Deletion of betaine-homocysteine S-methyltransferase in mice perturbs choline and 1-carbon metabolism, resulting in fatty liver and hepatocellular carcinomas - PubMed (original) (raw)

Deletion of betaine-homocysteine S-methyltransferase in mice perturbs choline and 1-carbon metabolism, resulting in fatty liver and hepatocellular carcinomas

Ya-Wen Teng et al. J Biol Chem. 2011.

Abstract

Betaine-homocysteine S-methyltransferase (BHMT) uses betaine to catalyze the conversion of homocysteine (Hcy) to methionine. There are common genetic polymorphisms in the BHMT gene in humans that can alter its enzymatic activity. We generated the first Bhmt(-/-) mouse to model the functional effects of mutations that result in reduced BHMT activity. Deletion of Bhmt resulted in a 6-fold increase (p < 0.01) in hepatic and an 8-fold increase (p < 0.01) in plasma total Hcy concentrations. Deletion of Bhmt resulted in a 43% reduction in hepatic S-adenosylmethionine (AdoMet) (p < 0.01) and a 3-fold increase in hepatic S-adenosylhomocysteine (AdoHcy) (p < 0.01) concentrations, resulting in a 75% reduction in methylation potential (AdoMet:AdoHcy) (p < 0.01). Bhmt(-/-) mice accumulated betaine in most tissues, including a 21-fold increase in the liver concentration compared with wild type (WT) (p < 0.01). These mice had lower concentrations of choline, phosphocholine, glycerophosphocholine, phosphatidylcholine, and sphingomyelin in several tissues. At 5 weeks of age, Bhmt(-/-) mice had 36% lower total hepatic phospholipid concentrations and a 6-fold increase in hepatic triacyglycerol concentrations compared with WT (p < 0.01), which was due to a decrease in the secretion of very low density lipoproteins. At 1 year of age, 64% of Bhmt(-/-) mice had visible hepatic tumors. Histopathological analysis revealed that Bhmt(-/-) mice developed hepatocellular carcinoma or carcinoma precursors. These results indicate that BHMT has an important role in Hcy, choline, and one-carbon homeostasis. A lack of Bhmt also affects susceptibility to fatty liver and hepatocellular carcinoma. We suggest that functional polymorphisms in BHMT that significantly reduce activity may have similar effects in humans.

PubMed Disclaimer

Figures

FIGURE 1.

FIGURE 1.

Confirmation of _Bhmt_−/− mice. A, Bhmt chimeric mice were generated using a gene targeting vector that removed exons 6 and 7 of the gene as described under “Experimental Procedures.” loxP, locus of X-over P1; frt, flippase recognition target; neo, neomycin cassette; flp, flippase recombination enzyme; cre, cyclization recombination enzyme. B, PCR analysis of genomic DNA isolated from Bhmt+/+, Bhmt+/−, and _Bhmt_−/− mice. The PCR product of the WT allele was 1600 bp, whereas the knock-out allele was 545 bp. C, hepatic BHMT activity was measured using a radiometric assay in Bhmt+/+ (black bar), Bhmt+/− (hatched bar), and Bhmt_−/− (white bar) mice. Data are presented as mean ± S.E., n = 4–8 per group. Different letters differ significantly (p < 0.01) by analysis of variance and Tukey-Kramer HSD tests. D, BHMT protein in liver from Bhmt+/+ and Bhmt_−/− mice were probed by Western blot analysis. The size of the BHMT protein is 45 kDa. E, body weights of Bhmt+/+ (black square), Bhmt+/− (gray square), and _Bhmt_−/− (white square) mice were measured using a scale from age 3.5 to 20 weeks old. Data are presented as mean ± S.E., n = 10–25 per group. *, p < 0.05, different from Bhmt+/+ by Student's t test.

FIGURE 2.

FIGURE 2.

_Bhmt_−/− mice have altered activities of enzymes involved in one-carbon metabolism. CHDH, GNMT, and PEMT activities were measured in 5-week-old Bhmt+/+ (black bar) and _Bhmt_−/− mice (white bar) mouse liver using radiometric assays. Data are presented as mean ± S.E., n = 6–8 per group. *, p < 0.05, different from Bhmt+/+ by Student's t test.

FIGURE 3.

FIGURE 3.

_Bhmt_−/− mice have fatty liver and reduced hepatic phospholipid concentrations. A, hepatic phospholipids of 5-week-old Bhmt+/+ (black bar) and _Bhmt_−/− (white bar) mice were analyzed by a thin layer chromatography-phosphate assay method. Data are presented as mean ± S.E., n = 5 per group. *, p < 0.01, different from Bhmt+/+ by Student's t test. B, morphology of liver from 5-week-old mice was shown by H&E staining at ×100 magnification. Scale bar = 50 μm. C, hepatic TAG of 5-week-old Bhmt+/+ (black bar) and _Bhmt_−/− (white bar) mice was extracted and quantitated by a colorimetric assay. Data are presented as mean ± S.E., n = 6–8 per group. *, p < 0.05, different from Bhmt+/+ by Student's t test. D, VLDL secretion rate of 5-week-old Bhmt+/+ (black square) and _Bhmt_−/− (white square) mice was determined by measuring the plasma TAG after lipoprotein lipase inhibitor injection. Data are presented as mean ± S.E., n = 6 per group. *, p < 0.05, different from Bhmt+/+ by Student's t test. PLs, phospholipids.

FIGURE 4.

FIGURE 4.

Bhmt deletion results in liver tumors at 1 year of age. A, visible hepatic tumors in 1-year-old _Bhmt_−/− male mice. The inspection of liver specimens at ×4 magnification revealed single or multiple nodules up to 8 mm in diameter. B, H&E images of tumor bearing livers at ×400 magnification, showing representative hepatocellular carcinoma and adenoma morphology. Scale bar, 50 μm. 1, regenerative hyperplastic nodule containing streams of proliferative oval and biliary cells; 2, hepatocellular adenoma (*, center of the nodule) well circumscribed with lobular structure absent, compressive borders, and eosinophilic hepatocytes; 3, hepatocellular carcinoma represented by a large mass of abnormal vacuolated hepatocytes, containing poorly demarcated areas of smaller hepatocytes with less vacuolization, amphophilic cytoplasm, and a streaming growth pattern (arrow); 4, hepatocellular carcinoma nodule of variably sized hepatocytes exhibiting cytomegaly with hyaline droplets, small hepatocytes with streaming growth pattern, oval cell proliferation, Ito cell proliferation between cords (unlabeled arrow), and infiltration of leukocytes.

FIGURE 5.

FIGURE 5.

Changes in one-carbon metabolism due to Bhmt deletion. Deletion of Bhmt resulted in changes in the concentrations of the metabolites or activities of enzymes involved in homocysteine and one-carbon metabolism in tissues.

References

    1. Feng Q., Kalari K., Fridley B. L., Jenkins G., Ji Y., Abo R., Hebbring S., Zhang J., Nye M. D., Leeder J. S., Weinshilboum R. M. (2011) Mol. Genet. Metab. 102, 126–133 - PMC - PubMed
    1. da Costa K. A., Kozyreva O. G., Song J., Galanko J. A., Fischer L. M., Zeisel S. H. (2006) FASEB J. 20, 1336–1344 - PMC - PubMed
    1. Boyles A. L., Billups A. V., Deak K. L., Siegel D. G., Mehltretter L., Slifer S. H., Bassuk A. G., Kessler J. A., Reed M. C., Nijhout H. F., George T. M., Enterline D. S., Gilbert J. R., Speer M. C. (2006) Environ. Health Perspect. 114, 1547–1552 - PMC - PubMed
    1. Weisberg I. S., Park E., Ballman K. V., Berger P., Nunn M., Suh D. S., Breksa A. P., 3rd, Garrow T. A., Rozen R. (2003) Atherosclerosis 167, 205–214 - PubMed
    1. Xu X., Gammon M. D., Zeisel S. H., Bradshaw P. T., Wetmur J. G., Teitelbaum S. L., Neugut A. I., Santella R. M., Chen J. (2009) FASEB J. 23, 4022–4028 - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources