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J.A. Haley Veterans’ Hospital Research Service and Department of
Internal Medicine (G.B., M.L.S., M.P.S., R.V.F.) University of
South Florida College of Medicine Tampa, Florida 33612
Department of Radiation Oncology (L.M.K.) Mayo Foundation
Rochester, Minnesota 55905
Hypertension-Endocrine Branch
(L.C., M.J.Q.) National Heart, Lung and Blood Institute
National Institutes of Health Bethesda, Maryland, 20892
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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, and the
translocation of epitope-tagged glucose transporter 4 (GLUT4) to the
plasma membrane, during insulin action in transiently transfected rat
adipocytes. Overexpression of wild-type PDK-1 provoked increases in the
activity of cotransfected hemagglutinin (HA)-tagged PKC- and
concomitantly enhanced HA-tagged GLUT4 translocation. Expression of
both kinase-inactive PDK-1 and an activation-resistant form of PKC-
that is mutated at Thr-410, the immediate target of PDK-1 in the
activation loop of PKC-, inhibited insulin-induced increases in both
HA-PKC- activity and HA-GLUT4 translocation to the same extent as
kinase-inactive PKC-. Moreover, the inhibitory effects of
kinase-inactive PDK-1 were fully reversed by cotransfection of
wild-type PDK-1 and partly reversed by wild-type PKC-, but not by
wild-type PKB. In contrast to the T410A PKC- mutant, an analogous
double mutant of PKB (T308A/S473A) that is resistant to PDK-1
activation had only a small effect on insulin-stimulated HA-GLUT4
translocation and did not inhibit HA-GLUT4 translocation induced by
overexpression of wild-type PDK-1. Our findings suggest that both PDK-1
and its downstream target, Thr-410 in the activation loop of PKC-,
are required for insulin-stimulated glucose transport. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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and -
via PI 3-kinase (1, 2, 3, 4, 5),
and these atypical PKCs appear to be required for insulin-stimulated
GLUT4 translocation and glucose transport in rat adipocytes (1, 2),
3T3/L1 adipocytes (3, 4), and L6 myotubes (5). Similarly, insulin
activates protein kinase B (PKB) via PI 3-kinase (6, 7), and, although
only a relatively small fraction of insulin-stimulated GLUT4
translocation in rat adipocytes appears to require PKB (8),
constitutively active PKB (8, 9, 10), like constitutively active PKC-
(1, 4, 5), provokes strong insulin-like effects on GLUT4 translocation
and/or glucose transport in rat adipocytes and 3T3/L1 adipocytes.
In addition to the above described similarities, PKB (11, 12) and
atypical PKCs (13, 14) may be activated by comparable or seemingly
related mechanisms that involve concomitant increases in
D3-PO4 polyphosphoinositides and action of a
3-phosphoinositide- dependent protein kinase-1 (PDK-1).
Accordingly, in conjunction with increases in D3-PO4
polyphosphoinositides, PDK-1 phosphorylates analogous sites in the
activation loops of both PKB and PKC-, viz., Thr-308 in
PKB (11, 12) and Thr-410 in PKC- (13, 14). Phosphorylation of these
sites by PDK-1 is thought to be required for and, indeed, may
facilitate other phosphorylations, including autophosphorylation, and
subsequent activation of PKB and PKC-. In the case of PKC-,
however, it has not been shown that PDK-1 is, in fact, required for 1)
insulin-induced increases in PKC- activity, or 2) insulin effects on
biological processes. Presently, we examined 1) the role of PDK-1 in
insulin-induced activation of PKC-, and 2) the importance of PDK-1
and its targets, Thr-308 in PKB and Thr-410 in PKC-, during insulin
stimulation of epitope-tagged GLUT4 translocation in transiently
transfected rat adipocytes.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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and 2, overexpression of wild-type (WT) PDK-1
in transiently transfected rat adipocytes provoked increases in the
translocation of hemagglutinin (HA)-tagged GLUT4 to the plasma membrane
that were approximately 55% of the increases observed with insulin
treatment. Insulin provoked further increases in HA-GLUT4 translocation
in cells overexpressing WT-PDK-1, presumably via activation of PI
3-kinase, increased availability of D3-PO4
polyphosphoinositides (see Refs. 3, 5, 15), and further activation
of PKC- (see Ref 14 . and below). In contrast to WT-PDK-1, expression
of kinase-inactive (KI) PDK-1 (Lys-110 mutated to Asn; see Ref. 13)
inhibited insulin-stimulated HA-GLUT4 translocation, to about the same
extent as KI-PKC- (Fig. 1), which is
known to inhibit this process (1, 2). As seen in Fig. 2, the inhibitory effect of KI-PDK-1 on
insulin-stimulated HA-GLUT4 translocation was reversed or prevented by
cotransfection of WT-PDK-1: this verified that inhibitory effects of
KI-PDK-1 were due to the specific mutation in its catalytic domain. Of
further note, the inhibitory effect of KI-PDK-1 on insulin-stimulated
HA-GLUT4 translocation was also partially reversed or prevented by
coexpression of WT-PKC- significantly, but not WT-PKB (Fig. 3): these, along with other findings (see
below), suggested that the inhibitory effect of KI-PDK-1 was more
dependent upon a failure of insulin to activate PKC-, as compared
with PKB.
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that is resistant to activation by PDK-1 (13, 14) inhibited
insulin-stimulated HA-GLUT4 translocation to the same extent as both
PDK-1 and KI-PKC-, i.e. by approximately 65% (Fig. 1
).
In comparison, expression of an analogous activation-resistant
double-mutant (T308A,S473A) form of PKB, which functions as a
dominant-negative for certain actions of insulin (but not glucose
transport) in 3T3/L1 adipocytes (16), inhibited insulin-stimulated
HA-GLUT4 translocation by only 20%; nevertheless, as discussed below,
this modest inhibitory effect of double-mutant PKB is similar to that
observed with KI-PKB in rat adipocytes (8). In comparing the effects of
these activation-resistant mutant forms of PKC- and PKB (see Figs. 1 and 4 and Ref 15), note that, relative to endogenous levels, increases
in the level of total PKB in cells transfected with the double PKB
mutant were, if anything, greater than the increases in total PKC-
observed in cells transfected with the T410A-PKC- mutant: thus, it
is clear that, relative to endogenous levels, there were marked
increases in levels of the expressed PKB double mutant in transfected
cells.
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.
For this purpose, we used an activation-resistant T410A mutant form of
PKC-, which is only weakly active and cannot be activated by
agonists (13), including, as we have recently confirmed, insulin
(not shown). As seen in Fig. 4, the T410A
mutant form of PKC- markedly inhibited the stimulatory
ef-fects of WT-PDK-1 on HA-GLUT4 translocation. The
analogous activation-resistant double-mutant (T308A,S473A) form of
PKB, which serves as effective dominant-negative signaling factor (see
Ref. 16), on the other hand, had only a small insignificant effect on
WT-PDK-1-stimulated HA-GLUT4 translocation (Fig. 4). In further support
of the possibility that PKC- serves as a downstream mediator during
the action of overexpressed WT-PDK-1, the cell-permeable myristoylated
PKC- pseudosubstrate inhibited WT-PDK-1-stimulated HA-GLUT4
translocation (Fig. 4). Note that the myristoylated PKC-
pseudosubstrate, which fully inhibits the activity of PKC- in
vitro (1), as well as the effects of insulin on glucose transport
and GLUT4 translocation (1, 2), also inhibits the activation of PKC-
by insulin in intact cells (our unpublished observations),
apparently by virtue of a requirement for autophosphorylation of
threonine-560 and other presently undefined sites after
PDK-1-dependent phosphorylation of threonine-410 in the activation loop
of PKC-. In contrast, the myristoylated PKC- pseudosubstrate does
not inhibit PKB activation or action (our unpublished
observations). From these findings, it may be surmised that 1) PKB
activation is not dependent upon PKC-, and that 2) inhibitory
effects of the PKC- pseudosubstrate on insulin- or PKD-1-stimulated
glucose transport and/or GLUT4 translocation cannot be explained by
inhibition of PKB.
The above-described findings suggested that PKC-, and/or PKC-,
since these atypical PKCs apparently function interchangeably in
supporting insulin-stimulated GLUT4 translocation in the rat adipocyte
(see Ref. 2), serves as a downstream effector(s) for PDK-1 during
insulin stimulation; in further support of this possibility, we found
that 1) transient expression of WT-PDK-1 markedly enhanced the activity
of coexpressed HA-tagged PKC- in control and insulin-treated
adipocytes (Fig. 5A), and 2) expression
of KI-PDK-1 inhibited the activation of HA-tagged PKC- by insulin
(Fig. 5, A and B). These findings provided clear evidence that PDK-1 is
required for insulin-induced activation of PKC-; they also provided
an explanation for the increase in HA-GLUT4 translocation caused by
overexpression of WT-PDK-1 in the absence of insulin (Figs. 1, 2, and 4), although, as is apparent, we did not observe a strict
proportionality between PKC- activation and HA-GLUT4 translocation
when effects of PDK-1 alone were compared with those of insulin. The
latter discrepancy could reflect a more generalized activation of
PKC- during simple PDK-1 overexpression, as opposed to more
compartmentalized increases in D3-PO4 polyphosphoinositides
and subsequent PKC- activation in specific pools, that probably
occur during insulin treatment. The discrepancy may also reflect the
participation of factors other than PI 3-kinase, PDK-1, PKC-, and
PKB during insulin-stimulated GLUT4 translocation.
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and
PKB allowed us to not only evaluate the role of PDK-1, but also to
directly compare the relative roles of two of its targets, PKC- and
PKB, in insulin-stimulated GLUT4 translocation. As is apparent, our
findings provided further independent support for the hypothesis (1, 2, 4) that PKC- and/or PKC- is/are required for insulin-stimulated
GLUT4 translocation in the rat adipocyte. PKB, on the other hand,
appeared to be required for only a small component of
insulin-stimulated GLUT4 translocation, as suggested by relatively mild
inhibitory effects of doubly mutated (T308A,S473A) PKB on
insulin-stimulated HA-GLUT4 translocation. Nevertheless, the presently
observed mild inhibitory effects of the activation-resistant PKB double
mutant were similar in magnitude to inhibitory effects of KI-PKB
observed previously (8), and it is therefore possible that a small
component of the insulin effect on GLUT4 translocation in the rat
adipocyte may require the activation of PKB. However, the situation in
3T3/L1 adipocytes may be different, as PKB was not found to be
required, even partially, for insulin-stimulated glucose transport in
these cells (16).
The strong activation of HA-PKC- caused by simple overexpression of
WT-PDK-1 was surprising, as it indicated that PDK-1 could act by mass
action without concomitant increases in PI 3-kinase activity and the
availability of D3-PO4 polyphosphoinositides. However,
overexpressed WT-PDK-1 may have amplified the effects of resting, but
nevertheless required levels of D3-PO4
polyphosphoinositides (see Ref. 14) and superimposed insulin effects on
HA-PKC- activity and HA-GLUT4 translocation, albeit smaller in cells
overexpressing WT-PDK-1, may reflect the activation of PI 3-kinase and
further increases in D3-PO4 polyphosphoinositides, followed
by activation of HA-PKC- and/or endogenous PKC-, particularly in
specific cellular locations that are important for GLUT4
translocation.
The above findings showed that expression of both the KI and the mutant
(T410A) forms of PKC-, as well as KI-PDK-1, inhibited
insulin-stimulated HA-GLUT4 translocation. A plausible mechanism for
these inhibitory effects was studied by examining the effects of these
inhibitory mutant kinases on the activation of intact PKC-. To this
end, we cotransfected plasmids encoding these inhibitory mutant
kinases, along with plasmid encoding HA-tagged PKC-. As seen in Fig. 5B, insulin-stimulated HA-PKC activity was inhibited to the extent of
80%, 50%, and 80% by expression of KI-PKC-, T410A-PKC-, and
KI-PDK-1, respectively. Importantly, basal HA-PKC activity was not
affected by coexpression of these inhibitory proteins, thus militating
against the possibility of nonspecific effects. Although the inhibitory
effects of these mutant proteins on insulin-stimulated HA-PKC-
enzyme activity did not match up perfectly with the inhibitory effects
on HA-GLUT4 translocation (cf. Figs. 1 and 5), it
nevertheless seemed plausible, if not likely, that the inhibition of
PKC- was an important mechanistic factor to explain the observed
inhibitory effects of KI-PKC-, T410A-PKC-, and KI-PDK-1 on
insulin-stimulated HA-GLUT4 translocation.
In contrast to the inhibitory effects of KI-PKC- and T410A-PKC-
on insulin-induced activation of both HA-tagged WT-PKC- and
HA-tagged GLUT4 translocation, the expression of these PKC mutants did
not inhibit the activation of AU1-tagged PKB by insulin (Fig. 6): clearly, the inhibitory effects of
these PKC- mutants could not be explained by changes in PKB
activity. On the other hand, as expected (13, 14), KI-PDK-1 inhibited
insulin-induced activation of AU1-PKB (Fig. 6).
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participates in insulin-induced translocation of GLUT4, there are
inherent caveats with this experimental approach, including 1) only a
small fraction of adipocytes are successfully transfected during
electroporation; 2) the expression of foreign mutated signaling
proteins in transfected cells is very large relative to the level of
endogenous wild-type proteins; 3) transfected epitope-tagged HA-GLUT4
may not be fully comparable to endogenous GLUT4; and 4) insulin effects
on HA-GLUT4 in cultured adipocytes are modest in comparison to insulin
effects on endogenous GLUT4 translocation and subsequent glucose
transport in fresh adipocytes (this largely reflects an artefactual
increase in basal activity during the overnight incubation of cultured
adipocytes). On the other hand, our findings with the present
transfection system are at least qualitatively similar to those
observed in studies of adenoviral transfer of KI-PKC- in 3T3/L1
adipocytes, in which insulin effects on endogenous GLUT4 translocation
and glucose transport were measured (4). Clearly, there is a need to
utilize various experimental approaches to test the hypothesis that PI
3-kinase/PDK-1-dependent activation of atypical PKCs, and/or ,
is required for insulin stimulation of glucose transport.
In summary, our findings show that PDK-1 and its immediate downstream
target, viz. the Thr-410 phosphorylation site in the
activation loop of PKC-, are required for 1) insulin-induced
activation of PKC-, and 2) insulin stimulation of GLUT4
translocation in the rat adipocyte. Further studies are needed to
determine whether insulin acutely regulates PDK-1 activity and/or
subsequent Thr-410 phosphorylation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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(FLAG-tagged); pCIS2/T308A, S473A-PKB; pCIS2/WT-PKB; pCDNA3/KI-PKC-
(HA-tagged); pCDNA3/WT-PKC-; or cDNA insert-free plasmids (vector
groups). Constructs for WT-PDK-1, KI-PDK-1, and T410A-PKC- were
kindly supplied by Dr. Alex Toker and are described more fully in Ref.
13 . The double-mutant form of PKB was made by site-directed mutagenesis
of the wild-type construct (8) using the MORPH mutagenesis kit
according to the manufacturer’s instructions (5' 
3', Inc.,
Boulder, CO). Mutagenic oligonucleotides 5'-CCA CTA TGA AGG
CAT TTT GCG GAA CGC CGG-3' and 5'-TTC CCC CAG TTC
GCC TAC TCG GCC AGT GGC ACA-3' created point
mutations T308A and S473A, as well as silent mutations that created a
new BglI site and disrupted an XmnI site.
Mutations were confirmed by direct sequencing. The pCDNA3/KI-PKC-
construct was described previously (1, 2, 3).
Cells were incubated overnight for 20–24 h to allow time for
expression of cDNA inserts (see Refs. 1, 2, 3). As shown in immunoblots
further described in the text, note that 1) HA-GLUT4 expression was not
altered by coexpression of various proteins; 2) there were large
increases in total cellular PDK-1, PKB, and PKC- in transfected
cells, as compared with cells transfected with vector alone (VEC); and
3) successfully transfected cells comprise only 5–10% of total cells,
and increases in expressed proteins in transfected cells are therefore
10–20 times greater than that suggested by simple inspection of
immunoblots of total cell lysates. Sources for antibodies used for
immunoblotting were: PDK-1 and PKB (Upstate Biotechnology, Inc., Lake Placid, NY); PKC-/ (Santa Cruz Biotechnology, Inc.), HA (Covance); and FLAG (Zymed Laboratories, Inc., South San Francisco, CA)].
After overnight incubation with plasmids, cells were washed and
incubated for 30 min in glucose-free Krebs Ringer phosphate (KRP)
medium containing 1% BSA, with or without 10 nM insulin as
indicated. Where indicated, some cells were treated with 50
µM myristoylated PKC- pseudosubstrate (MYR-PKC--PS)
[which inhibits PKC- and insulin-stimulated glucose transport (see
Ref. 1)] for 60 min before insulin treatment. After incubation, cell
surface HA-GLUT4 was measured, using mouse monoclonal anti-HA
antibodies (Covance, Berkeley, CA) and 125I-labeled sheep
antimouse IGG second antibody (Amersham Pharmacia Biotech), as described previously (1, 2).
To study HA-tagged PKC- activation, cells (0.8 ml 50% suspension)
were electroporated in the presence of 1 µg pCDNA3 containing cDNA
encoding HA-tagged PKC- (see Ref. 2), along with 7 µg pCDNA3
containing no insert (vector group) or cDNA insert encoding KI-PKC-,
T410A-PKC-, KI-PDK-1, or WT-PDK-1. After incubation for 20–24 h to
allow time for expression, cells were washed and incubated for 10 min
in glucose-free KRP medium with or without 10 nM insulin as
indicated. After incubation, cells were sonicated, and 400 µg cell
lysate protein were subjected to immunoprecipitation with mouse
monoclonal anti-HA antibodies (Covance), after which HA-PKC-
immunoprecipitates were collected (see Ref. 2), washed, and assayed for
PKC- enzyme activity as described previously (1, 2). Note that
expression and recovery of immunoprecipitable HA-PKC- was not
altered by coexpression of other proteins.
To study AU1-tagged PKB activation, cells (0.8 ml 50% suspension) were
electroporated in the presence of 3 µg pCDNA3 encoding AU1-tagged PKB
(cDNA prepared as in Ref. 17 and subcloned into pCDNA3), along with 7
µg pCDNA3 encoding KI-PKC-, T410A-PKC-, or KI-PDK-1. After
incubation for 20–24 h to allow time for expression, cells were washed
and incubated for 3 min with or without 10 nM insulin.
After incubation, cells were sonicated, and 200 µg cell lysate
protein were immunoprecipitated with mouse monoclonal anti-AU1 antibody
(Covance), after which AU1-PKB immunoprecipitates were collected and
assayed for enzyme activity as described previously (18).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by funds from the Department of Veterans Affairs Merit Review Program and NIH Research Grant 2R01DK-38079–09A1.
Received for publication April 28, 1999. Revision received June 16, 1999. Accepted for publication July 12, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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as a downstream
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M. L. Standaert, Y. Kanoh, M. P. Sajan, G. Bandyopadhyay, and R. V. Farese Cbl, IRS-1, and IRS-2 Mediate Effects of Rosiglitazone on PI3K, PKC-{lambda}, and Glucose Transport in 3T3/L1 Adipocytes Endocrinology, May 1, 2002; 143(5): 1705 - 1716. [Abstract] [Full Text] [PDF]
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 G. Bandyopadhyay, M. P. Sajan, Y. Kanoh, M. L. Standaert, M. J. Quon, R. Lea-Currie, A. Sen, and R. V. Farese PKC-{zeta} Mediates Insulin Effects on Glucose Transport in Cultured Preadipocyte-Derived Human Adipocytes J. Clin. Endocrinol. Metab., February 1, 2002; 87(2): 716 - 723. [Abstract] [Full Text] [PDF]
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S. K. Pandey, H.-J. He, A. Chesley, M. Juhaszova, M. T. Crow, and M. Bernier Wortmannin-Sensitive Pathway Is Required for Insulin-Stimulated Phosphorylation of Inhibitor {kappa}B{alpha} Endocrinology, February 1, 2002; 143(2): 375 - 385. [Abstract] [Full Text] [PDF]
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T. Teruel, R. Hernandez, and M. Lorenzo Ceramide Mediates Insulin Resistance by Tumor Necrosis Factor-{alpha} in Brown Adipocytes by Maintaining Akt in an Inactive Dephosphorylated State Diabetes, November 1, 2001; 50(11): 2563 - 2571. [Abstract] [Full Text] [PDF]
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A. Oku, M. Nawano, K. Ueta, T. Fujita, I. Umebayashi, K. Arakawa, T. Kano-Ishihara, A. Saito, M. Anai, M. Funaki, et al. Inhibitory effect of hyperglycemia on insulin-induced Akt/protein kinase B activation in skeletal muscle Am J Physiol Endocrinol Metab, May 1, 2001; 280(5): E816 - E824. [Abstract] [Full Text] [PDF]
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Y. Kanoh, G. Bandyopadhyay, M. P. Sajan, M. L. Standaert, and R. V. Farese Rosiglitazone, Insulin Treatment, and Fasting Correct Defective Activation of Protein Kinase C-{{zeta}}/{{lambda}} by Insulin in Vastus Lateralis Muscles and Adipocytes of Diabetic Rats Endocrinology, April 1, 2001; 142(4): 1595 - 1605. [Abstract] [Full Text]
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R. V. Farese Insulin-Sensitive Phospholipid Signaling Systems and Glucose Transport. Update II Experimental Biology and Medicine, April 1, 2001; 226(4): 283 - 295. [Abstract] [Full Text]
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G. Bandyopadhyay, Y. Kanoh, M. P. Sajan, M. L. Standaert, and R. V. Farese Effects of Adenoviral Gene Transfer of Wild-Type, Constitutively Active, and Kinase-Defective Protein Kinase C-{lambda} on Insulin-Stimulated Glucose Transport in L6 Myotubes Endocrinology, November 1, 2000; 141(11): 4120 - 4127. [Abstract] [Full Text] [PDF]
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