Exercise-enhanced activation in human skeletal muscle JENS
FRIIS
BAK
AND
OLUF
of glycogen synthase
PEDERSEN
Medical Endocrinological Department III, University Aarhus Amtssygehus, DK-8000 Aarhus C, Denmark
Clinic of Internal Medicine,
BAK, JENS FRIIS, AND OLUF PEDERSEN. Exercise-enhanced activation of glycogen synthase in human skeletal muscle. Am. J. Physiol. 258 (Endocrinol. Metab. 21): E957-E963, 1990.The present study was undertaken to elucidate aspects of the regulatory mechanisms leading to enhanced glucose metabolism and insulin sensitivity of muscle after physical exertion. Biopsies were obtained from the vastus lateralis muscle of healthy volunteers before and after 60 min of bicycle exercise at 60% of their maximal aerobic capacity. Insulin binding to wheat germ agglutinin-purified muscle insulin receptors as well as basal and insulin-stimulated receptor kinase activity toward an exogenous substrate were unaltered by exercise. Muscle glycogen levels diminished from 3.35 t 0.26 to 1.85 2 0.13 mg/ 100 mg muscle (P < 0.01) and the half-maximal activation constant of glycogen synthase for glucose 6-phosphate decreased from 0.62 & 0.05 to 0.25 * 0.02 mM (P < 0.001). Total glycogen synthase activity was unchanged. In the absence of phosphatase inhibitors, glucose 6-phosphate-independent glycogen synthase activity of the crude enzyme extract increased during in vitro incubation. The initial rate of activation (through dephosphorylations) of glycogen synthase was 0.18 t 0.06 vs. 0.37 t 0.03 Ugmin-lomg-’ protein before and after exercise, respectively (P c 0.02). The total as well as the glycogen-associated phosphoprotein phosphatase activity was, however, unaffected by exercise.
presently known about the in vivo regulation of the phosphatases. Both the total and glycogen-associated phosphatase-1 activity are reduced in rat skeletal muscle after 24 h of fasting or 38 h after streptozotocin-induced diabetes (29). Moreover, recent findings by Freymond et al. (10) suggest that glycogen synthase phosphatase activity in skeletal muscle is decreased in insulin-resistant subjects, thus contributing to the impaired responsiveness of glycogen synthase to insulin stimulation in these patients (10, 32). Still, it remains to be verified whether phosphatase activity in human skeletal muscle is modulated when glycogen levels are reduced in vivo as a result of a physical exercise bout. Thus the present study aimed to explore the effect of exercise on the regulatory mechanisms of carbohydrate metabolism in human skeletal muscle by studying solubilized and wheat germ agglutinin (WGA) -purified insulin receptors for insulin binding and insulin-stimulatable kinase activity, as well as the activities of glycogen synthase and the phosphoprotein phosphatases.
exercise; insulin receptor tase; human muscle
Subjects. Sixteen healthy nonobese students (13 males and 3 females) volunteered to participate in the study. Their mean age was 24 t 1 yr, and mean body mass index was 22.6 t 0.6 kg/m’. None were receiving any medication. The study was approved by the local ethical committee, and all subjects gave informed consent according to the Second Declaration of Helsinki. Protocol. Within 7 days before the exercise test, the maximal aerobic capacity (Vozmax) was estimated in each person by a standard incremental exercise test using a bicycle ergometer (1). Moreover, each subject was instructed not to perform any major physical exertion within 72 hr before the examination. The exercise test began at 8:00 A.M. after an overnight fast. Initially, a catheter was inserted in a forearm vein for blood sampling in the basal state and throughout the exercise test. Next, with the use of a Bergstrom biopsy needle, a muscle biopsy was obtained from the vastus lateralis muscle. Under local anesthesia (1% lidocaine) a small incision was made through the skin and muscle sheath 15-20 cm above the knee corresponding to the vastus lateralis. The biopsy needle was inserted and 200-250 mg muscle were aspirated. The muscle biopsies were immediately inspected, and blood and fat remnants were removed. Thus within 15 s the biopsy was deep-frozen in liquid nitrogen,
kinase;
glycogen synthase
phospha-
PHYSICAL EXERCISE, glucoseuptakeofcontracting muscle increases markedly despite low physiological concentrations of insulin. Several studies of the regulatory mechanisms of glucose metabolism in exercising muscle have been conducted in animals and humans. In the acute phase of an exercise bout, local insulin-independent factors account for the increased glucose metabolism and the enhanced insulin sensitivity (25, 26). The signaling mechanism for increased glucose turnover during and after an exercise bout is not fully understood. The postexercise increase in insulin sensitivity (2) may involve both the insulin receptor function as well as postreceptor pathways for glucose metabolism. The breakdown of glycogen stores might be one local initiator to enhance glucose metabolism in exercising muscle (2, 25). One of the postreceptor regulatory systems is the glycogen synthase, which controls glycogen synthesis and is strongly stimulated after muscular exercise (20). Different phosphoprotein phosphatases (phosphatase-1 and phosphatase-2) are able to activate glycogen synthase through dephosphorylations (16). However, very little is DURING
0193-1849/90
$1.50 Copyright
METHODS
0 1990 the American
Physiological
Society
E957
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where it was stored until the biochemical analyses were performed. Each subject then exercised for 60 min using a bicycle ergometer at a load corresponding to 60% of the individual V02 max as calculated from above. Every 15 min throughout the exercise, blood samples were collected. Within 10 min after termination of the exercise, a second muscle biopsy was obtained from the same thigh as before at a distance of -5 cm from the first incision, using exactly the same procedure. During the first 2 h of the recovery period, while the subject was still fasting, blood samples were collected every 30 min. All the assays for insulin receptor binding, kinase activity, glycogen synthase activity, and phosphatase activity in the pre- and postexercise muscle biopsies were run in parallel on the same day to eliminate the influence of any day-to-day variation. Preparation of solubilized insulin receptors. The muscle insulin receptors were solubilized and purified as previously described (la). Briefly, the frozen muscle biopsy was homogenized in an ice-cold buffer (1 ml/100 mg muscle) containing (in mM) 25 N-Z-hydroxyethylpiperazine-A/‘-2ethanesulfonic acid (HEPES), 4 EDTA, 10 NaF, 1 benzamidine, and 2 phenylmethylsulfonyl fluoride (PMSF) and also 9 X lo5 kallikrein inhibitor units/ 1 (KIU/l) aprotinin and 1% (vol/vol) Triton X-100, pH 7.4, using an ice-cooled glass homogenizer with a motordriven nylon pestle (2,000 revolutions/min). The homogenate was centrifuged at 3,200 g for 10 min at 4°C. From the supernatant 25 ~1 were saved for the glycogen synthase assay. The remaining supernatant was incubated during rotation end-over-end for 30 min at 4°C and then centrifuged at 150,000 g for 90 min. The 150,000-g supernatant (6 ml) was recycled three times on a 0.5 x l.Ocm WGA-Sepharose column. The column was washed at 4°C with 50 ml of a buffer containing 25 mM HEPES, 150 mM NaCl, and 0.05% (vol/vol) Triton X-100, pH 7.4. The receptor proteins were eluted from the WGA column with a buffer containing (in mM) 300 N-acetylD-glucosamine, 50 HEPES, 110 NaCl, 2.5 KCl, 1 CaC12, and 1 MgC12 and also 0.05% Triton X-100 and 10% (vol/ vol) glycerol, pH 7.6. Finally, PMSF and aprotinin were added to the eluate in final concentrations of 2 mM and 9 x 10” KIU/l, respectively. Insulin binding. Aliquots (30 ~1) of the wheat germ eluates were incubated in duplicate in a total volume of 250 ~1 in a buffer containing (final concentrations, in mM) 25 HEPES, 135 NaCl, 4.8 KCL, 1.7 MgSO,, 2.5 CaC12, and 1.2 sodium phosphate and also 1% (wt/vol) human albumin (pH 7.6) and 14 pM 1251-labeled insulin (kindly donated by NOVO Research Institute, Copenhagen, Denmark), with or without increasing concentrations of unlabeled insulin at 4°C for 18 h. Receptorbound insulin was precipitated by 25% (wt/vol) polyethylene glycol (PEG; mol wt 6,000) and bovine y-globulin (5). After centrifugation at 2,600 g for 10 min at 4”C, the precipitates were washed twice with 1 ml 12.5% (wt/vol) PEG and counted in a gamma counter (LKB-minigamma). Insulin binding data was analyzed by a Scatchard plot using the LIGAND program of Munson and Rodbard (22).
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Insulin receptor kinase assay. The ability of the insulin receptor to phosphorylate exogenous substrates was tested using the synthetic peptide poly( Glu4 Tyr’) (Sigma Chemical, St. Louis, MO) (4). Aliquots (120 ~1) of receptor preparations were preincubated in the absence and presence of 1O-11- 10B7 M insulin for 60 min at 21°C. Poly(Glu4 Tyr’) (20 ~1, final concentration 0.2 mg/ml) was added, and the phosphorylation reaction was initiated by adding 20 ~1 of (in mM) 4 MnC12, 8 MgC12, and 20 ATP and also -2 &i [T-~~P]ATP (New England Nuclear, Boston, MA). All tubes were incubated at 21°C for 30 min. The phosphorylations proceeded linearly under these conditions for >60 min (data not shown). The reaction was terminated by spotting 80 ~1 of the reaction mixture on 2-cm squares of Whatman 31ET filter paper and immersing the papers in 10% (wt/vol) trichloroacetic acid containing 10 mM sodium pyrophosphate. After extensive washing, the papers were counted for 32P in a liquid scintillation counter (Beckman). Insulin receptor kinase activity was normalized for the number of high-affinity insulin binding sites in the eluate, as calculated from the above-mentioned approach, and thus expressed as femtomoles phosphate incorporated into poly( Glu4 Tyr’) per femtomole insulin receptors per minute. Glycogen synthase activity and glycogen content. A 25~1 sample of the supernatant from the first centrifugation of the homogenate was diluted with 1,475 ~1 of (in mM) ice-cold 63 tris(hydroxymethyl)aminomethane (Tris) . HCl, 25 NaF, and 26 EDTA (pH 7.8). Thirty microliters of this enzyme extract were incubated in duplicate at 30°C with 60 ~1 of (in mM) 0.13 UDP-[UJ4C]glucose (New England Nuclear), 63 Tris . HCl, 25 NaF, 26 EDTA, and O-6.7 glucose 6-phosphate (G-6-P) and also 6.7 mg/ ml glycogen, (pH 7.8) (20). After 15 min, the reaction was stopped by spotting 75 ~1 of the mixture on Whatman 31ET filter papers, which after extensive washing with ethanol (66% vol/vol) were counted in a scintillation counter (Beckman) for 14C incorporated into glycogen. Protein determinations of the enzyme extract were performed using the Bio Rad dye reagent (Munich, FRG). Glycogen synthase activity was expressed as nanomoles UDP-glucose incorporated into glycogen per minute per milligram soluble protein in the homogenate. In the text, maximal activity refers to the glycogen synthase activity in the presence of 6.7 mM G-6-P. One unit (U) corresponds to the incorporation of 1 nmol UDP-glucose into glycogen per minute. Fractional velocities were calculated as glycogen synthase activity in the presence of subsaturating levels of G-6-P divided by glycogen synthase activity in the presence of 6.7 mM G-6-P. The concentration of G-6-P giving half-maximal stimulation of the glycogen synthase (A0.5 for G-6-P) was calculated using a Hill plot (20). Muscle glycogen was determined from a weighed muscle sample after acid hydrolysis (1 N HCl) at 100°C for 2 h (25). The concentration of hydrolyzed glucose residues was measured with a glucose oxidase method (Boehringer Mannheim, FRG). Conversion rate of endogenous glycogen synthase. Approximately 25 mg of muscle were used to determine the rate of activation of the endogenous glvcoeen svnthase. ” “4 ” d -----,
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EXERCISE
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i.e., the rate of conversion of the endogenous glycogen synthase from the G-6-P-dependent (D) form to the G6-P-independent (I) form. The muscle biopsy was homogenized in an ice-cooled buffer (230 ,ul buffer/l0 mg muscle) containing (in mM) 50 Tris . HCl, 10 EDTA, and 50 2mercaptoethanol, pH 7.8. The homogenate was centrifuged at 6,000 g for 20 min at 4°C. From the supernatant 100 ~1 were diluted with 75 ~1 buffer and then incubated at 30°C. The activation reaction was terminated at 0, 5, 10, 15, and 30 min by diluting 25 ~1 of the mixture in 975 ~1 of an ice-cold buffer containing (in mM) 50 Tris HCl, 20 EDTA, and 130 NaF, pH 7.8. Fluoride was able to inhibit this reaction totally, indicating that the activation of the glycogen synthase was a result of dephosphorylations. Glycogen synthase activity was measured in the absence and in the presence of 6.7 mM G-6-P as described above. The rate of conversion of the glycogen synthase was expressed as units of I-form of glycogen synthase produced per minute per milligram protein. Phosphatase activity. The phosphatase activity in muscle was measured with [32P]phosphorylase a as a substrate (6). To prepare [32P]phosphorylase a, 50 mg phosphorylase b were incubated with 1 mg phosphorylase kinase (both from Sigma), together with 1 mM (-0.4 mCi) [32P]ATP (New England Nuclear) for 60 min at 21°C in a buffer containing 50 (in mM) Tris HCl, 50 ,& glycerophosphate, 10 magnesium acetate, pH 8.2. The phosphorylated phosphorylase a was precipitated with ammonium sulfate (45% saturation) during centrifugation at 6,000 g for 15 min, at 4°C. The precipitate was washed once in a buffer containing (in mM) 50 Tris HCl, 1 EDTA, and 50 2-mercaptoethanol and also 45% ammonium sulfate, pH 7.0. Subsequently it was resuspended in a buffer containing (in mM) 50 Tris HCl, 1 EDTA, and 50 2-mercaptoethanol, pH 7.0. This suspension was dialyzed for 20 h against 2 liters of the same buffer. The crystals were precipitated by centrifugation at 1,700 g for 10 min at 4”C, and finally the [32P]phosphorylase a was resuspended in (in mM) 50 Tris. HCl, 250 NaCl, 1 EDTA, and 50 2-mercaptoethanol, pH 7.0 and stored at -2O’C. To measure phosphatase activity, -40 mg (wet weight) of the muscle biopsy were homogenized using the previously described technique in an ice-cold buffer (4 pl/mg muscle) containing (in mM) 4 EDTA, 1 benzamidine, 2 PMSF, and 14 Zmercaptoethanol, pH 7.2. The homogenate was centrifuged at 5,900 g, for 20 min at 4°C. From the resulting supernatant duplicates of 5 ~1 were incubated at 30°C with 50 ~1 [32P]phosphorylase a, and 50 ,ul of a buffer with (in mM) 4 EDTA, 20 imidazole, and 5 2-mercaptoethanol, with or without 0.1 mg glycogen, pH 7.5. After 5, 10, 15, and 25 min, aliquots of 25 ~1 were transferred to 800 ~1 10% (wt/vol) trichloroacetic acid. One hundred microliters of 2.5% (wt/vol) bovine serum albumin were added, and the proteins were precipitated by centrifugation at 1,400 g for 5 min. From the supernatants 500 ~1 were counted for released [32P]phosphate. The background radioactivity was determined in the absence of muscle enzyme extracts. It was stable and amounted to ~10% of the radioactivity released by the
IN
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E959
phosphatase after 10 min of incubation. To precipitate glycogen particles, 50 ~1 of the enzyme extract were ultracentrifuged at 150,000 g for 2 h at 4°C (29). The glycogen pellet was resuspended in a buffer (3 pl/mg of initial muscle weight) containing (in mM) 2.5 EDTA, 10 imidazol, 0.1 PMSF, 15 2-mercaptoethanol, and 0.1 benzamidine, pH 7.2. Glycogen-associated phosphatase activity was determined as described above. Phosphatase-1 accounts for the majority of the phosphatase activity in the muscle homogenate (16), and no efforts were exerted to eliminate any influence of the phosphatase-2A that might also be present in the cytosol of the muscle cells. Chemical quantities. Blood samples were analyzed for plasma insulin (NOVO radioimmunoassay kit for human insulin), plasma glucose (Merck enzymatic kit, Merck, Darmstadt, FRG), plasma free fatty acids (FFA; l7), plasma ketone bodies (3-hydroxybutyrate and acetic acid; 3l), and plasma lactate (15). StatisticaL methods. Differences before and after the exercise test were statistically analyzed with the paired t test. Variations in plasma concentrations of insulin and metabolites during the exercise test were tested for significance using a multiple analysis of variance (MANOVA) for repeated measurements. P values ~0.05 were considered statistically significant.
l
RESULTS
The variations in plasma concentrations of insulin (P < 0.005), FFA (P c O.OOl), lactate (P < 0.005), and ketone bodies (P c 0.025) during the exercise test are shown in Fig. 1. Throughout the test there were no variations in the levels of plasma glucose, which remained stable at 5.0 t 0.1 mM (Fig. 1). In muscle biopsies from ten subjects, the maximum insulin binding to the solubilized receptors (B,,,) was determined from Scatchard analysis of the binding data using the LIGAND computer program. However, the Scatchard plots were curvilinear (Fig. 2), and therefore the data were fitted to a one-site binding model under the assumption that the curvilinearity of the plotted raw data might be attributable to a nonspecific binding of the ligand. This nonspecific binding component was handled as a computer-fitted parameter (13), which amounted to 0.6 t 0.1 and 0.5 t 0.1% of the total added insulin before and after exercise, respectively [P = not significant (NS)] . After subtraction of this nonspecific binding component, B,,, was 60.9 t 5.6 before vs. 64.9 t 3.9 fmol/lOO mg muscle after exercise [P = NS; see Fig. 2 (inset)]. The insulin-stimulatable kinase activity of the insulin receptors was examined by measuring basal and insulinstimulated phosphorylation of the exogenous synthetic peptide poly(Glu4 Tyr’). The basal kinase activity was 1.86 t 0.17 vs. 1.61 t 0.10 fmol phosphate incorporated. fmol receptor-‘. min-l (P = NS) before and after exercise. On in vitro insulin stimulation there was a slightly lower kinase activity in the postexercise receptors (Fig. 3). This difference, however, did not attain statistical significance (P = 0.06). The recovery of soluble protein in the muscle homog-
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E960
EXERCISE
^r 5.0 5 14.0-
.-.-i-.-.-.-j--. I
-
GLYCOGEN
SYNTHASE
IN
HUMAN
MUSCLE
: I
: I
6.0-
AND
I
l
s 3.08 2 (3 2.0-
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60
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(fmol)
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Insulin
100 (fmol/lOO
150
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2. Scatchard plots of mean values of insulin binding data from wheat germ agglutinin-purified muscle insulin receptors. Muscle biopsies were obtained before (0) and after (0) exercise. ‘251-insulin (14 pM) was added, together with increasing amounts of unlabeled insulin, and bound insulin was precipitated with polyethylene glycol (see METHODS). Inset: corresponding computer-derived Scatchard plots calculated on assumption that 0.6 t 0.1 and 0.5 t 0.1% of total added insulin were subject to nonspecific binding. Means t SE, n = 10. FIG.
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2 LL g 0.2! n 0.0
I
c $B400-
:
3
*
.Bsoox m g200'; Y g100-
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0.1 Insulin
1 (nM)
10
100
FIG. 3. Kinase activity of wheat germ agglutinin-purified insulin receptors from skeletal muscle biopsies obtained before (0) and after (0) exercise. Peptide-incorporated 32P (Pi) was normalized to maximal insulin binding (B,,,) as calculated from Scatchard plots of insulin binding data (see METHODS). Means * SE, n = 10.
iii n O-
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TIME (hrs) FIG. 1. Metabolic profiles during exercise test. Blood samples obtained before, during, and after exercise were analyzed for plasma concentrations of (from top to bottom) glucose, insulin, free fatty acids (FFA), ketone bodies, and lactate. Exercise period (O-l h) is indicated by vertical dashed lines. Means t SE, n = 11.
enate was 65.6 t 2.5 and 64.0 t 1.6 mg protein/g wet muscle before and after exercise, respectively (NS), suggesting that the water content of the muscle biopsies did not change significantly during exercise. The glycogen content of the skeletal muscle biopsies diminished significantly from 3.35 t 0.26 to 1.85 k-O.13 mg/lOO mg muscle (P c 0.01) after the exercise bout. Glycogen synthase was activated as demonstrated by an increase in the fractional velocities (Fig. 4) and a significant reduction in A0.5for G-6-P from 0.62 t 0.05 to 0.25 t 0.02 mM (P c 0.001). Total glycogen synthase activity was unchanged by exercise and amounted to 42.8 t 2.6 vs. 43.6 t 2.6 U/mg protein before and after exercise. - -? respectively. ’ ” * To explore the mechanism of activation of the endogenous glycogen synthase, the crude enzyme extracts from six pairs of muscle biopsies were incubated for various
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EXERCISE
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IN
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r
0
0.01
0.1
1
[Glucose-6-phosphate]
10
(mM)
FIG. 4. Fractional velocities of glycogen synthase at various glucose 6-phosphate levels (O-6.7 mM) in muscle biopsies obtained before (0) and after (0) exercise. Means * SE, n = 10.
10
Incubation
v i
I
1
I
I
1
nI
1
0
5
10
15
20
25
30
35
Time
(min)
Incubation
(min)
FIG. 6. Phosphorylase phosphatase activity measured as release of phosphate from [“2P]phosphorylase a. Phosphatase activity was measured both in enzyme extracts of crude homogenate (A) and in glycogen particles (B) from muscle biopsies obtained before (0) and after (0) exercise test. Means t SE, n = 6.
0 1
O!
20
Time
FIG. 5. Conversion of glycogen synthase to its glucose 6-phosphateindependent form (I form) during in vitro incubation of crude enzyme extract from skeletal muscle biopsies in absence (00, solid line) and in presence of 130 mM NaF (VA, dashed line). Muscle biopsies were obtained before (OA) and after (OV) exercise. Means t SE, K! = 6.
periods of time in the presence and absence of fluoride (a serine phosphatase inhibitor). Fluoride was able to inhibit the activation process totally (Fig. 5). Glycogen synthase activity was determined repeatedly in the absence of G-6-P during 30 min of incubation. The rate of conversion of the muscle glycogen synthase to the I form was calculated from the slope of the initial part (the first 10 min) of the reaction curve as shown in Fig. 5. This rate increased significantly from 0.18 t 0.07 before exercise to 0.37 t 0.03 U. min-l . mg protein-l after exercise (P < 0.05). Thus the exercise-induced activation of glycogen synthase appeared to be a result of dephosphorylations. The difference in the rate of glycogen synthase activation disappeared after lo-15 min of incubation (Fig. 5). To further explore the activation of glycogen synthase, the phosphoprotein phosphatase activity in the muscle was measured. This was done by using [32P]phosphorylase a as a substrate. It appears from Fig. 6 that the phosphatase activity was linear only within the first 510 min of incubation. The postexercise phosphatase activity of the total enzyme extract of the muscle biopsy was very similar to the preexercise phosphatase activity (Fig. 6A ). Addition of exogenous glycogen (1 mg/ml) to
the reaction buffer did not interfere with the phosphatase reaction (data not shown). Moreover, in precipitated glycogen particles, the glycogen-associated phosphatase activity was also unaffected by physical exercise (Fig. W . DISCUSSION
Physical exercise leads to major changes in the carbohydrate metabolism of contracting muscle. Using a crude membrane preparation from skeletal muscle, insulin binding has been reported both to increase (30) and to decrease (3) during high-intensity exercise. In the present study, in human skeletal muscle, we show that the amount of soluble insulin receptors recovered from WGA columns was the same before and after exercise. The insulin-stimulated kinase activity of the insulin receptors was slightly lower after exercise. However, this difference did not attain statistical significance. In rat skeletal muscle, insulin binding and kinase activity of solubilized insulin receptors were also unchanged after an acute exercise bout (28). Likewise, in patients with insulin-dependent diabetes, 6 wk of moderate physical training did not change the muscle insulin receptor binding or kinase function (la). However, in rats heavy physical training for 4 wk increased the insulin receptor binding, whereas the insulin-stimulated kinase activity decreased correspondingly in the vastus intermedius muscle (9) and in the biceps femoris muscle (27). In contrast, no change was found in binding and kinase activity of insulin receptors from the tensor fascia lata muscles (27). Because catecholamines inhibit insulin receptor kinase activity (12, 23) this postexercise reduction in receptor kinase activity found in some muscles
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E962
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may be a result of the increased sympathoadrenergic activity during exercise. In our study the influence of acute exercise on muscle glycogen levels, glycogen synthase activity, and the rate of glycogen synthase activation, as well as the phosphatase activity was also examined. As expected, the total (maximal) activity of the glycogen synthase was unchanged after termination of the exercise bout. It is to be noted, however, that a postexercise increase in total glycogen synthase activity may occur after 24-48 h of recovery (20). Likewise, more chronic physical training increased the total activity of glycogen synthase in skeletal muscle from insulin-dependent diabetic patients (1 a.) The present results confirm several previous findings (2,8,20) that glycogen synthase is activated after muscle exercise, i.e., it becomes more sensitive to the allosteric activator G-6-P and thus it becomes stimulated in favor of glycogen synthesis. Regulation of glycogen synthase activity is generally believed to be a result of phosphorylation-dephosphorylation reactions. However, it remains to be shown whether glycogen synthase is actually dephosphorylated in vivo during or after glycogen-depleting exercise. The fact that fluoride was able to inhibit the in vitro activation of glycogen synthase provides indirect evidence that dephosphorylation processes were involved. Therefore, we measured the rate of activation of endogenous glycogen synthase in the crude enzyme extract simply by leaving out the phosphatase inhibitor (fluoride) in the buffers. Our results indicate that the initial rate of glycogen synthase conversion through dephosphorylations was nearly doubled after the exercise bout. The possibility exists that glycogen synthase had become a more suitable substrate for the phosphatases after exercise. However, if the more dephosphorylated forms of glycogen synthase should become more activable by phosphatases, we would expect the activation curve of glycogen synthase to show an upward concavity. This was not the case, as shown in Fig. 5. It was important, therefore, to consider whether there was a concomitant increase in the activity of the phosphatases that dephosphorylate and thereby activate glycogen synthase. The phosphoprotein phosphatase activity was analyzed by measuring the release of phosphate from an exogenous phosphorylase a. The arguments for using this substrate are several. First, protein phosphatase-1, also termed phosphorylase phosphatase, accounts for the majority of the potential phosphatase activity in rabbit skeletal muscle (16). Second, phosphatase-1 is the only protein phosphatase specifically associated with the protein-glycogen complex, where glycogen synthase is located (16, 18). Third, phosphorylase a is a much more well-defined substrate than glycogen synthase, which can be phosphorylated at multiple sites. Phosphoprotein phosphatase activity in exercised, glycogen-depleted muscles was identical to the preexercise activity both in the soluble enzyme extract of the crude muscle homogenate as well as in the precipitated glycogen particles. This is at variance with studies in vitro showing the ability of glycogen to inhibit the phosphatase, as determined both from measurements of the rate
IN
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of dephosphorylation of [32P]phosphorylase a (21) and from studies of the rate of activation of the D form of glycogen synthase (24). Because the present data suggest that activation of glycogen synthase during glycogen-depleting exercise does not involve an increased activity of the endogenous phosphatases, alternative mechanisms might be involved in the activation of glycogen synthase after acute exercise. Local factors in or among the glycogen particles may be speculated to account for the increased rate of glycogen synthase dephosphorylations. The fact that the glycogen-associated phosphatase activity was unchanged after glycogen depletion suggests that glycogen-linked phosphatases were not liberated together with the glucose residues after glycogen breakdown. Accordingly, a rearrangement of glycogen synthase enzymes and phosphatase-1 enzymes may take place within the glycogen particles, for example to bring glycogen synthase into a more favorable position for the phosphatase after glycogen depletion. Increased adrenergic activity during an exercise bout leads to a rapid increase in the concentration of adenosine 3’,5’-cyclic monophosphate (CAMP) in muscle (11). Epinephrine has been reported to promote the translocation of phosphatase-1 from glycogen to cytosol in rat skeletal muscle (29) through a CAMP-dependent protein kinase-mediated phosphorylation of the glycogen-binding G component of the glycogen-associated phosphatase-1 (14). Moreover, elevated muscle CAMP levels activate an inhibitor of phosphatase-1 (inhibitor-l) through a CAMP-dependent protein kinase (7). Therefore, the coexistence of increased glycogen synthase activity and increased adrenergic activity appear to be conflicting. Indeed, during muscle exercise the glycogen phosphorylase activity is increased (1 l), and glycogenolysis is more pronounced than glycogen synthesis. Glycogen synthase activation is probably primarily important when glycogen resynthesis is needed after exercise. Very recent data from Kida et al. (19) show an inactivation of glycogen synthase and a slower activation of an exogenous D form of glycogen synthase in enzyme extracts of human muscle biopsies obtained within a few seconds after termination of muscle contractions. However, more direct measurements of the phosphatase activity were not performed. The design of this study varied from ours in a number of respects. First, to provide a more activated form of glycogen synthase, patients were examined after a meal. Second, exercise consisted of exhaustive isometric contractions under anoxic conditions. Third, the duration of the exercise bout was -1 min. Unfortunately, glycogen levels were not measured. Most important, the above findings demonstrate that glycogen synthase activity can be rapidly changed during an exercise bout. From the present study on biopsies of human muscle obtained in the recovery phase of an acute exercise bout, we conclude that glycogen synthase is more activable through dephosphorylation reactions. This activation is not a result of an increased insulin-receptor binding or kinase function, not is it attributable to an increase in the total or the glycogen-associated phosphatase activity.
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EXERCISE
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Thus exercise-induced glycogen depletion is postulated to result in a rearrangement of the glycogen-linked enzymes to favor dephosphorylations of glycogen synthase. We thank Dr. Erik A. Richter for critical review of our manuscript and Lisbet Blak, Tove Skrumsager, and Pernille Sonne for excellent technical assistance. This study was supported by grants from the Danish Medical Research Council, Danish Diabetes Association, Aarhus Universitets Forskningsfond, NOVO Foundation, and Nordisk Insulin Foundation. Address reprint requests to J. F. Bak. Received
15 November
1989; accepted
in final
form
28 February
1990.
REFERENCES 1. ASTRAND, I. Aerobic work capacity in men and women with special reference to age. Acta Physiol. Stand. 49, Suppl. 169: 9-92, 1960. la.BAK, J. F., U. K. JACOBSEN, F. S. JORGENSEN, AND 0. PEDERSEN. Insulin receptor function and glycogen synthase activity in skeletal muscle biopsies from patients with insulin-dependent diabetes mellitus: effects of physical training. J. CZin. EndocrinoZ. Metab. 69: 158-164, 1989. 2 BOGARDUS, C., P. THUILLEZ, E. RAVUSSIN, B. VASQUEZ, M. NARIMIGA, AND S. AZHAR. Effect of muscle glycogen depletion on in vivo insulin action in man. J. CZin. Inuest. 72: 1605-1610, 1983. 3. BONEN, A., M. H. TAN, P. CLUNE, AND R. L. KIRBY. Effects of exercise on insulin binding to human muscle. Am. J. Physiol. 248 (EndocrinoZ. Metab. 11): E403-E408, 1985. 4. BRAUN, S., W. E. RAYMOND, AND E. RACKER. Synthetic tyrosine polymers as substrates and inhibitors of tyrosine-specific protein kinases. J. BioZ. Chem. 259: 2051-2054, 1984. 5. BURANT, C. F., M. K. TREUTELAAR, G. E. LANDRETH, AND M. G. BUSE. Phosphorylation of insulin receptors solubilized from rat skeletal muscle. Diabetes 33: 704-708, 1984. E. G. KREBS, AND E. H. FISCHER. 6. CHAN, C. P., S. J. MCNALL, Stimulation of protein phosphatase activity by insulin and growth factors in 3T3 cells. Proc. NatZ. Acad. Sci. USA 85: 6257-6261, 1988. 7. COHEN, P., J. G. FOULKES, C. F. B. HOLMES, G. A. NIMMO, AND N. K. TONKS. Protein phosphatase inhibitor-l and inhibitor-2 from rabbit skeletal muscle. Methods Enzymol. 159: 427-437, 1988. 8. DEVLIN, J. T., AND E. S. HORTON. Effects of prior high-intensity exercise on glucose metabolism in normal and insulin resistant men. Diabetes 34: 973-979, 1985. G. L., M. K. SINHA, AND J. F. CARO. Insulin receptor 9. DOHM, binding and protein kinase activity in muscles of trained rats. Am. J. Physdol. 252 (Endocrinol. Metab. 15): E170-E175, 1987. 10. FREYMOND, D., C. BOGARDUS, M. OKUBO, K. STONE, AND D. MOTT. Impaired insulin-stimulated muscle glycogen synthase activation in vivo in man is related to low fasting glycogen synthase phosphatase activity. J. CZin. Inuest. 82: 1503-1506, 1988. 11. GOLDFARB, A. H., J. F. BRUNO, AND P. J. BUCKENMEYER. Intensity and duration of exercise effects on skeletal muscle CAMP, phosphorylase, and glycogen. J. AppZ. Physiol. 66: 190-194, 1989. 12. HARING, H., D. KIRSCH, B. OBERMAIER, B. ERMEL, AND F. MACHICAO. Decreased tyrosine kinase activity of insulin receptor isolated from rat adipocytes rendered insulin-resistant by catecholamine treatment in vivo. Biochem. J. 234: 59-66, 1986. 13. HELMERHORST, E. The insulin-receptor interaction: is the kinetic approach for inferring negative-cooperative site-site interactions valid? Biochem. Biophys. Res. Commun. 147: 399-407, 1987. 14. HIRAGA, A., AND P. COHEN. Phosphorylation of the glycogenbinding subunit of protein phosphatase-1G by cyclic-AMP-depend-
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ent protein kinase promotes translocation of the phosphatase from glycogen to cytosol in rabbit skeletal muscle. Eur. J. Biochem. 161: 763-769, 1986. 15. HOHORST, H. J. Lactate analysis. In: Methods of Enzymatic Analysis, edited by H. U. Bergmeyer. New York: Academic, 1963, p. 266270. I. S., A. A. STEWART, AND P. COHEN. The protein 16. INGEBRITSEN, phosphatases involved in cellular regulation. Eur. J. Biochem. 132: 297-307,1983. determination of free fatty 17. ITAYA, K., AND M. UI. Calorimetric acids in biological fluids. J. Lipid. Res. 6: 16-22, 1968. 18. KHATRA, B. S. Purification of glycogen-bound high-molecularweight phosphoprotein phosphatase from rabbit skeletal muscle. Methods Enzymol. 159: 368-377, 1988. 19. KIDA, Y., A. KATZ, A. D. LEE, AND D. M. MOTT. Contractionmediated inactivation of glycogen synthase is accompanied by inactivation of glycogen synthase phosphatase in human skeletal J. 259: 901-904, 1989. 2. muscle. Biochem. KOCHAN, R. G., D. R. LAMB, S. A. LUTZ, C. V. PERRILL, E. M. REINMANN, AND K. K. SCHLENDER. Glycogen synthase activation in human skeletal muscle: effects of diet and exercise. Am. J. Physiol. 236 (Endocrinol. Metab. Gastrointest. Physiol. 5): E660E666, 1979. 21. MELLGREN, R. L., AND M. COULSON. Coordinated feedback regulation of muscle glycogen metabolism: inhibition of purified phosphorylase phosphatase by glycogen. Biochem. Biophys. Res. Commun. 114: 148-154,1983. 22. MUNSON, P. J., AND D. RODBARD. LIGAND: a versatile computerized approach for characterization of ligand-binding systems. AnaZ. Biochem. 107: 220-239,198O. 23. OBERMAIER, B., B. ERMEL, D. KIRSCH, J. MUSHACK, E. RATTENHUBER, E. BIEMER, F. MACHICAO, AND H. U. HARING. Catecholamines-and tumour promoting phorbolesters inhibit insulin receptor kinase and induce insulin resistance in isolated human adipocytes. Diabetologia 30: 93-99, 1987. 24. OKUBO, M., C. BOGARDUS, S. LILLIOJA, AND D. M. MOTT. Glucose6-phosphate stimulation of human muscle glycogen synthase phosphatase. Metabolism 37: 1171-1176, 1988. 25. RICHTER, E. A., L. P. GARETTO, M. N. GOODMAN, AND N. B. RUDERMAN. Enhanced muscle glucose metabolism after exercise: modulation by local factors. Am. J. Physiol. 246 (Endocrinol. Metub. 9): E476-E482, 1984. 26. RICHTER, E. A., T. PLOUG, AND H. GALBO. Increased muscle glucose uptake after exercise. No need for insulin during exercise. Diabetes 34: 1041-1048, 1985. 27. SANTOS, R. F., C. E. MONDON, G. M. REAVEN, AND S. AZHAR. Effects of exercise training on the relationship between insulin binding and insulin-stimulated tyrosine kinase activity in rat skeletal muscle. MetaboZism 38: 376-386, 1989. 28. TREADWAY, J. L., D. E. JAMES, E. BURCEL, AND N. B. RUDERMAN. Effect of exercise on insulin receptor binding and kinase activity in skeletal muscle. Am. J. Physiol. 256 (Endocrinol. Metab. 19): E138-E144,1989. 29. VILLA-M• RUZZI, E. Effects of streptozotocin-diabetes, fasting and adrenaline on phosphorylase phosphatase activities of rat skeletal muscle. MOL. Cell. EndocrinoZ. 47: 43-48, 1986. 30. WEBSTER, B. A., S. R. VIGNA, AND T. PAQUETTE. Acute exercise, epinephrine, and diabetes enhance insulin binding to skeletal muscle. Am. J. Physiol. 250 (Endocrinol. Metab. 13): E186-E197, 1986. 31. WILDENHOF, K. E. A micromethod for enzymatic determination of acetoacetate and 3-hydroxybutyrate in blood and urine. Stand. J. CZin. Lab. Invest. 25: 171-175, 1970. 32. YOUNG, A. A., C. BOGARDUS, D. WOLFE-LOPEZ AND D. M. MOTT. Muscle glycogen synthesis and disposition of infused glucose in humans with reduced rates of insulin-mediated carbohydrate storage. Diabetes 37: 303-308, 1988. l
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FRIIS
BAK
AND
OLUF
of glycogen synthase
PEDERSEN
Medical Endocrinological Department III, University Aarhus Amtssygehus, DK-8000 Aarhus C, Denmark
Clinic of Internal Medicine,
BAK, JENS FRIIS, AND OLUF PEDERSEN. Exercise-enhanced activation of glycogen synthase in human skeletal muscle. Am. J. Physiol. 258 (Endocrinol. Metab. 21): E957-E963, 1990.The present study was undertaken to elucidate aspects of the regulatory mechanisms leading to enhanced glucose metabolism and insulin sensitivity of muscle after physical exertion. Biopsies were obtained from the vastus lateralis muscle of healthy volunteers before and after 60 min of bicycle exercise at 60% of their maximal aerobic capacity. Insulin binding to wheat germ agglutinin-purified muscle insulin receptors as well as basal and insulin-stimulated receptor kinase activity toward an exogenous substrate were unaltered by exercise. Muscle glycogen levels diminished from 3.35 t 0.26 to 1.85 2 0.13 mg/ 100 mg muscle (P < 0.01) and the half-maximal activation constant of glycogen synthase for glucose 6-phosphate decreased from 0.62 & 0.05 to 0.25 * 0.02 mM (P < 0.001). Total glycogen synthase activity was unchanged. In the absence of phosphatase inhibitors, glucose 6-phosphate-independent glycogen synthase activity of the crude enzyme extract increased during in vitro incubation. The initial rate of activation (through dephosphorylations) of glycogen synthase was 0.18 t 0.06 vs. 0.37 t 0.03 Ugmin-lomg-’ protein before and after exercise, respectively (P c 0.02). The total as well as the glycogen-associated phosphoprotein phosphatase activity was, however, unaffected by exercise.
presently known about the in vivo regulation of the phosphatases. Both the total and glycogen-associated phosphatase-1 activity are reduced in rat skeletal muscle after 24 h of fasting or 38 h after streptozotocin-induced diabetes (29). Moreover, recent findings by Freymond et al. (10) suggest that glycogen synthase phosphatase activity in skeletal muscle is decreased in insulin-resistant subjects, thus contributing to the impaired responsiveness of glycogen synthase to insulin stimulation in these patients (10, 32). Still, it remains to be verified whether phosphatase activity in human skeletal muscle is modulated when glycogen levels are reduced in vivo as a result of a physical exercise bout. Thus the present study aimed to explore the effect of exercise on the regulatory mechanisms of carbohydrate metabolism in human skeletal muscle by studying solubilized and wheat germ agglutinin (WGA) -purified insulin receptors for insulin binding and insulin-stimulatable kinase activity, as well as the activities of glycogen synthase and the phosphoprotein phosphatases.
exercise; insulin receptor tase; human muscle
Subjects. Sixteen healthy nonobese students (13 males and 3 females) volunteered to participate in the study. Their mean age was 24 t 1 yr, and mean body mass index was 22.6 t 0.6 kg/m’. None were receiving any medication. The study was approved by the local ethical committee, and all subjects gave informed consent according to the Second Declaration of Helsinki. Protocol. Within 7 days before the exercise test, the maximal aerobic capacity (Vozmax) was estimated in each person by a standard incremental exercise test using a bicycle ergometer (1). Moreover, each subject was instructed not to perform any major physical exertion within 72 hr before the examination. The exercise test began at 8:00 A.M. after an overnight fast. Initially, a catheter was inserted in a forearm vein for blood sampling in the basal state and throughout the exercise test. Next, with the use of a Bergstrom biopsy needle, a muscle biopsy was obtained from the vastus lateralis muscle. Under local anesthesia (1% lidocaine) a small incision was made through the skin and muscle sheath 15-20 cm above the knee corresponding to the vastus lateralis. The biopsy needle was inserted and 200-250 mg muscle were aspirated. The muscle biopsies were immediately inspected, and blood and fat remnants were removed. Thus within 15 s the biopsy was deep-frozen in liquid nitrogen,
kinase;
glycogen synthase
phospha-
PHYSICAL EXERCISE, glucoseuptakeofcontracting muscle increases markedly despite low physiological concentrations of insulin. Several studies of the regulatory mechanisms of glucose metabolism in exercising muscle have been conducted in animals and humans. In the acute phase of an exercise bout, local insulin-independent factors account for the increased glucose metabolism and the enhanced insulin sensitivity (25, 26). The signaling mechanism for increased glucose turnover during and after an exercise bout is not fully understood. The postexercise increase in insulin sensitivity (2) may involve both the insulin receptor function as well as postreceptor pathways for glucose metabolism. The breakdown of glycogen stores might be one local initiator to enhance glucose metabolism in exercising muscle (2, 25). One of the postreceptor regulatory systems is the glycogen synthase, which controls glycogen synthesis and is strongly stimulated after muscular exercise (20). Different phosphoprotein phosphatases (phosphatase-1 and phosphatase-2) are able to activate glycogen synthase through dephosphorylations (16). However, very little is DURING
0193-1849/90
$1.50 Copyright
METHODS
0 1990 the American
Physiological
Society
E957
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E958
EXERCISE
AND
GLYCOGEN
SYNTHASE
where it was stored until the biochemical analyses were performed. Each subject then exercised for 60 min using a bicycle ergometer at a load corresponding to 60% of the individual V02 max as calculated from above. Every 15 min throughout the exercise, blood samples were collected. Within 10 min after termination of the exercise, a second muscle biopsy was obtained from the same thigh as before at a distance of -5 cm from the first incision, using exactly the same procedure. During the first 2 h of the recovery period, while the subject was still fasting, blood samples were collected every 30 min. All the assays for insulin receptor binding, kinase activity, glycogen synthase activity, and phosphatase activity in the pre- and postexercise muscle biopsies were run in parallel on the same day to eliminate the influence of any day-to-day variation. Preparation of solubilized insulin receptors. The muscle insulin receptors were solubilized and purified as previously described (la). Briefly, the frozen muscle biopsy was homogenized in an ice-cold buffer (1 ml/100 mg muscle) containing (in mM) 25 N-Z-hydroxyethylpiperazine-A/‘-2ethanesulfonic acid (HEPES), 4 EDTA, 10 NaF, 1 benzamidine, and 2 phenylmethylsulfonyl fluoride (PMSF) and also 9 X lo5 kallikrein inhibitor units/ 1 (KIU/l) aprotinin and 1% (vol/vol) Triton X-100, pH 7.4, using an ice-cooled glass homogenizer with a motordriven nylon pestle (2,000 revolutions/min). The homogenate was centrifuged at 3,200 g for 10 min at 4°C. From the supernatant 25 ~1 were saved for the glycogen synthase assay. The remaining supernatant was incubated during rotation end-over-end for 30 min at 4°C and then centrifuged at 150,000 g for 90 min. The 150,000-g supernatant (6 ml) was recycled three times on a 0.5 x l.Ocm WGA-Sepharose column. The column was washed at 4°C with 50 ml of a buffer containing 25 mM HEPES, 150 mM NaCl, and 0.05% (vol/vol) Triton X-100, pH 7.4. The receptor proteins were eluted from the WGA column with a buffer containing (in mM) 300 N-acetylD-glucosamine, 50 HEPES, 110 NaCl, 2.5 KCl, 1 CaC12, and 1 MgC12 and also 0.05% Triton X-100 and 10% (vol/ vol) glycerol, pH 7.6. Finally, PMSF and aprotinin were added to the eluate in final concentrations of 2 mM and 9 x 10” KIU/l, respectively. Insulin binding. Aliquots (30 ~1) of the wheat germ eluates were incubated in duplicate in a total volume of 250 ~1 in a buffer containing (final concentrations, in mM) 25 HEPES, 135 NaCl, 4.8 KCL, 1.7 MgSO,, 2.5 CaC12, and 1.2 sodium phosphate and also 1% (wt/vol) human albumin (pH 7.6) and 14 pM 1251-labeled insulin (kindly donated by NOVO Research Institute, Copenhagen, Denmark), with or without increasing concentrations of unlabeled insulin at 4°C for 18 h. Receptorbound insulin was precipitated by 25% (wt/vol) polyethylene glycol (PEG; mol wt 6,000) and bovine y-globulin (5). After centrifugation at 2,600 g for 10 min at 4”C, the precipitates were washed twice with 1 ml 12.5% (wt/vol) PEG and counted in a gamma counter (LKB-minigamma). Insulin binding data was analyzed by a Scatchard plot using the LIGAND program of Munson and Rodbard (22).
IN
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Insulin receptor kinase assay. The ability of the insulin receptor to phosphorylate exogenous substrates was tested using the synthetic peptide poly( Glu4 Tyr’) (Sigma Chemical, St. Louis, MO) (4). Aliquots (120 ~1) of receptor preparations were preincubated in the absence and presence of 1O-11- 10B7 M insulin for 60 min at 21°C. Poly(Glu4 Tyr’) (20 ~1, final concentration 0.2 mg/ml) was added, and the phosphorylation reaction was initiated by adding 20 ~1 of (in mM) 4 MnC12, 8 MgC12, and 20 ATP and also -2 &i [T-~~P]ATP (New England Nuclear, Boston, MA). All tubes were incubated at 21°C for 30 min. The phosphorylations proceeded linearly under these conditions for >60 min (data not shown). The reaction was terminated by spotting 80 ~1 of the reaction mixture on 2-cm squares of Whatman 31ET filter paper and immersing the papers in 10% (wt/vol) trichloroacetic acid containing 10 mM sodium pyrophosphate. After extensive washing, the papers were counted for 32P in a liquid scintillation counter (Beckman). Insulin receptor kinase activity was normalized for the number of high-affinity insulin binding sites in the eluate, as calculated from the above-mentioned approach, and thus expressed as femtomoles phosphate incorporated into poly( Glu4 Tyr’) per femtomole insulin receptors per minute. Glycogen synthase activity and glycogen content. A 25~1 sample of the supernatant from the first centrifugation of the homogenate was diluted with 1,475 ~1 of (in mM) ice-cold 63 tris(hydroxymethyl)aminomethane (Tris) . HCl, 25 NaF, and 26 EDTA (pH 7.8). Thirty microliters of this enzyme extract were incubated in duplicate at 30°C with 60 ~1 of (in mM) 0.13 UDP-[UJ4C]glucose (New England Nuclear), 63 Tris . HCl, 25 NaF, 26 EDTA, and O-6.7 glucose 6-phosphate (G-6-P) and also 6.7 mg/ ml glycogen, (pH 7.8) (20). After 15 min, the reaction was stopped by spotting 75 ~1 of the mixture on Whatman 31ET filter papers, which after extensive washing with ethanol (66% vol/vol) were counted in a scintillation counter (Beckman) for 14C incorporated into glycogen. Protein determinations of the enzyme extract were performed using the Bio Rad dye reagent (Munich, FRG). Glycogen synthase activity was expressed as nanomoles UDP-glucose incorporated into glycogen per minute per milligram soluble protein in the homogenate. In the text, maximal activity refers to the glycogen synthase activity in the presence of 6.7 mM G-6-P. One unit (U) corresponds to the incorporation of 1 nmol UDP-glucose into glycogen per minute. Fractional velocities were calculated as glycogen synthase activity in the presence of subsaturating levels of G-6-P divided by glycogen synthase activity in the presence of 6.7 mM G-6-P. The concentration of G-6-P giving half-maximal stimulation of the glycogen synthase (A0.5 for G-6-P) was calculated using a Hill plot (20). Muscle glycogen was determined from a weighed muscle sample after acid hydrolysis (1 N HCl) at 100°C for 2 h (25). The concentration of hydrolyzed glucose residues was measured with a glucose oxidase method (Boehringer Mannheim, FRG). Conversion rate of endogenous glycogen synthase. Approximately 25 mg of muscle were used to determine the rate of activation of the endogenous glvcoeen svnthase. ” “4 ” d -----,
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EXERCISE
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GLYCOGEN
SYNTHASE
i.e., the rate of conversion of the endogenous glycogen synthase from the G-6-P-dependent (D) form to the G6-P-independent (I) form. The muscle biopsy was homogenized in an ice-cooled buffer (230 ,ul buffer/l0 mg muscle) containing (in mM) 50 Tris . HCl, 10 EDTA, and 50 2mercaptoethanol, pH 7.8. The homogenate was centrifuged at 6,000 g for 20 min at 4°C. From the supernatant 100 ~1 were diluted with 75 ~1 buffer and then incubated at 30°C. The activation reaction was terminated at 0, 5, 10, 15, and 30 min by diluting 25 ~1 of the mixture in 975 ~1 of an ice-cold buffer containing (in mM) 50 Tris HCl, 20 EDTA, and 130 NaF, pH 7.8. Fluoride was able to inhibit this reaction totally, indicating that the activation of the glycogen synthase was a result of dephosphorylations. Glycogen synthase activity was measured in the absence and in the presence of 6.7 mM G-6-P as described above. The rate of conversion of the glycogen synthase was expressed as units of I-form of glycogen synthase produced per minute per milligram protein. Phosphatase activity. The phosphatase activity in muscle was measured with [32P]phosphorylase a as a substrate (6). To prepare [32P]phosphorylase a, 50 mg phosphorylase b were incubated with 1 mg phosphorylase kinase (both from Sigma), together with 1 mM (-0.4 mCi) [32P]ATP (New England Nuclear) for 60 min at 21°C in a buffer containing 50 (in mM) Tris HCl, 50 ,& glycerophosphate, 10 magnesium acetate, pH 8.2. The phosphorylated phosphorylase a was precipitated with ammonium sulfate (45% saturation) during centrifugation at 6,000 g for 15 min, at 4°C. The precipitate was washed once in a buffer containing (in mM) 50 Tris HCl, 1 EDTA, and 50 2-mercaptoethanol and also 45% ammonium sulfate, pH 7.0. Subsequently it was resuspended in a buffer containing (in mM) 50 Tris HCl, 1 EDTA, and 50 2-mercaptoethanol, pH 7.0. This suspension was dialyzed for 20 h against 2 liters of the same buffer. The crystals were precipitated by centrifugation at 1,700 g for 10 min at 4”C, and finally the [32P]phosphorylase a was resuspended in (in mM) 50 Tris. HCl, 250 NaCl, 1 EDTA, and 50 2-mercaptoethanol, pH 7.0 and stored at -2O’C. To measure phosphatase activity, -40 mg (wet weight) of the muscle biopsy were homogenized using the previously described technique in an ice-cold buffer (4 pl/mg muscle) containing (in mM) 4 EDTA, 1 benzamidine, 2 PMSF, and 14 Zmercaptoethanol, pH 7.2. The homogenate was centrifuged at 5,900 g, for 20 min at 4°C. From the resulting supernatant duplicates of 5 ~1 were incubated at 30°C with 50 ~1 [32P]phosphorylase a, and 50 ,ul of a buffer with (in mM) 4 EDTA, 20 imidazole, and 5 2-mercaptoethanol, with or without 0.1 mg glycogen, pH 7.5. After 5, 10, 15, and 25 min, aliquots of 25 ~1 were transferred to 800 ~1 10% (wt/vol) trichloroacetic acid. One hundred microliters of 2.5% (wt/vol) bovine serum albumin were added, and the proteins were precipitated by centrifugation at 1,400 g for 5 min. From the supernatants 500 ~1 were counted for released [32P]phosphate. The background radioactivity was determined in the absence of muscle enzyme extracts. It was stable and amounted to ~10% of the radioactivity released by the
IN
HUMAN
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E959
phosphatase after 10 min of incubation. To precipitate glycogen particles, 50 ~1 of the enzyme extract were ultracentrifuged at 150,000 g for 2 h at 4°C (29). The glycogen pellet was resuspended in a buffer (3 pl/mg of initial muscle weight) containing (in mM) 2.5 EDTA, 10 imidazol, 0.1 PMSF, 15 2-mercaptoethanol, and 0.1 benzamidine, pH 7.2. Glycogen-associated phosphatase activity was determined as described above. Phosphatase-1 accounts for the majority of the phosphatase activity in the muscle homogenate (16), and no efforts were exerted to eliminate any influence of the phosphatase-2A that might also be present in the cytosol of the muscle cells. Chemical quantities. Blood samples were analyzed for plasma insulin (NOVO radioimmunoassay kit for human insulin), plasma glucose (Merck enzymatic kit, Merck, Darmstadt, FRG), plasma free fatty acids (FFA; l7), plasma ketone bodies (3-hydroxybutyrate and acetic acid; 3l), and plasma lactate (15). StatisticaL methods. Differences before and after the exercise test were statistically analyzed with the paired t test. Variations in plasma concentrations of insulin and metabolites during the exercise test were tested for significance using a multiple analysis of variance (MANOVA) for repeated measurements. P values ~0.05 were considered statistically significant.
l
RESULTS
The variations in plasma concentrations of insulin (P < 0.005), FFA (P c O.OOl), lactate (P < 0.005), and ketone bodies (P c 0.025) during the exercise test are shown in Fig. 1. Throughout the test there were no variations in the levels of plasma glucose, which remained stable at 5.0 t 0.1 mM (Fig. 1). In muscle biopsies from ten subjects, the maximum insulin binding to the solubilized receptors (B,,,) was determined from Scatchard analysis of the binding data using the LIGAND computer program. However, the Scatchard plots were curvilinear (Fig. 2), and therefore the data were fitted to a one-site binding model under the assumption that the curvilinearity of the plotted raw data might be attributable to a nonspecific binding of the ligand. This nonspecific binding component was handled as a computer-fitted parameter (13), which amounted to 0.6 t 0.1 and 0.5 t 0.1% of the total added insulin before and after exercise, respectively [P = not significant (NS)] . After subtraction of this nonspecific binding component, B,,, was 60.9 t 5.6 before vs. 64.9 t 3.9 fmol/lOO mg muscle after exercise [P = NS; see Fig. 2 (inset)]. The insulin-stimulatable kinase activity of the insulin receptors was examined by measuring basal and insulinstimulated phosphorylation of the exogenous synthetic peptide poly(Glu4 Tyr’). The basal kinase activity was 1.86 t 0.17 vs. 1.61 t 0.10 fmol phosphate incorporated. fmol receptor-‘. min-l (P = NS) before and after exercise. On in vitro insulin stimulation there was a slightly lower kinase activity in the postexercise receptors (Fig. 3). This difference, however, did not attain statistical significance (P = 0.06). The recovery of soluble protein in the muscle homog-
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E960
EXERCISE
^r 5.0 5 14.0-
.-.-i-.-.-.-j--. I
-
GLYCOGEN
SYNTHASE
IN
HUMAN
MUSCLE
: I
: I
6.0-
AND
I
l
s 3.08 2 (3 2.0-
0
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:
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1 l I-
l\T : I
Insulin
100 (fmol/lOO
150
mg muscle)
2. Scatchard plots of mean values of insulin binding data from wheat germ agglutinin-purified muscle insulin receptors. Muscle biopsies were obtained before (0) and after (0) exercise. ‘251-insulin (14 pM) was added, together with increasing amounts of unlabeled insulin, and bound insulin was precipitated with polyethylene glycol (see METHODS). Inset: corresponding computer-derived Scatchard plots calculated on assumption that 0.6 t 0.1 and 0.5 t 0.1% of total added insulin were subject to nonspecific binding. Means t SE, n = 10. FIG.
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0.8-
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0.4-
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I
c $B400-
:
3
*
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0.1 Insulin
1 (nM)
10
100
FIG. 3. Kinase activity of wheat germ agglutinin-purified insulin receptors from skeletal muscle biopsies obtained before (0) and after (0) exercise. Peptide-incorporated 32P (Pi) was normalized to maximal insulin binding (B,,,) as calculated from Scatchard plots of insulin binding data (see METHODS). Means * SE, n = 10.
iii n O-
:
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TIME (hrs) FIG. 1. Metabolic profiles during exercise test. Blood samples obtained before, during, and after exercise were analyzed for plasma concentrations of (from top to bottom) glucose, insulin, free fatty acids (FFA), ketone bodies, and lactate. Exercise period (O-l h) is indicated by vertical dashed lines. Means t SE, n = 11.
enate was 65.6 t 2.5 and 64.0 t 1.6 mg protein/g wet muscle before and after exercise, respectively (NS), suggesting that the water content of the muscle biopsies did not change significantly during exercise. The glycogen content of the skeletal muscle biopsies diminished significantly from 3.35 t 0.26 to 1.85 k-O.13 mg/lOO mg muscle (P c 0.01) after the exercise bout. Glycogen synthase was activated as demonstrated by an increase in the fractional velocities (Fig. 4) and a significant reduction in A0.5for G-6-P from 0.62 t 0.05 to 0.25 t 0.02 mM (P c 0.001). Total glycogen synthase activity was unchanged by exercise and amounted to 42.8 t 2.6 vs. 43.6 t 2.6 U/mg protein before and after exercise. - -? respectively. ’ ” * To explore the mechanism of activation of the endogenous glycogen synthase, the crude enzyme extracts from six pairs of muscle biopsies were incubated for various
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EXERCISE
AND
GLYCOGEN
SYNTHASE
IN
HUMAN
E961
MUSCLE
r
0
0.01
0.1
1
[Glucose-6-phosphate]
10
(mM)
FIG. 4. Fractional velocities of glycogen synthase at various glucose 6-phosphate levels (O-6.7 mM) in muscle biopsies obtained before (0) and after (0) exercise. Means * SE, n = 10.
10
Incubation
v i
I
1
I
I
1
nI
1
0
5
10
15
20
25
30
35
Time
(min)
Incubation
(min)
FIG. 6. Phosphorylase phosphatase activity measured as release of phosphate from [“2P]phosphorylase a. Phosphatase activity was measured both in enzyme extracts of crude homogenate (A) and in glycogen particles (B) from muscle biopsies obtained before (0) and after (0) exercise test. Means t SE, n = 6.
0 1
O!
20
Time
FIG. 5. Conversion of glycogen synthase to its glucose 6-phosphateindependent form (I form) during in vitro incubation of crude enzyme extract from skeletal muscle biopsies in absence (00, solid line) and in presence of 130 mM NaF (VA, dashed line). Muscle biopsies were obtained before (OA) and after (OV) exercise. Means t SE, K! = 6.
periods of time in the presence and absence of fluoride (a serine phosphatase inhibitor). Fluoride was able to inhibit the activation process totally (Fig. 5). Glycogen synthase activity was determined repeatedly in the absence of G-6-P during 30 min of incubation. The rate of conversion of the muscle glycogen synthase to the I form was calculated from the slope of the initial part (the first 10 min) of the reaction curve as shown in Fig. 5. This rate increased significantly from 0.18 t 0.07 before exercise to 0.37 t 0.03 U. min-l . mg protein-l after exercise (P < 0.05). Thus the exercise-induced activation of glycogen synthase appeared to be a result of dephosphorylations. The difference in the rate of glycogen synthase activation disappeared after lo-15 min of incubation (Fig. 5). To further explore the activation of glycogen synthase, the phosphoprotein phosphatase activity in the muscle was measured. This was done by using [32P]phosphorylase a as a substrate. It appears from Fig. 6 that the phosphatase activity was linear only within the first 510 min of incubation. The postexercise phosphatase activity of the total enzyme extract of the muscle biopsy was very similar to the preexercise phosphatase activity (Fig. 6A ). Addition of exogenous glycogen (1 mg/ml) to
the reaction buffer did not interfere with the phosphatase reaction (data not shown). Moreover, in precipitated glycogen particles, the glycogen-associated phosphatase activity was also unaffected by physical exercise (Fig. W . DISCUSSION
Physical exercise leads to major changes in the carbohydrate metabolism of contracting muscle. Using a crude membrane preparation from skeletal muscle, insulin binding has been reported both to increase (30) and to decrease (3) during high-intensity exercise. In the present study, in human skeletal muscle, we show that the amount of soluble insulin receptors recovered from WGA columns was the same before and after exercise. The insulin-stimulated kinase activity of the insulin receptors was slightly lower after exercise. However, this difference did not attain statistical significance. In rat skeletal muscle, insulin binding and kinase activity of solubilized insulin receptors were also unchanged after an acute exercise bout (28). Likewise, in patients with insulin-dependent diabetes, 6 wk of moderate physical training did not change the muscle insulin receptor binding or kinase function (la). However, in rats heavy physical training for 4 wk increased the insulin receptor binding, whereas the insulin-stimulated kinase activity decreased correspondingly in the vastus intermedius muscle (9) and in the biceps femoris muscle (27). In contrast, no change was found in binding and kinase activity of insulin receptors from the tensor fascia lata muscles (27). Because catecholamines inhibit insulin receptor kinase activity (12, 23) this postexercise reduction in receptor kinase activity found in some muscles
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may be a result of the increased sympathoadrenergic activity during exercise. In our study the influence of acute exercise on muscle glycogen levels, glycogen synthase activity, and the rate of glycogen synthase activation, as well as the phosphatase activity was also examined. As expected, the total (maximal) activity of the glycogen synthase was unchanged after termination of the exercise bout. It is to be noted, however, that a postexercise increase in total glycogen synthase activity may occur after 24-48 h of recovery (20). Likewise, more chronic physical training increased the total activity of glycogen synthase in skeletal muscle from insulin-dependent diabetic patients (1 a.) The present results confirm several previous findings (2,8,20) that glycogen synthase is activated after muscle exercise, i.e., it becomes more sensitive to the allosteric activator G-6-P and thus it becomes stimulated in favor of glycogen synthesis. Regulation of glycogen synthase activity is generally believed to be a result of phosphorylation-dephosphorylation reactions. However, it remains to be shown whether glycogen synthase is actually dephosphorylated in vivo during or after glycogen-depleting exercise. The fact that fluoride was able to inhibit the in vitro activation of glycogen synthase provides indirect evidence that dephosphorylation processes were involved. Therefore, we measured the rate of activation of endogenous glycogen synthase in the crude enzyme extract simply by leaving out the phosphatase inhibitor (fluoride) in the buffers. Our results indicate that the initial rate of glycogen synthase conversion through dephosphorylations was nearly doubled after the exercise bout. The possibility exists that glycogen synthase had become a more suitable substrate for the phosphatases after exercise. However, if the more dephosphorylated forms of glycogen synthase should become more activable by phosphatases, we would expect the activation curve of glycogen synthase to show an upward concavity. This was not the case, as shown in Fig. 5. It was important, therefore, to consider whether there was a concomitant increase in the activity of the phosphatases that dephosphorylate and thereby activate glycogen synthase. The phosphoprotein phosphatase activity was analyzed by measuring the release of phosphate from an exogenous phosphorylase a. The arguments for using this substrate are several. First, protein phosphatase-1, also termed phosphorylase phosphatase, accounts for the majority of the potential phosphatase activity in rabbit skeletal muscle (16). Second, phosphatase-1 is the only protein phosphatase specifically associated with the protein-glycogen complex, where glycogen synthase is located (16, 18). Third, phosphorylase a is a much more well-defined substrate than glycogen synthase, which can be phosphorylated at multiple sites. Phosphoprotein phosphatase activity in exercised, glycogen-depleted muscles was identical to the preexercise activity both in the soluble enzyme extract of the crude muscle homogenate as well as in the precipitated glycogen particles. This is at variance with studies in vitro showing the ability of glycogen to inhibit the phosphatase, as determined both from measurements of the rate
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of dephosphorylation of [32P]phosphorylase a (21) and from studies of the rate of activation of the D form of glycogen synthase (24). Because the present data suggest that activation of glycogen synthase during glycogen-depleting exercise does not involve an increased activity of the endogenous phosphatases, alternative mechanisms might be involved in the activation of glycogen synthase after acute exercise. Local factors in or among the glycogen particles may be speculated to account for the increased rate of glycogen synthase dephosphorylations. The fact that the glycogen-associated phosphatase activity was unchanged after glycogen depletion suggests that glycogen-linked phosphatases were not liberated together with the glucose residues after glycogen breakdown. Accordingly, a rearrangement of glycogen synthase enzymes and phosphatase-1 enzymes may take place within the glycogen particles, for example to bring glycogen synthase into a more favorable position for the phosphatase after glycogen depletion. Increased adrenergic activity during an exercise bout leads to a rapid increase in the concentration of adenosine 3’,5’-cyclic monophosphate (CAMP) in muscle (11). Epinephrine has been reported to promote the translocation of phosphatase-1 from glycogen to cytosol in rat skeletal muscle (29) through a CAMP-dependent protein kinase-mediated phosphorylation of the glycogen-binding G component of the glycogen-associated phosphatase-1 (14). Moreover, elevated muscle CAMP levels activate an inhibitor of phosphatase-1 (inhibitor-l) through a CAMP-dependent protein kinase (7). Therefore, the coexistence of increased glycogen synthase activity and increased adrenergic activity appear to be conflicting. Indeed, during muscle exercise the glycogen phosphorylase activity is increased (1 l), and glycogenolysis is more pronounced than glycogen synthesis. Glycogen synthase activation is probably primarily important when glycogen resynthesis is needed after exercise. Very recent data from Kida et al. (19) show an inactivation of glycogen synthase and a slower activation of an exogenous D form of glycogen synthase in enzyme extracts of human muscle biopsies obtained within a few seconds after termination of muscle contractions. However, more direct measurements of the phosphatase activity were not performed. The design of this study varied from ours in a number of respects. First, to provide a more activated form of glycogen synthase, patients were examined after a meal. Second, exercise consisted of exhaustive isometric contractions under anoxic conditions. Third, the duration of the exercise bout was -1 min. Unfortunately, glycogen levels were not measured. Most important, the above findings demonstrate that glycogen synthase activity can be rapidly changed during an exercise bout. From the present study on biopsies of human muscle obtained in the recovery phase of an acute exercise bout, we conclude that glycogen synthase is more activable through dephosphorylation reactions. This activation is not a result of an increased insulin-receptor binding or kinase function, not is it attributable to an increase in the total or the glycogen-associated phosphatase activity.
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Thus exercise-induced glycogen depletion is postulated to result in a rearrangement of the glycogen-linked enzymes to favor dephosphorylations of glycogen synthase. We thank Dr. Erik A. Richter for critical review of our manuscript and Lisbet Blak, Tove Skrumsager, and Pernille Sonne for excellent technical assistance. This study was supported by grants from the Danish Medical Research Council, Danish Diabetes Association, Aarhus Universitets Forskningsfond, NOVO Foundation, and Nordisk Insulin Foundation. Address reprint requests to J. F. Bak. Received
15 November
1989; accepted
in final
form
28 February
1990.
REFERENCES 1. ASTRAND, I. Aerobic work capacity in men and women with special reference to age. Acta Physiol. Stand. 49, Suppl. 169: 9-92, 1960. la.BAK, J. F., U. K. JACOBSEN, F. S. JORGENSEN, AND 0. PEDERSEN. Insulin receptor function and glycogen synthase activity in skeletal muscle biopsies from patients with insulin-dependent diabetes mellitus: effects of physical training. J. CZin. EndocrinoZ. Metab. 69: 158-164, 1989. 2 BOGARDUS, C., P. THUILLEZ, E. RAVUSSIN, B. VASQUEZ, M. NARIMIGA, AND S. AZHAR. Effect of muscle glycogen depletion on in vivo insulin action in man. J. CZin. Inuest. 72: 1605-1610, 1983. 3. BONEN, A., M. H. TAN, P. CLUNE, AND R. L. KIRBY. Effects of exercise on insulin binding to human muscle. Am. J. Physiol. 248 (EndocrinoZ. Metab. 11): E403-E408, 1985. 4. BRAUN, S., W. E. RAYMOND, AND E. RACKER. Synthetic tyrosine polymers as substrates and inhibitors of tyrosine-specific protein kinases. J. BioZ. Chem. 259: 2051-2054, 1984. 5. BURANT, C. F., M. K. TREUTELAAR, G. E. LANDRETH, AND M. G. BUSE. Phosphorylation of insulin receptors solubilized from rat skeletal muscle. Diabetes 33: 704-708, 1984. E. G. KREBS, AND E. H. FISCHER. 6. CHAN, C. P., S. J. MCNALL, Stimulation of protein phosphatase activity by insulin and growth factors in 3T3 cells. Proc. NatZ. Acad. Sci. USA 85: 6257-6261, 1988. 7. COHEN, P., J. G. FOULKES, C. F. B. HOLMES, G. A. NIMMO, AND N. K. TONKS. Protein phosphatase inhibitor-l and inhibitor-2 from rabbit skeletal muscle. Methods Enzymol. 159: 427-437, 1988. 8. DEVLIN, J. T., AND E. S. HORTON. Effects of prior high-intensity exercise on glucose metabolism in normal and insulin resistant men. Diabetes 34: 973-979, 1985. G. L., M. K. SINHA, AND J. F. CARO. Insulin receptor 9. DOHM, binding and protein kinase activity in muscles of trained rats. Am. J. Physdol. 252 (Endocrinol. Metab. 15): E170-E175, 1987. 10. FREYMOND, D., C. BOGARDUS, M. OKUBO, K. STONE, AND D. MOTT. Impaired insulin-stimulated muscle glycogen synthase activation in vivo in man is related to low fasting glycogen synthase phosphatase activity. J. CZin. Inuest. 82: 1503-1506, 1988. 11. GOLDFARB, A. H., J. F. BRUNO, AND P. J. BUCKENMEYER. Intensity and duration of exercise effects on skeletal muscle CAMP, phosphorylase, and glycogen. J. AppZ. Physiol. 66: 190-194, 1989. 12. HARING, H., D. KIRSCH, B. OBERMAIER, B. ERMEL, AND F. MACHICAO. Decreased tyrosine kinase activity of insulin receptor isolated from rat adipocytes rendered insulin-resistant by catecholamine treatment in vivo. Biochem. J. 234: 59-66, 1986. 13. HELMERHORST, E. The insulin-receptor interaction: is the kinetic approach for inferring negative-cooperative site-site interactions valid? Biochem. Biophys. Res. Commun. 147: 399-407, 1987. 14. HIRAGA, A., AND P. COHEN. Phosphorylation of the glycogenbinding subunit of protein phosphatase-1G by cyclic-AMP-depend-
IN
HUMAN
MUSCLE
E963
ent protein kinase promotes translocation of the phosphatase from glycogen to cytosol in rabbit skeletal muscle. Eur. J. Biochem. 161: 763-769, 1986. 15. HOHORST, H. J. Lactate analysis. In: Methods of Enzymatic Analysis, edited by H. U. Bergmeyer. New York: Academic, 1963, p. 266270. I. S., A. A. STEWART, AND P. COHEN. The protein 16. INGEBRITSEN, phosphatases involved in cellular regulation. Eur. J. Biochem. 132: 297-307,1983. determination of free fatty 17. ITAYA, K., AND M. UI. Calorimetric acids in biological fluids. J. Lipid. Res. 6: 16-22, 1968. 18. KHATRA, B. S. Purification of glycogen-bound high-molecularweight phosphoprotein phosphatase from rabbit skeletal muscle. Methods Enzymol. 159: 368-377, 1988. 19. KIDA, Y., A. KATZ, A. D. LEE, AND D. M. MOTT. Contractionmediated inactivation of glycogen synthase is accompanied by inactivation of glycogen synthase phosphatase in human skeletal J. 259: 901-904, 1989. 2. muscle. Biochem. KOCHAN, R. G., D. R. LAMB, S. A. LUTZ, C. V. PERRILL, E. M. REINMANN, AND K. K. SCHLENDER. Glycogen synthase activation in human skeletal muscle: effects of diet and exercise. Am. J. Physiol. 236 (Endocrinol. Metab. Gastrointest. Physiol. 5): E660E666, 1979. 21. MELLGREN, R. L., AND M. COULSON. Coordinated feedback regulation of muscle glycogen metabolism: inhibition of purified phosphorylase phosphatase by glycogen. Biochem. Biophys. Res. Commun. 114: 148-154,1983. 22. MUNSON, P. J., AND D. RODBARD. LIGAND: a versatile computerized approach for characterization of ligand-binding systems. AnaZ. Biochem. 107: 220-239,198O. 23. OBERMAIER, B., B. ERMEL, D. KIRSCH, J. MUSHACK, E. RATTENHUBER, E. BIEMER, F. MACHICAO, AND H. U. HARING. Catecholamines-and tumour promoting phorbolesters inhibit insulin receptor kinase and induce insulin resistance in isolated human adipocytes. Diabetologia 30: 93-99, 1987. 24. OKUBO, M., C. BOGARDUS, S. LILLIOJA, AND D. M. MOTT. Glucose6-phosphate stimulation of human muscle glycogen synthase phosphatase. Metabolism 37: 1171-1176, 1988. 25. RICHTER, E. A., L. P. GARETTO, M. N. GOODMAN, AND N. B. RUDERMAN. Enhanced muscle glucose metabolism after exercise: modulation by local factors. Am. J. Physiol. 246 (Endocrinol. Metub. 9): E476-E482, 1984. 26. RICHTER, E. A., T. PLOUG, AND H. GALBO. Increased muscle glucose uptake after exercise. No need for insulin during exercise. Diabetes 34: 1041-1048, 1985. 27. SANTOS, R. F., C. E. MONDON, G. M. REAVEN, AND S. AZHAR. Effects of exercise training on the relationship between insulin binding and insulin-stimulated tyrosine kinase activity in rat skeletal muscle. MetaboZism 38: 376-386, 1989. 28. TREADWAY, J. L., D. E. JAMES, E. BURCEL, AND N. B. RUDERMAN. Effect of exercise on insulin receptor binding and kinase activity in skeletal muscle. Am. J. Physiol. 256 (Endocrinol. Metab. 19): E138-E144,1989. 29. VILLA-M• RUZZI, E. Effects of streptozotocin-diabetes, fasting and adrenaline on phosphorylase phosphatase activities of rat skeletal muscle. MOL. Cell. EndocrinoZ. 47: 43-48, 1986. 30. WEBSTER, B. A., S. R. VIGNA, AND T. PAQUETTE. Acute exercise, epinephrine, and diabetes enhance insulin binding to skeletal muscle. Am. J. Physiol. 250 (Endocrinol. Metab. 13): E186-E197, 1986. 31. WILDENHOF, K. E. A micromethod for enzymatic determination of acetoacetate and 3-hydroxybutyrate in blood and urine. Stand. J. CZin. Lab. Invest. 25: 171-175, 1970. 32. YOUNG, A. A., C. BOGARDUS, D. WOLFE-LOPEZ AND D. M. MOTT. Muscle glycogen synthesis and disposition of infused glucose in humans with reduced rates of insulin-mediated carbohydrate storage. Diabetes 37: 303-308, 1988. l
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