Protein metabolism in the choline-deficient rat. I. Effect of choline on serum proteins.
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High incidence of hepatocellular carcinomas induced by a choline deficient L-amino acid defined diet in rats.
The carcinogenicities of a choline deficient L-amino acid defined (CDAA) diet and a semipurified choline deficient diet were comparatively examined. A total of 60 male Fischer 344 rats, 6 weeks old, were divided into 5 experimental groups each consisting of 12 rats. Group 1 received the CDAA diet chronically to the end of the 52-week experiment while Group 2 was given the same diet for the first 24 weeks and then a basal diet for the following 28 weeks. Groups 3, 4, and 5 received a choline supplemented L-amino acid defined diet, the semipurified choline deficient diet, and a semipurified choline supplemented diet, respectively, throughout the experimental period. All surviving rats were subjected to complete macroscopic examination at Week 52. Histopathologically diagnosed hepatocellular carcinomas were induced in Group 1 at an incidence of 100%; multiple metastatic nodules were seen in the lungs of one of the animals. Hepatocellular carcinomas were also induced in Group 4 rats at a significantly lower incidence of 20%. No hepatocellular carcinomas were observed in rats in Groups 2, 3, and 4. The results indicate that the CDAA diet exerts more potent carcinogenicity for the livers of rats than does the semipurified choline deficient diet. However, limited exposure for 24 weeks may have not been sufficient for hepatocellular carcinoma induction by the CDAA diet at Week 52 although a high incidence of hyperplastic nodules and slight cirrhosis were evidence of persistent lesions.
Amino Acids↗
Oxidative damage lipid peroxidation in the kidney of choline-deficient rats.
Phosphatidylcholine is the most abundant phospholipid constituent of cell membranes and choline is a quaternary amine required for phosphatidylcholine synthesis. The impairment of membrane functions is considered as an indication of oxidative damage. In order to kinetically analyze the time course of the pathogenesis of renal necrosis following to choline deficiency in weanling rats, we determined markers of membrane lipid peroxidation (thiobarbituric acid reactive substances; TBARS and hydroperoxide-induced chemiluminescence (BOOH-CL) ) and studied the histopathological damage. Plasma TBARS (t(1/2) = 2.5 days) was an early indicator of systemic oxidative stress, likely involving liver and kidney. The levels of TBARS an BOOH-CL increased by 80% and by 183%, respectively, in kidney homogenates with t(1/2) = 1.5 days and 4 days, respectively. The levels of BOOH-CL were statistically higher in rats fed a choline-deficient diet at day 6, in a mixture of membranes (from plasmatic, smooth and rough endoplasmic reticulum and Golgi), in mitochondrial membranes and in lysosomal membranes. The results indicate that choline deficiency produces oxidative damage in kidney subcellular membranes. Necrosis involved mainly convoluted tubules and appeared with a t(1/2) = 5.5 days. An increase in the production of reactive oxygen species, triggered by NADH overproduction in the mitochondrial dysfunction associated with choline deficiency appears as one of the pathogenic mechanism of mitochondrial and cellular oxidative damage in choline-deficiency.
Animals↗
Effects of choline deficiency and methotrexate treatment upon liver folate content and distribution.
We examined the effects of feeding rats a choline deficient diet, of treating rats with low doses of methotrexate (MTX, 0.1 mg/kg, daily), and of combined choline deficiency and MTX treatment upon the content and distribution of folates in liver. We used a newly devised technique for analysis of folates which utilized affinity chromatography followed by high pressure liquid chromatography. Compared to control rats, total hepatic folate content decreased by 31% in the choline deficient rats, by 48% in the MTX treated rats, and by 60% in rats which were both choline deficient and treated with MTX. In extracts of livers from control rats, folates were present predominantly as penta (35%) and hexaglutamyl (52%) derivatives. The pteridine ring structure distribution of these folates was as follows: 48% 5-methyltetrahydrofolate, 14% formylated tetrahydrofolate, and 39% tetrahydrofolate. In choline deficient animals, there was a decrease in the relative concentration of pentaglutamyl folates and an increase in the relative concentration of heptaglutamyl folates. In livers from MTX treated animals, MTX-polyglutamates with 2-5 glutamate residues accumulated. The consequences of MTX treatment were: a) an elongation of the glutamate chains of the folates as the proportion of hepta- and octaglutamyl derivatives was increased relative to penta- and hexaglutamyl folates; b) the occurrence of unreduced folic acid; c) a decrease in the relative concentration of 5-methyltetrahydrofolate and an increase in the relative concentration of formylated tetrahydrofolate, and d) no change in the relative concentrations of tetrahydrofolate. In livers from animals that were both choline deficient and treated with MTX, the tetrahydrofolate concentrations were 50% of control while formylated tetrahydrofolate concentrations increased 3-fold. These data are discussed from the standpoint of the current understanding of mechanisms that regulate the elongation of the glutamic acid chains of folates and those that regulate folate dependent synthesis and utilization of one carbon unit.
Animals↗
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.
Animals↗
A choline-deficient diet exacerbates fatty liver but attenuates insulin resistance and glucose intolerance in mice fed a high-fat diet.
Liver fat accumulation is proposed to link obesity and insulin resistance. To dissect the role of liver fat in the insulin resistance of diet-induced obesity, we altered liver fat using a choline-deficient diet. C57Bl/6 mice were fed a low-fat (10% of calories) or high-fat (45% of calories) diet for 8 weeks; during the final 4 weeks, diets were either choline deficient or choline supplemented. In choline replete animals, high-fat feeding induced weight gain, elevated liver triglycerides (171%), hyperinsulinemia, and glucose intolerance. Choline deficiency did not affect body or adipose depot weights but amplified liver fat accumulation with high-fat diet (281%, P < 0.01). However, choline deficiency lowered fasting plasma insulin (from 983 +/- 175 to 433 +/- 36 pmol/l, P < 0.01) and improved glucose tolerance on a high-fat diet. In mice on 30% fat diet, choline deficiency increased liver mRNA levels of the rate-limiting enzyme in phosphatidylcholine synthesis and of enzymes involved in free fatty acid esterification, without affecting those of de novo lipogenesis or fatty acid oxidation. We conclude that liver fat accumulation per se does not cause insulin resistance during high-fat feeding and that choline deficiency may shunt potentially toxic free fatty acids toward innocuous storage triglyceride in the liver.
Animals↗
Expression of phosphatidylethanolamine N-methyltransferase-2 is markedly enhanced in long term choline-deficient rats.
When rats are fed a choline-deficient (CD) diet, acute fatty liver develops along with other biochemical changes. However, when choline deficiency is prolonged, the growth rate of CD rats is similar to that of control rats fed a choline-supplemented diet. Furthermore, CD rats maintain their levels of choline-containing lipids, such as phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin. The mechanism for this compensation in CD rats was investigated. We screened the major tissues for the activities of two important enzymes involved in the biosynthesis of phosphatidylcholine, CTP:phosphocholine cytidylyltransferase (CT) and phosphatidylethanolamine N-methyltransferase (PEMT). Only the livers of CD rats had higher specific enzyme activities of PEMT and CT than control animals. The amount of PEMT2, one of two PEMTs in liver, increased 5-fold in CD rats after 6 weeks on the CD diet. A similar increase in the level of PEMT2 mRNA suggested that this activation was due to enhanced expression of the PEMT2 gene in CD livers. The labeling of phosphatidylcholine in isolated hepatocytes from CD rats was consistent with the conversion of PE to PC being increased as a result of a higher expression of liver PEMT. We conclude that activation of PE methylation at the level of gene expression may be the mechanism by which CD rats compensate for the lack of dietary choline.
Animals↗
Changes in aldehyde dehydrogenase occurring during rat hepatocarcinogenesis induced by ethionine combined with dietary choline deficiency.
Chronic exposure to ethionine (0.05%) combined with dietary choline deficiency was used to study changes in aldehyde dehydrogenase (ALDH) activity during hepatocarcinogenesis in male Sprague-Dawley rats. Over a period of 43 weeks, animals were sacrificed at intervals and the ALDH phenotype of normal liver and any lesions was characterized by histochemical analysis, total activity assays, and gel electrophoresis, using propionaldehyde and nicotinamide adenine dinucleotide (NAD+) to detect normal liver ALDH activity and benzaldehyde and nicotinamide adenine dinucleotide phosphate (NADP+) for tumor-associated ALDH. In animals receiving ethionine plus choline deficiency, significant changes in ALDH were observed histochemically by 9 weeks, when there was a distinct shift in activity from its normal centrilobular pattern to a periportal distribution. The first NAD+- and NADP+-dependent ALDH-positive enzyme-altered foci were also seen at 9 weeks. There was no correlation between the ALDH and gamma-glutamyl transpeptidase phenotypes of an individual focus. Areas of cholangiofibrosis, cystic degeneration, and bile duct proliferation were distinctly ALDH negative. No significant changes in benzaldehyde and NADP+ ALDH activity were detectable by total activity assays or gel electrophoresis prior to the appearance of overt neoplasms at 26 weeks. No significant changes in ALDH activity occurred in animals receiving either ethionine or choline deficient diet alone. By histochemistry, total activity assays and gel electrophoresis, only 7 of the 28 (25%) of the hepatic neoplasms examined expressed the tumor-associated ALDH phenotype. An additional five neoplasms had barely detectable levels of benzaldehyde and NADP+ ALDH activity. These results are in striking contrast to changes in ALDH activity occurring during hepatocarcinogenesis induced by other protocols we have tested previously in which from 50 to 96% of all neoplasms were ALDH positive.
Aldehyde Dehydrogenase↗
Effect of choline deficiency on CTP: phosphocholine cytidylyltransferase and choline kinase activities in rat liver subcellular fractions.
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Animals↗
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.
Animals↗
Behavioral and neurochemical effects of a chronic choline-deficient diet in the rat.
Chronic administration of a choline-deficient diet for 20 days caused no modification in ACh levels in the cortex, hippocampus and olfactory system of the rat and an increase in the density (Bmax) of the muscarinic receptors in the hippocampus. The choline-deficient diet caused no modification in noradrenaline levels and a reduction in the density of alpha1-adrenoceptors in the cortex, hippocampus and olfactory system. This paper discusses the possibility that these neurochemical effects are correlated with the reduction in spontaneous memory decay which was observed in the staircase maze after 20 days of a choline-deficient diet.
Acetylcholine↗
Choline deficiency augments and antibody to tumor necrosis factor-alpha attenuates endotoxin-induced hepatic injury.
Alcoholic liver disease can be associated with hepatic choline deficiency and hepatic steatosis, abnormalities also observed in rats administered choline-deficient (CD) diets. Bacterial lipopolysaccharides (LPS) have been postulated to play a key role in this choline deficiency model of liver injury, and LPS hepatotoxicity is mediated to a major extent by the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). This study addressed the following questions: Does LPS administration exacerbate an in vivo liver injury induced by choline deficiency? If so, do CD rats have increased serum TNF-alpha concentrations and does pretreatment anti-TNF-alpha IgG attenuate this injury? Rats administered choline-sufficient (CS) or CD diets for 16 days were intravenously administered either saline or LPS. One group of CD rats also received a single dose of anti-TNF-alpha IgG before LPS administration. Changes in histology and serum transaminase levels were determined. Both liver histology and serum transaminases were unchanged in the CS group treated with LPS, compared with the CS group treated with saline (control group). However, compared with this control group, transaminases were 5- to 7-fold higher in saline-treated CD rats and 30- to 50-fold higher in LPS-treated CD rats. Livers of saline-treated CD rats had massive fatty infiltration, and no necrosis but livers of LPS-treated CD rats showed both extensive fatty infiltration and large areas of necrosis. Serum TNF-alpha concentrations in CD rats (saline or LPS treated) were significantly elevated, compared with levels in corresponding CS rats. Pretreatment with the anti-TNF-alpha IgG prevented hepatonecrosis in LPS-treated CD rats and lowered their serum transaminases by one-third. Thus, LPS administration exacerbated liver injury induced by choline deficiency, and this injury was probably partially mediated by TNF-alpha and attenuated by anti-TNF-alpha IgG.
Animals↗
Nicotine improves cognitive disturbance in rodents fed with a choline-deficient diet.
The effect of nicotine on learning disturbances was tested in rats. Rats were fed either a choline-enriched or a choline-deficient diet. Concentration of acetylcholine in the whole brain was significantly lower in rats fed with choline-deficient diet than rats fed with choline-enriched diet. Passive avoidance learning shows that rats on a choline-deficient diet showed significantly impaired learning compared to rats on a choline-enriched diet. Nicotine (0.04 mg/kg) administered intraperitoneally significantly potentiated learning in rats on a choline-deficient diet, as well as in rats on a choline-enriched diet. We, therefore, suggest that nicotine may potentiate learning in an acetylcholine-deprived brain.
Acetylcholine↗
Different response to choline deficiency of the serum ornithine carbamoyltransferase activity in four strains of rats.
Rats of the Donryu, Wistar, Fischer, and Sprague-Dawley strains were examined for the effects of choline deficiency on liver lipids, serum lipids, and serum ornithine carbamoyltransferase. The liver total lipid, triacylglycerol, cholesterol and phospholipid contents in the choline-deficient rats were significantly higher than those in choline-sufficient rats. The contents of total lipids and phospholipids in the liver of the Wistar and Fischer rats fed on a choline-deficient diet were significantly higher than those of the Donryu and Sprague-Dawley rats. The levels of triacylglycerol, cholesterol and phospholipids in the serum were significantly decreased by feeding with the choline-deficient diet. The serum ornithine carbamoyltransferase activity was increased in the Wistar and Fischer strains by feeding with the choline-deficient diet. The Wistar and Fischer strains were consequently the most sensitive to both lipid accumulation and liver lesions induced by the choline deficiency.
Animals↗
cAMP enhances Cx43 gap junction formation and function and reverses choline deficiency apoptosis.
Previously, it had been shown that acute choline deficiency (CD) induced apoptosis in cultured rat liver epithelial cells, whereas cells that are adapted to survive in low-choline-containing medium acquire resistance to CD apoptosis and undergo malignant transformation. Thus, understanding the mechanisms of action of CD could increase our understanding of the role of choline, an essential nutrient, in the process of malignant transformation. The present experiments were designed to test the hypothesis that CD might function as a pro-apoptotic trigger by altering the localization of connexin 43 gap junction protein and gap junctional intercellular communication (GJIC). Established liver epithelial cells (WB cells; Hep3B cells) were maintained in a defined, serum-free medium control (70 microM choline) or choline deficient medium (CD, 5 microM choline) and the localization of connexin 43 protein (Cx43) was studied by immunocytochemistry and Western blotting. In nontumorigenic WB cells, CD apoptosis was associated with retention of Cx43 in the golgi/ER region of the cytoplasm and decreased GJIC as measured using a preloading fluorescent dye transfer method (calcein AM/DiIC(18)). Cells maintained in CD in the presence of 8-bromoadenosine 3':5'-cyclic monophosphate exhibited restoration of Cx43 at the plasma membrane and increased GJIC and inhibition of apoptosis. These studies show that CD apoptosis in nontumorigenic liver epithelial cells is associated with alterations to Cx43 and GJIC and that an uncoupling of Cx43 localization and GJIC is related to resistance to CD apoptosis in transformed liver epithelial cells.
8-Bromo Cyclic Adenosine Monophosphate↗
Methotrexate effects on hepatic betaine levels in choline-supplemented and choline-deficient rats.
Groups of rats fed both choline-supplemented and choline-deficient diets were injected with methotrexate (MTX) at dose levels of 0.1 mg/kg/day and 0.2 mg/kg/day. Both doses produced lowered hepatic betaine levels in the choline-supplemented animals when compared with nontreated, pair-fed controls. Since betaine levels were very low in the livers of choline-deficient rats, MTX had no further effect on hepatic betaine in these animals. These data suggest that the betaine lowering seen in livers of choline-fed rats may be due to utilization as a means of compensating for the MTX-induced loss of N5 methyltetrahydrofolate for the vital methylation of homocysteine in methionine biosynthesis.
Aging↗
Dietary choline deficiency alters global and gene-specific DNA methylation in the developing hippocampus of mouse fetal brains.
The availability of choline during critical periods of fetal development alters hippocampal development and affects memory function throughout life. Choline deficiency during fetal development reduces proliferation and migration of neuronal precursor cells in the mouse fetal hippocampus and these changes are associated with modifications in the protein levels of some cell cycle regulators and early differentiation markers. We fed C57 BL/6 mouse dams diets deficient or normal in choline content from days 12 to 17 of pregnancy, and then collected fetal brains on embryonic day 17. Using laser-capture micro-dissection we harvested cells from the ventricular and subventricular zones of Ammon's horn and from the prime germinal zone of the dentate gyrus (hippocampus). In the ventricular and subventricular zones from the choline-deficient group, we observed increased protein levels for kinase-associated phosphatase (Kap) and for p15(INK4b) (two cell cycle inhibitors). In the dentate gyrus, we observed increased levels of calretinin (an early marker of neuronal differentiation). In fetal brain from mothers fed a choline-deficient diet, DNA global methylation was decreased in the ventricular and subventricular zones of Ammon's horn. We also observed decreased gene-specific DNA methylation of the gene (Cdkn3) that encodes for Kap, correlating with increased expression of this protein. This was not the case for p15(INK4b) or calretinin (Cdkn2b and Calb2, respectively). These data suggest that choline deficiency-induced changes in gene methylation could mediate the expression of a cell cycle regulator and thereby alter brain development.
Animals↗
Effect of choline deficiency on S-adenosylmethionine and methionine concentrations in rat liver.
Choline and C1 metabolism pathways intersect at the formation of methionine from homocysteine. Hepatic S-adenosylmethionine (AdoMet) concentrations are decreased in animals ingesting diets deficient in choline, and it has been suggested that this occurs because the availability of methionine limits AdoMet synthesis. If the above hypothesis is correct, changes in hepatic AdoMet concentrations should relate in some consistent manner to changes in hepatic methionine concentrations. Rats were fed on a choline-deficient or control diet for 1-42 days. Hepatic choline concentrations in control animals were 105 nmol/g, and decreased to 50% of control after the first 7 days on the choline-deficient diet. Hepatic methionine concentrations decreased by less than 20%, with most of this decrease occurring between days 3 and 7 of choline deficiency. Hepatic AdoMet concentrations decreased by 25% during the first week, and continued to decrease (in total, by over 60%) during each subsequent week during which animals consumed a choline-deficient diet. Hepatic S-adenosylhomocysteine (AdoHcy) concentrations increased by 50% when animals consumed a choline-deficient diet. AdoHcy is formed when AdoMet is utilized as a methyl donor. In summary, choline deficiency can deplete hepatic stores of AdoMet under dietary conditions that only minimally decrease the availability of methionine within liver. Thus decreased availability of methionine may not have been the only mechanism whereby choline deficiency lowers hepatic AdoMet concentrations. We suggest that increased utilization of AdoMet might also have occurred.
Animals↗