1 Division of Molecular Metabolism and Diabetes, Department of Internal Medicine, Tohoku University Graduate School of Medicine, Sendai, 980-8574; 2 Third Department of Internal Medicine, Yamaguchi University School of Medicine, Yamaguchi 755-8505; 3 Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Tokyo 113-8566; 4 Fourth Department of Medicine, Saitama Medical School, Saitama 350-0495; 5 Institute for Adult Disease, Asahi Life Foundation, Tokyo 160; 6 Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo 153-8904, Japan
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ABSTRACT |
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To investigate the role of
3-phosphoinositide-dependent protein kinase 1 (PDK1) in the
insulin-signaling pathway for glucose metabolism, wild-type (wt), the
kinase-dead (kd), or the plecstrin homology (PH) domain deletion
(PH) mutant of PDK1 was expressed using an adenovirus gene
transduction system in 3T3-L1 adipocytes. wt-PDK1 and kd-PDK1 were
found in both membrane and cytosol fractions, whereas
PH-PDK1, which
exhibited PDK1 activity similar to that of wt-PDK1, was detected
exclusively in the cytosol fraction. Insulin dose dependently activated
protein kinase B (PKB) but did not change atypical protein kinase C
(aPKC) activity in control cells. aPKC activity was not affected by
expression of wt-, kd-, or
PH-PDK1 in either the presence or the
absence of insulin. Overexpression of wt-PDK1 enhanced insulin-induced
activation of PKB as well as insulin-induced phosphorylation of
glycogen synthase kinase (GSK)3
/
, a direct downstream target of
PKB, although insulin-induced glycogen synthesis was not significantly enhanced by wt-PDK1 expression. Neither
PH-PDK1 nor kd-PDK1
expression affected PKB activity, GSK3 phosphorylation, or glycogen
synthesis. Thus membrane localization of PDK1 via its PH domain is
essential for insulin signaling through the PDK1-PKB-GSK3
/
pathway. Glucose transport activity was unaffected by expression of
wt-PDK1, kd-PDK1, or
PH-PDK1 in either the presence or the absence
of insulin. These findings suggest the presence of a signaling pathway
for insulin-stimulated glucose transport in which PDK1 to PKB or aPKC is not involved.
glycogen synthesis; glucose transport; GLUT4; protein kinase B; protein kinase C
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INTRODUCTION |
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INSULIN STIMULATES GLUCOSE UPTAKE into muscle and adipose tissues by redistribution of the insulin-responsive glucose transporter GLUT4 from intracellular stores to the plasma membrane. Insulin transmits its signals through a cell surface tyrosine kinase receptor, which stimulates multiple intracellular signaling events (49). Activated insulin receptors phosphorylate adapter proteins, members of the insulin receptor substrate family, which recruit and activate downstream effector molecules. Among the downstream proteins, activation of phosphatidylinositol 3-kinase (PI 3-kinase) is required for insulin's acute glucose metabolism-regulating actions, such as acceleration of glucose transport and glycogen synthesis.
PI 3-kinase (pharmacological) inhibitors, e.g., wortmannin and
LY-294002, reportedly completely inhibit the insulin-induced activation
of glycogen synthase in a variety of cell models (27, 35, 43,
45). Glycogen synthase kinase 3 (GSK3) plays an important role
in the regulation of glycogen synthesis via inhibitory phosphorylation
of glycogen synthase (22, 50). Two isoforms of GSK3,
GSK3 and -
, are broadly expressed and play multiple regulatory
roles in development and metabolism (40). GSK3 is constitutively active in cells and is transiently inhibited after insulin treatment (17). Insulin-induced inactivation of
GSK3 by insulin appears to be mediated by phosphorylation of GSK3 at Ser21 (
) or Ser9 (
) by protein kinase B
(PKB) (17), which was blocked by PI 3-kinase inhibitors.
Thus the PI 3-kinase-PKB-GSK3 pathway is proposed to mediate the
insulin-induced glycogen synthesis. On the other hand, it was
also reported that the insulin-specific stimulation of glycogen
synthase is mediated by activation of protein phosphatase 1 (PP1), a
Ser/Thr protein phosphatase. Insulin reportedly stimulates PP1,
followed by dephosphorylation/activation of glycogen synthase in
skeletal muscle and Swiss 3T3 cells, although the precise signaling
pathway is unknown (10, 38, 39, 46).
The PI 3-kinase inhibitors (15, 37) also reportedly inhibit insulin-stimulated glucose transport. In transfection studies, expressions of dominant negative mutants of PI 3-kinase (28, 32) have been shown to exert marked blocking effects on insulin-stimulated glucose transport and GLUT4 translocation in rat and 3T3-L1 adipocytes. Furthermore, overexpression of the wild-type (29) or constitutively active mutant (23, 51) of PI 3-kinase promoted glucose transport activity and GLUT4 translocation. Hence, these experiments collectively suggest that PI 3-kinase plays a central role in insulin-stimulated GLUT4 translocation. However, it is unclear how activation of PI 3-kinase relays the insulin signal to GLUT4 translocation. Recently, several pathways have been reported to be activated in response to stimulation with insulin and other growth factors downstream from PI 3-kinase. Among them, activations of Ser/Thr kinases, PKB (31), and atypical protein kinase C (aPKC) (8) are reportedly involved in insulin-stimulated glucose transport, although these claims are controversial (33, 56).
Activation of PKB- and PKC-
requires phosphorylation on
Thr308 and Thr410, respectively, by the Ser/Thr
kinase 3-phosphoinositide-dependent protein kinase 1 (PDK1)
(24). PDK1, which is expressed ubiquitously in human
tissue, is composed of an amino-terminal catalytic domain and a
noncatalytic carboxy-terminal tail containing a plecstrin homology (PH)
domain (3, 47). Recombinant PDK1 phosphorylates Thr308 of PKB-
directly in vitro, in a reaction that is
almost completely dependent on phosphatidylinositol 3,4,5-triphosphate
[PtdIns(3, 4, 5)P3] (3, 11).
However, increased PDK1 activity in response to growth factor
stimulation has not been observed.
Two recent reports have suggested that PDK1 is required for
insulin-stimulated translocation of epitope-tagged GLUT4 in rat adipocytes (7, 24), although the role of PDK1 in the
translocation of endogenous GLUT4 is unclear. Several recent reports
have raised the possibility that the behavior of overexpressed
epitope-tagged GLUT4 does not reflect that of the endogenous GLUT4
(1, 14, 41, 44, 53). In addition, the role of PDK1 in the
regulation of glycogen synthesis has not been studied. To investigate
the role of PDK1 in the insulin-signaling pathway involved in the regulation of glucose metabolism, such as acceleration of glucose transport and glycogen synthesis, wild-type PDK1 (wt-PDK1), the kinase-dead mutant (kd-PDK1), or the PH domain deletion mutant (PH-PDK1) of PDK1 was expressed using an adenovirus gene
transduction system in 3T3-L1 adipocytes. The effects of these mutants
on the pathway through glucose transport and glycogen synthesis were then examined.
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MATERIALS AND METHODS |
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Antibodies.
The antisera which recognized wild-type PKB and anti-GLUT4 antiserum
(29) were raised against synthetic peptides corresponding to residues 466-480 of rat PKB- and residues 495-509 of
rat GLUT4, respectively. The rabbit polyclonal anti-phospho-GSK3
/
(Ser21/9), anti-phospho-PKB (Thr308), and
anti-phospho-PKB (Ser473) antibodies were purchased from
New England Biolabs (Beverly, MA). Sheep polyclonal anti-PDK1
antibody for immunoprecipitation, which was raised against the PH
domain of PDK1, and anti-PDK1 antibody for immunoblotting, which was
raised against the full-length human PDK1, were purchased from Upstate
Biotechnology Institute (Lake Placid, NY). The mouse monoclonal
anti-GSK3
/
, the rabbit polyclonal anti-hemagglutinin (HA)
antibody, and the rabbit polyclonal anti-aPKC antibody were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell culture. 3T3-L1 fibroblasts were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% donor calf serum 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% fetal calf serum for 48 h. Cells were refed with DMEM supplemented with 10% fetal calf serum every other day for the following 4-10 days (29). More than 90% of the cells expressed the adipocyte phenotype.
Gene transduction.
3T3-L1 adipocytes were incubated in DMEM containing the recombinant
adenovirus, which encodes epitope-tagged PDK1, or kd-PDK1 (K111A), or
PH-PDK1 (59), at a multiplicity of infection of 200-300 pfu/cell for 1 h at 37°C. Growth medium was then
added. Experiments were performed 4-5 days after infection.
Exogenous protein expression was observed in >90% of 3T3-L1 adipocytes.
Immunoblotting. Cells, which had been serum starved and then incubated with or without 1-100 nM insulin were lysed and boiled in Laemmli buffer containing 10 mM dithiothreitol, subjected to SDS-polyacrylamide gel electrophoresis, and transferred onto nitrocellulose filters. The filters were incubated with the indicated antibodies and then with anti-rabbit, anti-mouse, or anti-sheep immunoglobulin G coupled to horseradish peroxidase. The immumoblots were visualized with an enhanced chemiluminescence detection kit (Amersham, Buckinghamshire, UK). The intensities of bands were quantified using an NIH Image 1.62 program. Values presented are means ± SD of three separate experiments.
Subcellular fractionation.
An abbreviated differential centrifugation procedure was used to obtain
the membrane and cytosol fractions from 3T3-L1 adipocytes expressing
wt-, kd-, or PH-PDK1. Cells were serum starved, incubated with or
without insulin, and homogenized in HES (20 mM HEPES, pH 7.4, 1 mM
EDTA, 250 mM sucrose), supplemented with 0.1 mM sodium orthovanadate
and 1 mM phenylmethylsulfonyl fluoride (PMSF). The homogenate was
centrifuged at 900 g for 10 min, and the resulting supernatant was centrifuged at 170,000 g for 75 min at
4°C, yielding the cytosol fraction as a supernatant. The pellet was
solubilized in ice-cold HES buffer containing 1% Triton X-100 and
fractionated by centrifugation at 175,000 g for 75 min at
4°C, yielding the membrane fraction as a supernatant.
PDK1 activity assay.
Cells were serum starved, incubated with or without 100 nM insulin for
5 min, and solubilized in ice-cold lysis buffer A (50 mM
Tris, pH 7.4, 100 mM NaCl, 10 mM EDTA, 10% glycerol, 1% Nonidet P-40,
1 mM sodium orthovanadate, 1 mM PMSF, 40 mM -glycerophosphate, and
50 mM NaF). Lysates were extracted by centrifugation at 15,000 g for 10 min followed by immunoprecipitation with anti-PDK1
antibody or anti-HA antibody. The Ser/Thr kinase activities of the
wt-PDK1 and its mutant were assayed in the immunoprecipitates, as
previously reported, by using a peptide sequence derived from the
activation loop of PKB-
(KTFCGTPEYLAPEVRR) as a substrate (21,
59).
aPKC activity assay.
Cells were serum starved, incubated with or without indicated
concentrations of insulin for 5 min, and solubilized in ice-cold lysis
buffer A. Lysates were extracted by centrifugation at 15,000 g for 10 min followed by immunoprecipitation with anti-aPKC
antibody. The immunoprecipitates were then assayed for the ability to
phosphorylate a PKC substrate peptide, namely
[Ser159]PKC-(153-164)-NH2
(Upstate Biotechnology), in reaction buffer containing 50 mM
Tris · HCl (pH 7.5), 5 mM MgCl2, 100 µM sodium orthovanadate, 100 µM sodium pyrophosphate, 1µM CaCl2,
1 mM NaF, 100 µM PMSF, and 50 µM [
-32P]ATP
(8).
PKB activity assay. Cells were serum starved, incubated with or without the indicated concentrations of insulin for 5 min, and solubilized in ice-cold lysis buffer A. Lysates were extracted by centrifugation at 15,000 g for 10 min followed by immunoprecipitation with anti-PKB antibody. The Ser/Thr kinase activity of the wild-type PKB was assayed in the immunoprecipitates as previously reported (17, 59) by using a peptide sequence derived from GSK3 (GRPRTSSFAEG) as a substrate (17).
Glycogen synthesis assay. 3T3-L1 adipocytes in a 12-well culture dish were serum starved overnight in DMEM containing 0.2% bovine serum albumin, and glucose-free incubation was performed for 45 min in Krebs-Ringer phosphate buffer (29). The cells were incubated for 15 min with or without the indicated concentration of insulin, and the reaction was initiated by the addition of [U-14C]glucose (2 µCi/sample) and glucose (5 mM final concentration). The assay was terminated after 1 h by washing with ice-cold PBS, and the cells were solubilized in 30% KOH (58). The glucose incorporation into glycogen was determined as described previously (26).
Glucose transport assay. 3T3-L1 adipocytes in a 12-well culture dish were serum and glucose starved as described for the glycogen synthesis assay. Cells were then incubated with or without the indicated concentration of insulin for 15 min, and 2-deoxy-D-[3H]glucose uptake was measured as described previously (29).
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RESULTS |
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We first studied the subcellular distribution of PDK1 expressed
exogenously using an adenovirus gene transduction system in 3T3-L1
adipocytes. HA-tagged wt-, kd-, or PH-PDK1 was expressed at similar
levels in 3T3-L1 adipocytes (Fig.
1A). Marked increases (~6.5-
to 7-fold) in expression levels of PDK1 proteins relative to endogenous
PDK1 were achieved, as demonstrated by immunoblotting with anti-PDK1
antibody (Fig. 1B). To examine intracellular localization of
wt- and mutant PDK1, these 3T3-L1 adipocytes were fractionated into
cytosol and membrane fractions. The GLUT1 glucose transporter, a
membrane protein, was detected almost exclusively in the membrane fraction but not detectable in the cytosol fraction (data not shown). As shown in Fig. 1C, immunoblotting with
anti-tag antibody revealed that wt-PDK1 and kd-PDK1 were found in both
membrane and cytosol fractions in the basal and insulin-stimulated
states. In contrast,
PH-PDK1 was detected almost exclusively in the
cytosol fraction but was barely detectable in the membrane fraction,
even in the insulin-stimulated state. These results demonstrate the PH
domain to be essential for membrane targeting of PDK1.
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Next, in 3T3-L1 adipocytes expressing wt-PDK1, kd-PDK1, or PH-PDK1,
the Ser/Thr kinase activity of PDK1 was measured in immunoprecipitates with anti-tag antibody (Fig.
2A) or antibody against the PH
domain of PDK1 [anti-PDK1(PH) antibody; Fig. 2B].
Overexpression of wt-PDK1 increased kinase activity 5.8-fold compared
with the endogenous PDK1 activity in the basal state (Fig.
2B). Insulin did not stimulate either endogenous or
exogenous wt-PDK1 activity (Fig. 2, A and B).
kd-PDK1 did not exhibit Ser/Thr kinase activity, as shown in Fig.
2A. In contrast,
PH-PDK1 exhibited Ser/Thr kinase
activity similar to that of wt-PDK1. Insulin did not affect
PH-PDK1
Ser/Thr kinase activity (Fig. 2A). As expected, the Ser/Thr
kinase activity of
PH-PDK1 was not detected in immunoprecipitates
with anti-PDK1 (PH) antibody (Fig. 2B). Expression of
neither kd-PDK1 nor
PH-PDK1 altered Ser/Thr kinase activity in
immunoprecipitates with anti-PDK1 (PH) antibody (Fig. 2B),
demonstrating that these mutants do not exert a dominant inhibitory
effect on the Ser/Thr kinase activity of endogenous PDK1.
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It has recently been reported that aPKC is one of the downstream
targets of PDK1 (16, 34). We therefore investigated the effects of expressing wt- or mutant PDK1 on aPKC activity. 3T3-L1 adipocytes overexpressing LacZ (control), wt-PDK1, kd-PDK1, or PH-PDK1 were stimulated with or without the indicated concentrations of insulin, and Ser/Thr kinase activity was measured in the
immunoprecipitates with anti-aPKC antibody (Fig.
3). In control cells, similar aPKC activities were observed in basal and insulin (1-100
nM)-stimulated states. Expression of wt-PDK1, kd-PDK1, or
PH-PDK1
did not alter aPKC activity in either the basal or the
insulin-stimulated state (Fig. 3). Thus insulin does not stimulate aPKC
activity in 3T3-L1 adipocytes, and wt-, kd-, or
PH-PDK1
overexpression has no effect on aPKC activity.
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PKB is another downstream target of PDK1. To investigate the effects of
PDK1 expression on PKB phosphorylation, we examined the phosphorylation
states of PKB with or without insulin stimulation in 3T3-L1 adipocytes
overexpressing LacZ (control), wt-PDK1, kd-PDK1, or PH-PDK1. As
shown in Fig. 4A, expression
of wt-PDK1 significantly enhanced insulin-induced PKB phosphorylation
at Thr308 ~1.7-fold, whereas expression of kd-PDK1 or
PH-PDK1 did not alter insulin-induced Thr308
phosphorylation of PKB compared with control cells. On the other hand,
expression of wt-PDK1, kd-PDK1, or
PH-PDK1 did not affect Ser473 phosphorylation of PKB with or without insulin
stimulation compared with control cells (Fig. 4B). To
further examine the effects of PDK1 protein expression on PKB, we
measured basal and insulin-stimulated PKB activity in
immunoprecipitates with anti-PKB antibody. In control cells, insulin
stimulated PKB activity dose dependently with a sixfold increase at 100 nM insulin. Overexpression of wt-PDK1 further enhanced insulin-induced
activation of PKB, which was more apparent at a low insulin
concentration (1 nM), whereas overexpression of wt-PDK1 did not affect
basal PKB activity (Fig. 4C). These findings suggest that
exogenously expressed wt-PDK1 activates PKB in the presence of insulin
but does not stimulate PKB activity without insulin, although the
activity of wt-PDK1 did not differ between the basal and
insulin-stimulated states (Fig. 2). In contrast, expression of kd-PDK1
did not significantly alter either basal or insulin-stimulated PKB
activity compared with the activities in control cells (Fig. 4).
Furthermore, expression of
PH-PDK1, which had Ser/Thr kinase
activity similar to that of wt-PDK1 (Fig. 2A), did not
affect either basal or insulin-stimulated PKB activity (Fig. 4). These
findings suggest that membrane localization of PDK1 via its PH domain
is important for activation of PKB in response to insulin.
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PI 3-kinase plays a central role in glucose metabolism, in response to
insulin, including such functions as acceleration of glycogen synthesis
and glucose transport. GSK3/
is one of the downstream targets of
PKB and is an upstream regulator of glycogen synthase. Activation of
PKB in response to insulin reportedly leads to phosphorylation of
GSK3
/
at Ser21/9 and thereby reduces its activity
(17). We first examined the effects of expressing wt-,
kd-, or
PH-PDK1 on the phosphorylation states of GSK3
/
at
Ser21/9 after 10 min of insulin stimulation. Expression
levels of endogenous GSK3
/
were not affected by overexpression of
wt-, kd-, or
PH-PDK1 in 3T3-L1 adipocytes (Fig.
5A). Insulin induced
phosphorylation of GSK3
/
at Ser21/9 in 3T3-L1
adipocytes expressing LacZ (control) in a dose-dependent manner (Fig.
5B). Overexpression of wt-PDK1 significantly
enhanced insulin-stimulated Ser21/9 phosphorylation of
GSK3
/
(Fig. 5B), whereas expression of kd-PDK1 or
PH-PDK1 did not significantly affect basal and insulin-stimulated phosphorylation states of GSK3
/
at Ser21/9 (Fig.
5B). The phosphorylation levels of GSK3
/
at
Ser21/9 (Fig. 5B) paralleled the PKB activities
(Fig. 4). Thus PDK1 is involved in the signaling pathway from insulin
receptor (IR) to GSK3
/
phosphorylation via PKB.
|
Next, we studied insulin-induced glycogen synthesis in 3T3-L1
adipocytes overexpressing LacZ (control), wt-PDK1, kd-PDK1, or
PH-PDK1 (Fig. 5C). In control cells, insulin dose
dependently stimulated glycogen synthesis. Overexpression of wt-PDK1
did not enhance the basal or insulin-stimulated glycogen synthesis
(Fig. 5C), despite the enhanced PKB activity and GSK3
/
phosphorylation by overexpression of wt-PDK1, which was more apparent
in low concentration of insulin (Figs. 4 and 5B). These
findings suggest that phosphorylation of GSK3
/
is not sufficient
for insulin-induced glycogen synthesis in 3T3-L1 adipocytes.
Furthermore, kd-PDK1 or
PH-PDK1 did not alter glycogen synthesis in
either the basal or the insulin-stimulated state (Fig. 5C).
PI 3-kinase also plays an important role in insulin-stimulated glucose
transport and translocation of GLUT4. To investigate the possible
involvement of PDK1 in this pathway, we examined the effect of
expressing the wt- and mutant PDK1 on insulin-stimulated glucose uptake
in 3T3-L1 adipocytes. 2-Deoxy-D-[3H]glucose
uptake in response to 1-100 nM insulin was measured in 3T3-L1
adipocytes expressing LacZ (control), wt-PDK1, kd-PDK1, or
PH-PDK1. Insulin stimulated 2-deoxyglucose uptake in a
dose-dependent manner in these cells, with a 10-fold increase at 100 nM
compared with the basal values (Fig. 6).
Basal and insulin-stimulated (1-100 nM) 2-deoxyglucose uptakes
were unaffected by expression of wt-, kd-, or
PH-PDK1 (Fig. 6). Thus
overexpression of wt-, kd-, or
PH-PDK1 has no influence on glucose
transport activity in 3T3-L1 adipocytes.
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DISCUSSION |
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In the present study, wt-PDK1 was active in the basal state, and
its activity was unaltered by insulin addition. Furthermore, expression
of wt-PDK1 enhanced insulin-induced PKB activation, but expression of
PH-PDK1, which exhibited Ser/Thr kinase activity similar to that
wt-PDK1, did not affect either basal or insulin-stimulated PKB
activity. These findings indicate that membrane localization of PDK1
via its PH domain is essential to insulin-induced PKB activation, which
is consistent with previous in vitro findings that
PH-PDK1 does not
activate PKB in lipid vesicles (20). PDK1 is unlikely
to be activated or inhibited by any extracellular signals tested to
date (55, 59). PtdIns(3,4)P2 and
PtdIns(3,4,5)P3, which are produced by growth
factor-activated PI 3-kinase, induced membrane localization of PKB via
its PH domain, followed by phosphorylations of PKB at
Thr308 and Ser473 by PDK1 and PDK2,
respectively (48), which are reportedly required for full
activation of PKB (2). In the present study, overexpession of wt-PDK1, but not
PH-PDK1, enhanced insulin-induced
Thr308 phosphorylation. Thus recruitment of PKB to the
membrane by insulin stimulation may allow PKB to be phosphorylated at
Thr308 by membrane-localized active PDK1, resulting in
activation of PKB. We cannot, however, rule out the possibility that a
PDK1-interacting protein, which is situated downstream from PI
3-kinase, enables PDK1 to phosphorylate PKB. Indeed, the interaction of
PDK1 with the PDK1-interacting fragment converts PDK1 into a kinase
that is capable of phosphorylating both Thr308 and
Ser473 of PKB and is directly activated by
PtdIns(3,4)P2 and
PtdIns- (3,4,5)P3 (4). PDK1 is
able to form complexes with various PKC family members
(34) and p70 S6 kinase (5, 42), and it is
also possible that these interactions modulate the PDK1 function. In
the present study, Ser473 phosphorylation was not affected
by wt-PDK1 or kd-PDK1 expression. No effects of wt-PDK1 or kd-PDK1
expression on Ser473 phosphorylation were observed either
in Chinese hamster ovary (CHO) cells, as reported previously
(59). Thus Ser473 phosphorylation appears to
occur through a different mechanism in vivo. We also reported that
phosphorylation of Thr308 of PKB, which is possibly
catalyzed by PP2A, was rapidly dephosphorylated in CHO cells, and that
kd-PDK1 has a dominant negative effect on dephosphorylation of
Thr308 (59). In contrast, the rapid
dephosphorylation of Thr308 or enhancement of
Thr308 phosphorylation induced by kd-PDK1 was not observed
in 3T3-L1 adipocytes. Thus the mechanism whereby Thr308 of
PKB is dephosphorylated apparently depends on cell types.
aPKC is another direct downstream target of PDK1 (16, 34)
that is reportedly involved in insulin-stimulated glucose transport (8, 33). However, in the present study, aPKC (- and
-isoforms) activity was not altered by either insulin addition or
the expression of wt-PDK1 or its mutants. To rule out the possibility
that the discrepancy between our results and those of other reports is attributable to a difference in the PKC activity assay system, we
employed several methods, including those described in previous reports
(8, 33). Activation of aPKC by insulin was not, however, observed (data not shown). In addition, enhancement of aPKC activity was observed in 3T3-L1 adipocytes with overexpression of the wild-type PKC-
or PKC-
by use of an adenovirus gene transduction system (53a), but the aPKC activity derived from exogenous aPKCs was not enhanced by insulin addition. Similar results were observed in L6
myoblasts expressing HA-tagged PKC-
(56). In contrast, when wild-type PKC-
or PKC-
was expressed in CHO cells stably expressing the IR (CHO-IR cells), insulin-induced activation of aPKC
activity was observed by use of the present kinase assay system
(53a). Recently, Balendran et al. (6) reported
that PKC-
in embryonic stem (ES) cells lacking PDK1
(PDK1
/
cells) is not phosphorylated at its T-loop
residue in contrast to that in PDK1+/+ ES cells. However,
they were unable to detect any significant activity of PKC-
after
the immunoprecipitation of PKC-
from PDK1+/+ or
PDK1
/
ES cells in either the basal state or after
stimulation with insulin-like growth factor I, serum, or
lysophosphatidic acid by use of the previously reported methods. Thus
the lack of aPKC activation by insulin in 3T3-L1 adipocytes is not due
to differences among kinase assay systems; rather, activation of aPKC
by insulin is likely to depend on the cell types or strains. In the
3T3-L1 adipocytes used in the present study, aPKC is unlikely to be
activated by insulin under the conditions in which insulin stimulates
glucose transport as well as GLUT4 translocation. In any case, aPKC is unlikely to be situated in the signaling pathway by which insulin stimulates glucose transport.
PKB reportedly phosphorylates GSK3 (17), resulting in the
inactivation of GSK3 in vitro (17). In addition, insulin
has been shown to rapidly inactivate GSK3 in several cell types
(12, 18, 19, 27, 35, 36, 57). The PI 3-kinase inhibitors, which block the inactivation of GSK3, also completely inhibit the
insulin-induced activation of glycogen synthase in a variety of cell
models (27, 35, 43, 45). Thus the PI 3-kinase-PKB-GSK3 pathway has been proposed to at least partially mediate insulin-induced glycogen synthesis. In the present study, the effects of expressing wt-PDK1, kd-PDK1, or PH-PDK1 on this pathway were examined in 3T3-L1
adipocytes. Although overexpression of wt-PDK1, but not of either
kd-PDK1 or
PH-PDK1, enhanced insulin-induced PKB activation and
phosphorylation of GSK3
/
at Ser21/9, which was more
apparent in low concentration of insulin, insulin-stimulated glycogen
synthesis was not affected by wt-PDK1 overexpression in 3T3-L1
adipocytes. These results are consistent with the recent findings that
expression of a membrane-targeted (constitutively active) form of PKB
failed to stimulate glycogen synthesis in 3T3-L1 adipocytes (31,
54). In addition, it was also reported that, in metabolically
mature 3T3-L1 adipocytes, the insulin-specific stimulation of glycogen
synthase is primarily mediated by PP1 activation rather than by GSK3
inactivation, whereas in 3T3-L1 preadipocytes, GSK3 inactivation is
likely to mediate the activation of glycogen synthase
(13). Thus these findings indicate that the PI
3-kinase-PKB-GSK3 signaling pathway is not sufficient for insulin-stimulated glycogen synthesis and that another major signaling pathway(s) mediating glycogen synthesis exists in 3T3-L1 adipocytes.
Whether PKB is involved in the signaling pathway from the IR through
GLUT4 translocation and glucose uptake is controversial. A PKB mutant
(PKB-AA), in which the phosphorylation sites (Thr308 and
Ser473) are replaced by alanines, reportedly lacks Ser/Thr
kinase activity and inhibits insulin-stimulated activation of
endogenous PKB (30). However, insulin-stimulated glucose
uptake in 3T3-L1 adipocytes was not affected by overexpression of
PKB-AA (30, 33). On the contrary, a kinase-inactive,
phosphorylation-deficient PKB construct with mutations of
Lys179Ala, Thr308Ala, and Ser473Ala
(PKB-AAA) behaved as a dominant negative inhibitor of insulin-dependent activation of cotransfected wild-type HA-tagged PKB. Furthermore, PKB-AAA almost entirely blocked the insulin-dependent increase in cell
surface myc-tagged GLUT4 in L6 myoblasts (56). In
addition, membrane-targeted (constitutively active) PKB reportedly
stimulates glucose uptake and GLUT4 translocation in 3T3-L1 adipocytes
(31), rat adipocytes (52), and L6 muscle
cells (25). In the present report, overexpression of
wt-PDK1, but not kd-PDK1 or PH-PDK1, enhanced insulin-induced PKB
activity at all insulin concentrations examined, especially the 1 nM
concentration. However, glucose uptake was unaffected by overexpression
of wt-PDK1 at any of the insulin concentrations examined, although
insulin-induced phosphorylation of GSK3
/
was enhanced by wt-PDK1
expression. These data indicate that an increase in PKB activity
induced by PDK1 overexpression is not sufficient for insulin-stimulated
glucose transport. This does not mean that PDK1 is not implicated in
the pathway for insulin-stimulated glucose transport but suggests the
presence of a signaling pathway in which PDK1 to PKB or aPKC is not
involved. Recently, it has been reported that insulin-stimulated GLUT4
translocation involves two independent pathways: a PI
3-kinase-dependent pathway and a PI 3-kinase-independent pathway
involving cobalamin (Cbl) and Cbl-associated protein (CAP)
(9). The PDK1-independent pathway, suggested in the
present study, seems to correspond to the Cbl-CAP pathway. However, an
unidentified pathway downstream from PI 3-kinase that does not involve
PDK1 might be also present, as we previously reported that
overexpression of wild-type PI 3-kinase in 3T3-L1 adipocytes induced
acceleration of glucose transport as well as GLUT4 translocation
(29). Further studies are required to clarify the pathway
mediating insulin-stimulated glucose transport.
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ACKNOWLEDGEMENTS |
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This work was supported by a Grant-in-Aid for Creative Research (10NP0201) and a Grant-in-Aid for Scientific Research (B2, 13470226) from the Ministry of Education, Science, Sports and Culture of Japan to Y. Oka.
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FOOTNOTES |
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Address for reprint requests and other correspondence: Y. Oka, Division of Molecular Metabolism and Diabetes, Dept. of Internal Medicine, Tohoku Univ. Graduate School of Medicine, Seiryo-Machi, Sendai, 980-8574, Japan (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.
First published February 19, 2002;10.1152/ajpendo.00486.2001
Received 31 October 2001; accepted in final form 1 February 2002.
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