AJP - Endo Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 283: E338-E345, 2002. First published March 12, 2002; doi:10.1152/ajpendo.00457.2001
0193-1849/02 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
283/2/E338    most recent
00457.2001v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (20)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tsuru, M.
Right arrow Articles by Oka, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tsuru, M.
Right arrow Articles by Oka, Y.
Vol. 283, Issue 2, E338-E345, August 2002

Role of PKC isoforms in glucose transport in 3T3-L1 adipocytes: insignificance of atypical PKC

Masatoshi Tsuru1, Hideki Katagiri2, Tomoichiro Asano3, Tetsuya Yamada1,2, Shigeo Ohno4, Takehide Ogihara3, and Yoshitomo Oka1,2

1 Third Department of Internal Medicine, Yamaguchi University School of Medicine, Ube, Yamaguchi 755-8505; 2 Division of Molecular Metabolism and Diabetes, Department of Internal Medicine, Tohoku University Graduate School of Medicine, Sendai 980-8574; 3 Faculty of Medicine, Department of Internal Medicine, University of Tokyo, Tokyo 113-8566; and 4 Department of Molecular Biology, Yokohama City University School of Medicine 3 - 9, Yokohama 236, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To elucidate the involvement of protein kinase C (PKC) isoforms in insulin-induced and phorbol ester-induced glucose transport, we expressed several PKC isoforms, conventional PKC-alpha , novel PKC-delta , and atypical PKC isoforms of PKC-lambda and PKC-zeta , and their mutants in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Endogenous expression and the activities of PKC-alpha and PKC-lambda /zeta , but not of PKC-delta , were detected in 3T3-L1 adipocytes. Overexpression of each wild-type PKC isoform induced a large amount of PKC activity in 3T3-L1 adipocytes. Phorbol 12-myristrate 13-acetate (PMA) activated PKC-alpha and exogenous PKC-delta but not atypical PKC-lambda /zeta . Insulin also activated the overexpressed PKC-delta but not PKC-alpha . Expression of the wild-type PKC-alpha or PKC-delta resulted in significant increases in glucose transport activity in the basal and PMA-stimulated states. Dominant-negative PKC-alpha expression, which inhibited the PMA activation of PKC-alpha , decreased in PMA-stimulated glucose transport. Glucose transport activity in the insulin-stimulated state was increased by the expression of PKC-delta but not of PKC-alpha . These findings demonstrate that both conventional and novel PKC isoforms are involved in PMA-stimulated glucose transport and that other novel PKC isoforms could participate in PMA-stimulated and insulin-stimulated glucose transport. Atypical PKC-lambda /zeta was not significantly activated by insulin, and expression of the wild-type, constitutively active, and dominant-negative mutants of atypical PKC did not affect either basal or insulin-stimulated glucose transport. Thus atypical PKC enzymes do not play a major role in insulin-stimulated glucose transport in 3T3-L1 adipocytes.

protein kinase C; glucose transport; insulin; phorbol 12-myristate 13-acetate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

REGULATION OF GLUCOSE HOMEOSTASIS is one of the most important actions of insulin. Insulin stimulates the translocation of GLUT4 glucose transporters from intracellular storage sites to the plasma membrane in muscle and adipose tissue, resulting in increased glucose uptake. Many intensive studies have focused on the cellular and molecular mechanisms responsible for these trafficking events (15, 39).

The involvement of protein kinase C (PKC) in glucose transport activation was originally recognized in studies using pharmacological agents. Phorbol 12-myristate 13-acetate (PMA), an activator of conventional and novel PKC, stimulates glucose transport activity (27) by inducing translocation of both GLUT1 and GLUT4 glucose transporters (1). In addition, insulin- and PMA-stimulated glucose transport activity was inhibited by staurosporine in isolated rat adipocytes (33, 41). These results suggest that PKC participates in activating glucose transport in adipocytes.

Several lines of evidence have indicated that phosphatidylinositol 3-kinase (PI 3-kinase) activation is important in insulin-stimulated glucose transport. The PI 3-kinase pharmacological inhibitors, such as wortmannin (37) and LY-294002 (12), and expression of the dominant-negative mutants of PI 3-kinase (18, 22, 25) reportedly markedly block insulin-stimulated glucose transport and GLUT4 translocation in rat and 3T3-L1 adipocytes. Furthermore, overexpression of wild-type PI 3-kinase tagged with the GLUT2 COOH-terminal domain (16) or the constitutively active mutant of PI 3-kinase (29) promoted glucose transport activity and GLUT4 translocation. These findings suggest a central role for PI 3-kinase in insulin-stimulated glucose transport.

Recently, several pathways have been reported to be activated by growth factor stimulation downstream from PI 3-kinase. Atypical PKC, consisting of PKC-zeta and PKC-lambda , which are not activated by diacylglycerol or phorbol ester, is one of such effectors of PI 3-kinase. Atypical PKC enzymes are activated in vitro in the presence of the products of PI 3-kinase and phosphatidylinositol triphosphate (32, 43), and are also activated by growth factor stimulation (2). Furthermore, two groups of investigators have reported that PKC-zeta or PKC-lambda played a major role in glucose transport activation by insulin in 3T3-L1 adipocytes (8, 26), L6 myocytes (6), and rat (42) and human (5) adipocytes, although they hypothesized that different atypical PKC isoforms were involved. In contrast, it was also reported that activation of PKC-delta , a novel PKC, but not an atypical PKC, is a major signal in insulin-induced glucose transport (10). Thus the necessity of activating atypical PKC in insulin-induced glucose transport is controversial.

In the present study, to elucidate the involvement of conventional, novel, and atypical PKCs in insulin-induced and PMA-induced glucose transport, we examined the effects of overexpressing several PKC isoforms and their mutants (PKC-alpha as a conventional PKC isoform, PKC-delta as a novel PKC isoform, and PKC-lambda and PKC-zeta as atypical PKC isoforms). 3T3-L1 adipocytes were transfected with these isoforms using an adenovirus-mediated gene transduction system.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Antibodies. The rabbit polyclonal anti-PKC-alpha antibody, anti-PKC-delta antibody, and anti-PKC-zeta antibody, which also reacts with the lambda -isoform, were purchased from Santa Cruz Biotechnology.

Cell culture. 3T3-L1 fibroblasts were maintained in DMEM containing 10% donor calf serum (GIBCO) in an atmosphere of 10% CO2 at 37°C. Two days after the fibroblasts had reached confluence, differentiation was induced by treating cells with DMEM containing 0.5 mM 3-isobutyl 1-methylxanthine, 4 µg/ml dexamethasone, and 10% FBS for 48 h. Cells were incubated with DMEM supplemented with 10% FBS every other day for the following 4-10 days. More than 90% of the cells expressed the adipocyte phenotype (23).

Expression constructs. The cDNAs encoding the entire rabbit PKC-alpha (35), its dominant-negative mutant (7) mouse PKC-delta (36), its dominant-negative mutant (19) mouse PKC-zeta (17), its dominant-negative mutant (9) mouse PKC-lambda (2), and its dominant-negative (2) and constitutively active (26) mutants were kindly provided by Dr. S. Ohno. Recombinant adenoviruses containing each PKC gene were constructed by homologous recombination between the expression cosmid cassette and the parental virus genome, as described previously (22, 30, 34).

Gene transduction. 3T3-L1 adipocytes were incubated with DMEM containing the adenovirus for 1 h at 37°C, and the growth medium was then added. Experiments were performed 60 h after the infection. Recombinant adenoviruses were applied at a multiplicity of infection (MOI) of 200-300 plaque-forming units (pfu)/cell, and 3T3-L1 adipocytes infected with the Adex1CalacZ virus (21), which encodes Escherichia coli lacZ, were used as a control, since Adex1CAlacZ gene expression was observed in >90% of 3T3-L1 adipocytes applied at an MOI of 200-300 pfu/cell on postinfection day 3 but did not affect glucose transport activity compared with noninfected cells, as reported previously (22, 23).

PKC activity assay. PKC activity was assayed using a peptide substrate, as previously described (8). 3T3-L1 cells cultured in 12-well plates were incubated with or without 1 µM insulin or 1.6 µM PMA for 10 min. Cells were homogenized in buffer containing 20 mM Tris · HCl (pH 7.5), 0.25 mM sucrose, 1.2 mM EGTA, 20 mM beta -mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium vanadate, 1 mM sodium pyrophosphate, 1 mM NaF, 1% Triton X-100, 0.5% Nonidet P-40, and 150 mM NaCl and then cleared of insoluble substances by centrifugation. PKC-alpha and PKC-delta were immunoprecipitated by incubation with polyclonal anti-PKC-alpha antibody or anti-PKC-delta antibody, respectively, for 16 h at 0-4°C. The immunoprecipitates were suspended in reaction buffer containing 50 mM Tris · HCl (pH 7.5), 1 mM NaHCO3, 5 mM MgCl2, and 1 mM PMSF and then were assayed for the ability to phosphorylate a PKC pseudosubstrate peptide, namely 40 µM [Ser25]PKC-(19-31) (GIBCO-BRL) in buffer containing 50 mM Tris · HCl (pH 7.5), 5 mM MgCl2, 100 µM sodium vanadate, 100 µM sodium pyrophosphate, 1 µM CaCl2, 1 mM NaF, 100 µM PMSF, and 50 µM [gamma -32P]ATP. To assay PKC-zeta /lambda activity, polyclonal anti-PKC-zeta antibody and [Ser159]PKC-epsilon -(153-164)-NH2 (Upstate Biotechnology) were used for immunoprecipitation and as the reaction substrate, respectively. Reactions were stopped with 5% acetic acid, and aliquots of the reaction mixture were spotted on P-81 filter paper (Whatman), washed in 5% acetic acid, and counted for 32P. The results were quantitated using an image analyzer BAS2000 (Fujix).

Glucose transport assay. 3T3-L1 adipocytes in 12-well plates were serum starved for 3 h in DMEM containing 0.2% BSA, followed by a 45-min glucose-free incubation in Krebs-Ringer phosphate buffer. Cells were then incubated with or without 1 µM insulin or 1.6 µM PMA for 15 min, and 0.1 mM 2-deoxy-D-[3H]glucose uptake was measured as described previously (22, 23).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Conventional PKC, PKC-alpha , has some involvement in PMA-induced, but not in insulin-induced, glucose transport. First, we expressed the wild-type and dominant-negative mutants of a conventional PKC isoform, PKC-alpha , to increase and suppress PKC-alpha activity, respectively, using an adenovirus-mediated gene transduction system. Immunoblotting with anti-PKC-alpha antibody showed that successful expression of wild-type and the mutated PKC-alpha was achieved in 3T3-L1 adipocytes by infection with the recombinant adenoviruses (Fig. 1A). PKC-alpha activity was measured in the immunoprecipitates with anti-PKC-alpha antibody (Fig. 1B). Consistent with the results obtained from the immunoblotting study, overexpression of the wild-type PKC-alpha produced an 18-fold increase in PKC-alpha activity in the basal state compared with that in control 3T3-L1 adipocytes that were infected with adenovirus recombined with the lacZ gene. Stimulation with insulin did not affect the endogenous or exogenously expressed PKC-alpha activity. In contrast, PMA increased both endogenous and exogenously expressed PKC-alpha activity by ~2.5-fold and 1.6-fold, respectively (Fig. 1B). Thus exogenous PKC-alpha was functionally expressed. In addition, expression of the dominant-negative mutant of PKC-alpha completely inhibited PKC-alpha activation by PMA while having no effect on endogenous PKC-alpha activity in the basal- and insulin-stimulated states (Fig. 1B). Thus expression of the mutant PKC-alpha resulted in exerting a dominant-negative effect on PKC-alpha activation by PMA.


View larger version (50K):
[in this window]
[in a new window]
  		Fig. 1.   Protein kinase C (PKC)-alpha expression levels and activities in the absence or presence of insulin or phorbol 12-myristate 13-acetate (PMA). A: expression levels of PKC-alpha proteins. Lysates from 3T3-L1 adipocytes (control) and from those expressing lacZ (control), wild-type (WT) PKC-alpha , or the dominant negative (DN) mutant of PKC-alpha were immunoblotted with anti-PKC-alpha antibody. B: control 3T3-L1 adipocytes and 3T3-L1 adipocytes expressing PKC-alpha WT or PKC-alpha DN were incubated with or without 1 µM insulin or 1.6 µM PMA for 10 min. Lysates were immunoprecipitated with anti-PKC-alpha antibody, and PKC-alpha activities in the immunoprecipitates were assayed, as described in MATERIALS AND METHODS. Values presented as means ± SD of 3 separate experiments.

To examine the effects of PKC-alpha activity on insulin- and PMA-induced glucose transport, we measured 2-deoxyglucose uptake in 3T3-L1 adipocytes overexpressing wild-type and dominant-negative PKC-alpha with or without insulin or PMA stimulation (Fig. 2). Insulin and PMA stimulated 2-deoxyglucose uptake by ~7.0-fold and 1.7-fold, respectively, in control (lacZ-expressing) 3T3-L1 adipocytes. Expression of the wild-type PKC-alpha in 3T3-L1 adipocytes produced a significant increase in glucose transport activity to ~1.9- and 2.2-fold in the basal and PMA-stimulated states, respectively. In contrast, expression of the dominant-negative mutant of PKC-alpha did not affect basal glucose transport activity, but a significant decrease was observed in the PMA-stimulated state. Neither the wild-type nor the dominant-negative mutant of PKC-alpha had an effect on insulin-stimulated glucose transport activity. These findings demonstrate that 1) PKC-alpha activation stimulates glucose transport activity, 2) PKC-alpha activation is involved in the PMA-stimulated glucose transport, although this effect is partial, and 3) PKC-alpha activation is not involved in the insulin-stimulated glucose transport activity.


View larger version (44K):
[in this window]
[in a new window]
  		Fig. 2.   Insulin- or PMA-stimulated 2-deoxy-D-glucose uptake in control and PKC-alpha WT or PKC-alpha DN overexpressing 3T3-L1-adipocytes. Uptake of 2-deoxy-D-glucose after incubation with or without 1 µM insulin or 1.6 µM PMA for 15 min was assayed in control 3T3-L1 adipocytes and 3T3-L1 adipocytes expressing PKC-alpha WT or PKC-alpha DN for 4 min. Data presented as means ± SD of 3 separate experiments.

Exogenously expressed PKC-delta induced a small increase in glucose transport activity in the basal, insulin-stimulated, and PMA-stimulated states. Next, we expressed the wild-type and the dominant-negative mutants of PKC-delta , a novel PKC enzyme, in 3T3-L1 adipocytes and measured PKC-delta activity and glucose transport activity in the presence or absence of insulin or PMA. Endogenous PKC-delta expression was very low, almost undetectable, in control 3T3-L1 adipocytes, whereas the exogenous expressions of the wild-type and dominant-negative mutants of PKC-delta were clearly detected, as shown by immunoblotting with anti-PKC-delta antibody (Fig. 3A). PKC-delta activity was measured in the immunoprecipitates with anti-PKC-delta antibody (Fig. 3B). Consistent with the results of the immunoblotting study, endogenous PKC-delta activity was almost undetectable in control 3T3-L1 adipocytes in either the basal, the insulin-stimulated, or the PMA-stimulated state. Overexpression of PKC-delta induced a large amount of PKC-delta activity in 3T3-L1 adipocytes. Insulin and PMA stimulated this activity by 1.3- and 2.4-fold, respectively. In contrast, expression of the dominant-negative mutant of PKC-delta did not lead to PKC-delta activity in either the basal, insulin-stimulated, or PMA-stimulated states (Fig. 3B).


View larger version (49K):
[in this window]
[in a new window]
  		Fig. 3.   PKC-delta expression levels and activities in the absence or presence of insulin or PMA. A: expression levels of PKC-delta proteins. Lysates from control 3T3-L1 adipocytes and from those expressing PKC-delta WT or PKC-delta DN were immunoblotted with anti-PKC-delta antibody. B: control 3T3-L1 adipocytes and 3T3-L1 adipocytes expressing PKC-delta WT or PKC-delta DN were incubated with or without 1 µM insulin or 1.6 µM PMA for 10 min. Lysates were immunoprecipitated with anti-PKC-delta antibody, and PKC-delta activities in the immunoprecipitates were assayed as described in MATERIALS AND METHODS. Values presented as means ± SD of 3 separate experiments.

We further assayed the 2-deoxyglucose uptake in 3T3-L1 adipocytes overexpressing the wild-type or dominant-negative PKC-delta (Fig. 4). Expression of the wild-type PKC-delta induced significant increases in glucose transport activity of ~2.0-, 1.3-, and 3.2-fold in the basal, insulin-, and PMA-stimulated states, respectively, compared with that in control 3T3-L1 adipocytes. In contrast, expression of the dominant-negative mutant of PKC-delta did not affect basal, insulin-stimulated, or PMA-stimulated glucose transport activity (Fig. 4). These findings demonstrate that a novel PKC could participate in PMA-induced and insulin-induced glucose transport activation, although endogenous expression of PKC-delta is below the detection limits.


View larger version (48K):
[in this window]
[in a new window]
  		Fig. 4.   Insulin- or PMA-stimulated 2-deoxy-D-glucose uptake in control and PKC-delta WT or PKC-delta DN overexpressing 3T3-L1 adipocytes. Uptake of 2-deoxy-D-glucose after incubation with or without 1 µM insulin or 1.6 µM PMA for 15 min was assayed in control 3T3-L1 adipocytes and 3T3-L1 adipocytes expressing PKC-delta WT or PKC-delta DN for 4 min. Data presented as means ± SD of 3 separate experiments.

Atypical PKC enzymes do not play a major role in insulin-stimulated glucose transport. Seemingly conflicting results were reported by two research groups who found that different isoforms of atypical PKC enzymes, PKC-zeta and PKC-lambda , are mainly involved in insulin-stimulated glucose transport. To determine which isoform plays the major role in glucose transport activation by insulin, we exogenously expressed the wild type or dominant-negative mutant of PKC-zeta or PKC-lambda in 3T3-L1 adipocytes, followed by measurement of atypical PKC activity and glucose transport activity with or without insulin or PMA stimulation.

First, to assess whether endogenous PKC-zeta /lambda was activated by PMA and insulin, the endogenous enzymes were immunoprecipitated from control 3T3-L1 adipocytes with a polyclonal anti-PKC-zeta antibody that also reacts with the PKC-lambda isoform. Immunoprecipitates from control 3T3-L1 adipocytes showed substantial activity above the background measured in irrelevant IgG immunoprecipitates. Immunoblotting study revealed a faint but detectable band of endogenous atypical PKCs (Figs. 5A and 6A). As expected, PMA stimulation of control 3T3-L1 adipocytes did not alter atypical PKC-zeta /lambda activities. In addition, contrary to previous reports, insulin stimulation did not activate the endogenous atypical PKCs (Figs. 5B and 6B).


View larger version (50K):
[in this window]
[in a new window]
  		Fig. 5.   PKC-zeta expression levels and activities in the absence or presence of insulin or PMA. A: expression levels of PKC-zeta proteins. Lysates from control 3T3-L1 adipocytes and from those expressing PKC-zeta WT or PKC-zeta DN were immunoblotted with anti-atypical PKC antibody. B: control 3T3-L1 adipocytes and 3T3-L1 adipocytes expressing PKC-zeta WT or PKC-zeta DN were incubated with or without 1 µM insulin or 1.6 µM PMA for 10 min. Lysates were immunoprecipitated with anti-PKC-zeta antibody, and PKC-zeta activities in the immunoprecipitates were assayed as described in MATERIALS AND METHODS. Values presented as means ± SD of 3 separate experiments.



View larger version (37K):
[in this window]
[in a new window]
  		Fig. 6.   PKC-lambda expression levels and activities in the absence or presence of insulin or PMA. A: expression levels of PKC-lambda proteins. Lysates from control 3T3-L1 adipocytes and from those expressing PKC-lambda WT or PKC-lambda DN were immunoblotted with anti-atypical PKC antibody. B: control 3T3-L1 adipocytes and 3T3-L1 adipocytes expressing PKC-lambda WT or PKC-lambda DN were incubated with or without 1 µM insulin or 1.6 µM PMA for 10 min. Lysates were immunoprecipitated with anti-PKC-zeta antibody, which also reacts with the lambda -isoform, as described in MATERIALS AND METHODS, and PKC-lambda activities in the immunoprecipitates were assayed as described in MATERIALS AND METHODS. Values presented as means ± SD of 3 separate experiments.

Exogenously overexpressed wild-type or the dominant-negative mutant of PKC-zeta or PKC-lambda was clearly detected by immunoblotting with anti-PKC-zeta antibody (Figs. 5A and 6A). Overexpression of atypical PKC-zeta or PKC-lambda resulted in increases in atypical PKC activity of ~10.4- and 4.1-fold, respectively, but atypical PKC was not activated by PMA. Insulin treatment did not significantly increase exogenously expressed atypical PKC activities (Figs. 5B and 6B). In addition, expression of dominant-negative mutants of PKC-zeta or PKC-lambda did not significantly alter atypical PKC activity in either the basal, insulin-stimulated, or PMA-stimulated state (Figs. 5B and 6B).

2-Deoxyglucose uptake was measured in 3T3-L1 adipocytes overexpressing the wild-type or dominant-negative PKC-zeta (Fig. 7) or PKC-lambda (Fig. 8) in the presence or absence of insulin or PMA. As expected, exogenous expression of these proteins did not influence PMA-stimulated glucose transport. Unexpectedly, overexpression of the wild-type PKC-zeta or PKC-lambda did not increase either basal or insulin-stimulated glucose transport activity. In addition, expression of dominant-negative mutants of these atypical PKCs did not influence insulin-stimulated glucose transport activity.


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 7.   Insulin- or PMA-stimulated 2-deoxy-D-glucose uptake in control and overexpressed PKC-zeta WT or PKC-zeta DN 3T3-L1 adipocytes. The uptake of 2-deoxy-D-glucose after incubation with or without 1 µM insulin or 1.6 µM PMA for 15 min was assayed in control 3T3-L1 adipocytes and 3T3-L1 adipocytes expressing PKC-zeta WT or PKC-zeta DN for 4 min. Data presented as means ± SD of 3 separate experiments.



View larger version (28K):
[in this window]
[in a new window]
  		Fig. 8.   Insulin- or PMA-stimulated 2-deoxy-D-glucose uptake in control and PKC-lambda WT or PKC-lambda DN overexpressing 3T3-L1 adipocytes. Uptake of 2-deoxy-D-glucose after incubation with or without 1 µM insulin or 1.6 µM PMA for 15 min was assayed in control 3T3-L1 adipocytes and 3T3-L1 adipocytes expressing PKC-lambda WT or PKC-lambda DN for 4 min. Data presented as means ± SD of 3 separate experiments.

Because glucose transport activity was not affected by overexpression of wild-type atypical PKC-lambda , we performed additional experiments using a constitutively active mutant of PKC-lambda to further increase atypical PKC activity. Expression of constitutively active PKC-lambda increased atypical PKC activity to ~33-fold (Fig. 9B), which was consistent with its expression level (Fig. 9A). However, expression of constitutively active PKC did not affect either basal or insulin-stimulated glucose transport activity (Fig. 9C). Thus endogenous atypical PKC activity was not increased by insulin treatment, and no evidence indicating involvement of atypical PKC in insulin-induced signaling stimulating glucose transport was obtained in experiments focusing on expression of wild-type, dominant-negative, and constitutively active atypical PKC. These data indicate that atypical PKC enzymes do not play a major role in insulin-stimulated glucose transport.


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 9.   Effects of expression of constitutively active PKC-lambda on atypical PKC activity and glucose transport activity. A: expression levels of PKC-lambda proteins. Lysates from control 3T3-L1 adipocytes and from those expressing constitutively active (CA) PKC-lambda were immunoblotted with anti-atypical PKC antibody. B: control 3T3-L1 adipocytes and 3T3-L1 adipocytes expressing PKC-lambda CA were incubated with or without 1 µM insulin for 10 min. Lysates were immunoprecipitated with anti-atypical PKC antibody, and PKC activities in the immunoprecipitates were assayed as described in tMATERIALS AND METHODS. Values presented as means ± SD of 3 separate experiments. C: uptake of 2-deoxy-D-glucose after incubation with or without 1 µM insulin or 1.6 µM PMA for 15 min was assayed in control 3T3-L1 adipocytes and 3T3-L1 adipocytes expressing PKC-lambda CA for 4 min. Data presented as means ± SD of 3 separate experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We expressed the wild-type and dominant-negative mutants of each PKC isozyme, PKC-alpha as a conventional PKC isoform, PKC-delta as a novel PKC isoform, and PKC-zeta and PKC-lambda representing atypical PKC isoforms. The present results show clearly that conventional PKC participates, to some extent, in PMA-stimulated but not in insulin-stimulated glucose transport activity. It was reported that stable expression of the wild-type or constitutively active forms of PKC-alpha , PKC-beta 1, and PKC-beta 2 failed to influence basal glucose transport in 3T3-L1 fibroblasts and adipocytes (8). However, in the present study, a small increase in glucose transport activity in the basal state was observed in PKC-alpha -overexpressing adipocytes. The PKC enzyme was transiently expressed using an adenovirus-mediated gene transduction system, and PKC activity and glucose transport activity were measured 2 days after the transfection; i.e., the period of expression of these PKC enzymes was much shorter than that of stable expression. Some unknown mechanisms compensating for increased PKC activities may operate in cells overexpressing conventional PKC enzymes, especially in stable transfectants. The discrepancy between the previous and present results may therefore be attributable to different periods of expression.

The results obtained for exogenously expressed PKC-delta appeared to be very similar to those obtained for PKC-alpha . However, the activity of endogenous PKC-delta was below the detectable level because of its extremely low expression level in 3T3-L1 adipocytes. There was no detectable increase in endogenous PKC-delta activity, even in the PMA-stimulated state. In primary cultures of rat skeletal muscle, PKC-delta reportedly mediates insulin-stimulated glucose transport (10) and regulates insulin receptor activity and routing (11). In 3T3-L1 adipocytes, although no information is available as to what novel PKC isozyme(s) is expressed, our exogenously expressed PKC-delta results suggest that another novel PKC isozyme(s), if present, could respond to PMA and contribute to PMA-stimulated glucose transport activity.

Insulin treatment did not activate endogenously or exogenously expressed PKC-alpha , whereas insulin activated exogenously expressed PKC-delta . This is consistent with reports that novel PKC is activated downstream from PI 3-kinase (31). In accordance with these results, the average value of insulin-stimulated glucose transport activity was greater (1.4-fold) in adipocytes overexpressing PKC-delta than in control adipocytes, although the difference did not reach statistical significance in four independent experiments. These findings suggest that conventional PKC is not involved in insulin-stimulated glucose transport but that novel PKC isoforms, if expressed in 3T3-L1 adipocytes, may contribute, to a limited extent, to insulin-stimulated glucose transport activity.

Two research groups have reported atypical PKC to be involved in insulin-stimulated glucose transport in adipocytes, although the two groups proposed the involvement of different atypical PKC isoforms. Standaert et al. (42) reported that inhibition of PKC-zeta activity by the PKC-zeta pseudosubstrate or Ro31-8220 paralleled inhibition of insulin-stimulated glucose transport in rat adipocytes. They also reported that expression of wild-type or constitutively active PKC-zeta stimulated translocation of coexpressed GLUT4 that was tagged with myc and that expression of dominant-negative PKC-zeta partially inhibited translocation of tagged GLUT4 in rat adipocytes. They thus suggest PKC-zeta to be a downstream effector of PI 3-kinase through insulin-stimulated GLUT4 translocation. Kotani et al. (26) reported, however, that overexpression of the constitutively active mutant of PKC-lambda activated glucose transport activity in 3T3-L1 adipocytes. In addition, they showed that overexpression of the dominant-negative mutant of PKC-lambda partially inhibited glucose transport activity, suggesting that PKC-lambda lies in the insulin signaling pathway responsible for regulating glucose uptake. In their study, overexpression of constitutively active PKC-lambda induced an ~600-fold increase in PKC-lambda activity, whereas insulin stimulated endogenous PKC-lambda activity by at most threefold. Despite this huge, nonphysiological increase in PKC-lambda activity with overexpression of the constitutively active mutant, glucose transport activity was rather smaller than that caused by insulin in control (noninfected) 3T3-L1 adipocytes. On the other hand, in the present study, a modest increase in atypical PKC activity, close to the physiological condition, was achieved. Expression of the wild-type PKC-zeta , PKC-lambda , and constitutively active PKC-lambda produced increases in atypical PKC activity to ~10-, 4-, and 33-fold, respectively, but these procedures did not affect glucose transport activity. Thus, under physiological conditions, an increase in atypical PKC activity is not sufficient for glucose transport activation. Furthermore, we detected no measurable activation of atypical PKC-lambda /zeta by insulin, whereas insulin fully activated (i.e., an ~9-fold increase) glucose transport activity. It is also noteworthy that exogenously expressed wild-type PKC-lambda /zeta was not significantly activated by insulin. On the contrary, when wild-type PKC-lambda was overexpressed in Chinese hamster ovary cells using an adenovirus-mediated gene transduction system, PKC-lambda was efficiently activated by insulin (data not shown), indicating that the adenovirus-mediated gene transduction system and our assay system for atypical PKC activity worked well and that insulin does not activate atypical PKC in 3T3-L1 adipocytes. Expression of dominant-negative mutants of atypical PKC enzymes did not influence insulin-stimulated glucose transport activity in the present study. In addition, we also observed that Go6983 (100 nM), which reportedly inhibits the activities of several PKC enzymes, including PKC-zeta , did not inhibit basal or insulin-stimulated glucose transport activity in 3T3-L1 adipocytes (data not shown). Taken together, these findings clearly show that activations of atypical PKC enzymes, PKC-lambda and PKC-zeta , do not play a major role in insulin signaling through glucose transport activation under physiological conditions. Recently, similar results were also reported in L6 myocytes, i.e., insulin did not significantly stimulate either endogenous atypical PKC-zeta /lambda or transfected hemagglutinin epitope-tagged PKC-zeta (44). Indeed, we observed that overexpression of constitutively active atypical PKC-lambda in undifferentiated L6 myoblasts using recombinant adenovirus increased basal glucose transport activity without altering basal or insulin-stimulated glucose transport activity in differentiated L6 myocytes (unpublished observation). Thus atypical PKC activation by insulin might depend on ambient factors such as the differentiation state of the cells, and activation of concomitant stimuli may also be important.

Regarding the mechanism whereby insulin stimulates glucose transport activity, 3-phosphoinositide-dependent protein kinase 1 (PDK1) and protein kinase B (PKB) have received considerable attention as signals downstream from PI 3-kinase activation. Several studies using the dominant-negative mutants of PKB were reported, but the effects on insulin-stimulated glucose transport and GLUT4 translocation were different among these mutants, such as the kinase-inactive mutant (14), the phosphorylation-deficient mutant (24), and the mutant having both mutations (44). It was recently reported that PDK1, which phosphorylates PKB and leads to its activation (3), also activates serine/threonine kinases, including atypical PKC (28), p70 (4, 40) and p90 (20) S6 kinase, cAMP-dependent protein kinase (13), and serum and glucocorticoid-inducible kinase (38). It is possible that each PKB mutant binds to PDK1 and affects PDK1 to activate these pathways in a different manner. Similarly, stable and long-term expression and extremely high levels of expression of atypical PKC or its mutants may also induce a feedback effect on upstream kinases, such as PDK1. The discrepancies among several reports may have arisen from the difference in periods and levels of expression of exogenous proteins. In the present study, by use of the adenovirus gene transduction system, transient physiological expression was achieved. Taken together, our results strongly suggest that atypical PKC isoforms do not play a major role in insulin-stimulated glucose transport under physiological conditions in 3T3-L1 adipocytes.


    ACKNOWLEDGEMENTS

This work was supported by Grant-in-Aid for Scientific Research no. 13470226 (to Y. Oka) and Creative Basic Research Grant no. 10NP0201 (to Y. Oka) from the Ministry of Education, Science, Sports and Culture of Japan.


    FOOTNOTES

Address for reprint requests and other correspondence: Y. Oka, Division of Molecular Metabolism and Diabetes, Department of Internal Medicine, Tohoku University Graduate School of Medicine, Seiryo-machi, Sendai, Miyagi 980-8574 (E-mail: oka{at}int3.med.tohoku.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

March 12, 2002;10.1152/ajpendo.00457.2001

Received 11 October 2001; accepted in final form 27 February 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ahmad, Z, Green FJ, Subuhi HS, and Watanabe AM. Autonomic regulation of type 1 protein phosphatase in cardiac muscle. J Biol Chem 264: 3859-3863, 1989[Abstract/Free Full Text].

2.   Akimoto, K, Takahashi R, Moriya S, Nishioka N, Takayanagi J, Kimura K, Fukui Y, Osada S, Mizuno K, Hirai S, Kazlauskas A, and Ohno S. EGF or PDGF receptors activate atypical PKClambda through phosphatidylinositol 3-kinase. EMBO J 15: 788-798, 1996[Web of Science][Medline].

3.   Alessi, DR, James SR, Downes CP, Holmes AB, Gaffney PR, Reese CB, and Cohen P. Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Balpha. Curr Biol 7: 261-926, 1997[Web of Science][Medline].

4.   Alessi, DR, Kozlowski MT, Weng QP, Morrice N, and Avruch J. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro. Curr Biol 8: 69-81, 1998[Web of Science][Medline].

5.   Bandyopadhyay, G, Sajan MP, Kanoh Y, Standaert ML, Quon MJ, Lea-Currie R, Sen A, and Farese RV. PKC-zeta mediates insulin effects on glucose transport in cultured preadipocyte-derived human adipocytes. J Clin Endocrinol Metab 87: 716-723, 2002[Abstract/Free Full Text].

6.   Bandyopadhyay, G, Standaert ML, Galloway L, Moscat J, and Farese RV. Evidence for involvement of protein kinase C (PKC)-zeta and noninvolvement of diacylglycerol-sensitive PKCs in insulin-stimulated glucose transport in L6 myotubes. Endocrinology 138: 4721-4731, 1997[Abstract/Free Full Text].

7.   Bandyopadhyay, G, Standaert ML, Kikkawa U, Ono Y, Moscat J, and Farese RV. Effects of transiently expressed atypical (zeta, lambda), conventional (alpha, beta) and novel (delta, epsilon) protein kinase C isoforms on insulin-stimulated translocation of epitope-tagged GLUT4 glucose transporters in rat adipocytes: specific interchangeable effects of protein kinases C-zeta and C-lambda. Biochem J 337: 461-470, 1999[Medline].

8.   Bandyopadhyay, G, Standaert ML, Zhao L, Yu B, Avignon A, Galloway L, Karnam P, Moscat J, and Farese RV. 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. J Biol Chem 272: 2551-2558, 1997[Abstract/Free Full Text].

9.   Berra, E, Diaz-Meco MT, Dominguez I, Municio MM, Sanz L, Lozano J, Chapkin RS, and Moscat J. Protein kinase C zeta isoform is critical for mitogenic signal transduction. Cell 74: 555-563, 1993[Web of Science][Medline].

10.   Braiman, L, Alt A, Kuroki T, Ohba M, Bak A, Tennenbaum T, and Sampson SR. Protein kinase Cdelta mediates insulin-induced glucose transport in primary cultures of rat skeletal muscle. Mol Endocrinol 13: 2002-2012, 1999[Abstract/Free Full Text].

11.   Braiman, L, Alt A, Kuroki T, Ohba M, Bak A, Tennenbaum T, and Sampson SR. Insulin induces specific interaction between insulin receptor and protein kinase C delta in primary cultured skeletal muscle. Mol Endocrinol 15: 565-574, 2001[Abstract/Free Full Text].

12.   Cheatham, B, Vlahos CJ, Cheatham L, Wang L, Blenis J, and Kahn CR. Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol Cell Biol 14: 4902-4911, 1994[Abstract/Free Full Text].

13.   Cheng, X, Ma Y, Moore M, Hemmings BA, and Taylor SS. Phosphorylation and activation of cAMP-dependent protein kinase by phosphoinositide-dependent protein kinase. Proc Natl Acad Sci USA 95: 9849-9854, 1998[Abstract/Free Full Text].

14.   Cong, LN, Chen H, Li Y, Zhou L, McGibbon MA, Taylor SI, and Quon MJ. Physiological role of Akt in insulin-stimulated translocation of GLUT4 in transfected rat adipose cells. Mol Endocrinol 11: 1881-1890, 1997[Abstract/Free Full Text].

15.   Czech, MP, and Corvera S. Signaling mechanisms that regulate glucose transport. J Biol Chem 274: 1865-1868, 1999[Free Full Text].

16.   Frevert, EU, and Kahn BB. Differential effects of constitutively active phosphatidylinositol 3-kinase on glucose transport, glycogen synthase activity, and DNA synthesis in 3T3-L1 adipocytes. Mol Cell Biol 17: 190-819, 1997[Abstract].

17.   Goodnight, J, Kazanietz MG, Blumberg PM, Mushinski JF, and Mischak H. The cDNA sequence, expression pattern and protein characteristics of mouse protein kinase C-zeta. Gene 122: 305-311, 1992[Web of Science][Medline].

18.   Haruta, T, Morris AJ, Rose DW, Nelson JG, Mueckler M, and Olefsky JM. Insulin-stimulated GLUT4 translocation is mediated by a divergent intracellular signaling pathway. J Biol Chem 270: 27991-27994, 1995[Abstract/Free Full Text].

19.   Hirai, S, Izumi Y, Higa K, Kaibuchi K, Mizuno K, Osada S, Suzuki K, and Ohno S. Ras-dependent signal transduction is indispensable but not sufficient for the activation of AP1/Jun by PKC delta. EMBO J 13: 2331-2340, 1994[Web of Science][Medline].

20.   Jensen, CJ, Buch MB, Krag TO, Hemmings BA, Gammeltoft S, and Frodin M. 90-kDa ribosomal S6 kinase is phosphorylated and activated by 3-phosphoinositide-dependent protein kinase-1. J Biol Chem 274: 27168-27176, 1999[Abstract/Free Full Text].

21.   Kanegae, Y, Lee G, Sato Y, Tanaka M, Nakai M, Sakaki T, Sugano S, and Saito I. Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase. Nucleic Acids Res 23: 3816-3821, 1995[Abstract/Free Full Text].

22.   Katagiri, H, Asano T, Inukai K, Ogihara T, Ishihara H, Shibasaki Y, Murata T, Terasaki J, Kikuchi M, Yazaki Y, and Oka Y. Roles of PI 3-kinase and Ras on insulin-stimulated glucose transport in 3T3-L1 adipocytes. Am J Physiol Endocrinol Metab 272: E326-E331, 1997[Abstract/Free Full Text].

23.   Katagiri, H, Asano T, Ishihara H, Inukai K, Shibasaki Y, Kikuchi M, Yazaki Y, and Oka Y. Overexpression of catalytic subunit p110alpha of phosphatidylinositol 3-kinase increases glucose transport activity with translocation of glucose transporters in 3T3-L1 adipocytes. J Biol Chem 271: 16987-16990, 1996[Abstract/Free Full Text].

24.   Kitamura, T, Ogawa W, Sakaue H, Hino Y, Kuroda S, Takata M, Matsumoto M, Maeda T, Konishi H, Kikkawa U, and Kasuga M. Requirement for activation of the serine-threonine kinase Akt (protein kinase B) in insulin stimulation of protein synthesis but not of glucose transport. Mol Cell Biol 18: 3708-3717, 1998[Abstract/Free Full Text].

25.   Kotani, K, Carozzi AJ, Sakaue H, Hara K, Robinson LJ, Clark SF, Yonezawa K, James DE, and Kasuga M. Requirement for phosphoinositide 3-kinase in insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Biochem Biophys Res Commun 209: 343-348, 1995[Web of Science][Medline].

26.   Kotani, K, Ogawa W, Matsumoto M, Kitamura T, Sakaue H, Hino Y, Miyake K, Sano W, Akimoto K, Ohno S, and Kasuga M. Requirement of atypical protein kinase clambda for insulin stimulation of glucose uptake but not for Akt activation in 3T3-L1 adipocytes. Mol Cell Biol 18: 6971-6982, 1998[Abstract/Free Full Text].

27.   Lee, LS, and Weinstein IB. Membrane effects of tumor promoters: stimulation of sugar uptake in mammalian cell cultures. J Cell Physiol 99: 451-460, 1979[Web of Science][Medline].

28.   Le Good, JA, Ziegler WH, Parekh DB, Alessi DR, Cohen P, and Parker PJ. Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1. Science 281: 2042-2045, 1998[Abstract/Free Full Text].

29.   Martin, SS, Haruta T, Morris AJ, Klippel A, Williams LT, and Olefsky JM. Activated phosphatidylinositol 3-kinase is sufficient to mediate actin rearrangement and GLUT4 translocation in 3T3-L1 adipocytes. J Biol Chem 271: 17605-17608, 1996[Abstract/Free Full Text].

30.   Miyake, S, Makimura M, Kanegae Y, Harada S, Sato Y, Takamori K, Tokuda C, and Saito I. Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome. Proc Natl Acad Sci USA 93: 1320-1324, 1996[Abstract/Free Full Text].

31.   Moriya, S, Kazlauskas A, Akimoto K, Hirai S, Mizuno K, Takenawa T, Fukui Y, Watanabe Y, Ozaki S, and Ohno S. Platelet-derived growth factor activates protein kinase C epsilon through redundant and independent signaling pathways involving phospholipase C gamma or phosphatidylinositol 3-kinase. Proc Natl Acad Sci USA 93: 151-155, 1996[Abstract/Free Full Text].

32.   Nakanishi, H, Brewer KA, and Exton JH. Activation of the zeta isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem 268: 13-16, 1993[Abstract/Free Full Text].

33.   Nishimura, H, and Simpson IA. Staurosporine inhibits phorbol 12-myristate 13-acetate- and insulin-stimulated translocation of GLUT1 and GLUT4 glucose transporters in rat adipose cells. Biochem J 302: 271-277, 1994[Medline].

34.   Niwa, H, Yamamura K, and Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108: 193-199, 1991[Web of Science][Medline].

35.   Ohno, S, Akita Y, Konno Y, Imajoh S, and Suzuki K. A novel phorbol ester receptor/protein kinase, nPKC, distantly related to the protein kinase C family. Cell 53: 731-741, 1988[Web of Science][Medline].

36.   Ohno, S, Mizuno K, Adachi Y, Hata A, Akita Y, Akimoto K, Osada S, Hirai S, and Suzuki K. Activation of novel protein kinases C delta and C epsilon upon mitogenic stimulation of quiescent rat 3Y1 fibroblasts. J Biol Chem 269: 17495-17501, 1994[Abstract/Free Full Text].

37.   Okada, T, Kawano Y, Sakakibara T, Hazeki O, and Ui M. Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin. J Biol Chem 269: 3568-3573, 1994[Abstract/Free Full Text].

38.   Park, J, Leong ML, Buse P, Maiyar AC, Firestone GL, and Hemmings BA. Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway. EMBO J 18: 3024-3033, 1999[Web of Science][Medline].

39.   Pessin, JE, Thurmond DC, Elmendorf JS, Coker KJ, and Okada S. Molecular basis of insulin-stimulated GLUT4 vesicle trafficking. Location! Location! Location! J Biol Chem 274: 2593-2596, 1999[Free Full Text].

40.   Pullen, N, Dennis PB, Andjelkovic M, Dufner A, Kozma SC, Hemmings BA, and Thomas G. Phosphorylation and activation of p70s6k by PDK1. Science 279: 707-710, 1998[Abstract/Free Full Text].

41.   Standaert, ML, Buckley DJ, Ishizuka T, Hoffman JM, Cooper DR, Pollet RJ, and Farese RV. Protein kinase C inhibitors block insulin and PMA-stimulated hexose transport in isolated rat adipocytes and BC3H-1 myocytes. Metabolism 39: 1170-1179, 1990[Web of Science][Medline].

42.   Standaert, ML, Galloway L, Karnam P, Bandyopadhyay G, Moscat J, and Farese RV. Protein kinase C-zeta as a downstream effector of phosphatidylinositol 3-kinase during insulin stimulation in rat adipocytes. Potential role in glucose transport. J Biol Chem 272: 30075-30082, 1997[Abstract/Free Full Text].

43.   Toker, A, Meyer M, Reddy KK, Falck JR, Aneja R, Aneja S, Parra A, Burns DJ, Ballas LM, and Cantley LC. Activation of protein kinase C family members by the novel polyphosphoinositides PtdIns-3,4-P2 and PtdIns-3,4,5-P3. J Biol Chem 269: 32358-32367, 1994[Abstract/Free Full Text].

44.   Wang, Q, Somwar R, Bilan PJ, Liu Z, Jin J, Woodgett JR, and Klip A. Protein kinase B/Akt participates in GLUT4 translocation by insulin in L6 myoblasts. Mol Cell Biol 19: 4008-4018, 1999[Abstract/Free Full Text].

Am J Physiol Endocrinol Metab 283(2):E338-E345
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society


This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
A. Carranza, P. L. Musolino, M. Villar, and S. Nowicki
Signaling cascade of insulin-induced stimulation of L-dopa uptake in renal proximal tubule cells
Am J Physiol Cell Physiol, December 1, 2008; 295(6): C1602 - C1609.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
T. Yuasa, R. Kakuhata, K. Kishi, T. Obata, Y. Shinohara, Y. Bando, K. Izumi, F. Kajiura, M. Matsumoto, and Y. Ebina
Platelet-Derived Growth Factor Stimulates Glucose Transport in Skeletal Muscles of Transgenic Mice Specifically Expressing Platelet-Derived Growth Factor Receptor in the Muscle, but It Does Not Affect Blood Glucose Levels
Diabetes, November 1, 2004; 53(11): 2776 - 2786.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
A. R. Shikhman, D. C. Brinson, and M. K. Lotz
Distinct pathways regulate facilitated glucose transport in human articular chondrocytes during anabolic and catabolic responses
Am J Physiol Endocrinol Metab, June 1, 2004; 286(6): E980 - E985.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. Nakayama, K. Mikoshiba, T. Yamamori, and K. Akagawa
Activation of Syntaxin 1C, an Alternative Splice Variant of HPC-1/Syntaxin 1A, by Phorbol 12-Myristate 13-Acetate (PMA) Suppresses Glucose Transport into Astroglioma Cells via the Glucose Transporter-1 (GLUT-1)
J. Biol. Chem., May 28, 2004; 279(22): 23728 - 23739.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
S. Perrini, A. Natalicchio, L. Laviola, G. Belsanti, C. Montrone, A. Cignarelli, V. Minielli, M. Grano, G. De Pergola, R. Giorgino, et al.
Dehydroepiandrosterone Stimulates Glucose Uptake in Human and Murine Adipocytes by Inducing GLUT1 and GLUT4 Translocation to the Plasma Membrane
Diabetes, January 1, 2004; 53(1): 41 - 52.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
M. Asghar, T. Hussain, and M. F. Lokhandwala
Overexpression of PKC-{beta}I and -{delta} contributes to higher PKC activity in the proximal tubules of old Fischer 344 rats
Am J Physiol Renal Physiol, December 1, 2003; 285(6): F1100 - F1107.
[Abstract] [Full Text]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
I. Talior, M. Yarkoni, N. Bashan, and H. Eldar-Finkelman
Increased glucose uptake promotes oxidative stress and PKC-{delta} activation in adipocytes of obese, insulin-resistant mice
Am J Physiol Endocrinol Metab, August 1, 2003; 285(2): E295 - E302.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
T. Imamura, J. Huang, I. Usui, H. Satoh, J. Bever, and J. M. Olefsky
Insulin-Induced GLUT4 Translocation Involves Protein Kinase C-{lambda}-Mediated Functional Coupling between Rab4 and the Motor Protein Kinesin
Mol. Cell. Biol., July 15, 2003; 23(14): 4892 - 4900.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
N. Brose and C. Rosenmund
Move over protein kinase C, you've got company: alternative cellular effectors of diacylglycerol and phorbol esters
J. Cell Sci., January 12, 2002; 115(23): 4399 - 4411.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
283/2/E338    most recent
00457.2001v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (20)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Tsuru, M.
Right arrow Articles by Oka, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tsuru, M.
Right arrow Articles by Oka, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online