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Effects of exercise and insulin on insulin signaling proteins in human skeletal muscle


Medicine & Science in Sports & Exercise: July 1999 - Volume 31 - Issue 7 - p 998-1004
Basic Sciences: Original Investigations

Effects of exercise and insulin on insulin signaling proteins in human skeletal muscle. Med. Sci. Sports Exerc., Vol. 31, No. 7, pp. 998-1004, 1999. Insulin and exercise independently increase glucose metabolism in muscle. Moreover, exercise training or a prior bout of exercise increases insulin-stimulated glucose uptake in resting skeletal muscle. The present study was undertaken to compare how physiological hyperinsulinemia and moderate intensity aerobic exercise affect the tyrosine phosphorylation state and activity of insulin signaling molecules in healthy, physically inactive volunteers. Subjects had biopsies of the vastus lateralis muscle before and immediately after 30 min of either hyperinsulinemia (euglycemic insulin clamp) or moderate-intensity exercise on a cycle ergometer (∼60% of O2max). Insulin receptor and IRS-1 tyrosine phosphorylation, association of the p85 regulatory subunit of PI 3-kinase with IRS-1, IRS-1 associated PI 3-kinase activity, and glycogen synthase activity were determined in muscle biopsy specimens taken from healthy subjects before and after insulin or exercise. Physiological hyperinsulinemia increased the rate of glucose disposal from 11.4 ± 1.5 to 25.6 ± 6.7 μmol·kg−1·min−1 (P < 0.01), insulin receptor and IRS-1 tyrosine phosphorylation (173 ± 19% and 159 ± 35% of basal values, respectively, P < 0.05), association of the p85 regulatory subunit of PI 3-kinase with IRS-1 (159 ± 10%, P < 0.05), and glycogen synthase fractional velocity (136 ± 11%, P < 0.01). Exercise also increased glucose disposal, from 10.4 ± 0.5 to 15.6 ± 1.7 μmol·kg−1·min−1 (P < 0.01) and glycogen synthase fractional velocity (253 ± 35% of basal, P < 0.01). The exercise-induced increase in glycogen synthase was greater than that due to insulin (P < 0.05). In contrast to insulin, exercise decreased tyrosine phosphorylation of the insulin receptor to 72 ± 10% of basal values (P < 0.05 vs basal and P < 0.05 vs insulin) and had no effect on IRS-1 tyrosine phosphorylation, or association of p85 with IRS-1. The exercise-induced decreased insulin receptor tyrosine phosphorylation could explain the well-known effect of exercise to enhance the sensitivity of muscle to insulin.

The Division of Diabetes, Department of Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, TX; and The Joslin Diabetes Center, Boston, MA

Submitted for publication June 1998.

Accepted for publication December 1998.

The excellent technical assistance of Andrea Barrentine and Jean Finlayson and the nursing aid of Patricia Diaz and Norma Ortiz are gratefully acknowledged. This study was supported in part by NIH grants R01DK47936 (LM) R01DK24092 (RAD), a General Clinical Research Center Grant RR01346 (General Clinical Research Center, Audie Murphy Memorial Veterans Hospital, San Antonio, Texas), and a Clinical Associate Physician award to Dr. Pendergrass. None of the authors have a conflict of interest with any company whose product may have been used in this study.

Address for correspondence: Lawrence J. Mandarino, Ph.D., Division of Diabetes, Department of Medicine, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, Texas 78284-7886. E-mail:

Dr. Pendergrass' current address is Department of Medicine, Section of Endocrinology, Tulane University Medical Center, New Orleans, LA.

Insulin exerts its effects through a complex array of intracellular signaling events. Binding of insulin to the alpha subunit of the insulin receptor produces a conformational change in the receptor that activates the tyrosine kinase in the beta subunit. This kinase phosphorylates tyrosine residues in the beta subunit, and these phosphorylated tyrosines serve as the key element of amino acid motifs that associate with cytosolic proteins such as IRS-1 and IRS-2 (31,32) and Shc (30). The IRS family of insulin receptor substrates in turn are phosphorylated on tyrosine residues that participate as recognition sites for proteins with SH2 domains, such as Grb2 and the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) (2,27). Binding of Grb2 induces association with Sos and activation of the mean arterial pressure (MAP) kinase (ERK1/2) cascade (24), whereas activation of PI 3-kinase leads to activation of Akt/PKB and mediates many of the metabolic effects of insulin (6,12,20,29).

Both exercise and muscle contraction increase insulin's metabolic actions in skeletal muscle (3,11,14,25), but the biochemical and physiological mechanisms responsible for this are unknown. It has been postulated that exercise enhances insulin signaling in some manner and thus increases insulin sensitivity, but few studies have examined this question and the published data are conflicting. In rats, tetanic muscle contraction does not activate any of the proteins that are known to be involved in insulin signaling, nor does it increase the subsequent ability of insulin to initiate signaling events (15). Likewise, voluntary running in rats has little or no effect on insulin signaling (16). In healthy humans, one-legged exercise increases the rate at which insulin activates insulin receptor tyrosine kinase, when the insulin is given immediately after exercise (34), and this could be involved in the ability of insulin to activate its signaling system. However, this finding may be related to increased muscle blood flow and insulin delivery that occurs with acute exercise and is unlikely to explain how a bout of exercise can increase insulin sensitivity even after some time, when blood flow has returned to resting levels. Moreover, the high intensity of one-legged exercise used in that study (34) is not likely to represent the kind of exercise performed on a regular basis by insulin resistant individuals with obesity or Type 2 diabetes mellitus. In addition, the insulin infusion used in that study was sufficient to produce plasma insulin concentrations of 100-120 μU·mL−1, which are at the upper end of the normal physiological range in humans.

The present study was undertaken to compare the effects of physiological hyperinsulinemia with those of moderate exercise on insulin signaling events in human skeletal muscle in vivo. The euglycemic, hyperinsulinemic clamp technique with muscle biopsies was used to compare the effects of a physiological concentration of plasma insulin (about 60 μU·mL−1) for 30 min with 30 min of moderate-intensity exercise (55-60% of O2max) on insulin signaling, glycogen synthase activity, and glucose disposal in healthy human volunteers.

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Subjects. Fourteen healthy subjects with a normal glucose tolerance test participated in the studies. Nine of these subjects (4 women and 5 men, aged 37 ± 4 yr, BMI = 26.9 ± 1.0 kg·m−2, O2max of 33 ± 4 mL·kg−1·min−1) received percutaneous needle biopsies of the vastus lateralis muscle before and immediately after 30 min of exercise, and five of the subjects (1 woman and 4 men, aged 31 ± 5 yr, BMI = 25.2 ± 2.4 kg·m−2) had muscle biopsies before and at the end of 30 min of a euglycemic, hyperinsulinemic clamp study. None of the subjects who participated in the euglycemic clamp study also had an exercise study. None of the subjects reported taking part in a regular exercise program, and none were taking any medications known to affect glucose metabolism. All subjects were instructed to refrain from exercise for 48 h before any study. Subjects gave their written, informed consent before participation, and the Institutional Review Board of the University of Texas Health Science Center at San Antonio approved the study protocol.

Exercise study design. On the first day of the study, O2max was determined using a cycle ergometer (Ergometrics 800S, Sensormedics, Inc., Yorba Linda, CA) and a Sensormedics 2900 Metabolic Measurement System (Sensormedics, Inc., Savi Park, CA) in the breath-by-breath mode. Heart rate and rhythm were monitored continuously using a MAX1 Stress System (Marquette Instruments, Milwaukee, WI). The anaerobic threshold was estimated using the V-slope method (4). Subjects exercised to maximum voluntary exhaustion, and all achieved a respiratory exchange ratio ≥ 1.10.

On a separate day at least 1 wk later, subjects reported to the General Clinical Research Center of the Audie Murphy Memorial Veterans Hospital at 7:00 a.m. after having consumed nothing but water for the preceding 12 h. A primed (25 μCi), continuous (0.25 μCi) infusion of 3-[3H]glucose was started to determine the rate of glucose disposal. A vein in the back of the hand was catheterized, and the hand was placed in a heated, Plexiglas box for sampling of arterialized blood. After resting for 90 min, a percutaneous biopsy of the vastus lateralis muscle was performed under local anesthesia, as described (23). The subjects rested for another 60 min and then exercised on a stationary cycle at 90% of anaerobic threshold heart rate (approximately 60% of O2max in untrained subjects) for 30 min. Arterialized blood was sampled for plasma insulin, glucose, lactate, and 3-[3H]glucose specific activity during the last 30 min of the basal period and at 5-10 min intervals during the exercise. Immediately after the exercise, a second muscle biopsy was performed in the opposite leg. Muscle biopsies were frozen in liquid nitrogen within 15 s and stored in a liquid nitrogen freezer until processing.

Euglycemic hyperinsulinemic clamp. Euglycemic insulin clamps were performed as previously described (10). Subjects reported to the General Clinical Research Center of the Audie Murphy Memorial Veterans Hospital at 7:00 a.m. after having consumed nothing but water for the preceding 12 h. A primed (25 μCi), continuous (0.25 μCi) infusion of 3-[3H]glucose was started to determine the rate of glucose disposal. A vein in the back of the hand was catheterized, and the hand was placed in a heated, Plexiglas box for sampling of arterialized blood. After resting for 90 min, a percutaneous biopsy of the vastus lateralis muscle was performed under local anesthesia. The subjects rested for another 60 min, and a primed, continuous infusion of insulin was begun at a rate of 40 mU·m−2·min−1. The plasma glucose was measured every 5 min and maintained constant by infusion of 20% dextrose. After 30 min, a second muscle biopsy was obtained from the other leg and the study was ended.

Muscle biopsy processing. Muscle biopsy specimens were homogenized while still frozen in an ice-cold buffer (10 μL·mg−2 tissue) consisting of final concentrations of: 50 mM HEPES, pH 7.6; 150 mM NaCl; 20 mM sodium pyrophosphate; 20 mM β-glycerophosphate; 10 mM NaF; 2 mM sodium orthovanadate; 2 mM EDTA; 1% Nonidet P-40; 10% glycerol; 2 mM phenylmethylsulfonyl fluoride; 1 mM MgCl2; 1 mM CaCl2; 10 μg·mL−1 leupeptin; and 10 μg·mL−1 aprotinin. A Polytron homogenizer (Brinkman Instruments, Westbury, NY) set on maximum speed for 30 s was used for homogenization. Homogenates were incubated on ice for 20 min and then centrifuged at 10,000 × g for 20 min at 4°C. Aliquots of the supernatants were used for all assays.

Antibodies. Polyclonal anti-pI 3-kinase p85 subunit, antic-terminal IRS-1, anti-insulin receptor β subunit, and monoclonal antiphosphotyrosine (4G10) antibodies were obtained from Upstate Biotechnology (Lake Placid, NY). Lysates of 3T3 L1, Jurkat, and A431 cell lines served as controls in immunoblotting and also were obtained from Upstate Biotechnology (Lake Placid, NY).

Immunoprecipitation. For immunoprecipitation, aliquots of the homogenate were diluted to 2 μg protein·μL−1 of homogenization buffer and incubated for 2 h on ice with specific antibodies. The amounts of protein used were as follows: for insulin receptor β subunit, 250 μg; for IRS-1, 250 μg; and for p85, 50 μg. Protein A sepharose 4B beads (Sigma Chemical Co., St. Louis, MO) were prepared by washing twice with 0.5% Tween-20, 0.05% SDS, and 0.1% bovine serum albumin and twice with homogenization buffer. Protein A beads were then added to the immunoprecipitation reaction, and incubation was continued for another 1.5 h at 4°C with rotation. The protein A beads-antibody complex was precipitated by brief centrifugation.

SDS polyacrylamide electrophoresis and immunoblotting. After denaturing by boiling with SDS-PAGE sample buffer containing 4% SDS and 8 M urea, proteins were separated on 7.5% polyacrylamide gels and blotted electrophoretically onto nitrocellulose paper. After blocking with Tris-buffered saline (TBS) containing nonfat dried milk and Tween-20 (20 mM tris, pH 7.5; 137 mM NaCl; 2.7 mM KCl; 0.2% Tween-20; and 3% nonfat dried milk), blots were incubated at 4°C with appropriate antibodies diluted in TBS-Tween-20 milk. After washing with TBS-Tween-20, the blot was incubated with peroxidase-conjugated antirabbit or antimouse IgG for 1 h at room temperature. The bands were visualized using a chemiluminescence detection system (ECL, Amersham Life Science, Buckinghamshire, UK) on x-ray film (X-OMAT-AR, Kodak, Rochester, NY). Images were digitized by scanning and band intensity was quantified using Image Tool Software.

Phosphatidylinositol 3-kinase activity assay. Immunoprecipitates were washed successively with: 1) phosphate-buffered saline (PBS) containing 0.5% NP-40, 100 μM sodium orthovanadate, 1 mM DTT in PBS; 2) 100 mM tris-HCl, pH 7.5, containing 500 mM LiCl2, 100 μM sodium vanadate, and 1 mM DTT; and 3) 100 mM tris-HCl, pH 7.5, containing 100 mM NaCl, 500 μM EDTA, 100 μM sodium vanadate, and 1 mM DTT. Washing was done one time each in buffers 1 and 2 and twice in buffer 3. Packed beads were suspended and incubated for 5 min in 20 μL of phosphatidylinositol (Avanti Polar Lipids, Alabaster, AL) dissolved (final concentration of 0.5 μg·mL−1) in 50 mM HEPES, pH 7.5 containing 1 mM EGTA, and 1 mM sodium phosphate at room temperature, with occasional shaking. The kinase reaction was started by the addition of 10 μL of 50 mM HEPES, pH 7.5, containing 1 mM EGTA, 1 mM sodium phosphate, 50 mM MgCl2, 200 μM ATP, and 0.8 μCi·μL−1 of γ-[32P] ATP (6000 Ci·mmol−1, DuPont NEN, Boston, MA). After 5 min of incubation at room temperature with vigorous shaking, the reaction was terminated by the addition of 15 μL of 4 N HCl and 130 μL of MeOH/CHCl3 (1:1, v/v). After brief centrifugation, 40 μL of the organic solvent layer was spotted onto a thin layer chromatography plate (Silica gel 60, Whatman, Hillsboro, OR) that had been activated with potassium oxalate and baking at 100°C. After separation of phosphoinositides in running solvent (CHCl3: MeOH:H2O:NH4OH, 60:47:11.3:2), plates were dried and exposed to x-ray film. Spots were scraped from the plates and radioactivity was counted using a liquid scintillation counter. Kinase activity was quantified using PI 3-kinase activity in a single lot of Jurkat cell lysate as a standard.

Glycogen synthase activity assay. GS activity was determined in soluble fractions of muscle extract using 0.1 and 10 mM glucose 6-phosphate (G-6-P), as described previously (23). GS fractional velocity (GSFV) was defined as the ratio of GS activities determined at 0.1 and 10 mM G-6-P. This ratio is increased by insulin infusion in humans and represents dephosphorylation and activation of GS.

Analytical techniques. Plasma insulin was determined by radioimmunoassay (DPC Inc., Los Angeles, CA). Blood lactate was determined on perchloric acid extracts of whole blood using an enzymatic technique (28). Plasma glucose specific activity was determined by the method of Somogyi and rates of glucose disposal were calculated using Steele's nonsteady state equation (9).

Statistics. Post- and pre-exercise values were compared using paired t-tests, or where data were not distributed normally, by a sign rank test (BMDP Statistical Software, Inc., Los Angeles, CA).

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Hormone and substrate concentrations. During the euglycemic clamp, plasma glucose concentrations were maintained constant (4.8 ± 0.28 mM), within 5% of basal values. During moderate intensity exercise, the plasma glucose concentration did not change (5.13 ± 0.11 mM basally, 5.25 ± 0.10 mM during exercise). Basal insulin concentrations, averaged over the 30 min before insulin infusion, were 60 ± pmol·L−1 in the subjects who had euglycemic clamps, and the insulin infusion increased the plasma insulin concentration to 414 ± 30 pmol·L−1 at the 30-min time point. In subjects who exercised, basal insulin concentrations for the 30-min period before exercise were 66 ± 12 pmol·L−1. Plasma insulin concentrations did not change at any time during the 30-min exercise period and averaged 60 ± 12 pmol·L−1. The blood lactate concentration rose promptly from a basal value of 0.60 ± 0.11 to 2.34 ± 0.4 mM 10 min after starting exercise and remained elevated throughout the exercise period, although it fell from the peak value to 1.49 ± 0.16 mM after 30 min of exercise. Lactate concentrations were not measured during the euglycemic clamps.

Glucose metabolism. The effects of insulin and exercise on total body glucose disposal are contrasted in Figure 1. Hyperinsulinemia increased the rate of glucose disposal from 11.4 ± 1.5 to 25.6 ± 6.7 μmol·kg−1·min−1 (P < 0.01), and exercise also increased glucose disposal, from 10.4 ± 0.5 to 15.6 ± 1.7 μmol·kg−1·min−1 (P < 0.01). The increase in glucose disposal caused by insulin was greater than that caused by exercise (P < 0.05).

Figure 1

Figure 1

Glycogen synthase activity. Both exercise and insulin increased GS0.1 activity and GSFV, without changing total glycogen synthase activity (GS10) (Table 1, Fig. 2). The magnitude of activation of GSFV (fold-stimulation over basal values) induced by exercise was significantly greater than with insulin (P < 0.05).



Figure 2-Effec

Figure 2-Effec

Insulin signaling elements. The effects of exercise or insulin on insulin receptor and IRS-1 tyrosine phosphorylation and association of p85 and PI 3-kinase activity with IRS-1 are shown in Table 2 and Figure 2. Physiological hyperinsulinemia increased tyrosine phosphorylation of the insulin receptor beta subunit to 173 ± 19% of the basal value (P < 0.01). All subsequent steps in insulin signaling were increased similarly. In contrast, exercise decreased insulin receptor β subunit tyrosine phosphorylation by about 25% (P < 0.038; Fig. 2) and had no significant effect on tyrosine phosphorylation of IRS-1, p85 associated with IRS-1, or PI 3-kinase activity associated with IRS-1. Neither insulin nor exercise had an effect on the amounts of insulin receptor β subunit, IRS-1, or p85 protein in muscle, as determined by immunoblot analysis (Table 2).



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It has been known for many years that exercise and insulin produce many of the same metabolic effects in muscle, including translocation of GLUT4 to the plasma membrane, stimulation of glucose uptake, and activation of glycogen synthase (3,13,17). The present study was undertaken to compare the effects of insulin and exercise on early insulin receptor signaling events in human skeletal muscle. The insulin and exercise interventions were comparable in duration and were chosen to represent physiologically relevant stimuli, similar to those encountered in daily life. The results show that physiological concentrations of insulin effectively activate the insulin signaling cascade, glycogen synthase activity, and glucose disposal within 30 min. Moderate exercise, on the other hand, decreased insulin receptor phosphorylation and did not activate the proximal steps in the insulin signaling cascade measured in this study. However, exercise increased whole body glucose disposal and was more effective than insulin in activating glycogen synthase. The exercise-induced increase in glucose disposal and glycogen synthase activity occurred in the presence of basal insulinemia.

With regard to insulin receptor tyrosine phosphorylation, insulin increased the receptor beta subunit phosphotyrosine content significantly within 30 min. This is consistent with a previous report that examined the time course of insulin activation of the receptor tyrosine kinase (26) and extends these prior observations by demonstrating that even a short, 30-min, exposure to physiological hyperinsulinemia increases the activity of the insulin receptor. This time course of increase and the plasma insulin level achieved in the present study closely approximate the increase in plasma insulin concentration, glycogen synthase activity, and whole body glucose disposal after ingestion of a mixed meal by healthy volunteers (37). These results also confirm that insulin, when given in vivo to healthy subjects, results in increased association of PI 3-kinase with IRS-1 (18), and extend those observations to physiological concentrations of hyperinsulinemia. To our knowledge, this is the first demonstration that exposure of human muscle in vivo to physiological concentrations of insulin for as short as 30 min activates the insulin signaling cascade and emphasizes the importance of these early insulin receptor signaling events to physiological stimuli that are encountered in everyday life.

Like insulin, exercise also increased the rate of glucose disposal and glycogen synthase activity, but it did so without exerting an effect on the insulin receptor signaling events measured in this study. It is noteworthy that, although the insulin-stimulated increase in glucose disposal was approximately twice as great as that caused by exercise, exercise increased glycogen synthase activity to a greater degree than did insulin. This suggests that in the immediate postexercise period (muscle samples were obtained within five min of stopping exercise), there is likely to be a shift in the intracellular partitioning of glucose metabolism compared to that which occurs during insulin infusion, with a greater proportion of glucose uptake being stored as glycogen. This speculation is consistent with previously published results (5,19) that demonstrate a rapid repletion of muscle glycogen after exercise.

Despite the marked exercise-induced activation of glycogen synthase and stimulation of glucose disposal, the latter presumably occurring by increased translocation of GLUT4 glucose transporters, neither insulin receptor phosphorylation nor early insulin signaling events were activated. These observations provide clear-cut evidence that the exercise-mediated increase in muscle glucose disposal does not require activation of the early events in the known insulin signaling cascade. This is consistent with earlier reports in rodents, using treadmill running or electrical stimulation to promote muscle contraction (15,16), and in humans, using more intense exercise (34). In fact, the present study shows that moderate exercise decreased, rather than increased, tyrosine phosphorylation of the insulin receptor. This occurred without a change in the plasma insulin concentration during exercise and suggests that exercise decreased the tyrosine kinase activity of the insulin receptor beta subunit or increased the activity of a known or uncharacterized tyrosine phosphatase. These results are similar to those previously reported in rats (15). It is tempting to speculate that a decrease in the basal level of tyrosine phosphorylation of the insulin receptor could sensitize insulin signaling to a subsequent stimulation by insulin. This would be consistent with the known sensitizing effects of both acute and chronic exercise on insulin-mediated glucose disposal in man (11). Regardless, the present findings demonstrate that exercise does not increase muscle glucose uptake or glycogen synthesis by a mechanism that includes an increase insulin receptor tyrosine kinase activity or any of the proximal steps in insulin signaling.

The biochemical mechanism by which muscle contraction increases glucose disposal remains unknown. Muscle contraction induces GLUT4 translocation to the sarcolemma or specifically, the t-tubules (13,17). Unlike insulin, this effect is insensitive to inhibition by wortmannin, implying that the exercise-induced GLUT4 translocation does not depend on PI 3-kinase activity (21,22), although there is one report that contraction-mediated GLUT4 translocation is blocked by higher doses of wortmannin, a PI 3-kinase inhibitor (35). Therefore, it is possible that in the present study there could have been an increase in PI 3-kinase activity in some subfraction of PI 3-kinase activity that was not measured in IRS-1 or p85 immunoprecipitates. Regardless, there is some evidence that the GLUT4 transporters that are translocated in response to exercise come from a different pool of transporters than those that are responsive to insulin (8). Other mechanisms may also come into play. Kinases such as MAP kinase (ERK1/2) or JNK (c-jun N-terminus kinase) (2) or protein kinase C (7,36) may play a role. Yet another possibility is that paracrine factors such as adenosine (33) are involved. Finally, Zierler and Wu (38) has promoted the idea that insulin promotes a phenomenon much like depolarization in insulin-sensitive muscle cells in vitro, resulting in shifts in potassium and sodium across the cell membrane. It is possible that cell depolarization associated with contraction may also produce insulin-like effects.

Exercise of moderate intensity and duration was used in the current study to mimic the kind of exercise performed by most people in everyday life. That the exercise was indeed moderate is documented by the modest increase in blood lactate concentrations to 2.5-3 mM. Moreover, the insulin concentrations achieved during the euglycemic clamp were similar to those reached after ingestion of a mixed meal. Therefore, we believe that the results of this study can be extrapolated to daily life and indicate that even moderate exercise produces profound effects on muscle glucose metabolism and glycogen synthase activity without altering the insulin signaling cascade. (Table 3)



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