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 PPARalpha 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-alpha (PPARalpha) 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 150 microM PPAR ligand Wy-14643 (WY) and A16:0. A second experiment tested the effects of albumin and insulin using 150 microM 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 150 microM of 16:0 or an ethanol control. For all experiments, cells were harvested at 0, 6, 12, 18, and 24 h posttreatment. In experiments 1 and 2, mRNA expression was assessed by quantitative PCR of selected PPARalpha target genes as well as PPARalpha coactivators (ACOX1, CPT1A, ACADVL, ACSL1, PPARA, PPARGC1A, LPIN1). In experiment 1, there was a linear increase in mRNA expression of CPT1A (approximately 500%) and ACSL1 (50 to 200%) by 6 h of incubation with both WY and A16:0. The LPIN1 mRNA increased by >100% by 6 h only with A16:0. Further, there was a linear increase in expression of PPARA (approximately 100%) with A16:0 through 24 h 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 150 microM free 16:0 as assessed by cell counts after 12 h 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 150 microM 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 PPARalpha target genes in MDBK cells.

Functional Role of PPARs in Ruminants: Potential Targets for Fine-Tuning Metabolism during Growth and Lactation

“…Beef cattle longissimus muscle and mammary gland had relatively modest expression of PPARA followed by the least expression in hoof corium, lung, rumen, MDBK, MAC-T, PMN, and placenta (Figure 1(a)). We and others have consistently detected expression of PPARA in liver [55–60] and in MDBK cells in which also its activity was confirmed [28, 36, 61]. Partly corroborating our data (Figure 1), this PPAR isotype has been detected in bovine endothelial cells [62], skeletal muscle [63], rumen [64], uterus [43, 65], and neutrophils [66].…”

“…An additional assay available today is the direct measurement of activation of PPAR isotypes after nuclear isolation by the presence of PPRE immobilized onto the bottom of cell culture wells; however, such assays have not been developed for ruminants [61]. The use of these techniques with greater sensitivity, precision, and reliance in ruminants has been scant [61]. Most of the studies performed in ruminants are based on measurements of changes in expression of genes or proteins after treatment with PPAR isotype-specific agonists.…”

“…Other in vitro studies were carried out in order to test the response to PPAR isotypes in two bovine cell lines (MDBK and MAC-T) with the purpose of determining PPAR α and PPAR γ target genes [26, 28, 36, 61]. Besides target genes, those studies also uncovered several biological functions of PPAR isotypes in ruminants.…”

Biosynthesis of milk fat, protein, and lactose: roles of transcriptional and posttranscriptional regulation

“…The two experiments differed in that, for Kadegowda and collaborators (108), the LCFA were not coupled with bovine serum albumin (BSA), and in Jacobs and collaborators, a calculated ratio of ϳ6:1 of LCFA/BSA was used. In addition, as previously discussed and demonstrated (222), the use of albumin, although more similar to the in vivo situation, can be problematic because BSA tends to retain with higher affinity the remaining fatty acids (212), making them less available to the cells. It is, however, interesting that Jacobs and collaborators were not able to detect the expression of PPARG in MAC-T cells.…”

The pro-inflammatory cytokine tumor necrosis factor α stimulates expression of the carnitine transporter OCTN2 (novel organic cation transporter 2) and carnitine uptake via nuclear factor-κB in Madin-Darby bovine kidney cells

“…Nevertheless, we are confident that MDBK cells, which are widely used to investigate mechanistic questions in bovine tissues (Bionaz et al, 2008;Thering et al, 2009), are suitable for addressing this specific question, because OCTN2 expression is regulated similarly, at least by PPARα, in different cell types including hepatocytes and kidney cells (Luci et al, 2006;Zhou et al, 2014). Thus, we expect the same mechanism of regulation by TNFα in liver cells.…”