Time-restricted feeding downregulates cholesterol biosynthesis program via RORγ-mediated chromatin modification in porcine liver organoids

Background: 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.Results: 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 cofactor p300, histone marks H3 K27 ac 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).Conclusions: 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.

Sodium butyrate alleviates deoxynivalenol-induced hepatic cholesterol metabolic dysfunction via RORγ-mediated histone acetylation modification in weaning piglets

Background:Cholesterol is an essential component of lipid rafts in cell plasma membrane,which exerts a hepatoprotective role against mycotoxin exposure in pigs,and cholesterol metabolism is vulnerable to epigenetic histone acetylation.Therefore,our present study aimed to investigate whether a histone deacetylase inhibitor(sodium butyrate [NaBu]) could protect the porcine liver from deoxynivalenol(DON) exposure by modulating cholesterol metabolism.Herein,we randomly divided 28 pigs into four groups,which were fed an uncontaminated basal diet,contaminated diet(4 mg DON/kg),uncontaminated diet supplemented with 0.2% NaBu or 4 mg/kg DON contaminated diet(4 mg DON/kg) supplemented with 0.2% NaBu for 28 d.Results:We found that the serum alanine transaminase(ALT),aspartate transaminase(AST),and alkaline phosphatase(ALP) were all increased in pigs exposed to DON,indicative of significant liver injury.Furthermore,the cholesterol content in the serum of DON-exposed pigs was significantly reduced,compared to the healthy Vehicle group.Transcriptome analysis of porcine liver tissues revealed that the cholesterol homeostasis pathway was highly enriched due to DON exposure.In which we validated by qRT-PCR and western blotting that the cholesterol program was markedly activated.Importantly,NaBu effectively restored parameters associated with liver injury,along with the cholesterol content and the expression of key genes involved in the cholesterol biosynthesis pathway.Mechanistically,we performed a ChIP-seq analysis of H3K27ac and showed that NaBu strongly diminished DON-increased H3K27ac genome-wide enrichment.We further validated that the elevated H3K27ac and H3K9ac occupancies on cholesterol biosynthesis genes were both decreased by NaBu,as determined by ChIP-qPCR analysis.Notably,nuclear receptor RORγ,a novel regulator of cholesterol biosynthesis,was found in the hyperacetylated regions.Again,a remarkable increase of RORγ at both mRNA and protein levels in DON-exposed porcine livers was drastically reduced by NaBu.Consistent with RORγ expression,NaBu also hindered RORγ transcriptional binding enrichments on these activated cholesterol biosynthesis genes like HMGCR,SQLE,and DHCR24.Furthermore,we conducted an in vitro luciferase reporter assay to verify that porcine RORγ directly bonds to the promoters of the above target genes.Conclusions:Collectively,our results demonstrate the utility of the natural product Na Bu as a potential anti-mycotoxin nutritional strategy for regulating cholesterol metabolism via RORγ-mediated histone acetylation modification.

Mechanism of Niacin Induced Hot Flushes and Suppression of Cholesterol

Niacin or nicotinic acid is a form of B3 vitamin prescribed at higher concentrations for the suppression of cholesterol levels. Supplemental doses may cause very little or no side effects. However, higher concentrations of niacin cause hot flushes for most people. Here we propose a biochemical mechanism of niacin induced hot flushes. Orally taken prescription doses of niacin are converted to NAD with the liberation of excess pyrophosphate which in turn releases energy in the form of heat (hot flushes through capsaicin receptor) by the action of pyrophosphatases. The excess pyrophosphate may suppress cholesterol biosynthesis through feedback mechanism. The pathways of NAD and cholesterol biosynthesis were discussed with refence to the production and function of pyrophosphate.

Study on the Effects of Reducing Blood Lipid by Flavonoids from Ampelopsis grossedentata on Hyperlipidemia Rats

In order to study the effects of Ampelopsis grossedentata on endogenous cholesterol biosynthesis and the effects of reducing blood lipid in hyperlipidemia rats,extraction of flavonoids from Ampelopsis grossedentata by using ethanol(TFAG),and the effects of TFAG on intracellular cholesterol synthesis were detected by amphotericin B-cell model;SD hyperlipidemia rat model was established by feeding high fat diet.A formulated medicine called Xuezhikang was used as a positive control,and TFAG of different doses were administered to the stomach for 30 d continuously to measure the indexes of heart,liver tissue homogenate and serum;part of the liver was taken for pathological observation.The results showed that TFAG could significantly inhibit the synthesis of cholesterol in cells.TFAG of different doses could significantly reduce the content of TC and TG in serum of hyperlipidemia rats,and increase the content of HDL;TC and TG in heart and liver were also decreased;besides,it could increase the content of SOD,CAT and GSH in the liver of hyperlipidemia rats,and reduce the content of MDA.The results of pathological section showed that TFAG could improve the damage degree of hepatocytes in hyperlipidemia rats,and the effect of high dose group was similar to that of Xuezhikang group.In general,TFAG has good antioxidant and reducing blood lipid effects,and can effectively inhibit liver steatosis.

Squalene epoxidase promotes colorectal cancer cell proliferation through accumulating calcitriol and activating CYP24A1-mediatedMAPK signaling

Background:Colorectal cancer(CRC)is one of the most malignant tumorswith high incidence,yet its molecular mechanism is not fully understood,hindering the development of targeted therapy.Metabolic abnormalities are a hallmark of cancer.Targeting dysregulated metabolic features has become an important direction for modern anticancer therapy.In this study,we aimed to identify a new metabolic enzyme that promotes proliferation of CRC and to examine the related molecular mechanisms.Methods:We performed RNA sequencing and tissue microarray analyses of human CRC samples to identify new genes involved in CRC.Squalene epoxidase(SQLE)was identified to be highly upregulated in CRC patients.The regulatory function of SQLE in CRC progression and the therapeutic effect of SQLE inhibitors were determined by measuring CRC cell viability,colony and organoid formation,intracellular cholesterol concentration and xenograft tumor growth.Themolecularmechanism of SQLE functionwas explored by combining transcriptome and untargeted metabolomics analysis.Western blotting and realtime PCR were used to assess MAPK signaling activation by SQLE.Results:SQLE-related control of cholesterol biosynthesis was highly upregulated in CRC patients and associated with poor prognosis.SQLE promoted CRC growth in vitro and in vivo.Inhibition of SQLE reduced the levels of calcitriol(active form of vitamin D3)and CYP24A1,followed by an increase in intracellular Ca2+concentration.Subsequently,MAPK signaling was suppressed,resulting in the inhibition of CRC cell growth.Consistently,terbinafine,an SQLE inhibitor,suppressed CRC cell proliferation and organoid and xenograft tumor growth.Conclusions:Our findings demonstrate that SQLE promotes CRC through the accumulation of calcitriol and stimulation of CYP24A1-mediated MAPK signaling,highlighting SQLE as a potential therapeutic target for CRC treatment.