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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go) 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).

 

    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Infection Efficiency (Fig. 2AGo)
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 12–13 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).

 
Dominant Negative ras Overexpression (Fig. 2BGo)
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. 3AGo)
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. 3Go). 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.

 
Effects of Adenovirus rasasn17 Infection on Map Kinase Activation (Fig. 3BGo)
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. 4Go)
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. 4Go) 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.

 
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. 5–6GoGo)
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. 5Go, 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. 5Go, 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.

 
Total PI3-kinase activity in cytosol and membranes after stimulation of adipocytes with insulin or EGF is shown in Fig. 6Go. 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 5Go and 6Go). 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. 6Go).

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{alpha} 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of a Recombinant Mutant rasasn17 Adenovirus (Fig. 1Go)
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 8–10 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 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 ({alpha}-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 [{gamma}-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 [{gamma}-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 {alpha}-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. Back

Received for publication July 3, 1996. Revision received September 20, 1996. Accepted for publication September 30, 1996.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Cheatham B, Kahn CR 1995 Insulin action and the insulin signaling network. Endocr Rev 16:117–142[Medline]
  2. Kodaki T, Woscholski R, Hallberg B, Rodriguez-Viciana P, Downward J, Parker PJ 1994 The activation of phosphatidylinositol 3-kinase by Ras. Curr Biol 4:798–806[Medline]
  3. Viciana-Rodriguez P, Warne PH, Dhand R, Vanhaesebroeck B, Gout I, Fry MJ, Waterfield MD, Downward J 1994 Phosphatidylinositol-3-OH kinase as a direct target of Ras. Nature 370:527–532[CrossRef][Medline]
  4. Hu Q, Klippel A, Muslin AJ, Fantl WJ, Williams LT 1995 Ras-dependent induction of cellular responses by constitutively active phosphatidylinositol-3 kinase. Science 268:100–102[Medline]
  5. Sjolander A, Yamamoto K, Huber BE, Lapetina EG 1991 Association of p21ras with phosphatidylinositol 3-kinase. Proc Natl Acad Sci USA 88:7908–7912[Abstract]
  6. Rodriguez-Viciana P, Warne PH, Vanhaesebroeck B, Waterfield MD, Downward J 1996 Activation of phosphoinositide 3-kinase by interaction with Ras and by point mutation. EMBO J 15:2442–2451[Abstract]
  7. Lazar DF, Wiese RJ, Brady MJ, Mastick CC, Waters SB, Yamauchi K, Pessin JE, Cuatrecasas P, Saltiel AR 1995 Mitogen-activated protein kinase kinase inhibition does not block the stimulation of glucose utilization by insulin. J Biol Chem 270:20801–20807[Abstract/Free Full Text]
  8. Kahn BB 1992 Facilitative glucose transporters: regulatory mechanisms and dysregulation in diabetes. J Clin Invest 89:1367–1374[Medline]
  9. Quon MJ, Chen H, Ing BL, Liu ML, Zarnowski MJ, Yonezawa K, Kasuga M, Cushman SW, Taylor SI 1995 Roles of -1-Phosphatidylinositol 3-Kinase and ras in regulating translocation of GLUT4 in transfected rat adipose cells. Mol Cell Biol 15:5403–5411[Abstract]
  10. Clarke J, Young P, Yonezawa K, Kasuga M, Holman G 1994 Inhibition of the translocation of GLUT1 and GLUT4 in 3T3–L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin. Biochem J 300:631–635[Medline]
  11. Cheatham B, Vlahos C, Cheatham L, Wang L, Blenis J, Kahn CR 1994 Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol Cell Biol 14:4902–4911[Abstract]
  12. Haruta T, Morris AJ, Rose DW, Nelson JG, Mueckler M, Olefsky JM 1995 Insulin-stimulated GLUT4 translocation is mediated by a divergent intracellular signaling pathway. J Biol Chem 270:27991–27994[Abstract/Free Full Text]
  13. Wiese RJ, Mastick CC, Lazar DF, Saltiel AR 1995 Activation of mitogen-activated protein kinase and phosphatidylinositol 3'-kinase is not sufficient for the hormonal stimulation of glucose uptake, lipogenesis, or glycogen synthesis is 3T3–L1 adipocytes. J Biol Chem 270:3442–3446[Abstract/Free Full Text]
  14. Navé BT, Haigh RJ, Hayward AC, Siddle K, Shepherd PR. Compartment-specific regulation of phosphoinositide 3-kinase by platelet-derived growth factor and insulin in 3T3-L1 adipocytes. Biochem J 313:55–60
  15. Herbst JJ, Andrews GC, Contillo LG, Singleton DH, Genereux PE, Gibbs EM, Lienhard GE 1995 Effect of the activation of phosphatidylinositol 3-kinase by a thiophosphotyrosine peptide on glucose transport in 3T3L1 adipocytes. J Biol Chem 270:26000–26005[Abstract/Free Full Text]
  16. Hausdorff SF, Frangioni JV, Birnbaum MJ 1994 Role of p21ras in insulin-stimulated glucose transport in 3T3–L1 adipocytes. J Biol Chem 269:21391–21394[Abstract/Free Full Text]
  17. Fingar DC, Birnbaum MJ 1994 A role for Raf-1 in the divergent signaling pathways mediating insulin-stimulated glucose transport. J Biol Chem 269:10127–10132[Abstract/Free Full Text]
  18. Manchester J, Kong X, Lowry OH, Lawrence Jr JC 1994 Ras signaling in the activation of glucose transport by insulin. Proc Natl Acad Sci USA 91:4644–4648[Abstract]
  19. Houseknecht KL, Zhu AX, Gnudi L, Hamann A, Zierath JR, Flier JS, Kahn BB 1996 Overexpression of H-Ras selectively in adipose tissue of transgenic mice: evidence for enhanced sensitivity to insulin. J Biol Chem 271:11347–11355[Abstract/Free Full Text]
  20. Kozma L, Baltensperger K, Klarlund J, Porras A, Santos E, Czech MP 1993 The Ras signaling pathway mimics insulin action on glucose transporter translocation. Proc Natl Acad Sci USA 90:4460–4464[Abstract]
  21. Benito M, Porras A, Nebreda AR, Santos E 1991 Differentiation of 3T3L1 fibroblast to adipocytes induced by transfection of ras oncogenes. Science 253:565–568[Medline]
  22. Sale EM, Atkinson PGP, Sale GJ 1995 Requirement of Map kinase for differentiation of fibroblast to adipocytes, for insulin activation of p90 S6 kinase and for insulin or serum stimulation of DNA synthesis. EMBO J 14:674–684[Abstract]
  23. Gnudi L, Zhu AX, Hamann A, Houseknecht KL, Flier JS, Kahn BB 1995 Obesity and divergent effects on insulin signaling in transgenic mice expressing a dominant negative mutant ras. Diabetes 44:20A[Abstract]
  24. Cai H, Szeberenyi J, Cooper GM 1988 Inhibition of NIH 3T3 cell proliferation by a mutant Ras protein with preferential affinity for GDP. Mol Cell Biol 8:3235–3243[Medline]
  25. Dorrestijn J, Ouwens DM, Berghe N Vd, Bos JL, Maassen JA 1996 Expression of a dominant negative ras mutant does not affect stimulation of glucose uptake and glycogen synthesis by insulin. Diabetologia 39:558–563[CrossRef][Medline]
  26. DePaolo D, Reusch JEB, Carel K, Bhuripanyo P, Leitner JW, Draznin B 1996 Functional interactions of phosphatidylinositol 3-kinase with GTPase-activating protein in 3T3–L1 adipocytes. Mol Cell Biol 16:1450–1457[Abstract]
  27. Becker TC, Noel RJ, Coats WS, Gomez-Foix AM, Alam T, Gerard RD, Newgard CB 1994 Use of recombinant adenovirus for metabolic engineering of mammalian cells. Methods Cell Biol 43:161–189[Medline]
  28. Gluzman Y, Reichl H, Solnic D 1982 Helper-free adenovirus type-5 vectors. In: Gluzman Y (ed) Eucaryotic Viral Vectors. Harbor Laboratory Press, Cold Spring Harbor, NY, pp 187–192
  29. Shepherd PR, Nave BT, Siddle K 1995 Insulin stimulation of glycogen synthesis and glycogen synthase activity is blocked by wortmannin and rapamycin in 3T3L1 adipocytes: evidence for the involvement of phosphoinositide 3-kinase and p70 ribosomal protein-S6 kinse. Biochem J 305:25–28[Medline]
  30. Hill DP, Wurst W 1993 Screening for novel pattern formation genes using gene trap approaches. Methods Enzymol 225:664–681[Medline]
  31. Gibbs JB 1995 Determination of guanine nucleotide bound to Ras in mammalian cells. Methods Enzymol 255:118–125[Medline]
  32. Chang PY, Goodyear LJ, Benecke H, Markuns JS, Moller DE 1995 Impaired insulin signaling in skeletal muscles from transgenic mice expression kinase-deficient insulin receptors. J Biol Chem 270:12593–12600[Abstract/Free Full Text]
  33. Lamphere L, Carpenter CL, Sheng ZF, Kallen RG, Lienhard GE 1994 Activation of PI 3-kinase in 3T3L1 adipocytes by association with insulin receptor substrate-1. Am J Physiol 266:E486–E494
  34. Kahn BB, Charron MJ, Lodish HF, Cushman SW, Flier JS 1989 Differential regulation of two glucose transporters in adipose cells from diabetic and insulin treated diabetic rats. J Clin Invest 84:404–411[Medline]
  35. Labarca C, Paigen K 1980 A simple rapid and sensitive DNA assay procedure. Ann Biochem Exp Med (Calcutta) 102:344–351