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Molecular and Cellular Biology, December 1998, p. 6971-6982, Vol. 18, No. 12
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Requirement of Atypical Protein Kinase C
for
Insulin Stimulation of Glucose Uptake but Not for Akt Activation in
3T3-L1 Adipocytes
Ko
Kotani,1
Wataru
Ogawa,1,*
Michihiro
Matsumoto,1
Tadahiro
Kitamura,1
Hiroshi
Sakaue,1
Yasuhisa
Hino,1
Kazuaki
Miyake,1
Wataru
Sano,1
Kazunori
Akimoto,2
Shigeo
Ohno,2 and
Masato
Kasuga1
Second Department of Internal Medicine, Kobe
University School of Medicine, Chuo-ku, Kobe
650-0017,1 and
Department of
Molecular Biology, Yokohama City University School of Medicine,
Kanazawa-ku, Yokohama 236-0004,2 Japan
Received 25 February 1998/Returned for modification 15 April
1998/Accepted 14 August 1998
 |
ABSTRACT |
Phosphoinositide (PI) 3-kinase contributes to a wide variety of
biological actions, including insulin stimulation of glucose transport
in adipocytes. Both Akt (protein kinase B), a serine-threonine kinase
with a pleckstrin homology domain, and atypical isoforms of protein
kinase C (PKC
and PKC
) have been implicated as downstream effectors of PI 3-kinase. Endogenous or transfected PKC
in 3T3-L1 adipocytes or CHO cells has now been shown to be activated by insulin
in a manner sensitive to inhibitors of PI 3-kinase (wortmannin and a
dominant negative mutant of PI 3-kinase). Overexpression of
kinase-deficient mutants of PKC
(
KD or 
NKD), achieved with the use of adenovirus-mediated gene transfer, resulted in inhibition of
insulin activation of PKC
, indicating that these mutants exert dominant negative effects. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to the plasma membrane, but not growth hormone- or hyperosmolarity-induced glucose uptake, were
inhibited by
KD or 
NKD in a dose-dependent manner. The maximal
inhibition of insulin-induced glucose uptake achieved by the dominant
negative mutants of PKC
was ~50 to 60%. These mutants did not
inhibit insulin-induced activation of Akt. A PKC
mutant that lacks
the pseudosubstrate domain (
PD) exhibited markedly increased
kinase activity relative to that of the wild-type enzyme, and
expression of 
PD in quiescent 3T3-L1 adipocytes resulted in the
stimulation of glucose uptake and translocation of GLUT4 but not in the
activation of Akt. Furthermore, overexpression of an Akt mutant in
which the phosphorylation sites targeted by growth factors are replaced
by alanine resulted in inhibition of insulin-induced activation of Akt
but not of PKC
. These results suggest that insulin-elicited signals
that pass through PI 3-kinase subsequently diverge into at least two
independent pathways, an Akt pathway and a PKC
pathway, and that the
latter pathway contributes, at least in part, to insulin stimulation of
glucose uptake in 3T3-L1 adipocytes.
 |
INTRODUCTION |
Phosphoinositide (PI) 3-kinase, a
lipid kinase composed of an SRC homology 2 (SH2) domain-containing
regulatory subunit and a 110-kDa catalytic subunit, catalyzes
phosphorylation of the D3 position of PIs (46, 48). This
enzyme was first identified complexed with SRC kinase and the middle T
antigen of polyomavirus and was later found to associate with various
tyrosine-phosphorylated proteins in response to stimulation of cells
with growth factors or cytokines (46, 48). Activation of PI
3-kinase, either by targeting of the enzyme to the plasma membrane
(27) or as a consequence of direct interaction between the
SH2 domain of the regulatory subunit and phosphorylated tyrosine
residues present within specific motifs (5), results in the
triggering of various important biological actions. Thus, with the use
of either a dominant negative protein that blocks the interaction
between PI 3-kinase and tyrosine-phosphorylated proteins (21, 33,
41) or pharmacological inhibitors of the enzyme, such as
wortmannin or LY294002 (12, 47), PI 3-kinase has been shown
to participate in intracellular trafficking, organization of the
cytoskeleton, cell growth and transformation, prevention of apoptosis,
cell differentiation, and several metabolic actions of insulin
(11, 17, 23, 24, 33, 41, 46, 48). However, relatively little
is known about the downstream effectors of PI 3-kinase that mediate
each of these biological effects.
Recently, two types of serine-threonine kinase have been shown to act
downstream of PI 3-kinase. One of these kinases is Akt (also known as
protein kinase B), which contains a pleckstrin homology domain. Several
growth factors induce rapid activation of Akt in intact cells (10,
16, 28). However, the mechanism of Akt activation in cells is not
fully understood. Phosphatidylinositol 3,4-bisphosphate, one of the
products of PI 3-kinase activity in vivo, binds directly to the
pleckstrin homology domain of Akt and stimulates kinase activity in
vitro (18, 28), suggesting that Akt may be directly
activated by this lipid in intact cells. On the other hand, activation
of Akt was shown to parallel its phosphorylation status, and
replacement of serine and threonine residues that are sites of
ligand-induced phosphorylation in the enzyme abolished its activation
(3, 31). These data, together with the identification of a
kinase that phosphorylates and activates Akt (4, 44),
suggest that Akt activity is regulated mainly by phosphorylation.
Whichever mechanism is primarily responsible for regulation of Akt, the
observation that pharmacological or molecular biological inhibitors of
PI 3-kinase prevent Akt activation in intact cells (10, 16, 26,
29) indicates that Akt acts downstream of PI 3-kinase.
The protein kinase C (PKC) family of serine-threonine kinases comprises
at least 11 members (37). One class of PKC isozymes, termed
atypical PKC, is distinct from other members of this family in several
respects. For example, the atypical PKC enzymes, consisting of PKC
and PKC
, are not activated by diacylglycerol or phorbol ester,
whereas members of the other two classes, conventional PKC and novel
PKC, are activated by these reagents both in vitro and in vivo (1,
2, 36-38, 40, 49). PKC
, also identified as an atypical PKC
isozyme, is the human counterpart of mouse PKC
(42).
Evidence suggests that atypical PKC is the second type of
serine-threonine kinase that acts downstream of PI 3-kinase. PKC
is
activated in vitro by phosphatidylinositol 3,4,5-trisphosphate (36), another product of PI 3-kinase activity
(46). When expressed in 3Y1 fibroblasts or HepG2 hepatoma
cells expressing various mutant platelet-derived growth factor
receptors, PKC
contributed to trans activation of the
tetradecanoyl phorbol acetate-responsive element in response to
platelet-derived growth factor or epidermal growth factor in a PI
3-kinase-dependent manner (2). Furthermore, the activity of
atypical PKC stimulated by either insulin or bacterial lipopolysaccharide was shown to be inhibited by either a
pharmacological inhibitor or a dominant negative mutant of PI 3-kinase
(22, 34, 43). All of these observations indicate that
atypical PKC isozymes are downstream effectors of PI 3-kinase.
Stimulation of glucose uptake into skeletal muscle and adipocytes is
one of the most important actions of insulin. Insulin-stimulated glucose uptake and translocation of the glucose transporter GLUT4 to
the plasma membrane, an essential step for glucose uptake in muscle and
adipocytes, were markedly attenuated by pharmacological inhibitors or a
dominant negative mutant of PI 3-kinase (13, 39, 41).
Furthermore, a constitutively active mutant of PI 3-kinase promoted
glucose uptake and translocation of GLUT4 in quiescent adipocytes
(20). These observations suggest a central role for PI
3-kinase in regulation of glucose transport. However, a downstream
effector of PI 3-kinase that mediates stimulation of glucose uptake has
not been identified.
Although Akt and atypical PKC act downstream of PI 3-kinase, it remains
unclear which actions of PI 3-kinase are mediated by which protein
kinase. It is also not known whether Akt and atypical PKC act in the
same signaling pathway or whether they transmit signals through
different pathways. To address these important questions, we have
investigated the roles of these protein kinases in insulin-stimulated
glucose uptake. We recently showed that a mutant Akt in which the sites
of ligand-induced phosphorylation were replaced by alanine acts in a
dominant negative manner (26). Because this mutant did not
affect insulin-induced glucose uptake (26), we have now
examined the possible role of atypical PKC as a downstream effector of
PI 3-kinase in this process. We have investigated the effects of
dominant negative mutants and a constitutively active mutant of PKC
in order to determine whether this enzyme participates in the
regulation of glucose uptake by insulin in 3T3-L1 adipocytes. We have
also investigated whether Akt and PKC
function in the same or
different signaling pathways.
 |
MATERIALS AND METHODS |
Cells and antibodies.
3T3-L1 preadipocytes were maintained
and induced to differentiate into adipocytes as described previously
(41). To establish CHO-IR cells that express (in addition to
human insulin receptors) tagged PKC
(CHO-IR/PKC
cells), we
transfected CHO-IR cells with both pSV40-high (which confers resistance
to hygromycin) and an SRD vector encoding T7 epitope-tagged mouse
PKC
(2). Transfected cells were selected and cloned as
described previously (25). CHO cells that express FLAG
epitope-tagged Akt (CHO-Akt cells) have been described previously
(26). To prepare a construct encoding mouse PKC
tagged
with the T7 epitope, we performed PCR with a sense primer (5'-AAG GCC
ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CCC AGC AGG ACC GAC), an
antisense primer (5'-GAG GTC GAA GTC TTG CAG CCC), and a full-length
mouse PKC
cDNA as the template. The resulting PCR product,
containing nucleotides 4 to 762 of PKC
cDNA fused with a DNA
sequence encoding the T7 epitope, was digested with PstI and
ligated to the PstI site of cDNA encoding mouse PKC
. The
resulting cDNA construct encoded T7 epitope-tagged mouse PKC
and was
subcloned into an SR
expression vector.
Polyclonal antibodies to PKC
(
190) and to PKC
(
170)
were generated against glutathione S-transferase (GST)
fusion proteins containing amino acids 190 to 240 of mouse PKC
or
amino acids 170 to 240 of mouse PKC
, respectively. A monoclonal
antibody (MAb) to PKC
(
CT), induced by a GST fusion protein
containing amino acids 397 to 558 of mouse PKC
, was obtained from
Transduction Laboratories. Polyclonal antibodies to PKC
(
CT),
generated in response to a peptide corresponding to the COOH terminus
of rat PKC
(amino acids 577 to 592), were obtained from GIBCO BRL. Polyclonal antibodies to PKC
(
197) that were generated in
response to a peptide corresponding to amino acids 197 to 213 of mouse PKC
were as described previously (1). Polyclonal
antibodies to Akt, induced by a GST fusion protein containing amino
acids 428 to 480 of rat Akt1, as well as a MAb (1F8) and polyclonal antibodies to GLUT4 were as described previously (41).
Antibodies to Akt2 and to Akt3 were obtained from Upstate Biotechnology.
RT-PCR.
Transcripts encoding PKC
or PKC
were detected
by reverse transcription (RT)-PCR analysis. Complementary DNA was
synthesized, with the use of a FastTrack 2.0 kit and a cDNA Cycle kit
(Invitrogen), from ~300 µg of polyadenylated RNA extracted from
3T3-L1 adipocytes or total RNA extracted from mouse brain. PCR was then
performed with 1/10 of the resulting cDNA as the template and with
primers that correspond to nucleotides 247 to 265 and 576 to 595 of
mouse PKC
cDNA or to nucleotides 145 to 164 and 700 to 719 of mouse PKC
cDNA. The amplification protocol comprised 30 cycles of
denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and
extension at 72°C for 1 min.
Construction of and infection with adenovirus vectors.
Various PKC
mutant constructs are shown in Fig.
1. Complementary DNAs encoding wild-type
mouse PKC
(
WT) and a kinase-deficient mutant in which glutamate
is substituted for Lys273 in the kinase domain (
KD) were
as described previously (2). To prepare constructs encoding

PD and 
PDKD, hemagglutinin (HA) epitope-tagged,
NH2-terminal-deletion mutants of PKC
, we performed PCR
with sense (5'-TTA GGT ACC ATG TAC CCA TAC GAT GTT CCG GAT TAC GCT AGC
CTC GCC AAA CGT TTC AAT AGG CGC) and antisense (5'-GAT ACC ACT CTC CCT
GGT) primers and wild-type mouse PKC
cDNA as a template. The
resulting PCR product, containing nucleotides 442 to 735 of PKC
cDNA
fused with a DNA sequence encoding the HA epitope, was digested with
EcoT22I and ligated to the EcoT22I site of cDNA
encoding either
WT or
KD. To construct 
NKD, we performed
PCR with sense (5'-TTA GGT ACC ATG TAC CCA TAC GAT GTT CCG GAT TAC GCT
AGC CTC TCG TCC AGT CTA GGT CTG CAG) and antisense (5'-GAT AGA ATG CAG
CCC GAC) primers and
KD cDNA as a template. The resulting product,
containing nucleotides 742 to 966 of PKC
cDNA fused with a DNA
sequence encoding the HA epitope, was digested with ClaI and
fused with the ClaI site of cDNA encoding
WT.
Complementary DNA encoding the wild-type or various mutant proteins was
subcloned into pAxCAwt (35), and adenovirus vectors
containing these cDNAs were generated by transfecting 293 cells with
the corresponding pAxCAwt plasmid together with DNA-terminal protein
complex (35), as described previously (41). The
resulting vectors were termed AxCA
WT, AxCA
KD, AxCA
PD,
AxCA
PDKD, and AxCA
NKD, respectively.

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FIG. 1.
Structures of various mutants of PKC , KD,
 NKD, and  PDKD contain a Lys273-to-Glu mutation
(K273E).  PD,  PDKD, and  NKD contain the HA epitope at
their truncated NH2 termini. The first and last amino acids
of each protein are numbered. PD, CR, and CD, pseudosubstrate,
cysteine-rich, and catalytic domains, respectively.
|
|
An adenovirus vector encoding a dominant negative mutant of PI 3-kinase
(AxCA

p85) was as described previously (
41). An
adenovirus
vector encoding a mutant Akt (AxCAAkt-AA) in which
the phosphorylation
sites (Thr
308 and Ser
473) targeted by growth
factors are replaced by alanine was as previously
described
(
26). CHO cells or 3T3-L1 adipocytes were infected
with
adenovirus vectors at the multiplicities of infection (MOIs)
(in PFU
per cell) indicated in Results, as described previously
(
41,
50). The cells were used in experiments 24 to 48 h after
infection.
After we submitted this paper, the wild-type mouse PKC

cDNA used to
construct the adenoviruses encoding the various mutant
proteins was
found to lack the nucleotides encoding the 47 NH
2-terminal
amino acids. Although the deleted amino acid sequence does not
contain
any known functional domains, we repeated all of the experiments
that
used the NH
2-terminally deleted

WT or

KD with
adenovirus
vectors encoding a full-length wild-type or a full-length
kinase-deficient
PKC

, and we obtained essentially identical results.
This further
suggests that the NH
2-terminal 47 amino acids
are not essential
for the signaling of insulin-induced glucose
uptake.
Kinase assays.
CHO cells or 3T3-L1 adipocytes were deprived
of serum for 16 to 20 h, incubated in the absence or presence of
insulin, and then immediately frozen with liquid nitrogen. For assay of
the kinase activity of endogenous or T7 epitope-tagged PKC
, the
frozen cells were lysed as described previously (2) and the
lysate was centrifuged (15,000 × g for 20 min). The
protein concentration in the resulting supernatants was determined with
the use of the bicinchoninic acid protein assay reagent (Pierce), and
equal amounts of protein were subjected to immunoprecipitation with
polyclonal antibodies or MAbs to PKC
or with antibodies to the T7
epitope tag. After washing twice with buffer A (50 mM MOPS
[morpholinepropanesulfonic acid]-HCl [pH 7.5], 0.5% Triton X-100,
10% glycerol, 0.1% bovine serum albumin, 5 mM EDTA, 5 mM EGTA, 20 mM
NaF, 50 mM
-glycerophosphate, 2 mM sodium orthovanadate, 2 mM
dithiothreitol, 1 µg of leupeptin per ml, and 2 mM
phenylmethylsulfonyl fluoride), once with buffer A containing 1 M NaCl,
and then once with a solution containing 20 mM Tris-HCl (pH 7.5), 5 mM
MgCl2, and 1 mM EGTA, the immunoprecipitates were incubated
for 14 min at 30°C with 0.4 µCi of [
-32P]ATP in a
reaction mixture (25 µl) containing 35 mM Tris-HCl (pH 7.5), 10 mM
MgCl2, 0.5 mM EGTA, 0.1 mM CaCl2, 40 µM
unlabeled ATP, and 30 µM myelin basic protein (MBP) as a substrate.
When indicated, phosphatidylserine (PS) (100 µg/ml) was also present in the reaction mixture.
For assay of the kinase activity of endogenous or FLAG-tagged Akt, the
frozen cells were lysed in a solution containing 50
mM HEPES-NaOH (pH
7.6), 150 mM NaCl, 1% Triton X-100, 1 mg of
bacitracin per ml, 1 mM
phenylmethylsulfonyl fluoride, 1 mM EDTA,
1 mM sodium orthovanadate, 10 mM NaF, and 30 mM sodium pyrophosphate.
The lysates were subjected to
immunoprecipitation with antibodies
to Akt or to FLAG. After three
washes with HEPES-buffered saline
(pH 7.5) containing 0.1% Triton
X-100, the immunoprecipitates
were incubated for 30 min at 30°C with
3.0 µCi of [

-
32P]ATP in a reaction mixture (30 µl)
containing 20 mM Tris-HCl
(pH 7.5), 10 mM MgCl
2, 25 µM
unlabeled ATP, 1 µM protein kinase
inhibitor, and 0.2 mg of histone
2B per ml as a
substrate.
All kinase reactions were terminated by the addition of sodium dodecyl
sulfate (SDS) sample buffer, and the samples were then
fractionated by
SDS-polyacrylamide gel electrophoresis. The radioactivity
incorporated
into substrates was determined with a Fuji BAS 2000
image
analyzer.
Glucose uptake and translocation of GLUT4.
Glucose uptake
was assayed as described previously (41). In brief, 3T3-L1
adipocytes were incubated for 16 h in Dulbecco's modified
Eagle's medium containing 5.6 mM glucose and 0.5% fetal bovine serum.
The cells were washed twice with DB buffer (140 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 1.5 mM KH2PO4, 8 mM
Na2HPO4 [pH 7.4], 0.5 mM MgCl2)
and then incubated with 100 nM insulin for 15 min, 0.5 µg of growth
hormone (GH) per ml for 10 min, or 300 mM sorbitol for 60 min. DB
buffer (1 ml) containing bovine serum albumin (1 mg/ml) and 0.1 mM
2-deoxy-D-[1,2-3H]glucose (1 µCi) was added
to each well, and after 5 min, the cells were washed and then
solublized with 0.1% SDS. The radioactivity incorporated into the
cells was measured with a liquid scintillation counter.
Translocation of GLUT4 to the plasma membrane was measured by the
plasma membrane lawn assay as previously described (
41).
In
brief, 3T3-L1 adipocytes cultured on coverslips were incubated
in a
hypotonic buffer and immediately disrupted by being placed
under an
ultrasonic microprobe. For antibody labeling, sonicated
cells were
fixed in 2% paraformaldehyde, and the lawn of plasma
membrane
fragments was prepared with antibodies to GLUT4 and tetramethyl
rhodamine isothiocyanate-labeled secondary antibodies. Samples
were
then examined with a fluorescence
microscope.
 |
RESULTS |
Expression of PKC
in 3T3-L1 adipocytes.
Atypical PKC
isozymes comprise PKC
and PKC
. To examine the relative abundances
of these two isoforms in 3T3-L1 adipocytes, we prepared antibodies
specific for each. T7 epitope-tagged PKC
or PKC
was transiently
expressed in COS7 cells, and cell lysates were subjected to
immunoprecipitation with antibodies either to PKC
(
190), to
PKC
(
170), or to the T7 epitope or with control serum. The
immunoprecipitates were then subjected to immunoblot analysis with
antibodies to T7 (Fig. 2A). Proteins of
~80 kDa reactive with antibodies to T7 were detected in the
immunoprecipitates prepared from the lysates expressing T7-tagged
PKC
with 
190 or with antibodies to T7 and in the
immunoprecipitates prepared from the lysates expressing T7-tagged
PKC
with 
170 or with antibodies to T7. These results indicate
that 
190 and 
170 specifically recognize PKC
and PKC
,
respectively. In contrast, polyclonal antibodies generated against a
peptide corresponding to the COOH terminus of rat PKC
(
CT)
detected both T7-tagged PKC
and PKC
(Fig. 2B), indicating that
these antibodies recognize both PKC
and PKC
.

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FIG. 2.
Expression of PKC , but not PKC , in 3T3-L1
adipocytes. (A) Specificity of antibodies to PKC and PKC . COS7
cells cultured in 6-cm-diameter dishes were transiently transfected,
with the use of Lipofectamine (Gibco), with 6 µg of SRD or SR
vectors encoding T7 epitope-tagged PKC or PKC . Cell lysates were
subjected to immunoprecipitation (IP) with antibodies to T7, to PKC
( 190), or to PKC ( 170) or with control serum (Cont), and
the resulting immunoprecipitates were subjected to immunoblot analysis
with antibodies to T7. (B) Recognition of both PKC and PKC by
antibodies generated in response to a peptide corresponding to the COOH
terminus of rat PKC ( CT). COS7 cells transiently transfected
as described for panel A were lysed and subjected to
immunoprecipitation with either antibodies to T7 or normal mouse
globulin (NMG). The resulting immunoprecipitates were then subjected to
immunoblot analysis with either antibodies to T7 (lower panel) or
 CT (upper panel). (C) Analysis of PKC and PKC protein
expression in mouse brain and 3T3-L1 adipocytes. Lysates prepared from
mouse brain or 3T3-L1 adipocytes were subjected to immunoprecipitation
with  190,  170, or control rabbit serum, and the
immunoprecipitates were subjected to immunoblot analysis with  CT.
(D) Analysis of PKC and PKC transcripts in mouse brain and 3T3-L1
adipocytes. PCR was performed with primers specific for mouse PKC or
PKC cDNA and with either the indicated concentration of full-length
PKC or PKC cDNA subcloned into the SRD vector (left panel) or
cDNA synthesized from RNA extracted from either mouse brain or 3T3-L1
adipocytes (right panel). Amplification products were analyzed by
agarose gel electrophoresis and ethidium bromide staining. Lane M,
molecular size standards. Data are representative of those from three
experiments.
|
|
When a cell lysate prepared from mouse brain was subjected to
immunoprecipitation with either


190,


170, or control serum
and the immunoprecipitates were then subjected to immunoblot analysis
with


CT, proteins of ~80 kDa were detected in the
immunoprecipitates
prepared with


190 or


170 but not in
those prepared with control
serum, suggesting that brain expresses both
PKC

and PKC

(Fig.
2C). In contrast, when 3T3-L1 adipocyte lysates
were subjected
to the same analysis, an ~80-kDa protein was detected
only in
the immunoprecipitate prepared with


190 (Fig.
2C). When
the
same membrane was probed with a MAb generated in response to a
GST
fusion protein containing amino acids 397 to 558 of mouse
PKC

(


CT), again an ~80-kDa protein was detected only in the
immunoprecipitate prepared with


190 (data not shown). These
results suggest that 3T3-L1 adipocytes express PKC

protein but
not
PKC
protein.
We also examined the expression of PKC

and PKC

at the mRNA level
by RT-PCR. PCR performed with specific oligonucleotide
primers based on
the sequence of either mouse PKC

or PKC

cDNA,
and with as little
as 0.6 pM PKC

or PKC

cDNA subcloned into
the SRD vector as a
template, yielded amplification products of
the expected size (~350
bp for PKC

and ~570 bp for PKC

) (Fig.
2D). When PCR was
performed with the same primer pairs and cDNA
that was synthesized from
RNA extracted from mouse brain, PCR
products of the expected size were
obtained with each set of primers.
However, with cDNA that was
synthesized from RNA extracted from
3T3-L1 adipocytes as the template,
a PCR product of the expected
size was obtained with the primers
corresponding to PKC

but not
with those corresponding to PKC

.
Thus, consistent with the results
of protein analysis, 3T3-L1
adipocytes contain PKC

mRNA but not
PKC
mRNA.
Activation of PKC
by insulin in a PI 3-kinase-dependent
manner.
3T3-L1 adipocytes were incubated for various times in the
absence or presence of 100 nM insulin, lysed, and subjected to
immunoprecipitation with control serum or polyclonal antibodies to
PKC
generated against a peptide corresponding to amino acids 197 to
213 of mouse PKC
(
197). These antibodies were previously shown
not to cross-react with PKC
(1). Kinase activity in the
immunoprecipitates was then assayed with MBP as a substrate. The kinase
activity precipitated by 
197 was markedly greater than that
precipitated by control serum (Fig. 3B).
Activation of PKC
was evident within 3 min of exposure of cells to
insulin; the activity was maximal (about three times that of the basal
value) at 5 min and remained increased at 10 min (Fig. 3A). Prior
treatment of the cells with 100 nM wortmannin or infection of the cells
with AxCA
p85, an adenovirus vector that encodes a dominant negative
mutant of PI 3-kinase (41), prevented insulin-induced
activation of PKC
(Fig. 3B).

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FIG. 3.
Stimulation of PKC activity by insulin in 3T3-L1 and
CHO cells. (A) Time course of insulin stimulation of PKC activity in
3T3-L1 adipocytes. 3T3-L1 cells cultured in 6-cm-diameter plates were
incubated in the presence of 100 nM insulin for the indicated times,
after which the cells were lysed and subjected to immunoprecipitation
with polyclonal antibodies to PKC ( 197). The resulting
immunoprecipitates were then assayed for kinase activity with MBP as a
substrate. Data are expressed relative to the activity at zero time.
(B) Effects of inhibitors of PI 3-kinase on insulin stimulation of
PKC activity in 3T3-L1 adipocytes. 3T3-L1 cells were incubated in
the absence or presence of 100 nM wortmannin for 20 min or were
infected at the indicated MOI (PFU per cell) with an adenovirus vector
encoding p85 (AxCA p85), and they were then incubated in the
absence or presence of 100 nM insulin for 5 min. Cells were then lysed
and subjected to immunoprecipitation (IP) with either  197 or
normal rabbit serum (NRS). Immunoprecipitates were assayed for PKC
activity as described for panel A. Data are expressed relative to the
activity of control  197 precipitates. (C) Insulin stimulation of
PKC activity in transiently transfected CHO-IR cells. CHO-IR cells
were transiently transfected with constructs encoding T7-tagged
wild-type PKC or KD. After 48 h, the cells were deprived of
serum, incubated in the absence or presence of 100 nM insulin for 3 min, and then lysed. The lysates were subjected to immunoprecipitation
with antibodies to T7, and the immunoprecipitates were subjected either
to immunoblot analysis with antibodies to T7 (upper panel) or to the
PKC kinase assay (lower two panels). (D) Effect of p85 on
insulin-stimulated PKC activity in CHO-IR/PKC cells.
CHO-IR/PKC cells were infected with AxCA p85 at the indicated MOI
(PFU per cell). Cells were then incubated in the absence or presence of
100 nM insulin for 3 min, after which cell lysates were subjected to
immunoprecipitation with antibodies to the T7 epitope and PKC
activity was assayed in the resulting precipitates. Data are expressed
relative to the activity of precipitates from uninfected cells not
exposed to insulin. Quantitative data in panels A, B, and D are
means ± standard errors from three experiments, and those in
panel C are means from two experiments.
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To confirm that insulin activates PKC

, we transiently transfected
CHO-IR cells with constructs encoding T7 epitope-tagged
wild-type
PKC

or a mutant PKC

in which Lys
273 in the kinase
domain was replaced by glutamate (

KD). The transfected
cells were
incubated in the absence or presence of insulin, lysed,
and subjected
to immunoprecipitation with antibodies to T7. Although
the amounts of
T7-tagged PKC

and

KD in the immunoprecipitates
were similar, the
precipitates prepared from the cells expressing
PKC

showed higher
kinase activity, and this activity was increased
about 1.8-fold in
response to insulin (Fig.
3C). These observations
suggest that the
kinase activity precipitated with antibodies
to T7 was attributable to
PKC

and not to some other kinase associated
with PKC

.
To investigate further the effect of insulin on PKC

activation, we
established a CHO cell line that stably expresses both
human insulin
receptors and T7 epitope-tagged mouse PKC

(CHO-IR/PKC
cells).
Exposure of CHO-IR/PKC

cells to insulin for 3 min resulted
in an
~2.5-fold increase in PKC

activity measured in immunoprecipitates
prepared with antibodies to the T7 epitope (Fig.
3D). This stimulation
was inhibited by infection of cells with AxCA

p85. These results
suggested that PKC

is activated by insulin in a PI
3-kinase-dependent
manner.
Dominant negative effects of kinase-defective mutants of PKC
on
insulin-induced activation of PKC
.
To investigate the roles of
PKC
in cells by specifically inhibiting the activity of the
endogenous enzyme, we have constructed adenovirus vectors that encode
dominant negative mutants of PKC
. Infection of cells with an
adenovirus (AxCA
KD) encoding
KD revealed that the mutant enzyme
was not activated either by insulin treatment in intact cells (Fig.
4A) or by addition of PS to the kinase
assay mixture (Fig. 4B); PS stimulated the activity of the wild-type enzyme ~2.5-fold. Infection of CHO-IR/PKC
cells with AxCA
KD resulted in inhibition of insulin-induced activation of PKC
(measured in immunoprecipitates prepared with antibodies to the T7
epitope) in an MOI-dependent manner (Fig.
5A), without an effect on the amount of
T7 epitope-tagged PKC
in the cells (data not shown).

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FIG. 4.
Effects of insulin in vivo and PS in vitro on the kinase
activities of various PKC mutants. (A) 3T3-L1 adipocytes cultured in
six-well plates were infected (or not) with adenovirus vectors encoding
 PD ( PD), KD (KD), or  NKD ( NKD) at an MOI of 150, 30, or 50 PFU/cell, respectively. The cells were then incubated in the
absence or presence of 100 nM insulin for 5 min, lysed, and subjected
to immunoprecipitation with a MAb to PKC . The immunoprecipitates
were assayed for PKC activity. (B) KB cells cultured in six-well
plates were infected (or not) with adenovirus vectors encoding WT
(WT), KD (KD),  PD ( PD), or  NKD ( NKD) at an MOI of
1, 3, 15, or 5 PFU/cell, respectively; these MOIs resulted in the
expression of similar amounts of PKC proteins. The cells were lysed,
the total lysates were subjected to immunoprecipitation with a MAb to
PKC , and the immunoprecipitates were assayed for PKC activity in
the absence or presence of PS (100 µg/ml). Data are means ± standard errors from three experiments.
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FIG. 5.
Effects of KD and  NKD on insulin-induced
activation of PKC . (A and B) CHO-IR/PKC cells were infected with
AxCA KD (A) or AxCA NKD (B) at the indicated MOI (PFU per cell),
incubated in the absence or presence of 100 nM insulin for 5 min, and
lysed. The total lysates were subjected to immunoprecipitation with a
MAb to the T7 epitope, and the immunoprecipitates were then assayed for
PKC activity. (C) 3T3-L1 adipocytes were infected with AxCA NKD
at the indicated MOI (PFU per cell), incubated in the absence or
presence of 100 nM insulin for 5 min, and lysed. The total lysates were
subjected to immunoblot analysis with a MAb to PKC (lower panel) or
to immunoprecipitation with polyclonal antibodies to PKC
( 197); the immunoprecipitates were then assayed for PKC
activity (upper panel). Quantitative data are expressed as fold
stimulation relative to uninfected cells not exposed to insulin and are
means ± standard errors from three experiments.
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|
Although these results indicated that

KD inhibits the activation of
transfected PKC

by insulin, we could not examine the
effect of this
mutant on endogenous PKC

activity because the
polyclonal
antibodies to PKC

(


197) also precipitate

KD. We
therefore
constructed an adenovirus vector (AxCA


NKD) that encodes
an
NH
2-terminally truncated version of

KD (


NKD);
because this
mutant does not possess the region of PKC

corresponding
to the
immunogen, it is not recognized by the polyclonal antibodies to
PKC

.


NKD was not activated either by insulin in vivo or by
PS
in vitro (Fig.
4). Infection of CHO-IR/PKC

cells with
AxCA


NKD
resulted in an MOI-dependent inhibition of
insulin-induced activation
of T7 epitope-tagged PKC

(Fig.
5B). The
extent of the inhibition
paralleled the extent of expression of the
mutant protein, and
the expression of the mutant protein did not affect
the amount
of T7 epitope-tagged PKC

(data not
shown).
We then investigated the effect of


NKD on endogenous PKC

activity in 3T3-L1 adipocytes. Fully differentiated 3T3-L1
adipocytes
were infected with AxCA


NKD at various MOIs and
incubated in
the absence or presence of insulin, after which
immunoprecipitates
prepared with polyclonal antibodies to PKC

were
assayed for PKC
activity. Insulin stimulation of endogenous PKC

activity in 3T3-L1
adipocytes was inhibited by


NKD in an
MOI-dependent manner,
being virtually abolished at an MOI of 100 PFU/cell (Fig.
5C);
again, the extent of inhibition paralleled the
extent of expression
of the mutant protein, and the expression of the
mutant protein
did not affect the amount of endogenous PKC

protein
(Fig.
5C).
Immunoblot analysis revealed that the amount of mutant
PKC

protein
in the cells infected with AxCA


NKD at an MOI of
100 PFU/cell
was 5 to 10 times that of endogenous PKC

protein.
Insulin-induced
stimulation of PI 3-kinase activity in 3T3-L1 cells,
measured
in immunoprecipitates prepared with antibodies to
phosphotyrosine,
was not inhibited by either

KD or


NKD (data
not shown). Thus,
both of these PKC

mutants exerted dominant
negative effects on
insulin-induced activation of PKC

.
Inhibition of insulin-stimulated glucose uptake and GLUT4
translocation by dominant negative mutants of PKC
.
We next
examined the effects of the dominant negative mutants of PKC
on
insulin-stimulated glucose uptake and translocation of GLUT4 in 3T3-L1
adipocytes. Cells were infected with either AxCA
KD or AxCA
NKD
and then assayed for insulin-induced glucose uptake. Overexpression of
either
KD or 
NKD resulted in a dose-dependent inhibition of
insulin-stimulated glucose uptake (Fig.
6A and B). Basal glucose transport was
not affected by infection of cells with the viruses encoding either of
the dominant negative mutants of PKC
(Fig. 6A and B). The amounts of
GLUT4 protein, assessed by immunoblot analysis, in infected and
noninfected cells were also similar (Fig.
7). Glucose uptake stimulated by either
GH or hyperosmolarity (300 mM sorbitol) was not affected by AxCA
KD (Fig. 6C).

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FIG. 6.
Effects of KD and  NKD on glucose uptake in
3T3-L1 adipocytes. (A and B) 3T3-L1 adipocytes were infected with
AxCA KD (A) or AxCA NKD (B) at the indicated MOI (PFU per cell)
and then incubated in the absence or presence of 100 nM insulin for 15 min. Cells were then assayed for glucose uptake. (C) 3T3-L1 adipocytes
were infected (or not) with AxCA KD at an MOI of 150 PFU/cell,
incubated in the absence or presence of GH (0.5 µg/ml) for 10 min or
300 mM sorbitol for 60 min, and then assayed for glucose uptake. Data
are means ± standard errors from three experiments.
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FIG. 7.
Effects of various PKC mutants on the amount of GLUT4
protein in 3T3-L1 adipocytes. Cells were infected (or not) with
AxCA KD, AxCA PD, AxCA NKD, or AxCA WT at an MOI of 150 PFU/cell, and total cell lysates were subjected to immunoprecipitation
with a MAb to GLUT4. The immunoprecipitates were then subjected to
immunoblot analysis with polyclonal antibodies to GLUT4.
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|
To confirm that the observed inhibition of insulin-induced glucose
uptake was due to inhibition of PKC

activity, we attempted
to
reverse the effect of the


NKD mutant by overexpressing the
wild-type enzyme. 3T3-L1 adipocytes were infected first with
AxCA


NKD
at an MOI of 100 PFU/cell, and, after 12 h, they
were infected
again with AxCA

WT, an adenovirus vector encoding
wild-type PKC

,
at different MOIs (Fig.
8). The cells were then assayed for
insulin-induced
glucose uptake. Infection of the cells with AxCA

WT
partially
reversed the inhibition of glucose uptake by


NKD in an
MOI-dependent
manner. The expression of


NKD protein was not
affected by the
second infection of the cells with AxCA

WT (data not
shown). These
results suggested that the inhibition of insulin-induced
glucose
uptake by


NKD is due to inhibition of PKC

activity.

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FIG. 8.
Effect of overexpression of wild-type PKC on the
inhibition of insulin-stimulated glucose uptake by  NKD. 3T3-L1
adipocytes were infected (or not) with AxCA NKD at an MOI of 100 PFU/cell and, after 12 h, with AxCA WT at the indicated MOI.
After an additional 36 h, the cells were assayed for glucose
uptake. Data are means ± standard errors from three
experiments.
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|
The effects of the dominant negative mutants of PKC

on
insulin-stimulated translocation of GLUT4 were examined by the plasma
membrane lawn assay. Exposure of noninfected 3T3-L1 adipocytes
to
insulin resulted in a marked increase in the amount of GLUT4
immunoreactivity in the plasma membrane, an effect that was
substantially
inhibited in cells infected with AxCA

KD (Fig.
9). An essentially
identical inhibitory
effect on insulin-induced translocation of
GLUT4 was observed in cells
infected with AxCA


NKD (data not
shown). These results suggested
that PKC

activity is required
for insulin-induced glucose uptake and
translocation of GLUT4.

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FIG. 9.
Effects of PKC mutants on translocation of GLUT4 to
the plasma membrane of 3T3-L1 adipocytes. Uninfected cells (A and B) or
cells that were infected with AxCA KD (C) or AxCA PD (D through
F) at an MOI of 150 PFU/cell were incubated in the absence (A through
E) or presence (F) of 100 nM wortmannin for 20 min and then in the
absence (A, D, and F) or presence (B, C, and E) of 100 nM insulin for 5 min. Plasma membrane fragments were then prepared for
immunofluorescence microscopy with antibodies to GLUT4 and tetramethyl
rhodamine isothiocyanate-labeled secondary antibodies. Data are
representative of those from at least three independent experiments.
|
|
Enhancement of glucose transport and GLUT4 translocation by a
constitutively active mutant of PKC
.
To investigate whether
activation of PKC
is capable of stimulating glucose uptake in 3T3-L1
adipocytes, we prepared an adenovirus vector that encodes a
constitutively active mutant of PKC
. Because the activity of PKC
enzymes is negatively regulated by the pseudosubstrate domain, we
constructed a mutant PKC
that lacks this domain (
PD). As
expected, the kinase activity of 
PD was markedly greater than
that of the wild-type enzyme, and this activity was little affected by
insulin treatment in vivo or PS in vitro (Fig. 4). Infection of 3T3-L1
adipocytes with a virus (AxCA
PD) that encodes this mutant
resulted in stimulation of glucose uptake in an MOI-dependent manner
(Fig. 10). The extent of the
stimulation paralleled the extent of expression of the mutant protein
(data not shown). At an MOI of 150 PFU/cell, the extent of the
stimulatory effect was similar to that achieved by 100 nM insulin
(Fig. 10). 
PDKD, a mutant PKC
that lacks both kinase
activity and the pseudosubstrate domain, did not stimulate glucose
uptake (data not shown), suggesting that the activation of sugar
transport by 
PD was due to its kinase activity and not to
nonspecific effects of viral infection. Furthermore, the amounts of
GLUT4 (Fig. 7) and of insulin-stimulated PI 3-kinase activity
precipitated with antibodies to phosphotyrosine (data not shown) were
not increased by expression of 
PD. Insulin did not increase
glucose uptake further in cells that had been infected with
AxCA
PD at an MOI of 150 PFU/cell, and treatment of such cells
with wortmannin had little effect on glucose uptake (Fig. 10).
Moreover, GLUT4 translocation, as assessed by the plasma membrane
lawn assay, was stimulated by 
PD, and this stimulation was not
affected by insulin or wortmannin (Fig. 9).

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FIG. 10.
Effect of  PD on glucose uptake in 3T3-L1
adipocytes. Cells were infected with AxCA PD at the indicated MOI
(PFU per cell) and incubated in the absence or presence of 100 nM
wortmannin for 20 min and then with or without 100 nM insulin for 10 min. Glucose uptake was then assayed. Data are means ± standard
errors from three experiments.
|
|
Localization of PKC
and Akt to different signaling
pathways.
Finally, we examined whether PKC
and Akt, both of
which are downstream effectors of PI 3-kinase, act in the same or
different signaling pathways. Insulin induced a fivefold increase in
Akt activity in 3T3-L1 adipocytes (Fig.
11A). Infection of cells with AxCA
KD
did not affect insulin-induced activation of Akt, even at an MOI of 150 PFU/cell, a dose sufficient to inhibit insulin-induced glucose uptake
by ~50% (Fig. 6A). Infection with AxCA
PD did not result in the
activation of Akt, even at an MOI of 150 PFU/cell (Fig. 11B), a virus
dose sufficient to activate glucose uptake to an extent similar to that
achieved by insulin (Fig. 10). Essentially similar results were
obtained with CHO cells (data not shown). These observations indicate
that Akt does not function downstream of PKC
.

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FIG. 11.
Localization of PKC and Akt to different signaling
pathways. (A and B) Effects of KD (A) and  PD (B) on Akt
activity in 3T3-L1 adipocytes. Cells were infected with AxCA KD (A)
or AxCA PD (B) at the indicated MOI (PFU per cell) and then
incubated in the absence or presence of 100 nM insulin for 10 min. Cell
lysates were subjected to immunoprecipitation with antibodies to Akt,
and the resulting precipitates were assayed for Akt kinase activity. (C
and D) Effects of a dominant negative mutant of Akt on insulin-induced
activation of Akt (C) and PKC (D) in CHO-Akt cells. Cells were
infected at the indicated MOI (PFU per cell) with an adenovirus vector
(AxCAAkt-AA) encoding Akt-AA, incubated in the absence or presence
of 100 nM insulin for 10 min, and lysed. Lysates were subjected to
immunoprecipitation with antibodies to either the FLAG epitope (C) or
PKC (D), and the resulting immunoprecipitates were assayed for Akt
or PKC activity, respectively. (E and F) Effects of Akt-AA on
insulin-induced activation of Akt2 (E) and PKC (F) in 3T3-L1
adipocytes. Cells were infected with AxCAAkt-AA at the indicated MOI
(PFU/cell), incubated in the absence or presence of 100 nM insulin for
3 min, and lysed. Lysates were subjected to immunoprecipitation with
antibodies to Akt2 (E) or to PKC (F), and the precipitates were
assayed for Akt and PKC activity, respectively. Data are means ± standard errors from three experiments.
|
|
We next investigated whether Akt contributes to the activation of
PKC

with the use of a dominant negative mutant of Akt.
We have
recently shown that a mutant Akt (Akt-AA) in which the
phosphorylation
sites (Thr
308 and Ser
473) targeted by growth
factors are replaced by alanine acts in a
dominant negative manner
(
26). Infection of CHO cells stably
expressing FLAG
epitope-tagged rat Akt1 (CHO-Akt cells) with an
adenovirus encoding
Akt-AA (AxCAAkt-AA) resulted in inhibition
of insulin-stimulated Akt
activity in an MOI-dependent manner;
at an MOI of 20 PFU/cell, ~70%
inhibition was achieved (Fig.
11C).
Insulin treatment of CHO-Akt cells
increased twofold the kinase
activity measured in immunoprecipitates
prepared with antibodies
to PKC

, and this stimulatory action of
insulin on PKC

activity
was not affected by expression of Akt-AA
(Fig.
11D).
We also tested the effect of Akt-AA on insulin stimulation of PKC

activity in 3T3-L1 adipocytes. We have previously shown
that infection
of these cells with AxCAAkt-AA at an MOI of 200
PFU/cell inhibited
insulin-induced activation of endogenous Akt
by ~90%
(
26). Because the antibodies to Akt used in our previous
study recognize Akt1, Akt2, and Akt3 (
26), it was suggested
that Akt-AA inhibits all three known isoforms of Akt. Because
Akt2 is a
major isoform of Akt in 3T3-L1 adipocytes (
11), we
directly
tested the effect of Akt-AA on insulin-stimulated Akt2
activity in
3T3-L1 adipocytes with the use of antibodies to Akt2.
These
antibodies did not recognize Akt-AA (data not shown), which
is
derived from rat Akt1. Insulin induced an approximately fivefold
increase in kinase activity present in immunoprecipitates prepared
with
the antibodies to Akt2 (Fig.
11E). The amount of kinase activity
in
immunoprecipitates prepared with antibodies to Akt3 was not
substantially greater than that present in precipitates prepared
with
control antibodies (data not shown). Infection of 3T3-L1
cells with
AxCAAkt-AA at an MOI of 200 PFU/cell inhibited insulin
stimulation of
Akt2 activity by ~80% (Fig.
11E). However, AxCAAkt-AA
had no effect
on insulin-stimulated PKC

activity (Fig.
11F), suggesting
that Akt
does not contribute to PKC

activation by
insulin.
The inhibitory effect of


NKD on insulin-stimulated glucose uptake
was partial (~50 to 60%) (Fig.
6B), whereas this mutant
almost
completely abolished the insulin-induced increase in PKC
activity
(Fig.
5C). These results may suggest the existence of
a redundant
pathway that mediates insulin stimulation of glucose
uptake. To examine
whether Akt is responsible for such a redundant
pathway, we finally
examined the effect of coexpression of

KD
and Akt-AA on
insulin-induced glucose uptake in 3T3-L1 adipocytes.
The inhibitory
effect of

KD on insulin stimulation of glucose
uptake in cells
infected with AxCAAkt-AA at an MOI of 200 PFU/cell
was similar to
that apparent in cells not infected with AxCAAkt-AA
(Fig.
12).

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FIG. 12.
Combined effects of Akt-AA and KD on insulin-induced
glucose uptake. 3T3-L1 adipocytes were infected with AxCA KD at the
indicated MOI, and after 8 h, they were infected (or not) with
AxCAAkt-AA at an MOI of 200 PFU/cell. After 40 h, the cells were
assayed for insulin-induced glucose uptake. Data are expressed as a
percentage of maximal insulin-induced glucose uptake and are means ± standard errors from three experiments.
|
|
 |
DISCUSSION |
Evidence suggests that stimulation of glucose transport by insulin
is mediated mainly by a pathway triggered by PI 3-kinase (12, 20,
21, 39, 41). Because both Akt and atypical PKC isozymes act
downstream of PI 3-kinase, it has been of interest to determine whether
insulin-stimulated glucose uptake is mediated through one of these
protein kinases or through an as-yet-unknown effector of the lipid
kinase. To address this question, we have now investigated the role of
an atypical PKC isoform, PKC
, in intact cells by specifically
inhibiting the activity of the endogenous enzyme. Overexpression of
kinase-deficient mutants of atypical PKC has been shown to inhibit
various biological actions, including activation of mitogen-activated
protein kinase, DNA synthesis, nuclear factor-
B-dependent
trans activation, and v-ras-induced transformation (7-9, 15), although it is not clear whether such mutants inhibit the activity of the endogenous enzymes.
We have now shown that expression of kinase-defective mutants of PKC
(
KD and 
NKD) inhibited insulin-induced activation of both
transfected and endogenous PKC
. Because the insulin-induced increase
in PI 3-kinase activity, measured in immunoprecipitates prepared with
antibodies to phosphotyrosine, was not affected by either of these
mutant proteins, it appears that they do not inhibit the insulin
receptor kinase or subsequent phosphorylation of IRS1; rather, they
prevent specific signaling downstream of PI 3-kinase. Normal activation
of Akt by insulin in cells expressing
KD also supports this
conclusion. These observations thus demonstrated that
KD and

NKD act in a dominant negative manner.
Overexpression of either
KD or 
NKD inhibited insulin
stimulation of glucose uptake and GLUT4 translocation in 3T3-L1
adipocytes. Basal glucose transport in cells expressing these mutants
was similar to that in uninfected cells. Furthermore, glucose uptake induced by either GH or hyperosmolarity attributable to sorbitol, both
of which promote glucose transport through a PI 3-kinase-independent mechanism (41), was not affected by
KD, suggesting that
KD inhibited insulin-induced glucose uptake not by an effect on a component of the transport machinery but by blocking specific signals
mediated through PKC
. Indeed, the amount of GLUT4 protein in
cells infected with AxCA
KD or AxCA
NKD was unchanged.
To investigate further the role of PKC
in glucose uptake and
translocation of GLUT4, we examined the effects of a constitutively active mutant of PKC
. The kinase activity of 
PD, a PKC
mutant that lacks the pseudosubstrate domain, is markedly greater than that of the wild-type enzyme. Infection of cells with AxCA
PD stimulated glucose uptake to an extent similar to that achieved by
insulin, without affecting the amount of GLUT4 protein or of insulin-stimulated PI 3-kinase activity precipitated with antibodies to
phosphotyrosine. The observation that insulin did not result in further
stimulation of glucose uptake in these cells suggests that the effects
of insulin and PKC
are not additive and that PKC
lies on the
insulin signaling pathway responsible for regulating glucose uptake.
The inability of wortmannin to inhibit glucose transport stimulated by

PD is consistent with the conclusion that PKC
acts downstream
of PI 3-kinase. These results, together with the ability of wild-type
PKC
to compensate for the inhibition of insulin-induced glucose
uptake by 
NKD, indicate that PKC
contributes to insulin
stimulation of glucose uptake.
Expression of Akt fused with a viral Gag protein or tagged with a
myristoylation signal sequence induced glucose uptake or translocation
of GLUT4 in adipocytes (30, 45). Although these observations
do not necessarily indicate that insulin signaling to glucose uptake is
mediated by Akt, it is important to determine whether Akt and PKC
participate in the same signaling pathway or whether they transmit
signals through different pathways. We have now shown that
KD did
not inhibit insulin-induced activation of Akt and that 
PD did not
increase basal Akt activity. These data indicate that the inhibitory
effect of
KD on insulin-stimulated glucose uptake is not mediated by
prevention of Akt activation and that 
PD stimulation of glucose
transport is not mediated by activation of Akt. Moreover, we have shown
that the inhibition of insulin-induced activation of Akt by a dominant
negative mutant of Akt (Akt-AA) did not affect insulin stimulation of
PKC
activity, indicating that insulin activation of PKC
is
independent of Akt activation.
Cong et al. (14) have shown that when isolated adipocytes
were transiently transfected with constructs encoding HA-tagged GLUT4
and a kinase-deficient mutant of Akt (Akt-K179A), the amount of
HA-tagged GLUT4 on the cell surface, in the absence or presence of
insulin, was ~20% less than that apparent in cells that were transfected with a control plasmid in addition to that encoding HA-tagged GLUT4, suggesting that Akt contributes to insulin stimulation of translocation of GLUT4 in these cells. However, we have recently found that overexpression of a similar kinase-deficient mutant of Akt
(Akt-K179D) affected neither insulin-stimulated Akt activity in
CHO cells (26) nor insulin-stimulated glucose uptake in
3T3-L1 adipocytes (32). We do not know the reason for this
discrepancy. It is possible that the effects of kinase-deficient
mutants of Akt may be different in different cells and tissues.
The reason why the dominant negative mutants of PKC
inhibited
insulin-stimulated glucose uptake by only ~50% in 3T3-L1 adipocytes, whereas 
NKD almost completely abolished the insulin-induced increase in PKC
activity precipitated with antibodies to PKC
, is
not clear. One possibility is that there is a redundant pathway that
mediates insulin stimulation of glucose uptake and that prevention of
signal transduction by the PKC
pathway is therefore not sufficient to block glucose uptake completely. It is not known which molecules might be responsible for such a redundant pathway; however, the participation of Akt is unlikely, given that expression of Akt-AA did
not exert an additive effect on the inhibition of glucose uptake by
KD. Calera et al. (11) have shown that the amount of Akt2
associated with GLUT4-containing vesicles was increased in response to
insulin. Although the physiological significance of this observation
remains to be elucidated, we cannot exclude the possibility that a
small increase in Akt activity in a specific compartment of the cell
may be sufficient to activate glucose transport fully. Another possible
explanation for the discrepancy between the extents of inhibition of
glucose uptake and of PKC
activity by the dominant negative mutants
of PKC
is that the activity of immunoprecipitated PKC
does not
completely reflect the activity of this enzyme in intact cells. It has
been suggested that interaction with lipids produced in response to
extracellular stimuli regulates the enzymatic activity of atypical PKC
(1, 36-38, 43). Thus, lipids essential for the activation
of PKC
in intact cells may have been removed, at least in part,
during immunoprecipitation, possible explaining the relatively small activation of the enzyme observed in the immunoprecipitates. This possibility is supported by our observation that PKC
immunoprecipitated from insulin-stimulated cells was further activated
by PS in vitro (32). Therefore, we cannot exclude the
possibility that
KD and 
NKD may not completely block the
activity of PKC
in intact cells, whereas the insulin-induced
increase in the activity of the immunoprecipitated enzyme appears to be abolished.
Two groups have reported that PKC
is expressed in 3T3-L1 adipocytes
on the basis of immunoblot analysis with antibodies generated in
response to a peptide corresponding to the COOH-terminal region of rat
PKC
(GFEYINPLLLSAEESV) (6, 19). This
amino acid sequence is highly homologous to the corresponding sequence
of mouse PKC
(GFEYINPLLMSAEECV). We have now shown
that antibodies induced by the same COOH-terminal peptide of PKC
(
CT) recognized mouse PKC
transiently expressed in COS7 cells.
Moreover, 
CT detected an ~80-kDa protein in immunoprecipitates
prepared from 3T3-L1 adipocyte lysates with antibodies to PKC
(
190) but not in those prepared with antibodies to PKC
(
170). These results, together with those of our RT-PCR analysis,
suggest that PKC
, but not PKC
, is expressed in 3T3-L1 adipocytes
and that the protein previously detected in 3T3-L1 adipocytes by the
antibodies to the PKC
peptide is actually PKC
.
Bandyopadhyay et al. (6) have shown that insulin-stimulated
glucose uptake was reduced by ~30% in 3T3-L1 adipocytes expressing a
kinase-deficient mutant of rat PKC
. We have found that
overexpression of a kinase-deficient mutant of PKC
inhibited the
insulin-induced increase in PKC
activity in 3T3-L1 adipocytes
(32), suggesting that the inhibition of glucose transport
observed by these investigators may be due to the inhibition of PKC
.
However, it is possible that PKC
also participates in insulin signal
transduction, because the same group recently reported that transient
expression of a kinase-defective mutant PKC
in rat adipocytes, or
incubation of the adipocytes with a peptide corresponding to the
pseudosubstrate domain of PKC
, inhibited insulin-induced
translocation of transfected GLUT4 or glucose transport, respectively
(43). Furthermore, it has been reported that
insulin-stimulated protein synthesis in hematopoietic cells was
modulated by transfection with a mutant PKC
cDNA (34). It
is thus important to determine which actions of insulin that are
dependent on PI 3-kinase are mediated by which isoform of atypical PKC
in different cells and tissues.
In summary, we have shown that PKC
is required for insulin
stimulation of glucose uptake, but not for Akt activation, and that a
dominant negative Akt mutant did not affect PKC
activity in 3T3-L1
adipocytes. These results suggest that insulin-elicited signals that
pass through PI 3-kinase are subsequently transmitted by at least two
independent pathways: an Akt pathway and a PKC
pathway. The
mechanism by which PKC
is activated by PI 3-kinase remains unclear.
Clarification of the mechanism by which PKC
is regulated should
increase our understanding of the mechanism by which signals from PI
3-kinase diverge and are transmitted by various downstream effectors,
subsequently resulting in a broad range of biological effects.
 |
ACKNOWLEDGMENTS |
This work was supported by a grant from the Ministry of
Education, Science, Sports, and Culture of Japan, a Grant-in-Aid for Research for the Future Program from the Japan Society for the Promotion of Science, a grant from the Uehara Memorial Foundation, a
grant for studies on the pathophysiology and complications of diabetes
from Tsumura Pharma Ltd. (to M.K.), and a grant from Suzuken Memorial
Foundation (to W.O.).
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Second
Department of Internal Medicine, Kobe University School of Medicine,
7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Phone:
81-78-341-7451. Fax: 81-78-382-2080. E-mail:
ogawa{at}med.kobe-u.ac.jp.
 |
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A new member of the third class in the protein kinase C family, PKC , expressed dominantly in an undifferentiated mouse embryonal carcinoma cell line and also in many tissues and cells.
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Molecular and Cellular Biology, December 1998, p. 6971-6982, Vol. 18, No. 12
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