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Department of Diabetes and Metabolism (M.F., H.K., T.O., M.Ab., N.S., Y.F., T.A.), Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo 113-8655, Japan; Department of Molecular Biology (Y.G.), Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan; Institute for Adult Disease (H.S., M.An., Y.On., H.O., M.K.), Asahi Life Foundation, Shinjuku-ku, Tokyo 160-0023, Japan; and Division of Molecular Metabolism and Diabetes (Y.Ok.), Department of Internal Medicine, Tohoku University Graduate School of Medicine, Seiryo-machi, Sendai 980-8574, Japan
Address all correspondence and requests for reprints to: Tomoichiro Asano, Department of Diabetes and Metabolism, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. E-mail: asano-tky{at}umin.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, which activates three different MAPKs [ERK, p38, and jun amino terminal kinase (JNK)], also induces insulin resistance. To better understand the respective roles of these three MAPK pathways in insulin signaling and their contribution to insulin resistance, constitutively active MAPK/ERK kinase (MEK)1, MAPK kinase (MKK6), and MKK7 mutants were overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated transfection procedure. The MEK1 mutant, which activates ERK, markedly down-regulated expression of the insulin receptor (IR) and its major substrates, IRS-1 and IRS-2, mRNA and protein, and in turn reduced tyrosine phosphorylation of IR as well as IRS-1 and IRS-2 and their associated phosphatidyl inositol 3-kinase (PI3K) activity. The MKK6 mutant, which activates p38, moderately inhibited IRS-1 and IRS-2 expressions and IRS-1-associated PI3K activity without exerting a significant effect on the IR. Finally, the MKK7 mutant, which activates JNK, reduced tyrosine phosphorylation of IRS-1 and IRS-2 and IRS-associated PI3K activity without affecting expression of the IR, IRS-1, or IRS-2. In the context of our earlier report showing down-regulation of glucose transporter 4 by MEK1-ERK and MKK6/3-p38, the present findings suggest that chronic activation of ERK, p38, or JNK can induce insulin resistance by affecting glucose transporter expression and insulin signaling, though via distinctly different mechanisms. The contribution of ERK is, however, the strongest. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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TNF is known to activate three different MAPKs—ERK, p38 MAPK (p38), and jun amino terminal kinase (JNK; Refs. 16, 17, 18)—and to contribute to insulin resistance under such pathophysiological conditions as obesity, cancer-induced cachexia, and bacterial infections (19, 20). For example, TNF reduces IRS-1 expression and insulin-induced IRS-1 tyrosine phosphorylation, effects that should each reduce insulin sensitivity (19, 21, 22, 23, 24). Moreover, we previously demonstrated the effects of chronic ERK, p38 or JNK activation on glucose transporter expression by selectively overexpressing the kinase situated immediately upstream from each MAPK. We found that chronic activation of either the MAPK/ERK kinase (MEK) 1-ERK or MAPK kinase (MKK) 6/3-p38 pathway leads to up-regulation of GLUT1 expression and down-regulation of GLUT4 expression, resulting in significantly increased basal glucose transport but decreased insulin-induced transport (25). Consistent with that finding, PD98059, a specific ERK inhibitor, restores insulin sensitivity in the presence of TNF (21, 26). On the other hand, recent evidence suggests that activated JNK phosphorylates a serine residue on IRS-1, thereby inhibiting its tyrosine phosphorylation (27). Thus, the precise roles played by ERK, p38, and JNK in mediating TNF-induced insulin resistance remain unclear.
In the present study, we used an adenovirus-mediated transfection procedure to overexpress three constitutively active MKK mutants in 3T3-L1 adipocytes and then evaluated the effects of the resultant MAPK activation (18, 28, 29, 30, 31) on the expressions of IR, IRS-1, and IRS-2, the degree of their tyrosine phosphorylations, and their associated PI3K activations. Our results indicate that, by acting via IR, IRS-1, and IRS-2, ERK has the strongest inhibitory effect on insulin signaling.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). We found that LASDSE-MEK1 significantly increased the level of ERK phosphorylation, whereas EE-MKK6 and DED-MKK7 had little effect. Similarly, exposure to stressful stimuli—epidermal growth factor (EGF), platelet-derived growth factor (PDGF)-B chain/B chain dimer (BB), and TNF—also increased ERK phosphorylation (Fig. 1A), although only TNF- induced increases persisted for at least 24 h. The effects of EGF and PDGF-BB were more transient, with levels of ERK phosphorylation returning to baseline within 8–24 h after stimulation (Fig. 1B).
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). EGF and PDGF-BB evoked transient phosphorylation of p38 that was detected within 10 min of their application (Fig. 2B). TNF again had a more pronounced and prolonged effect than EGF or PDGF-BB, evoking an elevation in p38 phosphorylation that persisted for at least 24 h.
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stimulation each induced prolonged phosphorylation of JNK, whereas EGF and PDGF-BB evoked transient and minimal JNK phosphorylation (Fig. 3).
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, EGF, and PDGF on Insulin-Induced Increases in Glucose Transport shows basal and insulin-stimulated 2-deoxy-[3H]glucose uptakes into 3T3-L1 adipocytes exposed to various stress-inducing stimuli for 24 h. Exposure to TNF for 24 h significantly elevated basal glucose transport in 3T3-L1 adipocytes but reduced the insulin-induced increase in glucose uptake. On the other hand, consistent with earlier reports (32, 33, 34), exposure to EGF or PDGF-BB had no significant effect on either basal or insulin-stimulated glucose uptake.
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on Expressions of IR and IRS or overexpressing a constitutively active MKK mutant. We found that overexpression of LASDSE-MEK1 markedly reduced expression of the IR and its major substrates, IRS-1 and IRS-2 proteins, by 82%, 97%, and 90%, respectively (Fig. 3
, A–C); overexpression of EE-MKK6 did not significantly affect IR protein expression but induced a more modest reduction than MEK1 in the expressions of IRS-1 and IRS-2 proteins, by 38% and 21%, respectively. Furthermore, DED-MKK7 overexpression had no significant effect on the expression of IR, IRS-1, or IRS-2 protein. Incubating cells with TNF for 24 h reduced the expressions of IR, IRS-1, and IRS-2 proteins by 63%, 72%, and 48%, respectively. To confirm that these changes are specific to each pathway, we employed two established inhibitors; PD98050, a specific ERK inhibitor, and SB203580, a specific p38 MAPK inhibitor. Expressions of IR, IRS-1, and IRS-2 proteins, reduced by MEK1, were restored upon incubation with PD98059. Those reduced by MKK6 were restored by incubation with SB203580. Furthermore, PD98059 normalized expressions of IR, IRS-1, and IRS-2 reduced by TNF, whereas SB203580 had no significant effect.
To better understand the mechanism underlying the diminished expression of IR and IRS proteins, levels of IR, IRS-1, and IRS-2 mRNA were assessed using ribonuclease (RNase) protection assays (Figs. 5 and 6, A–C). We found the IR mRNA level to be reduced, in the same manner as expression of its protein, by either MEK1 overexpression or TNF stimulation. We also found that, as compared with control LacZ, overexpression of either LASDSE-MEK1 or EE-MKK6 significantly reduced expression of both IRS-1 and IRS-2 mRNA, with the effect on IRS-1 being especially pronounced. Exposing cells to TNF reduced levels of both transcripts by approximately 50%. Apparently then, the observed declines in IRS-2 and IRS-2 proteins reflected the reduced availability of their respective mRNAs.
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on Insulin-Induced Tyrosine Phosphorylation of IR and IRS Proteins, A–C), we found that overexpression of LASDSE-MEK1 strongly inhibited tyrosine phosphorylation of the IR, IRS-1, and IRS-2 (by 52%, 81%, and 63%, respectively), as did exposure to TNF (by 51%, 68%, and 45%, respectively); EE-MKK6 did not significantly affect tyrosine phosphorylation of the IR or IRS-2, while having a small but significant effect on IRS-1 (a 29% inhibition). Similarly, overexpression of DED-MKK7 had no significant effect on tyrosine phosphorylation of IR while significantly inhibiting tyrosine phosphorylation of both IRS-1 and IRS-2, although the effect was not as pronounced (by 37% and 26%, respectively).
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on Insulin-Induced p85 (PI3K) Docking and PI3K Activity on docking of the p85 regulatory subunit of PI3K with IRS (Fig. 8, A and B) and on insulin-induced, IRS-associated PI3K activity (Fig. 9, A and B) were consistent with their effects on tyrosine phosphorylation. To evaluate the influence of these three MKK mutants on overall IR signaling, we determined total phosphotyrosine-phosphorylated protein associated PI3K activity in 3T3-L1 adipocytes infected with these mutants (Fig. 9C). Overexpression of LASDSE-MEK1 and incubation with TNF each significantly reduced the docking of p85 with IRS-1 (by 83% and 55%, respectively) and IRS-2 (by 72% and 49%, respectively), in turn reducing IRS-associated PI3K activity. Overexpression of DED-MKK7 had a smaller though still significant inhibitory effect on p85 docking and PI3K activities associated with both IRS-1 (by 28% and 30%, respectively) and IRS-2 (by 26% and 34%, respectively), whereas EE-MKK6 significantly affected only p85 docking to IRS-1 (by 29%) and the associated PI3K activity (by 33%). PI3K activity immunoprecipitated by the antiphosphotyrosine antibody was similarly reduced, as observed with IRS-1 or IRS-2, and the reduction was most marked with overexpression of LASDSE-MEK1.
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on Akt Expression and Insulin-Induced Akt Phosphorylation). Overexpression of LASDSE-MEK1 significantly reduced insulin-induced phosphorylation of Akt at Thr-308 (by 92%) and Ser-473 (by 69%), as did incubation with TNF (by 80% and 66%, respectively); EE-MKK6 and DED-MKK7 also reduced phosphorylation at both Thr-308 (by 58% and 41%, respectively) and Ser-473 (by 24% and 28%, respectively), though not to the degree seen with LASDSE-MEK1 or TNF (Fig. 10, A and B). Akt expression was not significantly affected by MAPK activation (Fig. 10C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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secreted from adipocytes reportedly impairs insulin activity in peripheral tissues (muscle and fat) as well as in the liver (22, 23, 35, 36). Consistent with those findings, treatment with thiazolidinediones, peroxisomal proliferator-activated protein-
agonists, suppresses TNF production by adipocytes and normalizes insulin sensitivity in obese insulin-resistant animals (35, 37); and disruption of the gene encoding TNF or its receptor improves insulin sensitivity in mice (38, 39).
As mentioned in the Introduction, stimulation with TNF reportedly induces three types of MAPK-ERK, p38, and JNK (16, 17, 18). With respect to regulation of glucose transporters, we previously showed that activation of the MEK1-ERK and MKK6/3-p38 pathways up-regulates the expression of GLUT1 but down-regulates that of GLUT4, resulting in increased basal glucose uptake and diminished insulin-sensitive uptake (25). Such regulation of GLUT gene expression by ERK and p38 is thought to be one possible mechanism underlying TNF-induced insulin resistance. However, effects on GLUT expression are certainly only part of the story, as chronic exposure to TNF also reduces IRS-1 expression, tyrosine phosphorylation, docking of the p85 regulatory subunit of PI3K, and IRS-associated PI3K activity (22, 23, 40, 41, 42, 43). One hypothesis possibly explaining this effect is that ERK plays a key role in TNF-induced suppression of insulin signaling. Consistent with this idea, treating cells with PD98059, an ERK inhibitor, restores insulin sensitivity in the presence of TNF (21, 26). On the other hand, it was recently suggested that JNK plays a critical role in TNF-induced insulin resistance via its association with IRS-1 and the resultant phosphorylation of IRS-1 at serine 307 (27). Serine-phosphorylated IRS-1 is reportedly resistant to being tyrosine-phosphorylated by the insulin receptor (41, 44, 45). Thus, whereas evidence from numerous earlier studies indicates that TNF induces insulin resistance, the precise mechanism, in particular the pathway via which the signal from the TNF receptor is transduced, remains unclear.
In the present study, to clarify the individual actions of each MAPK, a constitutively active mutant of the upstream kinase for each MAPK was overexpressed using an adenovirus transfection procedure. The effects of the resultant MAPK activation were comparable to those seen with TNF stimulation, suggesting them to be within physiological range and that activation of ERK, p38 or JNK can impair insulin signaling. In addition, to confirm that the effects of LASDSE-MEK1 and EE-MKK6 on the regulation of IR or IRS-proteins are truly mediated by the activation of ERK and p38 pathways, respectively, specific ERK and p38 inhibitors were used.
Activation of the MEK1-ERK pathway dramatically suppressed the expressions of IR, IRS-1, and IRS-2. As a result, insulin-induced tyrosine phosphorylation of IRS-1 and IRS-2, docking of the p85 regulatory subunit of PI3K, and PI3K activation were all markedly impaired. When the amounts of immunoprecipitated IRS-1 and IRS-2 were normalized, the PI3K activities associated with equal amounts of IRS-1 and IRS-2 were moderately increased. We speculate that the reductions in IRS-proteins might have increased the individual efficiencies of IRS-1 or IRS-2 molecules in associating with the IR and/or PI3K protein. At any rate, the PI3K activity reduction appears to mainly be attributable to the down-regulation of IR and IRS protein expressions. Taking our earlier report into consideration (25), activation of the MEK1-ERK pathway apparently induces severe insulin resistance by inhibiting expressions of both GLUT4 and insulin-signaling proteins.
Secondly, MKK6-p38 pathway activation only moderately reduced IRS-1 and IRS-2 expressions. When the amounts of immunoprecipitated IRS-1 and IRS-2 were normalized, the PI3K activities associated with equal amounts of IRS-1 and IRS-2 were slightly but not significantly up-regulated (by 10% and 30%, respectively). Consequently, the overall contribution of p38 to TNF-induced insulin resistance would presumably be smaller than that of ERK. Indeed, treating cells with SB203580, a p38-specific inhibitor, had little effect on TNF-induced insulin resistance, which suggests that the p38 pathway is not physiologically relevant (46).
Finally, MKK7-JNK pathway activation had no effect on the expression of either IRS-1 or IRS-2 but suppressed tyrosine phosphorylation of both and, in turn, suppressed activation of PI3K and Akt. Consistent with those findings, earlier reports showed JNK to associate with IRS-1 and phosphorylate serine residue-307 (27), thereby inhibiting tyrosine phosphorylation by the IR.
Several previous reports have shown the individual contributions of ERK, p38, and the JNK pathway to TNF-induced insulin resistance (26, 27, 46, 47, 48, 49, 50). However, this study has clearly demonstrated, by overexpressing active mutants of the respective upstream kinases, that ERK, p38 and JNK each have the potential to induce insulin resistance via distinctly different molecular mechanisms. Furthermore, our findings strongly suggest that the ERK contribution is much greater than those of p38 and JNK, based on our observation that ERK markedly suppresses the expressions of IR and IRS-proteins as well as that of GLUT4.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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was from Genzyme Transgenics Corp. (Cambridge, MA). Recombinant human EGF and human PDGF-BB were from Promega Corp. (Madison, WI) and Life Technologies, Inc. (Gaithersburg, MD), respectively. Inhibitors PD98059 and SB203580 were from Calbiochem (La Jolla, CA). Phosphatidylinositol was from Sigma (St. Louis, MO). Aluminum-backed silica gel thin layer chromatographic plates were from Merck (Darmstadt, Germany). [-32P]ATP and [-32P]uridine triphosphate were from ICN Biomedicals (Irvine, CA). 2-Deoxy-D-[3H]glucose, protein A-Sepharose 4 Fast Flow, and an enhanced chemiluminescence detection system were from Amersham Pharmacia Biotech (Arlington Heights, IL). All other reagents from commercial sources were of analytical grade.
Antibodies
Specific antibodies against IRS-1 and IRS-2 were prepared as described previously (51). An antibody against Akt was prepared by immunizing rabbits with a synthetic peptide derived from C-terminal amino acids 466–479 of mouse Akt. Anti-p85 antibody and antiphosphotyrosine antibody (4G10) were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-IRß antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibody and anti-phospho-JNK (Thr183/Tyr185) were from Cell Signaling Technology (Beverly, MA). Anti-phospho-p38 MAP kinase (Thr180/Tyr182) antibody, anti-phospho-Akt (Thr308) antibody and anti-phospho-Akt (Ser473) 4E2 monoclonal antibody were from New England Biolabs, Inc. (Beverly, MA).
Cell Culture
3T3-L1 fibroblasts were initially maintained in DMEM supplemented with 10% donor calf serum (Life Technologies, Inc.) under an atmosphere of 90% air/10% CO2 at 37 C. Differentiation was induced 2 d after the cells had reached confluence, by replacing the normal culture medium with DMEM supplemented with 0.5 mM 3-isobutyl-1-methylxanthine, 4 µg/ml dexamethasone (Sigma), and 10% fetal bovine serum (Life Technologies, Inc.) for 48 h. The cells were then incubated for an additional 4–10 d in DMEM supplemented with 10% fetal bovine serum; the medium was changed every other day. With this protocol, more than 90% of the cells expressed the adipocyte phenotype (7).
Construction of MAPK Mutants
Plasmids containing cDNAs encoding constitutively active MEK1 (LASDSE-MEK1), MKK6 (EE-MKK6), and MKK7 (DED-MKK7) mutants were prepared as follows. LASDSE-MEK1 was constructed by replacing serine-218 with aspartic acid, serine-222 with glutamic acid, and two critical leucines (Leu-11 and Leu-37) in the nuclear export signal sequence of Xenopus MAPKK with alanines (29). EE-MKK6 was constructed by substituting serine-207 and threonine-211 with glutamic acid (30). DED-MKK7 was constructed by replacing serine-287 with aspartic acid, threonine-291 with glutamic acid and serine-293 with aspartic acid.
Gene Transduction
The methods of constructing recombinant adenoviruses (52) and infecting 3T3-L1 adipocytes (5, 53) were previously described in detail. Briefly, the expression cosmid cassette pAdexCAwt was ligated to the cDNA encoding LacZ from Escherichia coli or to one of the aforementioned mutants. Thereafter, homologous recombination of the recombinant cosmid cassette and its parental virus genome was carried out, and 3T3-L1 cells were infected with the indicated adenovirus. Experiments were performed 48 h after infection. Therefore, recombinant adenoviruses were applied at a multiplicity of infection of 200–300 plaque-forming units/cell.
Immunoprecipitation and Immunoblotting
3T3-L1 adipocytes plated in 12-well tissue culture dishes were first incubated for 3 h in serum-free DMEM at 37 C, and then for an additional 5 min with or without 10-7 M insulin. The cells were then lysed at 4 C with ice-cold PBS (1 ml/well) containing 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 2 mM sodium orthovanadate, after which the insoluble material was removed by centrifugation at 15,000 x g for 10 min at 4 C. The cell lysates (1 ml, approximately 1 mg of total protein) were incubated with anti-IR, anti-IRS-1, or anti-IRS-2 antibody (10 µg) for 1 h and precipitated by incubation with 20 µl of protein A-Sepharose for 1 h. The resultant immunocomplexes were washed five times with the lysis buffer and boiled in 30 µl of Laemmli sample buffer containing 10 mM dithiothreitol. The proteins were resolved on 7.5% or 10% SDS-PAGE and transferred to nitrocellulose membranes (Schleicher \|[amp ]\| Schuell, Inc., Keene, NH). After blocking with TBS-T (10 mM Tris, pH 7.6; 150 mM NaCl; and 0.05% Tween-20) containing 3% BSA for 1 h, the membranes were incubated with the appropriate antibodies. The proteins were visualized by enhanced chemiluminescence using horseradish peroxidase-labeled antirabbit or mouse IgG (Amersham Pharmacia Biotech). In some experiments, the band intensities were quantified with a Molecular Imager GS-525 using Screen-CH (Bio-Rad Laboratories, Inc., Richmond, CA).
Glucose Transport Assay
3T3-L1 adipocytes in 24-well culture dishes were serum-starved overnight in DMEM containing 0.2% BSA, after which they were incubated for 45 min in glucose-free Krebs-Ringer phosphate buffer [in mM: 137 NaCl, 4.7 KCl, 10 sodium phosphate (pH 7.4), 0.5 MgCl2, and 1 CaCl2 (7)]. Basal and stimulated uptakes of 2-deoxy-D-[3H]glucose were then measured as described previously (54).
PI3K Assay
Isolated adipocytes were prepared, and some were stimulated with insulin as described above. The cells were then solubilized in ice-cold lysis buffer containing 20 mM Tris (pH 7.5), 137 mM NaCl, 1 mM CaCl2, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride and 100 µM sodium orthovanadate, after which the lysates were extracted, incubated with anti-IRS-1, anti-IRS-2 or antiphosphotyrosine antibody and precipitated as described above. PI3K activities in the immunoprecipitates were assayed as described previously (51).
RNA Extraction
Total cell RNA was prepared from 3T3-L1 adipocytes using an Isogen RNA isolation kit (Nippon Gene, Toyama, Japan). RNA concentrations were estimated based on absorbance at 260 nm; 10 µg of RNA from each sample were used for the RNase protection assays described below.
Preparation of Riboprobes
Riboprobes were synthesized as described previously (51). To obtain mouse IR, IRS-1, and IRS-2 cDNAs, polymerase chain reaction was performed based on the reported sequences using mouse cDNA libraries. The amplified fragments, which corresponded to nucleotides 2232–2386 of mouse IR cDNA, nucleotides 2200–2350 of mouse IRS-1 cDNA and nucleotides 3270–3420 of mouse IRS-2 cDNA were subcloned into a TA vector. The resultant plasmids were then linearized with HindIII and used for in vitro transcription.
RNase Protection Assay
RNase protection assays were carried out according to the manufacturer’s instructions (RPA III; Ambion, Inc., Austin, TX). Pooled samples containing 10 µg of total RNA from adipocytes were hybridized with the riboprobes for IR, IRS-1, and IRS-2. After treatment with RNase, the protected fragments were resolved on 5% polyacrylamide-urea gels and subjected to autoradiography; band intensities were determined using a Molecular Imager GS-525.
Statistical Analysis
Quantitative values were expressed as means ± SE. Statistical significance was tested using the Mann-Whitney’s U test. Values of P < 0.05 were considered statistically significant.
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FOOTNOTES |
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Received for publication April 5, 2002. Accepted for publication November 26, 2002.
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