Role of PKC isoforms in glucose transport in 3T3-L1
adipocytes: insignificance of atypical PKC
Masatoshi
Tsuru1,
Hideki
Katagiri2,
Tomoichiro
Asano3,
Tetsuya
Yamada1,2,
Shigeo
Ohno4,
Takehide
Ogihara3, and
Yoshitomo
Oka1,2
1 Third Department of Internal Medicine, Yamaguchi
University School of Medicine, Ube, Yamaguchi 755-8505;
2 Division of Molecular Metabolism and Diabetes,
Department of Internal Medicine, Tohoku University Graduate School of
Medicine, Sendai 980-8574; 3 Faculty of Medicine,
Department of Internal Medicine, University of Tokyo, Tokyo 113-8566;
and 4 Department of Molecular Biology, Yokohama City
University School of Medicine 3 - 9, Yokohama 236, Japan
 |
ABSTRACT |
To elucidate the involvement of protein
kinase C (PKC) isoforms in insulin-induced and phorbol ester-induced
glucose transport, we expressed several PKC isoforms, conventional
PKC-
, novel PKC-
, and atypical PKC isoforms of PKC-
and
PKC-
, and their mutants in 3T3-L1 adipocytes using an
adenovirus-mediated gene transduction system. Endogenous expression and
the activities of PKC-
and PKC-
/
, but not of PKC-
, were
detected in 3T3-L1 adipocytes. Overexpression of each wild-type PKC
isoform induced a large amount of PKC activity in 3T3-L1 adipocytes.
Phorbol 12-myristrate 13-acetate (PMA) activated PKC-
and exogenous
PKC-
but not atypical PKC-
/
. Insulin also activated the
overexpressed PKC-
but not PKC-
. Expression of the wild-type
PKC-
or PKC-
resulted in significant increases in glucose
transport activity in the basal and PMA-stimulated states.
Dominant-negative PKC-
expression, which inhibited the PMA
activation of PKC-
, decreased in PMA-stimulated glucose
transport. Glucose transport activity in the insulin-stimulated state
was increased by the expression of PKC-
but not of PKC-
. These
findings demonstrate that both conventional and novel PKC isoforms are involved in PMA-stimulated glucose transport and that other novel PKC
isoforms could participate in PMA-stimulated and insulin-stimulated glucose transport. Atypical PKC-
/
was not significantly activated by insulin, and expression of the wild-type, constitutively active, and
dominant-negative mutants of atypical PKC did not affect either basal
or insulin-stimulated glucose transport. Thus atypical PKC enzymes do
not play a major role in insulin-stimulated glucose transport in 3T3-L1 adipocytes.
protein kinase C; glucose transport; insulin; phorbol 12-myristate
13-acetate
 |
INTRODUCTION |
REGULATION OF GLUCOSE
HOMEOSTASIS is one of the most important actions of insulin.
Insulin stimulates the translocation of GLUT4 glucose transporters from
intracellular storage sites to the plasma membrane in muscle and
adipose tissue, resulting in increased glucose uptake. Many intensive
studies have focused on the cellular and molecular mechanisms
responsible for these trafficking events (15, 39).
The involvement of protein kinase C (PKC) in glucose transport
activation was originally recognized in studies using pharmacological agents. Phorbol 12-myristate 13-acetate (PMA), an activator of conventional and novel PKC, stimulates glucose transport activity (27) by inducing translocation of both GLUT1 and GLUT4
glucose transporters (1). In addition, insulin- and
PMA-stimulated glucose transport activity was inhibited by
staurosporine in isolated rat adipocytes (33, 41). These
results suggest that PKC participates in activating glucose transport
in adipocytes.
Several lines of evidence have indicated that phosphatidylinositol
3-kinase (PI 3-kinase) activation is important in insulin-stimulated glucose transport. The PI 3-kinase pharmacological inhibitors, such as
wortmannin (37) and LY-294002 (12), and
expression of the dominant-negative mutants of PI 3-kinase (18,
22, 25) reportedly markedly block insulin-stimulated glucose
transport and GLUT4 translocation in rat and 3T3-L1 adipocytes.
Furthermore, overexpression of wild-type PI 3-kinase tagged with the
GLUT2 COOH-terminal domain (16) or the constitutively
active mutant of PI 3-kinase (29) promoted glucose
transport activity and GLUT4 translocation. These findings suggest a
central role for PI 3-kinase in insulin-stimulated glucose transport.
Recently, several pathways have been reported to be activated by growth
factor stimulation downstream from PI 3-kinase. Atypical PKC,
consisting of PKC-
and PKC-
, which are not activated by diacylglycerol or phorbol ester, is one of such effectors of PI 3-kinase. Atypical PKC enzymes are activated in vitro in the presence of the products of PI 3-kinase and phosphatidylinositol triphosphate (32, 43), and are also activated by growth factor
stimulation (2). Furthermore, two groups of
investigators have reported that PKC-
or PKC-
played a major role
in glucose transport activation by insulin in 3T3-L1 adipocytes
(8, 26), L6 myocytes (6), and rat
(42) and human (5) adipocytes, although they
hypothesized that different atypical PKC isoforms were involved. In
contrast, it was also reported that activation of PKC-
, a novel PKC,
but not an atypical PKC, is a major signal in insulin-induced glucose transport (10). Thus the necessity of activating atypical
PKC in insulin-induced glucose transport is controversial.
In the present study, to elucidate the involvement of conventional,
novel, and atypical PKCs in insulin-induced and PMA-induced glucose
transport, we examined the effects of overexpressing several PKC
isoforms and their mutants (PKC-
as a conventional PKC isoform, PKC-
as a novel PKC isoform, and PKC-
and PKC-
as atypical PKC
isoforms). 3T3-L1 adipocytes were transfected with these isoforms using
an adenovirus-mediated gene transduction system.
 |
MATERIALS AND METHODS |
Antibodies.
The rabbit polyclonal anti-PKC-
antibody, anti-PKC-
antibody, and
anti-PKC-
antibody, which also reacts with the
-isoform, were
purchased from Santa Cruz Biotechnology.
Cell culture.
3T3-L1 fibroblasts were maintained in DMEM containing 10% donor calf
serum (GIBCO) in an atmosphere of 10% CO2 at 37°C. Two days after the fibroblasts had reached confluence, differentiation was
induced by treating cells with DMEM containing 0.5 mM 3-isobutyl 1-methylxanthine, 4 µg/ml dexamethasone, and 10% FBS for 48 h. Cells were incubated with DMEM supplemented with 10% FBS every other
day for the following 4-10 days. More than 90% of the cells expressed the adipocyte phenotype (23).
Expression constructs.
The cDNAs encoding the entire rabbit PKC-
(35), its
dominant-negative mutant (7) mouse PKC-
(36), its dominant-negative mutant (19) mouse
PKC-
(17), its dominant-negative mutant (9) mouse PKC-
(2), and its
dominant-negative (2) and constitutively active
(26) mutants were kindly provided by Dr. S. Ohno.
Recombinant adenoviruses containing each PKC gene were constructed by
homologous recombination between the expression cosmid cassette and the
parental virus genome, as described previously (22, 30,
34).
Gene transduction.
3T3-L1 adipocytes were incubated with DMEM containing the adenovirus
for 1 h at 37°C, and the growth medium was then added. Experiments were performed 60 h after the infection. Recombinant adenoviruses were applied at a multiplicity of infection (MOI) of
200-300 plaque-forming units (pfu)/cell, and 3T3-L1 adipocytes infected with the Adex1CalacZ virus (21), which encodes
Escherichia coli lacZ, were used as a control, since
Adex1CAlacZ gene expression was observed in >90% of 3T3-L1 adipocytes
applied at an MOI of 200-300 pfu/cell on postinfection day
3 but did not affect glucose transport activity compared with
noninfected cells, as reported previously (22, 23).
PKC activity assay.
PKC activity was assayed using a peptide substrate, as previously
described (8). 3T3-L1 cells cultured in 12-well plates were incubated with or without 1 µM insulin or 1.6 µM PMA for 10 min. Cells were homogenized in buffer containing 20 mM
Tris · HCl (pH 7.5), 0.25 mM sucrose, 1.2 mM EGTA, 20 mM
-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM
sodium vanadate, 1 mM sodium pyrophosphate, 1 mM NaF, 1% Triton X-100,
0.5% Nonidet P-40, and 150 mM NaCl and then cleared of insoluble
substances by centrifugation. PKC-
and PKC-
were
immunoprecipitated by incubation with polyclonal anti-PKC-
antibody
or anti-PKC-
antibody, respectively, for 16 h at 0-4°C.
The immunoprecipitates were suspended in reaction buffer containing 50 mM Tris · HCl (pH 7.5), 1 mM NaHCO3, 5 mM
MgCl2, and 1 mM PMSF and then were assayed for the ability
to phosphorylate a PKC pseudosubstrate peptide, namely 40 µM
[Ser25]PKC-(19-31) (GIBCO-BRL) in
buffer containing 50 mM Tris · HCl (pH 7.5), 5 mM
MgCl2, 100 µM sodium vanadate, 100 µM sodium
pyrophosphate, 1 µM CaCl2, 1 mM NaF, 100 µM PMSF, and
50 µM [
-32P]ATP. To assay PKC-
/
activity,
polyclonal anti-PKC-
antibody and
[Ser159]PKC-
-(153-164)-NH2
(Upstate Biotechnology) were used for immunoprecipitation and as the
reaction substrate, respectively. Reactions were stopped with 5%
acetic acid, and aliquots of the reaction mixture were spotted on P-81
filter paper (Whatman), washed in 5% acetic acid, and counted for
32P. The results were quantitated using an image analyzer
BAS2000 (Fujix).
Glucose transport assay.
3T3-L1 adipocytes in 12-well plates were serum starved for 3 h in
DMEM containing 0.2% BSA, followed by a 45-min glucose-free incubation
in Krebs-Ringer phosphate buffer. Cells were then incubated with or
without 1 µM insulin or 1.6 µM PMA for 15 min, and 0.1 mM
2-deoxy-D-[3H]glucose uptake was measured as
described previously (22, 23).
 |
RESULTS |
Conventional PKC, PKC-
, has some involvement in PMA-induced, but
not in insulin-induced, glucose transport.
First, we expressed the wild-type and dominant-negative mutants of a
conventional PKC isoform, PKC-
, to increase and suppress PKC-
activity, respectively, using an adenovirus-mediated gene transduction
system. Immunoblotting with anti-PKC-
antibody showed that
successful expression of wild-type and the mutated PKC-
was achieved
in 3T3-L1 adipocytes by infection with the recombinant adenoviruses
(Fig. 1A). PKC-
activity
was measured in the immunoprecipitates with anti-PKC-
antibody (Fig.
1B). Consistent with the results obtained from the
immunoblotting study, overexpression of the wild-type PKC-
produced
an 18-fold increase in PKC-
activity in the basal state compared
with that in control 3T3-L1 adipocytes that were infected with
adenovirus recombined with the lacZ gene. Stimulation with insulin did
not affect the endogenous or exogenously expressed PKC-
activity. In
contrast, PMA increased both endogenous and exogenously expressed
PKC-
activity by ~2.5-fold and 1.6-fold, respectively (Fig.
1B). Thus exogenous PKC-
was functionally expressed. In
addition, expression of the dominant-negative mutant of PKC-
completely inhibited PKC-
activation by PMA while having no effect
on endogenous PKC-
activity in the basal- and insulin-stimulated states (Fig. 1B). Thus expression of the mutant PKC-
resulted in exerting a dominant-negative effect on PKC-
activation
by PMA.

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Fig. 1.
Protein kinase C (PKC)- expression levels and
activities in the absence or presence of insulin or phorbol
12-myristate 13-acetate (PMA). A: expression levels of
PKC- proteins. Lysates from 3T3-L1 adipocytes (control) and from
those expressing lacZ (control), wild-type (WT) PKC- , or the
dominant negative (DN) mutant of PKC- were immunoblotted with
anti-PKC- antibody. B: control 3T3-L1 adipocytes and
3T3-L1 adipocytes expressing PKC- WT or PKC- DN were incubated
with or without 1 µM insulin or 1.6 µM PMA for 10 min. Lysates were
immunoprecipitated with anti-PKC- antibody, and PKC- activities
in the immunoprecipitates were assayed, as described in MATERIALS
AND METHODS. Values presented as means ± SD of 3 separate
experiments.
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|
To examine the effects of PKC-
activity on insulin- and PMA-induced
glucose transport, we measured 2-deoxyglucose uptake in 3T3-L1
adipocytes overexpressing wild-type and dominant-negative PKC-
with
or without insulin or PMA stimulation (Fig.
2). Insulin and PMA stimulated
2-deoxyglucose uptake by ~7.0-fold and 1.7-fold, respectively, in
control (lacZ-expressing) 3T3-L1 adipocytes. Expression of the
wild-type PKC-
in 3T3-L1 adipocytes produced a significant increase
in glucose transport activity to ~1.9- and 2.2-fold in the basal and
PMA-stimulated states, respectively. In contrast, expression of the
dominant-negative mutant of PKC-
did not affect basal glucose
transport activity, but a significant decrease was observed in the
PMA-stimulated state. Neither the wild-type nor the dominant-negative
mutant of PKC-
had an effect on insulin-stimulated glucose transport
activity. These findings demonstrate that 1) PKC-
activation stimulates glucose transport activity, 2) PKC-
activation is involved in the PMA-stimulated glucose transport,
although this effect is partial, and 3) PKC-
activation
is not involved in the insulin-stimulated glucose transport activity.

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Fig. 2.
Insulin- or PMA-stimulated 2-deoxy-D-glucose
uptake in control and PKC- WT or PKC- DN overexpressing
3T3-L1-adipocytes. Uptake of 2-deoxy-D-glucose after
incubation with or without 1 µM insulin or 1.6 µM PMA for 15 min
was assayed in control 3T3-L1 adipocytes and 3T3-L1 adipocytes
expressing PKC- WT or PKC- DN for 4 min. Data presented as
means ± SD of 3 separate experiments.
|
|
Exogenously expressed PKC-
induced a small increase in glucose
transport activity in the basal, insulin-stimulated, and PMA-stimulated
states.
Next, we expressed the wild-type and the dominant-negative mutants of
PKC-
, a novel PKC enzyme, in 3T3-L1 adipocytes and measured PKC-
activity and glucose transport activity in the presence or absence of
insulin or PMA. Endogenous PKC-
expression was very low, almost
undetectable, in control 3T3-L1 adipocytes, whereas the exogenous
expressions of the wild-type and dominant-negative mutants of PKC-
were clearly detected, as shown by immunoblotting with anti-PKC-
antibody (Fig. 3A). PKC-
activity was measured in the immunoprecipitates with anti-PKC-
antibody (Fig. 3B). Consistent with the results of the
immunoblotting study, endogenous PKC-
activity was almost
undetectable in control 3T3-L1 adipocytes in either the basal, the
insulin-stimulated, or the PMA-stimulated state. Overexpression of
PKC-
induced a large amount of PKC-
activity in 3T3-L1
adipocytes. Insulin and PMA stimulated this activity by 1.3- and
2.4-fold, respectively. In contrast, expression of the
dominant-negative mutant of PKC-
did not lead to PKC-
activity in
either the basal, insulin-stimulated, or PMA-stimulated states (Fig.
3B).

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Fig. 3.
PKC- expression levels and activities in the absence
or presence of insulin or PMA. A: expression levels of
PKC- proteins. Lysates from control 3T3-L1 adipocytes and from those
expressing PKC- WT or PKC- DN were immunoblotted with
anti-PKC- antibody. B: control 3T3-L1 adipocytes and
3T3-L1 adipocytes expressing PKC- WT or PKC- DN were incubated
with or without 1 µM insulin or 1.6 µM PMA for 10 min. Lysates were
immunoprecipitated with anti-PKC- antibody, and PKC- activities
in the immunoprecipitates were assayed as described in MATERIALS
AND METHODS. Values presented as means ± SD of 3 separate
experiments.
|
|
We further assayed the 2-deoxyglucose uptake in 3T3-L1 adipocytes
overexpressing the wild-type or dominant-negative PKC-
(Fig.
4). Expression of the wild-type PKC-
induced significant increases in glucose transport activity of ~2.0-,
1.3-, and 3.2-fold in the basal, insulin-, and PMA-stimulated states,
respectively, compared with that in control 3T3-L1 adipocytes. In
contrast, expression of the dominant-negative mutant of PKC-
did not
affect basal, insulin-stimulated, or PMA-stimulated glucose transport activity (Fig. 4). These findings demonstrate that a novel PKC could
participate in PMA-induced and insulin-induced glucose transport activation, although endogenous expression of PKC-
is below the detection limits.

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Fig. 4.
Insulin- or PMA-stimulated 2-deoxy-D-glucose
uptake in control and PKC- WT or PKC- DN overexpressing 3T3-L1
adipocytes. Uptake of 2-deoxy-D-glucose after incubation
with or without 1 µM insulin or 1.6 µM PMA for 15 min was assayed
in control 3T3-L1 adipocytes and 3T3-L1 adipocytes expressing PKC-
WT or PKC- DN for 4 min. Data presented as means ± SD of 3 separate experiments.
|
|
Atypical PKC enzymes do not play a major role in insulin-stimulated
glucose transport.
Seemingly conflicting results were reported by two research groups who
found that different isoforms of atypical PKC enzymes, PKC-
and
PKC-
, are mainly involved in insulin-stimulated glucose transport.
To determine which isoform plays the major role in glucose transport
activation by insulin, we exogenously expressed the wild type or
dominant-negative mutant of PKC-
or PKC-
in 3T3-L1 adipocytes,
followed by measurement of atypical PKC activity and glucose transport
activity with or without insulin or PMA stimulation.
First, to assess whether endogenous PKC-
/
was activated by PMA
and insulin, the endogenous enzymes were immunoprecipitated from
control 3T3-L1 adipocytes with a polyclonal anti-PKC-
antibody that
also reacts with the PKC-
isoform. Immunoprecipitates from control
3T3-L1 adipocytes showed substantial activity above the background
measured in irrelevant IgG immunoprecipitates. Immunoblotting study
revealed a faint but detectable band of endogenous atypical PKCs (Figs.
5A and
6A). As expected, PMA
stimulation of control 3T3-L1 adipocytes did not alter atypical
PKC-
/
activities. In addition, contrary to previous reports,
insulin stimulation did not activate the endogenous atypical PKCs
(Figs. 5B and 6B).

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Fig. 5.
PKC- expression levels and activities in the absence
or presence of insulin or PMA. A: expression levels of
PKC- proteins. Lysates from control 3T3-L1 adipocytes and from those
expressing PKC- WT or PKC- DN were immunoblotted with
anti-atypical PKC antibody. B: control 3T3-L1 adipocytes and
3T3-L1 adipocytes expressing PKC- WT or PKC- DN were incubated
with or without 1 µM insulin or 1.6 µM PMA for 10 min. Lysates were
immunoprecipitated with anti-PKC- antibody, and PKC- activities
in the immunoprecipitates were assayed as described in MATERIALS
AND METHODS. Values presented as means ± SD of 3 separate
experiments.
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Fig. 6.
PKC- expression levels and activities in the absence
or presence of insulin or PMA. A: expression levels of
PKC- proteins. Lysates from control 3T3-L1 adipocytes and from those
expressing PKC- WT or PKC- DN were immunoblotted with
anti-atypical PKC antibody. B: control 3T3-L1 adipocytes and
3T3-L1 adipocytes expressing PKC- WT or PKC- DN were incubated
with or without 1 µM insulin or 1.6 µM PMA for 10 min. Lysates were
immunoprecipitated with anti-PKC- antibody, which also reacts with
the -isoform, as described in MATERIALS AND METHODS, and
PKC- activities in the immunoprecipitates were assayed as described
in MATERIALS AND METHODS. Values presented as means ± SD of 3 separate experiments.
|
|
Exogenously overexpressed wild-type or the dominant-negative mutant of
PKC-
or PKC-
was clearly detected by immunoblotting with
anti-PKC-
antibody (Figs. 5A and 6A).
Overexpression of atypical PKC-
or PKC-
resulted in increases in
atypical PKC activity of ~10.4- and 4.1-fold, respectively, but
atypical PKC was not activated by PMA. Insulin treatment did not
significantly increase exogenously expressed atypical PKC activities
(Figs. 5B and 6B). In addition, expression of
dominant-negative mutants of PKC-
or PKC-
did not significantly
alter atypical PKC activity in either the basal, insulin-stimulated, or
PMA-stimulated state (Figs. 5B and 6B).
2-Deoxyglucose uptake was measured in 3T3-L1 adipocytes overexpressing
the wild-type or dominant-negative PKC-
(Fig.
7) or PKC-
(Fig.
8) in the presence or absence of
insulin or PMA. As expected, exogenous expression of these proteins did
not influence PMA-stimulated glucose transport. Unexpectedly,
overexpression of the wild-type PKC-
or PKC-
did not increase
either basal or insulin-stimulated glucose transport activity. In
addition, expression of dominant-negative mutants of these atypical
PKCs did not influence insulin-stimulated glucose transport activity.

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Fig. 7.
Insulin- or PMA-stimulated 2-deoxy-D-glucose
uptake in control and overexpressed PKC- WT or PKC- DN 3T3-L1
adipocytes. The uptake of 2-deoxy-D-glucose after
incubation with or without 1 µM insulin or 1.6 µM PMA for 15 min
was assayed in control 3T3-L1 adipocytes and 3T3-L1 adipocytes
expressing PKC- WT or PKC- DN for 4 min. Data presented as
means ± SD of 3 separate experiments.
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Fig. 8.
Insulin- or PMA-stimulated 2-deoxy-D-glucose
uptake in control and PKC- WT or PKC- DN overexpressing 3T3-L1
adipocytes. Uptake of 2-deoxy-D-glucose after incubation
with or without 1 µM insulin or 1.6 µM PMA for 15 min was assayed
in control 3T3-L1 adipocytes and 3T3-L1 adipocytes expressing PKC-
WT or PKC- DN for 4 min. Data presented as means ± SD of 3 separate experiments.
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|
Because glucose transport activity was not affected by
overexpression of wild-type atypical PKC-
, we performed additional experiments using a constitutively active mutant of PKC-
to further increase atypical PKC activity. Expression of constitutively active PKC-
increased atypical PKC activity to ~33-fold (Fig.
9B), which was consistent with
its expression level (Fig. 9A). However, expression of
constitutively active PKC did not affect either basal or
insulin-stimulated glucose transport activity (Fig. 9C).
Thus endogenous atypical PKC activity was not increased by insulin
treatment, and no evidence indicating involvement of atypical PKC in
insulin-induced signaling stimulating glucose transport was obtained in
experiments focusing on expression of wild-type, dominant-negative, and
constitutively active atypical PKC. These data indicate that atypical
PKC enzymes do not play a major role in insulin-stimulated glucose
transport.

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Fig. 9.
Effects of expression of constitutively active PKC- on
atypical PKC activity and glucose transport activity. A:
expression levels of PKC- proteins. Lysates from control 3T3-L1
adipocytes and from those expressing constitutively active (CA) PKC-
were immunoblotted with anti-atypical PKC antibody. B:
control 3T3-L1 adipocytes and 3T3-L1 adipocytes expressing PKC- CA
were incubated with or without 1 µM insulin for 10 min. Lysates were
immunoprecipitated with anti-atypical PKC antibody, and PKC activities
in the immunoprecipitates were assayed as described in tMATERIALS
AND METHODS. Values presented as means ± SD of 3 separate
experiments. C: uptake of 2-deoxy-D-glucose
after incubation with or without 1 µM insulin or 1.6 µM PMA for 15 min was assayed in control 3T3-L1 adipocytes and 3T3-L1 adipocytes
expressing PKC- CA for 4 min. Data presented as means ± SD of
3 separate experiments.
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 |
DISCUSSION |
We expressed the wild-type and dominant-negative mutants of each
PKC isozyme, PKC-
as a conventional PKC isoform, PKC-
as a novel
PKC isoform, and PKC-
and PKC-
representing atypical PKC
isoforms. The present results show clearly that conventional PKC
participates, to some extent, in PMA-stimulated but not in insulin-stimulated glucose transport activity. It was reported that
stable expression of the wild-type or constitutively active forms of
PKC-
, PKC-
1, and PKC-
2 failed to influence basal glucose transport in 3T3-L1 fibroblasts and adipocytes (8).
However, in the present study, a small increase in glucose transport
activity in the basal state was observed in PKC-
-overexpressing
adipocytes. The PKC enzyme was transiently expressed using an
adenovirus-mediated gene transduction system, and PKC activity and
glucose transport activity were measured 2 days after the transfection;
i.e., the period of expression of these PKC enzymes was much shorter
than that of stable expression. Some unknown mechanisms compensating for increased PKC activities may operate in cells overexpressing conventional PKC enzymes, especially in stable transfectants. The
discrepancy between the previous and present results may therefore be
attributable to different periods of expression.
The results obtained for exogenously expressed PKC-
appeared to be
very similar to those obtained for PKC-
. However, the activity of
endogenous PKC-
was below the detectable level because of its
extremely low expression level in 3T3-L1 adipocytes. There was no
detectable increase in endogenous PKC-
activity, even in the
PMA-stimulated state. In primary cultures of rat skeletal muscle,
PKC-
reportedly mediates insulin-stimulated glucose transport (10) and regulates insulin receptor activity and routing
(11). In 3T3-L1 adipocytes, although no information is
available as to what novel PKC isozyme(s) is expressed, our exogenously
expressed PKC-
results suggest that another novel PKC isozyme(s), if
present, could respond to PMA and contribute to PMA-stimulated glucose transport activity.
Insulin treatment did not activate endogenously or exogenously
expressed PKC-
, whereas insulin activated exogenously expressed PKC-
. This is consistent with reports that novel PKC is activated downstream from PI 3-kinase (31). In accordance with these
results, the average value of insulin-stimulated glucose transport
activity was greater (1.4-fold) in adipocytes overexpressing PKC-
than in control adipocytes, although the difference did not reach
statistical significance in four independent experiments. These
findings suggest that conventional PKC is not involved in
insulin-stimulated glucose transport but that novel PKC isoforms, if
expressed in 3T3-L1 adipocytes, may contribute, to a limited extent, to
insulin-stimulated glucose transport activity.
Two research groups have reported atypical PKC to be involved in
insulin-stimulated glucose transport in adipocytes, although the two
groups proposed the involvement of different atypical PKC isoforms.
Standaert et al. (42) reported that inhibition of PKC-
activity by the PKC-
pseudosubstrate or Ro31-8220 paralleled inhibition of insulin-stimulated glucose transport in rat adipocytes. They also reported that expression of wild-type or constitutively active PKC-
stimulated translocation of coexpressed GLUT4 that was
tagged with myc and that expression of dominant-negative
PKC-
partially inhibited translocation of tagged GLUT4 in rat
adipocytes. They thus suggest PKC-
to be a downstream effector of PI
3-kinase through insulin-stimulated GLUT4 translocation. Kotani et al. (26) reported, however, that overexpression of the
constitutively active mutant of PKC-
activated glucose transport
activity in 3T3-L1 adipocytes. In addition, they showed that
overexpression of the dominant-negative mutant of PKC-
partially
inhibited glucose transport activity, suggesting that PKC-
lies in
the insulin signaling pathway responsible for regulating glucose
uptake. In their study, overexpression of constitutively active PKC-
induced an ~600-fold increase in PKC-
activity, whereas insulin
stimulated endogenous PKC-
activity by at most threefold.
Despite this huge, nonphysiological increase in PKC-
activity
with overexpression of the constitutively active mutant, glucose
transport activity was rather smaller than that caused by insulin in
control (noninfected) 3T3-L1 adipocytes. On the other hand, in the
present study, a modest increase in atypical PKC activity, close to the
physiological condition, was achieved. Expression of the wild-type
PKC-
, PKC-
, and constitutively active PKC-
produced increases
in atypical PKC activity to ~10-, 4-, and 33-fold, respectively, but
these procedures did not affect glucose transport activity. Thus, under physiological conditions, an increase in atypical PKC activity is not
sufficient for glucose transport activation. Furthermore, we detected
no measurable activation of atypical PKC-
/
by insulin, whereas
insulin fully activated (i.e., an ~9-fold increase) glucose transport
activity. It is also noteworthy that exogenously expressed wild-type
PKC-
/
was not significantly activated by insulin. On the
contrary, when wild-type PKC-
was overexpressed in Chinese hamster
ovary cells using an adenovirus-mediated gene transduction system,
PKC-
was efficiently activated by insulin (data not shown), indicating that the adenovirus-mediated gene transduction system and
our assay system for atypical PKC activity worked well and that insulin
does not activate atypical PKC in 3T3-L1 adipocytes. Expression of
dominant-negative mutants of atypical PKC enzymes did not influence
insulin-stimulated glucose transport activity in the present study. In
addition, we also observed that Go6983 (100 nM), which reportedly
inhibits the activities of several PKC enzymes, including PKC-
, did
not inhibit basal or insulin-stimulated glucose transport activity in
3T3-L1 adipocytes (data not shown). Taken together, these findings
clearly show that activations of atypical PKC enzymes, PKC-
and
PKC-
, do not play a major role in insulin signaling through glucose
transport activation under physiological conditions. Recently, similar
results were also reported in L6 myocytes, i.e., insulin did not
significantly stimulate either endogenous atypical PKC-
/
or
transfected hemagglutinin epitope-tagged PKC-
(44). Indeed, we observed that overexpression of
constitutively active atypical PKC-
in undifferentiated L6 myoblasts
using recombinant adenovirus increased basal glucose transport activity
without altering basal or insulin-stimulated glucose transport activity
in differentiated L6 myocytes (unpublished observation). Thus atypical
PKC activation by insulin might depend on ambient factors such as the
differentiation state of the cells, and activation of concomitant
stimuli may also be important.
Regarding the mechanism whereby insulin stimulates glucose transport
activity, 3-phosphoinositide-dependent protein kinase 1 (PDK1) and
protein kinase B (PKB) have received considerable attention as signals
downstream from PI 3-kinase activation. Several studies using the
dominant-negative mutants of PKB were reported, but the effects on
insulin-stimulated glucose transport and GLUT4 translocation were
different among these mutants, such as the kinase-inactive mutant
(14), the phosphorylation-deficient mutant (24), and the mutant having both mutations
(44). It was recently reported that PDK1, which
phosphorylates PKB and leads to its activation (3), also
activates serine/threonine kinases, including atypical PKC
(28), p70 (4, 40) and p90 (20)
S6 kinase, cAMP-dependent protein kinase (13), and serum
and glucocorticoid-inducible kinase (38). It is possible
that each PKB mutant binds to PDK1 and affects PDK1 to activate these
pathways in a different manner. Similarly, stable and long-term
expression and extremely high levels of expression of atypical PKC or
its mutants may also induce a feedback effect on upstream kinases, such
as PDK1. The discrepancies among several reports may have arisen from
the difference in periods and levels of expression of exogenous
proteins. In the present study, by use of the adenovirus gene
transduction system, transient physiological expression was achieved.
Taken together, our results strongly suggest that atypical PKC isoforms
do not play a major role in insulin-stimulated glucose transport under
physiological conditions in 3T3-L1 adipocytes.
 |
ACKNOWLEDGEMENTS |
This work was supported by Grant-in-Aid for Scientific Research no.
13470226 (to Y. Oka) and Creative Basic Research Grant no. 10NP0201 (to
Y. Oka) from the Ministry of Education, Science, Sports and Culture of Japan.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: Y. Oka, Division of Molecular Metabolism and Diabetes, Department of Internal Medicine, Tohoku University Graduate School of Medicine, Seiryo-machi, Sendai, Miyagi 980-8574 (E-mail:
oka{at}int3.med.tohoku.ac.jp).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
March 12, 2002;10.1152/ajpendo.00457.2001
Received 11 October 2001; accepted in final form 27 February 2002.
 |
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