Hormonal regulation of homocysteine metabolism

Jacobs, Rene Lee (2002) Hormonal regulation of homocysteine metabolism. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

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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