Activation of Protein Kinase C (alpha , beta , and zeta ) by Insulin in 3T3/L1 Cells
TRANSFECTION STUDIES SUGGEST A ROLE FOR PKC-zeta IN GLUCOSE TRANSPORT*

(Received for publication, February 15, 1996, and in revised form, September 25, 1996)

Gautam Bandyopadhyay Dagger , Mary L. Standaert Dagger , LiMing Zhao Dagger , Bingzhi Yu §, Antoine Avignon , Lamar Galloway Dagger , Purushotham Karnam Dagger , Jorge Moscat par and Robert V. Farese Dagger

From the Dagger  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 par  Centro de Biologia Molecular "Servero Ochoa," Universidad Autonoma, Canto Blanco, 28049 Madrid, Spain

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

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-alpha and PKC-beta . 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-alpha , PKC-beta , and PKC-epsilon , but not PKC-zeta . Moreover, insulin provoked increases in the enzyme activity of immunoprecipitable PKC-zeta . In transfection studies, stable overexpression of wild-type or constitutively active forms of PKC-alpha , PKC-beta 1, and PKC-beta 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-zeta increased, and a dominant-negative mutant form of PKC-zeta decreased, basal and insulin-stimulated glucose transport in fibroblasts and adipocytes. These findings suggested that: (a) insulin activates PKC-zeta , as well as PKC-alpha and beta ; and (b) PKC-zeta is required for, and may contribute to, insulin effects on glucose transport in 3T3/L1 cells.


INTRODUCTION

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-zeta , 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-alpha and PKC-beta 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-zeta 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-alpha , PKC-beta 1, and PKC-beta 2 failed to influence basal or insulin-stimulated glucose transport, despite inhibiting insulin effects on glycogen synthesis; and (b) expression of wild-type PKC-zeta enhanced, and dominant-negative PKC-zeta inhibited, basal and insulin-stimulated glucose transport.


EXPERIMENTAL PROCEDURES

General Procedures

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 Activity

In 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 beta -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-alpha -(19-31)-NH2 (Life Technologies, Inc.) or [Ser159]PKC-epsilon -(153-164)-NH2 (Upstate Biotechnology, Inc.) in 100 µl of buffer containing 50 mM Tris/HCl (pH 7.5), 50 µM [gamma -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.

Assay of PKC-zeta Enzyme Activity

We have recently found2 that insulin provokes rapid increases in enzyme activity of PKC-zeta in specific immunoprecipitates (i.e. devoid of PKC-alpha , beta , delta , and epsilon ) prepared from total cell lysates of rat adipocytes. In virtually identical experiments, in 3T3/L1 adipocytes, PKC-zeta was immunoprecipitated by incubating polyclonal anti-PKC-zeta 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 beta -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-epsilon -(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-zeta pseudosubstrate (Ki = 10-20 µM).

Western Analysis of PKC and Other Proteins

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-alpha , PKC-epsilon , PKC-delta , and PKC-zeta were obtained from Life Technologies. In most experiments, we assayed total PKC-beta (i.e. PKC-beta 1+2) with antisera raised against a peptide sequence (conjugated to albumin or keyhole limpet hemocyanin) present in the V3 region common to PKC-beta 1 and PKC-beta 2 (see Refs. 9 and 10). In some experiments, we also used beta 1-specific and beta 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-alpha , PKC-beta 1+2, PKC-beta 1, PKC-beta 2, and PKC-zeta 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%.

Assays of PKC mRNA Levels

Methods for measurement of PKC-beta (1+2), PKC-beta 1, and PKC-beta 2 by ribonuclease protection assay have been described previously (12).

Transfection of PKC into 3T3/L1 Cells

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-beta 2 (rat), in which the expression of the cDNA insert is regulated by a mouse metallothionine promoter (see Ref. 13), and pMV7-PKC-alpha (mouse), pMV7-PKC-epsilon (rat), pMV7-PKC-delta (rat), pMV7-PKC-beta 1 (rat), and pMV12-PKC-beta 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-zeta were prepared in Dr. Moscat's laboratory (see Ref. 17). To prepare pCDNA3-PKC-beta 2 (rat), intact rat PKC-beta 2 cDNA insert was excised from the vector pTB701-PKC-beta 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-alpha , and constitutively active rat PKC-epsilon (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-zeta 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, PCDSRalpha , containing cDNAs for wild-type or constituitively active (deleted in pseudosubstrate sites) forms of bovine PKC-alpha and PKC-beta 2 (obtained from Dr. M. Muramatsu, University of Tokyo, Tokyo, Japan; see Ref. 19); these constructs were designated by Dr. Muramatsu as SRalpha PKCalpha (wild-type PKC-alpha ), SRalpha PKAC (constituitively active PKC-alpha ), SRalpha PKCbeta (wild-type PKC-beta 2), and SRalpha PKCbeta -Delta EE (constituitively active PKC-beta 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-zeta -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-zeta 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.

Glucose Transport and Glycogen Synthesis Assays

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.


RESULTS

Total PKC Enzyme Activity, Method I

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).


Fig. 1. Summary of effects of insulin (100 nM × 10 min) and TPA (500 nM × 10 min) on PKC enzyme activity (as per Method I) and immunoreactive PKC-alpha and/or PKC-beta in 3T3/L1 adipocytes (left) and fibroblasts (right). Hatched bars reflect changes in membrane fractions. Open bars reflect changes in cytosol fractions. Treatments (INS or TPA) are shown at bottom. Bars and parentheses indicate mean ± S.E. of n determinations. Asterisks indicate p < 0.05 (t test).
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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-alpha -(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-epsilon -(153-164)-NH2 as the substrate in Method II assays). Method II was not used in fibroblasts.

Table I.

Effects of phorbol ester pretreatment on insulin-induced changes in PKC activity in cytosol and membrane fractions of 3T3/L1 adipocytes

PKC was assayed by Method II, using [Ser159]PKC-varepsilon -(153-164)-NH2 as substrate. Values are means ± S.E. of five or six determinations. p values (in parentheses) were determined by t test. NS, not significant.
Pretreatment 32PO4 incorporated
Cytosol
Membrane
Control Insulin (p vs. control) Control Insulin (p vs. control)

pmol/mg protein/min
None 123  ± 20 164  ± 12  (NS) 96  ± 2 148  ± 12 (p < 0.005)
5 µM TPA (24 h) 37  ± 4 55  ± 5  (p < 0.025) 32  ± 5 50  ± 5 (p < 0.05)

The incorporation of 32PO4 in the above assays presumably reflected the sum of enzyme activities of all PKC isoforms, including alpha , beta , epsilon , and zeta  (see below). In order to gain insight into the question of whether insulin activates atypical PKCs, such as PKC-zeta , we took advantage of the fact that overnight treatment with TPA largely depleted PKC-alpha , beta , and epsilon , but had no effect on 70-kDa PKC-zeta (Fig. 2). (Note: unlike the situation in BC3H-1 myocytes (16), neither TPA nor insulin treatment altered PKC-alpha , PKC-beta 1, PKC-beta 2 or PKC-epsilon mRNA levels in 3T3/L1 adipocytes, and induction or retention of PKC-beta 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-epsilon -(153-164)-NH2, a preferred substrate for both PKC-epsilon and PKC-zeta (PKC-alpha and PKC-beta 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-zeta , which is distributed between cytosol and membrane fractions and is not translocated during agonist treatment.


Fig. 2. Changes in distribution of immunoreactive PKC-alpha , beta , epsilon , and zeta  in cytosol and membrane fractions during acute (500 nM × 10 min) and prolonged (5 µM × 24 h) TPA treatment (+) of 3T3/L1 adipocytes. Minus signs (-) indicate controls.
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Immunoprecipitable PKC-zeta Enzyme Activity

In order to test the possibility that insulin activated PKC-zeta , we studied enzyme activity in specific PKC-zeta immunoprecipitates and found that enzyme activity of PKC-zeta 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-alpha , beta , or epsilon  in the PKC-zeta immunoprecipitates and preimmune serum did not immunoprecipitate PKC-zeta (Fig. 3). Insulin did not affect the amount of PKC-zeta recovered in these precipitates (approximately 50%), and it may be surmised that insulin increased the specific enzyme activity of PKC-zeta , apparently through a factor or covalent modification that was retained during immunoprecipitation and assay procedures.2


Fig. 3. Effects of insulin on enzyme activity of immunoprecipitable PKC-zeta in 3T3/L1 adipocytes. Adipocytes were treated for 10 min with 100 nM insulin, and PKC-zeta immunoprecipitates were prepared and assayed as described under "Experimental Procedures." Incorporation values shown at right are mean ± S.E. of four determinations; p < 0.01, t test, control versus insulin. As shown at left, PKC-zeta immunoblots had no appreciable immunoreactive PKC-zeta , beta , or epsilon , and preimmune (PI) serum did not immunoprecipitate significant PKC-zeta . Dots (bullet ) and apparent Mr designations indicate levels of brain (Br) PKC standards blotted simultaneously with adipocyte (Ad) PKC-zeta immunoprecipitates. IGG, immunoreactive gamma globulin bands.
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Acute Changes in Immunoreactive PKC

In 3T3/L1 adipocytes, insulin provoked time-dependent decreases in cytosolic, and increases in membrane, PKC-alpha , and PKC-beta (Figs. 1 and 4). The changes in immunoreactive PKC-alpha and PKC-beta 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-alpha and PKC-beta were approximately one-half of those observed in adipocytes (summarized in Fig. 1). In contrast to PKC-alpha and PKC-beta , insulin had no consistent effect on the cellular distribution of either PKC-epsilon or the 70-kDa form of PKC-zeta in adipocytes (Fig. 4) and fibroblasts (data not shown). Acute (10 min) TPA treatment, on the other hand, stimulated the translocation of PKC-epsilon , as well as PKC-alpha and PKC-beta , but not 70-kDa PKC-zeta in adipocytes (Fig. 2). It may be noted in Figs. 2 and 4 that, in addition to the 70-kDa form of PKC-zeta , the anti-PKC-zeta 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-alpha , as it (a) mirrored changes in authentic PKC-alpha (measured by PKC-alpha antiserum), and (b) increased upon transfection of cells with plasmids containing the PKC-alpha cDNA insert (data not shown). Of further note, this 80-kDa protein was not recovered in PKC-zeta immunoprecipitates, which only contained 70-kDa PKC-zeta (Fig. 3). It may also be noted in Figs. 2 and 4 that the cytosolic PKC-beta 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-beta 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-beta 1, and it is of interest that, in other cell types, a similarly migrating PKC-beta has been reported to be poorly activated, as it apparently lacks key prerequisite phosphorylations (see Ref. 21).


Fig. 4. Time-dependent changes in the distribution of immunoreactive PKC-alpha , beta , epsilon , and zeta  in cytosol and membrane fractions during insulin treatment (100 nM) of 3T3/L1 adipocytes. Dots indicate 80-kDa mass markers.
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Effects of Cellular Differentiation on PKC Isoforms

There were approximately 50% decreases in the cellular concentrations of PKC-alpha and PKC-beta following differentiation of fibroblasts into adipocytes; PKC-epsilon , on the other hand, increased slightly in adipocytes, and PKC-zeta did not change appreciably during differentiation (data not shown). Immunoreactive PKC-delta was not detectable in either fibroblasts or adipocytes. As shown in Fig. 5, when untransfected cells were blotted with beta 1- and beta 2-specific antisera, PKC-beta 1 (75- and 80-kDa moieties) was readily detectable in fibroblasts and adipocytes, whereas PKC-beta 2 was poorly detectable, if at all.


Fig. 5. Transfection (Tx)-induced increases in PKC-beta 1 and PKC-beta 2 in 3T3/L1 adipocytes and fibroblasts. A, PKC-beta 1 and PKC-beta 2 levels in cell extracts of untransfected cells (-) and stably transfected cells (+) are shown side-by-side in each box. The antibodies used to develop these immunoblots are shown at the left. Dots indicate 80-kDa mass markers. B, ribonuclease protection assays for PKC-beta mRNA, using 32P-labeled riboprobes, as indicated at the left, that protect mRNA fragments common to both beta 1 and beta 2, or specific for either beta 1 or beta 2, in 20 µg of total RNA isolated from untransfected cells (-) or PKC-beta 2-transfected (+) fibroblasts (left) and adipocytes (right). C, down-regulation of transfected PKC-beta 2 in adipocytes. Cells were untransfected (-) or transfected with pMV12-PKC-beta 2 (+) or pMV12 empty vector (V), as indicated in lower (Tx) row, and treated (+) or not treated (-) for 20 h with 5 µM TPA, as indicated in the upper (Rx) row.
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Stable Expression of PKC

Stable transfection of cells with PKC-beta 1 and, even more strikingly, PKC-beta 2, resulted in sizable overexpression of these isoforms in both fibroblasts and adipocytes (Fig. 5). Transfection-induced increases in immunoreactive PKC-beta 2 were comparable using a variety of vectors, i.e. pMTH, pMV7, pMV12, and pCDNA3. Transfection-induced expression of PKC-beta 2 yielded primarily an 80-kDa moiety, whereas expression of PKC-beta 1 yielded 75-kDa, as well as 80-kDa, moieties (Fig. 5). Stable transfection of cells with cDNAs encoding PKC-alpha and PKC-zeta 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-beta 2 transfection, perhaps reflecting higher basal levels of the PKC-alpha and PKC-zeta isoforms. In contrast to PKC-alpha , PKC-beta , and PKC-zeta , we were not able to express PKC-delta or significantly overexpress PKC-epsilon , as judged by immunoblot analysis (data not shown).


Fig. 6. Transfection-induced increases in PKC-alpha (A) and PKC-zeta (B) in 3T3/L1 fibroblasts and adipocytes. -, untransfected cells; +, cells transfected with pMV7-PKC-alpha or pCDNA3-PKC-zeta .
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Table II.

Relative changes in immunoreactive PKC-zeta , GLUT1, and GLUT4 levels in PKC-zeta transfectants

Clones from Fig. 10 were examined for immunoreactive 70-kDa PKC-zeta , GLUT1, and GLUT4, and levels were quantitated with a Bio-Rad Molecular Imaging System.
Transfectant Fibroblast (mean ± S.E.)
Adipocyte (mean ± S.E.)
PKC-zeta GLUT1 PKC-zeta GLUT1 GLUT4

% control % control
Wild-type PKC-zeta 169  ± 10 107  ± 5 213  ± 11 98  ± 8 93  ± 8
Dominant-negative PKC-zeta 182  ± 23 147  ± 8 193  ± 40 107  ± 7 126  ± 15

In the case of PKC-beta 2, we verified that transfection resulted in specific increases in PKC-beta 2 mRNA (Fig. 5). We also verified that transfected PKC-beta 2 was down-regulated by overnight 5 µM TPA treatment (Fig. 5), and it may therefore be surmised that transfected PKC-beta 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-alpha and PKC-beta 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-alpha (i.e. the upper band in PKC-zeta blots whose identity as PKC-alpha was confirmed with anti-PKC-alpha antiserum) in PKC-zeta transfectants, regardless of whether they were wild-type or dominant-negative mutants. As will become apparent (see below), this co-expression of PKC-alpha could not explain observed changes in glucose transport experiments. In contrast to PKC-alpha , we did not observe changes in PKC-beta or PKC-epsilon levels in PKC-zeta transfectants.


Fig. 7. Effects of transfected PKC-alpha and PKC-beta on cytosolic (C) and membrane (M) PKC activity and insulin-stimulated glycogen synthesis in 3T3/L1 adipocytes. Cells were co-transfected with vectors alone (pCDSRalpha and pCDNA3), or vectors containing (in pCDSRalpha ) cDNAs encoding wild-type (WT) PKC-alpha (SRalpha PKCalpha ), constituitively active (CONST) PKC-alpha (SRalpha PKCAC), wild-type (WT) PKC-beta 2 (SRalpha PKCbeta ), or constituitively active (CONST) PKC-beta 2 (SRalpha PKCbeta Delta EE). CONTROL, untransfected cells; VECTORS, cells transfected with vectors not containing PKC inserts. PKC activity in unstimulated cells (upper panels) was assayed by Method II using [Ser25]PKC-alpha -(19-31)-NH2 as substrate. Insulin (100 nM) was used to stimulate glycogen synthesis ([14C]glucose conversion to glycogen), as indicated (+, stippled bars) at bottom of the lower panels (note low control (-) values). Values are mean ± S.E. of 4 clones, each assayed simultaneously in quadruplicate.
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Effects of PKC Expression on Glucose Transport

As shown in Fig. 8, there was little or no effect of overexpression of wild-type PKC-alpha , PKC-beta 1, or PKC-beta 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-alpha and PKC-beta 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-alpha (those obtained from Dr. Peter Parker) also failed to alter glucose transport responses (data not shown).


Fig. 8. Dose-dependent effects of insulin on [3H]2-deoxyglucose uptake in 3T3/L1 fibroblasts (left panels) and adipocytes (right panels) transfected with plasmids containing cDNAs encoding PKC-alpha (in PMV7) or PKC-zeta (in pCDNA3) (upper panels) and PKC-beta 1 or PKC-beta 2 (in pMV7, pMV12, or pMTH) (lower panels). Untransfected cells are indicated by CON and open circle . Results with cells transfected with empty vectors were not significantly different from control, and, for simplicity, are not shown. Values are mean ± S.E. of n clones, each assayed in quadruplicate at each insulin concentration. Note the differences in ordinate values in fibroblast and adipocyte experiments.
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Fig. 9. Effects of insulin on [3H]2-deoxyglucose uptake in 3T3/L1 fibroblasts (upper panel) and adipocytes (lower panel) transfected with plasmids containing cDNAs encoding wild-type or constituitively active PKC-alpha or PKC-beta . Cells were co-transfected with empty vectors (pCDSRalpha and pCNDA3), or vectors containing (in pCDSRalpha ) cDNAs encoding wild-type (WT) PKC-alpha (SRalpha PKCalpha ), constituitively active (CONSTIT) PKC-alpha (SRalpha PKCAC), wild-type (WT) PKC-beta 2 (SRalpha PKCbeta ), or constituitively active (CONSTIT) PKC-beta 2 (SRalpha PKCbeta Delta EE). CON, untransfected cells; VEC, cells transfected with vectors alone. Insulin concentrations (0, 5, and 100 nM) are indicated at bottom of panels. Values are mean ± S.E. of 4 fibroblast clones and 6-9 adipocyte clones, each assayed in quadruplicate at each insulin concentration. See Fig. 7 for PKC activities of adipocytes in these groups.
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In contrast to PKC-alpha and PKC-beta , the overexpression of wild-type PKC-zeta increased basal and insulin-stimulated glucose transport in fibroblasts (Figs. 8 and 10). In adipocytes (Fig. 10), overexpression of PKC-zeta 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-zeta inhibited basal and insulin-stimulated glucose transport in both fibroblasts and adipocytes (Fig. 10). As shown in Table II, immunoreactive PKC-zeta levels were increased approximately 2-fold in wild-type and dominant negative PKC-zeta transfectants, and observed changes in glucose transport in PKC-zeta 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-zeta ; this further suggested that TPA-resistant PKC enzyme activity largely reflected PKC-zeta activity, as it would be expected to be increased in wild-type overexpressers, but not with expression of catalytically inactive PKC-zeta . Further, since immunoreactive PKC-zeta 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-zeta 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-zeta 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-zeta overexpressers were equal to, if not greater than, the levels seen in controls. In contrast, in PKC-zeta dominant-negative transfectants, insulin effects on GLUT4 and GLUT1 appeared to be blunted (Fig. 12).


Fig. 10. Effects of insulin on [3H]2-deoxyglucose uptake in 3T3/L1 fibroblasts (upper panel) and adipocytes (lower panel) transfected with plasmids containing cDNAs encoding wild-type and dominant-negative forms of PKC-zeta . Cells were not transfected (CONTROL) or transfected with empty vector (pCDNA3) or vector containing cDNAs encoding wild-type (WT) or dominant-negative (DOM-NEG) PKC-zeta . Insulin concentrations (0, 5, and 100 nM) are indicated at bottom of panels. Values are mean ± S.E. of n clones, each assayed in quadruplicate at each insulin concentration. Asterisks indicate p < 0.05 (t test comparison to untransfected controls). See Table I for levels of immunoreactive PKC-zeta , GLUT1, and GLUT4 in these clones.
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Fig. 11. Effects of expression of wild-type (WT) and dominant-negative (DN) PKC-zeta on PKC enzyme activity (A) and levels of PKC-zeta immunoreactivity (B) in 3T3/L1 adipocytes. Cells were treated for 24 h with 5 µM TPA to down-regulate PKC-alpha , beta , delta , and epsilon , so that enzyme activity of membrane (MEM) and cytosol (CYT) fractions would primarily reflect PKC-zeta . PKC enzyme activity was then measured using Method II as described under "Experimental Procedures." Bars and parentheses indicate mean ± S.E. of n determinations.
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Fig. 12. Effects of transfection (TX)-induced overexpression of wild-type (WT) PKC-zeta (left, A) and expression of dominant-negative (DOM NEG) PKC-zeta (right, B) on the subcellular distribution of GLUT4 and GLUT1 glucose transporters between microsomes (MS) and plasma membranes (PM) in untreated (-) and insulin-treated (+) (100 nM × 20 min) 3T3/L1 adipocytes. 50 µg of PM and MS protein were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and blotted for GLUT4 and GLUT1. PKC-zeta WT overexpressers at left were directly compared to untransfected controls on the same blots. Similar changes were observed in three PKC-zeta WT overexpresser clones and two dominant-negative PKC-zeta clones.
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Effects of PKC-alpha and PKC-beta Expression on Glycogen Synthesis

In contrast to glucose transport, the expression of both wild-type and constituitively active forms of PKC-alpha and PKC-beta 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-alpha . Along these lines, it was of interest to find that overexpression of wild-type PKC-zeta , and the expression of dominant-negative PKC-zeta failed to alter insulin effects on glycogen synthesis (data not shown); this suggested that, in these PKC-zeta transfections, initial insulin signaling was intact, and inhibitory effects of PKC on glycogen synthesis were isoform-dependent.


DISCUSSION

We presently found that insulin provoked increases in membrane PKC enzyme activity and stimulated the translocation of immunoreactive PKC-alpha and PKC-beta 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-alpha and PKC-beta . However, the enzyme assays of total PKC presently used may also have reflected PKC-zeta , 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-alpha , beta , and epsilon  by overnight 5 µM TPA pretreatment. The latter finding suggested that insulin activated PKC-zeta , as well as PKC-alpha and beta , and, indeed, this was confirmed by finding that insulin provoked increases in enzyme activity of immunoprecipitable PKC-zeta .

Although we did not presently study the mechanism of PKC-zeta activation in 3T3/L1 cells, we have found,2 in rat adipocytes, that PKC-zeta is rapidly phosphorylated during insulin action, and both wortmannin and LY294002 inhibit insulin-induced activation of immunoprecipitable PKC-zeta . It therefore seems likely that PI 3-kinase activation is required for PKC-zeta activation, and we are currently trying to identify the kinase responsible for PKC-zeta phosphorylation.

The failure to observe a significant change in the subcellular distribution of PKC-epsilon in 3T3/L1 adipocytes during insulin treatment contrasts with observations in rat adipocytes (9). However, it should be noted that: (a) PKC-epsilon was more prevalent in membrane (relative to cytosol) fractions of 3T3/L1 cells; and (b) insulin activates, but does not translocate, PKC-epsilon in fetal chick neurons, apparently through a covalent modification (27). Thus, the failure to observe a translocation of PKC-epsilon 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-beta 2 in NIH3T3 cells that have low levels of endogenous insulin receptors (16). Presently, we were able to markedly increase PKC-beta 2 levels by stable transfection of 3T3/L1 cells that have relatively little or no endogenous PKC-beta 2. We were also able to overexpress both wild-type and constituitively active forms of PKC-alpha , PKC-beta 1, and PKC-beta 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-alpha and PKC-beta 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-alpha , beta 1, or epsilon , or induction of PKC-beta 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-alpha and PKC-beta 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-alpha and PKC-beta , the overexpression of PKC-zeta 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-zeta may have been activated, even in the absence of agonist addition. In adipocytes, although basal and submaximal insulin effects were enhanced by PKC-zeta , the maximal insulin effect was on the average, unchanged, perhaps reflecting a rate limitation caused by factors other than PKC-zeta , e.g. GLUT4 levels. Along the latter lines, in both fibroblasts and adipocytes, it seemed clear that observed alterations in glucose transport in PKC-zeta transfectants could not be explained by changes in total levels of GLUT1 or GLUT4. Moreover, in adipocytes overexpressing wild-type PKC-zeta , 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-zeta overexpression appeared to alter the translocation of both GLUT4 and GLUT1.

In keeping with the possibility that PKC-zeta may participate in the regulation of glucose transport, a dominant-negative form of PKC-zeta 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-zeta 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-zeta .

In summary, insulin activated PKC-zeta , as well as PKC-alpha and PKC-beta , in 3T3/L1 cells. Wherea, the stable expression of both wild-type and constitutively active forms of PKC-alpha , PKC-beta 1 and PKC-beta 2 failed to alter basal or insulin-stimulated glucose transport, the stable overexpression of PKC-zeta stimulated, and a dominant-negative form of PKC-zeta inhibited, basal and insulin-stimulated glucose transport in 3T3/L1 cells. Our findings therefore suggested that PKC-zeta may contribute to insulin-stimulated glucose transport in 3T3/L1 cells. Further studies will be required to test this hypothesis.


FOOTNOTES

*   This work was supported by funds from the Department of Veterans Affairs Merit Review Program and National Institutes of Health Grant DK38079. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1    The abbreviations used are: PKC, protein kinase C; DAG, diacylglycerol; cPKC, conventional PKC; nPKC, novel PKC; aPKC, atypical PKC; PI, phosphatidylinositol; TPA, 12-O-tetradecanoylphorbol-13-acetate; PMSF, phenylmethylsulfonyl fluoride; PS, phosphatidylserine; PAGE, polyacrylamide gel electrophoresis.
2    Insulin effects on PKC-zeta activation were found to be inhibited by both wortmannin and LY294002, suggesting dependence upon PI 3-kinase activation (M. L. Standaert, L. Galloway, P. Karnam, G. Bandyopadhyay, and R. V. Farese, submitted for publication).
3    G. Bandyopadhyay, M. L. Standaert, L. Zhao, B. Yu, A. Avignon, L. Galloway, P. Karnam, J. Moscat, and R. V. Farese, unpublished observations.

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