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H-ras Induces Glucose Uptake in Brown Adipocytes in an Insulin- and Phosphatidylinositol 3-Kinase-Independent Manner
EXPERIMENTAL CELL RESEARCH ARTICLE NO.
243, 274 –281 (1998)
EX984154
H-ras Induces Glucose Uptake in Brown Adipocytes in an Insulin- and Phosphatidylinositol 3-Kinase-Independent Manner Angela M. Valverde, Paloma Navarro, Manuel Benito, and Margarita Lorenzo1 Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University Complutense, 28040 Madrid, Spain
Fetal brown adipocytes (parental cells) expressed mainly Glut4 mRNA glucose transporter, the expression of Glut1 mRNA being much lower. At physiological doses, insulin stimulation for 15 min increased 3-fold glucose uptake and doubled the amount of Glut4 protein located at the plasma membrane. Moreover, phosphatidylinositol (PI) 3-kinase activity was induced by the presence of insulin in those cells, glucose uptake being precluded by PI 3-kinase inhibitors such as wortmannin or LY294002. H-raslys12-transformed brown adipocytes showed a 10-fold higher expression of Glut1 mRNA and protein than parental cells, Glut4 gene expression being completely down-regulated. Glucose uptake increased by 10-fold in transformed cells compared to parental cells; this uptake was unaltered in the presence of insulin and/or wortmannin. Transient transfection of parental cells with a dominant form of active Ras increased basal glucose uptake by 5-fold, no further effects being observed in the presence of insulin. However, PI 3-kinase activity (immunoprecipitated with anti-ap85 subunit of PI 3-kinase) remained unaltered in H-ras permanent and transient transfectants. Our results indicate that activated Ras induces brown adipocyte glucose transport in an insulin-independent manner, this induction not involving PI 3-kinase activation. © 1998 Academic Press Key Words: insulin resistance; H-ras transformation; Glut4/Glut1 mRNA and protein content; glucose uptake.
INTRODUCTION
Brown adipose tissue is specialized in nonshivering thermogenesis in neonates, responsible for heat production associated with the expression of the mitochondrial uncoupling protein 1 [1]. Brown adipose tissue differentiation also encompasses an adipogenic-program related to lipid synthesis and its accumulation results in a multilocular fat droplets phenotype [2]. In addition, it is well known that insulin is the main 1 To whom correspondence and reprint requests should be addressed. Fax: 34-91-3941779. E-mail: [email protected].
0014-4827/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
signal involved in lipogenesis and glucose, the main lipogenic substrate in fetal brown adipocytes [3,4]. Glucose transport in adipocytes is maintained mainly by the activity of the insulin-regulated glucose transporter Glut4, although the ubiquitous Glut1 glucose transporter is often expressed at appreciable levels together with Glut4 [reviewed in 5]. During fetal life, Glut1 is expressed at high levels in brown adipose tissue, although Glut4 mRNA expression greatly increased at the end of fetal development [2,6]. Moreover, insulin/IGF-I treatment for 24 h increases both glucose uptake and Glut4 mRNA expression in parallel to lipid content in 20-day fetal brown adipocytes in primary culture [4]. The mechanism by which acute insulin treatment in adipocytes increases glucose uptake involves signaling complexes containing IRS-1 and the p85/p110-type of phosphatidylinositol (PI) 3-kinase that mediates Glut4 glucose transporter redistribution to the plasma membrane [7,8]. Furthermore, inhibition of IGF-I-induced IRS-1-associated PI 3-kinase activity with wortmannin or LY294002 leads to an inhibition of IGF-I-induced glucose uptake in fetal brown adipocytes [9]. Insulin resistance, a smaller than normal response to a given amount of insulin, is a characteristic clinical feature of a number of disease states, such as obesity and non-insulin-dependent diabetes mellitus, and is associated with hyperglycemia, hyperinsulinemia, and hyperlipemia [10]. Several states of insulin resistance are related to receptor and postreceptor defects or alterations in insulin postreceptor signaling. In fact, prolonged insulin treatment of 3T3-L1 adipocytes results in insulin resistance [11]. Tumor necrosis factor-a also induced insulin resistance in cultured adipocytes by inhibiting insulin receptor autophosphorylation and IRS-1 tyrosine phosphorylation [12,13]. Moreover, deficiency in brown adipose tissue has been shown to result in the development of glucose intolerance and severe insulin resistance [14,15]. In a previous work we have examined the effect of brown adipocyte H-ras transformation (in the representative clone MB1.3.19 achieved by cotransfection with constructs of SV40LTAg and pMEXneo H-raslys12), in the insulin signal-
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ing cascade, compared with those events in primary cells [16,17]. We described an impairment in insulin signaling upstream from Ras as a consequence of a serine–threonine phosphorylation of the receptor; meanwhile downstream from Ras the signaling cascade was overstimulated regardless the presence of the hormone [17]. Accordingly, we have now examined in H-raslys12 transformed brown adipocytes the effect of insulin on glucose uptake and on the expression of Glut4 and Glut1 glucose transporters, compared to those events in the parental cells. Our results show a severe impairment of glucose uptake in response to insulin, the expression of Glut1 mRNA and protein being up-regulated and Glut4 mRNA and protein being down-regulated in H-ras transformed brown adipocytes. Moreover, H-ras dramatically induced glucose uptake, in a PI 3-kinase-independent manner. MATERIALS AND METHODS Materials. Insulin, wortmannin and BSA (fraction V, essentially fatty acid free) were from Sigma Chemical Co. (St. Louis, MO). LY294002 was purchased from Calbiochem (Calbiochem-Novabiochem International, La Jolla, CA). Fetal calf serum (FCS), phosphate-buffered saline (PBS), and culture medium were from Imperial Laboratories (Hampshire, UK). RNAzol B was from Biotecx Lab (Dallas, TX). Nylon membranes were GeneScreen (NEN Research Products, Boston, MA). Autoradiographic films were Kodak X-OMAT/AR (Eastman Kodak Co., Rochester, NY). 2-Deoxy-D[1-3H]glucose (11.0 Ci/mmol), [a-32P]dCTP (3000 Ci/mmol), [g-32P]dATP (3000 Ci/mmol), and the multiprimer DNA-labeling system kit were purchased from Amersham (Buckinghamshire, UK). All other reagents used were of the purest grade available. The cDNAs used as probes were Glut4 and Glut1 [18]. Rabbit polyclonal antibodies to the C-terminus of Glut4 and Glut1 were provided by Biogenesis (Biogenesis Ltd., United Kingdom). The transfection MBS mammalian transfection kit was from Stratagene (La Jolla, CA). Dr. E. Santos (NIH, Bethesda, MD) kindly provided the plasmid constructs used for transfection experiments (pMEXneo and pMEXneo H-raslys12). For immunoprecipitation the anti-ap85 subunit of PI 3-kinase mouse monoclonal antibody was the gift of Dr. J. Downward and P. Rodriguez-Viciana (Imperial Cancer Research Foundation, London, UK). The anti-ras monoclonal antibody (Y13-259) was purchased from Oncogene Science (Uniondale, NY). Cell culture. Parental brown adipocytes were obtained from interscapular brown adipose tissue of 20-day Wistar rat fetuses and isolated by collagenase dispersion as described [9,16]. Cells were plated at 1.5 3 106 cells/60-mm diameter tissue culture dishes for glucose uptake determination and at 4.5 3 106 cells/100-mm dishes for plasma membrane and RNA isolation or immunoprecipitation of proteins. Cultures were in minimal essential medium with Earle’s salts (MEM) supplemented with 10% FCS. After 4 – 6 h of culture at 37°C, cells were rinsed twice with PBS and 70% of the initial cells attached to the dish forming a monolayer. Cells were maintained for 20 h in a serum-free medium supplemented with 0.2% (w/v) BSA and further stimulated with insulin (1, 10, and 100 nM, as indicated in each case) for 5 or 15 min. H-ras-transformed brown adipocyte cell lines were established by the means of cotransfection of brown adipocyte primary cells with transforming H-ras gene pMEXneo (Hraslys12) in cooperation with SV40-LTAg [16,17]. The best-characterized H-ras transformed cell line clone (MB1.3.19) was plated at 0.35 3 106 cells/60-mm dishes for glucose uptake determination and at 1 3 106 cells/100-mm dishes for plasma membrane and RNA
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isolation or immunoprecipitation of proteins. Cells were grown in 10% FCS–DMEM with antibiotics and G418 (250 mg/ml) for selection of neoresistance marker to 80% confluence. Then, cells were starved for 20 h in serum-free MEM and subsequently stimulated for 5 or 15 min with various doses of insulin as indicated under Results and in the figure legends. Control cells were maintained in MEM in the absence of insulin. To analyze the effect of PI 3-kinase inhibitors on glucose uptake, cells were pretreated for 15 min with wortmannin or LY294002, at the doses previously described [9]. Both inhibitors were initially dissolved in dimethyl sulfoxide, and in all experimental series control cells were treated with the corresponding volumes of dimethyl sulfoxide. Glucose uptake determination. Cells growing in 60-mm-diameter dishes were serum deprived for 20 h and washed three times with ice-cold Krebs–Ringer-phosphate buffer (KRP) (135 mM NaCl, 5.4 mM KCl, 1.4 mM CaCl2, 1.4 mM MgSO4, and 10 mM sodium pyrophosphate, pH 7.4). Then, cells were incubated with 1 ml KRP buffer with or without insulin for 10 min at 37°C, and 2-deoxy-D[1-3H]glucose (500 nCi/ml) was added to this solution to a final concentration of 0.1 mM, the incubation being continued for 5 min at 37°C. Cells were then washed three times with ice-cold KRP buffer and solubilized in 1 ml of 1% SDS, as described [20]. The radioactivity of a 200 ml aliquot was determined in a scintillation counter. Glucose transport was determined in triplicate dishes from five independent experiments. In parallel dishes, proteins were determined by the Bradford dye method [21], using the Bio-Rad reagent (Bio-Rad, Richmond, CA) and BSA as the standard. Results are means 6 SEM (n 5 5) and are expressed as disintegration per minute per microgram of protein. Statistical significance was tested with a one-way analysis of variance followed by the protected least-significant difference test, where differences were accepted as significant at the P , 0.05 level. Subcellular fractionation and Western blotting. For preparation of plasma membrane fraction, cells from five dishes of 100 mm diameter were pooled for each experimental condition after harvesting with ice-cold PBS. Then, cells were homogenized in an ice-cold buffer containing 20 mM Hepes, 250 mM sucrose, 2 mM EGTA, 0.2 mM PMSF, 1 mM leupeptin, pH 7.4. Nuclei and unbroken cells were removed by centrifugation at 2000g for 10 min. Plasma membrane fraction was prepared by centrifugation of the supernatant in a Sorvall RC 5B superspeed centrifuge SS34 rotor, at 18,000g for 1 h and 4°C with. The membrane pellets were resuspended in homogenization buffer before storage at 220°C, and proteins were determined. To verify the preparation of plasma membrane, phosphodiesterase-1 activity was assayed under all the conditions. For Western blot analysis of Glut4 and Glut1, plasma membrane proteins (100 and 50 mg, respectively) after SDS–PAGE were transferred to Immobilon membranes, blocked using 5% nonfat dried milk in 10 mM Tris–HCl and 150 mM NaCl, pH 7.5, and incubated overnight with anti-Glut1 antibody (1:1000) or anti-Glut4 antibody (1:500) in 0.05% Tween 20, 1% nonfat dried milk in the buffer indicated above. Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL-Plus) Western blotting protocol (Amersham). Western blot analysis was performed from three independent experiments. RNA extraction and Northern blot analysis. Cells from 100-mmdiameter dishes were washed twice with ice-cold PBS and lysed directly with RNAzol B following the protocol supplied by the manufacturer for total RNA isolation [19]. Total cellular RNA (20 mg) was submitted to Northern blot analysis, i.e., electrophoresed on 0.9% agarose gels containing 0.66 M formaldehyde, transferred to GeneScreen TM membranes using a VacuGene blotting apparatus (LKBPharmacia) and cross-linked to the membranes by UV light. Hybridization was in 0.25 mM NaHPO4, pH 7.2, 0.25 M NaCl, 100 mg/ml denatured salmon sperm DNA, 7% SDS, and 50% deionized formamide, containing denatured 32P-labeled cDNA (106 cpm/ml) for 40 h, at 42°C, as described [4]. Complementary DNA labeling was carried out with [a-32P]dCTP to a specific activity of 109 cpm/mg of DNA by
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FIG. 1. Glucose uptake in parental (BA) and H-ras-transformed (MB1.3.19) brown adipocytes: insulin dose dependency. Cells were serum deprived for 20 h prior to the stimulation for 15 min with insulin, at the doses indicated. Glucose uptake was measured over the last 5 min of culture as described under Materials and Methods. Results are means 6 SEM (n 5 5) from five independent experiments and are expressed as disintegration per minute per microgram of protein. Statistical significance was tested with a one-way analysis of variance followed by the protected least-significant different test, where differences between values in the presence of insulin vs control is represented by (w); wP , 0.01.
using multiprimer DNA-labeling system kit. For serial hybridization with different probes, the blots were stripped and rehybridized subsequently as needed in each case. Membranes were subjected to autoradiography and relative densities of the hybridization signals were determined by densitometric scanning of the autoradiograms in a laser densitometer (Molecular Dynamics, Sunnyvale, CA). Northern blot analysis was performed from three independent experiments. PI 3-kinase activity. It was measured by in vitro phosphorylation of phosphatidylinositol as previously described [9]. Cells from 100mm-diameter dishes were incubated in the absence or presence of insulin for 5 min as indicated in the figure legends. After washing with ice-cold PBS cells were solubilized in lysis buffer containing 10 mM Tris–HCl, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF , 100 mM Na3VO4, 1% Triton X-100, 10 mg/ml leupeptin, 10 mg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.6. Lysates were clarified by centrifugation at 15,000g for 10 min at 4°C and proteins (600 mg) were immunoprecipitated with the anti-ap85 subunit of PI 3-kinase mouse monoclonal antibody or with the anti-p21-ras monoclonal antibody (Y13-259). The immunoprecipitates were washed successively as previously described [9]. To each pellet were added 25 ml of 1 mg/ml of L-a-phosphatidylinositol/ L-a-phosphatidyl-L-serine sonicated in 25 mM Hepes (pH 7.5) and 1 mM EDTA. The PI 3-kinase reaction was started by the addition of 100 nM [g-32P]ATP (10 mCi) and 300 mM ATP in 25 ml of 25 mM Hepes (pH 7.4), 10 mM MgCl2, and 0.5 mM EGTA. After 15 min at room temperature the reaction was stopped by the addition of 500 ml of CHCl3:methanol (1:2) in a 1% concentration of HCl plus 125 ml of chloroform and 125 ml of HCl (10 mM). The samples were centrifuged, and the lower organic phase was removed and washed once with 480 ml of methanol:100 mM HCl plus 2 mM EDTA (1:1). The organic phase was extracted, dried in vacuo, and resuspended in chloroform. Samples were applied to a silica gel thin-layer chromatography (TLC) plate (Merck). TLC plates were developed in propanol-1 and acetic acid 2 N; 65:35 (v/v), dried, visualized by autoradiography, and quantitated by scanning laser densitometry. Transfection conditions. Parental brown adipocytes (100-mm-diameter dishes) were transiently transfected using the calcium phosphate precipitation technique, with 15 mg of a fusion plasmid containing either transforming Ras DNA cloned in an eukaryotic expression vector (pMEXneo H-raslys12) or pMEXneo carrier DNA. After 4 h of incubation, cells were shocked with 3 ml of 15% glycerol for 2 min, washed, and then incubated 20 h in serum-free medium, as previously described [16]. At the end of the culture time, cells were
incubated for 5–15 min either in the absence or in the presence of 100 nM insulin for determination of PI 3-kinase activity or glucose uptake, as described above.
RESULTS
H-ras-Transformed Brown Adipocytes Showed Insulin-Independent Glucose Uptake We have studied the glucose uptake in response to different doses of insulin in both parental and H-ras transformed brown adipocytes as shown in Fig. 1. Cells were serum deprived for 20 h in MEM culture (containing 5 mM glucose) prior to the stimulation for 15 min with insulin, at the doses indicated, and glucose uptake was measured over the last 5 min of the incubation, as described under Materials and Methods. As shown in Fig. 1, 1 nM insulin significantly induced glucose uptake by 3-fold compared to basal levels observed in parental brown adipocytes. No further increase in glucose uptake was observed at higher doses of the hormone. Transformed brown adipocytes clone (MB1.3.19) showed a 30-fold higher basal glucose uptake compared to parental cells. However, no further effect was observed in the presence of insulin, regardless the dose used (Fig. 1). Glut4 and Glut1 Gene Expression in Parental and H-ras-Transformed Brown Adipocytes A second issue addressed in this study is the direct comparison of the distribution of Glut4 and Glut1 mRNA species in both parental and transformed cells, either under growing conditions (10% FCS–DMEM) or in 20-h serum-deprived cells in MEM culture untreated or treated for 15 min with 100 nM insulin (Fig. 2). Parental cells expressed both the insulin-dependent glucose transporter Glut4 and the basal glucose trans-
INDUCTION OF GLUCOSE UPTAKE BY H-ras
FIG. 2. Glut4 and Glut1 mRNA expression in parental (BA) and H-ras-transformed (MB1.3.19) brown adipocytes. Cells growing in FCS were serum deprived for 20 h prior to the stimulation for 15 min with 100 nM insulin. Total RNA (20 mg) was submitted to Northern blot analysis and hybridized with labeled Glut4 and Glut1 cDNAs. A final hybridization with the 18S rRNA cDNA was performed for normalization. Autoradiograms from representative experiments out of three are shown. Arrows indicate the position of Glut4 and Glut1 mRNAs.
porter Glut1 mRNA, although the expression of Glut1 mRNA was very low compared to Glut4 mRNA expression. No differences in the expression of both Glut4 and Glut1 mRNA were found under serum or serum-deprivation culture conditions. Although the presence of insulin for 24 h dramatically induced Glut4 mRNA expression in fetal brown adipocytes [4], short-term insulin treatment (15 min) does not affect Glut4 mRNA accumulation compared to serum-deprived cells as shown in Fig. 2. Besides the high expression of the canonical 2.8-kb Glut4 mRNA in parental cells, a second mRNA isoform of approximately 2.3 kb was apparent. Whether this isoform is the result of the existence of alternative polyadenylation sites and its contribution to the Glut4 protein synthesis remains to be established. In H-ras-transformed brown adipocytes we could not detect Glut4 mRNA expression, at least in total RNA (20 mg) Northern blot. However, the expression of Glut1 mRNA was 10-fold higher compared to parental cells, regardless the culture conditions. This overexpression of Glut1 mRNA in transformed cells could account for the increased glucose uptake observed in these cells. In addition to the mRNA accumulation, we studied the effect of insulin treatment on Glut4 and Glut1 protein distribution in plasma membrane, by Western blot analysis. Either parental cells or H-ras-transformed cells were serum deprived for 20 h (untreated cells) prior to the stimulation for 15 min with 100 nM insulin (Fig. 3). Then, cells were harvested and homogenized and plasma membrane fractions were obtained as described under Materials and Methods. Plasma membrane proteins were submitted to Western blot analysis and immunoblotted for Glut4 and Glut1 detection. For detection of Glut4, 100 mg of plasma membrane protein was loaded. In parental cells, insulin
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stimulation increased by twofold the amount of Glut4 protein in the plasma membrane compared to untreated cells; meanwhile Glut4 protein was undetectable in plasma membrane from H-ras-transformed cells regardless of insulin stimulation. Glut1 protein was immunodetectable in 50 mg of plasma membrane proteins from both parental and H-ras-transformed cells. However, H-ras-transformed cells expressed 10fold higher levels of Glut1 than that observed in parental cells, in agreement with the data of Glut1 mRNA shown above (Fig. 3). The amount of Glut1 in plasma membrane remained unmodified by treatment with insulin in either H-ras-transformed cell or parental cells (Fig. 3). Transient Transfection of H-raslys12 Gene-Induced Glucose Uptake in Parental Cells in an InsulinIndependent Manner To figure out if the overstimulated glucose uptake found in H-ras-transformed cells was due to the overexpression of the transforming H-ras gene, we transiently transfected parental cells with 15 mg of a fusion plasmid containing transforming Ras DNA cloned in an eukaryotic expression vector (pMEXneo H-raslys12) or with pMEXneo carrier DNA [16]. After the transfection, cells were cultured for 20 h in a serum-free medium and incubated either in the absence or in the presence of insulin (100 nM) for 15 min, glucose uptake being determined over the last 5 min of the incubation, as described above (Fig. 4). In cells transfected with pMEXneo carrier DNA, 100 nM insulin produced a similar stimulation of glucose uptake (threefold increase) as described in Figure 1. Transfection with pMEXneo H-raslys12 construct produced a fivefold increase in the basal glucose uptake, this effect being significantly higher than that produced by 100 nM insulin in cells transfected with pMEXneo. However, insulin stimulation of H-ras transiently transfected
FIG. 3. Glut4 and Glut1 protein content in parental (BA) and H-ras-transformed (MB1.3.19) brown adipocytes. Cells growing in FCS were serum deprived for 20 h (C) prior to the stimulation for 15 min with 100 nM insulin. Cells from five dishes of 100 mm diameter were pooled for each experimental condition, and plasma membrane fractions were obtained as described under Materials and Methods. Plasma membrane proteins (100 mg for Glut4 and 50 mg for GLUT1) were submitted to SDS–PAGE, blotted to nylon membrane, immunodetected with the anti-Glut4 and anti-Glut1 antibodies, and developed with ECL. Representative experiments out of three are shown. Arrows indicate the position of Glut4 and Glut1 proteins.
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cells did not produce any further stimulation on glucose uptake over that produced by H-ras (Fig. 4). These results clearly indicate that active H-ras transient transfection induced glucose uptake in parental cells, suggesting that the similar effect found in established H-ras-transformed brown adipocyte cell line was not due to a clonal variation. Glucose Uptake in H-ras-Transformed Brown Adipocytes Is PI 3-Kinase Independent The fact that glucose uptake is dramatically increased in H-ras-transformed brown adipocytes prompted us to investigate whether this effect could be blocked by PI 3-kinase inhibitors, such as wortmannin and LY294002 [22,23], at the doses previously shown that totally inhibited PI 3-kinase in parental cells [9]. Cells were serum deprived for 20 h and then pretreated for 15 min with 20 nM wortmannin or 10mM LY294002 or the corresponding volume of dimethyl sulfoxide, and subsequently incubated either in the absence or in the presence of insulin (100 nM) for 15 min, glucose uptake being determined over the last 5 min of the incubation, as described above. As shown in Table 1, wortmannin or LY294002 does not affect the basal glucose uptake observed in parental cells, but significantly inhibits insulin-induced glucose uptake in parental cells. However, wortmannin or LY294002 does not modify the
FIG. 4. Effect of active H-ras transfection on basal and insulininduced glucose uptake in parental brown adipocytes. Fetal brown adipocytes were transiently transfected with transforming Ras DNA vector (pMEXneo H-raslys12) or with carrier DNA (pMEXneo), as described under Materials and Methods. After transfection, cells were serum deprived for 20 h prior to the stimulation for 15 min with 100 nM insulin. Glucose uptake was measured over the last 5 min of culture. Results are means 6 SEM (n 5 5) from five independent experiments and are expressed as disintegration per minute per microgram of protein. Statistical significance was tested with a oneway analysis of variance followed by the protected least-significant different test, where differences between values in the presence of insulin and/or H-ras vs control is represented by (w) or between H-ras transfected cells 6 insulin vs insulin alone is represented by (*); wP , 0.01, *p , 0.05.
TABLE 1 Effect of PI 3-Kinase Inhibitors on Basal and Insulin-Induced Glucose Uptake in either Parental (BA) and H-rasTransformed (MB1.3.19) Brown Adipocytes Treatment None Wortmannin LY294002 Insulin Insulin1wortmannin Insulin1LY294002
BA
MB1.3.19
2.0 6 0.3 1.9 6 0.2 1.8 6 0.3 6.7 6 0.8✯ 2.1 6 0.3* 2.3 6 0.4*
61 6 7 59 6 8 54 6 7 60 6 7 56 6 6 59 6 8
Note. Cells were serum deprived for 20 h and then pretreated for 15 min with 20 nM wortmannin, 10 mM LY294002, or the corresponding volume of dimethyl sulfoxide and subsequently incubated either in the absence or in the presence of insulin (100 nM) for 15 min, glucose uptake being determined over the last 5 min of the incubation, as described under Materials and Methods. Results are means 6 SEM (n 5 5) from five independent experiments and are expressed as disintegration per minute per microgram of protein. Statistical significance was tested with a one-way analysis of variance followed by the protected least-significant different test, where differences between values in the presence of insulin vs. control is represented by (✯) and differences between values in the presence of PI 3-kinase inhibitor vs those in its absence is represented by (*); ✯,* P , 0.01.
higher basal glucose uptake observed in H-ras-transformed cells. Furthermore, no effect of PI 3-kinase inhibitors was detected either in insulin-treated transformed brown adipocytes (Table 1). Finally, we decided to investigate whether the overexpression of H-raslys12 could result in an increased PI 3-kinase activity in either transformed or transiently transfected parental brown adipocytes (Fig. 5). Parental cells were transiently transfected under the same conditions as described above. After transfection, cells were serum starved and subsequently stimulated with 100 nM insulin for 5 min and lysed, and 600 mg of protein was immunoprecipitated with the anti-ap85 regulatory subunit of PI 3-kinase monoclonal antibody. The resulting immune complexes were assayed for PI 3-kinase activity as described under Materials and Methods. As shown in Fig. 5A, parental brown adipocytes displayed almost undetectable PI 3-kinase activity in anti-ap85 immunoprecipitates under control conditions. Upon treatment with 100 nM insulin, the basal PI 3-kinase activity increased by eightfold. However, transient transfection with H-raslys12 did not modify the PI 3-kinase activity observed under basal conditions. Furthermore, stimulation with insulin in H-raslys12-transfected cells did not significantly increase PI 3-kinase activity compared to insulin-treated cells. Transformed cells (clone MB1.3.19), overexpressing constitutively high levels of active p21-ras protein, were serum starved for 20 h and subsequently stimulated with 100 nM insulin for 5 min and lysed, and 600 mg of protein was immunoprecipitated with the anti-
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immunoprecipitation with the anti-ras monoclonal antibody (Y13-259) either under basal conditions or under insulin stimulation (Fig. 5C). DISCUSSION
FIG. 5. PI 3-kinase activity in active H-ras overexpressing parental (BA) and transformed (MB1.3.19) brown adipocytes. (A) Parental brown adipocytes (BA) were transiently transfected with transforming Ras DNA vector (pMEXneo H-raslys12) or with carrier DNA (pMEXneo), and after transfection, cells were serum deprived for 20 h. Then, cells were stimulated for 5 min with 100 nM insulin or received an equivalent volume of solvent. Whole cell lysates 600 mg of protein) were subjected to immunoprecipitation with anti-ap85 monoclonal antibody. The immune complexes were washed and immediately used for an in vitro PI 3-kinase assay as described under Materials and Methods. The conversion of PI to PI phosphate PIP) in the presence of [g-32P]ATP was analyzed by thin-layer chromatography. (B) H-ras-transformed brown adipocytes (MB1.3.19) were serum deprived for 20 h prior to the stimulation for 5 min with 100 nM insulin or received an equivalent volume of solvent. Whole cell lysates (600 mg of protein) were subjected to immunoprecipitation with anti-ap85 monoclonal antibody and assayed for PI 3-kinase activity. (C) H-ras-transformed brown adipocytes were treated as in B and lysates (600 mg of protein) were subjected to immunoprecipitation with anti-ras monoclonal antibody (Y13-259) and assayed for PI 3-kinase activity. Representative experiments of three are shown.
ap85 monoclonal antibody. Transformed cells showed a low ap85-associated PI 3-kinase activity under basal conditions; this activity increased in response to 100 nM insulin (threefold) but in a lower extent than that observed in parental cells (Fig. 5B). Although we did not find an increased PI 3-kinase activity in anti-ap85immunoprecipitates from H-ras-overexpressing cells, since it has been established that p110 subunit of PI 3-kinase is an effector of the activated Ras protein [24], we used another approach in order to confirm these data, assaying PI 3-kinase activity directly in anti-p21ras immunoprecipitates from H-ras-transformed cells. No PI 3-kinase activity was observed in H-ras-transformed brown adipocytes after lysis and subsequent
Fetal brown adipocytes (parental cells) expressed very high levels of Glut4 mRNA glucose transporter, the expression of Glut1 mRNA being restricted to a minimum. Under physiological conditions, insulin induced glucose uptake, doubling the amount of Glut4 protein located in plasma membrane. Glut1 was much less abundant than Glut4 in plasma membrane, making its contribution to transport negligible. Moreover, insulin increased PI 3-kinase activity, and the inhibition of PI 3-kinase activity resulted in an impaired insulin-induced glucose uptake, suggesting that insulin induced glucose uptake in a PI 3-kinase-dependent manner in fetal brown adipocytes. Our results are consistent with the well-known requirement of PI 3-kinase activity for the movement of glucose transporters to the cell membrane in both white and brown adipose tissues [25,26]. H-raslys12-transformed brown adipocytes substantially increased the expression of the ubiquitous glucose transporter Glut1 mRNA and the amount of Glut1 protein in plasma membrane, the expression of Glut4 (either mRNA and protein) being down-regulated. Possibly this high Glut1 gene expression is a compensatory response for lack of Glut4 expression in this cell line. Under these conditions, the glucose uptake turned to be dramatically increased compared to parental cells, in an insulin-independent manner. To determine if this effect was due to the overexpression of transforming H-ras gene or to the clonal variation, we have reproduced these results by transient transfection of parental cells with transforming Ras DNA cloned in an eukaryotic expression vector [16]. H-raslys12 gene transiently transfected to parental cells increased basal glucose uptake to a higher extent that insulin alone, this effect of Ras on glucose uptake being insulin independent as found in H-ras-transformed cell lines. Regarding Ras effect on glucose transport, although some authors have proposed that the Ras signaling pathway mimics insulin action on Glut4 translocation in 3T3-L1 cells although decreasing by 95% Glut4 protein [27], other results indicate that p21-ras is not involved in insulin-stimulated glucose transport [28,29]. In fact, constitutive active p21-ras or Raf-1 either microinjected or transfected into 3T3-L1 cells greatly stimulated the expression of Glut1 in the absence of insulin and increased 40-fold the basal glucose uptake [28,30]. Our results either in parental brown adipocytes transiently transfected with active Ras or in permanently overexpressing H-ras-transformed cells are in agreement with those later results. Recently, an enhanced
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glucose uptake was found in K-ras-transformed fibroblasts associated with an increased N-glycosylation of Glut1 [31]. Whether or not this mechanism could be operating in H-ras-transformed brown adipocytes remains to be established. Because a growing number of studies indicate that Ras and PI 3-kinase signaling pathways may converge at the cytoplasm [24 –32], although others suggest that both pathways are independent in the insulin signaling in 3T3-L1 adipocytes [33], we decided to investigate whether the increased glucose uptake produced by overexpression of H-ras could be related to an increased PI 3-kinase activity. However, inhibition of PI 3-kinase activity with wortmannin did not decrease the higher basal glucose uptake observed in H-ras-transformed cells. Moreover, our results clearly demonstrate that ap85-associated-PI 3-kinase activity is not induced by H-ras overexpression in either transiently or permanently transfected cells. Furthermore, PI 3-kinase activity is not increased in anti-p21-ras immunoprecipitates from H-raslys12-transformed cells, a system where we have previously shown that the content of Ras.GTP form was constitutively very high [16]. Thus, H-ras induced glucose uptake in a PI 3-kinaseindependent manner. However, we cannot exclude the possibility that other Ras effectors, such as Ral GDP dissociation stimulator or protein kinase Cz, could be involved in the Ras-increased glucose uptake. Moreover, insulin stimulates ap85-associated PI 3-kinase activity in H-ras-overexpressing cells, glucose transport being no further stimulated by addition of the hormone. In conclusion, our results show that Ras overexpression strongly induces glucose uptake in brown adipocytes in an insulin-independent manner, this induction not involving PI 3-kinase activation. We are grateful for valuable reagents provided by Dr. Dr. J. Downward and P. Rodriguez-Viciana (Imperial Cancer Research Foundation, London, UK) and Dr. E. Santos (National Institutes of Health, Bethesda, MD). P.N. was the recipient of a fellowship from the Ministerio de Educacion y Cultura. Grant SAF96/0115 from the Comision Interministerial de Ciencia y Tecnologia, Spain, supported this work
REFERENCES Ailhaud, G., Grimaldi, P., and Negrel, R. (1992). Cellular and molecular aspects of adipose tissue development. Annu. Rev. Nutr. 12, 207–233. 2. Teruel, T., Valverde, A. M., Benito, M., and Lorenzo, M. (1995). Differentiation of rat brown adipocytes during late fetal development: role of insulin-like growth factor I. Biochem. J. 310, 771–776. 3. Lorenzo, M., Roncero, C., Fabregat, I., and Benito, M. (1988). Hormonal regulation of rat fetal lipogenesis in brown adipocyte primary cultures. Biochem. J. 251, 617– 620. 4. Teruel, T., Valverde, A. M., Benito, M., and Lorenzo, M. (1996). Insulin-like growth factor I and insulin induce adipogenic-re-
lated gene expression in fetal brown adipocyte primary cultures. Biochem. J. 319, 627– 632. 5.
Stephens, J. M., and Pilch, P. F. (1995). The metabolic regulation and vesicular transport of GLUT4, the major insulin-responsive glucose transporter. Endocr. Rev. 16, 529 –546.
6.
Santalucı´a, T., Camps, M., Castello, A., Mun˜oz, P., Nuel, A., Testar, X., Palacin, M., and Zorzano, A. (1992). Developmental regulation of GLUT1 (Erithroid/Hep G2) and GLUT4 (Muscle/ Fat) glucose transporter expression in rat heart, skeletal muscle, and brown adipose tissue. Endocrinology 130, 837– 846.
7.
Martin, S., Haruta, T., Morris, A. J., Klippel, A., Williams, L. T., and Olefsky, J. M. (1996). Activated phosphatidylinositol 3-kinase is sufficient to mediated actin rearrangement and GLUT4 translocation in 3T3-L1 adipocytes. J. Biol. Chem. 271, 17605– 17608.
8.
Guilherme, A., Klarlund, J. K, Krystal, G., and Czech, M. P. (1996). Regulation of phosphatidylinositol 3,4,5-trisphosphate 59-phosphatase activity by insulin. J. Biol. Chem. 271, 29533– 29536.
9.
Valverde, A. M., Lorenzo, M., Navarro, P., and Benito, M. (1997). Phosphatidylinositol 3-kinase is a requirement for insulin-like growth factor I-induced differentiation, but not for mitogenesis, in fetal brown adipocytes. Mol. Endocrinol. 11, 595– 607.
10.
Haring, H. U. (1991). The insulin receptor signaling mechanism and contribution to the pathogenesis of insulin resistance. Diabetologia 34, 848 – 861.
11.
Ricort, J. M., Tanti, J. F., Van Obberghen, E., and Le Marchand-Brustel, Y. (1995) Alterations in insulin signaling pathway induced by prolonged insulin treatment of 3T3-L1. Diabetologia 38, 1148 –1156.
12.
Feinstein, R., Kanety, H., Papa, M. Z., Lunenfeld, B., and Karasik, A. (1993). Tumor necrosis alpha suppresses insulininduced tyrosine phosphorylation of insulin receptor and its substrates. J. Biol. Chem. 268, 26055–26058.
13.
Hotamisligil, G. S., Murray, D. L., Choy, L. N., and Spiegelman, B. M. (1994). Tumor necrosis alpha inhibits signaling from the insulin receptor. Proc. Natl. Acad. Sci. USA 91, 4854 – 4858.
14.
Hamann, A., Benecke, H., Le Marchand-Brustel, Y., Susulic, V. S., Lowell, B. B., and Flier, J. S. (1995). Characterization of insulin resistance and NIDDM in transgenic mice with reduced brown fat. Diabetes 44, 1266 –1273.
15.
Hamann, A., Flier, J. S., and Lowell, B. B. (1995). Decreased brown fat markedly enhances susceptibility to diet-induced obesity, diabetes and hyperlipemia. Endocrinology 137, 22–29.
16.
Lorenzo, M,, Valverde, A. M., Teruel, T., Alvarez, A., and Benito, M. (1996). p21-ras induced differentiation-related gene expression in fetal brown adipocyte primary cells and cell lines. Cell Growth Differ. 7, 1251–1259.
17.
Valverde, A. M, Lorenzo, M., Teruel, T., and Benito, M. (1997). Alterations in the insulin signaling pathway induced by immortalization and H-ras transformation of brown adipocytes. Endocrinology 138, 3195–3206.
18.
Birnbaum, M. J. (1989). Identification of a novel gene encoding an insulin-responsive glucose transporter protein. Cell 57, 305– 315.
19.
Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thyocyanate–phenol– chloroform extraction. Anal. Biochem. 162, 156 –159.
20.
Rice, K. M., and Garner C. W. (1994). Correlation of the insulin receptor substrate-1 with insulin-responsive deoxyglucose transport in 3T3-L1 adipocytes. Biochem. Biophys. Res. Commun. 198, 523–530.
1.
INDUCTION OF GLUCOSE UPTAKE BY H-ras 21.
22.
23.
24.
25.
26.
27.
Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 –254. Okada, T., Kawano, Y., Sakakibara, Y., Hazeki, O., and Ui, J. (1994). Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and anti-lipolysis in rat adipocytes. J. Biol. Chem. 269, 3568 –3573. Vlahos, C. J., Matter, W. F., Hui, K. Y., and Brown, R. F. (1994). A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269, 5241–5248. Rodrigez-Viciana, P., Warne, P. H., Vanhaesebroeeck, B., Waterfield, M. D., and Downward, J. (1996). Phosphatidylinositol3-OH kinase as a direct target of Ras. EMBO J. 15, 2442–2451. Schimizu, Y., and Schimazu, T. (1994). Effects of wortmannin on increased glucose transport by insulin and norepinephrine in primary cultures of brown adipocytes. Biochem. Biophys. Res Commun. 202, 660 – 665. Omatsu-Kanbe, M., and Kitasato, H. (1992). Insulin and noradrenaline independently stimulate the translocation of glucose transporters from intracellular stores to the plasma membrane in mouse brown adipocytes. FEBS Lett. 314, 246 –250. Kozma, L., Baltenperger, K., Klarlund, J., Porras, A., Santos, E., and Czech, M. P. (1993). The Ras signaling pathway mimics insulin action on glucose transporter translocation. Proc. Natl. Acad. Sci. USA 90, 4460 – 4464.
Received December 15, 1997 Revised version received April 30, 1998
281
28.
Hausdorff, S. F., Frangioni, J. V., and Birnbaum, M. J. (1994). Role of p21ras in insulin-stimulated glucose transport in 3T3-L1 Adipocytes. J. Biol. Chem. 269, 21391–21394.
29.
Van den Berghe, N., Ouwens, D. M., Maaseen, J. A., van Mackelenbergh, M. G. H., Sips, H. C. M., and Krans, H. M. J. (1994). Activation of the Ras/mitogen-activated protein kinase signaling pathway alone is not sufficient to induce glucose uptake in 3T3-L1 adipocytes. Mol. Cell. Biol. 14, 2372– 2377.
30.
Fingar, D. C., and Birnbaum, M. J. (1994). A role for Raf-1 signaling pathways mediating insulin-stimulated glucose transport. J. Biol. Chem. 269, 10127–10132.
31.
Onetti, R., Baulida, J., and Bassols, A. (1997). Increased glucose transport in ras-transformed fibroblasts: A possible role for N-glycosylation of GLUT1. FEBS Lett. 407, 267–270.
32.
Hu, Q., Klippel, A., Muslin, A. J, Fantl, W. J., and Williams, L. T. (1995). Ras-dependent induction of cellular responses by constitutively active phosphatidylinositol-3 kinase. Science 268, 100 –102.
33.
Gnudi, L., Frever, E. U., Houseknecht, K. L., Erhardt, P., and Kahn. B. B. (1997). Adenovirus mediated gene transfer of dominant negative Rasasn17 in 3T3-L1 adipocytes does not alter insulin-stimulated phosphatidylinositol 3-kinase activity or glucose transport. Mol. Endocrinol. 11, 67–76.
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EX984154
H-ras Induces Glucose Uptake in Brown Adipocytes in an Insulin- and Phosphatidylinositol 3-Kinase-Independent Manner Angela M. Valverde, Paloma Navarro, Manuel Benito, and Margarita Lorenzo1 Department of Biochemistry and Molecular Biology, Faculty of Pharmacy, University Complutense, 28040 Madrid, Spain
Fetal brown adipocytes (parental cells) expressed mainly Glut4 mRNA glucose transporter, the expression of Glut1 mRNA being much lower. At physiological doses, insulin stimulation for 15 min increased 3-fold glucose uptake and doubled the amount of Glut4 protein located at the plasma membrane. Moreover, phosphatidylinositol (PI) 3-kinase activity was induced by the presence of insulin in those cells, glucose uptake being precluded by PI 3-kinase inhibitors such as wortmannin or LY294002. H-raslys12-transformed brown adipocytes showed a 10-fold higher expression of Glut1 mRNA and protein than parental cells, Glut4 gene expression being completely down-regulated. Glucose uptake increased by 10-fold in transformed cells compared to parental cells; this uptake was unaltered in the presence of insulin and/or wortmannin. Transient transfection of parental cells with a dominant form of active Ras increased basal glucose uptake by 5-fold, no further effects being observed in the presence of insulin. However, PI 3-kinase activity (immunoprecipitated with anti-ap85 subunit of PI 3-kinase) remained unaltered in H-ras permanent and transient transfectants. Our results indicate that activated Ras induces brown adipocyte glucose transport in an insulin-independent manner, this induction not involving PI 3-kinase activation. © 1998 Academic Press Key Words: insulin resistance; H-ras transformation; Glut4/Glut1 mRNA and protein content; glucose uptake.
INTRODUCTION
Brown adipose tissue is specialized in nonshivering thermogenesis in neonates, responsible for heat production associated with the expression of the mitochondrial uncoupling protein 1 [1]. Brown adipose tissue differentiation also encompasses an adipogenic-program related to lipid synthesis and its accumulation results in a multilocular fat droplets phenotype [2]. In addition, it is well known that insulin is the main 1 To whom correspondence and reprint requests should be addressed. Fax: 34-91-3941779. E-mail: [email protected].
0014-4827/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
signal involved in lipogenesis and glucose, the main lipogenic substrate in fetal brown adipocytes [3,4]. Glucose transport in adipocytes is maintained mainly by the activity of the insulin-regulated glucose transporter Glut4, although the ubiquitous Glut1 glucose transporter is often expressed at appreciable levels together with Glut4 [reviewed in 5]. During fetal life, Glut1 is expressed at high levels in brown adipose tissue, although Glut4 mRNA expression greatly increased at the end of fetal development [2,6]. Moreover, insulin/IGF-I treatment for 24 h increases both glucose uptake and Glut4 mRNA expression in parallel to lipid content in 20-day fetal brown adipocytes in primary culture [4]. The mechanism by which acute insulin treatment in adipocytes increases glucose uptake involves signaling complexes containing IRS-1 and the p85/p110-type of phosphatidylinositol (PI) 3-kinase that mediates Glut4 glucose transporter redistribution to the plasma membrane [7,8]. Furthermore, inhibition of IGF-I-induced IRS-1-associated PI 3-kinase activity with wortmannin or LY294002 leads to an inhibition of IGF-I-induced glucose uptake in fetal brown adipocytes [9]. Insulin resistance, a smaller than normal response to a given amount of insulin, is a characteristic clinical feature of a number of disease states, such as obesity and non-insulin-dependent diabetes mellitus, and is associated with hyperglycemia, hyperinsulinemia, and hyperlipemia [10]. Several states of insulin resistance are related to receptor and postreceptor defects or alterations in insulin postreceptor signaling. In fact, prolonged insulin treatment of 3T3-L1 adipocytes results in insulin resistance [11]. Tumor necrosis factor-a also induced insulin resistance in cultured adipocytes by inhibiting insulin receptor autophosphorylation and IRS-1 tyrosine phosphorylation [12,13]. Moreover, deficiency in brown adipose tissue has been shown to result in the development of glucose intolerance and severe insulin resistance [14,15]. In a previous work we have examined the effect of brown adipocyte H-ras transformation (in the representative clone MB1.3.19 achieved by cotransfection with constructs of SV40LTAg and pMEXneo H-raslys12), in the insulin signal-
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ing cascade, compared with those events in primary cells [16,17]. We described an impairment in insulin signaling upstream from Ras as a consequence of a serine–threonine phosphorylation of the receptor; meanwhile downstream from Ras the signaling cascade was overstimulated regardless the presence of the hormone [17]. Accordingly, we have now examined in H-raslys12 transformed brown adipocytes the effect of insulin on glucose uptake and on the expression of Glut4 and Glut1 glucose transporters, compared to those events in the parental cells. Our results show a severe impairment of glucose uptake in response to insulin, the expression of Glut1 mRNA and protein being up-regulated and Glut4 mRNA and protein being down-regulated in H-ras transformed brown adipocytes. Moreover, H-ras dramatically induced glucose uptake, in a PI 3-kinase-independent manner. MATERIALS AND METHODS Materials. Insulin, wortmannin and BSA (fraction V, essentially fatty acid free) were from Sigma Chemical Co. (St. Louis, MO). LY294002 was purchased from Calbiochem (Calbiochem-Novabiochem International, La Jolla, CA). Fetal calf serum (FCS), phosphate-buffered saline (PBS), and culture medium were from Imperial Laboratories (Hampshire, UK). RNAzol B was from Biotecx Lab (Dallas, TX). Nylon membranes were GeneScreen (NEN Research Products, Boston, MA). Autoradiographic films were Kodak X-OMAT/AR (Eastman Kodak Co., Rochester, NY). 2-Deoxy-D[1-3H]glucose (11.0 Ci/mmol), [a-32P]dCTP (3000 Ci/mmol), [g-32P]dATP (3000 Ci/mmol), and the multiprimer DNA-labeling system kit were purchased from Amersham (Buckinghamshire, UK). All other reagents used were of the purest grade available. The cDNAs used as probes were Glut4 and Glut1 [18]. Rabbit polyclonal antibodies to the C-terminus of Glut4 and Glut1 were provided by Biogenesis (Biogenesis Ltd., United Kingdom). The transfection MBS mammalian transfection kit was from Stratagene (La Jolla, CA). Dr. E. Santos (NIH, Bethesda, MD) kindly provided the plasmid constructs used for transfection experiments (pMEXneo and pMEXneo H-raslys12). For immunoprecipitation the anti-ap85 subunit of PI 3-kinase mouse monoclonal antibody was the gift of Dr. J. Downward and P. Rodriguez-Viciana (Imperial Cancer Research Foundation, London, UK). The anti-ras monoclonal antibody (Y13-259) was purchased from Oncogene Science (Uniondale, NY). Cell culture. Parental brown adipocytes were obtained from interscapular brown adipose tissue of 20-day Wistar rat fetuses and isolated by collagenase dispersion as described [9,16]. Cells were plated at 1.5 3 106 cells/60-mm diameter tissue culture dishes for glucose uptake determination and at 4.5 3 106 cells/100-mm dishes for plasma membrane and RNA isolation or immunoprecipitation of proteins. Cultures were in minimal essential medium with Earle’s salts (MEM) supplemented with 10% FCS. After 4 – 6 h of culture at 37°C, cells were rinsed twice with PBS and 70% of the initial cells attached to the dish forming a monolayer. Cells were maintained for 20 h in a serum-free medium supplemented with 0.2% (w/v) BSA and further stimulated with insulin (1, 10, and 100 nM, as indicated in each case) for 5 or 15 min. H-ras-transformed brown adipocyte cell lines were established by the means of cotransfection of brown adipocyte primary cells with transforming H-ras gene pMEXneo (Hraslys12) in cooperation with SV40-LTAg [16,17]. The best-characterized H-ras transformed cell line clone (MB1.3.19) was plated at 0.35 3 106 cells/60-mm dishes for glucose uptake determination and at 1 3 106 cells/100-mm dishes for plasma membrane and RNA
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isolation or immunoprecipitation of proteins. Cells were grown in 10% FCS–DMEM with antibiotics and G418 (250 mg/ml) for selection of neoresistance marker to 80% confluence. Then, cells were starved for 20 h in serum-free MEM and subsequently stimulated for 5 or 15 min with various doses of insulin as indicated under Results and in the figure legends. Control cells were maintained in MEM in the absence of insulin. To analyze the effect of PI 3-kinase inhibitors on glucose uptake, cells were pretreated for 15 min with wortmannin or LY294002, at the doses previously described [9]. Both inhibitors were initially dissolved in dimethyl sulfoxide, and in all experimental series control cells were treated with the corresponding volumes of dimethyl sulfoxide. Glucose uptake determination. Cells growing in 60-mm-diameter dishes were serum deprived for 20 h and washed three times with ice-cold Krebs–Ringer-phosphate buffer (KRP) (135 mM NaCl, 5.4 mM KCl, 1.4 mM CaCl2, 1.4 mM MgSO4, and 10 mM sodium pyrophosphate, pH 7.4). Then, cells were incubated with 1 ml KRP buffer with or without insulin for 10 min at 37°C, and 2-deoxy-D[1-3H]glucose (500 nCi/ml) was added to this solution to a final concentration of 0.1 mM, the incubation being continued for 5 min at 37°C. Cells were then washed three times with ice-cold KRP buffer and solubilized in 1 ml of 1% SDS, as described [20]. The radioactivity of a 200 ml aliquot was determined in a scintillation counter. Glucose transport was determined in triplicate dishes from five independent experiments. In parallel dishes, proteins were determined by the Bradford dye method [21], using the Bio-Rad reagent (Bio-Rad, Richmond, CA) and BSA as the standard. Results are means 6 SEM (n 5 5) and are expressed as disintegration per minute per microgram of protein. Statistical significance was tested with a one-way analysis of variance followed by the protected least-significant difference test, where differences were accepted as significant at the P , 0.05 level. Subcellular fractionation and Western blotting. For preparation of plasma membrane fraction, cells from five dishes of 100 mm diameter were pooled for each experimental condition after harvesting with ice-cold PBS. Then, cells were homogenized in an ice-cold buffer containing 20 mM Hepes, 250 mM sucrose, 2 mM EGTA, 0.2 mM PMSF, 1 mM leupeptin, pH 7.4. Nuclei and unbroken cells were removed by centrifugation at 2000g for 10 min. Plasma membrane fraction was prepared by centrifugation of the supernatant in a Sorvall RC 5B superspeed centrifuge SS34 rotor, at 18,000g for 1 h and 4°C with. The membrane pellets were resuspended in homogenization buffer before storage at 220°C, and proteins were determined. To verify the preparation of plasma membrane, phosphodiesterase-1 activity was assayed under all the conditions. For Western blot analysis of Glut4 and Glut1, plasma membrane proteins (100 and 50 mg, respectively) after SDS–PAGE were transferred to Immobilon membranes, blocked using 5% nonfat dried milk in 10 mM Tris–HCl and 150 mM NaCl, pH 7.5, and incubated overnight with anti-Glut1 antibody (1:1000) or anti-Glut4 antibody (1:500) in 0.05% Tween 20, 1% nonfat dried milk in the buffer indicated above. Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL-Plus) Western blotting protocol (Amersham). Western blot analysis was performed from three independent experiments. RNA extraction and Northern blot analysis. Cells from 100-mmdiameter dishes were washed twice with ice-cold PBS and lysed directly with RNAzol B following the protocol supplied by the manufacturer for total RNA isolation [19]. Total cellular RNA (20 mg) was submitted to Northern blot analysis, i.e., electrophoresed on 0.9% agarose gels containing 0.66 M formaldehyde, transferred to GeneScreen TM membranes using a VacuGene blotting apparatus (LKBPharmacia) and cross-linked to the membranes by UV light. Hybridization was in 0.25 mM NaHPO4, pH 7.2, 0.25 M NaCl, 100 mg/ml denatured salmon sperm DNA, 7% SDS, and 50% deionized formamide, containing denatured 32P-labeled cDNA (106 cpm/ml) for 40 h, at 42°C, as described [4]. Complementary DNA labeling was carried out with [a-32P]dCTP to a specific activity of 109 cpm/mg of DNA by
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FIG. 1. Glucose uptake in parental (BA) and H-ras-transformed (MB1.3.19) brown adipocytes: insulin dose dependency. Cells were serum deprived for 20 h prior to the stimulation for 15 min with insulin, at the doses indicated. Glucose uptake was measured over the last 5 min of culture as described under Materials and Methods. Results are means 6 SEM (n 5 5) from five independent experiments and are expressed as disintegration per minute per microgram of protein. Statistical significance was tested with a one-way analysis of variance followed by the protected least-significant different test, where differences between values in the presence of insulin vs control is represented by (w); wP , 0.01.
using multiprimer DNA-labeling system kit. For serial hybridization with different probes, the blots were stripped and rehybridized subsequently as needed in each case. Membranes were subjected to autoradiography and relative densities of the hybridization signals were determined by densitometric scanning of the autoradiograms in a laser densitometer (Molecular Dynamics, Sunnyvale, CA). Northern blot analysis was performed from three independent experiments. PI 3-kinase activity. It was measured by in vitro phosphorylation of phosphatidylinositol as previously described [9]. Cells from 100mm-diameter dishes were incubated in the absence or presence of insulin for 5 min as indicated in the figure legends. After washing with ice-cold PBS cells were solubilized in lysis buffer containing 10 mM Tris–HCl, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF , 100 mM Na3VO4, 1% Triton X-100, 10 mg/ml leupeptin, 10 mg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.6. Lysates were clarified by centrifugation at 15,000g for 10 min at 4°C and proteins (600 mg) were immunoprecipitated with the anti-ap85 subunit of PI 3-kinase mouse monoclonal antibody or with the anti-p21-ras monoclonal antibody (Y13-259). The immunoprecipitates were washed successively as previously described [9]. To each pellet were added 25 ml of 1 mg/ml of L-a-phosphatidylinositol/ L-a-phosphatidyl-L-serine sonicated in 25 mM Hepes (pH 7.5) and 1 mM EDTA. The PI 3-kinase reaction was started by the addition of 100 nM [g-32P]ATP (10 mCi) and 300 mM ATP in 25 ml of 25 mM Hepes (pH 7.4), 10 mM MgCl2, and 0.5 mM EGTA. After 15 min at room temperature the reaction was stopped by the addition of 500 ml of CHCl3:methanol (1:2) in a 1% concentration of HCl plus 125 ml of chloroform and 125 ml of HCl (10 mM). The samples were centrifuged, and the lower organic phase was removed and washed once with 480 ml of methanol:100 mM HCl plus 2 mM EDTA (1:1). The organic phase was extracted, dried in vacuo, and resuspended in chloroform. Samples were applied to a silica gel thin-layer chromatography (TLC) plate (Merck). TLC plates were developed in propanol-1 and acetic acid 2 N; 65:35 (v/v), dried, visualized by autoradiography, and quantitated by scanning laser densitometry. Transfection conditions. Parental brown adipocytes (100-mm-diameter dishes) were transiently transfected using the calcium phosphate precipitation technique, with 15 mg of a fusion plasmid containing either transforming Ras DNA cloned in an eukaryotic expression vector (pMEXneo H-raslys12) or pMEXneo carrier DNA. After 4 h of incubation, cells were shocked with 3 ml of 15% glycerol for 2 min, washed, and then incubated 20 h in serum-free medium, as previously described [16]. At the end of the culture time, cells were
incubated for 5–15 min either in the absence or in the presence of 100 nM insulin for determination of PI 3-kinase activity or glucose uptake, as described above.
RESULTS
H-ras-Transformed Brown Adipocytes Showed Insulin-Independent Glucose Uptake We have studied the glucose uptake in response to different doses of insulin in both parental and H-ras transformed brown adipocytes as shown in Fig. 1. Cells were serum deprived for 20 h in MEM culture (containing 5 mM glucose) prior to the stimulation for 15 min with insulin, at the doses indicated, and glucose uptake was measured over the last 5 min of the incubation, as described under Materials and Methods. As shown in Fig. 1, 1 nM insulin significantly induced glucose uptake by 3-fold compared to basal levels observed in parental brown adipocytes. No further increase in glucose uptake was observed at higher doses of the hormone. Transformed brown adipocytes clone (MB1.3.19) showed a 30-fold higher basal glucose uptake compared to parental cells. However, no further effect was observed in the presence of insulin, regardless the dose used (Fig. 1). Glut4 and Glut1 Gene Expression in Parental and H-ras-Transformed Brown Adipocytes A second issue addressed in this study is the direct comparison of the distribution of Glut4 and Glut1 mRNA species in both parental and transformed cells, either under growing conditions (10% FCS–DMEM) or in 20-h serum-deprived cells in MEM culture untreated or treated for 15 min with 100 nM insulin (Fig. 2). Parental cells expressed both the insulin-dependent glucose transporter Glut4 and the basal glucose trans-
INDUCTION OF GLUCOSE UPTAKE BY H-ras
FIG. 2. Glut4 and Glut1 mRNA expression in parental (BA) and H-ras-transformed (MB1.3.19) brown adipocytes. Cells growing in FCS were serum deprived for 20 h prior to the stimulation for 15 min with 100 nM insulin. Total RNA (20 mg) was submitted to Northern blot analysis and hybridized with labeled Glut4 and Glut1 cDNAs. A final hybridization with the 18S rRNA cDNA was performed for normalization. Autoradiograms from representative experiments out of three are shown. Arrows indicate the position of Glut4 and Glut1 mRNAs.
porter Glut1 mRNA, although the expression of Glut1 mRNA was very low compared to Glut4 mRNA expression. No differences in the expression of both Glut4 and Glut1 mRNA were found under serum or serum-deprivation culture conditions. Although the presence of insulin for 24 h dramatically induced Glut4 mRNA expression in fetal brown adipocytes [4], short-term insulin treatment (15 min) does not affect Glut4 mRNA accumulation compared to serum-deprived cells as shown in Fig. 2. Besides the high expression of the canonical 2.8-kb Glut4 mRNA in parental cells, a second mRNA isoform of approximately 2.3 kb was apparent. Whether this isoform is the result of the existence of alternative polyadenylation sites and its contribution to the Glut4 protein synthesis remains to be established. In H-ras-transformed brown adipocytes we could not detect Glut4 mRNA expression, at least in total RNA (20 mg) Northern blot. However, the expression of Glut1 mRNA was 10-fold higher compared to parental cells, regardless the culture conditions. This overexpression of Glut1 mRNA in transformed cells could account for the increased glucose uptake observed in these cells. In addition to the mRNA accumulation, we studied the effect of insulin treatment on Glut4 and Glut1 protein distribution in plasma membrane, by Western blot analysis. Either parental cells or H-ras-transformed cells were serum deprived for 20 h (untreated cells) prior to the stimulation for 15 min with 100 nM insulin (Fig. 3). Then, cells were harvested and homogenized and plasma membrane fractions were obtained as described under Materials and Methods. Plasma membrane proteins were submitted to Western blot analysis and immunoblotted for Glut4 and Glut1 detection. For detection of Glut4, 100 mg of plasma membrane protein was loaded. In parental cells, insulin
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stimulation increased by twofold the amount of Glut4 protein in the plasma membrane compared to untreated cells; meanwhile Glut4 protein was undetectable in plasma membrane from H-ras-transformed cells regardless of insulin stimulation. Glut1 protein was immunodetectable in 50 mg of plasma membrane proteins from both parental and H-ras-transformed cells. However, H-ras-transformed cells expressed 10fold higher levels of Glut1 than that observed in parental cells, in agreement with the data of Glut1 mRNA shown above (Fig. 3). The amount of Glut1 in plasma membrane remained unmodified by treatment with insulin in either H-ras-transformed cell or parental cells (Fig. 3). Transient Transfection of H-raslys12 Gene-Induced Glucose Uptake in Parental Cells in an InsulinIndependent Manner To figure out if the overstimulated glucose uptake found in H-ras-transformed cells was due to the overexpression of the transforming H-ras gene, we transiently transfected parental cells with 15 mg of a fusion plasmid containing transforming Ras DNA cloned in an eukaryotic expression vector (pMEXneo H-raslys12) or with pMEXneo carrier DNA [16]. After the transfection, cells were cultured for 20 h in a serum-free medium and incubated either in the absence or in the presence of insulin (100 nM) for 15 min, glucose uptake being determined over the last 5 min of the incubation, as described above (Fig. 4). In cells transfected with pMEXneo carrier DNA, 100 nM insulin produced a similar stimulation of glucose uptake (threefold increase) as described in Figure 1. Transfection with pMEXneo H-raslys12 construct produced a fivefold increase in the basal glucose uptake, this effect being significantly higher than that produced by 100 nM insulin in cells transfected with pMEXneo. However, insulin stimulation of H-ras transiently transfected
FIG. 3. Glut4 and Glut1 protein content in parental (BA) and H-ras-transformed (MB1.3.19) brown adipocytes. Cells growing in FCS were serum deprived for 20 h (C) prior to the stimulation for 15 min with 100 nM insulin. Cells from five dishes of 100 mm diameter were pooled for each experimental condition, and plasma membrane fractions were obtained as described under Materials and Methods. Plasma membrane proteins (100 mg for Glut4 and 50 mg for GLUT1) were submitted to SDS–PAGE, blotted to nylon membrane, immunodetected with the anti-Glut4 and anti-Glut1 antibodies, and developed with ECL. Representative experiments out of three are shown. Arrows indicate the position of Glut4 and Glut1 proteins.
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cells did not produce any further stimulation on glucose uptake over that produced by H-ras (Fig. 4). These results clearly indicate that active H-ras transient transfection induced glucose uptake in parental cells, suggesting that the similar effect found in established H-ras-transformed brown adipocyte cell line was not due to a clonal variation. Glucose Uptake in H-ras-Transformed Brown Adipocytes Is PI 3-Kinase Independent The fact that glucose uptake is dramatically increased in H-ras-transformed brown adipocytes prompted us to investigate whether this effect could be blocked by PI 3-kinase inhibitors, such as wortmannin and LY294002 [22,23], at the doses previously shown that totally inhibited PI 3-kinase in parental cells [9]. Cells were serum deprived for 20 h and then pretreated for 15 min with 20 nM wortmannin or 10mM LY294002 or the corresponding volume of dimethyl sulfoxide, and subsequently incubated either in the absence or in the presence of insulin (100 nM) for 15 min, glucose uptake being determined over the last 5 min of the incubation, as described above. As shown in Table 1, wortmannin or LY294002 does not affect the basal glucose uptake observed in parental cells, but significantly inhibits insulin-induced glucose uptake in parental cells. However, wortmannin or LY294002 does not modify the
FIG. 4. Effect of active H-ras transfection on basal and insulininduced glucose uptake in parental brown adipocytes. Fetal brown adipocytes were transiently transfected with transforming Ras DNA vector (pMEXneo H-raslys12) or with carrier DNA (pMEXneo), as described under Materials and Methods. After transfection, cells were serum deprived for 20 h prior to the stimulation for 15 min with 100 nM insulin. Glucose uptake was measured over the last 5 min of culture. Results are means 6 SEM (n 5 5) from five independent experiments and are expressed as disintegration per minute per microgram of protein. Statistical significance was tested with a oneway analysis of variance followed by the protected least-significant different test, where differences between values in the presence of insulin and/or H-ras vs control is represented by (w) or between H-ras transfected cells 6 insulin vs insulin alone is represented by (*); wP , 0.01, *p , 0.05.
TABLE 1 Effect of PI 3-Kinase Inhibitors on Basal and Insulin-Induced Glucose Uptake in either Parental (BA) and H-rasTransformed (MB1.3.19) Brown Adipocytes Treatment None Wortmannin LY294002 Insulin Insulin1wortmannin Insulin1LY294002
BA
MB1.3.19
2.0 6 0.3 1.9 6 0.2 1.8 6 0.3 6.7 6 0.8✯ 2.1 6 0.3* 2.3 6 0.4*
61 6 7 59 6 8 54 6 7 60 6 7 56 6 6 59 6 8
Note. Cells were serum deprived for 20 h and then pretreated for 15 min with 20 nM wortmannin, 10 mM LY294002, or the corresponding volume of dimethyl sulfoxide and subsequently incubated either in the absence or in the presence of insulin (100 nM) for 15 min, glucose uptake being determined over the last 5 min of the incubation, as described under Materials and Methods. Results are means 6 SEM (n 5 5) from five independent experiments and are expressed as disintegration per minute per microgram of protein. Statistical significance was tested with a one-way analysis of variance followed by the protected least-significant different test, where differences between values in the presence of insulin vs. control is represented by (✯) and differences between values in the presence of PI 3-kinase inhibitor vs those in its absence is represented by (*); ✯,* P , 0.01.
higher basal glucose uptake observed in H-ras-transformed cells. Furthermore, no effect of PI 3-kinase inhibitors was detected either in insulin-treated transformed brown adipocytes (Table 1). Finally, we decided to investigate whether the overexpression of H-raslys12 could result in an increased PI 3-kinase activity in either transformed or transiently transfected parental brown adipocytes (Fig. 5). Parental cells were transiently transfected under the same conditions as described above. After transfection, cells were serum starved and subsequently stimulated with 100 nM insulin for 5 min and lysed, and 600 mg of protein was immunoprecipitated with the anti-ap85 regulatory subunit of PI 3-kinase monoclonal antibody. The resulting immune complexes were assayed for PI 3-kinase activity as described under Materials and Methods. As shown in Fig. 5A, parental brown adipocytes displayed almost undetectable PI 3-kinase activity in anti-ap85 immunoprecipitates under control conditions. Upon treatment with 100 nM insulin, the basal PI 3-kinase activity increased by eightfold. However, transient transfection with H-raslys12 did not modify the PI 3-kinase activity observed under basal conditions. Furthermore, stimulation with insulin in H-raslys12-transfected cells did not significantly increase PI 3-kinase activity compared to insulin-treated cells. Transformed cells (clone MB1.3.19), overexpressing constitutively high levels of active p21-ras protein, were serum starved for 20 h and subsequently stimulated with 100 nM insulin for 5 min and lysed, and 600 mg of protein was immunoprecipitated with the anti-
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immunoprecipitation with the anti-ras monoclonal antibody (Y13-259) either under basal conditions or under insulin stimulation (Fig. 5C). DISCUSSION
FIG. 5. PI 3-kinase activity in active H-ras overexpressing parental (BA) and transformed (MB1.3.19) brown adipocytes. (A) Parental brown adipocytes (BA) were transiently transfected with transforming Ras DNA vector (pMEXneo H-raslys12) or with carrier DNA (pMEXneo), and after transfection, cells were serum deprived for 20 h. Then, cells were stimulated for 5 min with 100 nM insulin or received an equivalent volume of solvent. Whole cell lysates 600 mg of protein) were subjected to immunoprecipitation with anti-ap85 monoclonal antibody. The immune complexes were washed and immediately used for an in vitro PI 3-kinase assay as described under Materials and Methods. The conversion of PI to PI phosphate PIP) in the presence of [g-32P]ATP was analyzed by thin-layer chromatography. (B) H-ras-transformed brown adipocytes (MB1.3.19) were serum deprived for 20 h prior to the stimulation for 5 min with 100 nM insulin or received an equivalent volume of solvent. Whole cell lysates (600 mg of protein) were subjected to immunoprecipitation with anti-ap85 monoclonal antibody and assayed for PI 3-kinase activity. (C) H-ras-transformed brown adipocytes were treated as in B and lysates (600 mg of protein) were subjected to immunoprecipitation with anti-ras monoclonal antibody (Y13-259) and assayed for PI 3-kinase activity. Representative experiments of three are shown.
ap85 monoclonal antibody. Transformed cells showed a low ap85-associated PI 3-kinase activity under basal conditions; this activity increased in response to 100 nM insulin (threefold) but in a lower extent than that observed in parental cells (Fig. 5B). Although we did not find an increased PI 3-kinase activity in anti-ap85immunoprecipitates from H-ras-overexpressing cells, since it has been established that p110 subunit of PI 3-kinase is an effector of the activated Ras protein [24], we used another approach in order to confirm these data, assaying PI 3-kinase activity directly in anti-p21ras immunoprecipitates from H-ras-transformed cells. No PI 3-kinase activity was observed in H-ras-transformed brown adipocytes after lysis and subsequent
Fetal brown adipocytes (parental cells) expressed very high levels of Glut4 mRNA glucose transporter, the expression of Glut1 mRNA being restricted to a minimum. Under physiological conditions, insulin induced glucose uptake, doubling the amount of Glut4 protein located in plasma membrane. Glut1 was much less abundant than Glut4 in plasma membrane, making its contribution to transport negligible. Moreover, insulin increased PI 3-kinase activity, and the inhibition of PI 3-kinase activity resulted in an impaired insulin-induced glucose uptake, suggesting that insulin induced glucose uptake in a PI 3-kinase-dependent manner in fetal brown adipocytes. Our results are consistent with the well-known requirement of PI 3-kinase activity for the movement of glucose transporters to the cell membrane in both white and brown adipose tissues [25,26]. H-raslys12-transformed brown adipocytes substantially increased the expression of the ubiquitous glucose transporter Glut1 mRNA and the amount of Glut1 protein in plasma membrane, the expression of Glut4 (either mRNA and protein) being down-regulated. Possibly this high Glut1 gene expression is a compensatory response for lack of Glut4 expression in this cell line. Under these conditions, the glucose uptake turned to be dramatically increased compared to parental cells, in an insulin-independent manner. To determine if this effect was due to the overexpression of transforming H-ras gene or to the clonal variation, we have reproduced these results by transient transfection of parental cells with transforming Ras DNA cloned in an eukaryotic expression vector [16]. H-raslys12 gene transiently transfected to parental cells increased basal glucose uptake to a higher extent that insulin alone, this effect of Ras on glucose uptake being insulin independent as found in H-ras-transformed cell lines. Regarding Ras effect on glucose transport, although some authors have proposed that the Ras signaling pathway mimics insulin action on Glut4 translocation in 3T3-L1 cells although decreasing by 95% Glut4 protein [27], other results indicate that p21-ras is not involved in insulin-stimulated glucose transport [28,29]. In fact, constitutive active p21-ras or Raf-1 either microinjected or transfected into 3T3-L1 cells greatly stimulated the expression of Glut1 in the absence of insulin and increased 40-fold the basal glucose uptake [28,30]. Our results either in parental brown adipocytes transiently transfected with active Ras or in permanently overexpressing H-ras-transformed cells are in agreement with those later results. Recently, an enhanced
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glucose uptake was found in K-ras-transformed fibroblasts associated with an increased N-glycosylation of Glut1 [31]. Whether or not this mechanism could be operating in H-ras-transformed brown adipocytes remains to be established. Because a growing number of studies indicate that Ras and PI 3-kinase signaling pathways may converge at the cytoplasm [24 –32], although others suggest that both pathways are independent in the insulin signaling in 3T3-L1 adipocytes [33], we decided to investigate whether the increased glucose uptake produced by overexpression of H-ras could be related to an increased PI 3-kinase activity. However, inhibition of PI 3-kinase activity with wortmannin did not decrease the higher basal glucose uptake observed in H-ras-transformed cells. Moreover, our results clearly demonstrate that ap85-associated-PI 3-kinase activity is not induced by H-ras overexpression in either transiently or permanently transfected cells. Furthermore, PI 3-kinase activity is not increased in anti-p21-ras immunoprecipitates from H-raslys12-transformed cells, a system where we have previously shown that the content of Ras.GTP form was constitutively very high [16]. Thus, H-ras induced glucose uptake in a PI 3-kinaseindependent manner. However, we cannot exclude the possibility that other Ras effectors, such as Ral GDP dissociation stimulator or protein kinase Cz, could be involved in the Ras-increased glucose uptake. Moreover, insulin stimulates ap85-associated PI 3-kinase activity in H-ras-overexpressing cells, glucose transport being no further stimulated by addition of the hormone. In conclusion, our results show that Ras overexpression strongly induces glucose uptake in brown adipocytes in an insulin-independent manner, this induction not involving PI 3-kinase activation. We are grateful for valuable reagents provided by Dr. Dr. J. Downward and P. Rodriguez-Viciana (Imperial Cancer Research Foundation, London, UK) and Dr. E. Santos (National Institutes of Health, Bethesda, MD). P.N. was the recipient of a fellowship from the Ministerio de Educacion y Cultura. Grant SAF96/0115 from the Comision Interministerial de Ciencia y Tecnologia, Spain, supported this work
REFERENCES Ailhaud, G., Grimaldi, P., and Negrel, R. (1992). Cellular and molecular aspects of adipose tissue development. Annu. Rev. Nutr. 12, 207–233. 2. Teruel, T., Valverde, A. M., Benito, M., and Lorenzo, M. (1995). Differentiation of rat brown adipocytes during late fetal development: role of insulin-like growth factor I. Biochem. J. 310, 771–776. 3. Lorenzo, M., Roncero, C., Fabregat, I., and Benito, M. (1988). Hormonal regulation of rat fetal lipogenesis in brown adipocyte primary cultures. Biochem. J. 251, 617– 620. 4. Teruel, T., Valverde, A. M., Benito, M., and Lorenzo, M. (1996). Insulin-like growth factor I and insulin induce adipogenic-re-
lated gene expression in fetal brown adipocyte primary cultures. Biochem. J. 319, 627– 632. 5.
Stephens, J. M., and Pilch, P. F. (1995). The metabolic regulation and vesicular transport of GLUT4, the major insulin-responsive glucose transporter. Endocr. Rev. 16, 529 –546.
6.
Santalucı´a, T., Camps, M., Castello, A., Mun˜oz, P., Nuel, A., Testar, X., Palacin, M., and Zorzano, A. (1992). Developmental regulation of GLUT1 (Erithroid/Hep G2) and GLUT4 (Muscle/ Fat) glucose transporter expression in rat heart, skeletal muscle, and brown adipose tissue. Endocrinology 130, 837– 846.
7.
Martin, S., Haruta, T., Morris, A. J., Klippel, A., Williams, L. T., and Olefsky, J. M. (1996). Activated phosphatidylinositol 3-kinase is sufficient to mediated actin rearrangement and GLUT4 translocation in 3T3-L1 adipocytes. J. Biol. Chem. 271, 17605– 17608.
8.
Guilherme, A., Klarlund, J. K, Krystal, G., and Czech, M. P. (1996). Regulation of phosphatidylinositol 3,4,5-trisphosphate 59-phosphatase activity by insulin. J. Biol. Chem. 271, 29533– 29536.
9.
Valverde, A. M., Lorenzo, M., Navarro, P., and Benito, M. (1997). Phosphatidylinositol 3-kinase is a requirement for insulin-like growth factor I-induced differentiation, but not for mitogenesis, in fetal brown adipocytes. Mol. Endocrinol. 11, 595– 607.
10.
Haring, H. U. (1991). The insulin receptor signaling mechanism and contribution to the pathogenesis of insulin resistance. Diabetologia 34, 848 – 861.
11.
Ricort, J. M., Tanti, J. F., Van Obberghen, E., and Le Marchand-Brustel, Y. (1995) Alterations in insulin signaling pathway induced by prolonged insulin treatment of 3T3-L1. Diabetologia 38, 1148 –1156.
12.
Feinstein, R., Kanety, H., Papa, M. Z., Lunenfeld, B., and Karasik, A. (1993). Tumor necrosis alpha suppresses insulininduced tyrosine phosphorylation of insulin receptor and its substrates. J. Biol. Chem. 268, 26055–26058.
13.
Hotamisligil, G. S., Murray, D. L., Choy, L. N., and Spiegelman, B. M. (1994). Tumor necrosis alpha inhibits signaling from the insulin receptor. Proc. Natl. Acad. Sci. USA 91, 4854 – 4858.
14.
Hamann, A., Benecke, H., Le Marchand-Brustel, Y., Susulic, V. S., Lowell, B. B., and Flier, J. S. (1995). Characterization of insulin resistance and NIDDM in transgenic mice with reduced brown fat. Diabetes 44, 1266 –1273.
15.
Hamann, A., Flier, J. S., and Lowell, B. B. (1995). Decreased brown fat markedly enhances susceptibility to diet-induced obesity, diabetes and hyperlipemia. Endocrinology 137, 22–29.
16.
Lorenzo, M,, Valverde, A. M., Teruel, T., Alvarez, A., and Benito, M. (1996). p21-ras induced differentiation-related gene expression in fetal brown adipocyte primary cells and cell lines. Cell Growth Differ. 7, 1251–1259.
17.
Valverde, A. M, Lorenzo, M., Teruel, T., and Benito, M. (1997). Alterations in the insulin signaling pathway induced by immortalization and H-ras transformation of brown adipocytes. Endocrinology 138, 3195–3206.
18.
Birnbaum, M. J. (1989). Identification of a novel gene encoding an insulin-responsive glucose transporter protein. Cell 57, 305– 315.
19.
Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thyocyanate–phenol– chloroform extraction. Anal. Biochem. 162, 156 –159.
20.
Rice, K. M., and Garner C. W. (1994). Correlation of the insulin receptor substrate-1 with insulin-responsive deoxyglucose transport in 3T3-L1 adipocytes. Biochem. Biophys. Res. Commun. 198, 523–530.
1.
INDUCTION OF GLUCOSE UPTAKE BY H-ras 21.
22.
23.
24.
25.
26.
27.
Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248 –254. Okada, T., Kawano, Y., Sakakibara, Y., Hazeki, O., and Ui, J. (1994). Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and anti-lipolysis in rat adipocytes. J. Biol. Chem. 269, 3568 –3573. Vlahos, C. J., Matter, W. F., Hui, K. Y., and Brown, R. F. (1994). A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269, 5241–5248. Rodrigez-Viciana, P., Warne, P. H., Vanhaesebroeeck, B., Waterfield, M. D., and Downward, J. (1996). Phosphatidylinositol3-OH kinase as a direct target of Ras. EMBO J. 15, 2442–2451. Schimizu, Y., and Schimazu, T. (1994). Effects of wortmannin on increased glucose transport by insulin and norepinephrine in primary cultures of brown adipocytes. Biochem. Biophys. Res Commun. 202, 660 – 665. Omatsu-Kanbe, M., and Kitasato, H. (1992). Insulin and noradrenaline independently stimulate the translocation of glucose transporters from intracellular stores to the plasma membrane in mouse brown adipocytes. FEBS Lett. 314, 246 –250. Kozma, L., Baltenperger, K., Klarlund, J., Porras, A., Santos, E., and Czech, M. P. (1993). The Ras signaling pathway mimics insulin action on glucose transporter translocation. Proc. Natl. Acad. Sci. USA 90, 4460 – 4464.
Received December 15, 1997 Revised version received April 30, 1998
281
28.
Hausdorff, S. F., Frangioni, J. V., and Birnbaum, M. J. (1994). Role of p21ras in insulin-stimulated glucose transport in 3T3-L1 Adipocytes. J. Biol. Chem. 269, 21391–21394.
29.
Van den Berghe, N., Ouwens, D. M., Maaseen, J. A., van Mackelenbergh, M. G. H., Sips, H. C. M., and Krans, H. M. J. (1994). Activation of the Ras/mitogen-activated protein kinase signaling pathway alone is not sufficient to induce glucose uptake in 3T3-L1 adipocytes. Mol. Cell. Biol. 14, 2372– 2377.
30.
Fingar, D. C., and Birnbaum, M. J. (1994). A role for Raf-1 signaling pathways mediating insulin-stimulated glucose transport. J. Biol. Chem. 269, 10127–10132.
31.
Onetti, R., Baulida, J., and Bassols, A. (1997). Increased glucose transport in ras-transformed fibroblasts: A possible role for N-glycosylation of GLUT1. FEBS Lett. 407, 267–270.
32.
Hu, Q., Klippel, A., Muslin, A. J, Fantl, W. J., and Williams, L. T. (1995). Ras-dependent induction of cellular responses by constitutively active phosphatidylinositol-3 kinase. Science 268, 100 –102.
33.
Gnudi, L., Frever, E. U., Houseknecht, K. L., Erhardt, P., and Kahn. B. B. (1997). Adenovirus mediated gene transfer of dominant negative Rasasn17 in 3T3-L1 adipocytes does not alter insulin-stimulated phosphatidylinositol 3-kinase activity or glucose transport. Mol. Endocrinol. 11, 67–76.