Effects of betaine in a murine model of mild cystathionine-β-synthase deficiency

An elevated plasma concentration of homocysteine, a sulfur-containing amino acid derived from methionine, has been recognized as an independent risk factor for the development of vascular disease (Kang et aL, 1992). Methionine is adenylated by methionine adenosyltransferase to form S-adenosylmethionine, an important biological methyl donor. Numerous methyltransferases catalyze the transfer of a methyl group from S-adenosylmethionine to a methyl acceptor, producing a methylated product and S- adenosylhomocysteine which is subsequently hydrolysed to form adenosine and homocysteine. Homocysteine has several possible fates: 1) remethylation to form methionine via either the cobalamin-dependent methionine synthase (using N⁵-methyltetrahydrofolate as a methyl donor) or betaine:homocysteine methyltransferase (using betaine as a methyl donor); 2) catabolism by the transsulfuration pathway, ultimately forming cysteine; 3) export to the extracellular space. Two vitamin B₆-dependent enzymes comprise the transsulfuration pathway: cystathionine β-synthase, which condenses homocysteine with serine to form cystathionine, and cystathionine y-lyase, which cleaves cystathionine to cysteine, NH₄⁺ and α-ketobutyrate. -- Altered flux through the remethylation or transsulfuration pathways as a result of genetic mutations or impaired vitamin status has been shown to affect plasma homocysteine levels (Ubbink et al, 1993; Kluijtmans et al., 1999). In recent years it has also become apparent that certain hormones can affect homocysteine metabolism. It has been shown that hypothyroid patients tend to have elevated plasma homocysteine and that these levels are normalized when thyroid levels are restored by thyroxine treatment (Nedrebo et aL, 1998; Hussein et al, 1999). Altered homocysteine metabolism has been observed in diabetes mellitus. Diabetic patients (Types 1 and 2) with signs of kidney dysfunction (i.e. elevated creatinine levels) tend to have increased plasma homocysteine (Hultberg et al, 1991). However in the absence of renal dysfunction, Type 1 diabetic patients exhibit decreased plasma homocysteine relative to normal subjects (Robillon et al., 1994). In this thesis we investigated the hormonal regulation of homocysteine metabolism in rats in hope that we could illuminate a possible mechanism for altered plasma homocysteine levels in human hypothyroidism and Type 1 diabetes mellitus. -- In Chapter 3, hypothyroidism was induced in one study by addition of propythiouracil (PTU) to the drinking water for 2 weeks. In a second study, thyroidectomized and sham-operated rats were used with thyroid hormone replacement via mini-osmotic pumps. Unlike the human hypothyroid patients, both groups of hypothyroid rats exhibited decreased total plasma homocysteine (30% in PTU rats, 50% in thyroidectomized rats) versus their respective controls. Thyroid replacement normalized homocysteine levels in the thyroidectomized rat. Increased activities of the hepatic trans-sulfuration enzymes were found in both models of hypothryoidism. These results provide a possible explanation for the decreased plasma homocysteine concentrations. The hypothyroid rat cannot be used as a model to study homocysteine metabolism in hypothyroid patients. -- The purpose of our second study (Chapter 4) was to investigate homocysteine metabolism in a type 1 diabetic animal model and to examine whether insulin plays a role in its regulation. Diabetes was induced by intravenous administration of streptozotocin (100 mg/kg) to rats. Depending on the experiment, we observed a 30-70 % reduction in plasma homocysteine in the untreated diabetic rat. Treatment with insulin of the diabetic rat raised plasma homocysteine concentrations. Transsulfuration and remethylation enzymes were measured in both liver and kidney. We observed an increase in the activities of the hepatic transsulfuration enzymes (cystathionine β-synthase and cystathionine y-lyase) in the untreated diabetic rat. Insulin treatment normalized the activities of these enzymes. The renal activities of these enzymes were unchanged as was the proportion of plasma homocysteine metabolized by the kidney. These results suggest that insulin is involved in the regulation of plasma homocysteine concentrations by affecting the hepatic transsulfuration pathway. -- The increased hepatic cystathionine β-synthase activity in the untreated-diabetic rat was associated with elevated mRNA levels. Similar to its activity, cystathionine β-synthase mRNA levels were reduced by insulin administration. To further investigate the regulation of cystathionine β-synthase we incubated rat hepatoma cells (H4IIE) with various hormones. Cystathionine β-synthase mRNA, protein, and activity were induced in triamcinolone stimulated H4IIE cells. This induction was prevented by insulin incubation. CPT-cAMP, an analogue of cAMP, also induces cystathionine β-synthase mRNA levels in cultured cells. Co-incubation of insulin with CPT-cAMP prevents any increase in mRNA levels. These experiments provide evidence of the direct regulation of cystathionine β-synthase by insulin and its counter-regulatory hormones. -- Given the broad regulatory effects of glucagon on amino acid metabolism and the fact that plasma glucagon levels are often elevated in Type 1 diabetes we investigated the effect of glucagon on homocysteine metabolism in the rat (Chapter 5). Male Sprague Dawley rats were treated with glucagon (4 mg/kg/day in three divided doses) for 2 days while control rats received vehicle injections. Glucagon treatment resulted in a 30% decrease in total plasma homocysteine and increased hepatic activities of glycine N-methyltransferase, cystathionine β-synthase and cystathionine y-lyase. Enzyme activities of the remethylation pathway were unaffected. The 90% elevation in activity of cystathionine β-synthase was accompanied by a two-fold increase in its mRNA level. Hepatocytes prepared from glucagon-injected rats exported less homocysteine, when incubated with methionine, than did hepatocytes of saline-treated rats. Flux through cystathionine β-synthase was increased five-fold in hepatocytes isolated from glucagon-treated rats as determined by production of ¹⁴CO₂ and 1-¹⁴C-α-ketobutyrate from L-[1-¹⁴C]methionine. Methionine transport was elevated two-fold in hepatocytes isolated from glucagon-treated rats resulting in increased hepatic methionine levels. Hepatic concentrations of S-adenosylmethionine and S-adenosylhomocysteine, allosteric activators of cystathionine β-synthase, were also increased following glucagon treatment. These results indicate that glucagon can regulate plasma homocysteine through its effects on the hepatic transsulfuration pathway. -- This thesis provides clear evidence that glucagon lowers plasma homocysteine while insulin has the opposite effect. The mechanism for this reciprocal regulation has been outlined in detail. Taken together, it is clear that these metabolic hormones can be very important in controlling plasma homocysteine levels and that the liver is the site of this hormonal regulation.

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Disordered methionine/homocysteine metabolism in premature vascular disease. Its occurrence, cofactor therapy, and enzymology.

Arteriosclerosis and Thrombosis A Journal of Vascular Biology (1993)

Nicholas P.B. Dudman D. E. L. Wilcken Jun Wang Judith F Lynch D.J. Macey

Mild homocysteinemia occurs surprisingly often in patients with premature vascular disease. We studied the possible enzymatic sources of this mild hyperhomocysteinemia and the control of homocysteine levels in plasma by treatment of patients with the cofactors and cosubstrates of homocysteine catabolism. We assessed homocysteine metabolism in 131 patients who had premature disease in their coronary, peripheral, or cerebrovascular circulation by using a standard oral methionine-load test. Impaired homocysteine metabolism occurred in 28 patients. We assayed levels of the primary enzymes of homocysteine catabolism in cultured skin fibroblast extracts from 15 of these 28 patients. The patients' cystathionine beta-synthase levels (3.68 +/- 2.52 nmol/h per milligram of cell protein, mean +/- SD) were markedly depressed compared with those from 31 healthy adult control subjects (7.61 +/- 4.49, P < .001). The patients' levels of 5-methyltetrahydrofolate: homocysteine methyltransferase were normal. While betaine: homocysteine methyltransferase was not expressed in skin fibroblasts, 24-hour urinary betaine and N,N-dimethylglycine measurements were consistent with normal or enhanced remethylation of homocysteine by betaine: homocysteine methyltransferase in the 13 patients tested. When treated daily with choline and betaine, pyridoxine, or folic acid, there was a normalization of the postmethionine plasma homocysteine level in 16 of 19 patients. Our results indicate that mild homocysteinemia in premature vascular disease may be caused by either a folate deficiency or deficiencies in cystathionine beta-synthase activity. It does not necessarily involve deficiencies of either 5-methyltetrahydrofolate:homocysteine methyltransferase or betaine:homocysteine methyltransferase. Effective treatment regimens are also defined.

Homocystinuria

Betaine

Methionine synthase

Hyperhomocysteinemia

Choline

Transsulfuration

Transmethylation

10.1161/01.atv.13.9.1253

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Citations (209)

Metabolic regulatory properties of S-adenosylmethionine and S-adenosylhomocysteine

Clinical Chemistry and Laboratory Medicine (CCLM) (2007)

James D. Finkelstein

Abstract In mammalian liver, two intersecting pathways, remethylation and transsulfuration, compete for homocysteine that has been formed from methionine. Remethylation of homocysteine, employing either methyltetrahydrofolate or betaine as the methyl donor, forms a methionine cycle that functions to conserve methionine. In contrast, the transsulfuration sequence – cystathionine synthase and cystathionase – serves to irreversibly catabolize the homocysteine while synthesizing cysteine. The rate of homocysteine formation and its distribution between these two pathways are the sites for metabolic regulation and coordination. The mechanisms for regulation include both the tissue content and the kinetic properties of the component enzymes as well as the concentrations of their substrates and other metabolic effectors. Adenosylmethionine and adenosylhomocysteine are important regulatory metabolites and may use one or more mechanisms to affect the enzymes. Adenosylmethionine is a positive effector of its own synthesis, cystathionine synthase and glycine methyltransferase but impairs both homocysteine methylases. Thus, the concentration of adenosylmethionine may be self-regulatory in mammalian liver. By means of other enzymatic mechanisms, the hepatic concentration of adenosylhomocysteine, an index of homocysteine accumulation, is also self-regulated. These considerations pertain primarily to liver, which has the unique capacity to synthesize more adenosylmethionine in the presence of excess methionine. However, there are organ-specific patterns of methionine metabolism and its regulation. All tissues possess the methionine cycle with methyltetrahydrofolate as the methyl donor but only liver, kidney, pancreas, intestine and brain also contain the transsulfuration pathway. The limitation of adenosylmethionine concentrations may make adenosylhomocysteine a more significant metabolic regulator in extrahepatic tissues. However, estimates of regulatory changes based on determinations of the plasma concentrations of the two metabolites are of limited value and must be used with caution. In addition, the recent description of “cystathionine (CBS) domains” in proteins not involved with methionine metabolism raises the possibility that abnormal concentrations of the adenosyl metabolites may impact on other metabolic pathways. Clin Chem Lab Med 2007;45:1694–9.

Transsulfuration

Methionine synthase

Transmethylation

Methionine Adenosyltransferase

Cystathionine gamma-lyase

Metabolic pathway

Betaine

Catabolism

Cysteine Metabolism

10.1515/cclm.2007.341

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Citations (184)

Colloquium: Homocyst(e)ine, Vitamins and Arterial Occlusive Diseases Human Homocysteine Catabolismi Three Major Pathways and Their Relevance to Development of Arterial Occlusive Disease1'2

Nicholas P. B. DÃœDMAN Ross B. Gordon Paul A. DAWSOtf David E.L. Wilcken

Two separate metabolic pathways that methylate homocysteine to methionine are known in humans, utilizing, respectively, 5-methyltetrahydrofo- late and betaine as methyl donors. Deficiency of the folate-dependent methylation system is linked to hyper- homocysteinemia. Our data suggest that this deficiency leads to concurrent metabolic down-regulation of ho mocysteine transsulfuration that may contribute to hyp- erhomocysteinemia. By contrast, no instances have been reported of hyperhomocysteinemia resulting from deficiencies of betaine-dependent homocysteine meth ylation. Long-term betaine supplementation of 10 pa tients, who had pyridoxine-resistant homocystinuria and gross hyperhomocysteinemia due to deficiency of cystathionine 0-synthase activity, caused a substantial lowering of plasma homocysteine, which has now been maintained for periods of up to 13 years. Betaine had to be taken regularly because the effect soon disappeared when treatment was stopped. In conclusion, depressed activity of the transsulfuration pathway may contribute to hyperhomocysteinemia because of primarydeficien cies of enzymes of either the transsulfuration or of the folate-dependent methylation pathways. Stimulation of betaine-dependent homocysteine remethylation causes a commensurate decrease in plasma homocysteine that can be maintained as long as betaine is taken. J. Nutr. 126: 1295S-1300S, 1996.

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Hyperhomocysteinemia and Risk of Atherosclerosis, Cardiovascular Disease and Cancer: A Concise Update

Springer eBooks (2021)

Varna Taranikanti

Transmethylation

Methionine synthase

Hyperhomocysteinemia

Transsulfuration

Betaine

Homocystinuria

10.1007/978-3-030-57839-8_12

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Mice deficient in cystathionine beta synthase display altered homocysteine remethylation pathway

Molecular Genetics and Metabolism (2007)

Jean‐Marc Alberto Julien Hamelet Christophe Noll Sébastien Blaise Jean‐Pierre Bronowicki

Homocystinuria

Hyperhomocysteinemia

Methionine synthase

Betaine

10.1016/j.ymgme.2007.05.002

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Citations (11)

Homocysteine: Overview of Biochemistry, Molecular Biology, and Role in Disease Processes

Seminars in Vascular Medicine (2005)

Brian Fowler

Homocysteine is derived from the essential amino acid methionine and plays a vital role in cellular homeostasis in man. Homocysteine levels depend on its synthesis, involving methionine adenosyltransferase, S-adenosylmethionine-dependent methyltransferases such as glycine N-methyltransferase, and S-adenosylhomocysteine hydrolase; its remethylation to methionine by methionine synthase, which requires methionine synthase reductase, vitamin B12, and 5-methyltetrahydrofolate produced by methylenetetrahydrofolate reductase or betaine methyltransferase; and its degradation by transsulfuration involving cystathionine β-synthase. The control of homocysteine metabolism involves changes of tissue content or inherent kinetic properties of the enzymes. In particular, S-adenosylmethionine acts as a switch between remethylation and transsulfuration through its allosteric inhibition of methylenetetrahydrofolate reductase and activation of cystathionine β-synthase. Mutant alleles of genes for these enzymes can lead to severe loss of function and varying severity of disease. Several defects lead to severe hyperhomocysteinemia, the most common form being cystathionine β-synthase deficiency, with more than a hundred reported mutations. Less severe elevations of plasma homocysteine are caused by folate and vitamin B12 deficiency, and renal disease and moderate hyperhomocysteinemia are associated with several common disease states such as cardiovascular disease. Homocysteine toxicity is likely direct or caused by disturbed levels of associated metabolites; for example, methylation reactions through elevated S-adenosylhomocysteine.

Methionine synthase

Transsulfuration

Homocystinuria

Hyperhomocysteinemia

Methionine Adenosyltransferase

Cystathionine gamma-lyase

10.1055/s-2005-872394

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Citations (150)

Diet and Hyperhomocysteinemia Prevention

Springer eBooks (2021)

Farha Al Naabi