(Received for publication, February 15, 1996, and in revised form, September 25, 1996)
From the J. A. Haley Veterans Hospital Research
Service, and Departments of Internal Medicine and Biochemistry,
University of South Florida, Tampa, Florida 33612, the
§ Department of Biochemistry, China Medical University,
Shenyang, Liaoning Province 11001, People's Republic of China,
¶ Hopital Lapeyronie/Service des Maladies Metaboliques, 34295 Montpellier, France, and
Centro de Biologia Molecular "Servero
Ochoa," Universidad Autonoma, Canto Blanco, 28049 Madrid, Spain
We presently studied (a) insulin
effects on protein kinase C (PKC) and (b) effects of
transfection-induced, stable expression of PKC isoforms on glucose
transport in 3T3/L1 cells. In both fibroblasts and adipocytes, insulin
provoked increases in membrane PKC enzyme activity and membrane levels
of PKC- and PKC-
. However, insulin-induced increases in PKC
enzyme activity were apparent in both non-down-regulated adipocytes and
adipocytes that were down-regulated by overnight treatment with 5 µM phorbol ester, which largely depletes PKC-
,
PKC-
, and PKC-
, but not PKC-
. Moreover, insulin provoked
increases in the enzyme activity of immunoprecipitable PKC-
. In
transfection studies, stable overexpression of wild-type or
constitutively active forms of PKC-
, PKC-
1, and
PKC-
2 failed to influence basal or insulin-stimulated
glucose transport (2-deoxyglucose uptake) in fibroblasts and
adipocytes, despite inhibiting insulin effects on glycogen synthesis.
In contrast, stable overexpression of wild-type PKC-
increased, and
a dominant-negative mutant form of PKC-
decreased, basal and
insulin-stimulated glucose transport in fibroblasts and adipocytes.
These findings suggested that: (a) insulin activates
PKC-
, as well as PKC-
and
; and (b) PKC-
is
required for, and may contribute to, insulin effects on glucose
transport in 3T3/L1 cells.
3T3/L1 cells are useful models for studying insulin action, as they offer advantages of a transfectable cultured cell line and contain GLUT1 glucose transporters as fibroblasts, and acquire GLUT4, the major insulin-regulated glucose transporter, during differentiation. The signaling systems that are used by insulin to regulate glucose metabolism and other cellular functions in 3T3/L1 cells are, however, poorly understood. With respect to protein kinase C (PKC),1 some studies suggested that insulin does not activate this signaling system in either 3T3/L1 fibroblasts (1) or adipocytes (2), although in adipocytes, insulin was found to provoke an increase in cytosolic PKC activity (3), and, in fibroblasts, some insulin effects on the phosphorylation of eukaryotic initiation factors appeared to be PKC-dependent (4). In addition, studies with phorbol esters, as diacylglycerol (DAG) analogues that acutely activate and chronically deplete conventional PKCs (cPKCs) and novel PKCs (nPKCs), have suggested that such DAG-sensitive PKCs do not play a major role in insulin-stimulated glucose transport in 3T3/L1 cells (1, 3, 5). On the other hand, atypical PKCs (aPKCs) are not activated or depleted by DAG or phorbol esters, but may nevertheless be activated by other lipid signaling substances, e.g. polyphosphoinositides derived through the activation of phospatidylinositol (PI) 3-kinase, a process that is activated by insulin.
Because of seemingly conflicting reports on overall PKC activation in
3T3/L1 cells, and because of the paucity of studies on atypical PKCs,
such as PKC-, in insulin action, we addressed these questions using
several experimental approaches. Accordingly, we found that insulin
increased DAG production, and stimulated the translocation of PKC-
and PKC-
from the cytosol to the membrane fraction, in 3T3/L1 cells.
Perhaps, more interestingly, we found that insulin increased PKC enzyme
activity, not only in membrane preparations from 3T3/L1 cells
containing cPKCs, nPKCs, and aPKCs, but also in (a) membrane
and cytosolic preparations from adipocytes in which cPKCs and nPKCs
were largely depleted by phorbol ester treatment, and (b) in
PKC-
immunoprecipitates from adipocyte lysates. Moreover, we found
in stably transfected 3T3/L1 fibroblasts and adipocytes that:
(a) expression of wild-type and constituitively active forms
of PKC-
, PKC-
1, and PKC-
2 failed to
influence basal or insulin-stimulated glucose transport, despite
inhibiting insulin effects on glycogen synthesis; and (b)
expression of wild-type PKC-
enhanced, and dominant-negative PKC-
inhibited, basal and insulin-stimulated glucose transport.
3T3/L1 cells were obtained from American Type Culture Collection (Rockville, MD) and were cultured as described (6) to yield fibroblasts and differentiated adipocytes. For adipocytes, insulin was withdrawn 48 h prior to experimentation, and for both fibroblasts and adipocytes, medium was changed to serum-free Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 25 mM glucose for 3 h, and then to glucose-free Krebs-Ringer phosphate (KRP) buffer for 30 min prior to experimental use. Cells were treated with vehicle, insulin (Elanco) or 12-O-tetradecanoylphorbol-13-acetate (TPA; Sigma) for the indicated times, keeping the total incubation time constant for all samples.
Assays of Total PKC Enzyme ActivityIn Method I, PKC enzyme
activity was measured in extracts of control and insulin-treated 3T3/L1
cells that were incubated and subsequently assayed in parallel,
essentially as described previously in studies of rat adipocytes (7).
In brief, cells from two or three 100-mm plates of each treatment group
were pooled and homogenized in buffer I containing 0.25 M
sucrose, 1.2 mM EGTA, 0.1 mM
phenylmethylsulfonyl fluoride (PMSF), 20 µg/ml leupeptin, 20 mM -mercaptoethanol, and 20 mM Tris (pH
7.5). Cytosol and membrane fractions were obtained by centrifugation at
100,000 × g for 60 min. Membranes were
resuspended in buffer I supplemented to contain 1% Triton X-100, 5 mM EGTA, and 2 mM EDTA, and then cleared of
insoluble substances by centrifugation. Cytosol and Triton
X-100-solubilized membrane fractions from equal amounts (as per protein
determination) of control and insulin-treated cells were
chromatographed in parallel on FPLC Mono Q columns (Pharmacia
Biotech Inc.) and fractions were assayed (see Ref. 7) for
phosphatidylserine (PS)/diolein/Ca2+-dependent
phosphorylation of histone IIIs (all reagents from Sigma).
PKC activity was also analyzed by Method II, as described previously in
studies of BC3H-1 myocytes (8). In brief, cells were homogenized in
buffer containing 50 mM Tris/HCl (pH 7.5), 1 mM
NaHCO3, 5 mM MgCl2, 1 mM PMSF, 20 µg/ml aprotinin, and 20 µg/ml leupeptin.
Membranes and cytosol fractions were obtained by centrifugation at
100,000 × g for 60 min, and 5-10 µg of protein was
assayed for ability to phosphorylate PKC pseudosubstrate derivatives, namely 40 µM
[Ser25]PKC--(19-31)-NH2 (Life
Technologies, Inc.) or
[Ser159]PKC-
-(153-164)-NH2 (Upstate
Biotechnology, Inc.) in 100 µl of buffer containing 50 mM
Tris/HCl (pH 7.5), 50 µM [
-32P]ATP
(DuPont NEN), 5 mM MgCl2, 100 µM
sodium vanadate, 100 µM sodium pyrophosphate, 1 µM CaCl2, 1 mM NaF, and 100 µM PMSF. Reactions were stopped with 5% acetic acid, and
aliquots of the reaction mixture were spotted on P-81 filter papers,
washed in 5% acetic acid, and counted for 32P. In this
assay, membrane PKC enzyme activity is dependent upon endogenous lipid
and non-lipid co-factors. In experiments in which the cytosol was
assayed, PS (40 µg/ml) was also added to provide a phospholipid
milieu. These assays were conducted in the presence and absence of
peptide substrate to define substrate-dependent PKC
activity. 32PO4 incorporation in the absence of
substrate accounted for only 10-20% of that observed in presence of
substrate. In both methods of assay, RO 31-8220 (Roche; kindly supplied
by Dr. Geoff Lawton), a relatively specific PKC inhibitor, virtually
abolished the phosphorylation of added substrate.
We have recently
found2 that insulin provokes rapid
increases in enzyme activity of PKC- in specific immunoprecipitates
(i.e. devoid of PKC-
,
,
, and
) prepared from
total cell lysates of rat adipocytes. In virtually identical
experiments, in 3T3/L1 adipocytes, PKC-
was immunoprecipitated by
incubating polyclonal anti-PKC-
antiserum (Life Technologies, Inc.)
for 16 h at 0-4 °C with 1 mg of total cell lysate protein in
buffer containing 20 mM Tris/HCl (pH 7.5), 0.25 M sucrose, 1.2 mM EGTA, 20 mM
-mercaptoethanol, 1 mM PMSF, 20 µg/ml leupeptin, 20 µg/ml aprotinin, 1 mM sodium vanadate, 1 mM
sodium pyrophosphate, 1 mM NaF, Triton X-100 (1%), Nonidet
(0.5%), and 150 mM NaCl. Precipitates using non-immune serum were simultaneously prepared to determine blank values. Precipitates were collected on Protein A/G-Sepharose beads, washed and
suspended in 50 mM Tris/HCl (pH 7.5), 1 mM
NaHCO3, 5 mM MgCl2, 1 mM PMSF, 20 µg/ml aprotinin, and 20 µg/ml leupeptin,
and then assayed for 8 min at 30 °C as in Method II above, using 40 µM [Ser159]PKC-
-(153-164)-NH2 as substrate,
and 40 µg/ml PS. In these immunoprecipitate assays, enzyme reaction
rates are linear with respect to time, are dependent upon added PS, but
not diolein or Ca2+, and are fully inhibited by 100 µM PKC-
pseudosubstrate (Ki = 10-20 µM).
PKC isoforms in
cytosol and membrane fractions were assayed by immunoblotting as
described (9). In brief, equal amounts of protein from cytosol and
membrane fractions (prepared as described above in Method I PKC enzyme
assay and stored in Laemmli buffer) of control and insulin- or
TPA-treated 3T3/L1 cells were subjected to sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to
nitrocellulose membranes, and subsequently immunoblotted with anti-PKC
isoform-specific, polyclonal antisera. Antisera for PKC-, PKC-
,
PKC-
, and PKC-
were obtained from Life Technologies. In most
experiments, we assayed total PKC-
(i.e.
PKC-
1+2) with antisera raised against a peptide sequence
(conjugated to albumin or keyhole limpet hemocyanin) present in the
V3 region common to PKC-
1 and
PKC-
2 (see Refs. 9 and 10). In some experiments, we also
used
1-specific and
2-specific antisera kindly supplied by Dr. Susan Jaken. Epitope specificities of antisera were confirmed by showing that signals were lost when assays were conducted in the presence of immunizing peptide, and/or by reactivity with recombinant PKCs (see Refs. 9 and 11). Antisera specificities for
PKC-
, PKC-
1+2, PKC-
1,
PKC-
2, and PKC-
were also verified by overexpression
of plasmids containing cDNAs that encode each of these isoforms in
3T3/L1 cells (see below). Antisera for GLUT1 and GLUT4 were obtained
from East Acres Biochemicals. Immunodetection was accomplished by
antibody-associated alkaline phosphatase colorimetric staining as
described previously (9) or, in most cases, by chemiluminescence (ECL,
Amersham). Blots were scanned and quantified with an LKB laser
densitometer or, in most cases, with a Bio-Rad Chemiluminescence
Molecular Imaging System, and results were expressed relative to the
control(s), on the same blot, set at 100%.
Methods for measurement of
PKC- (1+2), PKC-
1, and
PKC-
2 by ribonuclease protection assay have been
described previously (12).
Four plasmid
eukaryotic expression vectors, pMTH, pMV7, pMV12, and pCDNA3
(Invitrogen), were used to transfect PKC isoforms. pMTH, pMV7, and
pCDNA3 vectors contain a neomycin resistance gene, and pMV12 is
identical to pMV7, except that the neomycin resistance gene is replaced
by a hygromycin resistance gene. pMTH-PKC-2 (rat), in
which the expression of the cDNA insert is regulated by a mouse
metallothionine promoter (see Ref. 13), and pMV7-PKC-
(mouse),
pMV7-PKC-
(rat), pMV7-PKC-
(rat), pMV7-PKC-
1
(rat), and pMV12-PKC-
2 (rat), in which inserts are
regulated by long terminal repeats of a Moloney murine sarcoma virus
(see Refs. 14 and 15), were kindly supplied by Dr. Harald Mischak (also see Ref. 16). pCDNA3 containing cDNAs encoding wild-type and dominant-negative (a lysine 281 to tryptophan point mutation in the
catalytic site) forms of rat PKC-
were prepared in Dr. Moscat's laboratory (see Ref. 17). To prepare pCDNA3-PKC-
2
(rat), intact rat PKC-
2 cDNA insert was excised from
the vector pTB701-PKC-
2 (kindly supplied by Dr. Y. Ono,
Kobe University, Kobe, Japan) through partial digestion of the vector
with EcoRI, and then introduced into pCDNA3 at the
EcoRI locus of the multiple cloning site of the vector.
Constitutively active (an alanine-25 to glutamate point mutation in the
pseudosubstrate region) and dominant-negative (point-mutated in the
ATP-binding site) forms of bovine PKC-
, and constitutively active
rat PKC-
(alanine 159 to glutamate point mutation in the
pseudosubstrate site) were kindly supplied by Drs. Peter Parker
(provided through Imperial Cancer Research Fund) (see Ref. 18) and Kirk
Ways (Eli Lilly Co., Indianapolis, IN) and were also excised and
ligated into pCDNA3. Rat PKC-
cDNA obtained from Dr. Ono was
also inserted into pCDNA3 and yielded identical results with the
construct prepared in Dr. Moscat's laboratory. Orientation of inserts
and integrity of coding regions were verified by restriction mapping.
In some experiments, cells were co-transfected with empty pCDNA3
vector (for neomycin resistance) and the eukaryotic expression vector,
PCDSR
, containing cDNAs for wild-type or constituitively active
(deleted in pseudosubstrate sites) forms of bovine PKC-
and
PKC-
2 (obtained from Dr. M. Muramatsu, University of
Tokyo, Tokyo, Japan; see Ref. 19); these constructs were designated by
Dr. Muramatsu as SR
PKC
(wild-type PKC-
), SR
PKAC
(constituitively active PKC-
), SR
PKC
(wild-type PKC-
2), and SR
PKC
-
EE (constituitively active
PKC-
2). Plasmids containing empty vectors or vectors
plus inserts were transfected in parallel into the same group of 3T3/L1
fibroblasts by LipofectAMINE treatment following instructions of the
supplier (Life Technologies, Inc.). Individual neomycin-resistant or
hygromycin-resistant clones were harvested and subsequently expanded,
along with untransfected controls, to produce fibroblasts and
adipocytes, as described above. Although not studied in great detail,
growth characteristics, overall differentiation and gross cell
morphology did not appear to be significantly altered in any of the
selected clones that are herein reported; also note that, as described
below, PKC-
-transfected adipocytes (a) had normal
complements of GLUT4, a characteristic component of differentiated
adipocytes, and (b) manifested normal glycogen synthesis
responses to insulin. It therefore seems likely that initial insulin
signaling responses were intact in PKC-
tranfectants. Transfection
efficiencies were judged by Western analysis of the expressed protein,
and, in some cases, as indicated, this was complemented by measurement
of mRNA levels and/or PKC enzyme activity.
As described above, controls and transfected clones were cultured and assayed for glucose transport and immunoreactive PKC in parallel. [3H]2-Deoxyglucose (DuPont NEN) uptake was determined as described (6). Results in clones expressing significant amounts of transfected PKC (see below) were subsequently pooled and compared as a group to parallel-assayed controls. Glycogen synthesis assays were conducted by incubating cells for 60 min in KRP buffer containing 5 mM glucose and [U-14C]D-glucose (DuPont NEN), with or without 100 nM insulin; after incubation, cells were homogenized in 4 N KOH, heated for 30 min at 100 °C, and labeled glycogen was trapped on filter paper, washed with cold ethanol, and counted for radioactivity.
PKC enzyme activity of
Triton X-100-solubilized membrane fractions of 3T3/L1 adipocytes eluted
from Mono Q columns as described previously (7), and most histone IIIs
phosphorylation reflected PS/diolein/Ca2+-dependent PKC activity. This
was confirmed by using other PKC substrates, e.g.
protamine-SO4, and 1 µM RO 31-8220 to inhibit PKC-dependent protein phosphorylation. Membrane fractions
from insulin-treated (100 nM × 10 min) 3T3/L1 adipocytes
were more active than control membrane fractions. In four separate
experiments in which the PKC-dependent activities in all
column fractions were summed, insulin provoked 2-fold increases in
total elutable PKC enzyme activity of Triton X-100-solubilized membrane
fractions (summarized in Fig. 1). In 3T3/L1 fibroblasts,
insulin also provoked increases in total-elutable membrane PKC
activity, which were less, namely approximately 50% increases above
control, than those provoked by insulin in 3T3/L1 adipocytes (Fig. 1).
In contrast to changes in membranes, we did not observe significant
differences in total elutable PKC enzyme activity in cytosolic
fractions of control and insulin-treated cells (data not shown).
Total PKC Enzyme Activity, Method II
We also assayed PKC
enzyme activity in whole membranes of adipocytes using Method II; in
this assay, exogenous lipid co-factors are not added, and PKC enzyme
activity is dependent upon PKC content and endogenous lipids and other
activators. As compared to Method I, insulin-induced increases in whole
membrane PKC enzyme activity were more modest in the Method II assay,
but, were, nevertheless, significant, namely control membranes, 34 ± 1.5 versus insulin-treated (100 nM × 10 min)
membranes, 46 ± 2 pmol of PO4 incorporated into [Ser25]PKC--(19-31)-NH2/min/mg of protein
(mean ± S.E.; n = 6; p < 0.005, t test) (also see Table I for results using
[Ser159]PKC-
-(153-164)-NH2 as the
substrate in Method II assays). Method II was not used in
fibroblasts.
|
The incorporation of 32PO4 in the above assays
presumably reflected the sum of enzyme activities of all PKC isoforms,
including ,
,
, and
(see below). In order to gain insight
into the question of whether insulin activates atypical PKCs, such as
PKC-
, we took advantage of the fact that overnight treatment with
TPA largely depleted PKC-
,
, and
, but had no effect on 70-kDa PKC-
(Fig. 2). (Note: unlike the situation in BC3H-1
myocytes (16), neither TPA nor insulin treatment altered PKC-
,
PKC-
1, PKC-
2 or PKC-
mRNA levels
in 3T3/L1 adipocytes, and induction or retention of
PKC-
2 was not observed after TPA treatment.) As shown in
Table I, in the absence of TPA pretreatment, insulin provoked a 54%
increase in membrane-dependent
32PO4 incorporation into
[Ser159]PKC-
-(153-164)-NH2, a preferred
substrate for both PKC-
and PKC-
(PKC-
and PKC-
are only 50 and 30% as effective; see Ref. 20). Moreover, after overnight TPA
pretreatment, there were 60-70% decreases in overall
32PO4 incorporation into this substrate,
presumably reflecting losses of cPKCs and nPKCs, but insulin-induced
increases in PKC activity nevertheless remained apparent in membranes
(56% increase) and, somewhat surprisingly, became more clearly evident
and statistically significant in cytosol (49% increase) fractions.
These results were in keeping with the possibility that insulin
activated TPA-resistant PKCs, such as PKC-
, which is distributed
between cytosol and membrane fractions and is not translocated during
agonist treatment.
Immunoprecipitable PKC-
In order to test
the possibility that insulin activated PKC-, we studied enzyme
activity in specific PKC-
immunoprecipitates and found that enzyme
activity of PKC-
immunoprecipitates was increased more than 2-fold
after treating adipocytes for 10 min with 100 nM insulin
(Fig. 3). As stated under "Experimental Procedures," there was little or no significant immunoreactive PKC-
,
, or
in the PKC-
immunoprecipitates and preimmune serum did not immunoprecipitate PKC-
(Fig. 3). Insulin did not affect the amount of PKC-
recovered in these precipitates (approximately 50%), and it
may be surmised that insulin increased the specific enzyme activity of
PKC-
, apparently through a factor or covalent modification that was
retained during immunoprecipitation and assay
procedures.2
Acute Changes in Immunoreactive PKC
In 3T3/L1
adipocytes, insulin provoked time-dependent decreases in
cytosolic, and increases in membrane, PKC-, and PKC-
(Figs. 1 and
4). The changes in immunoreactive PKC-
and PKC-
were maximal at 2-10 min of insulin treatment and then diminished at
20 min. In 3T3/L1 fibroblasts, the relative effects of insulin on the
translocation of PKC-
and PKC-
were approximately one-half of
those observed in adipocytes (summarized in Fig. 1). In contrast to
PKC-
and PKC-
, insulin had no consistent effect on the cellular distribution of either PKC-
or the 70-kDa form of PKC-
in
adipocytes (Fig. 4) and fibroblasts (data not shown). Acute (10 min)
TPA treatment, on the other hand, stimulated the translocation of PKC-
, as well as PKC-
and PKC-
, but not 70-kDa PKC-
in
adipocytes (Fig. 2). It may be noted in Figs. 2 and 4 that, in addition
to the 70-kDa form of PKC-
, the anti-PKC-
antiserum (from Life Technologies, Inc.) also cross-reacted with an 80-kDa moiety that translocated in response to acute insulin and TPA treatment (Figs. 2
and 4) and down-regulated with overnight, 5 µM TPA
treatment. This 80-kDa band is most likely cross-reacting PKC-
, as
it (a) mirrored changes in authentic PKC-
(measured by
PKC-
antiserum), and (b) increased upon transfection of
cells with plasmids containing the PKC-
cDNA insert (data not
shown). Of further note, this 80-kDa protein was not recovered in
PKC-
immunoprecipitates, which only contained 70-kDa PKC-
(Fig.
3). It may also be noted in Figs. 2 and 4 that the cytosolic PKC-
that translocated better in response to insulin treatment, and
down-regulated better in response to overnight TPA treatment, had an
apparent mass of 80 kDa on SDS-PAGE. In addition, the cytosol contained
a 75-kDa PKC-
moiety that was, in general, more plentiful, but less
responsive to acute insulin or TPA treatment, as compared to the 80-kDa
moiety. On the basis of overexpression studies (see below), this 75-kDa moiety appeared to be predominately a lower Mr
form of PKC-
1, and it is of interest that, in other cell
types, a similarly migrating PKC-
has been reported to be poorly
activated, as it apparently lacks key prerequisite phosphorylations
(see Ref. 21).
Effects of Cellular Differentiation on PKC Isoforms
There
were approximately 50% decreases in the cellular concentrations of
PKC- and PKC-
following differentiation of fibroblasts into
adipocytes; PKC-
, on the other hand, increased slightly in
adipocytes, and PKC-
did not change appreciably during
differentiation (data not shown). Immunoreactive PKC-
was not
detectable in either fibroblasts or adipocytes. As shown in Fig.
5, when untransfected cells were blotted with
1- and
2-specific antisera,
PKC-
1 (75- and 80-kDa moieties) was readily detectable
in fibroblasts and adipocytes, whereas PKC-
2 was poorly
detectable, if at all.
Stable Expression of PKC
Stable transfection of cells with
PKC-1 and, even more strikingly, PKC-
2,
resulted in sizable overexpression of these isoforms in both
fibroblasts and adipocytes (Fig. 5). Transfection-induced increases in
immunoreactive PKC-
2 were comparable using a variety of
vectors, i.e. pMTH, pMV7, pMV12, and pCDNA3.
Transfection-induced expression of PKC-
2 yielded
primarily an 80-kDa moiety, whereas expression of PKC-
1
yielded 75-kDa, as well as 80-kDa, moieties (Fig. 5). Stable
transfection of cells with cDNAs encoding PKC-
and PKC-
increased the levels of these PKCs approximately 2-3-fold (Fig.
6 and Table II), albeit to a lesser
relative degree than that observed with PKC-
2
transfection, perhaps reflecting higher basal levels of the PKC-
and
PKC-
isoforms. In contrast to PKC-
, PKC-
, and PKC-
, we were
not able to express PKC-
or significantly overexpress PKC-
, as
judged by immunoblot analysis (data not shown).
|
In the case of PKC-2, we verified that transfection
resulted in specific increases in PKC-
2 mRNA (Fig.
5). We also verified that transfected PKC-
2 was
down-regulated by overnight 5 µM TPA treatment (Fig. 5),
and it may therefore be surmised that transfected PKC-
2
was biologically active (also see below). In some cases, we verified
that there were increases in PKC enzyme activity in transfected cells,
e.g. as shown in Fig. 7, expression of normal PKC-
and PKC-
2 increased adipocyte membrane and
cytosol PKC enzyme activities substantially, and both enzyme activities
were further increased in adipocytes transfected with constituitively active forms of these PKCs. As shown in Fig. 6, and as reported by Ways
et al. (22), there were concomitant, but variable increases in 80-kDa PKC-
(i.e. the upper band in PKC-
blots
whose identity as PKC-
was confirmed with anti-PKC-
antiserum) in
PKC-
transfectants, regardless of whether they were wild-type or
dominant-negative mutants. As will become apparent (see below), this
co-expression of PKC-
could not explain observed changes in glucose
transport experiments. In contrast to PKC-
, we did not observe
changes in PKC-
or PKC-
levels in PKC-
transfectants.
Effects of PKC Expression on Glucose Transport
As shown in
Fig. 8, there was little or no effect of overexpression
of wild-type PKC-, PKC-
1, or PKC-
2, on
basal or insulin-stimulated glucose transport in either fibroblasts or
adipocytes. Similarly, the expression of other constructs encoding
either wild-type or constituitively active (pseudosubstrate-deleted)
forms of PKC-
and PKC-
2 failed to influence glucose
transport significantly, i.e. relative to untransfected
cells or cells transfected with empty vectors, in control or
insulin-stimulated fibroblasts and adipocytes (Fig. 9).
In other experiments (data not shown), the expression of point-mutated,
constituitively active and dominant-negative forms of PKC-
(those
obtained from Dr. Peter Parker) also failed to alter glucose transport
responses (data not shown).
In contrast to PKC- and PKC-
, the overexpression of wild-type
PKC-
increased basal and insulin-stimulated glucose transport in
fibroblasts (Figs. 8 and 10). In adipocytes (Fig. 10),
overexpression of PKC-
increased glucose transport in control and
submaximally stimulated cells, but the maximal insulin effect was not
changed significantly. Of further interest, a dominant-negative,
point-mutated form of PKC-
inhibited basal and insulin-stimulated
glucose transport in both fibroblasts and adipocytes (Fig. 10). As
shown in Table II, immunoreactive PKC-
levels were increased
approximately 2-fold in wild-type and dominant negative PKC-
transfectants, and observed changes in glucose transport in PKC-
transfectants could not be explained by changes in total GLUT1 and/or
GLUT4 levels. As shown in Fig. 11, PKC enzyme activity
in TPA-down-regulated adipocytes was 2-fold higher in cytosol fractions
of cells transfected with wild-type, but not dominant-negative,
PKC-
; this further suggested that TPA-resistant PKC enzyme activity
largely reflected PKC-
activity, as it would be expected to be
increased in wild-type overexpressers, but not with expression of
catalytically inactive PKC-
. Further, since immunoreactive PKC-
was increased by approximately 2-fold in both wild-type and
dominant-negative transfectants, it may also be surmised that the
specific enzyme activity of total PKC-
was decreased by 50% in
dominant-negative transfectants. As shown in Fig. 12,
in keeping with increased basal glucose transport activity, both GLUT4
and GLUT1 were more plentiful in plasma membranes and less plentiful in
microsomes in wild-type PKC-
overexpressers, as compared to
controls. Also, with insulin treatment, resultant GLUT4 and GLUT1
levels in plasma membranes (approximately 2-fold increases were seen)
of wild-type PKC-
overexpressers were equal to, if not greater than,
the levels seen in controls. In contrast, in PKC-
dominant-negative
transfectants, insulin effects on GLUT4 and GLUT1 appeared to be
blunted (Fig. 12).
Effects of PKC-
In contrast to glucose transport, the expression of
both wild-type and constituitively active forms of PKC- and
PKC-
2 led to inhibition of insulin-induced increases in
[U-14C]glucose incorporation into glycogen (Fig. 7). This
confirmed that these transfected PKCs were biologically, as well as
enzymatically (also see Fig. 7), active. The greatest inhibition
of glycogen synthesis was observed with constituitively active
PKC-
. Along these lines, it was of interest to find that
overexpression of wild-type PKC-
, and the expression of
dominant-negative PKC-
failed to alter insulin effects on glycogen
synthesis (data not shown); this suggested that, in these PKC-
transfections, initial insulin signaling was intact, and inhibitory
effects of PKC on glycogen synthesis were
isoform-dependent.
We presently found that insulin provoked increases in membrane PKC
enzyme activity and stimulated the translocation of immunoreactive PKC- and PKC-
to membrane fractions in 3T3/L1 adipocytes and fibroblasts. It therefore appeared that increases in membrane PKC
enzyme activity, at least partly, reflected increases in PKC-
and
PKC-
. However, the enzyme assays of total PKC presently used may
also have reflected PKC-
, which is activated by phosphatidic acid,
polyphosphoinositides, phosphatidylserine, and certain fatty acids
(23-25). Accordingly, insulin is known to activate PI 3-kinase in
3T3/L1 cells (26). In addition, insulin effects on PKC activity were
evident in both cytosol and membrane fractions of 3T3/L1 adipocytes
largely depleted of PKC-
,
, and
by overnight 5 µM TPA pretreatment. The latter finding suggested that
insulin activated PKC-
, as well as PKC-
and
, and, indeed,
this was confirmed by finding that insulin provoked increases in enzyme activity of immunoprecipitable PKC-
.
Although we did not presently study the mechanism of PKC- activation
in 3T3/L1 cells, we have found,2 in rat adipocytes, that
PKC-
is rapidly phosphorylated during insulin action, and both
wortmannin and LY294002 inhibit insulin-induced activation of
immunoprecipitable PKC-
. It therefore seems likely that PI 3-kinase
activation is required for PKC-
activation, and we are currently
trying to identify the kinase responsible for PKC-
phosphorylation.
The failure to observe a significant change in the subcellular
distribution of PKC- in 3T3/L1 adipocytes during insulin treatment contrasts with observations in rat adipocytes (9). However, it should
be noted that: (a) PKC-
was more prevalent in membrane (relative to cytosol) fractions of 3T3/L1 cells; and (b)
insulin activates, but does not translocate, PKC-
in fetal chick
neurons, apparently through a covalent modification (27). Thus, the
failure to observe a translocation of PKC-
does not necessarily mean that this isoform is not activated by insulin in 3T3/L1 cells.
Glucose transport effects of insulin have been reported to be increased
by transfection-induced expression of PKC-2 in NIH3T3 cells that have low levels of endogenous insulin receptors (16). Presently, we were able to markedly increase PKC-
2
levels by stable transfection of 3T3/L1 cells that have relatively
little or no endogenous PKC-
2. We were also able to
overexpress both wild-type and constituitively active forms of PKC-
,
PKC-
1, and PKC-
2 in 3T3/L1 cells.
Nevertheless, the expression of each of these PKC isoforms had little
or no effect on basal or insulin-stimulated glucose transport. Our
findings therefore suggested that these PKC isoforms were not
rate-limiting in insulin action, and, moreover, even when
constituitively activated to a degree quantitatively comparable to, or
greater than, that observed in insulin-treated cells (cf.
Figs. 1 and 7), expressed PKC-
and PKC-
2 were not sufficient to either initiate or potentiate glucose transport responses
in 3T3/L1 cells. Of further note, we did not observe significant
retention of PKC-
,
1, or
, or induction of
PKC-
2, during prolonged TPA treatment in 3T3/L1
adipocytes, and the full retention of insulin effects on glucose
transport in 3T3/L1 cells (see Ref. 1, 3, and 5, and this study)
suggests that these DAG-sensitive isoforms are not required for the
glucose transport effect of insulin in these cells.
In contrast to the failure of expression of normal and constitutively
active forms of PKC- and PKC-
to influence glucose transport,
overexpression of these PKCs was effective in inhibiting insulin-induced increases in glycogen synthesis in 3T3/L1 adipocytes. This finding is in keeping with other findings that suggest that DAG-responsive PKCs inhibit glycogen synthesis (28-30). However, it
should be noted that the presently observed inhibition of glycogen synthesis apparently does not reflect a generalized inhibition of
insulin receptor function, as indicated by the apparent intactness of
glucose transport responses in these PKC-enriched transfectants. PKC
must therefore inhibit only certain insulin-sensitive signaling factors, or, alternatively, more distal regulatory factors, or glycogen
synthase itself (see Refs. 28-30). Along these lines, it should also
be noted that PKC-dependent inhibition of glycogen synthase
may occur as a paradoxical restraining mechanism during insulin action,
as we have found that the PKC inhibitor, RO 31-8220, increases insulin
effects on glycogen synthesis in rat adipocytes and rat skeletal
muscle.3
In contrast to PKC- and PKC-
, the overexpression of PKC-
in
fibroblasts provoked increases in basal, submaximal, and maximal insulin-stimulated glucose transport. Although not entirely certain, the increase in basal transport may reflect that some of the expressed PKC-
may have been activated, even in the absence of agonist addition. In adipocytes, although basal and submaximal insulin effects
were enhanced by PKC-
, the maximal insulin effect was on the
average, unchanged, perhaps reflecting a rate limitation caused by
factors other than PKC-
, e.g. GLUT4 levels. Along the latter lines, in both fibroblasts and adipocytes, it seemed clear that
observed alterations in glucose transport in PKC-
transfectants could not be explained by changes in total levels of GLUT1 or GLUT4.
Moreover, in adipocytes overexpressing wild-type PKC-
, the observed
changes in glucose transport, both basally and in response to insulin,
appeared to reflect changes in the subcellular distribution of both
GLUT4 and GLUT1; thus, PKC-
overexpression appeared to alter the
translocation of both GLUT4 and GLUT1.
In keeping with the possibility that PKC- may participate in the
regulation of glucose transport, a dominant-negative form of PKC-
inhibited basal and insulin-stimulated glucose transport in both
fibroblasts and adipocytes. Here again, the inhibition of glucose
transport could not be readily explained by changes in total GLUT1
and/or GLUT4 levels, and blunted responses of both transporters
appeared to contribute to decreases in glucose transport. In addition,
the intactness of glycogen synthesis responses in dominant-negative
PKC-
transfectants suggested that initial insulin signaling systems
were intact in these cells. Nevertheless, further studies will be
needed to determine whether the observed inhibitory effects on glucose
transport were directly due to the dominant-negative action of the
expressed mutant PKC-
.
In summary, insulin activated PKC-, as well as PKC-
and PKC-
,
in 3T3/L1 cells. Wherea, the stable expression of both wild-type and
constitutively active forms of PKC-
, PKC-
1 and
PKC-
2 failed to alter basal or insulin-stimulated
glucose transport, the stable overexpression of PKC-
stimulated, and
a dominant-negative form of PKC-
inhibited, basal and
insulin-stimulated glucose transport in 3T3/L1 cells. Our findings
therefore suggested that PKC-
may contribute to insulin-stimulated
glucose transport in 3T3/L1 cells. Further studies will be required to
test this hypothesis.