(Received for publication, July 5, 1994; and in revised form, September 19, 1994)
From the
In this study, we examined the distribution of protein
serine/threonine phosphatase-1 (PP-1) and analyzed the effect of
insulin on PP-1 and its mechanism of activation in freshly isolated rat
adipocytes. The adipocyte particulate fraction (PF) constituted 80%
of cellular PP-1 activity, while PP-2A was entirely cytosolic. Insulin
rapidly stimulated PF PP-1 in a time- and dose-dependent manner
(maximum stimulation at 5 min with 4 nM insulin).
Immunoprecipitation of PF with an antibody against the site-1 sequence
of rabbit skeletal muscle glycogen-associated PP-1 (PP-1G) subunit
indicated that
40% of adipocyte PP-1 activity was due to PP-1G form
of the enzyme. Insulin stimulated PP-1G (120% over basal levels)
without affecting the other forms of PP-1 in the PF. Insulin activation
of PP-1 was accompanied by >2-fold increase in the phosphorylation
state of the 160-kDa regulatory subunit of PP-1. Stimulation of
p21
/mitogen-activated protein kinase pathway (MAP) with
GTP analogues also resulted in stimulation of PP-1 similar to insulin.
The insulin effect on MAP kinase and PP-1 activation was blocked by a
GTP antagonist, guanyl-5`-yl thiophosphate. The inhibitors of MAP
kinase activation (viz. cAMP agonists, SpcAMP and ML-9) also
blocked PP-1 stimulation by insulin. The time course of MAP kinase
activation preceded the phosphorylation of PP-1 regulatory subunit and
PP-1 activation. We conclude that insulin rapidly activates a
membrane-associated PP-1 in adipocytes, which may be similar to rabbit
skeletal muscle PP-1G, and the activation is mediated by
p21
/MAP kinase pathway.
The major physiological role of insulin is to control body fuel metabolism by promoting the uptake of glucose and the other nutrients and their conversion into glycogen, proteins, and lipids. Insulin stimulates these pathways by controlling the activation state of certain key enzymes of glycogen and lipid metabolism via phosphorylation and dephosphorylation mechanisms(1, 2, 3) .
Protein
phosphatase-1 (PP-1) ()is one of the four major types of
serine hreonine phosphatases in mammalian cells, with a widespread
tissue distribution and capability to dephosphorylate a variety of
regulatory proteins in vitro(4) . The active forms of
PP-1 are believed to be largely particulate, and their association with
subcellular structures is mediated by ``targeting subunits''
distinct from the catalytic subunit(5) . Thus, PP-1 exists as a
complex with other proteins that target it to particular subcellular
locations, modify its substrate specificity, and appear to regulate the
enzyme activity(5) . In the rabbit skeletal muscle, PP-1 is
found associated with glycogen particles (PP-1G), the sarcoplasmic
reticulum, the myofibrils, and the cytosolic inhibitor 2
protein(5, 6) . Insulin has been shown to promote
site-specific phosphorylation (site-1) of the 160-kDa regulatory
subunit (G subunit) of rabbit skeletal muscle PP-1(7) , an
event that is believed to activate PP-1 with subsequent
dephosphorylation (and activation) of glycogen synthase and
phosphorylase kinase (inactivation). Phosphorylation of site-1 is
catalyzed by an insulin-stimulated protein kinase, which is a mammalian
homologue of ribosomal S6 kinase II(8) . In contrast to
insulin, adrenalin via cAMP/protein kinase A inhibits the
mitogen-activated protein (MAP) kinase pathway and also stimulates
phosphorylation at site-2 on PP-1G subunit, which in turn causes
dissociation of PP-1C subunit from the G subunit and its release from
glycogen(9, 10) .
A number of recent studies with
transfected cell lines indicate that p21/MAP kinase
pathway plays a pivotal role in insulin's effect on
mitogenesis(11, 12, 13, 14, 15) .
However, the intracellular targets of MAP kinase have not been
completely defined. MAP kinase phosphorylates and activates the
transcription factors c-Myc, c-Jun, phospholipase A2, and the 90-kDa
protein serine/threonine kinase, Rsk-2 (16, 17) ,
resulting in the phosphorylation of the ribosomal protein S6. In the
skeletal muscle, PP-1G is phosphorylated and activated in response to
insulin by an insulin-stimulated protein kinase, which is a mammalian
homologue of Rsk-2(8) , thus providing a potential link between
tyrosine kinase-mediated MAP kinase activation and stimulation of
glycogen synthesis. In contrast to skeletal muscle, which has been
widely studied with respect to glycogen metabolism and PP-1 regulation,
no data exists in the literature on the regulation of PP-1 in freshly
isolated rat adipocytes.
In view of the recent reports demonstrating
the formation of p21 GTP and rapid activation of MAP
kinase cascade in response to insulin and other growth factors in
cultured 3T3 L1 adipocytes, in the present study, we have examined the
regulation of PP-1 by insulin in freshly isolated rat adipocytes,
identified the regulatory subunit of the enzyme that controls the
enzyme activity, and studied the contribution of PI3 kinase and
p21
/MAP kinase pathway in insulin's effect on PP-1.
To evaluate the contribution of PI3 kinase pathway, the recently
described specific inhibitor of PI3 kinase, wortmannin (18) was
used. The involvement of Ras or other GTP binding proteins in insulin
response was investigated by a) treatment of permeabilized rat
adipocytes with non-hydrolyzable GTP analogs, b) studying the effect of
GTP antagonist, GDP
S on insulin response, and c) blocking MAP
kinase and 90-kDa S6 kinase activation with SpcAMP, a cAMP agonist, and
ML-9, a myosin light chain kinase inhibitor.
Because PP-1 activity can be present
in its latent form(22) , the total PP-1 activity was estimated
by incubation of extracts with TPCK-treated trypsin (40 µg/ml
5 min at 37 °C). The reaction was stopped by the addition
of 3 volumes of STI (100 µg/ml) followed by centrifugation and
assay of enzyme activity in the supernatants.
P-Labeled
phosphorylase a was prepared by reacting
[
-
P]ATP with purified phosphorylase kinase
and phosphorylase b as described earlier(23) .
A second experiment was run
simultaneously in which cells were incubated with cold P for the indicated time; PF and CF were prepared in a buffer (same
as above except that sodium fluoride,
-glycerophosphate, and
sodium pyrophosphate were omitted), followed by immunoprecipitation
with site-1 antibody and the assay of enzyme activity in the
immunoprecipitates. Briefly, the cell lysates (100 µg of protein)
from above were precleared by incubation with rat IgG (5 µg/ml,
coupled to protein A-Sepharose) at 4 °C for 1 h. The supernatants
were immunoprecipitated with PP-1G subunit antibody (10 µg/ml) at
room temperature for 1 h, followed by treatment with 50 µl of
protein A-Sepharose CL6B (50% v/v) for 1 h. In some experiments, the
antibody was pre-incubated with the competing peptide before adding to
the cell lysates. The pellets were washed four times with lysis buffer
(see above) and resuspended in 40 µl of 2
SDS-sample
buffer. The samples were incubated at 37 °C for 10 min, followed by
centrifugation (10,000
g for 30 s) to pellet down the
Sepharose beads. Electrophoresis of the immunoprecipitates was
performed in 7.5% SDS-polyacrylamide gels followed by autoradiography (24) . The protein contents of PP-1 catalytic and regulatory
subunits were determined by immunoprecipitating unlabeled cell lysates
with G subunit antibody, followed by separation of immunoprecipitated
proteins on SDS-PAGE and Western blot analysis with PP-1C or PP-1G
subunit antibody. After the transfer of proteins to nitrocellulose
membranes, the membranes were probed with PP-1C or PP-1G subunit
antibody. The catalytic and the regulatory subunits of PP-1 were
identified by incubating with
I-protein A (0.2
µCi/ml) followed by autoradiography. The intensity of the signal
was quantitated by densitometric analysis of the autoradiograms as well
as by the ``cut and count'' technique.
For the measurement of enzyme activity in the immunoprecipitates of control and insulin-exposed cells, the immunoprecipitations were performed in the cold room at 4 °C as described above, except that the lysis buffer did not contain phosphatase inhibitors. The absence of phosphatase inhibitors might result in considerable dephosphorylation of PP-1G subunit, thereby resulting in an underestimation of insulin effect. Because all the steps were carried out in the cold room and the treatment groups were always compared with the controls, the magnitude of error is lesser than anticipated. The immune complexes were washed four times with wash buffer and resuspended in the same buffer containing 15 µg/ml site-1 peptide (against which the antibody was raised) and incubated at 4 °C for 1 h to release the bound enzyme from the immune complex. An aliquot of the supernatant was assayed for PP-1 activity as described above.
In the initial studies, the cellular distribution of protein
phosphatase-1 and -2A activities was examined in adipocyte particulate
and cytosolic fractions. Particulate fraction comprised of 75% of
cellular PP-1 activity, while 25% of PP-1 was present in the soluble
fraction. In contrast to PP-1, the contribution of PP-2A to the
membranous phosphatase activity was very small, and most of the PP-2A
activity was found in the cytosol (Fig. 1). Mild trypsin
treatment resulted in 40% increase in particulate PP-1 activity
toward phosphorylase a, while the cytosolic PP-1 and PP-2A
activities were not affected. Insulin treatment resulted in a rapid
time-dependent increase in the spontaneous particulate PP-1 activity
(50% increase over basal values within 5 min, with a return to basal
levels within 20-30 min) (Fig. 2) without affecting the
total (trypsin-released) PP-1. Insulin had no effect on cytosolic PP-1
or PP-2A activities (data not shown). Earlier studies by Villa-Moruzzi (27) had shown that insulin treatment of differentiated 3T3 L1
adipocytes in culture resulted in the stimulation of cytosolic PP-1
activity. More importantly, trypsin treatment of the cytosolic extracts
was required to detect significant insulin effect. The insulin effect
was attributed to stimulation of the FA/GSK-3. The present studies do
not confirm the above observations. While trypsin treatment of the
particulate fractions resulted in considerable increase in basal PP-1
activity toward phosphorylase a, the cytosolic PP-1 activity
was not increased in control or insulin-stimulated cells. The reason
for this discrepancy is not clear at present. It could be due to
cultured cells as opposed to the freshly isolated adipose cells used in
the present study.
Figure 1:
Distribution of
spontaneous and total (trypsinreleased) PP-1 and PP-2A activities in
the adipocyte subcellular fractions. Protein phosphatases were
extracted as detailed in the text, and subcellular fractions were
prepared and assayed for the phosphatase activities using P-labeled phosphorylase as a substrate. Results are the
mean ± S.E. of four independent experiments performed in
duplicates. 1, spontaneous activities; 2,
trypsin-released total activities.
Figure 2: Time course of PP-1 activation by insulin. Adipocytes were treated with 4 nM insulin for 0-30 min. PP-1 activity was measured in the particulate fractions. Results are the mean of four independent experiments ± S.E. performed in duplicate.
The stimulation of PP-1 activity by insulin was dose-dependent, with a maximal effect observed at 4 nM (Fig. 3). Higher concentrations of insulin were inhibitory. Although the molecular basis for the biphasic effect of insulin on PP-1 activity is not understood, these results are similar to the findings of Chan et al.(28) in 3T3-D1 fibroblasts.
Figure 3: Dose response of PP-1 activation by insulin. Adipocytes were treated with various doses of insulin for 5 min, followed by the assay of enzyme activity in the PF. Results are the mean of two independent experiments.
The activation of PP-1 by insulin could theoretically occur at the level of protein synthesis or by post-translational modification of the enzyme. Western blot analysis of particulate fractions with PP-1 catalytic subunit antibody revealed similar amounts of a 37-kDa catalytic domain in both control and insulin-treated adipocytes. The identity of 37-kDa protein as PP-1C subunit was verified by competition studies with a peptide corresponding to the 14-amino acid sequence at the COOH-terminal region of rabbit skeletal muscle PP-1C subunit (data not shown).
Because PP-1 is known to exist as a complex bound to different targeting subunits, the nature of the regulatory subunit of adipocyte PP-1 was examined. Adipocyte membranes were solubilized in 0.5% Triton X-100, and the proteins were subjected to SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and immunoblotted with an antibody raised against a peptide surrounding the site-1 sequence of rabbit skeletal muscle PP-1G. Earlier studies by Cohen et al.(5, 6) have suggested that the targeting subunit of PP-1 in the sarcoplasmic reticulum is similar to glycogen-bound PP-1G subunit, thereby raising the possibility that one targeting subunit might associate with different cell organelles. The results of immunoblot analysis indicate that adipocyte membranes contain a protein of molecular mass of 160 kDa that binds to PP-1G subunit antibody (Fig. 4, lane3). This protein was not detected in the cytosolic fraction (Fig. 4, lane4), and the binding of the antibody to this protein was blocked by the presence of peptide against which the antibody was made (Fig. 4, lane5). Further confirmation that the adipocytes contain a protein that might be the regulatory subunit of PP-1 came from experiments in which the solubilized membrane proteins were immunoprecipitated with PP-1G subunit antibody (Table 1). In control preparations, 40% of membranous PP-1 activity was immunoprecipitated by the antibody. Insulin treatment resulted in 120% increase in immunoprecipitable PP-1 activity while the supernatants did not show an insulin effect, suggesting that insulin activates PP-1G in the adipocytes without affecting the other forms of PP-1.
Figure 4: Detection of PP-1 regulatory subunit in the adipocyte particulate fractions by Western blot analysis with PP-1G subunit antibody. Lane1, molecular weight marker; lane2, rabbit skeletal muscle extract (10 µg of protein); lane3, adipocyte particulate fraction (50 µg of protein); lane4, cytosolic fraction; lane5, particulate fraction probed with PP-1G subunit antibody in the presence of antigen.
Since the activation of PP-1 by insulin in the skeletal muscle
involves phosphorylation of site-1 on the regulatory subunit of the
enzyme, experiments were performed comparing the phosphorylation status
of the putative regulatory subunit of adipocyte PP-1 in the control and
insulin-stimulated cells. In these experiments, adipocytes were
metabolically labeled with [P]orthophosphate for
2 h followed by treatment with insulin. The labeled proteins present in
the PF and CF were immunoprecipitated with PP-1G subunit antibody, and
the immunoprecipitates were subjected to SDS-PAGE followed by
autoradiography. Parallel experiments were performed on cells that were
treated with cold phosphate followed by immunoprecipitation and Western
blot analysis to quantitate the amount of the regulatory subunit. The
amount of
P incorporated into the regulatory subunit was
determined by densitometric analysis of the autoradiograms of
phosphorylated proteins. The extent of phosphorylation was quantitated
after normalizing for variations in the contents of proteins in the
immunoprecipitates. Insulin treatment resulted in a 2-fold increase in
the
P incorporation into 160-kDa protein (Fig. 5, lane2). This protein was absent in the cytosolic
fractions (Fig. 5, lanes3 and 4) and
it was not immunoprecipitated by the preimmune serum (Fig. 5, lanes5 and 6); the immunoprecipitation was
completely blocked by the competing peptide (Fig. 5, lanes7 and 8).
Figure 5:
Phosphorylation of adipocyte PP-1
regulatory subunit by insulin. P-Labeled adipocytes were
exposed to insulin (4 nM) for 5 min. Triton-solubilized
particulate or cytosolic fractions (100 µg of protein) were
precleared by treatment rat IgG (10 µg) precoupled to protein
A-Sepharose. The supernatants were incubated with PP-1G subunit
antibody (20 µg) precoupled to protein A-Sepharose for 1 h at room
temperature with constant mixing. The immunoprecipitates were washed
four times in lysis buffer, eluted with 2
LSB, followed by
separation on a 7.5% SDS-polyacrylamide gel and autoradiography. A
representative autoradiogram is shown. Lane1,
control PF; lane2, insulin-treated adipocyte PF; lanes3 and 4, CF from control and
insulin-treated adipocytes; lanes5 and 6,
adipocyte PF from control and insulin-treated cells immunoprecipitated
with preimmune serum; lanes7 and 8,
immunoprecipitates from control and insulin-treated adipocyte PFs in
the presence of excess antigen.
From the above results, it appears that in the intact adipose tissue, insulin stimulates PP-1 activity by increasing the phosphorylation on the regulatory subunit. Further studies are needed on a highly purified enzyme preparation to examine the sites and the amino acid residues that are phosphorylated in response to insulin and whether the activation state of PP-1 could be modulated by another phosphatase.
To understand the mechanism of
PP-1 activation by insulin and to determine the upstream activators of
this enzyme, the contribution of PI-3 kinase and Ras/MAP kinase
signaling pathways were examined. To investigate the role of PI3
kinase, cells were incubated with wortmannin, a specific inhibitor of
PI3 kinase. Pretreatment of cells with maximally effective
concentrations of wortmannin did not block insulin's effect on
PP-1 (Fig. 6). Studies by Klarlund et al.(29) have shown that incubation of permeabilized 3T3 L1
adipocytes in culture with the non-hydrolyzable GTP analogues results
in a rapid activation of MAP kinase and S6 kinases. To examine the role
of Ras/MAP kinase pathway, two complimentary approaches were used. In
the first approach, toxin-permeabilized adipocytes were exposed
to a non-hydrolyzable analogue of GTP, GTP
S, or a GTP antagonist,
GDP
S, in the presence or absence of insulin, and PP-1 activation
was monitored. In the second approach, MAP kinase activation was
blocked by treatment of cells with a cAMP agonist, SpcAMP, and ML-9, a
MAP kinase/S6 kinase inhibitor previously reported to inhibit insulin
effect on glucose transport(30) . If MAP kinase pathway is
indeed involved in insulin's effect on adipocyte PP-1, then
treatment with GTP
S should result in PP-1 stimulation. Treatment
of permeabilized cells with GTP
S for 5-10 min resulted in
30-40% increase in PP-1 activity (Fig. 7). The extent of
stimulation of the enzyme was comparable with insulin. Inclusion of
insulin with the GTP analogue did not further increase the stimulation
of PP-1. In contrast, presence of a GTP antagonist, GDP
S, during
insulin exposure completely blocked the effect of insulin on PP-1
activation (Fig. 7). Basal levels of the enzyme were not
affected by GTP antagonist or by the
toxin that was used to
permeabilize the cells to GTP analogues.
Figure 6: Effect of wortmannin, a PI3-kinase inhibitor on PP-1 activation. Adipocytes were exposed to wortmannin (1 µM) for 15 min prior to insulin treatment. PP-1 activity was measured in the PF. Results are the mean ± S.E. of three independent experiments.
Figure 7:
GTPS activates PP-1 in permeabilized
cells. Adipocytes were permeabilized by treatment with
toxin (15
µg/ml) for 10 min, the medium was removed, and adipocytes were
resuspended in fresh KRP buffer containing GTP
S (1 mM)
with and without insulin and incubated for 10 min. A second batch of
cells was incubated with GDP
S for 10 min followed by insulin
exposure for 5 min. Results are the mean ± S.E. of four
independent experiments performed in
duplicate.
To further confirm that MAP kinase activation indeed results in the activation of PP-1, the kinetics of stimulation of MAP kinase by insulin was studied using myelin basic protein as a substrate. Within 2 min of exposure to insulin, there was a 2-fold increase in MAP kinase activity, with a peak at 5 min and a return to basal levels in 10 min. The effect of insulin was dose-dependent, with a maximum response at 4 nM in 5 min. Treatment of cells with ML-9, a myosin light chain kinase inhibitor, not only blocked the insulin effect on MAP kinase (Fig. 8), it also inhibited PP-1 activation (Fig. 9). In contrast to ML-9, rapamycin, an immunosuppressant drug known to inhibit the 70-kDa S6 kinase I, had no effect on PP-1 stimulation by insulin (results not shown).
Figure 8: Inhibition of MAP kinase activation by ML-9, a myosin light chain kinase inhibitor. Adipocytes were incubated with ML-9 (100 µM) for 10 min followed by insulin (4 nM) treatment for 5 min. MAP kinase activity was assayed as described in the text. Results are the mean of four independent experiments ± S.E.
Figure 9: ML-9 also blocks insulin activation of PP-1. Results are the mean of four independent experiments ± S.E. performed in duplicate.
The results presented in this report indicate that 40% of
the membrane-associated PP-1 activity in rat adipocytes is complexed to
a protein that may be similar to the G subunit, which targets the
rabbit skeletal muscle PP-1 to glycogen protein particles. This
conclusion is based on immunoblotting and immunoprecipitation studies
with an antibody raised against site-1 sequence of rabbit skeletal
muscle PP-1G. The contribution of the other forms of PP-1 could not be
assessed in the present study due to lack of specific antibodies.
Studies by Hubbard et al.(31) have shown that
sarcovesicular PP-1 in rabbit skeletal muscle is similar to PP-1G,
suggesting that the G subunit may play a dual role in targeting PP-1 to
two different subcellular locations. A dual function for the G subunit
is further supported by the predicted amino acid sequence, which
contains a hydrophobic region near the COOH terminus that could serve
as a membrane-spanning or -anchoring domain(32) . The
demonstration of PP-1G/sarcovesicular PP-1-like subunit in adipocyte
membranes further reinforces the possibility that reversible targeting
by organelle-specific subunits may be a general but important
regulatory mechanism for controlling the location, substrate
specificity, and activity of PP-1. Insulin rapidly activates the
membrane-associated PP-1 in rat adipocytes in a time- and
dose-dependent manner. The insulin effect on PP-1 (40-50% above
basal values) was consistent and significant (p < 0.05).
Insulin did not influence the abundance of particulate PP-1 catalytic
subunit (data not shown), thereby suggesting that the observed increase
in the enzyme activity is due to activation of the enzyme rather than
an increase in the content of PP-1C subunit. Immunoprecipitation
studies indicated that insulin activates the PP-1G/sarcovesicular PP-1
form >2-fold without altering the other forms of the enzyme (Table 1). It should be noted, however, that PP-1 activity
measurements were performed using the conventional substrate
phosphorylase a. This may not be the ideal substrate for
adipocyte PP-1, as the major function of adipose tissue is to store
lipids in the form of triglycerides and to mobilize fat during certain
physiological and pathophysiological states when a cell is challenged
with excessive cAMP levels. Further studies are needed to identify the
ideal in vitro and in vivo substrates of adipocyte
PP-1.
Studies with P-labeled adipocyte membranes
suggest that insulin effect was mediated by increased phosphorylation
of the putative regulatory subunit of PP-1. Based on the results of the
kinetics of PP-1 activation, the dose response data and studies with
the inhibitors, viz. SpcAMP and ML-9, the effects of insulin
on PP-1 appear to be mediated via the activation of MAP kinase cascade,
which in turn phosphorylates and activates 90-kDa ribosomal S6 kinase
II. This enzyme has been previously reported to be homologous to
insulin-stimulated protein kinase isolated from the skeletal muscles of
rabbits(8) . Insulin-stimulated protein kinase activates PP-1
by increasing site-1 phosphorylation on its regulatory
subunit(7) . Recent studies from this laboratory have also
shown that insulin stimulates PP-1 activity in cultured rat skeletal
muscle cells by increasing phosphorylation of a 160-kDa regulatory
subunit of PP-1(25) . A similar mechanism appears to be
responsible for the activation of PP-1 by insulin in adipocyte
membranes.
The above findings are in agreement with the concept that a large proportion of PP-1 in many eukaryotic cells and tissues is particulate (4) . Recent in vitro studies with the bacterially expressed three isoforms of the catalytic subunit of PP-1 suggest that inhibitor 2 is critical for the correct folding of nascent PP-1C molecules, and it acts as a cytosolic reservoir of PP-1C molecules that could be directed to various subcellular locations when required, following the synthesis of specific targeting subunits(33) . The function of inhibitor 2 in the adipocytes cannot be determined from the results of present studies.
A number
of recent studies indicate that p21/MAP kinase pathway
plays a pivotal role in insulin's effects on mitogenesis (11, 12, 13, 14, 15) .
However, the role of p21
/MAP kinase in metabolic effects
of insulin on glucose transport and glycogen synthesis remains
controversial(34, 35, 36) . Given the fact
that in the rabbit skeletal muscle, PP-1 activation is due to
phosphorylation of site-1 of the G subunit by an insulin-stimulated
protein kinase, we began studies to identify the upstream activators of
PP-1 and the insulin signaling pathway(s) responsible for
phosphorylation and activation of PP-1. Since insulin rapidly activates
both PI3 kinase and MAP kinase pathways, experiments were designed to
evaluate the contribution of each of these pathways. As seen in Fig. 6, inhibition of PI3 kinase with a specific inhibitor,
wortmannin, did not block PP-1 activation. However, treatment of
adipocytes with a GTP antagonist, GDP
S, completely blocked insulin
stimulation of MAP kinase/S6 kinase (29) as well as PP-1 (Fig. 7). In addition, treatment of permeabilized adipocytes
with GTP
S mimicked insulin's effect on PP-1 activation as
well as MAP kinase activation reported earlier. The findings that PP-1
activation is mediated in rat adipocytes via the activation of Ras/MAP
kinase pathway is further supported by experiments with cAMP agonist,
SpcAMP. Treatment of adipocytes with SpcAMP not only blocked MAP kinase
activation by insulin but also inhibited insulin effect on PP-1
activation (data not shown). The antagonism between cAMP and MAP kinase
pathway in different cell systems is well documented (37, 38, 39, 40, 41) . The
present findings on inhibition of insulin's effect on PP-1 by a
cAMP agonist lend further support to the earlier observations of the
counterregulatory effects of adrenalin on insulin. Whether it is due to
site-specific phosphorylation on PP-1 as well as MAP kinase inhibition
is not known.
In summary, the present study indicates that insulin rapidly activates a membranous form of adipocyte PP-1 that is similar to PP-1G, and the activation is mediated via Ras/MAP kinase pathway.