Maternal choline supplementation in a rat model of periconceptional alcohol exposure: Impacts on the fetus and placenta.

BACKGROUND: Maternal choline supplementation in rats can ameliorate specific neurological and behavioral abnormalities caused by alcohol exposure during pregnancy. We tested whether choline supplementation ameliorates fetal growth restriction and molecular changes in the placenta associated with periconceptional ethanol exposure (PCE) in the rat. METHODS: Sprague Dawley dams were given either 12.5% ethanol (PCE) or 0% ethanol (Con) in a liquid diet from 4 days prior to 4 days after conception. At day 5 of pregnancy, dams were either placed on a standard chow (1.6 g choline/kg chow) or an intermediate chow (2.6 g choline/kg chow). On day 10 of pregnancy, a subset of the intermediate dams were placed on a chow further supplemented with choline (7.2 g choline/kg chow), resulting in 6 groups. Fetuses and placentas were collected on day 20 of pregnancy for analysis. RESULTS: Choline supplementation resulted in increased fetal weight at late gestation, ameliorating the deficits due to PCE. This was most pronounced in litters on a standard chow during pregnancy. Choline also increased fetal liver weight and decreased fetal brain:liver ratio, independent of alcohol exposure. Placental weight was reduced as choline levels in the chow increased, particularly in female placentas. This resulted in a greater ratio of fetal:placental weight, suggesting increased placental efficiency. Global DNA methylation in the placenta was altered in a sex-specific manner by both PCE and choline. However, the increased glycogen deposition in female placentas, previously reported in this PCE model, was not prevented by choline supplementation. CONCLUSIONS: Our results suggest that choline has the potential to ameliorate fetal growth restriction associated with PCE and improve placental efficiency following prenatal alcohol exposure. Our study highlights the importance of maternal nutrition in moderating the severity of adverse fetal and placental outcomes that may arise from prenatal alcohol exposure around the time of conception.

Periconceptional ethanol exposure induces a sex specific diuresis and increase in AQP2 and AVPR2 in the kidneys of aged rat offspring.

Maternal alcohol consumption can impair renal development and program kidney dysfunction in offspring. Given that most women who drink alcohol cease consumption upon pregnancy recognition, we aimed to investigate the effect of alcohol around the time of conception (PC:EtOH) on offspring renal development and function. Rats received a liquid diet ±12.5% v/v ethanol from 4 days before to 4 days after mating. At postnatal day 30, nephron number was assessed. Urine flow and electrolyte (Na, K, Cl) excretion was measured at 6 and 19 months and blood pressure at 12 months. At 19 months, kidneys were collected for gene and protein analysis and assessment of collecting duct length. At postnatal day 30, PC:EtOH offspring had fewer nephrons. At 6 months, PC:EtOH exposure did not alter urine flow nor affect blood pressure at 12 months. At 19 months, female but not male offspring exposed to PC:EtOH drank more water and had a higher urine flow despite no differences in plasma arginine vasopressin (AVP) concentrations. Aqp2 mRNA and Avpr2 mRNA and protein expression was increased in kidneys from female PC:EtOH offspring but collecting duct lengths were similar. Immunofluorescent staining revealed diffuse cytoplasmic distribution of AQP2 protein in kidneys from PC:EtOH females, compared with controls with apical AQP2 localization. PC:EtOH resulted in a low nephron endowment and in female offspring, associated with age-related diuresis. Changes in expression and cellular localization of AQP2 likely underpin this disturbance in water homeostasis and highlight the need for alcohol to be avoided in early pregnancy.

Periconceptional ethanol exposure alters the stress axis in adult female but not male rat offspring.

Ethanol consumption during pregnancy alters offspring hypothalamus-pituitary-adrenal (HPA) axis regulation. However, little is known about the outcomes of alcohol consumption confined to the periconceptional period. This study investigated the effects of periconceptional ethanol (PC:EtOH) exposure on corticosterone concentrations, response to restraint stress and gene expression of adrenal, hypothalamic, and hippocampal glucocorticoid-related pathways in rat offspring. Female Sprague-Dawley rats were treated with PC:EtOH (12.5% v/v EtOH liquid diet) or a control diet from four days before conception, until embryonic day 4. At 6 (adult) and 12-14 (aged) months of age, basal corticosterone concentrations were measured, while in a separate cohort of aged rats, blood pressure, heart rate, and plasma corticosterone concentrations were measured during a 30-minute restraint stress. Adrenal gland, hypothalamic and hippocampal tissue from aged rats were subjected to transcriptomic analysis. PC:EtOH exposure reduced basal plasma corticosterone concentrations in adult and aged female but not male offspring (p < .05). The corticosterone and pressor response were significantly reduced in aged PC:EtOH female offspring following restraint (p < .05). Expression of adrenal steroidogenesis genes (Mc2r, Cyp11a1, Cyp21a1, 11bhsd2, and Nr3c1) and hypothalamic genes (Crh, Crh-r1, Nr3c1, and Hsp90a1) was not affected by PC:EtOH. In aged female offspring exposed to PC:EtOH, adrenal mRNA expression of Hsp90a1 was significantly elevated, and within the hippocampus, mRNAs for glucocorticoid receptor (Nr3c1) and Hsp90a1 were increased (p < .05). This study supports the hypothesis that prenatal alcohol exposure programs sex-specific alterations in the HPA axis and provides the first evidence that the periconceptional period is a critical window for programing of this axis. Lay summary This study investigated the impact of alcohol consumption around the time of conception on offspring stress reactivity in a rat model. Offspring exposed to alcohol displayed altered cardiovascular responses to stress and had reduced circulating concentrations of the stress hormone corticosterone both under basal conditions and following a stressful challenge. This study also identified altered expression of key genes in an important part of the brain known to be involved in stress responsiveness; the hippocampus. If similar outcomes occur in humans, these results would suggest that alcohol consumption, even before a woman knows she is pregnant, may significantly impact stress-related outcomes in children.

Effects of periconceptional maternal alcohol intake and a postnatal high-fat diet on obesity and liver disease in male and female rat offspring.

The effects of maternal alcohol consumption around the time of conception on offspring are largely unknown and difficult to determine in a human population. This study utilized a rodent model to examine if periconceptional alcohol (PC:EtOH) consumption, alone or in combination with a postnatal high-fat diet (HFD), resulted in obesity and liver dysfunction. Sprague-Dawley rats were fed a control or an ethanol-containing [12.5% (vol/vol) EtOH] liquid diet from 4 days before mating until 4 days of gestation ( n = 12/group). A subset of offspring was fed a HFD between 3 and 8 mo of age. In males, PC:EtOH and HFD increased total body fat mass ( PPC:EtOH < 0.05, PHFD < 0.0001); in females, only HFD increased fat mass ( PHFD < 0.0001). PC:EtOH increased microvesicular liver steatosis in male, but not female, offspring. Plasma triglycerides, HDL, and cholesterol were increased in PC:EtOH-exposed males ( PPC:EtOH < 0.05), and LDL, cholesterol, and leptin (Lep) were increased in PC:EtOH-exposed females ( PPC:EtOH < 0.05). mRNA levels of Tnf-α and Lep in visceral adipose tissue were increased by PC:EtOH in both sexes ( PPC:EtOH < 0.05), and Il-6 mRNA was increased in males ( PPC:EtOH < 0.05). These findings were associated with reduced expression of microRNA-26a, a known regulator of IL-6 and TNF-α. Alcohol exposure around conception increases obesity risk, alters plasma lipid and leptin profiles, and induces liver steatosis in a sex-specific manner. These programmed phenotypes were similar to those caused by a postnatal HFD, particularly in male offspring. These results have implications for the health of offspring whose mothers consumed alcohol around the time of conception.

Maternal alcohol intake around the time of conception causes glucose intolerance and insulin insensitivity in rat offspring, which is exacerbated by a postnatal high-fat diet.

Alcohol consumption throughout pregnancy can cause metabolic dysregulation, including glucose intolerance in progeny. This study determined if periconceptional (PC) alcohol (12% v/v in a liquid diet) (PC:EtOH) consumed exclusively around conception results in similar outcomes in Sprague-Dawley rats. Control (C) rats were given a liquid diet containing no alcohol but matched to ensure equal caloric intake. PC maternal alcohol intake (from 4 days before conception until day 4 of gestation), resulted in offspring with elevated fasting plasma glucose (∼10-25%, P < 0.05), impaired glucose tolerance (P < 0.05), and decreased insulin sensitivity (P < 0.01) at 6 months of age. This was associated with increased hepatic gluconeogenesis and sex-specific alterations in peripheral protein kinase B (AKT) signaling. These changes were accompanied by increased mRNA expression of DNA methyltransferases (DNMTs) 1, 3a, and 3b (1.5- to 1.9-fold, P < 0.05) in fetal liver in late gestation, suggesting PC:EtOH may cause epigenetic changes that predispose offspring to metabolic dysfunction. Exposure to a postnatal (PN) high-fat and cholesterol diet (HFD) from 3 months of age caused hyperinsulinemia (∼2-fold increase, P < 0.001) and exacerbated the metabolic dysfunction in male offspring exposed to PC:EtOH but had no additive effects in females. Given many women may drink alcohol while planning a pregnancy, it is crucial to increase public awareness regarding the effects of alcohol consumption around conception on offspring health.

Impact of low dose prenatal ethanol exposure on glucose homeostasis in Sprague-Dawley rats aged up to eight months.

Excessive exposure to alcohol prenatally has a myriad of detrimental effects on the health and well-being of the offspring. It is unknown whether chronic low-moderate exposure of alcohol prenatally has similar and lasting effects on the adult offspring's health. Using our recently developed Sprague-Dawley rat model of 6% chronic prenatal ethanol exposure, this study aimed to determine if this modest level of exposure adversely affects glucose homeostasis in male and female offspring aged up to eight months. Plasma glucose concentrations were measured in late fetal and postnatal life. The pancreas of 30 day old offspring was analysed for ß-cell mass. Glucose handling and insulin action was measured at four months using an intraperitoneal glucose tolerance test and insulin challenge, respectively. Body composition and metabolic gene expression were measured at eight months. Despite normoglycaemia in ethanol consuming dams, ethanol-exposed fetuses were hypoglycaemic at embryonic day 20. Ethanol-exposed offspring were normoglycaemic and normoinsulinaemic under basal fasting conditions and had normal pancreatic ß-cell mass at postnatal day 30. However, during a glucose tolerance test, male ethanol-exposed offspring were hyperinsulinaemic with increased first phase insulin secretion. Female ethanol-exposed offspring displayed enhanced glucose clearance during an insulin challenge. Body composition and hepatic, muscle and adipose tissue metabolic gene expression levels at eight months were not altered by prenatal ethanol exposure. Low-moderate chronic prenatal ethanol exposure has subtle, sex specific effects on glucose homeostasis in the young adult rat. As aging is associated with glucose dysregulation, further studies will clarify the long lasting effects of prenatal ethanol exposure.