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Insulin Receptor Substrate-2-dependent Interleukin-4 Signaling in Macrophages Is Impaired in Two Models of Type 2 Diabetes Mellitus

2004; Elsevier BV; Volume: 279; Issue: 27 Linguagem: Inglês

10.1074/jbc.m404368200

ISSN

1083-351X

Autores

Matthew E. Hartman, Jason C. O’Connor, Jonathan P. Godbout, Kyle D. Minor, Valerie R. Mazzocco, Gregory G. Freund,

Tópico(s)

Immune cells in cancer

Resumo

We have shown previously that hyperinsulinemia inhibits interferon-α-dependent activation of phosphatidylinositol 3-kinase (PI3-kinase) through mammalian target of rapamycin (mTOR)-induced serine phosphorylation of insulin receptor substrate (IRS)-1. Here we report that chronic insulin and high glucose synergistically inhibit interleukin (IL)-4-dependent activation of PI3-kinase in macrophages via the mTOR pathway. Resident peritoneal macrophages (PerMΦs) from diabetic (db/db) mice showed a 44% reduction in IRS-2-associated PI3-kinase activity stimulated by IL-4 compared with PerMΦs from heterozygote (db/+) control mice. IRS-2 from db/db mouse PerMΦs also showed a 78% increase in Ser/Thr-Pro motif phosphorylation without a difference in IRS-2 mass. To investigate the mechanism of this PI3-kinase inhibition, 12-O-tetradecanoylphorbol-13-acetate-matured U937 cells were treated chronically with insulin (1 nm, 18 h) and high glucose (4.5 g/liter, 48 h). In these cells, IL-4-stimulated IRS-2-associated PI3-kinase activity was reduced by 37.5%. Importantly, chronic insulin or high glucose alone did not impact IL-4-activated IRS-2-associated PI3-kinase. Chronic insulin + high glucose did reduce IL-4-dependent IRS-2 tyrosine phosphorylation and p85 association by 54 and 37%, respectively, but did not effect IL-4-activated JAK/STAT signaling. When IRS-2 Ser/Thr-Pro motif phosphorylation was examined, chronic insulin + high glucose resulted in a 92% increase in IRS-2 Ser/Thr-Pro motif phosphorylation without a change in IRS-2 mass. Pretreatment of matured U937 cells with rapamycin blocked chronic insulin + high glucose-dependent IRS-2 Ser/Thr-Pro motif phosphorylation and restored IL-4-dependent IRS-2-associated PI3-kinase activity. Taken together these results indicate that IRS-2-dependent IL-4 signaling in macrophages is impaired in models of type 2 diabetes mellitus through a mechanism that relies on insulin/glucose-dependent Ser/Thr-Pro motif serine phosphorylation mediated by the mTOR pathway. We have shown previously that hyperinsulinemia inhibits interferon-α-dependent activation of phosphatidylinositol 3-kinase (PI3-kinase) through mammalian target of rapamycin (mTOR)-induced serine phosphorylation of insulin receptor substrate (IRS)-1. Here we report that chronic insulin and high glucose synergistically inhibit interleukin (IL)-4-dependent activation of PI3-kinase in macrophages via the mTOR pathway. Resident peritoneal macrophages (PerMΦs) from diabetic (db/db) mice showed a 44% reduction in IRS-2-associated PI3-kinase activity stimulated by IL-4 compared with PerMΦs from heterozygote (db/+) control mice. IRS-2 from db/db mouse PerMΦs also showed a 78% increase in Ser/Thr-Pro motif phosphorylation without a difference in IRS-2 mass. To investigate the mechanism of this PI3-kinase inhibition, 12-O-tetradecanoylphorbol-13-acetate-matured U937 cells were treated chronically with insulin (1 nm, 18 h) and high glucose (4.5 g/liter, 48 h). In these cells, IL-4-stimulated IRS-2-associated PI3-kinase activity was reduced by 37.5%. Importantly, chronic insulin or high glucose alone did not impact IL-4-activated IRS-2-associated PI3-kinase. Chronic insulin + high glucose did reduce IL-4-dependent IRS-2 tyrosine phosphorylation and p85 association by 54 and 37%, respectively, but did not effect IL-4-activated JAK/STAT signaling. When IRS-2 Ser/Thr-Pro motif phosphorylation was examined, chronic insulin + high glucose resulted in a 92% increase in IRS-2 Ser/Thr-Pro motif phosphorylation without a change in IRS-2 mass. Pretreatment of matured U937 cells with rapamycin blocked chronic insulin + high glucose-dependent IRS-2 Ser/Thr-Pro motif phosphorylation and restored IL-4-dependent IRS-2-associated PI3-kinase activity. Taken together these results indicate that IRS-2-dependent IL-4 signaling in macrophages is impaired in models of type 2 diabetes mellitus through a mechanism that relies on insulin/glucose-dependent Ser/Thr-Pro motif serine phosphorylation mediated by the mTOR pathway. The first member of the insulin receptor substrate (IRS) 1The abbreviations used are: IRS, insulin receptor substrate; Ch.Ins, chronic insulin; IFN, interferon; IGF, insulin-like growth factor; IL, interleukin; JAK, janus kinase; mTOR, mammalian target of rapamycin; PerMΦs, peritoneal macrophages; PI3-kinase, phosphatidylinositol 3-kinase; STAT, signal transducers and activators of transcription; TNF, tumor necrosis factor; TPA, 12-O-tetradecanoylphorbol-13-acetate. family, IRS-1, was initially discovered in Fao hepatoma cells as a tyrosine-phosphorylated substrate of the insulin receptor (1White M.F. Maron R. Kahn C.R. Nature. 1985; 318: 183-186Crossref PubMed Scopus (450) Google Scholar). In addition to insulin signaling, IRS proteins are integrally linked to intracellular signaling pathways initiated by IGF-I and the cytokines IL-2, 3, 4, 7, 9, 10, 13, 15 and IFN-α and IFN-γ (2Haddad T.C. Conover C.A. J. Biol. 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Leukocyte Biol. 2003; 73: 209-212Crossref PubMed Scopus (1439) Google Scholar). Recently, subacute chronic inflammation (36Pradhan A.D. Manson J.E. Rifai N. Buring J.E. Ridker P.M. JAMA. 2001; 286: 327-334Crossref PubMed Scopus (3399) Google Scholar, 37Greaves D.R. Gordon S. Trends Immunol. 2001; 22: 180-181Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) has been identified as a significant contributor to the complications associated with type 2 diabetes mellitus (38Plutzky J. Viberti G. Haffner S. J. Diabetes Complications. 2002; 16: 401-415Crossref PubMed Scopus (76) Google Scholar). Cardiovascular problems such as accelerated and exacerbated atherosclerosis appear especially responsive to the proinflammatory environment of diabetes (37Greaves D.R. Gordon S. Trends Immunol. 2001; 22: 180-181Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 39Ross R. Annu. Rev. Physiol. 1995; 57: 791-804Crossref PubMed Scopus (893) Google Scholar). The diabetic state is known to alter macrophage function resulting in increased lipoprotein lipase production and TNF-α release (40Sartippour M.R. Renier G. Diabetes. 2000; 49: 597-602Crossref PubMed Scopus (41) Google Scholar). In addition, hyperglycemia, through the generation of advanced glycation end products, increases macrophage secretion of IL-1 and TNF-α while enhancing intercellular adhesion molecule 1 expression and decreasing phagocytic activity (41Liu B.F. Miyata S. Kojima H. Uriuhara A. Kusunoki H. Suzuki K. Kasuga M. Diabetes. 1999; 48: 2074-2082Crossref PubMed Scopus (71) Google Scholar). Because the IRS-2/PI3-kinase pathway is common to both IL-4 and insulin signaling in macrophages, we wanted to demonstrate that IL-4-dependent IRS-2/PI3-kinase signal transduction was inhibited in diabetic macrophages and that the likely mechanism was a result of mTOR-dependent serine phosphorylation of IRS-2 on Ser/Thr-Pro motifs. Materials—The U937 promonocytic cell line was purchased from American Type Culture Collection (Rockville, MD). All cell culture reagents and chemicals were purchased from Sigma except as noted below. Fetal calf serum (0.05 ng/ml, 0.48 enzyme unit/ml of endotoxin) was purchased from Atlanta Biologicals (Norcross, GA). [γ-32P]ATP was purchased from PerkinElmer Life Sciences. Protein G-Sepharose and the ECL Western blotting analysis system were purchased from Amersham Biosciences. Silica Gel 60 thin layer chromatography plates were purchased from EM Science (Gibbstown, NJ). Bio-Rad protein reagent was purchased from Bio-Rad. Anti-IRS-1, anti-IRS-2, anti-p85, anti-JAK1, anti-JAK3, anti-mitotic protein monoclonal #2, antiphosphotyrosine, and antiphospho-STAT6 antibodies and TF-1 cell lysate were purchased from Upstate Biotechnology (Lake Placid, NY). Antiactin (C-2) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). IL-4 and rapamycin were purchased from Calbiochem. Nitrocellulose membrane was purchased from Osmotics (Westborough, MA). A One Touch Ultra® glucometer and glucose strips were purchased from Lifescan (Milpitas, CA). Sensitive rat insulin radioimmunoassay kit was purchased from Linco Research, Inc. (St. Charles, MO). Animals—All animal care and use were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NRC). 8- to 12-week-old db/db (C57BL/6J-leprdb/leprdb) mice and their agematched nondiabetic db/+ (C57BL/6J-leprdb/+) littermates were bred in-house from mice purchased from Jackson Laboratories (Bar Harbor, ME). Mice were housed in standard shoebox cages and given pelleted food (NIH 5K52; LabDiet, Purina Mills Inc., Brentwood, MO) and water ad libitum in a temperature - (72 °C) and humidity- (45–55%) controlled environment with a 12/12-h dark/light cycle (7:00 a.m.–7:00 p.m.). Blood Glucose and Serum Insulin Measurement—Blood was collected from the lateral saphenous vein of 8-week-old db/+ and db/db mice. Blood glucose levels were measured using a One Touch Ultra® glucometer per the manufacturer's instructions. For random and fasting blood glucose measurements, blood was collected at 9:00 a.m. from mice fed ad libitum or fasted overnight, respectively. Serum insulin levels were measured by radioimmunoassay according to the manufacturer's instructions. For random serum and fasting insulin levels, blood was collected as above, and serum was fractionated by centrifuging whole blood for 10 min at 16,000 × g. Peritoneal Macrophage (PerMΦ) Isolation—Mice were sacrificed by CO2 asphyxiation, and peritoneal cells were collected by peritoneal lavage using two 5-ml washes of ice-cold low glucose growth medium (glucose-free RPMI 1640 supplemented with 10% fetal calf serum, 1.0 g/liter glucose, 2 g/liter sodium bicarbonate, 110 mg/liter sodium pyruvate, 62.1 mg/liter penicillin, 100 mg/liter streptomycin, and 10 mm HEPES, pH 7.4). Macrophages where then isolated from the lavage fluid by adherence to plastic using the following procedure. Lavage cells were pelleted and resuspended in 5 ml of hypertonic red blood cell lysis buffer (142 mm NaCl, 1 mm KHCO3, 118 mm NaEDTA, pH 7.4) at room temperature for 4 min then mixed 1:1 with growth medium, pelleted, and resuspended in pure growth medium at 37 °C. Cells were plated at 5 × 105 cells/ml, and after 30 min plates were washed twice to remove nonadherent cells, resulting in ∼80% pure macrophages, confirmed by morphology (42Ceddia M.A. Woods J.A. J. Appl. Physiol. 1999; 87: 2253-2258Crossref PubMed Scopus (45) Google Scholar). PI3-Kinase Assays—PI3-kinase activity was assayed as described previously (43Godbout J.P. Cengel K.A. Cheng S.L. Minshall C. Kelley K.W. Freund G.G. Cell. Signal. 1999; 11: 15-23Crossref PubMed Scopus (14) Google Scholar). In brief, cells were treated as indicated and then lysed in ice-cold homogenization buffer (1% Triton X-100, 100 mm NaCl, 50 mm NaF, 1 mm phenylmethylsufonyl fluoride, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 2 mm sodium orthovanadate, 50 mm okadaic acid and 50 mm Tris, pH 7.4). Lysates were clarified, normalized to 1 mg of protein/1.5 μg of antibody, and immunoprecipitated for 2 h with either anti-IRS-1 or -2 antibody as indicated. PI3-kinase activity was assayed in immunoprecipitates as follows. Immune complexes were washed three times with 1% Triton X-100, 1 mm dithiothreitol, and phosphate-buffered saline, pH 7.4, and three times with 0.1 m NaCl, 1 mm dithiothreitol, and 10 mm Tris, pH 7.4. Kinase assays were initiated by the addition of 0.333 mg/ml sonicated l-α-phosphatidylinositol in 0.4 mm EGTA, 0.4 mm NaPO4, 8 μm [γ-32P]ATP (41.6 μCi/nmol), 5 mm MgCl2, and 20 mm HEPES, pH 7.1. The reactions were terminated after 15 min, and phospholipids were extracted in chloroform and separated by thin layer chromatography. Phospholipid reaction products were analyzed on a Molecular Dynamics Phosphorimager System (Sunnyvale, CA). Cell Culture/Insulin and Glucose Treatment—U937 cells were grown in low glucose growth medium. Cells were passaged 1:1 with fresh medium every 3 days. All cell counts were performed on a Coulter ZM (Miami, FL). For high glucose treatments, cells were resuspended at 5 × 105 cells/ml in low glucose growth medium with the addition of 3.5 g/liter glucose and grown for 48 h. For chronic insulin treatment, cells were resuspended at 5 × 105 cells/ml in low glucose growth medium with or without the addition of 3.5 g/liter glucose and grown for 48 h. During the last 18 h, 1 nm insulin was added. TPA Maturation of U937 Promonocytes to Macrophages—TPA maturation was performed as described previously (44Deszo E.L. Brake D.K. Cengel K.A. Kelley K.W. Freund G.G. J. Biol. Chem. 2001; 276: 10212-10217Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 45Deszo E.L. Brake D.K. Kelley K.W. Freund G.G. Cell. Signal. 2004; 16: 271-280Crossref PubMed Scopus (22) Google Scholar). In brief, U937 cells were pelleted and resuspended in growth medium containing 100 nm TPA and incubated for 3 h. Cells were then washed and plated at 5 × 105 cells/ml in TPA-free medium and allowed to mature for 2 days prior to experimentation. Maturation was confirmed by CD11b and CD86 staining using flow cytometry. Western Analysis—Western analysis was performed as described previously (13Cengel K.A. Freund G.G. J. Biol. Chem. 1999; 274: 27969-27974Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). In brief, 5 × 106 cells were lysed in 1 ml of ice-cold homogenization buffer. Lysates were clarified and normalized using Bio-Rad protein reagent. For immunoprecipitation, lysates were normalized to 1 mg of protein/1.5 μg of antibody and immunoprecipitated for 2 h. For whole cell lysates, proteins were normalized and were loaded at 250 μg/lane. Proteins were resolved by SDS-PAGE under reducing conditions and then electrotransferred to nitrocellulose. For membrane stripping and reprobing, nitrocellulose membranes were incubated at 100 °C for 10 min with a stripping buffer containing 2% SDS, 6.25 mm Tris-HCl, pH 6.8, and 0.704% (v/v) β-mercaptoethanol. Stripped membranes were washed extensively with 0.01% Tween 20 and Tris-buffered saline, pH 7.0, then blocked with 5% bovine serum albumin for 1 h. Blots were reprobed with primary antibody overnight at 4 °C. Immunoreactive proteins were visualized using the indicated primary antibodies and enhanced ECL reagents followed by autoradiography and densitometry. Statistical Analysis—Data are presented as the mean ± S.E. The significance of difference was determined by one-way analysis of variance. Statistical significance was denoted at p < 0.05. IL-4-activated IRS-2-associated PI3-Kinase Activity Is Impaired in PerMΦs from db/db Mice—We have shown previously that chronic insulin exposure-dependent serine phosphorylation of IRS-1 can block cytokine signaling in myeloma cells in vitro (13Cengel K.A. Freund G.G. J. Biol. Chem. 1999; 274: 27969-27974Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). To investigate whether macrophages from type 2 diabetic animals had impaired IRS-2-dependent cytokine signaling, we examined PerMΦs from db/db mice (Table I). Fig. 1A shows that PerMΦs from 8-week-old db/db mice treated with IL-4 (5.5 ng/ml, 15 min) had a 44% (db/+, 100 ± 11.8%; db/db, 56.0 ± 12.3%; p < 0.05) reduction in IRS-2-associated PI3-kinase activity compared with similarly treated PerMΦs from db/+ control mice. Activity of PI3-kinase associated with IRS-2 basally was not different in db/db and db/+ PerMΦs. Fig. 1, B and C, demonstrates that, when insulin (100 nm, 10 min) or IGF-I (10 ng/ml, 10 min) was used in place of IL-4, PerMΦ PI3-kinase activity associated with IRS-2 was also reduced by 34% (db/+, 100 ± 9.3%; db/db, 66.2 ± 11.3%; p < 0.05) and 39% (db/+, 100 ± 4.6%; db/db, 61.4 ± 4.0%; p < 0.01) in db/db mice, respectively. To determine the serine phosphorylation state of IRS-2 in db/db mouse PerMΦs, Western analysis was performed. Fig. 1D demonstrates that IRS-2 Ser/Thr-Pro motif phosphorylation was increased 78% in db/db mouse PerMΦs compared with PerMΦs from db/+ mice (db/+, 100 ± 2.2%; db/db, 177.9 ± 2.2%; p < 0.001). Importantly, IRS-2 mass from both db/db and db/+ mice was similar (Fig. 1E; db/+, 100 ± 20.67%; db/db, 99 ± 20.67%; p = not significant). The mass of PI3-kinase from db/db and db/+ PerMΦs was also equivalent (data not shown). Taken together these results indicate that PerMΦs from type 2 diabetic mice have a reduced ability to form active IRS-2/PI3-kinase complexes coupled with augmented basal Ser/Thr-Pro motif IRS-2 phosphorylation.Table IDb/db mice have elevated serum insulin and blood glucose levelsdb/+db/dbBody Wt (g)26.72 ± 0.7436.10 ± 1.34ap < 0.05.FBG (mg/dl)93.4 ± 11.07379.2 ± 46.9ap < 0.05.RBG (mg/dl)157.6 ± 17.42448.6 ± 38.67ap < 0.05.FSI (ng/ml)1.31 ± 0.042.26 ± 0.07ap < 0.05.RSI (ng/ml)2.04 ± 0.065.31 ± 0.09ap < 0.05.(0.356 nm)(0.927 nm)a p < 0.05. Open table in a new tab Chronic Insulin and High Glucose Synergize to Block IL-4-activated IRS-2-associated PI3-Kinase Activity—We have shown previously that maturing promonocytic U937 cells with TPA induces a macrophage phenotype (44Deszo E.L. Brake D.K. Cengel K.A. Kelley K.W. Freund G.G. J. Biol. Chem. 2001; 276: 10212-10217Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 45Deszo E.L. Brake D.K. Kelley K.W. Freund G.G. Cell. Signal. 2004; 16: 271-280Crossref PubMed Scopus (22) Google Scholar). Fig. 2A shows that when U937 cells were matured with 100 nm TPA IRS-2 was up-regulated 10-fold. IRS-1 expression was not detected in U937 cells prior to maturation and was barely detectable after maturation (data not shown). When matured U937 cells were treated with 100 nm insulin for 10 min, IRS-2-associated PI3-kinase activity increased 150-fold, whereas no specific change in IRS-1-associated PI3-kinase was observed (Fig. 2B). To investigate whether chronic insulin (1 nm, 18 h) and high glucose (4.5 g/liter, 48 h) could impair IRS-2-dependent IL-4 signaling, we examined IRS-2·PI3-kinase complexes in matured U937 cells. Fig. 2C demonstrates that chronic insulin + high glucose treatment led to a 37.5% (–Ch.Ins, 100 ± 3.3%; +Ch.Ins, 62.5 ± 3.3%; p < 0.01) reduction in peak IL-4-activated (5.5 ng/ml, 15 min) IRS-2-associated PI3-kinase activity. Importantly, IL-4-activated IRS-2-associated PI3-kinase activity was not affected by chronic insulin or high glucose alone (Fig. 2D). Fig. 2E shows that chronic insulin + high glucose reduced by 54% (–Ch.Ins, 100 ± 16.2%; +Ch.Ins, 45.9 ± 16.2%; p < 0.05) IL-4-dependent (5.5 ng/ml, 15 min) IRS-2 tyrosine phosphorylation and inhibited by 37% (–Ch.Ins, 100 ± 6.3%; +Ch.Ins, 62.8 ± 6.3%; p < 0.01) IL-4-dependent (5.5 ng/ml, 15 min) IRS-2/PI3-kinase p85 association. Insulin alone had no impact on the ability of IL-4 to induce IRS-2 tyrosine phosphorylation or p85 association (data not shown). These results indicate that chronic insulin + high glucose synergize to block IL-4 from inducing IRS-2 tyrosine phosphorylation thereby diminishing its subsequent ability to associate with PI3-kinase. JAK/STAT Signaling Activated by IL-4 Is Unaffected by Chronic Insulin + High Glucose—As Fig. 2 shows, IL-4-dependent IRS-2 tyrosine phosphorylation is inhibited in matured U937 cells treated with chronic insulin + high glucose. This finding indicates that the ability of IL-4 to activate JAK may be impaired. Fig. 3A demonstrates that when matured U937 cells were treated with chronic insulin + high glucose, IL-4-dependent (5.5 ng/ml, 15 min) JAK1 autophosphorylation was similar to IL-4-dependent JAK1 activation in cells treated with just high glucose. As in Fig. 2, chronic insulin alone had no effect by itself and did not alter the ability of IL-4 to induce JAK1 autophosphorylation (data not shown). Because IL-4 can also transduce its signal via JAK3 (46Johnston J.A. Bacon C.M. Riedy M.C. O'Shea J.J. J. Leukocyte Biol. 1996; 60: 441-452Crossref PubMed Scopus (96) Google Scholar), JAK3 was examined. Fig. 3B shows that JAK3 was not detected in matured U937 cells. Finally, to examine whether other JAK1 substrates aside from IRS-2 were impacted by chronic insulin + high glucose IL-4-dependent (5.5 ng/ml, 15 min) STAT6 tyrosine phosphorylation was examined. Fig. 3C demonstrates that like IL-4-dependent JAK1 autophosphorylation, chronic insulin + high glucose had no effect on IL-4-dependent tyrosine phosphorylation of STAT6. As above, insulin alone did not alter the ability of IL-4 to induce STAT6 phosphorylation (data not shown). Taken together these results indicate that chronic insulin and high glucose target the IRS-2/PI3-kinase arm of the IL-4 signaling pathway and not the JAK/STAT arm. The mTOR Pathway Is Required for the Chronic Insulin + High Glucose Effect on IL-4 Signaling—We have shown previously that mTOR-dependent serine phosphorylation of IRS-1 within Ser/Thr-Pro motifs inhibits IFN-α signaling (18Hartman M.E. Villela-Bach M. Chen J. Freund G.G. Biochem. Biophys. Res. Commun. 2001; 280: 776-781Crossref PubMed Scopus (34) Google Scholar). To determine whether IL-4-dependent IRS-2 associated PI3-kinase activation was regulated by mTOR, rapamycin inhibition studies were performed. Fig. 4A shows that chronic insulin (1 nm, 18 h) + high glucose (4.5 g/liter, 48 h) treatment of matured U937 cells increased Ser/Thr-Pro motif phosphorylation 92% (–Ch.Ins, 100 ± 14.2%; +Ch.Ins, 192 ± 14.2%; p < 0.01) over high glucose alone. Importantly, pretreatment of matured U937 cells with 1 nm rapamycin 30 min prior to the addition of insulin completely blocked the increase in IRS-2 Ser/Th

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