Studies On Regulation Of UGT1A9 And Propofol Metabolism By The Clock Gene Rev-erba

ObjectivesDiurnal rhythms（～24-hour cycle）are present in nearly all living organisms.REV-ERBais an essential component of the biological clock system.The nuclear receptor REV-ERBa（NR1D1）maintains the homeostasis of the biological clock system by regulating the core clock factor BMAL1.REV-ERBais implicated in regulating the metabolism of endogenous（e.g.,bilirubin,bile acids,thyroid hormone,and steroid hormones）and exogenous compounds（e.g.,drugs,carcinogens and environmental pollutants）.UGT1A9 is a critical member of the UGT enzyme family,which catalyzes the glucuronidation reaction of compounds.The expression of UGT1A9 showed a robust diurnal rhythm.It is unclear whether REV-ERBahas a regulatory effect on UGT1A9.Therefore,this study aims to characterize the diurnal rhythm of UGT1A9 in mouse liver and to determine the molecular mechanism underlying the rhythmicity.These findings clarified the effect of REV-ERBaon the metabolism of propofol（a substrate of UGT1A9）.Method1.The diurnal rhythmicity in the expression of UGT1A9 and DEC2 in the livers of wild-type（WT）and Rev-erba^-/-（knockout）mice at six time points was analyzed by using RT-qPCR and western blotting assays.RT-qPCR was used to explore the diurnal expression of Ugt1a9 in Hepa-1c1c7 cells stimulated by serum shock.2.The chronopharmacokinetics of propofol was studied by using wild-type and Rev-erba^-/-mice.The mice were given intraperitoneal injection of propofol（100 mg/kg）at different time points（ZT6 and ZT18）.Plasma was collected at specific time points（5,30,60,and 120 minutes）.The concentration of propofol and its metabolites in plasma were determined by UPLC-QTOF/MS analysis.Based on the non-compartmental analysis,the temporal variations of pharmacokinetic parameters of propofol and its metabolites in vivo were evaluated.Liver microsomal assays were used to assess the activity of UGT1A9 in the livers of wild-type and Rev-erba^-/-mice at different time points（ZT6 and ZT18）.3.RT-qPCR and western blotting assays were used to assess the efficiencies of Rev-erbaknockdown or overexpression at the m RNA and protein levels,and determine the expression of DEC2 and UGT1A9 m RNA（protein）in Hepa-1c1c7 cells.4.RT-qPCR and western blotting assays were used to assess the efficiencies of REV-ERBaknockdown or overexpression at the m RNA and protein levels,and determine the expression of UGT1A9 m RNA（protein）in Hep G2 cells.5.Promoter sequence analysis and luciferase reporter assays were used to determined the molecular regulatory mechanism.Dec2 trans-repressed Ugt1a9 via direct binding to an E-box–like motif in the gene promoter.Results1.Rev-erbaregulates the expression of hepatic UGT1A9.The hepatic UGT1A9 m RNA and protein in wild-type mice displayed a robust diurnal rhythm with a peak level at ZT6.Rev-erbaablation downregulated UGT1A9 m RNA and protein,and blunted its rhythm.Hepa-1c1c7 cells with a serum shock showed temporal oscillations in Ugt1a9 m RNA.2.Rev-erbaregulates UGT1A9-mediated glucuronidation of propofol.Rev-erbaablation downregulated UGT1A9 expression,and resulted in alterations in drug metabolism（glucuronidation）.According to in vitro metabolism assays,glucuronidation of propofol in wild-type liver microsomes was more extensive at ZT6 than ZT18,consistent with the diurnal pattern of UGT1A9 protein.Glucuronidation of propofol at both time points was reduced in liver microsomes of Rev-erba^-/-mice,and the time dependency was lost.We determined the metabolism and pharmacokinetics of propofol in wild-type and Rev-erba^-/-mice after drug administration at ZT6 or ZT18.Neither dosing time nor genotype affected the pharmacokinetic profile of propofol.Also,metabolism of propofol（formation of propofol glucuronide）was time-dependent,and the concentration of propofol glucuronide in the liver of wild-type mice was significantly higher when administered at ZT6 than ZT18.3.Rev-erbaregulates the expression of UGT1A9 in Hepa-1c1c7 and Hep G2 cells.The regulatory effects of Rev-erbaon UGT1A9 were additionally assessed using mouse hepatoma Hepa-1c1c7 cells.Overexpression of Rev-erbaled to a significant increase in Ugt1a9m RNA.Knockdown of Rev-erba（by siRNA）resulted in a reduction in Ugt1a9 m RNA.In line with the m RNA changes,overexpression and knockdown of Rev-erbacaused an increase and a decrease in UGT1A9 protein,respectively.Positive regulation effects of REV-ERBaon UGT1A9 were observed in human hepatoma Hep G2 cells.All these data confirmed REV-ERBaas a positive regulator of UGT1A9.4.Rev-erbaablation upregulates DEC2 expression in mouse liver.Hepatic Dec2 m RNA exhibited a robust diurnal rhythm in wild-type mice,peaking at ZT6.Dec2 m RNA was elevated and its rhythm was dampened（reflected by a decreased peak-to-valley ratio）in Rev-erba^-/-mice.DEC2 protein was rhythmically expressed in the liver of wild-type mice with a peak value at ZT14.Rev-erbaablation upregulated the protein level of DEC2and blunted its rhythm.Diurnal DEC2 protein was phase-shifted about 8 hours relative to its m RNA rhythm.5.Dec2 mediates Rev-erbato regulate the transcriptional regulation of Ugt1a9.Dec2 trans-repressed Ugt1a9 through direct binding to an E-box–like element in gene promoter.Transient transcription analysis were performed using an Ugt1a9 luciferase reporter plasmids（i.e.,Ugt1a9-Luc,-2000/+100 bp）.Co-transfection of Ugt1a9-Luc reporter with Dec2resulted in a significant decrease in the transcription activity,and the inhibitory effects of Dec2were dose-dependent.The Dec2 effects on Ugt1a9 transcription were lost when the E-box–like element was truncated or mutated.ConclusionHepatic UGT1A9 displays a diurnal rhythmicity in expression levels of protein,m RNA and glucuronidation activity in mice.Rev-erbagenerates and regulates rhythmic Ugt1a9 through periodical inhibition of Dec2,a transcriptional repressor of Ugt1a9.The findings have implications for understanding of diurnal clock–controlled drug metabolism and of metabolism-based chronotherapeutics.