Third Department of Internal Medicine (H.K., M.F., K.I., Y.F., T.O., M.F., T.A.) Faculty of Medicine, University of Tokyo, Tokyo 113, Japan; The Institute for Adult Disease (H.O., M.A., H.S., Y.O., M.K.), Asahi Life Foundation, Tokyo 160, Japan; and The Third Department of Internal Medicine (Y.O.), Yamaguchi University School of Medicine, Yamaguchi 755, Japan
Address all correspondence and requests for reprints to: Dr. Tomoichiro Asano, Third Department of Internal medicine, University of Tokyo 73-1, Hongo, Bunkyo-ku, Tokyo, Japan 113-8655. E-mail: asano-tky{at}umin.ac.jp
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ABSTRACT |
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In 3T3-L1 adipocytes, insulin-induced accumulations of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate were markedly suppressed by overexpression of wild-type PTEN with the N-terminal myristoylation tag, but not by that without the tag. On the contrary, the C124S mutants of PTEN enhanced insulin-induced accumulations of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. Interestingly, the phosphorylation level of Akt at Thr308 (Akt2 at Thr309), but not at Ser473 (Akt2 at Ser474), was revealed to correlate well with the accumulation of phosphatidylinositol 3,4,5-trisphosphate modified by overexpression of these PTEN proteins. Finally, insulin-induced increases in glucose transport activity were significantly inhibited by the overexpression of myristoylated wild-type PTEN, but were not enhanced by expression of the C124S mutant of PTEN. Therefore, in conclusion, 1) PTEN dephosphorylates both phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate in vivo, and the C124S mutants interrupt endogenous PTEN activity in a dominant-negative manner. 2) The membrane targeting process of PTEN may be important for exerting its function. 3) Phosphorylations of Thr309 and Ser474 of Akt2 are regulated differently, and the former is regulated very sensitively by the function of PTEN. 4) The phosphorylation level of Ser474, but not that of Thr309, in Akt2 correlates well with insulin-stimulated glucose transport activity in 3T3-L1 adipocytes. 5) The activity of endogenous PTEN may not play a major role in the regulation of glucose transport activity in 3T3-L1 adipocytes.
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INTRODUCTION |
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Recently, not only the producers of 3-phosphoinositides, but also proteins that destroy 3-phosphoinositides, have received attention, as they are also important for regulating the cellular content of 3-position phosphorylated phosphoinositides. SH2 domain-containing inositol 5'-phosphatase is known to have a specific catalytic activity, dephosphorylating 5-phosphate from PI(3, 4, 5)P3 (26). Another protein that has recently received considerable attention is phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Having strong and specific enzyme activity, PTEN dephosphorylates 3-phosphate from 3-phosphoinositides (27). The evidence that cells which have a mutated PTEN gene have increased cellular 3-phosphoinositide content (28, 29) and that they show tumorigenicity (30, 31, 32) indicates that the degradation of 3-phosphoinositides is necessary for maintaining normal cellular functions.
The critical roles of PI 3-kinase in insulin signal transduction are well established. In blocking PI 3-kinase activation with specific inhibitors or overexpressing the dominant-negative form of p85, various insulin effects, such as GLUT4 translocation and DNA synthesis, were shown to be inhibited (15, 24, 33). However, whether a 3-phosphoinositide degradation mechanism is involved in the regulation of insulin actions remains unclear. Herein, we demonstrate the roles of PTEN in the regulation of 3-position phosphorylated phosphoinositide metabolism in experiments using Sf-9 insect cells as well as 3T3-L1 adipocytes. In addition, the effects of PTEN on insulin-induced Akt phosphorylation and insulin-stimulated glucose uptake were investigated in 3T3-L1 adipocytes.
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RESULTS |
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Quantification of 3-Phosphoinositides from 3T3-L1
Adipocytes
Subsequently, wild-type PTEN and its mutants were
overexpressed in 3T3-L1 adipocytes by adenoviral infection. LacZ from
E. coli and p85
of PI 3-kinase were used as controls.
3T3-L1 adipocytes were infected with these adenoviruses, and
expressions of wild-type PTEN and its mutants were observed by
immunoblotting (Fig. 3A
). These cells
were prelabeled with [32P]-orthophosphate for
2 h and incubated with or without insulin. Then,
[32P]-labeled phosphoinositides from the
cells were deacylated and quantified using HPLC. The radioactivities of
the quantified glycerophosphoinositol 3,4-bisphosphate from
PI(3, 4)P2 and glycerophosphoinositol
3,4,5-trisphosphate from PI(3, 4, 5)P3 extracted from cells stimulated
for 1 min with insulin are shown in Fig. 3
, B and C, respectively.
Without insulin stimulation, the levels of
PI(3, 4)P2 and PI(3, 4, 5)P3
were very low even when catalytic-dead PTEN mutants were overexpressed.
Insulin stimulation markedly increased the synthesis of
PI(3, 4)P2 and PI(3, 4, 5)P3,
and the overexpression of myristoylated PTEN markedly attenuated
insulin-induced accumulations of both PI(3, 4)P2
and PI(3, 4, 5)P3. On the other hand, the effect of
wild-type PTEN was revealed to be far smaller than that of
myristoylated PTEN, and only a partial decrease in insulin-induced
synthesis of PI(3, 4)P2 was observed in cells
overexpressing wild-type PTEN. In addition, the effect of myristoylated
PTEN was very similar to that of overexpressed
p85
in terms of
the suppression of insulin-induced accumulations of
PI(3, 4)P2 and
PI(3, 4, 5)P3.
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Overexpression of PTEN Did Not Affect Insulin-Induced PI 3-Kinase
Activation
As shown in Fig. 3, overexpression of PTEN and its
mutants affects insulin-stimulated accumulations of
PI(3, 4)P2 and PI(3, 4, 5)P3.
To investigate whether overexpression of PTEN and its mutants affects
insulin-induced PI 3-kinase activation, we examined the in
vitro PI 3-kinase activities of antiphosphotyrosine antibody
immunoprecipitants from 3T3-L1 adipocytes overexpressing control LacZ
and PTEN proteins. As shown in Fig. 4
, neither PTEN nor its mutants altered PI 3-kinase activities in the
antiphosphotyrosine antibody immunoprecipitates, regardless of insulin
stimulation. In contrast, the overexpression of
p85
markedly inhibited insulin-induced PI 3-kinase activation, as we
reported previously (33).
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Effects of Wild-Type and Mutant PTEN on Akt
Phosphorylation
The phosphorylation and activation of Akt are reportedly regulated
by the lipid products of PI 3-kinase (reviewed in Refs.
39, 40, 41). Thus, we investigated the effects of
overexpressing wild-type and mutant PTEN proteins on Akt
phosphorylation in the presence or absence of insulin stimulation.
Using anti-Akt antibody, we confirmed that the endogenous Akt protein
level did not significantly change with the adenoviral expression of
LacZ, PTEN or its mutants, or p85
(Fig. 6A
). Figure 6
(panels B and C) shows the
phosphorylation levels of Akt, which were demonstrated with
antiphospho-Thr308-Akt antibody and antiphospho-Ser473-Akt antibody
(New England Biolabs, Beverly, MA), which recognize both
phospho-Thr308 of Akt1 and phospho-Thr309 of Akt2, and both
phospho-Ser473 of Akt1 and phospho-Ser474 of Akt2, respectively
(42). Akt2 expression in 3T3-L1 cells reportedly increases
during differentiation from fibroblasts into adipocytes, and in 3T3-L1
adipocytes, Akt2 is dominantly expressed as compared with Akt1
(42, 43). Thus, in 3T3-L1 adipocytes, antiphospho-Thr308
Akt antibody and antiphospho-Ser473 Akt antibody are considered to
detect mainly phospho-Thr309 and phospho-Ser474 of Akt2,
respectively.
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Interestingly, the effects of these overexpressed PTEN proteins on
insulin-induced phosphorylation were revealed to be much smaller for
Ser474 than for Thr309 of Akt2. While overexpression of myristoylated
PTEN significantly, although only partially, reduced Ser474
phosphorylation, overexpression of phosphatase-dead types did not
increase the Ser474 phosphorylation level. Overexpression of p85
strongly inhibited insulin-induced phosphorylation of both Thr309 and
Ser474.
Effects of Overexpressing PTEN and Its Mutants on
Insulin-Stimulated Glucose Uptake by 3T3-L1 Adipocytes
Finally, we investigated how PTEN and its mutants affect
insulin-induced glucose transport activation of 3T3-L1 adipocytes,
which is thought to be an action occurring downstream from PI 3-kinase
and presumably downstream from Akt activations (reviewed in Refs.
44 and 45). Overexpression of myristoylated
PTEN decreased 2-deoxyglucose uptake by insulin-stimulated 3T3-L1
adipocytes by 47%, while nonmyristoylated PTEN had no effect (Fig. 7). In addition, phosphatase-dead PTEN
mutants did not affect insulin-induced glucose transport activation,
despite enhancing insulin-induced accumulations of
PI(3, 4)P2 and PI(3, 4, 5)P3
and insulin-induced phosphorylation of the Thr309 of Akt2. In contrast
to the weak effect of PTEN,
p85
strongly inhibited the
insulin-induced increase in glucose transport activity, i.e.
by approximately 90%.
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DISCUSSION |
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In the field of insulin action, activation of PI 3-kinase has been
demonstrated to be essential for various insulin-induced glucose
metabolic functions including glucose uptake and glycogen synthesis as
well as insulin-induced mitogenesis. For example, it was shown that
overexpression of a catalytic subunit of PI 3-kinase, p110, induced
GLUT4 translocation to the cell surface resulting in increased glucose
uptake, irrespective of insulin stimulation (50), while
overexpression of the dominant negative form of the regulatory subunit,
p85
, inhibited this action (24, 33). Thus, taking the
above-mentioned facts and several reports concerning the oncogenic role
of PI 3-kinase and the tumor-suppressing property of PTEN into
consideration, it is reasonable to speculate that PTEN plays a role in
insulin-induced glucose metabolism as important as that of PI 3-kinase.
Indeed, one previous report (51) described overexpression
of PTEN as significantly attenuating insulin-induced GLUT4
translocation and glucose uptake in 3T3-L1 adipocytes. However, it
remains unclear whether or not endogenous PTEN is indeed involved in
the regulation of glucose metabolism. To answer this question, we
prepared wild-type as well as the dominant negative forms of PTEN
constructs with or without an N-terminal myristoylation tag.
In this study, first of all, we investigated whether overexpressing PTEN and its mutants affects cellular quantities of 3-phosphoinositides in Sf-9 cells as well as 3T3-L1 adipocytes. Several reports have shown that PTEN has strong and specific phosphatase activity for 3-phosphoinositides in vitro (27, 28, 36, 49), but few reports have focused on PTEN activity toward each type of 3-phosphoinositide in the physiological in vivo environment of cells. Our results confirmed that not only PI(3, 4, 5)P3, but also PI(3, 4)P2, is regulated by PTEN, while the cellular PI(3)P content was unaffected by PTEN and its mutants expressed in Sf-9 insect cells as well as 3T3-L1 adipocytes.
Contrary to our expectations, wild-type PTEN without the myristoylation tag did not markedly decrease 3-position phosphorylated phosphoinositides in Sf-9 cells, despite the very high level of expression induced by the baculovirus system. Similarly, the effect of overexpressing wild-type PTEN without the myristoylation tag in 3T3-L1 adipocytes was revealed to be minimal also in terms of the reductions in PI(3, 4)P2 and PI(3, 4, 5)P3, despite its overexpression level exceeding 50-fold as compared with that of endogenous PTEN.
Several studies have shown that, in PTEN-deficient cells, levels of 3-phosphoinositides are increased and that reintroduction of wild-type PTEN normalizes these levels (29). Thus, our results suggest that when some amount of PTEN protein comparable to that of endogenous PTEN exists, the effects on 3-phosphoinositides are already adequate and saturated at least in Sf-9 cells and 3T3-L1 adipocytes. On the other hand, PTEN with the myristoylation tag did significantly and strongly reduce the accumulations of PI(3, 4)P2 and PI(3, 4, 5)P3 in both Sf-9 cells and 3T3-L1 adipocytes. Several reports have suggested that PTEN exists mainly in the cytoplasm (37, 38). Although it has been speculated that PTEN moves to the membrane when activated (35, 36), this hypothesis has not yet been demonstrated. In this study, it was shown that the membrane-associated portions of wild-type and catalytic dead PTEN temporarily increased in response to insulin stimulation. On the other hand, in the case of myristoylated PTEN, a much higher level of overexpressed protein existed in the membrane fraction, even in the basal state, than in the case of nonmyristoylated PTEN. Thus, the myristoylated-PTEN used in this study is regarded as a constitutively active form of the PTEN mutant. Although the functional regulatory mechanism of endogenous PTEN is unclear at present, taking into consideration that the myristoylated PTEN functions as a constitutively active form, the membrane targeting process of PTEN is likely to be important. Further study is needed to elucidate the mechanism of PTEN translocation to the membranes.
The C124S point mutation of PTEN, which corresponds to the catalytic
pocket that grasps the inositol ring of 3-phosphoinositides
(36), has been identified in various tumor cells.
Overexpression of the C124S mutant of PTEN markedly enhanced the
increases in 3-phosphoinositides induced by p110 overexpression in
Sf-9 cells and by insulin stimulation in 3T3-L1 adipocytes. These
results indicate that this mutant protein is not only unable to
catalyze 3-phosphoinositides but also has an apparent dominant negative
effect against the activity of endogenous PTEN. A functional difference
between C124S mutants of PTEN with and without the N-terminal
myristoylation tag was observed for the phosphoinositide content 20 min
after insulin stimulation. Overexpression of the C124S mutant of PTEN
with, but not that without, the myristoylation tag maintained elevated
cellular PI(3, 4)P2 and
PI(3, 4, 5)P3 contents induced by insulin
stimulation for at least 20 min. As shown in Fig. 5
, C124S PTEN without
the myristoylation tag temporarily and rapidly moves to membranes after
insulin stimulation, similar to wild-type PTEN, but subsequently
returns to the cytoplasm. This is the most likely reason for the C124S
mutant of PTEN without the myristoylation tag not being able to
continue to exert a dominant-negative effect against endogenous PTEN,
unlike that with the N-terminal myristoylation tag. These results,
obtained with C124S mutants of PTEN, indicate that endogenous PTEN does
play a role in the dephosphorylation of PI(3, 4)P2
and PI(3, 4, 5)P3 in Sf-9 cells as well as 3T3-L1
adipocytes.
Much remains unknown regarding the pathway between PI 3-kinase activation and GLUT4 translocation. At present, Akt is regarded as a possible mediator of insulin-induced GLUT4 translocation (52), although this speculation has not been substantiated (44, 45). Akt is a serine/threonine kinase located downstream from PI 3-kinase, and its activation is reportedly mediated by phosphorylation at Thr308 (corresponding to Thr309 of Akt2) and Ser473 (corresponding to Ser474 of Akt2) (41). Phosphorylation of Thr308 is mediated by 3-phosphoinositide-dependent kinase 1 (PDK1), the activity of which is dependent upon PI(3, 4, 5)P3 or both PI(3, 4)P2 and (2) PI(3, 4, 5)P3. On the other hand, the kinase responsible for the phosphorylation of Ser473 has been suggested to be 3-phosphoinositide-dependent kinase 2 (PDK2), or possibly also PDK1 (53), but remains to be determined.
Comparing Fig. 3 with Fig. 6
, our data revealed a close relationship
between the cellular PI(3, 4, 5)P3 content and the
level of phosphorylation at Thr309 of Akt2. This correlation between
PI(3, 4, 5)P3 and Thr309 phosphorylation of Akt
agrees with the hypothesis that both Akt and PDK1 are recruited onto
membranes by PI(3, 4, 5)P3, where Akt is
phosphorylated by PDK1. However, interestingly, the
phosphorylation level of Ser474 of Akt2 did not show a good correlation
with the cellular content of either PI(3, 4)P2 or
(2) PI(3, 4, 5)P3. This discrepancy
appears to strongly support the hypothesis that phosphorylation of
Ser474 is mediated by mechanisms other than phosphorylation by PDK1,
although phosphorylation of Ser474 is likely to be mediated by the
activation of PI 3-kinase since overexpression of the dominant negative
form of the p85
regulatory subunit of PI 3-kinase inhibited
phosphorylation at both Thr309 and Ser474. Further study is necessary
to clarify the regulation of the phosphorylation level of Ser474 of
Akt.
In addition, a very important finding of the current study is that the phosphorylation of Ser474, but not that of Thr309, which is highly dependent on the synthesis of PI(3, 4, 5)P3, in Akt2 correlates well with an increase in glucose transport activity. Overexpression of PTEN with the N-terminal myristoylation tag suppresses insulin-induced phosphorylation of Thr309 and Ser474 in Akt2 to 21% and 40%, respectively, and decreases insulin-induced glucose uptake to 53%. Overexpression of the C124S mutant of PTEN enhanced neither the phosphorylation of Ser474 of Akt2 nor insulin-induced glucose uptake by 3T3-L1 adipocytes, despite a 1.7- to 4.1-fold increase in the synthesis of PI(3, 4, 5)P3 and also a 2.6- to 5.3-fold increase in phosphorylation of Thr309 of Akt2. Such phenomena were also observed in experiments employing stimulation with insulin concentrations lower than 100 nM (data not shown). Therefore, it may be important to determine which kinase phosphorylates Ser474 in Akt2 and which protein binds to this C-terminal portion of Akt, and we are currently performing experiments with these aims. However, since the dominant negative mutants of PTEN did not affect (enhance) glucose uptake in either the presence or the absence of insulin, endogenous PTEN may not play a major role in the regulation of glucose transport activity in 3T3-L1 adipocytes, despite endogenous PTEN clearly affecting the accumulations of PI(3, 4)P2 and PI(3, 4, 5)P3.
In conclusion, 1) PTEN dephosphorylates both PI(3, 4)P2 and PI(3, 4, 5)P3 in vivo, and C124S mutants interrupted endogenous PTEN activity in a dominant-negative manner. 2) Membrane targeting processes of PTEN may be important for this function. 3) Phosphorylations of Thr309 and Ser474 of Akt2 were regulated differently, and the former regulation was very sensitive to the function of PTEN. 4) The phosphorylation level of Ser474, but not that of Thr309, in Akt2 correlates well with insulin-stimulated glucose transport activity in 3T3-L1 adipocytes. 5) The activity of endogenous PTEN may not play a major role on the regulation of glucose transport activity in 3T3-L1 adipocytes.
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MATERIALS AND METHODS |
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DNA Constructs
PTEN cDNA of Rattus norvegicus was amplified by PCR
from its brain cDNA library with primers corresponding to sequences
already reported and cloned into pCR2.1 plasmid vector
(Invitrogen, San Diego, CA). The Cys124 to Ser mutant form
(C124S) of PTEN was constructed using PCR with mutagenic
oligonucleotides 5'-CCA AGG CTG GGA AAG GAC GGA CTG-3' and 5'-CCT TGG
AGT GAA TTG CTG CAA CAT G-3' as PCR primers and ligated with the newly
integrated StyI restriction enzyme site. Nucleotides for the
N-terminal FLAG tag and myristoylation signal peptide were added using
oligonucleotides 5'-ATG GAC TAC AAG GAC GAC GAT GAC AAG ACA GCC ATC ATC
AAA GAG-3' and 5'-ATG GGA TGT TGG TGT AGC AGC AAT CCC GAA GAC GAC GAC
GAC TAC AAG GAC GAC-3', respectively. All sequences were confirmed
using a 370A DNA sequencer (PE Applied Biosystems,
Norwalk, CT).
Cell Culture
Sf-9 insect cells were maintained in TC-100 medium (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% FBS
(Life Technologies, Inc.) at 27 C. Sf-9 cells were
harvested 48 h after baculoviral infection for subsequent
experiments. 3T3-L1 fibroblasts were maintained in DMEM containing 10%
donor calf serum (Life Technologies, Inc.) under an
atmosphere of 10% CO2/90% air at 37 C. Two days
after the fibroblasts reached confluence, differentiation was induced
by incubating these cells for 48 h in DMEM containing 0.5
mM 3-isobutyl-1-methylxanthine, 4 mg/ml dexamethasone, and
10% FBS. Thereafter, the cells were maintained in DMEM supplemented
with 10% FBS, which was renewed every other day. The cells were
infected with the indicated adenoviruses on day 3 after inducing
differentiation, and the experiments were conducted on day 6 when
>90% of cells expressed the adipocyte phenotype and GLUT4 was almost
fully expressed.
Antibodies
Antiphosphotyrosine antibody 4G10 was purchased from
Upstate Biotechnology, Inc. (Lake Placid, NY). The
anti-PTEN antibody was raised in rabbits against synthetic peptides
corresponding to 18 C-terminal amino acids (DPENEPFDEDQHSQITKV) of the
PTEN protein. Antibody was affinity purified on Affi-Gel-10
(Bio-Rad Laboratories, Inc., Hercules, CA) columns to
which the corresponding peptide had been coupled, and extensively
dialyzed against PBS (PBS) and concentrated with Centricon 30 (Amicon
Inc., Beverly, MA). Antibodies against Akt, the phospho-Thr308 and
phospho-Ser473 of Akt, were purchased from New England Biolabs, Inc. (Beverly, MA). Antibody against the C-terminal GLUT2 tag of
p110 was prepared as described previously (50, 54).
Gene Transduction
To obtain recombinant baculoviruses, all DNA constructs (Fig. 1)
were subcloned into pBacPAK8 and transfected with the parental viral
genome into Sf-9 insect cells as instructed by the manufacturer
(CLONTECH Laboratories, Inc., Palo, Alto, CA).
Baculoviruses overexpressing LacZ and the GLUT2-tagged p110
subunit
of PI 3-kinase were made as previously described (34).
Viral titers for protein overexpression were adjusted so that the
expression levels of the wild-type and mutant PTEN proteins were
similar, irrespective of co-overexpression of p110
. Similarly, the
expression levels of p110
were adjusted so as to be similar,
irrespective of co-overexpression of PTEN or other co-overexpressed
proteins. The experiments described below were executed 48 h after
baculoviral infection.
To obtain recombinant adenoviruses, each DNA construct (Fig. 1) was
subcloned into a pAdexCAwt cosmid cassette and transfected with the
parental viral genome into HEK293 cells as described previously
(50). Adenoviruses overexpressing LacZ and
p85
were
prepared as described previously (33, 34). In the case of
adenoviruses, maximal overexpression of myristoylation signal-tagged
mutants was lower than that of non-myristoylation-tagged mutants. To
maximize the effects of overexpressed PTEN mutants, the levels of
overexpressed proteins were adjusted so as to be similar in wild-type
PTEN and the C124S mutant, and in myr-wild-type PTEN and the myr-C124S
mutant. The expression level of nonmyristoylation mutants was
approximately 3 times that of myristoylation signal-added mutants in
the experiments described below. The subsequent experiments were
executed 72 h after adenoviral infection of 3T3-L1 adipocytes.
Western Blotting
Cells were washed with ice-cold PBS twice, and collected with
Laemmli sample buffer containing 100 mM dithiothreitol, and
then subjected to SDS-PAGE. Immunoblotting was performed with the
ECL-plus (for anti-phosphoThr308-Akt antibody immunoblotting) or the
ECL (for other antibodies) system, according to the manufacturers
instructions. The images of bands on films were scanned with a
film-scanner (EPSON, Nagano, Japan) and the intensities were quantified
by NIH Image.
In Vivo 32P-Labeling of
Phosphoinositides
Sf-9 cells infected with baculovirus were phosphate-starved with
phosphorus-free DMEM (Sigma) with 5 mM HEPES
adjusted to pH 6.2 for 18 h and then labeled with
[32P]-orthophosphate (0.5 mCi/ml) for
4 h. Thereafter, the cells were washed with ice-cold PBS,
collected with 750 µl of methanol-1 N HCl (1:1) solution,
and mixed well and the lipids were extracted by adding 380 µl of
chloroform.
Similarly, 3T3-L1 adipocytes infected with adenovirus were phosphate starved for 14 h with phosphorus-free DMEM, followed by serum starvation for 1 h. [32P]-orthophosphate (0.5 mCi/ml) was added, and the cells were cultured for an additional 2 h. After the labeling period, the cells were incubated with or without 100 nM insulin for the indicated times. The reaction was then terminated with an ice-cold PBS wash, followed by the addition of methanol-1 N HCl (1:1) and finally lipid extraction with chloroform.
HPLC Analysis
The extracted lipid was deacylated and subjected to
anion-exchange HPLC using a Partisphere strong anion-exchange column
(Whatman, Clifton, NJ) as described previously
(55). The radioactivity was detected with an on-line
radiochemical detector in the case of experiments using Sf-9 cells. The
samples from labeled 3T3-L1 adipocytes were subjected to HPLC analysis,
after which fractionation was carried out for every 20-sec elution from
the HPLC column and the radioactivity of each fraction was counted
using a liquid scintillation counter.
[32P]-labeled PI(3)P,
PI(3, 4)P2, and PI(3, 4, 5)P3
generated in vitro as described previously (34)
were deacylated and used as internal standards. Each sample and
standard was coinjected with the nonradioactive nucleotides ADP and ATP
for each HPLC analysis to confirm the peak of each phosphorylated
lipid.
PI 3-Kinase Activity Assay
3T3-L1 adipocytes infected with adenovirus were serum starved
for 3 h in DMEM containing 0.2% BSA. Then, the cells were
stimulated with 100 nM insulin for 15 min, washed once with
ice-cold PBS, and lysed with PBS containing 1% Nonidet P-40, 0.35
mg/ml phenylmethylsulfonyl fluoride, and 100 mM sodium
vanadate. Insoluble materials were eliminated by centrifugation, and
the supernatants were immunoprecipitated with antiphosphotyrosine
antibodies and protein G-Sepharose. The PI 3-kinase activity in the
immunoprecipitates was measured as described previously
(50).
Subcellular Fractionation of 3T3-L1 Adipocytes
The method of obtaining the total membrane fraction from 3T3-L1
adipocytes was as described previously (56) with some
modifications. 3T3-L1 adipocytes were serum-starved for 3 h in
DMEM containing 0.2% BSA and incubated with or without 100
nM insulin for the indicated times. Cells were washed twice
with ice-cold HES buffer (20 mM HEPES, pH 7.4, 1
mM EDTA, 22.5 mM sucrose), and then detached
from the dish using a rubber policemen and collected in 0.6 ml of
homogenizing buffer (HES buffer containing 1 mM sodium
vanadate, 1 mM phenylmethylsulfonyl fluoride, 40
mM ß-glycerophosphate, 50 mM sodium
fluoride). The cells were homogenized by passage through a 29-gauge
needle 16 times. One tenth of the suspension was set aside, sonicated,
and prepared for SDS-PAGE as the cell lysate. The remaining nine tenths
of the suspension was centrifuged at 600 x g for 10
min to remove mainly nuclei and mitochondria as the sediment. The
supernatant was centrifuged at 250,000 x g for 90 min
at 4 C to precipitate the total membrane particulate fraction. The
supernatant was removed, and the precipitate was resuspended with
homogenizing buffer and again centrifuged at 250,000 x
g for 90 min. After the supernatant had been removed, the
precipitant was lysed with 50 µl of lysis buffer (50
mM Tris-HCl, pH 7.4, 100 mM
NaCl, 10 mM EDTA, 10% glycerol, 1% Nonidet
P-40, 1 mM sodium vanadate, 1
mM phenylmethylsulfonyl fluoride, 40
mM ß-glycerophosphate, 50
mM sodium fluoride). The lysate was centrifuged
at 15,000 x g for 10 min, and the supernatant was
prepared for SDS-PAGE as the membrane fraction.
2-Deoxyglucose Uptake Assay
3T3-L1 adipocytes in a 12-well culture dish were serum starved
for 3 h in DMEM containing 0.2% BSA, and glucose-free incubation
was performed for 45 min in Krebs-Ringer phosphate buffer
(15). Cells were then incubated with or without 100
NM insulin for 15 min, and
2-deoxy-D-[3H]-glucose uptake
was measured as described previously (57).
Statistical Analysis
Data are expressed as means ± SE. Comparisons
were made using the unpaired Students t test;
P < 0.05 was considered a statistically significant
difference. The representative immunoblots from one experiment were
shown in some figures.
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FOOTNOTES |
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Received for publication September 27, 2000. Accepted for publication May 15, 2001.
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REFERENCES |
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