Induction of inflammation and insulin resistance by TNF-α treatment in C₂C₁₂ myotubes.

<p>(A) Differentiation of C₂C₁₂ myoblasts into myotubes was induced by replacing the growth medium with the differentiation medium (DMEM/ 2% horse serum). The levels of MyoD and Myogenin were measured by immunoblotting. α-tubulin was used as a loading control. (B-C) On Day 5 after differentiation, C₂C₁₂ cells were incubated with 2 ng/ml or 20 ng/ml TNF-α after starvation in serum-free DMEM as indicated. Cell lysates were subjected to immunoblot analysis. Bars and a line graph show densitometric quantitation of p-NF-κB (lower left), p-p38MAPK and p-ERK-1/2 (lower right) respectively. The values are expressed as the means ± SEM. Level of p-NF-κB after TNF-α stimulation was compared to that of untreated control. *,P<0.05; **,P<0.01. (D) Immunoblot assay for the nuclear translocation of NF-κB by TNF-α stimulation. Cells were treated with 2 ng/ml TNF-α and then the nuclear fraction was harvested and subjected to immunoblotting with antibodies to NF-κB and p-NF-κB. Histone was used as a loading control. (E) Induction of insulin resistance by TNF-α. Cells were treated with 100 nM insulin after pre-incubation with 2 or 20 ng/ml TNF-α in C₂C₁₂ myotubes for 2 h or 4 days. Insulin-induced p-Akt levels were measured by immunoblot analysis. Bars show densitometric quantitation of Akt phosphorylation. The values are expressed as the means ± SEM. *,P<0.05; **,P<0.01 versus TNF-α-untreated, insulin-stimulated control.