Hyperglucagonemia in rats results in decreased plasma homocysteine and increased flux through the transsulfuration pathway in liver

An elevated plasma level of homocysteine is a risk factor for the development of cardiovascular disease. The purpose of this study was to investigate the effect of glucagon on homocysteine metabolism in the rat. Male Sprague-Dawley rats were treated with 4 mg/kg/day (3 injections per day) glucagon 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 beta-synthase, and cystathionine gamma-lyase. Enzyme activities of the remethylation pathway were unaffected. The 90% elevation in activity of cystathionine beta-synthase was accompanied by a 2-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 beta-synthase was increased 5-fold in hepatocytes isolated from glucagon-treated rats as determined by production of (14)CO(2) and alpha-[1-(14)C]ketobutyrate from l-[1-(14)C]methionine. Methionine transport was elevated 2-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 beta-synthase, were also increased following glucagon treatment. These results indicate that glucagon can regulate plasma homocysteine through its effects on the hepatic transsulfuration pathway.     

Suppression of methionine-induced hyperhomocysteinemia by glycine and serine in rats

The hyperhomocysteinemia induced by a dietary addition of 1% methionine was significantly suppressed by the concurrent addition of 1% glycine or 1.4% serine to the same degree. The methionine-induced increase in the hepatic concentration of methionine metabolites was significantly suppressed by glycine and serine, but the hepatic cystathionine beta-synthase activity was not enhanced by these amino acids. When the methionine-supplemented diet was changed to the methionine plus glycine or serine diet, the plasma homocysteine concentration rapidly decreased during and after the first day. The hyperhomocysteinemia induced by an intraperitoneal injection with methionine was also suppressed by concurrent injection with glycine or serine, although the effect of serine was significantly greater than that of glycine. These results indicate that glycine and serine were effective for suppressing methionine-induced hyperhomocysteinemia: serine and its precursor glycine are considered to have elicited their effects mainly by stimulating cystathionine synthesis by supplying serine, another substrate for cystathionine synthesis.     

Increased plasma homocysteine concentration in rats from a low casein diet

Rats were fed on a 10% casein (10C) diet, 30% casein (30C) diet, 10C+0.5% methionine diet, or 30C+0.5% methionine diet for 14 d to investigate the relationship between the dietary protein level and plasma homocysteine concentration. The plasma homocysteine concentration was significantly higher in the rats fed on the 10C diet than in the rats fed on the 30C diet, and this phenomenon persisted even under the condition of methionine supplementation. The activity of hepatic cystathionine beta-synthase (CBS) was significantly lower in the rats fed on the 10% casein diets than in the rats fed on the 30% casein diets, irrespective of methionine supplementation. This is the first demonstration of a low-protein diet increasing the plasma homocysteine concentration in experimental animals. It is suggested that the decreased CBS activity might be associated, at least in part, with the hyperhomocysteinemia caused by the low-casein diet.     

Effect of dietary soybean protein level on the plasma homocysteine concentration in rats

There was an inverse correlation between the plasma homocysteine concentration and dietary protein level or protein intake when a soybean protein isolate (SPI) was used as a protein source for rats. The hepatic cystathionine beta-synthase activity increased in response to the dietary SPI level. The results suggest that a high-protein diet might be an effective means to lower the plasma homocysteine concentration, probably through enhancement of the homocysteine-metabolizing activity.     

High-Casein Diet Suppresses Guanidinoacetic Acid-Induced Hyperhomocysteinemia and Potentiates the Hypohomocysteinemic Effect of Serine in Rats

To determine the effect of dietary protein level on experimental hyperhomocysteinemia, rats were fed 10% casein (10C) and 40% casein (40C) diets with or without 0.5% guanidinoacetic acid (GAA) for 14 d. In addition, rats were fed 10C + 0.75% methionine (10CM) and 40C + 0.75% methionine (40CM) diets with or without 2.5% serine for 14 d to determine the relationship between the dietary protein level and intensity of the hypohomocysteinemic effect of serine. GAA supplementation markedly increased the plasma homocysteine concentration in rats fed with the 10C diet, whereas it did not increase the plasma homocysteine concentration in rats fed with the 40C diet. Although serine supplementation significantly suppressed the methionine-induced enhancement of plasma homocysteine concentration, the decreased plasma homocysteine concentration was significantly lower in rats fed with the 40CM diet than in rats fed with the 10CM diet. The hepatic cystathionine β-synthase and betaine-homocysteine S-methyltransferase activities were significantly higher in rats fed with the 40C or 40CM diet than in rats fed with the 10C or 10CM diet, irrespective of supplementation with GAA and serine. These results indicate that the high-casein diet was effective for both suppressing GAA-induced hyperhomocysteinemia and potentiating the hypohomocysteinemic effect of serine, probably through the enhanced activity of homocysteine-metabolizing enzymes.     

Cysteine supplementation decreases plasma homocysteine concentration in rats fed on a low-casein diet in rats

The effect of dietary supplementation with cysteine on the plasma homocysteine concentration was investigated in rats fed on 10% casein (10C) and 30% casein (30C) diets. The 10C diet significantly increased the plasma homocysteine concentration as compared with the 30C diet. The hyperhomocysteinemia induced by the 10C diet was significantly suppressed by cysteine supplementation even at a 0.3% level, whereas cysteine did not decrease the plasma homocysteine concentration when added to the 30C diet. In contrast, 0.3% methionine supplementation of the 10C diet tended to increase the plasma homocysteine concentration. Cysteine supplementation to rats fed on the 10C diet did not alter the plasma cysteine concentration and the hepatic activities of cystathionine beta-synthase and betaine:homocysteine S-methyltransferase, whereas it significantly decreased the hepatic concentrations of S-adenosylmethionine and betaine. These results suggest that cysteine supplementation might be effective for suppressing the hyperhomocysteinemia induced by a low-protein diet.     

Modulation of methyl group metabolism by streptozotocin-induced diabetes and all-trans-retinoic acid

The hepatic enzyme glycine N-methyltransferase (GNMT) plays a major role in the control of methyl group and homocysteine metabolism. Because disruption of these vital pathways is associated with numerous pathologies, understanding GNMT control is important for evaluating methyl group regulation. Recently, gluconeogenic conditions have been shown to modulate homocysteine metabolism and treatment with glucocorticoids and/or all-trans-retinoic acid (RA)-induced active GNMT protein, thereby leading to methyl group loss. This study was conducted to determine the effect of diabetes, alone and in combination with RA, on GNMT regulation. Diabetes and RA increased GNMT activity 87 and 148%, respectively. Moreover, the induction of GNMT activity by diabetes and RA was reflected in its abundance. Cell culture studies demonstrated that pretreatment with insulin prevented GNMT induction by both RA and dexamethasone. There was a significant decline in homocysteine concentrations in diabetic rats, owing in part to a 38% increase in the abundance of the transsulfuration enzyme cystathionine beta-synthase; treatment of diabetic rats with RA prevented cystathionine beta-synthase induction. A diabetic state also increased the activity of the folate-independent homocysteine remethylation enzyme betaine-homocysteine S-methyltransferase, whereas the activity of the folate-dependent enzyme methionine synthase was diminished 52%. In contrast, RA treatment attenuated the streptozotocin-mediated increase in betaine-homocysteine S-methyltransferase, whereas methionine synthase activity remained diminished. These results indicate that both a diabetic condition and RA treatment have marked effects on the metabolism of methyl groups and homocysteine, a finding that may have significant implications for diabetics and their potential sensitivity to retinoids.     

Folate and homocysteine metabolism in copper-deficient rats.

To investigate the effect of copper deficiency on folate and homocysteine metabolism, we measured plasma, red-cell and hepatic folate, plasma homocysteine and vitamin B-12 concentrations, and hepatic methionine synthase activities in rats. Two groups of male Sprague-Dawley rats were fed semi-purified diets containing either 0. 1 mg (copper-deficient group) or 9.2 mg (control group) of copper per kg. After 6 weeks of dietary treatment, copper deficiency was established as evidenced by markedly decreased plasma and hepatic copper concentrations in rats fed the low-copper diet. Plasma, red-cell, hepatic folate, and plasma vitamin B-12 concentrations were similar in both groups, whereas plasma homocysteine concentrations in the copper-deficient group were significantly higher than in the control group (P<0.05). Copper deficiency resulted in a 21% reduction in hepatic methionine synthase activity as compared to the control group (P<0.01). This change most likely caused the increased hepatic 5-methyltetrahydrofolate and plasma homocysteine concentrations in the copper-deficient group. Our results indicate that hepatic methionine synthase may be a cuproenzyme, and plasma homocysteine concentrations are influenced by copper nutriture in rats. These data support the concept that copper deficiency can be a risk factor for cardiovascular disease.     

Hyperhomocysteinemia induced by guanidinoacetic acid is effectively suppressed by choline and betaine in rats

Rats were fed 25% casein (25C) diets differing in choline levels (0-0.5%) with and without 0.5% guanidinoacetic acid (GAA) or 0.75% L-methionine for 7 d to determine the effects of dietary choline level on experimental hyperhomocysteinemia. The effects of dietary choline (0.30%) and betaine (0.34%) on GAA- and methionine-induced hyperhomocysteinemia were also compared. Dietary choline suppressed hyperhomocysteinemia induced by GAA, but not by methionine, in a dose-dependent manner. GAA-induced enhancement of the plasma homocysteine concentration was suppressed by choline and betaine to the same degree, but the effects of these compounds were relatively small on methionine-induced hyperhomocysteinemia. Dietary supplementation with choline and betaine significantly increased the hepatic betaine concentration in rats fed a GAA diet, but not in rats fed a methionine diet. These results indicate that choline and betaine are effective at relatively low levels in reducing plasma homocysteine, especially under the condition of betaine deficiency without a loading of homocysteine precursor.     

Homocysteine metabolism in children with Down syndrome: in vitro modulation

The gene for cystathionine beta-synthase (CBS) is located on chromosome 21 and is overexpressed in children with Down syndrome (DS), or trisomy 21. The dual purpose of the present study was to evaluate the impact of overexpression of the CBS gene on homocysteine metabolism in children with DS and to determine whether the supplementation of trisomy 21 lymphoblasts in vitro with selected nutrients would shift the genetically induced metabolic imbalance. Plasma samples were obtained from 42 children with karyotypically confirmed full trisomy 21 and from 36 normal siblings (mean age 7.4 years). Metabolites involved in homocysteine metabolism were measured and compared to those of normal siblings used as controls. Lymphocyte DNA methylation status was determined as a functional endpoint. The results indicated that plasma levels of homocysteine, methionine, S-adenosylhomocysteine, and S-adenosylmethionine were all significantly decreased in children with DS and that their lymphocyte DNA was hypermethylated relative to that in normal siblings. Plasma levels of cystathionine and cysteine were significantly increased, consistent with an increase in CBS activity. Plasma glutathione levels were significantly reduced in the children with DS and may reflect an increase in oxidative stress due to the overexpression of the superoxide dismutase gene, also located on chromosome 21. The addition of methionine, folinic acid, methyl-B(12), thymidine, or dimethylglycine to the cultured trisomy 21 lymphoblastoid cells improved the metabolic profile in vitro. The increased activity of CBS in children with DS significantly alters homocysteine metabolism such that the folate-dependent resynthesis of methionine is compromised. The decreased availability of homocysteine promotes the well-established "folate trap," creating a functional folate deficiency that may contribute to the metabolic pathology of this complex genetic disorder.     

Increased Plasma Homocysteine Concentration in Rats from a Low Casein Diet

Rats were fed on a 10% casein (10C) diet, 30% casein (30C) diet, 10C+0.5% methionine diet, or 30C+0.5% methionine diet for 14 d to investigate the relationship between the dietary protein level and plasma homocysteine concentration. The plasma homocysteine concentration was significantly higher in the rats fed on the 10C diet than in the rats fed on the 30C diet, and this phenomenon persisted even under the condition of methionine supplementation. The activity of hepatic cystathionine β-synthase (CBS) was significantly lower in the rats fed on the 10% casein diets than in the rats fed on the 30% casein diets, irrespective of methionine supplementation. This is the first demonstration of a low-protein diet increasing the plasma homocysteine concentration in experimental animals. It is suggested that the decreased CBS activity might be associated, at least in part, with the hyperhomocysteinemia caused by the low-casein diet.     

Methionine metabolism in mammals. Adaptation to methionine excess

We conducted a systematic evaluation of the effects of increasing levels of dietary methionine on the metabolites and enzymes of methionine metabolism in rat liver. Significant decreases in hepatic concentrations of betaine and serine occurred when the dietary methionine was raised from 0.3 to 1.0%. We observed increased concentrations of S-adenosylhomocysteine in livers of rats fed 1.5% methionine and of S-adenosylmethionine and methionine only when the diet contained 3.0% methionine. Methionine supplementation resulted in decreased hepatic levels of methyltetrahydrofolate-homocysteine methyltransferase and increased levels of methionine adenosyltransferase, betaine-homocysteine methyltransferase, and cystathionine synthase. We used these data to simulate the regulatory locus formed by the enzymes which metabolize homocysteine in livers of rats fed 0.3% methionine, 1.5% methionine, and 3.0% methionine. In comparison to the model for the 0.3% methionine diet group, the model for the 3.0% methionine animals demonstrates a 12-fold increase in the synthesis of cystathionine, a 150% increase in flow through the betaine reaction, and a 550% increase in total metabolism of homocysteine. The concentrations of substrates and other metabolites are significant determinants of this apparent adaptation.     

Effect of regulatory mutations of sulphur metabolism on the levels of cysteine- and homocysteine-synthesizing enzymes in Neurospora crassa

1. Regulation of four enzymes involved in cysteine and homocysteine synthesis, i.e. cysteine synthase (EC 4.2.99.8), homocysteine synthase (EC 4.1.99.10), cystathionine beta-synthase (EC 2.1.22) and gamma-cystathionase (EC 4.4.1.1) was studied in the wild type and sulphur regulatory mutants of Neurospora crassa. 2. Homocysteine synthase and cystathionine beta-synthase were found to be regulatory enzymes but only the former is under control of the cys-3 - scon system regulating several enzymes of sulphur metabolism, including gamma-cystathionase. 3. The results obtained with the mutants strongly suggest that homocysteine synthase plays a physiological role as an enzyme of the alternative pathway of methionine synthesis. Cysteine synthase activity was similar in all strains examined irrespective of growth conditions. 4. The sconc strain with derepressed enzymes of sulphur metabolism showed an increased pool of sulphur amino acids, except for methionine. Particularly characteristic for this pool is a high content of hypotaurine, a product of cysteine catabolism.     

Effect of D,L-propargylglycine on cystathionine metabolism in rats

Hepatic gamma-cystathionase activity at 12 h after the intraperitoneal injection decreased in proportion to the amount of D,L-propargylglycine administered, but hepatic cystathionine beta-synthase activity did not change. Contents of cystathionine in the liver increased gradually from 0.25 mg to 30 mg/200 g body weight in proportion to the amounts of D,L-propargylglycine injected; in the kidney, 0.5 mg to 10 mg; in the brain, 5 mg to 20 mg; in the serum, 0.25 mg to 30 mg. Contents of N-acetylcystathionine in the liver and kidney also increased in parallel with the accumulation of cystathionine.     

Cystathionine Accumulation in Various Regions of Brain of DL-Propargylglycine-Treated Rats

The contents of cystathionine and taurine, as well as cystathionine beta-synthase activity in various regions of the brains of normal and DL-propargylglycine-treated rats, were measured. The content of cystathionine in each region of brain increased gradually from 0.5 mg to 20 mg/200 g body weight in relation to the dose of DL-propargylglycine. Cystathionine was found to be unevenly distributed in brains of both normal and DL-propargylglycine-treated rats. On the other hand, the activity of cystathionine beta-synthase was evenly distributed in various regions of normal rat brain, and was unaltered following treatment of rats with DL-propargylglycine. The concentration of taurine was similarly unaffected by DL-propargylglycine injection.     

Unusual metabolism of sulfur-containing amino acids in rats treated with DL-propargylglycine

S-(2-Hydroxy-2-carboxyethyl)homocysteine, S-(3-hydroxy-3-carboxy-n-propyl)-cysteine, N-acylated S-(beta-carboxyethyl)cysteine, and N-acylated S-(3-hydroxy-3-carboxy-n-propyl) cysteine were excreted in the urine after DL-propargylglycine treatment. Cystathionine was also accumulated in several tissues of DL-propargylglycine-treated rats. N-Monoacetylcystathione was found in the liver of rats and was also detected in the kidney and serum. Cystathionine gamma-lyase activity in liver decreased to about 4% of that of control rats 24 h after the DL-propargylglycine injection, and alanine aminotransferase activity decreased to about 35% of that of control rats. On the other hand, aspartate aminotransferase and cystathionine beta-synthese activity did not show significant changes from those of control rats. The ability of normal tissues to synthesize cystathionine utilizing cystathionine beta-synthase was 1.98 +/- 0.40 mumol/min/g in liver, 0.61 +/- 0.13 in kidney, and 0.18 +/- 0.015 in brain. The maximal contents of cystathionine in rat tissues and the administered amounts of DL-propargylglycine agreed well with the ability to synthesize cystathionine in each tissue.     

The effect of diet and of methionine loading on activity of enzymes in the transulfuration pathway in sheep

In three experiments, activity of hepatic enzymes associated with metabolism of methionine through the transulfuration pathway were studied with respect to possible effects of diet and methionine infusion per abomasum. In experiment 1 no differences in methionine adenosyltransferase (MAT) or cystathionine lambda-lyase (CGL) were detected between lucerne and wheaten straw diets, or between effects of fasting for 48 h and 96 h after feeding lucrene chaff as opposed to fasting after feeding wheaten straw. Fasting for 96 h resulted in a trend toward increasing CGL and MAT specific activities on both diets. In experiment 2 MAT was depressed significantly by infusion of methionine at 1.4 g/day and to a greater extent by infusion at 4.2 g/day, whilst CGL was not significantly affected. In experiment 3 MAT specific activity decreased significantly in response to both levels of methionine supplementation. Betaine-homocysteine methyltransferase activity was increased by methionine infusion. CGL decreased in all treatments but there was a larger decrease in those animals receiving methionine infusion. No significant changes were observed in relation to other enzymes examined which included cystathionine beta-synthase and threonine dehydratase. These observations are consistent with the hypothesis that in sheep the increase in methionine in blood plasma which occurs when methionine is absorbed in increased amounts may be due to reduced entry into the transulfuration pathway because of a repression of MAT activity.     

Effects of dietary zinc deficiency on homocysteine and folate metabolism in rats

In rats, zinc deficiency has been reported to result in elevated hepatic methionine synthase activity and alterations in folate metabolism. We investigated the effect of zinc deficiency on plasma homocysteine concentrations and the distribution of hepatic folates. Weanling male rats were fed ad libitum a zinc-sufficient control diet (382.0 nmol zinc/g diet), a low-zinc diet (7.5 nmol zinc/g diet), or a control diet pair-fed to the intake of the zinc-deficient rats. After 6 weeks, the body weights of the zinc-deficient and pair-fed control groups were lower than those of controls, and plasma zinc concentrations were lowest in the zinc-deficient group. Plasma homocysteine concentrations in the zinc-deficient group (2.3 +/- 0.2 micromol/L) were significantly lower than those in the ad libitum-fed and pair-fed control groups (6.7 +/- 0.5 and 3.2 +/- 0.4 micromol/L, respectively). Hepatic methionine synthase activity in the zinc-deficient group was higher than in the other two groups. Low mean percentage of 5-methyltetrahydrofolate in total hepatic folates and low plasma folate concentration were observed in the zinc-deficient group compared with the ad libitum-fed and pair-fed control groups. The reduced plasma homocysteine and folate concentrations and reduced percentage of hepatic 5-methyltetrahydrofolate are probably secondary to the increased activity of hepatic methionine synthase in zinc deficiency.     

Contents of sulfur amino acids, and cystathionine beta-synthase and gamma-lyase activities in various tissues from agkistroden blomhoffi (mamushi)

The concentrations of sulfur-containing amino acids, taurine, cystathionine, methionine and cystine, as well as cystathionine beta-synthase and gamma-lyase activities in various tissues of Agkistrodon blomhoffi (mamushi) were measured. The concentration of taurine in examined tissues was greater than the concentration of other sulfur-containing amino acids. The concentration of cystathionine in various tissues was also much higher than those of methionine and cystine, but the concentration of cystathionine in the brain was lower than that of methionine. In all tissues examined in this study, cystathionine beta-synthase activity was much higher than that of cystathionine gamma-lyase. The ratios of cystathionine beta-synthase to gamma-lyase activities in various tissues were 5.6 to approximately 85.6. The concentration of sulfur-containing amino acids in muscle and skin divided into eight portions of the body were also determined. The concentrations of methionine and cystine in each portion of muscle and skin were almost the same, but the concentrations of taurine and cystathionine in each portion of the body were varied.     

Modulatory effect of curcumin on methionine-induced hyperlipidemia and hyperhomocysteinemia in albino rats

The present study was designed to investigate the antioxidant effect of curcumin on methionine-induced hyperlipidemia and hyperhomocysteinemia in Wistar rats (200-250 g) of either sex. The vehicle control rats were treated with 1% Tween 80 in normal saline (2 ml/kg, po) for 30 days. Hyperlipidemia and hyperhomocysteinemia was induced by methionine administration (1 g/kg, po) for 30 days. A significant increase in total cholesterol, triglycerides, low density lipoprotein cholesterol (LDL-C) and homocysteine levels in serum and thiobarbituric acid reactive substances (TBARS) levels in heart homogenates were observed with a concomitant decrease in serum high density lipoprotein (HDL-C) levels in pathogenic control (i.e. group II) rats, as compared to vehicle control (i.e. group I) rats. Further, curcumin (200 mg/kg, p.o.) treatment in methionine treated rats for 30 days significantly decreased the total cholesterol, triglycerides, LDL-C and homocysteine levels in serum and TBARS levels in heart homogenates and increased serum HDL-C levels, as compared to pathogenic control (i.e. group II) rats. The results of biochemical observations were supplemented by histopathological examination of rat's aortic section. The results of test drug were comparable to that obtained with folic acid (100 mg/kg, p.o.). The results suggest that curcumin has significant antihyperlipidemic and antihyperhomocysteinemic effect against methionine-induced hyperlipidemia and hyperhomocysteinemia in rats.