Environmental temperature and choline requirements in rats. II. Choline and methionine requirements for lipotropic activity

Young rats were fed choline-deficient diets and maintained at different environmental temperatures. The hepatic lipid level remained normal in rats at 2 degrees when 25 mg of choline per 100 g of food was fed; 50 mg of choline per 100 g food was required at 21 degrees and 100 mg of choline per 100 g food at 33 degrees to prevent excessive lipid accumulation. These values were equivalent to a mean daily intake per rat of 3 mg of choline at 2 degrees, 5.5 mg at 21 degrees, and 7 mg at 33 degrees respectively. When the growth rate was slower owing to a slight inadequacy of histidine in the basal choline-deficient diet, normal hepatic lipid was maintained by supplements of 50 mg of choline per 100 g food at 21 degrees and 33 degrees. Increasing the methionine content of the diet two- or three-fold from a basal value of 340 mg per 100 g food was as effective as 200 mg of choline per 100 g of food in lowering hepatic lipids at 2 degrees, 21 degrees, and 33 degrees.     

Evidence that the rate of phosphatidylcholine catabolism is regulated in cultured rat hepatocytes

The regulation of phosphatidylcholine (PC) catabolism has been studied in choline-deficient rat hepatocytes. Supplementation of choline-deficient hepatocytes, prelabeled with [3H]choline, with 100 microM choline increased the rate of PC catabolism by approx. 2-fold. The major product of PC degradation was glycerophosphocholine in both choline-deficient and choline-supplemented cells. Choline supplementation decreased the radioactivity recovered in lysoPC by 50%. This effect was accompanied by a 2-fold increase of labeled glycerophosphocholine. Comparable results were obtained when PC of the cells was prelabeled with [3H]methionine or [3H]glycerol. The activity of phospholipase A in cytosol, mitochondria and microsomes isolated from choline-deficient rat liver was similar to the activity in control liver, when determined with [3H]PC vesicles as the substrate. Measurement of the activity of phospholipase A with endogenously [3H]choline-labeled PC showed that the formation of lysoPC in mitochondria isolated form choline-supplemented cells was 40% lower than in choline-deficient cells. Alternatively, the formation of [3H]glycerophosphocholine and [3H]choline in microsomes from choline-supplemented cells was significantly higher (1.4-fold) than in microsomes from choline-deficient cells. These results suggest that the rate of PC catabolism is regulated in rat hepatocytes and that the concentration of PC might be an important regulatory factor.     

Evidence that remodeling of the fatty acids of phosphatidylcholine is regulated in isolated rat hepatocytes and involves both the sn-1 and sn-2 positions

The remodeling of the fatty acyl moieties of phosphatidylcholine (PC) has been studied in choline-deficient and choline-supplemented hepatocytes prepared from a choline-deficient rat. Choline-deficient hepatocytes were prelabeled with [Me-3H]choline for 30 min and subsequently incubated for up to 12 h in the presence or absence of choline. Analysis of the molecular species of PC from choline-deficient cells showed that, at the end of the pulse, approx. 75% of the label was incorporated into palmitate-containing species and only approx. 16% of the labeled species contained stearate. During the chase period there was a redistribution of label and after 12 h approx. 56% of the total radioactivity was associated with palmitate containing species and 37% was recovered in stearate-containing species. A similar distribution of radioactivity was observed in choline-supplemented cells. Measurement of the specific radioactivity of the major molecular species of PC was consistent with a precursor-product relationship between palmitate-containing species and stearate-containing species with arachidonate or linoleate on the sn-2 position. A model is presented which takes into account remodeling of both the sn-1 and sn-2 positions of PC.     

Choline deficiency causes translocation of CTP:phosphocholine cytidylyltransferase from cytosol to endoplasmic reticulum in rat liver

The choline-deficient rat liver has been chosen as a physiologically relevant model system in which to study the regulation of phosphatidylcholine biosynthesis. When 50-g rats were placed on a choline-deficient diet for 3 days, the activity of CTP:phosphocholine cytidylyltransferase (CT) was increased 2-fold in the microsomes and decreased proportionately in the cytosol. A low titer antibody to CT was obtained from chickens and used to identify the amount of CT protein in cytosol from rat liver. The amount of CT recovered from the choline-deficient cytosol was significantly less than in cytosol from choline-supplemented rats. When hepatocytes were prepared from choline-deficient livers, supplementation of the medium of the cells with choline caused CT to move from the membranes to cytosol within 1-2 h. The activity of another translocatable enzyme of glycerolipid metabolism, phosphatidate phosphohydrolase, was unchanged in cytosol from choline-deficient rat livers, and the microsomal activity of this enzyme was only minimally increased. When the livers were fractionated into endoplasmic reticulum and Golgi, there was a 2-fold increase in the activity on the endoplasmic reticulum from choline-deficient livers but no change in activity associated with Golgi. Thus, the increased association of CT with endoplasmic reticulum in choline-deficient livers appears to be specific to that subcellular fraction, and the subcellular location of other enzymes may not be affected.     

LECITHIN SYNTHESIS, INTRACELLULAR TRANSPORT, AND SECRETION IN RAT LIVER : IV. A Radioautographic and Biochemical Study of Choline-Deficient Rats Injected with Choline-3H

Injection of choline-³H into choline-deficient rats resulted in an enhanced incorporation of the label into liver lecithin, as compared to the incorporation of label into liver lecithin of normal rats. The results obtained with the use of different lecithin precursors indicate that in the intact liver cell, both in vivo and in vitro, exchange of choline with phosphatidyl-choline is not significant. The synthesis and secretion of lecithins by the choline-deficient liver compare favorably with the liver of choline-supplemented rats, when both are presented with labeled choline or lysolecithin as lecithin precursors. Radioautography of the choline-deficient liver shows that 5 min after injection of choline-³H the newly synthesized lecithin is found in the endoplasmic reticulum (62%), mitochondria (13%), and at the "cell boundary" (20%). The ratio of the specific activity of microsomal and mitochondrial lecithin, labeled with choline, glycerol, or linoleate, was 1.53 at 5 min after injection, but the ratio of the specific activity of phosphatidyl ethanolamine (PE), labeled with ethanolamine, was 5.3. These results indicate that lecithin and PE are synthesized mainly in the endoplasmic reticulum, and are transferred into mitochondria at different rates. The site of a precursor pool of bile lecithin was studied in the intact rat and in the perfused liver. Following labeling with choline-³H, microsomal lecithin isolated from perfused liver had a specific activity lower than that of bile lecithin, but the specific activity of microsomal linoleyl lecithin was comparable to that of bile lecithin between 30 and 90 min of perfusion. It is proposed that the site of the bile lecithin pool is located in the endoplasmic reticulum and that the pool consists mostly of linoleyl lecithin.     

Influence of Dietary Choline Availability and Neuronal Demand on Acetylcholine Synthesis by Rat Brain

The main objective of this study was to test the hypothesis that the chronic administration of choline supplements a bound pool of choline from which free choline can be mobilized and used to support acetylcholine synthesis when the demand for precursor is increased. For these experiments, brain slices from rats fed diets containing different amounts of choline were incubated in a choline-free buffer and acetylcholine synthesis was measured under resting conditions and in the presence of K+-induced increases in acetylcholine synthesis and release. Rats fed the choline-supplemented diet had circulating choline levels that were 52% greater than the controls, and striatal and cerebral cortical slices from this group produced significantly more free choline during the incubation than slices from the controls. However, the synthesis and release of acetylcholine by these tissues did not differ from those by controls, during either resting or K+-evoked conditions. In contrast, acetylcholine synthesis and release by striatal and hippocampal slices from choline-deficient rats, animals that had circulating choline levels that were 80% of control values, decreased significantly; the production of free choline by these tissues was also depressed. Results indicate that, despite an increased production of free choline by brain slices from choline-supplemented rats, the synthesis of acetylcholine was unaltered, even in the presence of an increased neuronal demand. In contrast, the choline-deficient diet led to a decreased release of free choline from bound stores and an impaired ability of brain to synthesize acetylcholine.     

Feedback regulation of CTP:phosphocholine cytidylyltransferase translocation between cytosol and endoplasmic reticulum by phosphatidylcholine

The mechanism for the increased association of CTP:phosphocholine cytidylyltransferase (CT) with membranes of hepatocytes derived from choline-deficient, compared with choline-supplemented rats, has been investigated. The cells were maintained in culture for 4 h in a choline- and methionine-deficient medium. (Methionine is required for synthesis of phosphatidylcholine (PC) via methylation of phosphatidylethanolamine.) Afterward, the cells were incubated +/- choline for various times up to 4 h. In the presence, but not in the absence, of choline there was a translocation of CT activity from membranes to cytosol. During this time period there was no change in the amounts of unesterified fatty acids or diacylglycerol recovered from the hepatocytes. In addition, there was no evidence for a difference in the incorporation of 32P into CT or other cytosolic proteins isolated from hepatocytes +/- choline. In contrast, there was a highly significant correlation between the concentration of PC in the membranes and the increased activity of CT in the cytosol (R = 0.98) and the decreased activity in the membranes (R = 0.93). The concentration of PC could alternatively be altered by incubation of the choline-deficient hepatocytes with methionine or lyso-PC. With either of these supplementations highly significant correlation coefficients were observed between the concentration of PC in membranes and decreased activity of CT in membranes or increased activity in cytosol. The concentration of PC was reduced in the endoplasmic reticulum, but not the Golgi membranes, isolated from choline-deficient compared with choline-supplemented livers. The data suggest that the amount of PC in the endoplasmic reticulum feedback regulates the amount of CT associated with this membrane.     

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.     

Effect of sodium chloride concentration on fluid-phase assembly and stability of the C3 convertase of the classical pathway of the complement system.

The specificity of the phospholipid head-group for feedback regulation of CTP: phosphocholine cytidylyltransferase was examined in rat hepatocytes. In choline-deficient cells there is a 2-fold increase in binding of cytidylyltransferase to cellular membranes, compared with choline-supplemented cells. Supplementation of choline-deficient cells with choline, dimethylethanolamine, monomethylethanolamine or ethanolamine resulted in an increase in the concentration of the corresponding phospholipid. Release of cytidylyltransferase into cytosol was only observed in hepatocytes supplemented with choline or dimethylethanolamine. The apparent EC50 values (concn. giving half of maximal effect) for cytidylyltransferase translocation were similar for choline and dimethylethanolamine (25 and 27 microM respectively). The maximum amount of cytidylyltransferase released into cytosol with choline supplementation (1.13 m-units/mg membrane protein) was twice that (0.62) observed with dimethylethanolamine. Supplementation of choline-deficient hepatocytes with NN'-diethylethanolamine, N-ethylethanolamine or 3-aminopropanol also did not cause release of cytidylyltransferase from cellular membranes. The translocation of cytidylyltransferase appeared to be mediated by the concentration of phosphatidylcholine in the membranes and not the ratio of phosphatidylcholine to phosphatidylethanolamine. The results provide further evidence for feedback regulation of phosphatidylcholine biosynthesis by phosphatidylcholine.     

Effects of chronic (dietary) choline availability on the transport of choline across the blood-brain barrier

The effects of dietary choline availability on the transport of choline across the blood-brain barrier (BBB) were investigated using the intracarotid injection technique. Maintenance of rats on choline-deficient, basal choline, or choline-supplemented diets for 28-32 days led to respective increases in blood levels of choline and correlative increases in the velocity of transport of choline measured using a buffer injectate. When serum from these rats was included in the injectate and transport determined in control animals, there was a marked inhibition of choline transport that was related to the concentration of choline in the diets. Results suggest that the activity of the choline carrier at the BBB is antagonized by an inhibitory substance in serum whose concentration or activity may be modified by chronic alterations in circulating levels of choline and whose presence may normally regulate the velocity of choline transport.     

Choline-deficiency fatty liver: impaired release of hepatic triglycerides

After intravenous injection of palmitate-1-(14)C to rats fed a choline-deficient (CD) or choline-supplemented (CS) diet for 15-18 hr, liver triglycerides became labeled very rapidly. In CS, but not in CD rats, there was a considerable loss, with time, of radioactivity from liver triglycerides. At the same time, significantly less radioactivity appeared in plasma triglycerides of CD rats than of CS animals. No difference was seen in the triglyceride content of microsomes isolated from the liver of rats fed the two diets. The lower radioactivity in plasma triglycerides of CD rats was essentially due to a lower level and specific activity of very low density lipoprotein triglycerides. After intravenous injection of Triton and labeled palmitate, considerably less radioactivity accumulated in plasma triglycerides and phospholipids of CD rats than of CS animals. Post-Triton hyperphospholipidemia was also less pronounced in CD rats. It was concluded that the fatty liver observed in CD rats results from an impaired release of hepatic triglycerides into plasma.     

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.     

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.     

Phosphatidylethanolamine levels and regulation of phosphatidylethanolamine N-methyltransferase

The activity of phosphatidylethanolamine (PE) N-methyltransferase in liver microsomes, measured using endogenous microsomal PE as a substrate, was elevated 2-fold in the choline-deficient state. However, methyltransferase activity assayed in the presence of a saturating concentration of phosphatidyl-N-mono-methylethanolamine or microsomal PE was unchanged by choline deficiency. Accompanying the increase in methyltransferase activity in liver homogenates and microsomes were increased PE concentrations and an increased PE to phosphatidylcholine ratio. The concentration of other phospholipids was unchanged. Immunoblot analysis of choline-deficient and choline-supplemented rat liver microsomes using a rabbit polyclonal anti-PE N-methyltransferase antibody revealed that the amount of enzyme protein was unaltered. The regulation of methyltransferase by PE levels was also investigated in cultured hepatocytes obtained from choline-deficient rat livers. Supplementation of deficient hepatocytes with 200 microM methionine resulted in a 50% reduction in cellular PE levels over a 12-h period. PE N-methyltransferase activity assayed with endogenous PE was also reduced by 50%, but phosphatidyl-N-monomethylethanolamine-dependent activity was unchanged. A 4-h supplementation with choline did not affect PE levels or methyltransferase activity. Either methionine or choline supplementation resulted in net synthesis of cellular phosphatidylcholine. Immunoblotting of membranes from methionine-supplemented hepatocytes revealed no change in enzyme protein, a further indication that enzyme mass was constitutive, and activity was regulated by the concentration of PE.     

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.     

Gestational choline deficiency causes global and Igf2 gene DNA hypermethylation by up-regulation of Dnmt1 expression

During gestation there is a high demand for the essential nutrient choline. Adult rats supplemented with choline during embryonic days (E) 11-17 have improved memory performance and do not exhibit age-related memory decline, whereas prenatally choline-deficient animals have memory deficits. Choline, via betaine, provides methyl groups for the production of S-adenosylmethionine, a substrate of DNA methyltransferases (DNMTs). We describe an apparently adaptive epigenomic response to varied gestational choline supply in rat fetal liver and brain. S-Adenosylmethionine levels increased in both organs of E17 fetuses whose mothers consumed a choline-supplemented diet. Surprisingly, global DNA methylation increased in choline-deficient animals, and this was accompanied by overexpression of Dnmt1 mRNA. Previous studies showed that the prenatal choline supply affects the expression of multiple genes, including insulin-like growth factor 2 (Igf2), whose expression is regulated in a DNA methylation-dependent manner. The differentially methylated region 2 of Igf2 was hypermethylated in the liver of E17 choline-deficient fetuses, and this as well as Igf2 mRNA levels correlated with the expression of Dnmt1 and with hypomethylation of a regulatory CpG within the Dnmt1 locus. Moreover, mRNA expression of brain and liver Dnmt3a and methyl CpG-binding domain 2 (Mbd2) protein as well as cerebral Dnmt3l was inversely correlated to the intake of choline. Thus, choline deficiency modulates fetal DNA methylation machinery in a complex fashion that includes hypomethylation of the regulatory CpGs within the Dnmt1 gene, leading to its overexpression and the resultant increased global and gene-specific (e.g. Igf2) DNA methylation. These epigenomic responses to gestational choline supply may initiate the long term developmental changes observed in rats exposed to varied choline intake in utero.     

Interaction between exercise training and cold acclimation in rats

Five groups of 10 rats were used. Group A included sedentary rats kept at 24 degrees C, group B exercised-trained rats and group C rats exposed at -15 degrees C for 2 h every day and kept at 24 degrees C for the remaining time. These 3 groups were kept on this regimen for 10 weeks. In addition group D was acclimated to cold (2 h.d-1 at -15 degrees C) for 6 weeks and subsequently deacclimated at 24 degrees C for 4 weeks. Group E was also acclimated to cold for 6 weeks and during the deacclimation, at 24 degrees C period which lasted 4 weeks, the animals were exercised 2 h per day. Following the 10 week experimental period all animals were sacrificed and DNA and protein content of the IBAT as well as its total mass were measured. The results show significant increases in the cold adapted group. Exercise training which had no effect on brown adipose tissue IBAT at room temperature, caused an accelerated reduction in weight, DNA and protein content of the BAT in rats previously acclimated to cold. In spite of this, the thermogenic response to noradrenaline was significantly enhanced in the group which exercised during the deacclimation period. It is suggested that tissues other than IBAT may explain this enhanced heat production capacity.     

Brown adipose tissue thermogenesis in T3-treated rats.

T4 treatment results in an inactivation of brown adipose tissue (BAT) which has been attributed to a reduced need of thermoregulatory heat production. Since T3 formation in brown adipocytes is governed by a type II T4 5'-deiodinase which is inhibited by T4, we analyzed the possibility that results obtained by T4 treatment were due to a lack of T3 in the tissue. Hyperthyroidism was induced in adult rats by administration of T3 (50 micrograms/kg body weight daily s.c.). Euthyroid and hyperthyroid rats were maintained at 23 degrees C or exposed at 6 degrees C for 3 weeks. Hyperthyroid rats at 23 degrees C showed an increase in BAT mass and in DNA and total lipids contents; however, BAT thermogenic activity was depressed. BAT from cold-exposed hyperthyroid rats showed the same mass and DNA content than at 23 degrees C, but it showed an increase in thermogenic activity, this increase being lower than in cold-exposed euthyroid rats. We conclude that high levels of T3 in BAT do not stimulate the thermogenic activity of the tissue. On the contrary, they inhibit it in response to lower requirements of facultative thermogenesis, both at 23 degrees C and at 6 degrees C.     

Nonshivering thermogenesis and cold resistance in rats under severe cold conditions.

Following either chronic exposure to 6 degrees C, or outdoor winter exposure, or chronic treatment with tyramine rats were exposed to -40 degrees C and their oxygen consumption and colonic temperature monitored. Fall in body temperature with time of exposure followed a sigmoid curve which had an inflection point around 32.9 degrees C. Both the time required for body temperature to reach this point and hypothermic resistance defined as the total O2 consumed up to the inflection time were useful indices of resistance to severe cold; Three days before the cold tests, capacity for norepinephrine-induced nonshivering thermogenesis was measured in all animals by examination of their metabolic response to tyraminemthe magnitude of response to tyramine correlated well with hypothermic resistance only for those rats chroncally treated with tyramine. It is concluded that it is impossible to predict with any reasonable degree of confidence the cold resistance of a rat from its tyramine response. In cold-acclimated rats, factors in addition to norepinephrine sensitivity are significantly involved in cold resistance and deserve further studies.