A complex combination of adult health-related disorders can originate from developmental events that occur in utero. The periconceptional period may also be programmable. We report on the effects of restricting the supply of specific B vitamins (i.e., B12 and folate) and methionine, within normal physiological ranges, from the periconceptional diet of mature female sheep. We hypothesized this would lead to epigenetic modifications to DNA methylation in the preovulatory oocyte and/or preimplantation embryo, with long-term health implications for offspring. DNA methylation is a key epigenetic contributor to maintenance of gene silencing that relies on a dietary supply of methyl groups. We observed no effects on pregnancy establishment or birth weight, but this modest early dietary intervention led to adult offspring that were both heavier and fatter, elicited altered immune responses to antigenic challenge, were insulin-resistant, and had elevated blood pressure-effects that were most obvious in males. The altered methylation status of 4% of 1,400 CpG islands examined by restriction landmark genome scanning in the fetal liver revealed compelling evidence of a widespread epigenetic mechanism associated with this nutritionally programmed effect. Intriguingly, more than half of the affected loci were specific to males. The data provide the first evidence that clinically relevant reductions in specific dietary inputs to the methionine/folate cycles during the periconceptional period can lead to widespread epigenetic alterations to DNA methylation in offspring, and modify adult healthrelated phenotypes. E vidence from both epidemiological studies in humans and direct intervention studies in animals indicates that altering key developmental processes in utero can predispose offspring to many late-onset diseases such as dyslipidemia, type II diabetes, and heart disease (1, 2). In this regard, the effects of gross nutrient or protein deficiencies in maternal diet during pregnancy are well documented (3), although little is known about the effects of specific nutrients or the timing and mechanistic basis of nutrient programming (4). Here we investigated the effects of restricting the supply of specific B group vitamins (i.e., vitamin B 12 and folate) and sulfur amino acids (in particular, methionine) from the diet of adult female sheep from 8 weeks preceding until 6 days after conception, within physiological ranges encountered in both sheep (5) and humans (i.e., within the 5th and 95th percentiles) (6, 7). These micronutrients are important intermediates and/or have specific regulatory functions in the linked methionine-folate cycles (5, 7). In rodents, maternal supraphysiological methyl group supply and a low-protein diet (50% control) offered throughout pregnancy altered DNA methylation of candidate genes (agouti, glucocorticoid receptor, and peroxisomal proliferator-activated receptor-␣) (8, 9), but the extent of methylation change in these or more clinically relevant diets is not known. Gametes and preimplantation emb...

Maternal nutrient restriction at specific stages of gestation has differential effects on fetal development such that the offspring are programmed to be at increased risk of adult disease. We investigated the effect of gestational age and maternal nutrition on the maternal plasma concentration of leptin and cortisol together with effects on fetal adipose tissue deposition plus leptin, IGF-I, IGF-II ligand, and receptor mRNA abundance near to term. Singleton bearing ewes were either nutrient restricted (NR; consuming 3.2-3.8 MJ/d of metabolizable energy) or fed to appetite (consuming 8.7-9.9 MJ/d) over the period of maximal placental growth, i.e. between 28 and 80 d gestation. After 80 d gestation, ewes were either fed to calculated requirements, consuming 6.7-7.5 MJ/d, or were fed to appetite and consumed 8.0-10.9 MJ/d. Pregnancy resulted in a rise in plasma leptin concentration by 28 d gestation, which continued up to 80 d gestation when fed to appetite but not with nutrient restriction. Plasma cortisol was also lower in NR ewes up to 80 d gestation, a difference no longer apparent when food intake was increased. At term, irrespective of maternal nutrition in late gestation, fetuses sampled from ewes NR in early gestation possessed more adipose tissue, whereas when ewes were fed to appetite throughout gestation, fetal adipose tissue deposition and leptin mRNA abundance were both reduced. These changes may result in the offspring of NR mothers being at increased risk of obesity in later life.

It is apparent from epidemiological studies that the timing of maternal nutrient restriction has a major influence on outcome in terms of predisposing the resulting offspring to adult obesity. The present review will consider the extent to which maternal age, parity and nutritional restriction at defined stages of gestation can have important effects on fat deposition and endocrine sensitivity of adipose tissue in the offspring. For example, in 1-year-old sheep the offspring of juvenile mothers have substantially reduced fat deposition compared with those born to adult mothers. Offspring of primiparous adult mothers, however, show increased adiposity compared with those born to multiparous mothers. These offspring of multiparous ewes show retained abundance of the brown adipose tissue-specific uncoupling protein 1 at 1 month of age. A stimulated rate of metabolism in brown fat of these offspring may act to reduce adipose tissue deposition in later life. In terms of defined windows of development that can programme adipose tissue growth, maternal nutrient restriction targetted over the period of maximal placental growth results in increased adiposity at term in conjunction with enhanced abundance of mRNA for the insulin-like growth factor-I and -II receptors. In contrast, nutrient restriction in late gestation, coincident with the period of maximal fetal growth, has no major effect on adiposity but results in greater abundance of specific mitochondrial proteins, i.e. voltage-dependent anion channel and/or uncoupling protein 2. These adaptations may increase the predisposal of these offspring to adult obesity. Increasing maternal nutrition in late gestation, however, can result in proportionately less fetal adipose tissue deposition in conjunction with enhanced abundance of uncoupling protein 1.

As the role of leptin in energy balance in neonate is unknown, we investigated the effect of acute (2 h) and chronic (7 days) administration of leptin (100 microg/day) on thermoregulation and mitochondrial protein abundance in adipose tissue. The concentration of uncoupling protein (UCP)1 and voltage-dependent anion channel (VDAC) located on the inner and outer mitochondrial membranes, respectively, were measured. Administration of leptin prevented the normal decline in colonic temperature over the first few hours and days after birth. It subsequently accelerated the loss of both mRNA and protein for UCP1 but had no effect on VDAC abundance. At seven days of age, colonic temperature was correlated strongly with both mRNA abundance and thermogenic potential of UCP1 in leptin-treated but not control lambs, indicating more effective use of UCP1 for heat production following leptin administration. Leptin had no effect on weight gain or adipose tissue deposition; at one day of age only, leptin mRNA was correlated positively with adipose tissue weight. In conclusion, leptin administration to neonatal lambs improves thermoregulation and promotes the loss of UCP1 in brown adipose tissue.

Maternal nutrient restriction at specific stages of gestation has differential effects on fetal development such that the offspring are programmed to be at increased risk of a range of adult diseases, including obesity. We investigated the effect of maternal nutritional manipulation through gestation on fetal adipose tissue deposition in conjunction with mRNA abundance for uncoupling protein (UCP)1 and 2, peroxisome proliferator-activated receptors (PPAR)alpha and gamma, together with long and short forms of the prolactin receptor (PRLR). Singleton-bearing ewes were either nutrient restricted (3.2-3.8 MJ day(-1) metabolizable energy) or fed to appetite (8.7-9.9 MJ day(-1)) over the period of maximal placental growth, i.e. between 28 and 80 d gestation. After 80 d gestation, ewes were either fed to calculated requirements, (6.7-7.5 MJ day(-1)), or to appetite (8.0-10.9 MJ day(-1)). At term, offspring of nutrient-restricted ewes possessed more adipose tissue, an adaptation that was greatest in those born to mothers that fed to requirements in late gestation. This was accompanied by an increased mRNA abundance for UCP2 and PPARalpha, an adaptation not seen in mothers re-fed to appetite. Maternal nutrition had no effect on mRNA abundance for UCP1, PPARgamma, or PRLR. Irrespective of maternal nutrition, mRNA abundance for UCP1 was positively correlated with PPARgamma and the long and short forms of PRLR, indicating that these factors may act together to ensure that UCP1 abundance is maximized in the newborn. In conclusion, we have shown, for the first time, differential effects of maternal nutrition on key regulatory components of fetal fat metabolism.

We investigated the influence of maternal nutritional enhancement during the second half of gestation on prolactin receptor (PRLR) abundance in fetal brown adipose tissue (BAT) and liver close to term (i.e. 141-144 d gestation). Ewes were provided with 100% (i.e. control; n = 8) or 150% (i.e. well-fed; n = 7) of their metabolic requirements from 80 to 144 d gestation. Crude plasma membranes were prepared from fetal BAT and hepatic tissue, and individual molecular weight isoforms for the long and short forms of the PRLR were detected by immunoblotting. Mitochondrial preparations were prepared from BAT to measure the amount of the BAT-specific mitochondrial uncoupling protein-1 and its thermogenic activity (i.e. guanosine 5'-diphosphate binding). Fetuses sampled from well-fed ewes were heavier (controls, 3927 +/- 196 g; well-fed, 4783 +/- 219 g; p = 0.01) but possessed less BAT per kilogram body weight (controls, 5.92 +/- 0.43 g/kg; well-fed, 3.85 +/- 0.19 g/kg; p = 0.001), which had a greater uncoupling protein-1 abundance (controls, 56 +/- 5% of reference; well-fed, 78 +/- 9% of reference; p < 0.01) and higher thermogenic activity (controls, 157 +/- 41 pmol guanosine 5'-diphosphate per milligram mitochondrial protein; well-fed, 352 +/- 36 pmol guanosine 5'-diphosphate per milligram mitochondrial protein; p < 0.01) than controls. Multiple isoforms of the long and short forms of the P1LR were detected in all tissues. BAT from well-fed fetuses had a higher abundance of the 15-kD isoform of the long form of the PRLR (controls, 1.6 +/- 0.4 densitometric units; well-fed, 16.3 +/- 2.0 densitometric units; p < 0.001). This isoform was not detected in hepatic tissue. Maternal nutrient intake had no effect on any other isoforms of the PRLR in BAT or liver. In conclusion, increasing the quantity of feed provided in late gestation acts to promote fetal weight and BAT maturation, the combination of which will enhance neonatal viability.

No abstract

The liver is a major metabolic and endocrine organ of critical importance in the regulation of growth and metabolism. Its function is determined by a complex interaction of nutritionally regulated counter-regulatory hormones. The extent to which hepatic endocrine sensitivity can be programed in utero and whether the resultant adaptations persist into adulthood is unknown and was therefore the subject of this study. Young adult male sheep born to mothers that were fed either a control diet (i.e.100% of total live weight-maintenance requirements) throughout gestation or 50% of that intake (i.e. nutrient restricted (NR)) from 0 to 95 days gestation and thereafter 100% of requirements (taking into account increasing fetal mass) were entered into the study. All mothers gave birth normally at term, the singleton offspring were weaned at 16 weeks, and then reared at pasture until 3 years of age when their livers were sampled. NR offspring were of similar birth and body weights at 3 years of age when they had disproportionately smaller livers than controls. The abundance of mRNA for GH, prolactin, and IGF-II receptors, plus hepatocyte growth factor and suppressor of cytokine signaling-3 were all lower in livers of NR offspring. In contrast, the abundance of the mitochondrial protein voltage-dependent anion channel and the pro-apoptotic factor Bax were up regulated relative to controls. In conclusion, maternal nutrient restriction in early gestation results in adult offspring with smaller livers. This may be mediated by alterations in both hepatic mitogenic and apoptotic factors.