Plasma leptin concentration in fetal sheep during late gestation: ontogeny and effect of glucocorticoids

The ontogeny and developmental control of plasma leptin concentration in the fetus are poorly understood. The present study investigated plasma leptin concentration in chronically catheterized sheep fetuses near term, and in neonatal and adult sheep. The effect of glucocorticoids on plasma leptin in utero was examined by fetal adrenalectomy and exogenous cortisol or dexamethasone infusion. In intact, untreated fetuses studied between 130 and 140 d (term, 145 +/- 2 d), plasma leptin concentration increased in association with the prepartum cortisol surge. Positive relationships were observed between plasma leptin in utero and both gestational age and plasma cortisol. Plasma leptin was also inversely correlated with fetal p(a)O(2). The ontogenic rise in plasma leptin was abolished by fetal adrenalectomy. In intact fetuses at 123-127 d, plasma leptin was increased by infusions of cortisol (3-5 mg kg(-1)d(-1), +127 +/- 21%) for 5 d and dexamethasone (45-60 microg kg(-1)d(-1), +268 +/- 61%) for 2 d. However, the cortisol-induced rise in plasma leptin was transient; by the fifth day of infusion, plasma leptin was restored to within the baseline range. These findings show that, in the sheep fetus, an intact adrenal gland is required for the normal ontogenic rise in plasma leptin near term. Furthermore, fetal treatment with exogenous and endogenous glucocorticoids increases circulating leptin concentration in utero.     

Dissociation of pulsatile cortisol and adrenocorticotropin secretion in fetal sheep during late gestation.

In the fetal sheep, plasma cortisol concentrations gradually increase in the last weeks of gestation and abruptly rise during the final 48-72 h preceding birth. To determine if these changes in mean circulating cortisol concentrations result from increased pulsatile secretion and are driven by changes in ACTH pulses, blood samples from five chronically catheterized fetuses were collected every 5 min for 2 h at 133 days gestation and every 4 days thereafter until delivery at 146 +/- 2 days. Volume was replaced after each blood sample and erythrocytes were returned every 20 min. Plasma cortisol and ACTH secretion were pulsatile in fetuses at all ages. Cortisol pulse frequency increased significantly with gestation from a mean of 2.2 pulses/2 h at 133 days to 4.8 pulses/2 h at 146 days. The interpulse interval (mean +/- SE) decreased between 133 and 146 days from 54 +/- 11 min to 23 +/- 3 min, respectively. Cortisol pulse amplitude increased significantly from 10 +/- 2 ng/ml at 133 days to 44 +/- 13 ng/ml at 146 days. In contrast to cortisol, ACTH pulse frequency (3 +/- 0.6 pulses/2 h) and amplitude (21 +/- 3 pg/ml) were similar at 133 days and 146 days. The coincidence of cortisol and ACTH pulses did not change between 133 and 146 days. Furthermore, the number of coincident pulses failed to exceed random associations (hypergeometric probability analysis) and could have occurred by chance alone (P values ranged from 0.11-0.63). A point by point comparison of cortisol and ACTH concentrations in fetal circulation indicate that only 36% of the variance in cortisol concentrations could be explained by variance in ACTH (cross-correlation analysis). These data suggest that fetal cortisol and ACTH secretion are pulsatile and that, as gestation advances, increases in constitutive cortisol pulse amplitude and frequency may not be predominantly driven by pulsatile changes in ACTH in the ovine fetal circulation near term.     

Oxytocin stimulates glucagon and insulin secretion in fetal and neonatal sheep

In adults of several species arginine vasopressin (AVP) and oxytocin (OT) stimulate pancreatic secretion of immunoreactive plasma glucagon (IRG). In fetal sheep AVP is an important stress hormone and may be simultaneously secreted with OT; however, their effects on IRG secretion are not known. We sought to determine if AVP and/or OT affected pancreatic IRG secretion in fetal and neonatal sheep. Either AVP or OT was infused for 30 min in chronically catheterized fetal and neonatal sheep, obtaining peripheral arterial and/or portal venous blood samples before; 10, 15, and 30 min during; and 15, 30, and 60 min after infusion for measurements of blood gases, hematocrit, IRG, immunoreactive plasma insulin (IRI) and plasma glucose. AVP did not affect IRG or IRI in fetal sheep (mean +/- SE, 133 +/- 1 days gestation), but small increases occurred in portal venous blood of lambs (2-49 days old). In contrast, OT (4.6 +/- 0.3 mU/min.kg; n = 12) increased fetal plasma IRG from 72 +/- 5 to 86 +/- 6 and 97 +/- 7 pg/ml (P less than 0.001) and IRI from 16 +/- 2 to 20 +/- 3 and 20 +/- 2 microU/ml (P less than 0.02) at 15 and 30 min, respectively; 157 +/- 11 microU OT/min.kg had no effect. In lambs (2-49 days old), 3.0 mU OT/min.kg increased arterial (n = 15) IRG from 139 +/- 19 to 367 +/- 43 and 483 +/- 76 pg/ml (P less than 0.01) and portal IRG (n = 8) from 167 +/- 39 to 341 +/- 72 and 502 +/- 148 pg/ml (P less than 0.01), respectively. Arterial and portal IRI also rose (P less than 0.01) from 36 +/- 4 to 82 +/- 12 and 105 +/- 32 microU/ml and from 29 +/- 5 to 65 +/- 13 and 51 +/- 7 microU/ml, respectively. Glucose was unchanged in all experiments. In fetal and neonatal sheep, AVP has minimal effects on IRG and IRI release. In contrast, OT increases both substantially; furthermore, there is a difference in fetal and neonatal responsiveness. OT may be important in modulating glucagon and insulin secretion during and after parturition.     

Regulation of basal adrenocorticotropin and cortisol secretion by arginine vasopressin in the fetal sheep during late gestation.

This study tested the hypothesis that arginine vasopressin (AVP) is involved in the regulation of basal ACTH secretion in the ovine fetus near term. In five fetuses challenged with AVP (1 microgram/ml, iv bolus) plasma ACTH concentrations increased to an 8-fold peak within 10 min of the preceding baseline (55 +/- 6 to 403 +/- 241 pg/ml). Cortisol in fetal circulation subsequently increased 2-fold (11 +/- 1 to 28 +/- 5 ng/ml) within 15 min of the AVP injection. The AVP-induced rise in plasma ACTH and cortisol concentrations was blocked when the fetus was pretreated with the AVP V1 receptor antagonist d(Ch2)5Tyr(Me)AVP. In a total of seven studies, antagonist (10 micrograms/kg estimated BW, iv bolus) was administered to three fetuses, aged 137-147 days gestation, followed 40 min later by the exogenous AVP challenge, as described above. After AVP antagonist treatment, basal ACTH and cortisol concentrations were not significantly different from the preinjection baseline levels (P greater than 0.05, by analysis of variance). Moreover, plasma ACTH and cortisol remained unchanged after the AVP challenge. To further define the role of endogenous AVP in basal ACTH and cortisol secretion, the AVP antagonist was administered (five studies in two fetuses) at 30-min intervals for a total of three injections per fetus. This extended AVP antagonist regimen also failed to alter fetal circulating concentrations of ACTH or cortisol (P greater than 0.05). Cortisol in the maternal circulation was not affected by any of the fetal AVP or AVP antagonist treatments. Lambs were born at 146 +/- 2 days gestation (n = 5), within the range for the normal duration of pregnancy. These data do not support the hypotheses that AVP is involved in the regulation of basal ACTH secretion in the fetal sheep during the 10 days preceding parturition. Rather, the ability of AVP antagonist to block the AVP-induced rise in plasma ACTH and cortisol in the fetus suggests that basal and stimulated ACTH secretion are under separate regulatory mechanisms.     

Third trimester binge ethanol exposure results in fetal hypercapnea and acidemia but not hypoxemia in pregnant sheep

BACKGROUND: The mechanisms by which maternal ethanol abuse during pregnancy causes neurodevelopmental injury in the fetus are not well understood. The purpose of this study was to use a chronically instrumented fetal sheep model system to determine if a binge pattern of ethanol exposure administered throughout the third trimester reduced fetal arterial partial pressure of oxygen (PaO2); a positive finding would support the hypothesis that fetal hypoxemia may play a role in mediating ethanol-related birth defects. METHODS: Pregnant ewes received saline or 0.75, 1.25, 1.5, or 1.75 g/kg of ethanol intravenously over 1 hr beginning on day 109 of gestation (term = 145 days) for 3 consecutive days per week followed by 4 days without exposure. The fetuses were surgically instrumented on day 113, and experiments were performed on days 118 or 132, the 6th and the 12th ethanol exposure, respectively. RESULTS: Ethanol infusions resulted in peak blood ethanol concentrations of 80.8 +/- 6.5, 182.5 +/- 13.5, 224.4 +/- 13.9, and 260.6 +/- 20.0 mg/dl +/- SEM (maternal) and 70.0 +/- 5.9, 149.7 +/- 9.0, 216.9 +/- 14.0, and 233.3 +/- 19.8 mg/dl +/- SEM (fetal) in response to the 0.75, 1.25, 1.5, and 1.75 g/kg doses, respectively. Maternal and fetal heart rate and maternal blood pressure increased whereas fetal blood pressure decreased in a dose-dependent manner in response to ethanol infusions. Maternal and fetal arterial pH decreased and arterial partial pressures of carbon dioxide increased in response to ethanol infusions. Maternal PaO2 decreased whereas fetal PaO2 did not change in response to ethanol infusions. CONCLUSIONS: A binge ethanol exposure paradigm, three consecutive days per week throughout the third trimester at ethanol doses that created blood ethanol concentrations commonly achieved by human ethanol abusers, resulted in changes in maternal and fetal heart rate, changes in blood pressure, hypercapnea, acidemia, and maternal, but not fetal, hypoxemia. We conclude that in an ovine model system, ethanol doses that create blood ethanol concentrations as high as 260 mg/dl do not result in fetal hypoxemia. Remaining issues to address with this model system are whether neurodevelopmental injuries that are associated with maternal ethanol abuse are mediated by a reduction in fetal cerebral blood flow, fetal hypercapnea, or acidemia.     

Surgical disconnection of the hypothalamus from the fetal pituitary abolishes the corticotrophic response to intrauterine hypoglycemia or hypoxemia in the sheep during late gestation.

We have investigated the ACTH and cortisol responses to acute episodes of hypoxemia or hypoglycemia in fetal sheep in which the hypothalamus and pituitary were surgically disconnected at between 112 and 123 days gestation. Before 130 days gestation, basal plasma concentrations of ACTH were significantly greater in the hypothalamo-pituitary disconnected (HPD) fetuses than in the intact fetal sheep (126-130 days; 105.0 +/- 11.4 ng/liter, HPD group; 64.0 +/- 9.5 ng/liter, intact group). After 130 days, however, there was no difference between plasma ACTH concentrations in the HPD (136-140 days; 154.7 +/- 16.7 ng/liter HPD group; 113.6 +/- 19.1 ng/liter, intact group) and intact fetal sheep, and in both groups the mean ACTH concentrations were significantly greater after 136 days gestation than before 130 days. In the HPD group, however, while the plasma ACTH concentrations were elevated there was no prepartum increase in the plasma concentrations of cortisol. A decrease in the fetal arterial blood PO2 by approximately 50% for 30 min between 123 and 132 days, stimulated a significant increase in fetal ACTH and cortisol concentrations in the intact but not in the HPD fetuses. In the intact group, plasma ACTH concentrations were also significantly increased (P less than 0.001) above control values (98.2 +/- 21.2 ng/liter) at 120 min after the start of an iv infusion of insulin (1 IU/60 min) (517.2 +/- 160.5 ng/liter) and were still elevated at 60 min after the end of the infusion period (1248.1 +/- 643.2 ng/liter). In the HPD fetuses, however, there was no significant change in plasma ACTH concentrations during or after the insulin infusion. In both the HPD and intact groups, there was a significant increase in plasma ACTH concentrations above control values (62.5 +/- 8.5 ng/liter intact; 135.0 +/- 30.6 ng/liter, HPD) after intrafetal administration of CRF (+10 min; 117.5 +/- 8.1 ng/liter, intact; 225.3 +/- 33.1 ng/liter, HPD) indicating that the secretory capacity of the pituitary corticotrophs was not reduced by the HPD procedure. Our results demonstrate that an intact hypothalamic-pituitary connection is required to generate a normal prepartum increase in fetal cortisol concentrations and is essential for an appropriate fetal pituitary-adrenal response to intrauterine hypoxemia and hypoglycemia.     

Regulation of aldosterone secretion during hypoxemia at sea level and moderately high altitude

The aldosterone and cortisol responses to small doses of ACTH (0.125, 0.25, 0.5, and 1.25 micrograms) after dexamethasone administration were measured in normal subjects at sea level while breathing room air (mean O2 saturation, 97 +/- 0.9%) and again while breathing hypoxic gas to lower the O2 saturation to 90%. A population of subjects matched for age and sex adapted to 3000 meters above sea level living in Colombia, South America, was also studied (mean O2 saturation, 94 +/- 0.7%). Hypoxemia, either induced at sea level or as a consequence of high altitude living, resulted in significant inhibition of aldosterone secretion after progressive administration of increasing doses of ACTH, but did not affect the cortisol response to ACTH. In addition, it was associated with higher plasma atrial natriuretic hormone levels. PRA declined only during acute hypoxemia induced at sea level and did not change during sea level normoxemia or high altitude living. Plasma sodium and potassium concentrations were no different in the three experimental conditions. We conclude that hypoxemia inhibits ACTH-stimulated aldosterone secretion and speculate that atrial natriuretic hormone may have mediated this effect.     

Direct hemoperfusion with polymyxin B-immobilized fiber improves shock and hypoxemia during endotoxemia in anesthetized sheep

This study evaluates the effect of direct hemoperfusion (DHP) using polymyxin B-immobilized fibers (PMX-F) as an extracorporeal blood filter on systemic hypotension and lung injury during endotoxemia Sheep were anesthetized, intubated, mechanically ventilated with 50% oxygen and connected to the DHP system between the right femoral artery and left jugular vein. Group 1 (n = 6)sheep were infused with 10 microg/kg Escherichia coli endotoxin over a 30 min period. At the same time, sheep underwent DHP with PMX-F (Toraymyxin: PMX-20R) for 2 h at a flow rate of 60 ml/h. Group 2 (n = 6) sheep were infused with the same dose of endotoxin and treated with a sham column, in the same manner as those in group 1. DHP with PMX-F significantly improved and restored systemic pressure and arterial oxygen tension in group 1 sheep, although these values never returned to the baseline levels of group 2 sheep. Pulmonary hypertension and leukocytopenia were observed after endotoxin infusion in both groups, but there were no significant differences between these values. DHP with PMX-F significantly decreased the elevation of plasma nitric oxide products. The treatment with PMX-F improves shock and deteriorated oxygenation during endotoxemia, probably through the suppression of nitric oxide production.     

Adrenal hypertrophy produced by 17alpha-esterified glucocorticoids in rat foetus

Glucocorticoids were injected sc into rat foetuses through the uterine wall on day 19 of intrauterine development; two days later the foetuses were removed and their adrenal glands were weighed. Some betamethasone derivatives produced marked adrenal hypertrophy in the foetuses. Structural characteristics of the effective compounds were 17alpha-esterification and 16beta-methylation. 9alpha-Halogenation and 21-esterification of glucocorticoids were not essential for producing the hypertrophy. Propionic acid at the C-17 position was more effective than valeric and acetic acids in producing the hypertrophy. The effect of betamethasone 17, 21-dipropionate was dose-responsive and prevented by simultaneous administration of betamethasone 21-disodium phosphate. Results obtained with decapitated or encephalectomized foetuses suggested that the hypertrophy produced was not due to a direct action of the glucocorticoids on the adrenal gland, but mediated by the hypothalamo-pituitary system.     

Neuropeptide Y in the sheep fetus: effects of acute hypoxemia and dexamethasone during late gestation

Plasma concentrations of neuropeptide Y (NPY) were measured in pregnant ewes and their fetuses under basal conditions and in response to acute hypoxemia during late gestation. The effects of fetal treatment with dexamethasone on these NPY responses were also examined. Under general anesthesia, 10 Welsh Mountain ewes and their fetuses were chronically instrumented between 117-120 days gestation (dGA; term is approximately 145 dGA) with vascular and amniotic catheters, and an ultrasonic probe around a femoral artery of each fetus. At 124 dGA, five fetuses were continuously infused i.v. with dexamethasone for 48 h at a rate of 1.73 +/- 0.16 microg x kg(-1) x h(-1) while the remaining five fetuses received vehicle at the same rate. At 126 dGA, 45 h from the onset of either infusion, 1 h of materno-fetal hypoxemia was induced by reducing maternal FiO2. During normoxia, maternal plasma NPY concentrations were three times those measured in fetal plasma in both groups. During hypoxemia, PaO2 fell to similar levels in the control and dexamethasone-treated groups in both mothers and fetuses. In control animals, there was a significant increase in the NPY concentration in fetal, but not maternal, plasma during hypoxemia. Fetal treatment with dexamethasone significantly enhanced the fetal NPY response to acute hypoxemia but had no effects on basal NPY levels in the fetal or maternal plasma or on the maternal response to acute hypoxemia. These data show: 1) differences between the maternal and fetal plasma NPY response to maternal inhalation hypoxia; 2) that NPY may play a role in mediating fetal defense responses to acute hypoxemia; and 3) that fetal exposure to glucocorticoids modifies the fetal plasma NPY response to acute hypoxemia.     

Dual role of glucocorticoids in suckling-induced prolactin secretion

The exact contribution of corticosteroids to the control of prolactin secretion in lactating rats is poorly understood. Therefore, the present studies were focused on the effect of adrenalectomy and dexamethasone treatment on the suckling-induced prolactin release. Animals were adrenalectomized on the 3rd day of lactation and tested on the 7th day of lactation. In adrenalectomized animals, the suckling stimulus failed to induce the characteristic increase in plasma prolactin levels. Dexamethasone pretreatment (400 μg/kg b.w. s.c. 24, 48, 72 h before testing) of adrenalectomized rats restored this prolactin response. The same treatment with dexamethasone given to control animals attenuated the suckling stimulus induced prolactin response. The present findings indicate that corticosteroids are essential for a basic prolactin response of lactating rats.     

Regulation of glucogenesis by thyroid hormones in fetal sheep during late gestation

The effects of thyroid hormone deficiency in utero on the fetal glucogenic capacity were investigated by measuring glucose production and hepatic levels of glycogen and gluconeogenic enzymes in normal sheep fetuses in the fed and fasted states during late gestation and in those made thyroid hormone deficient by fetal thyroidectomy (TX). In the fed state, fetal TX had no effect on glucose uptake, utilisation or production by the fetus. It also had no apparent effect on the glycogen content or activities of the key gluconeogenic enzymes in the fetal liver. In addition, fetal plasma concentrations of insulin, cortisol, adrenaline or noradrenaline were unaffected by fetal TX in the fed state. In contrast, the rates of fetal O(2) consumption and CO(2) production per kilogram fetal bodyweight were significantly lower in TX than in intact fetuses in the fed state (P<0.05). TX prevented fetal glucose production in response to maternal fasting for 48 h. It also abolished the normal decreases in the fetal glucose carbon oxidation fraction, the rate of CO(2) production from glucose carbon and in the fraction of the umbilical O(2) uptake used for glucose carbon oxidation that occur during fasting in intact fetuses. At the end of the fast, plasma noradrenaline concentrations and hepatic levels of glycogen, glucose 6-phosphatase, fructose diphosphatase and alanine aminotransferase were significantly lower in TX than in intact fetuses. These observations show that thyroid hormones are essential for glucogenesis in the sheep fetus during late gestation and suggest that these hormones act both on the hepatic glucogenic pathways and on the mechanisms activating glucogenesis in utero.     

Adrenal and intestinal secretion of catecholamines and neuropeptides during splanchnic artery occlusion shock

Plasma levels of catecholamines and neuropeptides (met-enkephalin, ME; neurotensin, NT; neuropeptide Y, NPY; peptide YY, PYY; vasoactive intestinal polypeptide, VIP; cholecystokinin, CCK; bombesin, BMB) were examined in the femoral artery (FA), adrenal vein (AD), and portal vein (PV), in eight cats under halothane anesthesia at baseline (S1), at the end of a 2-hr ligation period of the major splanchnic arteries (celiac trunk, superior and inferior mesenteric arteries) (S2), immediately (S3) and 30 min (S4) after splanchnic reperfusion, and after the administration of naloxone (1 mg/kg, i.v.) (S5). During S2, there was a significant increase in portal vein VIP levels, while the other variables (hemodynamics, hormone levels) remained unchanged. During early shock (S3), significant (10- to 30-fold) increases in adrenal secretion of all catecholamines, ME, NT, NPY, and PYY occurred, while VIP and PYY were significantly released into the PV, and two- to tenfold increases in femoral artery catecholamine and ME levels were observed. Later shock (S4) led to a further fivefold increase, compared to S3, in adrenal release of norepinephrine (NE), dopamine (DA), and ME. Following naloxone administration (S5), the adrenal medullary release of NE, epinephrine (EPI), DA, NT, and NPY was significantly (twofold) increased; however, the animals' hemodynamic situation did not improve.     

Mechanisms of hypoxemia during panendoscopy

Hypoxemia during esophagogastroduodenoscopy (EGD), or panendoscopy has been generally attributed to sedation. We studied 49 patients ranging in age from 17 to 71 years with normal or nearly normal lung function undergoing EGD to determine the effects of sedation and the effects of the endoscope on arterial oxygen saturation (SaO2). All patients received intravenous diazepam and 41 also received meperidine. EGD was delayed 10.7 +/- 7.5 min after intravenous diazepam administration in the 42 group 1 patients. Seven patients underwent EGD within 2 min of receiving intravenous diazepam (group 2). Ventilation decreased after diazepam, recovered, then decreased immediately after endoscope insertion in the group 1 patients. Periods of hypopnea, up to 39 s long, were observed during EGD. The average decrease in SaO2 was 4.0% after diazepam (p less than 0.0001). SaO2 returned to the pre-EGD level, then decreased 2.4% during EGD (p less than 0.0005). Maximum SaO2 decrease occurred 27 +/- 6 s after insertion of the endoscope then rapidly recovered. There was a linear correlation between the duration of hypopnea and maximum SaO2 decrease (r = 0.84, p less than 0.001). All group 2 patients experienced a period of hypopnea (13.3 +/- 9.6 s) and SaO2 declined 9.0%. The SaO2 decline was significantly greater in the group 2 subjects (p less than 0.0001). Our results confirm previous findings that intravenous sedation causes hypoventilation and hypoxemia. Moreover, hypoventilation and further arterial oxygen desaturation are caused by either the mechanical effect of the endoscope or a reflex stimulated by it.     

Effect of glucocorticoids on gastrin secretion in man.

In order to investigate the effect of glucocorticoids on gastrin secretion, plasma gastrin levels were measured by radioimmunoassay in patients with Cushing's syndrome and those treated with glucocorticoids. Fasting plasma gastrin levels were significantly higher in these patients than in normal subjects, with exaggerated response to food. Conversely, short-term treatment or intravenous infusion of glucocorticoids had no significant influence on gastrin secretion in normal subjects. The possible mechanism by which glucocorticoids cause hypergastrinaemia are discussed.