The Lysophospholipase PNPLA7 Controls Hepatic Choline and Methionine Metabolism.

The in vivo roles of lysophospholipase, which cleaves a fatty acyl ester of lysophospholipid, remained unclear. Recently, we have unraveled a previously unrecognized physiological role of the lysophospholipase PNPLA7, a member of the Ca2+-independent phospholipase A2 (iPLA2) family, as a key regulator of the production of glycerophosphocholine (GPC), a precursor of endogenous choline, whose methyl groups are preferentially fluxed into the methionine cycle in the liver. PNPLA7 deficiency in mice markedly decreases hepatic GPC, choline, and several metabolites related to choline/methionine metabolism, leading to various symptoms reminiscent of methionine shortage. Overall metabolic alterations in the liver of Pnpla7-null mice in vivo largely recapitulate those in methionine-deprived hepatocytes in vitro. Reduction of the methyl donor S-adenosylmethionine (SAM) after methionine deprivation decreases the methylation of the PNPLA7 gene promoter, relieves PNPLA7 expression, and thereby increases GPC and choline levels, likely as a compensatory adaptation. In line with the view that SAM prevents the development of liver cancer, the expression of PNPLA7, as well as several enzymes in the choline/methionine metabolism, is reduced in human hepatocellular carcinoma. These findings uncover an unexplored role of a lysophospholipase in hepatic phospholipid catabolism coupled with choline/methionine metabolism.

Hepatic phosphatidylcholine catabolism driven by PNPLA7 and PNPLA8 supplies endogenous choline to replenish the methionine cycle with methyl groups.

Choline supplies methyl groups for regeneration of methionine and the methyl donor S-adenosylmethionine in the liver. Here, we report that the catabolism of membrane phosphatidylcholine (PC) into water-soluble glycerophosphocholine (GPC) by the phospholipase/lysophospholipase PNPLA8-PNPLA7 axis enables endogenous choline stored in hepatic PC to be utilized in methyl metabolism. PNPLA7-deficient mice show marked decreases in hepatic GPC, choline, and several metabolites related to the methionine cycle, accompanied by various signs of methionine insufficiency, including growth retardation, hypoglycemia, hypolipidemia, increased energy consumption, reduced adiposity, increased fibroblast growth factor 21 (FGF21), and an altered histone/DNA methylation landscape. Moreover, PNPLA8-deficient mice recapitulate most of these phenotypes. In contrast to wild-type mice fed a methionine/choline-deficient diet, both knockout strains display decreased hepatic triglyceride, likely via reductions of lipogenesis and GPC-derived glycerol flux. Collectively, our findings highlight the biological importance of phospholipid catabolism driven by PNPLA8/PNPLA7 in methyl group flux and triglyceride synthesis in the liver.

Exploring disease-specific methylated CpGs in human male genital abnormalities by using methylated-site display-amplified fragment length polymorphism (MSD-AFLP).

The incidence of male reproductive system disorders, especially hypospadias, has been increasing in developed countries since the latter half of the 20th century. Endocrine-disrupting chemicals from the environment are considered to be involved in hypospadias onset through epigenetic alterations. This pilot study aimed to explore disease-specific methylated CpGs in human patient samples using the methylated-site display-amplified fragment length polymorphism (MSD-AFLP) technique developed by our research group [1]. We compared clinical samples from hypospadias and phimosis patients. Foreskin and blood samples were collected from one- to two-year-old patients with hypospadias (N = 3) and phimosis (N = 3) during surgical treatment. MSD-AFLP analysis showed significantly decreased CpG-methylation levels of genes such as MYH11 and increased CpG-methylation levels of genes such as PLA2G15 in hypospadias patients. Hierarchical clustering analysis showed that genes with significantly altered CpG levels were more markedly altered in DNA from blood than from foreskin. Because of the small number of samples, further investigation is necessary to elucidate the association between variations in CpG levels in foreskin and blood DNA and male genital abnormalities. However, our MSD-AFLP method appears to be a useful tool for exploring disease-specific methylated-CpGs in human epidemiological studies.

Does the prenatal bisphenol A exposure alter DNA methylation levels in the mouse hippocampus?: An analysis using a high-sensitivity methylome technique.

BACKGROUND: There is still considerable debate about the effects of exposure to bisphenol A (BPA) an endocrine disrupter at low doses. Recently, many studies using animal models have shown that prenatal BPA exposure induces behavioral and neuronal disorders due to epigenetic changes in the brain. However, striking evidence of epigenomic changes has to be shown. METHODS: To investigate whether low-dose BPA exposure in the fetal stage can alter CpG methylation levels in the central nervous system, the hippocampus of the inbred C57BL/6 J mouse as the target tissue was collected to detect alterations in CpG methylation levels using a highly sensitive method of genome-wide DNA methylation analysis, methylated site display-amplified fragment length polymorphism (MSD-AFLP). RESULTS: BPA showed the sex-hormone like effects on male reproductive organs. Although we examined the methylation levels of 43,840 CpG sites in the control and BPA (200 µg/kg/day)-treated group (6 mice per group), we found no statistically significant changes in methylation levels in any CpG sites. CONCLUSIONS: At least under the experimental condition in this study, it is considered that the effect of low-dose BPA exposure during the fetal stage on hippocampal DNA methylation levels is extremely small.

Detection of dioxin-induced demethylation of mouse Cyp1a1 gene promoter by a new labeling method for short DNA fragments possessing 5'-methylcytosine at the end.

Environmental factors stimulate alteration of DNA methylation level. Investigation of the genome-wide DNA methylation status is important for environmental health studies. We here designed a genomic DNA amplification and labeling protocol using a methylation-sensitive restriction enzyme HinP1 I. This method can specifically amplify genomic DNA fragments possessing methyl-CpG at the end. The fragments are a relatively short size and dominantly located on CpG-islands. By using the samples prepared by this method, a dioxin-induced change in the methylation level of the mouse Cyp1a1 promoter was successfully evaluated using oligonucleotide probes covalently bound onto a glass plate. The method developed in this paper would be useful for other genome-wide analysis platforms for the large scale epigenome-wide association studies (EWAS) including human epidemiological samples.

Expression of Xenobiotic Biomarkers CYP1 Family in Preputial Tissue of Patients with Hypospadias and Phimosis and Its Association with DNA Methylation Level of SRD5A2 Minimal Promoter.

Several epidemiological studies have suggested that the incidence of male reproductive organ malformations, including hypospadias or cryptorchidism, has increased due to fetal-stage exposure to environmental pollutants. However, the association of chemical exposure with the expression of target regulatory genes in the tissues of patients has not yet been reported. Because experimental approaches or clinical trials in human studies are limited, especially those using fetal and/or infants, it is difficult to obtain clear physiological evidence of mechanisms underlying male reproductive malformations. Thus, the lack of physiological evidence makes this issue controversial. We analyzed preputial tissues from patients with hypospadias (n = 23) and phimosis (n = 16). The atypical CYP1 family genes, CYP1A1 and CYP1B1, are potential biomarkers of environmental chemical exposure. We then compared the expression levels of CYP1A1 and CYP1B1 between hypospadias and phimosis samples by quantitative RT-PCR analysis. The mRNA expression levels of SRD5A2 and AR also were measured, because the androgen-related genes involved in the onset of disorders of male reproductive system. A significantly higher CYP1B1 expression level and a lower AR expression level were observed in the hypospadias groups than in the phimosis group. Positive correlations (P < 0.001) between the mRNA expression levels of the CYP1 family and SRD5A2 were found in patients with hypospadias but not in those with phimosis. Moreover, the methylation levels of the four genes were determined by bisulfite genomic sequencing. Although the SRD5A2 promoter region showed moderate methylation, no methylation was detected in CYP1A1, CYP1B1, or AR. There was no significant difference in SRD5A2 promoter methylation level between hypospadias and phimosis patients. Negative correlations were found between the methylation level of SRD5A2, especially at the - 221 Sp1 site, and the CYP1 family mRNA expression levels (CYP1A1, p = 0.002; CYP1B1, p = 0.007) in hypospadias patients, but not in phimosis patients. The significant positive association of mRNA expression level and the negative association of methylation level of the SRD5A2 gene with the mRNA expression levels of CYP1 family genes in the preputial tissue seem to indicate the chemical exposure of patients with hypospadias.

Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles.

BACKGROUND: It has been pointed out that environmental factors or chemicals can cause diseases that are developmental in origin. To detect abnormal epigenetic alterations in DNA methylation, convenient and cost-effective methods are required for such research, in which multiple samples are processed simultaneously. We here present methylated site display (MSD), a unique technique for the preparation of DNA libraries. By combining it with amplified fragment length polymorphism (AFLP) analysis, we developed a new method, MSD-AFLP. RESULTS: Methylated site display libraries consist of only DNAs derived from DNA fragments that are CpG methylated at the 5' end in the original genomic DNA sample. To test the effectiveness of this method, CpG methylation levels in liver, kidney, and hippocampal tissues of mice were compared to examine if MSD-AFLP can detect subtle differences in the levels of tissue-specific differentially methylated CpGs. As a result, many CpG sites suspected to be tissue-specific differentially methylated were detected. Nucleotide sequences adjacent to these methyl-CpG sites were identified and we determined the methylation level by methylation-sensitive restriction endonuclease (MSRE)-PCR analysis to confirm the accuracy of AFLP analysis. The differences of the methylation level among tissues were almost identical among these methods. By MSD-AFLP analysis, we detected many CpGs showing less than 5% statistically significant tissue-specific difference and less than 10% degree of variability. Additionally, MSD-AFLP analysis could be used to identify CpG methylation sites in other organisms including humans. CONCLUSION: MSD-AFLP analysis can potentially be used to measure slight changes in CpG methylation level. Regarding the remarkable precision, sensitivity, and throughput of MSD-AFLP analysis studies, this method will be advantageous in a variety of epigenetics-based research.

Synthesis and properties of a series of well-defined and polydisperse benzo[1,2-b:4,3-b']dithiophene oligomers.

Monodisperse and polydisperse oligomers of benzo[1,2-b:4,3-b']dithiophene (BDT) (1-14), including three types of oligomers with different spacers combining BDT units (direct linkage, vinylene spacers, and ethynylene spacers), were synthesized, and their thermal, optical, and electrochemical properties were investigated. The oligomers were synthesized using Suzuki, Stille, Wittig, and Sonogashira coupling reactions. All of the monodisperse oligomers showed high melting points and 5% weight loss temperatures (T(d) > 400 degrees C). The fluorescence maxima of all oligomers were red-shifted, and the emission colors varied from blue to yellow as the chain lengths-and thus the conjugation lengths-increased. The vinylene-bridged oligomers emitted at longer wavelengths than the direct-linked and ethynylene-bridged oligomers. UV-vis absorption spectra in toluene solution indicated an effective conjugation length of about six BDT units for polydisperse oligomer 5. Cyclic voltammetry measurement indicated that tetramer 3 had high electrochemical stability. Although tetramer 3 and vinylene-bridged tetramer 8 exhibited reversible oxidation waves, ethynylene-bridged tetramer 13 showed an irreversible oxidation process. Each type of monodisperse oligomer exhibited higher HOMO levels with increasing chain length.