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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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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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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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), 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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. 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. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 12–15 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 10–12. 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 10–12 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
108–109 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 12–15 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 60–80 cells were counted in each of three separate
experiments.
Overexpression of rasasn17 in 3T3L1
Adipocytes
After an overnight infection (12–15 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 15–20 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; 12–15 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).
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ACKNOWLEDGMENTS |
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-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 |
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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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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