Abstract
BackgroundTime-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.ResultsOur 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).ConclusionsOur 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.
Highlights
Time-restricted feeding (TRF) is a dieting strategy based on nutrients availability and diurnal rhythm, shown to improve lipid metabolism efficiency
Time-restricted feeding does not affect cell growth and survival in porcine liver organoids Given that organoids are more physiologically relevant than 2D monolayers cells, we developed the porcine liver organoids
Immunofluorescent staining showed that TRF did not affect the hepatocytes viability (Fig. 2a), which was further confirmed by quantification of adenosine triphosphate (ATP) presence in cells using a celltiter measurement, indicative of metabolically active cells (Fig. 2b, P > 0.05)
Summary
Time-restricted feeding (TRF) is a dieting strategy based on nutrients availability and diurnal rhythm, shown to improve lipid metabolism efficiency. It has been demonstrated that around 10–15% of all liver mRNA are expressed in a rhythmic fashion Many of these genes play a role in cholesterol and glucose metabolism [4, 5, 8] with one essential part being retinoic acid-related (RAR) orphan receptor (ROR) [4, 13, 14]. Both RORγ and RORα are involved in controlling hepatic circadian rhythmic expression of glucose genes, whereas mice deficient in RORγ showed improved insulin sensitivity and glucose tolerance, especially at daytime [14]. The role of RORγ in cholesterol biosynthesis in liver physiology of mammals and its relation to TRF remain unclear
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