Data_Sheet_2_Peroxisome Proliferator-Activated Receptor Activation in Precision-Cut Bovine Liver Slices Reveals Novel Putative PPAR Targets in Periparturient Dairy Cows.XLSX

Metabolic challenges experienced by dairy cows during the transition between pregnancy and lactation (also known as peripartum), are of considerable interest from a nutrigenomic perspective. The mobilization of large amounts of non-esterified fatty acids (NEFA) leads to an increase in NEFA uptake in the liver, the excess of which can cause hepatic accumulation of lipids and ultimately fatty liver. Interestingly, peripartum NEFA activate the Peroxisome Proliferator-activated Receptor (PPAR), a transcriptional regulator with known nutrigenomic properties. The study of PPAR activation in the liver of periparturient dairy cows is thus crucial; however, current in vitro models of the bovine liver are inadequate, and the isolation of primary hepatocytes is time consuming, resource intensive, and prone to errors, with the resulting cells losing characteristic phenotypical traits within hours. The objective of the current study was to evaluate the use of precision-cut liver slices (PCLS) from liver biopsies as a model for PPAR activation in periparturient dairy cows. Three primiparous Jersey cows were enrolled in the experiment, and PCLS from each were prepared prepartum (−8.0 ± 3.6 DIM) and postpartum (+7.7± 1.2 DIM) and treated independently with a variety of PPAR agonists and antagonists: the PPARα agonist WY-14643 and antagonist GW-6471; the PPARδ agonist GW-50156 and antagonist GSK-3787; and the PPARγ agonist rosiglitazone and antagonist GW-9662. Gene expression was assayed through RT-qPCR and RNAseq, and intracellular triacylglycerol (TAG) concentration was measured. PCLS obtained from postpartum cows and treated with a PPARγ agonist displayed upregulation of ACADVL and LIPC while those treated with PPARδ agonist had increased expression of LIPC, PPARD, and PDK4. In PCLS from prepartum cows, transcription of LIPC was increased by all PPAR agonists and NEFA. TAG concentration tended to be larger in tissue slices treated with PPARδ agonist compared to CTR. Use of PPAR isotype-specific antagonists in PCLS cultivated in autologous blood serum failed to decrease expression of PPAR targets, except for PDK4, which was confirmed to be a PPARδ target. Transcriptome sequencing revealed considerable differences in response to PPAR agonists at a false discovery rate-adjusted p-value of 0.2, with the most notable effects exerted by the PPARδ and PPARγ agonists. Differentially expressed genes were mainly related to pathways involved with lipid metabolism and the immune response. Among differentially expressed genes, a subset of 91 genes were identified as novel putative PPAR targets in the bovine liver, by cross-referencing our results with a publicly available dataset of predicted PPAR target genes, and supplementing our findings with prior literature. Our results provide important insights on the use of PCLS as a model for assaying PPAR activation in the periparturient dairy cow.

妊娠期与泌乳期（亦称围产期）转换过程中，奶牛所遭遇的代谢挑战在营养基因组学视角下备受关注。大量非酯化脂肪酸（NEFA）的动员导致肝脏NEFA摄取增加，其过剩可引发肝脂积累，最终导致脂肪肝。有趣的是，围产期NEFA激活了过氧化物酶体增殖物激活受体（PPAR），该转录调控因子具有已知的营养基因组学特性。因此，研究围产期奶牛肝脏中PPAR的激活至关重要；然而，目前牛肝的体外模型尚不完善，而原代肝细胞的分离耗时、资源密集且易于出错，分离出的细胞在数小时内便会丧失其典型的表型特征。本研究旨在评估从肝活检中获取的精密切割肝切片（PCLS）作为评估围产期奶牛PPAR激活的模型的可行性。实验中纳入了三头初产泽西牛，分别在产前（-8.0 ± 3.6 DIM）和产后（+7.7± 1.2 DIM）制备PCLS，并分别独立使用多种PPAR激动剂和拮抗剂进行处理：PPARα激动剂WY-14643和拮抗剂GW-6471；PPARδ激动剂GW-50156和拮抗剂GSK-3787；以及PPARγ激动剂罗格列酮和拮抗剂GW-9662。通过RT-qPCR和RNAseq技术检测基因表达，并测量细胞内三酰甘油（TAG）浓度。产后奶牛的PCLS经PPARγ激动剂处理后，ACADVL和LIPC的表达上调，而经PPARδ激动剂处理的PCLS则表现出LIPC、PPARD和PDK4表达增加。产前奶牛的PCLS中，LIPC的转录受到所有PPAR激动剂和NEFA的增强。与CTR相比，经PPARδ激动剂处理的组织切片中的TAG浓度有增大的趋势。在自体血清培养的PCLS中使用PPAR同型特异性拮抗剂未能降低PPAR靶点的表达，除PDK4外，后者被证实为PPARδ靶点。转录组测序显示，在调整了假发现率后的p值为0.2时，对PPAR激动剂的响应存在显著差异，其中PPARδ和PPARγ激动剂的影响最为显著。差异表达基因主要与参与脂质代谢和免疫反应的通路相关。在差异表达基因中，通过将我们的结果与公开可用的预测PPAR靶基因数据集进行交叉引用，并补充相关文献的研究，我们确定了91个基因作为牛肝中新的潜在PPAR靶点。我们的研究结果为PCLS作为评估围产期奶牛PPAR激活模型的利用提供了重要的见解。