The placental exposome, placental epigenetic adaptations and lifelong cardio-metabolic health.

The placental exposome represents the sum of all placental exposures, and through its influence on placental function can affect an individual's susceptibility to cardio-metabolic disease later in life. The placental exposome includes direct exposures during gestation, as well as those prior to gestation that affect the gametes or aspects of maternal physiology that influence placental function. This review will discuss the evidence for placental responses to environmental signals and its involvement in programming offspring health. A wide range of exposures may influence the placenta including maternal metabolic and endocrine status, nutrition, stress and toxins. Epigenetic changes within the placenta induced by these exposures may mediate persistent effects on placental function. Identifying which exposures are most influential in terms of placental function and offspring health is key to focusing future research and developing stratified and personalised interventions.

N-acetylcysteine, xCT and suppression of Maxi-chloride channel activity in human placenta.

INTRODUCTION: Placental oxidative stress features in pregnancy pathologies but in clinical trials antioxidant supplementation has not improved outcomes. N-acetylcysteine (NAC) stimulates glutathione production and is proposed as a therapeutic agent in pregnancy. However, key elements of N-acetylcysteine biology, including its cellular uptake mechanism, remains unclear. This study explores how the cystine/glutamate transporter xCT may mediate N-acetylcysteine uptake and how N-acetylcysteine alters placental redox status. METHODS: The involvement of xCT in NAC uptake by the human placenta was studied in perfused placenta and Xenopus oocytes. The effect of short-term N-acetylcysteine exposure on the placental villous proteome was determined using LC-MS. The effect of N-acetylcysteine on Maxi-chloride channel activity was investigated in perfused placenta, villous fragments and cell culture. RESULTS: Maternoplacental N-acetylcysteine administration stimulated intracellular glutamate efflux suggesting a role of the exchange transporter xCT, which was localised to the microvillous membrane of the placental syncytiotrophoblast. Placental exposure to a bolus of N-acetylcysteine inhibited subsequent activation of the redox sensitive Maxi-chloride channel independently of glutathione synthesis. Stable isotope quantitative proteomics of placental villi treated with N-acetylcysteine demonstrated changes in pathways associated with oxidative stress, apoptosis and the acute phase response. DISCUSSION: This study suggests that xCT mediates N-acetylcysteine uptake into the placenta and that N-acetylcysteine treatment of placental tissue alters the placental proteome while regulating the redox sensitive Maxi-chloride channel. Interestingly N-acetylcysteine had antioxidant effects independent of the glutathione pathway. Effective placental antioxidant therapy in pregnancy may require maintaining the balance between normalising redox status without inhibiting physiological redox signalling.

Vitamin D deficiency and glycemic control among patients with type 2 diabetes mellitus in Jazan City, Saudi Arabia.

Background: The prevalence of vitamin D deficiency (VDD) is predicted to be high in patients with type 2 diabetes mellitus (T2DM), but the exact figure is not known in Jazan, Saudi Arabia. Emerging data suggests that VDD plays a role in glycemic control. The aim of this study was to measure the prevalence of VDD among T2DM patients and to investigate its association with patients' characteristics and glycemic control in Jazan. Methods: This is an analytical cross-sectional study which recruited 309 patients with T2DM randomly from primary health care centers in Jazan. Logistic regression analysis was conducted to determine the VDD predictors and to examine the association of VDD and glycemic control. Results: The VDD prevalence was found to be 60.8% in patients with T2DM. Age, gender, diabetic retinopathy (DR), dyslipidemia, glycemic control, and obesity were significantly associated with VDD, and all except obesity were independent predictors of VDD. There was a significant negative correlation between 25-hydroxyvitamin D and HbA1c. VDD was a significant independent predictor of poor glycemic control after adjustment for hypertension, DR, diabetic neuropathy, type of diabetes medication, diabetes duration, and education level. Conclusion: In this Saudi Arabian population, VDD is highly prevalent in people with T2DM and is associated with poor glycemic control. Health education targeting patients with T2DM and national strategies regarding vitamin D fortification are needed to prevent VDD in Saudi Arabia. Earlier VDD diagnosis by health care providers may help to improve the outcome for patients with T2DM. Establishing the causal association between VDD and glycemic control and clarifying the biological role of vitamin D in T2DM are important aims for future studies.

Glutamate cycling may drive organic anion transport on the basal membrane of human placental syncytiotrophoblast.

The organic anion transporter OAT4 (SLC22A11) and organic anion transporting polypeptide OATP2B1 (SLCO2B1) are expressed in the basal membrane of the placental syncytiotrophoblast. These transporters mediate exchange whereby uptake of one organic anion is coupled to efflux of a counter-ion. In placenta, these exchangers mediate placental uptake of substrates for oestrogen synthesis as well as clearing waste products and xenobiotics from the fetal circulation. However, the identity of the counter-ion driving this transport in the placenta, and in other tissues, is unclear. While glutamate is not a known OAT4 or OATP2B1 substrate, we propose that its high intracellular concentration has the potential to drive accumulation of substrates from the fetal circulation. In the isolated perfused placenta, glutamate exchange was observed between the placenta and the fetal circulation. This exchange could not be explained by known glutamate exchangers. However, glutamate efflux was trans-stimulated by an OAT4 and OATP2B1 substrate (bromosulphothalein). Exchange of glutamate for bromosulphothalein was only observed when glutamate reuptake was inhibited (by addition of aspartate). To determine if OAT4 and/or OATP2B1 mediate glutamate exchange, uptake and efflux of glutamate were investigated in Xenopus laevis oocytes. Our data demonstrate that in Xenopus oocytes expressing either OAT4 or OATP2B1 efflux of intracellular [(14)C]glutamate could be stimulated by conditions including extracellular glutamate (OAT4), estrone-sulphate and bromosulphothalein (both OAT4 and OATP2B1) or pravastatin (OATP2B1). Cycling of glutamate across the placenta involving efflux via OAT4 and OATP2B1 and subsequent reuptake will drive placental uptake of organic anions from the fetal circulation.

Differential pathways to adult metabolic dysfunction following poor nutrition at two critical developmental periods in sheep.

Epidemiological and experimental studies suggest early nutrition has long-term effects on susceptibility to obesity, cardiovascular and metabolic diseases. Small and large animal models confirm the influence of different windows of sensitivity, from fetal to early postnatal life, on offspring phenotype. We showed previously that undernutrition in sheep either during the first month of gestation or immediately after weaning induces differential, sex-specific changes in adult metabolic and cardiovascular systems. The current study aims to determine metabolic and molecular changes that underlie differences in lipid and glucose metabolism induced by undernutrition during specific developmental periods in male and female sheep. Ewes received 100% (C) or 50% nutritional requirements (U) from 1-31 days gestation, and 100% thereafter. From weaning (12 weeks) to 25 weeks, offspring were then fed either ad libitum (CC, UC) or were undernourished (CU, UU) to reduce body weight to 85% of their individual target. From 25 weeks, all offspring were fed ad libitum. A cohort of late gestation fetuses were studied after receiving either 40% nutritional requirements (1-31 days gestation) or 50% nutritional requirements (104-127 days gestation). Post-weaning undernutrition increased in vivo insulin sensitivity, insulin receptor and glucose transporter 4 expression in muscle, and lowered hepatic methylation at the delta-like homolog 1/maternally expressed gene 3 imprinted cluster in adult females, but not males. Early gestational undernutrition induced lower hepatic expression of gluconeogenic factors in fetuses and reduced in vivo adipose tissue insulin sensitivity in adulthood. In males, undernutrition in early gestation increased adipose tissue lipid handling mechanisms (lipoprotein lipase, glucocorticoid receptor expression) and hepatic methylation within the imprinted control region of insulin-like growth factor 2 receptor in adulthood. Therefore, undernutrition during development induces changes in mechanisms of lipid and glucose metabolism which differ between tissues and sexes dependent on the period of nutritional restriction. Such changes may increase later life obesity and dyslipidaemia risk.

Developmental origins of the metabolic syndrome: body clocks and stress responses.

The prevalence of the metabolic syndrome, which represents a spectrum of metabolic and cardiovascular disorders, continues to increase at an alarming rate in contemporary society. Inadequate responses of an individual to environmental challenges such as unbalanced diet or lack of physical exercise during their life course has been recognised to increase risk of this pathological condition. Recent evidence suggests that this may involve alterations in the settings of the circadian clock system, which consists of oscillating molecular pacemakers found not only in the hypothalamic region of the brain but also in most peripheral tissues, and of the hypothalamic-pituitary-adrenal (HPA) axis which regulates stress responses. These two systems are now known to interact to produce an integrated response to environmental challenges. In this review, we highlight the importance of environmental cues during early development in establishing the homeostatic set-points of the circadian clock and HPA stress systems. These effects can operate within the normal range and are not in themselves pathological, but can nevertheless affect an individual's response to environmental challenges in adult life and thus their risk of the metabolic syndrome.

Sex- and age-specific effects of nutrition in early gestation and early postnatal life on hypothalamo-pituitary-adrenal axis and sympathoadrenal function in adult sheep.

The early-life environment affects risk of later metabolic disease, including glucose intolerance, insulin resistance and obesity. Changes in hypothalamo-pituitary-adrenal (HPA) axis and sympathoadrenal function may underlie these disorders. To determine consequences of undernutrition in early gestation and/or immediately following weaning on HPA axis and sympathoadrenal function, 2- to 3-year-old Welsh Mountain ewes received 100% (C, n = 39) or 50% nutritional requirements (U, n = 41) from 1-31 days gestation, and 100% thereafter. From weaning (12 weeks) to 25 weeks of age, male and female offspring were then either fed ad libitum (CC, n = 22; UC, n = 19) or were undernourished (CU, n = 17; UU, n = 22) such that body weight was reduced to 85% of their individual target, based on a growth trajectory calculated from weights taken between birth and 12 weeks. From 25 weeks, ad libitum feeding was restored for all offspring. At 1.5 and 2.5 years, adrenocorticotropic hormone (ACTH) and cortisol concentrations were measured at baseline and in response to corticotropin-releasing factor (CRF) (0.5 microg kg(1)) plus arginine vasopressin (AVP) (0.1 microg kg(1)). At 2.5 years, HPA axis and sympathoadrenal (catecholamine) responses to a transport and isolation stress test were also measured. In females, post-weaning undernutrition reduced pituitary output (ACTH) but increased adrenocortical responsiveness (cortisol:ACTH area under curve) during CRF/AVP challenge at 1.5 years and increased adrenomedullary output (adrenaline) to stress at 2.5 years. In males, cortisol responses to stress at 2.5 years were reduced in those with slower growth rates from 12 to 25 weeks. Early gestation undernutrition was associated with increased adrenocortical output in 2.5-year-old females only. Pituitary and adrenal responses were also related to adult body composition. Thus, poor growth in the post-weaning period induced by nutrient restriction has sex- and age-specific effects on HPA and sympathoadrenal function. With altered glucose tolerance previously reported in this model, this may have long-term detrimental effects on metabolic homeostasis and cardiovascular function.

Effects of pre- and periconceptional undernutrition on arterial function in adult female sheep are vascular bed dependent.

The nutritional environment during development and even prior to conception may contribute to cardiovascular risk. In mature adult female sheep, we investigated the effect of preconceptional and periconceptional maternal nutritional restriction on the vascular reactivity of arteries from four vascular beds supplying the heart, thorax, kidney and hindlimb. Welsh Mountain ewes received 100% of nutrient requirements throughout gestation (control group, C, n = 18), or 50% of nutrient requirements for 30 days prior to conception (preconceptional group, PRE, n = 20) or for 15 days either side of conception (periconceptional group, PERI, n = 31) and 100% thereafter. In 3.5-year-old female offspring, the left anterior descending coronary (LAD), left internal thoracic (LITA), right renal and second and third order femoral arteries were dissected and their reactivity was assessed by organ bath or wire myography. Vasoconstrictor responses were greater in both LAD and LITA from PERI offspring compared with C (P < 0.01), while vasoconstriction was unaffected by maternal diet in arteries from the renal and femoral circulations (P = n.s.). Endothelium-dependent and -independent vasodilatation was attenuated in third order femoral arteries of PRE and PERI groups compared with C (P < 0.05). Endothelium-independent vasodilatation was attenuated in both the LAD and renal arteries in the PERI group compared with C (P < 0.05). These data show that moderate maternal undernutrition either prior to or around conception affects vascular function of adult offspring. The effect depends on the timing of the insult, but also on the vascular bed studied and vessel hierarchy in the vascular tree.

The effect of maternal undernutrition in early gestation on gestation length and fetal and postnatal growth in sheep.

In utero undernutrition in humans may result in cardiovascular (CV), metabolic, and growth adaptations. In sheep, maternal nutrient restriction during pregnancy, without effects on fetal or birth weight, results in altered CV control in the offspring. Adjustment of gestation length after undernutrition could be a strategy to enhance postnatal health/survival. The aim of this study was to determine in sheep the effect of a 50% reduction in maternal nutrient intake [undernutrition group (U) versus 100%, control group (C)] during 1-31 d of gestation (dGA) on gestation length and offspring size. By 28 dGA, U ewes had gained less weight than C, and twin-bearing ewes had gained less weight than singleton-bearing ewes regardless of group (p<0.05). In different-sex twin pairs, maternal undernutrition resulted in longer gestation compared with C (146.5+/-0.6 versus 144.6+/-0.6 d, p<0.05). Increased weight gain by weaning (20.8+/-0.8 versus 17.9+/-0.8 kg, p<0.05) was observed in U male twins. These findings suggest that the strategy (i.e. growth rate or length of time in utero) adopted by the fetus to enhance immediate survival depends on offspring number and sex. This is likely to reflect the degree of constraint imposed on the fetus.

Mismatched pre- and postnatal nutrition leads to cardiovascular dysfunction and altered renal function in adulthood.

The early life environment has long-term implications for the risk of developing cardiovascular (CV) disease in adulthood. Fetal responses to changes in maternal nutrition may be of immediate benefit to the fetus, but the long-term effects of these adaptations may prove detrimental if nutrition in postnatal life does not match that predicted by the fetus on the basis of its prenatal environment. We tested this predictive adaptive response hypothesis with respect to CV function in sheep. We observed that a mismatch between pre- and postnatal nutrient environments induced an altered CV function in adult male sheep that was not seen when environments were similar. Sheep that received postnatal undernutrition alone had altered growth, CV function, and basal hypothalamo-pituitary-adrenal axis activity in adulthood. Prenatal undernutrition induced greater weight gain by weaning compared with the prenatal control diet, which may provide a reserve in the face of a predicted poor diet in later life. In an adequate postnatal nutrient environment (i.e., relatively mismatched), these offspring exhibited cardiac hypertrophy and altered CV function in adulthood. These data support the concept that adult CV function can be determined by developmental responses to intrauterine nutrition made in expectation of the postnatal nutritional environment, and that if these predictions are not met, the adult may be maladapted and at greater risk of CV disease. Our findings have substantial implications for devising strategies to reduce the impact of a mismatch in nutrition levels in humans undergoing rapid socio-economic transitions in both developing and developed societies.

Nutritional challenges during development induce sex-specific changes in glucose homeostasis in the adult sheep.

The early-life environment has implications for risk of adult-onset diseases, such as glucose intolerance, insulin insensitivity, and obesity, effects that may occur with or without reduced birth weight. We determined the consequences of nutrient restriction in early gestation and early postnatal life and their interactions on postnatal growth, body composition, and glucose handling. Ewes received 100% (C, n = 39) or 50% nutritional requirements (U, n = 41) from 1 to 31 days gestation and 100% thereafter. Male and female offspring (singleton/twin) from C and U ewes were then fed either ad libitum (CC n = 22, UC n = 19) or to reduce body weight to 85% of target from 12 to 25 wk of age (CU n = 17, UU n = 22) and ad libitum thereafter. At 1.5 and 2.5 yr, glucose handling was determined by area under the curve (AUC) for glucose and insulin concentrations following intravenous glucose (0.5 g/kg body wt). Insulin sensitivity was determined at 2.5 yr following intravenous insulin (0.5 IU/kg). In females, postnatal undernutrition reduced (P < 0.05) glucose AUC at both ages, regardless of prenatal nutrition. Postnatal undernutrition did not affect insulin secretion in females but enhanced insulin-induced glucose disappearance in singletons. Poor early postnatal growth was associated with increased fat in females. In males, glucose tolerance was unaffected by undernutrition despite changes in insulin AUC dependent on age, treatment, and single/twin birth. Nutrition in early postnatal life has long-lasting, sex-specific effects on glucose handling in sheep, likely due, in females, to enhanced insulin sensitivity. Improved glucose utilization may aid weight recovery but have negative implications for glucose homeostasis and body composition over the longer term.

Developmental regulation of hepatic and renal gluconeogenic enzymes by thyroid hormones in fetal sheep during late gestation.

Tissue glucose-6-phosphatase (G6P) and phosphoenolpyruvate carboxykinase (PEPCK) activities were investigated in sheep fetuses after experimental manipulation of thyroid hormone status. Increments in hepatic and renal G6P and PEPCK activities seen between 127-130 and 140-145 days of gestation (term, 145 +/- 2 days) were abolished when the normal prepartum rise in plasma triiodothyronine (T3), but not cortisol, was prevented by fetal thyroidectomy (TX). At 127-130 days, hepatic and renal G6P, and renal PEPCK, activities were similar in intact and TX fetuses; however, hepatic PEPCK was increased by TX. At 140-145 days, tissue G6P and PEPCK activities in TX fetuses were lower than in intact fetuses. In immature fetuses infused with cortisol (2-3 mg (kg body wt)-1 day-1) for five days, hepatic and renal enzyme activities were increased to those seen in mature fetuses near term. After five days of T3 infusion (8-12 microg (kg body wt)-1 day-1), G6P and PEPCK activities in the liver and kidney were greater than in saline-infused fetuses, but only renal G6P and PEPCK increased to the level seen close to term. Therefore, in fetal sheep, thyroid hormones are important for the prepartum rises in G6P and PEPCK activities in the liver and kidney and may mediate, in part, the maturational effects of cortisol.