Long-chain fatty acid effects on peroxisome proliferator-activated receptor-α-regulated genes in Madin-Darby bovine kidney cells: Optimization of culture conditions using palmitate

Abstract

Studying long-chain fatty acid (LCFA) effects on gene network expression in bovine cells could provide useful information for future practical applications. An optimized in vitro system that does not require tissue collection or cell isolation could fill a niche in the study of PPARα activity in ruminants. Specific aims were to optimize culture conditions in Madin-Darby bovine kidney (MDBK) cells to achieve maximal mRNA expression of known peroxisome proliferator-activated receptor-α (PPARα) target genes using palmitate (16:0) as a representative LCFA. Variables included length of incubation time, use of albumin-bound (4:1 molar proportion) 16:0 (A16:0), or addition of insulin. A first time-course experiment tested culturing cells in Dulbecco's modified Eagle's medium with 150μM PPAR ligand Wy-14643 (WY) and A16:0. A second experiment tested the effects of albumin and insulin using 150μM of 16:0 without albumin or insulin (−Alb/−Ins), 16:0 without albumin plus 5 mg/L of bovine insulin (−Alb/+Ins), A16:0 without insulin (+Alb/−Ins), or a control. A third experiment was a preliminary metabolic characterization of cells and assessed intracellular lipid droplet formation after treatment with 150μM of 16:0 or an ethanol control. For all experiments, cells were harvested at 0, 6, 12, 18, and 24h posttreatment. In experiments 1 and 2, mRNA expression was assessed by quantitative PCR of selected PPARα target genes as well as PPARα coactivators (ACOX1, CPT1A, ACADVL, ACSL1, PPARA, PPARGC1A, LPIN1). In experiment 1, there was a linear increase in mRNA expression of CPT1A (∼500%) and ACSL1 (50 to 200%) by 6h of incubation with both WY and A16:0. The LPIN1 mRNA increased by >100% by 6h only with A16:0. Further, there was a linear increase in expression of PPARA (∼100%) with A16:0 through 24h of incubation. In experiment 2, insulin increased, and coupling LCFA with albumin tended to delay the response in expression of CPT1A and ACSL1 to 16:0. Data indicated a toxic effect of 150μM free 16:0 as assessed by cell counts after 12h of incubation. In experiment 3, MDBK cells appeared to use glucose and AA as energy sources and were able to secrete triglycerides. In addition, MDBK cells cultured with 150μM of 16:0 had a substantial uptake of LCFA and synthesized intracellular lipid droplets. Overall, results indicated that a 6-h incubation with free LCFA and addition of insulin was suitable to detect marked effects on mRNA expression of PPARα target genes in MDBK cells.