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In vivo exercise followed by in vitro contraction additively elevates

subsequent insulin-stimulated glucose transport by rat skeletal muscle

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 doi:10.1152/ajpendo.00758.2009 298:999-1010, 2010. First published Feb 23, 2010;Am J Physiol Endocrinol Metab

and Gregory D. Cartee Katsuhiko Funai, George G. Schweitzer, Carlos M. Castorena, Makoto Kanzaki

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In vivo exercise followed by in vitro contraction additively elevatessubsequent insulin-stimulated glucose transport by rat skeletal muscle

Katsuhiko Funai,1 George G. Schweitzer,1 Carlos M. Castorena,1 Makoto Kanzaki,2

and Gregory D. Cartee1,3

1Muscle Biology Laboratory, School of Kinesiology and 3Department of Molecular and Integrative Physiology, Universityof Michigan, Ann Arbor, Michigan; and 2Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan

Submitted 21 December 2009; accepted in final form 17 February 2010

Funai K, Schweitzer GG, Castorena CM, Kanzaki M, Cartee GD.In vivo exercise followed by in vitro contraction additively elevatessubsequent insulin-stimulated glucose transport by rat skeletal muscle.Am J Physiol Endocrinol Metab 298: E999–E1010, 2010. Firstpublished February 23, 2010; doi:10.1152/ajpendo.00758.2009.—Thecellular mechanisms whereby prior exercise enhances insulin-stimu-lated glucose transport (GT) are not well understood. Previous studiessuggested that a prolonged increase in phosphorylation of Akt sub-strate of 160 kDa (AS160) may be important for the postexerciseincrease in insulin sensitivity. In the current study, the effects of invivo exercise and in vitro contraction on subsequent insulin-stimu-lated GT were studied separately and together. Consistent with resultsfrom previous studies, prior exercise resulted in an increase in AS160642Thr phosphorylation immediately after exercise in rat epitrochle-aris muscles, and this increase remained 3 h postexercise concomitantwith enhanced insulin-stimulated GT. For experiments with in vitrocontraction, isolated rat epitrochlearis muscles were electrically stim-ulated to contract in the presence or absence of rat serum. Asexpected, insulin-stimulated GT measured 3 h after electrical stimu-lation in serum, but not after electrical stimulation without serum,exceeded resting controls. Immediately after electrical stimulationwith or without serum, phosphorylation of both AS160 (detected byphospho-Akt substrate, PAS, antibody, or phospho-642Thr antibody)and its paralog TBC1D1 (detected by phospho-237Ser antibody) wasincreased. However, both AS160 and TBC1D1 phosphorylation hadreversed to resting values at 3 h poststimulation with or withoutserum. Increasing the amount of exercise (from 1 to 2 h) or electricalstimulation (from 5 to 10 tetani) did not further elevate insulin-stimulated GT. In contrast, the combination of prior exercise andelectrical stimulation had an additive effect on the subsequent increasein insulin-stimulated GT, suggesting that these exercise and electricalstimulation protocols may amplify insulin-stimulated GT throughdistinct mechanisms, with a persistent increase in AS160 phosphory-lation potentially important for increased insulin sensitivity afterexercise, but not after in vitro contraction.

insulin sensitivity; Akt substrate of 160 kDa; TBC1D1; TBC1D4;glucose transporter 4

A SINGLE BOUT OF EXERCISE leads to a subsequent increase ininsulin-dependent glucose transport that can last for hours afterexercise (6, 8, 10, 12, 21, 29, 31). The enhanced insulin-stimulated glucose transport postexercise occurs as a result ofgreater insulin-stimulated cell-surface GLUT4 localization(20), but the cellular mechanisms that lead to this event are notwell understood. Many studies have found that prior exercisedoes not amplify insulin effects on proximal insulin signalingsteps [e.g., insulin receptor tyrosine kinase activity, insulin

receptor substrate tyrosine phosphorylation, insulin receptorsubstrate-associated phosphatidylinositol 3-kinase activity, pro-tein kinase B (Akt) serine phosphorylation, and Akt activity] (3,13, 18, 20, 22, 36, 38, 39). These results suggested that exercisemight improve insulin sensitivity by altering an insulin signalingstep distal to Akt.

The first substrate of Akt to be linked to the regulation ofGLUT4 translocation was Akt substrate of 160 kDa (AS160;also known as TBC1D4) (23, 33). Under basal conditions,AS160’s active Rab GTPase-activating protein domain is be-lieved to restrain the exocytosis of intracellular GLUT4 storagevesicles of 3T3-L1 adipocytes (7, 9, 24, 32, 33). Insulin-stimulated phosphorylation of AS160 on specific Akt motifs,with 642Thr being especially important, appears to relieve thisrestraint and allow GLUT4 to be recruited to the cell surfacemembranes.

Bruss et al. (4) demonstrated that either insulin or in vitrocontractile activity leads to phosphorylation of AS160 in skel-etal muscle. Arias et al. (1) found that AS160 phosphorylationis also elevated in rat epitrochlearis muscle immediately afterin vivo exercise. Furthermore, the elevated AS160 phosphory-lation was still evident at 3–4 h postexercise, which led to thehypothesis that this prolonged effect on AS160 may be impor-tant for the enhanced insulin-stimulated glucose transport atthis time. Consistent with this idea, Funai et al. (15) found that,when rats were allowed to eat rat chow after exercise, both theenhanced AS160 phosphorylation and increased insulin-stim-ulated glucose transport were reversed to resting levels, but,when rats remained fasted postexercise, the elevated AS160phosphorylation persisted concomitant with enhanced insulin-stimulated glucose transport for as long as 27 h after exercise.A persistent elevation in AS160 phosphorylation has also beenobserved in human skeletal muscle several hours after acuteexercise, suggesting it may be important for the improvementin insulin sensitivity in humans after exercise (34, 37).

Electrically stimulated contraction of isolated skeletal mus-cle has been widely used as a valuable model for elucidatingthe mechanisms that regulate the increased glucose transportafter in vivo exercise. When isolated rat epitrochlearis musclesare stimulated to contract in the presence of rat serum, there isa substantial increase in the subsequent insulin-stimulatedglucose transport measured 3 h postcontraction, reminiscent ofthe results observed after in vivo exercise (12, 13, 16). How-ever, when isolated rat epitrochlearis muscles are electricallystimulated using an identical protocol in the absence of serum,there is an increase in insulin-independent glucose transportimmediately after contraction but no subsequent improvementin insulin-stimulated glucose transport at 3 h postcontraction(8, 12, 13, 16). AS160 phosphorylation is increased immedi-

Address for reprint requests and other correspondence: G. D. Cartee, Univ.of Michigan, School of Kinesiology, Rm. 4745F, 401 Washtenaw Ave., AnnArbor, MI 48109-2214 (e-mail: gcartee@umich.edu).

Am J Physiol Endocrinol Metab 298: E999–E1010, 2010.First published February 23, 2010; doi:10.1152/ajpendo.00758.2009.

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ately after contraction by isolated epitrochlearis muscles (4,14) but whether this phosphorylation persists for hours aftercontraction remains to be determined. Thus electrical stimula-tion of isolated skeletal muscles in the presence of serum(which leads to increased insulin-stimulated glucose transport)or in the absence of serum (which does not) provides anopportunity for probing the idea that a persistent increase inAS160 phosphorylation can lead to enhanced insulin sensitiv-ity.

Accordingly, we hypothesized that contraction induced byelectrical stimulation of isolated rat epitrochlearis muscleswould lead to increased AS160 phosphorylation immediatelypostcontraction, whether or not serum was present. We furtherhypothesized that the presence of serum during contractionwould be required for the persistent increase in AS160 phos-phorylation and insulin-stimulated glucose transport at 3 hpostelectrical stimulation. We also studied isolated musclesthat were electrically stimulated to contract after rats per-formed in vivo exercise. We hypothesized that, at 3 h post-electrical stimulation, the levels of AS160 phosphorylation andinsulin-stimulated glucose transport in these muscles would notdiffer from the levels in muscles studied after in vivo exercisewithout in vitro electrical stimulation. Data supporting thesehypotheses would be consistent with the idea that in vivoexercise and in vitro contraction in serum lead to increasedinsulin sensitivity by a similar mechanism that is dependent ona persistent increase in AS160 phosphorylation. In addition, weevaluated exercise and in vitro contraction effects on phosphory-lation of TBC1D1 (a paralog of AS160, also implicated inglucose transport regulation), GLUT4 abundance, and otherpotential modulators of insulin sensitivity [muscle glycogenconcentration and AMP-activated protein kinase (AMPK) ac-tivation].

METHODS

Materials. Serum from male Wistar rats (120–200 g, fasted for 12h) was purchased from Gemini Bio-Products (West Sacramento, CA).Human recombinant insulin was obtained from Eli Lilly (Indianapolis,IN). Reagents and apparatus for SDS-PAGE and immunoblottingwere purchased from Bio-Rad (Hercules, CA). Bicinchoninic acidprotein assay reagent (no. 23227), T-PER tissue protein extractionreagent (no. 78510), and West Dura Extended Duration Substrate (no.34075) were from Pierce Biotechnology (Rockford, IL). Anti-phos-pho-308ThrAkt (p308ThrAkt, no. 9275), anti-phospho-172ThrAMPK(p172ThrAMPK, no. 2531), anti-phospho-(Ser/Thr) Akt substrate (PAS,no. 9611), and goat anti-rabbit IgG horseradish peroxidase (HRP) con-jugate (no. 7074) were from Cell Signaling Technology (Danvers, MA).PAS was designed to recognize Akt phosphorylation motif peptidesequences (RXRXXpT/S). Total TBC1D1 and phospho-237Ser TBC1D1(p237SerTBC1D1) polyclonal antibody and were provided by Dr. MakotoKanzaki at Tohoku University (25). Anti-AS160 (no. 07–741), anti-phospho-642Thr AS160 (p642ThrAS160, no. 07–802), and protein Gagarose beads (no. 16–266) were from Upstate USA (Charlottesville,VA). 3-O-methyl-[3H]glucose (3-[3H]MG) was from Sigma-Aldrich.[14C]mannitol was from Perkin Elmer (Waltham, MA). Other reagentswere from Sigma-Aldrich and Fisher Scientific (Pittsburgh, PA).

Insulin concentration in serum. Rat serum purchased from GeminiBio-Products was submitted to the Chemistry Laboratory Core of theMichigan Diabetes Research and Training Center for the measure-ment of insulin concentration. The insulin concentration of serum (21�U/ml) was determined with a double-antibody radioimmunoassayusing an 125I-Human insulin tracer (Linco Research), a rat insulinstandard (Novo), a guinea pig anti-rat insulin first antibody (Linco

Research), and a sheep anti-guinea pig gamma globulin-polyethyleneglycol second antibody (MDRTC).

Animal treatment. Procedures for animal care were approved by theUniversity of Michigan Committee on Use and Care of Animals. MaleWistar rats (120–200 g; Harlan, Indianapolis, IN) were provided withrodent chow (Lab Diet; PMI Nutritional International, Brentwood,MO) and water ad libitum until 1700 the night before the experimentwhen their food was removed. On the following day between 0800 and1000, rats were randomly assigned to 1) resting (REST), 2) postelectricalstimulation (in vitro electrical stimulation of epitrochlearis muscles:PES), 3) postexercise (in vivo exercise: PEX), or 4) postexercise andelectrical stimulation (in vivo exercise followed by in vitro electricalstimulation: PEX � PES) groups. For all experiments, rats were anes-thetized with an intraperitoneal injection of pentobarbital sodium (50mg/kg wt). While rats were under deep anesthesia, both epitrochlearismuscles were rapidly removed by dissection.

In vitro electrical stimulation. Paired isolated epitrochlearis mus-cles were incubated in flasks containing either Krebs-Henseleit buffer(KHB) � 0.1% BSA � 8 mM glucose � 32 mM mannitol (solution1) or in serum for 30 min in a shaking water bath at 35°C. For allincubation steps, flasks were continuously gassed from above with95% O2-5% CO2. After 30 min, one of the paired muscles wastransferred to a vial containing identical media (REST). The contralat-eral muscle was attached to a glass rod and a force transducer(Radnoti, Litchfield, CT). Mounted muscles were incubated in mediaidentical to the first incubation step and were stimulated to contract(PES) as previously described (0.1 ms twitch, 100 Hz train for 10 s,10 trains, 10 min; Grass S48 Stimulator; Grass Instruments, Quincy,MA) (12). For the experiments immediately postelectrical stimulation(0 h REST and 0 h PES; Fig. 1A), muscles were either freeze-clampedor transferred to vials containing KHB � 0.1% BSA � 2 mMpyruvate � 36 mM mannitol (solution 2; 30°C, 10 min) beforeincubation with 3-MG. For the experiments 3 h postelectrical stimu-lation [3 h REST and 3 h PES; Fig. 2A and Supplemental Fig. S2, A(Supplemental data for this article can be found on the AmericanJournal of Physiology: Endocrinology and Metabolism website)],muscles were incubated according to the protocol previously used (12,16). Immediately after electrical stimulation, all muscles (regardlessof whether the previous incubation was with or without serum) weretransferred to vials containing solution 1 for a 5-min wash step at35°C. Muscles were then transferred to other vials containing solution1 for 3 h at 35°C. After 3 h, muscles were transferred to flaskscontaining solution 2 for 30 min at 30°C. During this step, for somemuscle pairs, solution 2 contained 50 �U/ml of insulin; for other musclepairs, solution 2 contained no insulin. After 30 min, all muscles wereincubated with 3-MG (see 3-MG incubation, muscle homogenization,and 3-MG transport determination).

Serum vs. insulin experiment. One experiment compared the effectof incubation of muscles in serum with incubation of muscles in 21�U/ml insulin (which equaled the insulin concentration in serum;Supplemental Fig. S1, A). Immediately after dissection, isolatedepitrochlearis muscles were placed in solution 1 with no insulin,solution 1 with 21 �U/ml of insulin, or serum for 30 min at 35°C.Muscles were then transferred to a second flask including the identicalmedia as in step 1 for 10 min before being freeze-clamped.

Increasing the amount of exercise or electrical stimulation. Todetermine if exercise and electrical stimulation protocols were max-imally effective, the effects of increasing the amount of exercise orelectrical stimulation on subsequent insulin-stimulated glucose trans-port were studied. For in vivo exercise (Fig. 3A), rats performed 1 or2 h swim exercise in a barrel filled with water (35°C) to a depth of�60 cm (6–8 rats/barrel) for 2 or 4 � 30 min bouts, with a 5-min restperiod between each bout. Immediately after exercise (1 or 2 h) orrest, both epitrochlearis muscles were rapidly removed and wereincubated in serum for 30 min, serum for 10 min, solution 1 for 5 min

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wash, solution 1 for 3 h rest, solution 2 (�insulin) for 30 min, andthen 10 min in a solution containing 3-MG as described below. Forin vitro electrical stimulation (Fig. 4A), both epitrochlearis muscleswere dissected from unexercised rats and were incubated in serumbefore and during rest or electrical stimulation (5 or 10 tetani),followed by incubation in solution 1 for 5 min wash, solution 1 for 3h rest, solution 2 (�insulin) for 30 min, and then 10 min in a solutioncontaining 3-MG as described below.

Additivity experiments. In experiments comparing the combinedeffects of exercise and electrical stimulation on the subsequent in-crease in insulin-stimulated glucose transport, four groups were stud-ied: REST, PES, PEX, and PEX � PES. Before anesthetization andmuscle incubation steps, rats in the PEX or PEX � PES groupsperformed 2 h swim exercise. Immediately after exercise or rest, bothepitrochlearis muscles were rapidly removed and were either frozenimmediately (0 h PEX or 0 h REST; Fig. 6A) or were electrically

stimulated to contract in vitro before being frozen (0 h PEX � PES or0 h PES; Fig. 6A). In other rats, immediately after exercise or rest,both epitrochlearis muscles were dissected out and incubated in serumbefore and during electrical stimulation (10 tetani) or rest, followed bya 5-min wash in solution 1, a 3-h incubation step in solution 1, and a30-min rinse in solution 2 (� insulin) before incubation with 3-MG asdescribed below (3 h REST, 3 h PES, 3 h PEX and 3 h PEX � PES;Fig. 5A).

3-MG incubation, muscle homogenization, and 3-MG transportdetermination. After incubation in solution 2, muscles were trans-ferred to flasks containing KHB � 0.1% BSA � 8 mM 3-MG(including 3-[3H]MG at a final specific activity of 0.25 mCi/mmol)and 2 mM mannitol (including [14C]mannitol at a final specific activityof 6.25 �Ci/mmol) with or without 50 �U/ml insulin at 30°C. After 10min, muscles were rapidly blotted, trimmed, freeze-clamped, and stored(�80°C) until processed.

Fig. 1. Postelectrical stimulation (in vitro electrical stimulation of epitrochlearis muscles) (PES) for 0 h vs. 0 h in the resting state (REST). Rat epitrochlearismuscles were incubated in either buffer or serum before and during in vitro electrical stimulation or resting control. A: experimental design. KHB,Krebs-Henseleit buffer. B: rate of 3-O-methylglucose (3-MG) transport. C: phosphorylated (p) 308Thr protein kinase B (Akt). D: p172Thr AMP-activated proteinkinase (AMPK). E: anti-phospho-(Ser/Thr) Akt substrate (PAS)-Akt substrate of 160 kDa (AS160). F: p642ThrAS160. G: PAS-TBC1D1. H: p237SerTBC1D1.Data were analyzed with 2-way ANOVA and the Student-Newman-Keuls post hoc test. *P � 0.05 (electrical stimulation effect; post hoc test); †P � 0.05 (serumeffect; post hoc test). Data are means � SE, n � 4–6 rats/group. Open bars, resting (0 h REST); filled bars, immediately after electrical stimulation (0 h PES).RU, relative units.

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Frozen muscles were homogenized in 1 ml ice-cold homogeniza-tion buffer (2 mM Na3VO4, 2 mM EDTA, 2 mM EGTA, 2.5 mMsodium pyrophosphate, 1 mM -glycerophosphate, 1 mM phenyl-methanesulfonyl fluoride, and 1 �g/ml leupeptin in T-PER) usingglass-on-glass tubes (Kontes, Vineland, NJ). Homogenates were sub-sequently rotated at 4°C for 1 h before being centrifuged (15, 000 g for10 min at 4°C). Aliquots of the supernatant from muscles used for the3-MG transport measurement were pipetted into vials with scintilla-tion cocktail for scintillation counting, and 3-MG transport wasdetermined as previously described (5). A portion of supernatant wasused to determine protein concentration according to the manufacturer’sinstructions (Pierce Biotechnology catalog no. 23227). The remainingsupernatant was stored at �80°C until further analyzed.

Immunoprecipitation. Homogenized muscle (200–300 �g protein)was precleared and immunoprecipitated with anti-AS160 or anti-TBC1D1 at 4°C with protein G-agarose beads. After gentle rotationovernight, the immunoprecipitation mix was centrifuged at 4,000 g,and the supernatant was aspirated. After washing (four times with 500�l PBS), the protein bound to the beads was eluted with 2� SDSloading buffer and boiled before loading on a polyacrylamide gel.

Immunoblotting. Samples (immunoprecipitates or homogenates)were boiled with SDS loading buffer, separated by PAGE, andelectrophoretically transferred to nitrocellulose. Samples were thenrinsed with Tris-buffered saline plus Tween (TBST; 0.14 mol/l NaCl,0.02 mol/l Tris base, pH 7.6, and 0.1% Tween), blocked with 5%nonfat dry milk in TBST for 1 h at room temperature, washed 3 � 5

Fig. 2. 3 h PES vs. 3 h REST. Rat epitrochlearis muscles were incubated in serum before and during in vitro electrical stimulation or resting control and weresubsequently incubated in buffer solution for 3 h. A: experimental design. B: rate of 3-MG transport. C: p308ThrAkt. D: p172ThrAMPK. E: PAS-AS160.F: p642ThrAS160. G: PAS-TBC1D1. H: p237SerTBC1D1. Data were analyzed with 2-way ANOVA and the Student-Newman-Keuls post hoc test. *P � 0.05(insulin effect; post hoc test); †P � 0.05 (postelectrical stimulation effect; post hoc test). Data are means � SE, n � 6–12/group. Open bars, resting in serumfollowed by 3 h incubation in buffer (3 h REST); filled bars, in vitro electrical stimulation in serum followed by 3 h incubation in buffer (3 h PES).

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min at room temperature, and treated with the primary antibodies(1:1,000 in TBST � 5% BSA) overnight at 4°C. Blots were thenwashed 3 � 5 min with TBST, incubated with the secondary antibody,goat anti-rabbit IgG HRP conjugate (1:20,000 in TBST � 5% milk),for 1 h at room temperature, washed again 3 � 5 min with TBST, anddeveloped with West Dura Extended Duration Substrate reagent.Protein bands were quantified by densitometry (Alpha Innotech, SanLeandro, CA). The mean values for basal muscles (REST withoutinsulin) on each blot were normalized to equal 1.0, and then allsamples on the blot were expressed relative to the normalized basalvalue.

Muscle glycogen concentration. Muscles used for measurement ofglycogen were weighed and then homogenized in ice-cold 0.3 Mperchloric acid. An aliquot of the homogenate was stored at �80°Cfor later determination of glycogen concentration by the amylogluco-sidase method (27).

Statistical analysis. Statistical analyses were performed usingSigma Stat version 2.0 (San Rafael, CA). Data were expressed asmeans � SE. P � 0.05 was considered statistically significant. Datafrom the REST vs. PES (0 h and 3 h) experiments were analyzed withtwo-way ANOVA (main effects of PES and insulin). Data from theserum vs. insulin experiment were analyzed using one-way ANOVA.

Data from experiments to examine the effects of increasing theamount of exercise or electrical stimulation were analyzed usingone-way ANOVA for muscles with and without insulin. Data from theadditivity experiments were analyzed with: 1) one-way ANOVA forthose muscles that were frozen immediately after exercise and/orelectrical stimulation (0 h PEX � PES) or 2) two-way ANOVA (maineffects of PEX and PES) for those muscles that were frozen 3 h afterexercise and/or electrical stimulation (3 h PEX � PES) for muscleswith and without insulin. For all ANOVA, the Student-Newman-Keuls post hoc test was used to identify the source of significantvariance. When data failed the Levene Median test for equal variance,the Kruskal-Wallis nonparametric ANOVA on ranks, was used withDunn’s post hoc test.

RESULTS

Immediately postelectrical stimulation. Glucose transportdetermined immediately after the electrical stimulation (0 hPES) was significantly greater (P � 0.05) compared with 0 hREST values, regardless of the incubation media (serum-freebuffer or serum) (Fig. 1B). Electrical stimulation (0 h PES) also

Fig. 3. Comparison of 1- or 2-h bout ofin vivo exercise. Following 1 h of exercisebout (2 � 30 min), 2 h of exercise bout (4 �30 min), or sedentary, rat epitrochlearis mus-cles were incubated in serum and were sub-sequently incubated in buffer solution for 3 h.A: experimental design. B: rate of 3-MGtransport; data for muscles incubated withoutinsulin and those incubated with insulin wereeach analyzed with 1-way ANOVA and theStudent-Newman-Keuls post hoc test. PEX,postexercise (in vivo exercise). *P � 0.05(significantly different from 3 h REST withinthe insulin-treated muscles). C: insulin (in-crease above basal, calculated by subtractingthe values for muscles incubated without in-sulin from the respective values of pairedmuscles incubated with insulin) for the rate of3-MG transport; data were analyzed with1-way ANOVA and the Student-Newman-Keuls post hoc test. *P � 0.05 (significantlydifferent from 3 h REST with insulin).D: p642ThrAS160; data for muscles incubatedwithout insulin and those incubated with insulinwere each analyzed with 1-way ANOVA and theStudent-Newman-Keuls post hoc test. *P � 0.05(significantly different from 3 h REST, within themuscles that were incubated without insulin);#P � 0.05 (significantly different from 3 h REST,among the muscles that were incubated with insu-lin). Data are means � SE, n � 6/group.

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resulted in significantly greater p308ThrAkt, p172ThrAMPK, PAS-AS160, p642ThrAS160, PAS-TBC1D1, and p237SerTBC1D1 com-pared with the 0 h REST group, regardless of the incubation media(serum-free buffer or serum) (P � 0.05; Fig. 1, C–H).

Muscles in both the 0 h REST and 0 h PES groups that wereincubated with serum vs. no serum during the initial incubationsteps were characterized by a small but significant (P � 0.05)increase in glucose transport (Fig. 1B). Incubation in serum hadno effect on p172ThrAMPK and p237SerTBC1D1 (Fig. 1, D andH). In contrast, muscles that were incubated in serum beforeand during electrical stimulation (or resting controls) hadgreater values for p308ThrAkt, PAS-AS160, and p642ThrAS160(P � 0.05) compared with the muscles that were incubated inserum-free buffer before and during the electrical stimulationstep (Fig. 1, C, E, and F). Incubation in serum resulted ingreater PAS-TBC1D1 (compared with incubation in serum-free buffer) in muscles that were not stimulated to contract(P � 0.05), but incubation in serum had no effect on PAS-TBC1D1 (compared with incubation in serum-free buffer) in

muscles that were stimulated to contract (Fig. 1G). Incubatingmuscles in insulin at a concentration equivalent to that in theserum (21 �U/ml) resulted in levels of p308ThrAkt, PAS-AS160, p642ThrAS160, and PAS-TBC1D1 that were greatercompared with muscles incubated in serum-free buffer (P �0.05) but not different from muscles incubated in serum (Sup-plemental Fig. S1). Thus the effects of serum alone on theseoutcomes could be accounted for by the effects produced bythe insulin concentration that was found in the serum.

Postelectrical stimulation (3 h) without serum. As previouslyreported (8, 12, 16), prior electrical stimulation in serum-freebuffer (3 h PES) had no effect on glucose transport in basal orinsulin-stimulated muscles (measured 3 h after electrical stimula-tion) compared with 3 h REST (Supplemental Fig. S2, B). Asexpected, prior electrical stimulation had no persistent effect onthe phosphorylation of any of the proteins studied (p308ThrAkt,p172ThrAMPK, PAS-AS160, p642ThrAS160, PAS-TBC1D1, andp237SerTBC1D1) in basal or insulin-stimulated muscles at 3 hPES compared with 3 h REST (Supplemental Fig. S2, C–H).

Fig. 4. Comparison of 5 or 10 tetani in vitroelectrical stimulation. Isolated rat epitrochle-aris muscles were incubated in serum beforeand during in vitro electrical stimulation (5 vs.10 tetani) or resting control and were subse-quently incubated in buffer solution for 3 h.A: experimental design. B: rate of 3-MG trans-port; data for muscles incubated without insu-lin and those incubated with insulin were eachanalyzed with 1-way ANOVA and the Stu-dent-Newman-Keuls post hoc test. *P � 0.05(significantly different from 3 h REST in theinsulin-treated muscles). C: insulin (increaseabove basal, calculated by subtracting the val-ues for muscles incubated without insulinfrom the respective values of paired musclesincubated with insulin) for the rate of 3-MGtransport; data were analyzed with 1-wayANOVA and the Student-Newman-Keuls posthoc test. *P � 0.05 (significantly differentfrom 3 h REST). D: p642ThrAS160; data formuscles incubated without insulin and thoseincubated with insulin were each analyzedwith 1-way ANOVA and the Student-New-man-Keuls post hoc test. Data are means �SE, n � 5–6/group.

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Fig. 5. 3 h postexercise � electrical stimulation(3 h PEX � PES). Following 2 h of exercisebout (4 � 30 min) or sedentary, isolated ratepitrochlearis muscles were incubated in serumbefore and during in vitro electrical stimulationor resting control and were subsequently incu-bated in buffer solution for 3 h. A: experimentaldesign. B: rate of 3-MG transport; data for mus-cles incubated without insulin and those incu-bated with insulin were each analyzed with2-way ANOVA and the Student-Newman-Keulspost hoc test. *P � 0.05 (significantly differentfrom 3 h REST with insulin); **P � 0.05 (sig-nificantly different from 3 h REST, 3 h PEX, and3 h PES muscles that were insulin treated).C: insulin (increase above basal, calculated bysubtracting the values for muscles incubatedwithout insulin from the respective values ofpaired muscles incubated with insulin) for therate of 3-MG transport; data were analyzed with2-way ANOVA and the Student-Newman-Keulspost hoc test. *P � 0.05 (significantly differentfrom 3 h REST); **P � 0.05 (significantlydifferent from 3 h REST, 3 h PEX, and 3 h PES).D: p642ThrAS160; data for muscles incubatedwithout insulin and those incubated with insulinwere each analyzed with 2-way ANOVA andthe Student-Newman-Keuls post hoc test. *P �0.05 (significantly different from 3 h REST,comparison within muscles that were incubatedwithout insulin); #P � 0.05 (significantly differ-ent from 3 h REST, comparison among mus-cles that were incubated with insulin). E: PAS-TBC1D1; data were analyzed with 2-wayANOVA and the Student-Newman-Keuls posthoc test. *P � 0.05 (significantly differentfrom 3 h REST). F: GLUT4 abundance. Dataare means � SE, n � 8–14/group.

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Insulin treatment (immediately before and during the incubationwith 3-MG) significantly elevated 3-MG transport, p308ThrAkt,PAS-AS160, p642ThrAS160, and PAS-TBC1D1 (P � 0.05; Sup-plemental Fig. S2, B–C and E–G) compared with no insulintreatment but had no effect on p172ThrAMPK or p237SerTBC1D1(Supplemental Fig. S2, D and H).

Postelectrical stimulation (3 h) with serum. As previouslyreported (12, 16), prior electrical stimulation in the presence ofserum (3 h PES) resulted in greater insulin-stimulated glucosetransport (measured 3 h after electrical stimulation) comparedwith 3 h REST (P � 0.05; Fig. 2B). In contrast, prior electricalstimulation had no persistent effect on the phosphorylation ofany of the proteins studied (p308ThrAkt, p172ThrAMPK, PAS-AS160, p642ThrAS160, PAS-TBC1D1, and p237SerTBC1D1)in basal or insulin-stimulated muscles at 3 h PES comparedwith 3 h REST (Fig. 2, C–H). Insulin treatment (immediatelybefore and during the incubation with 3-MG) significantly ele-vated 3-MG transport, p308ThrAkt, PAS-AS160, p642ThrAS160,and PAS-TBC1D1 (P � 0.05; Fig. 2, B–C and E–G) comparedwith no insulin treatment but had no effect on p172ThrAMPK orp237SerTBC1D1 (Fig. 2, D and H).

Increasing the amount of exercise or electrical stimulation.Increasing the amount of exercise (1 vs. 2 h) or electricalstimulation (5 vs. 10 tetani) did not result in higher values forthe subsequent p642ThrAS160 and insulin-stimulated glucosetransport (Figs. 3 and 4), providing evidence that the protocolused for each stimulus (exercise or electrical stimulation) forthe additivity experiments was maximally effective.

Additivity experiments. The purpose of these experimentswas to examine the possibility that prior in vitro contraction (in

serum) enhances insulin-stimulated glucose transport through amechanism distinct from that after exercise. Insulin-stimulatedglucose transport in muscles from rats that exercised immedi-ately before electrical stimulation of the isolated muscles (PEX �PES) was compared with that of muscles that were subjected toPEX or PES treatment alone (Fig. 5B). Total force productionduring electrical stimulation (11,102.3 � 755.5 vs. 8,969.8 �1,129.2 g·min�1·g muscle�1, P � 0.05), but not peak force(478.0 � 26.1 vs. 424.5 � 52.4 g/g muscle, P � 0.122), wassignificantly reduced in PEX � PES muscles compared with PESmuscles. Either exercise (3 h PEX; Fig. 5C) or electrical stimu-lation (3 h PES; Fig. 5C) induced a subsequent increase ininsulin-stimulated glucose transport, with no significant differ-ences between the groups. Importantly, muscles that underwentelectrical stimulation after being dissected from exercised rats (3 hPEX � PES; Fig. 5C) had greater insulin-stimulated glucose trans-port compared with muscles from the 3 h PEX or 3 h PES group. Thegreater insulin-stimulated glucose transport in 3 h PEX � PESmuscles was not accompanied by altered p642ThrAS160, PAS-TBC1D1, or GLUT4 abundance in the 3 h PEX � PES group vs. thePEX group (Fig. 5, D–F). Furthermore, glycogen concentration,p172ThrAMPK, and p642ThrAS160 were not different for the0 h PEX � PES group compared with the 0 h PES group(Fig. 6, B–D).

DISCUSSION

The results of this study demonstrated that in vivo exercisecauses a sustained increase in AS160 phosphorylation (PAS or642Thr) and a subsequently increased insulin-stimulated glu-

Fig. 6. Immediately after exercise � electri-cal stimulation (0 h PEX � PES). Following2 h of exercise bout (4 � 30 min) or seden-tary, isolated rat epitrochlearis muscles werefrozen immediately or were incubated in se-rum or serum-free buffer before and duringin vitro electrical stimulation or resting control andwere frozen immediately after. A: experimentaldesign. B: muscle glycogen concentration: datawere analyzed with Kruskal-Wallis nonparamet-ric 1-way ANOVA on ranks and the Dunn’s posthoc test. *P � 0.05 (significantly different from 0h REST). C: p172ThrAMPK; data were analyzedwith 1-way ANOVA and the Student-Newman-Keuls post hoc test. *P � 0.05 (significantlydifferent from 0 h REST); **P � 0.05 (signifi-cantly different from 0 h REST and 0 h PEX).D: p642ThrAS160; data were analyzed with 1-wayANOVA and the Student-Newman-Keuls posthoc test. *P � 0.05 (significantly different from 0h REST); **P � 0.05 (significantly different from0 h REST, 0 h PES-buffer, 0 h PEX, and 0 hPEX/PES-buffer). Data are means � SE, n �6–8/group.

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cose transport in skeletal muscle, confirming earlier researchthat implicated the persistent increase in AS160 phosphory-lation after exercise as a possible mechanism for the prolongedimprovement in insulin sensitivity (1, 15). These earlier obser-vations led to the hypothesis in the current study that in vitrocontraction by isolated skeletal muscle in the presence ofserum (a procedure that results in increased insulin-stimulatedglucose transport) (12, 13, 16), but not in vitro contraction inthe absence of serum (which does not enhance insulin-stimu-lated glucose transport) (8, 12, 13, 16), would be characterizedby persistently elevated AS160 phosphorylation. AlthoughAS160 phosphorylation (PAS and 642Thr) was increased im-mediately after in vitro contraction, the elevated AS160 phos-phorylation had reversed at 3 h postcontraction whether or notserum was present during the electrical stimulation. Thus, incontrast to in vivo exercise, increased AS160 phosphorylation(PAS or 642Thr) cannot account for the increased insulinsensitivity found at 3 h after contraction in serum. Finally,insulin-stimulated glucose transport determined for musclesthat had performed both in vivo exercise and in vitro electricalstimulation (PEX � PES) exceeded the values found in mus-cles after either in vivo exercise (PEX) or in vitro electricalstimulation (PES) alone. These results raised the possibilitythat the in vivo exercise and in vitro electrical stimulationprotocols may not have caused elevated insulin-stimulatedglucose transport via an identical mechanism.

To identify the mechanism for improved insulin sensitivityafter contraction in serum, which did not induce a sustainedincrease in AS160 PAS or 642Thr phosphorylation, we alsoevaluated the phosphorylation of TBC1D1, a paralog protein ofAS160. Similar to AS160, TBC1D1 in mouse (35) or rat (2, 14,15) skeletal muscle becomes phosphorylated in response toinsulin, in vitro contraction, or in vivo exercise. In the currentstudy, 237Ser phosphorylation of TBC1D1 was increased im-mediately after in vitro contraction with or without serum.However, at 3 h PES, TBC1D1 237Ser phosphorylation hadreversed to resting control values. TBC1D1 PAS phosphory-lation was also not elevated at 3 h postexercise. Thus theenhanced insulin sensitivity 3 h after in vitro contraction inserum or 3 h after in vivo exercise occurred in the absence ofelevated phosphorylation (PAS or 237Ser) of TBC1D1. Inter-estingly, Treebak et al. (37) reported that prior exercise byhumans that increased insulin sensitivity was accompanied bya persistent increase in phosphorylation of AS160 on 318Ser,341Ser, and 751Ser but not on 642Thr or PAS sites. The sustainedeffect of in vivo exercise or in vitro contraction on AS160phosphosites other than 642Thr has not been evaluated for ratskeletal muscle. It remains possible that in vitro contraction ofrat epitrochlearis muscles in serum and/or in vivo exercise wasaccompanied by a persistent increase in phosphorylation ofAS160 on one or more sites other than 642Thr.

The most important new finding was the additive effect ofin vivo exercise and in vitro electrical stimulation protocols onsubsequent insulin-stimulated glucose transport. The insulin-stimulated increase in glucose transport (insulin; calculatedby subtracting glucose transport in muscles incubated withoutinsulin from glucose transport in muscles incubated with insu-lin) in the PEX � PES group was significantly greater than thatof the PEX or PES group. Furthermore, the effect was essen-tially additive as evidenced by comparing the insulin value inthe PEX � PES group (0.362 �mol·g�1 ·10 min�1) with the

sum of the insulin in the PEX group (0.156 �mol·g�1 ·10min�1) plus the insulin in the PES group (0.179 �mol·g�1·10min�1). In contrast, increasing the amount of exercise (from 1 to2 h) or electrical stimulation (from 5 to 10 tetani) did not increasethe insulin-stimulated glucose transport, providing evidence that 1h exercise or 5 tetani were maximally effective exercise orelectrical stimulation protocols, respectively. In this context, theadditive effect of exercise and in vitro contraction on insulin-stimulated glucose transport was consistent with the idea thatthese protocols used distinct mechanisms to enhance insulinsensitivity.

Several possible scenarios might account for the additiveincrease in insulin-stimulated glucose transport. One possibil-ity would be that exercise and electrical stimulation recruiteddiscrete groups of muscle fibers. However, the tetanic stimu-lation protocol would be expected to recruit all muscle fibers,consistent with the nearly complete depletion of glycogen withelectrical stimulation. Glycogen concentration was not lower inthe PEX � PES group compared with the PES or PEX groups,arguing against exercise and electrical stimulation recruitingentirely separate groups of muscle fibers. A second possiblescenario would be if exercise compared with in vitro contrac-tion altered different molecular processes that regulate GLUT4traffic (e.g., exocytosis vs. endocytosis; recruitment vs. dock-ing, tethering, or insertion of GLUT4, etc.). Although theenhanced insulin-stimulated glucose transport in the rat ep-itrochlearis after in vivo exercise can be accounted for bygreater cell surface GLUT4 content (20), the specific steps ofGLUT4 vesicle trafficking that are influenced by either exer-cise or in vitro contraction are uncertain. Another putativescenario would be if discrete pools of GLUT4 were recruitedby insulin to the cell surface membranes after exercise vs.postcontraction, but it is currently not technically feasible toexperimentally test this idea in skeletal muscle.

We assessed several specific potential mechanisms for theadditive effects of exercise and in vitro contraction on insulinaction in the PEX � PES group. Increased GLUT4 proteinexpression can lead to enhanced insulin-stimulated glucosetransport in isolated skeletal muscle (19), but the additiveeffect on insulin-stimulated glucose transport was not becausethe PEX � PES group had greater GLUT4 abundance than anyof the other groups studied. It has been suggested that glycogendepletion may elevate insulin sensitivity (26), but the additiveeffect of in vivo exercise and in vitro contraction on theinsulin-stimulated glucose transport in the PEX � PES groupcompared with PEX or PES alone was not attributable todifferences in glycogen depletion. Finally, activation of AMPKhas been reported to be associated with a subsequent increasein insulin sensitivity (13), but the PES and PEX � PES groupshad similar levels of AMPK phosphorylation. It is notable thatthe sustained increase in p642ThrAS160 in the 3 h PEX � PESgroup was essentially the same as in the 3 h PEX group (Fig.5D), indicating that electrical stimulation did not reverse thepersistent increase in AS160 phosphorylation found after ex-ercise alone. Thus the prolonged increase in AS160 phosphory-lation in the PEX � PES group may have contributed to aportion of the enhanced insulin-stimulated glucose transport inthese muscles.

Which phosphosite(s) of TBC1D1 is(are) recognized by thePAS antibody, and which kinases are potentially relevant forchanges in PAS-TBC1D1? PAS can recognize TBC1D1 phos-

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phorylated on 596Thr (11), and insulin leads to increasedp596Thr by an Akt-dependent mechanism based on results withskeletal muscle, cells, or recombinant Akt in cell-free assays(11, 28, 30). Insulin was reported to not cause an increase ofp237Ser TBC1D1 in skeletal muscle or cultured cells (11, 28),consistent with the current results. Furthermore, in a cell-freeassay, Akt can phosphorylate TBC1D1 on p596Thr, but not onp237Ser. Accordingly, it seems likely that PAS-TBC1D1 ininsulin-stimulated muscle in this study is reflecting Akt-in-duced phosphorylation of TBC1D1 on 596Thr.

Interpreting the effects of contraction on PAS-TBC1D1 iscomplicated because: 1) in vitro contraction can activate bothAkt and AMPK in rat epitrochlearis muscle (4, 14); 2) in acell-free assay, either Akt or AMPK can increase PAS-TBC1D1 (35); and 3) in a cell-free assay, AMPK can phos-phorylate TBC1D1 on both 596Thr and 237Ser (11). Severallines of evidence support an important role for AMPK in thecontraction-stimulated increase in PAS-TBC1D1: 1) theAMPK inhibitor compound C eliminated contraction-stimu-lated PAS-TBC1D1 without reducing phosphorylation of Aktin isolated rat epitrochlearis muscles (14); 2) wortmannineliminated the contraction effect on phosphorylation of glyco-gen synthase kinase-3 (GSK3), an Akt substrate, without re-ducing the contraction effects on AMPK phosphorylation orPAS-TBC1D1 in isolated rat epitrochlearis muscles (14); and3) genetic disruption of AMPK signaling eliminated the con-traction-induced increases in PAS-TBC1D1, p237Ser, andp596Thr in isolated mouse skeletal muscle (28). However, itremains uncertain which TBC1D1 phosphosite(s) was(were) re-flected by PAS-TBC1D1 in the electrically stimulated muscles.

The prolonged effect of exercise on AS160 phosphorylationis rather remarkable, with elevated AS160 phosphorylationfound at 3 h after in vivo exercise in the current study and aslong as 27 h postexercise in an earlier publication (15). Thisresult would be expected if there were sustained effects ofexercise on the activity of AS160 kinases (increased) and/orAS160 phosphatases (decreased). However, neither of the key AS160kinases (Akt, as evidenced by p308ThrAkt, p473SerAkt, and Aktactivity; or AMPK, as evidenced by p172ThrAMPK) is acti-vated at 3 h postexercise (1, 15), indicating that their prolongedactivation was not responsible for sustained AS160 phosphory-lation. To the best of our knowledge, the persistent effect ofexercise on protein phosphatases has not been reported, butthere is not a prolonged postexercise increase in phosphory-lation of other Akt substrates (GSK3 or TBC1D1) or AMPKsubstrates (acetyl-CoA carboxylase or TBC1D1) (1, 15). Thespecific protein phosphatase(s) that regulate AS160 have notbeen identified, but, because of the similarity in the sequencesof AS160 and TBC1D1, it seems likely that there is overlapbetween those that dephosphorylate AS160 and TBC1D1.Thus the lack of sustained elevation in TBC1D1 phosphory-lation provides indirect evidence against a persistent, postex-ercise inhibition of the relevant protein phosphatase(s) thatregulate AS160 phosphorylation. Our current working hypoth-esis is that prior in vivo exercise causes a long-lasting increasein AS160 phosphorylation by altering the access of relevantkinases or phosphatases to AS160 rather than by persistentlymodifying the activation of these enzymes. Access might beregulated by altering the colocalization of AS160 with therelevant enzyme(s). Alternatively, AS160 might undergo post-translational modifications or bind to other proteins that affect

AS160’s susceptibility to the actions of these kinases or phos-phatases. Regardless of the cellular mechanism, it is notablethat, when muscles were dissected out immediately afterin vivo exercise and incubated in vitro for several hours, thelevel of AS160 phosphorylation remained increased above theresting control values. Thus persistence of the elevated AS160phosphorylation after exercise did not require that muscleremain in vivo. Gulve et al. (17) demonstrated a persistentincrease in insulin-stimulated glucose transport when rat ep-itrochlearis muscles were dissected out immediately after ex-ercise and then incubated for 3 h in the absence of serum. Wehave also found increased AS160 phosphorylation when mus-cles were dissected out of rats immediately postexercise andincubated for 3 h in the absence of serum (unpublished results).These findings indicate that the sustained effects of exercise onboth AS160 phosphorylation and insulin-stimulated glucosetransport can persist for several hours independent of the directinfluence of systemic factors (including continued neural andhumoral inputs). The isolated epitrochlearis muscle includesmultiple different cell types, so it remains possible that cellsother than myocytes may play some regulatory role in thepersistent elevation of AS160 phosphorylation and insulin-stimulated glucose transport.

The current study focused on the potential role of sustainedAS160 phosphorylation in the persistent increase in glucosetransport after exercise or in vitro contractions, and it was notdesigned to identify what factor(s) in serum is(are) essential forserum’s effect on postcontraction insulin sensitivity. Previ-ously, Gao et al. (16) provided strong evidence that the ele-vated insulin-stimulated glucose transport found in isolated ratepitrochlearis muscles 3 h postcontraction in serum depends ona serum protein with a mass �10 kDa. Although they did notidentify the specific protein, they found that neither insulin norIGF-I could recapitulate the ability of serum to enhance glu-cose transport 3 h after in vitro contraction.

AS160 phosphorylation was increased immediately aftereither in vivo exercise or in vitro contractions, but these twotreatments differed in their ability to induce a sustained in-crease in AS160 phosphorylation determined at 3 h posttreat-ment. There were numerous differences between the two treat-ments, including that in vivo exercise occurred in the presenceof many systemic factors that were absent during in vitrocontractions (e.g., intact efferent and afferent innervation andthe vascular delivery of hormones, fuels, and other molecules).Another major difference was the duration of the treatments: 1h for exercise vs. 5–10 min for in vitro contractions. Thetreatments were also fundamentally different with regard to themode and pattern of muscle recruitment: voluntary activationof motoneurons using an undefined recruitment pattern leadingto dynamic, in vivo contractions vs. electrically stimulated,static and tetanic contractions that were performed in vitro. Itis unclear which of these or the many other differences be-tween in vivo exercise and in vitro contractions were importantfor the protocols’ differing effects on sustained AS160 phos-phorylation and which might be relevant for the additiveeffects on insulin-stimulated glucose transport.

In conclusion, the increased insulin-stimulated glucose trans-port observed 3 h after in vivo exercise was accompanied byincreased AS160 phosphorylation (PAS or 642Thr), but notTBC1D1 PAS phosphorylation, supporting the idea that sustainedphosphorylation of AS160 plays a role in the improved postexer-

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cise insulin sensitivity. In contrast, the elevated insulin sensitivityafter in vitro electrical stimulation in serum was not because of aprolonged increase in phosphorylation (PAS or 642Thr) of AS160or TBC1D1 (PAS or 237Ser). Clearly, in vivo exercise and in vitrocontraction differ in their ability to induce a prolonged increase inAS160 phosphorylation. Furthermore, in vivo exercise andin vitro electrical stimulation had additive effects on subsequentinsulin-stimulated glucose transport, suggesting that exercise andin vitro contraction may also differ in the mechanisms that areresponsible for their effects on insulin sensitivity. If this interpre-tation proves to be correct, the identification of their respectivemechanisms will provide an opportunity for their distinct mech-anisms to be targeted either separately or together to opposeskeletal muscle insulin resistance.

GRANTS

This research was supported by National Institutes of Health GrantsAG-010026 and DK-071771 (to G. D. Cartee). This work utilized the Chem-istry Laboratory Core of the Michigan Diabetes Research and Training Centerfunded by DK-020572 from the National Institute of Diabetes and Digestiveand Kidney Diseases.

DISCLOSURES

No conflicts of interest are declared by the authors.

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