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

Abstract

We have shown previously that hyperinsulinemia inhibits interferon-␣-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⌽s) from diabetic (db/db) mice showed a 44% reduction in IRS-2-associated PI3-kinase activity stimulated by IL-4 compared with PerM⌽s from heterozygote (db/؉) control mice. IRS-2 from db/db mouse PerM⌽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.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-␣ and IFN-␥ (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 proteasomedependent 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...

Induction of triggering receptor expressed on myeloid cells 1 in murine resident peritoneal macrophages by monosodium urate monohydrate crystals

“…Resident peritoneal macrophages were isolated from male ICR mice (ages 6-8 weeks), as reported elsewhere (16). After washing with PBS, the cells were suspended in RPMI 1640 medium (Sigma) supplemented with 5% heat-inactivated fetal calf serum (HyClone, Logan, UT), 100 units/ml penicillin, and 100 g/ml streptomycin (Invitrogen, Carlsbad, CA).…”

High glucose attenuates insulin-induced VEGF expression in bovine retinal microvascular endothelial cells

“…Matthew and associates reported that chronic insulin and high glucose synergistically inhibit the interleukin-4-dependent activation of PI3-kinase in macrophages through the mTOR pathway. 29 Tahir reported that high glucose impaired receptor and insulin receptor substrate-1 phosphorylation by 34%. Phosphatidylinositol 3-kinase activity was decreased by 85%.…”

Augmented Lipopolysaccharide-Induced TNF-α Production by Peritoneal Macrophages in Type 2 Diabetic Mice Is Dependent on Elevated Glucose and Requires p38 MAPK

“…All animal care and use was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council) as we have described (18). B6.Cg-ϩLeprdb/ϩLeprolb (db/db) mice and their age-matched nondiabetic B6.Cg-Mϩ/ϩLeprdb (db/ϩ) littermates were bred in-house from mice purchased from The Jackson Laboratory.…”

“…PI3K activity was assayed as we have described previously (18). In brief, PerM were isolated on plastic, treated as indicated, and lysed in ice-cold homogenization buffer (1% Triton X-100, 100 mM NaCl, 50 mM NaF, 1 mM PMSF, 2 g/ml aprotinin, 2 g/ml leupeptin, 2 mM sodium orthovanadate, 50 mM okadaic acid, and 50 mM Tris (pH 7.4)).…”

“…Western blot analysis was performed as we have described previously (18). In brief, PerM were isolated on plastic, treated as indicated, and lysed in ice-cold homogenization buffer.…”