Apolipoprotein D Transgenic Mice Develop Hepatic Steatosis through Activation of PPAR? and Fatty Acid Uptake

Labrie, Marilyne; Lalonde, Simon; Najyb, Ouafa; Thiery, Maxime; Daneault, Caroline; Des Rosiers, Chrisitne; Rassart, Eric; Mounier, Catherine

Primary hepatocytes were isolated by in situ liver perfusion and collagenase digestion as previously described [44] . Briefly, mice were anaesthetized by intraperitoneal injection of pentobarbital and the portal vein was cannulated. The liver was then perfused with perfusion buffer (10 mM HEPES, 142mM NaCl, 6,7mM KCl; pH 7,85) containing 0,6mM EGTA and 1,5 U/ mL heparin and subsequently digested with 30 000U collagenase type I (Worthington) dissolved in 150 mL of perfusion buffer containing 5 mM calcium. Hepatic cells were gently released from the Glisson capsule and incubated for 1h at room temperature with 5X Wash solution consisting of DMEM/F12 (Life technologies, Gibco) with 10% fetal bovine serum (Life technologies, Gibco), 500 U/mL penicillin, 500 μg/mL streptomycin and 1,25 μg/mL Fungizone (Life Technologies) by an orbital shaker. 1x10 6 cells were seeded on collagen-pretreated plates (Corning Costar) in DMEM/F12 media containing 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. The next day, culture media was removed and renewed with serum-free DMEM/F12 containing the same antibiotics. The cells were starved for 48h prior to the experiments.