A Molecular Basis for Insulin Resistance

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...

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confidence: 92%

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

Hartman

O’Connor

Godbout

et al. 2004

“…Of these targets, IRS proteins have been studied widely as a major target in the TNF-induced insulin resistance. TNF-␣ has been consistently reported to inhibit insulin signaling by increasing serine phosphorylation of IRS proteins (11,(15)(16)(17). Serine phosphorylation of IRS proteins leads to IRS inhibition through at least two possibilities, which are inhibition of tyrosyl phosphorylation of IRS-1 (13,16,17), and proteasome-mediated degradation of IRS-1 (18 -21).…”

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confidence: 99%

Aspirin Inhibits Serine Phosphorylation of Insulin Receptor Substrate 1 in Tumor Necrosis Factor-treated Cells through Targeting Multiple Serine Kinases

Gao

Zuberi

Quon

et al. 2003

230

145

“…Many in vitro and in vivo studies (5)(6)(7)(8)(9)(10)(11)(12)(13) have shown that increased Ser/Thr phosphorylation of IRS-1, e.g. after treatment of cells with either activators of protein kinase C (PKC), Ser/Thr phosphatase inhibitors, high insulin concentrations, or activation of cellular stress pathways by tumor necrosis factor ␣ and other cytokines, inhibits the IR-mediated tyrosine phosphorylation of IRS-1, thereby affecting insulin signal transduction.…”

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confidence: 99%

Protein Kinase C-ζ-induced Phosphorylation of Ser318 in Insulin Receptor Substrate-1 (IRS-1) Attenuates the Interaction with the Insulin Receptor and the Tyrosine Phosphorylation of IRS-1

Moeschel¹,

Beck²,

Weigert³

et al. 2004

115

Insulin receptor substrate-1 (IRS-1) was recently identified as a novel upstream substrate for the insulin-activated protein kinase C (PKC)-. This interaction down-regulates insulin signal transduction under hyper-insulinemic conditions. To clarify the molecular mechanism of this feedback loop, we sought to identify the PKC-phosphorylation sites of IRS-1 and to investigate their biological significance. Upon incubation of recombinant IRS-1 fragments with PKC-, we identified Ser 318 of rat IRS-1 (Ser 323 in human IRS-1) as the major in vitro phosphorylation site (confirmed by mutation of Ser 318 to alanine). To monitor phosphorylation of Ser 318 in cellular extracts, we prepared a polyclonal phosphosite-specific antibody. The biological significance was studied in baby hamster kidney cells stably expressing the insulin receptor (BHK IR ). Using the phospho-Ser 318 -specific antibody we observed that insulin stimulates phosphorylation of Ser 318 in IRS-1, which is mediated, at least partially, by PKC-. Moreover, we found that the previously described insulin-stimulated, PKC--mediated inhibition of the interaction of IRS-1 with the insulin receptor and the reduced tyrosine phosphorylation of IRS-1 was abrogated by mutation of IRS-1 Ser 318 to alanine. These results, generated in BHK IR cells, suggest that phosphorylation of Ser 318 by PKC-might contribute to the inhibitory effect of prolonged hyperinsulinemia on IRS-1 function.Insulin resistance is associated with a variety of physiological and patho-physiological states, including type 2 diabetes, hypertension, glucose intolerance, and obesity; however, the molecular mechanisms that modulate insulin signaling under these conditions are difficult to resolve. One important early site of divergence in insulin signaling, which seems to be a relevant target for modulation of the signal, is insulin receptor substrate 1 (IRS-1) 1 (1). IRS-1 is a hydrophilic protein with an apparent molecular mass of 180 kDa containing a conserved pleckstrin homology domain located at the amino terminus, adjacent to a phosphotyrosine binding (PTB) domain (2). After insulin-stimulation, this domain interacts with the activated insulin receptor (IR), IRS-1 is subsequently tyrosine-phosphorylated, and the signal is transmitted further (1, 2).In addition to tyrosine phosphorylation, IRS-1 undergoes Ser/Thr phosphorylation at multiple sites (3, 4). Many in vitro and in vivo studies (5-13) have shown that increased Ser/Thr phosphorylation of IRS-1, e.g. after treatment of cells with either activators of protein kinase C (PKC), Ser/Thr phosphatase inhibitors, high insulin concentrations, or activation of cellular stress pathways by tumor necrosis factor ␣ and other cytokines, inhibits the IR-mediated tyrosine phosphorylation of IRS-1, thereby affecting insulin signal transduction.The atypical PKC-, which is activated by insulin, has been identified to transduce insulin signaling downstream from IRS-1 and phosphatidylinositol 3-kinase in mediating glucose uptake. However, as reported recently, pr...