Divergent effects of exercise on metabolic and mitogenic signaling pathways in human skeletal muscle

^a Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, SE-114 86, Sweden; the
^b Department of Human Biology, University College of Physical Education and Sports, Stockholm, SE 114 86, Sweden; the
^c Department of Clinical Physiology, Karolinska Hospital, SE 171 76, Sweden;
^d Department of Molecular Medicine, Karolinska Hospital, SE 171 76, Sweden; and the
^e Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California, 94305-5332, USA

	ABSTRACT

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The molecular signaling mechanisms by which muscle contractions lead to changes in glucose metabolism and gene expression remain largely undefined. We assessed whether exercise activates MAP kinase proteins (ERK1/2, SEK1, and p38 MAP kinase) as well as Akt and PYK2 in skeletal muscle from healthy volunteers obtained during and after one-leg cycle ergometry at ~70% VO_2max. Exercise led to a marked increase in ERK1/2 phosphorylation, which rapidly decreased to resting levels upon recovery. Exercise increased phosphorylation of SEK1 and p38 MAP kinase to a lesser extent than ERK1/2. In contrast to ERK1/2, p38 MAP kinase phosphorylation was increased in nonexercised muscle upon cessation of exercise. Phosphorylation of the transcription factor CREB was increased in nonexercised muscle upon cessation of exercise. Exercise did not activate Akt or increase tyrosine phosphorylation of PYK2. Thus, exercise has divergent effects on parallel MAP kinase pathways, of which only p38 demonstrated a systemic response. However, our data do not support a role of Akt or PYK2 in exercise/contraction-induced signaling in human skeletal. Activation of the different MAP kinase pathways by physical exercise appears to be important in the regulation of transcriptional events in skeletal muscle.—Widegren, U., Jiang, X.-J., Krook, A., Chibalin, A. V., Björnholm, M., Tally, M., Roth, R. A., Henriksson, J., Wallberg-Henriksson, H., Zierath, J. R. Divergent effects of exercise on metabolic and mitogenic signaling pathways in human skeletal muscle. FASEB J. 12, 1379–1389 (1998)

Key Words: insulin action • mitogen-activated protein kinase • Akt kinase • calcium-dependent tyrosine kinase

	INTRODUCTION

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INSULIN AND CONTRACTILE ACTIVITY are potent stimulators of glucose metabolism (1, 2) and gene expression in skeletal muscle (3, 4). Although the signaling pathway by which insulin elicits downstream metabolic and mitogenic responses is fairly well established (5), the molecular signaling mechanism by which muscle contractions lead to increased glucose transport, metabolism, and gene expression remains largely undefined.

Phosphatidylinositol 3 kinase (PI-3 kinase)² plays an essential role in insulin-stimulated glucose transport, since GLUT4 (insulin-responsive glucose transporter) translocation and glucose transport are inhibited after in vitro exposure of isolated skeletal muscle to specific inhibitors of PI-3 kinase (2, 6–9). In contrast, muscle contraction-induced GLUT4 translocation and glucose transport are mediated by a PI 3-kinase-independent mechanism (6–8). The serine/threonine kinase Akt (PKB/Rac), a downstream target of PI 3-kinase, has been implicated to play a role in growth factor signaling to glucose transport (10–18). We have shown insulin increases Akt phosphorylation in human skeletal muscle via a PI 3-kinase-dependent pathway (9). However, Akt can be activated by okadaic acid (17) or by osmotic shock (19) through PI 3-kinase-independent pathways known to regulate glucose transport (20, 21). Overexpression of a constitutively active membrane-bound form of Akt in 3T3-L1 adipocytes (16, 17) and L6 myotubes (18) directly promotes glucose transport and translocation of GLUT1 and GLUT4 to the plasma membrane. Thus, Akt may be a point of convergence for insulin- and contraction-induced signaling pathways to glucose transport.

Acute exercise in humans (22) and rodents (23, 24) or in situ contraction of skeletal muscle in rodents (25) leads to enhanced insulin sensitivity. Exercise training also leads to increased muscle mass (4, 26), improved glucose tolerance (27, 28), and increased glucose transport capacity (29). Indeed, enhanced glucose uptake with chronic exercise training may be related in part to increased protein expression of GLUT4 (28, 30, 31). Recently, the mitogen-activated protein (MAP) kinase has been identified as a possible signaling pathway by which exercise or muscle contraction may lead to increased expression of muscle proteins (32–34). Several parallel MAP kinase pathways (ERK/MAP kinase; SAPK/JNK, and p38 HOG) integrate intracellular signals from diverse extracellular stimuli, including growth factors and/or various forms of cellular stress, thereby regulating gene transcription and protein synthesis (3, 35, 36). Little is known about the effects of muscle contraction on the regulation of MAP kinase signaling pathways in skeletal muscle. Furthermore, the transcription factors that link the MAP kinase cascade to changes in gene expression after physical exercise have not been identified.

There is substantial evidence that exercise increases glucose transport largely by a mechanism involving increased cytosolic calcium (37–42). In 3T3-L1 adipocytes, osmotic shock-induced glucose transport is accompanied by stimulation of a calcium-dependent tyrosine kinase (CADTK) (21), a newly identified rat homologue to the human PYK2 (43). CADTK/PYK2 is phosphorylated on tyrosine residues in response to various stimuli that elevate intracellular calcium concentration, as well as by protein kinase C (PKC) activation. Thus, PYK2 may be involved in contraction-induced signaling pathways.

Here we show that ERK1/2, SEK1, and p38 MAP kinase phosphorylation is rapidly and transiently elevated in exercised muscle. Furthermore, p38 MAP kinase phosphorylation is also elevated in nonexercised muscle. Thus, systemic factors may play a role in p38 MAP kinase activation. The magnitude of SEK1 and p38 MAP kinase phosphorylation was less than that observed for ERK1/2. In contrast to effects on MAP kinase proteins, exercise did not result in activation of Akt kinase or increase PYK2 tyrosine phosphorylation. Thus, the extent to which different MAP kinase pathways are activated by physical exercise may influence transcriptional events in skeletal muscle.

	METHODS

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Subjects
Fifteen healthy young volunteers (six females and nine males) participated in the study (age: 25±1 year; weight: 71.5±2.7 kg; height: 176±2 cm, BMI: 23.1±0.6 kg/m²). Informed consent was obtained from each subject and the study protocol was approved by the Ethical Committee at Karolinska Institute and Karolinska Hospital. Maximal oxygen uptake (VO_2max) for one- and two-leg cycle ergometry was determined 3 days before the experimental protocol was undertaken. VO_2max was 2.8 ± 0.4 and 3.8 ± 0.5 l/min for one- and two-leg exercise, respectively.

Open muscle biopsy procedure to obtain insulin-stimulated human skeletal muscle
An open muscle procedure was performed in three of the individuals. Biopsies were obtained under local anesthesia from the vastus lateralis portion of the quadriceps femoris, as previously described (2). Isolated muscle strips were prepared, placed in vials containing Krebs-Henseleit buffer supplemented with 5 mM Hepes and 0.1% bovine serum albumin (radioimmunoassay grade; Sigma, St. Louis, Mo.), and incubated for 40 min in the absence or presence of insulin (60 nM) as described (2).

Exercise protocol and muscle biopsy procedure
Subjects performed one-leg cycle ergometry at a load corresponding to ~70% of one leg VO_2max. After local anesthesia, an incision (5 mm long/10 mm deep) was made in the skin and muscle fascia, and a muscle biopsy (20–100 mg) was obtained from the vastus lateralis portion of the quadriceps femoris by means of a Weil-Blakesley's conchotome. Each biopsy was removed from a separate incision site, 3 cm apart. Muscle specimens were obtained from the nonexercised leg at 0 and 63 min ( Fig. 1), from the exercised leg at 10, 30, or 60 min of cycle ergometry, and at 15 or 60 min postexercise (recovery). No more than three biopsies were obtained from each leg. Muscle tissue was immediately frozen in liquid nitrogen and stored at -80°C.

View larger version (41K): [in this window] [in a new window]   Figure 1. Schematic representation of the study design to obtain skeletal muscle after one-legged bicycle exercise. Arrows represent time at which biopsy sampling was performed. No more than three biopsies were obtained from the right (exercised) leg.

Substrate and hormonal determinations
Blood samples were collected from a venous catheter in a cubital vein ~2 min before removal of each muscle biopsy. Plasma glucose concentration was analyzed as described by Lowry and Passonneau (44), and plasma lactate concentration was analyzed as described by Bergmeyer (45). Serum insulin was analyzed by RIA (insulin RIA 100 kit; Pharmacia, Uppsala, Sweden). Serum insulin-like growth factor I (IGF-I) was determined by radioimmunoassay after separation of IGFs from insulin-like growth factor binding proteins (IGFBPs) by acid-ethanol extraction and cryoprecipitation. To minimize interference of remaining IGFBPs, des(1–3)-IGF-I was used as radioligand (46). Intra- and interassay CVs were 4 and 11%, respectively. Serum IGFBP-1 levels were determined as described by Póvoa et al. (47). Intra- and interassay CVs were 3 and 10%, respectively.

Muscle lysate preparation and immunoblot analysis
Portions of skeletal muscle biopsies (20–30 mg) were homogenized in ice-cold buffer (20 mM HEPES pH 7.4, 1 mM EDTA, 5 mM EGTA, 10 mM MgCl₂, 50 mM ß-glycerophosphate, 1 mM Na₃VO₄, 2 mM DTT, 1% Triton X-100, 2 µg/ml leupeptin, 5 µg/ml aprotinin, 40 µg/ml PMSF). Homogenates were rotated for 60 min at 4°C and centrifuged at 15,000 x g for 45 min at 4°C. The supernatant was removed and protein concentration was determined (Bio Rad Protein Kit, Richmond, Calif.). Aliquots (30 µg) of the supernatant were separated by sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS-PAGE) (10% resolving gel) and blocked overnight (5% milk in Tris-buffered saline with 0.1% Tween 20). Each membrane was stained with Ponceau S immediately after protein transfer to provide visual evidence that uniform loading and electrophoretic transfer of proteins from all lanes was achieved. Membranes were immunoblotted with phospho-specific rabbit polyclonal antibodies (New England Biolabs, Beverly Mass.), including phospho-specific ERK1/ERK2 (1:1000), p38 MAPK (1:500), SEK1 (1:1000), and CREB (1:500), as indicated in the figures. The membranes were washed in Tris-buffered saline with 0.1% Tween 20. Bound antibodies were detected with peroxidase-linked anti-rabbit IgG (1:2000; Amersham, Buckinghamshire, England), incubated at room temperature (1 h). Protein phosphorylation was visualized by enhanced chemiluminescence (Amersham, Arlington Heights, Ill.), and phosphorylated proteins were quantified by densitometry. After immunoblotting with phospho-specific ERK or p38 anitbodies, membranes were stripped according to conventional methods specified by the manufacturer of the ECL reagents (Amersham), and immunoblotted with pan-ERK or p38 (New England Biolabs), respectively, for total ERK or p38 protein. To ensure that significant amounts of signaling intermediates were not retained in the pellet from the 15,000 x g centrifugation, the pellet was extracted by homogenization in buffer (30 mM Hepes, pH 7.5, 2 M Urea, 3.7% SDS). Aliquots of pellet and supernatant were immunoblotted with polyclonal anti-pan-ERK or p38 antibodies. Insignificant amounts of ERK and p38 were detected from the pellet extracts.

Immunoprecipitation and PYK2 phosphorylation
Muscle specimens (20 mg) were homogenized in ice-cold buffer and centrifuged as described above. The supernatant (1 mg) was immunoprecipitated overnight (4°C) with antiphosphotyrosine (Signal Transduction Laboratories, Lexington Ky.) or anti-PYK2 (Upstate Biotechnology, Lake Placid, N.Y.) antibody coupled to protein A-Sepharose. Immunoprecipitates were washed as indicated (48), resuspended in Laemmli sample buffer with 20 mM DTT, and heated (95°C) for 6 min. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blocked. Membranes were incubated with either anti-phosphotyrosine (RC20: 1:4000; Signal Transduction Laboratories, Lexington, Ky.) or anti-PYK2 antibody (1:1000), washed, and incubated with secondary antibody.

Akt kinase activity
Muscle specimens were lysed and centrifuged as described above for PYK2 immunoprecipitations. Aliquots of supernatant (600 µg) were immunoprecipitated with anti-Akt- antibody and Akt kinase activity was measured against a peptide substrate (GRPRTSSFAEG) based on a motif from glycogen synthase kinase 3 (49), as described in detail previously (50, 51). The Akt- antibody was raised in rabbit against a fusion protein of the PH domain of human Akt- and GST. [³²P] Incorporation into the peptide substrate was determined by resolving the reaction products on a 40% acrylamide gel. The gel was visualized on a phosphoImager (Bio-Rad), and the band corresponding to the peptide substrate was quantitated. Results are presented as arbitrary phosphoimager units.

Statistical analysis
Differences between substrate and hormone concentration at rest and during exercise were analyzed by one-way analysis of variance (ANOVA). When the ANOVA resulted in a significant F-ratio (P<0.05), the Newman Keuls post hoc test was used to identify statistical differences.

	RESULTS

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Substrate and hormone concentration at rest, during exercise, and at recovery
Plasma glucose, lactate, and serum insulin levels were assessed at rest, during exercise, and at recovery ( Table 1). Plasma glucose levels were not altered during the exercise or recovery periods. As expected, plasma lactate concentrations were increased after 30 min of exercise (P<0.001). Between 30 and 60 min of exercise, lactate levels decreased significantly (P<0.01), but remained significantly elevated (P<0.01) compared to resting levels. During recovery (15 min postexercise), lactate levels were not significantly different from preexercise resting levels. Throughout exercise and recovery, serum insulin levels were steadily and significantly reduced compared to resting levels (P<0.001). Serum IGF-1 levels increased 10% with exercise (P<0.05) and returned to preexercise levels upon recovery. Serum IGFBP-1 levels were similar between rest and exercise.

Divergent effects of insulin and exercise on Akt kinase activity
We determined whether Akt was a convergence point for insulin- and contraction-induced signaling pathways to glucose transport. Previously we had shown that insulin induces a fivefold increase in Akt kinase activity in rat soleus muscle (50). Furthermore, in human skeletal muscle, insulin elicits a concentration-dependent increase in Akt kinase activity (51). Akt activity was assessed in human skeletal muscle incubated in the absence or presence of 60 nM insulin, or in muscle biopsies obtained at rest or immediately after 30 or 60 min of exercise ( Fig. 2). Insulin induced a profound fivefold increase in Akt activity (n=3). In contrast, acute muscle contraction via bicycle exercise had no effect on Akt kinase (n=3).

View larger version (26K): [in this window] [in a new window]   Figure 2. Effects of exercise on Akt kinase activity in human skeletal muscle. A) Representative phosphoimage of phosphorylated GSK peptide in human skeletal muscle. Muscle samples were obtained at rest (left vastus lateralis muscle) or after 30 min exercise (right vastus lateralis muscle). For basal and insulin-stimulated conditions, muscle samples were obtained by means of an open muscle biopsy and incubated (40 min) in vitro as described in Methods, in the absence or presence of 60 nM insulin. Akt kinase activity in muscle lysates was measured against a peptide substrate based on a motif from GSK3 as described in Methods. B) Quantification of results presented in panel A. Values are reported as arbitrary phosphoImager units. Representative results from n = three muscles per group.

Exercise stimulation of ERK1/2 phosphorylation
A previous report provided evidence that exercise leads to phosphorylation of ERK1/2 in skeletal muscle (33). Here we further characterized the effects of exercise on ERK1/2 phosphorylation in human skeletal muscle. ERK1/2 was assessed in skeletal muscle biopsies using an antibody to phosphorylated Thr202 and Tyr204. Phosphorylation of ERK1 on these sites is known to activate this enzyme (52). First, we used a one-leg bicycle protocol to determine whether exercise-induced phosphorylation of ERK1/2 was a result of local or systemic factors. Consistent with the work of Aronson and co-workers (33), we also show that acute exercise leads to a profound increase in ERK1/2 phosphorylation in skeletal muscle ( Fig. 3A). Exercise (30 min) resulted in a 31 ± 8-fold increase (n=6) in ERK1/2 phosphorylation in skeletal muscle. Furthermore, since the increase in ERK1/2 phosphorylation was observed only in the exercised leg, our results provide evidence that local rather than systemic factors mediate this effect. Second, we established a time course for exercise-induced phosphorylation or ERK1/2 ( Fig. 3B). After 10 min of exercise, a marked increase in ERK1/2 phosphorylation was noted, approaching 50% of the maximal effect. ERK1/2 phosphorylation was further increased at 30 min of exercise, and this effect was maintained throughout the exercise bout.

View larger version (32K): [in this window] [in a new window]   Figure 3. Effects of exercise on ERK1/2 kinase phosphorylation. A) Representative autoradiograph of phospho-specific ERK1/2 kinase immunoblotting of human skeletal muscle (top panel). pan-ERK immunoblot on the same membrane to show equal loading (bottom panel). Muscles were obtained from the left vastus lateralis muscle during rest (0 and 63 min) or from the right vastus lateralis muscle during exercise (30 and 60 min) and recovery (15 or 60 min). B) ERK1/2 kinase phosphorylation was assessed in muscle samples from six subjects. Values are reported as percentage of exercise effect at 30 min.

Muscle contraction is known to have a persistent effect on glucose transport that can be observed up to 3 h after exercise (23). We next determined whether exercise has a persistent effect on ERK1/2 kinase phosphorylation in skeletal muscle. Phosphorylation of ERK1/2 decreased dramatically upon cessation of exercise (15 min recovery). After 60 min recovery, ERK1/2 phosphorylation was completely restored to resting levels. Our results suggest that in contrast to what has been observed for glucose transport, exercise does not have a persistent effect on ERK1/2 phosphorylation in muscle.

To assess whether repeated muscle biopsy sampling altered ERK1/2 phosphorylation, we performed a series of experiments in nonexercised humans whereby two biopsies were obtained from the same leg. Nonexercised muscle samples were obtained at 0 and 30 min or at 0 and 63 min from different incision sites in the same leg (n=3). Repeated muscle biopsy sampling did not alter ERK1/2 phosphorylation (data not shown). Thus, increased ERK1/2 phosphorylation observed with exercise is not a direct effect of the biopsy procedure.

Exercise induced phosphorylation of SEK1
SEK1 is a potent activator of SAPK in vitro and in vivo and is highly expressed in skeletal muscle (53). Phosphorylation of p54/48 SAP kinase by SEK dramatically stimulates the ability of SAP kinase to phosphorylate protein substrates such as c-Jun. We assessed SEK1 phosphorylation in skeletal muscle by using a phospho-specific antibody to Thr223. Exercise led to significant phosphorylation of SEK1 in skeletal muscle ( Fig. 4A). The magnitude of this effect was not as profound as that observed for ERK1/2. At 30 min of exercise, SEK1 phosphorylation was increased 2.5 ± 0.4-fold (n=5) compared to resting levels ( Fig. 4B). At 15 min recovery, SEK1 phosphorylation was increased to a level similar to that observed at 30 min exercise (n=4), and this effect persisted into the 60 min recovery period (data not shown). We next tested whether increased SEK1 phosphorylation postexercise was related to repeated biopsy sampling. In multiple biopsies obtained from the same leg of nonexercised individuals, no appreciable increase in SEK1 phosphorylation was noted ( Fig. 4C).

View larger version (25K): [in this window] [in a new window]   Figure 4. Effects of exercise on SEK1 phosphorylation. A) Representative autoradiograph of phospho-specific SEK1 immunoblotting of human skeletal muscle. Muscle samples were obtained as described in Fig. 3A. B) SEK1 phosphorylation in muscle samples from five subjects. Values are reported as percentage of exercise effect at 30 min. C) Representative autoradiograph of phospho-specific SEK1 kinase phosphorylation in repeated biopsy samples. Muscles were obtained as described in Fig. 3A.

Exercise induced p38 MAP (HOG) kinase phosphorylation
We assessed p38 MAP kinase phosphorylation in skeletal muscle by using a phospho-specific antibody that detects phosphorylation at Thr 180 and Tyr 182 (54). Both MKK3 and SEK phosphorylate p38 on tyrosine and threonine residues at the sequence T*GY*, resulting in p38 activation. Here we show that acute exercise (30 min) leads to a 2.2 ± 0.7-fold increase (n=7) in p38 MAP kinase phosphorylation in skeletal muscle ( Fig. 5A). Phosphorylation of p38 MAP kinase was maintained throughout the exercise bout and persisted into the 15 min recovery period ( Fig. 5B). Sixty minutes after exercise (recovery), p38 MAP kinase phosphorylation returned to resting levels (data not shown). Since we noted pronounced phosphorylation of p38 MAP kinase in nonexercised muscle obtained at the cessation of exercise, we assessed whether this was directly related to repeated biopsy sampling. Similar to ERK1/2, repeated muscle biopsy sampling did not lead to increased p38 MAP kinase phosphorylation ( Fig. 5C).

View larger version (27K): [in this window] [in a new window]   Figure 5. Effects of exercise on p38 MAP (HOG) kinase phosphorylation. A) Representative autoradiograph of phospho-specific p38 MAP kinase immunoblotting of human skeletal muscle (top panel). p38 immunoblot on the same membrane to show equal loading (bottom panel). Muscle samples were obtained as described in Fig. 3A. B) p38 MAP kinase phosphorylation in muscle samples from seven subjects. Values are reported as percentage of exercise effect at 30 min. C) Representative autoradiograph of phospho-specific p38 MAP kinase phosphorylation in repeated biopsy samples. Samples were obtained as described in Fig. 3A.

Effect of exercise on CREB phosphorylation
We assessed whether acute exercise leads to increased phosphorylation of the transcription factor CREB. CREB binds the cyclic AMP response element (CRE) and activates transcription in response to a variety of extracellular signals including cAMP, increased intracellular Ca²⁺, as well as several growth factors (55–58). CREB phosphorylation at Ser133 was determined using a phospho-specific antibody ( Fig. 6A). CREB-Ser133 phosphorylation did not increase in exercised muscle ( Fig. 6B). However, four of five subjects demonstrated a substantial increase in CREB phosphorylation in nonexercised muscle immediately upon cessation of exercise (60 min) ( Fig. 6C).

View larger version (25K): [in this window] [in a new window]   Figure 6. Effects of exercise on CREB phosphorylation. A) Representative autoradiograph of phospho-specific CREB immunoblotting of human skeletal muscle. Muscle samples were obtained as described in Fig. 3A. B) CREB phosphorylation in muscle samples from five subjects. Values are reported as percentage of exercise effect at 30 min. C) Individual results for CREB phosphorylation in muscle biopsies obtained in the rested (nonexercised) left leg before (rest 0 min) and after (rest 63 min) exercise of the right leg.

Effect of exercise on tyrosine phosphorylation of CADTK/PYK2
PYK2 is the human homologue of the recently identified calcium-sensitive tyrosine kinase CADTK/CAKb (43, 59–61). Activation of PYK2 has been suggested to play a role in stress-induced activation of c-Jun amino-terminal kinase (JNK) (43), activation of MAP kinase by G-protein coupled receptors (62), and stimulation of glucose transport by osmotic shock (21). We observed no change in tyrosine phosphorylation of PYK2 in human skeletal muscle after 30 or 60 min of exercise ( Fig. 7). Since hyperosmosis leads to tyrosine phosphorylation of PYK2 (21), we prepared muscle lysate from rat epitrochlearis muscle incubated for 20 min in the absence or presence 600 mM mannitol as a positive control for PKY2 activity in our assay system. Hyperosmosis led to a two- to threefold increase in tyrosine phosphorylation of PYK2 in rat epitrochlearis muscle (n=3).

View larger version (12K): [in this window] [in a new window]   Figure 7. A) Representative autoradiograph of tyrosine phosphorylation of PYK2 in human skeletal muscle. Muscle samples were obtained under resting conditions or with exercise (30 or 60 min) as described in Methods. Muscle lysate was immunoprecipitated with anti-phosphotyrosine and immunoblotting with anti-PYK2. Representative autoradiograph for n = five subjects. B) Representative autoradiograph of tyrosine phosphorylation of PYK2 in rat epitrochlearis muscle. Muscles were incubated for 20 min in the absence (basal) or presence of 600 mM mannitol (HO; hyperosmosis) and processed exactly as described in Methods for human muscle.

	DISCUSSION

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Recently, muscle contraction through exercise was linked with the activation of MAP kinase in human skeletal muscle (33). Here we demonstrate that several proteins in the MAP kinase family, including ERK1/2, SEK1, and p38 MAP kinase, are phosphorylated in skeletal muscle in response to exercise. Furthermore, we provide the first evidence for differential regulation by systemic and local factors of these parallel MAP kinase signaling pathways in human skeletal muscle. Exercise leads to a profound increase in phosphorylation of ERK1/2. Moreover, the increase in ERK1/2 phosphorylation is limited to the exercised muscle, and with cessation of exercise, phosphorylation is markedly and rapidly decreased to resting levels. Exercise also induced a transient increase in SEK1 phosphorylation. In contrast to the profound but transient increase in ERK1/2 phosphorylation, exercise led to a smaller increase in p38 MAP kinase phosphorylation. The magnitude of this effect paralleled that of SEK1. SEK1 is able to phosphorylate and activate p38 MAP kinase, as well as a third member of the MAP kinase family, SAPK. p38 MAP kinase phosphorylation was also increased in the resting leg, indicating that systemic exercise-induced factors may mediate this effect. Thus, p38 MAP kinase responds to systemic factors associated with physical exercise, whereas ERK1/2 is stimulated by local factors within the exercised muscle. Taken together, ERK1/2, SEK1, and p38 MAP kinase appear to be differentially regulated with exercise.

The MAP kinase family constitutes a major and ubiquitous intracellular signaling system involved in the regulation of cell growth, differentiation, and survival. The ERK pathway is typically associated with growth responses (3, 5), whereas the SAPK pathway is associated with apoptosis and stress conditions such as ultraviolet light, treatment with protein synthesis inhibitors, or exposure to proinflammatory cytokines such as tumor necrosis factor or interleukin 1 (36, 53, 63–65). p38 MAP kinase is involved in a cascade of events controlling cellular responses to growth factors and in a variety of cellular stress including osmotic shock (54), heat shock (66), and inflammatory cytokines (67). Our results provide a possible signaling mechanism by which exercise regulates protein metabolism in skeletal muscle. Exercise has multiple effects on protein metabolism and gene expression. For example, the transiently increased net protein degradation in skeletal muscle is an acute response after exercise (26, 68), whereas increased protein synthesis and changes in gene expression are chronic responses to exercise (4, 26, 28, 30, 31, 69). Additional studies are warranted to establish whether contraction-activated MAP kinase phosphorylation is directly linked to acute protein degradation and/or chronic increase in gene transcription in skeletal muscle with exercise.

The transcription factor CREB is a critical regulator of intermediate early gene transcription (70). Phosphorylation of CREB at Ser133 regulates its ability to activate transcription in response to growth factors, cAMP, chemical stress, and Ca²⁺ (55, 57, 70, 71). Activation of CREB would provide a possible route for exercise-induced changes in protein expression. Although CREB phosphorylation was not altered by exercise, we provide the first evidence that CREB is phosphorylated in nonexercised muscle upon cessation of exercise. Thus, CREB phosphorylation may be activated by an exercise-induced systemic factor that has no effect in contracting muscle. Although IGF levels were increased with exercise, insulin levels were markedly decreased in parallel. Thus, increased IGF levels are not likely to account for changes in CREB phosphorylation.

Substantial evidence has accumulated to show insulin and exercise/muscle contractions activate glucose transport in skeletal muscle by two separate and distinct signaling pathways (1, 2, 6–8). Several key proteins have been identified in the insulin signaling pathway to glucose transport including the insulin receptor, IRS-1, and PI 3-kinase (5). Earlier studies demonstrate that neither the insulin receptor (72), IRS-1 (73), nor PI 3-kinase (6–8, 74) are involved in the acute effect of exercise/muscle contraction to activate glucose transport. Despite this, little is known of the molecular mechanism by which muscle contraction leads to enhanced glucose transport.

The serine/threonine kinase Akt (PKB/Rac) has been implicated to play a role in intracellular signaling to glucose transport (10–17). Our previous studies (9, 50, 51) and this report show that insulin is a potent stimulator of Akt activity in skeletal muscle. The major route of growth factor-stimulated Akt activity is via PI 3-kinase-dependent pathways (9–15). However, Akt can be activated by okadaic acid (17) or by osmotic shock (21) via PI 3-kinase-independent pathways known to regulate glucose transport in adipocytes (20, 21) and skeletal muscle (38, 74). More important, overexpression of Akt in 3T3-L1 adipocytes or L6 myotubes directly promotes glucose transport and translocation of GLUT1 and GLUT4 to the plasma membrane (16–18). Thus, we hypothesized that Akt may be a point of convergence for insulin- and contraction-induced signaling pathways to glucose transport. Our present results demonstrate that muscle contraction does not activate Akt, providing evidence that contraction activates glucose transport by an Akt-independent pathway. However, one cannot rule out the possibility that an exercise-induced activation of Akt, which is below the level of detection of our assay, may contribute to the exercise-induced activation of glucose transport.

There is growing evidence for a role of Ca²⁺ as a direct stimulator of gene expression via the MAP-kinase cascade. In cardiac myocytes (75) and vascular endothelial cells (76), MAP kinase is activated in response to mechanical stretch, and this may occur in part via a Ca²⁺-mediated mechanism (77). Thus, contraction-induced Ca²⁺ release may be one of the signals leading to activation of the MAP kinase cascade in skeletal muscle. The mechanism by which muscle contraction stimulates glucose transport involves the release of Ca²⁺ from the sarcoplasmic reticulum to the cytosol. Evidence that Ca²⁺ is an early and necessary signal required to elicit glucose transport via this pathway is supported by the following observations: 1) the greater amount of Ca²⁺ entering a muscle during contraction, the greater the increase in glucose transport (37); 2) incubation of muscle with caffeine or calmodulin inhibitors, at concentrations too low to induce muscle contraction, increases glucose transport (41–43); and 3) low concentrations of dantrolene (5 µM), an inhibitor of Ca²⁺ release from the sarcoplasmic reticulum, inhibit calcium- and hypoxia-mediated glucose transport, with no effect on insulin-stimulated glucose transport (40–42). In addition to changes in Ca²⁺, local hypoxia or a decrease in pH may also lead to activation of the MAP-kinase cascade. However, incubation of isolated rat epitrochlearis skeletal muscle in acidic KHB buffer (pH 6.6) or under hypoxic conditions for 30 min did not alter phosphorylation of ERK1/2, p38, or SEK1 (A. Krook and J. R. Zierath, unpublished observation).

Osmotic shock also stimulates glucose transport in adipocytes and skeletal muscle by an insulin-independent pathway (21, 38). Chen and co-workers (21) have shown that the recently identified soluble tyrosine kinase, CADTK/PYK2, is phosphorylated in 3T3L1 adipocytes after stimulation with osmotic shock, but not after insulin stimulation. Activated CADTK/PYK2 stimulates tyrosine phosphorylation of cytoskeletal proteins and mediates intracellular signal transduction. In several cell lines, including PC12 and GN4, CADTK/PYK2 activation regulates ERK1/2 activity (61, 62) and leads to JNK activation (43). Given that the muscle contraction-induced increase in glucose transport involves a Ca²⁺-mediated mechanism, CADTK/PYK2 appeared to be an attractive candidate for mediating exercise effects on either glucose transport and/or Map kinase activation. However, exercise did not lead to increased tyrosine phosphorylation of PYK2 in human skeletal muscle.

Acute muscle contraction is a potent stimulator of glucose transport, even in states of severe insulin resistance (1, 29, 78, 79). Furthermore, chronic exercise training leads to increased GLUT4 protein expression (28, 30, 31) and improved glucose tolerance even in older sedentary glucose-intolerant or mild diabetic individuals (27, 28). Thus, intense interest is now focused on defining the intracellular signaling pathways that link muscle contraction to acute increases in glucose transport and gene expression. This pathway has great therapeutic potential and may serve as an alternative mechanism to bypass the defect responsible for insulin resistance in skeletal muscle.

Here we provide new insight into the intracellular signaling pathways by which muscle contraction through exercise regulates metabolic and mitogenic responses. Our data do not support a role of Akt or PYK2 in exercise/contraction-induced signaling in human skeletal muscle. However, we show that exercise activates multiple MAP kinase pathways in skeletal muscle. These findings lend further support for an intercellular signaling network by which repeated muscle contraction through exercise may enhance gene expression in skeletal muscle. Furthermore, we show that exercise has divergent effects on parallel MAP kinase pathways and that the extent to which these different pathways are activated may influence transcriptional events in skeletal muscle.

Note added in proof: While this manuscript was under review, two other studies were published demonstrating that in vitro contraction of rodent muscle is not associated with increased Akt kinase activity (80, 81).

	ACKNOWLEDGMENTS

 
We thank Mrs. Gunilla Hedin for valuable technical assistance. This study was supported by grants from the Novo-Nordisk Foundation (J.H., H.W.-H., J.R.Z), the Swedish National Center for Research in Sports (J.H., H.W.-H., J.R.Z.), the Swedish Diabetes Association (M.T., H.W.-H., J.R.Z), Thurings Foundation, Tore Nilson's Foundation, Marcus and Amalia Wallenbergs' Foundation, Foundation for Strategic Research (J.R.Z.), the Swedish Medical Research Council (7917; J.H, 9517 and 11945; H.W.-H, 11823 and 12211; J.R.Z), and the National Institutes of Health grant DK 34926 (R.A.R.).

	FOOTNOTES

 
¹ Correspondence: Department of Clinical Physiology, Karolinska Hospital, SE-171 76 Stockholm, Sweden. E-mail: jrz{at}klinfys.ks.se

² Abbreviations: GLUT4, the insulin-responsive glucose transporter; PI, phosphatidylinositol; PKB, protein kinase B, rac, related to A and C protein kinase; MAP, mitogen-activated protein; ERK; extracellular signal-regulated kinase, SAPK, stress-activated protein kinase; SEK, SAPK/ERK kinase; JNK, jun amino-terminal kinase; CREB, cyclic AMP response element binding protein; CADTK/PYK2, calcium-dependent tyrosine kinase; KHB, Krebs-Henseleit buffer; GSK3, glycogen synthase kinase 3; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IRS-1, insulin receptor substrate 1, IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein.

Received for publication March 5, 1998. Accepted for publication May 5, 1998.

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