In vivo and in vitro bisphenol A exposure effects on adiposity.

In utero exposure to the ubiquitous plasticizer, bisphenol A (BPA) is associated with offspring obesity. As adipogenesis is a critical factor contributing to obesity, we determined the effects of in vivo maternal BPA and in vitro BPA exposure on newborn adipose tissue at the stem-cell level. For in vivo studies, female rats received BPA before and during pregnancy and lactation via drinking water, and offspring were studied for measures of adiposity signals. For in vitro BPA exposure, primary pre-adipocyte cell cultures from healthy newborns were utilized. We studied pre-adipocyte proliferative and differentiation effects of BPA and explored putative signal factors which partly explain adipose responses and underlying epigenetic mechanisms mediated by BPA. Maternal BPA-induced offspring adiposity, hypertrophic adipocytes and increased adipose tissue protein expression of pro-adipogenic and lipogenic factors. Consistent with in vivo data, in vitro BPA exposure induced a dose-dependent increase in pre-adipocyte proliferation and increased adipocyte lipid content. In vivo and in vitro BPA exposure promotes the proliferation and differentiation of adipocytes, contributing to an enhanced capacity for lipid storage. These findings reinforce the marked effects of BPA on adipogenesis and highlight the susceptibility of stem-cell populations during early life with long-term consequence on metabolic homeostasis.

Effects of maternal dexamethasone treatment on pancreatic ß cell function in the pregnant mare and post natal foal.

REASONS FOR PERFORMING STUDY: Synthetic glucocorticoids are used to treat inflammatory conditions in horses. In other pregnant animals, glucocorticoids are given to stimulate fetal maturation with long-term metabolic consequences for the offspring if given preterm. However, their metabolic effects during equine pregnancy remain unknown. OBJECTIVE: Thus, this study investigated the metabolic effects of dexamethasone administration on pregnant pony mares and their foals after birth. STUDY DESIGN: Experimental study. METHODS: A total of 3 doses of dexamethasone (200 µg/kg bwt i.m.) were given to 6 pony mares at 48 h intervals beginning at ≈270 days of pregnancy. Control saline injections were given to 5 mares using the same protocol. After fasting overnight, pancreatic ß cell responses to exogenous glucose were measured in the mares before, during and after treatment. After birth, pancreatic ß cell responses to exogenous glucose and arginine were measured in the foals at 2 and 12 weeks. RESULTS: In mares during treatment, dexamethasone but not saline increased basal insulin concentrations and prolonged the insulin response to exogenous glucose. Basal insulin and glucose concentrations still differed significantly between the 2 groups 72 h post treatment. Dexamethasone treatment significantly reduced placental area but had little effect on foal biometry at birth or subsequently. Foal ß cell function at 2 weeks was unaffected by maternal treatment. However, by 12 weeks, pancreatic ß cell sensitivity to arginine, but not glucose, was less in foals delivered by dexamethasone- than saline-treated mares. CONCLUSIONS: Dexamethasone administration induced changes in maternal insulin-glucose dynamics, indicative of insulin resistance and had subtle longer term effects on post natal ß cell function of the foals. The programming effects of dexamethasone in horses may be mediated partially by altered maternal metabolism and placental growth.

Effects of birth weight, sex and neonatal glucocorticoid overexposure on glucose-insulin dynamics in young adult horses.

In several species, adult metabolic phenotype is influenced by the intrauterine environment, often in a sex-linked manner. In horses, there is also a window of susceptibility to programming immediately after birth but whether adult glucose-insulin dynamics are altered by neonatal conditions remains unknown. Thus, this study investigated the effects of birth weight, sex and neonatal glucocorticoid overexposure on glucose-insulin dynamics of young adult horses. For the first 5 days after birth, term foals were treated with saline as a control or ACTH to raise cortisol levels to those of stressed neonates. At 1 and 2 years of age, insulin secretion and sensitivity were measured by exogenous glucose administration and hyperinsulinaemic-euglycaemic clamp, respectively. Glucose-stimulated insulin secretion was less in males than females at both ages, although there were no sex-linked differences in glucose tolerance. Insulin sensitivity was greater in females than males at 1 year but not 2 years of age. Birth weight was inversely related to the area under the glucose curve and positively correlated to insulin sensitivity at 2 years but not 1 year of age. In contrast, neonatal glucocorticoid overexposure induced by adrenocorticotropic hormone (ACTH) treatment had no effect on whole body glucose tolerance, insulin secretion or insulin sensitivity at either age, although this treatment altered insulin receptor abundance in specific skeletal muscles of the 2-year-old horses. These findings show that glucose-insulin dynamics in young adult horses are sexually dimorphic and determined by a combination of genetic and environmental factors acting during early life.

Glucocorticoid programming of intrauterine development.

Glucocorticoids (GCs) are important environmental and maturational signals during intrauterine development. Toward term, the maturational rise in fetal glucocorticoid receptor concentrations decreases fetal growth and induces differentiation of key tissues essential for neonatal survival. When cortisol levels rise earlier in gestation as a result of suboptimal conditions for fetal growth, the switch from tissue accretion to differentiation is initiated prematurely, which alters the phenotype that develops from the genotype inherited at conception. Although this improves the chances of survival should delivery occur, it also has functional consequences for the offspring long after birth. Glucocorticoids are, therefore, also programming signals that permanently alter tissue structure and function during intrauterine development to optimize offspring fitness. However, if the postnatal environmental conditions differ from those signaled in utero, the phenotypical outcome of early-life glucocorticoid receptor overexposure may become maladaptive and lead to physiological dysfunction in the adult. This review focuses on the role of GCs in developmental programming, primarily in farm species. It examines the factors influencing GC bioavailability in utero and the effects that GCs have on the development of fetal tissues and organ systems, both at term and earlier in gestation. It also discusses the windows of susceptibility to GC overexposure in early life together with the molecular mechanisms and long-term consequences of GC programming with particular emphasis on the cardiovascular, metabolic, and endocrine phenotype of the offspring.

HORSE SPECIES SYMPOSIUM: Glucocorticoid programming of hypothalamic-pituitary-adrenal axis and metabolic function: Animal studies from mouse to horse.

Adrenal glucocorticoids, such as cortisol, are essential for normal fetal development and for maintaining homeostasis in adults. Developmental studies in humans and other animals have shown that exposure to excess glucocorticoids during critical windows of perinatal development can program permanent changes in hypothalamic-pituitary-adrenal (HPA) axis function and metabolic function, with adverse implications for the long-term health of the exposed offspring. The current review compares the programming of postnatal HPA axis function and glucose homeostasis among different species overexposed perinatally to glucocorticoids, with emphasis on the horse. The potential role of epigenetic modification of genes involved in the regulation of HPA axis and metabolic function at cellular and molecular levels is also discussed.

Programmed regulation of rat offspring adipogenic transcription factor (PPARγ) by maternal nutrition.

We determined the protein expression of adipogenic transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ) and its co-repressor and co-activator complexes in adipose tissue from the obese offspring of under- and over-nourished dams. Female rats were fed either a high-fat (60% kcal) or control (10% kcal) diet before mating, and throughout pregnancy and lactation (Mat-OB). Additional dams were 50% food-restricted from pregnancy day 10 to term [intrauterine growth-restricted (IUGR)]. Adipose tissue protein expression was analyzed in newborn and adult male offspring. Normal birth weight Mat-OB and low birth weight IUGR newborns had upregulated PPARγ with variable changes in co-repressors and co-activators. As obese adults, Mat-OB and IUGR offspring had increased PPARγ with decreased co-repressor and increased co-activator expression. Nutritionally programmed increased PPARγ expression is associated with altered expression of its co-regulators in the newborn and adult offspring. Functional studies of PPARγ co-regulators are necessary to establish their role in PPARγ-mediated programmed obesity.

Epigenomics, gestational programming and risk of metabolic syndrome.

Epigenetic mechanisms are emerging as mediators linking early environmental exposures during pregnancy with programmed changes in gene expression that alter offspring growth and development. There is irrefutable evidence from human and animal studies that nutrient and environmental agent exposures (for example, endocrine disruptors) during pregnancy may affect fetal/newborn development resulting in offspring obesity and obesity-associated metabolic abnormalities (metabolic syndrome). This concept of 'gestational programming' is associated with alterations to the epigenome (nongenomic) rather than changes in the DNA sequence (genomic). Epigenetic alterations induced by suboptimal maternal nutrition/endocrine factors include DNA methylation, histone modifications, chromatin remodeling and/or regulatory feedback by microRNAs, all of which have the ability to modulate gene expression and promote the metabolic syndrome phenotype. Recent studies have shown tissue-specific transcriptome patterns and phenotypes not only in the exposed individual, but also in subsequent progeny. Notably, the transmission of gestational programming effects to subsequent generations occurs in the absence of continued adverse environmental exposures, thus propagating the cycle of obesity and metabolic syndrome. This phenomenon may be attributed to an extrinsic process resulting from the maternal phenotype and the associated nutrient alterations occurring within each pregnancy. In addition, epigenetic inheritance may occur through somatic cells or through the germ line involving both maternal and paternal lineages. Since epigenetic gene modifications may be reversible, understanding how epigenetic mechanisms contribute to transgenerational transmission of obesity and metabolic dysfunction is crucial for the development of novel early detection and prevention strategies for programmed metabolic syndrome. In this review we discuss the evidence in human and animal studies for the role of epigenomic mechanisms in the transgenerational transmission of programmed obesity and metabolic syndrome.

Neonatal glucocorticoid overexposure programs pituitary-adrenal function in ponies.

The present study tested the hypothesis that overexposure to endogenous glucocorticoids in neonatal life alters the reactivity of the hypothalamic-pituitary-adrenal (HPA) axis in ponies at 1 and 2 yr of age. Newborn foals received saline (0.9% NaCl, n = 8, control) or long-acting adrenocorticotropic hormone (ACTH1-24) (Depot Synacthen 0.125 mg intramuscularly twice daily, n = 9) for 5 d after birth to raise cortisol concentrations 5- to 6-fold. At 1 and 2 yr of age, HPA axis function was assessed by bolus administration of short-acting ACTH1-24 (1 µg/kg intravenous) and insulin (0.5 U/kg intravenous) to induce hypoglycemic on separate days. Arterial blood samples were taken at 5 to 30-min intervals before and after drug administration to measure plasma ACTH and/or cortisol concentrations. There were no differences in the basal plasma ACTH or cortisol concentrations or in the cortisol response to exogenous ACTH1-24 with neonatal treatment or age. At 1 and 2 yr of age, the increment in plasma ACTH but not cortisol at 60 min in response to insulin-induced hypoglycemia was greater in ponies treated neonatally with ACTH than saline (P < 0.05). Neonatal cortisol overexposure induced by neonatal ACTH treatment, therefore, alters functioning of the HPA axis in adult ponies.

Sex-associated differences in pancreatic ß cell function in healthy preweaning pony foals.

REASONS FOR PERFORMING STUDY: Pancreatic ß cells are responsive to a range of stimuli during early post natal life in healthy pony foals. However, little is known about whether these responses are sex-linked. OBJECTIVES: To determine pancreatic ß cell responses to the insulin secretagogues, glucose, arginine and tolbutamide, in fillies and colts during the first 3 months after birth. STUDY DESIGN: In vivo experiment examining sex differences in pancreatic ß cell function in foals. METHODS: Female (n = 8) and male (n = 5) pony foals were infused i.v. with glucose (0.5 g/kg bwt 40% dextrose), arginine (100 mg/kg bwt) or tolbutamide (10 or 20 mg/kg bwt) over 5 min, at 48 h intervals, to assess pancreatic ß cell function at ages 2 and 12 weeks. Blood samples (4 ml) were taken through a jugular catheter at -30, -15 and 0 min (immediately before) and 5, 15, 30, 45, 60, 90 and 120 min after glucose, arginine and tolbutamide administration for measurements of plasma glucose, α-amino-nitrogen and insulin concentrations. RESULTS: The maximum increment in plasma insulin concentration in response to glucose was significantly higher in female (395 ± 58 ng/l) than male (172 ± 37 ng/l, P<0.05) pony foals 2 weeks after birth and the area under the insulin curve was significantly greater in females at this age. At 12 weeks, the insulin increment in response to glucose was significantly greater in fillies 45 min post infusion. The ß cell responses to arginine and tolbutamide were not sex-linked at either age. CONCLUSIONS: These data show that in ponies, fillies have a greater ß cell response to glucose than colts in early post natal life. Since glucose clearance was unaffected by sex, the results suggest that fillies may be less insulin sensitive than colts shortly after birth. Innate sex differences in the secretion and action of insulin in early post natal life may influence tissue development and growth with potentially more long-term metabolic consequences.

Glucocorticoid overexposure in neonatal life alters pancreatic beta-cell function in newborn foals.

Studies in humans and animals have linked abnormal programming of adult tissue function to excess glucocorticoids during perinatal development. The current study investigated the hypothesis that physiological variations in glucocorticoid concentrations during early neonatal life of the foal alter the secretory responses of the pancreatic ß cells 2 and 12 wk after treatment. Spontaneously delivered foals received either saline or long-acting ACTH for 5 d from 1 d after birth to maintain an endogenous rise in cortisol concentrations. Starting at d 10, pancreatic ß cell function was studied using an intravenous (i.v.) glucose tolerance test, an i.v. arginine challenge, and an i.v. tolbutamide challenge. The maximum increment in plasma insulin achieved in response to exogenous glucose was less in ACTH-treated foals at both 2 and 12 wk of age (P<0.05). By 12 wk of age, developmental changes also occurred in the magnitude and biphasic pattern of glucose-stimulated insulin release. The area under the insulin curve during the early phase of insulin secretion (0 to 30 min) was not different between the 2- and 12-wk-old animals but was significantly greater during the later phase (30 to 120 min) at 12 wk than at 2 wk (P<0.05). Arginine infusion induced a brief 5 to 15 min increase in plasma concentrations of insulin that was not different in saline- and ACTH-treated foals. The ß-cell response to tolbutamide infusion was rapid and monophasic, and there was no difference (P>0.05) in the area under the insulin curve with treatment at 2 or at 12 wk. However, after tolbutamide, plasma insulin concentrations remained increased for a longer period in the ACTH-treated than in the saline-treated foals at 12 wk of age (P<0.05). Hence, this is the first study to show altered pancreatic ß-cell function after ACTH-induced glucocorticoid overexposure during early postnatal life in foals.

Hypothalamic-pituitary-adrenal axis function in pony foals after neonatal ACTH-induced glucocorticoid overexposure.

REASONS FOR PERFORMING THE STUDY: The effects of overexposure to glucocorticoids during early life of the foal on the subsequent HPA programming of the hypothalamic-pituitary-adrenal axis are unknown. OBJECTIVES: To test the hypotheses that excess glucocorticoid exposure in early life subsequently increases both basal plasma concentrations of cortisol and the adrenocortical responsiveness to exogenous adrenocorticotropic hormone (ACTH). METHODS: Foals received either saline (0.9% NaCl, n = 9) or long-acting ACTH (0.125 mg i.m. b.i.d., n = 6) for 5 days from Day 1 to increase endogenous cortisol concentrations. Long-term indwelling catheters were inserted under local anaesthesia into the jugular veins of foals aged 2 and 12 weeks. After recovery, short-acting ACTH1-24 was given as a single i.v. injection (2 microg/kg bwt) and blood samples were taken at 5-30 min intervals before and after ACTH administration to measure plasma cortisol concentrations. RESULTS: Basal plasma cortisol concentrations were higher in ACTH- than in saline-treated foals at age 3 weeks, but not at 13 weeks. There were no significant differences in either the time profile or the area under the cortisol curve in response to ACTH between the 2 groups. CONCLUSIONS: These data suggest that ACTH-induced overexposure to glucocorticoids during early post natal life of the foal does not have a programming effect on HPA axis function at 13 weeks. In foals, the effects of ACTH-induced overexposure to glucocorticoids, if any, may not become apparent until much later in life in a long-lived species such as the horse. POTENTIAL RELEVANCE: These studies suggest that clinical and other stressful conditions that raise plasma cortisol concentrations during early life are unlikely to programme cardiovascular and metabolic function in horses in the short term.

Fetal cardiovascular, metabolic and endocrine responses to acute hypoxaemia during and following maternal treatment with dexamethasone in sheep.

In sheep, direct fetal treatment with dexamethasone alters basal cardiovascular function and the cardiovascular response to acute hypoxaemia. However, in human clinical practice, dexamethasone is administered to the mother, not to the fetus. Hence, this study investigated physiological responses to acute hypoxaemia in fetal sheep during and following maternal treatment with dexamethasone in doses and at dose intervals used in human clinical practice. Under anaesthesia, 18 fetal sheep were instrumented with vascular and amniotic catheters, a carotid flow probe and a femoral flow probe at 118 days gestation (term ca 145 days). Following 6 days recovery at 124 days gestation, 10 ewes received dexamethasone (2 x 12 mg daily i.m. injections in saline). The remaining animals were saline-injected as age-matched controls. Two episodes of hypoxaemia (H) were induced in all animals by reducing the maternal F(IO2)for 1 h (H1, 8 h after the second injection; H2, 3 days after the second injection). In fetuses whose mothers received saline, hypoxaemia induced significant increases in fetal arterial blood pressure, carotid blood flow and carotid vascular conductance and femoral vascular resistance, significant falls in femoral blood flow and femoral vascular conductance and transient bradycardia. These cardiovascular responses were accompanied by a fall in arterial pH, increases in blood glucose and blood lactate concentrations and increased plasma concentrations of catecholamines. In fetuses whose mothers were treated with dexamethasone, bradycardia persisted throughout hypoxaemia, the magnitude of the femoral vasoconstriction, the glycaemic, lactacidaemic and acidaemic responses and the plasma concentration of neuropeptide Y (NPY) were all enhanced during H1. However, during H2, all of these physiological responses were similar to saline controls. In dexamethasone fetuses, the increase in plasma adrenaline was attenuated during H1 and the increase in carotid vascular conductance during hypoxaemia failed to reach statistical significance both during H1 and during H2. These data show that maternal treatment with dexamethasone in doses and intervals used in human obstetric practice modified the fetal cardiovascular, metabolic and endocrine defence responses to acute hypoxaemia. Furthermore, dexamethasone-induced alterations to these defences depended on whether the hypoxaemic challenge occurred during or following maternal dexamethasone treatment.

The effects of pregnancy on the cardiovascular response to acute systemic isocapnic hypoxia in conscious sheep.

OBJECTIVE: This study tested the hypothesis that pregnancy affects the cardiovascular responses to hypoxia by altering the outputs of the peripheral components of the stress system and independent of changes in P(a)CO(2). DESIGN: Comparison of cardiovascular and endocrine responses to acute isocapnic hypoxia between pregnant and non-pregnant ewes. SETTING: Experimental laboratory. SAMPLE: Fifteen pregnant (118 days of gestation; term is ca. 145 days) and 8 non-pregnant sheep. METHODS: Chronically instrumented pregnant and non-pregnant ewes were subjected to 1 hour normoxia, 1 hour of acute systemic isocapnic hypoxia and 1 hour recovery. MAIN OUTCOME MEASURES: Arterial blood pressure, heart rate, femoral blood flow and femoral vascular conductance were recorded continuously throughout and arterial blood samples were taken during normoxia, hypoxia and recovery for the measurement of blood gas, metabolic and endocrine status. RESULTS: Basal blood pressure and blood glucose and lactate concentrations were lower in pregnant animals (P < 0.05). In contrast, basal cardiovascular variables and plasma concentrations of noradrenaline, adrenaline, neuropeptide Y, adrenocorticotropic hormone (ACTH) and cortisol were similar in pregnant and non-pregnant ewes. During hypoxia similar reductions in P(a)O(2) occurred in pregnant and non-pregnant animals, without alterations in P(a)CO(2) or pH(a). In non-pregnant ewes, acute hypoxia induced a transient increase in arterial pressure and sustained tachycardia without significant changes in femoral haemodynamics. Pregnancy attenuated the cardiovascular response, significantly diminishing the magnitude of the increment in heart rate throughout the hypoxic challenge (P < 0.001). However, hypoxia did not induce significant changes in blood metabolites or in plasma concentrations of any stress hormone measured in either pregnant or non-pregnant animals. CONCLUSION: Pregnancy not only affects basal but also stimulated cardiovascular function in the mother. The diminished chronotropic response to hypoxia in pregnancy is mediated via mechanisms independent of changes in P(a)CO(2) or in plasma concentrations of hormones or metabolites associated with activation of the stress system.