Circadian Regulation Of Intestinal CES1 By The Clock Gene Bmal1

Objective:CES1（Carboxylesterase 1）is an enzyme involved in hydrolysis of ester-and amide-containing compounds,and plays a crucial role in the metabolism of numerous drug（e.g.,mycophenolate mofetil,oseltamivir and trandolapril）and endogenous substances（e.g.,cholesterol esters and triglycerides）.Intestinal metabolism is closely related to the efficacy and toxicity of oral drugs,which is mainly affected by the expression and activity of metabolic enzymes.Recent studies have shown that the expression of various drug-metabolizing enzymes（e.g.,CYP2B10,UGT1A9 and FMO5）displays a diurnal rhythm.However,the diurnal rhythm of CES expression remains largely unknown.As a core circadian clock component,BMAL1 plays an important role in regulating the diurnal rhythms of many genes.This study aimed to investigate the diurnal rhythms of intestinal CES1 expression and activity,and to explore the mechanism for regulation of CES1 by intestinal clock gene Bmal1.Methods:1.PCR amplification and agarose gel electrophoresis experiments were used to identify the genotype of intestine-specific Bmal1 knockout(Bmal1^{i KO})mice.RT-q PCR and Western blotting were used to detect the expression of CES in Bmal1^{i KO} and wide-type(Bmal1^fl/fl)mice at six time points（i.e.,ZT2,ZT6,ZT10,ZT14,ZT18 and ZT22）.2.Serum shock experiments were used to determine the rhythmic expression of CES1 in vitro.In addition,si RNA was used to verify the regulatory effect of BMAL1 on CES1 in CT26 and Caco-2 cell models.3.Mouse intestine microsomes were prepared by ultracentrifugation.Intestinal microsomal incubation assays were performed to detect CES1 activity in vitro.To assess the activity of CES1 in vivo,pharmacokinetic studies were performed with Bmal1^{i KO} and Bmal1^fl/fl mice after oral garage of MMF（50 mg/kg）at two different time points（i.e.,ZT6and ZT18）.Plasma and tissue samples were collected at predetermined time points.The concentrations of drugs were determined by UHPLC-MS/MS.4.The regulatory effects of BMAL1 on CES1 were verified by overexpression and knockdown of Bmal1/BMAL1 in CT26 and Caco-2 cells.5.Luciferase reporter assay and chromatin immunoprecipitation（Ch IP）assay were used to determine the mechanism for regulation of CES1 by BMAL1.Results:1.The diurnal rhythm of CES1 expression in mouse intestine.Four Ces1 genes（namely,Ces1d,Ces1e,Ces1f and Ces1g）and seven Ces2 genes（namely,Ces2a,Ces2b,Ces2c,Ces2e,Ces2f,Ces2g and Ces2h）were detected in the intestine of Bmal1^fl/fl mice.Ces1d m RNA and CES1 protein showed a robust diurnal rhythm with a peak level at ZT6.Intestinal Bmal1 ablation downregulated Ces1d m RNA and CES1 protein and disrupted its rhythm.2.The diurnal rhythm of Ces1d/CES1 expression in synchronized cells.We observed rhythmic expression of Ces1d in synchronized CT26 cells.Bmal1knockdown downregulated the expression of Ces1d and blunted its rhythm.Likewise,CES1 m RNA varied with the time in serum-shocked Caco-2 cells,and BMAL1 knockdown downregulated CES1 expression and blunted its rhythm.3.BMAL1 rhythmically regulates intestinal CES1 activity.Microsomal incubation assays indicated that CES1 activity in intestinal microsomes was higher at ZT6 than at ZT18.Intestinal ablation of Bmal1 down-regulated the microsomal CES1 activity at ZT6,but had no effect at ZT18.Pharmacokinetic studies indicated that MPA concentrations in the plasma of Bmal1^fl/fl mice was higher when administered at ZT6 than at ZT18.Intestinal Bmal1 ablation reduced the plasma levels of MPA and abolished its time-dependency.4.BMAL1 positively regulates the expression of CES1.Overexpression of Bmal1 led to significant increases in Ces1d m RNA and CES1protein,whereas knockdown of Bmal1 caused decreases in Ces1d m RNA and CES1protein in CT26 cells.Likewise,overexpression of BMAL1 led to an increase in CES1m RNA,whereas knockdown of BMAL1 caused a decrease in CES1 m RNA in Caco-2 cells.5.BMAL1 transcriptionally regulates Ces1d/CES1 via E-box elements.Sequence analysis of Ces1d promoter revealed two potential E-box elements.Dual luciferase reporter assay and Ch IP assay indicated that BMAL1 directly trans-activated Ces1d by binding to an E-box（1614/-1605 bp）in the promoter.In addition,BMAL1transcriptionally activated CES1 via direct binding to an E-box（-1669/-1658 bp）in the human CES1 promoter.Conclusion:In this study,we uncovered the diurnal rhythm in expression of intestinal CES1 and demonstrated a circadian control of BMAL1 on intestinal CES1.Mechanistically,BMAL1trans-activates Ces1d/CES1 through directly binding to the E-box elements in the gene promoters,impacting the metabolism of MMF.This study has implications for understanding the chronopharmacology of oral medications,which provides scientific foundation for optimizing the administration time of CES1 substrate drugs.