Abstract
Hyperhomocysteinemia (HHCY) is a consequence of impaired methionine/cysteine metabolism and is caused by deficiency of vitamins and/or enzymes such as cystathionine beta-synthase (CBS). Although HHCY is an important and independent risk factor for cardiovascular diseases that are commonly associated with hepatic steatosis, the mechanism by which homocysteine promotes the development of fatty liver is poorly understood. CBS-deficient (CBS(-/-)) mice were previously generated by targeted deletion of the Cbs gene and exhibit pathological features similar to HHCY patients, including endothelial dysfunction and hepatic steatosis. Here we show abnormal lipid metabolism in CBS(-/-) mice. Triglyceride and nonesterified fatty acid levels were markedly elevated in CBS(-/-) mouse liver and serum. The activity of thiolase, a key enzyme in beta-oxidation of fatty acids, was significantly impaired in CBS(-/-) mouse liver. Hepatic apolipoprotein B100 levels were decreased, whereas serum apolipoprotein B100 and very low density lipoprotein levels were elevated in CBS(-/-) mice. Serum levels of cholesterol/phospholipid in high density lipoprotein fractions but not of total cholesterol/phospholipid were decreased, and the activity of lecithin-cholesterol acyltransferase was severely impaired in CBS(-/-) mice. Abnormal high density lipoprotein particles with higher mobility in polyacrylamide gel electrophoresis were observed in serum obtained from CBS(-/-) mice. Moreover, serum cholesterol/triglyceride distribution in lipoprotein fractions was altered in CBS(-/-) mice. These results suggest that hepatic steatosis in CBS(-/-) mice is caused by or associated with abnormal lipid metabolism.
Highlights
Hyperhomocysteinemia (HHCY) is a consequence of impaired methionine/cysteine metabolism and is caused by deficiency of vitamins and/or enzymes such as cystathionine -synthase (CBS)
Atherosclerosis lesions are not observed in CBSϪ/Ϫ mice, a recent study has shown that the atherosclerosis was accelerated in CBS/apolipoprotein E doubledeficient mice compared with apolipoprotein E-deficient mice [16]
We report abnormal lipid metabolism in CBSϪ/Ϫ mouse liver and serum (e.g. decreased enzyme activities of hepatic thiolase and serum lecithin-cholesterol acyltransferase (LCAT), increased levels of hepatic and serum triglyceride (TG), and defective HDL maturation), which may account for the development of hepatic steatosis in the mutant mice, an animal model for severe HHCY
Summary
Northern Blot Analysis—Total RNA was isolated from various tissues of an 8-week-old C57BL/6 male mouse using TRIzol (Invitrogen), and 10 g of each RNA sample was separated on a 6% formaldehyde, 1% agarose gel. After the separated RNA was transferred to a Hybond-XL nylon membrane (Amersham Biosciences), the hybridization was performed as described previously [18]. Western Blot Analysis—Tissues were quickly removed from an 8-week-old C57BL/6 male, homogenized with a Teflon tissue grinder in ice-cold buffer (100 mM sodium phosphate, pH 7.8, and 1 mM phenylmethylsulfonyl fluoride), and sonicated with the Sonifier 450 (Branson Ultrasonics, Danbury, CT). The mouse CBS protein was detected with an anti-rat CBS polyclonal antibody (1:1000 dilution), a horseradish peroxidase-labeled anti-rabbit IgG antibody, and the ECL Western blotting system (Amersham Biosciences)
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