Increased in vivo phosphorylation of insulin receptor at serine 994 in the liver of obese insulin-resistance Zucker rats. Role of the serine/threonine kinase (TANK)-binding kinase 1

MUÑOZ MC; TOBLLI JE; GIANI JF; DOMINICI FP; TURYN D

Breckenridge, Colorado, United States of America

Simposio; Keystone Symposia. Diabetes Mellitus, Insulin Action and Resistance; 2008

Keystone Symposia

Insulin resistance has been associated with several disease processes such as hypertension, type 2 diabetes, central obesity and chronic low-grade inflammation. Multiple changes in serine/threonine and tyrosine phosphorylation, in different components of the insulin receptor (IR) signaling pathway, have been linked to modulation of IR activity and insulin resistance. However, the role of IR serine (Ser) phosphorylation in insulin resistance remains elusive. We have previously shown that in obese Zucker rats (OZR), the angiotensin II (AII) type-1 receptor blocker irbesartan (IRB) enhances the insulin signaling pathway leading to Akt activation in liver. This enhancement of the insulin signaling cascade was accompanied by a reduction in the phosphorylation of the IR at Ser residue 994. To determine which Ser kinases are involved in the phosphorylation of IR at Ser994, we used peptide array phosphorylation assays that revealed that TANK-binding kinase 1 (TBK-1) is able to phosphorylate the IR at this residue. TBK-1 is an IKK-related kinase that mediates IKK and NF-kB activation in response to TNF-a, phorbol ester and growth factors. To study the role of TBK-1 in the in vivo phosphorylation of IR Ser994, ten-weeks-old male OZR (fa/fa) and their age-matched lean controls (+/?) (LZR) were divided into three groups (n=8): OZR; OZR treated with IRB (six months) and LZR. The extent of phosphorylation of several components of the insulin signalling system in liver was assessed by immunoprecipitation followed by immunoblotting with phosphospecific antibodies. Additionally, liver AII and TNF-a levels as well as fat deposits were determined by immunohistochemistry and Oil red O respectively.  We found that OZR displayed an elevation of hepatic AII, TNF-a, and IL-6 levels, together with a high proportion of macro and microvesicular steatosis in hepatocytes, when compared with LZR. Following treatment with IRB, OZR showed a significant reduction (p< 0.01) in the immunostaining of TNF-a and IL-6, together with a reduction of hepatic steatosis compared with untreated-OZR (p<0.01). Moreover, insulin-stimulated IR tyrosine phosphorylation was increased by 48% in OZR after treatment with IRB (p< 0.01). This change correlated with a marked decrease in IR Ser994 phosphorylation (p<0.01). The hepatic TBK-1 abundance and the association of this enzyme with the IR were 4-fold increased in OZR (p<0.01 vs. LZR). Interestingly, chronic IRB treatment was associated with a reduction (4-fold)  in the hepatic abundance of TBK-1 and with a decrease (3-fold) in the TBK-1 associated to IR (p < 0.01 in both cases), suggesting that TBK-1 could be implicated in the insulin resistance mechanism in response to exacerbation of the renin-angiotensin system displayed by OZR.