Immortalized brown adipocyte cell lines have been generated from fetuses of mice deficient in the insulin-like growth factor I receptor gene (IGF-IR−/−), as well as from fetuses of wild-type mice (IGF-IR+/+). These cell lines maintained the expression of adipogenic- and thermogenic-differentiation markers and show a multilocular fat droplets phenotype. IGF-IR−/− brown adipocytes lacked IGF-IR protein expression; insulin receptor (IR) expression remained unchanged as compared with wild-type cells. Insulin-induced tyrosine autophosphorylation of the IR β-chain was augmented in IGF-IR–deficient cells. Upon insulin stimulation, tyrosine phosphorylation of (insulin receptor substrate-1) IRS-1 was much higher in IGF-IR−/− brown adipocytes, although IRS-1 protein content was reduced. In contrast, tyrosine phosphorylation of IRS-2 decreased in IGF-IR–deficient cells; its protein content was unchanged as compared with wild-type cells. Downstream, the association IRS-1/growth factor receptor binding protein-2 (Grb-2) was augmented in the IGF-IR−/− brown adipocyte cell line. However, SHC expression and SHC tyrosine phosphorylation and its association with Grb-2 were unaltered in response to insulin in IGF-IR–deficient brown adipocytes. These cells also showed an enhanced activation of mitogen-activated protein kinase (MAPK) kinase (MEK1/2) and p42/p44 mitogen-activated protein kinase (MAPK) upon insulin stimulation. In addition, the lack of IGF-IR in brown adipocytes resulted in a higher mitogenic response (DNA synthesis, cell number, and proliferating cell nuclear antigen expression) to insulin than wild-type cells. Finally, cells lacking IGF-IR showed a much lower association between IR or IRS-1 and phosphotyrosine phosphatase 1B (PTP1B) and also a decreased PTP1B activity upon insulin stimulation. However, PTP1B/Grb-2 association remained unchanged in both cell types, regardless of insulin stimulation. Data presented here provide strong evidence that IGF-IR–deficient brown adipocytes show an increased insulin sensitivity via IRS-1/Grb-2/MAPK, resulting in an increased mitogenesis in response to insulin.

Insulin and insulin-like growth factors (IGF-I and IGF-II) lead to a variety of biological effects in typical insulin target cells, such as adipocyte, hepatocyte, and myocyte. Insulin/IGF-I action is mediated by the insulin receptor (IR) and the IGF-I receptor (IGF-IR), respectively, which have very similar heterodimeric α2β2 structure and belong to the family of receptor tyrosine kinases (1). Upon ligand binding, insulin/IGF-IR undergoes autophosphorylation on tyrosine residues, which activates the intracellular tyrosine kinase of the β-subunit (2). This, in turn, stimulates the phosphorylation of cytoplasmic proteins, including the insulin receptor substrate (IRS) family (IRS-1, -2, -3, and -4) and SHC (37). Then, these phosphorylated IRS proteins bind proteins that contain Src homology 2 domains, such as the p85 regulatory subunit of phosphatidylinositol 3-kinase (8), growth factor receptor binding protein-2 (Grb-2), which links signaling via SOS to activation of the Ras complex (9), and protein tyrosine phosphatase SHP-2 (10) that lead to activation of various downstream signaling pathways. Like IRS proteins, SHC proteins are tyrosine phosphorylated upon IR activation and can associate with Grb-2 and subsequently activate the Ras/mitogen-activated protein kinase (MAPK) cascade (9,11).

Genetic engineering in mice has proved to be a powerful tool for studying the specificity, complementary, and redundancy in the signaling cascades by altering the structure or expression of a single gene or a set of genes. The lack of IGF-IR resulted in marked intrauterine growth retardation and several abnormalities of differentiation in various tissues, which led to early lethality around birth (12). Conversely, IR-deficient pups were nearly normal at birth but developed severe diabetes and ketosis, which led to death within 1 week (13). Mice that lack IRS-1 show a significant growth retardation and also a mild diabetes phenotype, indicating that this docking protein plays an important role in mediating growth and in the insulin action on peripheral tissues that regulate glucose uptake (14,15). However, mice that lack IRS-2 have no growth retardation but show a severe diabetic phenotype as a result of a β-cell defect, indicating that IRS-2 plays a crucial role in glucose homeostasis (16).

Brown adipose tissue is the main tissue involved in nonshivering thermogenesis in newborn animals and is responsible for heat production associated with the expression of the mitochondrial uncoupling protein-1 (UCP-1) (17). That brown adipocytes bear a high number of high-affinity endogenous IR and IGF-IR allowed us to study the role of insulin and IGF-I in proliferation (1820) and differentiation (21,22), as well as the balance between proliferation/differentiation processes in the insulin/IGF-I signaling pathways, under physiological conditions (23,24). The derivation of brown adipocyte cell lines from knockout mice that lack insulin action candidate genes have provided us new tools to dissect the insulin signaling pathways in vitro (25,26). The fact that brown adipocytes bear IR and IGF-IR has made greatly complex the dissection of the specific cellular effects mediated by either insulin or IGF-I. Thus, we generated immortalized cell lines from the fetal brown adipose tissue of IGF-IR–deficient and wild-type mice. In this study, we analyzed the effect of lacking IGF-IR on insulin signaling and insulin-induced growth response in IGF-IR–deficient brown adipocytes.

Materials.

FCS and culture media were obtained from Gibco (Gaithersburg, MD). Insulin and anti-mouse IgG-Agarose were from Sigma Chemical (St. Louis, MO). Protein A-agarose was from Roche Molecular Biochemicals (Mannheim, Germany). IGF-I was from Calbiochem-Novabiochem (La Jolla, CA). The antibodies against the IR β-subunit (sc-09), IGF-IR β-subunit (sc-713), Grb-2 (sc-255), and SHC (sc-967) were purchased from Santa Cruz Biotechnology (Palo Alto, CA). The monoclonal anti-Tyr(P) antibody (clone 4G10) was from Upstate Biotechnology (Lake Placid, NY). The polyclonal anti-SHC antibody was from Transduction Laboratories (Lexington, KY). The polyclonal antibodies against IRS-1 and IRS-2 were a gift from Dr. M.F. White (Joslin Diabetes Center, Boston, MA). The anti-phospho MAPK (Thr202/Tyr204), anti-phospho MEK1/2 (Ser217/221), anti-MEK, and anti-MAPK antibodies were purchased from New England Biolabs (Beverly, MA). The antiproliferating nuclear antigen (PCNA) antibody was from Roche Molecular Biochemicals (Mannheim, Germany). [α32P]-dCTP (3,000 Ci/mmol) and [3H]thymidine (2 μCi/ml; 1 μmol/l) were from Amersham (Aylesbury, U.K.). All other reagents used were of the purest grade available.

Generation of brown adipocyte cell lines.

Brown adipocytes were obtained from interscapular brown adipose tissue of 17.5–18.5 fetuses from 2 to 3 pregnant mice of normal genotype or from a pool of tissue of fetuses with body weight <0.5 g obtained from 2 to 3 pregnant mice IGF-IR+/− mated with male IGF-IR+/− and further submitted to collagenase dispersion as previously described (18). Then, cells were infected with the puromycin-resistance retroviral vector pBabe encoding SV 40 Large T antigen as described (25). After infection, fetal brown adipocytes were maintained in 20% FCS culture medium for 72 h, before selection with puromycin (1 μg/ml) for 1 week. These cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal serum (FS) and puromycin (1 μg/ml).

Immunoprecipitations.

Quiescent cells were treated without or with several doses of insulin, as indicated, and lysed at 4°C in 1 ml of a solution containing 10 mmol/l Tris/HCl, 5 mmol/l EDTA, 50 mmol/l NaCl, 30 mmol/l sodium pyrophosphate, 50 mmol/l NaF, 100 μmol/l Na3VO4, 1% Triton X-100, and 1 mmol/l phenylmethylsulfonyl fluoride, pH 7.6 (lysis buffer). Lysates were clarified by centrifugation at 15,000g for 10 min. After protein content determination, equal amounts of protein (500–600 μg) were immunoprecipitated at 4°C with the corresponding antibodies. The immune complexes were collected on Protein A-agarose or anti-mouse Ig-Agarose beads. Immunoprecipitates were washed with lysis buffer and extracted for 10 min at 95°C in 2× sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (200 mmol/l Tris/HCl, 6% SDS, 2 mmol/l EDTA, 4% 2-mercaptoethanol, and 10% glycerol, pH 6.8) and analyzed by SDS-PAGE.

Western blotting.

After SDS-PAGE, proteins were transferred to Immobilon membranes and were blocked using 5% nonfat dried milk or 3% BSA in 10 mmol/l Tris-HCl and 150 mmol/l NaCl (pH 7.5) and incubated overnight with several antibodies as indicated in 0.05% Tween-20, 10 mmol/l Tris-HCl, and 150 mmol/l NaCl (pH 7.5). Immunoreactive bands were visualized using the ECL Western blotting protocol (Amersham).

PTP1B activity.

Quiescent cells were treated without or with several doses of insulin as indicated. Cells were lysed in 50 mmol/l Tris-HCl, 0.15 mol/l NaCl, 1% Triton X-100, and 2 mmol/l EDTA (pH 7.5). Then cell lysates were applied through a Sephadex G-25 column to remove the free phosphate present in the lysates. After protein content determination, PTP1B phosphatase activity was determined by measuring phosphate release using a synthetic monophosphotyrosyl-containing peptide and the malachite green assay (Upstate Biotechnology).

Protein determination.

Protein determination was performed by the Bradford dye method (27), using the Bio-Rad reagent (Hercules, CA) and BSA as the standard.

RNA extraction and Northern blot analysis.

At the end of the culture time, cells were washed twice in ice-cold PBS and RNA was isolated as described (28). Total cellular RNA (10 μg) was submitted to Northern blot analysis, i.e., electrophoresed on 0.9% agarose gels containing 0.66 mol/l formaldehyde, transferred to GeneScreen membranes (NEN Life Science Products, Boston, MA), and cross-linked to the membranes by ultraviolet light. Hybridization was performed in 0.25 mmol/l NaHPO4 (pH 7.2), 0.25 mol/l NaCl, 100 μg/ml denatured salmon sperm DNA, 7% SDS, and 50% deionized formamide, containing denatured 32P-labeled cDNA (106 cpm/ml) for 24 h at 42°C. Fatty acid synthase (FAS) and 18S RNA cDNAs labeling was carried out with [α-32P] dCTP by using a multiprimer DNA-labeling system. FAS mRNA has been previously reported as an IGF-I target gene in fetal brown adipocytes (22). Membranes were subjected to autoradiography, and the relative densities of the hybridization signals were determined by densitometric scanning of the autoradiograms.

[3H]thymidine incorporation to DNA.

Cells were plated at 0.5 × 106/dish in 6-cm dishes in DMEM with 10% FCS. After 24 h, the medium was changed to DMEM with 0.05% insulin-free BSA and cells were further cultured for 24 h in the absence or presence of insulin (1, 10, and 100 nmol/l) or IGF-I (1 and 10 nmol/l). DNA synthesis was determined by [3H]thymidine incorporation (0.2 μCi/ml) over the last 4 h of culture (18). After two washes with ice-cold PBS, cells were lysed in 0.1% SDS. Trichloroacetate-precipitable DNA was then counted for incorporated radioactivity. All assays were performed in triplicate and expressed in cpm/dish.

Analysis of cell growth.

For cell growth experiments, wild-type and IGF-IR−/− brown adipocytes were plated at 0.5 × 106/dish in 6-cm dishes in DMEM with 10% FCS. After 24 h, the medium was changed to DMEM with 0.05% insulin-free BSA and cells were further cultured for 24 h in the absence or presence of insulin (1, 10, and 100 nmol/l). At that time, cells were trypsinized and cell number was determined in a FACScan flow cytometer (Becton Dickinson, San Jose, CA) or in a hemocytometer.

Fetal brown adipocytes from IGF-IR–deficient mice lack IGF-IR expression and autophosphorylation but express adipogenic and thermogenic markers.

Our first purpose in this work was to determine whether the immortalized brown adipocyte cell line derived from the IGF-IR knockout fetuses in fact lack IGF-IR expression. Cells were cultured under growing conditions (10% FS) to confluence, and then IGF-IR expression was analyzed by Western blotting. As shown in Fig. 1A, these cells (hereafter referred to as IGF-IR−/− cells) did not express endogenous IGF-IR β-chain as compared with the brown adipocyte cell line derived from the wild-type fetuses (hereafter referred to as IGF-IR+/+ cells) used as a positive control. We have previously shown that physiological doses of IGF-I (1–10 nmol/l) stimulate a rapid tyrosine autophosphorylation of the IGF-IR β-subunit in primary fetal brown adipocytes (23). On this basis, we next examined IGF-I–induced receptor phosphorylation in wild-type and IGF-IR–deficient cells. Quiescent cells (20 h serum-starved) were stimulated for 5 min with various doses of IGF-I and subsequently lysed. Then, equal amounts of protein were immunoprecipitated with the anti–IGF-IR β-chain antibody and analyzed by Western blotting with the anti-Tyr(P) antibody. Besides IGF-I (1–10 nmol/l) stimulated tyrosine autophosphorylation of its own receptor in IGF-IR+/+ cells, phosphorylation of the β-subunit in IGF-IR−/− cells was completely abolished (Fig. 1B).

The adipogenic marker FAS mRNA was detectable in growing brown adipocytes derived from the wild-type; the amount of FAS mRNA was 50% reduced in cells derived from the IGF-IR−/− animal (Fig. 1C). Moreover, the expression of malic enzyme and the thermogenic marker UCP-1, detected by immunoblotting, was not significantly different between the two cell lines. The morphology of both cell lines shown in Fig. 1D reveals multilocular fat droplets distribution specific to brown adipocytes.

IR expression and its tyrosine kinase activity in IGF-IR–deficient brown adipocytes.

Next, we addressed the expression of the IR and its tyrosine autophosphorylation upon insulin stimulation in IGF-IR–deficient brown adipocytes, as compared with wild-type cells. Both cell lines were cultured under growing conditions (10% FS) to confluence, and then IR α-chain protein content was analyzed by Western blotting. As shown in Fig. 2A, no differences in the expression of the insulin receptor α-chain were observed in the absence (IGF-IR−/−) or presence (IGF-IR+/+) of IGF-IR. To analyze further the tyrosine kinase activity of the brown adipocyte IR in both cell lines, we stimulated quiescent cells (20 h serum-starved) for 5 min with various doses of insulin and subsequently lysed. Then, equal amounts of protein were immunoprecipitated with the anti-IR β-chain antibody and analyzed by Western blotting with the anti-Tyr(P) antibody. As shown in Fig. 2B, insulin stimulated tyrosine phosphorylation of its cognate receptor in both cell lines. However, tyrosine phosphorylation of the IR in brown adipocytes lacking IGF-IR was much higher than that observed in wild-type brown adipocytes; maximal activity was elicited at 1 nmol/l insulin. In addition, we performed another experiment in IGF-IR−/− cells extending insulin stimulation to subnanomolar concentrations (Fig. 2B, bottom). Maximal β-chain tyrosine phosphorylation was elicited at 0.1 nmol/l concentration in IGF-IR–deficient brown adipocytes, as compared to the maximal effect in wild-type cells at 10 nmol/l insulin concentration.

Expression and tyrosine phosphorylation of IRS-1/IRS-2 upon insulin/IGF-I stimulation in wild-type and IGF-IR–deficient brown adipocytes.

Our next purpose was to investigate the ability of insulin to activate intracellular signaling pathways in these brown adipocyte cell lines. Regarding IRS proteins, we determined the expression of IRS-1 and IRS-2 and the ability of insulin/IGF-I to induce tyrosine phosphorylation of these molecules in IGF-IR–deficient brown adipocytes, as compared with wild-type cells. IGF-IR+/+ and IGF-IR−/− brown adipocytes were cultured in the presence of 10% FS to confluence and then serum-starved for 20 h. IRS-1 and IRS-2 protein content was analyzed by Western blot in both growing and quiescent cells. As shown in Fig. 3A, IRS-1 protein content was markedly reduced in growing and quiescent IGF-IR−/− cells as compared with the wild-type. However, IRS-2 protein expression remained unchanged (Fig. 3B). Then, we determined tyrosine phosphorylation of IRS-1/IRS-2 upon insulin or IGF-I stimulation. Quiescent IGF-IR+/+ and IGF-IR−/− brown adipocytes were stimulated with either insulin or IGF-I (10 nmol/l) for 5 min. At the end of the culture time, cells were lysed and equal amounts of total protein were immunoprecipitated with anti–IRS-1 or anti–IRS-2 antibodies. The resulting immune complexes were analyzed by Western blotting with the anti-Tyr(P) antibody. In wild-type brown adipocytes, both insulin and IGF-I induced a significant increase in tyrosine phosphorylation of IRS-1; the insulin effect was stronger than the effect of IGF-I at the same dose (10 nmol/l). Although IRS-1 expression was reduced in IGF-IR−/− brown adipocytes, insulin-stimulated IRS-1 tyrosine phosphorylation was higher in these cells as compared with the wild-type. As expected, IGF-IR−/− cells lacked the response to IGF-I in inducing IRS-1 tyrosine phosphorylation (Fig. 3A). Regarding IRS-2, insulin or IGF-I stimulation of IGF-IR+/+ brown adipocytes resulted in a marked increase in its tyrosine phosphorylation as compared with untreated cells; the effect of insulin was stronger than the effect of IGF-I. Although insulin stimulation of IGF-IR−/− cells increased IRS-2 tyrosine phosphorylation, this effect was lower than that observed in IGF-IR+/+ brown adipocytes. As expected, IGF-I stimulation failed to increase IRS-2 tyrosine phosphorylation in IGF-IR–deficient cells (Fig. 3B).

Insulin-induced IRS-1/2 association with Grb-2 in IGF-IR+/+ and IGF-IR−/− brown adipocytes.

To explore further the molecular consequences of the lack of IGF-IR on insulin signaling downstream of IRS in brown adipocytes, we analyzed the association of IRS-1/IRS-2 with the adapter protein Grb-2. Quiescent cells (20 h serum-starved) were stimulated for 5 min with various doses of insulin (1–100 nmol/l) and subsequently lysed. Then, equal amounts of protein were immunoprecipitated with the anti–Grb-2 antibody and analyzed by Western blotting with the anti-Tyr(P) antibody. As shown in Fig. 4, insulin stimulation of IGF-IR+/+ cells resulted in a dose-dependent presence of a 180- to 190-kDa band corresponding to Grb-2–associated IRS-1/2. It is interesting that the association of both IRS proteins with Grb-2 was higher in insulin-stimulated IGF-IR−/− cells; the maximal effect was elicited at 1 nmol/l insulin. When insulin stimulation of IGF-IR−/− brown adipocytes was extended to subnanomolar concentrations, we found maximal IRS-1/2 tyrosine phosphorylation at 0.1 nmol/l concentration (Fig. 4, bottom).

SHC expression and its tyrosine phosphorylation and the association with Grb-2 in IGF-IR–deficient cells.

We next investigated SHC expression and its downstream signaling in IGF-IR–deficient brown adipocytes. Both IGF-IR+/+ and IGF-IR−/− cells were cultured in the presence of 10% FS to confluence and then serum-starved for 20 h. SHC protein content was analyzed in growing and quiescent cells by Western blot. As shown in Fig. 5A, SHC expression was similar in both cell lines regardless of the absence or presence of IGF-IR. To study SHC tyrosine phosphorylation upon insulin stimulation, we stimulated quiescent IGF-IR+/+ and IGF-IR−/− brown adipocytes with various doses of insulin for 5 min. At the end of the culture time, cells were lysed and equal amounts of total protein were immunoprecipitated with the anti-SHC antibody. The resulting immune complexes were analyzed by Western blotting with the anti-Tyr(P) antibody. Insulin stimulation resulted in a similar increase in SHC tyrosine phosphorylation in both cell lines (Fig. 5B). As expected, IGF-IR−/− brown adipocytes did not increase SHC tyrosine phosphorylation upon IGF-I stimulation (results not shown). Next, we performed anti-SHC immunoprecipitations followed by anti–Grb-2 Western blotting in insulin-stimulated wild-type and IGF-IR−/− brown adipocytes. Figure 5B shows that SHC/Grb-2 association was markedly increased upon insulin stimulation of wild-type brown adipocytes as well as in IGF-IR–deficient cells, indicating that SHC expression and its signaling toward Ras/MAPK pathway upon insulin stimulation is unaffected in IGF-IR–deficient cells.

Insulin stimulation of MEK1/2 and MAPK in wild-type and IGF-IR–deficient brown adipocytes.

Activation of Ras/MAPK signaling cascade has been shown to be an essential requirement for insulin/IGF-I–induced brown adipocyte mitogenic responses (20). To assess the impact of the lack of IGF-IR expression on the final steps in this pathway, we determined insulin-induced MEK1/2 and p42/p44 MAPK phosphorylation in wild-type and IGF-IR−/− brown adipocytes. Quiescent (20 h serum-starved) IGF-IR+/+ and IGF-IR−/− brown adipocytes were stimulated with insulin (1–100 nmol/l) for 5 min. At the end of the culture time, cells were lysed, and equal amounts of total protein were submitted to SDS-PAGE followed by Western blotting with anti-phospho MEK1/2 and anti-phospho p42/p44 MAPK antibodies, respectively. Insulin induced both MEK1/2 phosphorylation and MAPK phosphorylation (Fig. 6, top) in wild-type brown adipocytes in a dose-dependent manner; maximal effect was elicited at 100 nmol/l insulin concentration. However, activation of both MEK1/2 and MAPK by insulin in IGF-IR−/− brown adipocytes was much higher than that observed in wild-type cells; the insulin response curve showed the maximal effect at 0.01 nmol/l concentration (Fig. 6, bottom).

Growth-promoting effect of insulin in wild-type and IGF-IR–deficient brown adipocytes.

Insulin/IGF-I are potent mitogens in fetal brown adipocyte primary cultures (1820). We then addressed whether the lack of IGF-IR in fetal brown adipose cells could affect the growth-promoting effect of insulin. Wild-type and IGF-IR−/− brown adipocyte cell lines were cultured for 24 h in serum-free medium in the absence or presence of various doses of insulin (1–100 nmol/l), and [3H]thymidine incorporation was determined during the last 4 h of culture. As shown in Fig. 7A, both immortalized fetal brown adipocyte cell lines showed an intrinsic mitogenic competence in the absence of insulin. Wild-type cells maximally increased approximately threefold [3H]thymidine incorporation in response to insulin in a dose-dependent manner. IGF-IR–deficient brown adipocytes also responded to insulin increasing DNA synthesis in a dose-dependent manner; the maximal effect was approximately fivefold at 100 nmol/l insulin concentration. As expected, IGF-I failed to stimulate [3H]thymidine incorporation into IGF-IR−/− brown adipocytes (results not shown). In addition to DNA synthesis experiments, we determined cell number increase upon 24 h of insulin treatment in both cell types. As shown in Fig. 7B, wild-type brown adipocytes nearly doubled cell number after 24 h of treatment with 100 nmol/l insulin. However, cells lacking IGF-IR showed a threefold increase in cell number, reaching the maximal effect at 1 nmol/l insulin concentration. PCNA, a nuclear protein required for cell cycle progression and cellular proliferation (19,29), was increased in the basal state in both cell lines (Fig. 7C). Upon insulin stimulation for 24 h, PCNA protein content increased in wild-type cells approximately threefold and in the IGF-IR–deficient cells approximately sevenfold; the maximal effect was elicited in both cases at 1 nmol/l insulin concentration.

PTP1B expression and its association with IR and IRS-1 in wild-type and IGF-IR–deficient brown adipocytes.

One possible mechanism to explain the increased insulin sensitivity of IGF-IR–deficient brown adipocytes is a decreased phosphatase activity. PTP1B expression was similar in both brown adipocyte cell lines regardless of the absence or presence of IGF-IR (Fig. 8A). Next, we investigated the association of PTP1B with IR and IRS-1 in both cell lines. To study this, we stimulated quiescent IGF-IR+/+ and IGF-IR−/− brown adipocytes with various doses of insulin for 5 min. At the end of the culture time, cells were lysed, and equal amounts of protein were immunoprecipitated with anti-PTP1B antibody. The resulting immune complexes were analyzed by Western blotting with the anti-IR and IRS-1 antibodies. As shown in Fig. 8B, insulin induced the association of PTP1B with both IR and IRS-1 in wild-type brown adipocytes; these effects were much lower in IGF-IR–deficient cells. Previous work demonstrated that PTP1B directly interacts with the SH3 domains of Grb-2 and that interaction enhanced IRS-1 dephosphorylation (30). Immunoprecipitation with anti-PTP1B antibody followed by Western blotting with anti–Grb-2 antibody revealed a stable interaction between these two proteins in both wild-type and IGF-IR−/− cells. However, this interaction did not change in insulin-stimulated cells. Finally, we assayed an IR phosphopeptide-associated PTP1B activity in wild-type and IGF-IR–deficient brown adipocytes upon insulin stimulation. As shown in Fig. 8C, insulin increased PTP1B activity by 2.5-fold in wild-type cells. However, no PTP1B activity was detected in insulin-stimulated IGF-IR−/− brown adipocytes.

During the past few years, our laboratory has established that quiescent fetal brown adipocytes constitute a suitable cell model in which to study the insulin/IGF-I signaling network under physiological conditions (2224). These cells bear a high number of high-affinity insulin and IGF-I binding sites per cell (18,21), which allow the unhampered investigation of the early events in the insulin/IGF-I action as well as the effect of these molecules on growth and differentiation of brown adipocytes. More recent, the advances in targeted mutagenesis of genes of the insulin and IGF-I signaling system in mice have provided new clues in the research of the functional differences among related molecules. In this regard, immortalized fetal brown adipocyte cell lines derived from the IRS-1–deficient mice showed an impairment in insulin-induced lipid synthesis as a result of a substantial reduction of Akt activation in response to the hormone (25). In addition, brown adipocytes lacking IRS-2 showed an impairment in Glut4 translocation and glucose uptake in response to insulin (26). In this study, we demonstrated that IGF-IR−/− brown adipocytes maintained the expression of the adipogenic- and thermogenic-differentiation markers. Although we observed a significant reduction in FAS mRNA content in IGF-IR−/− cells as compared with wild-type cells, they show a multilocular fat droplets phenotype typical of brown adipocytes, regardless of the process of immortalization. In addition, IGF-IR−/− brown adipocytes grow normally in the presence of 10% FS despite that other cell systems that lack IGF-IR, such as 3T3 fibroblasts, grow at a reduced rate in medium containing 10% serum (31).

IGF-I stimulated tyrosine phosphorylation of its receptor in wild-type cells but not in IGF-IR−/− cells. However, cells that lack IGF-IR are insulin target cells that allow us to study insulin signaling. In contrast, IGF-IR–deficient fibroblasts lack IR, which need to be overexpressed to study insulin effects (32). Our results indicate that the absence of IGF-IR and IGF-I signaling results in a marked increase in the sensitivity of the IR for its own ligand. It is interesting that restoration of IGF-I signaling by reintroduction of IGF-IR in IGF-IR−/− brown adipocytes did not result in diminution of insulin sensitivity (results not shown). Moreover, IGF-I at the physiological doses used in this study did not induce phosphorylation of the IR in wild-type and IGF-IR−/− brown adipocytes (results not shown). These results contrast with those obtained in IR-deficient hepatocytes, where insulin was able to stimulate IGF-IR phosphorylation (33). Likewise, Lamothe et al. (34) reported insulin effects through IGF-IR in IR-deficient fibroblasts. Thus, the absence of IGF-IR signaling in brown adipocytes can be compensated only by insulin through its own receptors.

IRS-1 and IRS-2, which are highly expressed in fetal brown adipocytes, act as multiple docking proteins by binding to downstream signal transduction molecules in response to activation by insulin or IGF-I (24). Despite the specific loss of IRS-1 expression in IGF-IR−/− brown adipocytes as compared with wild-type cells, we found an enhanced insulin-induced IRS-1 tyrosine phosphorylation. Conversely, the lack of IGF-IR resulted in a decrease of IRS-2 tyrosine phosphorylation in response to insulin without changes in its protein content. Thus, these data indicate that signaling from IR to IRS-1 or IRS-2 is altered in IGF-IR–deficient cells, resulting in an enhanced response to IRS-1 parallel to a decreased response to IRS-2. This evidence supports that IRS-1 and IRS-2 use different mechanisms to interact with the IR (3537), although a different cellular distribution of IRS-1 and IRS-2 cannot be ruled out. Conversely, either SHC expression, which has been shown to be increased in a chicken hepatoma cell line lacking IRS-1 (38), or its tyrosine phosphorylation in response to insulin was unaltered in IGF-IR–deficient cells.

We have previously shown that although both IRS-1/2 and SHC proteins can potentially connect the activated insulin/IGF-IR to the adapter protein Grb-2, IRS-1 and SHC are the predominant molecules that couple these receptors to activation of the Ras/MAPK cascade in brown adipocytes (24,39). However, we found an enhanced effect of insulin in IRS/Grb-2 association but not in SHC/Grb-2 association in IGF-IR−/− brown adipocytes as a consequence of the augmented signaling through IRS-1. Despite some evidence from several receptor systems indicating that SHC is the major docking molecule that connects cell surface receptors to p21 ras activation (4042), our results support that IRS-1 is the main protein that leads to Grb-2/Ras/MAPK signaling pathway in IGF-IR–deficient brown adipocytes. In fact, IGF-IR−/− brown adipocytes showed an increased phosphorylation of MEK and p42/44 MAPK and an increased mitogenesis in response to insulin, as compared with wild-type cells.

Cellular protein-tyrosine phosphatases play a crucial role in maintaining the steady-state phosphotyrosine content of proteins in the insulin action pathway (43). Among them, PTP1B has been shown extensively to be expressed in insulin-sensitive tissues and also to bind to and dephosphorylate IR (4446) and more recently IRS-1 (30). Thus, the increased insulin sensitivity and mitogenic response to insulin observed in brown adipocytes that lack IGF-IR is consistent with the loss of the association between IR, or IRS-1, and PTP1B. In fact, mice that lack PTP1B show an increased insulin sensitivity and also a loss of adiposity (47,48). Our results obtained in IGF-IR–deficient brown adipocytes seem to indicate that both IGF-IR expression and its signaling throughout development are probably required to increase the PTP1B cellular pool accessible to both IR or IRS-1. Consequently, the insulin signaling cascade involving the IRS-1/Grb-2/MAPK pathway is amplified in IGF-IR–deficient brown adipocytes and that might compensate the lack of IGF-I mitogenic stimuli. In addition, in brown adipocytes, there is a constitutive Grb-2/PTB-1B association as previously reported (30). This complex does not change between both types of brown adipocyte cell lines and is not further modulated by insulin stimulation. Thus, Grb-2-associated PTP1B represents a pool of PTP1B that does not contribute to the regulation of IGF-I/insulin signaling. Our results support the notion that free PTP1B plays an essential role in regulating the balance of insulin signaling. Overall, the increased insulin signaling and mitogenic response to insulin through its own receptor observed in brown adipocytes that lack IGF-IR may represent a compensatory mechanism by which IGF-IR−/− embryos can maintain fetal brown adipose tissue growth, despite the severe growth retardation observed in those mice (12).

In summary, data presented in this article in IGF-IR–deficient brown adipocytes provide a new insight in the insulin field, first by showing that the lack of IGF-IR is compensated by an increased insulin signaling and mitogenic response to insulin through IR/MAPK pathway and, second, by giving support to the fact that IRS-1—but not IRS-2 or SHC—and PTP1B are essential players involved in regulating the balance of postreceptor insulin signaling leading to mitogenesis.

FIG. 1.

Characterization of IGF-IR–deficient brown adipocytes. A: Wild-type (IGF-IR+/+) and IGF-I–deficient brown adipocytes (IGF-IR−/−) were cultured to confluence under growing conditions (10% FS), and then cells were lysed. Total protein (50 μg) was submitted to SDS-PAGE and analyzed by Western blotting with the anti–IGF-IR antibody. A representative experiment is shown. B: Quiescent (20 h serum-starved) IGF-IR+/+ and IGF-IR−/− brown adipocytes were stimulated with IGF-I (1–10 nmol/l) for 5 min. Control cells were cultured in the absence of IGF-I. At the end of the culture time, cells were lysed, and 600 μg total protein was immunoprecipitated with the anti–IGF-IR antibody. The resulting immune complexes were analyzed by Western blotting with the anti-Tyr(P) antibody. The results shown are representative of three experiments. C: Cells were cultured to confluence in the presence of DMEM–10% FS. Total RNA (10 μg) was isolated, submitted to Northern blot analysis, and hybridized with FAS and 18S RNA cDNAs. Total protein (50 μg) was submitted to SDS-PAGE and analyzed by Western blotting with the anti-malic enzyme and anti–UCP-1 antibodies. Representative autoradiograms are shown. The autoradiograms corresponding to three independent experiments were quantified by scanning densitometry. Results are expressed as arbitrary units of FAS mRNA and malic enzyme and UCP-1 protein content and are means ± SE. D: A representative photograph of each cell type is shown.

FIG. 1.

Characterization of IGF-IR–deficient brown adipocytes. A: Wild-type (IGF-IR+/+) and IGF-I–deficient brown adipocytes (IGF-IR−/−) were cultured to confluence under growing conditions (10% FS), and then cells were lysed. Total protein (50 μg) was submitted to SDS-PAGE and analyzed by Western blotting with the anti–IGF-IR antibody. A representative experiment is shown. B: Quiescent (20 h serum-starved) IGF-IR+/+ and IGF-IR−/− brown adipocytes were stimulated with IGF-I (1–10 nmol/l) for 5 min. Control cells were cultured in the absence of IGF-I. At the end of the culture time, cells were lysed, and 600 μg total protein was immunoprecipitated with the anti–IGF-IR antibody. The resulting immune complexes were analyzed by Western blotting with the anti-Tyr(P) antibody. The results shown are representative of three experiments. C: Cells were cultured to confluence in the presence of DMEM–10% FS. Total RNA (10 μg) was isolated, submitted to Northern blot analysis, and hybridized with FAS and 18S RNA cDNAs. Total protein (50 μg) was submitted to SDS-PAGE and analyzed by Western blotting with the anti-malic enzyme and anti–UCP-1 antibodies. Representative autoradiograms are shown. The autoradiograms corresponding to three independent experiments were quantified by scanning densitometry. Results are expressed as arbitrary units of FAS mRNA and malic enzyme and UCP-1 protein content and are means ± SE. D: A representative photograph of each cell type is shown.

FIG. 2.

IR expression and its tyrosine phosphorylation in IGF-IR–deficient cells. A: Wild-type (IGF-IR+/+) and IGF-I–deficient (IGF-IR−/−) brown adipocytes were cultured to confluence under growing conditions (10% FS) and then serum-starved for 20 h. Cells were lysed, and total protein (50 μg) was submitted to SDS-PAGE and analyzed by Western blotting with the anti-IR α-chain antibody. A representative experiment is shown. B: Top: Quiescent (20 h serum-starved) IGF-IR+/+ and IGF-IR−/− brown adipocytes were stimulated with insulin (1, 10, and 100 nmol/l) for 5 min. Control cells were cultured in the absence of the hormone. At the end of the culture time, cells were lysed and 600 μg of total protein was immunoprecipitated with the anti-IR β-chain antibody. The resulting immune complexes were analyzed by Western blotting with the anti-Tyr(P) antibody or the anti-IR β-chain antibody. Bottom: Quiescent IGF-IR−/− brown adipocytes were stimulated with insulin (0.01–10 nmol/l) for 5 min. Control cells were cultured in the absence of the hormone. At the end of the culture time, cells were lysed, and 600 μg total protein was immunoprecipitated with the anti-IR β-chain antibody. The resulting immune complexes were analyzed by Western blotting with the anti-Tyr(P) antibody or the anti-IR β-chain antibody. The autoradiograms corresponding to three independent experiments were quantified by scanning densitometry. Results are expressed as arbitrary units of phosphorylated IR β-chain and are means ± SE.

FIG. 2.

IR expression and its tyrosine phosphorylation in IGF-IR–deficient cells. A: Wild-type (IGF-IR+/+) and IGF-I–deficient (IGF-IR−/−) brown adipocytes were cultured to confluence under growing conditions (10% FS) and then serum-starved for 20 h. Cells were lysed, and total protein (50 μg) was submitted to SDS-PAGE and analyzed by Western blotting with the anti-IR α-chain antibody. A representative experiment is shown. B: Top: Quiescent (20 h serum-starved) IGF-IR+/+ and IGF-IR−/− brown adipocytes were stimulated with insulin (1, 10, and 100 nmol/l) for 5 min. Control cells were cultured in the absence of the hormone. At the end of the culture time, cells were lysed and 600 μg of total protein was immunoprecipitated with the anti-IR β-chain antibody. The resulting immune complexes were analyzed by Western blotting with the anti-Tyr(P) antibody or the anti-IR β-chain antibody. Bottom: Quiescent IGF-IR−/− brown adipocytes were stimulated with insulin (0.01–10 nmol/l) for 5 min. Control cells were cultured in the absence of the hormone. At the end of the culture time, cells were lysed, and 600 μg total protein was immunoprecipitated with the anti-IR β-chain antibody. The resulting immune complexes were analyzed by Western blotting with the anti-Tyr(P) antibody or the anti-IR β-chain antibody. The autoradiograms corresponding to three independent experiments were quantified by scanning densitometry. Results are expressed as arbitrary units of phosphorylated IR β-chain and are means ± SE.

FIG. 3.

Insulin effect on IRS-1/2 expression and tyrosine phosphorylation in wild-type and IGF-IR–deficient brown adipocytes. Wild-type and IGF-I–deficient brown adipocytes were cultured to confluence under growing conditions (10% FS) and then serum-starved for 20 h. Cells were lysed, and total protein (50 μg) was submitted to SDS-PAGE and analyzed by Western blotting with the corresponding antibodies against IRS-1 (A) and IRS-2 (B). Representative autoradiograms are shown. Quiescent (20 h serum-starved) wild-type (IGF-IR+/+) and IGF-IR–deficient (IGF-IR−/−) brown adipocytes were stimulated with insulin or IGF-I (10 nmol/l) for 5 min. At the end of the culture time, cells were lysed, and 600 μg total protein was immunoprecipitated with the anti–IRS-1 (A) or anti–IRS-2 (B) antibodies. The resulting immune complexes were analyzed by Western blotting with the anti-Tyr(P), anti–IRS-1, and anti–IRS-2 antibodies. The autoradiograms corresponding to three independent experiments were quantified by scanning densitometry. Results are expressed as arbitrary units of IRS-1 and IRS-2 tyrosine phosphorylation (normalized by the amount of total IRS-1 or IRS-2) and are means ± SE.

FIG. 3.

Insulin effect on IRS-1/2 expression and tyrosine phosphorylation in wild-type and IGF-IR–deficient brown adipocytes. Wild-type and IGF-I–deficient brown adipocytes were cultured to confluence under growing conditions (10% FS) and then serum-starved for 20 h. Cells were lysed, and total protein (50 μg) was submitted to SDS-PAGE and analyzed by Western blotting with the corresponding antibodies against IRS-1 (A) and IRS-2 (B). Representative autoradiograms are shown. Quiescent (20 h serum-starved) wild-type (IGF-IR+/+) and IGF-IR–deficient (IGF-IR−/−) brown adipocytes were stimulated with insulin or IGF-I (10 nmol/l) for 5 min. At the end of the culture time, cells were lysed, and 600 μg total protein was immunoprecipitated with the anti–IRS-1 (A) or anti–IRS-2 (B) antibodies. The resulting immune complexes were analyzed by Western blotting with the anti-Tyr(P), anti–IRS-1, and anti–IRS-2 antibodies. The autoradiograms corresponding to three independent experiments were quantified by scanning densitometry. Results are expressed as arbitrary units of IRS-1 and IRS-2 tyrosine phosphorylation (normalized by the amount of total IRS-1 or IRS-2) and are means ± SE.

FIG. 4.

Insulin effect on IRS-1/2 association with Grb-2 in wild-type and IGF-IR–deficient brown adipocytes. Top: Quiescent (20 h serum-starved) wild-type (IGF-IR+/+) and IGF-IR–deficient (IGF-IR−/−) brown adipocytes were stimulated with insulin (1, 10, and 100 nmol/l) for 5 min. Control cells were cultured in the absence of the hormone. Cell lysates were immunoprecipitated with the anti–Grb-2 antibody and analyzed by Western blotting with the anti-Tyr(P) and anti–Grb-2 antibodies. The position of IRS-1/2 is indicated by an arrowhead. Bottom: Quiescent IGF-IR−/− brown adipocytes were stimulated with insulin (0.001–10 nmol/l) for 5 min. Control cells were cultured in the absence of the hormone. At the end of the culture time, cells were lysed, and 600 μg total protein was immunoprecipitated with the anti–Grb-2 antibody. The resulting immune complexes were analyzed by Western blotting with the anti-Tyr(P) antibody or with the anti–Grb-2 antibody. The autoradiograms corresponding to three independent experiments were quantified by scanning densitometry. Results are expressed as arbitrary units of Grb-2–associated IRS-1/2 and are means ± SE.

FIG. 4.

Insulin effect on IRS-1/2 association with Grb-2 in wild-type and IGF-IR–deficient brown adipocytes. Top: Quiescent (20 h serum-starved) wild-type (IGF-IR+/+) and IGF-IR–deficient (IGF-IR−/−) brown adipocytes were stimulated with insulin (1, 10, and 100 nmol/l) for 5 min. Control cells were cultured in the absence of the hormone. Cell lysates were immunoprecipitated with the anti–Grb-2 antibody and analyzed by Western blotting with the anti-Tyr(P) and anti–Grb-2 antibodies. The position of IRS-1/2 is indicated by an arrowhead. Bottom: Quiescent IGF-IR−/− brown adipocytes were stimulated with insulin (0.001–10 nmol/l) for 5 min. Control cells were cultured in the absence of the hormone. At the end of the culture time, cells were lysed, and 600 μg total protein was immunoprecipitated with the anti–Grb-2 antibody. The resulting immune complexes were analyzed by Western blotting with the anti-Tyr(P) antibody or with the anti–Grb-2 antibody. The autoradiograms corresponding to three independent experiments were quantified by scanning densitometry. Results are expressed as arbitrary units of Grb-2–associated IRS-1/2 and are means ± SE.

FIG. 5.

Insulin effect on SHC tyrosine phosphorylation and its association with Grb-2 in wild-type and IGF-IR–deficient brown adipocytes. A: Wild-type (IGF-IR+/+) and IGF-I–deficient (IGF-IR−/−) brown adipocytes were cultured to confluence under growing conditions (10% FS) and then serum-starved for 20 h. Cells were lysed, and total protein (50 μg) was submitted to SDS-PAGE and analyzed by Western blotting with the anti-SHC antibody. A representative experiment is shown. B: Quiescent (20 h serum-starved) wild-type (IGF-IR+/+) and IGF-IR–deficient (IGF-IR−/−) brown adipocytes were stimulated with insulin (1–100 nmol/l) for 5 min. Control cells were cultured in the absence of the hormone. At the end of the culture time, cells were lysed, and 600 μg total protein was immunoprecipitated with the anti-SHC antibody. The resulting immune complexes were analyzed by Western blotting with the anti-Tyr(P), anti–Grb-2, and anti-SHC antibodies. The positions of p66, p52, and p46 SHC proteins and Grb-2 are indicated by an arrowhead. A representative experiment of three is shown.

FIG. 5.

Insulin effect on SHC tyrosine phosphorylation and its association with Grb-2 in wild-type and IGF-IR–deficient brown adipocytes. A: Wild-type (IGF-IR+/+) and IGF-I–deficient (IGF-IR−/−) brown adipocytes were cultured to confluence under growing conditions (10% FS) and then serum-starved for 20 h. Cells were lysed, and total protein (50 μg) was submitted to SDS-PAGE and analyzed by Western blotting with the anti-SHC antibody. A representative experiment is shown. B: Quiescent (20 h serum-starved) wild-type (IGF-IR+/+) and IGF-IR–deficient (IGF-IR−/−) brown adipocytes were stimulated with insulin (1–100 nmol/l) for 5 min. Control cells were cultured in the absence of the hormone. At the end of the culture time, cells were lysed, and 600 μg total protein was immunoprecipitated with the anti-SHC antibody. The resulting immune complexes were analyzed by Western blotting with the anti-Tyr(P), anti–Grb-2, and anti-SHC antibodies. The positions of p66, p52, and p46 SHC proteins and Grb-2 are indicated by an arrowhead. A representative experiment of three is shown.

FIG. 6.

Insulin activation of MAPK cascade in IGF-IR–deficient brown adipocytes. Top: Quiescent (20 h serum-starved) wild-type (IGF-IR+/+) and IGF-I–deficient (IGF-IR−/−) brown adipocytes were stimulated with insulin (1, 10, and 100 nmol/l) for 5 min. Control cells were cultured in the absence of the hormone. Then cells were lysed, and equal amounts of protein (50 μg) were submitted to SDS-PAGE followed by Western blot analysis with the anti-phospho MEK1/2 and anti-MEK1/2 and anti-phospho MAPK and MAPK antibodies. Bottom: Quiescent IGF-IR–deficient brown adipocytes were stimulated with insulin (0.001–10 nmol/l) for 5 min. Control cells were cultured in the absence of the hormone. Then cells were lysed, and equal amounts of protein (50 μg) were submitted to SDS-PAGE followed by Western blot analysis with the anti-phospho MEK1/2 and anti-MEK1/2 and anti-phospho MAPK and MAPK antibodies. The autoradiograms corresponding to three independent experiments were quantified by scanning densitometry. Results are expressed as arbitrary units of phosphorylated MEK1/2 and phosphorylated p42/p44 MAPK and are means ± SE.

FIG. 6.

Insulin activation of MAPK cascade in IGF-IR–deficient brown adipocytes. Top: Quiescent (20 h serum-starved) wild-type (IGF-IR+/+) and IGF-I–deficient (IGF-IR−/−) brown adipocytes were stimulated with insulin (1, 10, and 100 nmol/l) for 5 min. Control cells were cultured in the absence of the hormone. Then cells were lysed, and equal amounts of protein (50 μg) were submitted to SDS-PAGE followed by Western blot analysis with the anti-phospho MEK1/2 and anti-MEK1/2 and anti-phospho MAPK and MAPK antibodies. Bottom: Quiescent IGF-IR–deficient brown adipocytes were stimulated with insulin (0.001–10 nmol/l) for 5 min. Control cells were cultured in the absence of the hormone. Then cells were lysed, and equal amounts of protein (50 μg) were submitted to SDS-PAGE followed by Western blot analysis with the anti-phospho MEK1/2 and anti-MEK1/2 and anti-phospho MAPK and MAPK antibodies. The autoradiograms corresponding to three independent experiments were quantified by scanning densitometry. Results are expressed as arbitrary units of phosphorylated MEK1/2 and phosphorylated p42/p44 MAPK and are means ± SE.

FIG. 7.

Mitogenic response to insulin in wild-type and IGF-IR–deficient brown adipocytes. A: Wild-type (IGF-IR+/+) and IGF-IR–deficient (IGF-IR−/−) brown adipocytes were cultured for 24 h in a serum-free medium in either the absence or presence of insulin (1–100 nmol/l). DNA synthesis was determined by [3H]thymidine incorporation (0.2 μCi/ml) over the last 4 h of culture. After two washes with ice-cold PBS, cells were lysed and trichloroacetate-precipitable DNA was then counted for incorporated radioactivity. Results are expressed as cpm/dish and are means ± SE from six independent experiments, each one performed in triplicate. B: Cells were cultured for 24 h in either the absence or presence of various doses of insulin as described in A. At the end of the culture time, cells were trypsinized and counted. Results are expressed as fold increase in cell number and are means ± SE from six independent experiments, each one performed in duplicate. C: Cells were cultured for 24 h in either the absence or presence of various doses of insulin as described in A. At the end of the culture time, cells were lysed and total protein (50 μg) was submitted to SDS-PAGE and Western blotting with the anti-PCNA antibody. The autoradiograms corresponding to three independent experiments were quantified by scanning densitometry. Results are expressed as arbitrary units of PCNA expression and are means ± SE.

FIG. 7.

Mitogenic response to insulin in wild-type and IGF-IR–deficient brown adipocytes. A: Wild-type (IGF-IR+/+) and IGF-IR–deficient (IGF-IR−/−) brown adipocytes were cultured for 24 h in a serum-free medium in either the absence or presence of insulin (1–100 nmol/l). DNA synthesis was determined by [3H]thymidine incorporation (0.2 μCi/ml) over the last 4 h of culture. After two washes with ice-cold PBS, cells were lysed and trichloroacetate-precipitable DNA was then counted for incorporated radioactivity. Results are expressed as cpm/dish and are means ± SE from six independent experiments, each one performed in triplicate. B: Cells were cultured for 24 h in either the absence or presence of various doses of insulin as described in A. At the end of the culture time, cells were trypsinized and counted. Results are expressed as fold increase in cell number and are means ± SE from six independent experiments, each one performed in duplicate. C: Cells were cultured for 24 h in either the absence or presence of various doses of insulin as described in A. At the end of the culture time, cells were lysed and total protein (50 μg) was submitted to SDS-PAGE and Western blotting with the anti-PCNA antibody. The autoradiograms corresponding to three independent experiments were quantified by scanning densitometry. Results are expressed as arbitrary units of PCNA expression and are means ± SE.

FIG. 8.

PTP1B expression and its association with IR, IRS-1, and Grb-2 upon insulin stimulation in wild-type and IGF-IR–deficient brown adipocytes. A: Wild-type (IGF-IR+/+) and IGF-I–deficient (IGF-IR−/−) brown adipocytes were cultured to confluence under growing conditions (10% FS) and then serum-starved for 20 h. Cells were lysed, and total protein (50 μg) was submitted to SDS-PAGE and analyzed by Western blotting with the anti-PTP1B. A representative experiment is shown. B: Quiescent (20 h serum-starved) wild-type (IGF-IR+/+) and IGF-IR–deficient (IGF-IR−/−) brown adipocytes were stimulated with various doses of insulin (1–100 nmol/l) for 5 min. Control cells were cultured in the absence of the hormone. At the end of the culture time, cells were lysed, and 600 μg total protein was immunoprecipitated with PTP1B antibody. The resulting immune complexes were analyzed by Western blotting with the anti-IR, anti–IRS-1, anti–Grb-2, and anti-PTP1B antibodies. The positions of IR, IRS-1, Grb-2, and PTP1B are indicated by an arrowhead. The results shown are representative of three experiments. C: Quiescent (20 h serum-starved) wild-type (IGF-IR+/+) and IGF-IR–deficient (IGF-IR−/−) brown adipocytes were stimulated with various doses of insulin (10–100 nmol/l) for 5 min. At the end of the culture time, PTP1B phosphatase activity was measured as described in research design and methods. Results are expressed as percentage of stimulation of phosphatase activity in insulin-stimulated cells as compared with control cells and are means ± SE from three independent experiments.

FIG. 8.

PTP1B expression and its association with IR, IRS-1, and Grb-2 upon insulin stimulation in wild-type and IGF-IR–deficient brown adipocytes. A: Wild-type (IGF-IR+/+) and IGF-I–deficient (IGF-IR−/−) brown adipocytes were cultured to confluence under growing conditions (10% FS) and then serum-starved for 20 h. Cells were lysed, and total protein (50 μg) was submitted to SDS-PAGE and analyzed by Western blotting with the anti-PTP1B. A representative experiment is shown. B: Quiescent (20 h serum-starved) wild-type (IGF-IR+/+) and IGF-IR–deficient (IGF-IR−/−) brown adipocytes were stimulated with various doses of insulin (1–100 nmol/l) for 5 min. Control cells were cultured in the absence of the hormone. At the end of the culture time, cells were lysed, and 600 μg total protein was immunoprecipitated with PTP1B antibody. The resulting immune complexes were analyzed by Western blotting with the anti-IR, anti–IRS-1, anti–Grb-2, and anti-PTP1B antibodies. The positions of IR, IRS-1, Grb-2, and PTP1B are indicated by an arrowhead. The results shown are representative of three experiments. C: Quiescent (20 h serum-starved) wild-type (IGF-IR+/+) and IGF-IR–deficient (IGF-IR−/−) brown adipocytes were stimulated with various doses of insulin (10–100 nmol/l) for 5 min. At the end of the culture time, PTP1B phosphatase activity was measured as described in research design and methods. Results are expressed as percentage of stimulation of phosphatase activity in insulin-stimulated cells as compared with control cells and are means ± SE from three independent experiments.

This work was supported by grant PM97-0050 from the Ministerio de Educación y Cultura, Spain. We recognize the valuable technical skill of M. López.

1.
Ullrich A, Gray A, Tam AW, Yang-Feng T, Tsubokawa M, Collins C, Henzel W, Bon TL, Kathuria S, Chen E, Jacobs S, Francke U, Ramachandran J, Fujita-Yamaguchi Y: Insulin-like growth factor receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity.
EMBO J
5
:
2503
–2512,
1986
2.
Kasuga M, Karlsson FA, Kahn CR: Insulin stimulates the phosphorylation of the 95,000-dalton subunit of its own receptor.
Science
215
:
185
–187,
1982
3.
White MF, Kahn CR: The insulin signaling system.
J Biol Chem
269
:
1
–4,
1984
4.
White MF: The insulin signalling system and the IRS proteins.
Diabetologia
40
:
S2
–S17,
1987
5.
Lavan BE, Lane WS, Lienhard GE: The 60-kDa phosphotyrosine protein in insulin-treated adipocytes is a new member of the insulin receptor substrate family.
J Biol Chem
272
:
11439
–11443,
1997
6.
Lavan BE, Fantin WR, Chang ET, Lane WS, Keller SR, Lienhard GE: A novel 160-kDa phosphotyrosine protein in insulin-treated embryonic kidney cells is a new member of the insulin receptor substrate family.
J Biol Chem
272
:
21403
–21407,
1997
7.
Pelicci GL, Lanfrancone L, Grignani F, McGlade J, Cavallo F, Forni G, Nicoletti I, Pawson T, Pelicci PG: A novel transforming protein (SHC) with an SH2 domain is implicated in mitogenic signal transduction.
Cell
70
:
93
–104,
1992
8.
Backer JM, Myers MG Jr, Schoelson SE, Chin DJ, Sun XJ, Miralpeix M, Hu P, Margolis B, Skolnik EY, Schlessinger J, White MF: The phosphatidylinositol 3-kinase is activated by association with IRS-1 during insulin stimulation.
EMBO J
11
:
3469
–3479,
1992
9.
Skolnik EY, Lee CH, Batzer AG, Vicentini LM, Zhou M, Daly RJ, Myers MG Jr, Backer JM, Ullrich A, White MF, Schlessinger J: The SH2/SH3 domain-containing protein GRB-2 interacts with tyrosine phosphorylated IRS-1 and Shc: implications for insulin control of ras signalling.
EMBO J
12
:
1929
–1936,
1993
10.
Kuhne MR, Pawson T, Lienhard GE, Feng GS: The insulin receptor substrate 1 associates with the SH2-containing phosphotyrosine phosphatase Syp.
J Biol Chem
268
:
11479
–11481,
1993
11.
Rozakis-Adock M, McGlade J, Mbamala G, Pelicci G, Daly R, Li W, Batzer A, Thomas S, Brugge J, Pelicci PG, Schlessinger J, Pawson T: Association of the Shc and GRB2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases.
Nature
360
:
689
–692,
1992
12.
Liu JP, Backer J, Perkins AS, Robertson EJ, Efstradiatis A: Mice carrying null mutations of the genes encoding insulin-like growth factor I (IGF-I) and type 1 IGF-I receptor.
Cell
75
:
59
–72,
1993
13.
Joshi RL, Lamothe B, Cordonnier N, Mesbah K, Monthioux E, Jami J, Bucchini D: Targeted disruption of the insulin receptor gene in the mouse results in neonatal lethality.
EMBO J
15
:
1542
–1547,
1996
14.
Araki E, Lipes MA, Patti ME, Brüning JC, Haag BL 3rd, Johnson RS, Kahn CR: Alternative pathway of insulin signalling in mice made with targeted disruption of the IRS-1 gene.
Nature
372
:
186
–190,
1994
15.
Tamemoto H, Kadowaki T, Tobe K, Yagi T, Sakura H, Hayakawa T, Terauchi Y, Ueki K, Yaburagi Y, Satoh S, Sekihara H, Yoshiok S, Horikoshi H, Furuta Y, Ikawa Y, Kasuga M, Yazaki Y, Aizawa S: Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1.
Nature
372
:
182
–186,
1994
16.
Whithers DJ, Gutierrez JS, Towery H, Burks DJ, Ren JM, Previs S, Zhang Y, Bernal D, Pons S, Shulman GI, Bonner-Weir S, White MF: Disruption of IRS-2 causes type 2 diabetes in mice.
Nature
391
:
900
–903,
1998
17.
Ailhaud G, Grimaldi P, Nègrel R: Cellular and molecular aspects of adipose tissue development.
Ann Rev Nutr
12
:
207
–233,
1992
18.
Lorenzo M, Valverde AM, Teruel T, Benito M: IGF-I is a mitogen also involved in differentiation-related gene expression in fetal brown adipocytes.
J Cell Biol
123
:
1567
–1575,
1993
19.
Valverde AM, Lorenzo M, Teruel T, Benito M: cAMP inhibits IGF-I-induced proliferation in fetal rat brown adipocytes.
Exp Cell Res
218
:
305
–309,
1995
20.
Porras A, Alvarez AM, Valladares A, Benito M: P42/p44 mitogen-activated protein kinases activation is required for the insulin-like growth factor-I/insulin induced proliferation, but inhibits differentiation, in rat fetal brown adipocytes.
Mol Endocrinol
12
:
825
–834,
1998
21.
Teruel T, Valverde AM, Benito M, Lorenzo M: Insulin-like growth factor-I and insulin induced adipogenic-related gene expression in foetal brown adipocyte primary cultures.
Biochem J
319
:
627
–632,
1996
22.
Valverde AM, Lorenzo M, Navarro P, Benito M: 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,
1997
23.
Valverde AM, Teruel T, Lorenzo M, Benito M: Involvement of Raf-1 kinase and protein kinase Cζ in insulin-like growth factor-induced mitogenesis in brown adipocyte primary cultures.
Endocrinology
137
:
3832
–3841,
1996
24.
Valverde AM, Lorenzo M, Pons S, White MF, Benito M: IRS-1 and IRS-2 differential signaling in the insulin/IGF-I pathways in fetal brown adipocytes.
Mol Endocrinol
12
:
688
–697,
1998
25.
Valverde AM, Kahn CR, Benito M: Insulin signaling in IRS-1-deficient brown adipocytes: requirement of IRS-1 for lipid synthesis.
Diabetes
48
:
2122
–2131,
1999
26.
Fasshauer M, Klein J, Ueki K, Kriauciunas C, Benito M, White MF, Kahn CR: Essential role of IRS-2 in insulin stimulation of Glut4 translocation and glucose uptake in brown adipocytes.
J Biol Chem
275
:
25494
–25501,
2000
27.
Bradford M: A rapid and sensitive method for the quantitation of microgram quantities utilizing the principle of protein dye binding.
Anal Biochem
72
:
248
–254,
1976
28.
Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162
:
156
–159,
1987
29.
Baserga R: Growth regulation of the PCNA gene.
J Cell Sci
98
:
433
–436,
1991
30.
Goldstein BJ, Bittner-Kowalczyk A, White MF, Harbeck M: Tyrosine dephosphorylation and deactivation of insulin receptor substrate-1 by protein-tyrosine phosphatase 1B: possible facilitation by the formation of a ternary complex with the Grb-2 adaptor molecule.
J Biol Chem
11
:
4283
–4289,
2000
31.
Sell C, Rubini M, Rubin R, Liu JP, Efstratiadis A, Baserga R: Simian virus 40 large tumor antigen is unable to transform mouse embryonic fibroblasts lacking type 1 insulin-like growth factor receptor.
Proc Natl Acad Sci U S A
90
:
11217
–11221,
1993
32.
Morrione A, Valentinis B, Xu S-Q, Yumet G, Louvi A, Efstratiadis A, Baserga R: Insulin-like growth factor II stimulates cell proliferation through the insulin receptor.
Proc Natl Acad Sci U S A
94
:
3777
–3782,
1997
33.
Rother KI, Imai Y, Caruso M, Beguinot F, Formisano P, Accili D: Evidence that IRS-2 phosphorylation is required for insulin action in hepatocytes.
J Biol Chem
273
:
17491
–17497,
1998
34.
Lamothe B, Baudry A, Christoffersen CT, De Meyts P, Jami J, Bucchini D, Joshi RL: Insulin receptor-deficient cells as a new tool for dissecting complex interplay in insulin and insulin-like growth factors.
FEBS Lett
426
:
381
–385,
1998
35.
He W, Craparo A, Zhu Y, O’Neill TJ, Wang LM, Pierce JH, Gustafson TA: Interaction of insulin receptor substrate-2 (IRS-2) with the insulin and insulin-like growth factor I receptors.
J Biol Chem
271
:
11641
–11645,
1996
36.
Sawka-Verhelle D, Tartare-Decker S, White MF, Van Obberghen E: Insulin receptor substrate-2 binds to insulin receptor through its phosphotyrosine-binding domain and through a newly identified domain comprising amino acids 591–786.
J Biol Chem
271
:
5980
–5983,
1996
37.
Sawka-Verhelle D, Baron D, Mothe I, Filloux C, White MF, Van Obberghen E: Tyr624 and Tyr628 in insulin receptor substrate-2 mediated its association with the insulin receptor.
J Biol Chem
272
:
16414
–16420,
1997
38.
Taouis M, Dupont J, Gillet A, Derouet M, Simon J: Insulin receptor substrate-1 antisense expression in an hepatoma cell line reduces cell proliferation and induces overexpression of the Src homology 2 domain and collagen protein (SHC).
Mol Cell Endocrinol
137
:
177
–186,
1998
39.
Valverde AM, Mur C, Pons S, Alvarez A, White MF, Kahn CR, Benito M: Y895IRS-1/GRB-2 association mediates the insulin signaling involved in IRS-1-deficient brown adipocytes mitogenesis.
Mol Cell Biol
21
:
2269
–2280,
2001
40.
Sasaoka T, Rose DW, Jhun BH, Saltiel AR, Draznin B, Olefsky JM: Evidence for a functional role of Shc proteins in mitogenic signaling induced by insulin, insulin-like growth factor-1, and epidermal growth factor.
J Biol Chem
269
:
13689
–13694,
1994
41.
Sasaoka T, Ishiki M, Sawa T, Ishihara H, Takata Y, Imamura T, Usui I, Olefsky JM, Kobayashi M: Comparison in the insulin and insulin-like growth factor-1 mitogenic intracellular signaling pathways.
Endocrinology
137
:
4427
–4434,
1996
42.
Benjamin CW, Jones DA: Platelet-derived growth factor stimulates growth factor receptor binding protein-2 association with Shc in vascular smooth muscle cells.
J Biol Chem
269
:
30911
–30916,
1994
43.
Goldstein BJ, Ahmad F, Ding W, Li PM, Zhang WR: Regulation of the insulin signalling pathways by cellular protein-tyrosine phosphatases.
Mol Cell Biochem
182
:
91
–99,
1998
44.
Seely BL, Staubs PA, Reichart DR, Berhanu P, Milarski KL, Saltiel AR, Kusari J, Olefsky JM: Protein tyrosine phosphatase 1B interacts with the activated insulin receptor.
Diabetes
45
:
1379
–1385,
1996
45.
Bandyopadhyay D, Kusari A, Kenner KA, Liu F, Chernoff J, Gustafson TA, Kusari J: Protein-tyrosine phosphatase 1B complexes with the insulin receptor in vivo and is tyrosine-phosphorylated in the presence of insulin.
J Biol Chem
272
:
1639
–1645,
1997
46.
Dadke S, Kusari J, Chernoff J: Down-regulation of insulin signaling by protein-tyrosine phosphatase 1B is mediated by an N-terminal binding region.
J Biol Chem
275
:
23642
–23647,
2000
47.
Elchebly M, Payette P, Michaliszyn E, Cromlish W, Collins S, Loy AL, Normandin D, Cheng A, Himms-Hagen J, Chan CC, Ramachandran C, Gresser MJ, Tremblay ML, Kennedy BP: Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene.
Science
283
:
1544
–1548,
1999
48.
Klaman LD, Boss O, Peroni OD, Kim JK, Martino JL, Zabolotny JM, Moghal N, Lubkin M, Kim YB, Sharpe AH, Stricker-Krongrad A, Shulman GI, Neel BG, Kahn BB: Increased energy expenditure, decreased adiposity and tissue-specific insulin sensitivity in protein-tyrosine phosphatase 1B-deficient mice.
Mol Cell Biol
20
:
5479
–5489,
2000

Address correspondence and reprint requests to Manuel Benito, Departamento de Bioquímica y Biología Molecular, Centro Mixto CSIC/UCM, Facultad de Farmacia, Ciudad Universitaria, 28040 Madrid, Spain. E-mail: benito@eucmax.sim.ucm.es.

Received for publication 6 December 2000 and accepted in revised form 19 November 2001.

C.M. and A.M.V. contributed equally to this work. C.R.K. is a member of an advisory panel for Abbott Millenium.

BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; FAS, fatty acid synthase; FS, fetal serum; Grb-2, growth factor receptor–binding protein-2; IGF, insulin-like growth factor; IR, insulin receptor; IRS, insulin receptor substrate; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline; PTP1B, phosphotyrosine phosphatase 1B; UCP-1, uncoupling protein-1.