Palmitate-Induced Hepatocyte Dysfunction and DPP4i Modulation

The human hepatoma cell line HepG2 (American Type Culture Collection [ATCC], Manassas, VA, USA) was cultured in low glucose Dulbecco’s modified Eagle’s medium (DMEM; SH30021.01, HyClone Laboratories, Logan, UT, USA) supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin in a 5% CO₂ incubator at 37 °C. Cells were incubated in a humidified atmosphere at 37 °C and 5% CO₂ and cultured for 3 days to achieve 70% confluence before treatment with palmitate or DPP4i (teneligliptin). HepG2 cells were treated with palmitate (Sigma-Aldrich, 0.6 mM) for 18 h with or without pre-treatment with DPP4i (teneligliptin, 3 μM) for 6 h and were then analyzed. The mRNA and protein expression levels of DPP4, ER stress, and TRAIL-R2-mediated apoptosis markers were assessed. The mRNA levels of collagen1α1 and αSMA, the typical mesenchymal cell markers corresponding to the activated hepatocyte state that involves the deposition of extracellular matrix during liver fibrosis^{66 (link)}, were also analyzed. To analyze the effect of DPP4i on TRAIL-mediated apoptosis, HepG2 cells were incubated with the recombinant human TRAIL/TNFSF10 protein (TRAIL; Cat# 375-TL-010, R&D Systems) at concentrations of 50 and 100 ng/mL for 18 h, with or without pre-treatment with DPP4i (teneligliptin, 3 μM) for 6 h.