Serine Phosphorylation of Insulin Receptor Substrate 1 by Inhibitor κB Kinase Complex

Abstract

Insulin resistance contributes importantly to the pathophysiology of type 2 diabetes mellitus. One mechanism mediating insulin resistance may involve the phosphorylation of serine residues in insulin receptor substrate-1 (IRS-1), leading to impairment in the ability of IRS-1 to activate downstream phosphatidylinositol 3-kinase-dependent pathways. Insulin-resistant states and serine phosphorylation of IRS-1 are associated with the activation of the inhibitor B kinase (IKK) complex. However, the precise molecular mechanisms by which IKK may contribute to the development of insulin resistance are not well understood. In this study, using phosphospecific antibodies against rat IRS-1 phosphorylated at Ser 307 (equivalent to Ser 312 in human IRS-1), we observed serine phosphorylation of IRS-1 in response to TNF-␣ or calyculin A treatment that paralleled surrogate markers for IKK activation. The phosphorylation of human IRS-1 at Ser 312 in response to tumor necrosis factor-␣ was significantly reduced in cells pretreated with the IKK inhibitor 15 deoxy-prostaglandin J 2 as well as in cells derived from IKK knock-out mice. We observed interactions between endogenous IRS-1 and IKK in intact cells using a co-immunoprecipitation approach. Moreover, this interaction between IRS-1 and IKK in the basal state was reduced upon IKK activation and increased serine phosphorylation of IRS-1. Data from in vitro kinase assays using recombinant IRS-1 as a substrate were consistent with the ability of IRS-1 to function as a direct substrate for IKK with multiple serine phosphorylation sites in addition to Ser 312 . Taken together, our data suggest that IRS-1 is a novel direct substrate for IKK and that phosphorylation of IRS-1 at Ser 312 (and other sites) by IKK may contribute to the insulin resistance mediated by activation of inflammatory pathways.Many factors implicated in the development of insulin resistance such as TNF-␣ 1 (1, 2), free fatty acids (3, 4), and serine phosphatase inhibitors (5, 6) are able to activate the inhibitor B kinase (IKK) complex and its downstream effector, NFB (7-10). Interestingly, insulin-sensitizing drugs such as thiazolidinediones inhibit NFB activity (11). Adiponectin, a cytokine secreted by adipose cells whose plasma levels are negatively correlated with insulin resistance (12), inhibits IKK activity in cells (13). Moreover, diet-induced insulin resistance is ameliorated in IKK2-deficient mice (14). Because IKK and NFB are major components of the intracellular inflammatory pathway, a cross-talk between metabolic and inflammatory signaling pathways may play an important role in the development of insulin resistance and the pathophysiology of major public health problems such as diabetes and obesity. However, molecular mechanisms by which IKK may specifically interact with metabolic insulin signaling pathways are not well understood. IKK is a serine kinase that controls the activation of NFB, a ubiquitous transcription factor closely associated with inflammation (15-18). In addition to inflammation, NF...

Hereditary Postprandial Hypertriglyceridemic Rabbit Exhibits Insulin Resistance and Central Obesity

“…19,20 Increase in the secretion of tumor necrosis factor-␣ (TNF-␣), plasminogen activator Inhibitor-1 (PAI-1) from enlarged adipocyte are highly related to the development of obese and IR. [21][22][23][24] In our observation, adipocytes in PHT rabbit were larger than in JW rabbit histopathologically (data not shown). Therefore, it was considered that PHT rabbit has an abnormal secretion of adipocytokines from adipose tissue.…”

Free fatty acid-induced muscle insulin resistance and glucose uptake dysfunction: Evidence for PKC activation and oxidative stress-activated signaling pathways

“…To test this hypothesis in our model, we applied the proteomic approach to study the key protein components involved in the C2C12 insulin resistance model, following FFA chronic treatment. We used the comparative proteomic analysis to screen for different kinases (75), phosphatases [27] and phosphosite proteins [31]. The phosphosite screen clearly showed a significant increase in the phosphorylation of PKCα (122%), PKCβ (631%), PKCδ (351%), and PKCε (698%) isoforms in cells treated with oleate but only alpha (80%) and delta (51%) in cells pretreated with palmitate.…”

“…Serine 307 in IRS-1 occurs in response to TNF-α as a paralleled surrogate marker for IKK activation [31]. Consequently, IRS-1 Serine 307 is inducible leading to the inhibition of IRS-1 function by either IKK or c-jun N-terminal kinase (JNK) [32][33][34].…”

Lipid signalling in disease

“…Mounting evidence indicates that activation of c-Jun N-terminal kinase (JNK), IKK and conventional PKC is central to the development of insulin resistance in response to obesity -mediated by the aforementioned fatty acidinduced ER stress, ROS production, TLR activation and inflammatory signalling by TNF . JNK, IKK and PKC have all been reported to inhibit insulin action by phosphorylating IRS1 on serine [80][81][82] . Serine phosphorylation of IRS1 disrupts insulin-receptor signalling through several distinct mechanisms and blocks insulin action 83,84 .…”