior AICAR stimulation enhanced insulin-stimulated phosphorylation of TBC1D4 at Thr 649 and Ser 711 in wild-type muscle only. These phosphorylation events were positively correlated with glucose uptake. Our results provide evidence to support that AMPK is sufficient to increase skeletal muscle insulin sensitivity. Moreover, TBC1D4 phosphorylation may facilitate the effect of prior AMPK activation to enhance glucose uptake in response to insulin. 2

3 Page 3 of 38 Introduction The effect of insulin on skeletal muscle glucose uptake is increased in the period after a single bout of exercise. This phenomenon is observed in muscle from both humans and rodents (1 6) and may persist for up to 48 hours after exercise, depending on carbohydrate availability (7 9). Improved muscle insulin sensitivity post-exercise is mediated by one or several local contraction-induced mechanisms (1) involving both enhanced transport and intracellular processing of glucose. This period is characterized by increased GLUT4 protein abundance at the plasma membrane and enhanced glycogen synthase (GS) activity (11,12). These changes occur independent of global protein synthesis (13), including both total GLUT4 and GS protein content (4,11), and are independent of changes in proximal insulin signaling, including Akt activation (3,4,13 17). AMP-activated protein kinase (AMPK) is a heterotrimeric complex consisting of catalytic (α1/α2) and regulatory subunits (β1/β2 and γ1/γ2/γ3). Of the 12 heterotrimeric combinations, only 3 and 5 combinations have been found in human and mice skeletal muscle, respectively (18,19). AMPK is activated in response to various stimuli that increase cellular energy stress (e.g. metformin, hypoxia, hyperosmolarity, muscle contraction, and exercise) (2). With energy stress, intracellular concentrations of AMP and ADP accumulate. This activates AMPK allosterically and decrease the ability of upstream phosphatases to dephosphorylate Thr 172, which further increase AMPK phosphorylation and activity (21). Like exercise, 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR) increases AMPK activity in skeletal muscle (22) which partly mimics the metabolic changes observed during muscle contraction (23). 3

4 Page 4 of 38 TBC1D4 is involved in insulin-stimulated glucose transport in skeletal muscle (24) and is regulated via phosphorylation at multiple sites by Akt (25), thereby increasing translocation of GLUT4 to the plasma membrane. AMPK also targets TBC1D4, however, this does not seem to directly affect glucose uptake (26). As insulin (Akt) and exercise/aicar (AMPK) signaling pathways converge on TBC1D4, this may explain how exercise modulates insulin action to regulate glucose transport in skeletal muscle. Supporting this concept, TBC1D4 phosphorylation is elevated in skeletal muscle several hours after an acute bout of exercise in both rodents and humans, concomitant with increased insulin sensitivity on glucose uptake in the post-exercise period (15,16,27 3). Prior AICAR stimulation increases skeletal muscle insulin sensitivity (13). However, since AICAR exerts multiple AMPK-independent effects (31), the direct relationship between AMPK and muscle insulin sensitivity has not been established. Thus, the primary purpose of the present study was to determine whether AMPK directly regulates skeletal muscle insulin sensitivity on glucose uptake. We established an ex vivo protocol using mouse muscle to study insulin sensitivity after prior AICAR stimulation and tested the hypothesis that AMPK is necessary for the effect of AICAR to enhance insulin sensitivity. Furthermore, we evaluated TBC1D4 phosphorylation status, as this protein is a convergence point for insulin- and exercise-mediated signaling events. Research Design and Methods Animals / humans All experiments were approved by the Danish Animal Experimental Inspectorate and the regional animal ethics committee of Northern Stockholm and complied with the EU convention for protection of vertebra animals used for scientific purposes (Council of Europe 4

5 Page 5 of , Strasbourg, France, 1985). Except for wild-type (WT) mice (C57BL/6J, Taconic Denmark) used in Fig. 1, 3E and 8, the animals used in this study were muscle-specific kinase dead α 2 -AMPK (AMPK KD) (32), muscle-specific α 2 - and α 1 -AMPK double KO (AMPK mdko) (33) and γ 3 -AMPK KO mice (34) with corresponding WT littermates as controls. All mice in this study were female (24.3±.2 g) maintained on a 12:12 light-dark cycle (6: AM - 6: PM) with unlimited access to standard rodent chow and water. Serum was obtained from healthy young men in accordance with protocol approved by the Ethics Committee of Copenhagen (#H ) and complied with the ethical guidelines of the declaration of Helsinki II 2. Informed consent was obtained from all participating subjects before entering the study. Muscle incubations Fed animals were anesthetized by intraperitoneal injection of pentobarbital (1 mg/1 g body weight) before soleus and extensor digitorum longus (EDL) muscles were dissected and suspended in incubation chambers (Multi Myograph system; Danish Myo-Technology, Denmark) containing Krebs Ringer Buffer (KRB) [117 mm NaCl, 4.7 mm KCl, 2.5 mm CaCl 2, 1.2 mm KH 2 PO 4, 1.2 mm MgSO 4,.5 mm NaHCO 3 (ph 7.4)] supplemented with.1% BSA, 8 mm mannitol and 2 mm pyruvate. During the entire incubation period, the buffer was oxygenated with 95% O 2 and 5% CO 2 and maintained at 3 C. After 1 min of pre-incubation, muscles were incubated for 5 min in the absence or presence of 1 mm AICAR (Toronto Research Chemicals, Toronto, Canada) in 1% human serum from overnight fasted men. The use of serum is necessary to elicit an effect of AICAR on muscle insulin sensitivity (13). Soleus and EDL muscles were allowed to recover in the absence of AICAR in modified KRB supplemented with 5 mm glucose, 5 mm mannitol and.1% BSA 5

6 Page 6 of 38 for 4 (soleus) or 6 (EDL) hrs. During recovery, the medium was replaced once every hour to maintain an adequate glucose concentration. Subsequently, paired muscles from each animal were incubated for 3 min in KRB in the absence or presence of a submaximal (1 µu/ml) insulin concentration (Actrapid; Novo Nordisk, Denmark). Uptake of 2-deoxyglucose was measured during the last 1 min of the 3 min period by adding 1 mm [ 3 H]2-deoxyglucose (.56 MBq/ml) and 7 mm [ 14 C]mannitol (.167 MBq/ml) to the incubation medium. After incubation, muscles were harvested, washed in ice-cold KRB, quickly dried on filter paper and frozen in liquid nitrogen. Muscle processing Muscles were homogenized in 4 µl ice-cold buffer [1% glycerol, 2 mm sodium pyrophosphate, 1% NP-4, 2 mm PMSF, 15 mm sodium chloride, 5 mm HEPES, 2 mm β-glycerophosphate, 1 mm sodium fluoride, 1 mm EDTA, 1 mm EGTA, 1 µg/ml aprotinin, 3 mm benzamidine, 1 µg/ml leupeptin, and 2 mm sodium orthovanadate (ph 7.5)] for 2 x 3 s at 3 Hz using steel beads and a TissueLyzer II (Qiagen, Germany). Homogenates were rotated end-over-end for 1 hr before centrifugation at 16, g for 2 min. The supernatant (lysate) was collected, frozen in liquid nitrogen and stored at -8 C for later analyses. Glucose uptake measurements Glucose uptake was assessed by the accumulation of [ 3 H]2-deoxyglucose into muscle with the use of [ 14 C]mannitol (Perkin Elmer) as an extracellular marker. Radioactivity was measured on 25 µl lysate by liquid scintillation counting (Ultima Gold TM and Tri-Carb 291 TR, Perkin Elmer, MA) and related to the specific activity of the incubation buffer. 6

7 Page 7 of 38 SDS-PAGE and Western blot analyses Total protein abundance in muscle lysates was determined by the bicinchonic acid method (Pierce Biotechnology, IL). Muscle lysates were prepared in Laemmli buffer and heated for 1 min at 96 C. Equal amounts of protein were separated by SDS-PAGE on 5 or 7% self-cast gels and transferred to PVDF membranes using semidry-blotting. Membranes were blocked for 5-1 min in 2% skim milk or 3% BSA and probed with primary and secondary antibodies. Proteins with bound antibody were visualized with chemiluminescence (Millipore) using a digital imaging system (BioRad ChemiDoc MP). All membranes were stripped with buffer (1 mm 2-mecaptoethanol, 2% SDS, 62.5 mm Tris-HCl (ph 6.7)) and re-probed with new primary antibodies for detection of other phosphorylation sites on identical proteins or the corresponding total proteins. The stripping procedure was verified by re-incubating membranes with secondary antibodies for detection of possibly still bound primary antibody. Antibodies The following antibodies were from Cell Signaling Technology, MA: anti-phospho-ampk- Thr 172 (#2531), anti-phospho-acetyl-coa carboxylase (ACC) Ser 79 (#3661), anti-akt2 (D6G4) (#363), anti-phospho-akt-thr 38 (#9275), anti-phospho-akt-ser 473 (#9271), antiphospho-tbc1d1-thr 59 (#6927), anti-phospho-tbc1d4-ser 318 (#8619), anti-phospho- TBC1D4-Ser 588 (#873) and anti-phospho-tbc1d4-thr 642 (#8881). Anti-DYKDDDDK-Tag (FLAG-Tag) (F184, Sigma-Aldrich), anti-phospho-tbc1d1-ser 237 (#261452, Millipore), anti-tbc1d1 as previously described (35), anti-as16 (TBC1D4) (#7-741, Millipore), antiphospho-tbc1d4-ser 711 as previously described (26) and anti-ampk-α2 (SC-19131, Santa Cruz). Antibodies used for AMPK activity measurements were anti-ampk-γ3, anti-ampkα1 and anti-ampk-α2, all of which were kindly provided by prof. D. G. Hardie (University 7

8 Page 8 of 38 of Dundee, UK). AMPK activity assay Five different AMPK trimer complexes have been detected in mouse skeletal muscle: α2β2γ3, α2β1γ1, α2β2γ1, α1β1γ1 and α1β2γ1 (19). α2β2γ3-ampk activity was measured on γ3- AMPK IPs from 3 µg of muscle lysate using AMPK-γ3 antibody, G protein coupled agarose beads (Millipore) and IP buffer (5 mm NaCl, 1% Triton X-1, 5 mm sodium fluoride, 5 mm sodium-pyrophosphate, 2 mm Tris-base (ph 7.5), 5 µm PMSF, 2 mm DTT, 4 µg/ml leupeptin, 5 µg/ml soybean trypsin inhibitor, 6 mm benzamidine, and 25 mm sucrose). Samples were treated as previously described (19,36). In short, after overnight endover-end rotation at 4 C IPs were centrifuged for 1 min at 2, g and washed once in IP buffer, once in 6x assay buffer (24 mm HEPES, 48 mm NaCl, ph 7.) and twice in 3x assay buffer (1:1). The activity assay was performed for 3 min at 3 C in a total volume of 3 µl kinase mix (4 mm HEPES, 8 mm NaCl, 833 µm DTT, 2 µm AMP, 1 µm AMARA-peptide, 5 mm MgCl 2, 2 µm ATP, and 2 µci of [γ-33p]-atp (Perkin Elmer). The reaction was terminated by adding 1 µl 1% phosphoric acid. 2 µl of the reaction mix were spotted on P81 filter paper. These were subsequently washed 4 x 15 min in 1% phosphoric acid. AMPK activity was analyzed on dried filter paper using a Storm 85 PhosphorImager (Molecular Dynamics). The combined activity of α2β1γ1 and α2β2γ1 was measured on supernatants from the γ3-ampk IPs using the AMPK-α2 antibody for a second IP and the combined activity of α1β1γ1 and α1β2γ1 was measured on supernatants from the α2-ampk IPs using α1-ampk antibody for a third IP. In vivo gene electro-transfer 8

9 Page 9 of 38 TBC1D4 WT and TBC1D4 T649A and S711A DNA mutant constructs, containing T-to-A and S-to-A point mutations respectively, were commercially and individually synthesized from the gene encoding mouse TBC1D4 (GeneArt / Life-Technologies, Germany). All three constructs were subsequently subcloned into a p3xflag-cmv-9-1 vector using NotI and KpnI cloning sites before amplification in E. Coli TOP1 cells (Invitrogen). Plasmid DNA was extracted using an endotoxin-free Plasmid Mega Kit (Qiagen) and diluted in isotonic saline to a final concentration of 2 µg/µl. DNA (5 µg) was injected into tibialis anterior muscle 2 hours after hyaluronidase (Sigma-Aldrich) treatment (1 injection of 3 units/muscle, 1 unit/µl) and gene electro-transfer was performed as previously described (24). Seven days after gene electro-transfer, phosphorylation of TBC1D4 Thr 649 and Ser 711 was assessed in tibialis anterior muscle of anesthetized animals (8 mg pentobarbital/1 g body weight) in response to retro-orbital injection of either saline or insulin (1 U/kg). Ten minutes after injection, tibialis anterior muscle was removed, quickly frozen in liquid nitrogen and stored at -8 C for subsequent analysis. Statistics Statistical analyses were performed using Sigmaplot 11. (Systat Software Inc, Germany) and SPSS 2 (IBM Corporation) software. SPSS 2 was used for three-way ANOVA with repeated measures while all other analyses were performed using Sigmaplot 11.. Data are presented as means ± SEM. One-, two- or three-way ANOVAs with or without repeated measures was used to assess statistical differences, where appropriate. When a three-way interaction occurred (p<.5; Genotype x AICAR x ), a two-way ANOVA with repeated measures was used on each genotype (WT and KD or WT and mdko) in order to determine the site of interaction between AICAR and insulin (p<.5; AICAR x ). 9

10 Page 1 of 38 Main effect of genotype was included as text in figures. For post hoc testing, Student- Newman-Keuls test was used. Correlation analyses were performed by determination of Pearson product-moment correlation coefficient. Differences were considered statistically significant if p<.5. Results Prior AICAR stimulation increases insulin sensitivity in EDL, but not in soleus muscle. Acute AICAR stimulation increased glucose uptake and AMPK phosphorylation in both soleus and EDL muscle (Fig. 1A, B and F). We then determined the time point at which glucose uptake had reversed to basal levels in order to evaluate the effect of AICAR on insulin sensitivity. In WT soleus and EDL muscle glucose uptake reversed to basal levels after 4 and 6 hours of recovery from AICAR stimulation, respectively (Fig. 1C). Prior AICAR treatment increased the effect of a submaximal (1 µu/ml) insulin concentration to stimulate glucose uptake in EDL, but not in soleus muscle from WT mice (Fig. 1D and 1E). Based on these results, we chose to use only EDL muscle for subsequent experiments. Prior AICAR stimulation increases muscle insulin sensitivity in an AMPK-dependent manner. In order to clarify whether the effect of AICAR on insulin sensitivity is dependent on AMPK, we took advantage of the AMPK KD and AMPK mdko mouse models in which AMPK activity is decreased or ablated in skeletal muscle. Prior AICAR stimulation increased insulin sensitivity in isolated EDL muscle from WT littermates, but failed to increase insulin sensitivity in both transgenic models (Fig. 2A and B). The incremental increase in insulinstimulated glucose uptake (glucose uptake for insulin minus glucose uptake for basal) was significantly higher after prior AICAR stimulation in WT littermates only (Fig. 2C and D). 1

11 Page 11 of 38 AMPK activity and signaling. As AICAR acutely increases phosphorylation of AMPK and the downstream target ACC, we investigated whether this effect was maintained into recovery. Phosphorylation of AMPK and ACC was increased in EDL muscle previously stimulated with AICAR independent of genotype (Fig. 3A-D, H and I). We assume that the observed increase in ACC phosphorylation in muscle from both transgenic mouse models after prior AICAR treatment corresponds to AMPK-independent effects of AICAR on ACC phosphorylation or AMPK activation in non-muscle cells. However, prior AICAR treatment increased ACC phosphorylation to a greater extent in muscle from WT littermates compared to both transgenic models (although only significant in WT mice from the mdko model), indicating a maintained effect of prior AICAR stimulation on AMPK in muscle cells. Therefore, we measured AMPK activity in WT EDL muscle previously stimulated with AICAR. The combined activity of α1β1γ1 and α1β2γ1 was ~1.4-fold higher compared to unstimulated control muscle (p=.37), while α2β2γ3 activity was ~2.3-fold higher (p<.1) (Fig. 3E). In contrast, the combined activity of AMPK trimer complexes α2β2γ1 and α2β1γ1 was unchanged by prior AICAR treatment. This indicates a persistent effect of prior AICAR stimulation on specific AMPK trimer activity in particular α2β2γ3 activity. Prior AICAR stimulation increases muscle insulin sensitivity in an AMPK-γ3 dependent manner. A persistent increase in AMPK α2β2γ3 activity after AICAR stimulation prompted us to test the hypothesis, that the effect of AICAR to enhance muscle insulin sensitivity is mediated through the AMPK α2β2γ3 trimer complex. Indeed, prior AICAR stimulation failed to 11

12 Page 12 of 38 increase muscle insulin sensitivity in whole body γ 3 -AMPK KO mice (Fig. 3F and G). For unknown reasons, prior AICAR treatment still affected basal glucose uptake in WT littermates in this particular experiment suggesting that the acute effect of AICAR on glucose uptake was not fully reversed. Akt signaling. Prior AICAR stimulation potentially enhances muscle insulin sensitivity to stimulate glucose uptake by regulating proximal insulin-signaling proteins. To investigate this, we measured phosphorylation of Akt Thr 38 and Ser 473. did not further increase phosphorylation of Thr 38 and Ser 473 in muscle previously stimulated with AICAR compared to control muscle (Fig. 4A-F). TBC1D1 signaling. TBC1D1 is a closely related paralogue of TBC1D4 that is regulated by both AMPK and Akt, and regulates glucose transport (37 4). As AMPK increases phosphorylation of TBC1D1 Ser 231 in response to contraction and AICAR, and Akt increases phosphorylation of Thr 59 in response to insulin, we investigated whether changes in TBC1D1 phosphorylation occurred in parallel with the increase in muscle insulin sensitivity after prior AICAR stimulation. Phosphorylation of TBC1D1 Ser 231 was markedly increased in WT muscle previously stimulated with AICAR. Prior AICAR stimulation also modestly increased phosphorylation of TBC1D1 Ser 231 in muscle from AMPK KD and mdko mice (Fig. 5A, B, E and F). Furthermore, insulin increased phosphorylation of TBC1D1 Thr 59 in both mouse models independent of genotype (Fig 5C-F). However, in AMPK mdko mice and WT littermates, 12

13 Page 13 of 38 insulin-stimulated phosphorylation of TBC1D1 Thr 59 in prior AICAR-stimulated muscle was decreased compared to control muscle (Fig 5D). TBC1D4 signaling. TBC1D4 (like TBC1D1) has been identified as a substrate of both AMPK and Akt in skeletal muscle (25,26) and phosphorylation of TBC1D4 is critical for insulin-stimulated glucose uptake (24,41). In addition, TBC1D4 is phosphorylated at multiple sites in the post-exercise period in parallel with enhanced muscle insulin sensitivity (15,16,27 3). This indicates that regulation of muscle insulin sensitivity is linked to TBC1D4 phosphorylation. We found an increased effect of insulin on TBC1D4 Thr 649 and Ser 711 phosphorylation in muscle previously stimulated with AICAR compared to control muscle (Fig. 6A-D, I and J). Furthermore, this effect was dependent on AMPK, as no difference in insulin-mediated phosphorylation was observed between control and prior AICAR-stimulated muscle from either of the two AMPK transgenic models. Importantly, the effect of prior AICAR treatment was site-specific as insulin-stimulated phosphorylation of TBC1D4 Ser 324 and Ser 595 was unaffected (Fig. 6E-J) Glucose uptake correlates with TBC1D4 site-specific phosphorylation levels. To investigate whether AICAR/AMPK increases muscle insulin sensitivity through TBC1D4, we performed a correlation analysis between delta values (insulin minus basal) on muscle glucose uptake and TBC1D4 phosphorylation. We found that glucose uptake and phosphorylation of TBC1D4 Ser 711 was positively correlated in WT littermates from both AMPK mouse models (p<.1 and p<.1; Fig. 7A and B, respectively). Correlating data for glucose uptake and phosphorylation of TBC1D4 Thr 649 revealed a more scattered pattern that 13

14 Page 14 of 38 was positively correlated in WT littermates from the AMPK KD strain (p<.1; Fig. 7C), but not correlated in WT littermates from the AMPK mdko strain (p=.18; Fig. 7D). In addition, phosphorylation level of TBC1D4 Thr 649 and Ser 711 was positively and strongly correlated in WT littermates from both AMPK mouse models (p<.1; Fig. 7E and F). Phosphorylation level of TBC1D4 Thr 649 and Ser 711 may be causally linked. AMPK has been shown to regulate phosphorylation of TBC1D4 Ser 711 and in muscle overexpressing a 4P mutant of TBC1D4 (in which Ser 711 is not mutated) phosphorylation of Ser 711 is severely blunted (26). In order to investigate whether changes in phosphorylation level of TBC1D4 Ser 711 affects TBC1D4 Thr 649 phosphorylation and vice versa, TBC1D4- WT, TBC1D4-S711A and TBC1D4-T649A constructs were expressed in mouse tibialis anterior muscle by gene electro-transfer. increased phosphorylation of TBC1D4 Thr 649 in muscle expressing TBC1D4-WT or TBC1D4-S711A, but Thr 649 phosphorylation levels were significantly blunted in the latter (Fig. 8A and C). increased phosphorylation of TBC1D4 Ser 711 in muscle expressing TBC1D4-WT and this response was completely ablated in muscle expressing TBC1D4-T649A (Fig. 8B and C). Our results suggest, that the phosphorylation level of TBC1D4 Thr 649 and Ser 711 are mutually dependent on each other. Discussion Several lines of evidence implicate that AMPK activation regulates skeletal muscle insulin sensitivity. In C 2 C 12 myotubes, AICAR stimulation or hyperosmotic stress increases insulin sensitivity of which the latter effect is inhibited by the unspecific AMPK inhibitor compound C (43). Similarly, insulin sensitivity is increased in myotubes transfected with a constitutive active form of AMPKα, which is also suppressed by compound C (44). 14

15 Page 15 of 38 Furthermore, AICAR fails to increase insulin action in cells transfected with a dominant negative form of AMPKα (44). Collectively, our data and those obtained in cell culture systems (43,44) suggest AMPK plays an important role in mediating AICAR-induced increases in skeletal muscle insulin sensitivity to stimulate glucose transport. AICAR is taken up by the cell where it acts as an AMP mimetic thus potentially affecting multiple proteins regulated by AMP. Within recent years an increased number of AMPK-independent effects of AICAR have been described together with reports identifying AICAR as a modulator of enzymes such as glycogen phosphorylase, glucokinase, and phosphofructokinase (31). However, as AICAR did not increase insulin sensitivity in muscle from AMPK KD or mdko mice any possible AMPK-independent effect of AICAR does not seem to account for changes in glucose uptake in response to insulin. The improvement in insulin-stimulated glucose uptake in muscle previously stimulated with AICAR occurred independent of changes in proximal insulin signaling (Akt phosphorylation). This is consistent with earlier findings showing prior AICAR treatment does not increase either Akt phosphorylation or PI3K activity in rat skeletal muscle (13). Similar observations have been made in both human and rodent skeletal muscle after acute exercise (3,4,13,16,17). Based on this, the mechanism responsible for the AMPK-dependent increase in muscle insulin sensitivity likely involves signal transduction downstream of Akt, implicating a role for TBC1D1 or TBC1D4 We evaluated the phosphorylation status of key sites on TBC1D1 previously shown to increase in response to AICAR, muscle contraction, exercise or insulin (16,37,42,45). Phosphorylation of TBC1D1 Ser 231 was markedly increased in muscle from WT mice and only modestly increased in muscle from AMPK KD and mdko mice 6 hours after AICAR treatment. Conversely, phosphorylation of TBC1D1 Thr 59 was increased in response to 15

16 Page 16 of 38 insulin independent of genotype. Given that prior AICAR stimulation increased phosphorylation of TBC1D1 Ser 231 in both basal and insulin-stimulated muscle and insulin increased phosphorylation of TBC1D1 Thr 59 in all groups, our results suggest that phosphorylation of TBC1D1 Ser 231 and Thr 59 is not sufficient for regulating muscle insulin sensitivity in response to prior AICAR treatment. This is supported by findings of identical TBC1D1 Ser 231 phosphorylation and similar increases in insulin-stimulated PAS-TBC1D1 and Ser 59 phosphorylation in previously rested or exercised muscle from humans and rats (15,16,29). In addition to TBC1D1, we also analyzed phosphorylation status of TBC1D4 at multiple sites because it has been suggested to play a prominent role in regulating both insulin-stimulated glucose uptake and insulin action post exercise in skeletal muscle (24,27 29). Recent studies, using site-specific antibodies, suggest only phosphorylation of TBC1D4 Ser 711 is increased in mouse skeletal muscle in response to either exercise, AICAR or ex vivo muscle contraction (26,42). As AICAR-mediated phosphorylation of TBC1D4 Ser 711 is dependent on AMPK (26), the AMPK-dependent increase in insulin sensitivity after AICAR treatment may be mediated through changes in TBC1D4 Ser 711 phosphorylation during acute AICAR stimulation. Our data showing increased insulin action on Ser 711 phosphorylation in WT, but not in AMPK KD or mdko muscle previously stimulated with AICAR are consistent with this notion. In contrast to TBC1D4 Ser 711, phosphorylation of Thr 649 seems to be important for insulin-stimulated glucose uptake in mouse EDL muscle (26,41). However, this site is not regulated by acute AICAR treatment (26,46). Thus, the potentiated effect of insulin on TBC1D4 Ser 711 phosphorylation by prior AICAR treatment appears to mediate an enhanced AMPK-dependent phosphorylation of Thr 649, which may facilitate the increased effect of 16

17 Page 17 of 38 insulin on glucose uptake. Such relationship is supported by the correlative relationship between TBC1D4 Thr 649 and Ser 711 phosphorylation and in particular the strong relationship between Ser 711 phosphorylation and muscle glucose uptake. In addition, expression of the S711A TBC1D4 mutant decreased total phosphorylation of TBC1D4 Thr 649, whereas the T649A mutant severely decreased phosphorylation of Ser 711 both in the presence or absence of insulin. This clearly indicates interdependence between the two sites and supports a possible mechanism by which AMPK, through TBC1D4 Ser 711, regulates insulin action to stimulate glucose uptake. Previously it has been shown that discrepancies between Akt and TBC1D4 phosphorylation exist indicating that only a small fraction of the insulin signal is necessary for mediating full glucose uptake in response to insulin (47). This is also observed in the current study where phosphorylation of Akt Ser 38 and Thr 473 does not match either phosphorylation of TBC1D4 Thr 649 and Ser 711 or glucose uptake in prior AICAR stimulated WT muscle. However, in cases of normal insulin sensitivity (and perhaps increased) there seems to be a good correlation between plasma membrane GLUT4 and TBC1D4 phosphorylation (47). This indicates that glucose uptake and phosphorylation of TBC1D4 are associated as also indicated by the correlations in the present study. Besides a change in TBC1D4 phosphorylation it has been demonstrated that AICAR enhances insulin action in muscle cells by decreasing membrane cholesterol in an AMPKdependent manner (48). This seems plausible since AMPK has been shown to decrease the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the rate-limiting enzyme in cholesterol synthesis (49). We did not measure membrane cholesterol and therefore we cannot rule out that muscle insulin sensitivity after prior AICAR stimulation was affected by a change in membrane cholesterol content. 17

18 Page 18 of 38 The enhanced insulin-stimulated glucose uptake after AICAR treatment seems to be dependent on a persistent increase in muscle γ 3 -AMPK activity, as evidenced by AMPK activity measurements and insulin-stimulated glucose uptake in γ 3 -AMPK KO mice. Furthermore, prior AICAR stimulation failed to increase insulin sensitivity in mouse soleus muscle in which the α2β2γ3 complex represents <2% of all AMPK trimer complexes (19). In both human m. vastus lateralis (18) and mouse EDL muscle (19) the AMPK α2β2γ3 trimer complex accounts for one-fifth of all AMPK complexes, but its activity varies in regard to both AMPKα Thr 172 phosphorylation and total α 2- AMPK-associated activity (36). Of interest, AMPK-γ3 protein level is markedly decreased in skeletal muscle from trained humans (5), although insulin sensitivity in general is increased. Conversely, enhanced muscle insulin sensitivity after an acute bout of exercise seems to be lost in the trained state (51). Collectively, these results suggest that prior AICAR stimulation mimics the effect of exercise to enhance skeletal muscle insulin sensitivity possibly through an AMPK-γ3 dependent mechanism. In conclusion, prior AICAR stimulation is sufficient to enhance muscle insulin sensitivity. We provide evidence that this effect is likely mediated through AMPK signaling as AICAR failed to increase insulin sensitivity in skeletal muscle in which AMPK activity was blunted. Although we observed no change in proximal insulin signaling events, the enhanced insulin-stimulated glucose uptake observed after prior AICAR stimulation was associated and positively correlated with increased TBC1D4 Thr 649 and Ser 711 phosphorylation. This supports the idea, that prior activation of AMPK primes a pool of TBC1D4 to potentiate a subsequent effect of insulin to increase GLUT4 translocation to the cell surface and enhance glucose uptake. At present, we have not succeeded in establishing a mouse model for studying insulin sensitivity after prior muscle contraction. Therefore, future 18

19 Page 19 of 38 studies have to determine whether AMPK is also important for the enhanced insulin action after this intervention. As TBC1D4 signaling by insulin is potentiated after exercise in both human and rat skeletal muscle (15,16,29,3,52), our current working hypothesis is that exercise-induced increase in insulin sensitivity is also regulated via an AMPK-TBC1D4 signaling axis. Acknowledgements The authors acknowledge the assistance of Ann-Marie Petterson, Integrative Physiology group, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden. The authors also appreciate the kind donation of antibodies by D.G. Hardie (Division of Molecular Physiology, College of Life Sciences, University of Dundee, Scotland, U.K.) and L.J. Goodyear (Joslin Center and Harvard Medical School, Boston, MA, USA). Financial support This work was carried out as a part of the research programs "Physical activity and nutrition for improvement of health" funded by the University of Copenhagen (UCPH) Excellence Program for Interdisciplinary Research and the UNIK project: Food, Fitness & Pharma for Health and Disease (see supported by the Danish Ministry of Science, Technology and Innovation, and by 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 partially funded by an unrestricted donation from the Novo Nordisk Foundation ( This study was funded by the Danish Council for Independent Research Medical Sciences, the Novo 19

20 Page 2 of 38 Nordisk Foundation and the Lundbeck Foundation. Jonas T. Treebak was supported by a postdoctoral fellowship from The Danish Agency for Science, Technology and Innovation. Disclosure statement: The authors have nothing to disclose. Author contributions: Conception and design of research: R.K., J.T.T. and J.F.P.W. Performed experiments: R.K. and J.F. Performed analysis: R.K., J.B.B., L.L., B.V., P.S. and M.B. Interpreted results: All Drafted manuscript: R.K., J.T.T. and J.F.P.W. Edited and revised manuscript: All Read and approved final version: All Jørgen F.P. Wojtaszewski is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. References 1. Richter EA, Garetto LP, Goodman MN, Ruderman NB. Muscle glucose metabolism following exercise in the rat: increased sensitivity to insulin. J Clin Invest 1982;69(4): Richter EA, Mikines KJ, Galbo H, Kiens B. Effect of exercise on insulin action in human skeletal muscle. J Appl Physiol 1989;66(2): Wojtaszewski JF, Hansen BF, Kiens B, Richter E a. signaling in human skeletal muscle: time course and effect of exercise. 1997;46(11):

21 Page 21 of Wojtaszewski JF, Hansen BF, Gade, Kiens B, Markuns JF, Goodyear LJ, et al. signaling and insulin sensitivity after exercise in human skeletal muscle. 2;49(3): Cartee GD, Holloszy JO. Exercise increases susceptibility of muscle glucose transport to activation by various stimuli. Am J Physiol 199;258(2 Pt 1):E Frøsig C, Rose AJ, Treebak JT, Kiens B, Richter EA, Wojtaszewski JFP. Effects of endurance exercise training on insulin signaling in human skeletal muscle: interactions at the level of phosphatidylinositol 3-kinase, Akt, and AS16. 27;56(8): Cartee GD, Young D a, Sleeper MD, Zierath J, Wallberg-Henriksson H, Holloszy JO. Prolonged increase in insulin-stimulated glucose transport in muscle after exercise. Am J Physiol 1989;256(4 Pt 1):E Mikines KJ, Sonne B, Farrell PA, Tronier B, Galbo H. Effect of physical exercise on sensitivity and responsiveness to insulin in humans. Am J Physiol 1988;254(3 Pt 1):E Gulve EA, Cartee GD, Zierath JR, Corpus VM, Holloszy JO. Reversal of enhanced muscle glucose transport after exercise: roles of insulin and glucose. Am J Physiol 199;259(5 Pt 1):E Richter EA, Garetto LP, Goodman MN, Ruderman NB. Enhanced muscle glucose metabolism after exercise: modulation by local factors. Am J Physiol 1984;246(6 Pt 1):E Hansen PA, Nolte LA, Chen MM, Holloszy JO. Increased GLUT-4 translocation mediates enhanced insulin sensitivity of muscle glucose transport after exercise. J Appl Physiol 1998;85(4): Bogardus C, Thuillez P, Ravussin E, Vasquez B, Narimiga M, Azhar S. Effect of muscle glycogen depletion on in vivo insulin action in man. J Clin Invest 1983;72(5): Fisher JS, Gao J, Han D, Holloszy JO, Nolte LA. Activation of AMP kinase enhances sensitivity of muscle glucose transport to insulin. Am J Physiol Endocrinol Metab 22;282(1):E Bonen A, Tan MH, Watson-Wright WM. Effects of exercise on insulin binding and glucose metabolism in muscle. Can J Physiol Pharmacol 1984;62(12): Funai K, Schweitzer GG, Sharma N, Kanzaki M, Cartee GD. Increased AS16 phosphorylation, but not TBC1D1 phosphorylation, with increased postexercise insulin sensitivity in rat skeletal muscle. Am J Physiol Endocrinol Metab 29;297(1):E Funai K, Schweitzer GG, Castorena CM, Kanzaki M, Cartee GD. In vivo exercise followed by in vitro contraction additively elevates subsequent insulin-stimulated glucose transport by rat skeletal muscle. Am J Physiol Endocrinol Metab 21;298(5):E

22 Page 22 of Hamada T, Arias EB, Cartee GD. Increased submaximal insulin-stimulated glucose uptake in mouse skeletal muscle after treadmill exercise. J Appl Physiol 26;11(5): Wojtaszewski JF, Birk JB, Frøsig C, Holten M, Pilegaard H, Dela F. 5 AMP activated protein kinase expression in human skeletal muscle: effects of strength training and type 2 diabetes. J Physiol 25;564(Pt 2): Treebak JT, Birk JB, Hansen BF, Olsen GS, Wojtaszewski JFP. A activates AMPK beta1-containing complexes but induces glucose uptake through a PI3-kinasedependent pathway in mouse skeletal muscle. Am J Physiol Cell Physiol 29;297(4):C Kahn BB, Alquier T, Carling D, Hardie DG. AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab 25;1(1): Hardie DG, Ashford MLJ. AMPK: regulating energy balance at the cellular and whole body levels. Physiology (Bethesda) 214;29(2): Merrill GF, Kurth EJ, Hardie DG, Winder WW. AICA riboside increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle. Am J Physiol 1997;273(6 Pt 1):E Miyamoto L, Egawa T, Oshima R, Kurogi E, Tomida Y, Tsuchiya K, et al. AICAR stimulation metabolome widely mimics electrical contraction in isolated rat epitrochlearis muscle. Am J Physiol Cell Physiol 213;35(12):C Kramer HF, Witczak CA, Taylor EB, Fujii N, Hirshman MF, Goodyear LJ. AS16 regulates insulin- and contraction-stimulated glucose uptake in mouse skeletal muscle. J Biol Chem 26;281(42): Kramer HF, Witczak CA, Fujii N, Jessen N, Taylor EB, Arnolds DE, et al. Distinct signals regulate AS16 phosphorylation in response to insulin, AICAR, and contraction in mouse skeletal muscle. 26;55(7): Treebak JT, Taylor EB, Witczak CA, An D, Toyoda T, Koh H-J, et al. Identification of a novel phosphorylation site on TBC1D4 regulated by AMP-activated protein kinase in skeletal muscle. Am J Physiol Cell Physiol 21;298(2):C Arias EB, Kim J, Funai K, Cartee GD. Prior exercise increases phosphorylation of Akt substrate of 16 kda (AS16) in rat skeletal muscle. Am J Physiol Endocrinol Metab 27;292(4):E Treebak JT, Frøsig C, Pehmøller C, Chen S, Maarbjerg SJ, Brandt N, et al. Potential role of TBC1D4 in enhanced post-exercise insulin action in human skeletal muscle. Diabetologia 29;52(5):

23 Page 23 of Pehmøller C, Brandt N, Birk J. et al. Exercise Alleviates Lipid-Induced Resistance in Human Skeletal Muscle Signaling Interaction at the Level of TBC1 Domain Family Member ;61(11): Schweitzer GG, Arias EB, Cartee GD. Sustained postexercise increases in AS16 Thr642 and Ser588 phosphorylation in skeletal muscle without sustained increases in kinase phosphorylation. J Appl Physiol 212;113(12): Daignan-Fornier B & Pinson B. 5-Aminoimidazole-4-carboxamide-1-beta-Dribofuranosyl 5'-Monophosphate (AICAR), a Highly Conserved Purine Intermediate with Multible Effects. Metabolites 212;2(2): Mu J, Brozinick JT, Valladares O, Bucan M, Birnbaum MJ. A role for AMP-activated protein kinase in contraction- and hypoxia-regulated glucose transport in skeletal muscle. Mol Cell 21;7(5): Lantier L, Fentz J, Mounier R, Leclerc J, Treebak JT, Pehmøller C, et al. AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity. FASEB J 214;28(7): Barnes BR, Marklund S, Steiler TL, Walter M, Hjälm G, Amarger V, et al. The 5 -AMPactivated protein kinase gamma3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle. J Biol Chem 24;279(37): Chen S, Murphy J, Toth R, Campbell DG, Morrice NA, Mackintosh C. Complementary regulation of TBC1D1 and AS16 by growth factors, insulin and AMPK activators. Biochem J 28;49(2): Birk JB, Wojtaszewski JFP. Predominant alpha2/beta2/gamma3 AMPK activation during exercise in human skeletal muscle. J Physiol 26;577(Pt 3): Pehmøller C, Treebak JT, Birk JB, Chen S, Mackintosh C, Hardie DG, et al. Genetic disruption of AMPK signaling abolishes both contraction- and insulin-stimulated TBC1D1 phosphorylation and binding in mouse skeletal muscle. Am J Physiol Endocrinol Metab 29;297(3):E Vichaiwong K, Purohit S, An D, Toyoda T, Jessen N, Hirshman MF, et al. Contraction regulates site-specific phosphorylation of TBC1D1 in skeletal muscle. Biochem J 21;431(2): Frøsig C, Jensen TE, Jeppesen J, Pehmøller C, Treebak JT, Maarbjerg SJ, et al. AMPK and insulin action--responses to ageing and high fat diet. PLoS One 213;8(5):e An D, Toyoda T, Taylor EB, Yu H, Fujii N, Hirshman MF, et al. TBC1D1 regulates insulin- and contraction-induced glucose transport in mouse skeletal muscle. 21;59(6):

24 Page 24 of Chen S, Wasserman DH, MacKintosh C, Sakamoto K. Mice with AS16/TBC1D4- Thr649Ala knockin mutation are glucose intolerant with reduced insulin sensitivity and altered GLUT4 trafficking. Cell Metab 211;13(1): Treebak JT, Pehmøller C, Kristensen JM, Kjøbsted R, Birk JB, Schjerling P, et al. Acute exercise and physiological insulin induce distinct phosphorylation signatures on TBC1D1 and TBC1D4 proteins in human skeletal muscle. J Physiol 214;592(Pt 2): Smith JL, Patil PB, Fisher JS. AICAR and hyperosmotic stress increase insulin-stimulated glucose transport. J Appl Physiol 25;99(3): Ju J-S, Gitcho MA, Casmaer CA, Patil PB, Han D-G, Spencer SA, et al. Potentiation of insulin-stimulated glucose transport by the AMP-activated protein kinase. Am J Physiol Cell Physiol 27;292(1):C Taylor EB, An D, Kramer HF, Yu H, Fujii NL, Roeckl KSC, et al. Discovery of TBC1D1 as an insulin-, AICAR-, and contraction-stimulated signaling nexus in mouse skeletal muscle. J Biol Chem 28;283(15): Ducommun S, Wang HY, Sakamoto K, MacKintosh C, Chen S. Thr649Ala-AS16 knock-in mutation does not impair contraction/aicar-induced glucose transport in mouse muscle. Am J Physiol Endocrinol Metab 212;32(9):E Hoehn KL, Hohnen-Behrens C, Cederberg A, Wu LE, Turner N, Yuasa T, et al. IRS1- independent defects define major nodes of insulin resistance. Cell Metabolism 28;7(5): Habegger KM, Hoffman NJ, Ridenour CM, Brozinick JT, Elmerdorf JS. AMPK enhances insulin-stimulated GLUT4 regulation via lowering membrane cholesterol. Endocrinology 212;153(5): Carling D, Clarke PR, Zammit VA, Hardie DG. Purification and characterization of the AMP-activated protein kinase. Copurification of acetyl-coa carboxylase kinase and 3- hydroxy-3-methylglutaryl-coa reductase kinase activities. Eur J Biochem 1989;186(1-2): Frøsig C, Jørgensen SB, Hardie DG, Richter EA, Wojtaszewski JFP. 5 -AMP-activated protein kinase activity and protein expression are regulated by endurance training in human skeletal muscle. Am J Physiol Endocrinol Metab 24;286(3):E Mikines KJ, Sonne B, Tronier B, Galbo H. Effects of acute exercise and detraining on insulin action in trained men. J Appl Physiol 1989;66(2): Castorena CM, Arias EB, Sharma N, Cartee GD. Post-exercise Improvement in - Stimulated Glucose Uptake Occurs Concomitant with Greater AS16 Phosphorylation in Muscle from Normal and Resistant Rats. 214;(734):

25 Page 25 of 38 Figure Legends FIG. 1. Glucose uptake (A) and pampk Thr 172 (B) in soleus and EDL muscle in response to acute AICAR stimulation (5 min,.5-4 mm) (n=7-8). p<.5 vs. control ( mm) within muscle type. C: Glucose uptake in soleus and EDL muscle after 4 and 6 hours recovery from prior AICAR treatment (5 min, 1 mm), respectively (n = 8). D: Glucose uptake in EDL muscle incubated with or without insulin (1 µu/ml) 6 hours after prior AICAR treatment (5 min, 1 mm) (n = 24). p<.5 vs. basal control p<.5 vs. basal value within group; #p<.5 vs. response to insulin in control (interaction: x AICAR). E: Glucose uptake in soleus muscle incubated with or without insulin (1 µu/ml) 4 hours after prior AICAR stimulation (5 min, 1 mm) (n = 8). p<.1 main effect of insulin. Data were analyzed by one-way ANOVAs (A and B), paired t-tests (C) or two-way RM ANOVAs (D and E). Data are expressed as means ± SEM. F: Representative Western blot image. FIG. 2. Glucose uptake (A and B) or delta glucose uptake (insulin minus basal) (C and D) in EDL muscle from either AMPK KD (A and C) or AMPK mdko (B and D) mice and corresponding WT littermates incubated with or without insulin (1 µu/ml) 6 hours after prior AICAR treatment (5 min, 1 mm) (KD: n = 9-1, mdko: n = 12). Data are expressed as means ± SEM A: AICAR x x Genotype interaction (p<.1). p<.5 vs. basal value within genotype. #p<.1 vs. response to insulin in WT control (interaction: AICAR x ). B: AICAR x x Genotype interaction (p<.5). p<.1 vs. basal value within genotype. #p<.1 vs. response to insulin in WT control (interaction: AICAR x ). C: Data are extracted from the raw data given in (A). p<.1 vs. control within genotype. D: Data are extracted from the raw data given in (B). p<.5 vs. control within genotype. 25

26 Page 26 of 38 FIG. 3. pampk Thr 172 (A and C) and pacc Ser 212 (B and D) in EDL muscle from either AMPK KD (A and B) or AMPK mdko (C and D) mice and corresponding WT littermates incubated with or without insulin (1 µu/ml) 6 hours after prior AICAR treatment (5 min, 1 mm) (KD: n = 9-1, mdko: n = 12). Glucose uptake (F) or delta insulin-stimulated glucose uptake (insulin minus basal) (G) in EDL muscle from γ 3 -AMPK KO mice and corresponding WT littermates incubated with or without insulin (1 µu/ml) 6 hours after prior AICAR treatment (5 min, 1 mm) (n = 6-8). Data are expressed as means ± SEM A: indicates main effect of AICAR (p<.5). Main effect of genotype (p<.1). B: indicates main effect of AICAR (p<.1). Main effect of genotype (p<.1). C: AICAR x x Genotype interaction (p<.1). p<.1 vs. control within genotype. #p<.1 vs. response to AICAR in WT basal (interaction: AICAR x ) D: p<.1 vs. control value within genotype. p<.1 vs. response in WT (interaction: Genotype x AICAR). E: AMPK trimer specific activity in EDL muscle from C57BL/6J mice incubated with or without insulin (1 µu/ml) 6 hours after prior AICAR treatment (5 min, 1 mm) (n = 4-6). and indicates effect of AICAR within group (p=.37 and p<.1, respectively). F: AICAR x x Genotype interaction (p<.5). p<.1 vs. basal values within genotype; #p<.5 vs. response to insulin in WT control (interaction: AICAR x ). p<.1 control vs. AICAR. G: Data are extracted from the raw data given in (F). p<.1 vs. control within genotype. H and I: Representative Western blot images from AMPK KD and mdko studies, respectively. 26

27 Page 27 of 38 FIG. 4. pakt Thr 38 / Akt2 protein (A and C) and pakt Ser 473 / Akt2 protein (B and D) in EDL muscle from either AMPK KD (A and B) or AMPK mdko (C and D) mice and corresponding WT littermates incubated with or without insulin (1 µu/ml) 6 hours after prior AICAR treatment (5 min, 1 mm) (KD: n = 9-1, mdko: n = 12). Data are expressed as means ± SEM. A: indicates effect of insulin (p<.1). B: indicates effect of insulin (p<.1). indicates main effect of AICAR (p=.48). Main effect of genotype (p<.1). C: p<.1 vs. basal. #p<.5 vs. response to insulin in control (interaction: x AICAR). Main effect of genotype (p=.33). D: indicates effect of insulin (p<.1). p<.1 vs. response to insulin in WT (interaction: insulin x genotype). indicates genotype x AICAR interaction (p=.34). E and F: Representative Western blot images from AMPK KD and mdko studies, respectively. FIG. 5. ptbc1d1 Ser 231 / TBC1D1 protein (A and B) and ptbc1d1 Thr 59 / TBC1D1 protein (C and D) in EDL muscle from either AMPK KD (A and C) or AMPK mdko (B and D) mice and corresponding WT littermates incubated with or without insulin (1 µu/ml) 6 hours after prior AICAR treatment (5 min, 1 mm) (KD: n = 9-1, mdko: n = 12). Data are expressed as means ± SEM. A: p<.1 vs. control value within genotype. indicates genotype x AICAR interaction (p<.1). B: p<.1 vs. control value within genotype. indicates genotype x AICAR interaction (p=.1). # indicates insulin x AICAR interaction (p=.41). 27

28 Page 28 of 38 C: indicates effect of insulin (p<.1). Main effect of genotype (p<.1). D: p<.1 vs. basal. #p<.1 vs. response to insulin in control (interaction: x AICAR). E and F: Representative Western blot images from AMPK KD and mdko studies, respectively. FIG. 6. ptbc1d4 Thr 649 / TBC1D4 protein (A and B), ptbc1d4 Ser 711 / TBC1D4 protein (C and D), ptbc1d4 Ser 324 / TBC1D4 protein (E and F) and ptbc1d4 Ser 595 / TBC1D4 protein (G and H) in EDL muscle from either AMPK KD (A,C,E and G) or AMPK mdko (B,D,F and H) mice and corresponding WT littermates incubated with or without insulin (1 µu/ml) 6 hours after prior AICAR treatment (5 min, 1 mm) (KD: n = 8-1, mdko: n = 12). Data are expressed as means ± SEM. A: AICAR x x Genotype interaction (p<.5). p<.5 vs. basal value within genotype; #p<.5 vs. response to insulin in WT control (interaction: AICAR x ). B: AICAR x x Genotype interaction (p<.1). p<.5 vs. basal value within genotype; #p<.1 vs. response to insulin in WT control (interaction: AICAR x ). indicates effect of AICAR within basal (p<.1). C: AICAR x x Genotype interaction (p<.1). p<.1 vs. basal value within genotype; #p<.1 vs. response to insulin in WT control (interaction: AICAR x ). indicates effect of AICAR within basal (p<.5). D: AICAR x x Genotype interaction (p<.5). p<.5 vs. basal value within genotype; #p<.1 vs. response to insulin in WT control (interaction: AICAR x ). () indicates borderline effect of AICAR within basal (p=.5). E: indicates effect of insulin (p<.1). Main effect of genotype (p<.1) 28

29 Page 29 of 38 F+G: indicates effect of insulin (p<.1). H: indicates effect of insulin (p<.1). p<.5 vs. response to insulin in WT (interaction: x Genotype) I and J: Representative Western blot images from AMPK KD and mdko studies, respectively. FIG. 7. A and B show Pearson correlation between delta-insulin value (insulin minus basal) on glucose uptake and ptbc1d4 Ser 711 in WT littermates from KD and mdko mice, respectively. C and D show Pearson correlation between delta-insulin value (insulin minus basal) on glucose uptake and ptbc1d4 Thr 649 in WT littermates from KD and mdko mice, respectively. E and F show Pearson correlation between delta-insulin value (insulin minus basal) on ptbc1d4 Thr 649 and Ser 711 in WT littermates from KD and mdko mice, respectively. Sample size is n = R 2 and significance level are indicated in the respective panel. To visualize any bias due to grouping effect (control and prior-aicar) we also provide Pearson correlations (dashed lines) based on data from the individual groups (Control = open symbols, prior-aicar = closed symbols). FIG. 8. Phosphorylation level of TBC1D4 Thr 649 (A) and Ser 711 (B) in tibialis anterior muscle in response to retro-orbital injection of saline or insulin (1 U/kg, 1 min) 7 days after muscle gene electrotransfer of TBC1D4-WT, TBC1D4-T649A and TBC1D4-S711A. Upper bands in ptbc1d4 Ser 711 blot are unspecific and do not represent actual TBC1D4 protein. Total flagtagged TBC1D4 protein indicates expression level of the three constructs (n = 4-6). N.D: not detected. Data are expressed as means ± SEM. A and B: indicates effect of insulin within group (p<.5). #p<.5 vs. WT 29

30 Page 3 of 38 C: Representative Western blot image. 3

31 Page 31 of 38 Figure 1 A Glucose uptake [µmol / g protein / hr] Acute AICAR stimulation Soleus EDL B pampk Thr 172 Soleus EDL AICAR concentration [mm] AICAR concentration [mm] C Glucose uptake [µmol / g protein / hr] Glucose uptake post AICAR treatment Control Prior AICAR EDL D 25 # Glucose uptake [µmol / g protein / hr] SOLEUS 4 hr recovery EDL 6 hr recovery E 25 SOLEUS F Glucose uptake [µmol / g protein / hr] pampk&thr 172& AMPK-α 2& SOL&&EDL& SOL&&EDL&SOL&&EDL& SOL&EDL& SOL&&EDL& &.5& 1& 2& 4& AICAR&concentraAon&[mM] &

32 Page 32 of 38 Figure 2 A Glucose uptake [µmol / g protein / hr] Glucose uptake # Control Prior AICAR B 4 # Glucose uptake [µmol / g protein / hr] Glucose uptake AMPK KD AMPK mdko C Glucose uptake [µmol / g protein / hr] Delta Glucose uptake D Glucose uptake [µmol / g protein / hr] Delta Glucose uptake AMPK KD AMPK mdko

33 Figure 3 Page 33 of 38 pampk Thr172 pampk Thr172 A5 Main effect of genotype (p<.1) Main effect of genotype (p<.1) B 2. Control Prior AICAR AMPK KD AMPK mdko pacc Ser212 / ACC protein pacc Ser212 / ACC protein C 2.5 D 2.5 # AMPK KD AMPK mdko Glucose uptake 8 α2β2γ3 α2β2γ1 & α2β1γ1 α1β2γ1 & α1β1γ # 3 1 Control G 25 2 Glucose uptake [µmol / g protein / hr] F Glucose uptake [µmol / g protein / hr] AMPK activity [ pmol / min mg protein ] E Prior AICAR Delta Glucose uptake H WT' IB:'pAMPK'Thr172' AMPK γ3 KO mdko' IB:'pAMPK'Thr172' IB:'pACC'Ser212' IB:'AMPK<α2'' WT' ' ' ' ' IB:'pACC'Ser212' AMPK γ3 KO I KD' ' ' ' ' 2 IB:'ACC'' IB:'AMPK>α2'' IB:'ACC''

34 Figure 4 pakt Thr38 / Akt 2 protein pakt Thr38 / Akt 2 protein A 2 B4 3 1 Main effect of genotype (p<.1) Control Prior AICAR 15 Page 34 of AMPK KD # 2 1 WT' 2 KD' F ' ' ' ' IB:'pAkt'Thr38' IB:'Akt'2'' WT' mdko' ' ' ' ' IB:'pAkt'Thr38' IB:'pAkt'Ser473' AMPK KD 3 1 E 4 # 3 AMPK mdko D5 Main effect of genotype (p=.33) pakt Ser473 / Akt 2 protein pakt Ser473 / Akt 2 protein C 4 IB:'pAkt'Ser473' IB:'Akt'2'' AMPK mdko

35 Figure 5 Page 35 of 38 ptbc1d1 Ser231 / TBC1D1 protein ptbc1d1 Ser231 / TBC1D1 protein A 2.5 Control Prior AICAR B # D WT' 1. KD' F ' ' ' ' IB:'pTBC1D1'Ser231' IB:'pTBC1D1'Thr59' IB:'TBC1D1'' AMPK KD #.. # AMPK mdko ptbc1d1 Thr59 / TBC1D1 protein Main effect of genotype (p<.1) E AMPK KD ptbc1d1 Thr59 / TBC1D1 protein.. C 2.5 # 1.5 WT' mdko' ' ' ' ' IB:'pTBC1D1'Ser231' IB:'pTBC1D1'Thr59' IB:'TBC1D1'' AMPK mdko

36 ptbc1d4 Thr649 / TBC1D4 protein A5 3 B C4 AMPK KD AMPK mdko D4 # # ptbc1d4 Ser711 / TBC1D4 protein ptbc1d4 Ser711 / TBC1D4 protein # 6 Control Prior AICAR # 4 Page 36 of 38 ptbc1d4 Thr649 / TBC1D4 protein 1 () 2 1 AMPK KD ptbc1d4 Ser324 / TBC1D4 protein E3 Main effect of genotype (p<.1)¼ AMPK mdko ptbc1d4 Ser324 / TBC1D4 protein F AMPK KD AMPK mdko ptbc1d4 Ser595 / TBC1D4 protein ptbc1d4 Ser595 / TBC1D4 protein G3 H I WT' J KD' IB:'pTBC1D4'Thr649' IB:'pTBC1D4'Ser711' IB:'pTBC1D4'Ser324' IB:'pTBC1D4'Thr649' IB:'pTBC1D4'Ser711' IB:'pTBC1D4'Ser324' mdko' WT' ' ' ' ' IB:'pTBC1D4'Ser595' IB:'TBC1D4'' IB:'pTBC1D4'Ser595' AMPK KD ' ' ' ' IB:'TBC1D4'' AMPK mdko

37 Page 37 of 38 Figure 7 Δ Glucose uptake [µmol / g protein / hr] A R 2 =.56 p<.5 WT (KD) R 2 =.21 p=.18 R 2 =.71 p<.1 Control Prior AICAR B Δ Glucose uptake [µmol / g protein / hr] WT (mdko) R 2 =.28 p=.8 R 2 =.39 p=.1 R 2 =.28 p= Δ ptbc1d4 Ser Δ ptbc1d4 Ser 711 C 25 WT (KD) D 4 WT (mdko) Δ Glucose uptake [µmol / g protein / hr] R 2 =.16 p=.25 R 2 =.33 p= Δ ptbc1d4 Thr 649 R 2 =.51 p<.1 Δ Glucose uptake [µmol / g protein / hr] R 2 =.5 p=.48 R 2 =4.1E-5 p= Δ ptbc1d4 Thr 649 R 2 =.8 p=.18 Δ ptbc1d4 Thr 649 E WT (KD) R 2 =.74 p<.1 R 2 =.85 p<.1 R 2 =.89 p<.1 Δ ptbc1d4 Thr 649 F WT (mdko) R 2 =.55 p<.1 R 2 =.51 p<.1 R 2 =.31 p= Δ ptbc1d4 Ser Δ ptbc1d4 Ser 711

38 Figure 8 Page 38 of 38 ptbc1d4 Thr649 / TBC1D4 protein A # N.D. WT S711A T649A ptbc1d4 Ser711 / TBC1D4 protein B #.5 N.D. C WT( S711A( T649A( WT ptbc1d4(thr649( T649A ptbc1d4(ser711( S711A FLAG((TBC1D4)(