Increased Insulin Sensitivity in IGF-I Receptor–Deficient Brown Adipocytes

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.

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).

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.

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).

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

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.

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.

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.

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.

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.

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).

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).

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.

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).

Insulin/IGF-I are potent mitogens in fetal brown adipocyte primary cultures (18–20). 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.

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 (22–24). 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 (35–37), 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 (40–42), 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 (44–46) 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.

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.