Choline-deficient Rats Research Articles

Administration of Diethylnitrosamine in the Immediate Postnatal Period Coupled with Exposure to a Choline Deficient Diet Accelerates Hepatocarcinogenesis in the Rat

The objectives of this study were to determine if the combination of: i. neonatal administration of diethylnitrosamine and ii. subsequent feeding of a choline deficient diet could accelerate hepatic premalignancy in the rat. The presence and size of premalignant nodules and the activity of the liver cancer enzyme marker g-glutamyltranspeptidase (gGT) were used as indicators of hepatic premalignancy. Three day old Fisher 344 rats were administered sc, saline or diethylnitrosamine (DENA): 100 mg/Kg. These were divided into choline sufficient (CS) and choline deficient (CD) groups with diets introduced to dams. On postnatal day 21 rats were weaned onto CS or CD diets. Rats in choline sufficient saline-injected (CS), choline sufficient DENA-injected (CS+DENA), choline deficient saline-injected (CD) and choline deficient DENA-injected (CD+DENA) groups were assessed on day 86. Livers of CS and CS+DENA rats were normal in appearance. Livers of CD rats were paler; those of CD+DENA rats contained visible, large, tan liver nodules. Identical results were observed in both sexes. Liver gGT activity was low and comparable in rats of CS and CS+DENA groups. Relative to liver homogenate gGT activity in the CS group, liver homogenate gGT was increased in the CD group: 14.5 fold in males and 18 fold in females; and further increased in the CD+DENA group: 78 fold in males and 54 fold in females. Plasma g-glutamyltranspeptidase exhibited the same trends as liver. The CD+DENA group demonstrated the largest increase in activity: 158 fold in males; 199 fold in females over that in the CS group. Male and female rats belonging to CS and CS+DENA groups were euthyroid; those in CD and CD+DENA groups were hypothyroid. Compared to hormone levels in the CS group, in the CD group, decreases in T3 were: 27% in males and 13% in females; decreases in T4 were: 29% in males and 5.6% in females. Compared to hormone levels in the CS groups, in the CD+DENA groups, decreases in T3 were: 25% in males and 18% in males; decreases in T4 were: 31% in males and 25% in females. Plasma glucose levels were comparable in rats of CS and CS+DENA groups. Relative to these levels, plasma glucose levels in rats of CD and CD+DENA groups were decreased 30% in males and females. Plasma transaminase levels were low and comparable in all groups. The protocol developed accelerated hepatocarcinogenesis in the Fischer 344 rat. In CD+DENA male and female rats, it produced, in 86 days, visible, large hyperplastic lesions displaying high levels of gGT and high levels of plasma gGT that are consistent with being at a well developed advanced premalignant stage of hepatocarcinogenesis without any sign of toxicity.

Regeneration of liver with marked fatty change following partial hepatectomy in rats.

Resection of liver for primary and metastatic tumors and living donor liver transplantation has become a common clinical practice. The success of recovery depends on the regeneration and functions of the remnant liver. However, information on the regenerative potential of liver with steatosis and steatohepatitis, a common clinical problem in this country, is incomplete. Therefore, we evaluated regeneration after two-thirds partial hepatectomy (PH) in male F-344 rats with marked steatosis and mild steatohepatitis induced by feeding choline-deficient diet. Choline-deficient rats and control rats were killed at 24, 36, 48, 72, and 96 h after PH. Liver regeneration was determined by measuring mitotic activity and bromodeoxyuridine (BrdU) labeling in hepatocytes. Livers of rats maintained on the choline-deficient diet showed marked steatosis and mild steatohepatitis. In these animals the levels of serum and liver triacylglycerols (TG) were low and very high, respectively, when compared to controls. In control rats mitotic and BrdU labeling indices were maximal at 24 h followed by a rapid decline, whereas in choline-deficient rats both these indices increased significantly at 36 h and decreased gradually over the next 60 h. By 96 h the size of livers in both groups was comparable. The results of this study indicate that regeneration in the liver of rats with marked steatosis is not impaired.

Correlation between stellate cell activation and serum fibrosis markers in choline-deficient L-amino acid-defined diet-induced rat liver fibrosis.

The aim of this study was to investigate the association of stellate cell activation with serum fibrosis markers in a rat model of hepatic fibrosis prepared using a choline-deficient L-amino acid (CDAA) defined diet. CDAA diet administration resulted in increased liver hydroxyproline contents in a time-dependent manner with activated stellate cells, expressing alpha-smooth muscle actin (alpha-SMA) as well as increased serum concentrations of amino-terminal procollagen type III peptide (PIIIP) and the 7S fragment of type IV collagen. Hydroxyproline content of the liver showed a closer correlation with the serum 7S (r = 0.75, P < 0.01) concentration than with the serum PIIIP (r = 0.51, P < 0.01) concentration. The percent area of alpha-SMA-positive cells showed stronger correlation with the serum PIIIP concentration (r = 0.85, P < 0.01) than with the 7S concentration (r = 0.50, P < 0.01). These results indicate that the serum PIIIP concentration reflects the activity of fibrogenesis, while the serum 7S concentration reflects the accumulation of collagen fibers in the liver.

Choline supplementation during prenatal development reduces proactive interference in spatial memory

Previous research has demonstrated that increasing dietary choline during early development can have long-lasting effects on cholinergic (Ch) function that are correlated with improvement of spatial memory ability in rats. The present study is designed to further our understanding of these organizational changes in brain and behavior by examining the effects of spaced vs. massed trials. A third of the rats ( n=10) were supplemented with choline chloride prenatally by adding it to the drinking water of their dams. Another third were made deficient of choline during early development by removing choline from the dams diet. The remaining rats served as untreated controls. Postnatally, the offspring were maintained on a choline-sufficient diet and at 120 days of age they began 12-arm radial maze training. The maze data revealed two major effects of early choline availability: (1) Both choline-supplemented and choline-deficient rats performed more accurately than control littermates when trials were spaced. These differences in spatial ability did not appear to be a function of differential response or cue-use strategies. (2) Choline-supplemented rats showed little proactive interference when trials were massed; whereas control rats demonstrated moderate levels and choline-deficient rats exhibited high levels of proactive interference as a function of massed trials. These data suggest that the behavioral consequences of early dietary availability of choline may involve the modification of the discriminative abilities used to attend to stimuli that demarcate the end of one trial and the start of another as well as the capacity for remembering the locations that have been visited during a trial.

Choline availability to the developing rat fetus alters adult hippocampal long-term potentiation

Supplementation with choline during pregnancy in rats causes a long-lasting improvement of visuospatial memory of the offspring. To determine if the behavioral effects of choline are related to physiological changes in hippocampus, the effect of perinatal choline supplementation or deficiency on long-term potentiation (LTP) was examined in hippocampal slices of 6–8 and 12–14 month old rats born to dams consuming a control, choline-supplemented, or a choline-free diet during pregnancy. Stimulating and recording electrodes were placed in stratum radiatum of area CA1 to record extracellular population excitatory postsynaptic potentials (pEPSPs). To induce LTP, a theta-like stimulus train was generated. The amplitude of the stimulus pulses was set at either 10% or 50% of the stimulus intensity which had induced the maximal pEPSP slope on the input/output curve. We found that at both ages, a significantly smaller percentage of slices from perinatally choline-deficient rats displayed LTP after 10% stimulus intensity (compared with control and choline-supplemented rats), and a significantly larger percentage of slices from choline-supplemented rats displayed LTP at 50% stimulus intensity (compared with control and choline-deficient rats). Results reveal that alterations in the availability of dietary choline during discrete periods of development lead to changes in hippocampal electrophysiology that last well into adulthood. These changes in LTP threshold may underlie the observed enhancement of visuospatial memory seen after prenatal choline supplementation and point to the importance of choline intake during pregnancy for development of brain and memory function.

Homocysteine, B Vitamins, and Atherosclerosis

To the Editor: The conclusion by Folsom et al1 that raised homocysteine concentrations in the blood may be a consequence, not a cause, of coronary artery disease has serious clinical implications. As McCully2 pointed out in his recent editorial, arteriosclerosis in hyperhomocysteinemia may result from 3 different enzymatic abnormalities: deficiency of cystathionine synthase, which is a pyridoxal phosphate–dependent enzyme; deficiency of methyltetrahydrofolate homocysteine methyl transferase, which is a cobalamin-dependent enzyme; and deficiency of methylene tetrahydrofolate reductase, which is a folate-dependent enzyme. The last enzymatic abnormality explains the origin of arteriosclerosis observed in vitamin B6–deficient monkeys and choline-deficient rats, 2 important animal models in which hyperhomocysteinemia also leads to atherogenesis.2 Although I agree with Folsom et al1 that randomized trials are needed to better clarify the interrelationships of homocysteine, B vitamins, …

Prenatal dietary choline supplementation decreases the threshold for induction of long-term potentiation in young adult rats.

Choline supplementation during gestation in rats leads to augmentation of spatial memory in adulthood. We hypothesized that prenatal (E12-E17) choline supplementation in the rat would lead to an enhancement of hippocampal synaptic plasticity as assessed by long-term potentiation (LTP) at 3-4 mo of age. LTP was assessed blindly in area CA1 of hippocampal slices with first suprathreshold (above threshold for LTP generation in control slices) theta-burst stimulus trains. The magnitude of potentiation after these stimuli was not different between slices from control and prenatally choline supplemented animals. Next, threshold (reliably leading to LTP generation in control slices) or subthreshold theta-burst stimulus trains were applied to slices from control, prenatally choline-supplemented, and prenatally choline-deprived rats. Threshold level stimulus trains induced LTP in slices from both the control and choline-supplemented rats but not in those from the choline-deficient rats. Subthreshold stimulus trains led to LTP induction in slices from prenatally choline-supplemented rats only. These observations indicate that prenatal dietary manipulation of the amino acid, choline, leads to subsequent significant alterations of LTP induction threshold in adult animals.

Homocysteine, Folate, Vitamin B&lt;SUB&gt;6&lt;/SUB&gt;, and Cardiovascular Disease

The importance of hyperhomocysteinemia in the pathogenesis of arteriosclerosis was first recognized by study of vascular pathology in children with homocystinuria caused by 2 different enzymatic abnormalities of homocysteine metabolism. 1 Homocystinuria caused by deficiency of cystathionine synthase, a pyridoxal phosphate-dependent enzyme, is characterized by vascular abnormalities and frequent arterial and venous thromboses. 2 In 1968 a 2-month-old boy with a rare form of homocystinuria caused by deficiency of methyltetrahydrofolate homocysteine methyl transferase, a cobalamindependent enzyme, was discovered to have rapidly progressive arteriosclerosis. 1 Because of the different metabolic patterns caused by these 2 enzymatic abnormalities, it was suggested that homocysteine causes arteriosclerotic plaques by a direct effect on the cells and tissues of the arteries. A third enzyme abnormality leading to homocystinuria, deficiency of methylenetetrahydrofolate reductase, a folate-dependent enzyme, was found to cause arteriosclerotic plaques, 3 also supporting the conclusion that homocysteine is atherogenic. This interpretation explains the origin of arteriosclerosis observed in vitamin B6‐deficient monkeys and choline-deficient rats, 2 important animal models in which hyperhomocysteinemia also leads to atherogenesis. The atherogenic effect of homocysteine was subsequently demonstrated experimentally by parenteral and alimentary administration of the amino acid to rabbits 4,5 and baboons. 6

Choline Deficiency Augments and Antibody to Tumor Necrosis Factor‐α 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.

Simultaneous temporal processing is sensitive to prenatal choline availability in mature and aged rats.

Rats were trained at 2-4 months and at 24-26 months of age on a peak-interval timing procedure in which auditory and visual stimuli signaled two different fixed-interval schedules of reinforcement (15 and 30 s) that were presented simultaneously in a hierarchical fashion. Compared with control rats, increases in the probability of attention to the 15 s signal were observed for both the choline-supplemented and the choline-deficient rats. In contrast, an increase in attention to the 30 s signal was only observed for the choline-supplemented rats, whereas choline-deficient rats exhibited a decrease in attention that increased with age. Proportional rightward shifts in the remembered times of reinforcement emerged for the 24-26-month-old rats in the choline-deficient and control groups, but not in the choline-supplemented group. These results indicate that prenatal choline supplementation facilitates cognitive function across the lifespan, whereas prenatal choline deficiency impairs divided attention and accelerates age-related declines in temporal processing.

Effects of eicosapentaenoic acid on blood rheology in rats with fatty liver

A blood rheology study was conducted using Kikuchi's microchannel method in male Sprague Dawley rats with fatty liver induced by choline deficiency. Effects of eicosapentaenoic acid (EPA) on blood rheology were also evaluated. Male Sprague Dawley rats given normal feed served as the control. One group was given choline-deficient feed for 4 weeks (EPA (−) group), while another was given EPA (1000 mg/kg) once daily for 4 weeks together with choline-deficient feed (EPA (+) group). The microchannel passage time was determined using 100 mL of whole blood during observation under a microscope with a video camera. The passage time increased significantly in the EPA (−) group compared with the control group, while it was significantly shorter in the EPA (+) group compared with the EPA (−) group. Changes in platelet aggregation and leukocyte adhesion were less in the EPA (+) group than in the EPA (−) or the control groups. EPA supplementation appeared to correct blood flow changes induced by choline deficiency in rats.

Hepatic protein kinase C is not activated despite high intracellular 1,2- sn-diacylglycerol in obese Zucker rats

High intracellular 1,2,- sn-diacylglycerol (DAG) usually activates protein kinase C (PKC). In choline-deficient Fischer 344 rats, we previously showed that fatty liver was associated with elevated hepatic DAG and sustained activation of PKC. Steatosis is a sequelae of many liver toxins, and we wanted to determine whether fatty liver is always associated with accumulation of DAG with activation of PKC. Obese Zucker rats had 11-fold more triacylglycerol in their livers and 2-fold more DAG in their hepatic plasma membrane than did lean control Zucker rats. However, this increased diacylglycerol was not associated with translocation or activation of PKC in hepatic plasma membrane (activity in obese rats was 897 pmol/mg protein×min −1 vs. 780 pmol/mg protein×min −1 in lean rats). No differences in PKC isoform expression were detected between obese and lean rats. In additional studies, we found that choline deficiency in the Zucker rat did not result in activation of PKC in liver, unlike our earlier observations in the choline deficient Fischer rat. This dissociation between fatty liver, DAG accumulation and PKC activation in Zucker rats supports previous reports of abnormalities in PKC signaling in this strain of rats.

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.

Nascent VLDL phospholipid composition is altered when phosphatidylcholine biosynthesis is inhibited: evidence for a novel mechanism that regulates VLDL secretion

Previous work has shown that inhibition of phosphatidylcholine biosynthesis inhibits very low density lipoprotein (VLDL) secretion by causing a decrease in the number of particles in the Golgi but not in the endoplasmic reticulum of rat liver (Verkade et al. (1993) J. Biol. Chem. 268, 24990–24996). One explanation for this observation was that VLDL from choline deficient livers was degraded in a post-endoplasmic reticulum compartment. This hypothesis was supported by experiments in which choline deficient (CD) or choline supplemented (CS) rat hepatocytes were incubated ± Brefeldin A. In the presence of Brefeldin A, VLDL secretion was blocked, but no difference was observed in the degradation of apolipoprotein B (apoB) within the CD or CS cells. If increased catabolism of apoB were occurring in the endoplasmic reticulum of CD hepatocytes, enhanced degradation of apoB in CD cells might have been expected. Inhibition of phosphatidylcholine biosynthesis also caused decreases in the phosphatidylcholine content of membranes of the secretory pathway. The lipids of nascent VLDLs from the lumina of endoplasmic reticulum and Golgi prepared from CD rat liver were relatively enriched in phosphatidylethanolamine and depleted of phosphatidylcholine when compared to samples from CS liver. The changes in nascent VLDL phospholipid composition mimicked that of the organelle membranes from which the VLDLs were isolated. Possibly the phospholipid composition of the organelles is a factor in determining the final phospholipid composition of VLDLs. One hypothesis is that when phosphatidylcholine biosynthesis is impaired, nascent VLDL is assembled incorrectly and degraded by a quality control protease in a post-endoplasmic reticulum compartment.

Nicotinamide Megadosing Increases Hepatic Poly(ADP-Ribose) Levels in Choline-Deficient Rats

Previous work in our laboratory has shown that dietary megadoses of nicotinamide, used in the prevention of diabetes, cause increases in hepatic poly(ADP-ribose). Poly(ADP-ribose) is synthesized from NAD+ by a nuclear enzyme, poly(ADP-ribose)poly-merase, which is activated by DNA strand breaks. The nicotinamide-induced increase in poly(ADP-ribose) could result from an increase in substrate, NAD+, or the induction of strand breaks in DNA. Strand breaks may result from the depletion of single carbon groups, through the excretion of methylated derivatives of nicotinamide. To differentiate between these mechanisms, a 3 ՠ3 factorial experiment was conducted in which rats were fed diets containing various supplements of choline bitartrate (0, 2, 20 g/kg diet) and nicotinamide (0, 1, 2 g/kg diet). At the conclusion of treatments, blood NAD+ and liver lipid, NAD+ and poly(ADP-ribose) levels were determined. Choline deficiency caused the characteristic accumulation of fat in the liver at all levels of nicotinamide. In choline deficient rats, nicotinamide supplements further increased liver lipid concentration. Blood and liver NAD+ concentrations were increased by nicotinamide supplementation, irrespective of choline status. In contrast, liver poly(ADP-ribose) levels were increased by nicotinamide supplementation only in choline deficient rats. These results show that nicotinamide-induced increases in poly(ADP-ribose) levels appear to be dependent on decreased methyl donor status and suggest that adequate choline status is important for preventing some deleterious effects of nicotinamide treatment.

Lectin histochemistry of lipofuscin and certain ceroid pigments

Little is known at present about the saccharide components of lipofuscin (age pigment) and ceroid pigments in situ. The purpose of this study was, therefore, to study in detail the lectin reactivities of lipofuscin in neurons and cardiac myocytes of old humans and rats. In addition, those of diverse ceroid pigments found in human aortic atheromas, in the livers of choline-deficient rats, in the uteri of vitamin E-deficient rats and in the crushed epididymal fat pad of rats, are included. Cryostat and deparaffinized sections from all these tissues were either extracted with a solvent mixture of chloroform-methanol-water (10:10:3, v/v) and incubated with 7 different biotinylated lectins or left untreated. Delipidation was done in order to study whether it was possible to discriminate between the saccharide moieties of glycolipids and glycoproteins of lipofuscin and ceroid pigments in situ. Other similarly treated sections were used to study the autofluorescence, sudanophilia, acid-fastness and reactivity to PAS. The frequency and intensity of lectin binding and standard histochemical properties of all the pigments were evaluated semi-quantitatively and blind. The results indicated that mannose was in general the most consistently detected sugar residue in lipofuscin granules of humans and rats, and that this pigment may also contain acetylglucosamine, acetylgalactosamine, sialic acid, galactose and fucose. However, notable differences were found not only in the lipofuscin saccharide components of different cell types of humans and rats, but also in those in the same type of cells in both species. Although mannose was not detected in the hepatic ceroid of choline-deficient rats, this saccharide moiety was almost always present in the other ceroid pigments. Each of the ceroids also contained other types of saccharides although the frequency of the latter varied between different ceroid pigments. While lipofuscin and each of the ceroid pigments showed somewhat different lectin binding patterns, the variability in the frequency of reactivity to lectins suggests that these patterns may not be permanent but transient. In this sense, it appears that lectin histochemistry may not allow these pigments to be differentiated. Furthermore, the extractive procedures used in this study did not enable us to determine whether the saccharides detected in the pigments in situ corresponded to glycolipids or glycoproteins.

The effect of choline and myo-inositol on liver and carcass fat levels in aerobically trained rats.

Choline and myo-inositol are dietary supplements ingested under the premise that they facilitate the burning of stored fat. Choline and myo-inositol have been shown to prevent abnormal or excessive liver accumulation of cholesterol and triglycerides in choline and myoinositol deficient rats. The current study was designed to determine whether the consumption of choline and myoinositol by non-deficient aerobically trained rats affects the percent liver and carcass fat. Nineteen rats were trained aerobically for ten weeks then randomly assigned to an experimental group fed choline and myo-inositol mixed with their chow, or a control group fed only chow. Rats were sacrificed after 24 more days of aerobic training. Percent carcass and liver fat were determined by a lipid extraction procedure. There was a significant difference in the percent liver fat between groups, with the experimental group having less fat (6.69 +/- 2.23 vs 9.22 +/- 2.91 percent fat; r = 0.05). Percent carcass fat was not significantly different. There was a significant difference in the amount of weight gained during the 24 days of treatment, with the experimental group gaining less weight (5.1 +/- 9.2 vs 11.8 +/- 3.1 g; r < 0.05). The lack of an effect on percent carcass fat indicates that choline plus myoinositol supplements do not reduce adipose tissue mass but can inhibit weight gain while decreasing liver fat.

Choline and hepatocarcinogenesis in the rat.

Rats fed a choline deficient diet develop foci of enzyme-altered hepatocytes with subsequent formation of hepatic tumors. This is the only nutritional deficiency that, in itself, causes cancer. We suggested that carcinogenesis is triggered, in part, because of abnormalities in cell signals which regulate cell proliferation and cell death. Because choline deficient rats develop fatty liver (choline is needed for hepatic secretion of certain lipoproteins), we examined whether an important lipid second messenger involved in proliferative signaling, 1,2-sn-diacylglycerol, accumulated in liver and resulted in the prolonged activation of protein kinase C. We observed that 1,2-sn-diacylglycerol accumulated in the plasma membrane from the non-tumor portion of livers of rats fed a choline deficient diet, and that unsaturated free fatty acids, another activator of protein kinase C, also accumulated in deficient livers. Protein kinase C in the hepatic plasma membrane and nucleus of choline deficient rats was elevated for months; this is the only model system which exhibits such prolonged activation of protein kinase C. Premalignant, abnormal hepatic foci were detected only in the deficient rats, and 15% of deficient rats (none of the controls) had hepatocellular carcinoma at 1 year on the diet. In rats, an early event in choline deficiency is an increase in the rate of cell death. In liver from choline deficient rats, we observed an increase in the numbers of liver cells with fragmented DNA (characteristic of programmed cell death; apoptosis). We used a cell culture model (immortalized rat hepatocytes) to study the effects of choline deficiency on apoptosis. Liver cells grown in a choline deficient medium became depleted of choline, accumulated triacylglycerol and 1,2-sn-diacylglycerol, and had increased DNA fragmentation and other morphologic and biochemical changes associated with apoptosis. This model has great potential as a tool for studying the underlying link between choline deficiency and the regulation of the balance between cell proliferation and cell death. We suggest that choline deficiency altered the cell proliferation signals mediated by protein kinase C within liver, and altered cell apoptosis. These changes in cell signaling may be the triggering events which result in hepatic carcinogenesis.

Severe Folate Deficiency Causes Secondary Depletion of Choline and Phosphocholine in Rat Liver

It has previously been shown that choline deficiency causes depletion of hepatic folate concentration in rats. Two separate experiments were undertaken to investigate the converse phenomenon: whether folate deficiency would lead to depletion of hepatic choline. In Experiment 1, severe folate deficiency was induced in rats by feeding an amino acid-defined diet containing (per kg diet) 1.4 g choline, 0 mg folate and 10 g succinylsulfathiazole. Control rats were fed the same diet containing 8 mg folate/kg. After 4 wk, plasma and hepatic folate concentrations were significantly depleted in the severely folate-deficient rats compared with controls (P < 0.001), and hepatic choline and phosphocholine concentrations were 65 and 80% lower, respectively (P < 0.001). In Experiment 2, moderate folate deficiency was induced in rats by feeding the same diet as described above, but with the succinylsulfathiazole omitted. After 24 wk, significant systemic folate deficiency was present in the moderately folate-deficient rats compared with controls (P < 0.001). A modest reduction (36%, P = 0.087) in hepatic choline concentration was observed in the moderately folate-deficient rats compared with controls. No significant differences in hepatic phosphocholine concentrations were detected between the two groups. These results indicate that severe folate deficiency causes secondary hepatic choline deficiency in rats.

Restricting Food Intake Does Not Exacerbate the Effects of a Choline-Deficient Diet on Tissue Carnitine Concentrations in Rats

Previous reports in animals indicate that choline deficiency alters carnitine metabolism. Recent studies in humans suggest that choline deficiency occurs in individuals during long term total parenteral nutrition. Malnutrition is also a frequent complication in this population. We therefore examined the effect of restricting the intake of a choline-deficient diet on carnitine concentrations in plasma and tissues. Adult male rats were randomly assigned to one of four dietary regimens: control, choline deficient, restricted control (85% of control), or restricted choline deficient for 42-43 d. At the end of the experimental period, restricted animals weighed significantly less than their respective controls (P < 0.01). Liver weight relative to body weight and fat concentration were greater in choline-deficient animals (P < 0.01 and 0.001, respectively). Choline-deficient rats fed free access had elevated plasma carnitine concentration (P < 0.01). Urinary carnitine excretion was elevated in both groups of choline-deficient rats (P < 0.01), while liver, heart and muscle carnitine concentrations were lower than in controls (P < 0.05). Restricting dietary intake reduced plasma carnitine concentration in choline-deficient animals (P < 0.01), but did not alter tissue or urine carnitine concentrations in either group. Restricted, choline-deficient animals did not exhibit a worsening of the sequelae of choline deficiency. We conclude that choline deficiency alters carnitine concentrations in plasma and tissues and that restricting the intake of a choline-deficient diet does not alter this effect in tissues.