(Received for publication, July 24, 1995; and in revised form, October 2, 1995)
From the
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.
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) (
)kinase cascade
including MAP kinase kinase(9) , p44 (ERK1), and p42 (ERK2) MAP
kinases(10, 11, 12) , and the
90-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.
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 g for 10 min at 4 °C). Aliquots of the supernatant were removed
for determination of protein concentration(37) .
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, 50 mM
-glycerophosphate, 1 mM
Na
VO
, 2 mM dithiothreitol, 2 µg/ml
leupeptin, 5 µg/ml aprotinin, 40 µg/ml phenylmethylsulfonyl
fluoride, 1% Nonidet P-40), and immunoprecipitated with
-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
, 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).
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.
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 (
RSK2-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
-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.
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[C]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.
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
(-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.
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.
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.
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
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. ()
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.