Effects of choline deficiency and methotrexate treatment upon rat liver

Choline deficiency and treatment with methotrexate (MTX) both are associated with fatty infiltration of the liver. Choline, methionine, and folate metabolism are interrelated and converge at the regeneration of methionine from homocysteine. MTX perturbs folate metabolism, and it is possible that it also influences choline metabolism. We fed rats a choline deficient diet for 2 weeks and/or treated them with methotrexate (MTX; 0.1 mg/kg daily). Choline deficiency lowered hepatic concentrations of choline (to 43% control), phosphocholine (PCho; to 18% control), glycerophosphocholine (GroPCho; to 46% control), betaine (to 30% control), phosphatidylcholine (PtdCho; to 62% control), methionine (to 80% control), and S-adenosylmethionine (AdoMet; to 57% control), while S-adenosylhomocysteine (AdoHcy) and triacylglycerol concentrations increased (to 126% and 319% control, respectively). MTX treatment alone lowered hepatic concentrations of PCho (to 48% control), GroPCho (to 69% control), betaine (to 55% control), and AdoMet (to 75% control). The addition of MTX treatment to choline deficiency resulted in a larger decrease in AdoMet concentrations (to 75% control) and larger increases in AdoHcy and triacylglycerol concentrations (to 150% and 500% control, respectively) than was observed in choline deficiency alone. Livers from MTX-treated animals used radiolabeled choline to make the same metabolites as did livers from controls (most of the label was converted to PCho and betaine). In choline deficient animals, most of the labeled choline was converted to PtdCho. Therefore, MTX depleted hepatic PCho, GroPCho, and betaine by a mechanism that was different from that of choline deficiency. MTX increased the extent of fatty infiltration of the liver in choline deficient rats, and choline deficiency and MTX treatment damaged hepatocytes as measured by leakage of alanine aminotransferase activity. Our data are consistent with the hypothesis that the fatty infiltration of the liver associated with MTX treatment occurs because of a disturbance in choline metabolism.     

Methyl-group donors cannot prevent apoptotic death of rat hepatocytes induced by choline-deficiency

Choline-deficiency causes liver cells to die by apoptosis, and it has not been clear whether the effects of choline-deficiency are mediated by methyl-deficiency or by lack of choline moieties. SV40 immortalized CWSV-1 hepatocytes were cultivated in media that were choline-sufficient, choline-deficient, choline-deficient with methyl-donors (betaine or methionine), or choline-deficient with extra folate/vitamin B₁₂. Choline-deficient CWSV-1 hepatocytes were not methyl-deficient as they had increased intracellular S-adenosylmethionine concentrations (132% of control; P < 0.01). Despite increased phosphatidylcholine synthesis via sequential methylation of phosphatidylethanolamine, choline-deficient hepatocytes had significantly decreased (P < 0.01) intracellular concentrations of choline (20% of control), phosphocholine (6% of control), glycerophosphocholine (15% of control), and phosphatidylcholine (55% of control). Methyl-supplementation in choline-deficiency enhanced intracellular methyl-group availability, but did not correct choline-deficiency induced abnormalities in either choline metabolite or phospholipid content in hepatocytes. Methyl-supplemented, choline-deficient cells died by apoptosis. In a rat study, 2 weeks of a choline-deficient diet supplemented with betaine did not prevent the occurrence of fatty liver and the increased DNA strand breakage induced by choline-deficiency. Though dietary supplementation with betaine restored hepatic betaine concentration and increased hepatic S-adenosylmethionine/S-adenosylhomocysteine ratio, it did not correct depleted choline (15% of control), phosphocholine (6% control), or phosphatidylcholine (48% of control) concentrations in deficient livers. These data show that decreased intracellular choline and/or choline metabolite concentrations, and not methyl deficiency, are associated with apoptotic death of hepatocytes. J. Cell. Biochem, 64:196–208. © 1997 Wiley-Liss, Inc.     

Role of white adipose lipolysis in the development of NASH induced by methionine- and choline-deficient diet

Methionine- and choline-deficient diet (MCD) is a model for nonalcoholic steatohepatitis (NASH) in rodents. However, the mechanism of NASH development by dietary methionine/choline deficiency remains undetermined. To elucidate the early metabolic changes associated with MCD-NASH, serum metabolomic analysis was performed using mice treated with MCD and control diet for 3 days and 1  week, revealing significant increases in oleic and linoleic acids after MCD treatment. These increases were correlated with reduced body weight and white adipose tissue (WAT) mass, increased phosphorylation of hormone-sensitive lipase, and up-regulation of genes encoding carboxylesterase 3 and β2-adrenergic receptor in WAT, indicating accelerated lipolysis in adipocytes. The changes in serum fatty acids and WAT by MCD treatment were reversed by methionine supplementation, and similar alterations were detected in mice fed a methionine-deficient diet (MD), thus demonstrating that dietary methionine deficiency enhances lipolysis in WAT. MD treatment decreased glucose and increased fibroblast growth factor 21 in serum, thus exhibiting a similar metabolic phenotype as the fasting response. Comparison between MCD and choline-deficient diet (CD) treatments suggested that the addition of MD-induced metabolic alterations, such as WAT lipolysis, to CD-induced hepatic steatosis promotes liver injury. Collectively, these results demonstrate an important role for dietary methionine deficiency and WAT lipolysis in the development of MCD-NASH.     

Choline, an essential nutrient for humans

Choline is required to make essential membrane phospholipids. It is a precursor for the biosynthesis of the neurotransmitter acetylcholine and also is an important source of labile methyl groups. Mammals fed a choline-deficient diet develop liver dysfunction; however, choline is not considered an essential nutrient in humans. Healthy male volunteers were hospitalized and fed a semisynthetic diet devoid of choline supplemented with 500 mg/day choline for 1 wk. Subjects were randomly divided into two groups, one that continued to receive choline (control), and the other that received no choline (deficient) for three additional wk. During the 5th wk of the study all subjects received choline. The semisynthetic diet contained adequate, but no excess, methionine. In the choline-deficient group, plasma choline and phosphatidylcholine concentrations decreased an average of 30% during the 3-wk period when a choline-deficient diet was ingested; plasma and erthrocyte phosphatidylcholine decreased 15%; no such changes occurred in the control group. In the choline-deficient group, serum alanine aminotransferase activity increased steadily from a mean of 0.42 mukat/liter to a mean of 0.62 mukat/liter during the 3-wk period when a choline-deficient diet was ingested; no such change occurred in the control group. Other tests of liver and renal function were unchanged in both groups during the study. Serum cholesterol decreased an average of 15% in the deficient group and did not change in the control group. Healthy humans consuming a choline-deficient diet for 3 wk had depleted stores of choline in tissues and developed signs of incipient liver dysfunction. Our observations support the conclusion and choline is an essential nutrient for humans when excess methionine and folate are not available in the diet.     

Effect of methotrexate on long-chain fatty acid metabolism in liver of rats fed a standard or a defined, choline-deficient diet

The effect of methotrexate on lipids in serum and liver and key enzymes involved in esterification and oxidation of long-chain fatty acids were investigated in rats fed a standard diet and a defined choline-deficient diet. Hepatic metabolism of long-chain fatty acids were also studied in rats fed the defined diet with or without choline. When methotrexate was administered to the rats fed the standard diet there was a slight increase in hepatic lipids and a moderate reduction in the serum level. The palmitoyl-CoA synthetase activity and the microsomal glycerophosphate acyltransferase activity in the liver of rats were increased by methotrexate. The data are consistent with those where the liver may fail to transfer the newly formed triacylglycerols into the plasma with a resultant increase in liver triacylglycerol content and a decrease in serum lipid levels. Fatty liver of methotrexate-exposed rats can not be attributed simply to a reduction of fatty acid oxidation as the carnitine palmitoyltransferase activity was increased. The methotrexate response in the rats fed the defined choline-deficient diet was different. There was a reduction in both serum and hepatic triacylglycerol and the glycerophosphate acyltransferase and palmitoyl-CoA synthetase activities. The carnitine palmitoyltransferase activity was unchanged. Hepatomegaly and increased hepatic fat content, but decreased serum triacylglycerol, total cholesterol and HDL cholesterol were found to be related to the development of choline deficiency as the pleiotropic responses were almost fully prevented by addition of choline to the choline-deficient diet. Addition of choline to the choline-deficient diet normalized the total palmitoyl-CoA synthetase and carnitine palmitoyltransferase activities. In contrast to methotrexate exposure, choline deficiency increased the mitochondrial glycerophosphate acyltransferase activity. The data are consistent with those of where fatty liver induction of choline deficiency may be related to an enhanced esterification of long-chain fatty acids concomitant with a reduction of their oxidation.     

The active synthesis of phosphatidylcholine is required for very low density lipoprotein secretion from rat hepatocytes

Hepatocytes obtained from rats fed a choline-deficient diet for 3 days were cultured in a medium +/- choline (100 microM) or methionine (200 microM). We investigated how choline deficiency affected hepatic lipogenesis, apolipoprotein synthesis, and lipoprotein secretion. The mass of triacylglycerol and phosphatidylcholine secreted was increased about 3-fold and 2-fold, respectively, by the addition of either choline or methionine to the cultured cells. Similarly, a 3-fold stimulation in the secretion of [3H]triacylglycerol and [3H]phosphatidylcholine derived from [3H]oleate was observed after the addition of choline or methionine. Fractionation of secreted lipoproteins by ultracentrifugation revealed that the reduced secretion of triacylglycerol and phosphatidylcholine from choline-deficient cells was mainly due to impaired secretion of very low density lipoproteins (VLDL) (but not high density lipoproteins (HDL)). Fluorography of L-[4,5-3H]leucine-labeled lipoproteins showed a remarkable inhibition of VLDL secretion by choline deficiency. The addition of choline or methionine stimulated the synthesis of phosphatidylcholine and increased the cellular phosphatidylcholine levels to that in normal cells. While there was little effect of choline on the synthesis and amount of cellular phosphatidylethanolamine, the addition of methionine diminished cellular phosphatidylethanolamine levels. Choline deficiency did not change the rate of incorporation of L-[4,5-3H]leucine into cellular VLDL apolipoproteins, nor the rate of disappearance of radioactivity from L-[4,5-3H]leucine-labeled cellular apoB, apoE, and apoC. These results suggest that hepatic secretion of VLDL, but not HDL, requires active phosphatidylcholine biosynthesis. Secondly, the inhibitory effect of choline deficiency on VLDL secretion can be compensated by the methylation of phosphatidylethanolamine.     

S-Adenosyl-l-homocysteine in brain

Administration of methionine sulfoximine (MSO) to rats and mice significantly decreased cerebral levels ofS-adenosyl-l-homocysteine (AdoHcy). Concurrent administration of methionine prevented this decrease and, when methionine was given alone, significantly elevated AdoHcy levels resulted in both species. Regionally, AdoHcy levels varied from 20 nmol/g in rat cerebellum and spinal cord to about 60 nmol/g in hypothalamus and midbrain. MSO decreased AdoHcy in all regions tested except striatum, midbrain, and spinal cord. AdoMet/AdoHcy ratios (methylation index) varied from 0.48 in hypothalamus to 2.4 in cerebellum, and MSO administration decreased these ratios in all regions except hypothalamus. AdoHcy hydrolase activity was lowest in hypothalamus, highest in brainstem and, generally, varied inversely with regional AdoHcy levels. MSO decreased AdoHcy hydrolase activity in all regions except hypothalamus and spinal cord. Cycloleucine administration resulted in significantly decreased levels of mouse brain AdoHcy, whereas the administration of dihydroxyphenylalanine (DOPA) failed to affect AdoHcy levels. It is concluded that (a) cerebral AdoHcy levels are more tightly regulated than are those of AdoMet after MSO administration, (b) slight fluctuations of AdoHcy levels may be important in regulating AdoHcy hydrolase activity and hence AdoHcy catabolism in vivo, (c) the AdoMet/AdoHcy ratio reflects the absolute AdoMet concentration rather than the transmethylation flux, (d) the decreased AdoMet levels in midbrain, cortex, and striatum after MSO with no corresponding decrease in AdoHcy suggest an enhanced AdoMet utilization, hence an increased transmethylation in the MSO preconvulsant state.Supported by USPHS, NINCDS grant NS-06294.     

Deletion of betaine-homocysteine S-methyltransferase in mice perturbs choline and 1-carbon metabolism, resulting in fatty liver and hepatocellular carcinomas

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.     

Disruption of hepatic one-carbon metabolism impairs mitochondrial function and enhances macrophage activity in methionine–choline-deficient mice

One-carbon metabolism is a collection of metabolic cycles that supports methylation and provides one-carbon bound folates for the de novo synthesis of purine and thymidine nucleotides. The methylation of phosphatidylethanolamine to form choline has been extensively studied in the context of fatty liver disease. However, the role of one-carbon metabolism in supporting nucleotide synthesis during liver damage has not been addressed. The objective of this study is to determine how the disruption of one-carbon metabolism influences nucleotide metabolism in the liver after dietary methionine and choline restriction. Mice (n=8) were fed a methionine–choline-deficient or control diet for 3 weeks. We treated mice with the compound alloxazine (0.5 mg/kg), a known adenosine receptor antagonist, every second day during the final week of feeding to probe the function of adenosine signaling during liver damage. We found that concentrations of several hepatic nucleotides were significantly lower in methionine- and choline-deficient mice vs. controls (adenine: 13.9±0.7 vs. 10.1±0.6, guanine: 1.8±0.1 vs. 1.4±0.1, thymidine: 0.0122±0.0027 vs. 0.0059±0.0027 nmol/mg dry tissue). Treatment of alloxazine caused a specific decrease in thymidine nucleotides, decrease in mitochondrial content in the liver and exacerbation of steatohepatitis as shown by the increased hepatic lipid content and altered macrophage morphology. This study demonstrates a role for one-carbon metabolism in supporting de novo nucleotide synthesis and mitochondrial function during liver damage.     

Influence of dietary methionine level on the liver metallothionein mRNA level in rats

The effects of some methyl-containing compounds added to a choline-deficient diet on the metallothionein mRNA level in the rat liver were studied. The addition of choline or carnitine to the choline-deficient diet did not induce a gain in body weight, while the addition of either betaine or methionine to the choline-deficient diet, or of methionine to the choline-deficient diet with choline significantly increased the body weight. The metallothionein mRNA level in the liver of rats fed on the choline-deficient diet was similar to that of rats fed on the choline-deficient diet with choline, betaine or carnitine. However, the addition of methionine to the choline-deficient diet with or without choline caused a marked suppression in the metallothionein mRNA level in the liver. It is thus surmised that the metallothionein mRNA level in the liver might be regulated by the dietary content of methionine.     

Choline cannot be replaced by propanolamine in mice

Choline is an important nutrient for humans and animals. Animals obtain choline from the diet and from the catabolism of phosphatidylcholine made by phosphatidylethanolamine N-methyltransferase (PEMT). The unique model of complete choline deprivation is Pemt(-/-) mice that are fed a choline-deficient diet. This model, therefore, can be used for the examination of choline substitutes in mammalian systems. Recently, propanolamine was found to be a replacement for choline in yeast. Thus, we tested to see whether or not choline can be replaced by propanolamine in mice. Mice were fed a choline-deficient diet and supplemented with either methionine, 2-amino-propanol, 2-amino-isopropanol and 3-amino-propanol. We were unable to detect the formation of any of the possible phosphatidylpropanolamines. Moreover, none of them prevented liver damage, reduction of hepatic phosphatidylcholine levels or fatty liver induced in choline-deficient-Pemt(-/-) mice. These results suggest that choline in mice cannot be replaced by any of the three propanolamine derivatives.     

CHOLINE AND ACETYLCHOLINE IN RATS: EFFECT OF DIETARY CHOLINE

Abstract– The concentration of free choline in peripheral tissues (duodenum, heart, kidney, liver, stomach and plasma) of rats was found to be related to the amount of free choline in the diet. Under steady-state conditions, the concentration of free choline in plasma varied from a minimum of approx 6 nmol/ml (in rats fed a choline-deficient diet) to a maximum value not exceeding 21 nmol/ml. The concentration of plasma choline was elevated above 21 nmol/ml for a short time after parenteral administration of choline chloride or one of its precursors (CDP choline or phosphorylcholine), but was not affected by stress, endocrine manipulations, drug treatments or the time of day when rats were killed. The metabolism of intravenously administered [methyl-³H] choline was accelerated in peripheral tissues (except plasma) of choline-deficient rats, indicating that free choline is not preserved during choline deficiency by a reduction in its rate of turnover. Furthermore, the decrease in concentration of plasma choline that occurred in rats fed a choline-deficient diet was prevented by addition of deanol (dimethylaminoethanol) to the diet. These results indicate that free choline in peripheral tissues of rats is derived from both free choline in the diet, and from precursors of choline present within the diet. In contrast to the effects in peripheral tissues, the concentration of free choline in brain was not reduced by dietary deprivation of free choline; however, the increase in free choline that occurred when rats were decapitated was reduced in brains by deficiency of choline, suggesting a decrease in the concentration of esterified forms of cerebral choline. The concentration of acetylcholine was not reduced in the brain, duodenum, heart, kidney or stomach of 21-week old rats raised from birth on a choline-deficient diet, in the duodenum of rats given a choline-deficient diet for 1, 5 or 11 days, or in brains of rats deprived of free choline for 1 or 11 days. However, the rate of in vivo synthesis of ACh from [methyl-³H]choline was accelerated in cholinergic tissues that were depleted of free choline (i.e. duodenum, heart and stomach).     

Methyl group metabolism in sheep

1. Sheep have a very low intake of methyl nutrients in the post-ruminant state, due to the almost complete degradation of dietary choline by rumen microorganisms, the lack of dietary creatine and the relatively low content of methionine in microbial proteins. 2. Methylneogenesis provides a major source of labile methyl groups in post-ruminant sheep and impairment of the methylneogenesis leads to a marked reduction of the labile methyl pool. 3. S-Adenosylmethionine (AdoMet) metabolism via transmethylation is most active in sheep liver and pancreas and is regulated by the availability of methionine and intracellular ratios of AdoMet to S-adenosylhomocysteine (AdoHcy). 4. Adaptive mechanisms which arise as a consequence of the poor methyl nutrition in post-ruminant sheep are a marked reduction of labile methyl catabolism and an increase in the capacity of methylneogenesis.     

Reduction in VLDL, but not HDL, in plasma of rats deficient in choline

We have analyzed plasma lipoprotein levels in young male rats fed a choline-deficient diet for 3 days. We confirmed previous studies that choline deficiency promotes 6.5-fold accumulation of triacyglycerol in the liver (23.9 +/- 6.0 versus 3.69 +/- 0.92 mumol/g liver) and reduction of triacylglycerol concentration in plasma by 60% (0.17 +/- 0.04 versus 0.46 +/- 0.10 mumol/mL plasma). Agarose gel electrophoresis showed that the plasma very low density lipoprotein (VLDL) levels were reduced in choline-deficient rats, but the concentration of plasma high density lipoproteins (HDL) was not affected. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis of fractionated plasma lipoproteins revealed that the concentrations of apolipoproteins (apo) BH, BL, and E in VLDL from choline-deficient rats were 37.1, 11.0, and 37.2% of normal levels, respectively. In contrast, the amount of apo A-I, the major one in HDL, was almost unchanged. Correspondingly, there were decreased lipid (mainly phosphatidylcholine and triacylglycerol) levels in VLDL from choline-deficient rats, but no change in the levels of phosphatidylcholine, cholesterol, and cholesterol ester in HDL. There were similar levels of apo B and E (components of VLDL) in homogenates of livers from normal and choline-deficient rats, as determined by immunoblotting. These results support the hypothesis that choline deficiency causes reduction of VLDL, but not HDL, levels in plasma as a consequence of impaired hepatic VLDL secretion.     

Effects of methyl-deficient diets on methionine and homocysteine metabolism in the pregnant rat

Although the importance of methyl metabolism in fetal development is well recognized, there is limited information on the dynamics of methionine flow through maternal and fetal tissues and on how this is related to circulating total homocysteine concentrations. Rates of homocysteine remethylation in maternal and fetal tissues on days 11, 19, and 21 of gestation were measured in pregnant rats fed diets with limiting or surplus amounts of folic acid and choline at two levels of methionine and then infused with L-[1-(13)C,(2)H(3)-methyl]methionine. The rate of homocysteine remethylation was highest in maternal liver and declined as gestation progressed. Diets deficient in folic acid and choline reduced the production of methionine from homocysteine in maternal liver only in the animals fed a methionine-limited diet. Throughout gestation, the pancreas exported homocysteine for methylation within other tissues. Little or no methionine cycle activity was detected in the placenta at days 19 and 21 of gestation, but, during this period, fetal tissues, especially the liver, synthesized methionine from homocysteine. Greater enrichment of homocysteine in maternal plasma than placenta, even in animals fed the most-deficient diets, shows that the placenta did not contribute homocysteine to maternal plasma. Methionine synthesis from homocysteine in fetal tissues was maintained or increased when the dams were fed folate- and choline-deficient methionine-restricted diets. This study shows that methyl-deficient diets decrease the remethylation of homocysteine within maternal tissues but that these rates are protected to some extent within fetal tissues.     

Effect of choline deficiency on the enzymes that synthesize phosphatidylcholine and phosphatidylethanolamine in rat liver

Activities have been determined in subcellular fractions of livers from choline-deficient and normals rats for the enzymes that convert choline and ethanolamine to phosphatidylcholine and phosphatidylethanolamine respectively, that methylate phosphatidylethanolamine to yield phosphatidylcholine, and that oxidize choline to betaine. The activities of ethanolamine kinase, phosphoethanolamine cytidylyltransferase, and CDP-ethanolamine: 1,2-diacylglycerol phosphoethanolaminetransferase are not changed in the livers from choline-deficient rats for at least 18 days. Similarly, the activities of choline kinase and CDP-choline: 1,2-diacylglycerol phosphocholine transferase were unaffected by choline depletion. A decrease of 30-41% was observed, however, in the mitochondrial oxidation of choline to betaine. Also, the activity of the phosphocholine cytidylyltransferase was reduced in the choline-deficient livers to 60% olf the control values. The only observed increase in enzyme activity was a 62% elevation of the phosphatidylethanolamine-S-adenosylmethionine methyltransferase activity after 2 days of choline deficiency. This increased activity was maintained for at least 18 days of choline deprivation. The results suggest a lack of adaptive change in the levels of these phospholipid biosynthetic enzymes as a result of choline deficiency.     

The effect of S-adenosyl methionine on the TSH receptor function in human thyroid tissue: increase in binding of TSH and decrease in adenylate cyclase coupling

To determine the effects of S-Adenosyl methionine (AdoMet) on TSH receptor function and adenylate cyclase coupling, human thyroidal crude membrane fraction was pretreated with AdoMet and with S-Adenosyl homocysteine (AdoHcy), separately or in combination. 125I-TSH binding to the pretreated membrane and adenylate cyclase activity of the membrane were examined. In contrast to the reported effect of AdoMet on the decrease in GH binding to lactogenic receptor, AdoMet 0.5 mumoles/ml significantly increased the binding of TSH to the receptor by increasing the affinity of the binding, whereas it decreased the coupling of adenylate cyclase significantly. The effect of AdoMet was partially counteracted by the pretreatment of the membrane with AdoHcy. This effect of AdoMet is very similar to that of diamide previously reported. The result implies that the effect is due to an alteration in the tertiary structure of receptor protein triggered by methylation.     

Diet-dependent survival of protein repair-deficient mice

Protein L-isoaspartyl (D-aspartyl) O-methyltransferase (PCMT1) is a protein-repair enzyme, and mice lacking this enzyme accumulate damaged proteins in multiple tissues, die at an early age from progressive epilepsy and have an increased S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy) ratio in brain tissue. It has been proposed that the alteration of AdoMet and AdoHcy levels might contribute to the seizure phenotype, particularly as AdoHcy has anticonvulsant properties. To investigate whether altered AdoMet and AdoHcy levels might contribute to the seizures and thus the survivability of the repair-deficient mice, a folate-deficient amino acid-based diet was administered to the mice in place of a standard chow diet. We found that the low-folate diet significantly decreases the AdoMet/AdoHcy ratio in brain tissue and results in an almost threefold extension of mean life span in the protein repair-deficient mice. These results indicate that the increased AdoMet/AdoHcy ratio may contribute to the lowered seizure threshold in young PCMT1-deficient mice. However, mean survival was also extended almost twofold for mice on a control folate-replete amino acid-based diet compared to mice on the standard chow diet. Survival after 40 days was similar in the mice on the low- and high-folate amino acid-based diets, suggesting that the survival of older PCMT1-deficient mice is not affected by the higher brain AdoMet/AdoHcy ratio. Additionally, the surviving older repair-deficient mice have a significant increase in body weight when compared to age-matched normal mice, independent of the type of diet. This weight increase was not accompanied by an increase in consumption levels, indicating that the repair-deficient mice may also have an altered metabolic state.     

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.