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.

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 X min(-1) vs. 780 pmol/mg protein X 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.

Prolonged fasting in humans results in diminished plasma choline concentrations but does not cause liver dysfunction.

Choline is a major donor of methyl groups, a precursor for membrane synthesis, and a component of the neurotransmitter acetylcholine. Choline-deficient diets deplete humans of choline and cause hepatic dysfunction and steatosis. In this study we determined whether acute starvation also depletes choline, as indicated by changes in plasma choline or phosphatidylcholine. Healthy humans (n = 10) fasted for 7 d, ingesting only water and mineral-vitamin supplements. Their mean (+/- SEM) plasma choline concentration was 9.5 +/- 0.5 micromol/L at the start of the study and dropped to 7.8 +/- 0.3 micromol/L after 1 wk of fasting (P < 0.01). The plasma phosphatidylcholine concentration did not change significantly (2.2 +/- 0.1 mmol/L at the start of the study and 2.4 +/- 0.2 mmol/L after 1 wk of fasting). Capacity of the liver to secrete lipoproteins was not affected by prolonged fasting. The mean plasma concentration of low-density-lipoprotein cholesterol was 3.3 +/- 0.2 mmol/L (126 +/- 8 mg/dL) at the start of the study and 4.9 +/- 0.5 mmol/L (188 +/- 19 mg/dL) after 1 wk of fasting. Liver damage assessed by serum alanine aminotransferase activity occurred in only 1 of 10 subjects. We conclude that prolonged fasting in humans modestly diminished plasma choline but was not associated with signs of choline deficiency, such as perturbed lipoprotein secretion and liver damage.

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.

Evaluation of the potential of triethanolamine to alter hepatic choline levels in female B6C3F1 mice.

Triethanolamine (TEA), a widely used nongenotoxic alcohol-amine, has recently been reported to cause an increased incidence of liver tumors in female B6C3F1 mice, but not in males nor in Fischer 344 rats. Choline deficiency induces liver cancer in rodents, and TEA could compete with choline uptake into tissues. The potential of TEA to cause choline deficiency in the liver of these mice as a mode of tumorigenesis was investigated. Groups of female B6C3F1 mice were administered 0 (vehicle) or a maximum tolerated dosage (MTD) of 1000 mg/kg/day TEA (Trial I) and 0, 10, 100, 300, or 1000 mg/kg/day TEA (Trial II) in acetone vehicle via skin painting 5 days/week for 3 weeks. Female CDF(R) rats were also administered 0 or an MTD dosage of 250 mg/kg/day TEA (Trial II) in a similar manner. No clinical signs of toxicity were noted, and upon sacrifice, levels of hepatic choline, its primary storage form, phosphocholine (PCho), and its primary oxidation product, betaine, were determined. A statistically significant decrease in PCho and betaine, was observed at the high dosage (26-42%) relative to controls and a dose-related, albeit variable, decrease was noted in PCho levels. Choline levels were also decreased 13-35% at the high dose level in mice. No changes in levels of choline or metabolites were noted in treated rats. A subsequent evaluation of the potential of TEA to inhibit the uptake of (3)H-choline by cultured Chinese Hamster Ovary Cells revealed a dose-related effect upon uptake. It was concluded that TEA may cause liver tumors in mice via a choline-depletion mode of action and that this effect is likely caused by the inhibition of choline uptake by cells.

Choline availability alters embryonic development of the hippocampus and septum in the rat.

Choline availability in the diet during pregnancy alters fetal brain biochemistry with resulting behavioral changes that persist throughout the lifetime of the offspring. In the present study, the effects of dietary choline on cell proliferation, migration, and apoptosis in neuronal progenitor cells in the hippocampus and septum were analyzed in fetal brains at different stages of embryonic development. Timed-pregnant rats on day E12 were fed AIN-76 diet with varying levels of dietary choline for 6 days, and, on days E18 or E20, fetal brain sections were collected. We found that choline deficiency (CD) significantly decreased the rate of mitosis in the neuroepithelium adjacent to the hippocampus. An increased number of apoptotic cells were found in the region of the dentate gyrus of CD hippocampus compared to controls (5.5+/-0.7 vs. 1.9+/-0.3 apoptotic cells per section; p<0.01). Using a combination of bromodeoxyuridine (BrdU) labeling and an unbiased computer-assisted image analysis method, we found that modulation of dietary choline availability changed the distribution and migration of precursor cells born on E16 in the fimbria, primordial dentate gyrus, and Ammon's horn of the fetal hippocampus. CD also decreased the migration of newly born cells from the neuroepithelium into the lateral septum, thus indicating that the sensitivity of fetal brain to choline availability is not restricted to the hippocampus. We found an increase in the expression of TOAD-64 protein, an early neuronal differentiation marker, in the hippocampus of CD day E18 fetal brains compared to controls. These results show that dietary choline availability alters the timing of the genesis, migration, and commitment to differentiation of progenitor neuronal-type cells in fetal brain hippocampal regions known to be associated with learning and memory processes in adult brain.

Maternal dietary choline availability alters mitosis, apoptosis and the localization of TOAD-64 protein in the developing fetal rat septum.

Maternal changes in dietary choline availability alter brain biochemistry and hippocampal development in the offspring resulting in lifelong behavioral changes in the offspring. In order to better understand the relationship between maternal diet, brain cytoarchitecture and behavior, we investigated the effects of choline availability on cell proliferation, apoptosis and differentiation in the fetal rat brain septum. Timed-pregnant rats on day E12 were fed AIN-76 diet with varying levels of dietary choline for 6 days. We found that choline deficiency (CD) significantly decreased the rate of mitosis in the progenitor neuroepithelium adjacent to the septum. In addition, we found an increased number of apoptotic cells in the septum of CD animals compared to controls (3.5+/-0.5 vs. 1.7+/-0.5 apoptotic cells per section; p<0.05). However, CD had no effect on apoptosis in the indusium griseum (IG), a region of cortex dorsal to the septum. Using an unbiased image analysis method and a monoclonal antibody we found a decreased expression of the TOAD-64 kDa protein, a marker of commitment to neuronal differentiation during fetal development, in the dorsal lateral septum of CD animals. CD also decreased the expression of TOAD-64 kDa protein in the IG and cortical plate adjacent to the septum. These results show that dietary choline availability during pregnancy alters the timing of mitosis, apoptosis and the early commitment to neuronal differentiation by progenitor cells in regions of the fetal brain septum, as well as hippocampus, two brain regions known to be associated with learning and memory.

Apoptosis is induced by choline deficiency in fetal brain and in PC12 cells.

Treatment of rats with choline during critical periods in brain development results in long-lasting enhancement of spatial memory in their offspring. Apoptosis is a normal process during brain development, and, in some tissues, is modulated by the availability of the nutrient choline. In these studies, we examined whether availability of choline influences apoptosis in fetal brain and in the PC12 cell line derived from a rat pheochromocytoma. Timed-bred Sprague Dawley rats were fed a choline-deficient (CD), choline-control, or choline-supplemented (CS) diet for 6 days and, on embryonic day 18, fetal brain slices were prepared and apoptosis was assessed using terminal dUTP nucleotide end labeling (TUNEL) to detect DNA strand breaks and by counting of apoptotic bodies. TUNEL-positive cells were detected in 15.9% (P < 0.01), 8.7% and 7.2% of hippocampal cells from fetuses of dams fed the CD, control or CS diets, respectively. A similar inverse relationship between dietary intake of choline and TUNEL positive cells was detected in an area of cerebral cortex from these fetal brain slices. Counts of apoptotic bodies in fetal brain slices correlated inversely with choline intake of the mothers (6.2% (P < 0.01), 2.5% and 1.9% of hippocampal cells had apoptotic bodies in fetuses of dams fed the CD, control and CS diets, respectively). PC12 cells were grown in DMEM/F12 media supplemented with 70 microM choline or with 0 microM choline. The number of apoptotic bodies in PC12 cells increased when cells were grown in 0 microM choline medium (1.5%; P < 0.05) compared to 70 microM choline medium (0.55%). In PC12 cells, TUNEL labeling (DNA strand breaks) increased in choline deficient (13.5%, P < 0.05) compared to sufficient medium (5.0%). In addition, cleavage of genomic DNA-into 200 bp internucleosomal fragments was detected in choline-deficient cells. These results show that choline deficiency induces-apoptotic cell death in neuronal-type cells and in whole brain. We suggest that variations in choline availability to brain modulate apoptosis rates during development.

Exercise intensity-related responses of beta-endorphin and catecholamines.

Ten men and 10 women exercised on a bicycle ergometer for 20 min at 40, 60, and 80% maximal oxygen uptake (VO2max) to determine the relationship between plasma beta-endorphin, catecholamines, and exercise intensity. Compared to rest, plasma beta-endorphins were not significantly elevated during the 40 and 60% workloads (4.8 +/- 1.0 pmol.l-1 vs 3.8 +/- 0.7 and 6.3 +/- 0.9, respectively). In contrast, the 80% exercise significantly elevated endorphins to 16.1 +/- 4.0 pmol.l-1. Plasma norepinephrine concentrations were 0.30 +/- 0.04 ng.ml-1 at rest and increased with exercise intensity (40% = 0.60 +/- 0.05, 60% = 0.93 +/- 0.07, 80% = 2.00 +/- 0.14, VO2max = 2.55 +/- 0.14 ng.ml-1). Plasma epinephrine followed the same trend (rest = 0.07 +/- 0.01, 40% = 0.33 +/- 0.03, 60% = 0.49 +/- 0.02, 80% = 0.88 +/- 0.07, VO2max = 0.95 +/- 0.06 ng.ml-1). Norepinephrine was found to significantly correlate to endorphins (r = 0.499; P less than 0.02). Conversely, epinephrine was not correlated with beta-endorphin (r = 0.309; P greater than 0.05). The low correlation suggests a weak relationship between beta-endorphin and catecholamine responses during exercise. The results of this investigation suggest that the relationship between beta-endorphin and exercise intensity is curvilinear, with anaerobic activity producing the most significant endorphin response. It was also noted that the beta-endorphin response was not related to gender, but the amine response to exercise was gender-related, being greater for the men.

Cycloalkanones V: synthesis, distribution, and effects on triglyceride metabolism.

The 14-C-labeled 2,8-dibenzylcyclooctanone was synthesized to study its absorption, distribution, and excretion in rats. Maximum drug absorption from the GI tract occurred between 12 and 14 hr after administration. The major organs possessed maximum amounts of the drug in 1 hr, with the liver concentrating the most with 6.56% 14-C and the muscle mass reaching a maximum of 41% 14-C after 14 hr. The drug remained in the GI tract over the first 6 hr and was associated with the lipid and glycogen fractions. Eighty-seven percent was eliminated in the feces after 72 hr. 2,8-Dibenzylcyclooctanone caused a significant reduction in vitro of dihydroxyacetone phosphatase acyltransferase and sn-glycerol-3-phosphate acyltransferase, which is the proposed mechanism for the observed in vivo reduction of hepatic, intestinal, and serum triglycerides and total glycerolipids. In vivo administration of the drug resulted in a depression of liver acid phosphatidyl phosphatase, acid phosphatase and lipase, and adipose lipase. The drug increased the rates of excretion of exogenous cholesterol, palmitic acid, and progesterone.

Betaine in wine: answer to the French paradox?

In France, a diet high in saturated fat and cholesterol is associated with low coronary artery disease mortality and it may be that drinking wine is protective against ischemic heart disease. Recent studies suggest that high plasma homocysteine concentrations are an independent risk factor for coronary, cerebral and peripheral arterial occlusive diseases. One of several routes for metabolism of homocysteine involves methylation using betaine as the methyl donor. Betaine is often added to less expensive wine when beet sugar is used to increase alcohol content. We found that many commercial wines contain betaine; an average glass of wine contains approximately 3 mg betaine. This small amount is less than the dose used to lower homocysteine in patients with genetic forms of hyperhomocysteinemia, but we do not know whether humans with modest elevations of homocysteine would be influenced by this dose.

Diet, apoptosis, and carcinogenesis.

It is known that long-term withdrawal of choline from the diet induces hepatocellular carcinomas in animal models in the absence of known carcinogens. We hypothesize that a choline deficient diet (CD) alters the balance of cell growth and cell death in hepatocytes and thus promotes the survival of clones of cells capable of malignant transformation. When grown in CD medium (5 microM or 0 microM choline) CWSV-1 rat hepatocytes immortalized with SV40 large T-antigen underwent p53-independent apoptosis (terminal dUTP end-labeling of fragmented DNA; laddering of DNA in agarose gel). CWSV-1 cells which were adapted to survive in 5 microM choline acquired resistance to CD-induced apoptosis and were able to form hepatocellular carcinomas in nude mice. These adapted CWSV-1 cells express higher amounts of both the 32 kDa membrane-bound and 6 kDa mature form of TGF alpha compared to cells made acutely CD. Control (70 microM choline) and adapted cells, but not acutely deficient hepatocytes, could be induced to undergo apoptosis by neutralization of secreted TGF alpha. Protein tyrosine phosphorylation is known to protect against apoptosis. We found decreased EGF receptor tyrosine phosphorylation in acutely choline deficient CWSV-1 cells. TGF beta 1 is an important growth-regulator in the liver. CWSV-1 cells express TGF beta 1 receptors and this peptide induced cell detachment and death in control and acutely deficient cells. Hepatocytes adapted to survive in low choline were also resistant to TGF beta 1, although TGF beta 1 receptors and protein could be detected in the cytoplasm of these cells. The non-essential nutrient choline is important in maintaining plasma membrane structure and function, and in intracellular signaling. Our results indicate that acute withdrawal of choline induces p53-independent programmed cell death in hepatocytes, whereas cells adapted to survive in low choline are resistant to this form of apoptosis, as well as to cell death induced by TGF beta 1. Our results also suggest that CD may induce alterations (mutations?) in growth factor signaling pathways which may enhance cell survival and malignant transformation.

Neuroendocrine responses of type A individuals to exercise.

Ten Type A and 10 Type B individuals exercised for 20 minutes on a bicycle ergometer at 40%, 60%, and 80% of maximal capacity to determine if differences in neuroendocrine reactivity exist. Pre-exercise plasma concentrations of beta-endorphin and epinephrine were similar for Type As and Type Bs. Pre-exercise plasma levels of norepinephrine tended to be higher for the Type As (p less than 0.07). Post-exercise plasma epinephrine concentrations were similar for As and Bs for all trials. The 40% and 60% trials resulted in no differences in post-exercise norepinephrine and beta-endorphin levels for the Type As and Bs. Conversely, the 80% trials resulted in significantly greater norepinephrine and beta-endorphin concentrations for the Type As (p less than 0.05). Plasma serotonin levels at rest and during exercise were always lower for the Type As (p less than 0.05). These results suggest that our Type As had a greater neuroendocrine response to high-intensity exercise than our Type Bs. The greater reactivity and analgesia may allow the Type A person to suppress feelings of fatigue, thus enduring higher levels of exertion for longer periods of time.

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.

Choline deficiency-induced apoptosis in PC12 cells is associated with diminished membrane phosphatidylcholine and sphingomyelin, accumulation of ceramide and diacylglycerol, and activation of a caspase.

It is not well appreciated that nutritional status can modulate apoptosis, a process that eliminates unwanted or damaged cells. Choline is an essential nutrient, and its absence induces apoptosis. When PC12 cells were cultivated in a choline-free medium, apoptosis was induced (27.4% of cells apoptotic at 72 h as compared to 4.4% in control medium). In choline-free medium at 72 h, there was a 49% decrease in phosphatidylcholine concentration (P<0.01) and a 34% decrease in sphingomyelin concentration (P<0.01); however, there was no change in phosphatidylethanolamine concentration. Before detecting increased apoptosis in choline-deficient cells, we measured a significant increase in ceramide (218% control) and diacyglycerol (155% control) concentrations. The addition of a cell-permeable ceramide to cells in control medium induced apoptosis; however, adding a cell-permeable diacyglycerol did not induce apoptosis. Caspase is a common mediator of apoptosis, and choline deficiency-induced apoptosis was prevented completely by replacing choline or adding a caspase inhibitor into the medium within 48 h of initial choline deprivation. In those cells rescued by replacing choline at 36 h, the concentrations of phosphatidylcholine, sphingomyelin, ceramide, and diacyglycerol returned to levels of control cells. In those cells rescued by adding a caspase inhibitor at 36 h, the concentrations of sphingomyelin and ceramide returned to control levels, but the concentrations of phosphatidylcholine and diacyglycerol did not return to normal. We propose that availability of dietary factors (choline in this model) can modulate apoptosis. Mechanisms that we identify using this model may help us to explain why dietary choline influences brain development.

Glycerophosphocholine and phosphocholine are the major choline metabolites in rat milk.

Choline is a constituent of cell membranes, surfactant and acetylcholine and is also a major source of methyl groups for the regeneration of methionine from homocysteine. Previous analyses of rat, human and bovine milk measured only choline, phosphatidylcholine and sphingomyelin. Choline-containing compounds in milk from rats lactating for 15 d were measured by HPLC and gas chromatograph-mass spectrometry. In addition to the previously reported choline metabolites, substantial concentrations of glycerophosphocholine (3.7 mmol/L) and phosphocholine (653 mumol/L) were also detected. At 1 h after oral administration of [methyl-14C]choline to lactating rats, the major labeled metabolites were phosphocholine (91% of label in milk) and betaine (9%). Twenty-four hours after the dose, glycerophosphocholine was the major labeled metabolite (69% of label in milk). Rat mammary epithelial cells, in primary culture, synthesized and secreted phosphatidylcholine, phosphocholine, glycerophosphocholine and betaine. Thus, the mammary gland was able to synthesize the choline metabolites found in milk, but these metabolites may not be derived exclusively from uptake from maternal blood. We have established that the total choline concentration in rat milk is sevenfold higher than previously reported, with > 80% present as glycerophosphocholine and phosphocholine.

Estrogens reduce bone loss in the ovariectomized, lactating rat model.

Estrogen treatment of ovariectomized, lactating rats improved retention of bone mineral mass by 15-25% compared to ovariectomized, lactating rats receiving vehicle only. On the second day postpartum all lactating rats were ovariectomized and were placed along with age-matched non-mated controls on a whole-wheat flour-based diet with 0.1% calcium and 0.4% phosphorus. On day 6 postpartum estrogen treatment was begun with either implantation of a slow-release 17 beta-estradiol pellet or with the first of daily subcutaneous injections in sesame oil (vehicle). Increasing doses of estrogen resulted in decreased food consumption and decreased litter weight gain, both well-known effects of estrogens. Ovariectomized, lactating rats implanted with a slow-release pellet containing 0.35 mg 17 beta-estradiol had mean serum estradiol levels of 113.5 pg/ml. At the end of 21 days of lactation, femurs of dams with placebo pellets showed loss of 54% of bone ash weight compared with the non-mated controls versus only 42% loss by rats receiving estradiol treatment. Rats were also injected with estradiol benzoate in a sesame oil vehicle at 3 dose levels of 1.6, 5, or 16 micrograms/day. Only the 5 and 16 micrograms/day doses significantly improved retention of bone mineral mass during lactation (+17% and +18%, respectively, vs vehicle-injected, lactating rats). Estrone administered by subcutaneous injection also improved retention of bone during lactation; however, injection of 50 micrograms/day of estrone was required to produce an equivalent bone retention compared to 5 micrograms/day of estradiol. Thus, treatment of ovariectomized, lactating rats with estrogens results in a significant reduction of the loss of bone mineral mass associated with lactation.

Diet-hormone interrelationships in the rat.

Twenty-eight-day old male rats were fed, either ad libitum or in restricted amounts, isoenergetic diets containing 2, 5, 10, 15, 25, or 50% lactalbumin and 5, 11.9, or 21.1% fat for 8 weeks. They were then killed and the plasma levels of triiodothyronine (T3), thyroxine (T4), insulin, glucagon, corticosterone, norepinephrine (NE), and epinephrine (E) were measured. Dietary changes explained most of the variation in the plasma concentrations of T3, T4, insulin, and glucagon but less than 20% of the variation in the plasma concentrations of the adrenal hormones. Dietary protein level was directly related to plasma T4, insulin and corticosterone and inversely related to plasma T3, glucagon, NE, and E. Dietary fat level had its most significant effect on the plasma glucagon concentration to which it was inversely related whereas the most noteworthy effect of a low energy intake was to reduce plasma E and thereby to increase the NE/E ratio. A refeeding study confirmed the effects of dietary protein level on plasma hormone concentrations and showed that the changes in diet-hormone interrelationships in 12-week old male rats have been derived by multiple regression analyses of the data.

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)

Choline deficiency induces apoptosis in primary cultures of fetal neurons.

Treatment of rats with choline during brain development results in long-lasting enhancement of spatial memory whereas choline deficiency has the opposite effect. Changes in rates of apoptosis may be responsible. We previously demonstrated that choline deficiency induced apoptosis in PC12 cells and suggested that interruption of cell cycling due to a decrease in membrane phosphatidylcholine concentration was the critical mechanism. We now examine whether choline deprivation induces apoptosis in nondividing primary neuronal cultures of fetal rat cortex and hippocampus. Choline deficiency induced widespread apoptosis in primary neuronal cells, indicating that cells do not have to be dividing to be sensitive to choline deficiency. When switched to a choline-deficient medium, both types of cells became depleted of choline, phosphocholine and phosphatidylcholine, and in primary neurons neurite outgrowth was dramatically attenuated. Primary cells could be rescued from apoptosis by treatment with phosphocholine or lysophosphatidylcholine. As described previously for PC12 cells, an increase in ceramide (Cer) was associated with choline deficiency-induced apoptosis in primary neurons. The primary neuronal culture appears to be an excellent model to explore the mechanism whereby maternal dietary choline intake modulates apoptosis in the fetal brain.