Skeletal muscle contractile activity in vitro stimulates mitogen-activated protein kinase signaling

Tatsuya Hayashi, Michael F. Hirshman, Scott D. Dufresne, and Laurie J. Goodyear

Research Division, Joslin Diabetes Center, and Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02215


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Physical exercise is a potent stimulator of mitogen-activated protein (MAP) kinase signaling. To determine if this activation is secondary to systemic responses to exercise or due to muscle contractile activity per se, an isolated muscle preparation was developed. Contractile activity in vitro significantly increased p44MAPK and p42MAPK phosphorylation by 2.9- and 2.4-fold, respectively. Contraction-stimulated MAP kinase phosphorylation was not decreased in the presence of D-tubocurarine or calphostin C, suggesting that neither neurotransmitter release nor diacylglycerol-sensitive protein kinase C mediates the contraction-induced activation of this signaling cascade. However, PD-98059, an inhibitor of MAP kinase kinase (MEK), inhibited the contraction-induced increases in MAP kinase phosphorylation. PD-98059 did not alter contraction-induced increases in glucose uptake or glycogen synthase activity, demonstrating that MAP kinase signaling is not necessary for these important metabolic effects of contractile activity in skeletal muscle. These data suggest that contractile activity of the skeletal muscle fibers per se, and not responses to neurotransmitter release, hormones, or other systemic factors, is responsible for the stimulation of MAP kinase signaling with physical exercise.

exercise; soleus muscle; glucose transport; glycogen; mitogen-activated protein kinase kinase inhibitor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MAMMALIAN CELLS ARE continuously exposed to a complex array of hormones, growth factors, environmental stresses, and mechanical forces. The mechanism through which cells transduce these internal and external cues into intracellular responses likely involves the intricate regulation of multiple cascades of signaling molecules. The mitogen-activated protein (MAP) kinase has emerged as a critical signaling system in mammalian cells, and signaling through this cascade has been implicated in regulating numerous cellular functions (9, 27). All muscle cell types (smooth, cardiac, and skeletal) express components of the MAP kinase signaling pathway (3, 12, 14, 37), and numerous stimuli can activate this cascade of proteins in muscle cells. Of the different muscle cell types, the MAP kinase system in cardiac myocytes and vascular smooth muscle cells has been most extensively investigated. MAP kinase signaling occurs in response to both humoral factors and mechanical forces in these cells, activating transcription factors and putatively playing a role in cell proliferation and/or hypertrophy (12, 37). Compared with the intensive investigation of MAP kinase signaling in cardiac and smooth muscle cells, there has been limited study of this signaling cascade in adult skeletal muscle tissue, which is surprising given that the MAP kinase protein was originally purified from insulin-stimulated rabbit skeletal muscle (21).

Physical exercise is a potent stimulator of MAP kinase signaling in both rat (14) and human (3, 36) skeletal muscle. In human vastus lateralis muscle, bicycle exercise at an intensity of 70% of maximal oxygen consumption for 60 min significantly activated both MAP kinase isoforms as well as upstream and downstream molecules including Raf-1 kinase (Raf-1), MAP kinase kinase 1 (MEK1), and ribosomal S6 kinase 2 (RSK2) (3). The upstream signaling molecules that mediate this activation of the MAP kinase cascade with exercise are not known. One possibility is that MAP kinase signaling is a function of the exercise-induced increase in blood flow and the corresponding increase in delivery of humoral factors to the muscle, leading to activation of receptor-mediated signaling molecules. Alternatively, or in addition, the release of neurotransmitters at the neuromuscular junction in the contracting muscles could stimulate cell surface receptors. Because there is evidence for the release of autocrine and paracrine factors from contracting skeletal muscle fibers (18, 25, 30), these molecules could also play a role in stimulating MAP kinase signaling. Yet another possibility is that the contractile activity per se, independent of hormones, neurotransmitters, or autocrine and paracrine factors, increases MAP kinase signaling in skeletal muscle during physical exercise.

A single bout of physical exercise can have profound effects on a number of cellular functions including glucose uptake (17), glycogen metabolism (4), gene transcription (24), and protein synthesis (5). Given the robust stimulation of MAP kinase signaling that is observed during exercise, it is likely that this signaling cascade plays a role in mediating one or more of these biological responses. Recently, we reported that activation of MAP kinase and c-Jun NH2-terminal kinase signaling in response to muscle contractile activity in situ by sciatic nerve stimulation is associated with increased expression of c-jun and c-fos mRNA, suggesting that both pathways play roles in regulating early gene transcription in response to acute exercise (2). It is not known if MAP kinase signaling is involved in contraction-mediated alterations in glucose uptake or glycogen metabolism.

To determine if contractile activity per se can increase MAP kinase signaling in skeletal muscle, we have developed an in vitro contraction preparation using isolated rat soleus muscles. This preparation was also used to determine if MAP kinase signaling is necessary for contraction-stimulated increases in glucose transport and glycogen synthase activity. Our results demonstrate that contraction-activated MAP kinase signaling is not involved in the regulation of glucose transport and glycogen synthesis in skeletal muscle. Our results also provide the first evidence to suggest that the effects of muscle contraction on MAP kinase signaling occur independently of humoral, neural, or autocrine and paracrine factors.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Phosphospecific anti-MAP kinase antibody was provided by Quality Controlled Biochemicals (Hopkinton, MA). The inhibitors PD-98059 and calphostin C were purchased from Calbiochem (La Jolla, CA), and D-tubocurarine was from Sigma (St. Louis, MO). The 3-O-[3H]methyl-D-glucose, D-[14C]mannitol, and UDP-[14C]glucose were from New England Nuclear (Boston, MA). Enhanced chemiluminescence (ECL) reagents were from Transduction Laboratories (Lexington, KY), and anti-rabbit immunoglobulin-horseradish peroxidase-linked whole antibody was from Amersham (Arlington Heights, IL). Reagents for the protein assays were from Bio-Rad Laboratories (Hercules, CA), and all other chemicals, including the glucose (HK) reagent, were from Sigma.

Muscle preparation. Male Sprague-Dawley rats weighing 50-80 g obtained from Taconic (Germantown, NY) were fasted overnight before study. The rats were killed, and both soleus muscles were removed. Tendons from both ends of the muscle were tied with suture (silk 4-0) and mounted on a preincubation apparatus. The dissection and mounting procedures took ~2 min and were performed with extreme care to prevent any stretching or tearing of the muscle. The muscle was kept at resting length throughout the entire experimental procedures from the time of dissection to the final incubation.

For both the control ("sham") and contraction treatment, muscles were preincubated for 30-50 min in 4 ml of Krebs-Ringer bicarbonate (KRB) buffer (in mM: 117 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 24.6 NaHCO3) containing 2 mM sodium pyruvate (buffer A). At the end of the preincubation period the muscles were transferred to a contraction apparatus containing 7.5 ml of buffer A. Tension was recorded with a force transducer and chart recorder, and for both the muscles subjected to the sham treatment (sham-treated muscle) and muscles that were stimulated to contract, resting tension was set to 0.5 g. Contractions were produced by stimulating the muscle with a pulse generator with the following protocol: train rate = 0.033 s-1, train duration = 10 s, pulse rate = 100 Hz, duration = 0.1 ms, voltage = 50 V. The contraction protocol typically produced 10-15 g of tension as measured with the transducer and chart recorder. In preliminary experiments, contraction periods of 1-20 min were evaluated, with 10 min of contractions determined to be optimal for our experiments. For the sham-treated muscle, resting tension (0.5 g) was maintained for an equivalent time period. When added, PD-98059 (25 µM), calphostin C (0.5 µM), and D-tubocurarine (25 µM) were present during the entire experiment, whereas insulin (100 nM) was present during the last 30 min. PD-98059 and calphostin C were dissolved in DMSO and added to each buffer immediately before each experiment. The maximal concentration of DMSO was 0.1%, which did not affect any assay. The buffers were continuously gassed with 95% O2-5% CO2 and maintained at 30°C. After treatments, muscles were either immediately frozen in liquid nitrogen and subsequently used for studies of MAP kinase phosphorylation, glycogen content, and glycogen synthase activity or further incubated to determine rates of glucose transport.

Immunoblotting for MAP kinase. The phosphorylation of MAP kinase was assessed by immunoblotting muscle lysates with an antibody that recognizes only the phosphorylated forms of p44MAPK and p42MAPK. Muscles were homogenized in 20 mM HEPES (pH 7.4), 2 mM EGTA, 50 mM beta -glycerophosphate, 1 mM dithiothreitol, 1 mM Na3VO4, 1% Triton X-100, 10% glycerol, 10 µM leupeptin, 3 mM benzamidine, 5 µM pepstatin A, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride. Homogenates were rotated end over end for 60 min at 4°C and centrifuged at 15,000 g for 1 h at 4°C. The protein concentrations of the supernatants were determined by using BSA as the standard. Muscle proteins (100-150 µg) were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, and blocked in PBS containing 0.05% Tween 20 (PBS-T) and 5% nonfat milk for 1 h at room temperature. The membranes were incubated with the phosphospecific anti-MAP kinase antibody (1:2,000) in PBS-T and 2.5% dry milk for 3 h at room temperature or overnight at 4°C. Bound antibodies were detected with anti-rabbit immunoglobulin-horseradish peroxidase-linked whole antibody. The membranes were washed in PBS-T and then incubated with ECL reagents.

Glycogen synthase and glycogen content. Glycogen synthase activity was assayed by a method adapted from Thomas et al. (35). Muscles were homogenized in buffer containing 20 mM HEPES (pH 7.4), 2 mM EGTA, 50 mM NaF, 50 mM beta -glycerophosphate, 10 µM leupeptin, 3 mM benzamidine, and 10 µg/ml aprotinin and centrifuged at 15,000 g for 1 h at 4°C. The supernatants (60 µg of protein) were added to a reaction solution containing 50 mM Tris · HCl (pH 7.8), 5 mM EDTA, 6.7 mM UDP-[14C]glucose (100 µCi/mmol), 10 mg/ml glycogen, 50 mM beta -glycerophosphate, and 50 mM NaF in the absence or presence of 6.7 mM glucose 6-phosphate (G-6-P) at 30°C to measure G-6-P-independent (I-form) and total glycogen synthase activities. Reactions were terminated at 15 min by spotting the reaction mixture on filter papers, which, after extensive washing with 66% (vol/vol) ethanol, were counted in a scintillation counter for 14C incorporated into glycogen. Glycogen synthase activity was calculated as nanomoles of UDP-glucose incorporated into glycogen per minute per milligram of protein in the supernatant. Enzyme activity was also calculated as the ratio of the I-form activity to total activity. Muscle glycogen was determined from a weighed muscle sample after acid hydrolysis (2 M HCl) at 100°C for 2 h. The concentration of hydrolyzed glucose residues was measured with the glucose (HK) reagent.

Transport of 3-O-methyl-D-glucose. After contraction or sham treatment, muscles were transferred to a separate tube and incubated in 2.0 ml KRB buffer containing 1 mM 3-O-[3H]methyl-D-glucose (250 µCi/mmol) and 1 mM D-[14C]mannitol (167 µCi/mmol). Incubations were carried out in the absence or presence of insulin (100 nM) and/or PD-98059 (25 µM) for 10 min at 30°C. To terminate the transport reaction, muscles were dipped in KRB containing 80 µM cytochalasin B at 4°C. The muscles were blotted on filter paper, trimmed, frozen in liquid nitrogen, and stored at -80°C. Muscles were processed by being incubated in 300 µl of 1 M NaOH at 80°C for 10 min, and then the digested material was neutralized with 300 µl of 1 M HCl. The radioactivity in aliquots of the digested material was determined by scintillation counting for dual labels, and the extracellular and intracellular spaces were calculated. The transport rate was expressed as micromoles of 3-O-methyl-D-glucose incorporated into 1 ml of intracellular space per hour.

Statistical analysis. Data are presented as means ± SE. When comparing differences between two groups, statistical analysis was performed by using Student's t-test. Multiple comparisons were made by ANOVA using Fisher's least-significant difference test for post hoc analysis.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MAP kinase phosphorylation in isolated soleus muscles. A unique feature of the MAP kinase proteins is that full activation of the enzymes requires phosphorylation of threonine and tyrosine residues (pThr-Glu-pTyr). To determine the activated state of the MAP kinase isoforms in our muscles that were stimulated to contract, we used a phosphospecific MAP kinase antibody that recognizes the p42MAPK and p44MAPK isoforms only when both residues are phosphorylated (2, 3, 14). In preliminary experiments using hindlimb skeletal muscles, we have determined that an increase in MAP kinase dual phosphorylation correlates with MAP kinase activity measured by an immune complex kinase assay. Figure 1A shows MAP kinase phosphorylation in soleus muscles under four conditions: muscle immediately frozen on dissection, sham-treated muscle preincubated for 50 min followed by 10 min of resting tension, muscle preincubated for 50 min followed by 10 min of contraction, and muscle preincubated for 30 min followed by 30 min of incubation with 100 nM insulin. There was a small increase in MAP kinase phosphorylation in the sham-treated muscles compared with that in the muscles that were immediately frozen after dissection. Muscle contractile activity significantly increased both p44MAPK and p42MAPK phosphorylation by 2.9- and 2.4-fold, respectively, above the sham levels of phosphorylation (Fig. 1B). Insulin treatment also increased p44MAPK and p42MAPK phosphorylation by 2.6- and 2.3-fold, respectively. This degree of MAP kinase phosphorylation is comparable to that which has been observed with exercise and insulin treatment in vivo (14). These experiments demonstrate that there is a contraction-specific effect on MAP kinase phosphorylation and suggest that hormonal factors and increased blood flow are not major mediators of MAP kinase signaling with exercise.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 1.   Effects of contractile activity and insulin on mitogen-activated protein (MAP) kinase phosphorylation. Isolated soleus muscles were frozen immediately after dissection (Dis), after 60 min of incubation (Sham), after 50 min of preincubation and 10 min of contraction (Ctr), or after 30 min of preincubation and 30 min of incubation with 100 nM insulin (Ins). A: muscle proteins (100 µg) were resolved by SDS-PAGE (10%) and immunoblotted with an antibody that recognizes phosphorylated (activated) p44MAPK and p42MAPK. Both contraction and insulin significantly increased MAP kinase phosphorylation. B: graphs show quantification of p44MAPK (left) and p42MAPK (right) phosphorylation by scanning densitometry (n = 5-13/group). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. corresponding sham-treated muscle.

Effects of D-tubocurarine on basal and contraction-stimulated MAP kinase phosphorylation. Muscle contractile activity is initiated by a neural signal, leading to the release of acetylcholine from the motor nerve ending at the neuromuscular junction. To test the possibility that neurotransmitter release is required for the contraction-stimulated activation of MAP kinase, soleus muscles were stimulated to contract in the presence or absence of D-tubocurarine (25 µM), a neuromuscular junction blocker. Figure 2 shows that there was no effect of D-tubocurarine on either basal or contraction stimulated-MAP kinase phosphorylation. Tension development during contractile activity was not affected in the presence of D-tubocurarine (data not shown). Therefore, each muscle contraction is due to direct electrical stimulation of muscle cells.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 2.   Effects of calphostin C and D-tubocurarine on MAP kinase phosphorylation. Soleus muscles were frozen after 60 min of preincubation (Sham) or after 50 min of preincubation and 10 min of contraction (Ctr) in presence or absence of calphostin C (0.5 µM) or D-tubocurarine (25 µM). Muscle proteins (135 µg) were resolved by SDS-PAGE (10%) and immunoblotted with an antibody that recognizes phosphorylated (activated) p44MAPK and p42MAPK.

Effects of calphostin C on basal and contraction-stimulated MAP kinase phosphorylation. Previous studies have demonstrated that muscle contractile activity can increase protein kinase C (PKC) activity in rat skeletal muscle (8, 26). Pharmacological inhibition of PKC by calphostin C can block the activation of MAP kinase by hyperosmolality in opossum kidney cells (34) as well as mechanical load-induced MAP kinase activation in rat neonatal cardiac myocytes (38). Calphostin C is a cell-permeable, irreversible PKC inhibitor that interacts with the regulatory domain of the protein by competing at the binding site of diacylglycerol (DAG) and phorbol esters (19, 32). To determine if this PKC inhibitor affects contraction-stimulated MAP kinase activation, soleus muscles were preincubated for 50 min and stimulated to contract for 10 min in the presence or absence of calphostin C (0.5 µM). This dose of calphostin C has been shown to decrease glucose transport stimulation by the phorbol ester 12-deoxyphorbol 13-phenylacetate 20-acetate in isolated rat skeletal muscles (15). In the presence of calphostin C, muscle contractile activity increased MAP kinase phosphorylation to the same extent as in controls (Fig. 2). There was also no effect of calphostin C on basal MAP kinase phosphorylation.

Effects of the MEK inhibitor PD-98059 on contraction- and insulin-stimulated MAP kinase phosphorylation. To determine if the MEK inhibitor PD-98059 is effective in blocking contraction- and insulin-stimulated activation of MAP kinase, soleus muscles were preincubated, incubated, and stimulated to contract or treated with insulin in the absence or presence of PD-98059 (25 µM; Fig. 3). PD-98059 is a selective inhibitor of the phosphorylation and activation of MEK, which catalyzes the phosphorylation of MAP kinase on both threonine and tyrosine residues. The inhibitory effect appears to be due to the binding of the drug to MEK at a site that blocks access to activating enzymes and has been shown to prevent activation of MAP kinase and the subsequent phosphorylation of MAP kinase substrates both in vitro and in intact cells (1, 11). PD-98059 completely blocked both the contraction-induced (Fig. 3A) and insulin-induced (Fig. 3B) phosphorylation of both the p44MAPK and p42MAPK isoforms. Tension development during contractile activity was not affected by PD-98059 (data not shown).


View larger version (52K):
[in this window]
[in a new window]
 
Fig. 3.   Effects of PD-98059 [MAP kinase kinase (MEK) inhibitor] on MAP kinase phosphorylation. Soleus muscles were frozen after 50 min of preincubation and 10 min of contraction (A) and after 30 min of preincubation and 30 min of incubation with 100 nM insulin (B) in presence or absence of PD-98059 (PD; 25 µM). Dissected (Dis) samples were frozen immediately after dissection. Muscle proteins (150 µg) were resolved by SDS-PAGE (10%) and immunoblotted with an antibody that recognizes the phosphorylated (activated) p44MAPK and p42MAPK.

Effects of PD-98059 on contraction-stimulated glucose transport and glycogen synthase activity. To assess the role of MAP kinase signaling in glucose utilization, we measured the effects of PD-98059 on contraction-stimulated glucose transport and glycogen synthase activity. For the transport studies, soleus muscles were preincubated and stimulated to contract as described for the MAP kinase phosphorylation experiments and then incubated in the transport buffer containing 1 mM 3-O-[3H]methyl-D-glucose and 1 mM D-[14C]mannitol. When present, 25 µM PD-98059 was added to the buffer throughout the entire experiment. There was no effect of PD-98059 on basal rates of glucose transport (Fig. 4). In the absence of PD-98059, contractile activity increased transport by 2.2-fold above that in the treated muscles, and PD-98059 had no effect on the contraction-stimulated glucose transport (Fig. 4). Insulin treatment significantly increased 3-O-methyl-D-glucose (1.37 ± 0.07 µmol · ml-1 · h-1; P < 0.0001 vs. sham), and, similar to what was found for contractile activity, there was no effect of MEK inhibition on insulin-stimulated glucose transport (1.35 ± 0.04 µmol · ml-1 · h-1). These data demonstrate that the inhibition of MAP kinase signaling by PD-98059 has no effect on basal, contraction- or insulin-stimulated glucose transport in isolated rat skeletal muscle.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of PD-98059 on contraction-stimulated 3-O-methyl-D-glucose transport activity. Soleus muscles were incubated for 60 min (Sham) or were preincubated for 50 min and stimulated to contract for 10 min (Ctr) in presence or absence of PD-98059 (PD) and then incubated in Krebs-Ringer bicarbonate buffer containing 1 mM 3-O-[3H]methyl-D-glucose and 1 mM D-[14C]mannitol for 10 min. Radioactivity of each isotope was determined with a liquid scintillation analyzer, and extracellular and intracellular spaces were calculated (n = 15-27/group). ** P < 0.01 vs corresponding sham-treated group. NS, not significant.

PD-98059 also did not alter basal or contraction-stimulated glycogen synthase activity. Muscles were preincubated and stimulated to contract for 10 min, and glycogen synthase activity was measured in the absence or presence of 6.7 mM G-6-P. Contractile activity significantly increased G-6-P-independent glycogen synthase (I-form) activity (24.8 ± 3.1 vs. 17.3 ± 1.9 nmol · min-1 · mg-1; P < 0.05) without affecting total glycogen synthase activity (74.0 ± 3.3 vs. 72.1 ± 3.2 nmol · min-1 · mg-1). The ratio of the I-form activity to total activity was also increased by 30-40% in response to contractile activity, but these increases were not affected in the presence of PD-98059. Muscle glycogen content was decreased to a similar extent (15-20%) after contractile activity in the absence or presence of PD-98059 (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Physical exercise evokes a number of dynamic changes, many of which have the potential to alter signal transduction cascades in the contracting skeletal muscles. For example, exercise increases the concentrations in blood of several humoral factors that are known activators of MAP kinase signaling, including growth hormone, insulin-like growth factor I, catecholamines, and ANG II (13). These humoral factors may be effectively delivered to contracting muscles in response to the dramatic increase in muscle blood flow that occurs during exercise. Increased blood flow to the contracting muscles may also compensate for the decrease in insulin concentrations that results from exercise, preserving MAP kinase activity. To determine if humoral factors or increased blood flow is necessary for the activation of MAP kinase signaling in skeletal muscle, we studied contractile activity in isolated skeletal muscles. We found that contractile activity in the absence of humoral factors and increased blood flow increased MAP kinase phosphorylation and that the magnitude of the increase in phosphorylation was similar to that which occurs with exercise in vivo (3, 14). Our data also suggest that neurotransmitter activation is not necessary for MAP kinase signaling during exercise, because D-tubocurarine, a potent competitive inhibitor for the nicotinic acetylcholine receptor, had no effect on contraction-stimulated MAP kinase activation. Although we cannot directly test the possibility that an autocrine or paracrine factor is regulating MAP kinase signaling with muscle contraction, in preliminary experiments incubation of control (noncontracted) muscles with contraction-conditioned incubation media did not increase MAP kinase signaling (T. Hayashi and L. J. Goodyear, unpublished observations). Taken together, these data suggest that it is the contractile activity per se that increases MAP kinase signaling during physical exercise.

Contraction-stimulated MAP kinase activation presumably occurs through the sequential phosphorylation and activation of Raf-1 and MEK1, because these molecules are activated with the appropriate time course in response to muscle contraction (2). The upstream regulators of Raf-1 in contracting skeletal muscle have not been elucidated, although our recent studies have ruled out a role for secondary activation of tyrosine kinase receptors and insulin receptor substrate, Shc, or Grb2 molecules (29). Here, we determined if PKC is critical for contraction-stimulated MAP kinase activation by incubating and contracting isolated muscles in the absence or presence of the PKC inhibitor calphostin C. PKCalpha may activate the MAP kinase pathway through the direct phosphorylation of Raf-1 (20), and calphostin C can inhibit increases in MAP kinase activity induced by hyperosmolality in renal cells (34) and ANG II in cardiac myocytes (38). However, in rat skeletal muscle, we found no effect of the drug on contraction-stimulated MAP kinase signaling. Because calphostin C tends to show selectivity for conventional PKC isozymes (6) and only poorly inhibits atypical PKCs (23), our study rules out a role for DAG-sensitive PKCs in the activation of contraction-stimulated MAP kinase signaling.

Although it has long been known that physical exercise can result in a potent, insulin-independent stimulation of glucose uptake into contracting skeletal muscles, the intracellular signaling events that lead to this activation have not been fully elucidated (17). MAP kinase has been implicated in glucose transport regulation on the basis of the finding that microinjection of purified MAP kinase into Xenopus oocytes increases glucose transport (22). However, we found that pharmacological blockade of MAP kinase activation with PD-98059 did not alter rates of contraction-stimulated glucose transport. PD-98059 also had no effect on insulin-stimulated glucose transport in the isolated rat skeletal muscle, consistent with studies of insulin-stimulated glucose transport in cultured adipocytes (7, 16, 33) and skeletal muscle strips from human subjects (28). Although insulin and exercise increase glucose transport by distinct intracellular signaling molecules (17), neither stimulus requires MAP kinase signaling for normal activation of transport in skeletal muscle.

Rates of glycogenolysis in contracting muscle fibers are enhanced; this is followed by the rapid activation of glycogen synthase and the resynthesis of glycogen in the period immediately after contraction. Several years ago it was proposed that the MAP kinase signaling cascade regulates the insulin-stimulated activation of glycogen synthase, on the basis of the finding that RSK2 could phosphorylate and inactivate glycogen synthase kinase-3 (GSK-3) and could phosphorylate and activate the glycogen-bound form of type 1 protein phosphatase (PP1-G), two key regulators of insulin-stimulated glycogen synthase activity (10, 31). More recent studies have suggested that RSK2 does not play a regulatory role in glycogen synthase activation by insulin, and the physiological significance of RSK2 phosphorylation of GSK-3 and PP1-G is still not known. We tested the possibility that the exercise-induced activation of RSK2 functions to mediate glycogen synthesis in muscle. However, similar to what was found for insulin, our results demonstrate that MAP kinase signaling is not required for regulation of muscle glycogen metabolism in contracting skeletal muscle. Taken together, all of these data now provide overwhelming evidence that MAP kinase signaling is not involved in the acute regulation of glucose uptake or nonoxidative glucose disposal by insulin or exercise.

In summary, muscle contraction in vitro increased the phosphorylation of both the p44MAPK and p42MAPK isoforms. Signaling through the MAP kinase pathway is not necessary for the contraction-induced activation of glucose transport or glycogen synthase. Neither calphostin C, a PKC inhibitor, nor D-tubocurarine, a blocker of the neuromuscular junction, affected contraction-stimulated MAP kinase phosphorylation. We conclude that muscle contraction potently stimulates MAP kinase signaling in rat skeletal muscle without requiring systemic factors, nerve transmission, or activation of DAG-sensitive PKC. The in vitro contraction system will be a useful tool to study contraction-stimulated intracellular signaling cascades in skeletal muscle.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-42238 (to L. J. Goodyear). T. Hayashi was supported by the Manpei Suzuki Diabetes Foundation.


    FOOTNOTES

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

Address for reprint requests and other correspondence: L. J. Goodyear, Research Division, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215 (E-mail: laurie.goodyear{at}joslin.harvard.edu).

Received 9 March 1999; accepted in final form 12 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alessi, D. R., A. Cuenda, P. Cohen, D. T. Dudley, and A. R. Saltiel. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J. Biol. Chem. 270: 27489-27494, 1995[Abstract/Free Full Text].

2.   Aronson, D., S. D. Dufresne, and L. J. Goodyear. Contractile activity stimulates the c-Jun NH2-terminal kinase pathway in rat skeletal muscle. J. Biol. Chem. 272: 25636-25640, 1997[Abstract/Free Full Text].

3.   Aronson, D., M. A. Violan, S. D. Dufresne, D. Zangen, R. A. Fielding, and L. J. Goodyear. Exercise stimulates the mitogen-activated protein kinase pathway in human skeletal muscle. J. Clin. Invest. 99: 1251-1257, 1997[Abstract/Free Full Text].

4.   Bergstrom, J., and E. Hultman. Muscle glycogen synthesis after exercise: an enhancing factor localized to the muscle cells in man. Nature 210: 309-310, 1966[Medline].

5.   Booth, F. W., and P. A. Watson. Control of adaptations in protein levels in response to exercise. Federation Proc. 44: 2293-2300, 1985[Medline].

6.   Budworth, J., and A. Gescher. Differential inhibition of cytosolic and membrane-derived protein kinase C activity by staurosporine and other kinase inhibitors. FEBS Lett. 362: 139-142, 1995[Medline].

7.   Cheatham, B., C. J. Vlahos, L. Cheatham, L. Wang, J. Blenis, and C. R. Kahn. Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol. Cell. Biol. 14: 4902-4911, 1994[Abstract].

8.   Cleland, P. J., G. J. Appleby, S. Rattigan, and M. G. Clark. Exercise-induced translocation of protein kinase C and production of diacylglycerol and phosphatidic acid in rat skeletal muscle in vivo. Relationship to changes in glucose transport. J. Biol. Chem. 264: 17704-17711, 1989[Abstract/Free Full Text].

9.   Cobb, M. H., and E. J. Goldsmith. How MAP kinases are regulated. J. Biol. Chem. 270: 14843-14846, 1995[Free Full Text].

10.   Dent, P., A. Lavoinne, S. Nakielny, J. B. Caudwell, P. Watt, and P. Cohen. The molecular mechanism by which insulin stimulates glycogen synthesis in mammalian skeletal muscle. Nature 348: 302-308, 1990[Medline].

11.   Dudley, D. T., L. Pang, S. J. Decker, A. J. Bridges, and A. R. Saltiel. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. Acad. Sci. USA 92: 7686-7689, 1995[Abstract].

12.   Force, T., and J. V. Bonventre. Growth factors and mitogen-activated protein kinases. Hypertension 31: 152-161, 1998[Abstract/Free Full Text].

13.   Galbo, H. The hormonal response to exercise. Diabetes Metab. Rev. 1: 385-408, 1986[Medline].

14.   Goodyear, L. J., P.-Y. Chung, D. Sherwood, S. D. Dufresne, and D. E. Moller. Effects of exercise and insulin on mitogen-activated protein kinase signaling pathways in rat skeletal muscle. Am. J. Physiol. 271 (Endocrinol. Metab. 34): E403-E408, 1996[Abstract/Free Full Text].

15.   Hansen, P. A., J. A. Corbett, and J. O. Holloszy. Phorbol esters stimulate muscle glucose transport by a mechanism distinct from the insulin and hypoxia pathways. Am. J. Physiol. 273 (Endocrinol. Metab. 36): E28-E36, 1997[Abstract/Free Full Text].

16.   Haruta, T., A. J. Morris, D. W. Rose, J. G. Nelson, M. Mueckler, and J. M. Olefsky. Insulin-stimulated GLUT4 translocation is mediated by a divergent intracellular signaling pathway. J. Biol. Chem. 270: 27991-27994, 1995[Abstract/Free Full Text].

17.   Hayashi, T., J. F. Wojtaszewski, and L. J. Goodyear. Exercise regulation of glucose transport in skeletal muscle. Am. J. Physiol. 273 (Endocrinol. Metab. 36): E1039-E1051, 1997[Medline].

18.   Hellsten, Y., and U. Frandsen. Adenosine formation in contracting primary rat skeletal muscle cells and endothelial cells in culture. J. Physiol. (Lond.) 504: 695-704, 1997[Abstract].

19.   Kobayashi, E., K. Ando, H. Nakano, T. Iida, H. Ohno, M. Morimoto, and T. Tamaoki. Calphostins (UCN-1028), novel and specific inhibitors of protein kinase C. I. Fermentation, isolation, physico-chemical properties and biological activities. J. Antibiot. (Tokyo) 42: 1470-1474, 1989[Medline].

20.   Kolch, W., G. Heidecker, G. Kochs, R. Hummel, H. Vahidi, H. Mischak, G. Finkenzeller, D. Marme, and U. R. Rapp. Protein kinase C alpha activates RAF-1 by direct phosphorylation. Nature 364: 249-252, 1993[Medline].

21.   Lavoinne, A., E. Erikson, and E. Maller. Purification and characterization of the insulin-stimulated protein kinase from rabbit skeletal muscle; close similarity to S6 kinase II. Eur. J. Biochem. 199: 723-728, 1991[Abstract].

22.   Merrall, N. W., R. J. Plevin, D. Stokoe, P. Cohen, A. R. Nebreda, and G. W. Gould. Mitogen-activated protein kinase (MAP kinase), MAP kinase kinase and c-Mos stimulate glucose transport in Xenopus oocytes. Biochem. J. 295: 351-355, 1993[Medline].

23.   Mizuno, K., K. Noda, Y. Ueda, H. Hanaki, T. C. Saido, T. Ikuta, T. Kuroki, T. Tamaoki, S. Hirai, and S. Osada. UCN-01, an anti-tumor drug, is a selective inhibitor of the conventional PKC subfamily. FEBS Lett. 359: 259-261, 1995[Medline].

24.   Neufer, P. D., and G. L. Dohm. Exercise induces a transient increase in transcription of the GLUT-4 gene in skeletal muscle. Am. J. Physiol. 265 (Cell Physiol. 34): C1597-C1603, 1993[Abstract/Free Full Text].

25.   Reid, M. B. Role of nitric oxide in skeletal muscle: synthesis, distribution and functional importance. Acta Physiol. Scand. 162: 401-409, 1998[Medline].

26.   Richter, E. A., P. J. F. Cleland, S. Rattigan, M. G. Clark, and P. J. Cleland. Contraction-associated translocation of protein kinase C in rat skeletal muscle. FEBS Lett. 217: 232-236, 1987[Medline].

27.   Seger, R., and E. G. Krebs. The MAPK signaling cascade. FASEB J. 9: 726-735, 1995[Abstract/Free Full Text].

28.   Shepherd, P. R., B. T. Nave, J. Rincon, R. J. Haigh, E. Foulstone, C. Proud, J. R. Zierath, K. Siddle, and H. Wallberg-Henriksson. Involvement of phosphoinositide 3-kinase in insulin stimulation of MAP-kinase and phosphorylation of protein kinase-B in human skeletal muscle: implications for glucose metabolism. Diabetologia 40: 1172-1177, 1997[Medline].

29.   Sherwood, D. J., S. D. Dufresne, J. F. Markuns, B. Cheatham, D. E. Moller, D. Aronson, and L. J. Goodyear. Differential regulation of MAP kinase, p70S6K, and Akt by contraction and insulin in rat skeletal muscle. Am. J. Physiol. 276 (Endocrinol. Metab. 39): E870-E878, 1999[Abstract/Free Full Text].

30.   Stebbins, C. L., O. A. Carretero, T. Mindroiu, and J. C. Longhurst. Bradykinin release from contracting skeletal muscle of the cat. J. Appl. Physiol. 69: 1225-1230, 1990[Abstract/Free Full Text].

31.   Sutherland, C., I. A. Leighton, and P. Cohen. Inactivation of glycogen synthase kinase-3b by phosphorylation: new kinase connections in insulin and growth-factor signalling. Biochem. J. 296: 15-19, 1993[Medline].

32.   Tamaoki, T., and H. Nakano. Potent and specific inhibitors of protein kinase C of microbial origin. Biotechnology 8: 732-735, 1990[Medline].

33.   Tanti, J. F., T. Gremeaux, S. Grillo, V. Calleja, A. Klippel, L. T. Williams, E. Van Obberghen, and Y. Le Marchand-Brustel. Overexpression of a constitutively active form of phosphatidylinositol 3-kinase is sufficient to promote Glut 4 translocation in adipocytes. J. Biol. Chem. 271: 25227-25232, 1996[Abstract/Free Full Text].

34.   Terada, Y., K. Tomita, M. K. Homma, H. Nonoguchi, T. Yang, T. Yamada, Y. Yuasa, E. G. Krebs, S. Sasaki, and F. Marumo. Sequential activation of Raf-1 kinase, mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase by hyperosmolality in renal cells. J. Biol. Chem. 269: 31296-31301, 1994[Abstract/Free Full Text].

35.   Thomas, J. A., K. K. Schlender, and J. Larner. A rapid filter paper assay for UDPglucose-glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-glucose. Anal. Biochem. 25: 486-499, 1968[Medline].

36.   Widegren, U., X. J. Jiang, A. Krook, A. V. Chibalin, M. Bjornholm, M. Tally, R. A. Roth, J. Henriksson, H. Wallberg-Henriksson, and J. R. Zierath. Divergent effects of exercise on metabolic and mitogenic signaling pathways in human skeletal muscle. FASEB J. 12: 1379-1389, 1998[Abstract/Free Full Text].

37.   Yamazaki, T., I. Komuro, and Y. Yazaki. Molecular mechanism of cardiac cellular hypertrophy by mechanical stress. J. Mol. Cell. Cardiol. 27: 133-140, 1995[Medline].

38.   Zou, Y., I. Komuro, T. Yamazaki, R. Aikawa, S. Kudoh, I. Shiojima, Y. Hiroi, T. Mizuno, and Y. Yazaki. Protein kinase C, but not tyrosine kinases or Ras, plays a critical role in angiotensin II-induced activation of Raf-1 kinase and extracellular signal-regulated protein kinases in cardiac myocytes. J. Biol. Chem. 271: 33592-33597, 1996[Abstract/Free Full Text].


Am J Physiol Cell Physiol 277(4):C701-C707
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society