Knockout of PKC{alpha} Enhances Insulin Signaling Through PI3K

Michael Letiges, Markus Plomann, Mary L. Standaert, Gautam Bandyopadhyay, Mini P. Sajan, Yoshinori Kanoh and Robert V. Farese

Max-Planck Institute for Experimental Endocrinology (M.L.), 30625 Hannover, Germany; Institute for Biochemistry II (M.P.), Medical Faculty, University of Cologne, D-50931 Cologne, Germany; and James A. Haley Hospital (M.L.S., G.B., M.P.S., Y.K., R.V.F.), University of South Florida College of Medicine, Tampa, Florida 33612

Address all correspondence and requests for reprints to: Dr. Robert Farese, J. A. Haley Veterans Hospital, University of South Florida College of Medicine, 13000 Bruce B. Downs Boulevard, Tampa, Florida 33612. E-mail: rfarese{at}com1 med.usf.edu.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Insulin stimulates glucose transport and certain other metabolic processes by activating atypical PKC isoforms ({lambda}, {zeta}, {iota}) and protein kinase B (PKB) through increases in D3-polyphosphoinositides derived from the action of PI3K. The role of diacylglycerol-sensitive PKC isoforms is less clear as they have been suggested to be both activated by insulin and yet inhibit insulin signaling to PI3K. Presently, we found that insulin signaling to insulin receptor substrate 1-dependent PI3K, PKB, and PKC{lambda}, and downstream processes, glucose transport and activation of ERK, were enhanced in skeletal muscles and adipocytes of mice in which the ubiquitous conventional diacylglycerol-sensitive PKC isoform, PKC{alpha}, was knocked out by homologous recombination. On the other hand, insulin provoked wortmannin-insensitive increases in immunoprecipitable PKC{alpha} activity in adipocytes and skeletal muscles of wild-type mice and rats. We conclude that 1) PKC{alpha} is not required for insulin-stimulated glucose transport, and 2) PKC{alpha} is activated by insulin at least partly independently of PI3K, and largely serves as a physiological feedback inhibitor of insulin signaling to the insulin receptor substrate 1/PI3K/PKB/PKC{lambda}/{zeta}/{iota} complex and dependent metabolic processes.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PI3K AND ITS DOWNSTREAM effectors, atypical PKCs, {zeta} and {lambda} (1, 2, 3, 4, 5, 6), and protein kinase B (PKB) (7, 8, 9, 10), are thought to play important roles in insulin-stimulated glucose transport. In contrast, based on findings in studies with PKC inhibitors and phorbol ester-induced PKC down-regulation, diacylglycerol (DAG)-activated PKCs, including conventional PKCs, {alpha}, ß1, ß2, and {gamma}, and novel PKCs, {delta}, {epsilon}, {eta}, and {theta}, do not appear to be required for insulin-stimulated glucose transport in a number of cell types, including rat adipocytes (2, 4), rat skeletal muscles (5, 11), 3T3/L1 adipocytes (1, 5), L6 myotubes (5, 12), and BCH3–1 myocytes (5, 13). Moreover, based upon the activation of conventional and novel DAG-dependent PKCs by phorbol esters in intact cells, or by direct effects of PKC in vitro, one or more of these PKCs phosphorylate specific serine/threonine residues of the insulin receptor (14, 15, 16, 17) and insulin receptor substrate-1 (IRS-1) (18), and thereby impair insulin signaling to PI3K, which, in operating downstream of IRS-1, plays an important role in mediating insulin effects on glucose transport in adipocytes and skeletal muscles, as revealed in studies of knockout of the mouse IRS-1 gene (19, 20).

Other than for a limited amount of information obtained from PKC overexpression studies in cultured cells, there is relatively little information on the effects of specific DAG-dependent PKCs on insulin signaling to IRS-1, PI3K, PKC{zeta}/{lambda}, PKB, and the glucose transport system. In this regard, overexpression of PKC{alpha} (18, 21) and PKCß1 and PKCß2 (22) have been shown to inhibit insulin-induced activation of the insulin receptor and/or IRS-1-dependent activation of PI3K. However, it is uncertain whether the inhibitory effects of these overexpressed PKCs are attended by alterations in the activation of PKC{zeta}/{lambda}, PKB, glucose transport, and/or other distal targets, such as ERK, which apparently functions downstream of IRS-1 in mouse skeletal muscles (20) and downstream of PI3K and PKC{zeta}/{lambda} in rat adipocytes (23). It is also uncertain whether data derived from PKC overexpression studies truly reflect conditions that are comparable to those existing in cells containing physiological levels of these PKCs.

As another approach to evaluate the requirement for, or effects of, physiological levels of specific DAG-sensitive PKCs during insulin signaling to the glucose transport system, we have used gene-targeting methods and have reported that knockout of the mouse PKCß gene is attended by increases in insulin-stimulated glucose transport in isolated adipocytes, and increases in basal, as well as insulin-stimulated, glucose transport in isolated soleus muscles (24). These findings provided clear evidence that neither PKCß1 nor PKCß2 are required for insulin-stimulated glucose transport in mouse adipocytes and soleus muscles. Moreover, it was found that expression of wild-type PKCß1 inhibits the glucose transporter 4 (GLUT 4)/glucose transport system in mouse (24) and rat (4) adipocytes. However, these inhibitory effects of PKCß on the glucose transport system did not appear to be due to alterations in IRS-1-dependent PI3K activation (24).

Recently, we studied insulin-induced activation of IRS-1-dependent PI3K, PKB, PKC{lambda}, glucose transport, and ERK in adipocytes and skeletal muscles of mice in which the PKC{alpha} gene was knocked out by homologous recombination. Our findings suggest that PKC{alpha}, even at physiological levels, serves as a tonic endogenous inhibitor of IRS-1-dependent PI3K, PKB, and PKC{lambda} during insulin stimulation of glucose transport and ERK in mouse skeletal muscles and adipocytes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Targeted Disruption of the PKC{alpha} Gene
To elucidate the role of PKC{alpha} in insulin-stimulated glucose transport, we generated a targeted mutation in mouse by homologous recombination. For this, we constructed a targeting vector harboring a neomycin cassette (neo) insertion within the second exon of the PKC{alpha} gene (see below), thereby disrupting the transcription of the gene after homologous recombination. The targeting vector was electroporated into embryonic stem (ES) cells, and G418-resistent colonies were analyzed by Southern blot analysis for homologous recombination at the PKC{alpha} locus (Fig. 1AGo). Of 192 colonies screened, 30 showed the correct restriction fragment length polymorphism, resulting in a targeting frequency of 1 in 6. Further Southern blot analysis of six positive ES cell clones using different probes and restriction enzymes revealed a single and correct integration of the targeting vector (data not shown). Two of these were used for the generation of mutant mice through microinjection of PKC{alpha} +/- ES cells into NMRI albino mouse blastocysts to generate chimeras. Both ES cell lines resulted in germ line-transmitting male chimeras that subsequently were crossed to 129/SV females and gave rise to F1 heterozygous offspring on a pure 129 background. Intercrosses of such were used to establish a homozygote PKC{alpha}-deficient mouse line (Fig. 1BGo). To verify that the insertion of the targeting vector into the PKC{alpha} locus led to a null mutation, we performed RT-PCR analysis from several tissues using different primer pairs. In each case, PKC{alpha}-specific transcripts were undetectable (Fig. 1CGo and data not shown), indicating that the targeted mutations led to a null allele in the mouse. Despite the fact that PKC{alpha} is presumed to be the most ubiquitously expressed PKC isoform (among nine), the PKC{alpha}-deficient mouse appeared to be normal with regard to external characteristics, viability, and fertility.



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Figure 1. Targeted Mutation of the PKC{alpha} Locus in the Mouse

A, Restriction map of the PKC{alpha} locus (wild type, wt). The targeting vector was integrated into the endogenous locus by homologous recombination and gave rise to the mutant (mt) PKC{alpha} locus. B, BamHI; E, EcoRI; H, HindIII; P, PstI; X, XhoI; neo, neomycin selection cassette. B, Southern blot analysis of a litter derived from a heterozygote intercross. Genomic BamHI fragments were detected with a 5'-probe (probe A) indicating wt and mt alleles. The homozygote is represented in lane 4. Heterozygotes are represented in lanes 2, 3, 5, and 7. Lanes 1 and 6 are representative of wt mice. C, RT-PCR analysis of brain extracts generated from brain tissue of wt and mt mice.

 
Studies of Glucose Transport
Insulin-stimulated 2-deoxyglucose uptake was enhanced by 67% in isolated adipocytes, and by 108% in soleus muscles of PKC{alpha}-/- knockout mice, as compared with uptake in adipocytes and soleus muscles of corresponding littermate control PKC{alpha}+/+ mice (Fig. 2Go). In adipocytes of PKC{alpha}-/- knockout mice, the maximal effect of insulin was increased and the insulin dose-response curve was shifted to the left. Basal uptake of 2-deoxyglucose in adipocytes and soleus muscles, on the other hand, was comparable in PKC{alpha}-/- knockout and control PKC{alpha}+/+ mice.



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Figure 2. Effects of Knockout (KO) of PKC{alpha} on Insulin-Stimulated Glucose Transport in Adipocytes (A) and Soleus Muscles (B)

In panel A, adipocytes were prepared simultaneously from PKC{alpha}-/- (PKC{alpha} KO), and PKC{alpha}+/+ (wild-type) mice and incubated in triplicate for 30 min with indicated concentrations of insulin in four separate experiments. Uptake of [3H]2-deoxyglucose over 1 min was measured as described in Materials and Methods. In panel B, soleus muscles were prepared simultaneously from 14 PKC{alpha}+/+ and 13 PKC{alpha}-/- mice and incubated for 30 min with or without 100 nM insulin in three separate experiments. Uptake of [3H]2-deoxyglucose over 5 min was measured as described in Materials and Methods. Values are mean ± SE of (n) determinations. P was determined by t test comparison of peak insulin-stimulated mean values of PKC{alpha} knockouts vs. wild-type controls.

 
Studies of Activation of PI3K, PKB, PKC{lambda}, and ERK
Insulin-induced increases in the enzyme activities of IRS-1-dependent PI3K, PKB, PKC{lambda}, and ERK were increased in vastus lateralis muscles of PKC{alpha}-/- knockout mice, as compared with activities observed in muscles of corresponding littermate wild-type control PKC{alpha}+/+ mice (Fig. 3Go). Basal activities of PI3K, PKB, PKC{lambda}, and ERK, on the other hand, were virtually the same in vastus lateralis muscles of knockout and control mice.



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Figure 3. Effects of Knockout of PKC{alpha} on Insulin-Induced Activation of IRS-1-Dependent PI3K (A), PKB (B), PKC{lambda} (C), and ERK (D) in Vastus Lateralis Muscles

Wild-type PKC{alpha}+/+ control (CON) mice and PKC{alpha}-/- knockout (KO) mice were injected ip with vehicle or vehicle containing 0.001 U insulin per gram of body weight 15 min before killing. Muscles were harvested, homogenized, and subjected to immunoprecipitation and assays for IRS-1-dependent PI3K, PKC{lambda}, PKB, and ERK as described in Materials and Methods. Values are mean ± SE of (n) determinations. Inset shows a representative autoradiogram for labeling of phosphatidylinositol-3-PO4 in the PI3K assay.

 
Insulin-induced increases in the enzyme activity of IRS-1-dependent PI3K, PKB, and PKC{lambda} were also increased in adipocytes of PKC{alpha}-/- knockout mice (Fig. 4Go).



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Figure 4. Effects of Knockout of PKC{alpha} on Insulin-Induced Activation of IRS-1-Dependent PI3K (A), PKC{lambda} (C), and PKB (D) in Adipocytes

Adipocytes were simultaneously prepared from wild-type PKC{alpha}+/+ control mice and PKC{alpha}-/- knockout (KO) mice, and incubated for 10 min with or without 10 nM insulin, after which IRS-1, PKC{lambda}, and PKB were immunoprecipitated and assayed. Values are mean ± SE of (n) determinations. Shown in panel B are representative autoradiograms for alterations in labeling of phosphatidylinositol-3-PO4 in the PI3K assay and immunoblots for alterations in phosphoserine-473 in PKB (note correlation to changes in immunoprecipitable PKB enzyme activity).

 
Immunoblot Studies
Levels of immunoreactive IRS-1, IRS-2, p85{alpha}/PI3K subunit, phosphoinositide-dependent protein kinase 1 (PDK-1), PKB, PKC{lambda}, GLUT 1, and GLUT 4 in vastus lateralis muscles were essentially the same in PKC{alpha}-/- knockout mice and corresponding littermate control PKC{alpha}+/+ mice (Fig. 5Go). Slight differences in mobility of IRS-1 on SDS-PAGE were not consistently observed, but it is possible that the slightly slower migration of IRS-1 seen in Fig. 5Go may have been due to an increase in PKC{alpha}-dependent ser/thr phosphorylation. Despite increases in PKB enzyme activity, levels of immunoreactive phosphoserine-473-PKB were not significantly different in vastus lateralis muscles of insulin-stimulated PKC{alpha}-/- knockout and control PKC{alpha}+/+ mice (Fig. 5Go). As expected, PKC{alpha} was absent in muscles of PKC{alpha}-/- knockout mice (Fig. 5Go). Levels of GLUT1 and GLUT4 were the same in adipocytes of control PKC{alpha}+/+ mice and PKC{alpha}-/- knockout mice (data not shown).



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Figure 5. Levels of Immunoreactive IRS-1, IRS-2, PKC{alpha}, p85{alpha} Subunit of PI3K, PDK-1, PKB, Phosphoserine-473-PKB, PKC{lambda}, GLUT 1, and GLUT 4 in Vastus Lateralis Muscles of Wild-Type (WT) PKC{alpha}+/+ Mice and PKC{alpha}-/- Knockout (KO) Mice

Mice were treated with or without insulin as in Fig. 2Go. Shown here are blots representative of four or more determinations. Other than the absence of PKC{alpha}, there were no significant changes in levels of these substances in muscles of PKC{alpha} knockout mice.

 
Transfection Studies
The above studies suggested that endogenous PKC{alpha} in control PKC{alpha}+/+ mice inhibits insulin-stimulated glucose transport in skeletal muscles and adipocytes. In support of this suggestion, transient transfection of wild-type PKC{alpha} into adipocytes of PKC{alpha} knockout mice was attended by decreases in both basal and insulin-stimulated hemagglutin (HA)-GLUT 4 translocation to the plasma membrane (a convenient surrogate for glucose transport in transient transfection studies; see Materials and Methods) (Fig. 6Go). It should be noted that, after electroporation and overnight culturing, primary adipocytes are partially activated artefactually and show increased basal transport activity and decreased relative (i.e. to freshly incubated cells) effects of insulin on both glucose transport and GLUT 4 translocation (4). Maximal levels of insulin-stimulated transport/GLUT 4 translocation are, however, unaltered (4). The reason for this artefactual increase in basal translocation/transport activity is uncertain. Nevertheless, as seen in Fig. 6Go, like insulin-stimulated HA-GLUT 4 translocation activity, this basal translocation activity was inhibited by expression of PKC{alpha}.



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Figure 6. Effects of Expression of PKC{alpha} on Basal and Insulin-Stimulated Translocation of HA-GLUT 4 to the Plasma Membrane in Adipocytes of PKC{alpha}-/- Knockout (KO) Mice

Adipocytes were cotransfected with pCIS2 encoding HA-GLUT 4 along with pCDNA3 alone (Vector) or pCDNA3 encoding wild-type (WT) PKC{alpha} as described in Materials and Methods. After overnight incubation, adipocytes were incubated for 30 min in glucose-free KRP medium with or without 10 nM insulin, after which cell surface level of HA-GLUT 4 was measured as described in Materials and Methods. Values are mean ± SE of (n) determinations.

 
Serum Glucose and Insulin Levels
There were no significant differences in serum glucose and insulin levels in fed PKC{alpha}-/- knockout mice, as compared with fed control PKC{alpha}+/+mice: serum glucose in control PKC{alpha}+/+ mice, 187 ± 4 mg/dl (mean ± SE, n = 19); serum glucose in PKC{alpha}-/- knockout mice, 186 ± 3 mg/dl (mean ± SE, n = 19); serum insulin in control PKC{alpha}+/+ mice, 0.25 ± 0.03 ng/ml (mean ± SE, n = 15); and serum insulin in PKC{alpha}-/- knockout mice, 0.31 ± 0.05 ng/ml (mean ± SE, n = 15).

Effects of Insulin on Activity of PKC{alpha} and PKCß2 in Adipocytes and Skeletal Muscles
Because of the apparent inhibitory effects of PKC{alpha} (Refs. 18 and 21 and present findings) and PKCß (4, 22, 24) on insulin action, we used adipocytes isolated from wild-type mice and rats to determine whether these conventional PKCs are in fact activated by insulin, and, if so, by a mechanism comparable to, or different from, that used by insulin to activate atypical PKCs. As seen in Figs. 7Go and 8Go, insulin provoked increases in the activity of immunoprecipitable PKC{alpha} and PKCß2 in adipocytes of wild-type mice and rats. However, unlike PKC{zeta}/{lambda} activation (see Fig. 8Go), these increases in PKC{alpha} and PKCß2 activity were not inhibited by the PI3K inhibitor, wortmannin. It therefore appears that insulin activates atypical PKCs primarily via PI3K, and conventional PKCs largely independently of PI3K, in adipocytes of wild-type mice and rats.



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Figure 7. Effects of Insulin on Activity of PKC{alpha} (Panels A and B) and PKCß2 (Panels C and D) in Vastus Lateralis Muscles (Panels A and C) and Adipocytes (Panels B and D) of Wild-Type and PKC{alpha} Knockout Mice

In studies of the vastus lateralis muscles, mice were treated in vivo with 0.001 U insulin per gram of body weight given ip 15 min before animals were killed and muscles harvested. In studies of adipocytes in vitro, adipocytes were isolated and incubated in glucose-free KRP medium with or without 100 nM wortmannin for 15 min, and then treated with or without 10 nM insulin for 10 min. After experiments, cell lysates were prepared and PKC{alpha} and PKCß2 were immunoprecipitated and assayed as described in Materials and Methods. Values are mean ± SE of (n) determinations.

 


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Figure 8. Effects of Insulin on Activity of PKC{alpha} (Panels A and B) and PKCß2 (Panels C and D) and PKC{zeta}/{lambda} (Panels E and F) in Vastus Lateralis Muscles (Panels A, C, and E) and Adipocytes (Panels B, D, and F) in Wild-Type Rats

In studies of the vastus lateralis muscles, rats were treated in vivo with 0.001U insulin per g body wt given ip 15 min before animals were killed and muscles harvested. In studies of adipocytes in vitro, adipocytes were isolated and incubated in glucose-free KRP medium with or without 100 nM wortmannin for 15 min, and then treated with or without 10 nM insulin for 10 min. After experiments, cell lysates were prepared and PKC{alpha} and PKCß2 were immunoprecipitated and assayed as described in Materials and Methods. Values are mean ± SE of (n) determinations

 
As in adipocytes, insulin provoked increases in immunoprecipitable PKC{alpha} and PKCß2 activity in vastus lateralis skeletal muscles of wild-type mice and rats (Figs. 7Go and 8Go).

To be certain that insulin effects seen in immunoprecipitates prepared with anti-PKC{alpha} antiserum were truly reflective of PKC{alpha}, we simultaneously examined radioactivity in immunoprecipitates prepared from vastus lateralis muscles and adipocytes of wild-type and PKC{alpha} knockout mice. As seen in Fig. 7Go, relatively small amounts of radioactivity (i.e. counts per minute above that seen in the preimmune blanks) were recovered in these assays of PKC{alpha} immunoprecipitates prepared from vastus lateralis muscles and adipocytes of PKC{alpha} knockout mice. This nonspecific radioactivity amounted to approximately 10–20% of the radioactivity recovered in comparable immunoprecipitates prepared simultaneously from lysates of insulin-treated muscles and adipocytes of wild-type mice. Most importantly, unlike findings in PKC{alpha} assays of precipitates prepared from tissues of wild-type mice, there were no effects of insulin on this nonspecific radioactivity recovered in precipitates prepared from tissues of PKC{alpha} knockout mice. Although the source of this nonspecific radioactivity is uncertain (i.e. we did not detect other PKCs in PKC{alpha} immunoprecipitates), its presence, if anything, would have diminished, and caused an underestimation of, the relative effects of insulin on PKC{alpha} activity observed in immunoprecipitates prepared from wild-type mice. Accordingly, it could be argued that this nonspecific radioactivity observed in assays of PKC{alpha} knockout mice would have been a more appropriate blank (than routinely used preimmune serum immunoprecipitates) for assays of PKC{alpha} in wild-type tissues.

Although not shown here, we did not recover any nonspecific radioactivity at all in PKCß2 immunoprecipitates prepared from lysates of tissues of PKCß knockout mice.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
From our findings in PKC{alpha} knockout mice, it seems clear that, despite being activated by insulin, the conventional DAG/Ca2+-sensitive PKC, PKC{alpha}, is not required for insulin-stimulated glucose transport in adipocytes and skeletal muscle preparations of the mouse. This finding is similar to that observed in studies of knockout of the DAG/Ca2+-sensitive PKCß gene, viz., that PKCß is not required for insulin-stimulated glucose transport in these mouse tissues (24). Accordingly, it may be surmised from gene knockout studies, that both DAG/Ca2+-dependent conventional PKCs, {alpha} and ß, are not required for insulin-stimulated glucose transport in mouse adipocytes and skeletal muscles. This conclusion is in concert with findings in other studies of rat adipocytes (2), rat soleus muscles (5), mouse-derived 3T3/L1 adipocytes (1), rat-derived L6 myotubes (5, 12), and rat soleus muscles (5, 11) in which it was concluded, from use of relatively isoform-specific PKC inhibitors and phorbol ester-induced PKC down-regulation, that PKC{alpha} and PKCß are not required for insulin-stimulated glucose transport. On the other hand, this lack of dependence of insulin-stimulated glucose transport on conventional DAG-dependent PKCs contrasts with apparent dependence on atypical phosphatidylinositol-3,4,5-(PO4)3 (PIP3)-activated PKCs, {zeta} and/or {lambda}, as observed in rat adipocytes (2), 3T3/L1 adipocytes (1, 3), and L6 myotubes (5, 6).

In addition to finding that PKC{alpha} was not required for insulin-stimulated glucose transport, it was of interest to find that knockout of the PKC{alpha} gene was accompanied by substantial increases in insulin-stimulated glucose transport in isolated adipocytes and soleus muscle preparations. These increases in insulin-stimulated glucose transport activity in tissues of PKC{alpha} knockout mice were associated with, and most likely due to, increases in insulin-induced activation of IRS-1-dependent PI3K, and subsequent activation of PKB and/or PKC{lambda}. In keeping with the notion that IRS-1 is involved in these increases, insulin-induced activation of PKB in brown adipocytes (25) and liver (26), and insulin-induced activation of both PKB and PKC{lambda} in muscle and white adipocytes (Standaert, M. L., M. P. Sajan, Y. Kanoh, G. Bandyopadhyay, C. R. Kahn, and R. V. Farese, unpublished observations) are markedly diminished in these tissues of IRS-1 knockout mice. Thus, IRS-1-dependent PI3K appears to be a major regulator of PKB and PKC{lambda} in these tissues. On the other hand, we cannot rule out the possibility that increases in the activation of PI3K, PKB, and PKC{lambda} may have also resulted from alterations in the activation of other IRS family members, GAB1, or other factors that may, in conjunction with IRS-1, operate upstream of PI3K in muscle and adipose tissues of PKC{alpha} knockout mice. In any case, regardless of their upstream regulators, the presently observed increases in PKB and/or PKC{lambda} activation provide a plausible explanation for observed increases in insulin-stimulated glucose transport in adipocytes and soleus muscles of PKC{alpha} knockout mice.

It was also of interest to find that expression of wild-type PKC{alpha} in adipocytes of PKC{alpha}-/- knockout mice was attended by decreases in basal (but, as discussed above, probably artefactually partially stimulated by electroporation and overnight culturing) and, more importantly, insulin-stimulated GLUT 4 translocation. This inhibitory effect of expressed PKC{alpha} contrasts with that of expressed wild-type PKC{lambda} or PKC{zeta}, each of which enhances insulin effects on glucose transport and/or GLUT 4 translocation responses in rat adipocytes (2, 4), 3T3/L1 adipocytes (1, 3), rat skeletal muscles (27), and L6 myotubes (5, 6). Thus, it may be surmised that there are considerable differences in glucose transport and presumably other biological effects of atypical and conventional PKCs; this further implies that there are differences in substrates and/or cellular locations of these PKCs. In any event, the similarity of inhibitory effects of transiently transfected PKC{alpha} in adipocytes of PKC{alpha}-/- knockout mice to those of endogenous PKC{alpha} in adipocytes of control wild-type PKC{alpha}+/+ mice suggested that observed increases in signaling and glucose transport in muscles and adipocytes of PKC{alpha}-/- knockout mice can be attributed to the absence of endogenous levels of PKC{alpha} itself, rather than to other more chronic developmental alterations in signaling patterns that may arise during embryogenesis in PKC{alpha}-/- knockout mice.

Although it is clear that insulin activates atypical PKCs largely via PI3K-dependent increases in PIP3 [which enhances PDK-1-dependent loop phosphorylation and subsequent autophosphorylation, and relieves pseudosubstrate-dependent autoinhibition in atypical PKCs (28)], it has not been entirely clear how conventional PKCs, {alpha} and ß2, are activated by insulin. Whereas the PI3K inhibitor, wortmannin, completely or largely inhibits insulin-induced activation of immunoprecipitable PKC{zeta}/{lambda} in adipocytes (Ref. 2 and present results), wortmannin had little or no effect on insulin-induced activation of immunoprecipitable PKC{alpha} or PKCß2 in mouse and rat adipocytes. Thus, insulin effects on PKC{alpha} and PKCß may be largely dependent on activation of the de novo phospholipid/DAG synthesis pathway, which is known to be independent of PI3K (29). In this scenario, increases in de novo synthesis of DAG presumably provoked increases in autophosphorylation or other covalent modifications, or, alternatively, tight binding of an activating factor other than DAG, to account for observed increases in immunoprecipitable enzyme activity of these conventional PKCs, which were assayed in the presence of saturating amounts of the DAG analog, tetradecanoyl 13-phorbol acetate (TPA).

Regardless of the factor or mechanism responsible for PI3K-independent increases in the activity of immunoprecipitable PKC{alpha} and PKCß2, it may be surmised that one of the major functions of these conventional DAG-dependent PKCs is to serve as feedback inhibitors of insulin receptor signaling to IRS-1 (and possibly other IRS family members), PI3K, PKB, and PKC{lambda}, and thereby diminish insulin-stimulated glucose transport and other processes that are dependent on these signaling factors. Moreover, because insulin increased the activity of PKC{alpha} and PKCß2 in skeletal muscles and adipocytes, and, because increases in signaling to IRS-1, PI3K, PKC{lambda}, and PKB were evident in comparing PKC{alpha} knockout mice to wild-type mice, it may further be surmised that this feedback mechanism occurs in skeletal muscles and adipocytes in physiological circumstances, i.e. at ambient cellular PKC{alpha} levels and during insulin stimulation.

It was also of interest to find that insulin-induced increases in ERK in vastus lateralis skeletal muscles were enhanced by knockout of PKC{alpha}. This finding raised the possibility that enhanced activation of IRS-1, PI3K, PKB, and/or PKC{lambda} may have contributed to the observed increases in ERK activation in skeletal muscle. In keeping with this possibility, ERK activation by insulin in skeletal muscle is diminished in IRS-1 knockout mice (20), and PI3K and PKC{zeta}/{lambda} (but not PKB) are required for ERK activation by insulin, not only in rat adipocytes (23), but also in 3T3/L1 adipocytes and L6 myotubes (Bandyopadhyay, G., M. L. Standaert, M. P. Sajan, Y. Kanoh, and R. V. Farese, unpublished observations). On the other hand, wortmannin was not found to inhibit ERK activation in isolated rat soleus muscle preparations (30), and PI3K and its downstream effectors, PKB and PKC{lambda}, may not be required for ERK activation in this and possibly other skeletal muscles. If in fact PI3K and PKC{lambda} are not required for ERK activation in skeletal muscle, both the previously observed decrease in insulin-induced ERK activation in skeletal muscle in IRS-1 knockout mice (20), and the presently observed increase in insulin-induced activation of ERK in PKC{alpha} knockout mice, may reflect a requirement for IRS-1 to activate the ERK pathway independently of PI3K, in this tissue.

From the present findings, it may be surmised that conventional DAG-dependent PKCs can limit the activation of atypical PIP3-dependent PKCs, as well as IRS-1, PI3K, and PKB, during the action of insulin in adipose and muscle cells. This cross-talk between conventional PKCs and atypical PKCs is of particular interest, but the inhibition of insulin signaling via IRS-1/PI3K/PDK-1 is apparently not specific for conventional PKCs, as it also appears to occur in the PI3K-dependent activation and subsequent action of atypical PKCs (31). It therefore appears that the activation of PI3K and atypical PKCs, as well as conventional PKCs, can limit insulin-induced activation of IRS-1, PI3K, and its downstream effectors, PKB and atypical PKCs. These cross-talking and self-inhibiting negative feedback loops may be important to limit inordinate levels of metabolic signaling during sustained insulin action.

Finally, it was of interest to observe that the increases in insulin signaling to PI3K, PKB, and PKC{lambda}, and subsequent increases in insulin-stimulated glucose transport, particularly in skeletal muscles, were not accompanied by alterations in serum glucose or insulin levels. This may reflect the fact that knockout of the insulin receptor in mouse skeletal muscle may be attended by insulin resistance, without necessarily causing alterations in serum glucose or insulin levels, or overall systemic glucose intolerance (32). Moreover, other tissues, such as the liver, may be more important determinants of serum glucose and insulin levels than skeletal muscle. Along these lines, the failure to observe lower serum glucose or insulin levels in PKC{alpha}-/- knockout mice raises the possibility that there may have been a compensatory increase in hepatic glucose output to counteract increases in glucose transport in muscle.

In summary, knockout of the mouse PKC{alpha} gene was accompanied by increases in insulin-induced activation of IRS-1-dependent PI3K, PKB, PKC{lambda}, ERK, and 2-deoxyglucose uptake in both skeletal muscles and adipocytes. Moreover, unlike PI3K-dependent PKC{lambda} activation, insulin was found to activate PKC{alpha} and PKCß2 largely independently of PI3K. Our findings suggest that PKC{alpha} is not required for insulin-stimulated glucose transport, and, moreover, PKC{alpha}, even at physiological concentrations, apparently serves as an endogenous negative feedback inhibitor of insulin signaling through IRS-1, PI3K, PKB, and PKC{lambda} to the glucose transport system in both skeletal muscles and adipocytes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of PKC{alpha} Mutant Mice
To clone the mouse PKC{alpha} locus, a 129/Ola genomic phage library (obtained from Stratagene, La Jolla, CA) was screened using a full-length rat cDNA as a probe. Several phages were identified and purified to homogeneity and subsequently rescreened with a 5'-specific cDNA probe corresponding to exons 1–3. One phage was shown to harbor the first two exons of the PKC{alpha} gene and was used as follows for further cloning. A 13.0-kb PstI/BamHI genomic fragment was subcloned, and a neomycin cassette (neo) was introduced into the second exon of the gene. The targeting vector was linearized at a unique NotI site derived from the polylinker of the plasmid and introduced into E14 ES cells (33). After 10 d of selection with G418 (300 mg/ml), resistant ES cell clones were further analyzed by Southern blot analysis using probe A indicated in Fig. 1AGo. Targeted ES cells (PKC{alpha}+/-) were injected into NMRI albino mouse blastocysts to generate chimeras. Several chimeras gave germ line transmissions, which were used to establish the homozygous PKC{alpha}-deficient mouse line.

Genotyping
Genotypic characterization of recombinant ES cells and adult mice was done by Southern blot analysis of BamHI-digested genomic DNA. DNA was derived from adult tails and hybridized with a 5'-probe (probe A, Fig. 1AGo) distinguishing wild-type, heterozygote mutant and homozygote mutant alleles. The 5'-probe corresponds to a 1.2-kb PstI fragment recognizing an approximately 20.0-kb band in the wild type and a 9.0-kb band in the targeted allele.

RT-PCR Analyses
Total RNA was prepared from adult brains using TRIzol Life Technologies, Inc., Gaithersburg, MD) for RNA purification, an ExpeRT-PCR kit (Hybaid, Middlesex, UK) for cDNA synthesis, and Hotstart mix (QIAGEN, Chatsworth, CA) for PCR amplification. The following sets of primers were used to amplify specific PKC{alpha} transcripts: 1) Sense 1

5'-GCTGAGGCAGAAGAACGTGCATG-3' vs. Antisense 7

5'-GAAGTCATTCCGAGTCGTCCG-3' generating a 700-bp fragment; and 2) Sense 1

5'-GCTAGGCAGAAGAACGTGCATG-3' vs. Antisense 8

5'-GATCTTGATGGCTACAGTTCC-3' generating a 1,030-bp fragment.

Experimental Mice
Heterozygote PKC{alpha}-/+ chimeric germ line-transmitting mice were crossed into wild-type 129/SV mice, and male littermate offspring that were either homozygous for knockout (-/-) or full retention (+/+) of the PKC{alpha} gene were selected for experimental use. Note that the reporter gene construct and ES cells used for targeting were derived from 129/SV mice; thus, crossing chimeras into 129/SV mice yielded F1 heterozygous offspring on a pure 129/SV background. Consequently, wild-type PKC{alpha}+/+ and PKC{alpha}-/- knockout mice were genetically similar, except for the targeted gene. All mice were housed in the same controlled environment (alternating 12-h light and dark cycles) and fed the same diet for least 2 wk before experimental use.

Muscle Signaling Studies in Vivo
For studies of insulin-induced activation of PI3K, PKB, and PKC{lambda} in mouse skeletal muscle [which contains primarily PKC{lambda}, rather than PKC{zeta} (6)], mice, weighing approximately 30–35 g, were injected ip with 0.001 U insulin/gram of body weight 15 min before being killed. Vastus lateralis muscles were rapidly excised and homogenized (Polytron) in ice-cold buffer, which, for PKC{lambda} assays, contained 0.25 M sucrose, 20 mM Tris/HCl (pH, 7.5), 1.2 mM EGTA, 20 mM ß-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 µg/ml leupeptin,10 µg/ml aprotinin, 3 mM Na4P2O7, 3 mM Na3VO4, 3 mM NaF, and 1 µM L-arginine-microcystin. For PKB, PI3K, and ERK assays, the homogenizing buffer contained 50 mM Tris/HCl (pH, 7.5), 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 0.1% ß-mercaptoethanol, 50 mM NaF, 5 mM Na4P2O7, 10 mM ß-glycerophosphate, 1 mM PMSF, 10 µg/ml aprotinin, 20 µg/ml leupeptin, and 1 µM L-arginine-microcystin. After low-speed centrifugation for 10 min at 700 x g to remove unbroken cells, debris, nuclei and floating fat, 0.15 M NaCl, 1% Triton-X, and 0.5% Nonidet were added, and the resulting cell lysates were immunoprecipitated with antibodies that target 1) IRS-1 (rabbit polyclonal antiserum kindly supplied by Dr. Alan Saltiel, Parke-Davis Co., Kalamazoo, MI; or purchased from Santa Cruz Biotechnology, Inc., Santa Cruz, CA); 2) PKB [rabbit polyclonal antiserum obtained from Upstate Biotechnology, Inc., Lake Placid, NY]; 3) PKC{lambda} (rabbit polyclonal antiserum obtained from Santa Cruz Biotechnology, Inc. recognizes the C termini of both PKC{lambda} and PKC{zeta}) or ERK (rabbit polyclonal antiserum obtained from Santa Cruz Biotechnology, Inc.). Immunoprecipitates were collected on Sepharose-AG beads, and PI3K, PKB, PKC{lambda} and ERK were assayed for enzyme activity, as described below. Lysates were also used to measure levels of immunoreactive proteins, including, as another indicator of PKB activation, phosphoserine-473-PKB, as described below.

Adipocyte Signaling Studies in Vitro
Epididymal fat pads (pooled from at least five mice or two rats) were digested with collagenase as described (2, 24), and isolated adipocytes were incubated in glucose-free Krebs Ringer phosphate (KRP) medium for 10 min with or without 10 nM insulin. After incubation, cells were sonicated and lysates were subjected to immunoprecipitation and assayed for IRS-1-dependent PI3K, PKC{lambda}, PKB, PKC{alpha} and PKCß2 enzyme activity or, as another indicator of PKB activation, used to measure immunoreactive phosphoserine-473-PKB, as in muscle studies.

PKC{lambda}/{zeta} Activation
PKC{lambda}/{zeta} activity was measured as described previously (1, 2, 4). In brief, PKC{lambda}/{zeta} was immunoprecipitated with a rabbit polyclonal antiserum (Santa Cruz Biotechnology, Inc.) that recognizes the C termini of both PKC{lambda} and PKC{zeta} [however, mouse adipocytes contain mainly PKC{lambda}; see Ref. 34), collected on Sepharose-AG beads (Santa Cruz Biotechnology, Inc.), and incubated for 8 min at 30 C in 100 µl buffer containing 50 mM Tris/HCl (pH 7.5), 100 µM Na3VO4, 100 µM Na4 P2O7, 1 mM NaF, 100 µM PMSF, 4 µg phosphatidylserine (Sigma, St. Louis, MO), 50 µM [{gamma}-32P]ATP (NEN Life Science Products, Boston, MA), 5 mM MgCl2 and, as substrate, 40 µM serine analog of the PKC{epsilon} pseudosubstrate (Biosource Technologies, Inc., Camarillo, TX). After incubation, 32P-labeled substrate was trapped on P-81 filter papers and counted. Note that assays of knockout and wild-type samples were conducted simultaneously.

PKB Activation
PKB enzyme activity was measured using a kit obtained from Upstate Biotechnology, Inc., as described previously (6). In brief, PKB was immunoprecipitated with a rabbit polyclonal antiserum, collected on Sepharose-AG beads, and incubated as per directions in the PKB assay kit. PKB activation was also assessed by immunoblotting for phosphorylation of serine-473 (see below).

PI3K Activation
Immunoprecipitable IRS-1-dependent PI3K activity was measured as described previously (29).

ERK Activation
Immunoprecipitable ERK activity was measured as described previously (23).

Glucose Transport Studies
Adipocytes were prepared as described above in signaling studies. Soleus muscles were ligated at both ends, excised, and stretched to maintain resting length. Adipocytes and muscles were incubated for 30 min with or without 10 or 100 nM insulin, before measurement of [3H]2-deoxyglucose uptake over 1 min or 5 min, respectively, as described previously (1, 2, 4, 5).

Transfection Studies
Adipocytes were transiently cotransfected by electroporation of 0.4 ml cells in an equal volume of DMEM with 3 µg pCIS2 encoding HA-tagged (in an exofacial loop) GLUT 4 along with 7 µg pCDNA3 vector alone or pCDNA3 encoding wild-type PKC{alpha} as described previously (2, 4). After overnight incubation to allow time for expression, cells were washed and incubated in glucose-free KRP medium for 30 min with or without 10 nM insulin; next, 2 mM KCN was added to stop exocytotic and endocytotic phases of the translocation process, and cell surface HA-GLUT 4 was measured by incubation of intact KCN-stopped cells, first with mouse monoclonal anti-HA antibodies (Covance Laboratories, Inc., Berkeley, CA), and then with 125I-labeled rabbit antimouse-IgG secondary antibodies (Amersham Pharmacia Biotech, Arlington Heights, IL) as described previously (2, 4). Note that this cotransfection approach is based upon the assumption that simultaneous transfection of HA-GLUT 4 and PKC{alpha} leads to expression of both proteins largely in the same set of adipocytes; evidence that supports this assumption has been published previously (4).

Immunoblot Analyses
Western analyses were conducted as described previously (2, 4), using the following antibodies/antisera: 1) rabbit polyclonal anti-PKC{alpha}, PKCß1, PKCß2, PKC-{epsilon}, PKC{delta}, and anti-PKC{zeta}/{lambda} antisera (obtained from Santa Cruz Biotechnology, Inc.); 2) rabbit polyclonal anti-PKB antiserum (obtained from Upstate Biotechnology, Inc.); 3) rabbit polyclonal anti-phospho-S473-PKB antiserum (obtained from New England Biolabs, Inc., Beverly, MA); 4) rabbit polyclonal anti-GLUT 1 antiserum (kindly provided by Dr. Ian Simpson, NIH, Bethesda MD); 5) mouse monoclonal anti-GLUT 4 antibodies (obtained from Biogenesis, Poole, UK); 6) rabbit polyclonal anti-p85/PI3K antiserum (obtained from Upstate Biotechnology, Inc.); 7) rabbit polyclonal anti-3-phosphoinositide-dependent protein kinase-1 (PDK-1) antiserum (obtained from Upstate Biotecnology, Inc.); and 8) rabbit polyclonal anti-IRS-1 and anti-IRS-2 antisera (kindly supplied by Dr. Morris White).

Studies of Conventional PKC Activation in Adipocyte and Skeletal Muscles
PKC{alpha} and PKCß2 were immunoprecipitated from cell lysates with specific antisera obtained from Santa Cruz Biotechnology, Inc. and assayed as described above for PKC{lambda}, the only exception being that PKC{alpha} and PKCß2 assays were conducted in the presence of 1 mM CaCl2, 100 nM TPA (Sigma), and 40 µM PKC{alpha} pseudosubstrate containing serine rather than alanine (Biosource Technologies, Inc.) (also see Ref. 1 for assays of conventional PKCs). TPA (or DAG) was absolutely essential for observing PKC{alpha} and PKCß2 activity in these assays but was without effect in PKC{lambda}/{zeta} assays. As described below, wortmannin inhibited the activation of PKC{lambda}/{zeta}, but had little or no effect on the activation of PKC{alpha} and PKCß2. Blank values were determined from assays conducted with precipitates prepared with preimmune serum and were subtracted from immune serum values.


    ACKNOWLEDGMENTS
 


    FOOTNOTES
 
This work was supported by Deutsche Forschungsgemeinschaft Sta314/2–1 and KE246/7–2; NIH Grant RO1-DK-38079-11; and Department of Veterans Affairs Merit Review Program.

Abbreviations: DAG, Diacylglycerol; ES, embryonic stem; GLUT, glucose transporter; HA, hemagglutin; IRS, insulin receptor substrate; KRP, Krebs Ringer phosphate; PDK, phosphoinositide-dependent protein kinase; PIP3 phosphatidylinositol-3,4,5-(PO4)3; PKB, protein kinase B; PMSF, phenylmethylsulfonyl fluoride; TPA, tetradecanoyl 13-phorbol acetate.

Received for publication April 27, 2001. Accepted for publication December 21, 2001.


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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