The effects of TNFα and ceramide in insulin signaling in C2C12 myocytes (854.5)

Abstract

Both tumor necrosis factor alpha (TNFα) and non‐esterified fatty acids (e.g. ceramide) have been proposed to be crucial factors in the development of the insulin‐resistant state. Skeletal muscle is a key metabolic tissue and defects in insulin signaling in this tissue are central to the pathogenesis of diabetes. Here we determined the effect of TNFα and ceramide on the total protein expression and protein phosphorylation of key signaling molecules within the insulin signaling cascade in C2C12 myocytes after treatment with TNFα or with ceramide for 24 hours, 48 hours, and 72 hours, (+/‐ 1 hour Insulin) respectively. Treatment with TNFα resulted in a significant enhancement in total protein expression of Insulin Receptor β subunit (IRβ) both after 48 hours (p=0.0099) and 72 hours (p=0.0055). However, phosphorylation of IRβ (p‐IRβ) was significantly enhanced (p=0.0002) after treatment with TNFα for 48 hours (+ Insulin), but not observed after 72 hours of treatment. Interestingly, total expression of Protein Kinase B (Akt) and phosphorylated Akt (p‐Akt) was not significantly altered in any of the TNFα treatment groups. Treatment with ceramide resulted in no significant changes in total protein expression of IRβ. However, total p‐IRβ was significantly enhanced (p=0.0251) after treatment with ceramide for 48 hours (+ Insulin). This significant enhancement in p‐IRβ was not observed after 72 hours of treatment. Total protein expression of Akt was not significantly altered by ceramide treatment. Interestingly, p‐Akt was significantly increased (p=0.0251) after treatment with ceramide for 48 hours (+ Insulin). This significant enhancement in p‐Akt was not observed after 72 hours of treatment. These findings provide evidence of a differential potential for TNFα and ceramide to independently interfere with insulin signaling and to even alter protein expression.