Postexercise improvement in glucose uptake occurs concomitant with greater γ3-AMPK activation and AS160 phosphorylation in rat skeletal muscle

Abstract

A single exercise session can increase insulin-stimulated glucose uptake (GU) by skeletal muscle, concomitant with greater Akt substrate of 160 kDa (AS160) phosphorylation on Akt-phosphosites (Thr⁶⁴² and Ser⁵⁸⁸) that regulate insulin-stimulated GU. Recent research using mouse skeletal muscle suggested that ex vivo 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or electrically stimulated contractile activity-inducing increased γ3-AMPK activity and AS160 phosphorylation on a consensus AMPK-motif (Ser⁷⁰⁴) resulted in greater AS160 Thr⁶⁴² phosphorylation and GU by insulin-stimulated muscle. Our primary goal was to determine whether in vivo exercise that increases insulin-stimulated GU in rat skeletal muscle would also increase γ3-AMPK activity and AS160 site-selective phosphorylation (Ser⁵⁸⁸, Thr⁶⁴², and Ser⁷⁰⁴) immediately postexercise (IPEX) and/or 3 h postexercise (3hPEX). Epitrochlearis muscles isolated from sedentary and exercised (2-h swim exercise; studied IPEX and 3hPEX) rats were incubated with 2-deoxyglucose to determine GU (without insulin at IPEX; without or with insulin at 3hPEX). Muscles were also assessed for γ1-AMPK activity, γ3-AMPK activity, phosphorylated AMPK (pAMPK), and phosphorylated AS160 (pAS160). IPEX versus sedentary had greater γ3-AMPK activity, pAS160 (Ser⁵⁸⁸, Thr⁶⁴², Ser⁷⁰⁴), and GU with unaltered γ1-AMPK activity. 3hPEX versus sedentary had greater γ3-AMPK activity, pAS160 Ser⁷⁰⁴, and GU with or without insulin; greater pAS160 Thr⁶⁴² only with insulin; and unaltered γ1-AMPK activity. These results using an in vivo exercise protocol that increased insulin-stimulated GU in rat skeletal muscle are consistent with the hypothesis that in vivo exercise-induced enhancement of γ3-AMPK activation and AS160 Ser⁷⁰⁴ IPEX and 3hPEX are important for greater pAS160 Thr⁶⁴² and enhanced insulin-stimulated GU by skeletal muscle.

INTRODUCTION

Skeletal muscle accounts for up to 85% of insulin-mediated glycemic clearance, and glucose uptake is a key regulator of muscle glucose metabolism (15, 22). A single bout of exercise can 1) increase glucose uptake measured in isolated rat skeletal muscle in the absence of insulin shortly after completion of exercise, with most of this effect typically reversed by 3–4 h postexercise (10, 46); and 2) enhance insulin-stimulated glucose uptake, with this effect evident at ~2–4 h postexercise, and persisting for up to 48-h postexercise in rat muscle (6, 11, 45). Improved muscle insulin sensitivity for glucose uptake by skeletal muscle postexercise is secondary to an increased abundance of GLUT4 at cell surface membranes (25). However, the mechanisms leading to greater insulin-stimulated GLUT4 translocation and glucose uptake after acute exercise have not been fully elucidated. Many studies of human and rodent skeletal muscle have reported that prior acute exercise has little or no effect on proximal insulin-signaling steps (from insulin receptor binding to Akt activation) (5, 16, 18, 19, 52, 64), suggesting that exercise’s effect on insulin sensitivity may rely on modifications of more distal insulin signaling events.

The distal insulin signaling protein that has been most clearly linked to insulin-stimulated glucose uptake is the Rab GTPase activity protein Akt substrate of 160 kDa (AS160; also known as TBC1D4) (36). AS160 can be phosphorylated on multiple sites by Akt, including Thr⁶⁴² and Ser⁵⁸⁸ (7, 47). In adipocytes, point-mutation of one or both of these sites resulted in reduced insulin-induced GLUT4 translocation (47). In addition, insulin-stimulated mouse skeletal muscle overexpressing AS160 mutated to prevent phosphorylation of four Akt phosphomotifs (Ser³¹⁸, Ser⁵⁸⁸, Thr⁶⁴², and Ser⁷⁵¹) had reduced glucose uptake compared with control muscle (36). Furthermore, mice with a Thr⁶⁴⁹Ala-AS160 knockin mutation that prevented AS160 phosphorylation at Thr⁶⁴⁹ (equivalent to human Thr⁶⁴²) had reduced insulin-stimulated GLUT4 translocation and glucose uptake in skeletal muscle compared with muscle from wild-type (WT) mice (14). We found greater AS160 phosphorylation on Ser⁵⁸⁸ and Thr⁶⁴² in insulin-stimulated muscles several hours postexercise in isolated rat epitrochlearis muscle concomitant with enhanced insulin-stimulated glucose uptake (11, 18, 19, 48), implicating enhanced AS160 phosphorylation as an attractive candidate for mediating exercise’s effect on insulin sensitivity.

Because acute exercise does not increase subsequent insulin-stimulated Akt activation, it is important to identify other mechanisms for the increased AS160 phosphorylation and glucose uptake in insulin-stimulated muscles several hours postexercise. Recent results from Kjøbsted et al. (32, 34) have suggested a possible role in this regard for AMPK. AMPK is a heterotrimeric protein complex with a catalytic subunit (α1 or α2 isoform) and two regulatory subunits (β1 or β2 isoform; and γ1, γ2, or γ3 isoform). The γ3 isoform is selectively expressed by skeletal muscle, whereas γ1 and γ2 isoforms are expressed in multiple tissues (38). The γ1 and γ3 isoforms, but not the γ2 isoform, are reported to be associated with AMPK heterotrimeric complexes in mouse or human skeletal muscle (31). Previous research also indicated that, depending on the specific protocol, acute exercise can increase AMPK activity that is associated with γ1 and/or γ3 isoforms, but not the γ2 isoform in mouse and human skeletal muscle (31). Muscle γ3-AMPK activity is also increased by electrically stimulated contractions in mice (32, 57). Furthermore, either prior contractile activity or prior incubation with 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) that resulted in γ3-AMPK activation induced subsequently greater insulin-mediated muscle glucose uptake in WT control, but not in γ3-knockout mice (32, 34). These results indicated that γ3-AMPK was essential for the enhanced insulin-stimulated glucose uptake under these conditions. The phosphorylation of the AMPK phosphosite AS160 Ser⁷⁰⁴ (58), which corresponds to mouse Ser⁷¹¹, is increased immediately after exercise in human muscle (33, 57, 58) or contraction in mouse muscle (32). In addition, several hours after exercise, insulin-stimulated glucose uptake and AS160 phosphorylation on Ser⁵⁸⁸, Thr⁶⁴², and Ser⁷⁰⁴ are increased in human skeletal muscle (43, 59). Mutating AS160 Ser⁷⁰⁴ to Ala⁷⁰⁴, thereby preventing phosphorylation on this site, resulted in significantly lower insulin-mediated pAS160 Thr⁶⁴² in mouse skeletal muscle, suggesting that the ability to phosphorylate AS160 on Ser⁷⁰⁴ favors greater insulin-stimulated pThr⁶⁴² (34). Prior electrically stimulated contraction or incubation with AICAR caused greater AS160 pSer⁷⁰⁴ and increased insulin-stimulated glucose uptake several hours later in mouse skeletal muscle (32, 34). Taken together, these results suggest that enhanced phosphorylation of pSer⁷⁰⁴ by γ3-AMPK may play a role in the postexercise increase in insulin-stimulated phosphorylation on other AS160 sites, leading to greater insulin-stimulated glucose uptake in skeletal muscle.

Recent reviews have summarized the results of multiple studies that support the idea that the improved skeletal muscle insulin sensitivity after acute exercise may be triggered by greater γ3-AMPK, leading to elevated pAS160 Ser⁷⁰⁴, in turn, favoring subsequently enhanced insulin-mediated pAS160 Thr⁶⁴², which is related to greater insulin-stimulated glucose uptake by the previously exercised skeletal muscle (7, 31). However, no single study has assessed isoform-selective AMPK activity, site-specific AS160 phosphorylation on both Thr⁶⁴² and Ser⁷⁰⁴, and skeletal muscle glucose uptake determined both immediately postexercise and several hours postexercise. An important recent study by Kjöbsted et al. (32) that used electrically stimulated contraction rather than in vivo exercise included most of these measurements, but it did not assess either isoform-selective AMPK activity or AS160 Thr⁶⁴² phosphorylation immediately postcontraction. Although electrically stimulated muscle contraction is an invaluable model, it is not identical to in vivo exercise.

Research using either mice or rats has been especially valuable for pursuing the mechanisms that underlie improved insulin sensitivity after exercise. We have primarily focused on studying insulin-stimulated glucose uptake in rat epitrochlearis muscle for several reasons, including: 1) we have established an exercise protocol that provides for robust improvement in insulin-stimulated glucose uptake in this muscle; 2) there is extensive data on the influence of exercise, contraction, AICAR, and/or insulin on AS160 phosphorylation in this muscle; and 3) there is a great deal of information about a variety of physiological conditions on glucose uptake and insulin signaling in this muscle (e.g., aging, high-fat diet, and calorie restriction) (3, 4, 8, 11, 18, 19, 41, 42, 48, 49, 51, 62). Although many studies have demonstrated that acute exercise can elevate subsequent glucose uptake and phosphorylation of AS160 Ser⁵⁸⁸ and Thr⁶⁴² in insulin-stimulated skeletal muscle from rats (11, 18, 19, 48), none of these studies assessed isoform-selective AMPK activity or pAS160 Ser⁷⁰⁴. Accordingly, important aspects of the experimental design of the current study included 1) using in vivo exercise rather than electrically stimulated contraction, 2) assessing insulin-independent glucose uptake immediately postexercise and both insulin-independent and insulin-stimulated GU at 3 h post-exercise, 3) measuring exercise effects on γ1- and γ3-AMPK activation and AS160 Ser⁷⁰⁴ phosphorylation at both timepoints, and 4) determining exercise effects on key insulin signaling events (Akt Ser⁴⁷³ and Thr³⁰⁸ phosphorylation, and AS160 Ser⁵⁸⁸ and Thr⁶⁴² phosphorylation) at both timepoints. Our hypothesis was that acute exercise by rats would result in subsequently greater γ3-AMPK activation, AS160 Ser⁵⁸⁸, Thr⁶⁴², and Ser⁷⁰⁴ phosphorylation and glucose uptake in epitrochlearis muscles both immediately postexercise and 3 h postexercise.

MATERIALS AND METHODS

Materials

All of the chemicals were obtained from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Hanover Park, IL), unless otherwise noted. The apparatus and reagents for SDS-PAGE and nonfat dry milk (no. 170-6404) were obtained from Bio-Rad Laboratories (Hercules, CA). Pierce MemCode reversible protein stain kit (no. 24585), bicinchoninic acid protein assay (no. 23225), tissue protein extraction reagent (TPER; no. 78510), protein G magnetic beads (no. 10004D) and DynaMag-2 magnet (no. 12321D) were obtained from Thermo Fisher Scientific (Waltham, MA). Anti-phospho Akt Ser⁴⁷³ (pAkt^Ser473; no. 9271), anti-phospho Akt Thr³⁰⁸ (pAkt^Thr308; no. 13038), anti-Akt (no. 4691), anti-phospho AS160 Thr⁶⁴² (pAS160^Thr642; no. 8881), anti-phospho AS160 Ser⁵⁸⁸ (pAS160^Ser588; no. 8730), anti-phospho AMPKα Thr¹⁷² (pAMPKα^Thr172; no. 2531), anti-AMP-activated protein kinase-α (AMPKα; no.5831), anti-acetyl CoA carboxylase (ACC; no. 3676), anti-phospho ACC Ser⁷⁹ (pACC^Ser79; no. 3661), and anti-rabbit IgG horseradish peroxidase conjugate (no. 7074) were obtained from Cell Signaling Technology (Danvers, MA). Skeletal muscle expresses two isoforms of ACC. ACC1 has low expression in skeletal muscle and is phosphorylated by AMPK on ACC⁷⁹, and ACC2 is the predominant ACC isoform expressed by skeletal muscle and is phosphorylated by AMPK on Ser²¹² (1). Because the pACC^Ser79 antibody (no. 3661) detects both pACC1 Ser⁷⁹ and pACC2 Ser²¹² (40), we refer to results with this antibody hereafter as pACC Ser^79/212. Anti-phospho AS160 Ser⁷⁰⁴ (pAS160^Ser704) was provided by Dr. Laurie Goodyear (Joslin Diabetes Center and Harvard Medical School) (58). This antibody’s phosphospecificity was confirmed by analysis of muscles expressing either WT AS160 or an AS160-S704A mutant (58). Anti-AMP-activated protein kinase-γ3 (γ3-AMPK) was provided by Dr. David Thomson (Brigham Young University) (26). Anti-Akt substrate of 160 kDa (AS160; no. ABS54), P81 phosphocellulose squares (no. 20-134) and enhanced chemiluminescence Luminata Forte Western HRP Substrate (no. WBLUF0100) were purchased from EMD Millipore (Billerica, MA). [γ-³²P]-ATP, 2-deoxy-d-[³H]-glucose ([³H]-2-DG) and [¹⁴C]-mannitol were from Perkin Elmer (Boston, MA). Liquid scintillation cocktail (no. 111195-CS) was obtained from Research Products International (Mount Prospect, IL).

Animal treatment.

Animal care procedures were approved by the University of Michigan Committee on Use and Care of Animals. All methods were performed in accordance with the guidelines from the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (Bethesda, MD). Male Wistar rats (7–8 wk old; mean weight 255.5 ± 5.8 g; n = 76; Charles River Laboratories, Boston, MA) were housed (23–24°C) and maintained on a 12:12-h light-dark cycle (lights out at 1700) with unlimited access to standard rodent chow (Laboratory Diet no. 5L0D; LabDiet, St. Louis, MO) and water. Rats were fasted at ~1700 on the night before the terminal experiment. The following morning at ~0700, rats were assigned to sedentary or exercised groups by simple randomization. Exercised rats swam in a barrel filled with water (~45 cm depth, six rats swimming at a time) for 4 × 30-min bouts, with a 5-min rest period between each bout. The water temperature was maintained between 34°C and 35°C throughout the exercise. Other rats served as time-matched sedentary controls with each experiment.

Muscle dissection and incubation.

Rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg ip body wt), either immediately postexercise (IPEX) or 3–4 h postexercise (3hPEX) along with time-matched sedentary rats. For the IPEX experiment, following loss of pedal withdrawal reflex, both epitrochlearis muscles from each rat were rapidly dissected out. One muscle was trimmed, freeze-clamped using aluminum clamps cooled to the temperature of liquid N₂, and stored at −80°C until processed for further analysis. The contralateral muscle was placed in a vial containing 2 ml Krebs Henseleit (KHB) supplemented with 0.1% BSA, 2 mM sodium pyruvate, 6 mM mannitol (solution 1) for 10 min. Muscles were then transferred to another vial containing 2 ml KHB/BSA, 1 mM 2-DG (with final specific activity of 2.25 mCi/mmol [³H]-2-DG), and 9 mM mannitol (with final specific activity of 0.022 mCi/mmol [¹⁴C]-mannitol) (solution 2) for 15 min. For all incubation steps, the vials were shaken at 45 oscillations per minute and continuously gassed (95% O₂-5% CO₂) in a heated (35°C) water bath.

For the 3hPEX experiment, exercising rats were dried and returned to their cages without food following the final exercise bout. Both epitrochlearis muscles were dissected out from anesthetized exercised rats at 3–4 h after completion of exercise along with dissection of muscles from time-matched sedentary controls. After dissection, epitrochlearis muscles were incubated for 30 min at 35°C in vials containing 2 ml of solution 1 with either no insulin (basal) or a submaximally effective concentration of insulin (0.6 nM). After this initial incubation, the muscles were incubated for 20 min at 35°C in another vials containing 2 ml of solution 2 with the same insulin concentration as in the previous incubation step.

To determine whether increased insulin-stimulated glucose uptake was also observed in another forelimb muscle, we dissected out extensor digiti quinti proprius (EDQP) muscles from rats at 3hPEX and time-matched sedentary controls. EDQP muscles were incubated under conditions identical to those used for epitrochlearis muscles. Other EDQP muscles were processed and analyzed for myosin heavy chain expression, as previously described (12, 54).

For incubated muscles from both IPEX and 3hPEX experiments, after their final incubation step, muscles were blotted on filter paper moistened with ice-cold KHB, trimmed, freeze-clamped using aluminum tongs cooled in liquid N₂, and stored at −80°C for later processing and analysis.

Muscle lysate processing.

Frozen muscles were weighed, homogenized in 1 ml ice-cold lysis buffer using a TissueLyser II homogenizer (Qiagen, Valencia, CA). For muscle lysates used for determination of AMPK γ3 activity, the lysis buffer contained 10% glycerol, 20 mmol/l sodium pyrophosphate (NaPP), 1% NP-40, 2 mmol/l phenylmethylsulfonyl fluoride (PMSF), 150 mmol/l sodium chloride (NaCl), 50 mmol/l HEPES (pH 7.5), 20 mmol/l β-glycerophosphate, 10 mmol/l sodium fluoride, 1 mmol/l EDTA, 1 mmol/l EGTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 2 mmol/l sodium vanadate. In muscle lysates used for immunoblotting, the lysis buffer contained T-PER tissue protein extraction reagent (no. PI-78510; Thermo Scientific, Rockford, IL) supplemented with 1 mM EDTA, 1 mM EGTA, 2.5 mM NaPP, 1 mM sodium vanadate, 1 mM β-glycerophosphate, 1 µg/ml leupeptin, and 1 mM PMSF. Homogenates were rotated for 1 h at 4°C before centrifugation (15,000 g for 15 min at 4°C). The supernatants were transferred to microfuge tubes and stored at −80°C for subsequent analyses. Protein concentration was measured using the bicinchoninic acid procedure.

2-Deoxy-d-glucose uptake.

Aliquots of the supernatants (200 μl) from muscle lysates were pipetted into a vial together with scintillation cocktail. A scintillation counter (PerkinElmer) was used to determine the ³H and ¹⁴C disintegrations per minute. [³H]-2-DG uptake was calculated as previously described (9, 24).

γ1-AMPK and γ3-AMPK activity assay.

The specificity of the antibodies used to immunoprecipitate γ1-AMPK and γ3-AMPK isoforms was confirmed (Fig. 1). AMPK activity was determined as previously described (34). Briefly, muscle lysates (600 μg protein for γ1-AMPK or 300 µg protein for γ3-AMPK) were rotated at 4°C overnight with an antibody that specifically recognizes either γ1-AMPK (1:1,000) or γ3-AMPK (1:1,000). Then 50 μl of protein G-magnetic beads were added to the mixture, rotated for 2 h at 4°C. DynaMag-2 magnet was used to pellet the protein G immunocomplex. Each immunopellet was washed once in buffer A [50 mmol/l NaCl, 1% Triton X-100, 50 mmol/l sodium fluoride, 5 mmol/l sodium-pyrophosphate, 20 mmol/l Tris-base (pH 7.5), 500 μmol/l PMSF, 2 mmol/l dithiothreitol (DTT), 4 μg/ml leupeptin, 4 μg/ml aprotinin, and 250 mmol/l sucrose], once in 6× assay buffer (240 mmol/l HEPES, 480 mmol/l NaCl, pH 7.0), and twice in 3× assay buffer. Then the activity assay was performed in 30 μl of kinase mix buffer [40 mmol/l HEPES, pH 7.5, 80 mmol/l NaCl, 800 μmol/l DTT, 200 μmol/l AMP, 100 μmol/l AMARA peptide, 5 mmol/l magnesium chloride (MgCl₂), 200 μmol/l ATP, and 10 µCi of [γ-³²P]-ATP] for 30 min at 30°C. The reaction was stopped by the addition of 10 μl of 1% phosphoric acid. Next, 40 µl of supernatant was transferred to phosphocellulose paper. After 4 × 15 min washing with 1% phosphoric, the phosphocellulose paper was dried for 5 min and placed in the vials containing scintillation cocktail for scintillation counting. Results were expressed relative to the normalized mean of all the samples from each experiment.

Fig. 1.

Validation of AMPK isoform-specific antibodies for immunoblot (IB) and immunoprecipitation (IP). A: IB analysis was performed with 40 μg protein extract from gastrocnemius muscles of wild-type (WT) and AMPK γ1 knockout (KO-γ1) mice, and rat epitrochlearis muscle (Rat-EPI). B: endogenous AMPK γ1 complex were immunoprecipitated using the specific AMPK γ1 antibody from 600 μg extract protein from rat epitrochlearis muscle, and then performed IB with AMPK γ1 and γ3 antibody. C: IB analysis was performed with 40 μg protein extract from rat epitrochlearis muscle (Rat-EPI), and gastrocnemius muscles from WT and AMPK γ3 knockout (KO-γ3) mice. D: endogenous AMPK γ3 complexes were immunoprecipitated using the specific AMPK γ3 antibody from 300 μg extract protein from rat epitrochlearis muscles (EPI), and gastrocnemius muscles from WT and AMPK γ3 knockout (KO-γ3) mice, and then performed IB with AMPK γ3 and γ1 antibody.

Immunoblotting.

An equal amount of protein from muscle lysates was mixed with 6× Laemmli buffer, boiled for 5 min, subjected to SDS-PAGE, and transferred to polyvinylidene difluoride membranes. Equal loading was confirmed using the MemCode protein stain. Membranes were blocked with BSA or nonfat milk (5% in TBST, Tris-buffered saline, pH 7.5 plus 0.1% Tween-20) for 1 h at room temperature and incubated with appropriate concentrations of primary (1:1,000) and secondary antibodies (1:20,000), subjected to enhanced chemiluminescence and quantified by densitometry (AlphaView; ProteinSimple, San Jose, CA). Results were expressed relative to the normalized average of all the samples on the blot.

Statistical analysis.

Comparisons between two groups were performed using Student’s t-test. The main effects of insulin (basal or insulin) and exercise (sedentary or 3 h postexercise) were identified using two-way ANOVA, and the Tukey test was used for post hoc analysis to identify the source of significant variance (SigmaPlot version 13.0; Systat Software, San Jose, CA). Data lacking normal distribution and/or equal variance were mathematically transformed to achieve normality and equal variance before running two-way ANOVA.

RESULTS

2-Deoxy-d-glucose uptake.

For the immediately IPEX group compared with the sedentary (SED) control group, insulin-independent glucose uptake was significantly increased (P < 0.01; Fig. 2A). In the 3 h postexercise (3hPEX) experiment, there was a significant main effect of insulin (insulin > no insulin, P < 0.001) and exercise (3hPEX > SED; P < 0.001, Fig. 2B) for glucose uptake. Post hoc analysis indicated that insulin-independent glucose uptake was significantly greater (P < 0.001) for 3hPEX compared with SED values. Insulin-stimulated muscles had greater glucose uptake than paired muscles incubated without insulin for both SED (P < 0.05) and 3hPEX (P < 0.01) groups. Furthermore, glucose uptake by insulin-stimulated muscles from the 3hPEX group significantly exceeded (P < 0.01) values for the SED controls.

Fig. 2.

A: rates of 2-deoxy-d-glucose (2-DG) uptake in epitrochlearis muscles of immediately postexercise (IPEX) and time-matched sedentary (SED) rats. Data were analyzed by Studentʼs t-test. *P < 0.01, IPEX vs. SED group. Values are expressed as means ± SE; n = 8 per group. B: 2-DG uptake in paired epitrochlearis muscles, from 3 h postexercise (3hPEX) and time-matched SED rats, incubated without or with a submaximally effective insulin dose. Data were analyzed by two-way ANOVA, and Tukey post hoc analysis was performed to identify the source of significant variance. There were significant main effects of insulin (P < 0.001) and exercise (P < 0.001) on 2-DG uptake. ‡P < 0.001, 3hPEX vs. SED group without insulin; *Insulin vs. no insulin in both the SED (P < 0.05) and the 3hPEX (P < 0.01) groups; †P < 0.01, 3hPEX vs. SED group with insulin. Values are expressed as means ± SE; n = 12 per group.

γ1-AMPK and γ3-AMPK activity.

The antibodies used for γ1-AMPK and γ3-AMPK were specific for their respective isoforms (Fig. 1). There was no significant exercise effect on γ1-AMPK activity at either IPEX or 3hPEX (Fig. 3, A and B). γ3-AMPK activity was approximately fourfold greater (P < 0.01; Fig. 3C) for the IPEX group compared with the SED controls. In the 3hPEX experiment, there was a significant effect of exercise (3hPEX > SED; P < 0.01, Fig. 3D) for γ3-AMPK activity. Post hoc analysis demonstrated that γ3-AMPK activity was significantly greater for 3hPEX compared with SED values either without insulin (P < 0.001) or with insulin (P < 0.05).

Fig. 3.

A: γ1-AMPK activity in epitrochlearis muscles from immediately postexercise (IPEX) and time-matched sedentary (SED) rats; n = 4 per group. B: γ1-AMPK activity in epitrochlearis muscles, from 3 h postexercise (3hPEX) and time-matched SED rats, incubated with a submaximally effective insulin dose; n = 5 per group. C: γ3-AMPK activity in epitrochlearis muscles of IPEX and time-matched SED rats. *P < 0.01, IPEX vs. SED group, n = 7 per group. D: γ3-AMPK activity in epitrochlearis muscles, from 3hPEX and time-matched SED rats, incubated with a submaximally effective insulin dose. *P < 0.01, 3hPEX vs. SED group; n = 9 per group. Data were analyzed by Studentʼs t-test. Values are expressed as means ± SE.

Immunoblotting.

Equal loading of samples was confirmed based on the MemCode protein stain (not shown). For all of the phosphorylated proteins, the data were expressed as a ratio of the phosphorylated-to-total protein values.

Total protein abundance.

There was no significant exercise effect on total abundance of AMPKα, γ1-AMPK, γ3-AMPK, AS160, or ACC at IPEX (Fig. 4). In the 3hPEX experiment, there were small (15%), but significant, main effects of insulin (insulin > no insulin; P < 0.05) and exercise (SED > 3hPEX; P < 0.05) on AS160 abundance (Fig. 5E). Post hoc analysis indicated AS160 abundance in the SED muscles with insulin exceeded SED muscles without insulin (P < 0.05) and exceeded 3hPEX muscles with insulin (P < 0.05). Neither insulin nor exercise significantly altered the total protein abundance of Akt, AMPKα, γ1-AMPK, γ3-AMPK, or ACC (Fig. 5).

Fig. 4.

A–E: total AMPKα, γ1-AMPK, γ3-AMPK, AS160, and acetyl CoA carboxylase (ACC) abundance, in epitrochlearis muscles of immediately postexercise (IPEX) and time-matched SED rats. Data were analyzed by Studentʼs t-test. Values are expressed as means ± SE; n = 6–14 per group.

Fig. 5.

A–F: total AMPKα, γ1-AMPK, γ3-AMPK, Akt, AS160, and acetyl CoA carboxylase (ACC) abundance in paired epitrochlearis muscles, from 3 h postexercise (3hPEX) and time-matched sedentary (SED) rats, incubated without or with a submaximally effective insulin dose. *Insulin vs. no insulin in the SED (P < 0.05); SED vs. 3hPEX with insulin (P < 0.05). Data were analyzed by two-way ANOVA, and Tukey post hoc analysis was performed to identify the source of significant variance. Values are expressed as means ± SE; n = 6–12.

AMPKα phosphorylation.

The pAMPKα^Thr172/AMPKα value was significantly greater for the IPEX group compared with the SED controls (P < 0.001, Fig. 6A). For pAMPKα^Thr172/AMPKα ratio in the 3hPEX experiment, there was a significant main effect of exercise (3hPEX > SED; P < 0.05, Fig. 6C).

Fig. 6.

A: phosphorylated AMPKα^Thr172/AMPKα in epitrochlearis muscles of immediately postexercise (IPEX) and time-matched sedentary (SED) rats. Data were analyzed by Studentʼs t-test. *P < 0.001, IPEX vs. SED group. Values are expressed as means ± SE; n = 14 per group. B: representative immunoblots at IPEX. C: phosphorylated AMPKα^Thr172/AMPKα in paired epitrochlearis muscles, from 3 h postexercise (3hPEX) and time-matched SED rats, incubated without or with a submaximally effective insulin dose. Data were analyzed by two-way ANOVA, and Tukey post hoc analysis was performed to identify the source of significant variance. There was a significant main effect of exercise (P < 0.05) on phosphorylated AMPKα^Thr172/AMPKα. Values are expressed as means ± SE; n = 6 per group. D: representative immunoblots at 3hPEX.

ACC phosphorylation.

The pACC^Ser79/212/ACC value was significantly greater for the IPEX group compared with the SED controls (P < 0.05, Fig. 7A). For pACC^Ser79/212/ACC in the 3hPEX experiment, there were no significant insulin or exercise effects (Fig. 7C).

Fig. 7.

A: phosphorylated acetyl CoA carboxylase (ACC)^Ser79/212/ACC in epitrochlearis muscles of immediately postexercise (IPEX) and time-matched sedentary (SED) rats. Data were analyzed by Studentʼs t-test. *P < 0.05, IPEX vs. SED group. Values are expressed as means ± SE; n = 6 per group. B: representative immunoblots at IPEX. C: phosphorylated ACC^Ser79/212/ACC in paired epitrochlearis muscles, from 3 h postexercise (3hPEX) and time-matched SED rats, incubated without or with a submaximally effective insulin dose. Data were analyzed by two-way ANOVA. Values are expressed as means ± SE; n = 6 per group. D: representative immunoblots at 3hPEX.

Akt phosphorylation.

For pAkt^Ser473/Akt in the 3hPEX experiment, there were significant main effects of insulin (insulin > no insulin, P < 0.001) and exercise (3hPEX > SED; P < 0.05, Fig. 8A). Post hoc analysis indicated that insulin-treated muscles had a greater pAkt^Ser473/Akt than paired muscles incubated without insulin for both SED (P < 0.001) and 3hPEX (P < 0.001) groups. There was a nonsignificant trend (P = 0.053) for 3hPEX values to be slightly greater than SED values from insulin-stimulated muscles. For pAkt^Thr308/Akt, there was a significant main effect of insulin (insulin > no insulin, P < 0.001, Fig. 8B). Post hoc analysis indicated that insulin-treated muscles had a greater pAkt^Thr308/Akt than paired muscles incubated without insulin for both SED (P < 0.001) and 3hPEX (P < 0.001) groups.

Fig. 8.

A: phosphorylated Akt^Ser473/Akt. B: phosphorylated Akt^Thr308/Akt in paired epitrochlearis muscles, from 3 h postexercise (3hPEX) and time-matched sedentary (SED) rats, incubated without or with a submaximally effective insulin dose. Data were analyzed by two-way ANOVA, and Tukey post hoc analysis was performed to identify the source of significant variance. There were significant main effects of insulin (P < 0.001) and exercise (P < 0.05) on phosphorylated Akt^Ser473/Akt, as well as a significant main effect of insulin (P < 0.001) on phosphorylated Akt^Thr308/Akt. *P < 0.001; insulin vs. no insulin in both the SED and the 3hPEX groups. Values are expressed as means ± SE; n = 12 per group. C: representative immunoblots.

AS160 phosphorylation.

The values for pAS160^Ser704/AS160 (P < 0.0001, Fig. 9A), pAS160^Ser588/AS160 (P < 0.01, Fig. 9B) and pAS160^Thr642/AS160 (P < 0.05, Fig. 9C) were significantly greater for the IPEX group compared with the SED controls. For pAS160^Ser704/AS160 in the 3hPEX experiment, there were significant main effects of insulin (insulin > no insulin; P < 0.05) and exercise (3hPEX > SED; P < 0.001, Fig. 10A). Post hoc analysis indicated that insulin-treated muscles had a greater pAS160^Ser704/AS160 than paired muscles incubated without insulin for the 3hPEX (P < 0.05) group. The pAS160^Ser704/AS160 value was significantly greater for 3hPEX compared with the SED value either without insulin (P = 0.05) or with insulin (P < 0.001). For pAS160^Ser588/AS160, there was a significant main effect of insulin (insulin > no insulin; P < 0.001, Fig. 10B). Post hoc analysis detected that insulin-treated muscles had greater pAS160^Ser588/AS160 than paired muscles incubated without insulin for both SED (P < 0.001) and 3hPEX (P < 0.01) groups. For pAS160^Thr642/AS160, there were significant main effects of insulin (insulin > no insulin; P < 0.001) and exercise (3hPEX > SED; P < 0.05, Fig. 10C). Post hoc analysis indicated that insulin-treated muscles had a greater pAS160^Thr642/AS160 than paired muscles incubated without insulin for both SED (P < 0.001) and 3hPEX (P < 0.001) groups. In addition, pAS160^Thr642/AS160 was significantly greater (P < 0.05) for 3hPEX compared with SED values with insulin.

Fig. 9.

A: phosphorylated AS160^Ser704/AS160. B: phosphorylated AS160^Ser588/AS160. C: phosphorylated AS160^Thr642/AS160, in epitrochlearis muscles from immediately postexercise (IPEX) and time-matched sedentary (SED) rats. Data were analyzed by Studentʼs t-test. *P < 0.0001 for phosphorylated AS160^Ser704, P < 0.01 for phosphorylated AS160^Ser588, and P < 0.05 for phosphorylated AS160^Thr642 IPEX vs. SED group. Values are expressed as means ± SE; n = 14 per group. D: representative immunoblots.

Fig. 10.

A: phosphorylated AS160^Ser704/AS160 in paired epitrochlearis muscles, from 3hPEX and time-matched sedentary (SED) rats, incubated without or with a submaximally effective insulin dose. There were significant main effects of insulin (P < 0.05) and exercise (P < 0.001) on phosphorylated AS160^Ser704/AS160. ‡P = 0.050, 3 h postexercise (3hPEX) vs. SED group without insulin. *P < 0.05, insulin vs. no insulin in the 3hPEX group. †P < 0.001, 3hPEX vs. SED group with insulin. B: phosphorylated AS160^Ser588/AS160 in paired epitrochlearis muscles, from 3hPEX and time-matched SED rats, incubated without or with a submaximally effective insulin dose. There was a significant main effect of insulin (P < 0.001) on phosphorylated AS160^Ser588/AS160. *P < 0.01, insulin vs. no insulin in both the SED and the 3hPEX groups. C: phosphorylated AS160^Thr642/AS160 in paired epitrochlearis muscles, from 3hPEX and time-matched SED rats, incubated without or with a submaximally effective insulin dose. There were significant main effects of insulin (P < 0.001) and exercise (P < 0.05) on phosphorylated AS160^Thr642/AS160. *P < 0.001, insulin vs. no insulin in both the SED and the 3hPEX groups. †P < 0.05, 3hPEX vs. SED group with insulin. Data were analyzed by two-way ANOVA, and Tukey post hoc analysis was performed to identify the source of significant variance. Values are expressed as means ± SE; n = 9–12. D: representative immunoblots.

EDQP results.

The fiber type composition of the EDQP (6.1 ± 0.01% type I, 14.0 ± 0.01% type IIA, 26.7 ± 0.01% type IIX, and 53.2 ± 0.02% type IIB; n = 4) was very similar to the values for the epitrochlearis (8% type I, 13% type IIA, 28% type IIX, and 51% type IIB) (12). There were no significant exercise-induced increases in glucose uptake or AS160 phosphorylation (Ser⁷⁰⁴/AS160, Ser⁵⁸⁸/AS160, or Thr⁶⁴²/AS160) determined at 3hPEX (Fig. 11).

Fig. 11.

2-DG uptake and AS160 phosphorylation in paired extensor digiti quinti proprius (EDQP) muscles, from 3hPEX and time-matched SED rats, incubated without or with a submaximally effective insulin dose. A: 2-DG uptake. There was a significant main effect of insulin (P < 0.01) on 2-DG uptake. *Insulin vs. no insulin in both the SED (P < 0.05) and the 3hPEX (P < 0.05) groups. B: phosphorylated AS160Ser⁷⁰⁴/AS160. C: phosphorylated AS160Ser⁵⁸⁸/AS160. D: AS160Thr⁶⁴²/AS160. There were significant main effects of insulin (P < 0.001) on phosphorylated AS160Ser⁵⁸⁸/AS160 and phosphorylated AS160Thr⁶⁴²/AS160. *P < 0.05; insulin vs. no insulin in both the SED and the 3hPEX groups. Data were analyzed by two-way ANOVA, and Tukey post hoc analysis was performed to identify the source of significant variance. Values are expressed as means ± SE; n = 6 per group. E: representative immunoblots.

DISCUSSION

The current study investigated signaling events involved in the improvement in insulin sensitivity found after acute in vivo exercise. Consistent with earlier research (3, 11, 19, 48), prior exercise resulted in elevated insulin-stimulated glucose uptake at 3hPEX. The most important new results were that exercise leading to subsequently increased pAS160 Thr⁶⁴² and glucose uptake in insulin-stimulated rat epitrochlearis muscles also resulted in 1) increased γ3-AMPK activity, but not increased γ1-AMPK activity, compared with sedentary values at both IPEX and 3hPEX; 2) increased pAS160 Ser⁷⁰⁴ compared with sedentary values at IPEX; 3) greater Ser⁷⁰⁴ vs. sedentary values at 3hPEX in muscles incubated without insulin; and 4) greater pAS160 Ser⁷⁰⁴ vs. sedentary values in insulin-stimulated muscles. These data using in vivo exercise support and extend the idea, previously based largely on research using AMPK activation by AICAR or electrically stimulated contractions (32, 34) that activation of γ3-AMPK leading to greater pAS160 Ser704 may be linked to subsequently greater insulin-stimulated AS160 Thr⁶⁴² phosphorylation and glucose uptake in skeletal muscle.

The improved insulin-stimulated glucose uptake 3hPEX occurred independent of exercise-induced changes in proximal insulin signaling at the level of phosphorylation of Akt Thr³⁰⁸ and Akt Ser⁴⁷³ consistent with several earlier studies (11, 16, 29, 55). Earlier research in humans has also demonstrated increased postexercise insulin-stimulated glucose uptake by skeletal muscle without concomitantly improved Akt activation (43, 64). The current results also implicate a mechanism located downstream of Akt for the increased insulin-stimulated glucose uptake.

AS160 is a distal insulin signaling protein that has been convincingly linked to insulin-stimulated glucose uptake (14, 28, 36, 47). Greater AS160 phosphorylation on Thr⁶⁴² in insulin-stimulated muscles from the 3hPEX group compared with sedentary controls is consistent with earlier research (4, 11, 18, 19, 48). The lack of a significant increase in AS160 Ser⁵⁸⁸ phosphorylation in insulin-stimulated muscles at 3hPEX differs from some earlier studies that found greater AS160 Ser⁵⁸⁸ phosphorylation in insulin-stimulated rat skeletal muscle at 3–4 h postexercise (11, 48), although a recent study (4) reported a nonsignificant trend for greater AS160 Ser⁵⁸⁸ phosphorylation in insulin-stimulated rat muscle 3hPEX. The explanation for the differing results for Ser⁵⁸⁸ phosphorylation among these studies is uncertain. The current results indicate that greater phosphorylation on Thr⁶⁴² was accompanied by improved insulin sensitivity. In this context, it is notable that an AS160 mutation that prevented phosphorylation only on Thr⁶⁴² was sufficient to induce insulin resistance (14, 47), suggesting that modifying Thr⁶⁴² phosphorylation can alter insulin-stimulated glucose uptake.

Because greater AS160 Thr⁶⁴² phosphorylation in insulin-stimulated skeletal muscle several hours after exercise is potentially crucial for improving insulin sensitivity, it would be valuable to identify the mechanism underlying this outcome. The postexercise increase in Thr⁶⁴² phosphorylation was accompanied by greater Ser⁷⁰⁴ phosphorylation of AS160. Ser⁷⁰⁴ phosphorylation does not appear to be directly required for insulin-stimulated glucose uptake based on earlier research demonstrating that mutation of Ser⁷⁰⁴ to Ala⁷⁰⁴ did not alter insulin-stimulated glucose uptake in skeletal muscle from sedentary mice (58). However, Kjobsted et al. (34) recently reported that increased AS160 phosphorylation on Ser⁷⁰⁴, part of a consensus AMPK motif, can modify the extent of insulin-stimulated AS160 phosphorylation on Thr⁶⁴². In this context, it is significant that Ser⁷⁰⁴ phosphorylation was enhanced several hours after in situ muscle contractions or ex vivo AICAR treatment in mouse extensor digitorum longus (EDL) muscle, and that greater Ser⁷⁰⁴ phosphorylation was accompanied by greater Thr⁶⁴² phosphorylation under these conditions (32, 34, 58). In addition, many studies have reported that AS160 phosphorylation on Ser⁷⁰⁴ is increased IPEX in human vastus lateralis muscle (30, 33, 37, 57, 58). However, Ser⁷⁰⁴ phosphorylation had not been previously evaluated after in vivo exercise in rodent skeletal muscles. The IPEX increase in Ser⁷⁰⁴ phosphorylation in the current study was similar to the IPEX effects previously reported in earlier studies of human skeletal muscle. Furthermore, in rat epitrochlearis muscles incubated in the absence of insulin, Ser⁷⁰⁴ phosphorylation for the 3hPEX group exceeded values for the sedentary controls. In addition, Ser⁷⁰⁴ phosphorylation in insulin-stimulated muscles of 3hPEX rats were greater than the values for sedentary controls. The persistently elevated Ser⁷⁰⁴ phosphorylation 3hPEX was similar to earlier results demonstrating greater Ser⁷⁰⁴ phosphorylation for exercised vs. the contralateral unexercised muscle at ~4 to 5 h after a one-legged exercise session by healthy humans undergoing a hyperinsulinemic euglycemic clamp (43). The current results for greater Ser⁷⁰⁴ phosphorylation in rat epitrochlearis IPEX and 3hPEX align with the observations from earlier research in human vastus lateralis muscle postexercise and mouse EDL muscle postcontraction.

A great deal of research supports the idea that AMPK activation can positively modify insulin sensitivity (16, 17, 34, 35). Fisher et al. (16) reported that the prior incubation of isolated rat epitrochlearis muscles with the AMPK activator AICAR resulted in greater insulin-stimulated glucose uptake 3 h after AICAR treatment. Similarly, Kjobsted et al. (34) recently found that incubating isolated EDL muscles from WT mice with AICAR induced subsequently greater insulin-stimulated glucose uptake. However, this insulin-sensitizing effect was not found in isolated EDL muscles from γ3-AMPK-null mice, providing compelling evidence for a link between γ3-AMPK and AICAR-induced elevation in insulin-stimulated glucose uptake (34). Further support that γ3-AMPK activation can benefit insulin sensitivity was provided by the observation that in mouse EDL muscle, γ3-AMPK activity was increased 3 h after in situ contraction concomitant with enhanced insulin-stimulated AS160 Ser⁷⁰⁴ phosphorylation, AS160 Thr⁶⁴² phosphorylation and glucose uptake (32). It is notable that prior in situ contraction by γ3-AMPK-null mice did not induce increased AS160 Ser⁷⁰⁴ phosphorylation in mouse EDL muscle 3 h postcontraction, demonstrating that γ3-AMPK was essential for the contraction-induced increase in Ser⁷⁰⁴ phosphorylation (32). Oki et al. (41) recently reported that prior incubation of rat epitrochlearis muscles with AICAR resulted in subsequently greater AS160 Ser⁷⁰⁴ and Thr⁶⁴² phosphorylation concomitant with enhanced insulin-stimulated glucose uptake. Taking together these earlier findings with the current results in which exercise increased γ3-AMPK activity, but not γ1-AMPK activity, provides important new support for the hypothesis that the exercise-induced increase in γ3-AMPK activity may be involved in the enhanced insulin-stimulated glucose uptake determined at 3hPEX in the current study.

In addition to γ1-AMPK and γ3-AMPK activity, we assessed two other indices of AMPK activation, AMPK Thr¹⁷² phosphorylation and ACC Ser^79/212 phosphorylation. AMPK Thr¹⁷² phosphorylation was increased IPEX, consistent with earlier studies that evaluated the rat epitrochlearis after a similar exercise protocol (3, 48, 49), but the two previous studies that also assessed AMPK Thr¹⁷² phosphorylation at 3 to 4 h postexercise did not detect a persistent increase in AMPK Thr¹⁷² phosphorylation at these later timepoints (3, 48). Similarly, AMPK Thr¹⁷² phosphorylation was not elevated 5–7 h postexercise in human vastus lateralis muscle (43). Although AMPK Thr¹⁷² was not increased 3–7 h postexercise in these earlier studies, they reported either greater AS160 Thr⁶⁴² phosphorylation (43, 48) or greater AS160 phosphorylation using a phospho-Akt substrate antibody that likely recognized Thr⁶⁴² (3). Pehmøller et al. (43) also detected greater AS160 Ser⁷⁰⁴ phosphorylation at ~5–7 h postexercise without greater AMPK Thr¹⁷² phosphorylation. In contrast to these earlier results, a small, but statistically significant main effect of exercise on AMPK Thr¹⁷² phosphorylation remained detectable at 3hPEX in the current study. Taking these results together, increased AMPK Thr¹⁷² phosphorylation does not reliably predict greater pAS160 Ser⁷⁰⁴ several hours postexercise. Phosphorylation of ACC on Ser^79/212 was increased IPEX, but not 3hPEX in the current study. The lack of correspondence between ACC Ser^79/212 phosphorylation and γ3-AMPK activity at 3hPEX suggests that γ3-AMPK is not responsible for phosphorylation on this site. Assessing γ3-AMPK activity provided insights that would have been missed by evaluating only total AMPK Thr¹⁷² or ACC Ser^79/212 phosphorylation.

Which kinase(s) was(were) responsible for the observed IPEX elevation in AS160 phosphorylation on Ser⁵⁸⁸, Thr⁶⁴², and Ser⁷⁰⁴? Several lines of evidence argue against Akt being the relevant kinase for the IPEX effect. Neither Akt1 nor Akt2 phosphorylated AS160 on Ser⁷⁰⁴ in a cell-free assay (58), and using the same exercise protocol as the current study, we previously observed no increase in Akt Ser⁴⁷³ or Thr³⁰⁸ phosphorylation in rat epitrochlearis IPEX (3, 48). Recombinant Akt, p90 ribosomal S6 kinase 1 (RSK1), and serum- and glucocorticoid-induced protein kinase 1 (SGK1) phosphorylated AS160 Ser⁵⁸⁸ and Thr⁶⁴² in cell-free assays (20), but we previously observed no effect of this exercise protocol on phosphorylation of regulatory sites on SGK1 or RSK1 in rat epitrochlearis muscle (48). Mouse skeletal muscle expressing constitutively active CaMKKα was not characterized by greater Ser⁵⁸⁸ or Thr⁶⁴² phosphorylation than WT control muscle (27). Purified PKCζ did not phosphorylate AS160 on Ser⁷⁰⁴ in a cell-free assay (58). In contrast to the mostly negative results for several other kinases, multiple lines of evidence implicate AMPK as the leading candidate for the IPEX effects on AS160 phosphorylation. Purified AMPK phosphorylated AS160 on Ser⁷⁰⁴ in a cell-free assay (58). In addition, purified AMPK strongly phosphorylated AS160 Ser⁵⁸⁸ and weakly phosphorylated AS160 Thr⁶⁴² in a cell-free assay (20). Contraction increased Ser⁷⁰⁴ phosphorylation in the skeletal muscle of WT, but not AMPKα2 kinase-dead mice (58). Incubating HEK-293 cells with AICAR (an AMPK activator) elevated AS160 Ser⁵⁸⁸ phosphorylation (20). In the current study, we observed greater γ3-AMPK activity, but unaltered γ1-AMPK activity. Taking together our current results with earlier results, γ3-AMPK is implicated in the mechanism for elevated phosphorylation of AS160 on Ser⁷⁰⁴ and Ser⁵⁸⁸ in the IPEX rats. The observed increase in Ser⁷⁰⁴ phosphorylation would be predicted to favor the observed greater Thr⁶⁴² phosphorylation in the IPEX rats. However, a role for other unidentified kinases cannot be ruled out for the IPEX-induced increase in these AS160 phosphosites.

An obvious consideration when identifying the relevant kinases to phosphorylate protein substrates is that the kinase and substrates must be colocalized for phosphorylation to occur. Previous research has addressed the influence of insulin on the subcellular localization of Akt with AS160 in adipocytes and skeletal muscle (21, 65). However, the possibility that exercise might alter subcellular localization of Akt, AS160, or other relevant kinases remains to be explored.

AS160 phosphorylation depends on the balance between the opposing activities of the relevant protein kinases and protein phosphatases. We have performed a series of studies to understand the processes regulating AS160 dephosphorylation (4, 48, 50, 53). Some of the key results of these studies included the identification of protein phosphatase 1α (PP1α) in the dephosphorylation of AS160 Thr⁶⁴² and Ser⁵⁸⁸ in skeletal muscle (53), and demonstration that acute exercise slowed the dephosphorylation rate of Thr⁶⁴² and Ser⁵⁸⁸ in skeletal muscle lysates using a cell-free assay (4). It will be important to determine whether acute exercise alters PP1α activity and if the exercise effects on site-selective AS160 dephosphorylation in muscle lysates are also observed with intact skeletal muscle.

AS160 and its paralog protein, TBC1D1, share 50% identity, and each includes a GTPase-activating protein (GAP) domain (with 79% identity), a calmodulin-binding domain and two PTB domains. AS160 has multiple canonical Akt phosphorylation motifs, whereas TBC1D1 has only TBC1D1 Thr⁵⁹⁶, which corresponds to AS160 Thr⁶⁴² (13). Contraction-mediated glucose uptake was reduced in mouse skeletal muscle overexpressing TBC1D1 with a mutation that prevented phosphorylation on four phosphosites, including the AMPK phosphomotif Ser²³⁷ (2, 60). In contrast, overexpression of TBC1D1 with a mutation of Thr⁵⁹⁶ to prevent phosphorylation did not alter insulin-stimulated glucose uptake in mouse muscle (2). Furthermore, TBC1D1-null rats had normal insulin-stimulated GLUT4 translocation in skeletal muscle and normal in vivo insulin sensitivity, but reduced contraction-stimulated GLUT4 translocation and glucose uptake in muscle (63). These results implicate TBC1D1 in the regulation of contraction’s effect on insulin-independent glucose uptake in skeletal muscle, and they suggest TBC1D1 is likely relevant for the increased insulin-independent glucose uptake that was evident IPEX in the current study. However, the available data argue against TBC1D1 being a major determinant of insulin-stimulated glucose uptake. Importantly, exercise protocols that resulted in increased insulin-stimulated glucose uptake and AS160 Thr⁶⁴² phosphorylation in skeletal muscle from rats (19, 29) 3 or 27 h postexercise or humans (43) ~5–7 h postexercise did not induce concomitantly greater TBC1D1 phosphorylation 3 to 27 h after exercise. In contrast to extensive evidence linking AS160 phosphorylation to improved insulin sensitivity after exercise, substantial evidence argues against TBC1D1 phosphorylation being the mechanism for this exercise effect.

Many previous studies by various researchers using a wide variety of exercise protocols have found that acute exercise can improve insulin sensitivity in multiple species, including humans, rats, mice, sheep, and dogs (10, 23, 39, 43–45, 49, 61, 64). Similar to the compelling evidence that improved insulin sensitivity is a hallmark of exercise, using the same exercise protocol as the current study, we have consistently found a postexercise increase in insulin-stimulated glucose uptake by isolated rat epitrochlearis muscle (3, 4, 11, 19, 48, 49). In the context of the extensive literature linking exercise to improved insulin sensitivity, it seems likely that the increased insulin-stimulated glucose uptake in the epitrochlearis is an exercise-specific consequence of our protocol rather than a nonspecific stress response. However, to address this idea, we also assessed the effect of the same exercise protocol on insulin-stimulated glucose uptake by the EDQP, a forelimb muscle with a fiber-type profile very similar to the epitrochlearis (12). The exercise protocol did not improve insulin-stimulated glucose uptake in the EDQP, arguing against the idea that the enhanced insulin-stimulated glucose uptake in the epitrochlearis was attributable to a nonspecific response.

In 1982, Erik Richter made the seminal observation that a single exercise session can improve insulin sensitivity for glucose uptake by skeletal muscle (45). Twenty-five years later, Arias et al. (3) provided the initial evidence implicating greater phosphorylation of AS160 on Akt phosphosites as a plausible mechanism for this exercise-induced outcome. During the subsequent decade, multiple studies have reinforced the premise of AS160ʼs role in postexercise effects on insulin sensitivity in rat and human skeletal muscle (4, 11, 18, 19, 29, 43, 48, 56). However, the specific cellular events responsible for greater site-selective AS160 phosphorylation have remained elusive. Recently, Kjobsted et al. (32, 34) made the novel proposal that γ3-AMPK activation leads to subsequently greater phosphorylation of AS160 on Ser⁷⁰⁴, which, in turn, favors greater insulin-stimulated AS160 phosphorylation on Thr⁶⁴². The experimental support for this intriguing idea was largely based on results for mouse muscle after ex vivo exposure to AICAR or in situ electrically stimulated contractions rather than on in vivo exercise. Accordingly, the current results are significant because they provide support for this hypothesis using a very well-established in vivo exercise protocol that leads to increased insulin-stimulated glucose uptake in rat skeletal muscle. Going forward, it will be important to test whether this model can explain the relationship between exercise and insulin sensitivity under physiological conditions. For example, is this proposed model relevant for the improved insulin-stimulated glucose uptake after exercise by insulin-resistant individuals (6)? Is the nonuniform effect of acute exercise on insulin-stimulated glucose uptake across different muscle fiber types (8) related to fiber-type-specific exercise effects on γ3-AMPK activity and/or site-selective AS160 phosphorylation? The relationship between sustained AS160 phosphorylation and insulin-stimulated glucose uptake after in vivo exercise is based exclusively on research in males, so it will also be essential to perform similar experiments in females.

GRANTS

This research was supported by grants from the National Institutes of Health (DK-71771 to G. D. Cartee and DK-101043 to L. J. Goodyear).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

H.W. and G.D.C. conceived and designed research; H.W., E.B.A., and M.W.P. performed experiments; H.W. and M.W.P. analyzed data; H.W. and G.D.C. interpreted results of experiments; H.W. prepared figures; H.W. and G.D.C. drafted manuscript; H.W., E.B.A., M.W.P., L.J.G., and G.D.C. edited and revised manuscript; H.W., E.B.A., M.W.P., L.J.G., and G.D.C. approved final version of manuscript.