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Molecular and Cellular Biology, June 1999, p. 4008-4018, Vol. 19, No. 6
Programme in Cell Biology, The Hospital for
Sick Children, Toronto, Ontario M5G 1X8,1
Department of Biochemistry, The University of Toronto, Toronto,
Ontario M5S 1A8,2 and Experimental
Therapeutics, Ontario Cancer Institute, Toronto, Ontario M5G
2M9,3 Canada
Received 5 November 1998/Returned for modification 15 December
1998/Accepted 22 March 1999
L6 myoblasts stably transfected with a GLUT4 cDNA harboring an
exofacial myc epitope tag (L6-GLUT4myc myoblasts) were used to study
the role of protein kinase B alpha (PKB Translocation of GLUT4 from an
intracellular compartment to the plasma membrane largely accounts for
the stimulation of glucose transport by insulin in skeletal muscle
(16, 31, 38), cardiac muscle (48), and adipose
cells (23, 24). Two insulin-responsive cell lines also
express this transporter: L6 rat skeletal myotubes (34, 40)
and 3T3-L1 mouse adipocytes (24). Transfection of a
molecularly engineered form of this transporter containing an exofacial
epitope tag between the first and second transmembrane domains allows
for the detection of surface transporters in intact cells. GLUT4
molecules with an exofacial epitope tag have been heterologously
expressed in rat adipose cells (44, 51), 3T3-L1 adipocytes
(26), CHO cells (12, 26), H9c2 cardiomyocytes (55), and rat 3Y1 cells (22). We have recently
shown that stable expression of GLUT4myc in L6 myoblasts (L6-GLUT4myc
myoblasts) mimics the response to insulin seen with endogenous GLUT4 in
differentiated myotubes (29, 60).
Insulin-induced translocation of GLUT4 to the plasma membrane requires
the activity of phosphatidylinositol (PI) 3-kinase (47) in
rat adipocytes (43, 45), 3T3-L1 adipocytes (8, 9, 21,
27, 39, 51), L6 muscle cells (53), and rat skeletal
muscle (62). Moreover, treatment of intact 3T3-L1 adipocytes with a cell-permeant PI 3,4,5-triphosphate [PI
(3,4,5)-P3] compound, which is converted into a product of
PI 3-kinase once inside the cell, partly rescued the inhibition of
insulin-stimulated glucose transport by wortmannin (25). It
is unclear how the lipid products of PI 3-kinase relay the insulin
signal to the glucose transporters, but the serine/threonine kinase
protein kinase B (PKB)/Akt interacts with the lipid products of PI
3-kinase (19), and activation of PKB/Akt by insulin is
prevented by inhibitors of PI 3-kinase (1). To date, three
isoforms of PKB/Akt have been identified: PKB Recent reports have suggested that activation of PKB/Akt may mediate
the stimulation of glucose transport by insulin, since stable
overexpression of wild-type PKB In the present study, we examined the possible role of PKB in the
stimulation of GLUT4 translocation by insulin in muscle cells. L6
myoblasts expressing GLUT4myc were transiently transfected with a
PKB Materials.
Minimum essential medium Plasmids, cells, and transfections.
GLUT4myc cDNA was
constructed by inserting the human c-myc epitope (14 amino acids) into
the first ectodomain of GLUT4, subcloned into the pCXN2 vector, and
stably transfected into L6 myoblasts (26, 29). The open
reading frame of the bovine PKB cDNA was cloned into the eukaryotic
expression vector pcDNA3 (Invitrogen) and in the process was fused to
an N-terminal HA tag (recognized by the HA.11 antibody). HA-PKB K179A
was obtained by mutagenesis as described earlier (6) and was
employed as a template for additional point mutations using the
pALTER-1 plasmid (Promega). The oligonucleotides 5'-TCC CCC AGT TCG ACT
ACT CGG CTA GCG CGA CGG CC-3' and 5'-TCC CCC AGT TCG CCT ACT CGG CTA
GCG CGA CGG CC-3' were used to insert the T308A and S473A changes,
respectively. Mutations were confirmed by DNA sequencing. The construct
pcDNA3 GagPKB (6) was a kind gift from Paul J. Coffer
(University Hospital, Utrecht, The Netherlands). The construct
pSG5p85
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Protein Kinase B/Akt Participates in GLUT4
Translocation by Insulin in L6 Myoblasts
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
)/Akt1 in the insulin-induced
translocation of GLUT4 to the cell surface. Surface GLUT4myc was
detected by immunofluorescent labeling of the myc epitope in
nonpermeabilized cells. Insulin induced a marked translocation of
GLUT4myc to the plasma membrane within 20 min. This was prevented by
transient transfection of a dominant inhibitory construct of phosphatidylinositol (PI) 3-kinase (
p85). Transiently transfected cells were identified by cotransfection of green fluorescent protein. A
constitutively active PKB, created by fusion of a viral Gag protein
at its N terminus (GagPKB), increased the cell surface density of
GLUT4myc compared to that of neighboring nontransfected cells. A
kinase-inactive, phosphorylation-deficient PKB/Akt1 construct with
the mutations K179A (substitution of alanine for the lysine at position
179), T308A, and S473A (AAA-PKB) behaved as a dominant-negative
inhibitor of insulin-dependent activation of cotransfected wild-type
hemagglutinin (HA)-tagged PKB. Furthermore, AAA-PKB markedly inhibited
the insulin-induced phosphorylation of cotransfected BAD, demonstrating
inhibition of the endogenous PKB/Akt. Under the same conditions,
AAA-PKB almost entirely blocked the insulin-dependent increase in
surface GLUT4myc. PKB with alanine substitutions T308A and S473A
(AA-PKB) or K179A (A-PKB) alone was a less potent inhibitor of
insulin-dependent activation of wild-type HA-PKB or GLUT4myc
translocation than was AAA-PKB. Cotransfection of AAA-PKB with a
fourfold DNA excess of HA-PKB rescued insulin-stimulated GLUT4myc
translocation. AAA-PKB did not prevent actin bundling (membrane
ruffling), though this response was PI 3-kinase dependent. Therefore,
it is unlikely that AAA-PKB acted by inhibiting PI 3-kinase signaling.
These results outline an important role for PKB/Akt1 in the
stimulation of glucose transport by insulin in muscle cells in culture.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
, -
, and -
(Akt1,
-2, and -3) (17). In skeletal muscle and L6 muscle cells,
PKB and PKB, but not PKB, are stimulated by insulin
(59). Full activation of PKB/Akt by insulin requires hierarchical phosphorylation on two residues, Thr308 (Thr309 and Thr305
in the case of PKB and -, respectively) and Ser473 (Ser474 in the
case of PKB; PKB lacks an equivalent site) by
3-phosphoinositide-dependent protein kinase 1 (PDK-1) and PDK-2,
respectively (1-3, 14, 50).
/Akt1 or constitutively active
mutants of PKB/Akt1 increased glucose transport and translocation of
GLUT4 to levels similar to or greater than those achieved with insulin
in rat adipocytes (52), 3T3-L1 adipocytes (33,
56), and L6 muscle cells (20, 56). Stimulation of
glucose uptake was also observed in 3T3-L1 adipocytes expressing a
conditionally active membrane-targeted PKB/Akt1-mutant estrogen
receptor fusion protein that displays PKB activity only upon addition
of tamoxifen (32). Finally, treatment of isolated rat
adipocytes with insulin increased the presence of PKB/Akt2 protein
on immunopurified GLUT4 vesicles (7, 37), leading to
phosphorylation of GLUT4 vesicle proteins (37). Definitive
proof that PKB/Akt participates in insulin-dependent GLUT4
translocation, however, requires demonstration that ablation of the
kinase activity precludes the arrival of GLUT4 at the cell surface. Two
recent reports approached this question in rat adipocytes or 3T3-L1
adipocytes but yielded opposite results. In the first study, transient
overexpression of a kinase-inactive mutant of PKB/Akt1 (with a
substitution of alanine for lysine at position 179 [K179A]) in
isolated rat adipocytes reduced the sensitivity and the maximal
response of the GLUT4 response to insulin (11). The second
study showed that adenovirus-driven overexpression of a
phosphorylation-deficient PKB/Akt1 mutant (T308A S473A) did
not alter the stimulation of glucose transport and GLUT4 translocation
by insulin, despite markedly inhibiting endogenous PKB/Akt1
activation (30). Hence, the necessity for the stimulation of
GLUT4 translocation in two adipose cell types is controversial.
/Akt1 construct that combines the mutations seen in the
kinase-inactive and phosphorylation-deficient mutants of PKB/Akt1. We demonstrate that this construct has a dominant inhibitory action and
that it markedly inhibits insulin-induced GLUT4 translocation without
inhibiting insulin-stimulated, PI 3-kinase-dependent actin remodeling.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
, fetal bovine serum,
and other tissue culture reagents were purchased from Life
Technologies/GIBCO (Burlington, Ontario, Canada). Human insulin
(Humulin R) was obtained from Eli Lilly Canada Inc. (Toronto, Ontario,
Canada). pcDNA3 was purchased from Invitrogen. pEGFP was purchased from
Clontech (Palo Alto, Calif.). pEBG-mBAD and antibodies to BAD
(Bcl-associated death promoter) and phospho(Ser136)-BAD proteins were
purchased from New England Biolabs (Mississauga, Ontario, Canada).
Effectene transfection kits and plasmid DNA purification columns were
purchased from Qiagen (Mississauga, Ontario, Canada). Restriction
enzymes, ligase, and polymerase were purchased from New England
Biolabs. Oligonucleotides were purchased from Life Technologies/GIBCO. Protein A-Sepharose and protein G-Sepharose were from Pharmacia (Uppsala, Sweden). Polyclonal anti-Akt1 (raised to 20 C-terminal amino
acids), rabbit polyclonal anti-protein kinase C zeta (anti-PKC
; raised to 20 C-terminal amino acids), and monoclonal anti-myc (9E10)
antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz,
Calif.). Anti-hemagglutinin (anti-HA) antibody (HA.11) was purchased
from Babco (Berkeley, Calif.). Indocarbocyanine (Cy3)-conjugated goat
anti-mouse immunoglobulin G (IgG) and horseradish peroxidase
(HRP)-conjugated secondary antibodies were obtained from Jackson
ImmunoResearch (West Grove, Pa.). PKB/Akt substrate peptide (Crosstide)
was from Upstate Biotechnology (Lake Placid, N.Y.). Enhanced
chemiluminescence (ECL) reagent and [-32P]ATP (6,000 Ci/mmol) were purchased from Amersham (Oakville, Ontario, Canada).
Wortmannin and myelin basic protein were obtained from Sigma (St.
Louis, Mo.). Microcystin and okadaic acid were from Biomol (Plymouth
Meeting, Pa.). Rhodamine-phalloidin and Antifade mounting compound were
purchased from Molecular Probes (Leiden, The Netherlands). All
electrophoresis equipment and polyvinylidene difluoride membranes were
purchased from Bio-Rad (Mississauga, Ontario, Canada). All other
reagents were of the highest analytical grade.
SH2-N, commonly referred to as p85, the
dominant-negative mutant of the type I PI 3-kinase (46), was
a kind gift from Julian Downward (Imperial Cancer Research Fund, United
Kingdom), and the cDNA insert was subcloned into pcDNA3 for
experimentation. The wild-type, HA-tagged PKC construct subcloned
into plasmid pCDNA3 was a kind gift from Robert Farese (University of
South Florida).
supplemented with 10% fetal bovine serum in a
humidified atmosphere containing 5% CO2 and 95% air at
37°C (41). Transfections were performed in six-well plates according to the Effectene product manual (Qiagen). L6 myoblasts were
seeded at a density of 2 × 105 cells/well and
incubated overnight. DNA complexes were made at an 8:1 enhancer/DNA
ratio in all cases. However, in transfections that utilized less than 1 µg of DNA per well, the Effectene reagent was used at 6 µl per
condition. DNA was introduced to the cells at the start of the day for
5 h and then removed, and the cells were maintained for ca.
another 43 h until experimentation. For single-cell analysis of
GLUT4myc translocation or actin rearrangements, PKB or p85
constructs were cotransfected with 0.4 µg of pEGFP into L6-GLUT4myc
myoblasts grown on coverslips as indicated in the figure legends. For
PKB/Akt activity assays or expression of glutathione
S-transferase (GST)-BAD protein, transfections were also
performed in six-well plates as described in the figure legends.
Immunoprecipitation and assay of PKB/Akt kinase activity.
Immunoprecipitation of HA-PKB and the kinase assay were performed as
described previously (49). Cells were disrupted by addition
of a lysis buffer containing 50 mM HEPES (pH 7.6), 150 mM NaCl, 10%
(vol/vol) glycerol, 1% (vol/vol) Triton X-100, 30 mM sodium
pyrophosphate, 10 mM NaF, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 mM Na3VO4, 1 mM
dithiothreitol (DTT), and 100 nM okadaic acid. For each condition, HA
antibody (3 µg) was precoupled (16 h) to a mixture of protein A- and
protein G-Sepharose beads. These anti-HA-bead complexes were washed
twice with phosphate-buffered saline (PBS) and once with lysis buffer
(4°C). HA-PKB was immunoprecipitated by incubating 200 µg of total
cellular protein with the anti-HA-bead complexes for 2 to 3 h
with constant rotation (4°C). HA-PKB immunocomplexes were washed four
times with 1 ml of wash buffer (25 mM HEPES [pH 7.8], 10%
[vol/vol] glycerol, 1% [vol/vol] Triton X-100, 0.1% [wt/vol]
bovine serum albumin [BSA], 1 M NaCl, 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, 1 µM microcystin, and 100 nM okadaic acid) and twice with 1 ml of kinase buffer (50 mM Tris-HCl [pH 7.5],
10 mM MgCl2, and 1 mM DTT). The immunocomplexes were
incubated with constant agitation for 30 min at 30°C with 30 µl of
reaction mixture (kinase buffer containing 5 µM ATP, 2 µCi of
[-32P]ATP, and 100 µM Crosstide). Following the
reaction, 30 µl of the supernatant was transferred onto Whatman p81
filter paper and washed four times for 10 min each time with 3 ml of
175 mM phosphoric acid and once with distilled water for 5 min. The
filters were air dried and then subjected to liquid scintillation counting.
Immunoprecipitation and assay of atypical PKC activity.
Immunoprecipitation of PKC and assay of its kinase activity were
performed as described above for PKB with some modifications. Two types
of assays were used: immunoprecipitation of endogenous PKC from L6
myotubes, and immunoprecipitation of wild-type HA-tagged PKC
expressed in L6 myoblasts (generated by transfecting 0.4 µg of
HA-PKC or pcDNA3 vector into L6 myoblasts and assaying activity
48 h later). Cell lysates were prepared from transfected or
untransfected cells by using the lysis buffer described above. To
isolate wild-type PKC, anti-PKC antibody (C-20) or an irrelevant rabbit IgG was precoupled to protein A-Sepharose beads by incubating 2 µg of antibody per condition with 40 µl of the protein A-Sepharose beads (100 mg/ml) for a minimum of 2 h. To isolate HA-tagged
PKC, anti-HA antibody was precoupled to a 1:1 mixture of protein A- and protein G-Sepharose beads. Antibody-bead complexes were washed twice with ice-cold PBS and once with ice-cold lysis buffer. PKC was
isolated and washed as described above for Akt. The PKC
immunocomplex was then incubated with constant agitation for 10 min at
30°C with 30 µl of reaction mixture (kinase buffer containing 25 µM ATP, 5 µCi of [-32P]ATP, and 5 µM myelin
basic protein). Following the reaction, 30 µl of the supernatant was
transferred onto Whatman p81 filter paper and washed four times for 10 min each time with 3 ml of 175 mM phosphoric acid and once with
distilled water for 5 min. The filters were air dried and then
subjected to liquid scintillation counting.
Immunoblotting. Equal amounts of proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene difluoride filters as described previously (49). Immunoreactive bands were visualized either with HRP-conjugated sheep anti-mouse IgG for monoclonal antibodies or with horseradish peroxidase-conjugated goat anti-rabbit IgG for polyclonal antibodies (as indicated in the figure legends), using an ECL detection technique. Developed films were scanned and quantitated by using NIH Image software (National Institutes of Health, Bethesda, Md.). Cellular protein content was measured by the bicinchoninic acid method (5).
Indirect immunofluorescence and measurement of GLUT4myc translocation. Subconfluent L6-GLUT4myc myoblasts were grown on 25-mm-diameter glass coverslips in six-well plates and transfected as described above. Cells were deprived of serum in culture medium for 5 h prior to incubation with 100 nM insulin (20 min, 37°C). The following steps were performed at 4°C unless indicated otherwise. Cells were rinsed once with PBS prior to being incubated with 3% paraformaldehyde in PBS for 3 min and then PBS plus 100 mM glycine for 10 min. The cells were blocked with PBS containing 5% goat serum and 3% BSA (PBS-BSA plus GS) for 30 min. To detect GLUT4myc, coverslips were incubated with anti-myc antibody (9E10, 1:100) for 60 min, rinsed four times with PBS-BSA plus GS, and incubated with secondary antibody (Cy3-conjugated goat anti-mouse IgG, 1:1,000) for 30 min. The cells were rinsed four to six times in PBS and fixed in 3% paraformaldehyde for 30 min while shifting from 4°C to room temperature. The cells were incubated with PBS plus 100 mM glycine for 10 min, rinsed with PBS, and then rinsed with water before the coverslips were mounted and immediately analyzed by fluorescence microscopy with a Leica TCS 4D laser confocal fluorescence microscope.
Actin filament visualization. Subconfluent L6-GLUT4myc myoblasts were grown on 25-mm-diameter glass coverslips in six-well plates and transfected as described above. Cells were deprived of serum in culture medium for a total of 5 h prior to incubation with 100 nM insulin (5 min, 37°C) or, where indicated, 100 nM wortmannin (15-min pretreatment before addition of insulin and maintained during the insulin incubation). The cells were rinsed once with PBS (4°C) and then fixed with 3% paraformaldehyde for 30 min at room temperature followed by a 10-min wash in PBS plus 100 mM glycine. The cells were permeabilized with 0.1% (vol/vol) Triton X-100 in PBS for 10 min and rinsed twice with PBS. The cells were incubated with 0.2 U of rhodamine-phalloidin for 30 min prior to being rinsed four to six times with PBS and then with water before being mounted and immediately analyzed by fluorescence microscopy with a Leica inverted fluorescence microscope.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
AAA-PKB acts as a dominant-negative inhibitor of PKB/Akt1.
A dominant-negative PKB/Akt1 was created by substituting alanine
residues at the two major regulatory phosphorylation sites of
PKB/Akt1 (Thr308 and Ser473) and the phosphate transfer
residue in the catalytic site (Lys179) (AAA-PKB) (25a). To
demonstrate that AAA-PKB was kinase inactive, HA epitope-tagged AAA-PKB
and wild-type PKB (HA-AAA-PKB and HA-PKB, respectively) were
transiently expressed in L6-GLUT4myc myoblasts, independently. After
48 h, cells were either treated with 100 nM insulin for 10 min or
left untreated. HA-AAA-PKB and HA-PKB were immunoprecipitated from cell
lysates with an anti-HA epitope monoclonal antibody, and the samples
were processed for an in vitro PKB/Akt kinase assay. HA-PKB
immunoprecipitated from insulin-treated cells showed a mean increase in
kinase activity ± standard error (SE) of 18-fold ± 3-fold
relative to untreated (basal) cells (Fig.
1A). In contrast, the kinase activity
associated with HA-AAA-PKB was not elevated by insulin, as predicted
(Fig. 1A).
|
/Akt1.
BAD, a proapoptotic protein, is one of only a few well-characterized
substrates of PKB. Growth factors stimulate phosphorylation of BAD on
Ser136 by a PKB-dependent mechanism (13, 42), and this
response can be monitored with antibodies that specifically recognize
phosphorylated Ser136 on BAD. L6-GLUT4myc myoblasts were transiently
transfected with a GST-BAD fusion construct. Insulin treatment led to a
rapid elevation in Ser136 phosphorylation of BAD (by 5.5-fold ± 0.4-fold) (Fig. 2). Cotransfection of
AAA-PKB and GST-BAD markedly diminished the ability of insulin to
stimulate Ser136 phosphorylation of BAD (attaining an increase in
phosphorylation of only 2.2-fold ± 0.8-fold). This suggests that
AAA-PKB can effectively inhibit the activation of endogenous PKB
molecules by insulin. Together, the results shown in Fig. 1 and 2
indicate that AAA-PKB is a dominant-negative inhibitor of the
activation of wild-type PKB by insulin.
|
) or treated
with 100 nM insulin (+) for 10 min. Cells were lysed in
detergent-containing buffer as described in Materials and Methods and
then immunoblotted (IB) with anti-BAD (Characterization of a single-cell assay for GLUT4myc translocation in L6 myoblasts. L6-GLUT4myc myoblasts were used to establish a single-cell assay for GLUT4 translocation. The myc epitope is on the first extracellular segment of GLUT4, facilitating recognition of cell surface transporters by extracellular labeling without the need to perform subcellular fractionation, exofacial photolabeling, or plasma membrane lawn preparation. We recently reported that L6-GLUT4myc myoblasts respond to insulin by translocating GLUT4myc to the plasma membrane in a wortmannin-sensitive manner (60). Insulin caused a twofold increase in cell surface content of GLUT4myc and in glucose transport activity in L6-GLUT4myc myoblasts (60). Moreover, in these cells about 50% of the GLUT4myc protein was located intracellularly in a unique compartment enriched in the insulin-regulated aminopeptidase vp165 and relatively depleted of GLUT1 (56a). Thus, in L6 myoblasts, ectopic expression of GLUT4 generates an insulin-responsive phenotype.
To determine the effects of transient transfection of various cDNA constructs on the translocation of GLUT4myc to the cell surface, cotransfection with green fluorescent protein (GFP) cDNA was performed to facilitate recognition of transfected cells. Essentially, its use alleviated the need to permeabilize the cells to identify the expression of the transfected proteins. However, preliminary experiments were necessary to rule out the possibility that GFP expression itself interferes with insulin-stimulated GLUT4 translocation. Transfection of GFP alone (Fig. 3A, lower panels) did not alter cell surface GLUT4myc levels in either unstimulated cells (Fig. 3A, upper left panel) or cells treated with 100 nM insulin for 20 min (upper right panel) compared to untransfected neighboring cells. We next determined that GFP expression was a good indicator of coexpression of other DNA constructs. Equal amounts of GFP and HA-PKB DNA constructs were cotransfected into L6 myoblasts and processed for indirect immunofluorescence of the HA epitope. The left panel in Fig. 3B indicates the total number of cells present in the entire field of one representative experiment. The other panels in Fig. 3B illustrate that all of the GFP-positive cells (middle panel) in this field of cells were also positive for HA-PKB expression (right panel).
|
Inhibition of insulin-stimulated GLUT4 translocation by
dominant-negative PI 3-kinase in L6-GLUT4myc myoblasts.
Expression
of a dominant-negative construct of the p85 regulatory subunit of PI
3-kinase lacking the region that binds to the p110 catalytic subunit
(p85) inhibits insulin-induced GLUT4 translocation in rat fat
cells and 3T3-L1 adipocytes (35, 45). Transient transfection
of p85 in L6 myoblasts did not alter the level of cell surface
GLUT4myc in untreated (basal) cells compared to that in
untransfected neighboring cells (Fig. 3C, panel a). However,
p85 transfection completely blocked the insulin-stimulated translocation of GLUT4myc to the cell surface (Fig. 3C, panel b). This
experiment confirmed the dependence of GLUT4myc translocation on PI
3-kinase activity in L6 myoblasts.
A constitutively active PKB/Akt1 stimulates GLUT4myc
translocation.
Expression of constitutively active forms of
PKB/Akt1 in L6 muscle cells results in an increase in glucose
transport and in the amount of GLUT4 present at the plasma membrane
(20, 56). Here we coexpressed GFP with a constitutively
active PKB/Akt1 that has a viral Gag protein with its N-terminal
membrane-targeting myristoylation motif fused in frame to the N
terminus of PKB (6). The resulting PKB protein is located
almost exclusively at the plasma membrane and has a very high basal
activity level (57). Expression of GagPKB augmented the cell
surface density of GLUT4myc in the absence of insulin treatment (Fig.
3D, panel a) compared to the surrounding, untransfected cells. Insulin
treatment of cells expressing GagPKB slightly increased the
translocation of GLUT4myc to the cell surface compared to neighboring,
untransfected cells (Fig. 3D, panel b). Expression of wild-type,
HA-tagged PKB/Akt1 had no effect on either basal or
insulin-dependent surface exposure of GLUT4 (results not shown).
Expression of AAA-PKB inhibits GLUT4 translocation.
The
elevation of surface GLUT4myc levels in the L6 myoblasts expressing
GagPKB suggested that this kinase might be part of the signal
transduction pathway utilized by insulin to stimulate glucose
transport. To further investigate the role of PKB/Akt1 in the
stimulation of glucose transport by insulin, cells were transiently
transfected with HA-AAA-PKB, the dominant-negative inhibitor of PKB
activation by insulin. The effect of HA-AAA-PKB on insulin-induced
translocation of GLUT4 to the cell surface is illustrated in Fig.
4 (upper panels). The transfected cells were identified by their expression of GFP (shown in the lower panels).
HA-AAA-PKB had no detectable effect on the basal amount of GLUT4myc on
the cell surface compared to that of the surrounding, untransfected
cells (Fig. 4a). However, insulin could not stimulate GLUT4myc
translocation in cells that expressed HA-AAA-PKB compared to that of
the surrounding, untransfected cells (Fig. 4b).
|
AAA-PKB is a more effective inhibitor of GLUT4 translocation than
AA-PKB or A-PKB.
Two kinase-inactive mutants of PKB/Akt1 have
been suggested to have dominant inhibitory activity over the activation
of endogenous PKB by insulin or other growth factors. One of these
mutants has a K179A substitution (A-PKB) and has been referred to as
kinase dead since it cannot bind ATP or phosphorylate substrates
(11, 18). A nonactivatable mutant was also generated by
alanine substitutions T308A and S473A (AA-PKB) (30). This
mutant cannot receive activating inputs from the upstream kinases PDK-1
and PDK-2, and so it cannot be stimulated by growth factor receptor
signals. We compared the effectiveness of A-PKB and AA-PKB in
inhibiting the stimulation of GLUT4myc translocation and the activation
of HA-PKB by insulin in L6-GLUT4myc myoblasts with that of AAA-PKB.
L6-GLUT4myc myoblasts were transfected with 0.4 µg of either AAA-PKB,
AA-PKB, or A-PKB cDNA and then processed for GLUT4myc translocation as
described in the legend to Fig. 4. The intensity of the fluorescent
label of cell surface GLUT4myc was quantitated by using NIH Image
software. The pixel intensity of GLUT4myc staining per cell was
measured in similar numbers of transfected and nontransfected cells. A value of 100% was assigned to nontransfected, insulin-stimulated cells
within each field. The pixel intensity of the transfected cells in the
same field was then calculated as a fraction of this value. At least
three experiments using each of the PKB/Akt mutants were quantitated,
and the averaged results are shown in Fig.
5A. The pixel intensity in parallel
coverslips of unstimulated cells transfected with pcDNA3 and GFP only
was also measured and is indicated by the dotted line. Insulin caused a
1.9-fold increase in GLUT4myc above that in unstimulated cells (Fig.
5A). This degree of stimulation was not affected by the transfection of
pcDNA3. AAA-PKB significantly reduced the ability of insulin to recruit GLUT4myc to the cell surface, so that the hormone caused only a
0.6-fold increase (above that in unstimulated cells). Transfected AAA-PKB was significantly more effective than either AA-PKB or A-PKB at
inhibiting GLUT4 translocation. In fact, neither AA-PKB nor A-PKB
affected the increase in surface GLUT4myc in response to insulin
relative to untransfected or pcDNA3-transfected cells (Fig. 5A).
|
Coexpression of wild-type PKB with AAA-PKB restores GLUT4
translocation.
To further investigate whether the inhibitory
action of AAA-PKB was realized through its action on the PKB pathway,
cells were cotransfected with HA-AAA-PKB and HA-PKB at a DNA ratio of 1:4, with the rationale that excess wild-type PKB would restore insulin-stimulated GLUT4 translocation. The results of these
experiments are shown in Fig. 6. Cells
expressing GFP are identified by arrowheads. As seen above, insulin
could not stimulate the translocation of GLUT4myc in cells that
expressed HA-AAA-PKB (Fig. 6a). However, HA-AAA-PKB had no inhibitory
effect on insulin-induced GLUT4myc translocation in cells that were
cotransfected with four times more HA-PKB cDNA (Fig. 6b). These data
strongly support the hypothesis that AAA-PKB prevented GLUT4myc
translocation by inhibiting insulin activation of endogenous PKB.
|
PI 3-kinase-dependent actin reorganization is not affected by
AAA-PKB.
It is conceivable that the inhibition of GLUT4
translocation was a consequence of the ability of AAA-PKB to sequester
PI 3-kinase products, independent of its ability to inhibit PKB
activation. To examine this possibility, we determined the effect of
AAA-PKB on insulin-induced actin filament reorganization, a phenomenon that requires activation of PI 3-kinase in L6 muscle cells and 3T3-L1
adipocytes (39, 54). By comparison, the reorganization of
the actin cytoskeleton stimulated by platelet-derived growth factor is
also PI 3-kinase dependent but is also known to be PKB independent
in porcine aortic endothelial cells (15), pig aortic endothelial cells (61), and SKF5 neuroectodermal cells
(58). To explore the relationship between PKB/Akt and actin
reorganization in muscle cells, L6-GLUT4myc myoblasts cotransfected
with HA-AAA-PKB and GFP were treated for 5 min with insulin or were
left untreated. Cellular filamentous actin was then labeled with
rhodamine-conjugated phalloidin. Under basal conditions, phalloidin
labeled a large number of distinct actin filaments (Fig.
7a). Insulin treatment resulted in a
dramatic reorganization of actin, manifested as extensive bundling of
actin structures at the cell periphery and in distinctive actin spikes
within the cells (Fig. 7b, small arrowheads). In transfected cells
(indicated by large arrowheads), p85 had no effect on the
organization of actin in unstimulated cells but was able to completely
block the insulin-stimulated bundling of actin at the cell periphery
and the formation of actin spikes (Fig. 7a and b). Cells expressing
HA-AAA-PKB (Fig. 7c and d, arrowheads) did not have altered actin
filament organization in unstimulated cells. Nor did HA-AAA-PKB
interfere with insulin-induced actin bundling compared with that
observed in untransfected neighboring cells (Fig. 7c and d). The actin
filament reorganization induced by insulin was prevented by wortmannin
in all cells regardless of the presence or absence of AAA-PKB (Fig. 7e
and f). These results demonstrate that activation of PKB/Akt1 is not
required for actin bundling and furthermore suggest that the inhibitory
actions of AAA-PKB on GLUT4myc translocation are due to the ability of
this mutant to prevent the activation of endogenous PKB and are not attributable to inhibition of PI 3-kinase.
|
Measurement of atypical PKC activity.
L6 muscle cells express
atypical protein PKC (28) as detected with a polyclonal
antibody that also reacts with the 
isoform. To assess whether
PKC was activated by insulin, the endogenous enzyme was
immunoprecipitated from parental L6 myotubes with the same antibody. In
vitro kinase activity of the immunoprecipitates toward myelin basic
protein was measured. Immunoprecipitates from control cells showed
substantial activity above the background measured in irrelevant-IgG
immunoprecipitates. The background activity was only 7% of the
activity in PKC immunoprecipitates. Immunoprecipitates from L6
myotubes treated with insulin for 5 min did not show any further
increase in PKC activity relative to immunoprecipitates from control
cells (control, 1.00; insulin, 0.96 ± 0.08 [mean ± SE of
data from six independent experiments]). The PKC activity of
HA-tagged wild-type PKC transfected into L6-GLUT4myc myoblasts was
also determined. The transfected cells were incubated with insulin for
5 min, and HA-tagged proteins were immunoprecipitated for in vitro
PKC activity assays. As in the case of the endogenous kinase, the
exogenous enzyme was already active in immunoprecipitates from control
cells (the background amounted to only 25% of the activity) and
insulin stimulation did not increase the activity further (control,
1.00; insulin, 0.98 ± 0.005 [mean ± standard deviation of
two independent experiments]). Similar results were obtained when
cells were incubated with insulin for up to 15 min. Thus, contrary to a
previous report (4), we could not demonstrate
insulin-dependent activation of PKC in parental L6 myotubes or in
L6-GLUT4myc myoblasts.
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Inhibition of GLUT4myc translocation by AAA-PKB.
The PI
3-kinase dependence of insulin stimulation of glucose transporter
translocation has been clearly demonstrated in a variety of
insulin-responsive tissues and cell lines (47). How the PI
3-kinase lipid products mediate this translocation is still a point of
debate. Recent evidence indicates that the PH domain of the
serine/threonine kinase PKB/Akt1 binds PI (3,4)-P2 and PI (3,4,5)-P3 in membranes and that full stimulation of
this enzyme by insulin requires phosphorylation by the upstream kinases
PDK1 and PDK2 (2, 3, 50). Hence, the question of whether
PKB/Akt1 activation is required for the stimulation of glucose
uptake by insulin and other metabolic actions of the hormone has been
raised. Processes such as amino acid uptake, glycogen synthesis, and
glucose uptake were elevated in cells expressing constitutively or
conditionally active versions of PKB/Akt1 (20, 32, 33,
52). The use of a conditionally active PKB/Akt1 chimera was
the most informative of these approaches because the kinase could be
activated by tamoxifen with a time course closely resembling that of
the activation of the endogenous PKB/Akt1 by insulin
(32). Although these studies provide important insight into
the function of PKB/Akt1, they do not directly prove the
participation of PKB/Akt1 in the insulin-dependent regulation of
these metabolic functions.
/Akt1 in
the regulation of insulin-stimulated GLUT4 translocation in L6-GLUT4myc
myoblasts by transiently expressing the AAA-PKB/Akt1 mutant, which
cannot be phosphorylated by PDK1 or PDK2 and is itself kinase inactive.
We demonstrated that in L6-GLUT4myc myoblasts this mutant acts as a
dominant-negative inhibitor of insulin-stimulated PKB/Akt1, since it
could block the activation of HA-tagged wild-type PKB/Akt1 in
cotransfection experiments as well as insulin-stimulated phosphorylation of ectopically expressed BAD protein. These two approaches are complementary; the first demonstrates that AAA-PKB can
completely inhibit activation of PKB when the mutant is in excess of
the wild-type enzyme, while the second approach shows that AAA-PKB can
markedly inhibit the activation of endogenous PKB under a defined set
of transfection conditions. Therefore, the AAA-PKB mutant was tested
for its ability to interfere with insulin-dependent GLUT4myc
translocation under the same conditions. Measuring the
insulin-stimulated arrival of GLUT4myc at the cell surface, we observed
marked inhibition of this action of insulin in cells transfected with
AAA-PKB (Fig. 4 and 5A). Importantly, expression of excess wild-type
PKB could reverse the inhibitory effect of AAA-PKB (Fig. 6). This
latter result suggests that the AAA-PKB construct did not exert
nonspecific effects, e.g., irreversibly binding to upstream or
downstream signaling molecules and blocking their function.
To underscore the suggestion that AAA-PKB does not inhibit PI 3-kinase
activation or signaling, we analyzed the effects of AAA-PKB on the
insulin-dependent rearrangement of actin filaments in L6 myoblasts.
Insulin stimulates actin bundling under the plasma membrane (referred
to as membrane ruffling) in a PI 3-kinase-dependent manner
(39). In this study, we showed that inhibition of PI 3-kinase by wortmannin or by expression of a dominant-negative p85 inhibitory molecule of PI 3-kinase abolished
insulin-stimulated actin rearrangements in L6-GLUT4myc myoblasts.
Recently, actin rearrangements associated with membrane ruffling were
shown to be PKB/Akt independent in other cells treated with
platelet-derived growth factor (15, 58, 61). We observed
that AAA-PKB did not prevent the wortmannin-sensitive actin bundling
elicited by insulin. These results extend PKB independence to
insulin-induced actin bundling and demonstrate that PI 3-kinase
functions normally in the presence of AAA-PKB. Thus, AAA-PKB likely
prevented GLUT4myc translocation by inhibiting the activation of
endogenous PKB/Akt.
Effect of other PKB mutants on GLUT4 translocation. Attempts to inhibit the endogenous PKB/Akt activation and assess glucose transport regulation were made in two recent studies with different types of adipose cells. In the first study, cDNA encoding kinase-inactive A-PKB was introduced into rat adipocytes by electroporation and its effects on the appearance of cotransfected HA-tagged GLUT4 were tested after 20 h (11). In the second study, a cDNA encoding AA-PKB was introduced into 3T3-L1 adipocytes by adenovirus gene transfer and its effects were tested after 48 h (30). A-PKB caused a decrease in the sensitivity of insulin-stimulated HA-GLUT4 translocation and a partial (20%) reduction in the maximal mobilization of HA-GLUT4 to the cell surface of rat adipocytes (11). In contrast, AA-PKB did not prevent the stimulation of glucose transport by insulin or the translocation of the endogenous GLUT4 to the cell surface, despite preventing stimulation of protein synthesis by insulin (30). Unfortunately, due to experimental limitations, the former study could not demonstrate that the A-PKB mutant prevented PKB/Akt activation in rat adipocytes (11). On the other hand, AA-PKB was shown to significantly inhibit a large portion of the endogenous PKB/Akt activity on the basis of the reduced kinase activity remaining in the supernatant of lysates from cells transfected with HA-AA-PKB following the quantitative removal of HA-AA-PKB by immunoprecipitation (30). While this evidence is compelling, it is important to point out that approximately 15% of the insulin-stimulated activity of the endogenous PKB remained even at the highest dose of AA-PKB infection in that study (30). This represented a threefold increase in PKB activity above basal values. It is conceivable that this residual activation of PKB sufficed to stimulate glucose transport. The results from our study support this possibility. AA-PKB caused about an 80% inhibition of cotransfected HA-PKB but did not inhibit GLUT4myc translocation in L6 GLUT4myc myoblasts (Fig. 5). A more drastic inhibition of HA-PKB activity by AAA-PKB correlated with a substantial inhibition of GLUT4myc translocation. A-PKB was even less effective than AA-PKB at inhibiting HA-PKB activity, and like AA-PKB it was also unable to inhibit GLUT4myc translocation. We interpret these collective results to indicate that PKB activity may have to be inhibited substantially in order to result in an inhibition of GLUT4 translocation.
How is it envisaged that different PKB mutant forms have different dominant-negative effects? PKB forms multimeric complexes (possibly trimers) that are held in an inactive conformation by intermolecular interactions (10). PKB has no autokinase activity; its activation depends on phosphorylation of Thr308 and Ser473 by PDK1 and PDK2, which releases the inhibitory effect on a partnering PKB molecule (10). We predict that AAA-PKB would function as a complete inhibitor when two molecules of AAA-PKB complex with one wild-type PKB molecule. In this model, a complex consisting of two molecules of the other mutant, AA-PKB, and one wild-type PKB molecule could still retain up to one-third of its activity. That is, PDK phosphorylation of the wild-type PKB molecule in such a complex would derepress the activity of one of the AA-PKBs, allowing it to engage substrates. The mechanism described above lends support to the conclusion that the different PKB mutants reported in the literature and in the present study lead to different degrees of inhibition of wild-type PKB and that only when a nearly complete inhibition of the endogenous PKB is achieved is glucose transport inhibited. In addition, it is possible that different cell types have different levels of requirement for PKB in the stimulation of glucose transport, and part of this may be due to the requirement for specific PKB isoforms. In L6 muscle cells and skeletal muscle, the PKB/Akt2 isoform is not activated by insulin to
any measurable extent (59), whereas the PKB/Akt2 isoform
is more robustly activated by insulin than is PKB/Akt1 in rat
adipocytes (59). In addition to inhibiting the endogenous
PKB/Akt1 in 3T3-L1 adipocytes to a large but incomplete degree, the
AA-PKB mutant reduced the endogenous activity of PKB/Akt2 also to an
incomplete degree (a twofold stimulation in response to insulin was
still observed) (36). Clearly, sorting out the cell-specific, isoform-specific, and activity threshold requirements of
PKB will be essential to our understanding of the precise mechanism of
action of insulin.
While the manuscript was under review, Kasuga and colleagues reported
compelling evidence that a dominant-negative version of the atypical
PKC reduced the insulin-stimulated glucose uptake in 3T3-L1 cells by
50% (36). The possible contribution of atypical PKC to
GLUT4 translocation in L6-GLUT4myc myoblasts remains to be determined.
However, we were unable to show significant stimulation by insulin of
either endogenous atypical PKC/ or transfected HA-tagged PKC,
even though a significant level of basal activity was measured. These
results may underscore cell type-dependent engagements of the PKB and
PKC pathways.
It is quite possible that both types of targets of PI 3-kinase, namely,
the PKBs and atypical PKCs, contribute to the full translocation of
GLUT4 and stimulation of glucose uptake. The relative participation of
each pathway may be cell context dependent, and this context may
include the relative amounts and proportions of the different isoforms
within the two families. Ambient factors such as the state of
differentiation of the cell or tissue and the action of concomitant
stimuli may also be important. It is conceivable that PKBs and PKCs
provide the cells with a certain degree of redundancy and that their
interplay confers fine tuning of the insulin response.
In conclusion, the results presented here strongly suggest that
PKB/Akt1 participates in the stimulation of GLUT4 translocation in
muscle cells. Conversely, stimulation of actin remodeling by insulin,
which requires activation of PI 3-kinase, could be dissociated from the
activation of PKB/Akt1. The PKB/Akt1 mutant described here has
potent dominant inhibitory actions that will be useful in explorations
of the regulation of other physiological responses.
| |
ACKNOWLEDGMENTS |
|---|
This work was supported by a grant from the Medical Research Council of Canada to A.K. (MT7307). Q.W. was supported by a fellowship from the Eli Lilly/Banting & Best Diabetes Centre Research Personnel Awards Program.
We thank Gary Sweeney for helpful discussions. We are also grateful to
Paul Coffer for the GagPKB construct, Julian Downward for the p85
construct, Robert Farese for the Ha-PKC construct, and Richard Roth
for advice on the PKB in vitro kinase assay.
Q.W. and R.S. contributed equally to this study.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Programme in Cell Biology, The Hospital for Sick Children, 555 University Ave., Toronto, ON M5G 1X8, Canada. Phone: (416) 813-6392. Fax: (416) 813-5028. E-mail: amira{at}sickkids.on.ca.
| |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Beeson, M., Sajan, M. P., Dizon, M., Grebenev, D., Gomez-Daspet, J., Miura, A., Kanoh, Y., Powe, J., Bandyopadhyay, G., Standaert, M. L., Farese, R. V.
(2003). Activation of Protein Kinase C-{zeta} by Insulin and Phosphatidylinositol-3,4,5-(PO4)3 Is Defective in Muscle in Type 2 Diabetes and Impaired Glucose Tolerance: Amelioration by Rosiglitazone and Exercise. Diabetes
52: 1926-1934
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Katome, T., Obata, T., Matsushima, R., Masuyama, N., Cantley, L. C., Gotoh, Y., Kishi, K., Shiota, H., Ebina, Y.
(2003). Use of RNA Interference-mediated Gene Silencing and Adenoviral Overexpression to Elucidate the Roles of AKT/Protein Kinase B Isoforms in Insulin Actions. J. Biol. Chem.
278: 28312-28323
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Gual, P., Gonzalez, T., Gremeaux, T., Barres, R., Le Marchand-Brustel, Y., Tanti, J.-F.
(2003). Hyperosmotic Stress Inhibits Insulin Receptor Substrate-1 Function by Distinct Mechanisms in 3T3-L1 Adipocytes. J. Biol. Chem.
278: 26550-26557
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Sakoda, H., Gotoh, Y., Katagiri, H., Kurokawa, M., Ono, H., Onishi, Y., Anai, M., Ogihara, T., Fujishiro, M., Fukushima, Y., Abe, M., Shojima, N., Kikuchi, M., Oka, Y., Hirai, H., Asano, T.
(2003). Differing Roles of Akt and Serum- and Glucocorticoid-regulated Kinase in Glucose Metabolism, DNA Synthesis, and Oncogenic Activity. J. Biol. Chem.
278: 25802-25807
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Patel, N., Rudich, A., Khayat, Z. A., Garg, R., Klip, A.
(2003). Intracellular Segregation of Phosphatidylinositol-3,4,5-Trisphosphate by Insulin-Dependent Actin Remodeling in L6 Skeletal Muscle Cells. Mol. Cell. Biol.
23: 4611-4626
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Jiang, Z. Y., Zhou, Q. L., Coleman, K. A., Chouinard, M., Boese, Q., Czech, M. P.
(2003). Insulin signaling through Akt/protein kinase B analyzed by small interfering RNA-mediated gene silencing. Proc. Natl. Acad. Sci. USA
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Niu, W., Huang, C., Nawaz, Z., Levy, M., Somwar, R., Li, D., Bilan, P. J., Klip, A.
(2003). Maturation of the Regulation of GLUT4 Activity by p38 MAPK during L6 Cell Myogenesis. J. Biol. Chem.
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Brozinick, J. T. Jr, Roberts, B. R., Dohm, G. L.
(2003). Defective Signaling Through Akt-2 and -3 But Not Akt-1 in Insulin-Resistant Human Skeletal Muscle: Potential Role in Insulin Resistance. Diabetes
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Asante-Appiah, E., Kennedy, B. P.
(2003). Protein tyrosine phosphatases: the quest for negative regulators of insulin action. Am. J. Physiol. Endocrinol. Metab.
284: E663-E670
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Krieg, T., Landsberger, M., Alexeyev, M. F., Felix, S. B., Cohen, M. V., Downey, J. M.
(2003). Activation of Akt is essential for acetylcholine to trigger generation of oxygen free radicals. Cardiovasc Res
58: 196-202
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Chavez, J. A., Knotts, T. A., Wang, L.-P., Li, G., Dobrowsky, R. T., Florant, G. L., Summers, S. A.
(2003). A Role for Ceramide, but Not Diacylglycerol, in the Antagonism of Insulin Signal Transduction by Saturated Fatty Acids. J. Biol. Chem.
278: 10297-10303
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Greene, M. W., Sakaue, H., Wang, L., Alessi, D. R., Roth, R. A.
(2003). Modulation of Insulin-stimulated Degradation of Human Insulin Receptor Substrate-1 by Serine 312 Phosphorylation. J. Biol. Chem.
278: 8199-8211
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Martin, S., Vincent, J.-P., Mazella, J.
(2003). Involvement of the Neurotensin Receptor-3 in the Neurotensin-Induced Migration of Human Microglia. J. Neurosci.
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Park, D.-W., Kim, J.-R., Kim, S.-Y., Sonn, J.-K., Bang, O.-S., Kang, S.-S., Kim, J.-H., Baek, S.-H.
(2003). Akt as a Mediator of Secretory Phospholipase A2 Receptor-Involved Inducible Nitric Oxide Synthase Expression. J. Immunol.
170: 2093-2099
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Tremblay, F., Lavigne, C., Jacques, H., Marette, A.
(2003). Dietary Cod Protein Restores Insulin-Induced Activation of Phosphatidylinositol 3-Kinase/Akt and GLUT4 Translocation to the T-Tubules in Skeletal Muscle of High-Fat-Fed Obese Rats. Diabetes
52: 29-37
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Long, W., Barrett, E. J., Wei, L., Liu, Z.
(2003). Adrenalectomy enhances the insulin sensitivity of muscle protein synthesis. Am. J. Physiol. Endocrinol. Metab.
284: E102-E109
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Stolovich, M., Tang, H., Hornstein, E., Levy, G., Cohen, R., Bae, S. S., Birnbaum, M. J., Meyuhas, O.
(2002). Transduction of Growth or Mitogenic Signals into Translational Activation of TOP mRNAs Is Fully Reliant on the Phosphatidylinositol 3-Kinase-Mediated Pathway but Requires neither S6K1 nor rpS6 Phosphorylation. Mol. Cell. Biol.
22: 8101-8113
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Farhang-Fallah, J., Randhawa, V. K., Nimnual, A., Klip, A., Bar-Sagi, D., Rozakis-Adcock, M.
(2002). The Pleckstrin Homology (PH) Domain-Interacting Protein Couples the Insulin Receptor Substrate 1 PH Domain to Insulin Signaling Pathways Leading to Mitogenesis and GLUT4 Translocation. Mol. Cell. Biol.
22: 7325-7336
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Standaert, M. L., Ortmeyer, H. K., Sajan, M. P., Kanoh, Y., Bandyopadhyay, G., Hansen, B. C., Farese, R. V.
(2002). Skeletal Muscle Insulin Resistance in Obesity-Associated Type 2 Diabetes in Monkeys Is Linked to a Defect in Insulin Activation of Protein Kinase C-{zeta}/{lambda}/{iota}. Diabetes
51: 2936-2943
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Shiojima, I., Yefremashvili, M., Luo, Z., Kureishi, Y., Takahashi, A., Tao, J., Rosenzweig, A., Kahn, C. R., Abel, E. D., Walsh, K.
(2002). Akt Signaling Mediates Postnatal Heart Growth in Response to Insulin and Nutritional Status. J. Biol. Chem.
277: 37670-37677
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Berwick, D. C., Hers, I., Heesom, K. J., Moule, S. K., Tavare, J. M.
(2002). The Identification of ATP-citrate Lyase as a Protein Kinase B (Akt) Substrate in Primary Adipocytes. J. Biol. Chem.
277: 33895-33900
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Konrad, D., Bilan, P. J., Nawaz, Z., Sweeney, G., Niu, W., Liu, Z., Antonescu, C. N., Rudich, A., Klip, A.
(2002). Need for GLUT4 Activation to Reach Maximum Effect of Insulin-Mediated Glucose Uptake in Brown Adipocytes Isolated From GLUT4myc-Expressing Mice. Diabetes
51: 2719-2726
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Kim, Y.-B., Shulman, G. I., Kahn, B. B.
(2002). Fatty Acid Infusion Selectively Impairs Insulin Action on Akt1 and Protein Kinase C lambda /zeta but Not on Glycogen Synthase Kinase-3. J. Biol. Chem.
277: 32915-32922
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Webster, C. R. L., Srinivasulu, U., Ananthanarayanan, M., Suchy, F. J., Anwer, M. S.
(2002). Protein Kinase B/Akt Mediates cAMP- and Cell Swelling-stimulated Na+/Taurocholate Cotransport and Ntcp Translocation. J. Biol. Chem.
277: 28578-28583
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Hori, H., Sasaoka, T., Ishihara, H., Wada, T., Murakami, S., Ishiki, M., Kobayashi, M.
(2002). Association of SH2-Containing Inositol Phosphatase 2 With the Insulin Resistance of Diabetic db/db Mice. Diabetes
51: 2387-2394
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Tsuru, M., Katagiri, H., Asano, T., Yamada, T., Ohno, S., Ogihara, T., Oka, Y.
(2002). Role of PKC isoforms in glucose transport in 3T3-L1 adipocytes: insignificance of atypical PKC. Am. J. Physiol. Endocrinol. Metab.
283: E338-E345
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Ducluzeau, P.-H., Fletcher, L. M., Welsh, G. I., Tavare, J. M.
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Kanzaki, M., Pessin, J. E.
(2002). Caveolin-associated Filamentous Actin (Cav-actin) Defines a Novel F-actin Structure in Adipocytes. J. Biol. Chem.
277: 25867-25869
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Farese, R. V.
(2002). Function and dysfunction of aPKC isoforms for glucose transport in insulin-sensitive and insulin-resistant states. Am. J. Physiol. Endocrinol. Metab.
283: E1-E11
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Shiojima, I., Walsh, K.
(2002). Role of Akt Signaling in Vascular Homeostasis and Angiogenesis. Circ. Res.
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Chen, H. C., Bandyopadhyay, G., Sajan, M. P., Kanoh, Y., Standaert, M., Farese, R. V. Jr., Farese, R. V.
(2002). Activation of the ERK Pathway and Atypical Protein Kinase C Isoforms in Exercise- and Aminoimidazole-4-carboxamide- 1-beta -D-riboside (AICAR)-stimulated Glucose Transport. J. Biol. Chem.
277: 23554-23562
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