Zhang Kexin, Li Hao, Xin Zimeng, Li Yanwei, Wang Xiaolong, Hu Yun, Liu Haoyu, Cai Demin
College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, PR China.
Institute of Epigenetics and Epigenomics, Yangzhou University, Yangzhou, 225009, PR China.
J Anim Sci Biotechnol. 2020 Nov 2;11(1):106. doi: 10.1186/s40104-020-00511-9.
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.
限时进食（TRF）是一种基于营养物质可用性和昼夜节律的节食策略，已被证明可提高脂质代谢效率。我们之前已经证明，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，RORγ在猪肝脏生理学中对TRF的功能作用尚未确定，这主要是由于缺乏功能模型和分子工具。在本研究中，我们建立了猪肝脏类器官，并在白天的光照时段对其进行10小时的营养限制供应。
我们的结果表明，与对照组相比，TRF方案并未改变猪肝脏类器官中的肝细胞生理学，包括细胞活力、半胱天冬酶3/7酶活性以及细胞增殖的基因特征均未改变（P>0.05）。此外，我们发现TRF在mRNA和蛋白质水平上均下调了肝脏CHO生物合成程序，同时猪肝脏类器官中的细胞CHO含量也降低了（P<0.05）。通过对先前的ChIP-seq数据进行无偏生物信息分析和ChIP-qPCR验证，我们发现RORγ是12种靶向核受体（NRs）中对TRF有反应的主要转录因子（P<0.05）。这可能是通过RORγ直接结合MVK基因（编码甲羟戊酸激酶）实现的。最后，我们表明，与TRF载体对照组相比，在TRF处理的猪肝脏类器官中，RORγ激动剂和过表达增强了辅因子p300、组蛋白标记H3K27ac和H3K4me1/2以及RNA聚合酶II（Pol-II）在MVK基因座处的富集（P<0.05）。
我们的研究结果表明，TRF在猪肝脏类器官中触发了CHO生物合成基因座处RORγ介导的染色质重塑，并进一步改善了脂质代谢。
