©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Insulin Stimulation of Mitogen-activated Protein Kinase, p90, and p70 S6 Kinase in Skeletal Muscle of Normal and Insulin-resistant Mice
IMPLICATIONS FOR THE REGULATION OF GLYCOGEN SYNTHASE (*)

(Received for publication, July 24, 1995; and in revised form, October 2, 1995)

Pi-Yun Chang (1) Yannick Le Marchand-Brustel (2) Lynn A. Cheatham (1) David E. Moller (1)(§)

From the  (1)Department of Medicine, Beth Israel Hospital and Department of Cellular and Molecular Physiology, Harvard Medical School, Boston, Massachusetts 02215 and (2)Faculte de Medicine, INSERM Unité 145, Nice, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Skeletal muscles from mice stimulated with insulin in vivo were used to evaluate relationships between the insulin receptor tyrosine kinase, mitogen-activated protein (MAP) kinase, p90, p70 S6 kinase (p70), and glycogen synthase. Two models of insulin resistance were also evaluated: (a) transgenic mice with a severe insulin receptor defect and (b) gold thioglucose (GTG) mice (obesity with minimal insulin receptor dysfunction). In normal mice, insulin stimulated MAP kinase (6-fold), p90 (RSK2, 5-fold), p70 (10-fold), and glycogen synthase (30-50% increase in fractional velocity). In transgenic mice, stimulation of MAP kinase and RSK2 were not detectable, whereas activation of p70 and glycogen synthase were preserved. In GTG mice, activation of MAP kinase, RSK2, p70, and glycogen synthase were impaired. Since p70 and glycogen synthase were correlated, rapamycin was used to block p70, and glycogen synthase activation was unaffected in normal mice; however, it was partially impaired in transgenic mice. Conclusions: (a) stimulation of p70 and glycogen synthase are selectively preserved in muscles with a severe insulin receptor kinase defect, indicating signal amplification in pathways leading to these effects; (b) MAP kinase-RSK2 and p70 activation are impaired in obese mice, suggesting multiple loci for postreceptor insulin resistance; (c) glycogen synthase was dissociated from MAP kinase and RSK2, indicating that they are not required for this effect of insulin; and (d) p70 is not essential for glycogen synthase activation, but it may participate in redundant signaling pathways leading to this effect of insulin.


INTRODUCTION

Important metabolic and mitogenic physiologic effects result from activation of the insulin receptor tyrosine kinase which leads to processes that are largely mediated by net increases or decreases in Ser/Thr phosphorylation of multiple proteins. Thus, insulin stimulates phosphorylation of ribosomal S6, ATP citrate-lyase, acetyl-CoA carboxylase, and others(1, 2) . Net dephosphorylation of glycogen synthase, pyruvate kinase, pyruvate dehydrogenase, hormone-sensitive lipase, and hydroxylmethylglutaryl CoA reductase serve to regulate the function of these enzymes(1) . The importance of Ser/Thr phosphorylation/dephosphorylation is highlighted by the effects of the phosphatase inhibitor, okadaic acid, which mimics the effect of insulin to stimulate glucose transport(3, 4, 5) , but also blocks insulin's ability to promote metabolic biologic effects(5, 6) .

Insulin-stimulated Ser/Thr kinases include casein kinase II (1) and p70 S6 kinase (p70)(7, 8) . Furthermore, there is ample evidence showing that insulin stimulation activates components of the mitogen-activated protein (MAP) (^1)kinase cascade including MAP kinase kinase(9) , p44 (ERK1), and p42 (ERK2) MAP kinases(10, 11, 12) , and the approx90-kDa S6 kinases (p90 or RSK)(13, 14, 15) . Several lines of investigation suggest that p70 (via phosphorylation of ribosomal protein S6), MAP kinase (via phosphorylation of RSK, transcription factors, and proteins which modulate translation initiation), and RSK (via phosphorylation of transcription factors) have important roles in insulin-regulated cell proliferation and differentiation(16, 17, 18, 19) .

Skeletal muscle is a major target tissue for insulin action and muscle glycogen synthesis accounts for a substantial portion of in vivo insulin-mediated glucose disposal(20, 21) . Furthermore, specific defects in the activation by insulin of skeletal muscle glycogen synthase, the rate-limiting enzyme, have been described in humans with insulin-resistant states including non-insulin-dependent diabetes mellitus(22, 23, 24, 25) . Recently, a compelling link between insulin's activation of MAP kinase and RSK, and the activation of glycogen synthase in skeletal muscle has been put forth. Thus, Lavoinne and co-workers (14, 26) purified an insulin-sensitive protein kinase from rabbit skeletal muscle that appeared to be capable of activation of protein phosphatase 1 by phosphorylation of a specific serine residue on the protein phosphatase 1 G-subunit. Importantly, activated protein phosphatase 1 catalyzes the dephosphorylation of glycogen synthase (and phosphorylase b kinase) resulting in increased glycogen synthesis(26) . Furthermore, insulin-sensitive protein kinase could be activated in vitro by MAP kinase, and microsequencing of tryptic insulin-sensitive protein kinase peptides revealed 100% identity to a specific RSK isoform (RSK or RSK2)(14, 15) . Several studies also suggest that insulin-stimulated RSK2 (as well as p70) may inactivate glycogen synthase kinase-3 by phosphorylation, further contributing to the activation of glycogen synthase(27, 28, 29, 30) . In addition, rapamycin, a drug which specifically blocks p70 activation(18, 31) , reportedly inhibited insulin-stimulated glycogen synthase activity in cultured adipocytes(32) .

Despite the physiologic importance of muscle and extensive scrutiny of MAP kinase, RSK, and p70 in cultured cell systems, little is known of the potential for defects involving these enzymes in muscle insulin resistance or of their importance for the regulation of glycogen synthase in this tissue. In the present studies, we characterized the ability of insulin, administered in vivo to mice, to promote the activation of skeletal muscle p42/p44 MAP kinase, RSK2, and p70S6k in relation to activation of the insulin receptor kinase and glycogen synthase. In addition to studying these effects in muscle from normal mice, two distinctly different models of insulin resistance were employed: 1) transgenic mice with muscle-specific overexpression of dominant-negative insulin receptors which have a specific defect in muscle insulin receptor function (33) and 2) gold thioglucose (GTG)-obese mice, a well characterized model of insulin resistance which was previously shown to have impaired insulin-mediated activation of muscle glycogen synthase(34) . To specifically address the hypothesis that p70 may be required for insulin-mediated activation of muscle glycogen synthase, rapamycin was used to block the in vivo activation of this enzyme.


EXPERIMENTAL PROCEDURES

Animals

Transgenic mice (FVB-NJ strain) with muscle-specific overexpression of a mutant human insulin receptor (Ala Thr) were generated as described previously(33) . Equal numbers of 16-20-week-old homozygous transgenic mice or age- and sex-matched control FVB-NJ mice were used for each experiment. Obese mice were generated by injection of GTG as described previously(35) . Mean body weight of GTG-obese mice used in the present study was 70.7 ± 1.8 g (versus 45.2 ± 0.5 g for lean controls). After 6 h of fasting, plasma insulin levels in GTG-obese mice were 13.9 ± 2.8 ng/ml versus 0.84 ± 0.1 for lean mice, p < 0.0005); plasma glucose levels were 8.17 ± 0.4 mM for obese versus 6.44 ± 0.2 for lean, p < 0.005). GTG-obese and age-matched lean male Swiss-Albino mice were studied at age 24 weeks. Plasma insulin and glucose levels were measured as described previously(36) .

Methods for Insulin Administration

Food was withdrawn 6 h (GTG-obese mice and controls) or 12 h (FVB-NJ transgenic and control mice) before experiments. Mice were anesthetized with Avertin (Aldrich) and placed on a warm surface followed by intravenous injection (tail vein) with insulin (5 milliunits/g of body weight) or saline. This insulin dose was previously determined to result in maximal activation of skeletal muscle insulin receptor tyrosine kinase(37) . At the indicated time points, gluteal and/or gastrocnemius muscles were rapidly removed, snap-frozen in liquid nitrogen, and pulverized while frozen. In order to prevent hypoglycemia and the activation of counter-regulatory mechanisms, experiments where muscles were removed >5 min after insulin (or saline) injection included a simultaneous intravenous injection of propranolol (1 mg/kg) followed 5 min later (insulin-stimulated mice) by intraperitoneal injection of glucose (2 mg/g of body weight). Plasma glucose levels were measured before and 15 min after insulin injection to ensure that normoglycemia persisted. In some experiments, mice also received an intraperitoneal injection of rapamycin (Calbiochem) 5 min before insulin or saline administration.

Samples of powdered frozen muscles were homogenized and solubilized in ice-cold buffers (noted below) using a polytron or sonicator. Particulate matter was removed from muscle lysate/homogenate by centrifugation (12,000 times g for 10 min at 4 °C). Aliquots of the supernatant were removed for determination of protein concentration(37) .

Measurement of Skeletal Muscle Insulin Receptor Tyrosine Kinase Activity

Insulin receptor tyrosine kinase activity was measured using aliquots of solubilized muscle proteins and poly-Glu-Tyr as a substrate after immobilization on microtiter wells coated with a non-species-specific anti-insulin receptor antibody (AB-3, Oncogene Science, Uniondale, NY) as described previously(33) .

p42 MAP Kinase Phosphorylation and In-gel MAP Kinase Assay

The phosphorylation state of p42 MAP kinase (ERK2) was assessed by electrophoretic mobility which was detected by immunoblotting with anti-MAP kinase antibody (alpha-C2, provided by John Blenis, Harvard Medical School) as described previously(37) . Autoradiograms were analyzed using a scanning densitometer and Imagequant software (Molecular Dynamics). Individual peaks corresponding to phosphorylated or dephosphorylated p42 MAP kinase were separately integrated followed by calculation of the ratio of phosphorylated/dephosphorylated p42 MAP kinase.

In-gel MAP kinase activity was determined using a modification of methods described by Kameshita and Fujisawa (38) as follows. Samples of muscle lysate (37) containing 500 µg of solubilized protein were denatured in 0.5% SDS for 5 min at 90 °C, diluted to 0.1% SDS with buffer A (20 mM HEPES, pH 7.0, 5 mM EDTA, 10 mM EGTA, 10 mM MgCl(2), 50 mM beta-glycerophosphate, 1 mM Na(3)VO(4), 2 mM dithiothreitol, 2 µg/ml leupeptin, 5 µg/ml aprotinin, 40 µg/ml phenylmethylsulfonyl fluoride, 1% Nonidet P-40), and immunoprecipitated with alpha-C2 antibody coupled to Trisacryl protein A-Sepharose beads (Pierce). Immune complexes were washed thrice with buffer B (50 mM Tris-HCl, pH 7.4, 300 mM NaCl, 1.0% deoxycholate, 1.0% Triton X-100, 5 mM EDTA, 10% glycerol, 0.1% SDS), and once with buffer B containing 10 mM rather than 300 mM NaCl. Washed immune complexes were eluted in SDS-PAGE sample buffer and resolved on SDS-PAGE containing 0.4 mg/ml myelin basic protein (MBP, Sigma). As described previously(38) , SDS was removed from the gels followed by denaturation and renaturation. Renatured gels were incubated in buffer C (40 mM HEPES, pH 8.0, 10 mM MgCl(2), 0.1 mM EGTA, 2 mM dithiothreitol), followed by kinase reactions with buffer C containing 5.0 µCi/ml of [-P]ATP(38) . Gels were washed extensively with 5% trichloroacetic acid containing 1% sodium pyrophosphate and then dried. MBP phosphorylation at molecular weights corresponding to both p42 and p44 MAP kinases was detected by autoradiography and quantitated using a PhosphorImager (Molecular Dynamics).

Assessment of p90 (RSK2) Activation

Aliquots of solubilized muscle proteins (500 µg in buffer A) were immunoprecipitated with 4 µg of affinity-purified polyclonal antiserum (alphaRSK2-PCT, provided by S. Pelech, Kinetek Biotechnology, Vancouver, BC) raised against a C-terminal peptide (CNRNQSPVLEPVGRS) in RSK2 (39) coupled to Trisacryl protein A beads. Immunoprecipitates were washed thrice with buffer D (20 mM Tris-HCl, pH 7.2, and 0.5 M LiCl) and once with buffer E (20 mM Tris-HCl, pH 7.2, and 10 mM NaCl). Immune complexes were then incubated in 30 µl of buffer C containing [-P]ATP (50 µM final, 10 µCi/reaction) with or without S6 substrate peptide (RRRLSSLRA; Upstate Biotechnology, Inc., Lake Placid, NY) for 20 min at 30 °C. Reactions that included substrate were terminated by spotting onto phosphocellulose paper (Whatman P-81). After washing in 1% phosphoric acid, filters were subjected to Cerenkov counting. For RSK2 autokinase activity, reactions without substrate were terminated by the addition of 10 µl of 3times sample buffer followed by SDS-PAGE and subsequent autoradiography. Conditions for RSK2 immunoblotting were as described above for MAP kinase except that 0.5 µg/ml alphaRSK2-PCT was used as the primary antibody.

Assessment of p70 Activation

Polyclonal antisera raised against N-terminal residues 20-39 (alpha-p70-N2) or C-terminal residues 502-525 (alpha-p70-C) of p70 alpha1 (40) were provided by J. Blenis, Harvard Medical School, Boston. Immunoblotting of p70 was performed as described above for MAP kinase except that alpha-p70-C was used as the primary antibody. For p70 kinase assays, solubilized muscle proteins (500 µg) in buffer F (50 mM Tris-HCl, pH 7.5, 1.0% Triton X-100, 5 mM EGTA, 10 mM MgCl(2), 50 mM beta-glycerophosphate, 1 mM Na(3)VO(4), 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each of aprotinin and leupeptin) were immunoprecipitated with alpha-p70-N2. Immunoprecipitates were washed once with buffer G (10 mM Tris-HCl, pH 7.2, 100 mM NaCl, 0.5% SDS, 1% Nonidet P-40, 1 mM Na(3)VO(4), 1 mM EDTA, 2 mM dithiothreitol), once with buffer G containing 1 M rather than 100 mM NaCl, and once with buffer H (50 mM Tris-HCl, pH 7.2, 150 mM NaCl). Immune complexes were incubated in buffer C with [-P]ATP (50 µM final, 20 µCi/reaction) and S6 protein derived from 40 S ribosomes (41) for 20 min at 30 °C. Samples were then separated by 12% SDS-PAGE followed by autoradiography and PhosphorImager analysis.

Glycogen Synthase Assay

Samples of frozen muscle (100 mg) were homogenized in 0.4 ml of buffer I (50 mM Tris-HCl, pH 8, 5 mM EDTA, 100 mM KF). Glycogen synthase activity was measured (in duplicate for each sample) according to the method of Thomas et al.(42) using 4 mM uridine diphosphoglucose in the absence (active form) or presence (total activity) of 10 mM glucose 6-phosphate (G6P). Activity was measured using UDP[^14C]glucose and was expressed as nanomoles of glucose incorporated into glycogen/min/mg of protein. For each sample, an activity ratio (fractional velocity) was then calculated (activity in the absence of G6P/total activity).


RESULTS

Impairment of the Insulin Receptor Tyrosine Kinase Proportionately Impairs Insulin-stimulated MAP Kinase

As we have previously observed(37) , additional experiments showed that overexpression of mutant Thr human insulin receptors in transgenic mice resulted in marked impairment of net muscle insulin receptor tyrosine kinase activity after in vivo administration of a maximally effective insulin dose (Fig. 1A). We previously used an electrophoretic mobility shift approach to show that the phosphorylation of p42 and p44 MAP kinases in response to insulin was impaired in the skeletal muscle of mutant insulin receptor transgenic mice(37) . In order to determine enzyme activity, an in-gel MBP kinase assay was employed after immunoprecipitation of MAP kinase from solubilized muscle proteins (Fig. 1B). Although insulin stimulated MAP kinase by 6-fold in control mouse muscle, MAP kinase activation in transgenic muscle was severely impaired (<2-fold, Fig. 1, B and C).


Figure 1: Insulin-stimulated receptor tyrosine kinase and MAP kinase activity in control versus mutant insulin receptor transgenic mice. A, insulin receptor tyrosine kinase activity toward poly-Glu-Tyr was determined using gluteal muscles obtained after intravenous insulin (+) or saline(-) injection of control or transgenic (TG) mice. The results are expressed as -fold of control basal; each value represents mean ± S.E. of data from six to eight mice. * = transgenic versus insulin-stimulated control, p < 0.005. B, in-gel MAP kinase assay. This example autoradiogram shows MBP phosphorylation by p42 and p44 MAP kinases after immunoprecipitation from solubilized gluteal muscle proteins obtained 5 min after in vivo administration of insulin (+) or saline(-) to control or transgenic (TG) mice. C, quantitation of muscle MAP kinase. Insulin-stimulated p42 and p44 MAP kinase activity was determined as described above, followed by quantitation of MBP phosphorylation by PhosphorImager. The results are expressed as -fold of control basal; each value represents mean ± S.E. of data from six to eight mice. * = transgenic versus insulin-stimulated control, p < 0.04.



Impairment of the Insulin Receptor Kinase Has Discordant Effects on Insulin-stimulated Activation of RSK2 and p70

RSK2 is the specific p90 isoform which has been implicated in insulin-mediated regulation of glycogen synthase(15) . Thus, an antibody (alphaRSK2-PCT) raised against a RSK2 peptide sequence was employed. Immunoblotting of mouse muscle proteins with alphaRSK2-PCT revealed a prominant approx90-95 kDa band consistent with the molecular size of RSK2. Furthermore, other experiments showed that alphaRSK2-PCT reacts with recombinant RSK2 (but not with RSK1 or RSK3) expressed in COS cells. (^2)Since RSK is known to undergo autophosphorylation coincident with kinase activation(17) , we performed autokinase reactions using RSK2 immunoprecipitates. Fig. 2A shows that RSK2 autophosphorylation was stimulated severalfold by insulin in control muscle with no evidence for stimulation in transgenic muscle. RSK2 activity was also assessed using an immune complex kinase assay; a peptide corresponding to amino acids 231-239 of ribosomal S6 protein was used as the substrate since it is avidly phosphorylated by RSK(17) . As depicted in Fig. 2B, insulin stimulated RSK2 by 5-fold in control muscles, whereas no significant activation of RSK2 was observed in muscles from transgenic mice. Since activation of p70 correlates with the appearance of slower migrating species (phosphorylation) detected by immunoblotting after SDS-PAGE(43) , this approach was used to initially characterize insulin stimulation of muscle p70. Preliminary experiments showed that maximal stimulation in control mice occurred 15-20 min following insulin injection and with doses geq5 milliunits/g; furthermore, p70 phosphorylation in response to a maximally effective insulin dose appeared to be normal in muscles from mutant insulin receptor transgenic mice (not shown). Insulin-stimulated activation of p70 was then determined using muscle samples which had also been used to measure both insulin receptor tyrosine kinase (Fig. 1A) and RSK2 (Fig. 2B). As shown in Fig. 2C, insulin stimulated the activity of p70 (toward S6 protein) by approx10-fold in both control and transgenic muscle.


Figure 2: Insulin stimulation of p90 (RSK2) and p70 activity in control versus transgenic mice. A, RSK2 autokinase activity. This example autoradiogram shows results obtained with four control and four transgenic mice. Solubilized gluteal muscle proteins obtained 15 min after in vivo insulin (+) or saline(-) injection, were immunoprecipitated with anti-RSK2 (alphaRSK2-PCT) followed by an in vitro autokinase assay and subsequent SDS-PAGE. Arrows indicate 95-kDa protein bands which correspond to the RSK2 p90 isoform. Similar results were obtained with six additional mice from each group. B, muscle RSK2 activity. RSK2 was immunoprecipitated from solubilized muscle proteins as described above followed by in vitro immune complex kinase assays using S6 peptide as the substrate. The results are expressed as -fold of control basal. Each value represents mean ± S.E. of data obtained from 12 mice. * = transgenic versus insulin-stimulated control, p < 0.0002. C, muscle p70 activity. After in vivo insulin or saline, p70 was immunoprecipitated from solubilized gluteal muscle proteins using alpha-p70-N2 followed by in vitro immune complex kinase assays using ribosomal S6 protein as the substrate. Phosphorylation of S6 was quantitated by PhosphorImager after SDS-PAGE. Each value represents the mean ± S.E. of data obtained from six mice. Similar results were obtained with nine additional mice from each group.



A Maximally Effective Insulin Dose Normally Activates Glycogen Synthase in Transgenic Muscle

In order to correlate the marked defect in RSK2 (and MAP kinase) activation with potential impairment of insulin-stimulated glycogen synthase, muscles from control or transgenic mice were removed 15 min after insulin or saline injection and assayed. Glycogen synthase activity was measured in the presence or absence of the allosteric activator G6P using a saturating uridine diphosphoglucose concentration. In response to a maximally effective insulin dose (5 milliunits/g), the stimulated fractional velocity (as measured by the activity ratio -G6P/+G6P) was 1.43-fold of basal in muscles from both control and transgenic mice (Fig. 3). The extent of insulin stimulation we observed was comparable to previous results obtained with muscle from normal mice (34) or from humans(23) . Thus, the overexpression of mutant insulin receptors in muscle failed to exert transdominant effects on insulin-mediated activation of either P70 or glycogen synthase.


Figure 3: Insulin stimulation of glycogen synthase activity in control versus transgenic muscles. Samples of homogenized muscles were derived from control or transgenic (TG) mice 20 min after in vivo insulin (+) or saline(-) injection. Glycogen synthase activity was measured using 4 mM uridine diphospho[^14C]glucose in the presence or absence of G6P. Results are expressed as the glycogen synthase activity ratio (-G6P/+G6P). Each value represents mean ± S.E. of data obtained from 13 mice (17 muscles: 13 gluteal and 4 gastrocnemius). No differences were observed between the two muscles which were studied.



Insulin Receptor, p42 MAP Kinase, RSK2, and p70 Activation in Muscle from Obese Insulin-resistant Mice

Since prolonged fasting is known to substantially ameliorate the insulin resistance associated with this model(36) , mice were fasted for only 6 h prior to insulin stimulation. Net levels of maximal insulin-stimulated receptor kinase activity were slightly lower in obese mice. Thus, insulin stimulated muscle receptor kinase by 3.4 ± 0.3-fold in lean versus only 2.5 ± 0.2-fold in obese mice ( p = 0.01, n = 25 each) (Fig. 4A). In contrast, insulin-stimulated phosphorylation of p42 MAP kinase (as assessed by electrophoretic mobility shift assay) was severely impaired in muscle from obese mice (Fig. 4, B and C). Thus, insulin stimulated a 4-fold increase in lean muscle p42 MAP kinase phosphorylation versus no significant stimulation in muscle from obese mice (Fig. 4C). Similar results were obtained with the in-gel MBP kinase assay using MAP kinase immunoprecipitates from a smaller number of samples (not shown). Net protein levels of p42 and p44 MAP kinases were not altered by obesity. Evaluation of muscle RSK2 activation revealed that obesity was associated with a similarly severe defect in insulin stimulation of this enzyme (Fig. 5A). In contrast to the normal activation of p70 by insulin in muscles from transgenic mice, obesity was associated with a 56% decrease in insulin-stimulated activity compared to lean controls (Fig. 5B). No change in the expression of p70 was evident in muscle from obese mice as assessed by immunoblotting (not shown).


Figure 4: Effect of obesity on insulin-stimulated receptor tyrosine kinase and MAP kinase activity. A, lean or GTG-obese mice were fasted for 6 h followed by in vivo insulin (+) or saline(-) injection. Muscles were removed after 5 min followed by measurement of insulin receptor tyrosine kinase activity. The results are expressed as -fold of control basal; each value represents the mean ± S.E. of data obtained from 12 mice (24 muscles: 12 gluteal and 12 gastrocnemius). B, p42 MAP kinase activation was measured by immunoblotting with anti-MAP kinase (alpha-C2) in order to detect an electrophoretic mobility shift with insulin stimulation. The example autoradiogram shows results obtained with gluteal muscles from two lean and two obese mice 5 min after stimulation with insulin (+) or saline(-). Bands corresponding to dephosphorylated (lower) or phosphorylated (upper) p42 MAP kinase are shown. C, quantitation of p42 MAP kinase phosphorylation was achieved by densitometry of immunoblot autoradiograms obtained as described above. After calculation of a ratio (phosphorylated/dephosphorylated p42 MAP kinase) for each sample, results were expressed as -fold of control basal. Each value represents the mean ± S.E. of data obtained from 12 mice (24 muscles: 12 gluteal and 12 gastrocnemius). * = obese versus insulin-stimulated lean mice, p < 0.01. No differences were observed between the two muscles studied.




Figure 5: Effect of obesity on insulin-stimulated RSK2 and P70 activity. A, RSK2 immune complex kinases assays were performed as noted in legend to Fig. 2B. The results are expressed as -fold of control basal. Each value represents the mean ± S.E. of data obtained from six mice (12 muscles: 6 gluteal and 6 gastrocnemius). * = obese versus insulin-stimulated lean mice, p < 0.005. B, P70 activity was measured as described in legend to Fig. 2D. The results are expressed as -fold of control basal. Each value represents the mean ± S.E. of data obtained from 12 muscles in each group (six gluteal and six gastrocnemius). * = obese versus insulin-stimulated lean mice, p < 0.05. No differences were observed between the two muscles studied.



Insulin Stimulation of Glycogen Synthase in Muscle from Obese Insulin-resistant Mice

Using soleus muscles isolated from ad libitum fed GTG-obese mice incubated in vitro, we previously observed a substantial defect in insulin stimulation of glycogen synthase activity(34) . After 6 h of fasting, there was no significant reduction in maximal insulin-stimulated glycogen synthase activity measured in either gluteal or gastrocnemius muscles from GTG-obese mice used in the present study (Fig. 6). However, the insulin-stimulated increment in synthase activity was impaired in both muscles studied. Thus, insulin-stimulated synthase fractional velocity was 1.32 ± 0.05-fold of basal in lean gluteal muscles versus 1.10 ± 0.11 in obese gluteal muscles (p = 0.05); in gastrocnemius muscles, insulin-stimulated values from lean mice were 1.42 ± 0.06-fold of basal versus 1.16 ± 0.05 for obese mice (p = 0.03). Therefore, obesity was associated with a 50-60% decrease in insulin's ability to augment glycogen synthase activity.


Figure 6: Insulin-stimulated glycogen synthase activity in lean versus GTG-obese mice. Glycogen synthase activity was measured using gluteal (solid bars) and gastrocnemius (hatched bars) muscles as described in the legend to Fig. 3. The results are expressed as the glycogen synthase activity ratio. Each value represents the mean ± S.E. of data obtained from six gluteal or eight gastrocnemius muscles.



Activation of p70 Is Not Required for Insulin Stimulation of Muscle Glycogen Synthase

Given the correlation between normal activation of p70 and glycogen synthase in muscles from transgenic mice as well as the suggestion that p70 may be required for insulin-stimulation of glycogen synthase in adipocytes(32) , we used rapamycin to block the activation of p70in vivo. As shown in Fig. 7, A and B, pretreatment of normal mice with rapamycin 5 min prior to insulin injection completely abolished the activation of p70 toward S6. However, rapamycin had no effect on insulin stimulation of RSK2 as predicted (Fig. 7C). The same muscle samples were analyzed to determine the effect of p70 blockade on insulin stimulation of glycogen synthase. As shown in Fig. 7D, rapamycin also had no effect on insulin's ability to stimulate glycogen synthase.


Figure 7: Effect of rapamycin on insulin-stimulated p70, RSK2, and glycogen synthase activity in normal mouse muscle. 5 min prior to in vivo administration of insulin, control FVB-NJ mice received an intraperitoneal injection of 25 µg of rapamycin or saline. Gluteal muscles were removed 20 min following intravenous insulin (or saline) injections. A, this example autoradiogram shows basal and insulin-stimulated S6 phosphorylation in p70 immunoprecipitates in the absence or presence of rapamycin. B, quantitation of p70 activity. Immune complex kinase assays were conducted as described above and in legend to Fig. 2D. C, RSK2 activity was measured using immune complexes as described in legend to Fig. 2B. D, glycogen synthase activity was measured as described in legend to Fig. 3. The results for p70 and RSK2 are expressed as fold of basal; results for glycogen synthase are expressed as the activity ratio. Each value represents mean ± S.E. of data obtained from six mice.



Rapamycin Partially Impairs Insulin-stimulated Glycogen Synthase in Muscles from Mice with Impaired Insulin Receptor Function

Although rapamycin was apparently unable to impinge on insulin stimulation of glycogen synthase in normal mice, we sought to determine whether p70 is important when other signaling pathways have also been suppressed. Thus, we used rapamycin in transgenic mice where elements of the MAP kinase cascade were markedly impaired. As shown in Fig. 8, the ability of insulin to augment glycogen synthase fractional velocity in both gluteal and gastrocnemius muscles derived from mutant insulin receptor transgenic mice was partially reduced by rapamycin. The insulin-stimulated increment in synthase fractional velocity was reduced by 30% in gluteal muscles and by 50% in gastrocnemius. As in normal mice, p70 activation in muscle was also abolished by rapamycin treatment (not shown). Therefore, in the context of impaired insulin receptor function when MAP kinase and RSK2 activation are also defective, p70 appears to participate in insulin's regulation of muscle glycogen synthase.


Figure 8: Effect of rapamycin on insulin-stimulated glycogen synthase activity in muscles from mutant insulin receptor transgenic mice. Rapamycin treatment of transgenic mice was performed as described in the legend to Fig. 7. Twenty min after injection of insulin or saline, gluteal (solid bars) and gastrocnemius (hatched bars) muscles were removed, homogenized, and glycogen synthase activity was measured as described in the legend to Fig. 3. Each value represents the mean ± S.E. of data obtained from 8 mice. * = insulin-stimulated (+) rapamycin versus insulin-stimulated gluteal muscle(-) rapamycin, p < 0.03,** = insulin-stimulated (+) rapamycin versus insulin-stimulated gastrocnemius muscle(-) rapamycin, p < 0.003




DISCUSSION

Numerous studies using cultured cell systems indicate that insulin potently activates MAP kinases, p90 and p70 (reviewed in Kyriakis and Avruch(17) ). However, there is a relative paucity of knowledge regarding the characterization of these enzymes or their potential physiologic roles in skeletal muscle. Two previous reports demonstrated that insulin stimulates p42/p44 MAP kinase activation in rat skeletal muscle(44, 45) . Although RSK2 was purified from insulin-stimulated rabbit skeletal muscle(14, 15) , insulin stimulation of p70 in skeletal muscle has not been previously characterized. In the present studies, we used muscles derived from insulin-stimulated intact mice to evaluate the relationships between stimulation of the insulin receptor tyrosine kinase, activation of these Ser/Thr kinases, and glycogen synthase. We also assessed the potential for defects involving these enzymes in two distinct models of insulin resistance; and we directly addressed the potential role of p70 in insulin-mediated regulation of muscle glycogen synthase. A summary of our findings is presented in Table 1. In experiments where two muscles (gluteal and gastrocnemius) were separately analyzed, parallel results were obtained.



It is clear from the data obtained using transgenic mice with overexpression of dominant-negative insulin receptors in muscle, that in vivo activation of both MAP kinase and RSK2 is dependent upon the insulin receptor tyrosine kinase. The fact that MAP kinase and RSK2 were comparably impaired in muscle from transgenic mice is also consistent with the view that MAP kinase is the predominant upstream activator of RSK(16) . Indeed, we recently obtained data demonstrating that blockade of MAP kinase kinase in COS cells prevents in vivo growth factor-mediated activation of transfected RSK2. (^3)

The pathway(s) responsible for p70 activation are independent of ras (and therefore divergent from MAP kinase and RSK)(46) . Since activation of p70 is wortmannin sensitive and is not mediated by receptor tyrosine kinases which lack phosphatidylinositol-3 (PI-3) kinase binding sites, PI-3-kinase has been implicated as a requisite upstream element(43) . Thus, it was surprising that insulin normally mediated activation of p70 in muscles where the insulin receptor tyrosine kinase is markedly impaired and where severe defects in IRS-1 phosphorylation and PI-3-kinase activation were previously observed (37) . However, a recent study suggests that blockade of PI-3-kinase via a different approach (dominant negative 85-kDa subunit of PI-3-kinase) does not affect insulin-stimulated p70 activation(47) . Interestingly, Myers et al. reported that p70 was substantially activated by insulin in 32D cells transfected with IRS-1 alone, whereas significant insulin-mediated PI-3-kinase (or MAP kinase) was not evident without overexpression of both IRS-1 and insulin receptors(48) . Using a maximally effective insulin dose, we also found that glycogen synthase activation in muscles from transgenic mice was preserved. One possible interpretation of these unexpected results is that insulin stimulation of p70 and glycogen synthase is independent of the insulin receptor tyrosine kinase per se. Pharmacologic inhibition of PI-3-kinase also reportedly impairs insulin-mediated glycogen synthase activation in cultured cells(32, 49) , although insulin stimulation of glycogen synthase in CHO cells was not affected by PI-3-kinase inhibition via overexpression of dominant-negative PI-3-kinase 85-kDa subunit(49) . Since insulin stimulation of glycogen synthase and p70 appear to share common features, a more likely explanation is that pathways leading to activation of p70 and glycogen synthase in skeletal muscle involve signal amplification (potentially occurring at similar steps beyond the stimulation of PI-3-kinase) and both effects are therefore selectively sensitive to insulin stimulation.

Importantly, we were able to dissociate the stimulation of glycogen synthase (which was preserved) from activation of MAP kinase and RSK2 (which were abolished) in skeletal muscles from transgenic mice. Furthermore, no detectable insulin stimulation of RSK2 was present in muscles from GTG-obese mice although partial insulin stimulation of glycogen synthase was still evident. These results indicate that RSK2 activation is not essential for insulin-mediated activation of muscle glycogen synthase. This conclusion is supported by the results of several additional recent studies. Although stimulation of adipocytes with epidermal growth factor (or platelet-derived growth factor) activates MAP kinase and RSK2, glycogen synthase activity (50) or glycogen synthesis (51) are relatively unaffected. Thus, activation of MAP kinase and RSK2 is apparently not sufficient for stimulation of glycogen synthase in these cells. Moreover, pharmacologic inhibition of MAP kinase kinase (with attendant MAP kinase inhibition) failed to impair insulin stimulation of glycogen synthase in cultured fat or muscle cells(52) . The above findings do not exclude the possibility that RSK2 might function as part of a redundant pathway (as we suggest may be true for p70; see below).

The potential for defects involving insulin-responsive Ser/Thr kinases in physiologic states of insulin resistance has not been adequately explored. Like other animal models (and humans) with insulin resistance, GTG-obese mice are characterized by a marked defect in insulin-stimulated muscle glucose uptake(35) . Muscles from these mice also display modest impairment of insulin receptor tyrosine kinase and more substantial impairment of insulin-stimulated IRS-1 phosphorylation and PI-3-kinase activation(36, 53, 54) . In the current study, severe defects involving MAP kinase and RSK2 that were greater than the modest decrease in insulin receptor kinase function were observed. Furthermore, the substantial impairment of insulin-mediated p70 activation was unexpected given that p70 stimulation was selectively preserved in muscles from transgenic mice. In the skeletal muscle of obese insulin resistant mice at least two loci for postreceptor defects can therefore be implicated: one involving steps leading from the insulin receptor to MAP kinase and RSK (e.g. SHC or IRS-1 phosphorylation which may also result in impaired PI-3-kinase); and one involving additional steps upstream from the activation of p70. Although the defects involving p70(55) or elements of the MAP kinase cascade (51, 52) per se are not likely to account for the impaired insulin-mediated muscle glucose uptake, further study of the potential link between PI-3-kinase and p70 may lead to the discovery of new signaling molecules which are critical for glucose transport.

Correlations between (a) the selective preservation of p70 and glycogen synthase in transgenic mice and (b) partial defects, involving both p70 and glycogen synthase in GTG-obese mice, suggested that p70 might have a role in mediating insulin's stimulation of glycogen synthase in muscle. The potential involvement of p70 was supported by Shepherd et al.(32) who recently reported that rapamycin antagonizes insulin activation of glycogen synthase in cultured adipocytes. In contrast, other investigators failed to see an effect of rapamycin on synthase activation using isolated rat adipocytes (50) or CHO cells(49) . In order to establish whether p70 is necessary for stimulation of glycogen synthase by insulin in muscle, we achieved complete and specific blockade of p70 activation by in vivo pretreatment of intact mice with rapamycin. By dissociating the activation of p70 from glycogen synthase stimulation, we have clearly demonstrated that p70 is not required for this effect of insulin in muscle. Although p70 can phosphorylate and inactivate glycogen synthase kinase-3 in vitro(27) , more recent data suggests that in vivo activation of p70 is not required for insulin-mediated regulation of glycogen synthase kinase-3 in cultured L6 myotubes(56) . Thus, a potential molecular link between p70 and glycogen synthase activation has also been refuted.

The fact that rapamycin was able to partially impair insulin-mediated activation of glycogen synthase in transgenic muscle represents an intriguing finding. These data lead us to the following conclusion. Although selective blockade of p70 has no effect on synthase activation in muscle from normal mice, the data in transgenic muscle suggest that p70 may represent a redundant pathway which does participitate in mediating this effect of insulin. Skeletal muscles from transgenic mice are characterized by severe defects in insulin-stimulation of PI-3-kinase (37) , MAP kinase, and RSK2 (as well other components not yet evaluated). In this context, insulin-mediated activation of glycogen synthase may be (at least partially) dependent upon p70. It is important to recognize that there are at least three shortcomings of studies which rely on the use of pharmacologic inhibitors to evaluate the involvement of selected signaling molecules in mediating discrete effects of insulin: the inhibitor may not be specific; the inhibition may not be complete; the biologic effect may be mediated by multiple pathways acting in concert. Data from our own studies as well as an emerging body of knowledge based on the study transgenic knockout mice and other systems lead us to believe that multiple (and often redundant) signaling pathways participate in mediating certain effects of insulin.


FOOTNOTES

*
This work was supported in part by National Institutes of Health NIDDK Grant RO1 45878 (to D. E. M.), the Boston Obesity and Nutrition Center (NIH-P30DK46200), and NATO Grant CRG 931483 (to Y. M.-B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Endocrinology, R80T-100, Merck Research Laboratories, P. O. Box 2000, Rahway, NJ 07065. Fax: 908-594-5700.

(^1)
The abbreviations used are: MAP, mitogen-activated protein; GTG, gold thioglucose; G6P, glucose 6-phosphate; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; MBP, myelin basic protein; PI-3, phosphatidylinositol-3; IRS-1, insulin receptor substrate-1.

(^2)
C. Bjørbæk and D. Moller, unpublished results.

(^3)
Y. Zhao and D. Moller, unpublished results.


ACKNOWLEDGEMENTS

We are especially grateful to John Blenis (Harvard Medical School) for providing valuable reagents and advice. We also thank Steven Pelech (Kinetic Biotechnology, Vancouver, BC) for the RSK2 antibody, Neil Ruderman (Boston University) and Barbara B. Kahn and Jeffrey S. Flier (Beth Israel Hospital) for their careful reading of the manuscript.


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