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.
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ABSTRACT |
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INTRODUCTION |
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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/
, PKB, and the glucose transport system. In this regard, overexpression of PKC
(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
/
, 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
/
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, glucose transport, and ERK in adipocytes and skeletal muscles of mice in which the PKC
gene was knocked out by homologous recombination. Our findings suggest that PKC
, even at physiological levels, serves as a tonic endogenous inhibitor of IRS-1-dependent PI3K, PKB, and PKC
during insulin stimulation of glucose transport and ERK in mouse skeletal muscles and adipocytes.
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RESULTS |
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Effects of Insulin on Activity of PKC and PKCß2 in Adipocytes and Skeletal Muscles
Because of the apparent inhibitory effects of PKC (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. 7
and 8
, insulin provoked increases in the activity of immunoprecipitable PKC
and PKCß2 in adipocytes of wild-type mice and rats. However, unlike PKC
/
activation (see Fig. 8
), these increases in PKC
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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To be certain that insulin effects seen in immunoprecipitates prepared with anti-PKC antiserum were truly reflective of PKC
, we simultaneously examined radioactivity in immunoprecipitates prepared from vastus lateralis muscles and adipocytes of wild-type and PKC
knockout mice. As seen in Fig. 7
, relatively small amounts of radioactivity (i.e. counts per minute above that seen in the preimmune blanks) were recovered in these assays of PKC
immunoprecipitates prepared from vastus lateralis muscles and adipocytes of PKC
knockout mice. This nonspecific radioactivity amounted to approximately 1020% 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
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
knockout mice. Although the source of this nonspecific radioactivity is uncertain (i.e. we did not detect other PKCs in PKC
immunoprecipitates), its presence, if anything, would have diminished, and caused an underestimation of, the relative effects of insulin on PKC
activity observed in immunoprecipitates prepared from wild-type mice. Accordingly, it could be argued that this nonspecific radioactivity observed in assays of PKC
knockout mice would have been a more appropriate blank (than routinely used preimmune serum immunoprecipitates) for assays of PKC
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.
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DISCUSSION |
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In addition to finding that PKC was not required for insulin-stimulated glucose transport, it was of interest to find that knockout of the PKC
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
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
. 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
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
in these tissues. On the other hand, we cannot rule out the possibility that increases in the activation of PI3K, PKB, and PKC
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
knockout mice. In any case, regardless of their upstream regulators, the presently observed increases in PKB and/or PKC
activation provide a plausible explanation for observed increases in insulin-stimulated glucose transport in adipocytes and soleus muscles of PKC
knockout mice.
It was also of interest to find that expression of wild-type PKC in adipocytes of PKC
-/- 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
contrasts with that of expressed wild-type PKC
or PKC
, 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
in adipocytes of PKC
-/- knockout mice to those of endogenous PKC
in adipocytes of control wild-type PKC
+/+ mice suggested that observed increases in signaling and glucose transport in muscles and adipocytes of PKC
-/- knockout mice can be attributed to the absence of endogenous levels of PKC
itself, rather than to other more chronic developmental alterations in signaling patterns that may arise during embryogenesis in PKC
-/- 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, and ß2, are activated by insulin. Whereas the PI3K inhibitor, wortmannin, completely or largely inhibits insulin-induced activation of immunoprecipitable PKC
/
in adipocytes (Ref. 2 and present results), wortmannin had little or no effect on insulin-induced activation of immunoprecipitable PKC
or PKCß2 in mouse and rat adipocytes. Thus, insulin effects on PKC
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 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
, 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
and PKCß2 in skeletal muscles and adipocytes, and, because increases in signaling to IRS-1, PI3K, PKC
, and PKB were evident in comparing PKC
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
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. This finding raised the possibility that enhanced activation of IRS-1, PI3K, PKB, and/or PKC
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
/
(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
, may not be required for ERK activation in this and possibly other skeletal muscles. If in fact PI3K and PKC
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
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, 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
-/- 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 gene was accompanied by increases in insulin-induced activation of IRS-1-dependent PI3K, PKB, PKC
, ERK, and 2-deoxyglucose uptake in both skeletal muscles and adipocytes. Moreover, unlike PI3K-dependent PKC
activation, insulin was found to activate PKC
and PKCß2 largely independently of PI3K. Our findings suggest that PKC
is not required for insulin-stimulated glucose transport, and, moreover, PKC
, even at physiological concentrations, apparently serves as an endogenous negative feedback inhibitor of insulin signaling through IRS-1, PI3K, PKB, and PKC
to the glucose transport system in both skeletal muscles and adipocytes.
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MATERIALS AND METHODS |
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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. 1A) 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 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-/+ 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
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
+/+ and PKC
-/- 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 in mouse skeletal muscle [which contains primarily PKC
, rather than PKC
(6)], mice, weighing approximately 3035 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
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
(rabbit polyclonal antiserum obtained from Santa Cruz Biotechnology, Inc. recognizes the C termini of both PKC
and PKC
) or ERK (rabbit polyclonal antiserum obtained from Santa Cruz Biotechnology, Inc.). Immunoprecipitates were collected on Sepharose-AG beads, and PI3K, PKB, PKC
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, PKB, PKC
and PKCß2 enzyme activity or, as another indicator of PKB activation, used to measure immunoreactive phosphoserine-473-PKB, as in muscle studies.
PKC/
Activation
PKC/
activity was measured as described previously (1, 2, 4). In brief, PKC
/
was immunoprecipitated with a rabbit polyclonal antiserum (Santa Cruz Biotechnology, Inc.) that recognizes the C termini of both PKC
and PKC
[however, mouse adipocytes contain mainly PKC
; 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 [
-32P]ATP (NEN Life Science Products, Boston, MA), 5 mM MgCl2 and, as substrate, 40 µM serine analog of the PKC
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 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
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, PKCß1, PKCß2, PKC-
, PKC
, and anti-PKC
/
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 and PKCß2 were immunoprecipitated from cell lysates with specific antisera obtained from Santa Cruz Biotechnology, Inc. and assayed as described above for PKC
, the only exception being that PKC
and PKCß2 assays were conducted in the presence of 1 mM CaCl2, 100 nM TPA (Sigma), and 40 µM PKC
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
and PKCß2 activity in these assays but was without effect in PKC
/
assays. As described below, wortmannin inhibited the activation of PKC
/
, but had little or no effect on the activation of PKC
and PKCß2. Blank values were determined from assays conducted with precipitates prepared with preimmune serum and were subtracted from immune serum values.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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