The effects of TNF‐alpha and Ceramide on Insulin Receptor signaling and its localization to Caveolae in C2C12 Myocytes

Abstract

TNF‐α and ceramide are associated with the etiology of insulin resistance. There is evidence for the involvement of caveolae membrane rafts in the modulation of insulin signaling and glucose uptake. Thus, we propose to elucidate the signaling mechanisms by which TNF‐α and ceramide induce insulin resistance in C2C12 myocytes and the role of caveolins in the organization of this pathway within the membrane. We hypothesized that exposure of C2C12 myocytes to TNF‐α and ceramide will result in altered insulin signaling, decreased glucose uptake, and translocation of the insulin signaling cascade away from caveolae. C2C12 myocytes were treated with TNF‐α, ceramide, and insulin, individually and in combination. The treated cells were subjected to Western blot analysis for insulin receptor‐β (IR‐β), phosphorylated IR (p‐IR), Protein Kinase B (PKB or Akt), phosphorylated Akt (p‐Akt), Cav‐3, Glut‐4, and actin (as loading control). The total protein expression of IR, Cav‐3, and Glut‐4 was unaltered with treatment. However, there was a significant increase in the total expression of Akt in cells treated with TNF‐α. In addition, there was a significantly higher activation of Akt and IR in cells treated with TNF‐α after 48 hours exposure. This data suggests that the effect of TNF‐α on insulin signaling in a result of a cross‐talk mechanism. This data will further the identification of novel signaling mechanisms leading to insulin resistance as a prerequisite to improved diagnosis and therapeutic treatments for diabetes.