MEK/ERK and PI3K/Akt signaling pathways regulate IL-6 production. Total and phosphorylated ERK1/2 (A) or total and phosphorylated Akt (B) were analyzed by Western blotting of cellular extracts from control (-) and insulin-stimulated (+) THP-1 cells that were pre-treated with vehicle (DMSO), MEK1/2 inhibitor (U0126), or PI3K inhibitor (LY294002). Western blots representative of three independent experiments are shown. IL-6 (C) and TNF-α (D) mRNA expression determined by qRT-PCR in THP-1 cells treated for 1 hour as in (A) followed by stimulation for 30 minutes with 5 ng/ml insulin (+ insulin) or vehicle (- insulin), in the presence of vehicle (DMSO), and then stimulated with palmitate (250 μM) or fatty acid-free BSA, for an additional 24 hours. Values are means ± SE (n = 3) expressed relative to the expression level of these cytokines in BSA-treated cells.