Department of Medicine and Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284
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ABSTRACT |
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Insulin and exercise potently stimulate glucose metabolism and gene transcription in vivo in skeletal muscle. A single bout of exercise increases the rate of insulin-stimulated glucose uptake and metabolism in skeletal muscle in the postexercise period. The nature of the intracellular signaling mechanisms that control responses to exercise is not known. In mammalian tissues, numerous reports have established the existence of the mitogen-activated protein (MAP) kinase signaling pathway that is activated by a variety of growth factors and hormones. This study was undertaken to determine how a single bout of exercise and physiological hyperinsulinemia activate the MAP kinase pathway. The euglycemic-hyperinsulinemic clamp and cycle ergometer exercise techniques combined with percutaneous muscle biopsies were used to answer this question. In healthy subjects, within 30 min, insulin significantly increased MAP kinase [isoforms p42MAPK and p44MAPK (ERK1 and ERK2)] phosphorylation (141 ± 2%, P < 0.05) and activity (177 ± 5%, P < 0.05), and the activity of its upstream activator MEK1 (161 ± 16%, P < 0.05). Insulin also increased the activity of the MAP kinase downstream substrate, the p90 ribosomal S6 kinase 2 (RSK2) almost twofold (198 ± 45%, P < 0.05). In contrast, a single 30-min bout of moderate-intensity exercise had no effect on the MAP kinase pathway activation from MEK to RSK2 in muscle of healthy subjects. However, 60 min of exercise did increase extracellular signal-related kinase activity. Therefore, despite similar effects on glucose metabolism after 30 min, insulin and exercise regulate the MAP kinase pathway differently. Insulin more rapidly activates the MAP kinase pathway.
insulin; exercise; mitogen-activated protein kinase
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INTRODUCTION |
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INSULIN AND EXERCISE STIMULATE glucose metabolism and gene transcription in vivo in skeletal muscle. In vivo, insulin promptly induces glucose transporter (GLUT-4) translocation to the sarcolemma and increases glucose transport and glucose phosphorylation (5, 11), alters hexokinase activity or subcellular redistribution (43), and increases glycogen synthesis and glycogen synthase activity (4, 23, 42) and pyruvate dehydrogenase and glucose oxidation (30). Insulin also increases the expression of a variety of genes. Likewise, exercise increases muscle glucose transport and glycogen synthase activity and alters gene expression (12, 31, 34).
Insulin produces its effects by binding to the insulin receptor and
starting a cascade of events that begins with activation of the insulin
receptor -subunit tyrosine kinase activity, which first
phosphorylates the
-subunit of the insulin receptor itself. Tyrosine
phosphorylation sites on the receptor serve as recognition motifs for
the association of a variety of proteins with the insulin receptor,
including insulin receptor substrate-1 (IRS-1) (41). IRS-1, for
example, is phosphorylated in turn by the receptor tyrosine kinase and
serves as a key docking protein for a variety of other proteins,
including the regulatory subunits of phosphatidylinositol 3-kinase (PI
3-kinase) and the Grb2/Sos complex (17, 39). PI 3-kinase mediates most
of the metabolic effects of insulin and its downstream signaling events
(22, 38). Shc and Grb2/Sos link insulin receptor signaling to
activation of the mitogen-activated protein (MAP) kinase (MAPK) isoform
p42MAPK/p44MAPK (ERK1/ERK2) cascade, which is
not required for insulin's metabolic effects but mediates mitogenic
signaling (26). MAP kinase pathway activity is increased when Grb2/Sos
is recruited to the plasma membrane, where Sos increases the rate of
exchange of GTP for GDP on Ras, which in turn activates Raf (MAP/ERK
kinase kinase). This leads to activation of MEK, a
serine/threonine-tyrosine kinase that phosphorylates and activates ERK1
and ERK2. ERK2 is the predominant isoform in skeletal muscle. One of
the downstream elements phosphorylated by extracellular signal-related
kinases (ERKs) is p90 ribosomal S6 kinase (RSK2). The
signaling mechanisms by which exercise alters glucose metabolism and
gene expression are less well characterized. Muscle contraction and
voluntary exercise increase MAP kinase activity in rodents (19) and
human volunteers (1), and it is possible that this pathway is involved
in some of the metabolic or gene expression effects of exercise.
Because insulin and muscle contraction bring about many of the same metabolic effects, some investigators have speculated that exercise might share some common signaling elements with insulin receptor signaling (19, 20). However, in rats, neither tetanic contraction nor voluntary wheel running activated insulin receptor signaling (19). In a like manner, vigorous exercise in healthy young volunteers did not increase the magnitude of subsequent insulin stimulation of insulin receptor signaling, but it did increase the tyrosine phosphorylation of IRS-1 and the rate of activation of IRS-1-associated PI 3-kinase and glucose clearance (45). These studies suggest that insulin and exercise make use of distinctly different signaling mechanisms to activate glucose metabolism and gene expression.
Many of the studies that have characterized insulin receptor signaling
elements have been performed in vitro (9, 13, 16, 17, 32), whereas
exercise signaling has been studied mostly in vivo. Several
investigations have addressed questions of insulin receptor signaling
in vivo in humans, however (1, 8, 45). Hyperinsulinemia during a
euglycemic clamp experiment increased insulin receptor tyrosine kinase
activity, IRS-1 tyrosine phosphorylation, and the association of PI
3-kinase activity with IRS-1 (45). In humans, the results of another
study show that cycle exercise increased MAP kinase (ERK) activity. The
present study was undertaken to compare the effects of physiological
hyperinsulinemia with those of moderate exercise on systemic glucose
disposal, muscle glycogen synthase activity, and MAP kinase signaling
activity. Specifically, in the present study, the effects of
physiological hyperinsulinemia during a euglycemic clamp were compared
with those of moderate exercise (~60% of
O2 max). Systemic
glucose disposal was measured using tritiated glucose, and percutaneous muscle biopsies were performed before and at the end of 30 min of
insulin or exercise to assay glycogen synthase activity, MEK1, ERK
activity and phosphorylation, and RSK2 activity.
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METHODS AND MATERIALS |
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Subjects.
A total of 19 healthy volunteers participated in these studies; their
characteristics are given in Table 1. Seven
subjects participated in the insulin study, and twelve took part in the exercise study. All subjects had normal glucose tolerance, were healthy, and were not taking medications known to affect glucose metabolism. Subjects were instructed not to engage in exercise for 48 h
before being studied. The Institutional Review Board of the University
of Texas Health Science Center at San Antonio approved the protocol,
and each subject gave written consent before participating. Insulin
receptor signaling through the PI 3-kinase pathway has been reported
for five of the seven subjects having a euglycemic clamp and for all
ten of the subjects who participated in the exercise study (24).
Glycogen synthase activities and glucose disposal rates for these
subjects have been reported previously (24).
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Study design.
The study consisted of two experimental protocols. Protocol 1 was performed to determine the extent of activation of ERK1/ERK2 by
insulin in vivo in skeletal muscle of healthy lean subjects. A group of
seven healthy lean subjects was studied. Subjects reported to the
General Clinical Research Center (GCRC) at 0800. An antecubital vein
was catheterized with a 20-gauge catheter for infusion of insulin and
glucose. A primed (25 µCi) continuous infusion of [3-3H]glucose (0.25 µCi/min) was started, and
2 h were allowed for isotopic equilibration. After subjects had rested
for 60 min in bed, percutaneous biopsy of the vastus lateralis muscle
was performed under local anesthesia (29). The subject was allowed to
rest in bed for another 60 min to allow washout of any catecholamine effects of the muscle biopsy. Immediately after the muscle biopsy, euglycemic hyperinsulinemic clamps were performed as previously described (14). An insulin infusion (40 mU · m2 · min
1)
was started, arterialized blood glucose concentrations were measured
every 5 min, and blood glucose was clamped at 5 mM with an infusion of
20% dextrose through the antecubital vein. After 30 min of insulin
infusion, a second percutaneous muscle biopsy was taken from the
opposite leg, and the insulin infusion was then stopped. All muscle
biopsy specimens, which ranged in weight from 75 to 200 mg, were
rapidly blotted free of blood and frozen in liquid nitrogen in a cell
freezer until analysis. At the end of the study after insulin was
stopped, to prevent hypoglycemia, the subject was given a meal, and
glucose was infused until the normal plasma glucose concentration was
maintained. Activation of glycogen synthase served as a biochemical
marker for insulin's effects.
Materials. Phosphospecific ERK1/ERK2 rabbit polyclonal antibody raised against the dually phosphorylated Thr-Glu-Tyr region within the catalytic core of the active form of the MAP kinase enzymes was purchased from Promega (Madison, WI). Murine ERK-2 antibody, produced in rabbit immunized with a recombinant murine MAP kinase (p42ERK), a glutathione-S-transferase (GST)-MAPK (inactive) anti-RSK2 antibody, and 3R S6 RSK substrate peptide were purchased from Upstate Biotechnology, (Lake Placid, NY). An anti-MEK1 monoclonal antibody was obtained from Transduction Laboratories (Lexington, KY). [32P]ATP was purchased from NEN Life Science Products (Boston, MA). Protein A-agarose, protein G-agarose, myelin basic protein (MBP), and all other chemicals were purchased from Sigma Chemical (St. Louis, MO).
Muscle processing.
Muscle biopsies were homogenized in ice-cold lysis buffer containing 50 mM HEPES (pH 7.6), 150 mM NaCl, 2 mM EDTA, 10% glycerol, 20 mM
-glycerophosphate, 20 mM sodium pyrophosphate, 10 mM sodium fluoride
(NaF), 2 mM sodium orthovanadate (Na3VO4), 1 mM
CaCl2, 1 mM MgCl2, 1 mM
phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin, 10 µg/ml
aprotinin, and 1% NP-40. Homogenates were centrifuged at 15,000 g for 1 h at 4°C, and muscle debris was removed. Protein
concentrations in crude homogenates were estimated by the Lowry method
(27). The supernatant was stored at
80°C until used.
Western blotting. Muscle protein (250 µg) was solubilized in SDS sample buffer, boiled for 5 min, loaded onto a 10% SDS-polyacrylamide gel, subjected to electrophoresis, and transferred to nitrocellulose membranes. The membranes were then blocked in TBST (20 mmol/l Tris · HCl, pH 7.5, 150 mmol/l NaCl, 0.05% Tween-20) containing nonfat dried milk for 1 h at room temperature. The membranes were incubated at 4°C overnight with rabbit polyclonal phosphospecific ERK1/ERK2 antibody (recognizes both phosphorylated ERK1 and ERK2) at 1:2,000 dilution in blocking buffer. After being washed three times (5 min each) in TBST, the membranes were incubated with secondary antibody (goat anti-rabbit coupled to horseradish peroxidase; Amersham, Arlington Heights, IL) in TBST in a dilution of 1:1,500 and incubated for an additional 1 h at room temperature. The membranes were then washed three times in TBST and developed with the enhanced chemiluminescence detection system according to the manufacturer's protocol (Amersham). The autoradiographs were subjected to scanning densitometry, and the densities of products were quantified by an imaging densitometer fitted with Molecular Analyst Software.
Kinase assays. ERK2 activity assays were performed as described previously (35). An aliquot of human muscle protein (250 µg) was incubated with 10 µl of anti-murine ERK2 polyclonal antibody at 4°C overnight and adsorbed to 80 µl of 50% slurry of protein A-agarose beads for an additional 2 h. The immune complexes were washed three times with ice-cold lysis buffer and twice with kinase reaction buffer [100 mM Tris · HCl, pH 7.5, 40 mM magnesium acetate, 0.4 mM EGTA, 0.4 mM orthovanadate, 2 mM dithiothreitol (DTT)]. After the washes, the immunoprecipitates were suspended in 40 µl of kinase reaction buffer containing 20 µM ATP, 10 µCi/sample of [32P]ATP (6,000 mCi/mmol), and 0.25 mg/ml MBP as substrate. The suspension was incubated with agitation at 30°C for 45 min. The reaction was terminated by transferring 25-µl aliquots onto P-81 phosphocellulose paper discs and washed four times (5 min each, 300 ml/wash) in 0.75% H3PO4. The discs were washed once with acetone and air-dried, and the 32P incorporated into MBP was measured by liquid scintillation counting. Kinase activity termed "specific" was determined by subtracting the radioactivity detected in the absence of substrate from that detected in the presence of substrate. To ensure efficient and equal immunoprecipitation of samples and controls, Western blotting was performed on a portion of the immunoprecipitates, and the radioactivity count was normalized for ERK2 content.
MEK1 activity was assayed as previously described, with some modifications (2). Muscle protein (250 µg) was incubated with 1.25 µg/tube of anti-MEK1 monoclonal antibody at 4°C for 3 h followed by incubation with 50 µl of protein G-agarose beads for an additional 2 h. Immunoprecipitates were washed twice with lysis buffer and twice with MEK1 kinase buffer (25 mM HEPES, pH 7.5, 10 mM MgCl2, 2 mM DTT). After the washes, the immune complexes were resuspended in 80 µl of MEK1 kinase buffer containing 50 µM ATP, 10 µCi/sample of [32P]ATP (6,000 mCi/mmol), and a recombinant kinase-inactive mouse GST-MAPK (ERK2) (1.4 µg/tube) as substrate. The suspension was incubated with agitation for 30 min at 30°C and was terminated by addition of 40 µl of SDS sample buffer. Products were boiled for 5 min and resolved on 10% SDS-PAGE. The gel was dried and the phosphorylated GST-ERK2 (62 kDa) bands were quantified by PhosphorImager and ImageQuant Software (Molecular Dynamics). RSK activity assays were performed as previously described (2). Aliquots of 400 µg of muscle protein were precleared with protein A-agarose beads for 30 min at 4°C and then incubated with 4 µg of polyclonal anti-RSK2 antibody for 2 h at 4°C, followed by incubation with 80 µl of 50% slurry of protein A-agarose beads for an additional 2 h. The immune complexes were washed twice with lysis buffer, twice with LiCl buffer (500 mM LiCl, 100 mM Tris · HCl, pH 7.6, and 0.1% Triton X-100, 1 mM DTT), and once with RSK kinase buffer (30 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mM EGTA, 1 mM DTT). The immune complexes were resuspended in 50 µl of RSK kinase buffer containing 50 µg of 3R S6 peptide as substrate (final concentration 0.2 µg/µl), 50 µM ATP, and 10 µCi/sample of [32P]ATP (6,000 mCi/mmol). Reactions were incubated with agitation at 30°C for 15 min and were terminated by adding 10 µl of stopping solution containing 0.6% HCl, 1 mM ATP, and 1% BSA. The suspensions were then centrifuged at 10,000 rpm for 5 min at 4°C. After centrifugation, aliquots of the supernatant (20 µl) were spotted onto P-81 phosphocellulose paper discs in duplicate and washed four times in 0.75% H3PO4 forGlycogen synthase assay.
A portion of the muscle biopsy specimen was homogenized in a buffer
consisting of 50 mM potassium phosphate, pH 7.4; 2 mM DTT; 2 mM EDTA;
20 mM NaF; 10 µg/ml leupeptin; 10 µg/ml soybean trypsin inhibitor;
20 µg/ml p-aminobenzamidine; 70 µg/ml
N--p-tosyl-L-lysine chloromethyl ketone;
and 170 µg/ml PMSF. The homogenates were centrifuged at 13,000 g, and glycogen synthase activity was assayed in the soluble
fractions with 0.1 and 10 mM glucose 6-phosphate (G-6-P).
Glycogen synthase fractional velocity was calculated as the ratio of
the activity determined using 0.1 mM G-6-P to that determined
using 10 mM G-6-P (30).
Statistical analysis. All data are expressed as means ± SE. A one-tailed paired t-test was used to test for differences between basal and insulin or postexercise points as appropriate, with P < 0.05 considered to be statistically significant.
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RESULTS |
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Whole body glucose disposal and glycogen synthase activity.
The effect of exercise (all 10 subjects) and insulin (5 of 7 subjects)
on glucose disposal and glycogen synthase activity has been reported
previously (24). Insulin increased the rate of glucose disposal from
2.05 ± 0.27 basally to 4.6 ± 1.2 mg · kg1 · min
1
after 120 min (P < 0.05). Exercise also increased glucose
disposal from 1.87 ± 0.08 basally to 2.81 ± 0.30 mg · kg
1 · min
1
(P < 0.01) after 30 min, but this effect was significantly
less than that of insulin. In contrast, exercise increased glycogen synthase activity (GS) more than did insulin. GS0.1
increased from 0.27 ± 0.03 to 0.66 ± 0.10 nmol · min
1 · mg
protein
1 after 30 min of exercise
(P < 0.01) but from 0.33 ± 0.07 to 0.47 ± 0.12 nmol · min
1 · mg
protein
1 after insulin (P < 0.05).
Stimulation of MAP kinase signaling components by insulin and
exercise in human muscle biopsies.
To determine the extent of insulin- and exercise-induced
phosphorylation of ERK1/ERK2 in vivo in normal human skeletal muscle, we first obtained muscle biopsies from healthy subjects immediately before and after 30 min of insulin infusion or moderate exercise. Aliquots of muscle protein were separated by 12% SDS-PAGE and immunoblotted with a phosphospecific ERK1/ERK2 antibody that recognizes only the dual-phosphorylated 42 and 44 kDa ERK proteins. The
phosphorylated bands visualized by the ECL system on the immunoblots
were quantified by scanning densitometry. The results are shown in Fig.
1A. ERK2 (the most abundant isoform
in human muscle) phosphorylation increased by 30 min of insulin
infusion (141 ± 2%, P < 0.05). In contrast, 30 min of
moderate exercise had no effect on ERK1/ERK2 phosphorylation (Fig.
1B). To examine whether insulin-induced ERK phosphorylation is
accompanied by an increase in its kinase activity, we performed an ERK
kinase activity assay using the substrate MBP in
anti-immunoprecipitates of muscle samples taken from all subjects.
Insulin significantly increased ERK2 activity (177 ± 5%, P < 0.05; Fig. 2A). The ability of
anti-ERK2 immunoprecipitates to increase 32P incorporation
into MBP before and after exercise was also measured (Fig. 2B).
The results showed that 30 min of exercise did not increase ERK2
activity above basal level. To determine whether exercise of greater
duration increased ERK2 activity, two subjects had muscle biopsies
before and after 60 min of exercise, and ERK phosphorylation and
activity were assayed as described above. Sixty minutes of exercise
increased ERK phosphorylation and activity in both of these subjects
(Fig. 3). ERK phosphorylation normalized to
ERK2 protein content averaged 0.465 before and 0.921 density units/unit
protein after exercise. This was similar to the effect of 30 min of
insulin on ERK phosphorylation.
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DISCUSSION |
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Exercise and insulin both increase glucose transporter translocation,
glucose transport, and glycogen synthase activity, and stimulate
systemic glucose disposal in vivo in humans (5, 11, 15, 33, 34).
Because the effects of insulin and exercise are so similar, it has been
theorized that there are common elements in the pathways that signal
the responses to these stimuli. Since more details of the insulin
receptor signaling system have become known, some investigators have
hypothesized that exercise might use some of the elements of the
insulin signaling pathway. Goodyear et al. (19) used
electrically stimulated muscle contraction and insulin injection, as
well as voluntary running, to address this question in rats. Insulin
injection rapidly increased muscle insulin receptor tyrosine
phosphorylation and activated PI 3-kinase, but muscle contraction did
not affect these events (19). Goodyear et al. (18) also examined MAP
kinase pathway signaling stimulation by treadmill running and insulin
in rats. Exercise increased ERK1/ERK2 phosphorylation, JNK (c-Jun
NH2-terminal kinase) activity, and RSK-2 activity within 10 min, and this increase was sustained for 60 min (19). Insulin also
increased ERK phosphorylation and RSK-2 activity, but not JNK activity
(19). Two studies in humans reported that exercise increases MAP kinase
pathway (ERK) activity. Aronson et al. (2) showed that 60 min of
exercise at a workload corresponding to 70%
O2 max increased ERK
phosphorylation as well as Raf-1, MEK1, and RSK2 activities. Widegren
et al. (44) showed that 30 min of exercise activates ERK,
stress-activated protein kinase/ERK kinase 1, and p38 MAP kinase, but
not protein kinase B (PKB). One-legged exercise was also used in those
studies to demonstrate that the effect of exercise on ERK
phosphorylation was local rather than systemic (2). However, activation
of p38 MAP kinase also occurred in the rested leg (44). Other recent studies also failed to find an effect of muscle contraction on signaling mediated through PKB (6, 28).
The present study was undertaken to compare how insulin and exercise
activate the MAP kinase signaling pathway in humans. To compare these
stimuli, an attempt was made to make them physiologically similar. The
same duration, 30 min, was used for both. The insulin infusion produced
plasma insulin concentrations well within the physiological range. The
exercise was moderate in intensity (60% of
O2 max) and was chosen
to mimic the intensity and duration of an exercise bout that would be
typical for the average nonathlete. Although different subjects took
part in the insulin and exercise protocols, the two groups were similar
with regard to gender composition, body mass index, and age, and no
subject in any group engaged in any regular exercise. Therefore, these
groups could be expected to have similar responses to either exercise
or insulin, and it is unlikely that the difference between activation
of the MAP kinase pathway with insulin vs. exercise is due to
differences between subjects. Despite this, 30 min of exercise did not
activate the MAP kinase pathway, but 30 min of insulin significantly
increased MEK, ERK, and RSK2 activity. However, when the duration of
exercise was increased to 60 min, ERK activity was approximately
doubled. This confirms the results of previous studies (2). We conclude from these results that insulin more acutely increases the MAP kinase
pathway than does exercise.
It is also conceivable that the ability of exercise to stimulate MAP kinase activity after 60 min is due to increased sensitivity to low insulin concentrations that may be sufficient to produce an increase in ERK activity and phosphorylation. Although theoretically possible, the plasma insulin concentrations after 60 min of exercise are unlikely to have been high enough to produce any activation of MAP kinase pathway activity. Although they were not measured in this study, it is likely that the plasma insulin concentrations fell during exercise (24).
The present results also show that exercise-induced activation of glucose uptake and metabolism are not dependent upon activation of the MAP kinase pathway. This is consistent with the results of recent studies in rodents (46). However, it still could have been hypothesized that changes in gene expression brought about by exercise were mediated by the MAP kinase pathway. Nevertheless, at least with a single 30-min bout of exercise, the MAP kinase pathway was not activated, so it could not be responsible for exercise-induced changes in gene expression. However, the time response for activation of the MAP kinase pathway by exercise may have implications for any training responses that might be mediated by the MAP kinase pathway. It could be predicted from these results that for responses that depended upon MAP kinase pathway activation, an exercise duration >30 min/session might be required. These results provide further evidence that, even though insulin and exercise produce many of the same metabolic and gene expression events in skeletal muscle, they do not share the same proximal signaling pathways. Presumably, downstream convergence points in these signaling pathways exist, but they remain to be discovered.
These results show that some of the distal metabolic effects of these two stimuli are similar in direction but differ quantitatively. For instance, both stimuli increase glucose disposal, but within 30 min, insulin is more effective at this. Both increase glycogen synthase activity, but exercise is more effective than insulin in this respect. The lack of correspondence between the magnitude of the effects of insulin and exercise on glycogen synthase activity and glucose disposal suggests that a given increase in the activity of this enzyme is not always sufficient to produce the same magnitude of increase in glucose disposal. This may bear on the question of whether glycogen synthase is a rate-determining step in glucose uptake (36). Although overexpression of glycogen synthase in mice increases glucose uptake in skeletal muscle (25), studies using NMR spectroscopy have led to the conclusion that glucose transport, rather than glycogen synthase, exerts greater control over glucose disposal (37). These differences may indicate that, under different conditions, both glucose transport and the activity of glycogen synthase may be relatively more or less important for determining the rate of glucose uptake. Another point of note with respect to glycogen synthase is that, although 30 min of exercise did not activate the MAP kinase pathway, glycogen synthase activity was markedly increased. This activation is sustained for long periods (24); therefore, it is unlikely that exercise signals glycogen synthase through the MAP kinase pathway, consistent with recent results in rats (46).
The fact that a physiological concentration of insulin led to MAP kinase activation within 30 min indicates that this is a physiologically relevant phenomenon. The results of in vitro studies suggest that the effects of insulin on glucose metabolism are independent of MAP kinase pathway activity, because they are unaffected by either a dominant negative Ras inhibitor (16) or a pharmacological inhibitor of MEK, PD-098059 (26). Therefore, the physiological role of MAP kinase pathway activation by insulin is unclear at present. However, it is possible that, in muscle in vivo, insulin stimulation of the MAP kinase pathway might mediate the ability of insulin to suppress protein breakdown (40). It is also possible that the MAP kinase pathway can desensitize or "dampen" insulin signaling through the PI 3-kinase pathway. Results of in vitro experiments show that ERK can phosphorylate IRS-1 on serine residues and potentially inhibit this pathway (13).
In summary, the results of this study show that the same duration (30 min) of physiological hyperinsulinemia and moderate exercise has different effects on the ERK1/ERK2 pathway, including the downstream signaling element RSK2. Therefore, the metabolic or gene expression effects of 30 min of exercise cannot be mediated through the MAP kinase pathway in humans in vivo. Activation of the MAP kinase pathway occurs more rapidly in response to insulin than to exercise.
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ACKNOWLEDGEMENTS |
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The authors gratefully acknowledge the technical assistance of Andrea Barrentine, Kathy Camp, Gilbert Gomez, Jean Finlayson, Cindy Munoz, and Sheila Taylor. The excellent nursing assistance of Patricia Wolff and Norma Ortiz is also acknowledged.
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FOOTNOTES |
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This study was supported by a grant from the American Diabetes Association (L. J. Mandarino), and National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK-47936 to L. J. Mandarino and NIH, Division of Research Resources Grant RR-01346 to the General Clinical Research Center, Audie Murphy Veterans Affairs Hospital.
Present address: M. Pendergrass, Dept. of Medicine, Tulane University Medical School, New Orleans, LA; K. Maezono, Ajinomoto Co., Inc., Yokohama, Japan.
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. Mandarino, Division of Diabetes, Department of Medicine, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7886 (E-mail: mandarino{at}uthscsa.edu).
Received 12 April 1999; accepted in final form 17 December 1999.
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