In Skeletal Muscle Advanced Glycation End Products (AGEs) Inhibit Insulin Action and Induce the Formation of Multimolecular Complexes Including the Receptor for AGEs

Angela Cassese,Iolanda Esposito,Francesca Fiory,Alessia P.M Barbagallo,Flora Paturzo,Paola Mirra,Luca Ulianich,Ferdinando Giacco,Claudia Iadicicco,Angela Lombardi,Francesco Oriente,Emmanuel Van Obberghen,Francesco Beguinot,Pietro Formisano,Claudia Miele

doi:10.1074/jbc.m801698200

Abstract

Chronic hyperglycemia promotes insulin resistance at least in part by increasing the formation of advanced glycation end products (AGEs). We have previously shown that in L6 myotubes human glycated albumin (HGA) induces insulin resistance by activating protein kinase Calpha (PKCalpha). Here we show that HGA-induced PKCalpha activation is mediated by Src. Coprecipitation experiments showed that Src interacts with both the receptor for AGE (RAGE) and PKCalpha in HGA-treated L6 cells. A direct interaction of PKCalpha with Src and insulin receptor substrate-1 (IRS-1) has also been detected. In addition, silencing of IRS-1 expression abolished HGA-induced RAGE-PKCalpha co-precipitation. AGEs were able to induce insulin resistance also in vivo, as insulin tolerance tests revealed a significant impairment of insulin sensitivity in C57/BL6 mice fed a high AGEs diet (HAD). In tibialis muscle of HAD-fed mice, insulin-induced glucose uptake and protein kinase B phosphorylation were reduced. This was paralleled by a 2.5-fold increase in PKCalpha activity. Similarly to in vitro observations, Src phosphorylation was increased in tibialis muscle of HAD-fed mice, and co-precipitation experiments showed that Src interacts with both RAGE and PKCalpha. These results indicate that AGEs impairment of insulin action in the muscle might be mediated by the formation of a multimolecular complex including RAGE/IRS-1/Src and PKCalpha.

Highlights

advanced glycation end products (AGEs) and Insulin Resistance decrease the incidence of type 1 diabetes in non-obese diabetic mice [18] as well as the formation of atherosclerotic lesions in diabetic apolipoprotein E-deficient mice [19]
In this work we report that human glycated albumin (HGA) and dietary AGEs induce the formation of a complex including receptor for AGE (RAGE), protein kinase C (PKC)␣, and Src in L6 cells and in skeletal muscle from insulin-resistant high AGE diet (HAD)-fed mice, respectively
Role of HGA-activated Src in PKC␣ Activation in L6 Skeletal Muscle Cells—To investigate the molecular mechanisms of PKC␣ activation by AGEs, HGA-induced PKC␣ activity was measured in L6 cells treated with different pharmacological inhibitors to selectively block ERK1/2, phosphatidylinositol 3-kinase, and Src

Summary

EXPERIMENTAL PROCEDURES

General—Media, sera, antibiotics for cell culture, and the Lipofectamine reagent were from Invitrogen (Invitrogen). The cells were incubated for the appropriated times with 0.1 mg/ml of HGA or non-glycated human serum albumin (HA) as a control. The human glycated and nonglycated albumin preparations were tested for carboxymethyllysine (CML) concentrations and the extent of lysine and arginine modifications as already described [31]. Upon immunoprecipitation with anti-PKC␣ or anti-RAGE antibodies, PKC activity was assayed using the SignaTECT PKC assay system (Promega) according to the manufacturer’s instructions as described in Oriente et al [35]. Mice were sacrificed after 24 h by cervical dislocation, and muscle samples were collected rapidly and homogenized as previously reported [33]. For analyzing glucose utilization by skeletal muscle, an intravenous injection of 1␮Ci of the non-metabolizable glucose analog 2-deoxy-D-[1-3H]glucose (GE Healthcare) and an intraperitoneal injection of insulin (0.75 milliunits/kg of body weight) were administered to random-fed mice. The total area under the curve for glucose response during the insulin tolerance tests and the glucose tolerance tests was calculated by the trapezoidal method

RESULTS

68 Ϯ 10 ND

DISCUSSION