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.
Highlights
Betaine-homocysteine S-methyltransferase (BHMT) activity is found at high levels in the liver and kidney, and low levels in the brain, lenses, and other human tissues
Hcy is converted to methionine by BHMT or by methionine synthase, the latter of which uses methyltetrahydrofolate as the methyl homocysteine; AdoMet, S-adenosylmethionine; AdoHcy, S-adenosylhomocysteine; PtdCho, phosphatidylcholine; PtdEth, phosphatidylethanolamine; PCho, phosphocholine; GPCho, glycerophosphocholine; PtdIns, phosphatidylinositol; PtdSer, phosphatidylserine; BUN, plasma urea nitrogen; THF, tetrahydrofolate; nonesterified fatty acids (NEFA), nonesterified fatty acid; TAG, triacylglycerol; LDH, lactate dehydrogenase; ALT, alanine transaminase; CK, creatinine kinase; HDL-C, high density lipoprotein cholesterol; VLDL, very low density lipoprotein; ␥GT1, ␥-glutamyltransferase 1; HCC, hepatocellular carcinoma
BhmtϪ/Ϫ mice were validated by assaying hepatic BHMT activity (Fig. 1C) and Western blot (Fig. 1D)
Summary
Generation of BhmtϪ/Ϫ Mice—The gene targeting vector was designed to remove exons 6 and 7, the zinc-binding domains of the gene (Fig. 1A). Bhmt knockout mice were generated by crossing the Neo deleted mice with Cre deleters (number 003724, Jackson Laboratory), which expressed Cre recombinase that recognized loxP sites and deleted exons 6 and 7. Western blot analysis of BHMT was performed in liver homogenate as previously described [10]. BHMT activity was measured as the conversion of [14C]betaine to [14C]methionine and [14C]dimethylglycine as previously described [11]. CHDH activity was measured as the conversion of [14C]choline to [14C]betaine as previously described [12] using high pressure liquid chromatography (HPLC) on a Varian ProStar HPLC system (PS-210, Varian Inc., Palo Alto, CA) with a Pecosphere Silica column, 4.6 ϫ 83 mm (PerkinElmer Life Sciences) and a Berthold LB506 C-1 radiodetector (Oak Ridge, TN). Whole liver was sectioned serially, and multiple sections per sample were examined by 2 veterinary pathologists from the Department of Laboratory Animal Medicine, UNC, Chapel Hill. Statistics—Statistical differences were determined using analysis of variance, Tukey-Kramer HSD, and Student’s t test JMP version 6.0 (SAS Institute, Cary, NC) and reported as mean Ϯ S.E
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