Time-restricted feeding (TRF) is a dieting strategy based on nutrients availability and diurnal rhythm, shown to improve lipid metabolism efficiency. We have demonstrated previously that retinoic acid-related (RAR) orphan receptor (ROR) γ is the primary transcription factor controlling cholesterol (CHO) biosynthesis program of animals. However, the functional role of RORγ in liver physiology of pigs in response to TRF has not been determined, largely due to the lack of functional models and molecular tools. In the present study, we established porcine liver organoids and subjected them to restricted nutrients supply for 10-h during the light portion of the day. Our results showed that TRF regimen did not alter hepatocyte physiology, including unchanged cell viability, caspase 3/7 enzyme activity and the gene signature of cell proliferation in porcine liver organoids, compared to the control group (P > 0.05). Furthermore, we found that TRF downregulated the hepatic CHO biosynthesis program at both mRNA and protein levels, along with the reduced cellular CHO content in porcine liver organoids (P < 0.05). Using unbiased bioinformatic analysis of a previous ChIP-seq data and ChIP-qPCR validation, we revealed RORγ as the predominant transcription factor that responded to TRF, amongst the 12 targeted nuclear receptors (NRs) (P < 0.05). This was likely through RORγ direct binding to the MVK gene (encoding mevalonate kinase). Finally, we showed that RORγ agonists and overexpression enhanced the enrichment of co-factor p300, histone marks H3K27ac and H3K4me1/2, as well as RNA polymerase II (Pol-II) at the locus of MVK, in TRF-porcine liver organoids, compared to TRF-vector control (P < 0.05). Our findings demonstrate that TRF triggers the RORγ-mediated chromatin remodeling at the locus of CHO biosynthesis genes in porcine liver organoids and further improves lipid metabolism.

中文翻译：

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时间受限的进食通过猪肝脏类器官中的RORγ介导的染色质修饰来下调胆固醇的生物合成程序

限时喂养（TRF）是一种基于营养物质可利用性和昼夜节律的节食策略，可提高脂质代谢效率。先前我们已经证明，视黄酸相关（RAR）孤儿受体（ROR）γ是控制动物胆固醇（CHO）生物合成程序的主要转录因子。然而，由于缺乏功能模型和分子工具，RORγ在猪对TRF的肝脏生理中的功能尚未确定。在本研究中，我们建立了猪肝类器官，并在一天中较轻的部分使它们受到有限的营养供应10小时。我们的结果表明，TRF方案不会改变肝细胞的生理状况，包括不变的细胞活力，与对照组相比，猪肝类器官中半胱天冬酶3/7酶的活性和细胞增殖的基因特征（P> 0.05）。此外，我们发现TRF在mRNA和蛋白质水平上均下调了肝CHO生物合成程序，并降低了猪肝类器官中的细胞CHO含量（P <0.05）。使用先前ChIP-seq数据的无偏生物信息学分析和ChIP-qPCR验证，我们揭示了RORγ是12个靶向核受体（NRs）中对TRF响应的主要转录因子（P <0.05）。这可能是由于RORγ直接与MVK基因结合（编码甲羟戊酸激酶）。最后，我们发现RORγ激动剂和过度表达增强了辅因子p300，组蛋白标记H3K27ac和H3K4me1 / 2的富集，与TRF载体对照相比，TRF猪肝类器官中MVK位置的RNA聚合酶II（Pol-II）（Pol-II）（P <0.05）。我们的研究结果表明TRF触发猪肝类器官中CHO生物合成基因的基因座上的RORγ介导的染色质重塑，并进一步改善脂质代谢。