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Mechanism for Differential Effect of Protein-Tyrosine Phosphatase 1B on Akt Versus Mitogen-Activated Protein Kinase in 3T3-L1 Adipocytes

Division of Endocrinology and Metabolism (S.S., K.E., K.S., A.K.), Department of Medicine, Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan; Gonda (Goldschmied) Diabetes Center (M.B.-A.), Division of Endocrinology, Diabetes and Hypertension, Department of Medicine, University of California, Los Angeles, and the West Los Angeles Veterans Administration Medical Center, Los Angeles, California 90095

Address all correspondence and requests for reprints to: Hiroshi Maegawa, M.D., Ph.D., Division of Endocrinology and Metabolism, Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan. E-mail: maegawa{at}belle.shiga-med.ac.jp.

	Abstract

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We investigated the effect of overexpression of protein-tyrosine phosphatase 1B (PTP1B) on insulin signaling in 3T3-L1 adipocytes. Overexpression of a wild-type PTP1B in L1 adipocytes as well as in L6 myocytes, led to a profound decrease in insulin-stimulated phosphorylation of MAPK. Even though the decrease in insulin receptor substrate protein-1 (IRS-1) phosphorylation was identical with that seen in L6 myocytes, overexpression of wild-type PTP1B in L1 adipocytes was associated with modest impairment of insulin-stimulated Akt phosphorylation in addition to a small, but significant, attenuation in insulin-stimulated glucose uptake, when compared with a phosphatase-negative mutant. Regarding the relatively small effect on Akt phosphorylation, we obtained identical results in rat 1 fibroblasts overexpressing human insulin receptor, suggesting that the higher expression levels of insulin receptor and IRS-1 might be responsible. With regard to the large effect on MAPK phosphorylation, we found that PTP1B overexpression led to the impaired phosphorylation of both IRS-1 and Shc, resulting in a decrease in their association with Grb2. Furthermore, phosphorylation of Shc stimulated by platelet-derived growth factor was also attenuated, without any change in its receptors, suggesting that PTP1B directly regulates Shc phosphorylation. These data demonstrate that PTP1B negatively regulates insulin signaling in the MAPK cascade to a much greater extent than the Akt pathway in some cell lines, especially in L1 adipocytes.

	Introduction

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AFTER INSULIN BINDS to its own receptor, the insulin receptor (IR) is tyrosine phosphorylated, and its tyrosine kinase activity is activated. The activated IR binds to the insulin receptor substrate proteins (IRSs) and Shc protein. These docking proteins are also phosphorylated on tyrosine residues. The tyrosine-phosphorylated IRSs and Shc protein then activate their downstream signaling pathways, such as phosphatidylinositol-3 kinase (PI-3 kinase) and the MAPK cascade (1). Because tyrosine phosphorylation is essential for insulin signal transduction, the balance of enzyme activity between protein tyrosine kinases and protein tyrosine phosphatases (PTPases) seems critical for mediation of insulin’s effects and in the pathogenesis of insulin-resistant states.

Several lines of evidence demonstrate that PTPase activity is abnormally up-regulated in adipose tissue, skeletal muscle, and liver in insulin-resistant states, and that there is an inverse relationship between PTPase activity and insulin sensitivity in human and rodent models, even though the controversial findings exist (2, 3). Regarding candidate PTPases for the regulation of the insulin signaling pathway, a role for the tandem-domain transmembrane enzymes, leukocyte antigen related (LAR), and leukocyte common antigen-related phosphatase (LRP)/receptor PTP- and the intracellular single domain enzymes, protein-tyrosine phosphatase 1B (PTP1B) and SHP2, have been postulated (4, 5).

In particular, PTP1B directly interacts with the activated IR and exhibits the highest specific activity to IRS-1 (6, 7). We reported that high glucose conditions impaired the insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 due to the increased expression and activity of PTP1B in rat 1 fibroblasts expressing human IR (HIRc) (8, 9). Furthermore, we recently reported that overexpression of PTP1B in cell culture models of insulin target tissues such as L6 myocytes and Fao hepatoma cells led to impaired insulin-stimulated glucose metabolism (10). Moreover, mice lacking the PTP1B gene show increased insulin sensitivity and resistance to high-fat feeding induced obesity, together with enhanced insulin-induced tyrosine-phosphorylation of the IR and IRS-1 in insulin target tissues (11, 12). However, the increased tyrosine-phosphorylation of IR and IRS was observed in muscle and liver tissues, but not in adipose tissue. Thus, the role of PTP1B in adipose tissue may differ from that in muscle and liver tissues.

In the current study, we employed the adenovirus-mediating gene transfer technique, and analyzed the effect of PTP1B overexpression on insulin signaling in 3T3-L1 adipocytes, and compared it with that seen in other cell lines such as L6 myocytes and HIRc cells. Furthermore, we investigated the mechanism for the inhibitory effects of PTP1B on the MAPK cascade, because the MAPK cascade was found to be more sensitive than the PI-3 kinase cascade. Our data demonstrate that PTP1B negatively regulates insulin signaling, but that the MAPK cascade is much more sensitive to its actions than the Akt pathway in some cell lines, especially in 3T3-L1 adipocytes.

	Materials and Methods

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Materials
Porcine insulin was kindly provided by Eli Lilly (Indianapolis, IN). Anti-PTP1B antibody, antibody to p85 subunit of PI-3 kinase, and anti-IRS-1 antibody were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Antiphospho-Akt antibody and antiphospho-MAPK antibody were from New England Biolabs, Inc. (Beverly, MA). Anti-Shc antibody and anti-Grb2 antibody, horseradish peroxidase-conjugated phosphotyrosine antibody (RC20H) and insulin receptor antibody were from Transduction Laboratories, Inc. (Lexington, KY). Horseradish peroxidase-linked antirabbit and antimouse antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). DMEM and fetal calf serum (FCS), were obtained from Life Technologies, Inc. (Carlsbad, CA). All radioisotopes were obtained from NEN Life Science Products (Boston, MA). XAR-5 film was obtained from Eastman Kodak Co. (Rochester, NY). All other reagents and chemicals were purchased from Sigma (St. Louis, MO).

Cell culture
3T3-L1 preadipocytes were grown and maintained in high glucose DMEM containing 50 U/ml of penicillin, 50 µg/ml of streptomycin, and 10% FCS in a 10% CO₂ environment. The cells were allowed to grow for 2 d post confluency and were then differentiated by addition of the same medium containing isobutylmethylxanthine (500 µM), dexamethasone (25 µM), and insulin (1 µM) for 3 d and in medium containing insulin for an additional 3 d. The medium was then changed every 3 d until the cells were fully differentiated, typically by 15 d. Before experimentation, the adipocytes were trypsinized and reseeded in the appropriate culture dishes. L6 myoblasts were grown and maintained in MEM- containing 50 U/ml of penicillin, 50 µg/ml of streptomycin, and 10% FCS in a 5% CO₂ environment. The cells were reseeded in the appropriate culture dishes and, after reaching subconfluency, the medium was changed to MEM- containing 2% FCS. The medium was then changed every 2 d until the cells were fully differentiated, typically by 5 d. The Ad-E1A-transformed human embryonic kidney cell line 293 was cultured in high glucose DMEM containing 50 U/ml of penicillin, 50 µg/ml of streptomycin, and 10% FCS in a 5% CO₂ environment. Fao hepatoma cells and HIRc cells were maintained in DMEM.

Preparation of recombinant adenovirus
The recombinant adenovirus containing the cDNA encoding the PTP1B wild-type (WT) and cysteine²¹⁵/serine²¹⁵ mutant (MT) were isolated by homologous recombination with two plasmids, pACCMVpLpA and pJM17 as described previously (10, 13). The recombinant plasmid, pAC-PTP1B-WT or pAC-PTP1B-MT, and pJM17 were purified and cotransfected into 293 cells. Because 293 cells were originally derived from adenovirus transformation, the missing E1 gene function of pJM17 was provided in transmission. The resulting recombinant viruses containing the PTP1B-WT and PTP1B-MT were denoted as Ad5-PTP1BWT and Ad5-PTP1B-MT respectively, and were replication defective (at least in cells lacking the E1 region of adenovirus) but fully infectious.

Cell treatment
3T3-L1 adipocytes were infected at a multiplicity of infection (MOI) of 10–40 plaque formation units/cell for 16 h with stocks of either a control recombinant adenovirus (Ad5-ctrl) containing the cytomegalovirus promoter, pUC 18 polylinker, a fragment of the Simian virus 40 genome or the recombinant adenovirus containing PTP1B WT (Ad5-PTP1BWT) and Ad5-PTP1B-MT. Tranfected cells were incubated for 56 h at 37 C in 10% CO₂ and high glucose DMEM with 2% heat-inactivated serum, followed by incubation in the starvation media required for the assay. L6 myocytes were infected at 10–50 MOI for 1 h, and Fao cells were at 10–20 MOI for 1 h. The cells were incubated for 56 h at 37 C in 5% CO₂ and appropriate medium with 2% heat-inactivated serum, followed by incubation in the starvation media required for the assay. The efficiency of adenovirus-mediated gene transfer was approximately 90%, as measured by immunocytochemistry.

Western blotting
Ad5-ctrl, Ad5-PTP1B-WT, or Ad5-PTP1B-MT-infected cells were starved for 16 h in regular glucose DMEM with 0.05% FCS. The cells were stimulated with 16.7 nM insulin for 5–10 min at 37 C and lysed in a solubilizing buffer containing 20 mM Tris, 1 mM EDTA, 140 mM NaCl, 1% Nonidet P-40, 50 U/ml of aprotinin, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonylfluoride, 50 mM NaF (pH 7.5) for 30 min at 4 C. The cell lysates were centrifuged to remove insoluble materials. For Western blot analysis, whole-cell lysates (20 µg protein per lane) were denatured by boiling in Laemmli sample buffer containing 100 mM dithiothreitol and resolved by SDS-PAGE. Gels were transferred to nitrocellulose by electroblotting in Towbin buffer containing 20% methanol. For immunoblotting, membranes were blocked and probed with specified antibodies. Blots were then incubated with horseradish peroxidase-linked second antibody followed by chemiluminescence detection, according to the manufacturer’s instructions (Amersham Pharmacia Biotech, Arlington Heights, IL).

Measurement of PTPase activity
Differentiated 3T3-L1 adipocytes were infected with Ad5-PTP1B-WT or Ad5-PTP1B-MT at the indicated MOI for 16 h and grown in medium containing heat-inactivated serum (2%) for 72 h. PTPase activity was measured using p-nitrophenyl phosphate (pNPP) as substrate according the modified method of Venable et al. (14). In briefly, transfected cells were lysed with the buffer [20 mM Tris, pH 7.5, containing 140 mM NaCl, 1 mM EDTA, 1% (vol/vol) Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin]. Equal aliquots of the cell extracts (100 µg of total protein in 200 µl of the buffer) were incubated with 1 µg of anti-PTP1B antibody at 4 C for 1 h with constant rotation. Then, 30 µl of Protein A/G Plus-agarose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added, and the incubation was continued for an additional 2 h. The immunoprecipitant was collected by centrifugation at 14,000 x g for 5 min. The pellets were washed three times in the buffer (20 mM Tris, pH 7.5; 10 mM dithiothreitol) and then incubated with 50 µl of the buffer containing 10 mM pNPP for 1 h at 37 C. Reactions were terminated by the addition of 1 M NaOH. PTP1B activity was then determined by absorbance at 405 nm.

2-Deoxyglucose transport
The procedure for glucose transport was a modification of a previously described method (15). Differentiated 3T3-L1 adipocytes were infected with Ad5-PTP1B-WT or Ad5-PTP1B-MT at the 50 MOI for 16 h, and grown in medium containing heat-inactivated serum (2%) for 72 h. Cells were then incubated in FCS and glucose free MEM- in the absence (basal) or presence of indicated concentrations of insulin for 1 h at 37 C. Glucose uptake was determined in duplicate or triplicate at each time point after the addition of 10 µl substrate (2-[³H]deoxyglucose or L-[³H]glucose; 0.1 µCi, final concentration 0.01 mM) to provide a concentration at which cell membrane transport is rate limiting. The value for L-glucose was subtracted to correct each sample for the contributions of diffusion and trapping.

Statistics
The values are expressed as mean ± SEM, unless otherwise stated. Scheffé’s multiple comparison test was used to determine the significance of any differences among more than three groups. P < 0.05 was considered significant.

	Results

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Expression of WT and MT PTP1B in 3T3-L1 adipocytes
Differentiated 3T3-L1 adipocytes were infected with recombinant adenovirus expressing WT or MT PTP1B as described in Materials and Methods. Both WT and MT PTP1B were expressed in a dose-dependent manner (Fig. 1A). As shown in Fig. 1B, PTPase activity in the anti-PTP1B antibody immunoprecipitant was also increased in a dose-dependent manner in the case of overexpression of WT PTP1B but was unchanged in PTP1B mutant, as previously described (13).

View larger version (19K): [in this window] [in a new window]   Figure 1. Expression and PTPase activity of PTP1B-WT and PTP1B-MT in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were infected with Ad5-ctrl (ctrl), Ad5-PTP1B-WT, or Ad5-PTP1B-MT at the indicated MOI for 16 h, respectively. After 72 h, the cells were lysed, and analyzed by SDS-PAGE followed by Western blotting with anti-PTP1B antibody (A). PTPase activity in the anti-PTP1B antibody immunoprecipitant was also measured as pNPP as substrate (B). Each bar shows the mean fold-increase ± SE (n = 3) of both expression level (A) and PTPase activity (B) as compared with that in control cells (ctrl).

View larger version (32K): [in this window] [in a new window]   Figure 2. Effects of PTP1B overexpression on tyrosine-phosphorylation of IR and IRS-1, insulin-induced association of IRS-1 with p85 of PI-3 kinase, and insulin-stimulated glucose uptake. Differentiated 3T3-L1 adipocytes were infected with Ad5-ctrl (ctrl), Ad5-PTP1B-WT (WT), or Ad5-PTP1B-MT at 50 MOI for 16 h. After 56 h, the cells were starved for 16 h, and stimulated with or without 16.7 nM insulin for 5 min. Cells were then lysed and immunoprecipitated with anti-IR antibody or anti-IRS-1 antibody. Immunocomplexes were analyzed by Western blotting with either phosphotyrosine antibody (RC20H) (A and B, upper panel), or anti-p85 antibody, respectively (B). The membrane was reblotted with the corresponding antibody. Each Western blot is a representative of at least three independent experiments. Differentiated 3T3-L1 adipocytes were infected with Ad5-ctrl (ctrl), Ad5-PTP1B-WT (WT), or Ad5-PTP1B-MT (MT) at 50 MOI for 16 h. After 72 h, cells were incubated in MEM- in with or without 16.7 nM insulin for 1 h at 37 C. 2-Deoxy-glucose uptake was then measured (C). The graph shows mean ± SE of five experiments.

Differential effects of PTP1B expression on insulin-stimulated Akt and MAPK phosphorylation
Expression of WT PTP1B was associated with only a minor effect on insulin-stimulated Akt phosphorylation in 3T3-L1 adipocytes (Fig. 3, A and B), even though tyrosine phosphorylation of the IR and IRS-1, and IRS-1 association with p85 subunit was decreased by 44.0 ± 10.0% (P < 0.05; Fig. 2, A and B). On the other hand, expression of WT PTP1B inhibited the insulin-stimulated phosphorylation of MAPK (18.2 ± 7.6% of control, P < 0.01; Fig. 3, A and B). Conversely, the expression of mutant PTP1B had no effect on MAPK phosphorylation (data not shown).

View larger version (40K): [in this window] [in a new window]   Figure 3. Different effects of PTP1B overexpression on insulin-stimulated Akt and MAPK phosphorylation. 3T3-L1 adipocytes, L6 myocytes and HIRc cells were infected with Ad5-ctrl (ctrl), or Ad5-PTP1B-WT (WT), at 50, 20, or 1 MOI for 16 h or 1 h, respectively. Following infection, cells were serum-starved (16 h), and incubated with or without 16.7 nM insulin for 10 min. Total cell lysates (20 µg) were subjected to SDS-PAGE and immunoblotted with either antiphospho-specific Akt antibody or antiphospho-specific MAPK antibody (A). Phosphorylation levels were quantified by NIH image. The solid bars show the mean percentage ± SE (n = 3) of phosphorylated levels in the insulin-stimulated control cells (open bar, B). In L6 myocytes overexpressing WT PTP1B, insulin-induced IRS-1 phosphorylation and association with Grb2 were assessed by Western blotting (C). In HIRc cells overexpressing WT PTP1B, insulin-induced Shc phosphorylation and association with Grb2 were assessed by Western blotting (D). Each Western blot is a representative of three independent experiments.

In L6 myocytes, overexpression of PTP1B inhibited insulin-stimulated IRS-1 phosphorylation (to 43.0 ± 15.2% of control, P < 0.05), resulting in a decreased association of IRS-1 with Grb2 (31.0 ± 5.1% of control, P < 0.01; Fig. 3C). In HIRc cells, overexpression of PTP1B inhibited insulin stimulated Shc phosphorylation (to 66.9 ± 6.8% of control, P < 0.05), resulting in a 51.8 ± 12.3% decrease (P < 0.05) in association of Shc with Grb2 (Fig. 3D).

Effects of PTP1B expression on platelet-derived growth factor (PDGF)-stimulated Shc phosphorylation and MAPK phosphorylation
To investigate direct effect of PTP1B expression on Shc phosphorylation, L6 myocytes were stimulated by PDGF, and Shc phosphorylation was measured, because we have previously observed that PTP1B overexpression had no effects on PDGF receptor phosphorylation in L6 myocytes (10). After PDGF stimulation, levels of tyrosine-phosphorylation of its receptors were unchanged, and PDGF-stimulated Akt phosphorylation was not affected by PTP1B expression. However, PDGF-stimulated Shc phosphorylation was decreased by PTP1B overexpression, leading to the attenuation of MAPK phosphorylation (Fig. 4).

View larger version (52K): [in this window] [in a new window]   Figure 4. PDGF stimulated Shc phosphorylation and MAPK phosphorylation. L6 myocytes were infected with Ad5-ctrl (ctrl) or Ad5-PTP1B-WT (WT) at 20 MOI for 1 h. After stimulation with PDGF (30 ng/ml), tyrosine-phosphorylation levels of PDGF receptors and Shc protein and phosphorylation levels of Akt and MAPK were assessed by Western blotting.

View larger version (42K): [in this window] [in a new window]   Figure 5. Expression levels of IR, IRS-1, and PTP1B protein. Expression levels of IR (A), IRS-1 (B), and endogenous PTP1B (C) in 3L3-L1 adipocytes, L6 myocytes, Fao hepatoma cells, and HIRc cells were quantified by Western blotting using corresponding antibodies. Each Western blot is representative of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the current study, we overexpressed a WT and a Cys²¹⁵/Ser²¹⁵ mutant PTP1B in 3T3-L1 adipocytes by using adenovirus-mediated gene transfer to analyze the effect of PTP1B on insulin signaling and action. In 3T3-L1 adipocytes, PTP1B-WT overexpression showed almost complete inhibition of insulin-stimulated MAPK phosphorylation, but small inhibitory effects on insulin-stimulated Akt phosphorylation and glucose transport. On the other hand, we reported that PTP1B overexpression dramatically blocked insulin-stimulated MAPK phosphorylation, Akt phosphorylation, and glycogen synthesis in L6 myocytes and Fao cells (10). Thus, our observation also supports the hypothesis that PTP1B exerts differing effects on insulin signaling in different cell types.

In 3T3-L1 adipocytes, overexpression of WT PTP1B decreased levels of tyrosine phosphorylation of the IR and IRS-1 by about 50%, and inhibited the association between IRS-1 and the p85 subunit of PI-3 kinase equally as well as in L6 myocytes. However, insulin-stimulated Akt phosphorylation and glucose transport were affected to a relatively small degree. Venable et al. (14) reported that overexpression of PTP1B inhibited insulin-induced tyrosine-phosphorylation of the IR and IRS-1 by about 50% but did not affect glucose transport in 3T3-L1 adipocytes. In contrast to their study, we overexpressed a phosphatase-dead mutant of PTP1B as a control in the current study. Because this mutant PTP1B has been shown to be substrate trapping (5), overexpression of mutant PTP1B demonstrated some dominant negative effects on insulin signaling, but almost no effects on insulin-stimulated glucose uptake. Thus, compared with mutant PTP1B, we were able to detect a small but significant decrease in insulin-stimulated glucose uptake in 3T3-L1 adipocytes overexpressing WT PTP1B.

One possible explanation for this dissociation between 3T3-L1 adipocytes and L6 myocytes is that tyrosine phosphorylation of the IR and IRSs may be impaired to a different degree. That is, in 3T3-L1 adipocytes, the IR and IRS-1 proteins are more abundant. Hence, even PTP1B overexpression led to decreased tyrosine-phosphorylation of signaling molecules such as IR and IRS-1. The remaining IRS-1 associated with p85 subunit of PI-3 kinase may be sufficient for Akt activation because full activation of PI-3 kinase may not be needed for its downstream effects. But this was not the case in L6 myocytes with a relatively small number of signaling molecules (Fig. 5, A and B). Supporting this idea, we obtained identical findings in HIRc cells, in which a large amount of signaling molecules is expressed. In the case of the PDGF receptor, it has been reported that its expression is an important determinant of Erk activation (16). We also reported that the cellular expression level of PKC- is a determinant of the insulin-responsiveness of cJun N-terminal kinase activation (17). Another explanation is that PTP1B may not play an important role in 3T3-L1 adipocytes, but that other PTPases such as PTP and/or LAR may do so. However, we observed a relatively large amount of PTP1B in these cells (Fig. 5C). Therefore, this explanation seems to be unlikely. Alternatively, it has been reported that PTP1B anchors to endoplasmic reticulum and is activated after release into cytosol by truncation of its COOH terminus (18, 19), and that the intracellular localization of PTP1B is critical for its actions (20). Thus, the localization of overexpressed PTP1B may be different in each cell line. Further studies are needed to clarify this issue. Finally, PTP1B activity and content may be regulated in a different manner in adipose tissue and skeletal muscle (21). Moreover, even in adipose tissue, it has been reported that PTPase activity differs between omental and sc fat tissue (22). Thus, it is possible that the role and regulation of PTP1B may be different in each tissue subtype.

Association of the p85 subunit of PI-3 kinase with IRS-1 is important to mediate the insulin signal to the Akt pathway (1). On the other hand, the association of Grb2 with IRS-1 and Shc protein is critical to mediate insulin signaling to the MAPK cascade (1). In cells overexpressing PTP1B, tyrosine-phosphorylation of both IRS-1 and Shc were inhibited, resulting in impaired association of IRS-1 with Grb2 and Shc. Furthermore, it is reported that Grb2 promotes the formation of a stable protein complex between tyrosine-phosphorylated IRS-1 and PTP1B (7). Therefore, the Grb2 binding site of IRS-1 may be more sensitive to dephosphorylation by PTP1B, than the p85 binding site. Moreover, PDGF-stimulated Shc phosphorylation and MAPK activity were also impaired by PTP1B overexpression, suggesting that PTP1B might act directly on Shc protein. Because it is reported that Shc is the signaling molecule predominantly responsible for coupling IR to activation of the MAPK cascade (23), the impairment of Shc phosphorylation is presumably mainly responsible for the attenuation of MAPK cascade activation seen in cells overexpressing PTP1B.

In conclusion, our data demonstrate that PTP1B negatively regulates insulin signaling but that the MAPK cascade is much more sensitive to this regulation than proteins in the Akt pathway in some cell lines, especially 3T3-L1 adipocytes.

	Acknowledgments

 
We are grateful to Dr. J. M. Olefsky (University of California, San Diego, CA), Dr. A. Klip (The Hospital For Sick Children, Toronto, Canada), and Dr. C. R. Kahn (Joslin Diabetes Center, Boston, MA) for donating 3T3-L1, HIRc, L6 and Fao cells, respectively.

	Footnotes

 
This work was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports and Culture, Japan (to H.M.). M.B.-A. was supported in part by a Merit Review award from the U.S. Veterans Administration Research Service and by funding from the Gonda Family Endowment.

Abbreviations: FCS, Fetal calf serum; HIRc cell, rat 1 fibroblasts overexpressing human insulin receptor; IR, insulin receptor; IRS, insulin receptor substrate; LAR, leukocyte antigen-related phosphatase; LRP, leukocyte common antigen-related phosphatase; MOI, multiplicity of infection; MT, mutant; PDGF, platelet-derived growth factor; PI-3 kinase, phosphatidylinositol-3 kinase; pNPP, p-nitrophenyl phosphate; PTPase, protein tyrosine phosphatase; PTP1B, protein-tyrosine phosphatase 1B; WT, wild-type.

Received May 15, 2002.

Accepted for publication August 27, 2002.

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