Christopher Torrens

Feeding pregnant rats a protein-restricted diet persistently alters the methylation of specific cytosines in the hepatic PPARα promoter of the offspring

Induction of an altered phenotype by prenatal under-nutrition involves changes in the epigenetic regulation of specific genes. We investigated the effect of feeding pregnant rats a protein-restricted (PR) diet with different amounts of folic acid on the methylation of individual CpG dinucleotides in the hepatic PPAR alpha promoter in juvenile offspring, and the effect of the maternal PR diet on CpG methylation in adult offspring. Pregnant rats (five per group) were fed 180 g/kg casein (control) or 90 g/kg casein with 1 mg/kg folic acid (PR), or 90 g/kg casein and 5 mg/kg folic acid (PRF). Offspring were killed on postnatal day 34 (five males and females per group) and day 80 (five males per group). Methylation of sixteen CpG dinucleotides in the PPAR alpha promoter was measured by pyrosequencing. Mean PPAR alpha promoter methylation in the PR offspring (4.5 %) was 26 % lower than controls (6.1 %) due to specific reduction at CpG dinucleotides 2 (40 %), 3 (43 %), 4 (33 %) and 16 (48 %) (P < 0.05). There was no significant difference in methylation at these CpG between control and PRF offspring. Methylation of CpG 5 and 8 was higher (47 and 63 %, respectively, P < 0.05) in the PRF offspring than control or PR offspring. The methylation pattern in day 80 PR offspring was comparable to day 34 PR offspring. These data show for the first time that prenatal nutrition induces differential changes to the methylation of individual CpG dinucleotides in juvenile rats which persist in adults.

Dietary protein restriction of pregnant rats in the F0 generation induces altered methylation of hepatic gene promoters in the adult male offspring in the F1 and F2 generations.

Epidemiological studies and experimental models show that maternal nutritional constraint during pregnancy alters the metabolic phenotype of the offspring and that this can be passed to subsequent generations. In the rat, induction of an altered metabolic phenotype in the liver of the F1 generation by feeding a protein-restricted diet (PRD) during pregnancy involves the altered methylation of specific gene promoters. We therefore investigated whether the altered methylation of PPARalpha and glucocorticoid receptor (GR) promoters was passed to the F2 generation. Females rats (F0) were fed a reference diet (180 g/kg protein) or PRD (90 g/kg protein) throughout gestation, and AIN-76A during lactation. The F1 offspring were weaned onto AIN-76A. F1 females were mated and fed AIN-76A throughout pregnancy and lactation. F1 and F2 males were killed on postnatal day 80. Hepatic PPARalpha and GR promoter methylation was significantly (P<0 x 05) lower in the PRD group in the F1 (PPARalpha 8 %, GR 10 %) and F2 (PPARalpha 11 %, GR 8 %) generations. There were trends (P<0 x 1) towards a higher expression of PPARalpha, GR, acyl-CoA oxidase and phosphoenolpyruvate carboxykinase (PEPCK) in the F1 and F2 males, although this was significant only for PEPCK. These data show for the first time that the altered methylation of gene promoters induced in the F1 generation by maternal protein restriction during pregnancy is transmitted to the F2 generation. This may represent a mechanism for the transmission of induced phenotypes between generations

Dietary protein restriction in pregnancy induces hypertension and vascular defects in rat male offspring.

It is established that dietary protein restriction of pregnant rats results in their offspring developing hypertension. However, to date no studies have investigated peripheral vascular function of offspring using the low protein model. Therefore, the aim of the study was to assess isolated resistance artery function from adult male offspring of control (C, 18% casein) and protein-restricted (PR, 9% casein) pregnant dams at two different ages. The birthweight of PR offspring did not significantly differ from that of C offspring. Systolic blood pressure was significantly elevated in PR compared with C (p < 0.05). Maximal vascular contraction to phenylephrine and the thromboxane analog U46619 were similar in C and PR offspring at postnatal d 87 and 164. Relaxation induced by the endothelium-dependent vasodilators acetylcholine or bradykinin was significantly reduced in the PR group (p < 0.05). Relaxation to the endothelium-independent vasodilator sodium nitroprusside and phosphodiesterase type 3 inhibitor cilostamide was less in the PR offspring compared with C (p < 0.01). Dietary protein restriction in pregnancy induces hypertension and vascular dysfunction in male offspring. Abnormalities in the nitric oxide-cGMP pathway may explain the defect in endothelium-dependent and -independent relaxation. Reduced vasodilation may be a potential mechanism underlying the elevated systolic blood pressure observed in this model.

Folate Supplementation During Pregnancy Improves Offspring Cardiovascular Dysfunction Induced by Protein Restriction

Dietary protein restriction in the rat compromises the maternal cardiovascular adaptations to pregnancy and leads to raised blood pressure and endothelial dysfunction in the offspring. In this study we have hypothesized that dietary folate supplementation of the low-protein diet will improve maternal vascular function and also restore offspring cardiovascular function. Pregnant Wistar rats were fed either a control (18% casein) or protein-restricted (9% casein) diet ±5 mg/kg folate supplement. Function of isolated maternal uterine artery and small mesenteric arteries from adult male offspring was assessed, systolic blood pressure recorded, and offspring thoracic aorta levels of endothelial nitric oxide (NO) synthase mRNA measured. In the uterine artery of late pregnancy dams, vasodilatation to vascular endothelial growth factor was attenuated in the protein-restricted group but restored with folate supplementation, as was isoprenaline-induced vasodilatation (P<0.05). In male offspring, protein restriction during pregnancy led to raised systolic blood pressure (P<0.01), impaired acetylcholine-induced vasodilatation (P<0.01), and reduced levels of endothelial NO synthase mRNA (P<0.05). Maternal folate supplementation during pregnancy prevented this elevated systolic blood pressure associated with a protein restriction diet. With folate supplementation, endothelium-dependent vasodilatation and endothelial NO synthase mRNA levels were not significantly different from either the control or protein-restricted groups. Maternal folate supplementation of the control diet had no effect on blood pressure or vasodilatation. This study supports the hypothesis that folate status in pregnancy can influence fetal development and, thus, the risks of cardiovascular disease in the next generation. The concept of developmental origins of adult disease focuses predominately on fetal life but must also include a role for maternal cardiovascular function.

Low protein diet fed exclusively during mouse oocyte maturation leads to behavioural and cardiovascular abnormalities in offspring

Early embryonic development is known to be susceptible to maternal undernutrition, leading to a disease‐related postnatal phenotype. To determine whether this sensitivity extended into oocyte development, we examined the effect of maternal normal protein diet (18% casein; NPD) or isocaloric low protein diet (9% casein; LPD) restricted to one ovulatory cycle (3.5 days) prior to natural mating in female MF‐1 mice. After mating, all females received NPD for the remainder of gestation and all offspring were litter size adjusted and fed standard chow. No difference in gestation length, litter size, sex ratio or postnatal growth was observed between treatments. Maternal LPD did, however, induce abnormal anxiety‐related behaviour in open field activities in male and female offspring (P < 0.05). Maternal LPD offspring also exhibited elevated systolic blood pressure (SBP) in males at 9 and 15 weeks and in both sexes at 21 weeks (P < 0.05). Male LPD offspring hypertension was accompanied by attenuated arterial responsiveness in vitro to vasodilators acetylcholine and isoprenaline (P < 0.05). LPD female offspring adult kidneys were also smaller, but had increased nephron numbers (P < 0.05). Moreover, the relationship between SBP and kidney or heart size or nephron number was altered by diet treatment (P < 0.05). These data demonstrate the sensitivity of mouse maturing oocytes in vivo to maternal protein undernutrition and identify both behavioural and cardiovascular postnatal outcomes, indicative of adult disease. These outcomes probably derive from a direct effect of protein restriction, although indirect stress mechanisms may also be contributory. Similar and distinct postnatal outcomes were observed here compared with maternal LPD treatment during post‐fertilization preimplantation development which may reflect the relative contribution of the paternal genome.

Mismatched pre- and postnatal nutrition leads to cardiovascular dysfunction and altered renal function in adulthood

The early life environment has long-term implications for the risk of developing cardiovascular (CV) disease in adulthood. Fetal responses to changes in maternal nutrition may be of immediate benefit to the fetus, but the long-term effects of these adaptations may prove detrimental if nutrition in postnatal life does not match that predicted by the fetus on the basis of its prenatal environment. We tested this predictive adaptive response hypothesis with respect to CV function in sheep. We observed that a mismatch between pre- and postnatal nutrient environments induced an altered CV function in adult male sheep that was not seen when environments were similar. Sheep that received postnatal undernutrition alone had altered growth, CV function, and basal hypothalamo–pituitary–adrenal axis activity in adulthood. Prenatal undernutrition induced greater weight gain by weaning compared with the prenatal control diet, which may provide a reserve in the face of a predicted poor diet in later life. In an adequate postnatal nutrient environment (i.e., relatively mismatched), these offspring exhibited cardiac hypertrophy and altered CV function in adulthood. These data support the concept that adult CV function can be determined by developmental responses to intrauterine nutrition made in expectation of the postnatal nutritional environment, and that if these predictions are not met, the adult may be maladapted and at greater risk of CV disease. Our findings have substantial implications for devising strategies to reduce the impact of a mismatch in nutrition levels in humans undergoing rapid socio-economic transitions in both developing and developed societies.

Glycine rectifies vascular dysfunction induced by dietary protein imbalance during pregnancy

Protein restriction in rat pregnancy programmes the development of elevated systolic blood pressure and vascular dysfunction in the offspring. A recent study has shown that hypertension is reversed by maternal glycine supplementation. Whether this protective effect is exerted directly on the embryo and fetus, or indirectly via effects on the mother, is unknown although we have previously shown abnormalities in the maternal vasculature. We tested the hypothesis that dietary glycine repletion would reverse endothelial dysfunction in protein‐restricted pregnant rat dams using wire myography. Impaired acetylcholine‐ (P < 0.01) and isoprenaline‐induced (P < 0.05) vasodilatation in isolated mesenteric arteries (MA) from protein‐restricted pregnant dams was accompanied by reduced vascular nitric oxide (NO) release (P < 0.05). Dietary glycine supplementation reversed vascular dysfunction in MA (P < 0.05) and improved NO release thus potentially protecting the maternal circulation. The impaired NO release in the MA of low protein diet dams was not accompanied by reduced eNOS mRNA expression, suggesting that eNOS activity was altered. Protein restriction did not alter the vascular function of a conduit artery, the thoracic aorta. These results provide evidence that adequate provision of glycine, a conditionally essential amino acid in pregnancy, may play a role in the vascular adaptations to pregnancy, protecting the fetus from abnormal programming of the cardiovascular system.

Maternal protein restriction in the rat impairs resistance artery but not conduit artery function in pregnant offspring

Dietary protein restriction during gestation has been shown to produce vascular dysfunction in pregnant rats and hypertension in their offspring. However, no studies have to date examined the effects of such ‘programming’ on the vascular function of female offspring when they in turn become pregnant. We have therefore studied isolated conduit and resistance artery function from pregnant female offspring of control (C, 18 % casein) and protein‐restricted (PR, 9 % casein) pregnant dams. There were no differences in birth weight, weight gain during pregnancy, litter size, fetal weight, placental weight, fetal : placental weight ratio or organ weights between the C and PR groups. In isolated mesenteric arteries, the vasodilatation in response to the endothelial‐dependent vasodilator acetylcholine (ACh) and the β‐adrenoceptor agonist isoprenaline was decreased in the PR group, while there were no differences in the constriction in response to potassium (125 mm) or the α1‐adrenoceptor agonist phenylephrine (PE). No differences in any responses were seen in the isolated thoracic aorta. We conclude that dietary protein restriction in pregnancy programmes vasodilator dysfunction in isolated resistance arteries of female offspring when they become pregnant, but does not affect conduit arteries.

Transmission of raised blood pressure and endothelial dysfunction to the F2 generation induced by maternal protein restriction in the F0, in the absence of dietary challenge in the F1 generation.

We have previously demonstrated that maternal protein restriction during pregnancy leads to raised blood pressure and endothelial dysfunction in the offspring (F1). Here we show that these characteristics are transmitted to the F2 offspring through the maternal line, in the absence of any additional challenges to the F1. Female Wistar rats were fed either a control (18% casein) or protein-restricted diet (PR; 9% casein) throughout pregnancy. Female F1 offspring, maintained on standard chow postpartum, were mated with breeding males to produce F2 progeny. Systolic blood pressure (SBP) in male F2 offspring was assessed by tail-cuff plethysmography at age 100 d and vascular function of small mesenteric arteries by wire myography at age 80 and 200 d. SBP was raised in PR F2 offspring compared with controls (control 122.1 (SEM 2.3) mmHg, n 7; PR 134.7 (SEM 3.2) mmHg, n 6; P<0.01) and endothelial function, assessed by vasodilatation to acetylcholine, was impaired at both age 80 d (% maximal response: control 89.7 (SEM 2.6), n 14; PR 72.7 (SEM 4.4), n 15; P<0.01) and 200 d (effective concentration equal to 50% of maximum (pEC50): control 7.67 (SEM 0.10), n 10; PR 7.33 (SEM 0.07), n 8; P<0.05). The present study demonstrates that both raised blood pressure and endothelial dysfunction are passed via the maternal line to grand-offspring in the absence of any additional dietary challenges to their F1 mothers. Risk factors for chronic disease may therefore be heritable by non-genomic processes.

Maternal fat intake in rats alters 20:4n-6 and 22:6n-3 status and the epigenetic regulation of Fads2 in offspring liver

Poor prenatal nutrition, acting through epigenetic processes, induces persistent changes in offspring phenotype. We investigated the effect of maternal fat intake on polyunsaturated fatty acid (PUFA) status and on the epigenetic regulation of Fads2, encoding Δ6 desaturase (rate limiting in PUFA synthesis), in the adult offspring. Rats (n=6 per dietary group) were fed either 3.5% (w/w), 7% (w/w) or 21% (w/w) butter or fish oil (FO) from 14 days preconception until weaning. Offspring (n=6 males and females per dietary group) were fed 4% (w/w) soybean oil until postnatal day 77. 20:4n-6 and 22:6n-3 levels were lower in liver phosphatidylcholine (PC) and phosphatidylethanolamine and plasma PC (all P<.0001) in offspring of dams fed 21% than 3.5% or 7% fat regardless of type. Hepatic Fads2 expression related inversely to maternal dietary fat. Fads2 messenger RNA expression correlated negatively with methylation of CpGs at −623, −394, −84 and −76 bases relative to the transcription start site (all P<.005). Methylation of these CpGs was higher in offspring of dams fed 21% than 3.5% or 7% fat; FO higher than butter. Feeding adult female rats 7% fat reduced 20:4n-6 status in liver PC and Fads2 expression and increased methylation of CpGs −623, −394, −84 and −76 that reversed in animals switched from 7% to 4% fat diets. These findings suggest that fat exposure during development induces persistent changes, while adults exhibit a transient response, in hepatic PUFA status in offspring through epigenetic regulation of Fads2. Thus, epigenetic regulation of Fads2 may contribute to short- and long-term regulation of PUFA synthesis.