Phosphoinositide 3-Kinase Is Required for Insulin-Induced but Not for Growth Hormone- or Hyperosmolarity-Induced Glucose Uptake in 3T3-L1 Adipocytes
Hiroshi Sakaue,
Wataru Ogawa,
Masafumi Takata,
Shoji Kuroda,
Ko Kotani,
Michihiro Matsumoto,
Motoyoshi Sakaue,
Shoko Nishio,
Hikaru Ueno and
Masato Kasuga
Second Department of Internal Medicine (H.S., W.O., M.T., S.K.,
K.K., M.M., M.S., M.K.), Kobe University School of Medicine, Kobe
650, Japan,
Molecular Cardiology Unit (S.N., H.U.), Kyushu
University School of Medicine, Fukuoka 812-82, Japan
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ABSTRACT
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The regulatory mechanism of glucose uptake in
3T3-L1 adipocytes was investigated with the use of recombinant
adenovirus vectors encoding various dominant negative proteins.
Infection with a virus encoding a mutant regulatory subunit of
phosphoinositide (PI) 3-kinase that does not bind the 110-kDa
catalytic subunit (
p85) inhibited the insulin-induced increase
in PI 3-kinase activity coprecipitated by antibodies to phosphotyrosine
and glucose uptake in a virus dose-dependent manner. Overexpression of
a dominant negative RAS mutant in which Asp57
is replaced with tyrosine (RAS57Y) or of a dominant negative SOS mutant
that lacks guanine nucleotide exchange activity (
SOS) abolished the
insulin-induced increase in mitogen-activated protein kinase activity,
but had no effect on PI 3-kinase activity or glucose uptake. Although
GH and hyperosmolarity attributable to 300 mM
sorbitol each promoted glucose uptake and translocation of glucose
transporter (GLUT)4 to an extent comparable to that of insulin, these
stimuli triggered little or no association of PI 3-kinase activity with
tyrosine-phosphorylated proteins. Overexpression of
p85 or treatment
of cells with wortmannin, an inhibitor of PI 3-kinase activity, had no
effect on glucose uptake or translocation of GLUT4 stimulated by GH or
hyperosmolarity. Moreover, overexpression of
SOS or RAC17N also did
not affect the increase in glucose uptake induced by these stimuli. A
serine/threonine kinase Akt, a constitutively active mutant of which
was previously shown to stimulate glucose uptake, is activated by
insulin, GH, and hyperosmolarity to
4-fold,
2.1-fold, and
2.3-fold over basal level, respectively. These results suggest that
insulin-induced but neither GH- or hyperosmolarity-induced glucose
uptake is PI 3-kinase-dependent, and neither RAS nor RAC is required
for glucose uptake induced by these stimuli in 3T3-L1 adipocytes.
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INTRODUCTION
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Stimulation of glucose uptake is one of the most important
short-term actions of insulin. Although the effects of insulin on
various biological activities are mediated by activation of the insulin
receptor tyrosine kinase (1), the mechanism by which receptor
activation increases glucose uptake is not fully understood.
Phosphoinositide (PI) 3-kinase is thought to play a pivotal role in
this process. This lipid kinase consists of a 110-kDa catalytic subunit
(p110) and an 85-kDa regulatory subunit (p85) that contains SRC
homology 2 and 3 domains (2, 3). In response to insulin, insulin
receptor substrate (IRS)-1, the best characterized substrate of the
insulin receptor kinase, undergoes tyrosine phosphorylation and binds
to p85 through its SRC homology 2 domains (3), an interaction that is
thought to be crucial for activation of the enzyme (2, 3).
The importance of PI 3-kinase in glucose uptake was revealed with the
use of a dominant negative mutant of p85 (4) and wortmannin, a fungal
metabolite that inhibits the catalytic activity of p110 at nanomolar
concentrations (5). Wortmannin prevents the insulin-induced increase in
glucose uptake in various cells and tissues (3, 5). However, wortmannin
inhibits not only the catalytic subunit of PI 3-kinase but also
mammalian Vps34 homolog (also known as
phosphatidylinositol-specific PI 3-kinase) (6), p110
(also known
as G protein ß
subunit-activated PI 3-kinase) (7), a novel PI
4-kinase (8), phospholipase A2 (9), and mTOR/FRAP, a
putative target of rapamycin (10), indicating that wortmannin is not a
specific inhibitor of PI 3-kinase. Although the synthetic agent
LY294002 inhibits the catalytic activity of PI 3-kinase and
insulin-induced glucose uptake at concentrations 100 times those of
wortmannin (5, 11), it is not known whether PI 3-kinase is the only
target of this reagent.
When overexpressed in cultured cells, a mutant p85, which lacks the
binding site for p110, inhibited the association of endogenous PI
3-kinase with IRS-1 (4). In these cells, both insulin-induced glucose
uptake and the translocation of glucose transporters (GLUTs), which is
thought to be essential for glucose uptake (12), were markedly
attenuated (4), indicating that the association of the lipid kinase
with IRS-1 is required for this process. Although overexpression of the
mutant p85, which we have termed
p85, might be expected to inhibit
PI 3-kinase activity more specifically than pharmacological approaches,
the effect of this protein on glucose uptake has been examined only in
Chinese hamster ovary (CHO) cells, a fibroblast cell line. Fibroblasts
express GLUT1 glucose transporters, which are less sensitive to insulin
than the GLUT4 glucose transporters that are expressed by the
physiological target cells of insulin (12). Furthermore, these two
glucose transporters reside in different vesicles (13). The regulation
of glucose transport in fibroblasts may thus differ from that in
adipocytes or muscle cells. Although we have shown that microinjection
of
p85 or transient transfection with
p85 cDNA inhibits GLUT4
translocation in adipocytes (14, 15), it is not known whether the
association of PI 3-kinase with IRS-1 is required for glucose uptake in
the physiological target tissues of insulin.
Whether RAS and the signaling pathways initiated by this GTPase plays a
role in insulin regulation of glucose uptake is controversial.
Introduction of a constitutively active RAS was shown to promote
translocation of GLUT4 (12, 16), an effect that might be mediated by PI
3-kinase because activated RAS stimulates PI 3-kinase activity in
intact cells (17). On the other hand, inhibition of RAS activation by a
dominant negative mutant had no effect on insulin-induced translocation
of GLUT4 or glucose transport in adipocytes (18, 19). Another
complicating factor in evaluating the role of RAS-mitogen-activated
protein (MAP) kinase pathway in glucose uptake is that insulin-induced
MAP kinase activity is inhibited by wortmannin (20), suggesting that
the effect of wortmannin on glucose uptake may be partly mediated by
the RAS-MAP kinase pathway.
In addition to insulin, a variety of extracellular stimuli, including
GH, hydrogen peroxide, vanadium compounds, and inhibitors of serine or
threonine phosphatases such as okadaic acid (21, 22, 23, 24), stimulates
glucose uptake or GLUT4 translocation. Furthermore, cellular stresses
such as hypoxia, increase in heat, pH, hyperosmolarity, or contraction
(25, 26, 27, 28) also stimulate glucose uptake. Although it is less well
characterized than insulin-induced glucose uptake, the molecular
mechanism of insulin-independent glucose uptake is also of interest
because muscle contraction, which promotes glucose uptake in the
absence of insulin (27, 28), is thought to be important in reducing
plasma glucose concentrations in individuals with diabetes.
With the use of recombinant adenovirus vectors to introduce dominant
negative molecules into differentiated mouse 3T3-L1 adipocytes, we have
now investigated the roles of PI 3-kinase, RAS, and RAC in regulating
glucose transport induced by GH, hyperosmolarity, and insulin.
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RESULTS
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Transient expression of dominant negative proteins in cultured
cells is widely used to block a specific signaling pathway. Although
3T3-L1 adipocytes are a good model with which to study the mechanism of
glucose transport because of their high responsiveness to insulin, an
efficient method for achieving transient gene expression in these cells
has not been established. To introduce exogenous genes into the
cultured adipocytes, we used the replication-defective recombinant
adenovirus vector, Adex (29). We first tested the efficiency of gene
transfer with the use of Adex encoding ß-galactosidase (AdexCALacZ).
Six to 10 days after differentiation, the adipocytes were infected with
AdexCALacZ at various multiplicity of infections (MOIs). After 48
h, the cells were subjected to ß-galactosidase staining or immunoblot
analysis with antibodies to ß-galactosidase. Cytochemistry revealed
that the extent of ß-galactosidase expression increased in an
MOI-dependent manner, so that >95% of cells were positive for
staining at an MOI of 30 (Fig. 1
, A and B).
Immunoblot analysis also revealed the MOI-dependent increase in
ß-galactosidase expression (Fig. 1C
). These results demonstrated a
high efficiency of gene transfer with Adex.

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Figure 1. Expression of ß-Galactosidase from the AdexCALacZ
Vector in 3T3-L1 Adipocytes
Cells were infected with AdexCALacZ at the indicated MOIs and, after
48 h, subjected to ß-galactosidase staining [macroscopic view
(A), x400 magnification (B)] or immunoblot analysis with antibodies
to ß-galactosidase (C). One-twentieth of the lysate from a 6-cm plate
was applied to each lane in panel C.
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We therefore prepared Adexes that encode a mutant regulatory
subunit of PI 3-kinase that lacks the binding site for the 110-kDa
catalytic subunit (
p85), a mutant RAS in which Asp57 is
replaced by tyrosine (RAS57Y), a mutant SOS that lacks guanine
nucleotide exchange activity (
SOS), and a mutant RAC in which
Ser17 is replaced by asparagine (RAC17N) and
that exhibits an affinity for GDP 100 times that for GTP (30).
These viruses were termed AdexCA
p85, AdexCAHRAS57Y, AdexCA
SOS,
and AdexCARAC17N, respectively.
We first evaluated whether these dominant negative molecules block
specific signaling pathways in 3T3-L1 adipocytes. When 3T3-L1
adipocytes were infected with various Adexes and subjected to
immunoblot analyses after 48 h, all the introduced genes were
expressed efficiently in an MOI-dependent manner (Fig. 2A
, Fig. 3A
).
Infection with AdexCA
p85 inhibited insulin-induced PI 3-kinase
activity that was precipitated with antibodies to phosphotyrosine in an
MOI-dependent manner, with almost 95% inhibition apparent at an MOI of
30 (Fig. 2B
). Essentially the same results were obtained when PI
3-kinase activity was precipitated with antibodies to IRS-1 (data
not shown). The ability of
p85 to block the PI 3-kinase pathway was
confirmed by investigating the effect of
p85 on insulin-induced
glucose uptake. Infection with AdexCA
p85 inhibited insulin-induced
glucose uptake in an MOI-dependent manner, with inhibition of almost
60% apparent at an MOI of 30 (Fig. 4
).
AdexCA
SOS, AdexCAHRAS57Y, and AdexCARAC17N did not affect
insulin-induced PI 3-kinase activity (Fig. 3B
) or glucose uptake (Fig. 5E
and data not shown), indicating that the
suppression of glucose uptake by infection with AdexCA
p85 was not
due to nonspecific effects of virus infection but to specific
inhibition of the PI 3-kinase path-way. On the other hand,
overexpression of
SOS, which we have previously shown inhibits the
acti-vation of RAS by insulin in CHO cells (31), as well as
overexpression of RAS57Y, attenuated insulin-induced MAP kinase
activity in 3T3-L1 adipocytes (Fig. 3C
), whereas infection with
AdexCA
p85 or AdexCARAC17N did not affect MAP kinase activity (Figs. 2C
and 3B
). Furthermore, infection of 3T3-L1 adipocytes with
AdexCARAC17N, at an MOI of 30, completely inhibited insulin-induced
membrane ruffling (data not shown), which is known to be regulated by
RAC (30). These observations indicate that all the introduced genes
with the use of Adex were capable of inhibiting specific signaling
pathways in differentiated adipocytes as expected.

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Figure 3. Effects of Overexpression of SOS, RAS57Y, and
RAC17N on Insulin-Induced Activation of PI 3-Kinase and MAP Kinase in
3T3-L1 Adipocytes
A, Expression of SOS, RAS57Y, and RAC17N. 3T3-L1 adipocytes were
infected with AdexCA SOS, Adex-CAHRAS57Y, or AdexCARAC17N at the
indicated MOIs. Forty-eight hours after infection, the cells were lysed
and the total lysates were subjected to immunoblot analysis with
antibodies to SOS, to RAS, or to HA (for RAC17N). One-twentieth of the
lysate from a 6-cm plate was applied to each lane. B, PI 3-kinase
assay. The virus-infected cells were incubated in the absence or
presence of 0.1 µM insulin for 5 min, after which PI
3-kinase activity was precipitated with antibodies to phosphotyrosine
and assayed. Data are representative of three independent experiments.
C, MAP kinase assay. The virus-infected cells were incubated with or
without 0.1 µM insulin for 10 min, after which
immunoprecipitates prepared with antibodies to MAP kinase were assayed
for MAP kinase activity. Data are means ± SE from
three experiments.
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Figure 4. Effects of Overexpression of p85 on
Insulin-Stimulated Glucose Uptake in 3T3-L1 Adipocytes
Cells were infected with AdexCA p85 at the indicated MOI. Forty-eight
hours after infection, the cells were treated with or without insulin,
and 2-deoxy[3H]glucose uptake was assayed. Data are
means ± SE from at least three experiments.
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Figure 5. Roles of PI 3-Kinase Activity and SOS in Glucose
Uptake Induced by GH or Hyperosmolarity in 3T3-L1 Adipocytes
A, Effects of GH and hyperosmolarity on PI 3-kinase activity. 3T3-L1
adipocytes were incubated in the absence or presence of 0.1
µM insulin for 5 min, GH (0.5 µg/ml) for 5 min, or 300
mM sorbitol for 30 min, after which PI 3-kinase activity
was precipitated with antibodies to phosphotyrosine and assayed. B, Time course of GH-stimulated PI 3-kinase activity.
3T3-L1 adipocytes were incubated with GH (0.5 µg/ml) for the
indicated times, after which PI 3-kinase activity was assayed in
immunoprecipitates prepared with antibodies to phosphotyrosine. Data in
panels A and B are representative of three independent experiments. C
and D, Effects of p85 (C) and wortmannin (D) on glucose uptake
stimulated by insulin, GH, or hyperosmolarity. 3T3-L1 adipocytes were
infected with or without AdexCA p85 (C) or preincubated in the
absence or presence of 100 nM wortmannin for 20 min. The
cells were subsequently exposed to insulin, GH, or sorbitol at the
above concentrations and 2-deoxy[3H]glucose uptake was
assayed as described in Materials and Methods. Data are
means ± SE from at least three experiments. E,
Effects of SOS or RAC17N on glucose uptake stimulated by insulin,
GH, or hyperosmolarity. 3T3-L1 adipocytes were infected with either
AdexCA SOS or AdexCARAC17N at an MOI of 30. Forty-eight hours after
infection, the cells were treated with insulin, GH, or sorbitol, and
2-deoxy[3H]glucose uptake was assayed. Data are
means ± SE from at least three independent
experiments.
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With the use of the various mutant proteins described above, we
investigated the molecular mechanisms of glucose uptake induced by GH
and sorbitol hyperosmolarity, both of which are known to stimulate
glucose uptake, and compared them with that by insulin. When 3T3-L1
cells were treated with GH or sorbitol, glucose uptake was stimulated
5- to 6-fold, similar to the effect of insulin (Fig. 5
, CE). Maximal
insulin stimulation with either GH or sorbitol did not result in
further increase in glucose uptake (insulin alone, 32,575 ± 2,944
cpm; insulin plus GH, 30,725 ± 1,504 cpm; insulin plus
hyperosmolarity, 28,678 ± 2,342 cpm; data are means ±
SE from three experiments). Instead, glucose uptake
achieved by insulin plus either GH or sorbitol was slightly decreased
as compared with that achieved by insulin alone. Similar reduction was
previously observed with isolated adipocytes treated with insulin plus
sorbitol (26).
Sorbitol had no effect on PI 3-kinase activity precipitated with
antibodies to phosphotyrosine (Fig. 5A
). GH, which induces tyrosine
phosphorylation of IRS-1 and the association of PI 3-kinase with IRS-1
in isolated adipocytes (32) and 3T3-F442 adipocytes (33), the latter of
which are closely related to 3T3-L1 adipocytes, stimulated PI 3-kinase
activity in a time-dependent manner (Fig. 5B
). However, the extent of
PI 3-kinase activation induced by GH was <10% of that achieved with
insulin (Fig. 5A
), whereas the extent of GH-induced glucose uptake was
similar to that induced by insulin. These data suggest that the
association of PI 3-kinase with tyrosine-phosphorylated protein is not
required for glucose uptake induced by GH or hyperosmolarity.
To test this hypothesis, we examined the effect of overexpression of
p85 on glucose uptake induced by these stimuli. Infection with
AdexCA
p85 at an MOI of 30, at which concentration the virus
inhibited insulin-induced glucose uptake by
60%, had no effect on
glucose uptake induced by GH or sorbitol (Fig. 5C
). Pretreatment of
uninfected cells with wortmannin at a concentration of 100
nM abolished insulin-induced glucose uptake but had no
effect on glucose uptake induced by GH or hyperosmolarity (Fig. 5D
).
Because translocation of GLUT4 is an essential step to promote glucose
uptake in adipocytes, we next investigated the effect of
p85 on
translocation of GLUT4 by photoaffinity labeling of membrane surface
GLUT4 with the use of 2-N-4-(1-azi-2, 2,
2-trifluoroethyl)benz-oyl-1,3-bis-(D-mannos-4-yloxy)-2-propylamine
(ATB-BMPA) (28, 34). Insulin as well as GH and hyperosmolarity
treatment dramatically induced translocation of GLUT4 to the plasma
membrane in 3T3-L1 adipocytes (Fig. 6
).
Overexpression of
p85 inhibited GLUT4 translocation induced by
insulin by
60% whereas translocation of GLUT4 promoted by neither
sorbitol nor GH was affected by
p85 (Fig. 6
), consistent with the
data of glucose uptake. Involvement of PI 3-kinase to translocation of
GLUT 4 by various stimuli was also examined with plasma membrane lawn
assay. Insulin, GH, and sorbitol treatment provoked an increase
in immunoreactivity of GLUT4 in the plasma membrane lawn prepared from
3T3-L1 adipoicytes (Fig. 7
, B, D, and E).
Pretreatment of cells with wortmannin inhibited the increase in the
immunoreactivity induced by insulin (Fig. 7C
), whereas the increase in
the immunoreactivity promoted by either GH or hyperosmolarity was not
affected by wortmannin (Fig. 7
, E and G). These observations confirmed
that PI 3-kinase is not required for glucose uptake stimulated by GH or
sorbitol.

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Figure 6. Effects of Overexpression of p85 on
Translocation of GLUT4 in 3T3-L1 Adipocytes
Cells were infected with AdexCA p85 at the indicated MOI. Forty-eight
hours after infection, the cells were incubated with or without insulin
(A), GH (B), or sorbitol (C). Translocation of GLUT4 to the plasma
membrane was assayed by ATB-[2-3H]BMPA surface labeling
as described in Materials and Methods. Data are
means ± SE from at least three experiments.
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Figure 7. Effects of Wortmannin on Immunofluorescence
Labeling of GLUT4 in Plasma Membrane Lawn Prepared from 3T3-L1
Adipocytes
Cells were incubated in the absence (A, B, D, and F) or presence (C, E,
and G) of 100 nM wortmannin for 20 min and in the absence
(A) or presence of insulin (B and C), GH (D and E), or sorbitol (F and
G), after which plasma membrane fragments were prepared for
immunofluorescence microscopy with antibodies to GLUT4 (1F8) and
TRITC-labeled secondary antibodies. Data are representative of at least
three independent experiments.
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We also examined whether signaling pathways mediated through RAS or RAC
are involved in glucose uptake stimulated by GH or hyperosmolarity.
Overexpression of
SOS or RAC17N had no effect on glucose uptake
induced by GH, hyperosmolarity, or insulin (Fig. 5E
). Similar results
were obtained with a virus that encodes RAS57Y (data not shown). These
observations indicate that the signaling pathways initiated by RAS or
RAC do not mediate the activation of glucose uptake by these stimuli in
3T3-L1 adipocytes.
Finally, we examined whether these stimuli activate serine/threonine
kinase Akt, a constitutively active mutant of which has been shown to
promote glucose uptake as well as translocation of GLUT4 in 3T3-L1
adipocytes (35). Insulin stimulated Akt activity to
4-fold, and GH
or sorbitol stimulated to
2.1- and
2.3-fold over basal level,
respectively (Fig. 8
).

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Figure 8. Effects of Insulin, GH, or Sorbitol on Akt Activity
in 3T3-L1 Adipocytes
3T3-L1 adipocytes were treated with or without 0.1 µM
insulin for 5 min, GH (0.5 µg/ml) for 5 min, or 300 mM
sorbitol for 30 min, after which cells were lysed and precipitated with
polyclonal antibodies against Akt. The precipitates were then assayed
for Akt kinase activity. Data are means ± SE from
four independent experiments.
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DISCUSSION
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The 3T3-L1 cell line is a useful model for investigating the
metabolic actions of insulin. Under appropriate conditions, the
originally fibroblast-like cells acquire many characteristics of
adipocytes, including accumulation of lipids, expression of GLUT4 and
insulin receptors, and an increased sensitivity of glucose uptake to
insulin (12). Many investigators have studied insulin signaling in
3T3-L1 adipocytes by introducing exogenous genes into the preadipocytes
(12, 35, 36, 37). However, with such an approach, the exogenous genes may
affect the adipogenesis, given that insulin is a potent adipogenic
stimulus. Indeed, a recent report has shown that introduction of a
constitutively active mutant of Akt, a serine/threonine kinase known to
be activated by insulin, into 3T3-L1 preadipocytes accelerates
differentiation and enhances expression of GLUT1 protein (35).
Circumvention of this problem requires a method for gene transfer into
differentiated adipocytes. Despite the fact that vaccinia
virus-mediated gene transfer is effective in a certain cell lines (38),
we have found that 3T3-L1 adipocytes are not a good target for the
vaccinia system. We therefore attempted to introduce exogenous genes
into the differentiated adipocytes with the use of
replication-defective recombinant adenovirus vectors, which have proved
effective for gene transfer, especially in quiescent, differentiated
cells and tissues (29).
We have prepared adenovirus vectors that encode dominant negative
mutants of PI 3-kinase, RAS, SOS, and RAC. We previously reported that
overexpression of
p85 in fibroblasts attenuates insulin-induced
glucose uptake as well as translocation of GLUT1 (4), and
microinjection of
p85 inhibits insulin-induced translocation of
GLUT4 in adipocytes (14). We have shown that overexpression of
p85
with the use of adenovirus vector inhibited insulin-induced increase in
PI 3-kinase precipitated with antiphosphotyrosine antibodies as well as
glucose uptake in 3T3-L1 adipocytes in an MOI-dependent manner. By
immunoblot analysis, we compared the amount of GLUT4 and insulin
receptor in adipocytes that had been infected with or without
AdexCA
p85 and found that no detectable change in the amount of these
proteins was detected within 48 h after infection (data not
shown).
SOS or RAS57Y inhibited MAP kinase activity induced by
insulin without affecting PI 3-kinase activity precipitated by
antibodies to phosphotyrosine or insulin-induced glucose uptake.
Furthermore, overexpression of RAC17N blocked insulin-induced membrane
ruffling. All these data indicate that gene transfer with the use of
Adex is a useful method to investigate signal transduction in
differentiated adipocytes.
Although it has been reported that wortmannin inhibits insulin-induced
activation of MAP kinase (20), overexpression of
p85 did not affect
MAP kinase activity. These data may suggest that some uncharacterized
wortmannin-sensitive molecule may lie upstream of MAP kinase. Recently,
p110
, the catalytic activity of which is known to be inhibited by
wortmannin, was shown to regulate MAP kinase activation stimulated by
heterotrimeric G protein-coupled receptor (7), suggesting that p110
or a similar enzyme that can transmit signals to MAP
kinase may also be involved in insulin-induced MAP kinase
activation.
A number of stimuli other than insulin promote glucose uptake in
various cells and tissues, including fat and muscle cells. Although
some of these stimuli induce glucose uptake or GLUT4 translocation in a
wortmannin-insensitive manner (25, 26, 28), it is not yet known whether
GH and hyperosmolarity stimulate glucose uptake or GLUT4 translocation
via a PI 3-kinase-dependent mechanism. We have now shown that, in
3T3-L1 adipocytes, GH and hyperosmolarity increased glucose transport
and translocation of GLUT4 to a similar extent as insulin. The
observation that these stimuli induced little or no association of PI
3-kinase with tyrosine-phosphorylated proteins, together with the
failure of
p85 and wortmannin to inhibit stimulated glucose uptake
and translocation of GLUT4, indicates that GH and hyperosmolarity
promote glucose uptake via a pathway (or pathways) that bypasses or is
completely independent of PI 3-kinase.
Maximal insulin stimulation with neither GH nor hyperosmolarity
resulted in a further increase in glucose uptake. In isolated muscle,
the combined effects of contraction, which stimulates glucose uptake
via a wortmannin-insensitive mechanism, and maximal insulin stimulation
on glucose uptake are additive (28), suggesting that signaling pathways
of glucose uptake elicited by contraction in muscle and by either GH or
by sorbitol in adipocytes may be different.
Although earlier reports have suggested that RAS and RAC are not
involved in regulatory mechanism of glucose uptake stimulated by
insulin (18, 19, 39), it has not yet been determined whether these
GTP-binding proteins regulate glucose uptake elicited by other stimuli.
We thus investigated the effects of
SOS and RAC17N on sorbitol- or
GH-induced glucose uptake. Both sorbitol and GH, the latter of which is
known to activate RAS-MAP kinase cascade (40), do not seem to require
RAS activation for glucose uptake because overexpression of
SOS did
not affect glucose uptake. RAC is known to regulate stress-activated
protein kinase pathway, p38 MAP kinase pathway, and p21-activated
protein kinases (41, 42, 43). Hyperosmolarity as well as various cellular
stresses including heat shock, UV, ionizing radiation, and oxidative
stress are known to activate the stress-activated protein kinase and
p38 MAP kinase pathways (41). Furthermore, insulin activates p38 MAP
kinase as well as p21-activated protein kinase in L6 myotube (44). We
do not know whether any of these protein kinases are activated by
sorbitol or GH in 3T3-L1 adipocytes, but the failure of RAC17N to block
glucose uptake indicates that signals initiated by RAC activation may
not be involved in glucose uptake stimulated by hyperosmolarity or
GH.
Recently, Kohn et al. (35) have shown that overexpression of
a membrane-targeted mutant of Akt induces glucose uptake and
translocation of GLUT4 in quiescent 3T3-L1 adipocytes. Akt was
originally shown to be activated by various growth factors via
wortmannin-sensitive mechanisms; however, it was later found to be
activated by hyperosmolarity, heat-shock (45), or ß-adrenergic
agonists (46). Moreover, activation of Akt by these stimuli is not
sensitive to wortmannin (45, 46). We thus investigated the effects of
GH and hyperosmolarity on Akt activity in 3T3-L1 adipocytes. Although
the extent of stimulation is lower than that by insulin, both GH and
sorbitol activate Akt in the adipocytes. Furthermore, we have recently
found that pervanadate, which is known to stimulate glucose uptake,
also activates Akt in 3T3-L1 adipocytes (S. Kuroda, H. Sakaue, W.
Ogawa, and M. Kasuga, unpublished observation). These data do not
necessarily indicate that GH or sorbitol stimulates glucose transport
through Akt activation. On the contrary, the relatively weak activation
of Akt by GH or hyperosmolarity implies that signaling mechanisms in
addition to Akt may be involved in glucose uptake by these stimuli.
Selective inhibition of Akt activity, which might be achieved by
introduction of dominant inhibitory mutants of Akt (47) by the Adex
system, may be useful to elucidate a role of Akt in glucose uptake by
various stimuli.
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MATERIALS AND METHODS
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Cell Culture, Antibodies, and Reagents
3T3-L1 preadipocytes maintained under an atmosphere of 7.5%
CO2 in DMEM containing 25 mM glucose and
supplemented with 10% heat-inactivated calf serum, 2 mM
L-glutamine, penicillin (50 U/ml), and streptomycin sulfate
(50 µg/ml) were induced to differentiate into adipocytes as described
previously (14). Monoclonal antibodies to ß-galactosidase, to
phosphotyrosine (PY20), and to the hemaggulutinin (HA) epitope tag
(12CA5) were obtained from Promega (Madison, WI), Transduction Labs
(Lexington, KY), and Boehringer Mannheim (Indianapolis, IN),
respectively. Monoclonal antibodies to p85 (F12) and to IRS-1 (1D6) as
well as polyclonal antibodies to MAP kinase (
C92), prepared against
a synthetic peptide corresponding to residues 350 to 367 of rat
p44MAPK, were as described previously (48, 49). Antibodies
to mSOS1 (31), to H-RAS (50), to Akt (45), to GLUT4 (14), and to RAC
were gifts from D. Bowtell (University of Melbourne, Melbourne,
Australia), T. Tanaka (Kure Hospital, Hiroshima, Japan), U. Kikkawa
(Biosignal Research Center, Kobe University, Kobe, Japan), D. James
(University of Queensland, Brisbane, Australia), and Eisai
Pharmaceutical Co. (Tokyo, Japan), respectively. Wortmannin (Sigma, St.
Louis, MO) was dissolved in dimethyl sulfoxide, stored at -20 C in the
dark, and diluted with distilled water immediately before addition to
cells. Recombinant human GH was kindly provided by Novo-Nordisk
(Copenhagen, Denmark).
Construction of and Infection with Adenovirus Vectors
Bovine p85
that lacks the binding site for the 110-kDa
catalytic subunit of PI 3-kinase (
p85), mSOS1 that lacks the
catalytic domain for guanine nucleotide exchange activity (
SOS), and
H-RAS in which Asp57 is replaced by tyrosine (RAS57Y) were
as described previously (4, 31, 51, 52). HA epitope-tagged RAC17N (with
asparagine substituted for Ser17) was produced as described
previously (53). Recombinant adenovirus vectors were generated by
cloning the cDNAs into pAxCAwt (54), which contains the CAG promoter
(55), and cotransfection into 293 cells with DNA-TPC, as described
previously (29). Protein-encoding viruses were screened by immunoblot
analysis and cloned by limiting dilution. Adenovirus vectors were
propagated by a standard procedure and then purified and titrated as
described previously (56). An adenovirus vector encoding RAS57Y
(AdexHRAS57Y) was as described (52), and adenovirus vector encoding the
lacZ gene (AdexCALacZ) (54) was a gift from I. Saito (Tokyo University,
Tokyo, Japan). 3T3-L1 adipocytes were infected with Adexs at the
indicated MOI (MOI = plaque-forming units per cell) in DMEM
containing 10% FBS. The virus-containing medium was replaced with
fresh medium after 6 h, and the cells were used for various
experiments 48 h after infection.
ß-Galactosidase Staining
3T3-L1 adipocytes infected with AdexCALacZ were washed twice
with PBS and fixed with 0.25% glutaraldehyde at 4 C for 10 min. After
washing four times with PBS, the cells were stained with PBS containing
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (1
mg/ml), 5 mM K3Fe(CN)6, 5
mM K4(CN)6, and 2 mM
MgCl2.
PI 3-Kinase Assay
3T3-L1 adipocytes were incubated in serum-free DMEM for 16
h, treated with various reagents, and then immediately frozen with
liquid nitrogen. The cells were lysed in a lysis buffer described
previously (38, 48), and insoluble materials were removed by
centrifugation. The supernatants were subjected to immunoprecipitation
with either PY20 or antibodies against IRS-1, the immunoprecipitates
were washed, and PI 3-kinase activity was assayed in the
immunoprecipitates as described previously (38, 48). Lysate from a 6-cm
plate was used for each precipitation.
MAP Kinase and Akt Kinase Assay
MAP kinase assays (48) and Akt kinase assays (45) were performed
as described with the following modifications. 3T3-L1 adipocytes were
incubated in serum-free DMEM for 16 h, treated with various
reagents, and then immediately frozen with liquid nitrogen. For MAP
kinase assays, the cells were lysed in a solution containing 20
mM Tris-HCl (pH 7.5), 60 mM
ß-glycerophosphate, 10 mM MgCl2, 0.1
mM NaF, 10 mM EGTA, 10 mM sodium
pyrophosphate, 1% Nonidet P-40, 2 mM dithiothreitol, 1
mM phenylmethylsulfonyl fluoride, and 1 mM
sodium orthovanadate. For Akt kinase assays, the cells were lysed as
described (45). The lysates were centrifuged and the resultant
supernatants were subjected to immunoprecipitation either with anti-MAP
kinase antibodies, or anti-Akt kinase antibodies. After washing three
times with HEPES-buffered saline (pH 7.5) containing 0.1% Triton
X-100, the immunoprecipitates were incubated with 0.5 µCi of
[32P]ATP in a reaction mixture containing 20
mM Tris-HCl (pH 7.5), 10 mM MgCl2,
25 µM ATP, 1 µM PKI, and myelin basic
protein (0.5 mg/ml) or Histon 2B (0.2 mg/ml) for MAP kinase assays or
Akt kinase assays, respectively. After 15 min at 30 C, reactions ware
terminated by the addition of SDS sample buffer. The samples were
fractionated on a 15% SDS-polyacrylamide gel, and the radioactivity
incorporated into either myelin basic protein or Histon 2B was
determined with a Fuji BAS 2000 image analyzer (Fiji Film, Tokyo,
Japan). Lysate from a 6-cm plate was used for each precipitation.
Glucose Uptake
3T3-L1 adipocytes cultured in six-well plates were incubated for
16 h in DMEM containing 5.6 mM glucose and 0.5% FBS.
The cells were washed twice with DB buffer [140 mM NaCl,
2.7 mM KCl, 1 mM CaCl2, 1.5
mM KH2PO4, 8 mM
Na2HPO4, (pH 7.4), 0.5 mM
MgCl2] and incubated with the indicated concentrations of
insulin for 15 min, GH for 10 min, or sorbitol for 60 min. One
milliliter of DB buffer containing BSA (1 mg/ml) and 0.1 mM
2-deoxy-D-[1, 2-3H]glucose (1 µCi) was
added to each well, and, after 5 min, the cells were washed three times
with ice-cold DB buffer containing BSA (1 mg/ml) and 100 nM
phloretin and then solublized with 0.1% SDS. The radioactivity
incorporated into the cells was measured by liquid scintillation
counting.
Photoaffinity Labeling of GLUT4 in Plasma Membrane
Photoaffinity labeling of GLUT4 was performed as described
previously (28, 34) with the following modifications. 3T3-L1 adipocytes
cultured in 35-mm dishes were incubated for 16 h in DMEM
containing 5.6 mM glucose and 0.5% FBS. The cells were
washed twice with KRH buffer (136 mM NaCl, 4.7
mM KCl, 1.25 mM CaCl2, 1.25
mM MgCl2, 10 mM HEPES, pH 7.4) and
incubated with the indicated concentrations of insulin for 15 min, GH
for 10 min, or sorbitol for 60 min. The buffer was removed and replaced
by 200 µl of KRH buffer containing 500 µCi of
ATB-[2-3H]BMPA, kindly provided by G. Holman, University
of Bath, Bath, U.K.). The dishes were irradiated for 1 min using a
Rayonet photochemical reactor (The Southern New England Ultraviolet Co,
Branford, CT) with 300-nm lamps. The irradiated cells were washed with
KRH buffer and solubilized in 1 ml of lysis buffer containing 2%
nonaethyleneglycol dodecyl ether (C12E9), 5
mM sodium phosphate, 5 mM EDTA, pH 7.2, and
with proteinase inhibitors aprotinin, pepstatin, leupeptin, each at 1
µg/ml. The lysates were centrifuged and the supernatants were
subjected to immunoprecipitation with a monoclonal anti-GLUT4 antibody
(1F8). After washing four times with PBS containing 0.2%
C12E9, the labeled GLUT4 was released from the
immunoprecipitates with 10% SDS, 6 M urea, and 10%
mercaptoethanol and subjected to 10% SDS-PAGE. Gels were stained with
Coomassie blue and sliced by lane into 8-mm slices. The slices were
dried and solubilized in 30% H2O2 and 2%
ammonium hydroxide, and radioactivity was quantified in a liquid
scintillation counter. Labeled GLUT4 was quantified by integrating the
area under the 3H peak and subtracting the average
background radioactivity in the gel.
Plasma Membrane Lawn Assay and Analysis of Membrane Ruffling
GLUT4 translocation to the plasma membrane was measured by the
plasma membrane lawn assay as previously described (14). In brief,
3T3-L1 adipocytes cultured on coverslips were washed in PBS and treated
with 0.5 mg/ml poly L-lysine in PBS. Cells were incubated
in a hypotonic buffer (1/3 x KHMgE buffer (70 mM KCl,
30 mM HEPES, 5 mM MgCl2, 3
mM EGTA, pH 7.5), and immediately broken open by placing
under an ultrasonic microprobe in KHMgE buffer containing 0.1
mM phenylmethylsulfonyl fluoride and 1 mM
dithiothreitol. For antibody labeling, sonicated cells were fixed in
2% paraformaldehyde, and the lawn of plasma membrane fragments was
prepared with antibodies to GLUT4 (1F8) and tetramethyl rhodamine
isothiocyanate (TRITC)-labeled secondary antibodies. For analysis of
filamentous actin, 3T3-L1 adipocytes fixed with PBS containing 3.7%
paraformaldehyde were permeabilized with 0.2% Triton X-100 for 1 min
at 25 C and stained with TRITC-labeled phalloidin for 60 min at 25 C.
After washing with PBS, the cells were mounted in 90% glycerol
resolved in PBS containing 1 mg/ml p-phenylenediamine. Samples were
examined with a fluorescence microscope (model Axiophot, Zeiss, Jena,
Germany).
 |
ACKNOWLEDGMENTS
|
---|
We thank Dr. J. Miyazaki for the CAG promoter; I. Saito for
pAxCAwt, DNA-TPC, and technical advice on the production of adenovirus
vectors; G. Holman for ATB-BMPA and technical advice on the
photoaffinity labeling of GLUT4; T. Tanaka, D. Bowtell, U. Kikkawa, and
D. James for antibodies.
Supported by grants from the Ministry of Education, Science, and
Culture of Japan, Monbusho International Scientific Research Program,
and Otsuka Pharmaceutical Co., Ltd. (to M. K.).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Waturu Ogawa, Second Department of Internal Medicine, Kobe University School of Medicine, 75-1 Kusunoki-cho, Chuo-ku, Kobe 650, Japan.
Received for publication February 18, 1997.
Revision received May 28, 1997.
Accepted for publication May 29, 1997.
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