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Endocrinology Vol. 138, No. 8 3195-3206
Copyright © 1997 by The Endocrine Society

ARTICLES

Alterations in the Insulin Signaling Pathway Induced by Immortalization and H-ras Transformation of Brown Adipocytes1

Angela M. Valverde, Margarita Lorenzo, Teresa Teruel2 and Manuel Benito

Department of Biochemistry and Molecular Biology, Institute of Biochemistry, and Faculty of Pharmacy, University Complutense, 28040 Madrid, Spain

Address all correspondence and requests for reprints to: Dr. Manuel Benito, Department of Biochemistry and Molecular Biology, Institute of Biochemistry, and Faculty of Pharmacy, University Complutense, 28040 Madrid, Spain.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In fetal brown adipocyte primary cultures, insulin rapidly (at 5 min) induced tyrosine phosphorylation of the insulin receptor ß-subunit; this effect was maximal at physiological concentrations (1 nM). Insulin also stimulated insulin receptor substrate-1 tyrosine phosphorylation and subsequently activated phosphatidylinositol 3-kinase. Moreover, a 3-fold increase in the Ras.GTP active form and a 6-fold increase in Raf-1 kinase activity were induced after insulin stimulation. An immortalized brown adipocyte cell line (by permanent simian virus 40 large T antigen and pMEXneo cotransfection) showed a reduced maximal responsiveness to insulin in the same range of insulin concentrations studied (1–100 nM). Transformed brown adipocyte cell line (by permanent simian virus 40 large T antigen and pMEXneo H-raslys12 cotransfection) developed insulin resistance upstream from Ras, showing an impairment in the insulin receptor autophosphorylation, and in insulin receptor substrate-1 tyrosine phosphorylation and its association with phosphatidylinositol 3-kinase upon treatment with 1 nM insulin, although insulin receptor number and affinity (Kd) remained unaltered. This lack of effect was ameliorated upon treatment with higher insulin concentrations, in a dose-dependent manner. However, downstream from Ras, events such as formation of the Ras.GTP active form, and Raf-1 kinase and 12-O-tetradecanoylphorbol-13-acetate response element-chloramphenicol transferase (transiently transfected) activities were overstimulated, compared with those in primary and immortalized cells, in an insulin-independent manner. Wheat-germ lectin-purified receptors from H-raslys12-transformed brown adipocytes showed a marked phosphorylation in the basal state, which was suppressed by serine-threonine phosphatase pretreatment. Moreover, alkaline phosphatase pretreatment restored the tyrosine kinase activity of the receptor in response to insulin. We conclude that the decreased tyrosine autophosphorylation rate of the insulin receptor from H-raslys12-transformed brown adipocytes is a consequence of its basal serine/threonine phosphorylation, resulting in severe insulin resistance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
INSULIN resistance is a characteristic clinical feature of a number of disease states, such as obesity and noninsulin-dependent diabetes mellitus, and is associated with hyperglycemia, hyperinsulinemia, and hyperlipemia (reviewed in 1 . Several states of insulin resistance are related to receptor and postreceptor defects, such as naturally occurring mutations in the primary sequence of the insulin receptor that result in decreased insulin binding (2), a decrease in the number of insulin receptor molecules expressed on the plasma membrane of the target cells (3, 4), or alterations in insulin postreceptor signaling. In fact, several steps linking the insulin receptor with its final nuclear actions have been found to be defective in the liver and muscle of streptozotocin-induced diabetic animals (5, 6, 7). In addition, insulin receptor substrate-1 (IRS-1) phosphorylation and phosphatidylinositol (PI) 3-kinase activity were reduced in glucocorticoid-induced insulin resistance in the liver and in hepatoma cells (8, 9). On the other hand, alterations in the insulin signaling at the level of IRS-1 tyrosine phosphorylation, PI 3-kinase and mitogen-activated protein kinase activities have been described after prolonged insulin treatment of 3T3-L1 adipocytes (10). This chronic insulin resistance is mediated by a proteolytic fragment of the insulin receptor that inhibits the autophosphorylation of the receptor (11). Moreover, Tumor necrosis factor {alpha} also induced insulin resistance in cultured adipocytes by inhibiting insulin receptor autophosphorylation and IRS-1 phosphorylation (12, 13). All these data suggest that alterations in the early steps of the insulin signaling pathway may play an important role in several states of insulin resistance.

Brown adipose tissue is the main tissue involved in nonshivering thermogenesis in neonates, which is responsible for the heat production associated with the expression of mitocondrial uncoupling protein (14, 15). Brown adipose tissue differentiation also encompasses an adipogenic program related to lipid synthesis, and its accumulation results in a multilocular fat droplet phenotype (15, 16). In addition, it is well known that insulin is the main signal involved in brown adipocyte lipogenesis through its induction of genetic expression of the lipogenic enzymes (16, 17, 18, 19). However, the molecular mechanisms by which insulin actions are initiated in this tissue have not yet been described. Moreover, a deficiency of brown adipose tissue has been shown to result in the development of glucose intolerance and severe insulin resistance (20, 21). Recently, several stable and permanent brown adipocyte cell lines have been obtained by cotransfection with immortalizing simian virus 40 large T antigen (SV40-LTAg) and pMEXneo or mutated H-ras gene (pMEXneo H-raslys12) (22). These cell lines maintained specific properties of brown adipocytes, such as expression of the tissue-specific marker uncoupling protein. In a previous work we have characterized two series of clonal lines: MB4.9.X (achieved by cotransfection with constructs of SV40-LTAg and pMEXneo) and MB1.3.X (achieved by cotransfection with constructs of SV40-LTAg and pMEXneo H-raslys12), which displayed immortalized and transformed phenotypes, respectively (23). Accordingly, in the present paper we have examined the effect of brown adipocyte immortalization (in the representative clone MB4.9.2) or H-ras transformation (in the representative clone MB1.3.19) on the very early events upstream and downstream from Ras in the insulin signaling cascade compared with those events in primary cells. Our results show that the maximal responsiveness to insulin is reduced in immortalized brown adipocytes compared with primary cells. Moreover, mutated H-ras-transformed brown adipocytes developed a marked insulin resistance upstream from Ras as a consequence of a serine-threonine phosphorylation of the receptor; meanwhile, downstream from Ras, the signaling cascade was overstimulated regardless of the presence of the hormone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
FCS and culture medium were obtained from Imperial Laboratories (Hampshire, UK). Insulin and antimouse IgG-agarose were purchased from Sigma Chemical Co. (St. Louis, MO). Protein A-agarose was purchased from Boehringer Mannheim (Mannheim, Germany). The antiinsulin receptor monoclonal antibody (Ab-3) and the anti-ras monoclonal antibody (Y13–259) were purchased from Oncogene Science (Uniondale, NY). Py72 monoclonal anti-Tyr(P) antibody and {alpha}p85 mouse monoclonal antibody were gifts from Dr. E. Rozengurt and J. Sinnet-Smith and Drs. J. Downward and P. Rodriguez-Viciana, respectively (Imperial Cancer Research Foundation, London, UK). For IRS-1 immunoprecipitations, a rabbit polyclonal antibody was the gift of Dr. R. Kahn (Joslin Diabetes Center, Boston, MA), and another anti-IRS-1 rabbit polyclonal antibody was purchased from Upstate Biotechnology (Lake Placid, NY). The 4G10 anti-Tyr(P) monoclonal antibody was also obtained from Upstate Biotechnology. For Raf-1 immunoprecipitations, a rabbit polyclonal anti Raf-1 antiserum and the recombinant catalytically inactive MEK were gifts from Dr. S. Gutkind (NIH, Bethesda, MD). The antibodies to phosphoserine and phosphothreonine were purchased from Zymed (San Francisco, CA). Wheat-germ lectin coupled to Sepharose (WGA) was obtained from Pharmacia LKB (Uppsala, Sweden). Immobilized alkaline phosphatase was prepared by reacting 2 ml Affi-Gel 10 from Bio-Rad (Richmond, CA) with 5000 U alkaline phosphatase from Sigma Chemical Co. [{gamma}-32P]ATP (3000 Ci/mmol), [14C]chloramphenicol (54 mCi/mmol), and [125I]insulin (80 µCi/µg) were obtained from Amersham (Aylesbury, UK). The calcium phosphate mammalian transfection kit was purchased from Stratagene (La Jolla, CA). All other reagents used were of the purest grade available.

Cell culture
Brown adipocyte primary cells were obtained from interscapular brown adipose tissue of 20-day-old Wistar rat fetuses and isolated by collagenase dispersion as previously described (17). Cells were plated at 3.5 x 106 cells/100-mm tissue culture plate in MEM supplemented with 10% FCS to allow cell attachment to the plastic surface of the plates. After 4–6 h of culture at 37 C, cells were rinsed twice with PBS, and 80% of the initial cells were attached. Cells were maintained in 10% FCS-MEM until 80% confluence was reached. At this time cells were cultured for an additional 20 h in serum-free medium and subsequently stimulated for 5 min with various doses of insulin or in the absence of insulin as a control for cellular quiescence.

Brown adipocyte cell lines were established by means of cotransfection of brown adipocyte primary cells with constitutive constructs containing a neomycin resistance marker (pMEXneo) along with immortalizing SV40 LTAg (clones MB4.9.2, MB4.9.3, and MB4.9.6) or with transforming H-ras gene (pMEXneo H-raslys12) in cooperation with SV40-LTAg (clones MB1.3.19, MB1.3.12, and MB1.3.25) (22, 23). All of the cell lines were plated at 1 x 106 cells/100-mm plates and grown in 10% FCS-DMEM with antibiotics and G418 (250 µg/ml) for selection of the neo resistance marker to 80% confluence. Then cells were starved for 20 h in serum-free DMEM and subsequently stimulated for 5 min with various doses of insulin as indicated in Results and in the figure legends. Control cells were maintained in DMEM in the absence of insulin.

[125I]Insulin binding
Cells cultured for 20 h in a serum-free medium were incubated for 3 h at 20 C with 0.03 nM [125I]insulin in 1 ml binding buffer containing 25 mM HEPES-PBS and 1 mg/ml BSA in the absence or presence of graded concentrations of unlabeled insulin. Triplicate dishes were used for each data point. At the end of incubation, monolayers were rinsed with either ice-cold PBS-BSA or ice-cold 0.3 M sodium acetate, pH 4.5 (containing 0.15 M NaCl), then rinsed twice with PBS-BSA and dissolved in 0.1 N NaOH-1% SDS-2% Na2CO3, as previously described (24). Radioactivity was counted in a Packard {gamma}-counter (Downers Grove, IL). The radioactivity associated with the cells submitted to an acid wash representing internalized [125I]ligand was negligible. Total binding in the absence of competing ligand was approximately 5% of the radioactivity added in all cell types studied. Nonspecific binding was defined as the radioactivity that remained bound in the presence of 1000 nM unlabeled ligand and represented approximately 10% of the total binding. Bound vs. free plots, molecular masses, and number of binding sites per cell (calculated from the Scatchard plots) were derived from three separated experiments.

Immunoprecipitations
After 20 h of serum starvation, primary fetal brown adipocytes or brown adipocyte cell lines were treated with various doses of insulin for 5 min as indicated and lysed at 4 C in 1 ml of a solution containing 10 mM Tris-HCl, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 100 µM Na3VO4, 1% Triton X-100, and 1 mM phenylmethylsulfonylfluoride, pH 7.6 (lysis buffer). Lysates were clarified by centrifugation at 15,000 x g for 10 min, and the supernatants were transferred to a fresh tube. After determination of protein content, equal amount of protein (600 µg for primary brown adipocytes and 600 µg to 3 mg for brown adipocyte cell lines) were immunoprecipitated at 4 C with the monoclonal antibodies antiinsulin receptor (Ab-3), anti-Tyr(P) (Py72), and anti-{alpha}p85 or with a polyclonal antibody against a C-terminal peptide of IRS-1. The immune complexes were collected on antimouse IgG-agarose beads or, in the case of IRS-1 antibody, on protein A-agarose beads. Immunoprecipitates were washed three times with lysis buffer, extracted for 10 min at 95 C in 2 x SDS-PAGE sample buffer (200 mM Tris-HCl, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, and 10% glycerol, pH 6.8), and analyzed by SDS-PAGE as described in Results and in the figure legends.

Western blotting
After SDS-PAGE, proteins were transferred to Immobilon membranes. Membranes were blocked using 5% nonfat dried milk in 10 mM Tris-HCl and 150 mM NaCl, pH 7.5, and incubated overnight with several antibodies, as indicated, in 0.05% Tween-20, 1% nonfat dried milk in 10 mM Tris-HCl, and 150 mM NaCl, pH 7.5. Immunoreactive bands were visualized using the ECL Western blotting protocol (Amersham, Arlington Heights, IL).

Insulin receptor autophosphorylation assay
Insulin receptor autophosphorylation was measured as previously described (25). The anti-Tyr(P) or anti-insulin receptor immune complexes were incubated in 20 µl buffer containing 20 mM HEPES, 3 mM MnCl2, 10 mM MgCl2, and 20 µCi [{gamma}-32P]ATP (in a final concentration of 5 µM) for 15 min at room temperature. The complexes were washed twice with cold PBS and then resuspended in 2 x SDS-PAGE sample buffer and analyzed by SDS-PAGE. The separated proteins were dried in the gel, and the incorporation of [32P]phosphate into protein was visualized by autoradiography and quantitated by scanning laser densitometry (Molecular Dynamics densitometer, Sunnyvale, CA).

PI 3-kinase activity
PI 3-kinase activity was measured by in vitro phosphorylation of PI as described (26). Fetal brown adipocytes and brown adipocyte cell lines were incubated in the absence or presence of insulin as indicated in the figure legends. After washing with ice-cold PBS, cells were solubilized in lysis buffer containing 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonylfluoride. Lysates were clarified by centrifugation at 15,000 x g for 10 min at 4 C, and proteins were immunoprecipitated with anti-IRS-1 polyclonal antibody. The immunoprecipitates were washed successively in PBS containing 1% Triton X-100 and 100 µM Na3VO4 (twice); in 100 mM Tris (pH 7.5) containing 0.5 M LiCl, 1 mM EDTA, and 100 µM Na3VO4 (twice); and in 25 mM Tris (pH 7.5) containing 100 mM NaCl and 1 mM EDTA (twice). To each pellet were added 25 µl 1 mg/ml L-{alpha}-PI/L-{alpha}-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 [{gamma}-32P]ATP (10 µCi) and 300 µM ATP in 25 µl 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 µl CHCl3-methanol (1:2) in a 1% concentration of HCl plus 125 µl chloroform and 125 µl HCl (10 mM). The samples were centrifuged, and the lower organic phase was removed and washed once with 480 µl 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 TLC plate (Merck, Rahway, NJ). TLC plates were developed in propanol-1 and acetic acid (2 N; 65:35, vol/vol), dried, visualized by autoradiography, and quantitated by scanning laser densitometry.

Raf kinase activity
The activation of Raf-1 was measured by the phosphorylation of recombinant, catalytically inactive MEK in immunoprecipitates of Raf-1 antibodies, as described (27). After stimulation, cells were washed twice with ice-cold PBS and lysed in 500 µl lysis buffer containing 20 mM HEPES (pH 7.5), 10 mM EGTA, 2.5 mM MgCl2, 40 mM ß-glycerol phosphate, 1% Nonidet P-40, 2 mM Na3VO4, 1 mM phenylmethylsulfonylfluoride, and 1 mM dithiothreitol. Proteins from equal quantities of cell lysates were immunoprecipitated with 10 µl Raf-1 polyclonal antiserum for 2 h, and protein A-agarose beads were added for the last 45 min. Immune complexes were washed three times with ice-cold PBS containing 1% Nonidet P-40; once with 100 mM Tris (pH 7.4) and 0.5 M LiCl; and once in kinase buffer containing 12.5 mM MOPS (3-[N-morpholino]propane-sulfonic acid) (pH 7.5), 12.5 mM ß-glycerol phosphate, 7.5 mM MgCl2, 10 mM MnCl2, 0.5 mM EGTA, 0.5 mM NaF, and 0.5 mM Na3VO4. The kinase reaction was performed in 30 µl kinase buffer supplemented with 10 µCi of [{gamma}-32P]ATP, 50 µM ATP, and 1 µg recombinant, catalytically inactive MEK for 25 min at 30 C and was terminated by the addition of 4 x SDS-PAGE sample buffer followed by boiling at 95 C for 5 min. Samples were resolved in 10% SDS-PAGE, and gels were dried and subjected to autoradiography.

GTP/GDP binding to p21-ras
Serum-deprived cells were incubated for 4 h in phosphate-free DMEM and then labeled overnight in the same medium with 100 µCi/ml [32P]orthophosphate. After stimulation, cells were washed with ice-cold PBS and then lysed and scraped in 1 ml of a lysis buffer containing 50 mM Tris-HCl, 100 mM NaCl, 1 mM EGTA, 5 mM MgCl2, 1 mM dithiothreitol, 1% Triton X-114, 1 mM phenylmethylsulfonylfluoride, and 10 µg/ml leupeptin/aprotinin, pH 7.5. Mature p21-ras was separated from nonprocessed p21-ras by a Triton X-114 phase split as described (28). p21-ras was immunoprecipitated from the detergent phase with the monoclonal antibody Y13–259. Bound nucleotides were eluted from immunoprecipitates and analyzed by ascending TLC. Radioactivity corresponding to GDP and GTP was quantitated by phosphorimaging (Bio-Rad Laboratories, Richmond, CA). The percentage of GTP in relation to the total nucleotide pool (GTP plus GDP) was calculated, taking into account the different phosphorus contents of GTP and GDP, with the formula, % GTP = GTP x 100/GDP x 1.5 + GTP.

Transient transfections
Each 100-mm culture dish was transiently transfected using the calcium phosphate precipitation technique with 10 µg of a fusion plasmid containing the chloramphenicol acetyltransferase (CAT) reporter gene under the control of five activating protein-1 (AP-1) binding sites arranged in tandem (29) or with 20 µg transforming Ras DNA cloned in an eukaryotic expression vector (pMEXneo H-raslys12) (22). After 4 h of incubation, cells were shocked with 3 ml 15% glycerol for 2 min, washed, and then incubated 24 h in serum-free medium, as previously described (23). Cells were then lysed as described above for receptor purification or stimulated with various doses of insulin for an additional 24 h. Finally, cells were washed with cold PBS, CAT activity was determined by incubating 50 µl cell extracts with 0.25 µCi [14C]chloramphenicol-0.5 mM acetyl coenzyme A in 0.25 M Tris (pH 7.8) at 37 C for 12 h, and then samples were submitted to ascending TLC and subjected to autoradiography.

Purification and phosphorylation of the insulin receptor
Serum-deprived cells were solubilized in lysis buffer as described above. Solubilized receptors were added to 1 ml WGA-Sepharose and rotated end over end for 2 h at room temperature. After extensive washing with a buffer containing 50 mM Tris (pH 7.4), 0.05% Triton X-100, 100 mM NaCl, 2.5 mM KCl, and 1 mM CaCl2, receptors were eluted with 300 µl of the same buffer supplemented with 0.3 M N-acetylglucosamine.

WGA-purified receptors (10 µg protein) were incubated for 1 h at 4 C with 500 U immobilized alkaline phosphatase to remove phosphate before performing phosphorylation reactions. The supernatant was separated from the phosphatase beads by centrifugation and subsequently used in phosphorylation assays as previously described (30). Parallel samples were prepared by incubation of the purified receptors with alkaline phosphatase, which was inactivated by boiling for 30 min.

Portions containing 10 µg WGA-purified receptors (pretreated with active or inactive alkaline phosphatase) were preincubated for 1 h at room temperature with 100 nM insulin. The phosphorylation reaction was performed as indicated. After 15 min on ice, phosphorylation was stopped, and receptors were immunoprecipitated with the antiinsulin receptor antibody or the py72 anti-Tyr(P) antibody and separated by SDS-PAGE. The separated proteins were dried in the gel, and the incorporation of [32P]phosphate into protein was visualized by autoradiography and quantitated by scanning laser densitometry (Molecular Dynamics densitometer).

Protein determination
Protein determination was performed by the Bradford dye method (31), using the Bio-Rad reagent (Bio-Rad) and BSA as the standard.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of insulin on insulin receptor tyrosine autophosphorylation in primary fetal brown adipocytes and in immortalized and H-raslys12-transformed brown adipocyte cell lines
Isolated primary fetal brown adipocytes were grown in 10% FCS until 80% confluence was reached and then serum starved for 20 h. Cells were then incubated in the absence or presence of various concentrations of insulin for 5 min at 37 C, after which whole cell lysates (600 µg protein) were subjected to immunoprecipitation with the Py72 anti-Tyr(P) antibody. The immunoprecipitates were assayed for protein kinase activity as described in Materials and Methods. The presence of 1 nM insulin in the culture medium caused a marked increase in the tyrosine phosphorylation of a band of approximately 95 kDa, corresponding to the Mr of the ß-subunit of the insulin receptor, compared with that in nontreated cells (Fig. 1AGo). The level of tyrosine phosphorylation of the 95-kDa band was maximal at 1 nM insulin and did not change significantly when higher concentrations of insulin were added (10 or 100 nM; Fig. 1BGo). To investigate the pattern of tyrosine-phosphorylated proteins in immortalized or H-raslys12-transformed brown adipocytes, clones MB4.9.2 (representative of the MB4.9.X cell lines) and MB1.3.19 (representative of the MB1.3.X cell lines) were grown in the presence of 10% FCS until 80% confluence was reached. Then, cells were cultured for an additional 20 h in a serum-free medium and subsequently stimulated with various doses of insulin for 5 min at 37 C, after which whole cell lysates (600 µg protein) were assayed as described above. As shown in Fig. 1Go (A and B), immortalized brown adipocytes (clone MB4.9.2) reached maximal insulin receptor ß-chain tyrosine autophosphorylation at 1 nM insulin, although these cells showed a reduced maximal responsiveness to insulin compared to that of primary cells. In addition, H-raslys12-transformed brown adipocytes (clone MB1.3.19) lacked the insulin-induced tyrosine phosphorylation of the insulin receptor ß-chain at 1 nM insulin, and only a minimal amount of the 95-kDa phosphotyrosine band was detectable in these cells upon treatment with 100 nM insulin.



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  		Figure 1. Tyrosine phosphorylation induced by insulin in primary, immortalized, and H-ras-transformed brown adipocytes. A, Quiescent primary fetal brown adipocytes (BAT), immortalized brown adipocytes (clone MB4.9.2), and H-raslys12-transformed brown adipocytes (clone MB1.3.19) were incubated for 5 min at 37 C with various doses of insulin. Control cells (C) received an equivalent volume of solvent. Cells were then lysed, and immunoprecipitates (600 µg protein from both primary brown adipocytes and cell lines) were prepared using the monoclonal anti-Tyr(P) antibody Py72 and assayed for protein kinase activity. The proteins phosphorylated in the immune complexes were separated by SDS-PAGE, and gels were dried and subjected to autoradiography. The position of the ß-chain of the insulin receptor is indicated by an arrowhead. The positions of mol wt markers (x10-3) are shown on the left. A representative experiment is shown. B, The corresponding autoradiograms were quantitated by scanning densitometry. Results are expressed as arbitrary units of insulin receptor tyrosine autophosphorylation activity and are the mean ± SEM of three independent experiments.

 
Alternatively, autophosphorylation of the insulin receptor was studied by direct immunoprecipitation with the antiinsulin receptor ß-subunit antibody (Fig. 2Go). Thus, primary fetal brown adipocytes and clones MB4.9.2 and MB1.3.19 were stimulated with various doses of insulin for 5 min at 37 C, extracted as described in Materials and Methods and immunoprecipitated with the antiinsulin receptor monoclonal antibody, and then the resulting immune complexes were analyzed by Western blotting with the 4G10 anti-Tyr(P) antibody. As shown in Fig. 2Go, constitutive tyrosine phosphorylation of the ß-chain was not observed in either primary cells or immortalized or H-raslys12-transformed cell lines. However, 5-fold more protein loading (3 mg) than that needed for primary cells was required for immunodetection of the tyrosine-phosphorylated insulin receptor in response to insulin in both cell lines. Thus, immortalized cells reached the maximal insulin receptor ß-subunit tyrosine autophosphorylation at 1 nM, and this tyrosine phosphorylation in response to insulin was lower than that found in primary cells. These results were consistent with those of in the in vitro kinase assay (Fig. 1Go). However, transformed cells suppressed the insulin receptor ß-subunit tyrosine phosphorylation in response to 1 nM insulin; this lack of response was slightly ameliorated in response to higher insulin concentrations in a dose-dependent manner (Fig. 2Go).



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  		Figure 2. Immunoblot of insulin receptor with antiphosphotyrosine antibody. Primary (BAT), immortalized (clone MB4.9.2), and H-raslys12-transformed (clone MB1.3.19) brown adipocytes were incubated for 5 min at 37 C with various doses of insulin. Control cells (C) received an equivalent volume of solvent. Cells were then lysed, and immunoprecipitates (600 µg protein from primary brown adipocytes and 3 mg protein from cell lines) were prepared using the monoclonal antiinsulin receptor antibody. The immune complexes were separated by SDS-PAGE, followed by transfer of proteins to Immobilon and Western blotting with 4G10 anti-Tyr(P) antibody. Specific proteins were detected by ECL. The position of the ß-chain of the insulin receptor is indicated by an arrowhead. The positions of mol wt markers (x10-3) are shown on the left. The results shown are representative of at least three independent experiments.

 
As an additional step, we determined whether the alteration in the insulin receptor autophosphorylation described above could be due to a reduced number of insulin receptors after immortalization or H-raslys12 transformation of brown adipocytes. However, quantitation of the insulin receptors on the surface of primary brown adipocytes and immortalized (MB4.9.X) and H-ras-transformed (MB1.3.X) brown adipocyte cell lines by radioligand binding using [125I]insulin revealed an equivalent number of specific insulin-binding sites per cell with a similar affinity (Kd) in all cell types studied (Fig. 3Go).



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  		Figure 3. [125I]Insulin specific binding in primary, immortalized, and H-ras-transformed brown adipocytes. Primary (BAT), immortalized (clones MB4.9.X), and H-raslys12-transformed (clones MB1.3.X) brown adipocytes, after culture for 20 h in serum-free medium, were incubated for 3 h at 20 C with [125I]insulin in both the absence and presence of graded concentrations of unlabeled ligand for receptor binding analysis. Bound vs. free plots are the mean ± SEM (n = 9) from three independent brown adipocyte primary cultures or from three independent clones of the MB4.9.X and MB1.3.X series. Molecular masses and binding sites were calculated from the corresponding Scatchard plots as described in Materials and Methods.

 
Effect of insulin on IRS-1 phosphorylation and its association with PI 3-kinase in primary, immortalized, and H-ras-transformed brown adipocytes
The next step was to compare the effect of insulin on inducing tyrosine phosphorylation of IRS-1 and its association with the enzyme PI 3-kinase in primary brown adipocytes and immortalized or H-raslys12-transformed brown adipocyte cell lines. After 20 h of serum starvation, primary fetal brown adipocytes were incubated with various doses of insulin for 5 min at 37 C, and lysates were prepared. The cell lysates (600 µg protein) were then immunoprecipitated with an anti-{alpha}p85 monoclonal antibody as described in Materials and Methods, and the immune complexes were subjected to Western blotting analysis with the 4G10 anti-Tyr(P) antibody. As shown in Fig. 4Go (left panel), there was a significant increase in the tyrosine phosphorylation of the 160- to 185-kDa band compared with that in control cells, corresponding to the IRS-1 associated with p85-PI 3-kinase upon addition of 1 nM insulin to quiescent fetal brown adipocytes. The level of tyrosine phosphorylation of this band did not change significantly when higher doses of insulin were added (10 and 100 nM). Interestingly, in fetal brown adipocytes, p85-PI 3-kinase was not phosphorylated in tyrosine residues in response to insulin. Furthermore, we could not detect the 95-kDa band corresponding to the tyrosine-phosphorylated insulin receptor ß-subunit in these anti-p85 immuno-precipitates. As we could not detect the 160- to 185-kDa band corresponding to phosphorylated IRS-1 in the immunoprecipitates from clones MB4.9.2 and MB1.3.19 after loading 600 µg protein (data not shown), we immunoprecipitated a 5-fold higher protein content (3 mg). As shown in Fig. 4Go (right panel), we observed a maximal tyrosine phosphorylation of IRS-1 induced by insulin at 1 nM in immortalized brown adipocytes (clone MB4.9.2), although this effect was much lower than that observed in primary cells. However, in H-ras-transformed brown adipocytes (clone MB1.3.19), IRS-1 tyrosine phosphorylation was suppressed by 1 nM insulin; this lack of effect was ameliorated at higher insulin concentrations in a dose-dependent manner. Thus, a half-maximal effect was elicited at 10 nM, and a maximal effect occurred at 100 nM. Furthermore, none of these cell lines showed the p85-PI 3-kinase tyrosine-phosphorylated band in response to insulin.



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  		Figure 4. Phosphorylation of IRS-1 and its association with PI 3-kinase induced by insulin in primary, immortalized, and H-ras-transformed brown adipocytes. Primary (BAT), immortalized (clone MB4.9.2), and H-raslys12-transformed (clone MB1.3.19) brown adipocytes were incubated for 5 min at 37 C with various doses of insulin. Control cells (C) received an equivalent volume of solvent. Cells were then lysed, and immunoprecipitates (600 µg protein from primary brown adipocytes and 3 mg protein from cell lines) were prepared using the monoclonal antibody against {alpha}p85. The immune complexes were analyzed by SDS-PAGE followed by transfer of proteins to Immobilon and Western blotting with 4G10 anti-Tyr(P) antibody. Specific proteins were detected by ECL. The position of IRS-1 is indicated by an arrowhead. The positions of the mol wt markers (x10-3) are shown on the left. Lower panel, Equal amounts of protein from primary (BAT), immortalized (clone MB4.9.2), and H-raslys12-transformed (clone MB1.3.19) brown adipocytes were submitted to SDS-PAGE followed by Western blot analysis with the anti-IRS-1 antibody. Specific proteins were detected by ECL, and the position of IRS-1 is indicated. All results shown are representative of at least three independent experiments.

 
A possible explanation for the decrease in the IRS-1 tyrosine phosphorylation observed in both immortalized and H-ras-transformed brown adipocytes compared with that in primary cells would be decreased IRS-1 expression. To assess this, equals amount of protein from primary, MB4.9.2, and MB1.3.19 cells were submitted to direct Western blot analysis with the anti-IRS-1 antibody. As shown in the lower panel of Fig. 4Go, IRS-1 levels were similar in the three cell types studied.

Effect of insulin on IRS-1-associated PI 3-kinase activity in primary, immortalized, and H-ras-transformed brown adipocytes
We also investigated whether PI kinase activity was associated with the IRS-1 immunoprecipitates in response to insulin in primary and immortalized or transformed brown adipocytes. After serum starvation, cells were stimulated with various doses of insulin for 5 min and extracted as described in Materials and Methods, and 600 µg protein (from both primary brown adipocytes and cell lines) were immunoprecipitated with an anti-IRS-1 antibody. The resulting immune complexes were assayed for PI 3-kinase activity. As shown in Fig. 5Go, primary brown adipocytes displayed almost undetectable PI 3-kinase activity in IRS-1 immunoprecipitates under control conditions. Upon treatment with 1 nM insulin, the basal PI 3-kinase activity increased by 8-fold. No further increase was observed at higher concentrations of insulin (10 and 100 nM). MB4.9.2 or MB1.3.19 cell lines also displayed a very low basal PI 3-kinase activity (Fig. 5Go). When clone MB4.9.2 was stimulated with 1 nM insulin, there was a 3-fold increase in PI 3-kinase activity, which was significantly lower that than observed in primary cells with the same dose of the hormone. Furthermore, no changes in PI 3-kinase activity were observed when higher doses of insulin were added (10 and 100 nM). Transformed cells (clone MB1.3.19) showed a very low IRS-1-associated PI 3-kinase activity in response to 1 nM insulin; this effect was ameliorated by higher insulin concentrations in a dose-dependent manner. Thus, a half-maximal effect (1.5-fold) was elicited at 10 nM, and a maximal effect (3-fold) occurred at 100 nM insulin (Fig. 5Go).



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  		Figure 5. PI 3-kinase activity in anti-IRS-1 immunoprecipitates from insulin-treated primary, immortalized, and H-ras-transformed brown adipocytes. Quiescent primary fetal brown adipocytes (BAT) and clones MB4.9.2 and MB1.3.19 were cultured as described in Materials and Methods. Cells were then stimulated for 5 min with various doses of insulin. Control cells (C) received an equivalent volume of solvent. Whole cell lysates (600 µg protein from both primary cells and cell lines) were subjected to immunoprecipitation with anti-IRS-1 antibody. The immune complexes were washed and immediately used for an in vitro phosphatidylinositol kinase assay as described in Materials and Methods. The conversion of PI to PI phosphate in the presence of [{gamma}-32P]ATP was analyzed by TLC. Results are representative of at least three independent experiments.

 
Effect of insulin on the formation of Ras.GTP in primary, immortalized, and H-ras-transformed brown adipocytes
The importance of p21-ras in the insulin signaling pathway prompted us to investigate the percentage of the p21-Ras.GTP active form in brown adipocytes after insulin stimulation. As shown in Table 1Go, after 20 h of serum starvation, primary fetal brown adipocytes showed 20% of the Ras.GTP-active form. Moreover, the percentage of Ras.GTP increased by 3-fold after 5-min treatment with 1 nM insulin. Immortalized brown adipocytes (clone MB4.9.2) constitutively showed 30% of the Ras.GTP-active form, and this percentage doubled after 5-min treatment with 1 nM insulin. However, in H-raslys12-transformed brown adipocytes we found a higher percentage of constitutive p21-Ras.GTP-active form (66%) than in primary or immortalized brown adipocytes, and this percentage remained unchanged after insulin stimulation at either 1 nM (Table 1Go) or higher doses (results not shown).


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  		Table 1. Effect of insulin on the percentage of Ras.GTP in primary, immortalized, and H-ras-transformed brown adipocytes

 
Effect of insulin on Raf-1 kinase activity in primary, immortalized, and H-ras-transformed brown adipocytes
As an additional step, we determined the effect of insulin on Raf-1 kinase activation, as the immediate event downstream Ras activation described in the insulin signaling cascade. Serum-starved cells were stimulated with various doses of insulin and subsequently lysed as described in Materials and Methods. Proteins from cell lysates (600 µg) were immunoprecipitated with Raf-1 antiserum and assayed for Raf-1 kinase activity in the immune complex. The results presented in Fig. 6Go showed a marked increase (6-fold) in MEK phosphorylation in primary brown adipocytes treated for 5 min with insulin (1 nM) with respect to control cells without any further increase at higher insulin doses. Immortalized brown adipocytes (MB4.8.2) showed a maximal 2-fold increase in Raf-1 kinase activity upon treatment with the same dose of insulin (1 nM) compared with control cells. However, in transformed cell lines (MB1.3.19), a higher constitutive MEK phosphorylation was found, and this activity was insulin independent.



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  		Figure 6. Insulin activation of Raf-1 kinase in primary, immortalized, and H-ras-transformed brown adipocytes. Quiescent primary fetal brown adipocytes (BAT) and brown adipocyte cell lines (clones MB4.9.2 and MB1.3.19) were incubated for 5 min with various doses of insulin. Control cells (lane C) were cultured in the absence of the hormone. Cells were lysed, and proteins (600 µg from both primary cells and cell lines) were immunoprecipitated with anti-Raf-1 antiserum. The resulting immune complexes were assayed for MEK phosphorylation as described in Materials and Methods. The position of the catalytically inactive MEK is indicated by an arrowhead. The migration of the mol wt markers (x10-3) is indicated on the left.

 
Effect of insulin on transactivation of the 12-O-tetradecanoylphorbol-13-acetate response element (TRE)-CAT fusion gene in brown adipocyte cell lines
Finally, we studied the role of insulin in brown adipocyte cell lines at the nuclear level. To do this, we carried out transient transfections with plasmids containing the TRE-CAT fusion gene (five tandem repeats of binding sites for AP-1 transcription factors) (29). Thus, changes in CAT enzyme activity in response to insulin were used to monitor AP-1-regulated gene expression. Primary brown adipocytes showed a very low transfection efficiency with this plasmid. However, immortalized and H-ras-transformed brown adipocytes showed higher transfection efficiencies and were used to measure CAT activity. As shown in Fig. 7Go, insulin treatment for 24 h stimulated CAT activity by 2-fold in immortalized brown adipocytes (clone MB4.9.2); this effect was maximal at 1 nM. However, H-raslys12-transformed brown adipocytes (clone MB1.3.19) showed a higher constitutive CAT activity than immortalized cells, and this activity was insulin independent.



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  		Figure 7. Stimulation of TRE-CAT activity by insulin. Immortalized (clone MB4.9.2) and H-raslys12-transformed (clone MB1.3.19) brown adipocytes were transiently transfected with 10 µg TRE-CAT fusion gene. Upon transfection, cells were cultured for 24 h in serum-free medium and subsequently stimulated with various doses of insulin for an additional 24 h. At the end of the culture period, CAT activity was assayed, and acetylated chloramphenicol was identified by autoradiography. A representative experiment of three is shown.

 
Effect of alkaline phosphatase treatment on insulin receptor phosphorylation
To define the molecular mechanism of altered insulin signaling in mutated H-ras-transformed brown adipocytes, we determined the basal level of the insulin receptor tyrosine/serine-threonine phosphorylation in these cells. WGA-purified proteins from serum-deprived MB1.3.19 cells were incubated with active and inactive immobilized alkaline phosphatase. Then, the phosphorylation reaction was performed as described in Materials and Methods, and the insulin receptors were immunoprecipitated with either the antiinsulin receptor antibody or the Py72 anti-Tyr(P) antibody. As shown in Fig. 8AGo, H-raslys12-transformed brown adipocytes showed a marked insulin receptor ß-chain phosphorylation in the basal state, which was only detected by immunoprecipitation with the antiinsulin receptor antibody, whereas no tyrosine phosphorylation of the ß-subunit occurred after immunoprecipitation of the insulin receptor with the Py72 anti-Tyr(P) antibody. When WGA-purified proteins were preincubated with immobilized active alkaline phosphatase before performing the in vitro kinase reaction, phosphorylation of the insulin receptor (previously detected by immunoprecipitation with the antiinsulin receptor antibody) was totally abolished. Then, WGA-purified proteins from serum-deprived MB1.3.19 cells were stimulated with 100 nM insulin, and the in vitro kinase assay was performed. When WGA-purified proteins were immunoprecipitated with the antiinsulin receptor antibody, a band corresponding to the phosphorylated insulin receptor was observed. This band was similar to that observed in unstimulated cells. However, when the immunoprecipitation was performed with the Py72 anti-Tyr(P) antibody, the band corresponding to the tyrosine-phosphorylated receptor after insulin stimulation was almost undetected, as shown in the experiments performed with whole cell lysates (Figs. 1Go and 2Go). Finally, WGA-purified proteins were incubated with immobilized alkaline phosphatase before stimulation with 100 nM insulin and in vitro kinase assay. As shown in Fig. 8AGo, there was a 3-fold increase in tyrosine phosphorylation [detected by immunoprecipitation with the Py72 anti-Tyr(P) antibody] of insulin receptors preincubated with active alkaline phosphatase, suggesting that removal of the basal serine-threonine-bound phosphates restores tyrosine phosphorylation of the insulin receptor ß-subunit upon stimulation with insulin.



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  		Figure 8. Effect of treatment with alkaline phosphatase on receptor autophosphorylation. Equal amounts of WGA-purified proteins (10 µg) from serum-deprived H-raslys12-transformed brown adipocytes (MB1.3.19; A) or primary cells (BAT; B) were incubated with active or inactive alkaline phosphatase (AP) as indicated in Materials and Methods. Control or 100 nM insulin-stimulated incubations were submitted to an in vitro kinase assay as previously described, and insulin receptors were immunoprecipitated with the antiinsulin receptor antibody (IR) or with the anti-TyrP antibody (Py72). C, WGA-purified receptors from serum-deprived immortalized brown adipocytes (MB4.9.2) or immortalized cells transiently transfected with transforming Ras DNA (pMEXneoH-raslys12) were incubated with active or inactive alkaline phosphatase, stimulated with 100 nM insulin, and submitted to an in vitro kinase assay. Insulin receptors were immunoprecipitated with the Py72 anti-TyrP antibody. D, Equals amount of protein (600 µg) from primary, immortalized, and H-ras-transformed brown adipocytes were immunoprecipitated with the antiinsulin receptor antibody. The immune complexes were analyzed by SDS-PAGE followed by transfer of proteins to Immobilon and Western blotting with a mixture of anti-Pser/Pthr antibodies. Immunoreactive proteins were detected by ECL. The results shown in each panel are representative of at least three independent experiments.

 
To establish whether the basal serine-threonine phosphorylation of the insulin receptor was specific to Ras-transformed cells, we performed control experiments with the purified receptors from serum-deprived primary brown adipocytes. As shown in Fig. 8BGo, we did not find phosphorylation of the ß-chain in the parenteral cells after performing the in vitro kinase assay and immunoprecipitation under the experimental conditions described in Fig. 8AGo. Indeed, pretreatment of the purified receptors from primary cells with active alkaline phosphatase did not suppress the high tyrosine phosphorylation of the receptor observed in the presence of insulin, regardless of the antibody used for the immunoprecipitation. Similar results were obtained with WGA-purified receptors from MB4.9.2 immortalized cells (results not shown).

To further rule out possible effects of clonal selection, we transiently transfected the immortalized brown adipocyte cell line (MB4.9.2) with the pMEXneoH-raslys12 construct. WGA-purified receptors from MB4.9.2 and MB4.9.2H-raslys12 cells were stimulated with 100 nM insulin. Then, the in vitro kinase assay was performed, and the insulin receptors were immunoprecipitated with the Py72 anti-Tyr(P) antibody. As shown in Fig. 8CGo, the presence of insulin induced tyrosine autophosphorylation of the insulin receptor of the immortalized cells; this effect was much less than that in the primary cells (as shown in Figs. 1Go and 2Go). Interestingly, this band was suppressed when these cells were transiently transfected with the pMEXneoH-raslys12 construct. Furthermore, pretreatment of the WGA-purified receptors from MB4.9.2H-raslys12 cells with alkaline phosphatase before insulin stimulation partly restored the tyrosine phosphorylation of the insulin receptor that was lost in H-raslys12-transfected cells (Fig. 8CGo).

Finally, to show direct evidence of the constitutive insulin receptor serine-threonine phosphorylation induced by H-ras transformation of brown adipocytes, we performed an antiphosphoserine/threonine Western blot analysis of insulin receptor immunoprecipitates from the three cell types studied. As shown in Fig. 8DGo, after immunoprecipitation of serum-deprived primary, immortalized, and H-raslys12-transformed brown adipocytes followed by immunodetection with antiphosphoserine/threonine antibodies, we could only detect a single band corresponding to the insulin receptor ß-chain in the lane corresponding to H-raslys12-transformed cells (MB1.3.19).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although major advances have been made in recent years, the exact molecular events linking the insulin receptor tyrosine kinase to its final mitogenic/differentiation actions remain poorly understood. In the present study, we have characterized the very early events of the insulin signaling cascade in primary fetal rat brown adipocytes, a cellular model in which insulin is the main signal involved in the adipogenic program, by inducing the genetic expression of lipogenic enzymes (15, 16, 17, 18, 19). Insulin receptor ß-chain tyrosine autophosphorylation is the earliest known intracellular event in the insulin pathway (32). Under our experimental conditions, we did not find any basal insulin receptor ß-chain tyrosine autophosphorylation in quiescent brown adipocytes. Indeed, when a low concentration of insulin (1 nM) was added to serum-starved cells, the maximal increase in ß-chain tyrosine autophosphorylation was observed. This result indicates that fetal brown adipocytes offer a suitable cell system in which to study the mechanism of insulin action under physiological conditions, in contrast with other studies using in vitro cell systems that have been performed with higher insulin doses. IRS-1 tyrosine phosphorylation has been identified as a novel early event in the actions of both insulin and insulin-like growth factor I as well as those of cytokines and GH, acting as an adaptor protein that binds to the SH2 domains of the p85 subunit of PI 3-kinase (reviewed in Refs. 33 and 34). Quiescent primary brown adipocytes did not show IRS-1 tyrosine phosphorylation or IRS-1-associated activation of PI 3-kinase. These effects were maximally induced by low insulin concentrations (1 nM). Interestingly, we did not detect tyrosine phosphorylation of p85 and p110 PI 3-kinase subunits in the anti-p85 immune complexes. It is well established that stimulation of PI 3-kinase enzymatic activity is mediated through its association with phosphorylated IRS-1 (35, 36). Accordingly, fetal rat brown adipocytes showed an important increase in PI 3-kinase enzymatic activity in anti-IRS-1 immunoprecipitates upon stimulation with a low dose of insulin. In addition, Ras proteins play a crucial role in the insulin signaling pathway, and activation of Ras to its GTP form has been described upon stimulation with a variety of growth factors, including insulin and IGF-I. In this respect, insulin increased by 3-fold the percentage of Ras in its Ras.GTP-active form in primary brown adipocytes. Moreover, downstream from Ras, several serine/threonine kinases have been involved; among them, Raf-1 kinase resulted activated upon treatment with 1 nM insulin, indicating that stimulation of the RAS/serine-threonine kinase cascade leads to the adipogenic and thermogenic differentiation in fetal brown adipocytes, as recently demonstrated by transient transfections experiments (23).

As an additional step, we have examined the insulin signaling pathway in a representative SV40-LTAg immortalized brown adipocyte cell line (clone MB4.9.2) compared with that in primary brown adipocytes. After immortalization, brown adipocytes respond to insulin at the same doses as parenteral cells, but the maximal responsiveness was significantly lower in all steps studied, including insulin receptor ß-chain tyrosine autophosphorylation, p85-PI 3-kinase-associated IRS-1 phosphorylation, IRS-1-associated PI 3-kinase activation, and Raf-1 kinase activation in immortalized cells. As the number of insulin receptors per cell, its molecular mass, and the insulin sensitivity remain unaltered compared with those of parenteral cells, all of these results indicate only a reduced maximal responsiveness to insulin either upstream or downstream from Ras in immortalized brown adipocyte cell lines.

We also compared the insulin signaling pathway in a H-raslys12-transformed brown adipocyte cell line (MB1.3.19) with that in primary or immortalized brown adipocytes (MB4.9.2). Mutated H-raslys12-transformed brown adipocytes suppressed tyrosine autophosphorylation of the insulin receptor in response to low concentrations of insulin (1 nM) compared with primary or immortalized brown adipocytes; this impairment of insulin responsiveness was slightly ameliorated at higher insulin concentrations in a dose-dependent manner. Consistently, the p85-PI 3-kinase-associated tyrosine phosphorylations of IRS-1 and PI 3-kinase activation were almost suppressed in response to low concentrations of insulin. This effect was also ameliorated by stimulation at higher insulin concentrations, in a dose-dependent manner. These results provide additional evidence of the severe insulin resistance induced by Ras transformation in brown adipocytes. The dramatic inhibition of insulin receptor autophosphorylation in response to insulin cannot be accounted for by a constitutive tyrosine-phosphorylated state of the insulin receptor, the presence of a lower number of insulin-binding sites, or altered insulin receptor affinity. Thus, mutant p21-Ras by itself or through constitutive activated proteins downstream from Ras, as discussed below, could be inhibiting insulin receptor ß-chain tyrosine autophosphorylation in response to insulin by a feedback mechanism.

One of the mechanisms proposed to cause inhibition of the tyrosine kinase activity of the insulin receptor is the serine-threonine phosphorylation of the insulin receptor ß-subunit. In this regard, covalent modification of the insulin receptor by protein kinase A or C has been shown to reduce the intrinsic tyrosine kinase activity of the insulin receptor in response to insulin, resulting in insulin resistance (37, 38, 39). Treatment of WGA-purified receptors with alkaline phosphatase suggests that in the basal state (20 h of serum starvation), there is a serine-threonine phosphorylation of the insulin receptor in the H-raslys12-transformed brown adipocyte cell line. Importantly, removal of serine-threonine-bound phosphate before insulin stimulation and in vitro phosphorylation increased the tyrosine kinase activity of the insulin receptor in response to insulin, restoring the tyrosine kinase activity virtually lost in the Ras-transformed cells. This effect of Ras has been reproduced in immortalized cells by transient transfection with mutated H-raslys12 construct, ruling out possible effects of clonal selection. These results demonstrate that the constitutive serine-threonine phosphorylation of the insulin receptor only seen in the MB1.3.19 cell line is specifically induced by mutated H-ras. A possible candidate that might mediate this effect could be a cytosolic serine-threonine kinase directly activated downstream from Ras. In this regard, protein kinase C{zeta}, which has been described as a target of Ras (40), is highly expressed in MB1.3.19 cells (data not shown). However, we cannot exclude the possibility that a serine-threonine kinase associated with the insulin receptor could be involved. Further experimental work will be required to address this important issue.

Attenuations in insulin receptor protein tyrosine kinase activity due to increased serine phosphorylation have been recently described in patients with polycystic ovary syndrome (41). Indeed, women with polycystic ovary syndrome developed insulin resistance and glucose intolerance. Thus, H-ras-transformed brown adipocytes inoculated into nude mice might provide a new model in which to study the insulin resistance linked to alterations in the early events of insulin postreceptor signaling caused by serine-threonine phosphorylation of the insulin receptor in malignant cells.

As expected, H-ras-transformed brown adipocytes expressed a very high percentage of the Ras.GTP-active form compared with primary or immortalized brown adipocytes. This percentage was insulin independent. Consistently, an event downstream from Ras, such as Raf 1-kinase activity, was constitutively stimulated in the transformed brown adipocytes in an insulin-independent manner. These results provide additional support to our previous statement that constitutive activated proteins downstream from Ras may be inducing a serine-threonine phosphorylation on the insulin receptor by a feedback mechanism, resulting in severe insulin resistance. Finally, we have extended our investigations regarding the alterations in the insulin signaling caused by H-ras transformation toward to the nucleus. Previous experiments performed in a differentiated adipocyte cell line have established AP-1 transcription factors, which bind to the TRE found in enhancers of various genes, as mediators for insulin-regulated gene expression (42). In this respect, our data show that insulin significantly trans-activates the expression of the TRE-CAT fusion gene transiently transfected in immortalized brown adipocytes. However, in H-raslys12-transformed brown adipocytes, we observed a higher constitutive TRE-CAT activity, and this activity remained unchanged upon insulin treatment. These results confirm that the constitutive activation of events downstream from Ras in the cytosol by mutated H-ras transformation also occurs at the nuclear level. Thus, we found that overstimulation downstream from Ras results in mutated H-ras transformation regardless of the presence of insulin.

In conclusion, our results show that immortalization of brown adipocytes causes a decrease in the maximal responsiveness to insulin compared with that of parenteral cells. In addition, transformation of brown adipocytes by mutated H-ras gives rise to severe insulin resistance (at the early postreceptor events upstream from Ras) in the insulin signaling pathway. However, downstream from Ras, several events are overstimulated, suggesting a possible feedback mechanism causing an impairment of the insulin receptor by serine-threonine phosphorylation, resulting in severe insulin resistance upstream from Ras. Accordingly, Ras-transformed brown adipocytes provide a new insight into the insulin signal transduction linked to an insulin receptor defect caused by its serine-threonine phosphorylation.


   Acknowledgments
 
We are grateful for the valuable reagents provided by Drs. E. Rozengurt, J. Downward, P. Rodriguez-Viciana, and J. Sinnet-Smith (Imperial Cancer Research Foundation, London, UK); Dr. R. Kahn (Joslin Diabetes Center, Boston, MA); and Dr. S. Gutkind (NIH, Bethesda, MD). We thank Dr. J. M. Carrascosa (Universidad Autonoma, Madrid, Spain) for his help and advice.


   Footnotes
 
1 This work was supported by Grant SAF96/0115 from the Comision Interministerial de Ciencia y Tecnologia, Spain. Back

2 Recipient of a fellowship from the Comunidad Autonoma de Madrid. Back

Received December 4, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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