Si-Ni-San reverses dietary fat absorption defects in a murine model of depression.

Depression is often associated with fatigue/energy loss. However, we lack a detailed understanding of the factors explaining this association. In this study, we uncovered that depressed mice have a defect in fat absorption, resulting in weight loss and reduced circulating lipid levels. Si-Ni-San (SNS), a basic formula of traditional Chinese medicine (TCM) for the treatment of depression, was found to not only alleviate depression-like behaviors, but also reverse the weight loss and dietary fat absorption of depressed mice. We found that SNS improved body weight and circulating lipid levels of depressed mice by up-regulating proteins [such as FFA uptake protein (CD36), TAG synthesis proteins (GPAT3, MOGAT2, DGAT1 and DGAT2) and chylomicron packaging proteins (MTP and APOB)] in the fat absorption pathway. Furthermore, cell-based results conducted with LPS-stimulated mouse MODE-K and human Caco-2 cells support that SNS, as well as Sinensetin (SIN) and Nobiletin (NOB), the two active components of the formula, have a role in regulating lipid absorption. Mechanistic studies revealed that SNS reverses body weight and fat absorption defects of depressed mice in part through the NR1D1/BMAL1/DGAT2 axis. These findings advance our understanding of the crosstalk between depression and energy loss, highlight the importance of gut function in disease management, and provide a basis for the application of SNS in the clinical treatment of depression and related disorders.

CRY1/2 regulate rhythmic CYP2A5 in mouse liver through repression of E4BP4.

CYP2A5, an enzyme responsible for metabolism of diverse drugs, displays circadian rhythms in its expression and activity. However, the underlying mechanisms are not fully established. Here we aimed to investigate a potential role of CRY1/2 (circadian clock modulators) in circadian regulation of hepatic CYP2A5. Regulatory effects of CRY1/2 on CYP2A5 were determined using Cry1-null and Cry2-null mice, and validated using AML-12, Hepa1-6 and HepG2 cells. CYP2A5 activities both in vivo and in vitro were assessed using coumarin 7-hydroxylation as a probe reaction. mRNA and protein levels were detected by qPCR and western blotting, respectively. Regulatory mechanism was studied using a combination of luciferase reporter assays, chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP). We found that ablation of Cry1 or Cry2 in mice reduced hepatic CYP2A5 expression (at both mRNA and protein levels) and blunted its diurnal rhythms. Consistently, these knockouts showed decreased CYP2A5 activity (characterised by coumarin 7-hydroxylation) and a loss of its time-dependency, as well as exacerbated coumarin-induced hepatotoxicity. Cell-based assays confirmed that CRY1/2 positively regulated CYP2A5 expression and rhythms. Based on combined luciferase reporter, ChIP and Co-IP assays, we unraveled that CRY1/2 interacted with E4BP4 protein to repress its inhibitory effect on Cyp2a5 transcription and expression. In conclusion, CRY1/2 regulate rhythmic CYP2A5 in mouse liver through repression of E4BP4. These findings advance our understanding of circadian regulation of drug metabolism and pharmacokinetics.

Role of circadian clock in the chronoefficacy and chronotoxicity of clopidogrel.

BACKGROUND AND PURPOSE: The role of circadian locomotor output cycles kaput (CLOCK) in regulating drug chronoefficacy and chronotoxicity remains elusive. Here, we aimed to uncover the impact of CLOCK and dosing time on clopidogrel efficacy and toxicity. EXPERIMENTAL APPROACH: The antiplatelet effect, toxicity and pharmacokinetics experiments were conducted with Clock-/- mice and wild-type mice, after gavage administration of clopidogrel at different circadian time points. The expression levels of drug-metabolizing enzymes were determined by quantitative polymerase chain reaction (qPCR) and western blotting. Transcriptional gene regulation was investigated using luciferase reporter and chromatin immunoprecipitation assays. KEY RESULTS: The antiplatelet effect and toxicity of clopidogrel in wild-type mice showed a dosing time-dependent variation. Clock ablation reduced the antiplatelet effect of clopidogrel, but increased clopidogrel-induced hepatotoxicity, with attenuated rhythms of clopidogrel active metabolite (Clop-AM) and clopidogrel, respectively. We found that Clock regulated the diurnal variation of Clop-AM formation by modulating the rhythmic expression of CYP1A2 and CYP3A1, and altered clopidogrel chronopharmacokinetics by regulation of CES1D expression. Mechanistic studies revealed that CLOCK activated Cyp1a2 and Ces1d transcription by directly binding to the enhancer box (E-box) elements in their promoters, and promoted Cyp3a11 transcription through enhancing the transactivation activity of albumin D-site-binding protein (DBP) and thyrotroph embryonic factor (TEF). CONCLUSIONS AND IMPLICATIONS: CLOCK regulates the diurnal rhythmicity in clopidogrel efficacy and toxicity through regulation of CYP1A2, CYP3A11 and CES1D expression. These findings may contribute to optimizing dosing schedules for clopidogrel and may deepen understanding of the circadian clock and chronopharmacology.

Recent advances in circadian-regulated pharmacokinetics and its implications for chronotherapy.

Dependence of pharmacokinetics and drug effects (efficacy and toxicity) on dosing time has long been recognized. However, significant progress has only recently been made in our understanding of circadian rhythms and their regulation on drug pharmacokinetics, efficacy and toxicity. This review will cover the relevant literature and a series of publications from our work summarizing the effects of circadian rhythms on drug pharmacokinetics, and propose that the influence of circadian rhythms on pharmacokinetics are ultimately translated into therapeutic effects and side effects of drugs. Evidence suggests that daily rhythmicity in expression of drug-metabolizing enzymes and transporters necessary for drug ADME (absorption, distribution, metabolism and excretion) are key factors determining circadian pharmacokinetics. Newly discovered mechanisms for circadian control of the enzymes and transporters are covered. We also discuss how the rhythms of drug-processing proteins are translated into circadian pharmacokinetics and drug chronoefficacy/chronotoxicity, which has direct implications for chronotherapy. More importantly, we will present perspectives on the challenges that are still needed for a breakthrough in translational research. In addition, knowledge of the circadian influence on drug disposition has provided new possibilities for novel pharmacological strategies. Careful application of pharmacokinetics-based chronotherapy strategies can improve efficacy and reduce toxicity. Circadian rhythm-mediated metabolic and transport strategies can also be implemented to design drugs.

Silicon-Controlled Rectifier Embedded Diode for 7 nm FinFET Process Electrostatic Discharge Protection.

A new silicon-controlled rectifier embedded diode (SCR-D) for 7 nm bulk FinFET process electrostatic discharge (ESD) protection applications is proposed. The transmission line pulse (TLP) results show that the proposed device has a low turn-on voltage of 1.77 V. Compared with conventional SCR and diode string, the proposed SCR-D has an additional conduction path constituting by two additional inherent diodes, which results in a 1.8-to-2.2-times current surge capability as compared with the simple diode string and conventional SCR with the same size. The results show that the proposed device meets the 7 nm FinFET process ESD design window and has already been applied in actual circuits.

Deficiency of intestinal Bmal1 prevents obesity induced by high-fat feeding.

The role of intestine clock in energy homeostasis remains elusive. Here we show that mice with Bmal1 specifically deleted in the intestine (Bmal1iKO mice) have a normal phenotype on a chow diet. However, on a high-fat diet (HFD), Bmal1iKO mice are protected against development of obesity and related abnormalities such as hyperlipidemia and fatty livers. These metabolic phenotypes are attributed to impaired lipid resynthesis in the intestine and reduced fat secretion. Consistently, wild-type mice fed a HFD during nighttime (with a lower BMAL1 expression) show alleviated obesity compared to mice fed ad libitum. Mechanistic studies uncover that BMAL1 transactivates the Dgat2 gene (encoding the triacylglycerol synthesis enzyme DGAT2) via direct binding to an E-box in the promoter, thereby promoting dietary fat absorption. Supporting these findings, intestinal deficiency of Rev-erbα, a known BMAL1 repressor, enhances dietary fat absorption and exacerbates HFD-induced obesity and comorbidities. Moreover, small-molecule targeting of REV-ERBα/BMAL1 by SR9009 ameliorates HFD-induced obesity in mice. Altogether, intestine clock functions as an accelerator in dietary fat absorption and targeting intestinal BMAL1 may be a promising approach for management of metabolic diseases induced by excess fat intake.

Clock gene Bmal1 controls diurnal rhythms in expression and activity of intestinal carboxylesterase 1.

OBJECTIVES: We aimed to characterize diurnal rhythms in CES1 expression and activity in mouse intestine, and to investigate a potential role of the core clock gene Bmal1 in generating diurnal rhythms. METHODS: The regulatory effects of intestinal Bmal1 on diurnal CES1 expression were assessed using intestine-specific Bmal1 knockout (Bmal1iKO) mice and colon cancer cells. The relative mRNA and protein levels were determined by qPCR and Western blotting, respectively. Metabolic activity of CES1 in vitro and in vivo were determined by microsomal assays and pharmacokinetic studies, respectively. Transcriptional gene regulation was investigated using luciferase reporter assay. KEY FINDINGS: Total CES1 protein varied significantly according to time of the day in wild-type (Bmal1fl/fl) mice, peaking at ZT6. Of detectable Ces1 genes, Ces1d mRNA displayed a robust diurnal rhythm with a peak level at ZT6, whereas mRNAs of Ces1e, 1f and 1g showed no rhythms in wild-type mice. Loss of intestinal Bmal1 reduced the levels of total CES1 protein and Ces1d mRNA, and blunted their diurnal rhythms in mice. In vitro microsomal assays indicated that intestinal metabolism of mycophenolate mofetil (MMF, a known CES1 substrate) was more extensive at ZT6 than at ZT18. ZT6 dosing of MMF to wild-type mice generated a higher systemic exposure of mycophenolic acid (the active metabolite of MMF) as compared with ZT18 dosing. Intestinal ablation of Bmal1 down-regulated CES1 metabolism at ZT6, and abolished its time-dependency both in vitro and in vivo. Furthermore, Ces1d/CES1 rhythmicity and positive regulation of Ces1d/CES1 by BMAL1 were confirmed in CT26 and Caco-2 cells. Mechanistically, BMAL1 trans-activated Ces1d/CES1 probably via binding to the E-box elements in the gene promoters. CONCLUSIONS: Bmal1 controls diurnal rhythms in expression and activity of intestinal CES1. Our findings have implications for understanding the crosstalk between circadian clock and xenobiotic metabolism in the intestine.

Dysregulation of REV-ERBα impairs GABAergic function and promotes epileptic seizures in preclinical models.

To design potentially more effective therapies, we need to further understand the mechanisms underlying epilepsy. Here, we uncover the role of Rev-erbα in circadian regulation of epileptic seizures. We first show up-regulation of REV-ERBα/Rev-erbα in brain tissues from patients with epilepsy and a mouse model. Ablation or pharmacological modulation of Rev-erbα in mice decreases the susceptibility to acute and chronic seizures, and abolishes diurnal rhythmicity in seizure severity, whereas activation of Rev-erbα increases the animal susceptibility. Rev-erbα ablation or antagonism also leads to prolonged spontaneous inhibitory postsynaptic currents and elevated frequency in the mouse hippocampus, indicating enhanced GABAergic signaling. We also identify the transporters Slc6a1 and Slc6a11 as regulators of Rev-erbα-mediated clearance of GABA. Mechanistically, Rev-erbα promotes the expressions of Slc6a1 and Slc6a11 through transcriptional repression of E4bp4. Our findings propose Rev-erbα as a regulator of synaptic function at the crosstalk between pathways regulating the circadian clock and epilepsy.

Targeted inhibition of Rev-erb-α/ß limits ferroptosis to ameliorate folic acid-induced acute kidney injury.

BACKGROUND AND PURPOSE: Acute kidney injury (AKI) is a common and critical illness, resulting in severe morbidity and a high mortality. There is a considerable interest in identifying novel molecular targets for management of AKI. We investigated the potential role of the circadian clock components Rev-erb-α/ß in regulation of ferroptosis and AKI. EXPERIMENTAL APPROACH: AKI model was established by treating mice with folic acid. Regulatory effects of Rev-erb-α/ß on AKI and ferroptosis were determined using single-gene knockout (Rev-erb-α-/- and Rev-erb-ß-/- ) mice, incomplete double-knockout (icDKO, Rev-erb-α+/- Rev-erb-ß-/- ) mice and cells with erastin-induced ferroptosis. Targeted antagonism of Rev-erb-α/ß to alleviate AKI and ferroptosis was assessed using the small-molecule antagonist SR8278. Transcriptional gene regulation was investigated using luciferase reporter, mobility shift and chromatin immunoprecipitation assays. KEY RESULTS: Loss of Rev-erb-α or Rev-erb-ß reduced the sensitivity of mice to folic acid-induced AKI and eliminated the circadian time dependency in disease severity. This coincided with less extensive ferroptosis, a main cause of folic acid-induced AKI. Moreover, icDKO mice were more resistant to folic acid-induced AKI and ferroptosis as compared with single-gene knockout mice. Supporting this, targeting Rev-erb-α/ß by SR8278 attenuated ferroptosis to ameliorate folic acid-induced AKI in mice. Rev-erb-α/ß promoted ferroptosis by repressing the transcription of Slc7a11 and HO1 (two ferroptosis-inhibitory genes) via direct binding to a RORE cis-element. CONCLUSION AND IMPLICATIONS: Targeted inhibition of Rev-erb-α/ß limits ferroptosis to ameliorate folic acid-induced AKI in mice. The findings may have implications for improved understanding of circadian clock-controlled ferroptosis and for formulating new strategies to treat AKI.

Circadian Clock Component Rev-erbα Regulates Diurnal Rhythm of UDP-Glucuronosyltransferase 1a9 and Drug Glucuronidation in Mice.

UDP-glucuronosyltransferases (UGTs) are a family of phase II enzymes that play an important role in metabolism and elimination of numerous endo- and xenobiotics. Here, we aimed to characterize diurnal rhythm of Ugt1a9 in mouse liver and to determine the molecular mechanisms underlying the rhythmicity. Hepatic Ugt1a9 mRNA and protein displayed robust diurnal rhythms in wild-type mice with peak levels at zeitgeber time (ZT) 6. Rhythmicity in Ugt1a9 expression was confirmed using synchronized Hepa-1c1c7 cells. We observed time-varying glucuronidation (ZT6 > ZT18) of propofol, a specific Ugt1a9 substrate, consistent with the diurnal pattern of Ugt1a9 protein. Loss of Rev-erbα (a circadian clock component) downregulated the Ugt1a9 expression and blunted its rhythm in mouse liver. Accordingly, propofol glucuronidation was reduced and its dosing time dependency was lost in Rev-erbα -/- mice. Dec2 (a transcription factor) was screened to be the potential intermediate that mediated Rev-erbα regulation of Ugt1a9. We confirmed Rev-erbα as a negative regulator of Dec2 in mice and in Hepa-1c1c7 cells. Based on promoter analysis and luciferase reporter assays, it was found that Dec2 trans-repressed Ugt1a9 via direct binding to an E-box-like motif in the gene promoter. Additionally, regulation of Ugt1a9 by Rev-erbα was Dec2-dependent. In conclusion, Rev-erbα generates and regulates rhythmic Ugt1a9 through periodical inhibition of Dec2, a transcriptional repressor of Ugt1a9. Our study may have implications for understanding of circadian clock-controlled drug metabolism and of metabolism-based chronotherapeutics. SIGNIFICANCE STATEMENT: Hepatic Ugt1a9 displays diurnal rhythmicities in expression and glucuronidation activity in mice. It is uncovered that Rev-erbα generates and regulates rhythmic Ugt1a9 through periodical inhibition of Dec2, a transcriptional repressor of Ugt1a9. The findings may have implications for understanding of circadian clock-controlled drug metabolism and of metabolism-based chronotherapeutics.

Bmal1 regulates circadian expression of cytochrome P450 3a11 and drug metabolism in mice.

Metabolism is a major defense mechanism of the body against xenobiotic threats. Here we unravel a critical role of Bmal1 for circadian clock-controlled Cyp3a11 expression and xenobiotic metabolism. Bmal1 deficiency decreases the mRNA, protein and microsomal activity of Cyp3a11, and blunts their circadian rhythms in mice. A screen for Cyp3a11 regulators identifies two circadian genes Dbp and Hnf4α as potential regulatory mediators. Cell-based experiments confirm that Dbp and Hnf4α activate Cyp3a11 transcription by their binding to a D-box and a DR1 element in the Cyp3a11 promoter, respectively. Bmal1 binds to the P1 distal promoter to regulate Hnf4α transcriptionally. Cellular regulation of Cyp3a11 by Bmal1 is Dbp- and Hnf4α-dependent. Bmal1 deficiency sensitizes mice to toxicities of drugs such as aconitine and triptolide (and blunts circadian toxicity rhythmicities) due to elevated drug exposure. In summary, Bmal1 connects circadian clock and Cyp3a11 metabolism, thereby impacting drug detoxification as a function of daily time.

Circadian sensitivity to the cardiac glycoside oleandrin is associated with diurnal intestinal P-glycoprotein expression.

The cardiac glycoside oleandrin is a main active constituent of the botanical anti-cancer drug candidate PBI-05204, an extract of Nerium oleander. Here, we aimed to determine the circadian sensitivity of mice to oleandrin, and to investigate the role of intestinal P-gp in generating rhythmic drug toxicity. Toxicity and pharmacokinetic experiments were performed with wild-type, Bmal1iKO (intestine-specific Bmal1 knockout) and Bmal1fl/fl (control littermates of Bmal1iKO) mice. The cardiac toxicity (reflected by plasma CK-MB, LDH and cTn-I levels) varied significantly with the times of drug dosing in wild-type mice (a lower toxicity at ZT10 and more severe at ZT2/22). Dosing at ZT2 generated a higher drug exposure than ZT10, supporting a lower toxicity at ZT10. Intracellular accumulation of oleandrin (2.5-10 µM) was reduced in MDCKⅡ-MDR1 than in parental cells. MDR1 overexpression decreased the cell sensitivity to oleandrin toxicity. The net flux ratio (MDCKⅡ-MDR1 versus parental cells) was 2.9 for oleandrin. These data indicated oleandrin as a P-gp substrate. Both mdr1a mRNA and P-gp protein oscillated with the times of the day in small intestine of Bmal1fl/fl mice. Intestinal ablation of Bmal1 down-regulated mdr1a mRNA and P-gp protein, and abrogated their rhythms. Likewise, Bmal1 silencing led to down-regulated mdr1a mRNA and to a loss of its rhythmicity in serum-shocked CT26 cells. Based on luciferase reporter assays, Bmal1 regulated rhythmic mdr1a transcription through the clock output genes Hlf and E4bp4. Intestinal ablation of Bmal1 exacerbated oleandrin toxicity and enhanced drug exposure. Moreover, time dependency of toxicity and drug exposure were lost in Bmal1iKO mice. In conclusion, diurnal intestinal P-gp is a critical factor influencing daily oleandrin exposure and toxicity. Our findings have implications in minimizing oleandrin (and possibly Nerium oleander) toxicity and improving drug efficacy via dosing time optimization.

The Molecular Mechanism Regulating Diurnal Rhythm of Flavin-Containing Monooxygenase 5 in Mouse Liver.

Flavin-containing monooxygenase 5 (FMO5) is a phase I enzyme that plays an important role in xenobiotic metabolism. Here, we aimed to characterize diurnal rhythms of Fmo5 expression and activity in mouse liver and to investigate the potential roles of clock genes (Bmal1, Rev-erbα, and E4bp4) in the generation of diurnal rhythms. Fmo5 mRNA and protein showed robust diurnal rhythms, with peak values at zeitgeber time (ZT) 10/14 and trough values at ZT2/22 in mouse liver. Consistently, a diurnal rhythm was observed for in vitro microsomal Baeyer-Villiger oxidation of pentoxifylline (PTX), a specific reaction catalyzed by Fmo5. Pharmacokinetic studies revealed a more extensive Baeyer-Villiger oxidation of PTX at dosing time of ZT14 than at ZT2, consistent with the diurnal pattern of Fmo5 protein. Fmo5 expression was downregulated and its rhythm was blunted in Bmal1 -/- and Rev-erbα -/- mice. Positive regulation of Fmo5 by Bmal1 and Rev-erbα was confirmed in primary mouse hepatocytes and/or Hepa1-6 cells. Furthermore, Fmo5 expression was upregulated and its rhythm was attenuated in E4bp4 -/- mice. Negative regulation of Fmo5 by E4bp4 was validated using primary mouse hepatocytes. Combined luciferase reporter and chromatin immunoprecipitation assays demonstrated that Bmal1 (a known Rev-erbα activator) activated Fmo5 transcription via direct binding to an E-box (-1822/-1816 bp) in the promoter, whereas E4bp4 (a known Rev-erbα target gene) inhibited Fmo5 transcription by binding to two D-boxes (-1726/-1718 and -804/-796 bp). In conclusion, circadian clock genes control diurnal expression of Fmo5 through transcriptional actions on E-box and D-box cis-elements. SIGNIFICANCE STATEMENT: Hepatic Fmo5 displayed diurnal rhythmicities in expression and activity in mice. We uncovered the molecular mechanism by which the rhythmic Fmo5 expression was generated. Fmo5 promoter presents E-box and D-box binding elements for transcriptional actions from circadian clock proteins such as Bmal1, E4bp4, and Dbp. These findings have implications for understanding clock-controlled drug metabolism and for facilitating the practice of chronotherapeutics.

The Circadian Clock Gene Bmal1 Controls Intestinal Exporter MRP2 and Drug Disposition.

The intestinal exporter MRP2 plays an important role in disposition and elimination of a wide range of drugs. Here, we aimed to clarify the impact of circadian clock on intestinal MRP2, and to determine the molecular mechanisms for generation of diurnal MRP2 expression. Methods: The regulatory effects of Bmal1 on intestinal MRP2 expression were assessed using intestine-specific Bmal1 knockout (Bmal1iKO ) mice and colon cancer cells. The relative mRNA and protein levels were determined by qPCR and Western blotting, respectively. Everted gut sac, cell viability and in situ intestinal perfusion experiments were performed to evaluate intestinal efflux of the MRP2 substrate methotrexate (MTX). Toxicity and pharmacokinetic experiments were performed with Bmal1iKO mice and control littermates (Bmal1fl/fl mice) after oral gavage of MTX. Transcriptional gene regulation was investigated using luciferase reporter, mobility shift and chromatin immunoprecipitation (ChIP) assays. Results: Bmal1iKO mice were generated by inter-crossing the mice carrying a Bmal1 exon 8 floxed allele (Bmal1fl/fl ) with Villin-Cre mice. Intestinal MRP2 expression exhibited a diurnal oscillation in Bmal1fl/fl mice with a zenith value at ZT6. Bmal1 ablation caused reductions in Mrp2 mRNA and protein levels [as well as in transport activity (measured by MTX)], and blunted their diurnal rhythms. Intestinal ablation of Bmal1 abrogated circadian time-dependency of MTX pharmacokinetics and toxicity. Bmal1/BMAL1 regulation of rhythmic Mrp2/MRP2 expression was also confirmed in the colon cancer CT26 and Caco-2 cells. Based on a combination of luciferase reporter, mobility shift and ChIP assays, we found that Dbp activated and E4bp4 repressed Mrp2 transcription via specific binding to a same D-box (-100/-89 bp) element in promoter region. Further, Bmal1 directly activated the transcription of Dbp and Rev-erbα through the E-boxes, whereas it negatively regulated E4bp4 via the transcriptional repressor Rev-erbα. Positive regulation of Mrp2 by Rev-erbα was also observed, and attained through modulation of E4bp4. Conclusion: Bmal1 coordinates temporal expressions of DBP (a MRP2 activator), REV-ERBα (an E4BP4 repressor) and E4BP4 (a MRP2 repressor), generating diurnal MRP2 expression.

Reverse Erythroblastosis Virus α Antagonism Promotes Homocysteine Catabolism and Ammonia Clearance.

Metabolic homeostasis of amino acids is essential for human health. Here, we aimed to investigate a potential role for the clock component reverse erythroblastosis virus α (Rev-erbα) in circadian regulation of amino acid metabolism. RNA-seq with Rev-erbα-/- mice showed expression changes in genes involved in amino acid metabolism, particularly, the urea cycle and methionine metabolism. Rev-erbα ablation increased hepatic mRNA, protein, and enzymatic activity of betaine homocysteine methyltransferase (Bhmt), cystathionine ß-synthase (Cbs), and cystathionine γ-lyase (Cth) and decreased the levels of plasma and liver homocysteine in mice. Cell-based assays confirmed negative regulation of these three genes by Rev-erbα. Combined luciferase reporter, mobility-shift, and chromatin immunoprecipitation assays identified Rev-erbα as a transcriptional repressor of Bhmt, Cbs, and Cth. Rev-erbα ablation or antagonism alleviated chemical-induced hyperhomocysteinemia in mice. This was accompanied by elevated expressions of Bhmt, Cbs, and Cth. Moreover, Rev-erbα ablation or antagonism promoted urea production and ammonia clearance. Of urea cycle-related genes, arginase 1 (Arg1), ornithine transcarbamylase (Otc), and carbamoyl-phosphate synthase 1 (Cps1) expressions were up-regulated in Rev-erbα-/- mice. Negative regulation of these urea cycle genes by Rev-erbα was validated using cell-based experiments. Mechanistic studies revealed that Rev-erbα inhibited CCAAT-enhancer-binding protein α transactivation to repress the transcription of Arg1, Cps1, and Otc. Conclusion: Rev-erbα antagonism alleviates hyperhomocysteinemia and promotes ammonia clearance. Targeting Rev-erbα represents an approach for the management of homocysteine- and ammonia-related diseases.

The nuclear receptor Shp regulates morphine withdrawal syndrome via modulation of Ugt2b expression in mice.

In this study, we aimed to investigate a potential role of small heterodimer partner (Shp, a nuclear receptor) in regulation of morphine withdrawal syndrome and to determine the mechanisms thereof. Somatic opiate withdrawal and pharmacokinetic experiments were performed with wild-type (WT) and Shp knockout (Shp-KO) mice. Regulatory effects of Shp on Ugt2b expression were assessed in vitro (using mouse hepatoma Hepa1-6 cells) and in vivo (using Shp-KO mice). Ugt2b mRNA and protein expressions were determined by qPCR and Western blotting, respectively. Microsomal Ugt2b activity was measured with morphine and chloramphenicol. Luciferase reporter, promoter analysis and chromatin immunoprecipitation assays were performed to identify the Hnf1α- and Rev-erbα-binding sites in Ugt2b36 promoter. Protein-protein interactions were explored using co-immunoprecipitation assays. Shp ablation exacerbated morphine withdrawal syndrome in mice. Furthermore, systemic and liver exposures of morphine were elevated in Shp-KO mice due to reduced metabolism. Down-regulation of morphine metabolism was supported by down-regulated expressions of Ugt2b genes in Shp-KO mice. Regulation of Ugt2b genes by Shp was confirmed in mouse hepatoma Hepa1-6 cells. Moreover, Shp positively regulated Ugt2b36 expression through repression of Dec2 and Rev-erbα, two negative regulators of Ugt2b36 enzyme. Rev-erbα repressed Ugt2b36 transcription via direct binding to a specific response element (located at -30/-15 bp) in promoter region of Ugt2b36, whereas Dec2 acted on Ugt2b36 expression via suppression of Hnf1α-transactivation of Ugt2b36 gene. In conclusion, Shp regulated morphine withdrawal syndrome via modulation of Ugt2b expression and detoxification capacity. Targeting Shp may represent a novel approach for management of morphine dependence.

The nuclear receptor Rev-erbα participates in circadian regulation of Ugt2b enzymes in mice.

Circadian clock is known to modulate phase I metabolism, however whether and how the phase II enzymes UDP-glucuronosyltransferases (UGTs) are regulated by circadian clock are largely unknown. In this study, we aimed to investigate a potential role of the clock gene Rev-erbα in regulation of Ugt2b enzymes. Ugt2b mRNA and protein expression in mouse livers were determined at a 4-h interval around the clock. Ugt2b activity was probed using morphine as a specific substrate. Regulation of Ugt2b by Rev-erbα was investigated using mouse hepatoma Hepa-1c1c7 cells and Rev-erbα knock-out (Rev-erbα-/-) mice. Luciferase reporter, mobility shift and chromatin immunoprecipitation (ChIP) assays were performed to identify the Rev-erbα binding site in Ugt2b36 promoter. Circadian variations in hepatic mRNA expression were observed for six Ugt2b genes (Ugt2b1, Ugt2b5, Ugt2b35, Ugt2b36, Ugt2b37, and Ugt2b38) in mice. Likewise, the total Ugt2b protein showed a circadian fluctuation. Glucuronidation of morphine (an Ugt2b substrate) both in vitro and in vivo was dosing-time dependent. Morphine glucuronidation was more extensive at the dosing time of ZT2 than at ZT14 consistent with the Ugt2b protein levels. Furthermore, Rev-erbα knockdown significantly increased Ugt2b mRNA and protein in Hepa-1c1c7 cells, whereas Rev-erbα overexpression or activation down-regulated Ugt2b expression. Moreover, Rev-erbα ablation in mice up-regulated the mRNA and protein expression of Ugt2b and blunted Ugt2b rhythmicity in the liver. In addition, Rev-erbα repressed the transcription of Ugt2b36 through specific binding to the -30 to -18 bp of promoter region based on a combination of luciferase reporter, mobility shift and ChIP assays. In summary, the clock gene Rev-erbα negatively regulates the expressions of Ugt2b genes, contributing to their circadian variations.

Small Heterodimer Partner Regulates Circadian Cytochromes p450 and Drug-Induced Hepatotoxicity.

The role of small heterodimer partner (SHP) in regulation of xenobiotic detoxification remains elusive. Here, we uncover a critical role for SHP in circadian regulation of cytochromes P450 (CYPs) and drug-induced hepatotoxicity. Methods: The mRNA and protein levels of CYPs in the livers of wild-type and SHP-/- mice were measured by quantitative real-time polymerase chain reaction and Western blotting, respectively. Regulation of CYP by SHP was investigated using luciferase reporter, mobility shift, chromatin immunoprecipitation, and/or co-immunoprecipitation assays. Results: The circadian rhythmicities of xenobiotic-detoxifying CYP mRNAs and proteins were disrupted in SHP-deficient mice. Of note, SHP ablation up-regulated Cyp2c38 and Cyp2c39, whereas it down-regulated all other CYP genes. Moreover, SHP regulated the expression of CYP genes through different mechanisms. SHP repressed Lrh-1/Hnf4α to down-regulate Cyp2c38, E4bp4 to up-regulate Cyp2a5, Dec2/HNF1α axis to up-regulate Cyp1a2, Cyp2e1 and Cyp3a11, and Rev-erbα to up-regulate Cyp2b10, Cyp4a10 and Cyp4a14. Furthermore, SHP ablation sensitized mice to theophylline (or mitoxantrone)-induced toxicity. Higher level of toxicity was correlated with down-regulated metabolism and clearance of theophylline (or mitoxantrone). In contrast, SHP ablation blunted the circadian rhythmicity of acetaminophen-induced hepatotoxicity and alleviated the toxicity by down-regulating Cyp2e1-mediated metabolism and reducing formation of the toxic metabolite. Toxicity alleviation by SHP ablation was also observed for aflatoxin B1 due to reduced formation of the toxic epoxide metabolite. Conclusion: SHP participates in circadian regulation of CYP enzymes, thereby impacting xenobiotic metabolism and drug-induced hepatotoxicity.

Identification of poliumoside metabolites in rat plasma, urine, bile, and intestinal bacteria with UPLC/Q-TOF-MS.

Poliumoside is representative of phenylethanoid glycosides, which are widely found in many plants. Poliumoside is also regarded as the main active component of Callicarpa kwangtungensis Chun (CK), though its oral bioavailability in rat is extremely low (0.69%) and its in vivo and in vitro metabolism has not yet been systematically investigated. In the present study, an ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) method was employed to identify the metabolites and investigate the metabolic pathways of poliumoside in rat after oral administration 1.5 g·kg-1 of poliumoside. As a result, a total of 34 metabolites (30 from urine, 17 from plasma, and 4 from bile) and 9 possible metabolic pathways (rearrangment, reduction, hydration, hydrolyzation, dehydration, methylation, hydroxylation, acetylation, and sulfation) were proposed in vivo. The main metabolite, acteoside, was quantified after incubated with rat intestinal bacteria in vitro. In conclusion, the present study systematically explored the metabolites of poliumoside in vivo and in vitro, proposing metabolic pathways that may be significant for further metabolic studies of poliumoside.

The Clock Protein Bmal1 Regulates Circadian Expression and Activity of Sulfotransferase 1a1 in Mice.

Sulfotransferase 1a1 (Sult1a1) is a phase II enzyme that contributes extensively to metabolism and detoxification of various drugs and chemicals. Here we aimed to investigate a potential role of the clock protein Bmal1 (brain and muscle Arnt-like protein-1) in circadian regulation of Sult1a1 in mice. The regulatory effects of Bmal1 on Sult1a1 were assessed both in vivo (using Bmal1- deficient mice) and in vitro (using both normal and serum-shocked Hepa-1c1c7 cells). The relative mRNA and protein levels of Sult1a1 in the cells or mouse livers were measured by RT-qPCR and Western blotting, respectively. Sulfation activities of two Sult1a1 substrates (i.e., p-nitrophenol and galangin) were determined using mouse liver S9 fractions. Transcriptional regulation of Sult1a1 by Bmal1 was investigated using luciferase reporter, electrophoretic mobility shift (EMSA), and chromatin immunoprecipitation (ChIP) assays. We first showed that hepatic Sult1a1 was rhythmically expressed at both mRNA and protein levels (higher expressions during the night than the daytime). Consistently, the liver sulfation activities toward two Sult1a1 substrates were circadian time dependent with a higher activity at ZT14 than at ZT2. Furthermore, deletion of Bmal1 in mice blunted the circadian rhythmicity of hepatic Sult1a1 (with reduced expression levels). Likewise, Bmal1 positively regulated Sult1a1 expression in conventionally cultured Hepa-1c1c7 cells, and Bmal1 knockdown blunted expression rhythmicity of Sult1a1 in serum-shocked Hepa-1c1c7 cells. A combination of promoter analysis, EMSA and ChIP assays revealed that Bmal1 stimulated Sult1a1 transcription through its specific binding to the-571- to -554-bp region (an E-box element) in the promoter. In conclusion, Bmal1 activated the transcription of Sult1a1 and controlled circadian expression and activity of the enzyme.