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J Appl Physiol 101: 1368-1376, 2006. First published June 29, 2006; doi:10.1152/japplphysiol.00416.2006
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Increased submaximal insulin-stimulated glucose uptake in mouse skeletal muscle after treadmill exercise

Taku Hamada, Edward B. Arias, and Gregory D. Cartee

Muscle Biology Laboratory, Division of Kinesiology, University of Michigan, Ann Arbor, Michigan

Submitted 7 April 2006 ; accepted in final form 21 June 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The primary purpose of this study was to determine the effect of prior exercise on insulin-stimulated glucose uptake with physiological insulin in isolated muscles of mice. Male C57BL/6 mice completed a 60-min treadmill exercise protocol or were sedentary. Paired epitrochlearis, soleus, and extensor digitorum longus (EDL) muscles were incubated with [3H]-2-deoxyglucose without or with insulin (60 µU/ml) to measure glucose uptake. Insulin-stimulated glucose uptake for paired muscles was calculated by subtracting glucose uptake without insulin from glucose uptake with insulin. Muscles from other mice were assessed for glycogen and AMPK Thr172 phosphorylation. Exercised vs. sedentary mice had decreased glycogen in epitrochlearis (48%, P < 0.001), soleus (51%, P < 0.001), and EDL (41%, P < 0.01) and increased AMPK Thr172 phosphorylation (P < 0.05) in epitrochlearis (1.7-fold), soleus (2.0-fold), and EDL (1.4-fold). Insulin-independent glucose uptake was increased 30 min postexercise vs. sedentary in the epitrochlearis (1.2-fold, P < 0.001), soleus (1.4-fold, P < 0.05), and EDL (1.3-fold, P < 0.01). Insulin-stimulated glucose uptake was increased (P < 0.05) ~85 min after exercise in the epitrochlearis (sedentary: 0.266 ± 0.045 µmol·g–1·15 min–1; exercised: 0.414 ± 0.051) and soleus (sedentary: 0.102 ± 0.049; exercised: 0.347 ± 0.098) but not in the EDL. Akt Ser473 and Akt Thr308 phosphorylation for insulin-stimulated muscles did not differ in exercised vs. sedentary. These results demonstrate enhanced submaximal insulin-stimulated glucose uptake in the epitrochlearis and soleus of mice 85 min postexercise and suggest that it will be feasible to probe the mechanism of enhanced postexercise insulin sensitivity by using genetically modified mice.

glucose transport; exertion; insulin sensitivity; Akt; AMP-activated protein kinase

A SINGLE EXERCISE BOUT HAS several effects on skeletal muscle glucose transport, including an increase in insulin-independent glucose transport during exercise. This effect begins to reverse relatively rapidly after completion of exercise, with most of the effect lost within 1–4 h postexercise in isolated rat skeletal muscle (42, 47). A second, more persistent effect, found in rats (5, 7, 8, 10, 24, 35, 42) and humans (36, 43, 46), is improved muscle glucose uptake with a submaximally effective, physiological insulin dose. This effect has often been observed at ~1–4 h postexercise, but under some circumstances the enhanced glucose uptake with submaximal insulin can persist as long as 48 h after completion of the exercise (8, 33). Prior exercise can also enhance glucose transport with supraphysiological, maximally effective insulin concentrations in rat and human skeletal muscle (5, 8, 42). The effects of physiological and supraphysiological insulin do not always coincide, suggesting that they may be regulated by distinct cellular events (5, 8).

The availability of genetically modified animals has opened new opportunities for understanding the mechanisms that underlie physiological processes, including enhanced insulin action after exercise. Because the mouse is the most commonly used mammalian species for transgenic and knockout research models, it is important to understand the effects of exercise on glucose transport in murine skeletal muscle. Results of earlier studies have indicated that a single exercise bout by mice can lead to a subsequent increase in insulin-stimulated glucose transport of isolated skeletal muscle with a supraphysiological insulin concentration (25, 45). However, these studies did not examine a submaximally effective, physiological insulin concentration.

Bonen and colleagues (2, 3) evaluated the effects of acute exercise (60-min treadmill running) on postexercise (~85 min postexercise) glucose uptake by isolated soleus and extensor digitorum longus (EDL) muscles from mice using a range of insulin concentrations, including no insulin and submaximally and maximally effective insulin. Data were statistically compared between exercised and sedentary groups by analysis of variance across the range of insulin concentrations. For both muscles, there was a significant main effect of exercise, with glucose uptake greater for exercised compared with sedentary controls. However, no statistical comparison between exercised and sedentary groups was described for a given submaximal insulin dose. Furthermore, they did not determine the insulin-stimulated increase above basal glucose uptake by subtracting the glucose uptake value for muscles without insulin from the glucose uptake value for paired muscles with insulin. Without accounting for the insulin-independent effect of prior exercise, it is not possible to quantitatively assess the insulin-stimulated increase in glucose uptake after exercise.

Accordingly, the primary aim of the present study was to determine the effect of prior exercise on submaximal insulin-stimulated glucose uptake in mouse skeletal muscles, including the soleus and EDL. We also studied the epitrochlearis, a muscle from mice that is suitable for measurement of in vitro glucose uptake (11, 18) and that has been commonly used for studies of postexercise insulin sensitivity in rats. To account for the exercise effects on both insulin-independent and insulin-dependent glucose uptake, we studied paired muscles from each animal (one incubated without insulin and the other with submaximally effective insulin) so that we could determine the insulin-stimulated glucose uptake.

The cellular processes that initiate and modulate improved insulin sensitivity after exercise are not well understood, but there is evidence in rat skeletal muscle that activation of AMP-activated protein kinase (AMPK) and/or depletion of muscle glycogen during exercise may be involved in triggering or modulating other cellular processes that are subsequently involved in enhanced postexercise insulin action (15, 28, 31). These putative relationships have not been assessed in mice. In addition, measurement of muscle AMPK phosphorylation and glycogen concentration can provide an indirect marker of the skeletal muscles that are recruited during exercise. Therefore, we also determined the effect of exercise on skeletal muscle AMPK phosphorylation and glycogen concentration.

Previous studies have found that acute exercise does not appear to improve proximal insulin signaling steps, including insulin receptor binding or phosphorylation, submaximal insulin-stimulation of the tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), or submaximal insulin-stimulation of IRS1-phosphatidyl inositol 3-kinase (PI3K) (2, 21, 43, 44, 46). Akt [also called protein kinase B (PKB)] is downstream of PI3K and important for insulin-stimulated glucose transport. Prior exercise by mice (45) resulted in increased activation of Akt in skeletal muscle with supraphysiological insulin levels. Akt activity is increased by phosphorylation on two sites (Ser473 and Thr308), and maximal activation of Akt requires phosphorylation on both sites (26, 38, 39). One (41) of three studies (40, 41, 43) that evaluated Akt Ser473 phosphorylation in human skeletal muscle during a euglycemic clamp at physiological insulin found increased Akt activation after acute exercise compared with the sedentary condition. To our knowledge, the effect of prior exercise on Akt Thr308 phosphorylation in muscles stimulated with a physiological insulin concentration has not been reported. Therefore we also determined whether prior exercise would enhance submaximal insulin-stimulated Akt serine or threonine phosphorylation in the soleus, EDL, and epitrochlearis of mice.

We hypothesized that after exercise by mice there would be an increase in submaximal insulin-stimulated glucose uptake accompanied by an increased threonine and/or serine phosphorylation of Akt and that these effects would be found only in muscles that had exercise-induced glycogen depletion and activation of AMPK.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Materials.   Human recombinant insulin was obtained from Eli Lilly and (Indianapolis, IN). 2-Deoxy-[3H]glucose and [14C]mannitol were purchased from Perkin-Elmer (Boston, MA). Reagents and apparatus for SDS-PAGE, nonfat dry milk, and nitrocellulose membranes were obtained from Bio-Rad Laboratories (Hercules, CA). A bicinchoninic acid assay kit for total protein determination and SuperSignal WestDura Extended Duration Substrate for immunodetection were purchased from Pierce Biotechnology (Rockford, IL). Anti-phospho-AMPK (Thr172; catalog no. 2531), anti-total AMPK{alpha} (which recognizes both {alpha}1 and {alpha}2; catalog no. 2532), anti-phospho-Akt (Ser473, catalog no. 9271; and Thr308, catalog no. 9275), and anti-total Akt (catalog no. 9272) were purchased from Cell Signaling Technology (Beverly, MA). Secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG) was purchased from Upstate Biotechnology (Lake Placid, NY). 125I-insulin (catalog no. 9011) and anti-insulin antibody (catalog no. 1013K) were purchased from Linco Research (St. Charles, MO). Other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Animals.   Animal care was approved by the University of Michigan Animal Care and Use Committee. Male C57BL/6 mice, purchased from Harlan (Indianapolis, IN), were housed in plastic cages (4 mice per cage) in an environmentally controlled room maintained at 22–24°C and were maintained on a 12:12-h light-dark cycle (0600:1800) and provided a standard diet (Lab Diet, PMI Nutrition International, Brentwood, MO) and water ad libitum.

Treadmill exercise protocol.   All mice (7–8 wk old) were familiarized with treadmill (Columbus Instruments, Columbus, OH) running for 10 min on 2 consecutive days at 0900 to 1000 (first day at 12–22 m/min; second day at 12–25 m/min). After each familiarization bout, the mice were returned to their cages with free access to food and water. At 0900 to 1000 on the morning after the second familiarization bout, the mice were randomly assigned to either a sedentary control or exercised group. The exercise protocol consisted of 60 min of running (15–25 m/min at 0% slope; Table 1). The approximate percentages of maximum O2 uptake required for untrained mice running at the speeds used in this exercise protocol, based on the prediction equation of Fernando et al. (14), would be expected to be ~80% at 15 m/min, ~86% at 20 m/min, and ~91% at 25 m/min. All of the exercised mice completed the 60-min treadmill protocol. After the exercise, mice were immediately anesthetized by an intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt). Paired epitrochlearis, soleus, and EDL muscles were dissected out. Epitrochlearis, soleus, EDL, and gastrocnemius muscles from some mice were immediately freeze-clamped with aluminum tongs cooled to the temperature of liquid nitrogen and stored at –80°C until processed as described below for AMPK phosphorylation or glycogen concentration. The time elapsed between completion of exercise and the freezing of muscles for AMPK analysis was 8.5 ± 0.1 min for epitrochlearis, 9.5 ± 0.1 min for soleus, 10.4 ± 0.1 min for EDL, and ~11 min for gastrocnemius. The time elapsed between completion of exercise and the freezing of the contralateral muscles for glycogen was 12.5 ± 0.1 min for epitrochlearis, 13.4 ± 0.1 min for soleus, 14.3 ± 0.1 min for EDL, and ~15 min for gastrocnemius. Muscles from other mice were incubated in vitro after dissection and used for measurement of glucose uptake (insulin-independent glucose uptake was determined at ~30 min and ~40 min postexercise, insulin dose response in muscles from sedentary mice, or glucose uptake with or without submaximal insulin determined ~85 min postexercise) or for phosphorylation of Akt (with or without submaximal insulin, determined ~85 min postexercise). Blood was collected from the descending vena cava of some mice immediately after skeletal muscles were dissected out.


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  		Table 1. Treadmill exercise protocol

 
In vitro muscle incubation.   Because the insulin-independent effect of prior exercise can begin to reverse rapidly upon cessation of exercise, the muscles from some mice underwent only a brief (5 min) initial incubation before being transferred to a second flask that included radiolabeled 2-deoxyglucose for assessment of glucose uptake. The time elapsed between completion of exercise and onset of in vitro muscle incubation (required for anesthesia and muscle dissections) was 8.8 ± 0.6 min for epitrochlearis, 9.7 ± 0.6 min for soleus, and 10.6 ± 0.6 min for EDL. Some muscles were initially incubated for 5 min in glass vials containing 1.5 ml of Krebs-Henseleit buffer (KHB) supplemented with 0.1% BSA, 2 mM sodium pyruvate, and 6 mM mannitol in the absence of insulin (buffer 1). For other muscles, the duration of the initial incubation step (in buffer 1) was increased to 15 min to minimize any residual effect of in vivo exposure to insulin on subsequent in vitro glucose uptake. Throughout all of the muscle incubations, vials were placed in a heated (35°C), shaking water bath and continuously gassed from above with 95% O2-5% CO2. After the initial incubation step (5 min or 15 min), the muscles were transferred to a second vial including radiolabeled 2-deoxyglucose for 15 min, as described below.

We aimed to study insulin-stimulated glucose uptake using an insulin dose that elicited less than a half-maximal increase in insulin-stimulated glucose uptake in muscles from sedentary mice because using an insulin dose that induced a greater insulin effect might obscure our ability to discern an exercise-induced increase in submaximal insulin-stimulated glucose uptake. To identify an appropriate submaximal insulin dose, muscles dissected from sedentary mice were initially incubated in glass vials containing 1.5 ml of buffer 1 supplemented with 0, 60, 120, or 20,000 µU/ml insulin for 30 min at 35°C and transferred to second vial for measurement of 2-deoxyglucose uptake as described below.

After we determined that 60 µU/ml induced 33–39% of maximal insulin-stimulated glucose uptake in each muscle, we measured glucose uptake with or without this insulin concentration in isolated muscles from other sedentary and exercised (60-min protocol) mice. Paired muscles were dissected out immediately postexercise or at a similar time in sedentary mice. One muscle from each pair was incubated without insulin, and the contralateral muscle was incubated with insulin (60 µU/ml) in glass vials containing 1.5 ml of buffer 1 for 60 min at 35°C. Muscles were then transferred to a second flask and incubated as described below to measure 2-deoxyglucose uptake, after which the muscles were freeze-clamped (~85 min after completion of the exercise).

To assess the possible role of Akt in postexercise insulin action, paired epitrochlearis, soleus, and EDL muscles were dissected out from other sedentary and exercised mice. One muscle from each pair was incubated without insulin, and the contralateral muscle was incubated with 60 µU/ml insulin during two incubation steps: step 1 was 60-min incubation with buffer 1, and step 2 was 15-min incubation in KHB supplemented with BSA, 1 mM 2-deoxyglucose, 9 mM mannitol, and the same insulin concentration as step 1. After the second incubation step, muscles were freeze-clamped (~85 min after completion of the exercise) and stored at –80°C until analyzed for phosphorylation of Akt Ser473 and Akt Thr308.

Measurement of 2-deoxyglucose uptake.   After the initial incubation period, muscles used for 2-deoxyglucose uptake were transferred to a second glass vial containing KHB supplemented with 0.1% BSA, 1 mM 2-deoxyglucose (including 2-deoxy-[3H]glucose, 6 mCi/mmol), 9 mM mannitol (including [14C]mannitol, 0.053 mCi/mmol), and the same insulin concentration as the preceding step. Muscles were incubated at 35°C for 15 min with a gas phase of 95% O2 and 5% CO2. After 15 min, muscles were rapidly blotted on ice-cold filter paper, trimmed, freeze clamped, and stored at –80°C until processed. Frozen muscles were weighed and then homogenized in 0.3 M perchloric acid. The homogenate was centrifuged (15,000 g for 15 min), and aliquots of supernatant were quantified for 3H and 14C by use of a liquid scintillation counter. Glucose uptake was determined as previously described (4).

Immunoblotting.   Frozen muscles were transferred to prechilled glass tubes and homogenized in ice-cold lysis buffer (0.5 ml) containing 20 mM Tris (pH 7.4), 150 mM NaCl, 1% Igepal, 1 mM activated Na3VO4 (19), 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM beta-glycerophosphate, and 1 µg/ml leupeptin. Homogenates were transferred to microfuge tubes, rotated for ~1 h at 4°C, and then centrifuged at 15,000 g for 15 min. Protein concentrations of the supernatants were determined by the bicinchoninic acid assay. Before immunoblotting, proteins were resolved on a 10% SDS-PAGE gel (75 µg protein for phospho-AMPK Thr172; 60 µg protein for phospho-Akt Ser473 or phospho-Akt Thr308). Resolved proteins were transferred to nitrocellulose at a constant current of 100 mA in electrotransfer buffer (25 mM Tris, 0.2 M glycine, and 20% methanol) overnight at 4°C. Nitrocellulose membranes were incubated in blocking solution [Tris-buffered saline (TBS) with 0.1% Tween 20 (TBST) and 5% nonfat dry milk] for 1 h at room temperature. Blots were washed with TBST and then incubated with the appropriate primary antibody for ~5 h at room temperature (anti-phospho-AMPK Thr172) or overnight at 4°C (anti-phospho-Akt Ser473 or Thr308). Blots were washed with TBST and then incubated with a secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG) for ~1 h at room temperature. Blots were washed of excess antibody with TBST and then incubated with SuperSignal substrate for immunodetection. Immunoreactive protein was quantified by densitometry using AlphaEase FC Software (Alpha Innotech, San Leandro, CA). The mean value for sedentary samples (without insulin for Akt analysis) on each immunoblot, expressed in densitometry units relative to total protein, was adjusted to equal 1.0, and each sample value was expressed relative to the adjusted mean value for the sedentary control.

Total AMPK{alpha} or total Akt protein was also determined using the blots that had been immunoblotted for phosphorylated AMPK (Thr172) or Akt (Ser473 and Thr308), respectively. The blots were first washed with TBS and then incubated with stripping buffer (2% SDS, 62.5 mM Tris pH 6.7, and 100 mM 2-mercaptoethanol) at 50°C for 30 min. Blots were washed with TBS and then immunoblotting was performed with total AMPK{alpha} or total Akt antibody using the protocol described above.

Muscle glycogen concentration.   Muscles were weighed and then homogenized in ice-cold 0.3 M perchloric acid. An aliquot of the homogenate was stored at –80°C for later determination of glycogen concentration by the amyloglucosidase method (32) and expressed as micromoles per gram of muscle wet weight.

Serum insulin concentration.   Blood collected from mice was allowed to clot for 30 min and centrifuged (2,000 g for 15 min), and the resultant serum was collected and stored at –20°C until analyzed for insulin concentration by radioimmunoassay essentially as described by Linco Research, Limit of sensitivity for the assay is 1 µU/ml. Interassay and intra-assay variabilities are 11.5 and 3.2%, respectively, at 28.5 µU/ml.

Statistical analysis.   Statistical analyses were done using Sigma Stat version 2.0 (San Rafael, CA). Data are expressed as means ± SE. One-way ANOVA was used to determine significant differences in glucose uptake for the insulin dose-response experiment, and a Tukey's post hoc test was to identify the source of significant variance. For all other comparisons, Student's unpaired t-test was used to analyze differences between two groups (sedentary vs. exercised). A P value <0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Muscle glycogen concentration.   Glycogen concentration was significantly reduced for exercised vs. sedentary mice in the epitrochlearis (48% decrease, P < 0.001), soleus (51% decrease, P < 0.001), and EDL (41% decrease, P < 0.01; Fig. 1). Gastrocnemius glycogen concentration was also decreased (47% decrease, P < 0.005) for exercised (7.7 ± 0.4 µmol/g, n = 7) compared with sedentary (14.4 ± 1.7 µmol/g, n = 7) mice.


Figure 1
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  		Fig. 1. Glycogen concentration for epitrochlearis, soleus, and extensor digitorum longus (EDL) muscles from sedentary (SED) and ~12 min postexercise (12min PEX) mice. Values are means ± SE for 8 muscles per group. 12min PEX are significantly different from SED, *P < 0.001 and **P < 0.01.

 
AMPK Thr172 phosphorylation.   AMPK Thr172 phosphorylation was significantly increased for exercised compared with sedentary values in the epitrochlearis (1.7-fold, P < 0.05), soleus (2.0-fold, P < 0.05), and EDL (1.4-fold, P < 0.05; Fig. 2). There was no significant difference between postexercise and sedentary groups for total AMPK{alpha} in epitrochlearis, soleus, or EDL muscles (data not shown). AMPK Thr172 phosphorylation in the gastrocnemius was also significantly (4.8-fold, P < 0.05) greater for exercised (4.8 ± 1.2 arbitrary units, n = 3) compared with sedentary (1.0 ± 0.5 arbitrary units, n = 3) mice.


Figure 2
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  		Fig. 2. Immunoblot analysis of AMPK Thr172 phosphorylation for epitrochlearis, soleus, and EDL muscles from SED and ~10 min postexercise (10min PEX) mice. Values are means ± SE for 7–8 muscles per group. AMPK phosphorylation is expressed relative to the average of SED values (1.0) on each blot. Representative immunoblots are shown. 10min PEX are significantly different from SED, *P < 0.05.

 
Serum insulin concentration.   Serum insulin concentration tended to be lower for exercised (17.3 ± 4.4 µU/ml, n = 3) compared with sedentary (31.2 ± 8.2 µU/ml, n = 3) mice, but the difference did not achieve statistical significance (P = 0.210).

Insulin-independent glucose uptake 30 and 40 min postexercise.   Muscles from exercised mice that underwent a 5-min initial incubation step began their 15-min incubation with radiolabeled 2-deoxyglucose ~15 min after completion of exercise, so the results are an indication of glucose uptake from ~15 to 30 min postexercise (referred to as 30 min postexercise). Insulin-independent glucose uptake at 30 min postexercise compared with sedentary controls was significantly increased in the epitrochlearis (20% increase, P < 0.001), soleus (40% increase, P < 0.05), and EDL (30% increase, P < 0.01; Fig. 3). To minimize any possible residual effect of in vivo insulin exposure on subsequent in vitro glucose uptake, muscles from other sedentary and exercised mice (n = 3 per group) underwent a longer (15 min) initial incubation period before incubation with radiolabeled 2-deoxyglucose (referred to as 40 min postexercise). Despite the threefold longer preincubation period, the insulin-independent glucose uptake remained elevated above sedentary control values in the 40 min postexercise group (epitrochlearis, sedentary = 0.421 ± 0.050 µmol·g–1·15 min–1 vs. 40 min postexercise = 0.612 ± 0.04, P < 0.05; soleus, sedentary = 0.463 ± 0.03 vs. 40 min postexercise = 0.584 ± 0.03, P < 0.05; EDL, sedentary = 0.391 ± 0.03 vs. 40 min postexercise = 0.549 ± 0.04, P < 0.05).


Figure 3
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  		Fig. 3. Effect of treadmill exercise on insulin-independent glucose uptake in isolated epitrochlearis, soleus, and EDL muscles ~30 min postexercise (30min PEX) compared with sedentary SED controls. Data are mean ± SE for 4 muscles per group. 30min PEX are significantly different from SED, *P < 0.001, **P < 0.05, and ***P < 0.01.

 
Insulin dose-response for muscles from sedentary mice.   We characterized the insulin dose response for glucose uptake in isolated muscles from sedentary mice (Fig. 4). Glucose uptake with 120 µU/ml compared with 20,000 µU/ml insulin was not significantly different for any of the muscles (epitrochlearis, soleus, or EDL). This plateau is indicative of attaining a maximal insulin effect. Glucose uptake with 60 µU/ml of insulin was greater than basal in the epitrochlearis (P < 0.05), soleus (P < 0.05), and EDL (P = 0.078). Thus we used 60 µU/ml as a submaximally effective concentration of insulin in the subsequent experiment to examine the postexercise insulin-stimulated glucose uptake.


Figure 4
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  		Fig. 4. Insulin dose response (0, 60, 120, and 20,000 µU/ml) for glucose uptake in isolated epitrochlearis (A), soleus (B), and EDL (C) from sedentary mice. Data are means ± SE for 4 muscles per group. *P < 0.05, **P < 0.001, ***P < 0.01 vs. 0 µU/ml. §P < 0.05, {dagger}P < 0.001 vs. 60 µU/ml.

 
Postexercise insulin-stimulated glucose uptake.   Muscles from exercised mice were incubated with radiolabeled 2-deoxyglucose ~70–85 min after completion of the exercise (this group is referred to as 85 min postexercise). Glucose uptake without insulin (Fig. 5, A and B) in the epitrochlearis or soleus muscles was not significantly different in the 85 min postexercise group compared with sedentary controls. However, there was a persistent effect of prior exercise on glucose uptake in the EDL without insulin at this time (85 min postexercise was 45% greater than sedentary, P < 0.05, Fig. 5C). Glucose uptake in muscles incubated with insulin was significantly higher for the 85 min postexercise compared with sedentary epitrochlearis, soleus, and EDL muscles (P < 0.05, Fig. 5). Calculated insulin-stimulated glucose uptake ({Delta} insulin) was significantly higher in the 85 min postexercise vs. sedentary group in the epitrochlearis (56% increase, P < 0.05, Fig. 5A) and the soleus (240% increase, P < 0.05, Fig. 5B), demonstrating increased postexercise submaximal insulin-stimulated glucose uptake. However, there was no significant difference for insulin-stimulated glucose uptake in the EDL of 85 min postexercise vs. sedentary mice (P = 0.434, Fig. 5C).


Figure 5
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  		Fig. 5. Glucose uptake (without or with 60 µU/ml insulin) in isolated epitrochlearis (A), soleus (B), and EDL (C) muscles from SED and ~85 min postexercise (85min PEX) mice. Paired muscles were studied: 1 muscle was incubated without insulin, and the contralateral muscle was incubated with insulin (60 µU/ml). {Delta} Insulin was calculated for paired muscles by subtracting glucose uptake for the muscle without insulin from glucose uptake for the contralateral muscle with insulin. Data are means ± SE for 6–15 muscles per group. 85min PEX are significantly different from SED, *P < 0.05.

 
Akt phosphorylation.   Akt Ser473 and Thr308 phosphorylation was not significantly different between 85 min postexercise and sedentary groups for epitrochlearis, soleus, or EDL muscles incubated with or without insulin (Fig. 6). Insulin-stimulated phosphorylation of Akt (calculated by subtracting basal values from values for paired muscles incubated with insulin) was also not significantly different for 85 min postexercise vs. sedentary groups (data not shown). There was also no significant difference between 85 min postexercise and sedentary groups for total Akt in epitrochlearis, soleus, or EDL muscles incubated without or with insulin (data not shown).


Figure 6
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  		Fig. 6. Akt Ser473 (A–C) and Thr308 (D–F) phosphorylation in isolated epitrochlearis (A and D), soleus (B and E), and EDL (C and F) muscles. Paired muscles from SED or 85min PEX were studied. One muscle from each mouse was incubated without insulin, and the contralateral muscle was incubated with insulin (60 µU/ml). Values for Akt Ser473 and Akt Thr308 phosphorylation are expressed relative to the average of SED values (1.0) on each blot. Representative immunoblots are shown. Data are means ± SE for 7–8 muscles per group.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The most important results in this study were that, in mice, 1) treadmill exercise resulted in significantly decreased glycogen concentration and increased AMPK Thr172 phosphorylation in epitrochlearis, soleus, and EDL muscles; 2) insulin-independent glucose uptake was significantly increased ~30 min after exercise in these muscles; 3) submaximal insulin-stimulated glucose uptake was increased ~85 min after exercise in epitrochlearis and soleus, but not EDL muscles; and 4) the increased insulin-stimulated glucose uptake was not accompanied by an exercise-induced enhancement in the phosphorylation of Akt (Ser473 and Thr308) compared with sedentary controls.

Our primary goal was to determine whether acute treadmill exercise by mice would lead to a subsequent elevation in insulin-stimulated glucose uptake with a submaximally effective insulin dose. The results clearly demonstrated this effect in isolated soleus and epitrochlearis muscles based on the calculated {Delta} insulin using paired muscles. Furthermore, at ~85 min postexercise, there was no residual effect of acute exercise on glucose uptake without insulin (insulin-independent glucose uptake) in the epitrochlearis or soleus muscles, so their increased glucose uptake with insulin can be attributed to enhanced insulin action. In this context, it is notable that the EDL, which did not have a significant increase in calculated insulin-stimulated glucose uptake at 85 min postexercise, was the only muscle studied that still had a significant exercise effect on insulin-independent glucose uptake at this time. Indeed, the insulin-independent glucose uptake at 85 min postexercise had not declined from the value found 30 min postexercise. This finding is reminiscent of results for the rat epitrochlearis muscle after exercise, in which a significant increase in the insulin-stimulated glucose uptake with submaximal insulin was evident only after most of the acute stimulatory effect of exercise on insulin-independent glucose uptake had worn off (42). It is possible that insulin-stimulated glucose uptake by the EDL becomes elevated at some later time, when the insulin-independent effect of exercise on glucose uptake has disappeared.

Only two previous studies have assessed the effect of acute exercise on subsequent glucose uptake by isolated mouse skeletal muscles (soleus and EDL) with a submaximally effective insulin concentration (2, 3). Neither of these studies calculated the insulin-stimulated glucose uptake using paired muscles or statistically compared the exercised to the sedentary group at a given submaximal insulin concentration. However, in both studies, the glucose uptake value for the soleus with submaximal insulin appeared to be greater postexercise compared with sedentary controls. In contrast, the glucose uptake with submaximal insulin in the EDL did not appear to be increased in one of these previous studies (2), and it was unclear whether there was an exercise effect in the EDL with submaximal insulin in the other study (3). Thus our results are consistent with and extend these earlier findings by providing unambiguous evidence for increased insulin-stimulated glucose uptake after exercise in mouse soleus and epitrochlearis muscles with a physiological insulin concentration.

The treadmill exercise protocol used in this study resulted in a significant reduction in glycogen concentration, increase in phosphorylation of AMPK, and increase in insulin-independent glucose uptake for epitrochlearis, soleus, and EDL muscles. These results provide evidence that each of the muscles was effectively recruited to contract by this exercise protocol. It has previously been suggested that each of these events that occur during exercise may play a role in the increase in insulin-stimulated glucose uptake after exercise.

Many studies have demonstrated that glycogen-depleting exercise is characterized by enhanced insulin action postexercise (5, 13, 17, 34, 35, 42). Furthermore, Nolte et al. (31) reported that injection of rats with a pharmacological dose of epinephrine resulted in muscle glycogen depletion and a subsequent increase in insulin-stimulated glucose uptake by isolated muscles. However, Fisher et al. (15) found that incubation of isolated rat skeletal muscle with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside and rat serum in vitro led to a subsequent increase in insulin-stimulated glucose uptake without any glycogen depletion, and we recently found that several in situ contraction protocols that induced a substantial decrease in glycogen concentration did not lead to a subsequent increase in insulin-stimulated glucose transport by isolated rat skeletal muscle (28). Our present results in mice, together with these previous findings in rats, are consistent with the idea that glycogen depletion may play a role in enhanced insulin-stimulated glucose uptake, but it is not sufficient for this effect in either species.

Vigorous exercise that leads to activation of AMPK is also characterized by subsequent enhancement of insulin action. Fisher et al. (15) found that incubation of rat skeletal muscles with 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (a compound taken up by muscle and converted to ZMP, which, in turn, activates AMPK) induced a subsequent (3.5 h later) increase in insulin-stimulated glucose uptake. This observation suggested that the transient increase in AMPK phosphorylation found during and shortly after exercise may play a role in triggering other cellular events that subsequently result in the increase in insulin-stimulated glucose uptake found after exercise. Our present results with the mouse EDL after treadmill exercise and previous results with rat epitrochlearis after in situ contractions (28) demonstrate that activation of muscle AMPK, even together with muscle glycogen depletion, may be necessary but is not sufficient for a subsequent increase in insulin sensitivity.

Several lines of evidence indicate that the exercise-induced increase in glucose uptake observed 30 min postexercise in muscles incubated without insulin was attributable to increased insulin-independent glucose uptake rather than being caused by a residual effect of in vivo exposure to insulin. Consistent with results from previous studies (16, 22), the serum insulin concentration tended to be lower for exercised compared with sedentary mice, and this result would not favor greater in vitro glucose uptake in the postexercise group by an insulin-dependent mechanism. Furthermore, the low, residual insulin concentration in the extracellular space of isolated muscles would be diluted several hundredfold when the muscles were placed in 1.5 ml of incubation solution, which would be expected to greatly reduce any persistent effect of in vivo insulin. Finally, a threefold increase in the duration of the first incubation step (from 5 to 15 min) before exposure to radiolabeled 2-deoxyglucose would be expected to eliminate, or at least substantially attenuate, any residual effect of in vivo insulin. However, the exercise effect on glucose uptake rate persisted in muscles that were incubated without insulin for 15 min before initiating measurement of glucose uptake, providing evidence that this exercise effect was not insulin dependent.

The exercise-induced increase in insulin-independent glucose uptake is secondary to increased GLUT4 translocation from the cell interior to the cell surface membranes (9), and the reversal of the elevated glucose uptake is the result of the internalization of the GLUT4. Holloszy (24) hypothesized that, after exercise, this GLUT4 moves to an intracellular compartment that is highly susceptible to subsequent recruitment by insulin. This idea is consistent with our present results. In muscles in which insulin-independent glucose uptake, and presumably insulin-independent GLUT4 translocation to the cell surface, had completely reversed at 85 min postexercise (i.e., soleus and epitrochlearis), we found postexercise insulin-stimulated glucose uptake was increased, whereas in the EDL, in which postexercise insulin-independent glucose uptake had not reversed at this time, insulin-stimulated glucose uptake was not enhanced.

The cellular mechanism leading to the postexercise increase in insulin-stimulated glucose uptake in skeletal muscle has eluded identification. Previous studies have not assessed insulin-stimulated muscles for the effect of prior exercise on Akt Thr308 phosphorylation, which is required for full activation of Akt. However, neither Akt Ser473 nor Akt Thr308 phosphorylation of insulin-stimulated muscles were different in 85 min postexercised and sedentary groups. Skeletal muscle expresses abundant levels of Akt 1 and Akt 2, and the latter appears to be more important for insulin-stimulated glucose uptake (1, 23, 29). It remains possible that exercise has an Akt isoform-specific effect, or it may act on distal signaling steps, such as AS160, an Akt substrate that has been linked to insulin-stimulated GLUT4 translocation (27, 37). Alternatively, exercise may amplify another signaling step, such as atypical PKC, that is linked to GLUT4 translocation (12) or make GLUT4 vesicles more susceptible to an unchanged insulin signal (24).

In isolated (6, 7, 20) and perfused (35) skeletal muscles from rats, the relative increase in insulin-stimulated glucose uptake can be ~50 to 300%, similar to the results for isolated mouse epitrochlearis (56%) and soleus (240%) muscles in the present study. The effect of acute exercise on in vivo insulin sensitivity of mice has apparently not been reported. However, a single exercise bout by rats can lead to a subsequent increase in insulin-stimulated glucose disposal in vivo (30). Prior exercise by humans also results in increased glucose uptake of insulin-stimulated skeletal muscle in vivo (36, 43). As with isolated mouse or rat skeletal muscle, the magnitude of the exercise-induced increase in glucose uptake in vivo for rats or humans during a hyperinsulinemic clamp after acute exercise can be substantial (≥100%). It would be important to measure the effect of acute exercise on in vivo glucose disposal in mice. It would also be valuable to determine the metabolic fate of the increased glucose uptake after exercise in mice (i.e., glycogen synthesis, glucose oxidation, and lactate formation).

In summary, an acute treadmill exercise protocol that led to decreased glycogen concentration, increased AMPK phosphorylation, and increased insulin-independent glucose uptake in mouse epitrochlearis, soleus, and EDL muscles also induced a subsequent increase in insulin-stimulated glucose uptake with submaximal insulin in epitrochlearis and soleus but not EDL muscles. Increased insulin-stimulated glucose uptake could not be attributed to an increase in submaximal insulin-stimulated phosphorylation of Akt Ser473 or Thr308 in skeletal muscle from exercised compared with sedentary control animals. The present results provide the first clear demonstration that acute exercise can improve submaximal insulin-stimulated glucose uptake in mouse skeletal muscle. These findings suggest that, in future experiments, it will be feasible to use genetically modified mice to assess the specific mechanisms that underlie enhanced postexercise insulin sensitivity in skeletal muscle.


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This research was supported by National Institutes of Health AG010026 [GenBank] (to G. D. Cartee) and the Michigan Diabetes Research and Training Center (National Institutes of Health DK20572). T. Hamada was a recipient of a fellowship of the Nakatomi Foundation.


   ACKNOWLEDGMENTS
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We thank Robyn E. Odzark for assistance in the glycogen assay.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. D. Cartee, Univ. of Michigan, Division of Kinesiology, Rm. 3040E, 401 Washtenaw Ave., Ann Arbor, MI 48109-2214 (e-mail: gcartee{at}umich.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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