Lucy R. Green

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.

Peri-implantation and late gestation maternal undernutrition differentially affect fetal sheep skeletal muscle development

Poor prenatal nutrition is associated with a greater risk of adult glucose intolerance and insulin insensitivity in the offspring. Skeletal muscle is the primary tissue for glucose utilization, and insulin resistance in muscle is the earliest identifiable abnormality in the pre‐diabetic patient. We investigated the effect of early and late gestation undernutrition on structure and markers of growth and glucose metabolism regulation in the fetal triceps brachii (TB, slow‐ and fast‐twitch myofibres) and soleus (slow‐twitch myofibres) muscles. Pregnant sheep were fed 100% nutrient requirements (C, n= 8) or a restricted diet peri‐implantation (PI, n= 9; 40%, 1–31 days gestation (dGA) (term ∼147)) or in late gestation (L, n= 6; 50%, 104–127 dGA). At 127 ± 1 dGA we measured myofibre and capillary density in the fetal TB and soleus muscles, and mRNA levels in the TB of insulin receptor (InsR), glucose transporter‐4 (GLUT‐4) and type 1 insulin‐like growth factor receptor (IGF‐1R). Total myofibre and capillary densities were lower in the TB, but not the soleus, of PI and L fetuses. The predominant effect in the L group was on slow‐twitch myofibres. In TB, InsR, GLUT‐4 and IGF‐1R mRNA levels were greater in L group fetuses. Our finding of reduced myofibre density is consistent with a redistribution of resources at the expense of specific peripheral tissues by early and late gestation undernutrition which may be mediated by a decrease in capillary density. The increase in key regulatory components of glucose uptake following late gestation undernutrition may constitute a short‐term compensation to maintain glucose homeostasis in the face of fewer type I (insulin‐sensitive) myofibres. However, together these adaptations may influence the risk of later metabolic disease and thus our findings have implications for future strategies aimed at improving maternal diet.

The role of nitric oxide synthesis in cardiovascular responses to acute hypoxia in the late gestation sheep fetus.

1. In fetal sheep (123‐129 days gestation) we investigated the effect of acute isocapnic hypoxia (Pa,O2, 12 +/‐ 0.6 mmHg) on the fetal heart rate (FHR), mean systemic arterial blood pressure (MAP), carotid blood flow (CBF), femoral blood flow (FBF), carotid vascular resistance (CVR) and femoral vascular resistance (FVR) with the infusion of either the nitric oxide synthase (NOS) inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME) or saline vehicle. 2. During normoxia, CBF was lower (P < 0.05) and MAP, FVR and CVR were higher with L‐NAME than with vehicle infusion (P < 0.01, P < 0.05 and P < 0.01, respectively). FHR fell 15 min after the onset of L‐NAME infusion (P < 0.05). During hypoxia in both groups, FHR showed an initial rapid fall (P < 0.05) and subsequent return to prehypoxic levels, and there was a fall in FBF (P < 0.01). MAP increased during hypoxia with vehicle (P < 0.05) but not L‐NAME infusion: thus MAP was similar during hypoxia in the two groups. The rebound tachycardia seen during recovery in the vehicle group (P < 0.01) was not evident in the L‐NAME group. The rise in CBF and fall in CVR during hypoxia with vehicle (P < 0.01 and P < 0.05, respectively) was absent with L‐NAME infusion. FVR rose during hypoxia in both groups (P < 0.05). 3. Thus NOS inhibition alters basal systemic vascular tone in the late gestation fetus. The rise in CBF and fall in CVR during hypoxia is absent with NOS inhibition.

Effect of nutritional restriction in early pregnancy on isolated femoral artery function in mid-gestation fetal sheep

Unbalanced maternal nutrition affects fetal endocrine and cardiovascular systems, sometimes accompanied by changes in growth, although this is usually in late gestation. We determined the effect of moderate restriction for the first half of gestation of maternal dietary protein, or of total calorific intake on isolated resistance artery function of mid‐gestation fetal sheep. Welsh Mountain ewes were nutritionally restricted by 30 % of the recommended nutrient intake (globally restricted) or 30 % of the recommended protein intake (protein‐restricted), compared to control ewes fed 100 % of recommended nutrient intake, for ~12 days prior to conception and for the subsequent 70 days of gestation. At mid‐gestation, fetal and placental weights were similar in all dietary groups. In isolated femoral arteries, the response curve to noradrenaline was reduced in protein‐restricted group fetuses (P < 0.05). Maximal relaxation (P < 0.01) and sensitivity (P < 0.05) to acetylcholine were markedly reduced in protein‐restricted group fetuses, and to a smaller extent in globally restricted group fetuses (response curve, P < 0.05). The dilator response (P < 0.05) and sensitivity (P < 0.05) to the α2 agonist UK14304 was lower in protein‐, but not in globally restricted group fetuses. The response (P < 0.05) and sensitivity (P < 0.05) to the nitric oxide donor sodium nitroprusside were reduced in protein‐restricted group fetuses compared to controls. Our data show that dietary imbalance, in particular restricted protein, of the ewe can produce blunting of endothelial‐dependent and ‐independent relaxation in systemic arteries from the mid‐gestation fetus. These changes may precede perturbed late‐gestation fetal and postnatal cardiovascular control.

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.

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 μg kg−1) plus arginine vasopressin (AVP) (0.1 μg 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.

Angiotensin II and cardiovascular chemoreflex responses to acute hypoxia in late gestation fetal sheep

1 In six intact and nine carotid sinus denervated (CSD) fetal sheep (125–128 days gestation) we measured heart rate (FHR), mean systemic arterial blood pressure (MAP), femoral and carotid blood flows (FBF and CBF), and femoral and carotid vascular resistances (FVR and CVR). Three experiments were conducted on successive days: normoxia followed by acute isocapnic hypoxia (Pa,O2 to ca 12 mmHg) with infusion of vehicle (HV experiment), the same protocol but with infusion of the angiotensin converting enzyme (ACE) inhibitor, captopril (HC experiment), and normoxia alone with captopril infusion (NC experiment). Plasma angiotensin II concentration ([AII]) was measured in these fetuses, and in a separate group of fetuses (n= 5) that were infused with the nitric oxide (NO) synthesis inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME) or saline vehicle. 2 During normoxia, cardiovascular parameters and plasma [AII] were unaltered by captopril infusion, apart from a fall in MAP (NC experiment only, P < 0.05) and FHR (HC experiment only, P < 0.05) in intact and CSD fetuses, respectively. No differences were observed between intact and CSD groups. 3 At the onset of hypoxia the rapid initial fall in FHR and rise in FVR was attenuated in CSD fetuses. In all fetuses FHR returned towards prehypoxic levels as hypoxia continued. In contrast, during hypoxia with vehicle infusion (HV experiment) plasma [AII] rose to a similar level in intact and CSD fetuses. 4 In both intact and CSD fetuses, the rise in [AII] during hypoxia was blocked by captopril or L‐NAME infusion. In CSD, but not intact, fetuses infused with captopril the rise in MAP was absent, and the fall in FBF and rise in FVR did not reach significance during hypoxia. 5 Thus, during normoxia CSD alone, or combined with ACE inhibition, does not consistently alter basal cardiovascular control in the late gestation fetus. The rise in [AII] during hypoxia is not mediated by carotid reflexes but may involve NO‐dependent mechanisms. In intact fetuses, AII does not appear to be pivotal in cardiovascular control during hypoxia. It is only when carotid reflex mechanisms are removed that a role for AII in the regulation of MAP and peripheral blood flow during hypoxia becomes apparent. These findings lend weight to the idea of multiple mechanisms of fetal cardiovascular control during hypoxia.

Programming of endocrine mechanisms of cardiovascular control and growth

Several epidemiologic studies have linked size at birth to health in adult life. One school of thought centers on the part that periconceptual or intrauterine events play in this relationship. The idea is that an event, or several events, during critical periods of development can program, or permanently alter, fetal physiology resulting in altered size at birth and subsequent adult disease, including that of the cardiovascular system. Maternal diet or body composition at the time of conception can influence placental development and subsequent transfer of nutrients and substrates to the fetus. Alterations to the maternal diet or body composition throughout gestation are then seen as challenges that are superimposed on this backdrop of periconceptual events. One task is to find an animal model that replicates the major features of the epidemiologic data:for adult cardiovascular disease this would be alteredfetal size and the development of postniatal hypertension. In addition, a critical issue is to investigate the mechanisms underlying this Fetal Origins of Adult Disease hypothesis. The multiple mechanisms that constitutefetal cardiovascular responses to hypoxia in lategestation at neuronal, endocrine, and local tissue levels have been studied extensively, and there is evidence from several diferent experimental paradigms that these control mechanisms can be progranmmed by intrauterine challenges. This review synthesizes the current knowledge in this area and considers how the programming of cardiovascular control relates to fetal growth.

Adaptation of cardiovascular responses to repetitive umbilical cord occlusion in the late gestation ovine fetus

1 The impact of repeated umbilical cord occlusion on the normal maturation of fetal heart rate (FHR) and mean arterial pressure (MAP) and the cardiovascular responses to successive umbilical cord occlusion was investigated over a 21 day period in the latter part of gestation. 2 Fifteen chronically instrumented sheep (control group n = 6; occlusion group n = 9) were studied for 21 days (113‐133 days of gestation, term = 145 days) with umbilical cord occlusions (90 s duration) performed every 30 min for 1‐4 h each day. On days 1, 9 and 18, FHR, FHR variation and MAP were monitored continuously and fetal arterial blood gases, pH and metabolites were measured at predetermined intervals. The baroreflex response to 75‐100 μg phenylephrine (i.v.) was tested on days 1 and 18. 3 Basal FHR decreased (ΔFHR: control, 34.6 ± 3.6 beats min−1; occlusion, 36.9 ± 2.7 beats min−1) and MAP increased (ΔMAP: control, 3.1 ± 1.7 mmHg; occlusion, 5.2 ± 2.1 mmHg) to a similar extent in control and occlusion groups between days 1 and 21 of the study. There was a small decline in FHR variation over the 21 day study in occlusion, but not control, group fetuses. 4 The magnitude of the fall in FHR decreased and the rise in MAP increased, despite similar changes in blood gases in response to umbilical cord occlusion, over the course of the 21 day study. Despite a significant decline in the ratio of ΔFHR to ΔMAP on days 9 and 18 compared to day 1, there was no difference between control and occlusion groups in baroreflex sensitivity. However ΔFHR/ΔPO2, an index of chemoreceptor sensitivity, had decreased by day 9 and 18 compared to day 1. 5 The cardiovascular responses to umbilical cord occlusion are altered with repetitive occlusions during the latter part of gestation, with a decrease in ΔFHR/ΔMAP, which does not involve changes in baroreflex sensitivity, but may involve changes in chemoreceptor sensitivity. However, repeated umbilical cord occlusion appears to have no impact on baseline cardiovascular control since there was no change in the normal maturational decrease in FHR and rise in MAP.