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Exercise does not alter subcellular localization, but increases phosphorylation of insulin-signaling proteins in human skeletal muscle

¹Center for Physical Activity and Nutrition, School of Exercise and Nutrition Sciences, Deakin University and ²Department of Physiology, The University of Melbourne, Melbourne, Australia

Submitted 12 July 2005 ; accepted in final form 20 September 2005

	ABSTRACT

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The subcellular localization of insulin signaling proteins is altered by various stimuli such as insulin, insulin-like growth factor I, and oxidative stress and is thought to be an important mechanism that can influence intracellular signal transduction and cellular function. This study examined the possibility that exercise may also alter the subcellular localization of insulin signaling proteins in human skeletal muscle. Nine untrained males performed 60 min of cycling exercise (67% peak pulmonary O₂ uptake). Muscle biopsies were sampled at rest, immediately after exercise, and 3 h postexercise. Muscle was fractionated by centrifugation into the following crude fractions: cytosolic, nuclear, and a high-speed pellet containing membrane and cytoskeletal components. Fractions were analyzed for protein content of insulin receptor, insulin receptor substrate (IRS)-1 and -2, p85 subunit of phosphatidylinositol 3-kinase, Akt, and glycogen synthase kinase-3 (GSK-3). There was no significant change in the protein content of the insulin signaling proteins in any of the crude fractions after exercise or 3 h postexercise. Exercise had no significant effect on the phosphorylation of IRS-1 Tyr⁶¹² in any of the fractions. In contrast, exercise increased (P < 0.05) the phosphorylation of Akt Ser⁴⁷³ and GSK-3/ Ser^9/21 in the cytosolic fraction only. In conclusion, exercise can increase phosphorylation of downstream insulin signaling proteins specifically in the cytosolic fraction but does not result in changes in the subcellular localization of insulin signaling proteins in human skeletal muscle. Change in the subcellular protein localization is therefore an unlikely mechanism to influence signal transduction pathways and cellular function in skeletal muscle after exercise.

insulin receptor substrate; intracellular signaling pathway

Although it is well established that IRS proteins are regulated via phosphorylation on multiple tyrosine and/or serine/threonine residues (20), a growing body of evidence indicates that the subcellular localization of IRS proteins also plays an important role in mediating insulin signaling (21). IRS protein localization within the cell may influence different cellular functions and in doing so provide a level of biological specificity. Although the subcellular localization of IRS proteins is not absolutely defined, these proteins have been identified with the plasma membrane, in the nucleus (18, 26), and in a high-speed pellet (HSP) fraction, which suggests an association with the cytoskeleton (2, 8). Furthermore, in response to various stimuli, such as insulin (1–3, 7), insulin-like growth factor I (IGF-I; see Ref. 18) and oxidative stress (19), IRS proteins can also undergo changes in subcellular localization.

Exercise or muscle contraction influences many cellular functions that are similar to those also regulated by insulin, such as muscle glucose uptake, glycogen synthesis, and amino acid uptake. The effect of exercise on these cellular functions is generally thought to be mediated by insulin-independent signal transduction pathways (15). The majority of previous studies indicate that acute exercise or muscle contraction, per se, does not influence the phosphorylation and activity of the insulin receptor, IRS-1, IRS-2, or other proximal proteins in the classical insulin signaling pathway in skeletal muscle (5, 6, 17, 23, 28). However, it should be noted that insulin signaling proteins were measured only in skeletal muscle whole cell lysates. These previous studies do not rule out the possibility that, at the subcellular level, exercise may induce novel relocalization or spatial rearrangement of IRS proteins or other proteins in the insulin signaling pathway similar to insulin, (1–3, 7), IGF-I (18), and oxidative stress (19). It is possible that exercise-mediated changes in the subcellular localization of insulin signaling proteins may alter protein-protein interactions and subsequently influence IRS-mediated signal transduction pathways and cellular function in skeletal muscle (25). The aim of this study, therefore, was to determine whether an acute bout of exercise can result in changes in the subcellular localization of insulin signaling proteins in human skeletal muscle.

	MATERIALS AND METHODS

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Subjects. Nine healthy, active, but untrained males (21 ± 2 yr, 82.5 ± 11.5 kg, 181 ± 9 cm; means ± SE) volunteered to serve as subjects for the experiment. Subjects demonstrated no major contraindications to exercise, as determined by a detailed medical questionnaire. Before giving written consent to participate, subjects were provided with both written and verbal information regarding the purpose, nature, and potential risks associated with the research study. The study protocol was approved by the Deakin University Human Research Ethics Committee.

Preexperimental protocol. All subjects underwent an incremental exercise test to volitional fatigue on an electromagnetically braked cycle ergometer (Lode, Groningen, The Netherlands) to determine their peak pulmonary oxygen uptake (O_{2 peak}). O_{2 peak} was determined as the average oxygen uptake rate over the last minute of exercise before exhaustion (O_{2 peak} = 47.1 ± 5.3 ml·kg^–1·min^–1). Subjects were instructed to refrain from physical activity, apart from that required for daily living, for 3 days before the trial. A standard diet (14 MJ, 80% of total energy as carbohydrate) was provided for the subjects to consume the day before the exercise trial, and subjects also abstained from alcohol, tobacco, and caffeine. Subjects presented to the laboratory on the morning of the trial, having fasted for 10–12 h overnight.

Experimental protocol. All subjects undertook a single bout of cycling exercise for 60 min at 67 ± 5% O_{2 peak}. After the exercise bout, subjects remained in the laboratory for 3 h and rested on a bed. Before the commencement of exercise, a catheter was inserted in a forearm vein for blood sampling. Blood was sampled at rest; at 10, 30, and 60 min of exercise; and then every 30 min during the 3-h recovery period. Muscle samples were obtained from the vastus lateralis using the percutaneous needle biopsy technique modified for suction (4) at rest (0 min), immediately after exercise (60 min), and after 3 h of recovery (240 min). Muscle samples were removed and immediately frozen in liquid nitrogen.

Blood analysis. Blood samples were transferred to lithium heparin tubes to prevent clotting and were then spun. The resulting supernatant was removed and stored at –20°C for later analysis. Plasma samples were analyzed for glucose and lactate using an automated method (EML 105; Radiometer, Copenhagen, Denmark). Plasma insulin concentrations were measured by RIA (Linco Research, St. Charles, MO).

Subcellular fractionation protocol. Muscle samples were homogenized in a 1:4 wt/vol ice-cold homogenization buffer (50 mM Tris, pH 7.8, 10 mM EDTA, pH 8.0, 100 mM NaF, 2 mM Na₃VO₄, 1 mM sodium pyrophosphate, 250 µM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) with a hand-held homogenizer (Polytron-Aggregate; Kinematica, Switzerland). Subcellular fractions were isolated by a method of differential centrifugation as previously described by Clark et al. (3), with the exception that SDS was used as a detergent to liberate proteins in the HSP rather than Triton X. IRS proteins have generally been considered to localize within cytosol, membrane, and cytoskeleton fractions (21), but recently, IRS proteins were detected in the nucleus, where they are thought to play an important role in the regulation of gene expression and cell growth and size (18, 26). Given that we have recently demonstrated in a separate study that exercise can induce translocation of proteins, in particular AMP-activated protein kinase, to a nuclear fraction in human skeletal muscle (11), a nuclear fraction was also isolated in the current study using the method previously described by McGee et al. (11). In summary, whole cell homogenates were initially spun for 10 min at 1,500 g (Beckman Ultracentrifuge). The resulting pellet was resuspended in 350 µl of homogenization buffer supplemented with detergents (10% glycerol, 1% Triton X, 50 mM MgCl) to liberate nuclear components from contractile and fibrous tissue and kept on ice for 30 min before being spun for 5 min at 3,500 g (Biofuge, Heraeus, Germany). The resulting supernatant was removed, snap-frozen in liquid nitrogen, and stored at –80°C as the nuclear fraction. Supernatant from the 1,500-g step was transferred to new tubes and spun for 60 min at 200,000 g (Beckman Ultracentrifuge) to obtain a HSP. Supernatant was removed, snap-frozen, and stored at –80°C as the cytosolic fraction. The resulting HSP, which contained both cytoskeletal and membrane components, was resuspended in homogenizing buffer supplemented with detergent (1% SDS) to liberate proteins from the cytoskeleton and kept on ice for 60 min before being snap-frozen and stored at –80°C. Characterization of the crude fractions was verified by immunoblotting, with the nuclear, cytosol, and HSP fractions found to be enriched for nuclear histone 1 (Santa Cruz Biotechnology), glyceraldehyde-3-phosphate dehydrogenase (Covance Research Products), and actin (Sigma) and GLUT4 (Santa Cruz Biotechnology), respectively (data not shown).

Immunoblotting. Total protein concentration for each fraction was determined (BCA Assay Kit; Pierce, Rockford, IL) using BSA as the standard. Proteins were separated and identified using SDS-PAGE. Samples (50–100 µg) were loaded onto 8% acrylamide SDS-PAGE gels with a molecular-weight marker (Precision Plus Protein Standards; Bio-Rad), before undergoing electrophoresis for 60 min at 180 volts. After electrophoresis, proteins were transferred to a nitrocellulose membrane by using a wet transfer protocol for 60–90 min at 100 volts. Membranes were blocked for 1–2 h [5% BSA, 1x Tris-buffered saline (TBS), 0.01% Tween, and 0.01% NaN₃] before being exposed overnight at 4°C to the following primary antibodies (1:1,000) in buffer (5% BSA in TBS, 0.5% Tween, and 0.01% NaN₃): polyclonal anti-insulin receptor -subunit, anti-IRS-1 and -2, anti-p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase), anti-Akt/protein kinase B (PKB) and anti-glycogen synthase kinase-3 (GSK-3; Upstate Biotechnology), polyclonal phosphospecific antibodies for anti-IRS-1 phospho-Tyr⁶¹² (Biosource), anti-Akt phospho-Ser⁴⁷³, and anti-GSK-3/ phospho-Ser^21/9 (Cell Signaling). After incubation with the primary antibodies, membranes were washed in 1x TBS-Tween (TBST) and exposed to an appropriate horseradish peroxidase-labeled secondary antibody (1:2,000 in skim milk powder and 1x TBST) for 60 min. Membranes were exposed to chemiluminescence substrates (Perkin-Elmer Life Sciences, Boston, MA) and exposed on a Kodak Image Station 440CF (NEN Life Science Products). Specific bands of interest were identified and quantified using Kodak 1D Image Analysis Software (Eastman Kodak, Rochester, NY).

Calculation and statistical analysis. Standards were included in all immunoblotting, and interassay variation was accounted for by normalizing data to control samples. All data are expressed as means ± SE. Statistical analysis was undertaken using a one-way ANOVA. When ANOVA revealed significant differences, further analysis was performed using the Student-Newman-Keuls post hoc test. The level of significance was set at P < 0.05.

	RESULTS

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Exercise. Subjects exercised at a mean power output of 162 ± 18 watts for 60 min, requiring an oxygen uptake of 31.5 ± 4.5 ml·kg^–1·min^–1 or 67 ± 5% O_{2 peak}. The respiratory exchange ratio and ventilation averaged 0.96 ± 0.02 and 64.1 ± 2.7 l/min, respectively, during exercise. Plasma glucose levels did not change significantly from resting levels (5.6 ± 0.7 mmol/l) for the duration of exercise and the 3-h recovery period. Plasma insulin levels significantly decreased from resting levels of 76.7 ± 11.9 to 42.8 ± 5.8 pmol/l after 60 min of exercise, but, within 30 min after completion of exercise, plasma levels had returned to resting levels. Plasma lactate significantly increased from rest (1.3 ± 0.3 mmol/l) to an average during exercise of 5.6 ± 0.7 mmol/l. After exercise, plasma lactate levels rapidly returned to resting levels within the first 30 min of the recovery period.

Subcellular localization. Exercise resulted in no significant changes in the protein content of the insulin signaling proteins (insulin receptor, IRS-1 and -2, p85 subunit of PI 3-kinase, Akt, and GSK-3) in any of the crude skeletal muscle fractions (cytosol, HSP, and nuclear) either immediately after exercise or 3 h postexercise compared with rest (Figs. 1–3). Insulin receptor could not be detected by immunoblotting in the nuclear fraction (Figs. 1 and 2).

View larger version (78K): [in this window] [in a new window]   Fig. 1. A: protein content of insulin receptor (IR), insulin receptor substrate (IRS)-1, IRS-2, p85 subunit of PI 3-kinase (p85), protein kinase B (Akt), and glycogen synthase kinase-3 (GSK-3) in the cytosol, high-speed pellet (HSP), and nuclear fractions of human skeletal muscle before (0 min), immediately after (60 min), and 3 h after (240 min) cycling exercise. B: phosphorylation (p) of IRS-1 Tyr612, Akt Ser473, and GSK-3 Ser9/21 in the cytosol, HSP, and nuclear fractions of human skeletal muscle before (0 min), immediately after (60 min), and 3 h after (240 min) cycling exercise. Insulin receptor was not detected in the nuclear fraction. Blots are representative of group mean data.

View larger version (12K): [in this window] [in a new window]   Fig. 3. Protein content of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase; p85), Akt, and GSK-3 in the cytosol, HSP, and nuclear fractions of human skeletal muscle before (0 min), immediately after (60 min), and 3 h after (240 min) cycling exercise. Results are not directly comparable between the different fractions. Values are means ± SE (n = 9). Black bar denotes exercise.

View larger version (11K): [in this window] [in a new window]   Fig. 2. Protein content of IR, IRS-1, and IRS-2 in the cytosol, HSP, and nuclear fractions of human skeletal muscle before (0 min), immediately after (60 min), and 3 h after (240 min) cycling exercise. IR receptor was not detected in the nuclear fraction. Results are not directly comparable between the different fractions. Values are means ± SE (n = 9). Black bar denotes exercise.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Phosphorylation of Akt Ser473 and GSK-3 Ser9/21 in the cytosol, HSP, and nuclear fractions of human skeletal muscle before (0 min), immediately after (60 min), and 3 h after (240 min) cycling exercise. Akt Ser473 was not detected in the HSP fraction. Results are not directly comparable between the different fractions. Values are means ± SE (n = 9). Black bar denotes exercise. *Denotes significantly different from 0 min.

	DISCUSSION

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With a technique of differential centrifugation, human skeletal muscle sampled at rest, immediately after an acute bout of moderate to intense cycling exercise, and 3 h postexercise was separated into the crude fractions (cytosol, nuclear, and HSP containing membrane and cytoskeletal components). The results demonstrate that exercise does not alter the content of IRS-1 and -2 or other proteins in the insulin signaling pathway, including insulin receptor, p85 subunit of PI 3-kinase, Akt, and GSK-3 in skeletal muscle subcellular fractions, either immediately or 3 h after exercise. Thus a change in the subcellular localization of insulin signaling proteins is an unlikely mechanism to influence downstream signal transduction pathways and subsequent cellular function in skeletal muscle.

Although the content of insulin signaling proteins did not change in the skeletal muscle fractions after exercise, it is possible that protein activation (phosphorylation and protein-protein interactions) may have been differentially altered by exercise in the subcellular fractions. Previous studies have demonstrated that activation of insulin receptor, IRS proteins, and associated PI 3-kinase activity are not influenced by exercise or muscle contraction, per se (5, 6, 17, 23, 28). However, in these studies, measurements were made in whole cell lysates where localized changes in the activation of individual proteins may be diluted and too small to be detected. By use of the percutaneous needle biopsy technique in humans, the size of a skeletal muscle biopsy sample is limited, and as such in the present study it was not possible to fully assess total tyrosine and/or serine phosphorylation and the activity of IRS proteins in each of the skeletal muscle fractions. However, with the use of an antibody raised against a specific IRS-1 phosphorylation motif (Tyr⁶¹²), a residue that is known to interact with the p85 regulatory subunit of PI 3-kinase, a small but nonsignificant increase in the phosphorylation of IRS-1 Tyr⁶¹² in the cytosol fraction could be detected immediately after exercise and 3 h after exercise, although no change was detected in the HSP fraction. In support of previous research (5, 6, 17, 23, 28), exercise, per se, does not appear to affect the activation of IRS-1, but it should be noted that IRS-1 is known to contain a number of phosphorylation sites, in addition to Tyr⁶¹², that can act as on and/or off switches to recruit various downstream signaling proteins (20). It cannot be completely ruled out that exercise per se may influence the localized activation of IRS-1 through specific phosphorylation on other individual, or any combination of, tyrosine/serine residues.

In contrast to the IRS proteins and insulin receptor, exercise or muscle contraction can increase serine phosphorylation and activity of Akt and GSK-3 in whole cell lysate from human skeletal muscle (14, 26). Akt, or protein kinase B, is a serine/threonine kinase that, in response to various stimuli, is activated rapidly via translocation to the plasma membrane where it is phosphorylated. After activation, Akt dissociates from the membrane and subsequently phosphorylates numerous downstream target proteins in both the nucleus and cytoplasm, including GSK-3 (10). In the present study, the protein content of Akt and GSK-3 was not altered by exercise in any of the skeletal muscle fractions. However, in the cytosol fraction only, exercise significantly increased the phosphorylation of Akt Ser⁴⁷³. A similar response was also measured for GSK-3 in the cytosol fraction only, with GSK-3 serine phosphorylation significantly increased immediately and 3 h after exercise. The physiological significance of increased phosphorylation of Akt and GSK-3 in response to exercise in the cytosol fraction only is unknown, although it may represent a level of biological control required to ensure regulation of specific exercise-mediated cellular functions. In rat skeletal muscle, contraction increases Akt Ser⁴⁷³ phosphorylation via a PI 3-kinase dependent mechanism (16), but Akt can also be activated in some cell systems by mechanisms that are independent of PI3-kinase, such as an increase in intracellular calcium (27). Whether this regulatory mechanism could also occur in contracting skeletal muscle, and perhaps more specifically in a cytosolic fraction only, is an area for further research. Alternatively, it cannot be completely ruled out that, in the HSP and nuclear fractions, Akt and GSK-3 were activated by exercise more rapidly and transiently than in the cytosol fraction and that methods employed in this study were not sensitive enough to detect significant increases in phosphorylation.

In the present study, GSK-3 serine phosphorylation was increased immediately and 3 h after exercise in the cytosol fraction only. One of the primary functions of GSK-3 is thought to be the regulation of glycogen synthase. An increase in GSK-3 serine phosphorylation deactivates the GSK-3 protein, which subsequently acts to increase glycogen synthase activity. However, in response to exercise, it is not entirely clear as to whether glycogen synthase is a direct physiological substrate of GSK-3 in skeletal muscle (14, 16, 24). GSK-3 may play a role in other cellular functions, including fuel metabolism, gene transcription, cell division, and survival. Interestingly, a study in rat skeletal muscle examining the subcellular localization and translocation of glycogen synthase in response to either high or low glycogen levels (manipulated through diet and exercise) could not detect glycogen synthase protein and activity in the cytosol fraction (12). Cellular localization and activation of glycogen synthase was not measured in the present study, and direct comparison with the study by Nielsen et al. (12) is further complicated by differences in the muscle fractionation techniques and exercise intervention employed. However, the potential differences in cellular localization and activation between GSK-3 and glycogen synthase highlight an important area for further research examining protein-protein interactions and regulation at the subcellular level to explain how exercise may mediate specific cellular responses in skeletal muscle.

In conclusion, an acute bout of moderate to intense cycling exercise can increase phosphorylation of downstream signaling proteins (Akt and GSK-3), specifically in the cytosolic fraction, but does not result in changes in the subcellular localization of IRS proteins or other proteins in the classical insulin signaling pathway in human skeletal muscle. As such, a change in the subcellular localization of insulin signaling proteins is an unlikely mechanism to influence downstream signal transduction pathways and subsequent cellular function in skeletal muscle after exercise.

	GRANTS

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This study was supported by Grant DP-0450338 from the Australian Research Council (K. F. Howlett and M. Hargreaves).

	ACKNOWLEDGMENTS

 
We acknowledge the excellent medical assistance provided by Dr. Andrew Garnham, School of Exercise and Nutrition Sciences, Deakin University, and the advice of Dr. Sean McGee, School of Exercise and Nutrition Sciences, Deakin University, with the nuclear extraction protocol.

	FOOTNOTES

 

Address for reprint requests and other correspondence: K. Howlett, School of Exercise and Nutrition Sciences, Deakin University, Burwood, Victoria 3125, Australia (e-mail: kirsten.howlett{at}deakin.edu.au)

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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