Placental expression of the obesity-associated gene FTO is reduced by fetal growth restriction but not by macrosomia in rats and humans.

Genetic variants in the FTO (fat mass- and obesity-associated) gene have the highest association of all obesity-associated genes. Its placental expression was shown to relate to birth weight, suggesting that it may participate in the control of fetal weight gain. To gain more insight into the implication of FTO in fetal growth, we measured its placental expression in samples including extremes of abnormal fetal growth, such as after intrauterine growth restriction (IUGR) or macrosomia in both rats and humans. In rats, fetal growth was modulated by maternal nutritional modifications. In humans, placental villi were collected from pathological pregnancies (i.e. with IUGR or fetal macrosomia). Placental FTO mRNA expression was reduced by IUGR but was not significantly affected by macrosomia in either rats or humans. Our data suggest that placental FTO may participate in interactions between the in utero environment and the control of fetal growth under IUGR conditions by modulating epigenetic processes.

Tissue-specific programming expression of glucocorticoid receptors and 11 beta-HSDs by maternal perinatal undernutrition in the HPA axis of adult male rats.

Maternal undernutrition leads to intrauterine growth retardation and predisposes to the development of pathologies in adulthood. The hypothalamo-pituitary-adrenal axis is a major target of early-life programming. We showed previously that perinatal maternal 50% food restriction leads to hypothalamo-pituitary-adrenal axis hyperactivity and disturbs glucocorticoid feedback in adult male rats. To try to better understand these alterations, we studied several factors involved in corticosterone sensitivity. We showed that unlike the restricted expression of 11 beta-HSD2 mRNA, the 11 beta-HSD1, glucocorticoid, and mineralocorticoid receptor genes are widely distributed in rat. In contrast to the hypothalamus, we confirmed that maternal undernutrition modulates hippocampal corticosterone receptor balance and leads to increased 11 beta-HSD1 gene expression. In the pituitary, rats exhibited a huge increase in both mRNA and mineralocorticoid receptor binding capacities as well as decreased 11 beta-HSD1/11 beta-HSD2 gene expression. Using IN SITU hybridization, we showed that the mineralocorticoid receptor gene was expressed in rat corticotroph cells and by other adenopituitary cells. In the adrenal gland, maternal food restriction decreased 11beta-HSD2 mRNA. This study demonstrated that maternal food restriction has both long-term and tissue-specific effects on gene expression of factors involved in glucocorticoid sensitivity and that it could contribute, via glucocorticoid excess, to the development of adult diseases.

Effect of natriuretic peptides on in vitro stimulated adrenocorticotropic hormone release and pro-opiomelanocortin mRNA expression by the fetal rat pituitary gland in late gestation.

OBJECTIVE AND METHODS: We investigated the effects of individual natriuretic peptides (atrial natriuretic peptide, ANP; brain natriuretic peptide, BNP, and C-type natriuretic peptide, CNP) on rat corticotropin-releasing factor stimulated adrenocorticotropic hormone (ACTH) secretion by the pituitary gland of 21-day-old rat fetuses in vitro and on pro-opiomelanocortin gene expression using in situ hybridization. RESULTS: Graded concentrations of ANP, BNP, or CNP (10(-10), 10(-9), and 10(-8) mol/l) induced a log dose dependent inhibition of ACTH secretion induced by rat corticotropin-releasing factor (10(-10) mol/l). These natriuretic peptides showed equipotent effects on a molar basis. Moreover, ANP, BNP, or CNP at 10(-10) mol/l reduced significantly the pituitary pro-opiomelanocortin mRNA expression. In addition, the immunoreactive ANP, BNP, and CNP cells were localized in the anterior lobe, but not in the intermediate lobe of the fetal pituitary gland. CONCLUSIONS: These data suggest that the fetal pituitary gland may be both a source and a target for natriuretic peptides that might control ACTH synthesis and release via an endocrine and/or paracrine mechanism. The natriuretic peptides could participate, as well as glucocorticoids, in the control of the corticotropin-stimulating activity of the fetal rat in late gestation.

Atrial natriuretic peptide and aldosterone secretions, and atrial natriuretic peptide-binding sites in kidneys and adrenal glands of pregnant and fetal rats in late gestation in response to a high-salt diet.

OBJECTIVE: This study aimed at determining, in the term pregnant rat, whether maternal and fetal plasma atrial natriuretic peptide (ANP) concentrations were modified in response to an oral sodium load, and to investigate whether any changes in plasma concentrations were able to modify the density and affinity of the different ANP-binding site subtypes in maternal and fetal kidneys and adrenal glands. METHODS: Pregnant rats kept in metabolic cages were divided into two groups. The normal sodium diet group had free access to rat chow and tap water whereas the high sodium diet group received 1% NaCl as drinking water for 10 consecutive days from day 11 to day 21 of gestation with free access to standard rat chow. Pregnant rats from both groups were killed by decapitation on day 21 of gestation. The plasma ANP and aldosterone concentrations were determined by RIA. The density and affinity of ANP receptors were determined in the maternal and fetal adrenal glands and kidneys. RESULTS: In the pregnant rats on the high-salt diet, the sodium and water intakes, as well as the urine volume and sodium excretion, were significantly higher than in the control group. After 10 days of high-salt intake, water and sodium retentions were not significantly different in the two groups, indicating that the pregnant rats were able to excrete excess salt. The high sodium intake did not change the body weight of the pregnant rats but did increase the body weight of the fetal rats. Maternal and fetal hematocrits remained unchanged in both groups, the high sodium intake did not modify plasma sodium concentration in the maternal rats but increased that of the fetuses, indicating an accumulation of sodium in the fetal rats. The dietary sodium intake did not change the plasma ANP concentrations but significantly decreased the plasma aldosterone concentrations in both the maternal and fetal rats. In response to the high-salt diet, the density and affinity of total ANP, ANPb and ANPc receptors were not altered in the maternal isolated renal glomeruli or the adrenal zona glomerulosa membranes or the fetal adrenal gland and kidney membrane preparations. CONCLUSION: These results suggest that ANP is not involved in the regulation of water and electrolyte balance in maternal and fetal rats during salt-loaded intake.

Effects of water deprivation on atrial natriuretic peptide secretion and density of binding sites in adrenal glands and kidneys of maternal and fetal rats in late gestation.

The effects of water deprivation for 3 days were studied in pregnant rats and their fetuses on day 21 of gestation. Maternal water deprivation induced a significant decrease of the body weight in both maternal and fetal rats. This weight loss was accompanied by significant increases in plasma osmolality and haematocrit in both maternal and fetal rats. Similarly, dehydration significantly decreased plasma atrial natriuretic peptide (ANP) concentrations and increased plasma aldosterone concentrations in maternal and fetal rats. Water-deprived maternal rats presented a significant increase in total ANP receptor density in isolated renal glomeruli and adrenal zona glomerulosa membranes. This increase was due to a significant increase in ANPc receptor density in both renal glomeruli and adrenal zona glomerulosa. The densities of total ANP, ANPb and ANPc receptors in fetal kidneys and adrenal glands were not affected by maternal dehydration. These results suggest that the dehydrated maternal rat is able to up-regulate the number of its ANP receptors in its kidneys and adrenal glands, in response to a decrease in plasma ANP concentrations. In contrast, the fetal rat does not seem to be able to regulate its own ANP receptors in response to maternal dehydration, in spite of a decrease in plasma ANP concentrations.

Molecular forms of atrial natriuretic peptide in atria and plasma from fetal and adult female rats.

Molecular forms of atrial natriuretic peptide (ANP) were determined by chromatography on Sephadex G50 fine coupled with a radioimmunoassay for rANP(1-28) in left and right atrial extracts and acidified plasmas from 17-, 19- and 21-day-old fetuses and adult female rats. Chromatographic analyses revealed two immunoreactive peaks of rANP with an apparent molecular weight of about 25 and 12 kD, respectively, in all atrial extracts from fetal and adult rats. Only one immunoreactive peak of rANP with an apparent molecular weight of about 3 kD was detected in all fetal plasmas. The 25- and 12-kD ANP molecular forms isolated from fetal atrial tissue and incubated with thrombin for 30 min at 37 degrees C were converted to ANP material with a molecular weight of about 3 kD. These results suggest that the two high molecular weight forms stored in fetal atrial tissue should be precursors of circulating rANP molecules, and the rat fetus should possess the same posttranslational processing of the precursor molecule as the adult rat as early as day 17 of gestation.

Characteristics and developmental changes of ANP-binding sites in rat adrenal glands during the perinatal period.

The binding of rANP(1-28) to receptors was studied on crude adrenal membranes from fetal rats between day 17 of gestation and term and also neonatal rats between weeks 1 and 4. The binding assays were carried out using 125I-rANP(1-28) as radioligand incubated with membrane preparations (2 mg/ml) for 90 min at 22 degrees C. The binding was specific, saturable and reversible. The Scatchard analysis of the binding data revealed a single class of binding sites of high affinity (kd approximately 10(-10) mol/l) which did not change significantly at all stages of development studied. The binding sites presented a higher affinity for ANP analogues which contained the C-terminal phenylalanine arginine residue. The number of ANP receptors expressed per adrenal increased regularly in fetal and neonatal rats and the perinatal evolution of these concentrations of ANP receptors was related to the increase in the size of the adrenals. When the concentrations of ANP receptors was expressed per microgram DNA, the concentrations of ANP receptors were higher in neonatal rats than in fetal rats and reflected the number of receptors per cell. These results suggest that these binding sites mediate the biological actions of ANF in the adrenal gland during the perinatal period.

Effects of rat corticotrophin-releasing factor, arginine vasopressin and oxytocin on the secretions of adrenocorticotrophic hormone and corticosterone in the fetal rat in late gestation: in vivo and in vitro studies.

The effects of rat corticotrophin-releasing factor (rCRF), arginine vasopressin (AVP) and oxytocin (OT) were investigated in vivo in 21-day-old rat fetuses injected through the umbilical vein and in vitro on perifused anterior pituitary glands from 21-day-old rat fetuses. In vivo, rCRF (1.25 pmol.50 microliter-1.fetus-1), AVP (5 pmol.50 microliter-1.fetus-1) alone and rCRF in association with AVP or oxytocin (12.5 pmol.50 microliter-1.fetus-1) increased plasma adrenocorticotrophic hormone (ACTH) and corticosterone levels only 30 min after the start of injection. During the first 10 min of the sampling period, the injection of these peptides alone or in combination and the injection of saline decreased the plasma ACTH concentration, which was lower than that of uninjected fetuses, but had no effect on the plasma corticosterone concentration. In vitro, the release of ACTH by perifused anterior pituitary glands was increased strongly by rCRF (4 pmol/0.5 ml) but only slightly by AVP (92 pmol/0.5 ml) and oxytocin (198 pmol/0.5 ml). Arginine vasopressin and oxytoxin potentiated the release of ACTH stimulated by rCRF in vitro but not in vivo. Our results suggest that rCRF is the major peptide that controls ACTH secretion in the fetal rat at term. In conclusion, the rise of the ACTH level observed only 30 min after injection of rCRF or AVP suggests the existence of a factor able to inhibit the ACTH response after injection of these peptides. This factor might be elicited by the blood volume expansion.

Effect of blood volume expansion on basal plasma atrial natriuretic factor and adrenocorticotropic hormone secretions in the fetal rat at term.

The effect of a blood volume expansion (1-1.5% of body weight) on atrial natriuretic factor (ANF) and adrenocorticotropic hormone (ACTH) secretions were investigated in 21-day-old rat fetuses injected intravenously through the umbilical vein with 50 microliters of isotonic saline. Basal ANF and ACTH concentrations were determined in the plasma with specific radioimmunoassays over a 30-min observation period. The basal plasma ANF concentration increased rapidly 1 min after blood volume expansion and then decreased to the control value 5 min later. The basal plasma ACTH concentration decreased during the first 10 min after injection and then increased to the control value 30 min after intravascular volume load. The results suggest that the fetal rat in late gestation is able to respond to blood volume expansion by increasing ANF secretion.

Effects of corticotropin-releasing factor, arginine vasopressin and oxytocin on the polymorphism of plasma adrenocorticotropic hormone in the rat fetus in late pregnancy.

The effects of rat corticotropin-releasing factor (rCRF, 1.25 pmol/50 microliters/fetus), arginine vasopressin (AVP, 5 pmol/50 microliters/fetus) and oxytocin (OT, 12.5 pmol/50 microliters/fetus) alone or in association were investigated in 21-day-old rat fetuses injected intravenously through the umbilical vein. Blood samples were collected 15 and 30 min after injection for the determination of corticosterone concentration and the different plasma molecular adrenocorticotropic hormone (ACTH) forms isolated by chromatography on Sephadex G50 fine. All the plasma samples chromatographed 15 and 30 min after injection of the different peptides showed 3 different molecular ACTH forms: big ACTH (> 20,000 molecular weight), intermediate ACTH (= 13,000) and little ACTH (= 4,500). The injection of rCRF or AVP alone and rCRF in association with AVP or OT increased the concentrations of big ACTH 15 min and little ACTH 30 min after injection. The injection of OT alone or in association with AVP did not change the concentration of the 3 molecular ACTH forms 15 and 30 min after injection. The rise of big ACTH 15 min after injection was not associated with a significant increase in plasma corticosterone concentration, whereas the increase in little ACTH 30 min later enhanced plasma corticosterone concentration. Our results suggest that rCRF or AVP alone and rCRF in association with AVP or OT injected intravenously in the fetal rat produced a selective release of the molecular ACTH forms and the increase in the plasma corticosterone concentration occurred when the proportion of little ACTH which is the predominant ACTH form in the fetal rat was enhanced.

Effects of Estrous Cycle and of in vivo Unilateral or Bilateral Adrenal Demedullation on the Distribution of Norepinephrine and Epinephrine Between Different Zones of the Adrenal.

Abstract The distribution of norepinephrine (NE) and epinephrine (E) between the capsule/glomerular zone and the remainder of the adrenal was studied in the adult female rat. Both catecholamines were present in these two parts of the gland. The concentration of E was higher than that of NE. In the capsule/glomerular zone the catecholamine concentrations were more than twenty to thirty times lower than in the inner part of the gland. The circulating levels of catecholamines were always very low. The present data also support very weak or no changes in catecholamine concentrations in both parts of the adrenal during the different stages of the estrous cycle. The plasma levels of both aldosterone and corticosterone, like those of catecholamines, did not vary significantly throughout the estrous cycle. One week after unilateral or bilateral demedullation, both E content and concentration were reduced in the whole capsule/glomerular zone of the adrenal although the NE content did not change. The reduction of NE concentration could be related to the drastic weight increase of this part of the gland on the operated side. Moreover, unilateral demedullation was unable to significantly modify the plasma levels of both E and NE. In contrast bilateral demedullation suppressed circulating E and induced a significant reduction (about 50%) of NE plasma level. The present data suggest: 1) an extra-adrenal origin for the NE innervation of the capsule/glomerular zone of the adrenal cortex, and 2) a dual origin for E in the capsule/glomerular zone; part of E could arise from the adrenal medulla and part from an extra-adrenal site.

The role of vasoactive intestinal polypeptide in the inhibition of antral and pyloric electrical activity in rabbits.

The nature of the neurotransmitter released by intramural non-cholinergic non-adrenergic inhibitory neurones and the type of vagal afferents involved in the inhibition of antral and pyloric electrical activity induced by vagal afferent stimulation were investigated in conscious rabbits in which both splanchnic nerves had previously been cut. The inhibitory effect of duodenal distension was reversed by bilateral thoracic vagotomy. Either intravenous or intra-aortic infusions of vasoactive intestinal polypeptide (VIP) inhibited the electrical activity of both antrum and pylorus for several minutes. Electrical stimulation of afferent vagal fibres and duodenal distension both inhibited antral and pyloric activity and produced a significant increase of portal plasma VIP concentration. Numerous VIP-immunoreactive fibres were found to be present in the muscular layers of the pylorus. The possibility that this form of inhibition is mediated by VIPergic fibres is discussed as is the likely involvement of vagal afferent fibres in the case of the response to duodenal distension.

Sympathetic control of antral and pyloric electrical activity in the rabbit.

The effects of section and stimulation of the sympathetic nerve trunk on gastric motility were investigated in conscious and decorticate rabbits. In conscious animals after section of the abdominal splanchnic nerve, rhythm of antral and pyloric bursts was enhanced, becoming more regular, and the period of arrest of the rhythmic bursts, which was usually observed at the end of inflation of the antrum in intact rabbits, was shortened to 33.6 +/- 4.0 s from 112.2 +/- 14.6 s observed before the sympathetic nerve transection. Adrenergic agonists, phenylephrine (100 micrograms/kg), clonidine (5 micrograms/kg) and salbutamol (1 mg/kg) inhibited antral and pyloric activity. In decorticate rabbits the major effect of stimulation of the peripheral or the central end of the thoracic sympathetic trunk was inhibition; this was seen both with the spontaneous and vagally induced e.m.g. activity of the antrum and pylorus. Inhibition induced by stimulation of sympathetic efferents was abolished by beta-blocking agents and that induced by stimulation of the sympathetic afferents disappeared after alpha-adrenergic block. Significance of a dual control of the gastric motility by the sympathetic nerve was discussed.

Inhibition of antral and pyloric electrical activity by vagal afferent stimulation in the rabbit.

The effects of stimulation of cervical vagal afferents on the electromyographic activity (e.m.g.) of the gastric antrum and pylorus have been studied both in conscious and decorticate, curarized rabbits. In the latter group, stimulation at 10-15 Hz (0.3-0.4 mA; 5-7 ms duration) inhibited both the antral and pyloric e.m.g. in 21 rabbits, elicited an excitatory response in two more but produced no change in the remaining 13 animals. The inhibitory response was not affected by guanethidine (1 mg/kg, i.v.), phentolamine (1 mg/kg, i.v.) or propranolol (0.1 mg/kg, i.v.) and persisted in rabbits in which the contralateral cervical vagus and both splanchnic nerves had been cut, the coeliac ganglia and both adrenal glands had been extirpated. It was only abolished by combined section of the spinal cord at the level of C1, C2 and the contralateral vagus in the neck. In conscious rabbits, vagal stimulation at 2-5 Hz (0.4 mA; 0.5-1.0 ms duration) consistently inhibited antral and pyloric e.m.g. This inhibitory response persisted in the presence of phentolamine, propranolol and naloxone (0.25-1.00 mg/kg), and in adrenalectomized rabbits with cut splanchnic nerves and extirpated coeliac ganglia. However, it was completely abolished by section of both vagi above the diaphragm. It is concluded that (1) the vagal afferents responsible for the inhibition of the gastric e.m.g. are already included in the abdominal vagus nerves, (2) this inhibition of gastric e.m.g. is mediated by non-adrenergic fibers, one group of them emerging from the thoracic spinal cord to join the thoracic vagi via the stellate ganglia.

Neural control of electrical gastric activity in response to inflation of the antrum in the rabbit.

The respective roles of the extrinsic innervation (vagal and splanchnic) and of the myenteric plexus in the control of gastric motility were investigated in rabbits by quantifying the effect of cutting the extrinsic nerves on the electrical response of the antrum and pylorus to inflation of the antrum. In intact animals the frequency of bursts during distension was significantly increased above the frequency during the 30 min preceding inflations. The interval from the end of step inflation to the first burst recorded after inflation was significantly longer than the mean interval between bursts in the 30 min preceding inflation, whereas no such difference was observed following ramp inflation. Both atropine and hexamethonium abolished the responses to distension. The basal rhythm of bursts recorded on the antrum and pylorus was markedly reduced during the first 3 days after vagotomy: the mean values of the frequency in response to distension were significantly less than those in intact animals, whereas the first interval after inflation was significantly increased. Thereafter no such differences were encountered. The only change after section of the splanchnic nerves was a significant reduction in the first interval after inflation, resulting in rapid restoration of the initial rhythm at the end of inflation. In vagotomized animals with cut splanchnic nerves, the basal electrical activity of the antrum was not affected but the responses to step inflations were much greater; the frequency of bursts during distension and the first interval after inflation were both significantly increased. Section of the vagal afferent fibres changed neither the frequency of the basal rhythm nor the responses to distension. These results indicate that the tonic excitatory effect mediated by vagal efferents on the basal rhythm of the antrum is of central origin and independent of visceral inputs. Local mechanisms within the peripheral plexus of neurones could be responsible for the enhanced responses to step inflations, which would normally be adjusted by the central mechanisms through the extrinsic innervation. Possible effects of changes in the sensitivity of the post-ganglionic neurones are discussed.

Specific effects of thoracic vagotomy on the electrical activity of the gastric antrum and pylorus in rabbits.

The electrical activity of the antrum and the pylorus was recorded in rabbits, with chronically implanted electrodes. Effects of food intake were studied before and after bilateral vagotomy (b.v.), ventral vagotomy (v.v.) or dorsal vagotomy (d.v.) performed just above the diaphragm. In fasted animals, the electrical activity was characterized by bursts of potentials at a frequency of 1.93 +/- 0.09/min, initiated 45 mm or 20 mm from the pylorus in the greater and the lesser curvature respectively. The velocity of propagation of the bursts increased from 5-6 mm/s in the proximal antrum to 12-25 mm/s in the last 10 mm of the antrum along the greater curvature and from 2-4 mm/s to 5-15 mm/s along the lesser curvature. Feeding led to a rapid increase in the frequency of both antral and pyloric bursts up to 4.01 +/- 0.10/min, lasting 30-60 min after a large meal but only 15 min when the amount of food was restricted. The behaviour of the rabbits was not significantly affected by bilateral or ventral vagotomy. In contrast, dorsal vagotomy resulted in a drastic decrease of food intake. The mean frequency (fo) of bursts before meals was significantly reduced after vagotomy in v.v. animals (1.49 +/- 0.11/min, P less than 0.02) and in d.v. animals (1.34 +/- 0.08/min, P less than 0.001) but not in b.v. or sham-operated rabbits. For the first 3 d after surgery, the frequency (f) of bursts during the 30 min after the beginning of meals was significantly different from fo and reduced relative to f before nerve sections (1.9 +/- 0.11/min and 2.32 +/- 0.20/min respectively in b.v. and v.v. animals). In sham-operated animals, the value of f was closely similar to f before surgery. The effects of meals on the frequency of bursts were again similar to the effects in intact animals within 4-6 d for v.v. animals and 6-9 d for b.v. animals. In the pylorus, a different pattern for electrical activity, which consisted of trains of spikes at a frequency of 0.3/s, was superimposed on the bursts and was most marked in d.v. animals. The spiking activity was not affected by atropine, phentolamine or propranolol. The role of the vagus nerve in the motile responses of the antrum to food intake is discussed. These results indicate the existence of a non-cholinergic, non-adrenergic innervation of the pylorus which is involved in the opening of the sphincter and appears to be mediated by the dorsal vagal trunk.

[Influence of the vagus nerve on the electromyographic activity of the antropyloric region in the rabbit]. / Influence du nerf vague sur l'activité électromyographique de la région antropylorique chez le Lapin.

The electromyographic activity of the stomach antrum and pylorus was recorded in rabbits in acute experimental conditions. Stimulation of the distally cut end of a cervical vagus induced an increase of EMG activity. These results confirm the existence of motor efferent fibers in the vagus. Stimulation of the central end of the vagus inhibited the spontaneous or evoked EMG activity. It is likely that the efferent fibers stimulated by the afferent volley were preganglionic, synapsing with inhibitory myenteric neurons.