Adenovirus-Mediated Gene Transfer of Dominant Negative Rasasn17 in 3T3L1 Adipocytes Does Not Alter Insulin-Stimulated PI3-Kinase Activity or Glucose Transport
Luigi Gnudi1,
Ernst U. Frevert,
Karen L. Houseknecht,
Peter Erhardt and
Barbara B. Kahn
The Division of Endocrinology (L.G., E.U.F., K.L.H.,
B.B.K.) Department of Medicine at Harvard Medical School and
Beth Israel Hospital and The Dana Farber Cancer Institute
(P.E.) Boston, Massachusetts 02215
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ABSTRACT
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Recent studies suggest that the ras-map kinase and
PI3-kinase cascades converge. We sought to determine whether PI3-kinase
is downstream of ras in insulin signaling in a classic insulin target
cell. We generated a recombinant adenovirus encoding dominant negative
ras by cloning the human H-ras cDNA with a ser to asn substitution at
amino acid 17 (rasasn17) into the pACCMVpLpA
vector and cotransfecting 293 cells with the pJM17 plasmid containing
the adenoviral genome. Efficiency of gene transfer was assessed by
infecting fully differentiated 3T3L1 adipocytes with a recombinant
adenovirus expressing ß-galactosidase (ß-gal); greater than 70% of
cells were infected. Infection of adipocytes with
rasasn17 resulted in 10-fold greater expression
than endogenous ras. This high efficiency gene transfer allowed
biochemical assays. Insulin stimulation of ras-GTP formation was
inhibited in rasasn17-expressing cells. Map
kinase gel mobility shift revealed that insulin (1
uM) or epidermal growth factor (100 ng/ml)
resulted in the appearance of a hyperphosphorylated species of p42 map
kinase in uninfected cells and those expressing ß-gal but not in
cells expressing rasasn17. In contrast, insulin
increased IRS-1-associated PI3-kinase activity approximately 10-fold in
control cells and high level overexpression of
rasasn17 did not impair this effect. Similarly,
insulin and epidermal growth factor activation of total (no
immunoprecipitation) PI3-kinase activity in both cytosol and total
cellular membranes and insulin stimulation of glucose transport were
not affected by expression of dominant negative ras. Thus,
adenovirus-mediated gene transfer is effective for studying insulin
signaling in fully differentiated insulin target cells. Inhibition of
ras activation abolishes insulin-stimulated phosphorylation of map
kinase but does not affect insulin stimulation of PI3-kinase activity.
In normal cell physiology, PI3-kinase does not appear to be downstream
of ras in mediating the actions of insulin.
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INTRODUCTION
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The pleiotrophic effects of insulin on mitogenic and metabolic
processes are mediated by a complex network of intracellular signaling
pathways (see Ref.1). Recent evidence suggests cross-talk among these
pathways. Insulin activates the intrinsic tyrosine kinase activity of
the insulin receptor, which results in the phosphorylation of
substrates such as insulin-responsive substrate-1 (IRS1), IRS2, and Shc
(1). These proteins act as docking molecules for other proteins via SH2
domains. Shc, IRS1, and IRS2 interact with the adaptor protein GRB2,
which is preassociated with a guanine nucleotide exchange factor, SOS.
This leads to activation of the low molecular weight GTP-binding
protein p21-ras, which in turn stimulates a cascade of phosphorylation
events (1). IRS1 also binds SH2 domains that are present in the
p85-regulatory subunit of PI3-kinase, leading to the activation of the
p110 catalytic subunit (1). A growing number of studies indicate that
the ras-map kinase and PI3-kinase-signaling pathways may converge
(2, 3, 4, 5, 6), and a site has been identified on the effector domain of ras
where p110 binds (3). Several lines of evidence suggest that PI3-kinase
can activate ras (4); other studies indicate that ras may activate
PI3-kinase (3, 5), and PI3-kinase has been hypothesized to be an
important mediator of downstream effects of ras (5). Although a
specific inhibitor of map kinase kinase (MEK) does not block activation
of PI3-kinase by insulin (7), this may not be surprising since the
direct physical interaction between ras and p110 is proximal to the
activation of MEK and may not require activation of more distal steps
in the map kinase cascade.
One of the most important actions of insulin is the stimulation of
glucose uptake into adipose cells and muscle, which occurs by eliciting
the translocation of GLUT4, the major insulin-regulatable glucose
transporter, from intracellular vesicles to the plasma membrane (8).
Recent studies have attempted to define the role of the PI3-kinase and
the ras-map kinase pathways in insulin stimulation of glucose
transport. Studies using the PI3-kinase inhibitors wortmannin (9, 10)
and LY294002 (11) or a dominant negative p85 subunit (9, 12)
demonstrate an important role of PI3-kinase in insulin stimulation of
GLUT4 translocation. However, stimulation of PI3-kinase activity with
growth factors such as platelet-derived growth factor (13, 14), with a
thiophosphotyrosine peptide (15), or with cytokines such as IL-4 which,
similar to insulin, activates IRS-1, minimally affects GLUT4
translocation. This indicates that not all modes of activation of
PI3-kinase are sufficient to stimulate glucose transport.
Investigations of the role of ras in the stimulation of glucose
transport by insulin have led to conflicting results. Studies with a
specific MEK inhibitor (7) or in which dominant negative ras (16),
activated ras (12, 16), activated raf (17), or neutralizing ras
antibodies were microinjected (or transfected) into 3T3-L1 adipocytes
(16) showed no effect on GLUT4 translocation. However, microinjection
of the same antibody into cardiac myocytes decreased insulin-stimulated
glucose transport (18). Overexpression of wild type ras (19) or
constitutively active (9, 20) ras stimulates GLUT4 translocation in the
absence of insulin in primary rat and mouse adipocytes and 3T3-L1
adipocytes, and overexpression of wild type ras also increases the
sensitivity for insulin stimulation of GLUT4 translocation and glucose
transport (19).
Inconsistencies in these results may be due to methodological
limitations of all the experimental approaches used. The studies are
complicated because effects of insulin on glucose transport and
metabolism that are relevant to normal physiology can be studied only
in terminally differentiated adipocytes or myocytes, since these are
the only cells that express the insulin-regulatable glucose
transporter, GLUT4, and that contain the cellular elements necessary
for normal trafficking of GLUT4. Use of standard transfection
techniques in these terminally differentiated cells results in low
efficiency of gene transfer (9). Establishing stable lines is
complicated by the fact that cells must be transfected before
differentiation when changes in the expression of a signaling molecule
can alter expression of other genes, including ones involved in
differentiation (21, 22) PI3-kinase activation (23), and glucose
transport (16, 20, 23). Furthermore, clonal selection of
differentiating cells can result in selection of cells with altered
rates of differentiation or metabolism. Microinjection studies are
limited by the fact that single cells are used, quantitation is
complex, the intracellular concentration of protein or antibody is
unknown, and transport or kinase assays cannot be performed. Although
meaningful data have been reported with all of these techniques, the
data are not entirely consistent, especially as they pertain to the
role of ras in insulin-stimulated glucose transport.
Therefore, we adapted the adenovirus gene transfer technique (Fig. 1
)
so that it could be used in 3T3L1 adipocytes. We aimed
to elucidate the relationship between the PI3-kinase and the ras-map
kinase pathways in whole cell physiology, as well as to study the role
of ras in the activation of glucose transport by insulin. We sought a
gene transfer system that 1) could achieve high efficiency and be
amenable to biochemical assays, 2) could be introduced in terminally
differentiated cells so as not to alter the differentiation process,
and 3) would be rapid enough to prevent chronic compensatory changes in
the cell. We generated a recombinant adenovirus encoding dominant
negative rasasn17 (24) with which we infected fully
differentiated 3T3-L1 adipocytes. We achieved high efficiency
(percentage of cells showing gene expression) and high level
rasasn17 expression. Dominant negative ras expression
resulted in inhibition of ras-GTP formation and map kinase activation
and no effects on either insulin-stimulated glucose transport or
insulin activation of PI3-kinase. Thus, activation of the ras-map
kinase cascade is not necessary for maximal stimulation of PI3-kinase
activity by insulin.

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Figure 1. Schematic Representation of the Strategy Used to
Generate Adv rasasn17 Recombinant Adenovirus
Rasasn17 cDNA was cloned into the vector pACCMV.pLpA. This
vector was contransfected with the replication competent pJM17 vector
into 293 cells. The resulting recombinant virus contains a
transcription unit consisting of the cytomegalovirus promoter/enhancer,
the mutant ras cDNA, and a polyadenylation cassette (see
Materials and Methods for details).
Numbers refer to viral map units (mu = 360 bp).
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RESULTS
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Infection Efficiency (Fig. 2A
)
Histochemical staining for ß-galactosidase (ß-gal)
(dark) showed that greater than 70% of differentiated 3T3-L1
adipocytes had successful gene transfer after an overnight infection
with recombinant virus encoding ß-gal. In 3T3L1 adipocytes, 1 and
4 h incubation with the recombinant adenovirus produced no
apparent infection. Only longer exposure times of 13, 15, or 24 h
were effective.

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Figure 2. Adenovirus-Mediated Gene Delivery Results in High
Efficiency (A) and High Level (B) Gene Expression in 3T3-L1 Adipocytes
A, Histochemical staining for ß-gal (dark) after
infection with recombinant adenovirus encoding ß-gal. Staining is
perinuclear due to nuclear localization signal in the construct. Cells
were infected with viral concentration of
108-109 plaque-forming units/ml for 13 h.
The ß-gal assay was performed 1213 h after infection as described
in Materials and Methods. B, High level expression of
p21-rasasn17 protein after overnight infection of
differentiated 3T3L1 adipocytes with the recombinant adenovirus
expressing the rasasn17 cDNA. Western blotting of cell
lysates was performed as described in Materials and
Methods (40 µg of protein per lane). Levels of ras expression
in cells infected with the ß-gal recombinant adenovirus were
comparable to the levels in uninfected control cells (not shown).
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Dominant Negative ras Overexpression (Fig. 2B
)
The level of ras expression was many times higher in
differentiated 3T3L1 adipocytes expressing dominant negative ras
compared with endogenous ras levels in uninfected adipocytes. No
overexpression of ras was detected in cells infected with the
recombinant adenovirus encoding ß-gal (not shown).
Effects of Adenovirus rasasn17
Infection on ras-GTP Formation (Fig. 3A
)
The dominant negative effect of rasasn17 was
demonstrated by investigating the effect on insulin stimulation of
ras-GTP formation. In uninfected control cells, insulin increased
ras-GTP levels 3.1 ± 0.71-fold over unstimulated cells
[mean ± SEM, n = 2, expressed as the ratio of
ras-GTP/(ras-GDP + ras-GTP)]. The effect of insulin was similar in
cells infected with adenovirus encoding ß-gal: 2.73 ± 0.87-fold
over basal, n = 2. However, expression of rasasn17
prevented insulin stimulation of ras-GTP formation (Fig. 3
). For
technical reasons, absolute values from multiple experiments could not
be combined, and a representative experiment is shown.

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Figure 3. Dominant Negative Effect of rasasn17
A, Insulin stimulation of GTP loading of ras in control adipocytes
expressing ß-gal and in adipocytes expressing rasasn17.
Fully differentiated 3T3-L1 adipocytes were infected overnight with
adenoviruses and then serum starved and labeled with
[32P] orthophosphate for 16 h as described in
Materials and Methods. Cells were treated with or
without 1 µM insulin for 5 min and lysed. Ras was
immunoprecipitated, the pellets were resuspended and centrifuged, and
the released nucleotides were separated by polyethyleneimine-cellulose
TLC. Results were quantitated by PhosphorImager and are expressed as
percent [32P]GTP-ras/([32P]GTP-ras +
[32P]GDP-ras). This is representative of two separate
experiments. B, Effect of dominant negative ras on the phosphorylation
of p42 map kinase. Map kinase mobility shift assay was performed on
uninfected 3T3L1 adipocytes and cells infected with the recombinant
ß-gal or rasasn17 adenovirus. Cells were serum starved
overnight (0.1% calf serum) and then treated for 5 min with either
insulin (1 µM) or EGF (100 ng/ml). Cytosol was prepared,
subjected to SDS-PAGE, transferred to nitrocellulose, and immunoblotted
with map kinase antiserum as described in Materials and
Methods. Twenty micrograms of cytosolic protein were loaded on
each lane. The upper band of the doublet appearing at 42 kDa after
stimulation with insulin or EGF represents a hyperphosphorylated form
of p42 map kinase. B, Basal; I, Insulin 1 µM; E, EGF 100
ng/ml. Data are representative of two separate infections. Parallel
results were also observed in cell membranes.
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Effects of Adenovirus rasasn17 Infection on
Map Kinase Activation (Fig. 3B
)
The dominant negative effect of rasasn17 was further
demonstrated by absence of map kinase activation as assessed by
electrophoretic mobility shift. In the basal state (absence of growth
factors), p42 and p44 map kinase were present as single bands in
uninfected control cells and cells infected with adenovirus ß-gal or
adenovirus rasasn17. In the control and ß-gal-infected
cells, stimulation with insulin or epidermal growth factor (EGF)
resulted in the appearance of another species with slower
electrophoretic mobility representing increased phosphorylation of p42
map kinase. EGF was a more potent stimulator than insulin. Increased
phosphorylation of p42 map kinase was not seen in the
rasasn17-infected cells in response to insulin or EGF. This
indicates that rasasn17 exerted a dominant negative effect
to inhibit activation of map kinase. Results using cell lysates were
consistent with those using cytosol.
Effects of Adenovirus rasasn17 Infection on
the Expression of Glut1 and Glut4 and on 2-Deoxyglucose Transport in
3T3L1 Adipocytes (Fig. 4
)
Because overexpression of wild type or activated ras results in
increased Glut1 expression (16) and chronic overexpression of
rasasn17 in adipocytes of transgenic mice can result in
decreased Glut1 and Glut4 expression (23), we measured levels of Glut1
and Glut4 in total membranes from uninfected cells and cells
infected with ß-gal or rasasn17. No significant
differences were observed in Glut1 or Glut4 protein levels (not shown)
or in 2-deoxyglucose transport in the absence or presence of 100
nM insulin (Fig. 4
) in 3T3L1 adipocytes that were
uninfected or infected with recombinant adenovirus expressing either
ß-gal or rasasn17.

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Figure 4. Lack of Effect of Dominant Negative ras on Glucose
Transport
2-Deoxyglucose transport was performed in uninfected (control) 3T3L1
adipocytes or cells infected with recombinant adenovirus encoding
ß-gal or rasasn17 cDNA as described in Materials
and Methods. Glucose transport was performed in triplicate in
two different experiments. Results are means ± SEM
expressed as a percent of basal transport in uninfected cells. No
significant differences were found in the amount of protein or DNA per
well.
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Effects of Adenovirus rasasn17 Infection on
the Expression of IRS-1 and the p85 Subunit of PI3-Kinase and on
PI3-Kinase Activity in 3T3L1 Adipocytes (
Figs. 56
)
Because alterations in PI3-kinase activity immunoprecipitated by
IRS-1 could result from changes in the expression of the p85 subunit of
PI3-kinase or of IRS-1, we immunoblotted cell lysates for these
proteins. No differences were observed in the amount of the p85 subunit
of PI3-kinase in 3T3L1 adipocytes that were uninfected or infected with
the recombinant adenovirus expressing either ß-gal or dominant
negative ras (Fig. 5
, panel A, top).
Similarly, IRS-1 expression was unchanged by rasasn17
(panel A, bottom) or by adenovirus-ß-gal infection (not
shown). There were also no differences in the amount of IRS-1 in the
pellet after the IRS-1 immunoprecipitation (not shown). Insulin
stimulated IRS-1-immunoprecipitable PI3-kinase activity by
10-fold
in uninfected 3T3L1 adipocytes (Fig. 5
, B and C). There were no
significant effects of expression of ß-gal or rasasn17 on
basal or insulin-stimulated (100 nM) PI3-kinase
activity.

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Figure 5. Dominant Negative rasasn17 Does Not
Alter IRS-1-Associated P13 Kinase Activity in 3T3-L1 Adipocytes
A, p85 and IRS-1 protein levels in total lysate (60 µg of protein per
lane) of 3T3L1 adipocytes that were uninfected (control) or infected
with recombinant adenovirus expressing ß-gal and rasasn17cDNA. Immunoblotting was carried out as described in
Materials and Methods. These data are representative of
two separate experiments with duplicate or quadruplicate wells for each
condition. Levels of IRS-1 in cells infected with the ß-gal
recombinant adenovirus are comparable to levels in the uninfected cells
(not shown). B, Effects of dominant negative ras on stimulation of
phosphatidylinositol 3-kinase by insulin. PI3-kinase activity was
assayed in 3T3L1 adipocytes that were uninfected (control) or infected
with recombinant adenovirus expressing ß-gal or rasasn17
cDNA. Equal amounts of cell lysate protein from basal and
insulin-treated 3T3L1 adipocytes were subjected to immunoprecipitation
with an IRS-1 antiserum (see Materials and Methods). For
each experiment, each condition was performed in duplicate for control
and adenovirus expressing ß-gal cells and in triplicate for
adenovirus expressing the rasasn17 cells. This
autoradiogram is representative of four separate PI3-kinase assays on
four separate cell infections. (-, basal; +, insulin 100
nM) (PI(3)P, phosphoinositol 3-phosphate). C,
Quantification of PI3-kinase activity by phosphorimaging. Results are
means ± SEM for four separate experiments.
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Figure 6. Effects of Dominant Negative ras on Stimulation of
Total (not Immunoprecipitated) PI3-Kinase Activity by Insulin and EGF
PI3-kinase activity was assayed in 3T3L1 adipocytes that were
uninfected (control) or infected with recombinant adenovirus encoding
ß-gal or rasasn17 cDNA. For measurement of cytosolic and
membrane-associated PI3-kinase activity, cells were stimulated with 100
nM insulin for 10 min or with 100 ng/ml EGF for 2.5 min.
Cells were homogenized and membranes and cytosol were prepared and
PI3-kinase activity was determined as described in Materials and
Methods. Adenosine (200 µM) was added to
membranes to reduce PI4 kinase activity so it did not overlap the
PI3-kinase signal on the TLC plate. Equal volumes of cytosol and
membranes were assayed; therefore, the results in cytosol and membranes
represent the same number of cells. Phosphoinositol 3-phosphate was
quantitated by phosphorimaging.
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Total PI3-kinase activity in cytosol and membranes after stimulation of
adipocytes with insulin or EGF is shown in Fig. 6
. Basal
PI3-kinase activity was increased 3- to 4.5-fold in cells infected with
either ß-gal or rasasn17. There was a further increase
with insulin and a smaller increase with EGF. The stimulation
(i.e. increment over basal) in cells infected with
rasasn17 was similar to that in cells infected with
ß-gal. Thus, there was no effect of dominant negative ras on total
PI3-kinase activity stimulated by insulin or EGF.
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DISCUSSION
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Growing evidence demonstrates that the ras-map kinase and
PI3-kinase pathways converge. Studies from one group show that ras
stimulates PI3-kinase activity (3, 6) whereas other studies using a
constitutively active PI3-kinase indicate that some cellular effects of
PI3-kinase depend on ras (4). Rasasn17 has been shown to
have a dominant negative effect on signaling via the ras-map kinase
pathway. The mechanism for decreased activation of ras is thought to
result from the ability of rasasn17 to bind ras exchange
factor(s) with high affinity, thus reducing the availability of the
exchange factor(s) for the formation of ras-GTP. Since the interaction
of the p110 subunit of PI3-kinase with the effector domain of ras has
been shown to be GTP dependent (3), rasasn17 should have a
dominant negative effect on this interaction. Therefore, we used this
mutant ras to determine whether activation of ras is important in the
effects of insulin and EGF on activation of PI3-kinase activity. Our
results demonstrate that rasasn17 abrogates the
insulin-stimulated formation of ras-GTP and the activation of map
kinase but has no effect on PI3-kinase activation. This indicates that
ras-initiated signaling is not necessary for insulin-stimulated
activation of IRS-1-associated PI3-kinase activity or of total
PI3-kinase activity in membranes or cytosol (Figs. 5
and 6
). We also
studied activation of PI3-kinase by EGF because of the possibility that
IRS-1-independent PI3-kinase activity could be affected. Although EGF
has a smaller stimulatory effect on PI3-kinase activity than insulin in
3T3L1 adipocytes, EGF stimulation was also unaffected by
rasasn17 (Fig. 6
).
In IRS-1-immunoprecipitated samples, viral infection had no effect on
PI3-kinase activity. Viral infection modestly increased basal
PI3-kinase activity in nonimmunoprecipated samples. Therefore, effects
with the ß-gal virus are the appropriate control, and both
insulin-stimulated and EGF-stimulated PI3-kinase activities are similar
in rasasn17-expressing cells compared with control cells
expressing ß-gal.
Studies of the signaling pathways involved in insulin action on
metabolic processes in classical insulin target cells have led to
conflicting results most likely due to limitations of all approaches
used, as delineated in the Introduction. Therefore, we adapted the
adenovirus gene transfer technique so that it could be used in 3T3L1
adipocytes. We were able to achieve more than 70% infection efficiency
after 1215 h exposure to the virus. With this technique we have
confirmed that inhibition of the ras map kinase cascade does not affect
insulin-stimulated glucose transport. A recent study using vaccinia
virus to express dominant negative ras in 3T3L1 adipocytes showed no
effect on glucose transport or on glycogen synthesis (25). However,
effects of dominant negative ras on PI3-kinase activation were not
studied. The adenovirus delivery system has advantages over
vaccinia-mediated gene delivery since the replication-deficient
adenovirus does not abort protein synthesis, and cells remain healthy
for many days after adenovirus infection. Thus, this technique will be
useful for further studies of signaling initiated by insulin and other
growth factors.
Two recent studies have investigated potential mechanisms for the
interaction of ras and PI3-kinase. One group demonstrated that in
3T3-L1 adipocytes, PI3-kinase inhibits GTPase-activating protein,
allowing the insulin signal to fully activate p21ras via
stimulation of guanine nucleotide exchange activity of SOS (26).
Another study identified the site of interaction between the effector
domain of ras and p110
and p110ß isoforms of PI3-kinase (6). A
point mutation in this region blocks the ability of ras to activate
PI3-kinase. Furthermore, the effect of ras to increase PI3-kinase
enzymatic activity appears to be synergistic with the effect of
tyrosine phosphopeptide binding to p85. Thus, a model is suggested in
which the PI3-kinase receives regulatory signals through two domains in
its amino-terminal region (6). One comes from tyrosine phosphoproteins
and possibly from SH3 domains of Src family kinases and from Rho family
proteins, through p85 and its interaction with the first 150 amino
acids of p110 (6). The other signal comes from the direct interaction
of Ras-GTP with the neighboring region of p110.
In our current study we demonstrate that, in 3T3-L1 adipocytes,
the activation of ras does not appear to be necessary for full
stimulation of PI3-kinase activity by insulin or EGF. This may be due
to cell type or growth factor specificity. Support for the possible
cell type specificity of interactions between the ras and PI3-kinase
pathways comes from the recent observation that the inhibitory effect
of PI3-kinase on GTPase-activating protein is adipocyte-specific (26).
It is still possible that PI3-kinase may receive regulatory signals
through multiple pathways including ras, but in adipocytes that are
stimulated by these growth factors (insulin and EGF), the input derived
from direct interaction with GTP-ras does not appear to be necessary
for activation of the lipid kinase. In conclusion, using this effective
means of DNA delivery in terminally differentiated insulin-target
cells, this study demonstrates that activation of ras in adipocytes is
not required for full activation of PI3-kinase by insulin or EGF.
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MATERIALS AND METHODS
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Generation of a Recombinant Mutant
rasasn17 Adenovirus (Fig. 1
)
A recombinant adenovirus expressing a dominant negative ras was
generated by cloning the human H-ras cDNA, with a lysine to asparagine
substitution at amino acid position 17 (gift of G. Cooper, Dana Farber
Cancer Institute), into the multiple cloning site of the vector
pACCMVpLpA (gift of C. Newgard, University of Texas) (27). This vector
was developed (28) by modification of the pAC vector, by replacement of
a region of the adenovirus genome between map units 1.3 and 9.1 with
the cytomegalovirus (CMV) promoter, a cloning cassette, and the SV40
genome that includes the small intron of the t antigen and the
polyadenylation signal. The p21-H-rasasn17 cDNA was
isolated from the plasmid pMT-M17 (gift of G. Cooper); the resulting
0.7-kb XbaI-PstI fragment was subcloned to the
XbaI-PstI sites of the pGem-3zf vector
polylinker. The H-rasasn17 cDNA was then isolated using the
BamHI-PstI sites and cloned into the PBSII-SK±
vector. The corresponding BamHI-SalI fragment was
then finally cloned into the corresponding sites of the pACCMVpLpA
polylinker with the generation of pACCMVpLpA-rasasn17. The
pACCMVpLpA-rasasn17 vector and the pJM17 vector (gift of C.
Newgard) were cotransfected into 80% confluent 293 cells using the
CaPO4-DNA coprecipitation technique with Modified Bovine
Serum (Stratagene kit. no. 200388, La Jolla, CA). After 810 days, the
death of the 293 cells indicated that a new recombinant virus coding
for the p21-H-rasasn17 protein had been generated. The
viral DNA was then extracted from 293 cells and confirmed by Southern
blot technique using the rasasn17 cDNA as a probe. A single
clone of recombinant adenovirus was isolated through serial dilution
using a plaque assay. The expansion of the recombinant adenovirus was
performed as previously described with 293 cells (27), and the virus
was subsequently concentrated on a cesium chloride gradient and
desalted in a desalting column (PD 10, Sephadex column, Pharmacia,
Piscataway, NJ). The concentration of the recombinant adenovirus was
assessed based on the absorbance at 260 nm and on limiting-dilution
plaque assay (27).
Cell Culture and Differentiation
3T3L1 fibroblasts were grown in DMEM (GIBCO Laboratories, Grand
Island, NY), at high glucose (450 mg/dl), and 10% calf serum (GIBCO
Laboratories). At confluence cells were differentiated (day 0) with
10% FBS (GIBCO Laboratories), insulin (870 nM),
dexamethazone (0.25 µM) (Sigma, St. Louis, MO), and
isobutylmethylxanthine (0.5 mM) (Sigma, St. Louis, MO). At
day 3 the media was changed to DMEM with high glucose (450 mg/dl) and
10% FBS (GIBCO Laboratories), and this media was replaced every other
day. Cells were used for experiments at day 1012. Only plates in
which 95% of the cells had reached adipocyte morphology were used
(29).
Infection Efficiency of 3T3L1 Adipocytes
Differentiated 3T3L1 adipocytes were infected at day 1012 for
1, 4, 13, 15, or 24 h in 1 ml of DMEM with either 0.1% calf serum
or 10% FBS with a recombinant adenovirus expressing ß-gal (gift of
C. Newgard) (27) at a final concentration of
108109 plaque-forming units/ml, determined by
limiting dilution assay in 293 cells. No toxic effect of the
recombinant adenovirus was evident by inspection of the differentiated
adipocytes or by assessing glucose transport, map kinase activation,
and PI3-kinase activity in ß-gal infected cells. The efficiency of
transfection was assessed by fixing the cells with 0.2% glutaraldehyde
(Sigma, St. Louis, MO), 6 mM EDTA, and 2.4 mM
MgCl2 in PBS and evaluating for expression of ß-gal
enzyme activity after staining with 5-bromo-4-chloro-3-indolyl
ß-D-galactopyranoside (X-gal stain) (Sigma). After 1- to
4-h infections, cells were washed free of virus and incubated in DMEM
with 10% FBS for a total of 1215 h after initiation of infection.
After 13- to 24-h infections, cells were washed free of virus and
immediately fixed. After three washes with 2 mM
MgCl2, 0.02% NP40 in PBS, fixed cells were incubated at
least 3 h at 37 C with X-gal as described (30). To estimate the
infection efficiency we calculated the ratio between the cells
expressing ß-gal (blue stain) and uninfected ones (unstained).
Between 6080 cells were counted in each of three separate
experiments.
Overexpression of rasasn17 in 3T3L1
Adipocytes
After an overnight infection (1215 h), cells were washed with
PBS twice, and a crude lysate was obtained with 1 mM HEPES,
1 mM EDTA, 5 mM EGTA, 10 mM
MgCl2, 50 mM ß-glycerolphosphate, 1
mM Na3VO4, 2 mM
dithiothreitol, 40 µg/ml phenylmethylsulfonylfluoride, 4 µg/ml
leupeptin, 1% NP-40. The lysate was run on a 10% slab polyacrylamide
gel, transferred to nitrocellulose, and immunoblotted with a monoclonal
antibody against human p21-H-ras (Oncogene Science, Uniondale, NY).
Briefly, blots were blocked [3% skim milk in Tris-buffered-saline
(TBS) with 0.1% Tween-20] for 2 h at room temperature after
which blots were incubated with ras antibody (1:50 dilution in blocking
solution) for 3 h at room temperature. Blots were washed in TBS,
0.1% Tween-20, and incubated with secondary antibody that is
conjugated to horseradish peroxidase. Bands were visualized with
enhanced chemiluminescence (ECL, Amersham, Little Chalfont, UK) and
quantified by densitometry.
Stimulation of ras-GTP Formation
GTP loading of ras was performed as described (31). Briefly,
adipocytes were incubated in phosphate-free DMEM supplemented with
0.1% calf serum and 0.5 mCi/ml [32P]orthophosphate for
16 h. Cells were then treated with or without 1 uM
insulin for 5 min and lysed on the plate with 1 ml lysis buffer (20
mM Tris-HCL pH 7.4, 150 mM NaCl, 1
mM MgCl2, 1% Triton-X-100) containing 1 µg
anti-Ras monoclonal antibody Y13-259 (Oncogene Science). Lysates were
scraped into Eppendorf tubes and rocked at 4 C for 2 h. The
extracts were then centrifuged, and the supernatants were added to
protein A Sepharose precoupled to goat anti-rat secondary antibody.
Samples were rocked for another 2 h and washed five times with
lysis buffer and once with PBS. Pellets were resuspended in 1
M KH2PO4 (pH 3.4) and incubated at
85 C for 3 min. Samples were then centrifuged, and the released
nucleotides were separated on polyethyleneimine-cellulose TLC plate
(Sigma). Plates were developed in 1 M
KH2PO4 (pH 3.4) and 32P
incorporated into GTP and GDP was quantified by PhosphorImager
Molecular Dynamics, Sunnyvale, CA). Results are expressed as percent
[32P]GTP-ras/([32P]GTP-ras +
[32P]GDP-ras).
Map Kinase Mobility Shift Assay
During an overnight incubation in DMEM-0.1% calf serum,
differentiated 3T3L1 adipocytes were left uninfected or were infected
with ß-gal or rasasn17 recombinant adenovirus. In the
morning, the cells were stimulated for 5 min with insulin (1
µM) or EGF (100 ng/ml) and immediately washed twice with
ice-cold PBS. Three hundred microliters of a buffer consisting of 50
µM ß-glycerolphosphate pH 7.3, 1.5 mM EGTA,
0.1 mM Na3VO4, 1 mM
dithiothreitol, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1
mM benzamidine were added to a 35-mm diameter plate. Cells
were then scraped, sonicated for 1520 sec, and centrifugated at
100,000 x g for 20 min at 4 C. The supernatant was run
on a slab 10% polyacrylamide SDS gel and transferred to nitrocellulose
paper. The phosphorylation state of p42 map kinase (ERK2) was detected
by immunoblotting with an anti-map kinase antibody (
-C2, provided by
J. Blenis, Harvard Medical School) as described previously (32). A
peroxidase-conjugated second antibody was used as a detection
system with chemiluminescence (ECL, Amersham, Arlington Heights,
IL).
PI3-Kinase Assay
Cells were infected overnight with ß-gal or
p21-H-rasasn17 recombinant adenovirus in DMEM high glucose
(450 mg/dl) with 0.1% calf serum; 1215 h later, cells were
stimulated for 10 min with 100 nM insulin. The medium was
then aspirated, and cells were washed twice with PBS and solubilized in
lysis buffer (600 µl for two 35-mm plates). The lysis buffer for
IRS-1-associated PI3-kinase activity had the following composition: 40
mM HEPES, 135 mM NaC1, 10 mM NaPP,
2 mM Na3VO4, 10 mM NaF,
2 mM EDTA, 2 mM phenylmethylsulfonylfluoride, 5
µg/ml leupeptin, 1.5% NP-40, 10% glycerol, 2 mM
KH2PO4, 5 mM NaHCO3,
0.5 mM CaCl2, 0.5 mM
MgSO4. The lysate was briefly vortexed, centrifuged at
12,000 x g for 5 min at 4 C to pellet any insoluble
materials, and transferred to a siliconized 1.5-ml Eppendorf tube for
immunoprecipitation. Seven hundred to 750 µg of proteins from the
lysate were immunoprecipitated with 20 µl anti-IRS-1 antibody (gift
of Morris White, Joslin Diabetes Center) for 90 min at 4 C;
subsequently, 80 µl of a 1:1 slurry of protein A Sepharose in PBS
were added to each tube, and this was incubated for 2 h at 4 C.
Tubes were then microfuged for 2 min to pellet beads;
immunoprecipitation efficiency was tested with IRS-1 Western blotting
of the supernatant (not shown). Beads were then washed twice with 20
mM HEPES, 100 mM NaCl, and 1 mM
Na3VO4; 40 µl of 20 mM HEPES, pH
7.5, 180 mM NaCl were added to the beads (40 µl), and
this suspension was incubated with [
-32P]ATP and
phosphatidylinositol for 5 min. The reaction was interrupted with 1
N HCl, and the inositol phospholipids were extracted with
chloroform-methanol (1:1). PI-monophosphate in the organic phase was
separated by TLC on aluminum-backed silica gel 60 plates (EM
Separations, Gibbstown, NJ) pretreated with a solution containing 25
mM
trans-1,2-diaminocyclohexane-N',N',N',N'-tetraacetic
acid (Sigma), 66% (vol/vol) ethanol, and 0.06 N NaOH in
the solvent system consisting of 37.5% (vol/vol) methanol, 30%
(vol/vol) chloroform, 22.5% (vol/vol) pyridine (Sigma), 1.33%
(vol/vol) formic acid, 1 M boric acid, and 8.5
mM butylated hydroxytoluene (Sigma) (33). PI-monophosphate
was detected by autoradiography and quantitated with a
Phos-phorImager.
For measurement of total (not immunoprecipitated) cytosolic and
membrane-associated PI3-kinase activity, cells were stimulated with 100
nM insulin for 10 min or with EGF, 100 ng/ml, for 2.5 min.
Cells were homogenized in lysis buffer (20 mM Tris-Cl, 140
mM NaCl, 10% glycerol, pH 7.4, with 1 mM
sodium orthovanadate, 2 µg/ml aprotinin and leupeptin, 0.5
mM dithiothreitol), and the homogenate was centrifuged for
1 h at 200,000 x g, to yield total membrane and
cytosolic fractions. Aliquots of membranes or cytosol in a total volume
of 25 µl lysis buffer were brought to room temperature for 5 min and
then mixed with 25 µl of a lipid/ATP solution containing 500 µg/ml
PI, 80 uM ATP, 0.8 uCi/µl [
-32P]ATP
(3000 Ci/mmol, New England Nuclear, Boston, MA), 20 mM
HEPES, pH 7.5, 50 mM NaCl, 12.5 mM
MgCl2, and 0.015% NP-40. To inhibit some of the PI4-kinase
activity in membrane fractions so it did not overlap the PI3-kinase
signal, adenosine was added to a final concentration of 200
uM. The reaction was stopped after 5 min by addition of 80
µl 1 N HCL, and the phospholipids were extracted and TLC
was performed as described above.
Immunoblotting for Glucose Transporters, IRS-1 and p85
Immunoblotting was performed on total membranes or cell lysates
for GLUT1 and GLUT4 and on crude lysates for IRS1 and p85 subunit of
PI3-kinase. The membranes and lysates were run on slab 10% SDS
polyacrylamide gels, transferred to nitrocellulose, and immunoblotted
with specific antibodies (courtesy of Bernard Thorens, University of
Lausanne, Lausanne, Switzerland, for GLUT1; Howard Haspel, Wayne State,
Detroit, MI, for GLUT4; Morris White, Joslin Diabetes Center, Boston,
MA, for IRS-1; Kurt Auger, Harvard Medical School, Boston, MA, for p85
subunit of PI3-kinase). GLUT1 and GLUT4 were immunoblotted as
previously described (34). For IRS-1 blotting, membranes were blocked
(3% BSA in TBS with 0.01% Tween-20) overnight at 4 C, after which
blots were incubated with IRS-1 antiserum (1:300 dilution in 1% skim
milk, TBS-0.01% Tween-20) for 2 h at room temperature. Blots were
washed in TBS-0.01% Tween-20 and incubated with a secondary antibody
conjugated to horseradish peroxidase. For p85, blots were blocked with
5% skim milk and 0.5% BSA in PBS supplemented with 0.2% Tween-20 at
room temperature for 1 h. Subsequently, blots were incubated with
antisera raised to the carboxyl terminal of p85 at a 1:5000 dilution in
1% skim milk, PBS, 0.2% Tween-20, and 0.02% NaN3 for
3 h at room temperature. Bands were visualized with ECL and
quantified by densitometry.
Glucose Transport in Differentiated 3T3L1 Adipocytes
Cells were infected for 12 h overnight in DMEM-10% FBS. In
the morning the cells were stepped down in DMEM (low glucose: 100
mg/dl) with no serum for 3 h at 37 C, 5% CO2. Cells
were subsequently washed in PBS three times, and glucose transport was
performed in 1 ml of glucose-free MEM. Insulin (0 or 100
nM) was added for 30 min followed by the addition of 100
µM 2-deoxyglucose with 0.33 µCi/well of
[3H]2-deoxyglucose (Amersham, UK). Transport was
performed for 10 min with gentle shaking in a water bath at 37 C.
Transport was then stopped with the addition of 1 ml phloretin (Sigma,
St. Louis, MO) solution in PBS (82 mg/liter). Cells were then washed
with PBS for three times and dryed at 37 C for 30 min. Subsequently,
cells were solubilized with 1 ml of 1 N NaOH .
2-Deoxyglucose incorporated into the cells was measured in an aliquot
of 400 µl after the addition of 50 µl concentrated HCl and 4 ml
scintillation fluid in a ß-counter. The remainder of the suspension
was used for DNA assay (35).
 |
ACKNOWLEDGMENTS
|
---|
We thank H. Haspel for the Glut4 antiserum, B. Thorens for Glut1
antiserum, K. Auger for the p85 antiserum, J. Blenis for the
-C2 map
kinase antiserum, M. White for IRS-1 antiserum, G. Cooper for the
rasasn17 cDNA and C. Newgard for the pACCMVpLpA and pJM17
plasmids and for the ß-gal recombinant adenovirus. We are grateful to
C. Newgard and R. Noel for invaluable advice on the adenovirus
technique, G. Cooper for helpful discussions, and P. R. Shepherd and C.
Carpenter for helpful comments on the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Barbara B. Kahn, M.D., Beth Israel Hospital, 330 Brookline Avenue, Research North, Room 325, Boston, Massachusetts 02215.
This work was supported by NIDDK/NIH Grants DK-43051 and DK-45874. Dr.
Gnudi is the recipient of the Juvenile Diabetes Foundation
International fellowship. Dr. Houseknecht is the recipient of a US
Department of Agriculture fellowship. Dr. Frevert was supported by the
Deutsche Forschungsgemeinschaft and Physician Scientist Award AG00294
from NIA/NIH.
1 Current address: Department of Clinical Medicine, Padova University,
Padova, Italy. 
Received for publication July 3, 1996.
Revision received September 20, 1996.
Accepted for publication September 30, 1996.
 |
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