We have previously shown that lambs delivered by caesarean section 1 week prematurely become hypothermic due to reduced brown adipose tissue function in conjunction with low plasma concentrations of cortisol and thyroid hormones. The present study therefore aimed to determine whether maternal dexamethasone (a synthetic corticosteroid) administration could improve thermoregulation in premature lambs to the extent that they become similar to term lambs. Lambs were either delivered by caesarean section into a warm (30 degrees C; WD) or cool (15 degrees C; CD) ambient temperature at 140 days of gestation, 2 days after maternal dexamethasone treatment, or at 146 days for controls. During the first 30 min of life the decline in colonic temperature was greater in dexamethasone treated lambs compared with controls delivered into the same ambient temperature. All lambs then restored colonic temperature although this adaptation took longest in dexamethasone treated lambs CD but these subsequently attained highest plateau colonic temperatures. Oxygen consumption, breathing frequency and plasma free fatty acid concentrations were highest in dexamethasone treated lambs CD. There were no differences in plasma thyroid hormones between groups, but cortisol concentrations were lower in dexamethasone treated lambs irrespective of delivery temperature. Analysis of brown adipose tissue samples at 6 h of life demonstrated that dexamethasone treated lambs WD had more uncoupling protein and, in both dexamethasone treated and control lambs, uncoupling protein content was higher in lambs CD compared with those WD. An effect of ambient temperature on thermogenic activity was only observed in the dexamethasone treated group. It is concluded that maternal dexamethasone treatment can significantly improve thermoregulation after birth following premature delivery by caesarean section. As a consequence, dexamethasone treated lambs delivered 1 week prematurely do not remain hypothermic and have higher or similar colonic temperatures compared with untreated lambs born 1-2 days before term.
I Clarke, L Heasman and ME Symonds
SJ Bernard, I Yuen, C McMillen, ME Symonds and PC Owens
Leptin mRNA was measured in adipose tissue of fetal sheep by reverse transcription polymerase chain reaction (RTPCR). Abundance of leptin mRNA relative to b-actin mRNA in fetal perirenal adipose tissue increased (P<0.02) with gestation, being higher at 144 d (0.73 +/- 0. 10, n=5) than at 90-91 d (0.40 +/- 0.08, n=6) or 125 d (0.40 +/- 0. 04, n=5) gestation (term approximately 147- 150 d). There was a positive relationship between relative abundance of leptin mRNA (y) and fetal body weight (x)between 90 and 144 d gestation (r 2 =0.27, P<0.01). The slope of the linear dependence of leptin mRNA on fetal weight was 15-fold greater (P<0.001) at 90-91d (y = 2.81x - 1.1, n=6, r 2 =0.71, P<0.025) than between 125-144 d gestation (y = 0.195x - 0.15, n=16, r 2 =0.39, P<0.01). Thus the leptin synthetic capacity of fetal adipose tissue appears to increase in late gestation but this is accompanied by constraint of its sensitivity to fetal body weight. We hypothesise that leptin synthesis in fetal adipose tissue is related to fetal nutrient supply and growth rate.
ME Symonds, A Mostyn, S Pearce, H Budge and T Stephenson
In the fetus, adipose tIssue comprises both brown and white adipocytes for which brown fat is characterised as possessing the unique uncoupling protein (UCP)1. The dual characteristics of fetal fat reflect its critical role at birth in providing lipid that is mobilised rapidly following activation of UCP1 upon cold exposure to the extra-uterine environment. A key stage in the maturation of fetal fat is the gradual rise in the abundance of UCP1. For species with a mature hypothalamic-pituitary axis at birth there is a gradual increase in the amount and activity of UCP1 during late gestation, in conjunction with an increase in the plasma concentrations of catecholamines, thyroid hormones, cortisol, leptin and prolactin. These may act individually, or in combination, to promote UCP1 expression and, following the post-partum surge in each hormone, UCP1 abundance attains maximal amounts.Adipose tIssue grows in the fetus at a much lower rate than in the postnatal period. However, its growth is under marked nutritional constraints and, in contrast to many other fetal organs that are unaffected by nutritional manipulation, fat mass can be significantly altered by changes in maternal and, therefore, fetal nutrition. Fat deposition in the fetus is enhanced during late gestation following a previous period of nutrient restriction up to mid gestation. This is accompanied by increased mRNA abundance for the receptors of IGF-I and IGF-II. In contrast, increasing maternal nutrition in late gestation results in less adipose tIssue deposition but enhanced UCP1 abundance. The pronounced nutritional sensitivity of fetal adipose tIssue to both increased and decreased maternal nutrition may explain why the consequences of an adverse nutritional environment persist into later life.
BS Yuen, IC McMillen, ME Symonds and PC Owens
Leptin mRNA was measured in adipose tissue of fetal sheep by reverse transcription polymerase chain reaction (RTPCR). Abundance of leptin mRNA relative to beta-actin mRNA in fetal perirenal adipose tissue increased (P<0.02) with gestation, being higher at 144 d (0.73+/-0. 10, n=5) than at 90-91 d (0.40 +/- 0.08, n=6) or 125 d (0.40 +/- 0. 04, n=5) gestation (term approximately 147- 150 d). There was a positive relationship between relative abundance of leptin mRNA (y) and fetal body weight (x) between 90 and 144 d gestation (r(2) =0.27, P<0.01). The slope of the linear dependence of leptin mRNA on fetal weight was 15-fold greater (P<0.001) at 90-91d (y = 2.81x - 1.1, n=6, r(2) =0.71, P<0.025) than between 125-144 d gestation (y = 0.195x - 0.15, n=16, r(2) =0.39, P<0.01). Thus the leptin synthetic capacity of fetal adipose tissue appears to increase in late gestation but this is accompanied by constraint of its sensitivity to fetal body weight. We hypothesise that leptin synthesis in fetal adipose tissue is related to fetal nutrient supply and growth rate.
A Mostyn, S Pearce, H Budge, M Elmes, AJ Forhead, AL Fowden, T Stephenson and ME Symonds
The present study examined the extent to which the late gestation rise in fetal plasma cortisol influenced adipose tIssue development in the fetus. The effect of cortisol on the abundance of adipose tIssue mitochondrial proteins on both the inner (i.e. uncoupling protein (UCP)1) and outer (i.e. voltage-dependent anion channel (VDAC)) mitochondrial membrane, together with the long and short forms of the prolactin receptor (PRLR) protein and leptin mRNA was determined. Perirenal adipose tIssue was sampled from ovine fetuses to which (i) cortisol (2-3 mg/day for 5 days) or saline was infused up to 127-130 days of gestation, and (ii) adrenalectomised and intact controls at between 142 and 145 days of gestation (term=148 days). UCP1 protein abundance was significantly lower in adrenalectomised fetuses compared with age-matched controls, and UCP1 was increased by cortisol infusion and with gestational age. Adrenalectomy reduced the concentration of the long form of PRLR, although this effect was only significant for the highest molecular weight isoform. In contrast, neither the short form of PRLR, VDAC protein abundance or leptin mRNA expression was significantly affected by gestational age or cortisol status. Fetal plasma triiodothyronine concentrations were increased by cortisol and with gestational age, an affect abolished by adrenalectomy. When all treatment groups were combined, both plasma cortisol and triiodothyronine concentrations were positively correlated with UCP1 protein abundance. In conclusion, an intact adrenal is necessary for the late gestation rise in UCP1 protein abundance but cortisol does not appear to have a major stimulatory role in promoting leptin expression in fetal adipose tIssue. It remains to be established whether effects on UCP1 protein are directly regulated by cortisol alone or mediated by other anabolic fetal hormones such as triiodothyronine.
JM Brameld, A Mostyn, J Dandrea, TJ Stephenson, JM Dawson, PJ Buttery and ME Symonds
We investigated the influence of maternal dietary restriction between days 28 and 80 of gestation followed by re-feeding to the intake of well-fed ewes up to 140 days of gestation (term is 147 days) in sheep, on expression of mRNA for insulin-like growth factor (IGF)-I, IGF-II and growth hormone receptor (GHR) in fetal liver and skeletal muscle. Singleton bearing ewes either consumed 3.2-3.8 MJ/day of metabolisable energy (ME) (i.e. nutrient restricted - approximately 60% of ME requirements, taking into account requirements for both ewe maintenance and growth of the conceptus) or 8.7-9.9 MJ/day (i.e. well fed - approximately 150% of ME requirements) between days 28 and 80 of gestation. All ewes were then well fed (150% of ME requirements) up to day 140 of gestation and consumed 8-10.9 MJ/day. At days 80 and 140 of gestation, five ewes were sampled from each group and fetal tissues taken. There was no difference in fetal body weight or liver weights between groups at either sampling date, or skeletal muscle (quadriceps) weight at 140 days. IGF-I mRNA abundance was lower in livers of nutrient-restricted fetuses at day 80 of gestation (nutrient restricted 2.35; well fed 3.70 arbitrary units), but was higher than well-fed fetuses at day 140 of gestation, after 60 days of re-feeding (restricted/re-fed 4.27; well fed 2.83;s.e.d. 0.98 arbitrary units, P=0.061 for dietxage interaction). IGF-II mRNA abundance was consistently higher in livers of nutrient-restricted fetuses (80 days: nutrient restricted 7.78; well fed 5.91; 140 days: restricted/re-fed 7.23; well fed 6.01;s.e.d. 1.09 arbitrary units, P=0.061 for diet). Nutrient restriction had no effect on hepatic GHR mRNA abundance, but re-feeding of previously nutrient-restricted fetuses increased GHR mRNA compared with continuously well-fed fetuses (80 days: nutrient restricted 70.6; well fed 75.1; 140 days: restricted/re-fed 115.7; well fed 89.4;s.e.d. 10.13 arbitrary units, P=0.047 for dietxage interaction). In fetal skeletal muscle, IGF-I mRNA abundance was not influenced by maternal nutrition and decreased with gestation age (P<0.01). IGF-II mRNA abundance was higher in skeletal muscle of nutrient-restricted fetuses compared with well-fed fetuses at day 80 of gestation (nutrient restricted 16.72; well fed 10.53 arbitrary units), but was lower than well-fed fetuses after 60 days of re-feeding (restricted/re-fed 7.77; well fed 13.72;s.e.d. 1.98 arbitrary units, P<0.001 for dietxage interaction). There was no effect of maternal nutrition or gestation age on fetal skeletal muscle GHR expression. In conclusion, maternal nutrient restriction in early to mid gestation with re-feeding thereafter results in alterations in hepatic and skeletal muscle expression of IGF-I, IGF-II and/or GHR in the fetus which may subsequently relate to altered organ and tissue function.
H Budge, A Mostyn, V Wilson, A Khong, AM Walker, ME Symonds and T Stephenson
The present study determines whether maternal administration of prolactin (PRL) to dams promotes the abundance of the brown adipose tissue-specific uncoupling protein-1 (UCP1) in fetal and neonatal rat pups. Recombinant PRL (24 micro g/kg per day), or an equivalent volume of saline, were infused into dams (n=19 per group) throughout pregnancy from 12 h after mating. Interscapular brown adipose tissue was sampled either from fetuses at 19.5 days of gestation (term=21.5 days) or from neonatal rat pups at approximately 18 h after birth. The abundance of UCP1 was determined by immunoblotting on adipose tissue samples from individual pups and pooled from groups of pups. This analysis was complemented by immunocytochemistry on representative adipose tissue samples. Maternal PRL infusion resulted in a greater abundance of UCP1 in fetal rats at 19.5 days of gestation (control: 97.2+/-8.4% reference; PRL: 525.6+/-74.4% reference; P<0.001) and in neonates 18 h after birth. In contrast, the abundance of the outer mitochondrial membrane protein voltage-dependent anion channel was unaffected by PRL. Neonatal adipose tissue sampled from pups born to PRL-infused dams possessed fewer lipid droplets, but more UCP1, as determined by immunocytochemistry. Fetal, but not maternal, plasma leptin concentrations were also increased by maternal PRL administration. In conclusion, as rats are altricial, and the potential thermogenic activity of brown adipose tissue develops over the first few days of postnatal life, these changes prior to, and at the time of, birth implicate PRL in fetal and neonatal adipose tissue maturation.