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Originally published In Press as doi:10.1074/jbc.M404368200 on April 27, 2004

J. Biol. Chem., Vol. 279, Issue 27, 28045-28050, July 2, 2004
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Insulin Receptor Substrate-2-dependent Interleukin-4 Signaling in Macrophages Is Impaired in Two Models of Type 2 Diabetes Mellitus*

Matthew E. Hartman{ddagger}, Jason C. O'Connor{ddagger}, Jonathan P. Godbout{ddagger}, Kyle D. Minor{ddagger}, Valerie R. Mazzocco{ddagger}, and Gregory G. Freund{ddagger}§

From the Departments of {ddagger}Animal Sciences and §Pathology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Received for publication, April 20, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have shown previously that hyperinsulinemia inhibits interferon-{alpha}-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 (PerM{Phi}s) from diabetic (db/db) mice showed a 44% reduction in IRS-2-associated PI3-kinase activity stimulated by IL-4 compared with PerM{Phi}s from heterozygote (db/+) control mice. IRS-2 from db/db mouse PerM{Phi}s 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The first member of the insulin receptor substrate (IRS)1 family, IRS-1, was initially discovered in Fao hepatoma cells as a tyrosine-phosphorylated substrate of the insulin receptor (1). 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-{alpha} and IFN-{gamma} (2–10). Importantly, serine phosphorylation of IRS-1 blocks insulin, IGF-I, and cytokine signaling through IRS-1 (11–15) and appears critical to the initiation of proteasome-dependent IRS-1 degradation (16, 17). We have shown that chronic insulin in the presence of high glucose leads to serine phosphorylation of IRS-1 through an mTOR-dependent mechanism and that this renders IRS-1 a poorer substrate for JAK1 (18). In addition, we have shown that serine phosphorylation targets IRS-1 for proteasome-dependent degradation in L6 muscle cells (19). However, these same mechanisms have not been investigated in relation to IRS-2-dependent cytokine signaling.

IRS-2 is expressed in a wide variety of tissues and appears, in IRS-1 knock-out mice, to duplicate many of the functions of IRS-1 (20–22). Although IRS-1 is most important to insulin action in skeletal muscle, IRS-2 appears to be required for insulin/IGF-I and, importantly, cytokine signaling in liver, muscle, fat, pancreatic {beta} cells, B cells, T cells, and macrophages (9, 23–25). IRS-2 knock-out studies in mice have shown that IRS-2 is important for neuroendocrine function, and a lack of IRS-2 leads to enhanced neointima formation (26, 27). Furthermore, IRS-2 is necessary for IL-4-induced proliferation and differentiation of T cells (28). Sequence alignment shows a high degree of homology between IRS-1 and IRS-2, including the presence of two Src homology 2 domain binding sites for the p85 subunit of PI3-kinase (20). As with IRS-1, serine phosphorylation appears to regulate IRS-2 tyrosine phosphorylation negatively. Removal of basal serine phosphorylation of IRS-2 increases IGF-I-stimulated tyrosine phosphorylation of IRS-2, and hyperphosphorylation of IRS-2 on serine residues impairs insulin receptor-dependent IRS-2 tyrosine phosphorylation (29). In addition, Ser/Thr phosphorylation of IRS-2 induced by either TNF-{alpha} or prolonged insulin exposure results in a reduced ability of IRS-2 to interact with the juxtamembrane region of the insulin receptor (15).

The IL-4 receptor is expressed ubiquitously on monocytes and macrophages (30, 31), and, as with other class I cytokine receptors (hematopoietin receptor family), the IL-4 receptor lacks intrinsic kinase activity and requires receptor-associated kinases for initiation of intracellular signaling (32, 33). Binding of IL-4 to its receptor leads to JAK1 and JAK3 activation. One or both of these tyrosine kinases phosphorylate the IL-4{alpha} chain on residues Tyr497, Tyr575, Tyr603, Tyr631, and Tyr713. Tyr497 is within the insulin/IL-4 receptor motif and is responsible for IRS-2 recruitment (34). IL-4 is a potent anti-inflammatory cytokine and leads to an "alternative activation phenotype" in macrophages. This IL-4-dependent macrophage activation results in an absence of nitric oxide production, generation of IL-10 and IL-1 receptor antagonist, and suppressive activity directed toward T cells (35).

Recently, subacute chronic inflammation (36, 37) has been identified as a significant contributor to the complications associated with type 2 diabetes mellitus (38). Cardiovascular problems such as accelerated and exacerbated atherosclerosis appear especially responsive to the proinflammatory environment of diabetes (37, 39). The diabetic state is known to alter macrophage function resulting in increased lipoprotein lipase production and TNF-{alpha} release (40). In addition, hyperglycemia, through the generation of advanced glycation end products, increases macrophage secretion of IL-1 and TNF-{alpha} while enhancing intercellular adhesion molecule 1 expression and decreasing phagocytic activity (41). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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). [{gamma}-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 x g.

Peritoneal Macrophage (PerM{Phi}) 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 x 105 cells/ml, and after 30 min plates were washed twice to remove nonadherent cells, resulting in ~80% pure macrophages, confirmed by morphology (42).

PI3-Kinase Assays—PI3-kinase activity was assayed as described previously (43). 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-{alpha}-phosphatidylinositol in 0.4 mM EGTA, 0.4 mM NaPO4, 8 µM [{gamma}-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 x 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 x 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 (44, 45). 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 x 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 (13). In brief, 5 x 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) {beta}-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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
IL-4-activated IRS-2-associated PI3-Kinase Activity Is Impaired in PerM{Phi}s 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 (13). To investigate whether macrophages from type 2 diabetic animals had impaired IRS-2-dependent cytokine signaling, we examined PerM{Phi}s from db/db mice (Table I). Fig. 1A shows that PerM{Phi}s 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 PerM{Phi}s from db/+ control mice. Activity of PI3-kinase associated with IRS-2 basally was not different in db/db and db/+ PerM{Phi}s. 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, PerM{Phi} 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 PerM{Phi}s, Western analysis was performed. Fig. 1D demonstrates that IRS-2 Ser/Thr-Pro motif phosphorylation was increased 78% in db/db mouse PerM{Phi}s compared with PerM{Phi}s 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/+ PerM{Phi}s was also equivalent (data not shown). Taken together these results indicate that PerM{Phi}s 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.


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  		TABLE I
Db/db mice have elevated serum insulin and blood glucose levels

Weight (Wt), fasting blood glucose (FBG), random blood glucose (RBG), fasting serum insulin (FSI), and random serum insulin (RSI) were determined. Results represent the average of five mice ± S.E.

 



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  		FIG. 1.
IL-4-activated IRS-2-associated PI3-kinase activity is impaired in PerM{Phi}s from db/db mice. A, PerM{Phi}s were isolated from db/+ and db/db mice as described under "Experimental Procedures." PerM{Phi}s were then treated (+) or not (–) with 5.5 ng/ml IL-4 for 15 min, and PI3-kinase activity was assayed in IRS-2 immunoprecipitates. Results are representative of three independent experiments. B, PerM{Phi}s were isolated from db/+ and db/db mice as described under "Experimental Procedures." PerM{Phi}s were then treated (+) or not (–) with 100 nM insulin (Ins) for 10 min, and PI3-kinase activity was assayed in IRS-2 immunoprecipitates. Results are representative of three independent experiments. C, PerM{Phi}s were isolated from db/+ and db/db mice as described under "Experimental Procedures." PerM{Phi}s were then treated (+) or not (–) with 10 ng/ml IGF-I for 10 min, and PI3-kinase activity was assayed in IRS-2 immunoprecipitates. Results are representative of three independent experiments. D, PerM{Phi}s were isolated from db/+ and db/db mice as described under "Experimental Procedures." Ser/Thr-Pro motif phosphorylation (pS/T-P) was measured in IRS-2 immunoprecipitates by Western analysis with an anti-pS/T-P antibody (upper panel). Membranes were stripped and reprobed with an anti-IRS-2 antibody (lower panel) as described under "Experimental Procedures." Results are representative of three independent experiments. E, PerM{Phi}s were isolated from db/+ and db/db mice as described under "Experimental Procedures." IRS-2 (upper panel) and {beta}-actin (lower panel) were measured in whole cell lysates by Western analysis using an anti-IRS-2 and anti-{beta}-actin antibody, respectively. Results are representative of three independent experiments.

 
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 (44, 45). 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.



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  		FIG. 2.
Chronic insulin and high glucose synergize to block IL-4-activated IRS-2-associated PI3-kinase activity. A, IRS-2 was detected by Western analysis using an anti-IRS-2 antibody from TPA-matured (Mat) and -unmatured (Con) U937 cells. Results are representative of three independent experiments. B, matured U937 cells were treated (+) or not (–) with 100 nM insulin (Ins) for 10 min. PI3-kinase activity was measured in IRS-1 and IRS-2 immunoprecipitates. Results are representative of three independent experiments. C, matured U937 cells were grown in high glucose medium (4.5 g/liter) for 48 h. During the last 18 h, cells were treated (+) or not (–) with 1 nM insulin (Ch.Ins). Cells were then stimulated with 5.5 ng/ml IL-4 for the indicated times. PI3-kinase activity was measured in IRS-2 immunoprecipitates. Results are representative of three independent experiments. D, matured U937 cells were grown in low glucose medium (1 g/liter) (Con, Ins) or high glucose medium (4.5 g/liter) (Glc) for 48 h. During the last 18 h, cells were treated with (Ins) or without (Con, Glc) 1 nM insulin. Cells were then stimulated with 5.5 ng/ml IL-4 for the indicated times. PI3-kinase activity was measured in IRS-2 immunoprecipitates. Results are representative of three independent experiments. E, matured U937 cells were grown in high glucose medium (4.5 g/liter) for 48 h. During the last 18 h, cells were treated (+) or not (–) with 1 nM insulin. Cells were then stimulated with (+) or without (–) 5.5 ng/ml IL-4 for 15 min. Phosphotyrosine (pY), PI3-kinase p85 (p85), and IRS-2 were detected by Western analysis in IRS-2 immunoprecipitates using an anti-pY, anti-p85, and anti-IRS-2 antibody, respectively. Results are representative of three independent experiments.

 
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 (46), 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.



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  		FIG. 3.
JAK/STAT signaling activated by IL-4 is unaffected by chronic insulin + high glucose. A, matured U937 cells were grown in high glucose medium (4.5 g/liter) for 48 h. During the last 18 h, cells were treated (+) or not (–) with 1 nM insulin (Ch.Ins). Cells were then stimulated (+) or not (–) with 5.5 ng/ml IL-4 for 15 min. Phosphotyrosine (pY) was detected by Western analysis in JAK1 immunoprecipitates using an anti-pY antibody (upper panel). JAK1 was detected by Western analysis in whole cell lysates using an anti-JAK1 antibody (lower panel). Results are representative of three independent experiments. B, matured U937 cells were grown in high glucose medium (4.5 g/liter) for 48 h. During the last 18 h, cells were treated (+) or not (–) with 1 nM insulin. Cells were then stimulated (+) or not (–) with 5.5 ng/ml IL-4 for 15 min. JAK3 was detected by Western analysis in whole cell lysates using an anti-JAK3 antibody. TF-1 cell lysates were used as a positive control for the JAK3 antibody. Results are representative of three independent experiments. C, matured U937 cells were grown in high glucose medium (4.5 g/liter) for 48 h. During the last 18 h, cells were treated (+) or not (–) with 1 nM insulin. Cells were then stimulated (+) or not (–) with 5.5 ng/ml IL-4 for 15 min. Phospho-STAT6 (pSTAT6) was detected by Western analysis in whole cell lysates using an anti-pSTAT6 antibody. Results are representative of three independent experiments.

 
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-{alpha} signaling (18). 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/Thr-Pro motif phosphorylation (Rap, +Ch.Ins, 16.2 ± 2.5%; p < 0.001 with +Ch.Ins above). In addition, insulin-independent Ser/Thr-Pro motif phosphorylation was also inhibited by rapamycin (Rap, –Ch.Ins, 18.4 ± 2.5%; p < 0.001 with –Ch.Ins above). The mass of IRS-2 was unchanged by the addition of rapamycin in the presence or absence of insulin. Similar to Figs. 2 and 3, 18 h of chronic insulin treatment in the presence of low glucose (1 g/liter) did not result in increased phosphorylation of IRS-2 Ser/Thr-Pro motifs (Fig. 4B) (–Ch.Ins, 100 ± 4.65%; +Ch.Ins, 102.8 ± 4.65%; p = not significant). Fig. 4C, like Fig. 1, demonstrates that IL-4-dependent IRS-2-associated PI3-kinase activity was reduced by 41.3% (–Ch.Ins, 100 ± 7.8%; +Ch.Ins, 58.7 ± 7.8%; p < 0.01) in matured U937 cells treated with chronic insulin + high glucose. When cells were pretreated with 1 nM rapamycin for 30 min prior to the chronic insulin addition, IL-4-dependent IRS-2-associated PI3-kinase activity was restored (Rap, –Ch.Ins, 195.3 ± 39.9%; +Ch.Ins, 178.1 ± 39.9%; p = not significant). The mass of p85 was unaffected by rapamycin pretreatment, and, as in Fig. 1, chronic insulin treatment had no effect on IL-4-activated PI3-kinase activity in the presence of low glucose (data not shown). Taken together, these results indicate that the mTOR pathway mediates the suppressive effect of chronic insulin + high glucose on IL-4-dependent IRS-2-associated PI3-kinase activation.



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  		FIG. 4.
The mTOR pathway is required for the chronic insulin + high glucose effect on IL-4 signaling. A, matured U937 cells were grown in high glucose medium (4.5 g/liter) for 48 h. During the last 18 h, cells were treated (+) or not (–) with 1 nM insulin (Ch.Ins). 30 min before the addition of insulin, cells were pretreated (+) or not (–) with 1 nM rapamycin (Rap). Ser/Thr-Pro motif phosphorylation (pS/T-P) was detected by Western analysis in IRS-2 immunoprecipitates using an anti-pS/T-P antibody (upper panel). IRS-2 was detected by Western analysis in whole cell lysates using an anti-IRS-2 antibody (lower panel). Results are representative of three independent experiments. B, matured U937 cells were grown in low glucose medium (1 g/liter) for 48 h. During the last 18 h, cells were treated (+) or not (–) with 1 nM insulin. Ser/Thr-Pro motif phosphorylation was detected by Western analysis in IRS-2 immunoprecipitates using an anti-pS/T-P antibody (upper panel). IRS-2 was detected by Western analysis in whole cell lysates using an anti-IRS-2 antibody (lower panel). Results are representative of three independent experiments. C, matured U937 cells were grown in high glucose medium (4.5 g/liter) for 48 h. During the last 18 h, cells were treated (+) or not (–) with 1 nM insulin. 30 min before the addition of insulin cells were pretreated (+) or not (–) with 1 nM rapamycin. After insulin treatment, cells were then stimulated (+) or not (–) with 5.5 ng/ml IL-4 for 15 min. PI3-kinase activity was measured in IRS-2 immunoprecipitates (upper panel). PI3-kinase p85 (p85) was detected by Western analysis in whole cell lysates using an anti-p85 antibody (lower panel). Results are representative of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Serine phosphorylation has emerged as an important mechanism for counterregulating insulin-dependent IRS-1/PI3-kinase signaling in diabetes (15, 47–50). Serine phosphorylation of IRS-1 occurs constitutively in the cell and is enhanced further by hyperinsulinemia (47, 48), hyperglycemia (51), and proinflammatory cytokines (52, 53). Numerous serine/threonine kinases have been shown to phosphorylate IRS-1 in vitro, including mTOR (18). Serine phosphorylation of IRS-1 can inhibit insulin receptor-dependent tyrosine phosphorylation of IRS-1 (11, 12, 14, 15, 54) and targets IRS-1 to the proteasome for degradation (16, 17, 19). Interestingly, it is not as well recognized that the same mechanisms that result in insulin resistance can, in turn, lead to cytokine resistance through impaired IRS-2 signaling. As Fig. 1 demonstrates, we examined IRS-2/PI3-kinase signaling in PerM{Phi}s from db/db mice, which have both marked hyperglycemia and hyperinsulinemia (Table I). These experiments showed a 44% reduction in IRS-2-associated PI3-kinase activity in response to IL-4 as well as reduced insulin and IGF-I-stimulated IRS-2-associated PI3-kinase activity. Coupled to this reduction in IL-4-dependent IRS-2-associated PI3-kinase activity in db/db mice was a 78% increase in IRS-2 Ser/Thr-Pro motif phosphorylation. Importantly, IRS-2 mass was unaffected in PerM{Phi}s from db/db mice compared with db/+ mice. These data are notable because they are the first to show that cytokine signaling mediated by IRS-2 is reduced in cells from diabetic animals. These results were somewhat unexpected in that reduced IRS-1-dependent insulin signaling is thought to be a result of proteasomal loss of IRS-1 subsequent to its serine phosphorylation (16, 17, 19), but here we show that IRS-2 mass in PerM{Phi}s is unchanged in diabetic db/db mice. These findings indicate that IRS-2 mass loss is not critical to blunted IL-4/IRS-2/PI3-kinase signaling. This is not entirely surprising because diabetes-dependent proteasomal loss of IRS-2 may be tissue-specific, and we have shown in vitro that insulin-dependent inhibition of IFN-{alpha}/IRS-1/PI 3 kinase signaling does not depend on IRS-1 mass loss (13, 18).

New work by Pirola et al. (55) shows that in L6 muscle cells, prolonged insulin treatment leads to a reduction in IRS-2 protein levels which is dependent on the PI3-kinase/mTOR pathway. In primary skeletal muscle cells from patients with impaired glucose tolerance, however, IRS-2 protein levels were unaffected by high glucose, although insulin-dependent IRS-2 tyrosine phosphorylation and associated PI3-kinase activity were decreased significantly (56). In adipocytes, high glucose treatment when combined with insulin results in a near complete loss of IRS-2 expression (57). Additionally, high glucose in human aortic endothelial cells has been shown to reduce IRS-2 expression (58). To determine the role of insulin and glucose in IL-4/IRS-2/PI3-kinase signaling in PerM{Phi}s, we examined matured U937 cells grown in medium enriched with both insulin and glucose. Fig. 2 shows that blunted IL-4-dependent IRS-2-associated PI3-kinase activation was only present when matured U937 cells were exposed to both chronic insulin and high glucose. These results are consistent with our previous in vitro findings in myeloma cells where we demonstrated that 1 nM insulin blunted IFN-{alpha}-dependent IRS-1-associated PI3-kinase activation in cells grown in high glucose (4.5 g/liter) medium (13, 18). In our previous work, however, we did not test low glucose conditions to determine its role in blocking cytokine signaling. Our new studies indicate that exposure to elevated glucose concentrations is as critical as insulin is to inducing IRS Ser/Thr-Pro motif phosphorylation and down-regulating IRS-2-dependent PI3-kinase interactions.

As we (18) and others (59, 60) have shown, mTOR phosphorylates proteins within Ser/Thr-Pro motifs. Therefore, to demonstrate that chronic insulin + high glucose-dependent blunting of IL-4/IRS-2/PI3-kinase signaling was the result of mTOR, rapamycin inhibition studies were performed. Fig. 4 shows that rapamycin blocks chronic insulin + high glucose-dependent IRS-2 Ser/Thr-Pro motif phosphorylation and reverses the effect that chronic insulin + high glucose has on IL-4/IRS-2/PI3-kinase signal transduction. It is important to note that rapamycin is considered a highly specific inhibitor, especially at the nanomolar concentrations that were used in this study. Unlike most kinase inhibitors, rapamycin has a unique mechanism that requires the binding of a coreceptor (FKBP12), and it targets a domain unique to mTOR situated outside of the kinase domain (61–65). Grolleau et al. (66) have demonstrated that treatment of Jurkat T cells with 20 nM rapamycin for 3 days results in altered expression of 16 proteins; however, none of these proteins is a serine kinase. In addition, the experiments run by Grolleau et al. (66) demonstrated that a number of kinases, such as members of the mitogen-activated protein kinase cascade, were unaffected by rapamycin treatment. When Davies et al. (67) examined the ability of rapamycin to inhibit various kinases, they found that even at 1 µM, none of the 24 kinases on their panel was affected. In this study, we used a concentration of rapamycin 20–1,000-fold lower than that used in the above two reports. These findings indicate that 1 nM rapamycin is specific for mTOR, and because Ser/Thr-Pro motifs are thought to be consensus sites for mTOR (59, 60), it is likely that the rapamycin effects we demonstrate are mediated directly by mTOR. In addition, chronic insulin + high glucose had no impact on IL-4-dependent JAK1/STAT6 signaling (Fig. 3). Taken together these findings indicate that chronic insulin + high glucose, acting through the mTOR pathway, does not prevent IL-4 from activating JAK1 but does disrupt the ability of IRS-2 from acting as a JAK1 substrate. This work is supported by our previous studies where we demonstrated that serine-phosphorylated IRS-1 was a poorer substrate for JAK1 (13) and that mTOR, but not p70s6k, mediated this effect (18). Also, because PerM{Phi}s from db/db mice have increased IRS-2 Ser/Thr-Pro motif phosphorylation compared with PerM{Phi}s from db/+ mice, and the mass of IRS-2 in PerM{Phi}s from both of these animal types is the same, our data buttress the idea that cytokine-dependent IRS-2/PI3-kinase signaling is perturbed in vivo by a mechanism dependent on IRS-2 serine phosphorylation and not IRS-2 proteasomal mass loss.

Finally, IL-4 plays a key role in inhibiting proinflammatory cytokine production in activated macrophages (68, 69). Prediabetes and type 2 diabetes mellitus are characterized by subacute chronic inflammation where individuals show elevated serum levels of acute phase proteins such as C-reactive protein and fibrinogen (70) and cytokines such as TNF-{alpha} (71) and IL-6 (70, 72). Although TNF-{alpha} is produced mainly by adipose tissue in obese prediabetic and diabetic individuals (71, 73), a critical source for the proinflammatory cytokines IL-6 and IL-1{beta} is activated macrophages (74). Furthermore, these macrophage-generated cytokines are potent inducers of liver-produced C-reactive protein and fibrinogen. How chronic inflammation develops in type 2 diabetes mellitus is unclear, but here we suggest that macrophage resistance to anti-inflammatory cytokines may be a potential mechanism. Currently, the role of IL-4/IRS-2/PI3-kinase signaling in macrophages is undefined. We (75, 76) have shown that during monocyte development the IL-4/IRS-2/PI3-kinase pathway is important to cell survival and growth, but in mature and activated macrophages this role for IL-4 has not been shown, and we have not observed it (data not shown). Although further research is needed to determine how disruption of IL-4/IRS-2/PI3-kinase signaling impacts macrophage dependent innate immunity in diabetes, our findings here show that the IRS-2 arm of the IL-4 pathway is inhibited in two models of type 2 diabetes mellitus by a mechanism that relies on insulin + glucose-dependent Ser/Thr-Pro motif phosphorylation mediated by the mTOR pathway.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant DDK064862 (to G. G. F.) and grants from the American Heart Association, Dallas, Texas (to G. G. F. and J. C. O.), the University of Illinois Agricultural Experiment Station (to G. G. F.), and the American Diabetes Association, Alexandria, Virginia (to M. E. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Pathology, College of Medicine, 506 South Mathews, University of Illinois at Urbana-Champaign, Urbana, IL 61801. Tel.: 217-244-8839; Fax: 217-244-5617; E-mail: freun{at}uiuc.edu.

1 The 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; PerM{Phi}s, peritoneal macrophages; PI3-kinase, phosphatidylinositol 3-kinase; STAT, signal transducers and activators of transcription; TNF, tumor necrosis factor; TPA, 12-O-tetradecanoylphorbol-13-acetate. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Jie Chen (Department of Cell and Structural Biology, University of Illinois, Urbana) for sharing her expertise in rapamycin and mTOR.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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