Membrane-targeted Phosphatidylinositol 3-Kinase Mimics Insulin
Actions and Induces a State of Cellular Insulin Resistance*
Katsuya
Egawa
§,
Prem M.
Sharma
,
Naoki
Nakashima
¶,
Yi
Huang
,
Evana
Huver
,
Gerry R.
Boss
, and
Jerrold
M.
Olefsky
**
From the
Department of Medicine, Division of
Endocrinology and Metabolism, and the Whittier Diabetes Institute,
the
Department of Medicine, University of California, San Diego,
La Jolla, California 92093, and ** Veterans Administration Research
Service, San Diego, La Jolla, California 92161
 |
ABSTRACT |
Phosphatidylinositol (PI) 3-kinase
plays an important role in various insulin-stimulated biological
responses including glucose transport, glycogen synthesis, and protein
synthesis. However, the molecular link between PI 3-kinase and these
biological responses is still unclear. We have investigated whether
targeting of the catalytic p110 subunit of PI 3-kinase to cellular
membranes is sufficient and necessary to induce PI 3-kinase dependent
signaling responses, characteristic of insulin action. We overexpressed Myc-tagged, membrane-targeted p110
(p110CAAX), and wild-type p110
(p110WT) in 3T3-L1 adipocytes by adenovirus-mediated gene
transfer. Overexpressed p110CAAX exhibited
~2-fold increase in basal kinase activity in p110 immunoprecipitates, that further increased to ~4-fold with insulin. Even at this
submaximal PI 3-kinase activity, p110CAAX fully
stimulated p70 S6 kinase, Akt, 2-deoxyglucose uptake, and Ras, whereas,
p110WT had little or no effect on these downstream effects.
Interestingly p110CAAX did not activate MAP
kinase, despite its stimulation of p21ras. Surprisingly,
p110CAAX did not increase basal glycogen
synthase activity, and inhibited insulin stimulated activity,
indicative of cellular resistance to this action of insulin.
p110CAAX also inhibited insulin stimulated, but
not platelet-derived growth factor-stimulated mitogen-activated protein
kinase phosphorylation, demonstrating that the
p110CAAX induced inhibition of
mitogen-activated protein kinase and insulin signaling is specific, and
not due to some toxic or nonspecific effect on the cells. Moreover,
p110CAAX stimulated IRS-1 Ser/Thr
phosphorylation, and inhibited IRS-1 associated PI 3-kinase activity,
without affecting insulin receptor tyrosine phosphorylation, suggesting
that it may play an important role as a negative regulator for insulin
signaling. In conclusion, our studies show that membrane-targeted PI
3-kinase can mimic a number of biologic effects normally induced by
insulin. In addition, the persistent activation of PI 3-kinase induced
by p110CAAX expression leads to desensitization
of specific signaling pathways. Interestingly, the state of cellular
insulin resistance is not global, in that some of insulin's actions
are inhibited, whereas others are intact.
 |
INTRODUCTION |
One of the major physiological functions of insulin is to
stimulate glucose transport into insulin-sensitive tissues by eliciting translocation of the major insulin responsive glucose transporter, GLUT4 from an intracellular compartment to the plasma membrane (1, 2).
However, the signaling events that mediate insulin-stimulated glucose
transport and GLUT4 translocation are poorly understood. Binding of
insulin to its receptor results in receptor autophosphorylation and
activation of the receptor tyrosine kinase, followed by tyrosine phosphorylation of several intermediate proteins, such as, insulin receptor substrates: IRS-1, 2, 3, 4, and the adaptor protein, Shc
(3-5). Tyrosine-phosphorylated insulin receptor substrates and the
insulin receptor itself, then bind to Src homology 2 (SH2)1 domain containing
proteins, which further propagates downstream signals.
Phosphatidylinositol (PI) 3-kinase, a dual protein and lipid kinase is
one such signaling molecule (6). It consists of an 85-kDa regulatory
subunit and a 110-kDa catalytic subunit. Several isoforms of the
regulatory subunit, p55
, p55PIK, p85
, p85
, and two isoforms of
the catalytic subunit, p110
and p110
, have been identified. The
p85 subunit is composed of an NH2-terminal Src homology 3 (SH3) domain and two SH2 domains (7). The SH2 domains flank the region
where p110 associates with p85. The SH2 domains interact with
phosphotyrosine residues leading to activation of the p110 catalytic
subunit. The p110 subunit phosphorylates phosphoinositides at the
3'-position of the inositol ring to generate phosphoinositides 3-P,
3,4-P2, and 3,4,5-P3 (7). PI 3-kinase also phosphorylates proteins on
serine/threonine residues (8-10).
Ras is another signaling protein downstream of IRS-1 and Shc. After
insulin stimulation, tyrosine phosphorylated Shc, and IRS-1, to a
lesser extent, interact with another SH2 domain containing adaptor
protein-Grb2, which is pre-associated with Sos, a guanine nucleotide
exchange factor that promotes the formation of the active GTP-bound
state of Ras (11). Stimulated Ras then activates a cascade of protein
serine/threonine kinases, which include Raf, MEK, and the MAP kinases.
Although PI 3-kinase and Ras appear to be on separate pathways
branching from IRS-1, both have been implicated in pathways that
mediate the mitogenic actions of insulin, whereas the metabolic effects
of insulin are primarily activated by PI 3-kinase dependent steps.
Several lines of evidence indicate that activation of PI 3-kinase by
insulin is required for GLUT4 translocation. For example, the PI
3-kinase inhibitors, wortmannin and LY 294002 prevent GLUT4 translocation and stimulation of glucose transport in rat and 3T3-L1
adipocytes (12-14). Dominant-negative mutants of the 85-kDa subunit of
PI 3-kinase can also inhibit GLUT4 translocation in response to insulin
(15-17). However, several other observations suggest that, although
necessary, PI 3-kinase activation is not sufficient to promote glucose
transporter translocation. Indeed, growth factors such as
platelet-derived growth factor (PDGF) can stimulate PI 3-kinase as
efficiently as insulin, but have only a minor effect on GLUT4
translocation (18, 19). Similarly, interleukin 4, which induces
tyrosine phosphorylation of IRS-1 and PI 3-kinase activation, does not
stimulate GLUT4 translocation in L6 myoblasts (18). Furthermore,
subcellular fractionation analyses indicates that insulin, unlike other
growth factors, stimulates PI 3-kinase activity not only in the plasma
membrane fraction but also in the low density microsomal compartment
(20-22) and possibly even in GLUT4 containing subfractions of the low density microsomal of adipocytes (23, 24). Thus, it appears that
insulin-mediated subcompartmentalization of PI 3-kinase may be unique
and might be key to the specificity of the effect of insulin on glucose transport.
The aim of this study was to determine whether targeting of PI 3-kinase
catalytic subunit to membranous structures is sufficient to trigger
signaling events downstream of PI 3-kinase. This allows us to directly
study PI 3-kinase-regulated cellular processes in the absence of
insulin and to determine whether PI 3-kinase activation is sufficient
to trigger signaling events specific for insulin. Furthermore, it
avoids potential problems associated with the use of PI 3-kinase
inhibitors in elucidating the actions of this enzyme. We, and, others
have recently demonstrated that increased PI 3-kinase activity induced
by expression of a constitutively active p110 subunit (p110*) can
induce GLUT4 translocation (25), but it stimulates glucose transport
only partially in the absence of insulin (26). In contrast, our
membrane localized form of the p110 subunit of PI 3-kinase resulted in
activation of downstream mitogenesis effects in COS-7 cells (27). Since
gene transfer in 3T3-L1 adipocytes by conventional methods is
inefficient, in the current experiments we utilized,
adenovirus-mediated, high efficiency gene transfer procedures (26, 28,
29), and created an adenoviral vector containing the p110-
subunit
of PI 3-kinase incorporating a CAAX box at the COOH terminus
in order to target the p110 subunit to cellular membranes. Our studies
showed that expression of the membrane-targeted p110 subunit of PI
3-kinase in 3T3-L1 adipocytes were sufficient to induce PI 3-kinase
dependent downstream signaling events, including glucose transport.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Porcine insulin was kindly provided by Lilly.
Phospho-specific MAP kinase antibody and phospho-specific p70 S6 kinase
antibody were from New England Biolabs, Inc. Anti-human S6 kinase and
anti-IRS-1 antibody were from Upstate Biotechnology Incorporated (UBI).
c-Myc antibody (9E10), Akt antibody (C-20), p110
-CT antibody, H-Ras antibody, and horseradish peroxidase-linked anti-rabbit, mouse, and
goat antibodies were from Santa Cruz Biotechnology, Inc. Erk-1 antibody
and PY20H were from Transduction. Dulbecco's modified Eagle's medium
(DMEM) and fetal calf serum (FCS) were obtained from Life Technologies.
All radioisotopes were obtained from NEN Life Science Products Inc.
(Boston, MA). XAR-5 film was obtained from Eastman-Kodak (Rochester,
NY). All other reagents and chemicals were purchased from Sigma.
Cell Culture--
3T3-L1 cells were grown and maintained in DMEM
high glucose medium containing 50 units of penicillin/ml, 50 µg of
streptomycin/ml, and 10% FCS in a 10% CO2 environment.
The cells were allowed to grow 2 days postconfluency, then
differentiated as described earlier (29). Prior to experimentation, the
adipocytes were trypsinized and reseeded in the appropriate culture
dishes. The Ad-E1A-transformed human embryonic kidney cell line 293 was
cultured in DMEM high glucose medium containing 50 units of
penicillin/ml, 50 units of streptomycin/ml, and 10% FCS in a 5%
CO2 environment.
Plasmid Construction--
pSG5-p110WT, and
pSG5-p110CAAX were obtained as described
previously (30). The 9E10 epitope was inserted at the amino terminus of
bovine p110
cDNA, and the CAAX motif (CKCVLS) was
inserted at the COOH terminus for pSG5-p110CAAX.
To get pAC-p110WT and pAC-p110CAAX,
p110WT and p110CAAX DNA digested
with BamHI were cloned into pACCMVpLpA (vector containing an
ampicillin selection maker), and digested with EcoRI to
determine the direction.
Preparation of Recombinant Adenovirus--
The recombinant
adenoviruses containing the cDNA encoding the
p110CAAX or p110WT were isolated by
homologous recombination with two plasmids, pACCMVpLpA (31) and pJM17
(32) as described previously (29). The recombinant plasmids,
pAC-p110CAAX or pAC-p110WT, and
pJM17 were purified and co-transfected into 293 cells. Since 293 cells
were originally derived from adenovirus transformation, the missing E1
gene function of pJM17 is provided in trans. The resulting
recombinant viruses containing the p110CAAX or
p110WT are denoted as Ad5-p110CAAX
or Ad5-p110WT, respectively, and are replication defective
(at least in cells lacking the E1 region of adenovirus), but fully infectious.
Detection of Recombinant Ad5-p110CAAX and
Ad5-p110WT or Wild-type Virus in Cell Culture Medium by PCR
Amplification of Viral DNA--
DNA templates for PCR were extracted
from the supernatant of the culture medium of the 293 cells that were
infected with each plaque isolates/viruses at a multiplicity of
infection (m.o.i.) of 50 plaque-forming units/ml. A multiplex PCR was
performed on 1/10 dilution of virus DNA using E1A and E2B
region-specific primers (33) and analyzed for the presence of
recombinant or wild-type adenovirus. The presence of
p110CAAX and p110WT cDNA inserts
in the recombinant virus was confirmed by PCR analysis of viral DNA
with p110
cDNA specific primers. One clone of each of the
recombinant viruses was amplified further in 293 cells.
Cell Treatment--
3T3-L1 adipocytes were transduced at a
m.o.i. of 1-40 plaque-forming units/cell for 16 h with stocks of
either a control recombinant adenovirus (Ad5-CT) containing the
cytomegalovirus promoter, pUC 18 polylinker, and a fragment of the SV40
genome, the recombinant adenoviruses
Ad5-p110CAAX or Ad5-p110WT.
Transduced cells were incubated for 48 h at 37 °C in 10%
CO2 and DMEM high glucose medium with 2% heat-inactivated
serum, followed by incubation in the starvation media required for the
assay. The efficiency of adenovirus-mediated gene transfer was
approximately 90% as measured by immunocytochemistry. The survival of
the differentiated 3T3-L1 adipocytes was unaffected by incubation of
cells with the different adenovirus constructs since the total cell
protein remained the same in infected or uninfected cells.
Western Blotting--
3T3-L1 adipocytes uninfected or infected
with Ad5-p110CAAX, Ad5-p110WT, or
Ad5-CT were starved for 16 h in DMEM regular glucose media with
0.05% FCS. The cells were stimulated with 100 ng of insulin/ml for
5-30 min at 37 °C and lysed in a solubilizing buffer containing 20 mM Tris, 1 mM EDTA, 140 mM NaCl,
1% Nonidet P-40, 50 units of aprotinin/ml, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl
fluoride, 50 mM sodium fluoride, pH 7.5, for 30 min at
4 °C. The cell lysates were centrifuged to remove insoluble
materials. For Western blot analysis, whole cell lysates (20-80 µg
of protein per lane) were denatured by boiling in Laemmli sample buffer
containing 100 mM dithiothreitol and resolved by SDS-PAGE.
Gels were transferred to nitrocellulose by electroblotting in Towbin
buffer containing 0.02% SDS and 20% methanol. For immunoblotting,
membranes were blocked and probed with specified antibodies. Blots were
then incubated with horseradish peroxidase-linked second antibody
followed by chemiluminescence detection, according to the
manufacturer's instructions (Pierce).
PI 3-Kinase Assay--
3T3-L1 adipocytes were infected with
Ad5-p110CAAX, Ad5-p110WT, or Ad5-CT
at the indicated m.o.i.s for 16 h at 37 °C and grown in medium
containing heat-inactivated serum (2%) for 52 h. Serum-starved (16 h) cells were incubated in the absence (basal) or presence of
insulin (100 ng/ml) for 10 min, washed once with ice-cold
phosphate-buffered saline, lysed, and subjected to immunoprecipitation
(300-500 µg of total protein) with antibodies to p110
-CT, c-Myc
(2 µg), or IRS-1 (4 µg) overnight at 4 °C. Immune complexes were
precipitated from the supernatant with protein G plus (Santa Cruz) or
protein A and washed as described (34). The washed immune complexes were incubated with phosphatidylinositol (Avanti) and
[
-32P]ATP (3000 Ci/mmol) for 10 min at room
temperature. Reactions were stopped with 20 ml of 8 N HCl
and 160 ml of CHCl3:methanol (1:1) and centrifuged, and the
lower organic phase was removed and applied to a silica gel thin-layer
chromatography (TLC) plate (Merck) which had been coated with 1%
potassium oxalate. TLC plates were developed in
CHCl3:CH3OH:H2O:NH4OH
(60:47:11.3:2), dried, and visualized, and quantitated on a Molecular
Dynamics PhosphorImager.
2-Deoxyglucose Transport--
The procedure for glucose
transport was a modification of the methods described by Klip et
al. (35). Differentiated 3T3-L1 adipocytes were infected with
Ad5-p110CAAX, Ad5-p110WT, or Ad5-CT
at the indicated m.o.i.s for 16 h at 37 °C and grown in medium
containing heat-inactivated serum (2%) for 72 h. Serum and
glucose-deprived cells were incubated in MEM in the absence (basal) or
presence of 100 ng of insulin/ml for 1 h at 37 °C. Glucose
uptake was determined in duplicate or triplicate at each point after
the addition of 10 µl of substrate (2-[3H]deoxyglucose
or L-[3H]glucose; 0.1 µCi, final
concentration 0.01 mmol/liter) to provide a concentration at which cell
membrane transport is rate-limiting. The value for
L-glucose was subtracted to correct each sample for the
contributions of diffusion and trapping.
Glycogen Synthase Activity--
Glycogen synthase activity was
determined as described previously (36). Differentiated 3T3-L1
adipocytes were infected with Ad5- p110CAAX,
Ad5-p110WT, or Ad5-CT at 40 m.o.i. for 16 h at
37 °C and grown in medium containing heat-inactivated serum (2%)
for 72 h. The cells were serum and glucose-starved in DMEM no
glucose, 0.1% BSA, 2 mM pyruvate medium for 3 h, then
stimulated with or without 200 ng of insulin/ml for 30 min in 5 mM glucose containing medium. Cells were washed with
ice-cold phosphate-buffered saline three times, scraped in the buffer
containing 50 mM Tris-HCl, 10 mM EDTA, 100 mM potassium fluoride, pH 7.4, and sonicated. After
centrifugation, protein concentration was measured. 10 µg of protein
was used to determine the ability to stimulate incorporation of
[14C]UDP-glucose into glycogen in the presence and
absence of glucose 6-phosphate.
Ras GTP/GDP Assay--
Differentiated 3T3-L1 adipocytes were
infected with Ad5-p110CAAX or Ad5-CT at 30 m.o.i. for 16 h at 37 °C and grown in medium containing
heat-inactivated serum (2%) for 48 h. Following 24 h serum
starvation, the cells were stimulated with or without insulin (100 ng/ml) for 10 min, washed with phosphate-buffered saline, scraped, and
frozen at
70 °C immediately. Frozen cell pellets were extracted in
ice-cold RIPA buffer (50 mM Hepes, pH 7.4, 10 mM MgCl2, 150 mM NaCl, 1% Nonidet
P-40, 0.5 mM phenylmethylsulfonyl fluoride, and 10 µg/ml
aprotinin, leupeptin, and pepstatin) by shaking for 5 min at 4 °C.
The resulting cell extracts were centrifuged at 10,000 × g for 2 min. The supernatants were divided in half and
either 3 µg of the anti-Ras antibody Y13-259 (Santa Cruz
Biotechnology) or 3 µg of rat IgG (Cappel) were added. To both
samples, goat anti-rat IgG and protein G-agarose were added as well as
NaCl, SDS, and deoxycholate to final concentrations of 500 mM, 0.05%, and 0.5%, respectively. The samples were
shaken gently for 1 h at 4 °C and then the immunoprecipitates
were washed 4 times in RIPA buffer containing 500 mM NaCl,
0.05% SDS, and 0.5% deoxycholate and 2 times in 20 mM
Tris-PO4, pH 7.4. The washed immunoprecipitates were
resuspended in 30 µl of 5 mM Tris-PO4, pH
7.4, 2 mM dithiothreitol, 2 mM EDTA (TED
buffer), heated to 100 °C for 3 min, cooled on ice, and centrifuged
at 10,000 × g for 2 min. The immunoprecipitates were
washed with an additional 15 µl of TED buffer which was combined with
the first 30 µl of TED buffer and GTP and GDP were measured as
described below.
GTP was converted to ATP using NDP kinase and ADP with the ATP measured
in the luciferase/luciferin system according to the following
reactions,
where PPi is pyrophosphate. This assay is sensitive to 1 fmol of
GTP and was performed essentially as described previously (37).
GDP was converted to GTP using pyruvate kinase and phosphoenolpyruvate
with the GTP measured as described above,
Because the final product is again emitted light, this assay is
also sensitive to 1 fmol. The reaction mixture was incubated for 30 min
at 30 °C and contained in a final volume of 15 µl of 50 mM glycine, pH 7.8, 10 mM dithiothreitol, 8 mM MgSO4, 50 µM phosphoenolpyruvate, 3 milliunits of pyruvate kinase and 5 µl of
sample or GDP standard. It should be noted that this assay measures the
sum of GTP + GDP; thus, the amount of GTP in the sample must be
subtracted from the amount of GTP + GDP to yield the amount of GDP. DNA
was measured by a standard fluorescence method using the fluorescent
dye bisbenzimidazole and protein was measured by the Bradford method.
The amounts of GDP and GTP in the samples are determined from standard
curves prepared with each set of samples and the data are expressed as
femtomoles of GTP or GDP per microgram of DNA or milligram of protein
in the cell lysate.
 |
RESULTS |
Expression of Ad5-p110CAAX and
Ad5-p110WT in 3T3-L1 Adipocytes
Western Blot of Myc-tagged Proteins--
The differentiated 3T3-L1
adipocytes were infected with recombinant adenoviruses expressing the
membrane-localized p110CAAX and the wild-type
p110WT at 40 m.o.i. for 16 h and protein
expression was examined 72 h later by Western blotting.
Immunoblotting was performed against the Myc-tagged epitope present at
the amino terminus of both the recombinant constructs. Specific bands
appeared at ~110 kDa corresponding to the
p110CAAX and the p110WT proteins
expressed in infected 3T3-L1 adipocytes (Fig.
1, lanes 2 and 3).
The level of expression of both the proteins was similar.

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Fig. 1.
Western blot analysis to determine expression
of p110CAAX and p110WT proteins in
3T3-L1 adipocytes. The differentiated 3T3-L1 adipocytes were
uninfected ( ) or infected with Ad5-p110CAAX
(lane 2) or Ad5-p110WT (lane 3) at
40 m.o.i. for 16 h and total cell lysates were prepared from
uninfected and infected cells, 72 h later, as described under
"Experimental Procedures." Equal amounts of protein (80 µg) were
resolved by SDS-PAGE, electrophoretically transferred to nitrocellulose
membrane, and blotted with anti-Myc antibody (9E10). Immunoreactive
bands were detected by enhanced chemiluminescence for the transduced
Myc-tagged proteins indicated by the arrow on the
right side.
|
|
PI 3-Kinase Activity--
The membrane-localized
p110CAAX and the wild-type p110WT
overexpressing 3T3-L1 adipocytes were incubated with or without insulin for 10 min and the PI 3-kinase activity was measured in anti-Myc and
anti-p110
immunoprecipitates. A representative experiment utilizing
anti-p110
immunoprecipitates is shown in Fig.
2A and quantitation of data
from seven separate experiments is shown in Fig. 2B, where
the PI 3-kinase activity is expressed as percent of the basal activity
(observed in unstimulated, Ad5-p110CAAX infected
cells). Overexpression of the membrane-localized
p110CAAX protein resulted in a ~2-fold
increase in p110
-associated PI 3-kinase activity when infected at
40 m.o.i. in the absence of insulin (Fig. 2B). Insulin
treatment of these cells further increased PI 3-kinase activity up to
4-fold (Fig. 2B), whereas, the cells infected at the same
m.o.i. with control adenovirus elicited only 2.5-fold increase in PI
3-kinase activity upon insulin stimulation. In contrast, the wild-type
p110WT overexpression induced only a modest, but a
dose-dependent elevation of the p110
-associated PI
3-kinase activity in the absence of insulin. Upon insulin treatment,
the level of p110
-associated PI 3-kinase activity increased
further, up to 2.5-fold in the p110WT expressing cells,
similar to that observed in uninfected or control infected cells.
Preincubation with 1 µM wortmannin blocked the p110-associated PI 3-kinase activity in the membrane-localized p110CAAX overexpressing 3T3-L1 adipocytes.

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Fig. 2.
Effects of overexpression of
p110CAAX and p110WT proteins on PI
3-kinase activity in 3T3-L1 adipocytes. Cells were uninfected ( )
or infected with Ad5-CT (Ctrl),
Ad5-p110CAAX (p110caax), or
Ad5-p110WT (p110WT) at the indicated m.o.i. for
16 h at 37 °C and grown in medium containing heat inactivated
serum (2%) for 60 h. Following infection, the cells were serum
starved (16 h), incubated with or without 1 µM wortmannin
(Wort.), stimulated with or without 100 ng of insulin/ml for
10 min, lysed, and subjected to immunoprecipitation with antibodies to
p110 . The washed immunoprecipitates were assayed for PI 3-kinase
activity with PI as substrate, and the labeled PI-3 phosphate product
(PI-3P) was resolved by thin-layer chromatography and
visualized by autoradiography. In A, data from a
representative experiment is shown. B shows mean ± S.E. of seven experiments and the data is expressed as percentage of
the maximal activity (=100%) observed in unstimulated
Ad5-p110CAAX-infected cells.
|
|
Biological Effects of p110CAAX
Ser/Thr Phosphorylation of Akt in 3T3-L1 Adipocytes--
Akt is a
serine/threonine kinase downstream of PI 3-kinase which is activated by
serine/threonine phosphorylation. (39). Akt has been implicated as a
mediator of several metabolic effects of insulin, including GLUT4
translocation, glucose uptake, and glycogen synthase activation.
Therefore, we determined whether PI 3-kinase can activate Akt, using
the gel mobility shift assay. Cell lysates from 3T3-L1 adipocytes
infected with increasing m.o.i. of the membrane-localized
p110CAAX and the wild-type p110WT
expressing adenoviruses, were analyzed by SDS-PAGE followed by Western
blotting with anti-Akt antibody (Fig. 3).
The retarded gel mobility indicates serine/threonine phosphorylation
and activation of Akt. Overexpression of the
p110CAAX led to a significant increase in Akt
activation, in a dose-dependent manner (Fig. 3, lanes
7 and 8). The extent of Akt activation by p110CAAX at 40 m.o.i. was comparable to
that observed with insulin alone (Fig. 3, lanes 2 and
8). Further addition of insulin had a modest additive effect
on Akt activation (lane 9). In contrast, p110WT
overexpression did not activate Akt (lanes 3-5). Expression
of an empty adenoviral vector, Ad5-CT, did not affect Akt activity either in the basal or insulin stimulated state (data not shown). The
activation of Akt with insulin or with p110CAAX
was completely inhibitable by treatment with wortmannin (Fig. 3,
lanes 6 and 10), demonstrating that PI 3-kinase
is necessary for Akt activation.

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Fig. 3.
Effects of overexpression of
p110CAAX and p110WT on Akt
activation in 3T3-L1 adipocytes. Cells were uninfected (( );
lanes 1 and 2) or infected with
Ad5-p110WT (p110WT; lanes 3-6), or
Ad5-p110CAAX (p110caax; lanes 7-10)
at the indicated m.o.i. for 16 h. Serum-starved (16 h) cells were
pretreated with 1 µM wortmannin (Wort., lanes
6 and 10) for 30 min, and incubated in the absence or
presence of insulin (100 ng/ml) for 30 min (lanes 2, 5, and
9), lysed, and subjected to SDS-PAGE, and immunoblotted with
anti-Akt antibody. Ser/Thr-phosphorylated Akt was detected by a
retarded migration of the enzyme (pAkt). The Western blot is a
representative of 10 independent experiments.
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|
p70 S6 Kinase Activation--
It has been shown that p70 S6
kinase, another serine/threonine kinase is downstream of PI 3-kinase
and Akt and that activation of PI 3-kinase and/or Akt is necessary for
p70 S6 kinase activation. We, therefore, examined the ability of the
membrane-localized p110CAAX to activate p70 S6
kinase by using both a mobility shift assay and the phospho-specific
antibody that detects p70 S6 kinase only when it is phosphorylated at
Thr421/Ser424. The effect of
p110CAAX overexpression on p70 S6 kinase
activation is parallel to that observed for Akt activation (Fig.
4). p110CAAX
overexpression led to an insulin-independent activation of p70 S6
kinase in a dose-dependent manner (Fig. 4, upper
panel, lanes 9-11). Insulin treatment had a small additive effect
on p70 S6 kinase stimulation (lane 12). p110WT
overexpression showed a modest effect to stimulate p70 S6 kinase mobility, but the extent of activation was much less than that exhibited by the p110CAAX protein or by insulin
(lanes 4-6). The inhibitors rapamycin or wortmannin
prevented insulin or p110CAAX stimulation of p70
S6 kinase. The phospho-specific p70 S6 kinase blot showed a similar
pattern of p70 S6 kinase activation as observed with the mobility shift
assay (Fig. 4, lower panel).

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Fig. 4.
Effects of overexpression of
p110CAAX and p110WT on p70 S6 kinase
activation in 3T3-L1 adipocytes. Cells were uninfected (( ),
lanes 1-3) or infected with Ad5-p110WT
(p110WT, lanes 4-8), or Ad5-p110CAAX
(p110caax, lanes 9-13) at the indicated m.o.i. for 16 h. Serum-starved (16 h) cells were pretreated with 1 µM
wortmannin (Wort., lanes 8 and 13) or 20 nM rapamycin (Rapa., lane 1) for 30 min, and
incubated in the absence or presence of insulin (100 ng/ml) for 30 min
(lanes 1, 3, 7, and 12). Total cell lysates (30 µg) were subjected to SDS-PAGE and immunoblotted with p70 S6 kinase
antibody (upper panel) or phospho-specific p70 S6 kinase
antibody (lower panel). The Western blot is representative
of six independent experiments.
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Glucose Transport Stimulation--
3T3-L1 adipocytes were infected
with Ad5-p110CAAX, Ad5-p110WT, or
Ad5-CT and 2-[3H]deoxyglucose uptake was measured 86 h later, after treating cells with or without 100 ng/ml insulin for
1 h. In uninfected cells, insulin stimulated glucose uptake by
~8-fold, and this was not affected by Ad5-CT infection (Fig.
5). In contrast, the basal 2-deoxyglucose
uptake in cells overexpressing p110CAAX was
elevated by 4-8-fold, compared with the basal uptake measured in cells
infected with the empty adenoviral vector (Ad5-CT). Thus, glucose
transport in cells expressing p110CAAX was
almost the same as that observed after insulin treatment alone (Fig.
5). In contrast, 2-deoxyglucose uptake in 3T3-L1 adipocytes infected
with p110WT was comparable with the uninfected cells, or
with cells infected with the empty adenoviral vector, and was further
stimulated up to 8-fold by insulin treatment. Thus, membrane-targeted
p110CAAX mimics insulin-induced glucose
transport activity in 3T3-L1 adipocytes, and overexpression of
p110WT was unable to stimulate glucose uptake in the
absence of insulin. Pretreatment with wortmannin inhibited
p110CAAX and insulin-induced glucose transport.
In addition, p110CAAX and p110WT
overexpression did not have any effect on the expression levels of Glut
4, compared with the CT infected cells (data not shown).

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Fig. 5.
2-Deoxyglucose uptake in 3T3-L1
adipocytes infected with increasing m.o.i. of adenoviruses expressing
p110CAAX and p110WT.
Differentiated 3T3-L1 adipocytes were infected with Ad5-CT
(ctrl), Ad5-p110WT (p110WT), or Ad5-
p110CAAX (p110caax) at the indicated
m.o.i. for 16 h at 37 °C and grown in medium containing
heat-inactivated serum (2%) for 72 h. Serum and glucose-deprived
cells were pretreated with 1 µM wortmannin
(Wort.) for 30 min in -DMEM, and incubated in the absence
or presence of 100 ng of insulin/ml for 1 h at 37 °C. Cells
were then washed with glucose-free medium and 2-deoxyglucose uptake was
measured. Each measurement was performed in duplicate or triplicate.
Data are the mean ± S.E. of 10 different observations and each
value was corrected for protein concentration.
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p21ras Activation--
To investigate whether
activated PI 3-kinase could stimulate the Ras pathway, we examined the
effects of membrane-targeted p110CAAX on basal
and insulin-induced p21ras GTP activity in 3T3-L1 adipocytes.
Insulin led to ~1.7-fold increase p21ras GTP activity in
Ad5-CT infected cells (Fig. 6).
Expression of the membrane-targeted p110CAAX
increased the level of p21ras GTP to the same extent as
insulin, and insulin treatment of the p110CAAX
expressing cells had no further effect.

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Fig. 6.
Membrane-targeted
p110CAAX elevates insulin-independent levels of
GTP-bound Ras in 3T3-L1 adipocytes. Differentiated 3T3-L1
adipocytes were infected with Ad5-CT (ctrl) or
Ad5-p110CAAX (p110caax) at 30 m.o.i. in medium containing heat-inactivated serum (2%) for 16 h.
Following infection, cells were serum starved (16 h), incubated in the
absence or presence of insulin (100 ng/ml) for 10 min, lysed, and
immunoprecipitated with the anti-Ras antibody, Y13-259. Then, GTP-Ras
and GDP-Ras were measured as described under "Experimental
Procedures." Results are expressed as mean ± S.E. of percentage
of GTP-bound Ras (% GTP) from five independent experiments. Percent
GTP was determined as GTP-Ras/(GTP-Ras + GDP-Ras) × 100.
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|
These results were reproducible with another method that utilizes
electroporation technique (38) to incorporate
[
-32P]GTP in 3T3-L1 adipocytes infected with control
or membrane-targeted p110CAAX adenoviruses to
measure p21ras GTP activity. Using this system, we found that
insulin stimulated p21ras GTP activity about 1.3-fold in
Ad5-CT-infected cells, in contrast to the 1.7-fold stimulation
described above. However, both the methods showed similar results that
the membrane-targeted PI 3-kinase stimulates Ras (data not shown).
p110CAAX Induces Cellular Insulin
Resistance
p110CAAX Stimulates Ser/Thr Phosphorylation
of IRS-1 and Inhibits Its Function--
It has been reported that PI
3-kinase phosphorylates serine/threonine residues of IRS-1 (9); we,
therefore, examined the effect of p110CAAX on
IRS-1 by the gel mobility shift assay. p110CAAX
expression caused a mobility shift of IRS-1 in the absence of insulin
without tyrosine phosphorylation, indicating phosphorylation of Ser/Thr
residues (Fig. 7A). In
addition, p110CAAX expression inhibited
insulin-stimulated IRS-1 tyrosine phosphorylation, without affecting
insulin receptor tyrosine phosphorylation (Fig. 7B).

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Fig. 7.
Membrane-targeted
p110CAAX stimulates Ser/Thr Phosphorylation of
IRS-1 and inhibits IRS-1 associated PI 3-kinase activity.
Differentiated 3T3-L1 adipocytes were infected with Ad5-CT
(ctrl) or Ad5-p110CAAX
(p110caax) at 40 m.o.i. in medium containing
heat-inactivated serum (2%) for 16 h. Following infection, cells
were serum starved (16 h), incubated in the absence or presence of
insulin (100 ng/ml) for 5 (A and B) or 10 (C) min. Total cell lysates (20 µg) were subjected to
SDS-PAGE and immunoblotted with IRS-1 antibody (A) or PY20H
(B). The Western blot is representative of three independent
experiments. The cell lysates were divided into three, and subjected to
immunoprecipitation with antibodies to p110 , IRS-1, or Myc
(C). After Myc antibody immunoprecipitation, the
supernatants were collected, and immunoprecipitated with IRS-1
antibody. The washed immunoprecipitates were assayed for PI 3-kinase
activity with PI as substrate, and the labeled PI-3 phosphate product
(PI-3P) was resolved by thin-layer chromatography and
visualized by autoradiography. C shows mean ± S.E. of
three experiments and the data is expressed as percentage of the
maximal activity (=100%) observed in insulin-stimulated,
Ad5-CT-infected cells. , Ad5-Ctrl; , Ad5-CAAX.
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To investigate whether this serine/threonine phosphorylation altered
IRS-1 function, we measured the PI 3-kinase activity associated with
IRS-1. As shown in Fig. 7C, expression of
p110CAAX resulted in ~40% inhibition of
insulin-stimulated PI 3-kinase activity in IRS-1 antibody
immunoprecipitates. This is in contrast to the ~45-55% increase in
p110CAAX induced PI 3-kinase activity observed
in p110
antibody immunoprecipitates (Fig. 7C). The
p110CAAX induced inhibition of insulin
stimulated IRS-1 associated PI 3-kinase activity was also observed in
the cell lysates immunodepleted of the c-Myc immune complexes (Fig.
7C).
Glycogen Synthase Activity--
Insulin led to an about 2.5-fold
increase in glycogen synthase activity measured in control 3T3-L1
adipocytes infected with the empty adenoviral vector, Ad5-CT (Fig.
8). In cells infected with
p110CAAX, the basal level of glycogen synthesis
was decreased by ~30% compared with control cells, while
insulin-stimulated glycogen synthase activity was completely inhibited.
In cells expressing p110WT, basal glycogen synthase
activity was unchanged and insulin led to an about 1.5-fold
stimulation; this degree of stimulation was less than that observed in
control cells, but the differences did not reach statistical
significance.

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Fig. 8.
Membrane-targeted
p110CAAX prevents insulin-induced glycogen
synthase activity in 3T3-L1 adipocytes. Differentiated 3T3-L1
adipocytes were infected with Ad5-CT (Ctrl),
Ad5-p110WT (p110WT), or
Ad5-p110CAAX (p110caax) at 40 m.o.i. for 16 h at 37 °C and grown in medium containing
heat-inactivated serum (2%) for 72 h. The cells were serum and
glucose starved in DMEM, 0.1% BSA, 2 mM sodium pyruvate
for 3 h, then stimulated with or without 200 ng of insulin/ml for
30 min in 5 mM glucose containing medium. Following which,
the cells were scraped, sonicated, and centrifuged. The ability of the
supernatant to stimulate incorporation of UDP-glucose into glycogen was
determined in the presence and absence of glucose 6-phosphate. Results
are expressed as mean ± S.E. of percentage of glycogen synthase
index (% GSI) from three independent experiments, and each
observation was performed in duplicate. Percent GSI was determined as:
(activity without glucose 6-phosphate/activity with G-6-P) × 100.
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MAP Kinase Phosphorylation--
We investigated whether the Ras
activation induced by p110CAAX expression was
accompanied by activation of the downstream effectors Erk1 and 2. This
was accomplished in uninfected cells and Ad5- p110CAAX-infected cells by assessing MAP kinase
activation using a phospho-specific MAP kinase antibody (Fig.
9, upper panel). Insulin
increased MAP kinase activation in uninfected and Ad5-CT-infected cells
by ~15-fold. Expression of the membrane-targeted
p110CAAX had no effect on the basal MAP kinase
phosphorylation state, and almost completely inhibited insulin-induced
phosphorylation and activation of both p44 and p42 MAP kinase (Fig. 9,
lane 5). In contrast, PDGF stimulated MAP kinase
phosphorylation was unchanged by p110CAAX
expression (Fig. 9, lane 6). Expression of MAP kinase, as
assessed by Western blotting with a polyclonal anti-Erk-1 antibody that recognizes both nonphosphorylated and phosphorylated forms,
demonstrated that the protein levels were not altered by infection
with either Ad5-CT or Ad5-p110CAAX (Fig. 9,
lower panel).

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Fig. 9.
Membrane-targeted
p110CAAX prevents insulin-induced MAP kinase
phosphorylation in 3T3-L1 adipocytes. Differentiated 3T3-L1
adipocytes were uninfected ( ) or infected with Ad5-CT
(ctrl) or Ad5-p110CAAX
(p110caax) at 10 m.o.i. in medium containing
heat-inactivated serum (2%) for 16 h. Following infection, cells
were serum starved (16 h), incubated in the absence or presence of
insulin (100 ng/ml, I) or PDGF (20 ng/ml, P) for
10 min, lysed, subjected to SDS-PAGE, and immunoblotted with
phospho-specific MAP kinase antibody (upper panel). The
membrane was stripped and reblotted with anti-Erk-1 antibody
(lower panel). The Western blots are representative of five
independent experiments.
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 |
DISCUSSION |
In this study, we have used adenoviral-mediated gene transfer to
assess whether targeting of the catalytic p110 subunit of PI 3-kinase
to cellular membranes by incorporating a CAAX box at the
COOH terminus (p110CAAX) is sufficient to induce
PI 3-kinase dependent signaling responses, characteristic of insulin
action, in 3T3-L1 adipocytes. We show that when appropriately targeted,
even modest levels of PI 3-kinase are sufficient to trigger full
activation of the downstream serine/threonine kinases, Akt and p70 S6
kinase, and also causes stimulation of glucose transport equal to the
effect of insulin. Surprisingly, insulin-mediated glycogen synthase
activity was completely blocked in cells expressing
p110CAAX. Furthermore,
p110CAAX stimulated serine/threonine
phosphorylation of IRS-1, and inhibited IRS-1 associated PI 3-kinase
activity. Another major finding is that the membrane-localized PI
3-kinase activity was sufficient to mimic insulin-induced formation of
GTP-bound p21ras. Last, we found that expression of
p110CAAX led to inhibition of insulin-mediated
MAP kinase activation, whereas PDGF-mediated MAP kinase activation was
unaffected. These results lead to several predictions and conclusions.
p110CAAX Mimics Insulin Actions--
We
demonstrate that membrane-targeted p110
(p110CAAX) promotes insulin-independent PI
3-kinase activity and is sufficient to maximally stimulate glucose
uptake, in a wortmannin-sensitive manner. The level of 2-deoxyglucose
uptake achieved in response to p110CAAX
expression was comparable to that seen in insulin-stimulated, control
adipocytes, whereas, non-targeted wild-type p110
(p110WT) had only a slight effect on 2-deoxyglucose uptake.
Since, the p110 subunit of PI 3-kinase contains a COOH-terminal
membrane targeting farnesylation sequence, it seems likely that the
overexpressed p110CAAX protein results in
increased PI 3-kinase activity predominantly in membrane fractions,
similar to insulin stimulation of endogenous PI 3-kinase. This implies
that glucose uptake is not merely a function of the amount of PI
3-kinase present, but that its appropriate membrane localization is
critical as well. This conclusion is quite consistent with other kinds
of studies in the literature. For example, PDGF, as well as other
growth factors, can stimulate PI 3-kinase in 3T3-L1 adipocytes, equally
well as insulin, but only insulin leads to glucose transport
stimulation (18, 19). These findings suggested that insulin induced
subcompartmentalization of PI 3-kinase is necessary for metabolic
signaling. Consistent with this, Frevert and Khan (26) showed that
co-expression of both the catalytic p110
subunit and the inter-SH2
region of the p85 regulatory subunit of PI 3-kinase in 3T3-L1
adipocytes led to a much higher level of PI 3-kinase activity than seen
with insulin stimulation alone, but it had only a partial effect to stimulate glucose transport without insulin. In addition, when PI
3-kinase was activated by thiophosphorylated peptides, corresponding to
the phosphotyrosine binding motif of the p85 subunit of PI 3-kinase,
only a minor effect on Glut 4 translocation was observed (40). Tanti
et al. (41) co-transfected rat adipose cells by electroporation with epitope-tagged Glut 4 and with either a
constitutively active (p110*) or a kinase inactive form of
p110kd (41). Co-transfection with the active version of
p110* resulted in stimulation of epitope-tagged Glut 4 translocation,
similar to insulin, and these workers also found that the p110* was
localized to the same intracellular compartment as the endogenous PI
3-kinase. Taken together with our current results, these studies
support the conclusion that active PI 3-kinase is sufficient to
stimulate glucose transport activity, only if it is targeted to the
proper subcellular membranous compartment.
We further examined other targets of insulin action which are thought
to be downstream of PI 3-kinase. Akt is a serine/threonine kinase that
is activated by insulin. It is activated by a dual mechanism involving
the binding of PI-(3,4)-P2 to its PH domain, as well as by
serine/threonine phosphorylation by one or more Akt kinases, which may,
themselves, be stimulated by the lipid products of PI 3-kinase (42).
Several lines of evidence suggest that Akt functions downstream of PI
3-kinase, e.g. insulin stimulated Akt kinase activity is
inhibitable by wortmannin, a PI 3-kinase specific inhibitor, and PDGF
receptor mutants that fail to activate PI 3-kinase, also fail to
activate Akt. Overexpression of a constitutively active Akt in 3T3-L1
adipocytes results in increased glucose uptake and Glut 4 translocation
in the absence of insulin (43). Consistent with these findings, our
data show that Akt activation is dependent on PI 3-kinase activity, and
that insulin and p110CAAX induced Akt activation
is inhibitable by wortmannin, indicating that Akt activation is
dependent on PI 3-kinase enzymatic function.
We also determined whether PI 3-kinase-mediated Akt activation would
lead to p70 S6 kinase stimulation, since it has been shown that p70 S6
kinase is stimulated by constitutively active Akt (8) and blocked by
inhibitors of PI 3-kinase (12, 44). Indeed, we found that
overexpression of the membrane-targeted p110CAAX
led to activation of p70 S6 kinase, which was completely inhibitable by
wortmannin (Fig. 4). Our results are supported by the study of Weng
et al. (44), which showed that transfection of a
constitutively active form of PI 3-kinase (p110*) into 293 cells
resulted in a 20-30-fold increase in cellular PI 3-kinase activity,
that resulted in activation of p70 S6 kinase by phosphorylation at
Thr-252. Wortmannin resulted in selective dephosphorylation at Thr-252 concomitant with inhibition of p70 S6 kinase activity. Furthermore, Klippel et al. (27) reported that in COS-7 cells, expression of membrane-localized p110 is sufficient to trigger downstream responses, characteristic of insulin action, including stimulation of
Akt and p70 S6 kinase. Their study further adds that these responses
can also be triggered by expression of p110*, that is cytosolic, but
exhibits a high specific activity. However, they observed maximum
activation of downstream responses in cells expressing the
membrane-localized p110. Thus, insulin treatment activates and targets
PI 3-kinase to specific membrane compartments, and this action is
mimicked by p110CAAX, which is sufficient to
trigger downstream responses characteristic of insulin action,
including stimulation of Akt, p70 S6 kinase, and glucose transport.
The role of PI 3-kinase in Ras-mediated signaling is unclear. An
association between p21ras and PI 3-kinase was first
demonstrated by co-immunoprecipitation in insulin and insulin-like
growth factor-1 stimulated, Ras-transformed epithelial cells by
Sjolander et al. (45). Subsequently, Ras was shown to bind
in vitro to the p110 subunit of PI 3-kinase by
Rodriguez-Viciana et al. (46). However, the relative
position of PI 3-kinase with respect to Ras is confusing. Conflicting
data exists suggesting that PI 3-kinase could be upstream, downstream, or independent of Ras. These alternate results are perhaps related to
cell-type differences. Rodriguez-Viciana et al. (30)
reported that a point mutation of the p110 subunit of PI 3-kinase at
the Ras-GTP binding site elevated PI 3-kinase activity in COS cells, and the interaction of Ras-GTP, but not Ras-GDP, with PI 3-kinase led
to an increase in its enzymatic activity (30). These data suggest that
Ras is upstream of PI 3-kinase. However, our data are consistent with
the idea that PI 3-kinase is upstream and can activate Ras (Fig. 6). We
find that membrane-targeted activated PI 3-kinase activates
p21ras, resulting in increased formation of p21ras GTP,
equal to the effect of insulin. This interpretation is in agreement
with earlier data from our own laboratory, in which we reported that
microinjection of dominant-negative PI 3-kinase, or PI 3-kinase
inhibitory antibodies, into rat fibroblasts inhibited insulin-induced
Fos induction, which was rescued by activated (T-24) Ras (47).
Similarly, studies by Hu et al. (48) suggest that Ras is
downstream of PI 3-kinase because transfection of constitutively active
PI 3-kinase resulted in Fos induction, which was blocked by both
dominant-negative Ras and Raf. They also found elevated levels of
GTP-bound Ras in cells transfected with constitutively active PI
3-kinase.
Cellular Insulin Resistance Induced by
p110CAAX--
Interestingly, we found that
p110CAAX did not mimic all of insulin's
actions, and, in some cases led to a decrease in insulin signaling
indicating a partial, and selective insulin resistant state. For
example, we found that p110CAAX did not mimic
the effect of insulin to stimulate glycogen synthesis. Not only did
p110CAAX expression fail to enhance basal
glycogen synthase activity, but it completely inhibited the ability of
insulin to stimulate glycogen synthesis. Activation of glycogen
synthase by insulin involves a coordinated response, including
phosphorylation induced inactivation of glycogen synthase kinase 3 (GSK3) and activation of protein phosphatase 1, by phosphorylation of
its G subunit (pp1G) (49). It has been suggested that GSK3 is a
downstream target of Akt, which, in turn, is dependent on PI 3-kinase
activity. Constitutively active Akt inhibits insulin's ability to
stimulate glycogen synthesis in 3T3-L1 adipocytes (43, 50, 51), and our
data also show that activation of Akt by the membrane-localized p110CAAX is not sufficient to cause glycogen
synthase activation in 3T3-L1 adipocytes. In theory, activated PI
3-kinase and Akt should inactivate GSK3 by phosphorylation leading to
stimulation of glycogen synthase activity, whereas we, and others, show
that p110CAAX or constitutively active Akt
inhibits insulin effects on this enzyme (43, 50, 51). However, it has
been shown recently that GSK3 expression is either very low, or absent
in 3T3-L1 adipocytes (52-54). Therefore, a role for GSK3 in our
results is problematic. Perhaps the low (or absent) expression of GSK3
explains why p110CAAX does not stimulate
glycogen synthase by itself. An alternate pathway for glycogen synthase
activation involves pp1, which has been suggested to be downstream of
the IRS-1/Shc-MAP kinase pathway by some investigators (55), but a
number of reports have indicated that this is not the case (56). In
addition, earlier results show that the MEK inhibitor PD098059 does not
lead to a decrease in insulin stimulation of glycogen synthesis (57).
Thus, a role for MAPK in the regulation of glycogen synthesis seems
unlikely. Another possibility is that IRS-1 directly or through its
interacting proteins, but independent of PI 3-kinase, might be involved
in the inhibition of glycogen synthase activity. Indeed, we find that
membrane-targeted p110CAAX serine/threonine
phosphorylates IRS-1, which is inhibitable by wortmannin. This in turn
prevents IRS-1 tyrosine phosphorylation and downstream signaling (Fig.
7).
Although the precise mechanisms underlying the
p110CAAX induced resistance are unknown, the
current results provide some interesting insights. First, despite the
fact that p110CAAX stimulated Ras activation, it
had no effect to stimulate MAP kinase phosphorylation, indicating a
blockade of MAP kinase activation at a site downstream of Ras.
Furthermore, in p110CAAX expressing cells,
insulin had no effect to stimulate MAP kinase phosphorylation, compared
with a robust stimulation in control cells. Since insulin is thought to
stimulate MAP kinase activation by activation of Ras (11), these
findings also point to a post-Ras blockade of the MAP kinase pathway.
On the other hand, p110CAAX expression did not
inhibit PDGF-stimulated MAP kinase phosphorylation, and this is
consistent with the interpretation that PDGF can lead to MAP kinase
activation through a Ras-dependent as well as a non-Ras
dependent pathway (58), and we would propose that expression of
p110CAAX inhibits only the
Ras-dependent input into MAP kinase. These findings also
demonstrate that the p110CAAX induced inhibition
of MAP kinase and insulin signaling is specific, and not due to some
toxic or nonspecific effect on the cells.
Taken together, our results are consistent with the view that
p110CAAX expression inhibits the actions of
insulin at a step distal to Ras activation, leading to inhibition of
MAP kinase, and, possibly, glycogen synthase activation. Importantly,
the cellular insulin resistance induced by
p110CAAX in these cells is not global. Thus,
p110CAAX expression stimulated AKT as well as
p70 S6 kinase phosphorylation, and insulin had a further effect when
added to p110CAAX expressing cells. This would
argue that this model of cellular insulin resistance is rather unique,
in that some of the insulin signaling pathways are inhibited, whereas,
others are intact. The fact that persistent activation of PI 3-kinase
leads to desensitization of subsequent downstream events is reminiscent
of the fact that hyperinsulinemia (either in vitro or
in vivo) will also lead to a state of cellular insulin
resistance. However, hyperinsulinemia-induced insulin resistance
affects all of insulin's actions, whereas, persistent PI 3-kinase
activation selectively inhibits specific insulin signaling. Since
insulin's biologic effects are pleiotrophic with engagement of
multiple divergent signaling pathways, further study of these cells may
enhance our understanding of which signaling pathways connect to which
biologic effects.
In summary, our studies show that PI 3-kinase activity can mimic a
number of biologic effects normally induced by insulin, but that
membrane targeting of this enzyme is necessary for activation of these
events. In addition, the persistent activation induced by
p110CAAX expression leads to desensitization of
specific signaling pathways. Interestingly, the state of cellular
insulin resistance is not global, in that some of insulin's actions
are inhibited, whereas others are intact.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Christopher B. Newgard for
providing the adenovirus plasmids and James G. Nelson for providing
differentiated 3T3-L1 adipocytes. We thank Matt Hickmann for help in
establishing the glycogen synthase assay.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Research Grant R01 DK 36651 (to J. M. O.).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.
§
Supported by the Uehara Memorial Foundation.
¶
Supported by the Manpei Suzuki Diabetes Foundation.

To whom correspondence should be addressed: Dept. of Medicine
(0673), University of California, San Diego, 9500 Gilman Dr., La Jolla,
CA 92093-0673. Tel.: 619-534-6651; Fax: 619-534-6653.
 |
ABBREVIATIONS |
The abbreviations used are:
SH2, Src homology
domain 2;
PI, phosphoinositide;
SH3, Src homology domain 3;
PDGF, platelet-derived growth factor;
DMEM, Dulbecco's modified Eagle's
medium;
FCS, fetal calf serum;
PCR, polymerase chain reaction;
m.o.i., multiplicity of infection;
PAGE, polyacrylamide gel electrophoresis;
GSK3, glycogen synthase kinase 3.
 |
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