Biomedical subjects

S H Zeisel

Publications and source records attributed to S H Zeisel.

At least 37 records · Page 2Linked to original sources

PubMed1997

Choline deficiency selects for resistance to p53-independent apoptosis and causes tumorigenic transformation of rat hepatocytes.

The mechanisms which drive initiated cells to progress to form carcinomas are poorly understood. CWSV-1 rat hepatocytes, in which p53 protein is inactivated by SV40 large T antigen, respond by inducing p53-independent apoptosis when acutely switched to medium containing low choline (16% apoptotic at 48 h in 5 microM choline) as compared with controls (1% apoptotic at 48 h in 70 microM choline). The rate of apoptosis was inversely correlated with cellular phosphatidylcholine content. Choline deficiency (CD)-induced apoptosis is probably mediated by TGFbeta1 and reactive oxygen species, since immunoneutralization of TGFbeta1 in the medium or treatment with N-acetylcysteine (an antioxidant) or addition of neocuproine (a transition metal chelator) prevented CD-induced apoptosis. CWSV-1 hepatocytes could be gradually adapted to survive in 5 microM choline. CD-adapted cells had increased membrane phosphatidylcholine concentrations (compared with acute CD cells). Adapted cells acquired relative resistance to CD-induced apoptosis (7% of adapted cells compared with 19% of non-adapted cells were apoptotic at 48 h in 5 microM choline). They also became relatively resistant to another p53-independent form of apoptosis (TGFbeta1-induced). CD-adapted hepatocytes developed increased capability for anchorage-independent growth and formed tumors when transplanted into nude mice; passage-matched control hepatocytes did not possess these properties. Cell transformation was dependent on exposure to the selective pressure of CD apoptosis, as we observed that when CD apoptosis was inhibited with an antioxidant during adaptation, cells did not become anchorage independent. Acquisition by p53-deficient cells of resistance to p53-independent inducers of apoptosis (CD, TGFbeta1 and reactive oxygen species) may leave cells without another important apoptotic defensive barrier and may be responsible for the progression of initiated cells to frank carcinomas.

Animals↗

PubMed1997

Choline deficiency causes increased localization of transforming growth factor-beta1 signaling proteins and apoptosis in the rat liver.

Dietary restriction is known to decrease cell proliferation and increase apoptosis in the liver; however, the role of withdrawal of single dietary factors on cells of the liver is less well understood. In this study, we investigated the effects of short-term choline deficiency (CD; also for choline deficient) on cell survival, proliferation and the expression of proteins related to the transforming growth factor-beta1 (TGF-beta1) growth-inhibitory signaling pathway in the liver. In animals fed a CD diet for 6 weeks, classical apoptotic bodies were detected in 0.28 +/- 0.04% of hepatocytes in CD livers compared to 0.096 +/- 0.006% of hepatocytes in control rats fed a choline-sufficient (CS) diet. These classical apoptotic cells exhibited DNA fragmentation when probed with an in situ end-labeling immunohistochemical method; TUNEL-positive nuclei were also seen in hepatocytes in CD livers which had accumulated large amounts of lipid, consistent with the known DNA-damaging effects of CD. In CS control livers, TGF-beta1 protein was found only in bile duct epithelium and nonparenchymal-type cells, and not in hepatocytes. However, the majority of hepatocytes in the CD liver expressed high levels of TGF-beta1 protein, as well as TGF-beta1 receptor types I and II. Nuclear localization of p27Kip1 protein, which may link TGF-beta1 expression to apoptosis, showed a 10-fold increase in CD hepatocytes (4.1 +/- 1.1 vs. 0.35 +/- 0.04% of cells) compared to controls. In addition, there was a 5-fold increase (0.54 +/- 0.031 vs. 0.011 +/- 0.007% of cells) in the mitotic index in CD-compared control livers. We conclude that feeding a CD diet for 6 weeks induces apoptosis in hepatocytes in the whole rat liver and that this form of cell death appears to be mediated, in part, by TGF-beta1 and related proteins.

Activin Receptors, Type I↗

PubMed1997

Choline: essential for brain development and function.

Pregnancy and lactation are periods when maternal reserves of choline are depleted. At the same time, the availability of choline for normal development of brain is critical. Variations in choline intake by mothers influence memory performance in their offspring. The mechanisms for this potent effect of choline are not yet elucidated but may involve changes in acetylcholine synthesis, transmembrane signal transduction, or regulation of apoptosis. These observations are important for the obstetrician and pediatrician as they consider the ideal dietary intake for mothers and their children.

Acetylcholine↗

PubMed1996

Choline and choline esters in human and rat milk and in infant formulas.

Large amounts of choline are required in neonates for rapid organ growth and membrane biosynthesis. Human infants derive much of their choline from milk. In our study, mature human milk contained more phosphocholine and glycerophosphocholine than choline, phosphatidylcholine, or sphingomyelin (P < 0.01). Previous studies have not recognized that phosphocholine and glycerophosphocholine exist in human milk. Concentrations of choline compounds in mature milk of mothers giving birth to preterm or full-term infants were not significantly different. Infant formulas also contained choline and choline-containing compounds. In infant formulas derived from soy or bovine milk, unesterified choline, phosphocholine, glycerophosphocholine, phosphatidylcholine, and sphingomyelin concentrations varied greatly. All infant formulas contained significantly less phosphocholine than did human milk. Soy-derived formulas contained significantly less glycerophosphocholine (P < 0.01) and phosphocholine (P < 0.01) and more phosphatidylcholine (P < 0.01) than did human or bovine milk or bovine milk-derived infant formulas. Rat milk contained greater amounts of glycerophosphocholine (almost 75% of the total choline moiety in milk) and phosphocholine than did human milk. When dams were provided with either a control, choline-deficient, or choline-supplemented diet, milk composition reflected the choline content of the diet. Because there are competing demands for choline in neonates, it is important to ensure adequate availability through proper infant nutrition. Although the free choline moiety is adequately provided by infant formulas and bovine milk, reevaluation of the concentrations of other choline esters, in particular glycerophosphocholine and phosphocholine, may be warranted.

Adolescent↗

PubMed1996

Choline deficiency induces apoptosis in SV40-immortalized CWSV-1 rat hepatocytes in culture.

Immortalized CWSV-1 rat hepatocytes, in which p53 protein is inactivated by SV40 large T antigen, had increased numbers of cells with strand breaks in genomic DNA (terminal dUTP end labeling) when grown in 0 Micron choline (67-73% of cells) than when grown in 70 Micron choline (2-3% of cells). Internucleosomal fragmentation of DNA (DNA ladders) was detected in cells grown with 5 Micron and 0 Micron choline for 72h. Cells treated with 0 or 5 Micron choline for 72h detached from the substrate in high numbers (58% of choline deficient cells vs. 1.4% of choline sufficient cells detached) exhibited a high incidence of apoptosis (apoptotic bodies were seen in 55-75% of cells; 67-73% had DNA strand breaks), and an absence of mitosis and proliferating cell nuclear antigen (PCNA) expression. Cells undergoing DNA fragmentation had functioning mitochondria. At 24h, cells grown in 0 or 5 Micron choline synthesize DNA more rapidly than those grown in 70 Micron choline. By 72h, the cells grown in 0 or 5 Micron choline were forming DNA much more slowly than control cells (assessed by thymidine incorporation, PCNA expression, and mitotic index). Western blot analysis showed that p53 in the nucleus of cells was detected in direct association with SV40 T-antigen, and was therefore likely to be inactive. We conclude that choline deficiency kills CWSV-1 hepatocytes in culture by inducing apoptosis via what may be a p53-independent process, and that this process begins in viable cells before they detach from the culture dish.

Animals↗

PubMed1996

Choline. A nutrient that is involved in the regulation of cell proliferation, cell death, and cell transformation.

Choline deficiency causes hepatocyte proliferation, apoptosis and transformation. Thus, it is an excellent model in which to study the molecular mechanisms underlying these processes. Several interesting questions can be addressed. What is the first event that begins the cells on the path towards transformation? Is it triggered by some autocrine factor produced in choline depleted cells? Does it involve alteration of DNA structure with subsequent apoptosis, compensatory cell proliferation, and enhanced survival of preneoplastic cells? Is there a specific choline deficiency signal which triggers apoptosis, with subsequent compensatory cell proliferation in a methyl-deficient environment causing hypomethylation of DNA? Does this result in abnormal transcription of genes with resulting transformation? Or is the activation of PKC the first event? PKC-mediated cell proliferation might then be balanced by down regulation of growth factor response, withdrawal of which causes apoptosis. The ensuing high rate of cell turnover might result in the survival and replication of preneoplastic cells. Multiple alternative variations of these questions exist. Whatever the critical first event is, our models also allow us to ask about molecular differences between cells that pass through these early events and those that do not. At first glance, choline deficiency may seem to be an artificial situation that might rarely occur in nature. However, the answers to some of the above questions will help us to understand how changes in gene expression and the signaling pathways that are fundamental for many cell functions, might be involved in liver cell proliferation, death and transformation.

Animals↗

PubMed1995

Nutrients, signal transduction and carcinogenesis.

Choline phospholipids play major roles in cellular regulation in addition to their essential function as structural components of membranes and lipoproteins. The unique functions of choline phospholipids as hormones (platelet activating factor, 1-alkyl, 2-acetylphosphatidylcholine, PAF) and sources (phosphatidylcholine, sphingolipids) of second messengers (sphingosine, diacylglycerol, lysophospholipids, arachidonic acid and its metabolites) may explain how dietary choline influences normal physiological processes as well as a diverse group of pathological processes, including carcinogenesis.

Animals↗

PubMed1995

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.

Animals↗

PubMed1995

Radioactive choline metabolism in guinea pig gallbladder. Is there measurable acetylcholine release?

Acetylcholine may be released from gallbladder intrinsic nerves in response to cholecystokinin stimulation. This study characterized metabolites of [14C]choline produced in the gallbladder and released during incubation, with or without cholecystokinin-octapeptide. Radiolabeled [14C]choline was applied to the mucosal or muscle surface of intact guinea pig gallbladders in an organ bath. After radiolabeling, gallbladders were incubated with or without the contractile agonist cholecystokinin-octapeptide. Metabolites of [14C]choline were identified in gallbladder tissue and incubation buffers using HPLC and thin-layer chromatography. The major metabolites of [14C]choline were betaine and phosphocholine. [14C]Phosphocholine was incorporated slowly into [14C]phosphatidylcholine. [14C]Choline was released into buffers during incubation. [14C]Acetylcholine constituted less than 1% of radiolabel in the gallbladder. There was no identifiable [14C]acetylcholine released in buffers. Cholecystokinin-octapeptide did not affect choline metabolism. These studies showed that choline in the gallbladder is metabolized along pathways similar to those in the liver. Gallbladders released mostly choline, rather than acetylcholine, even during hormonally induced contraction.

Acetylcholine↗

PubMed1995

Effects of prolonged (1 year) choline deficiency and subsequent re-feeding of choline on 1,2-sn-diradylglycerol, fatty acids and protein kinase C in rat liver.

Rats fed a choline-deficient diet develop foci of enzyme-altered hepatocytes with subsequent formation of hepatic tumors. They also develop fatty livers, because choline is needed for hepatic secretion of lipoproteins. We have previously reported that 1,2-sn-diradylglycerol accumulates in the livers of rats fed a choline-deficient diet for 1-27 weeks, and that protein kinase C activity in the hepatic plasma membrane is elevated during that time (da Costa et al., J. Biol. Chem., 268, 2100-2105, 1993). In the present study, we examined the changes that occur in rat liver at 52 weeks of choline deficiency and determined whether these changes were reversible when choline was returned to the diet of the deficient animals for 1 or 16 weeks. At 52 weeks, non-tumor liver samples from the experimental animals had increased 1,2-sn-diradylglycerol concentrations in the lipid droplets compared with control animals. Plasma membrane 1,2-sn-diradylglycerol levels in the liver did not differ between the two groups, but an age-related increase in membrane 1,2-sn-diradylglycerol concentrations was observed. Unsaturated free fatty acids, another activator of protein kinase C, accumulated in the deficient livers. Protein kinase C activity associated with the plasma membrane remained significantly elevated at 52 weeks in deficient livers. Hepatic foci expressing gamma-glutamyltranspeptidase were detected only in the deficient rats (0.83% of liver volume) and 15% of these rats had hepatocellular carcinoma at 1 year on the diet. At 53 weeks (1 week after choline was returned to the deficient group), 1,2-sn-diradylglycerol concentrations in the lipid droplets and hepatic free fatty acids had dropped to control levels. By 68 weeks (16 weeks of re-feeding choline), the membrane protein kinase C activity had returned to normal. At this time, 14% of the experimental animals had hepatocellular carcinoma. We suggest that choline deficiency altered the protein kinase C-mediated signal transduction within liver and this contributed to hepatic carcinogenesis in these animals.

Aging↗

PubMed1995

Choline distribution and metabolism in pregnant rats and fetuses are influenced by the choline content of the maternal diet.

Choline supplementation of pregnant rats between d 12 and 17 of pregnancy permanently enhances the spatial memory of offspring; however, the mechanism is unknown. We examined the effect of choline supplementation on metabolism of orally ingested choline by nonmated rats and pregnant rats and their fetuses. We studied the metabolism of an acute oral dose of 14C-choline chloride in pregnant and nonmated rats with and without choline supplementation (25 mmol/L choline chloride in water) on d 12-17 of pregnancy. During the first 2 h after oral dosing, plasma radiolabeled choline was detectable, whereas plasma choline metabolites contributed little to total radioactivity at any time. The pattern of accumulation of label in placentas was similar in all groups. Fetal tissues (i.e., brain, liver and carcass remnant) contained primarily 14C-phosphatidylcholine and 14C-phosphorylcholine. Also, we examined the fetal tissue distribution of isotopically labeled (deuterated) choline derived from the diet and from the dietary choline supplement. The distribution patterns for radiolabeled choline metabolites in fetuses of supplemented dams accumulated significantly (P < 0.01) more of their total choline and its metabolites than fetuses of control dams during d 12-17 of gestation (50 vs. 20%). In fetuses from supplemented dams, betaine concentrations were greater than in fetuses from control dams in all organs assayed (by 36-57%). Phosphorylcholine concentrations in brain of fetuses from supplemented dams were also greater. These experiments identify potential metabolites of choline that might mediate the observed effects on brain development in the rats.

Administration, Oral↗

PubMed1995

Pregnancy and lactation are associated with diminished concentrations of choline and its metabolites in rat liver.

Choline is an important nutrient that is actively transported from mother to fetus across the placenta and from mother to infant across the mammary gland. Thus, pregnancy and lactation are times when dietary requirements for choline may be increased. Pregnant rats eating AIN-76A diet (with and without choline) for 6 d (d 12-18 gestation) were compared with nonmated female and male rats eating the same diets. Similarly, lactating rats were compared with nonmated female rats, both groups eating these same diets for 25 d (gestation d 12-postpartum d 15). We measured choline and choline metabolites in livers on the last day of feeding. Nonmated female rats, eating the control diet, had higher hepatic choline metabolites concentrations than did male rats (choline, 98%; betaine, 96%; and phosphorylcholine, 55% higher), pregnant rats (phosphorylcholine, 47%; and betaine, 42% higher) or lactating rats (phosphorylcholine, 49%; phosphatidylcholine, 37%; and betaine, 273% higher). We found that nonmated females eating a choline deficient diet had only a modest diminution (33%) of the labile choline metabolite PCho in liver, compared with similar rats eating a control diet. When compared with similar rats fed a choline-adequate diet, pregnant rats fed a choline-deficient diet had significantly great diminution of hepatic phosphorylcholine (83% lower) than did nonmated females. Liver phosphorylcholine was only 12% lower than in controls in nonmated females fed the deficient diet for the same 25-d period. Lactating rats were the most sensitive to choline deficiency, with liver phosphorylcholine 88% lower than in similar rats fed control diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

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.

Animals↗

PubMed1994

Lecithin and choline in human health and disease.

Choline is involved in methyl group metabolism and lipid transport and is a component of a number of important biological compounds including the membrane phospholipids lecithin, sphingomyelin, and plasmalogen; the neurotransmitter acetylcholine; and platelet activating factor. Although a required nutrient for several animal species, choline is not currently designated as essential for humans. However, recent clinical studies show it to be essential for normal liver function. Additionally, a large body of evidence from the fields of molecular and cell biology shows that certain phospholipids play a critical role in generating second messengers for cell membrane signal transduction. This process involves a cascade of reactions that translate an external cell stimulus such as a hormone or growth factor into a change in cell transport, metabolism, growth, function, or gene expression. Disruptions in phospholipid metabolism can interfere with this process and may underlie certain disease states such as cancer and Alzheimer's disease. These recent findings may be appropriate in the consideration of choline as an essential nutrient for humans.

Animals↗

PubMed1994

Choline and human nutrition.

Choline is crucial for sustaining life. It modulates the basic signaling processes within cells, is a structural element in membranes, and is vital during critical periods in brain development. Choline metabolism is closely interrelated with the metabolism of methionine and folate. We believe that the normal human diet provides sufficient choline to sustain healthy organ function. However, vulnerable populations may become choline deficient, including the growing infant, the pregnant or lactating woman, the cirrhotic, and the patient fed intravenously. Further studies of choline requirements in these groups are required.

Animals↗

PubMed1993

Characterization of choline metabolism and secretion by human placental trophoblasts in culture.

Choline is an essential nutrient for fetal development and may be utilized to form phospholipids such as phosphatidylcholine and sphingomyelin; to synthesize the neurotransmitter, acetylcholine; and to donate methyl groups after being oxidized to betaine. Since the majority of choline required for fetal growth must be transported by the placenta from the maternal circulation, we examined the ability of isolated human trophoblasts to metabolize choline and to release choline and its metabolites into culture medium. Cytotrophoblasts were isolated from normal, full-term human placentas and incubated with [14C]choline for 3 h; the cells were washed to remove extracellular radiolabel, and the changes in intracellular and medium choline pools were followed for an additional 24 h. During the incubation, choline rapidly reached steady state intracellularly and label was incorporated into betaine, phosphocholine, cytidylyldiphosphocholine, phosphatidylcholine, glycerophosphocholine, lysophosphatidylcholine, and sphingomyelin. All labeled choline metabolites in cells, except glycerophosphocholine, decreased at 6 and 27 h of incubation (3 and 24 h, respectively, after labeled choline was removed), and labeled metabolites appeared in media. By 24 h after labeled choline was removed, the major labeled metabolites in the media were choline (82%), betaine (11%), and glycerophosphocholine (5%). Small amounts of phosphatidylcholine (1%), and lysophosphatidylcholine (1%) were found. Acetylcholine was a very minor choline metabolite in these cells. When placental cells were incubated for 66 h after isolation, they formed syncytiotrophoblasts, which incorporated labeled choline into metabolites in a similar pattern to cytotrophoblasts. These data indicate that isolated trophoblast cells can metabolize choline to form all of its major metabolites and that several metabolites are released to the medium in significant amounts. Thus, our data suggest that the major metabolite supplied to the fetus may be choline, but that betaine and glycerophosphocholine may also be vehicles for transfer of choline equivalents from mother to fetus.

Betaine↗

PubMed1993

Choline phospholipids: signal transduction and carcinogenesis.

Phospholipids act as vital elements in transmembrane signaling. Agonist-induced hydrolysis of phosphatidylinositides has been established as a major mechanism for transmitting messages into the interior of cells via protein phosphorylation cascades, ultimately regulating gene transcription. There is a growing body of evidence that choline phospholipids (phosphatidylcholine, sphingomyelin, and their metabolites) also are important mediators and modulators of transmembrane signaling. These functions may explain how choline phospholipids influence normal physiological processes as well as a diverse group of pathological processes.

Animals↗

PubMed1993

Accumulation of 1,2-sn-diradylglycerol with increased membrane-associated protein kinase C may be the mechanism for spontaneous hepatocarcinogenesis in choline-deficient rats.

Choline deficiency, via deprivation of labile methyl groups, is associated with a greatly increased incidence of hepatocarcinoma in experimental animals. This dietary deficiency also causes fatty liver, because choline is needed for hepatic secretion of lipoproteins. We hypothesized that fatty liver might be associated with the accumulation of 1,2-sn-diradylglycerol and subsequent activation of protein kinase C. Several lines of evidence indicate that cancers might develop secondary to abnormalities in protein kinase C-mediated signal transduction. We observed that rats fed a choline-deficient diet for 1, 6, or 27 weeks had increased hepatic concentrations of 1,2-diradylglycerol. At 1 and 6 weeks, hepatic plasma membrane from choline-deficient rats had increased concentrations of 1,2-sn-diacylglycerol and 1-alkyl, 2-acylglycerol, with the latter accounting for 20-26% of membrane 1,2-sn-diradylglycerol (as compared with only 2-5% in controls). Protein kinase C activity was increased in hepatic plasma membrane at 1 week of choline deficiency. By Western blotting there was an increase in the amount of protein kinase C zeta and a decrease in the amount of protein kinase C delta in liver at 1 week. By 6 weeks of choline deficiency, hepatic plasma membrane and cytosolic protein kinase C (PKC) activities were increased significantly, with increased amounts of hepatic plasma membrane protein kinase C alpha, and delta detected by Western blotting. Glycogen synthase activity in liver was diminished after 1 week of choline deficiency; this enzyme is inhibited by PKC-mediated phosphorylation. We suggest that choline deficiency perturbed PKC-mediated transmembrane signaling within liver and that this contributed to the development of hepatic cancer in these animals.

Animals↗