T Stephenson

Timing of nutrient restriction and programming of fetal adipose tissue development

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.

The influence of maternal nutrient restriction in early to mid-pregnancy on placental and fetal development in sheep

Placental weight is a primary factor determining size at birth in many species. In sheep, placental weight peaks at approximately mid-gestation, with structural remodelling occurring over the second half of pregnancy to meet the increasing nutritional demands of the growing fetus. Numerous factors influence placental growth and development in sheep, and many workers (see Kelly, 1992) have investigated the role of maternal nutrition as a regulator of placental and fetal size. We have studied the effects of feeding ewes approximately 50% of their recommended energy requirements during early to mid-pregnancy on fetal and placental indices measured at mid-gestation (i.e. 80 d) and close to term (i.e. 145 d). Maternal nutrient restriction is associated with a reduction in placental weight at 80 d, but an increase in placental weight at 145 d of gestation, compared with ewes fed adequately in early pregnancy. No significant effect on fetal weight was observed at either 80 or 145 d gestation, although differences in body dimensions and the insulin-like growth factor-1 axis were found in lambs from nutrient-restricted ewes delivered close to term. Maternal nutrition during pregnancy plays a pivotal role in the regulation of fetal and placental development in sheep, and therefore has the potential to influence both short- and longer-term health outcomes.

Ontogeny and nutritional manipulation of mitochondrial protein abundance in adipose tissue and the lungs of postnatal sheep

The present study examined the ontogeny of mitochondrial protein abundance in adipose tissue and lungs over the first month of life in the sheep and the extent to which this may be altered by maternal undernutrition during the final month of gestation. The ontogeny of uncoupling protein (UCP), voltage-dependent anion channel (VDAC) and cytochrome c abundance were determined in adipose tissue and lungs sampled from near-term fetuses and young sheep aged 4 h, 1, 7 and 30 d. In adipose tissue, the abundance of UCP1, VDAC and cytochrome c all peaked at 1 d of age and then decreased by 30 d of age, at which stage the brown adipose tissue-specific UCP1 was no longer detectable but UCP2 was clearly abundant. For the lungs, however, UCP2 and VDAC abundance both peaked 7 d after birth and then decreased by 30 d of age. During postnatal development, therefore, a marked change in mitochondrial protein abundance occurs within both adipose tissue and lungs. Maternal nutrient restriction had no effect on lamb growth or tissue weights at 30 d of age but was associated with increased abundance of UCP2 and VDAC but not cytochrome c in both adipose tissue and lungs. These mitochondrial adaptations within both adipose tissue and the lungs of offspring born to previously nutrient-restricted mothers may compromise adipose tissue and lung function during periods of environmental stress.

Influence of relative size at birth on growth and glucose homeostasis in twin lambs during juvenile life

The effect of differences in size at birth on growth and glucose homeostasis between female twin lambs during juvenile life was examined. Twenty-six sets of twins were entered into the study, of which ten were used for organ sampling at birth and 16 were studied over the first year of life. Eleven sets were defined as being mismatched for birthweight as the weight difference between twins was >25%, with light lambs weighing 4.1 +/- 0.3 kg and heavy lambs weighing 5.1 +/- 0.1 kg. All remaining twins were matched in bodyweight, weighing 4.6 +/- 0.5 kg. During the rapid period of juvenile growth (i.e. one, three and six months of age) and following stabilization of bodyweight (i.e. 12 months of age) glucose tolerance tests were performed by intravenously injecting 0.8 mg kg(-1) bodyweight glucose. This was followed the next day with an insulin tolerance test, performed by intravenously injecting 0.08 units kg(-1) bodyweight insulin. At birth there were no differences in organ weight as a fraction of total bodyweight between matched and mismatched twins, but the ratio of liver to brain weight was lower in light compared with heavy twins. Light lambs remained lighter than their twins up to six months of age, and crown-rump length was also shorter. At one and three months of age there were no differences in basal plasma glucose concentrations between the groups, but glucose tolerance was greater in light compared with heavy lambs at one and six months of age. Insulin tolerance was greater in light compared with matched lambs at one and six months of age. In conclusion, it has been shown that size at birth of one twin in relation to its co-twin is one factor determining glucose regulation during postnatal life. This not only affects glucose and insulin tolerance but also growth over the first six months of age.

The impact of presenting problem based guidelines for children with medical problems in an accident and emergency department

Aims: To evaluate the impact of presenting problem based guidelines in managing children with either diarrhoea (with or without vomiting) or seizure (with or without fever). Methods: This prospective observational study with an intervention was based on a paediatric accident and emergency (A&E) department in Nottingham. All patients (either GP or self referred) were acute attenders aged 0–15 years, with a medical presenting problem during 4 months in the spring of 1997 and 1999. Five hundred and thirty-one diarrhoea attendances (292 before guideline implementation and 239 after) and 411 seizure attendances (212 before guideline implementation and 199 after) were recorded. Evidence based and consensus ratified guidelines developed for the study were implemented using care pathway documentation. Process (documentation, time in the department, investigations, treatment) and outcome (admission to hospital, returns to A&E) data were collected from case notes. Results: The percentage of children investigated with blood tests fell significantly (haematology requests in diarrhoea presentations from 11% to 4%, biochemistry in seizure presentations from 29% to 17%). Intravenous infusions in diarrhoea presenters fell (9% to 1%), and more appropriate oral fluids were used. Management time in A&E was reduced (diarrhoea presenters: median of 55–40 minutes, seizure presenters: 80–55 minutes, but remained static for other presenting problems). Marked improvements in documentation were seen. Admission rates for diarrhoea attenders increased (27% to 34%) but remained the same for seizure (69% v 73%) Conclusions: The implementation of a presenting problem based guideline as a care pathway was associated with improvements in the quality of care by: improved documentation; reduced invasive investigations; more appropriate treatment, and reduced time spent in A&E.

Nutritional manipulation between early to mid-gestation: effects on uncoupling protein-2, glucocorticoid sensitivity, IGF-I receptor and cell proliferation but not apoptosis in the ovine placenta.

In sheep, modest maternal nutrient restriction (NR) over the period of rapid placental growth restricts placentome growth and results in offspring in which glucocorticoid action is enhanced. Therefore, this study investigated the placental effects of early to mid-gestational NR on glucocorticoid receptor (GR), 11beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2), uncoupling protein-2 (UCP2), and IGF type-I receptor (IGF-IR) mRNA abundance together with cell proliferation and apoptosis as determined histologically, and the mitochondrial proteins voltage-dependent anion channel and cytochrome c that are involved in apoptosis. Placenta was sampled at 80 and 140 days gestation (dGA; term ~147 dGA). NR was imposed between 28 and 80 days gestation when control and nutrient-restricted groups consumed 150 or 60% respectively of their total metabolizable energy requirements. All mothers were then fed to requirements up to term. Total fetal placentome weights were decreased by NR at 80 dGA but were heavier at 140 dGA following 60 days of nutritional rehabilitation. GR and UCP2 mRNA abundance increased whilst 11betaHSD2 mRNA decreased with gestational age. NR persistently up-regulated GR and UCP2 mRNA abundance. 11betaHSD2 mRNA was reduced by NR at 80 dGA but increased near to term. IGF-IRmRNA abundance was only decreased at 80 dGA. Placental apoptosis and mitochondrial protein abundance were unaffected by NR, whereas cell proliferation was markedly reduced. In conclusion, placental UCP2 and local glucocorticoid action are affected by the gestational nutritional status and may result in the offspring showing enhanced glucocorticoid sensitivity, thereby predisposing them to disease in later life.

Effect of maternal dexamethasone treatment and ambient temperature on prolactin receptor abundance in Brown adipose and hepatic tissue in the foetus and new-born lamb.

We investigated the influence of maternal dexamethasone treatment and ambient temperature on prolactin receptor (PRLR) abundance in brown adipose tissue (BAT) and hepatic tissue from foetuses and 6‐h‐old lambs delivered by caesarean section. Lambs were either delivered into a warm (30 °C; WD) or cool (15 °C; CD) ambient temperature at 140 days gestation, 2 days after dexamethasone treatment, or at 146 days gestation for controls. Uncoupling protein‐1 (UCP1) content of BAT was higher in dexamethasone‐treated groups compared to controls. A range of tissue‐specific PRLR isoforms was detected. For the long form of PRLR in BAT these isoforms had molecular weights of 66, 54, 34 and 19 kD compared with 88, 76, 66, 58, 54 and 48 kD in liver. In BAT, isoforms of the short form of PRLR had molecular weights of 66, 62, 54, 48, 33 and 31 kD compared with 82, 66, 56, 54, 48, 40 and 33 kD in liver. Dexamethasone treatment in CD lambs resulted in higher abundance of the 54 kD isoform of the short form of PRLR in liver, whilst in BAT dexamethasone resulted in a greater abundance of the 48 kD isoform of the short form, and lower abundance of the 66 kD isoform of the long form of PRLR, compared to controls. A negative correlation (r2=0.52) was observed between abundance of 66 kD isoform for the long form of PRLR and UCP1, compared with positive correlations (r2=0.58–0.60) for the abundance of the 54 and 48 kD isoforms for the short form of PRLR and UCP1. In conclusion, maternal dexamethasone treatment 1 week before term alters the abundance of PRLR isoforms in a tissue‐specific manner. This response is dependent on ambient temperature after birth and may provide a critical endocrine signal for maximising non‐shivering thermogenesis.

Experimental Evidence for Long-Term Programming Effects of Early Diet

Nutritional manipulation targeted at specific periods of embryo or placental development can result in substantial changes in fetal organ development despite no effects on fetal weight. In particular, kidney and fat mass are greater in nutrient restricted offspring in conjunction with higher mRNA abundance for leptin, insulin-like growth factors I/II and glucocorticoid receptors. As young adults, nutrient restricted offspring exhibit a blunting of the cardiovascular baroreflex. They also demonstrate increased plasma leptin following sympathetic stimulation, not observed in controls, indicating resetting of adipocyte sensitivity to stress. In conclusion, global nutrient restriction confined to periods of early development programmes adult physiology in a manner that may predispose to later disease given the appropriate environmental stimuli.

The Effects of Pregnancy and Maternal Nutrition on the Maternal Renin‐Angiotensin System in Sheep

Physiological changes occurring in the mother during pregnancy can determine the outcome of pregnancy in terms of birthweight and neonatal viability. Maternal adaptations include plasma volume expansion linked to enhanced activity of the renin‐angiotensin system (RAS). The present study was designed to determine whether these changes occur very early in gestation, and the extent to which maternal nutrient restriction may compromise the maternal RAS. Using sheep, we have investigated the effects of pregnancy per se, maternal nutrient restriction and later restoration of maternal diet on maternal body weight, plasma volume and plasma renin concentration (PRC), and angiotensinogen (Aogen) and arginine vasopressin (AVP) concentration. During the period of placental growth (i.e. 28‐80 days gestation) ewes were fed either a nutrient‐restricted (NR) diet or were well fed (WF). NR ewes consumed between 3.2 and 3.8 MJ day−1 of metabolisable energy (ME) which is close to 60% of requirements taking into account the ME required for both ewe maintenance and growth of the conceptus in order to produce a 4.5 kg lamb at term. WF ewes consumed 150% of ME requirements. Restoration of maternal diet between 80 and 140 days gestation (i.e. fed to satiety and consuming between 8 and 10.9 MJ day−1, which is close to 150% of ME requirements) followed previous nutrient restriction. Between pre‐conception and 28 days gestation, plasma volume increased in conjunction with a decline in PRC and Aogen concentration. During the period of nutrient restriction ewe body weight did not increase and plasma volume was lower in NR than WF ewes. During this time there was no effect of maternal nutrition on PRC; however, Aogen concentration was lower in the NR group. From 80 days gestation following the rise in food intake for previously NR ewes, greater increases in ewe body weight, plasma volume and PRC occurred up to term compared with ewes that were well fed throughout gestation. Plasma AVP concentration was not significantly affected by either maternal nutrition or gestational age. In conclusion, the stimulus of moderately severe maternal nutrient restriction evoked smaller rises in maternal weight, plasma volume and Aogen concentration than occurred in ewes that were well fed throughout gestation. Following the restoration of maternal diet after 80 days gestation, PRC gradually rose to peak at term. These adaptations in the maternal RAS during the critical period of placental growth may have long‐term effects on fetal development.

Effect of Postnatal Age and a β3‐Adrenergic Agonist (Zeneca D7114) Administration on Uncoupling Protein‐1 Abundance in the Lamb

We examined the effect of time after birth and β3‐adrenergic agonist (Zeneca D7114) administration on uncoupling protein‐1 (UCP1) abundance and thermoregulation in the lamb. Forty twin lambs, all born normally at term, were maintained at a cold ambient temperature of between 3 and 8 °C. At 0.5, 1.75, 5.25, 11.25 and 23.25 h after birth eight sets of twins were fed 20 ml of formula milk ± 10 mg kg−1 of β3‐adrenergic agonist, and 45 min after feeding brown adipose tissue (BAT) was sampled. Colonic temperature was measured and BAT analysed for UCP1 abundance, GDP‐binding to mitochondrial protein (i.e. thermogenic activity) and catecholamine content. Colonic temperature declined between 1.25 and 6 h from 40.2 °C to 39.2 °C and then increased to 39.8 °C at 12 h, but increased after feeding at all ages. UCP1 abundance increased from 1.25 h after birth, to peak at 2 h after birth in controls, compared with 6 h after birth in β3‐adrenergic agonist‐treated lambs. The level of GDP‐binding to mitochondrial protein did not change significantly with age but was increased by β3‐adrenergic agonist treatment. The noradrenaline (norepinephrine) content of BAT increased between 1.25 and 12 h after birth, irrespective of β3‐adrenergic agonist administration. The total weight of perirenal BAT plus its lipid, protein and mitochondrial protein content declined over the first 6 h of life. UCP1 development continues over the first 24 h of neonatal life, and can be manipulated by β3‐adrenergic agonist administration. This may represent one method of improving thermoregulation in newborn lambs.