Ontogeny and tissue-specific regulation of ghrelin mRNA expression suggest that ghrelin is primarily involved in the control of extraendocrine functions in the rat

Ghrelin is a 28-amino-acid gastric peptide that potently stimulates growth hormone (GH) secretion in vivo and in vitro. Ghrelin-expressing cells have been found in the oxyntic region of the stomach and in the arcuate nucleus of the hypothalamus. The aim of this work was to investigate the regional distribution and developmental changes in ghrelin mRNA levels in the pituitary, hypothalamus and gastrointestinal (GI) tract of the rat using a semiquantitative RT-PCR assay. We also describe the effects of ghrelin immunoneutralization in late gestation and those resulting from induction of an isolated GH deficiency in adult rats. Ghrelin mRNA was already expressed in the fetus by embryonic day 12 (E12), by E17 most of ghrelin mRNA was in the trunk. At E17, in situ hybridization did not reveal a clear expression of ghrelin mRNA in fetal stomach but showed high ghrelin mRNA levels in the placenta. In the pituitary gland, levels of ghrelin mRNA were high after birth but declined significantly with puberty, whereas in the hypothalamus they were barely detectable at birth and remained very low at all subsequent time points tested. In the GI tract, ghrelin mRNA levels were high from birth to 270 days of life. Immunoneutralization of ghrelin at E16 had no effect on survival or development. Rats showed normal somatotropic function, ghrelin expression and onset of puberty. In young adult rats, passive immunization against GHRH did not affect ghrelin mRNA levels in the pituitary, hypothalamus and stomach. Only a 72-hour fasting period induced a significant increase in ghrelin mRNA levels in the stomach, but not in the pituitary and hypothalamus. These results strongly indicate that ghrelin is an important GI hormone expressed early in life and primarily sensitive to nutritional status.     

Ghrelin tissue distribution: comparison between gene and protein expression

Ghrelin, the natural ligand of the GH secretagogue (GHS) receptor, was originally isolated from the stomach and detected in several tissues, but a systematic study of its tissue distribution has not been performed. In the present investigation, we evaluated ghrelin gene expression (by RT-PCR technique) and ghrelin protein concentration (by enzyme immunoassay technique) in tissues obtained from control rats as well as in rats subjected to 48-h fasting. The ghrelin gene was expressed in stomach, small intestine, brain, cerebellum, pituitary, heart, pancreas, salivary gland, adrenal, ovary and testis, with maximum expression occurring in the stomach, while no significant expression was detected by standard RT-PCR in liver, lung, kidney and skeletal muscle. Ghrelin protein was detected in stomach, small intestine, brain, cerebellum, pituitary, lung, skeletal muscle pancreas, salivary gland, adrenal, ovary and testis, at concentrations ranging from 0.05 to 1.43 ng/mg of homogenate protein (the highest concentration occurred in the lung, followed by the brain). Ghrelin was not detectable in the heart, liver and kidney. Therefore, gene and protein expression were dissociated. Fasting did not produce significant changes in ghrelin gene expression, while the distribution of ghrelin between different tissues was significantly modified: protein concentration increased in the brain, cerebellum, lung and salivary gland, while it decreased in the stomach.     

The inhibition of gastric ghrelin production by food intake in rats is dependent on the type of macronutrient

Ghrelin is a peptide mainly produced by the stomach that increases food intake and body weight. Ghrelin expression increases with fasting and is diminished by re-feeding, but although the expression of this hormone is regulated by the feeding state, the relation with diet composition is not yet well established. We have studied the inhibitory effect of the intake of two different macronutrients (fat and carbohydrates) on ghrelin production by the stomach in fasted rats, as well as the relation with another important signal in the regulation of energy balance, leptin. Ghrelin mRNA expression in the gastric mucosa was determined by Northern blotting, and leptin mRNA expression was determined by Northern blotting in the adipose tissue and by RT-PCR in the stomach; circulating and gastric concentrations of ghrelin and leptin were measured by enzyme immunosorbent assay and ELISA, respectively. Our results showed an increase in ghrelin mRNA levels in response to 14 h of fasting. Food intake for 20 min after the fast produced a decrease in ghrelin mRNA expression that was recovered in 45 min in rats that ate the fat diet, whereas levels remained low when rats ate the carbohydrate diet. Serum ghrelin followed a similar tendency. The decrease in ghrelin expression by feeding was associated with an increased expression of gastric leptin only when animals ate carbohydrates. We conclude that the inhibition of ghrelin production by the stomach after re-feeding of fasted rats is dependent on diet composition and can be related to the different satiating capacity of the ingested macronutrients, which is higher for carbohydrates than fat.     

Ghrelin与胎儿及新生儿生长发育和能量代谢的关系

Ghrelin是生长激素释放激素受体的内源性配体,在胎儿及新生儿生长发育和能量代谢过程中发挥重要作用.胎儿期血循环中ghrelin来源于自身分泌(主要是胰腺,胃分泌较少)和胎盘,既有辛酰基化又有去酰基化ghrelin存在,可直接刺激细胞增殖、骨生长,诱导神经管发生.脐血ghrelin水平明显高于母血,并与胎盘重量、胰岛素样生长因子-1、出生体重及身长呈负相关.新生儿期ghrelin主要由胃黏膜细胞分泌,新生儿肠内营养物质的摄入促进胃肠ghrelin分泌细胞的成熟和分泌.新生儿血清总ghrelin水平高于脐血,并与出生体重、身长呈负相关,小于胎龄儿血ghrelin水平高于正常胎龄儿和大于胎龄儿.     

Gastric leptin, but not estrogen and somatostatin, contributes to the elevation of ghrelin mRNA expression level in fasted rats

Ghrelin, an endogenous ligand for the GH secretagog receptor, is predominantly produced in the stomach. It has been reported that endogenous ghrelin levels are increased by fasting and decreased after refeeding. It has also been reported that estrogen upregulates ghrelin expression and production and that somatostatin inhibits ghrelin secretion, whereas leptin has a paradoxical effect. Recently, several studies have shown that estrogen, somatostatin, and leptin are produced in the stomach, but the direct effects of these gastric hormones on ghrelin expression in a fasting state remain obscure. In this study, we examined the mRNA expression levels of gastric ghrelin, aromatase (estrogen synthetase), leptin and somatostatin, and concentrations of stomach leptin and portal vein 17beta-estradiol in fasted male rats. After 48 h of fasting, although gastric ghrelin mRNA level was significantly increased, both gastric leptin mRNA level and leptin content were decreased. Further, refeeding of fasted rats resulted in a decrease in ghrelin expression level and an increase in leptin expression level. On the other hand, gastric estrogen and somatostatin levels did not change after fasting. In vitro studies revealed that leptin dose-dependently inhibited ghrelin expression and also inhibited estrogen-stimulated ghrelin expression. Moreover, ghrelin cells were found to be tightly surrounded by leptin cells. RT-PCR analysis clearly showed that long and short forms of the leptin receptor are expressed in the rat stomach. These results strongly suggest that an elevated gastric ghrelin expression level in a fasting state is regulated by attenuated restraint from decreased gastric leptin level.     

Molecular forms of hypothalamic ghrelin and its regulation by fasting and 2-deoxy-d-glucose administration

Ghrelin, an endogenous ligand for the GH secretagogue receptor, is a hormone expressed in stomach and other tissues, such as hypothalamus, testis, and placenta. This hormone acts at a central level to stimulate GH secretion and food intake. Little is known, however, about the molecular forms and physiological roles of ghrelin within the hypothalamus. In this report, we detail the molecular forms, mRNA expression patterns, and peptide contents of ghrelin within the rat hypothalamus. Using the combination of reverse-phase HPLC and ghrelin-specific RIA, we determined that the rat hypothalamus contains both n-octanoyl-modified and des-acyl ghrelins. Fasting for 24 and 48 h significantly decreased ghrelin mRNA expression in the hypothalamus to 24% and 28% of control values, respectively. Both n-octanoyl-modified and des-acyl ghrelin content in the hypothalamus decreased after 24 and 48 h of fasting. These results contrast the changes in gastric ghrelin after fasting, which decreased in content despite increased mRNA expression. Two hours after injection of 2-deoxy-d-glucose (2-DG), a selective blocker of carbohydrate metabolism, ghrelin peptide levels also decreased. Thus, induction of glucoprivic states, such as fasting and 2-DG treatment, decreased ghrelin gene expression and peptide content within the hypothalamus.     

Characterization of turkey and chicken ghrelin genes, and regulation of ghrelin and ghrelin receptor mRNA levels in broiler chickens

Ghrelin, a peptide hormone produced by the stomach in mammals, stimulates growth hormone release and food intake. Recently, ghrelin was identified and characterized in chicken proventriculus and shown to stimulate growth hormone release but inhibit feed intake. The purpose of this work was to identify and further characterize the ghrelin gene in chickens and in turkeys. Using molecular cloning techniques we have sequenced cDNAs corresponding to chicken (White Leghorn) and turkey ghrelin mRNAs. A total of 844 (chicken) or 869 (turkey) bases including the complete coding regions (CDS), and the 5'- and 3'-untranslated regions (UTRs) were determined. Nucleotide sequence (CDS) predicted a 116 amino acid precursor protein (preproghrelin) for both the chicken and the turkey that demonstrated complete conservation of an N-terminal 'active core' (GSSF) including a serine (position 3 of the mature hormone) known to be a modification (acylation) site important for ghrelin bioactivity. Additional nucleotide sequence was found in the 5'-UTRs of both Leghorn and turkey cDNAs that was not present in broilers or the red jungle fowl. The turkey ghrelin gene, sequenced from genomic DNA templates, contained five exons and four introns, a structure similar to mammalian and chicken ghrelin genes. Ghrelin was highly expressed in proventriculus with much lower levels of expression in other tissues such as pancreas, brain, and intestine. RT-PCR was used to quantify ghrelin mRNA levels relative to 18S rRNA in 3-week-old male broiler chickens. The level of ghrelin mRNA increased in proventriculus in response to fasting but did not decline with subsequent refeeding. Plasma ghrelin levels did not change significantly in response to fasting or refeeding and did not appear to reflect changes in proventriculus ghrelin mRNA levels. Ghrelin mRNA levels declined in broiler pancreas after a 48 h fast and increased upon refeeding. Expression of the gene encoding the receptor for ghrelin (growth hormone secretagogue receptor, GHS-R) and a variant form was detected in a variety of tissues collected from 3-week-old male broiler chickens possibly suggesting autocrine/paracrine effects. These results offer new information about the avian ghrelin and ghrelin receptor genes and the potential role that this system might play in regulating feed intake and energy balance in poultry.     

Ghrelin in neonatal rats: distribution in stomach and its possible role

Ghrelin, a 28 amino acid peptide, has recently been isolated from the rat stomach as an endogenous ligand for the GH secretagogue receptor. The fact that administration of ghrelin, centrally or peripherally, stimulates both food intake and GH secretion suggests that stomach ghrelin has an important role in the growth of rats. We used immunohistochemistry and radioimmunoassay to determine the age at which ghrelin-immunostained cells begin to appear in the rat stomach. Ghrelin-immunoreactive cells were found to be expressed in the fetal stomach from pregnancy day 18. The number of ghrelin-immunoreactive cells in the fetal stomach increased as the stomach grew. The amount of ghrelin in the glandular part of the rat stomach also increased, in an age-dependent manner, from the neonatal stage to adult. Eight hours of milk restriction significantly decreased the ghrelin concentration in the stomachs of 1-week-old rats, and increased the ghrelin concentration in their plasma. Administration of ghrelin to 1- and 3-week-old rats increased plasma GH concentrations. The daily subcutaneous administration of ghrelin to pregnant rats from day 15 to day 21 of pregnancy caused an increase in body weight of newborn rats. In addition, daily subcutaneous administration of ghrelin to neonatal rats from birth advanced the day of vaginal opening from day 30.7+/-0.94 to day 27.9+/-0.05. These results suggest that ghrelin may be involved in neonatal development.     

Influence of visceral adiposity on ghrelin secretion and expression in rats during fasting

Although circulating ghrelin levels correlate inversely with adiposity at baseline, little is known about the effect of percent visceral adipose tissue value (PVATV) on ghrelin expression and secretion in response to fasting. Our study demonstrated that ghrelin increased with 24-h fasting in rats with the lowest PVATV (less than 6%), after 3 days in rats with intermediate PVATV (6-9%) and 5 days in rats with the highest PVATV (greater than 9%). Ghrelin mRNA in the stomach was increased after 3 days in low-PVATV (5.8+/-0.9%) rats but not in high-PVATV (14+/-1.6%) rats. Therefore, both ghrelin secretion and mRNA were delayed in response to fasting in rats with increased visceral fat. In rats matched for PVATV, but with different body weights, the fasting induced similar levels of increased ghrelin while in rats with different PVATV ghrelin secretion was different in response to fasting, even when body weights were matched in two groups. These data suggested that the initial PVATV, not lean mass, was related to the pattern of plasma ghrelin in response to fasting in rats.     

Ghrelin, a new gastrointestinal endocrine peptide that stimulates insulin secretion: enteric distribution, ontogeny, influence of endocrine, and dietary manipulations.

Ghrelin, an endogenous ligand for the GH secretagogue receptor was characterized recently from extracts of rat stomach. We describe the enteric distribution of ghrelin, ontogeny of stomach ghrelin gene expression, effects of dietary and endocrine manipulations, and vagotomy on stomach ghrelin mRNA and peptide levels and secretion in the rat. Ghrelin expression was examined by Northern blotting. Tissue and plasma ghrelin levels were measured by RIA. A gradient of ghrelin production occurs in the rat gastrointestinal tract with the highest ghrelin expression and peptide levels in the mucosal layer of the stomach-fundus and the lowest levels in the colon. Ghrelin was not detectable in the fetal stomach and increased progressively after birth especially during the second and third postnatal weeks. Plasma ghrelin levels also increased in parallel with stomach ghrelin levels postnatally. Exogenous GH treatment decreased stomach ghrelin expression significantly. A high-fat diet decreased plasma ghrelin levels, whereas a low-protein diet increased plasma ghrelin levels significantly. Intravenous administration of ghrelin stimulates gastrin and insulin secretion. Our findings indicate that ghrelin is an important stomach hormone sensitive to nutritional intake; ghrelin may link enteric nutrition with secretion of GH, insulin, and gastrin.     

Ghrelin is an appetite-stimulatory signal from stomach with structural resemblance to motilin

BACKGROUND & AIMS: : Ghrelin, an endogenous ligand for growth hormone secretagogue receptor, was recently identified in the rat stomach. We examined the effects of the gastric peptide ghrelin on energy balance in association with leptin and vagal nerve activity. METHODS: : Food intake, oxygen consumption, gastric emptying, and hypothalamic neuropeptide Y (NPY) messenger RNA expression were measured after intra-third cerebroventricular or intraperitoneal injections of ghrelin in mice. The gastric vagal nerve activity was recorded after intravenous administration in rats. Gastric ghrelin gene expression was assessed by Northern blot analysis. Repeated coadministration of ghrelin and interleukin (IL)-1 beta was continued for 5 days. RESULTS: : Ghrelin exhibited gastroprokinetic activity with structural resemblance to motilin and potent orexigenic activity through action on the hypothalamic neuropeptide Y (NPY) and Y(1) receptor, which was lost after vagotomy. Ghrelin decreased gastric vagal afferent discharge in contrast to other anorexigenic peptides that increased the activity. Ghrelin gene expression in the stomach was increased by fasting and in ob/ob mice, and was decreased by administration of leptin and IL-1 beta. Peripherally administered ghrelin blocked IL-1 beta-induced anorexia and produced positive energy balance by promoting food intake and decreasing energy expenditure. CONCLUSIONS: : Ghrelin, which is negatively regulated by leptin and IL-1 beta, is secreted by the stomach and increases arcuate NPY expression, which in turn acts through Y(1) receptors to increase food intake and decrease energy expenditure. Gastric peptide ghrelin may thus function as part of the orexigenic pathway downstream from leptin and is a potential therapeutic target not only for obesity but also for anorexia and cachexia.     

Gender and gonadal influences on ghrelin mRNA levels in rat stomach

OBJECTIVE: The recently isolated endogenous GH secretagogue, named ghrelin, is a gastric peptide of 28 amino acids with an n-octanoylation in the serine 3 that confers the biological activity to this factor. Ghrelin has been shown to directly stimulate GH release in vivo and in vitro and to be involved in the regulation of gastric acid secretion and motility. In the present work we have studied gender and gonadal dependency of ghrelin mRNA expression in rat stomach. DESIGN AND METHODS: We analysed ghrelin mRNA expression in rat stomach by Northern blot analysis. We also examined the effect of gonadal steroid deprivation on ghrelin mRNA expression. RESULTS AND CONCLUSIONS: The results obtained showed clearly that ghrelin gastric mRNA expression increased with age in young rats (up to 90 days old) but exhibited no significant sex difference at each age tested. Ghrelin mRNA levels were lowest at postnatal day 9, reaching a stable level of expression at day 40 in both female and male rats, although the increase in female rats appears much more gradual than that in males. Moreover, neither ovariectomy nor orchidectomy significantly modified ghrelin mRNA gastric levels in adult rats. In conclusion, these data indicate that ghrelin mRNA expression is associated with age and that a progressive increase is present from the perinatal period up to a stable level after puberty. Gonadal hormones did not alter ghrelin mRNA levels. Taken together, these data showed that ghrelin mRNA levels in young rats are age but not gender dependent, and are not influenced by gonadal steroids.     

Ghrelin, a novel placental-derived hormone

Ghrelin, a GH-releasing acylated peptide, has been recently identified from the rat stomach. The purified peptide consists of 28 amino acids in which the serine 3 residue is n-octanoylated. Here we show that ghrelin messenger RNA and ghrelin peptide are present in the human as well as in rat placentae. In human placenta, ghrelin was detected by PCR at both first trimester and after delivery. While ghrelin was not detected by immunohistochemistry in human placenta at term, it was easily identified by immunohistochemistry at first trimester being mainly expressed in cytotrophoblast cells and scarcely in syncytiotrophoblast ones. Ghrelin was also identified in a human choriocarcinoma cell line, the BeWo cells. Ghrelin was found, by immunohistochemistry, in the cytoplasm of labyrinth trophoblast of rat placenta, whereas other placental cell types seems to be negative for ghrelin immunostaining. Moreover, placental ghrelin messenger RNA, in pregnant rats, showed a characteristic profile of expression being practically undetectable during early pregnancy, with a sharp peak of expression at day 16 and decreasing in the latest stages of gestation. In conclusion, ghrelin has been detected in human and rat placenta showing a pregnancy-related time course of expression. Whether placenta-derived ghrelin is involved in the modulation of GH release, or placental cell growth and differentiation remains to be established.     

Maternal ghrelin plays an important role in rat fetal development during pregnancy

Ghrelin, an acylated peptide serving as an endogenous ligand for GH secretagogue receptor (GHS-R), was originally isolated from rat and human stomach. In this study, we report the critical role of maternal ghrelin in fetal development. High levels of ghrelin receptor (GHS-R) mRNA were detected in various peripheral fetal tissues beginning at embryonic d 14 and lasting until birth. Fetal GHS-R expression was also confirmed in fetal tissues by immunohistochemistry. Autoradiography revealed that both des-acyl ghrelin and acyl ghrelin bind to fetal tissues. Chronic treatment of mothers with ghrelin resulted in a significant increase in birth weight in comparison to newborns from saline-treated mothers. Even when maternal food intake after ghrelin treatment was restricted through paired feeding, significant stimulation of fetal development still occurred. Conversely, active immunization of mothers against ghrelin decreased fetal body weight during pregnancy. A single ghrelin injection into the mother increased circulating ghrelin levels in the fetus within 5 min of injection, suggesting that maternal ghrelin transits easily to the fetal circulation. High levels of des-acyl ghrelin were detected in fetal blood and amniotic fluid. Both acylated and des-acyl ghrelin increased [3H]thymidine and 5-bromo-2'-deoxyuridine incorporation of cultured fetal skin cells in a dose-dependent manner, and calcium-imaging analysis revealed that acyl and des-acyl ghrelin increased the Ca2+ influx in discrete cultured fetal skin cells, respectively. These results indicate that maternal ghrelin regulates fetal development during the late stages of pregnancy.     

Role of ghrelin in streptozotocin-induced diabetic hyperphagia

Ghrelin, an endogenous ligand for the growth hormone (GH) secretagogue receptor, was originally purified from the rat stomach. We have previously reported that central administration of ghrelin increases food intake and body weight. To investigate the role of ghrelin in the hyperphagic response to uncontrolled diabetes, adult male rats were studied 14 days after administration of streptozotocin (STZ) or vehicle. STZ-treated diabetic rats were markedly hyperphagic. This hyperphagia was accompanied by hyperglycemia, hypoinsulinemia, and reduced plasma GH levels. Treatment of diabetic rats with insulin reversed these changes. Plasma ghrelin concentrations in untreated diabetic rats were significantly higher than in control rats and were normalized by insulin treatment. The ghrelin gene expression in the stomach was also higher in STZ diabetic rats than in control rats, but this difference was not significant. In contrast, plasma leptin was markedly reduced in STZ diabetic rats. This reduction in plasma leptin levels was reversed by insulin treatment. In addition, hypothalamic NPY mRNA levels were increased in STZ-treated diabetic rats and were reversed by insulin treatment. Furthermore, the hyperphagia was partially reversed by the administration of a ghrelin-receptor antagonist. Therefore, we conclude that the elevated plasma ghrelin levels, along with decreased plasma leptin levels, could contribute to the diabetic hyperphagia in part by increasing hypothalamic NPY. This is the first report to show the pathophysiological significance of ghrelin in diabetes.     

Nutritional regulation of insulin-like growth factor-binding protein gene expression in the ovine fetus and pregnant ewe.

The factors controlling the synthesis and degradation of the insulin-like growth factor-binding proteins (IGFBPs) during pregnancy are poorly understood. To clarify the roles of nutritional factors in the regulation of fetal and maternal IGFBP production, we examined the effects of fasting, refeeding, and glucose administration on plasma IGFBP concentrations and hepatic IGFBP mRNA levels in fetal lambs and pregnant ewes (n = 24). Maternal fasting for 3 days in late gestation stimulated a 50-100% increase in maternal plasma BP-1 concentrations (P < 0.05) and a 2- to 3-fold increase in fetal plasma BP-1 (P < 0.05), as determined by densitometric analysis of Western ligand blots. Fasting also stimulated a 40-70% increase in maternal plasma BP-2 concentrations (P < 0.05), but had no significant effect on fetal plasma BP-2 levels. Levels of hepatic BP-1 mRNA in the fetus and pregnant ewe during fasting paralleled plasma BP-1 levels, suggesting that fasting modulates fetal and maternal plasma BP concentrations at least in part through effects on hepatic gene expression. The effects of fasting on both mRNA and plasma levels of BP-1 and BP-2 were reversed by 3 days of refeeding and were prevented by glucose infusion during fasting. When ewes were made hyperglycemic by the infusion of hypertonic glucose, plasma BP-1 and BP-2 concentrations varied inversely with blood glucose concentrations. In addition, hyperglycemia reduced maternal liver BP-1 and BP-2 mRNA levels and fetal BP-1 mRNA levels by 50-65%. Direct administration of hypertonic glucose to the fetus decreased fetal plasma BP-1 levels acutely and reduced fetal BP-1 mRNA levels by 57%, but had no effect on fetal plasma BP-2 or fetal hepatic BP-2 mRNA levels. These findings indicate that glucose and other nutritional factors regulate gene expression and plasma levels of BP-1 and BP-2 in the pregnant ewe and BP-1 in the fetal lamb. The changes in expression of these IGFBPs during fasting and hyperglycemia may play roles in adaptation of the pregnant mother and fetus to metabolic stress.     

Maternal adaptations to pregnancy in spontaneously hypertensive rats: leptin and ghrelin evaluation

Leptin and/or ghrelin, initially thought to be considered messengers of energy metabolism, are now considered to play a role in normal and complicated pregnancy. In this study, pregnant, spontaneously hypertensive rats (SHR) have been used to evaluate, for the first time, the modification of leptin and ghrelin both at serum and tissue levels. In SHR, we evaluate plasma leptin level and tissue protein expression in both placenta and adipose tissue at the end of gestation (day 20) versus normotensive Wistar-Kyoto (WKY) animals. The expression of functional leptin receptor (Ob-Rb) in peripheral tissues and in the hypothalamus was evaluated. Moreover, we measured plasma ghrelin level and its mRNA expression in the stomach and placenta. SHR strain presented significantly lower plasma leptin levels when compared with those found in pregnant or not WKY controls. Interestingly, in the placenta, leptin gene expression was higher in SHR than normotensive WKY. Moreover, we demonstrated a resistance to the effects of leptin via 'downregulation' of hypothalamic receptors in pregnant SHR. Conversely, SHR presented significantly higher ghrelin plasma levels when compared with those found in pregnant or not WKY. However, we observed that ghrelin level in the stomach of SHR did not change during pregnancy, and on the opposite, mRNA ghrelin in the placenta of SHR was lower than that of normotensive rats, suggesting a different production of this hormone in the fetal-placental unit. These data gain further insight into metabolic hormone modifications observed in a model of pre-existing hypertension associated with pregnancy.     

Ontogeny of immunoreactive insulin in the fetal bovine pancreas

The aim of this study was to characterize the development of immunoreactive insulin (IRI) in the fetal bovine pancreas. Pancreatic IRI was acid extracted, and both pancreatic and serum IRI were quantitated by RIA. The amount of pancreatic IRI per wet tissue wt in first trimester fetuses was similar to that in the adult animal (8.2 +/- 0.7 and 5.9 +/- 1.7 U/g pancreas, respectively). IRI increased progressively during gestation, attaining 39.2 +/- 6.5 U/g pancreas in the third trimester, 7-fold higher than that in the adult. When pancreatic IRI concentrations were standardized for protein content of the extracts, a decrease was noted between the midsecond and third trimesters. This is most likely the result of dilution of the endocrine portion of the pancreas by the rapidly growing exocrine pancreas. IRI was also detectable in fetal sera from all three trimesters. In contrast to the profile for pancreatic concentrations of IRI, serum concentrations remained constant throughout gestation at approximately 20 microU/ml. Poly(A+)RNA was isolated from adult and fetal pancreata, and the relative levels of preproinsulin mRNA were assessed by DNA/RNA filter hybridization. There was a 2- to 3-fold increase in the relative level of preproinsulin mRNA in fetal pancreata between the first and second trimesters which was maintained through the third trimester. In the adult pancreas, preproinsulin mRNA levels were similar to those in the first trimester fetus. This profile for the ontogeny of pancreatic preproinsulin mRNA was similar to that for pancreatic IRI (units per pancreas) during fetal maturation. We conclude that in the bovine fetus: the endocrine pancreas synthesizes IRI during all three trimesters of development; pancreatic (units per g pancreas), but not serum, concentrations of IRI increase progressively as development proceeds; and the ontogeny of preproinsulin mRNA is paralleled by that of pancreatic IRI (units per pancreas).