Fig. 2.

Inhibition of 12/15-lipoxygenase (12/15-LO) activity attenuates tunicamycin-induced ER stress and insulin resistance. 3T3-L1 adipocytes were treated with 5 μM tunicamycin or DMSO solvent control for 24 h (A–C) with or without a 2-h pretreatment and coincubation with 0.1 μM of the 12/15-LO inhibitor cynnamyl 1–3,4-dihydroxy-α-cyanocinnamate (CDC) or 10 mM of the chemical chaperone 4-phenylbutyric acid (PBA). mRNA (A) and protein measurements (B) of ER stress markers and 12/15-LO were examined. C: 12(S)-HETE was measured in medium cultured with treated 3T3-L1 adipocytes by ELISA. D: 3T3-L1 adipocytes were treated with 5 μM tunicamycin or DMSO solvent control for 4 h and stimulated with 10 nM insulin for 10 min, and protein measurements of key insulin signaling markers were examined. The mRNA measurements were done by RT-PCR and protein measurements done by Western blot analysis; the data were normalized to total actin, and the fold changes in expression were calculated relative to DMSO control. Western blots are shown, and separate panels for each antibody represent the same exposure from the same gel (B and D). All data represent means ± SE; n = 3. Statistics were performed comparing each treatment with control as well as tunicamycin with tunicamycin + CDC/PBA; only statistically significant results are shown. *P < 0.05, †P < 0.02, and #P < 0.001 vs. control unless otherwise indicated.