Effect of maternal periconceptional undernutrition in sheep on cortisol regulation in offspring from mid-late gestation, through to adulthood.

Introduction: Maternal periconceptional undernutrition (PCUN) alters fetal hypothalamic-pituitary-adrenal axis (HPAA) function and placental glucocorticoid metabolism in sheep. The effects of PCUN on HPAA function in adult life are not known. We investigated the effects of PCUN on fetal adrenal development across gestation and on cortisol regulation in adult offspring. Methods: Ewes were undernourished from 61 days before to 30 days after conception ('PCUN') or fed ad libitum ('N'). mRNA expression in the fetal adrenal gland of ACTH receptor (ACTHR), steroidogenic acute regulatory protein (STAR), cytochrome P450 17A1 (CYP17A1), 11beta-hydroxysteroid-dehydrogenase type 2 (11ßHSD2), insulin-like growth factor-2 (IGF2), and in the fetal hippocampus of 11ßHSD1, 11ßHSD2, mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) was determined at 50 (adrenal only), 85, 120 and 131 days of gestation (term=148 days). In adult offspring (≥ 3 years, N; 10 female, 5 male, PCUN; 10 female, 10 male) a combined arginine vasopressin (AVP, 0.1 µg/kg) and corticotropin-releasing hormone (CRH, 0.5 µg/kg) challenge and a metyrapone (40 mg/kg) challenge were undertaken. mRNA expression of ACTHR, STAR and CYP17A1 were determined in adult adrenals. Results: Fetal adrenal STAR, CYP17A1 and IGF2 mRNA expression were not different between groups in early gestation but were higher in PCUN than N at 131 days' gestation (all p<0.01). PCUN reduced fetal hippocampal MR and GR mRNA expression by 50% at 85 day, but not in later gestation. Adult offspring plasma cortisol responses to AVP+CRH or metyrapone were not different between groups. Plasma ACTH response to AVP+CRH was lower in PCUN males but ACTH response to metyrapone was not different between groups. Adult adrenal ACTHR, STAR, and CYP17A1 mRNA expression were not affected by PCUN. Conclusions: We conclude that the effects of PCUN on fetal HPAA function that became apparent in late gestation, are not reflected in adrenal cortisol secretion in mid-adulthood.

Sexually dimorphic changes in the endocrine pancreas and skeletal muscle in young adulthood following intra-amniotic IGF-I treatment of growth-restricted fetal sheep.

Fetal growth restriction (FGR) is associated with decreased insulin secretory capacity and decreased insulin sensitivity in muscle in adulthood. We investigated whether intra-amniotic IGF-I treatment in late gestation mitigated the adverse effects of FGR on the endocrine pancreas and skeletal muscle at 18 mo of age. Singleton-bearing ewes underwent uterine artery embolization between 103 and 107 days of gestational age, followed by 5 once-weekly intra-amniotic injections of 360-µg IGF-I (FGRI) or saline (FGRS) and were compared with an unmanipulated control group (CON). We measured offspring pancreatic endocrine cell mass and pancreatic and skeletal muscle mRNA expression at 18 mo of age (n = 7-9/sex/group). Total α-cell mass was increased ∼225% in FGRI males versus CON and FGRS males, whereas ß-cell mass was not different between groups of either sex. Pancreatic mitochondria-related mRNA expression was increased in FGRS females versus CON (NRF1, MTATP6, UCP2), and FGRS males versus CON (TFAM, NRF1, UCP2) but was largely unchanged in FGRI males versus CON. In skeletal muscle, mitochondria-related mRNA expression was decreased in FGRS females versus CON (PPARGC1A, TFAM, NRF1, UCP2, MTATP6), FGRS males versus CON (NRF1 and UCP2), and FGRI females versus CON (TFAM and UCP2), with only MTATP6 expression decreased in FGRI males versus CON. Although the window during which IGF-I treatment was delivered was limited to the final 5 wk of gestation, IGF-I therapy of FGR altered the endocrine pancreas and skeletal muscle in a sex-specific manner in young adulthood.NEW & NOTEWORTHY Fetal growth restriction (FGR) is associated with compromised metabolic function throughout adulthood. Here, we explored the long-term effects of fetal IGF-I therapy on the adult pancreas and skeletal muscle. This is the first study demonstrating that IGF-I therapy of FGR has sex-specific long-term effects at both the tissue and molecular level on metabolically active tissues in adult sheep.

Effects of maternal periconceptional undernutrition in sheep on offspring glucose-insulin axis function into adulthood.

Maternal periconceptional undernutrition (PCUN) affected fetal pancreatic maturation in late gestation lambs and impaired glucose tolerance in 10-month-old sheep. To examine the importance of the timing of maternal undernutrition around conception, a further cohort was born to PCUN ewes [undernourished for 61 d before conception (PreC), 30 d after conception (PostC), or 61 d before until 30 d after conception (PrePostC)], or normally fed ewes (Control) (n = 15-20/group). We compared glucose tolerance, insulin secretion, and sensitivity at 36 months of age. We also examined protein expression of insulin signalling proteins in muscle from these animals and in muscle from a fetal cohort (132 d of gestation; n = 7-10/group). Adult PostC and PrePostC sheep had higher glucose area under the curve than Controls (P = 0.07 and P = 0.02, respectively), whereas PreC sheep were similar to Controls (P = 0.97). PostC and PrePostC had reduced first-phase insulin secretion compared with Control (P = 0.03 and P = 0.02, respectively). PreC was similar to Control (P = 0.12). Skeletal muscle SLC2A4 protein expression in PostC and PrePostC was increased 19%-58% in fetuses (P = 0.004), but decreased 39%-43% in adult sheep (P = 0.003) compared with Controls. Consistent with this, protein kinase C zeta (PKCζ) protein expression tended to be increased in fetal (P = 0.09) and reduced in adult (P = 0.07) offspring of all PCUN ewes compared with Controls. Maternal PCUN alters several aspects of offspring glucose homeostasis into adulthood. These findings suggest that maternal periconceptional nutrition has a lasting impact on metabolic homeostasis of the offspring.

Twin Conception in Sheep Leads to Impaired Insulin Sensitivity and Sexually Dimorphic Adipose Tissue and Skeletal Muscle Phenotypes in Adulthood.

Twins are often born small and early and have increased risk of obesity and diabetes later in life. Twin conception in sheep, regardless of whether the pregnancy continues as twins or is reduced to singleton in early gestation, alters offspring growth trajectory and body composition in young adulthood. We hypothesized that twin conception would result in insulin resistance in adulthood, with insulin-resistant adipose tissue and skeletal muscle phenotypes. At 3 years of age, body weight was not different among singletons, twins, and reductions; females weighed less than males. Singletons were leaner than reductions, with twins intermediate. Twins and reductions had decreased insulin sensitivity compared with singletons (singletons: mean [standard error of the mean]: 4.75 [0.4], twins: 3.34 [0.3], reductions: 3.67 [0.2] mg·I µU-1·kg-1·min-1, P < .01). There were no group differences in adipocyte size, adipose tissue, or circulating tumor necrosis factor α, monocyte chemoattractant protein 1, or interleukin 6 concentrations. In males, omental and subcutaneous adipose SLC2A4 was 1.5- to 2.0-fold greater in twins and reductions than in singletons ( P < .01) and SLC2A1 was greater in reductions than in singletons. Skeletal muscle IRS-1 was decreased in male twins but increased in female twins, compared to singletons ( P ≤ .01), with no effect on reductions in either sex. Skeletal muscle SLC2A4 was decreased in female twins and reductions but elevated in male twins and reductions compared to singletons ( P ≤ .01). We conclude that adult twin insulin resistance is not due to adipose tissue phenotype, but potentially phenotypic effects in skeletal muscle, and obesity is a result of twin conception per se with its origins in early gestation.

Neonatal milk supplementation in lambs has persistent effects on growth and metabolic function that differ by sex and gestational age.

The perinatal environment has a major influence on long-term health and disease risk. Preterm birth alters early-life environment and is associated with altered metabolic function in adulthood. Whether preterm birth per se or the early nutritional interventions used to support growth in preterm infants underpins this association is unknown. Lambs born preterm, following dexamethasone induction of labour, or spontaneously at term were randomised to receive nutrient supplementation, analogous to the milk fortifier used clinically or water as a control for the first 2 weeks after birth. Thereafter, nutrition was not different between groups. Growth was monitored, and the glucose-insulin axis function was assessed in juvenile (4 months) and adult life (14 months). Early nutrition influenced adult metabolic function and body composition to a greater extent than preterm birth. In supplemented females, arginine-stimulated insulin secretion was increased in preterm but reduced in term-born juveniles compared with controls (repeated-measures ANOVA P<0·01). In supplemented preterm males, adult weight, ponderal index (PI) and fasting insulin concentrations were elevated compared with preterm controls (weight, 75 (sem 3) v. 69 (sem 2) kg; PI, 48·0 (sem 2·1) v. 43·7 (sem 1·7) kg/m3; fasting insulin, 0·19 (sem 0·02) v. 0·10 (sem 0·02) ng/ml). Conversely, supplemented term-born males had reduced adult weight, PI and fasting insulin concentrations compared with term-born controls (weight, 64 (sem 2) v. 70 (sem 2) kg; PI, 44·4 (sem 1·8) v. 48·2 (sem 1·7) kg/m3; fasting insulin, 0·09 (sem 0·02) v. 0·14 (sem 0·02) ng/ml; all group×supplement interactions P<0·05). Adult metabolic health may reflect both gestational age at birth and early nutrition. Human studies are urgently needed to investigate the adult sex-specific health implications of neonatal nutritional strategies.

Brief neonatal nutritional supplementation has sex-specific effects on glucose tolerance and insulin regulating genes in juvenile lambs.

BACKGROUND: The nutritional plane and composition during fetal life can impact upon growth and epigenetic regulation of genes affecting pancreatic ß-cell development and function. However, it is not clear whether ß-cell development can be altered by nutritional factors or growth rate after birth. We therefore investigated the effect of neonatal nutritional supplements on growth, glucose tolerance, and pancreatic development in lambs. METHODS: Newborn lambs were randomized to daily nutritional supplements, calculated to increase macronutrient intake to a similar degree as human breast milk fortifier, or an equivalent volume of water, for 2 wk while continuing to suckle ewe milk. Intravenous glucose tolerance test (IVGTT) was performed at 4 mo of age, and pancreata collected for molecular analysis. RESULTS: Supplemented lambs had slower weight gain than controls. In supplemented lambs, insulin response to IVGTT was increased in males but decreased in females, compared to same sex controls, and was unrelated to growth rate. mRNA expression of key genes in ß-cell development showed sexually dimorphic effects. Epigenetic change occurred in the promotor region of PDX1 gene with decreased suppression and increased activation marks in supplemented lambs of both sexes. CONCLUSION: Nutritional interventions in early life have long-term, sex-specific effects on pancreatic function.

Nutritional regulation of fetal growth.

Fetal growth is largely regulated by nutritional supply. The placenta is responsible for fetal nutrient supply for much of pregnancy, but in early pregnancy nutrition is histiotrophic. Both placental size and efficiency, and fetal growth, may be affected by maternal nutritional state before and during very early pregnancy. In contrast, manipulating maternal nutrition during later stages of pregnancy has a smaller than expected effect on fetal growth. Maternal nutrition before and during early pregnancy also has a greater effect on gestation length than maternal nutrition later in pregnancy, suggesting that nutritional status may regulate both fetal growth trajectory and gestation length and that these two outcomes may be linked. Thus, determination of the nutritional factors regulating fetal growth, and potentially postnatal growth and body phenotype, may lie with the maternal nutritional status even before conception.

Antenatal corticosteroid exposure at term increases adult adiposity: an experimental study in sheep.

Clinical practice guidelines for elective cesarean section at early-term gestation (37-38 weeks) recommend antenatal corticosteroids to reduce neonatal respiratory morbidity. However, the long-term health implications for offspring exposed to corticosteroids at term are unknown and may differ from the effects of preterm corticosteroid exposure. We therefore randomized singleton-bearing ewes (n = 64) to receive a clinically relevant dose of corticosteroids at term or no treatment. Body composition was assessed in adult offspring using dual-energy X-ray absorptiometry. Relative to skeletal size female, but not male, offspring of steroid-treated ewes had increased weight and a greater fat mass than controls (relative weight: 49.1 ± 1.1 vs. 52.9 ± 1.2 kg/m², p = 0.02; relative fat mass: 5.4 ± 0.7 vs. 3.4 ± 0.7 kg/m², p = 0.04). Whether corticosteroid exposure at early-term gestation increases adult adiposity in humans is unknown and needs further investigation.

Unpredictable feeding impairs glucose tolerance in growing lambs.

Irregular eating is associated with insulin resistance and metabolic disease in adults but may affect young, growing children differently. We investigated the metabolic effects of unpredictable feeding in female juvenile lambs randomly assigned to receive, for six weeks, maintenance feed given twice daily in equal portions (Control Group, C; n = 24) or the same weekly feed amount in aliquots of variable size at unpredictable times (Unpredictable Group, U; n = 21). Intravenous glucose tolerance tests (IVGTT), insulin tolerance tests (ITT), and measurement of diurnal plasma cortisol concentrations were performed pre and post the dietary intervention. Groups were compared using t test and RM ANOVA. Weight gain was similar in both groups (C 18 ± 2%; U 16 ± 2% of initial body weight). Glucose area under the curve (AUC) was unchanged in C (AUC pre 818 ± 34, post 801 ± 33 mmol.min.l(-1)), but increased by 20% in U (pre 830 ± 25, post 1010 ± 19 mmol.min.l(-1); p<0.0001), with an inadequate insulin response to glucose load (log(AUC insulin first 40 minutes) post intervention C 1.49 ± 0.04 vs U 1.36 ± 0.04 ng.min.ml(-1); p = 0.03). Insulin tolerance and diurnal variation of plasma cortisol concentrations were not different between groups. Unpredictable feeding impairs insulin response to glucose in growing lambs despite high quality food and normal weight gain. Irregular eating warrants investigation as a potentially remediable risk factor for disordered glucose metabolism.

Periconceptional undernutrition in sheep affects adult phenotype only in males.

Periconceptional undernutrition (PCUN) in sheep alters fetal growth and metabolism and postnatal growth regulation, but effects on adult body composition are unknown. We investigated the effects of PCUN on adult phenotype. Singleton lambs of ewes fed normally (N, n = 17) or undernourished before (UN-61-0 d, n = 23), before and after (UN-61-30 d, n = 19), or after (UN-2-30d, n = 17) mating (d0) were weighed at birth, 12 weeks, and intermittently to adulthood. At the age of 3-4 years, body composition was assessed by dual-emission X-ray absorptiometry followed by postmortem examination. Compared with N animals, male, but not female, offspring of all UN groups had greater % fat mass (all UN versus N: 9 ± 1 versus 2 ± 1%, P < 0.001) and perirenal fat (544 ± 36 versus 222 ± 44 g, P = 0.002), and proportionately smaller hearts (4.5 ± 0.1 versus 5.2 ± 0.2 g·kg(-1)), lungs (9.1 ± 0.2 versus 10.6 ± 0.5 g·kg(-1)), and adrenals (0.06 ± 0.002 versus 0.08 ± 0.003 g·kg(-1)). UN males also had larger testes (726 ± 21 versus 545 ± 32 g, P = 0.007), but UN females had smaller ovaries (2.7 ± 0.08 versus 3.4 ± 0.4 g, P = 0.01). Changes were independent of birth weight or postnatal growth velocity. Brief PCUN has sex-specific effects on adult phenotype, predominantly affecting males, which may contribute to adverse metabolic outcomes.

Effects of periconceptional undernutrition on maternal taurine concentrations in sheep.

Taurine has an important role in numerous physiological processes, including many aspects of fetal development such as development of the pancreas and brain, and requirements increase during pregnancy. Periconceptional undernutrition has long-term effects on pancreas and brain function of the offspring, but the effects on maternal taurine economy are unknown. We, therefore, studied the effects of different periods of periconceptional undernutrition on maternal plasma and urine taurine concentrations before and during pregnancy. Four groups of singleton-bearing ewes were studied (n 10-11): controls fed ad libitum, and groups undernourished from 60 d before until mating (PreC), from 2 d before mating until 30 d after mating (PostC) or from 60 d before until 30 d after mating (Pre+PostC). In PreC ewes, plasma taurine concentrations remained at control levels for the first 30 d, and then decreased through the remainder of undernutrition, but recovered by 30 d after mating; urinary taurine excretion was low at mating, but recovered similarly. In PostC ewes, plasma taurine concentrations recovered after 2 weeks despite ongoing undernutrition; urinary taurine excretion had recovered by 30 d after mating. Pre+PostC ewes followed the same pattern as PreC for the first 60 d, but plasma taurine concentrations and urinary excretion recovered slowly, and did not reach the control levels until 97 d. These data suggest that different periods of mild periconceptional undernutrition in sheep have different but substantial effects on maternal taurine homoeostasis. These effects may be one mechanism by which maternal periconceptional undernutrition alters development of the offspring with implications for adult health.

Animal studies of the effects of early nutrition on long-term health.

Small size at birth is associated with increased risk of a variety of common chronic diseases in adulthood. Numerous experimental studies in animals have supported the observations in humans, demonstrating that changes in nutrition in early life can lead to altered long-term health. Importantly, these effects can be independent of size at birth, and can depend on the interaction between nutritional events before and after birth. Both macro- and micronutrient intake are important. Furthermore, these effects may vary according to the nature, timing, severity and duration of the nutritional insult. This review provides examples from animal studies of evidence of these long-term effects, and some possible underlying mechanisms whereby nutrition in early life can affect long-term health.

Periconceptional events perturb postnatal growth regulation in sheep.

Periconceptional undernutrition and twin conception alter intrauterine growth and metabolism and are associated with later adverse metabolic outcomes. The contribution of postnatal growth to these outcomes is less well defined. We investigated whether maternal periconceptional undernutrition or twin conception altered postnatal growth regulation in ways that could lead to metabolic disease. Single and twin offspring of ewes undernourished (UN) from 61 d before until 30 d after mating, fed to achieve and maintain 10-15% weight loss (UN), were compared with offspring of maintenance-fed controls (N). At 2 h and 1, 6, and 12 wk after birth, lambs were weighed and plasma hormone and metabolite concentrations analyzed. Milk intake, measured by deuterium oxide dilution, was inversely related to birth weight only in N singles, although twins had the greatest postnatal growth velocity. Positive associations were seen between milk intake, growth velocity, and leptin concentrations in N, but not UN, offspring. We conclude that periconceptional undernutrition alters the relationships between regulators of postnatal growth, including nutrient intake and key hormonal axes, in both singles and twins without affecting size at birth or postnatal growth velocity. Dissociation of growth from its key regulators is one possible mechanism underlying adverse metabolic outcomes after periconceptional undernutrition.

Undernutrition before mating in ewes impairs the development of insulin resistance during pregnancy.

OBJECTIVE: To assess the effects of periconceptional undernutrition on maternal adaptation of insulin-dependent metabolism during pregnancy. METHODS: Romney ewes were randomly assigned to receive normal nutrition (n=12) or undernutrition before (from 60 days before until mating, n=7), after (2 days before to 30 days after mating, n=6) or before and after mating (from 60 days before to 30 days after mating, n=10). Insulin sensitivity was measured by hyperinsulinaemic-euglycaemic clamp at 65 days of gestation (term=147 days). Lamb growth rate was measured in late gestation by chronically implanted growth catheters and weight at 133 days of gestation. RESULTS: Ewes undernourished before or both before and after mating failed to develop the insulin resistance of pregnancy seen in normally nourished ewes. Ewes undernourished after mating showed intermediate insulin sensitivity. This was not related to plasma concentrations of pregnancy-related hormones, but was related to insulin kinetics. There was an inverse relationship between insulin sensitivity and fetal growth, with ewes that were most insulin sensitive having smaller, more slowly growing lambs (highest compared with lowest tertile for insulin sensitivity: fetal weight 3.5+/-0.3 compared with 4.5+/-0.1 kg, P=.02; growth rate 2.0+/-0.2 compared with 2.6+/-0.2 mm.day-1, P=.05). CONCLUSION: Maternal undernutrition before conception impairs adaptation of insulin-related metabolism during pregnancy in ways that affect fetal growth. This suggests a key mechanism whereby prepregnancy nutritional status influences pregnancy outcome.

Do alterations in placental 11beta-hydroxysteroid dehydrogenase (11betaHSD) activities explain differences in fetal hypothalamic-pituitary-adrenal (HPA) function following periconceptional undernutrition or twinning in sheep?

Periconceptional undernutrition (UN) in sheep accelerates fetal hypothalamic-pituitary-adrenal (HPA) axis activation, resulting in preterm birth. In contrast, twin conception suppresses fetal HPA function and delays prepartum HPA activation. We hypothesized that these dissimilar effects on fetal HPA activity result from different influences of maternal glucocorticoid (GC) on maturation of the fetal HPA axis, mediated via different activities of placental 11beta-hydroxysteroid dehydrogenase (11betaHSD) isozymes. We examined the effects of twinning and maternal periconceptional UN from 60 days before until 30 days after mating on the ontogeny of placental 11betaHSD-1 and -2 enzyme activities. At day 85 of gestation, placental 11betaHSD-2 activity was lower in UN than in normally nourished (N) fetuses (P < .05) and was higher in twins than in singletons (P < .05). Furthermore, placental 11betaHSD-1 activity was not different between nutritional groups but was higher in twins than in singletons (P = .01). At day 85, fetal plasma cortisol (P < .001) and cortisone (P < .001) concentrations were lower in UN than in N fetuses, but the cortisol to cortisone ratio was higher in UN than in N fetuses (P = .01). There was no effect of fetus number on plasma cortisol or cortisone concentrations or on the ratio of cortisol to cortisone at day 85. Therefore, periconceptional UN and twinning may result in the alterations of placental 11betaHSD isozyme activities at particular times during gestation. Changes in these activities during critical periods of fetal development could affect transplacental transfer or placental generation of GCs that reach the fetus, potentially influencing the timing of activation of the fetal HPA axis, fetal maturation, and hence the development and health later in life.

Periconceptional undernutrition of ewes impairs glucose tolerance in their adult offspring.

Maternal undernutrition throughout pregnancy can have long-term effects on the health of adult offspring. Undernutrition around the time of conception alters growth, metabolism, and endocrinology of the sheep fetus, but the impact on offspring after birth is largely unknown. We determined the effect of maternal periconceptional undernutrition in sheep on glucose tolerance in the offspring before and after puberty. Undernourished (UN) ewes were fed individually to maintain weight loss of 10-15% bodyweight from 61 d before until 30 d after mating. Offspring (24 UN, 30 control) underwent an i.v. glucose tolerance test at 4 and 10 mo of age. Glucose tolerance was similar in both groups at 4 mo. Insulin area under the curve increased by 33% between 4 and 10 mo (101 +/- 8 versus 154 +/- 12 ng x min x mL(-1), p < 0.0001). At 10 mo, UN offspring had a 10% greater glucose area under the curve than controls (809 +/- 22 versus 712 +/- 20 mM x min, p < 0.01), a reduced first phase insulin response (p = 0.003) which was particularly apparent in females and in singletons, and a decreased insulin:glucose ratio (p = 0.01). We conclude that maternal undernutrition around the time of conception results in impaired glucose tolerance in postpubertal offspring.

Nutritional programming of adult disease.

Intrauterine and early neonatal life is a period of physiological plasticity, during which environmental influences may produce long-term effects. Both undernutrition and overnutrition during this period have been shown to change disease risk in adulthood. These effects are influenced by the type, timing and duration of inappropriate nutrition and by the previous nutritional environment and may not be reflected in changes in body size. An understanding of the interaction between nutrient imbalance and alteration of gene expression is likely to be the key to optimising future health outcomes.