Fig. 1.
Effects of palmitate, palmitoleate, and select MUFAs on proinflammatory signaling in L6 myotubes. (A, B) L6 myotubes (A) were treated with fatty acid-free BSA (vehicle control) or BSA conjugated to PA (0.75 mM) or PO (0.75 mM) for times indicated. Muscle cells (B) were incubated for 16 h with the fatty acids at the indicated concentrations. Cells were lysed and lysates subjected to immunoblot analysis using antibodies against IKKα/β Ser180/181, IκBα, and ERK 1/2 Thr202/Tyr204. (C) L6 myotubes were incubated in the absence or presence of 0.75 mM palmitate and/or palmitoleate at concentrations indicated for 16 h prior to lysis and immunoblotting for ERK 1/2 Thr202/Tyr204, IKKα/β Ser180/181, IκBα, and GAPDH. The cellular abundance of IκBα was quantified and expressed relative to GAPDH, which was used a gel loading control. (D, E) Alternatively, following treatment in the absence or presence of 0.75 mM palmitate and/or 0.75 mM palmitoleate, RNA was extracted from L6 myotubes and (D) IL-6 and (E) CINC-1 mRNA were determined by real time qPCR. Bars represent mean ± SEM from three separate experiments, and asterisks signify a significant difference from the untreated control values (P < 0.05). (F) Effect of different fatty acids upon proinflammatory signaling was assessed. L6 myotubes were treated for 16 h with 0.75 mM lauric acid (C12:0), 0.75 mM palmitate (C16:0), or 0.1 mM stearic acid (C18:0) alone or in combination with 0.75 mM palmitoleate (C16:1) or 0.75 mM oleate (C18:1). Lysates were immunoblotted to assess the phosphorylation status of IKKα/β and ERK 1/2 and total protein abundance of IκBα. Equal gel loading was ascertained by immunoblotting with an antibody against total ERK 1/2 and GAPDH. The blots are representative of three separate experiments.