Natural Compounds that Modulate Circadian Rhythms.

Circadian rhythms of innate 24 h cycles comprise well-conserved biological phenomena from cyanobacteria to mammalian. They are driven by light and regulated by clock genes that work as transcription factors and control the expression of many other genes and physiological functions in the cells. The expression of ~ 40% of protein-coding genes shows 24 h oscillation patterns in mice, implying their importance in normal body functions. Indeed, the physiological and behavioural rhythmicity generated through clock genes-mediated multiple mechanisms affects the quality of life at large. Disrupted circadian rhythmicity is associated with several kinds of diseases. For example, cancer cells show abnormal expression patterns for circadian rhythm genes that have been shown to regulate oncogenesis, drug responses, and disease prognosis. Furthermore, the modern globalisation of human lifestyle and business and social activities have disrupted innate circadian rhythm, resulting in a variety of diseases through disrupted humoral, immunological, and neuronal pathways. Safe and sustainable modulation of circadian rhythm has become a prevalent need that warrants basic and interventional research, as well as clinical investigations. Although traditional systems of medicine suggest some natural compounds with circadian rhythmmodulating potential, most of these have not been validated in laboratory or clinical studies. Reliable read-outs of the effects of test compounds on circadian rhythmicity have been limited by the availability of live cell assays. We have, herein, provided an overview of living cell-embedded real- time reporter gene assays designed for screening compounds that modulate circadian rhythm, and discussed the potential of some natural compounds for circadian rhythm modulation as validated by cell-based assay systems, and their role in disease therapeutics.

Hydroxy-ß-sanshool isolated from Zanthoxylum piperitum (Japanese pepper) shortens the period of the circadian clock.

We showed that an ethanol extract from Zanthoxylum piperitum can shorten the circadian rhythm at the cellular level and that this activity was due to hydroxy-ß-sanshool, a secondary metabolite in this plant. An ethanol extract of Z. piperitum was repeatedly fractionated using solid phase extraction and reverse-phase HPLC, then the circadian rhythms of cells to which the fractions were loaded were monitored using real-time reporter gene assays. We purified one HPLC peak and identified it as hydroxy-ß-sanshool using liquid chromatography (LC)-precision-mass spectrometry (MS). This compound shortened the period of Bmal1 and Per2 at the cellular level. Incubation cells for 24 h with hydroxy-ß-sanshool resulted in upregulated Per2 promoter activity. Hydroxy-ß-sanshool also dose-dependently upregulated expression of the clock genes Bmal1, Per1, Per2 and Cry1 and the clock-controlled oxidative stress responsive genes Gpx1and Sod2.

Withanolide Derivative 2,3-Dihydro-3ß-methoxy Withaferin-A Modulates the Circadian Clock via Interaction with RAR-Related Orphan Receptor α (RORa).

Withanolide derivatives have anticancer, anti-inflammatory, and other functions and are components of Indian traditional Ayurvedic medicine. Here, we found that 2,3-dihydro-3ß-methoxy withaferin-A (3ßmWi-A), a derivative of withaferin-A (Wi-A) belonging to a class of withanolides that are abundant in Ashwagandha (Withania somnifera), lengthened the period of the circadian clock. This compound dose-dependently elongated circadian rhythms in Sarcoma 180 cancer cells and in normal fibroblasts including NIH3T3 and spontaneously immortalized mouse embryonic fibroblasts (MEF). Furthermore, 3ßmWi-A dose-dependently upregulated the mRNA expression and promoter activities of Bmal1 after dexamethasone stimulation and of the nuclear orphan receptors, Rora and Nr1d1, that comprise the stabilization loop for Bmal1 oscillatory expression. We showed that 3ßmWi-A functions as an inverse agonist for RORa with an IC50 of 11.3 µM and that 3ßmWi-A directly, but weakly, interacts with RORa (estimated dissociation constant [Kd], 5.9 µM). We propose that 3ßmWi-A is a novel modulator of circadian rhythms.

Coelenterazine Analogue with Human Serum Albumin-Specific Bioluminescence.

A synthetic luciferin comprising an imidazopyrazinone core, named HuLumino1, was designed to generate specific bioluminescence with human serum albumin (HSA) in real serum samples. HuLumino1 was developed by attaching a methoxy-terminated alkyl chain to C-6 of coelenterazine and by eliminating a benzyl group at C-8. HSA levels were quantified within 5% error margins of an enzyme-linked immunosorbent assay without the need for any sample pretreatments because of the high specificity of HuLumino1.

ß-Arrestin 1 (ARRB1) serves as a molecular marker of the peripheral circadian rhythm.

The control of the circadian rhythm is important for health because it regulates physiological functions and is associated with health hazards. We aimed to identify a circadian biomarker of health status in human saliva, since collecting saliva is non-invasive, straightforward, and cost-effective. Among 500 genes potentially controlled by the salivary clock identified using chromatin immunoprecipitation (ChIP) assays, 22 of them showed reasonable transcriptional responses according to a DNA array in a salivary model system. Among these 22 genes, ARRB1, which is expressed in human salivary glands, was also expressed in model HSG cells at the transcriptional and translational levels. The profile of ARRB1 expression in human saliva was circadian, suggesting that ARRB1 could serve as a candidate circadian biomarker in saliva. We compared ARRB1 with other biomarkers in salivary samples from jet-lagged individuals. The circadian profile of ARRB1 reflected the time lag more than the profile of melatonin, whereas the profiles of cortisol and α-amylase did not reflect the time lag. Overall, these findings suggest that salivary ARRB1 could serve as a candidate biomarker that could be used to monitor the internal body clock.

Epigenetic regulation of the circadian clock: role of 5-aza-2'-deoxycytidine.

We have been investigating transcriptional regulation of the BMAL1 gene, a critical component of the mammalian clock system including DNA methylation. Here, a more detailed analysis of the regulation of DNA methylation of BMAL1 proceeded in RPMI8402 lymphoma cells. We found that CpG islands in the BMAL1 and the PER2 promoters were hyper- and hypomethylated, respectively and that 5-aza-2'-deoxycytidine (aza-dC) not only enhanced PER2 gene expression but also PER2 oscillation within 24 h in RPMI8402 cells. That is, such hypermethylation of CpG islands in the BMAL1 promoter restricted PER2 expression which was recovered by aza-dC within 1 day in these cells. These results suggest that the circadian clock system can be recovered through BMAL1 expression induced by aza-dC within a day. The RPIB9 promoter of RPMI8402 cells, which is a methylation hotspot in lymphoblastic leukemia, was also hypermethylated and aza-dC gradually recovered RPIB9 expression in 3 days. In addition, methylation-specific PCR revealed a different degree of aza-dC-induced methylation release between BMAL1 and RPIB9 These results suggest that the aza-dC-induced recovery of gene expression from DNA methylation is dependent on a gene, for example the rapid response to demethylation by the circadian system, and thus, is of importance to clinical strategies for treating cancer.

Harmine lengthens circadian period of the mammalian molecular clock in the suprachiasmatic nucleus.

The circadian clock is a cell-autonomous endogenous system that generates circadian rhythms in the behavior and physiology of most organisms. We previously reported that the harmala alkaloid, harmine, lengthens the circadian period of Bmal1 transcription in NIH 3T3 fibroblasts. Clock protein dynamics were examined using real-time reporter assays of PER2::LUC to determine the effects of harmine on the central clock in the suprachiasmatic nucleus (SCN). Harmine significantly lengthened the period of PER2::LUC expression in embryonic fibroblasts, in neuronal cells differentiated from neuronal progenitor cells and in SCN slices obtained from PER2::LUC mice. Although harmine did not induce the transient mRNA expression of clock genes such as Per1, Per2 and Bmal1 in embryonic fibroblasts, it significantly extended the half-life of PER2::LUC protein in neuronal cells and SCN slices. Harmine might lengthen the circadian period of the molecular clock by increasing PER2 protein stability in the SCN.

Thiazolidinediones are potent inducers of fibroblast growth factor 21 expression in the liver.

Fibroblast growth factor 21 (FGF21) is an effective metabolic regulator of glucose and lipid homeostasis in the context of insulin resistance, glucose intolerance and dyslipidemia in diabetic rodents and monkeys, and peroxisome proliferator-activated receptor α (PPARα) directly induces FGF21 expression in the rodent liver. Recent findings suggest that the effects and regulation of FGF21 qualitatively differ between rodents and humans. Here, we examined the effects of PPARα and PPARγ agonists on FGF21 mRNA expression in the mouse liver and in cultured hepatocytes. Intraperitoneal injection of both bezafibrate and pioglitazone induced FGF21 mRNA expression in the mouse liver. Rosiglitazone and pioglitazone as well as bezafibrate significantly induced FGF21 mRNA expression in cultured mouse hepatocytes. On the other hand, both rosiglitazone and pioglitazone significantly induced, whereas bezafibrate did not affect FGF21 mRNA expression in the human liver carcinoma cell line HepG2. Bezafibrate significantly induced pyruvate dehydrogenase kinase 4 mRNA expression, suggesting that HepG2 cells are sensitive to bezafibrate like the mouse liver. Our findings suggest that PPARγ-activating antidiabetic drugs such as rosiglitazone and pioglitazone induce FGF21 expression in mouse and human hepatocytes, and that PPARγ rather than PPARα might play an important role in human FGF21 production.

PPARγ activation induces acute PAI-1 gene expression in the liver but not in adipose tissues of diabetic model mice.

Insulin resistance in patients with type II diabetes has recently been treated with thiazolidinediones, a class of peroxisome proliferator-activated receptor γ (PPARγ) agonists. However, these compounds are possibly associated with a significant increase in the risk of cardiovascular events. We examined the effect of the PPARγ agonist rosiglitazone on the expression of plasminogen activator inhibitor-1 (PAI-1) that is the primary inhibitor of fibrinolysis in the liver of diabetic mice and cultured mouse and human hepatocytes. Concentrations of plasma PAI-1 and levels of its mRNA expression in the liver were significantly elevated in accordance with hepatic PPARγ1 and PPARγ2 mRNA accumulation in genetically diabetic db/db mice. An intraperitoneal injection of rosiglitazone significantly increased plasma PAI-1 concentrations in parallel with hepatic, but not with adipose mRNA levels in db/db mice, and did not affect these parameters in wild-type mice. Rosiglitazone as well as the PPARα agonist bezafibrate significantly induced PAI-1 mRNA expression in cultured mouse hepatocytes. Furthermore, both rosiglitazone and pioglitazone significantly induced, whereas bezafibrate did not affect PAI-1 mRNA expression in the human liver carcinoma cell line HepG2. The transient induction of PAI-1 gene expression mediated by PPARγ in the fatty liver might be involved in the increased risk of cardiovascular events associated with thiazolidinediones in diabetic patients through decreasing fibrinolytic activity.

Conserved amino acid residues in C-terminus of PERIOD 2 are involved in interaction with CRYPTOCHROME 1.

We investigated the amino acid sequences of rat PERIOD2 (rPER2) that are required for interaction with CRYPTOCHROME1 (CRY1) to understand the molecular mechanism of the circadian clock. Co-immunoprecipitation assays using various C-terminal fragments of rPER2 with internal deletions revealed that amino acid residues 1179-1198 are necessary for interaction with CRY1. To identify precisely which amino acid residues are responsible for the interaction, we substituted alanine for residues conserved among PER isoforms and species. We found that more than three mutations of conserved PER2 residues impaired not only binding to CRY1 but also subsequent nuclear translocation, although mutations of non-conserved residues did not affect interaction with CRY1. Thus, the conserved amino acid residues of 1179-1198 in PER2 are apparently responsible for binding to CRY1.

A sensitive internal standard method for the determination of melatonin in mammals using precolumn oxidation reversed-phase high-performance liquid chromatography.

A sensitive and accurate internal standard method to determine melatonin in mammalian tissues and physiological fluids has been described. This method includes the oxidation of melatonin to a highly fluorescent compound, N-[(6-methoxy-4-oxo-1,4-dihydroquinolin-3-yl)methyl]acetamide (6-MOQMA), and the determination of 6-MOQMA by a reversed-phase HPLC system. For the accurate and reliable determination, several melatonin analogs were designed and utilized as the internal standards, and ethyl and isopropyl analogs (having the corresponding alkyl group via the amide bond of melatonin instead of the methyl group) were found to be promising. Using these internal standards, highly accurate and sensitive determination could be accomplished using rat pineal gland samples, and the clear circadian rhythms are demonstrated. This method was also successfully applied to the determination of melatonin in a small amount (20 microL) of human saliva.

Novel stable fluorophore, 6-methoxy-4-quinolone, with strong fluorescence in wide pH range of aqueous media, and its application as a fluorescent labeling reagent.

6-Methoxy-4-quinolone (6-MOQ, 1), an oxidation product derived from 5-methoxyindole-3-acetic acid, is a novel fluorophore, which has several useful characteristics for biomedical analysis. Compound 1 has strong fluorescence with a large Stokes' shift in aqueous media, and the maximum fluorescence excitation and emission wavelengths are 243 nm and 374 nm, respectively. The molar absorptivity at the maximum excitation wavelength and fluorescence quantum yield in aqueous 10% (v/v) methanol are 32 600 L mol(-1) cm(-1) and 0.38, respectively. The fluorescence intensity of 1 is scarcely affected by changing the medium pH, showing strong fluorescence from pH 2.0 to 11.0. In addition, 1 is highly stable against light and heat, and no degradation was observed at 60 degrees C for 3 days with exposure to daylight. As a fluorescent labeling reagent, [(6-methoxy-4-oxo-1,4-dihydroquinolin-3-yl)methyl]amine (6-MOQ-NH2, 2) was synthesized, and determination of carboxylic acids was demonstrated; 50 pmol of standard propionic acid and isobutyric acid were derivatized, and the obtained S/N ratios for 10 fmol (injection amount) of these two acids were 206 and 164, respectively.

Determination of endogenous melatonin in the individual pineal glands of inbred mice using precolumn oxidation reversed-phase micro-high-performance liquid chromatography.

The amount of endogenous melatonin in the individual pineal glands of inbred mice has been determined using reversed-phase micro-high-performance liquid chromatography after precolumn oxidation of melatonin to a compound having strong fluorescence. The fluorescent compound was identified as N-[(6-methoxy-4-oxo-1,4-dihydroquinolin-3-yl)methyl]acetamide. The excitation and emission wavelengths of this compound are 245 and 380 nm, respectively, and the fluorescence intensity is 6.8 times greater than that of melatonin. Molar absorptivity and fluorescence quantum yield of this compound are 46,300[L mol(-1)cm(-1)] and 0.31 (245 nm), respectively. The lower quantification limit of melatonin in biological samples using this precolumn oxidation method is 200 amol, and the calibration curve of spiked melatonin is linear from 200 amol to 50 fmol (r>0.999). The sensitivity of the present method is almost 10 times higher than that of the previous method. The values of endogenous melatonin obtained for ICR, C57BL, BALB/c, and AKR mice are 4.7, 6.1, 7.4, and 18.8 fmol/pineal gland, respectively. The amounts of endogenous pineal melatonin of these strains had not been clearly reported due to the poor enzymatic activities for melatonin biosynthesis; this is the first report that clearly demonstrates the existence of endogenous melatonin in these inbred mice.