Gastric Ghrelin Cells Research Articles

DOCK4 regulates ghrelin production in gastric X/A-like cells.

Ghrelin, a gastric hormone, provides a hunger signal to the central nervous system to stimulate food intake. Ghrelin also modulates neuroinflammatory and apoptotic processes. Dedicator of cytokinesis 4 (DOCK4), a guanine nucleotide exchange factor (GEF), isinvolvedinthe regulationofneuronal polarization and axon regeneration. However, the effect of DOCK4 on ghrelin production has not been explored. The expression of DOCK4 in human and mouse stomach was examined by immunohistochemical staining. The synthesis and secretion of ghrelin in Dock4 null mice were evaluated by real-time quantitative PCR, Western blot and ELISA. The effects of DOCK4 on ghrelin production in mHypoE-42 cells were measured by real-time quantitative PCR and Western blot. We showed that DOCK4 was expressed in both human and mouse gastric ghrelin cells. The mRNA and protein levels of gastric ghrelin, as well as ghrelin secretion, were remarkably diminished in Dock4 null mice. Furthermore, we showed that overexpression of Dock4 significantly stimulated ghrelin expression, while siRNA knockdown of endogenous Dock4 resulted in a marked decrease of ghrelin in mHypoE-N42 cells. Our results identify DOCK4 as a critical regulator for ghrelin production in gastric X/A-like cells.

Gastric ghrelin cells in obese patients are hyperactive.

Distribution and activity of ghrelin cells in the stomach of obese subjects are controversial. We examined samples from stomachs removed by sleeve gastrectomy in 49 obese subjects (normoglycemic, hyperglycemic and diabetic) and quantified the density of ghrelin/chromogranin endocrine cells by immunohistochemistry. Data were compared with those from 13 lean subjects evaluated by gastroscopy. In 44 cases (11 controls and 33 obese patients) a gene expression analysis of ghrelin and its activating enzyme ghrelin O-acyl transferase (GOAT) was performed. In 21 cases (4 controls and 17 obese patients) the protein levels of unacylated and acylated-ghrelin were measured by ELISA tests. In 18 cases (4 controls and 14 obese patients) the morphology of ghrelin-producing cells was evaluated by electron microscopy. The obese group, either considered as total population or divided into subgroups, did not show any significant difference in ghrelin cell density when compared with control subjects. Inter-glandular smooth muscle fibres were increased in obese patients. In line with a positive trend of the desacylated form found by ELISA, Ghrelin and GOAT mRNA expression in obese patients was significantly increased. The unique ghrelin cell ultrastructure was maintained in all obese groups. In the hyperglycemic obese patients, the higher ghrelin expression matched with ultrastructural signs of endocrine hyperactivity, including expanded rough endoplasmic reticulum and reduced density, size and electron-density of endocrine granules. A positive correlation between ghrelin gene expression and glycemic values, body mass index and GOAT was also found. All obese patients with type 2 diabetes recovered from diabetes at follow-up after 5 months with a 16.5% of weight loss. Given the known inhibitory role on insulin secretion of ghrelin, these results suggest a possible role for gastric ghrelin overproduction in the complex architecture that takes part in the pathogenesis of type 2 diabetes.

The Effect of High-Fat Diet-Induced Obesity on the Expression of Nutrient Chemosensors in the Mouse Stomach and the Gastric Ghrelin Cell.

The stomach is the primary source of the orexigenic and adiposity-promoting hormone, ghrelin. There is emerging evidence on the nutrient-mediated modulation of gastric ghrelin secretion. However, limited information is available on gastric nutrient-sensing mechanisms in high-fat diet (HFD)-induced obesity. This study investigated the impact of HFD-induced obesity on the expression of nutrient chemosensors in mouse stomach, particularly ghrelin cells. Male C57BL/6 mice were fed either a standard laboratory diet (SLD) or HFD for 12 weeks. The expression of ghrelin, enzymes involved in ghrelin production (PC1/3, GOAT) and nutrient chemosensors (CD36, FFAR2&4, GPR93, CaSR, mGluR4 and T1R3) was determined by quantitative RT-PCR in the mouse corpus and antrum. Immunohistochemistry assessed the protein expression of CaSR and ghrelin in the corpus and antrum. Antral mRNA levels of CaSR and PC1/3 were increased in HFD compared to SLD mice, while mRNA levels of all other nutrient chemosensors examined remained unchanged. CaSR immunolabelling was observed in the gastric antrum only. Nearly 80% of antral ghrelin cells expressed CaSR, with a similar cell density and co-expression in SLD and HFD mice. In conclusion, HFD-induced obesity increased CaSR mRNA expression in mouse antrum. However, the high antral co-expression of CaSR and ghrelin was unaltered in HFD compared to SLD mice.

Nutrient-sensing components of the mouse stomach and the gastric ghrelin cell.

The ability of the gut to detect nutrients is critical to the regulation of gut hormone secretion, food intake, and postprandial blood glucose control. Ingested nutrients are detected by specific gut chemosensors. However, knowledge of these chemosensors has primarily been derived from the intestine, while available information on gastric chemosensors is limited. This study aimed to investigate the nutrient-sensing repertoire of the mouse stomach with particular emphasis on ghrelin cells. Quantitative RT-PCR was used to determine mRNA levels of nutrient chemosensors (protein: G protein-coupled receptor 93 [GPR93], calcium-sensing receptor [CaSR], metabotropic glutamate receptor type 4 [mGluR4]; fatty acids: CD36, FFAR2&4; sweet/umami taste: T1R3), taste transduction components (TRPM5, GNAT2&3), and ghrelin and ghrelin-processing enzymes (PC1/3, ghrelin O-acyltransferase [GOAT]) in the gastric corpus and antrum of adult male C57BL/6 mice. Immunohistochemistry was performed to assess protein expression of chemosensors (GPR93, T1R3, CD36, and FFAR4) and their co-localization with ghrelin. Most nutrient chemosensors had higher mRNA levels in the antrum compared to the corpus, except for CD36, GNAT2, ghrelin, and GOAT. Similar regional distribution was observed at the protein level. At least 60% of ghrelin-positive cells expressed T1R3 and FFAR4, and over 80% expressed GPR93 and CD36. The cellular mechanisms for the detection of nutrients are expressed in a region-specific manner in the mouse stomach and gastric ghrelin cells. These gastric nutrient chemosensors may play a role modulating gastrointestinal responses, such as the inhibition of ghrelin secretion following food intake.

Ghrelin plasma levels, gastric ghrelin cell density and bone mineral density in women with rheumatoid arthritis.

Generalized bone loss can be considered an extra-articular manifestation of rheumatoid arthritis (RA) that may lead to the occurrence of fractures, resulting in decreased quality of life and increased healthcare costs. The peptide ghrelin has demonstrated to positively affect osteoblasts in vitro and has anti-inflammatory actions, but the studies that correlate ghrelin plasma levels and RA have contradictory results. We aimed to evaluate the correlation between total ghrelin plasma levels, density of ghrelin-immunoreactive cells in the gastric mucosa, and bone mineral density (BMD) in twenty adult women with established RA with 6 months or more of symptoms (mean age of 52.70±11.40 years). Patients with RA presented higher ghrelin-immunoreactive cells density in gastric mucosa (P=0.008) compared with healthy females. There was a positive relationship between femoral neck BMD and gastric ghrelin cell density (P=0.007). However, these same patients presented a negative correlation between plasma ghrelin levels and total femoral BMD (P=0.03). The present results indicate that ghrelin may be involved in bone metabolism of patients with RA. However, the higher density of ghrelin-producing cells in the gastric mucosa of these patients does not seem to induce a corresponding elevation in the plasma levels of this peptide.

Immunohistochemistry evidence for gastric ghrelin cells hypertrophy in obese patients

Ghrelin cells are endocrine cells of the gastrointestinal tract mainly present in the glands of the stomach. They produce the “hunger hormone” that promotes appetite and adiposity in animal and human models. In response to these anabolic effects, several elements have suggested the influence of ghrelin on the regulation of metabolic functions and the development of obesity-related disorders. However, its physiologic significance is quite complex and some of the effects of ghrelin are still debated in the literature. Recent evidence suggests that ghrelin influences glucose homeostasis through the modulation of insulin secretion and insulin receptor signalling. T2 diabetes is the most frequent metabolic disorder associated with obesity. To date, the most effective treatment of obesity is bariatric surgery and one of the most used techniques is sleeve gastrectomy. It consists in the resection of 25% of the stomach producing a neo anatomy in which this organ becomes a tube-like structure, lacking the ghrelinrich part. Forty-seven percent of T2 diabetic obese patients treated by sleeve gastrectomy rescue their diabetic condition just after surgery and before any weight reduction. This clinical evidence clearly connects this new morphology of the stomach with the positive metabolic improvement and suggests that ghrelin could play a key role in this striking effect. We studied the gastric mucosa of the fundus of sleeve gastrectomized T2 diabetic-obese patients (n.5) by immunohistochemistry with Anti- Ghrelin antibody to verify the ghrelin cell morphology, immunoreactivity and density. Anti- Chromogranin A antibody was used to detect the whole endocrine population of gastric glands. We used the normal part of the gastric fundus removed from patients (n.3) suffering for gastric cancer, as control. Ghrelin receptor has been shown on peripheral nerve fibers of the autonomous nervous system, and because a direct innervation of Langerhans islets by intestinal neurons have been shown in the past, we extended our immunohistochemistry analyses to neuronal antigens in order to study the nervous network of gastric mucosae in these surgical samples. Results show that ghrelin cells in T2 diabetic obese patients represented a consistent percentage (48.4% vs 38% in controls) of chromogranin A immunoreactive cell population. Furthermore, ghrelin cells were hypertrophic and intensely immunostained when compared with those found in gastric mucosae of controls. Neuronal specific enolase immunoreactivity was clearly enhanced in the gastric mucosae of obese patients. These data suggest that the stomach of T2 diabetic obese patients increase ghrelin content and that sleeve gastrectomy could have an important metabolic role by anatomical reduction of this hormone. Furthermore, our result suggest a possible paracrine effect on the peripheral neuronal population that could also be implicated in this complex and striking clinical effect of bariatric surgery.

G protein-coupled receptor 120 signaling regulates ghrelin secretion in vivo and in vitro

Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, is produced predominantly in the stomach. It has been reported that endogenous ghrelin levels are increased by fasting and decreased immediately after feeding and that fasting-induced ghrelin release is controlled by the sympathetic nervous system. However, the mechanisms of plasma ghrelin decrement after feeding are poorly understood. Here, we studied the control of ghrelin secretion using ghrelin-producing cell lines and found that these cells express high levels of mRNA encoding G-protein coupled receptor 120 (GPR120). Addition of GW-9508 (a GPR120 chemical agonist) and α-linolenic acid (a natural ligand for GPR120) inhibited the secretion of ghrelin by ∼50 and 70%, respectively. However, the expression levels of preproghrelin and ghrelin O-acyltransferase (GOAT) mRNAs were not influenced by GW-9508. In contrast, the expression levels of prohormone convertase 1 were decreased significantly by GW-9508 incubation. Moreover, we observed that the inhibitory effect of GW-9508 on ghrelin secretion was blocked by a small interfering RNA (siRNA) targeting the sequence of GPR120. Furthermore, pretreatment with GW-9508 blocked the effect of the norepinephrine (NE)-induced ghrelin elevation in ghrelin cell lines. In addition, we showed that GW-9508 inhibited ghrelin secretion via extracellular signal-regulated kinase activity in ghrelin cell lines. Finally, we found that GW-9508 decreased plasma ghrelin levels in mice. These results suggest that the decrease of ghrelin secretion after feeding is induced partially by long-chain fatty acids that act directly on gastric GPR120-expressing ghrelin cells.

Seven transmembrane G protein-coupled receptor repertoire of gastric ghrelin cells

The molecular mechanisms regulating secretion of the orexigenic-glucoregulatory hormone ghrelin remain unclear. Based on qPCR analysis of FACS-purified gastric ghrelin cells, highly expressed and enriched 7TM receptors were comprehensively identified and functionally characterized using in vitro, ex vivo and in vivo methods. Five Gαs-coupled receptors efficiently stimulated ghrelin secretion: as expected the β1-adrenergic, the GIP and the secretin receptors but surprisingly also the composite receptor for the sensory neuropeptide CGRP and the melanocortin 4 receptor. A number of Gαi/o-coupled receptors inhibited ghrelin secretion including somatostatin receptors SSTR1, SSTR2 and SSTR3 and unexpectedly the highly enriched lactate receptor, GPR81. Three other metabolite receptors known to be both Gαi/o- and Gαq/11-coupled all inhibited ghrelin secretion through a pertussis toxin-sensitive Gαi/o pathway: FFAR2 (short chain fatty acid receptor; GPR43), FFAR4 (long chain fatty acid receptor; GPR120) and CasR (calcium sensing receptor). In addition to the common Gα subunits three non-common Gαi/o subunits were highly enriched in ghrelin cells: GαoA, GαoB and Gαz. Inhibition of Gαi/o signaling via ghrelin cell-selective pertussis toxin expression markedly enhanced circulating ghrelin. These 7TM receptors and associated Gα subunits constitute a major part of the molecular machinery directly mediating neuronal and endocrine stimulation versus metabolite and somatostatin inhibition of ghrelin secretion including a series of novel receptor targets not previously identified on the ghrelin cell.

Expression of Serum Retinol Binding Protein and Transthyretin within Mouse Gastric Ghrelin Cells

Ghrelin is an orexigenic peptide hormone produced mainly by a distinct group of dispersed endocrine cells located within the gastric oxyntic mucosa. Besides secreted gene products derived from the preproghrelin gene, which include acyl-ghrelin, desacyl-ghrelin and obestatin, ghrelin cells also synthesize the secreted protein nesfatin-1. The main goal of the current study was to identify other proteins secreted from ghrelin cells. An initial gene chip screen using mRNAs derived from highly enriched pools of mouse gastric ghrelin cells demonstrated high levels of serum retinol-binding protein (RBP4) and transthyretin (TTR), both of which are known to circulate in the bloodstream bound to each other. This high expression was confirmed by quantitative RT-PCR using as template mRNA derived from the enriched gastric ghrelin cell pools and from two ghrelin-producing cell lines (SG-1 and PG-1). RBP4 protein also was shown to be secreted into the culture medium of ghrelin cell lines. Neither acute nor chronic caloric restriction had a significant effect on RBP4 mRNA levels within stomachs of C57BL/6J mice, although both manipulations significantly decreased stomach TTR mRNA levels. In vitro studies using PG-1 cells showed no effect on RBP4 release of octanoic acid, epinephrine or norepinephrine, all of which are known to act directly on ghrelin cells to stimulate ghrelin secretion. These data provide new insights into ghrelin cell physiology, and given the known functions of RBP4 and TTR, support an emerging role for the ghrelin cell in blood glucose handling and metabolism.

Intravenous injection of urocortin 1 induces a CRF2 mediated increase in circulating ghrelin and glucose levels through distinct mechanisms in rats

Urocortins (Ucns) injected peripherally decrease food intake and gastric emptying through peripheral CRF2 receptors in rodents. However, whether Ucns influence circulating levels of the orexigenic and prokinetic hormone, ghrelin has been little investigated. We examined plasma levels of ghrelin and blood glucose after intravenous (iv) injection of Ucn 1, the CRF receptor subtype involved and underlying mechanisms in ad libitum fed rats equipped with a chronic iv cannula. Ucn 1 (10μg/kg, iv) induced a rapid onset and long lasting increase in ghrelin levels reaching 68% and 219% at 0.5 and 3h post injection respectively and a 5-h hyperglycemic response. The selective CRF2 agonist, Ucn 2 (3μg/kg, iv) increased fasting acyl (3h: 49%) and des-acyl ghrelin levels (3h: 30%) compared to vehicle while the preferential CRF1 agonist, CRF (3μg/kg, iv) had no effect. <!-- no-mfc -->Ucn 1's<!-- /no-mfc --> stimulatory actions were blocked by the selective CRF2 antagonist, astressin2-B (100μg/kg, iv). Hexamethonium (10mg/kg, sc) prevented Ucn 1-induced rise in total ghrelin levels while not altering the hyperglycemic response. These data indicate that systemic injection of Ucns induces a CRF2-mediated increase in circulating ghrelin levels likely via indirect actions on gastric ghrelin cells that involves a nicotinic pathway independently from the hyperglycemic response.

Postprandial inhibition of gastric ghrelin secretion by long-chain fatty acid through GPR120 in isolated gastric ghrelin cells and mice

Ghrelin is a gastric peptide hormone that controls appetite and energy homeostasis. Plasma ghrelin levels rise before a meal and fall quickly thereafter. Elucidation of the regulation of ghrelin secretion has been hampered by the difficulty of directly interrogating ghrelin cells diffusely scattered within the complex gastric mucosa. Therefore, we generated transgenic mice with ghrelin cell expression of green fluorescent protein (GFP) to enable characterization of ghrelin secretion in a pure population of isolated gastric ghrelin-expressing GFP (Ghr-GFP) cells. Using quantitative RT-PCR and immunofluorescence staining, we detected a high level of expression of the long-chain fatty acid (LCFA) receptor GPR120, while the other LCFA receptor, GPR40, was undetectable. In short-term-cultured pure Ghr-GFP cells, the LCFAs docosadienoic acid, linolenic acid, and palmitoleic acid significantly suppressed ghrelin secretion. The physiological mechanism of LCFA inhibition on ghrelin secretion was studied in mice. Serum ghrelin levels were transiently suppressed after gastric gavage of LCFA-rich lipid in mice with pylorus ligation, indicating that the ghrelin cell may directly sense increased gastric LCFA derived from ingested intraluminal lipids. Meal-induced increase in gastric mucosal LCFA was assessed by measuring the transcripts of markers for tissue uptake of LCFA, lipoprotein lipase (LPL), fatty acid translocase (CD36), glycosylphosphatidylinositol-anchored HDL-binding protein 1, and nuclear fatty acid receptor peroxisome proliferator-activated receptor-γ. Quantitative RT-PCR studies indicate significantly increased mRNA levels of lipoprotein lipase, glycosylphosphatidylinositol-anchored HDL-binding protein 1, and peroxisome proliferator-activated receptor-γ in postprandial gastric mucosa. These results suggest that meal-related increases in gastric mucosal LCFA interact with GPR120 on ghrelin cells to inhibit ghrelin secretion.

Transgenic overexpression of intraislet ghrelin does not affect insulin secretion or glucose metabolism in vivo

Whereas ghrelin is produced primarily in the stomach, a small amount of it is produced in pancreatic islets. Although exogenous administration of ghrelin suppresses insulin secretion in vitro or in vivo, the role of intraislet ghrelin in the regulation of insulin secretion in vivo remains unclear. To understand the physiological role of intraislet ghrelin in insulin secretion and glucose metabolism, we developed a transgenic (Tg) mouse model, rat insulin II promoter ghrelin-internal ribosomal entry site-ghrelin O-acyl transferase (RIP-GG) Tg mice, in which mouse ghrelin cDNA and ghrelin O-acyltransferase are overexpressed under the control of the rat insulin II promoter. Although pancreatic desacyl ghrelin levels were elevated in RIP-GG Tg mice, pancreatic ghrelin levels were not altered in animals on a standard diet. However, when Tg mice were fed a medium-chain triglyceride-rich diet (MCTD), pancreatic ghrelin levels were elevated to ∼16 times that seen in control animals. It seems likely that the gastric ghrelin cells possess specific machinery to provide the octanoyl acid necessary for ghrelin acylation but that this machinery is absent from pancreatic β-cells. Despite the overexpression of ghrelin, plasma ghrelin levels in the portal veins of RIP-GG Tg mice were unchanged from control levels. Glucose tolerance, insulin secretion, and islet architecture in RIP-GG Tg mice were not significantly different even when the mice were fed a MCTD. These results indicate that intraislet ghrelin does not play a major role in the regulation of insulin secretion in vivo.

Regulation of gastric hormones by systemic rapamycin

The mammalian target of rapamycin (mTOR), an evolutionarily conserved serine–threonine kinase, is an intracellular fuel sensor critical for cellular energy homeostasis. Gastrointestinal endocrine cells play a vital role in the regulation of energy balance by secreting hormones that inform the brain about energy supply. Here we showed the localization of mTOR signaling molecules in more than 90% of gastric ghrelin cells and 36 ± 3% of gastrin cells, while no somatostatin-positive cell showed phospho-S6K1 immunoreactivity. Inhibition of mTOR significantly stimulated expression of gastric ghrelin mRNA and protein, and the concentration of plasma ghrelin (2.06 ± 0.34 ng/ml vs. 12.53 ± 3.9 ng/ml, p < 0.05), inhibited gastrin synthesis and secretion (75.01 ± 6.71 pg/ml vs. 54.04 ± 3.65 pg/ml, p < 0.05), but had no effect on somatostatin production (165.2 ± 25.07 pg/ml vs. 178.9 ± 29.14 pg/ml, p = 0.73). Gastric mTOR is a gastric sensor whose activity is linked to the differential regulation of gastric hormone production and release.

Methyl Donor Deficiency Affects Fetal Programming of Gastric Ghrelin Cell Organization and Function in the Rat

Methyl donor deficiency (MDD) during pregnancy influences intrauterine development. Ghrelin is expressed in the stomach of fetuses and influences fetal growth, but MDD influence on gastric ghrelin is unknown. We examined the gastric ghrelin system in MDD-induced intrauterine growth retardation. By using specific markers and approaches (such as periodic acid-Schiff, bromodeoxyuridine, homocysteine, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling, immunostaining, reverse transcription-polymerase chain reaction), we studied the gastric oxyntic mucosa cellular organization and ghrelin gene expression in the mucosa in 20-day-old fetuses and weanling pups, and plasma ghrelin concentration in weanling rat pups of dams either normally fed or deprived of choline, folate, vitamin B6, and vitamin B12 during gestation and suckling periods. MDD fetuses weighed less than controls; the weight deficit reached 57% at weaning (P < 0.001). Both at the end of gestation and at weaning, they presented with an aberrant gastric oxyntic mucosa formation with loss of cell polarity, anarchic cell migration, abnormal progenitor differentiation, apoptosis, and signs of surface layer erosion. Ghrelin cells were abnormally located in the pit region of oxyntic glands. At weaning, plasma ghrelin levels were decreased (-28%; P < 0.001) despite unchanged mRNA expression in the stomach. This decrease was associated with lower body weight. Taken together, these data indicate that one mechanism through which MDD influences fetal programming is the remodeling of gastric cellular organization, leading to dysfunction of the ghrelin system and dramatic effects on growth.

Colocalization of ghrelinO-acyltransferase and ghrelin in gastric mucosal cells

Ghrelin is a peptide hormone with many known functions, including orexigenic, blood glucose-regulatory, and antidepressant actions, among others. Mature ghrelin is unique in that it is the only known naturally occurring peptide to be posttranslationally modified by O-acylation with octanoate. This acylation is required for many of ghrelin's actions, including its effects on promoting increases in food intake and body weight. GOAT (ghrelin O-acyltransferase), one of 16 members of the MBOAT family of membrane-bound O-acyltransferases, has recently been identified as the enzyme responsible for catalyzing the addition of the octanoyl group to ghrelin. Although the initial reports of GOAT have localized its encoding mRNA to tissues known to contain ghrelin, it is as yet unclear whether the octanoylation occurs within ghrelin-producing cells or in neighboring cells. Here, we have performed dual-label histochemical analysis on mouse stomach sections and quantitative PCR on mRNAs from highly enriched pools of mouse gastric ghrelin cells to demonstrate a high degree of GOAT mRNA expression within ghrelin-producing cells of the gastric oxyntic mucosa. We also demonstrate that GOAT is the only member of the MBOAT family whose expression is highly enriched within gastric ghrelin cells and whose whole body distribution mirrors that of ghrelin.

Characterization of a novel ghrelin cell reporter mouse

Ghrelin is a hormone that influences many physiological processes and behaviors, such as food intake, insulin and growth hormone release, and a coordinated response to chronic stress. However, little is known about the molecular pathways governing ghrelin release and ghrelin cell function. To better study ghrelin cell physiology, we have generated several transgenic mouse lines expressing humanized Renilla reniformis green fluorescent protein (hrGFP) under the control of the mouse ghrelin promoter. hrGFP expression was especially abundant in the gastric oxyntic mucosa, in a pattern mirroring that of ghrelin immunoreactivity and ghrelin mRNA. hrGFP expression also was observed in the duodenum, but not in the brain, pancreatic islet, or testis. In addition, we used fluorescent activated cell sorting (FACS) to collect and partially characterize highly enriched populations of gastric ghrelin cells. We suggest that these novel ghrelin-hrGFP transgenic mice will serve as useful tools to better understand ghrelin cell physiology.

Early onset of ghrelin production in a marsupial

Ghrelin regulates appetite in mammals and can stimulate growth hormone (GH) release from the pituitary. In rats and humans, ghrelin cells appear in the stomach during late fetal life. Nevertheless, the role of ghrelin in early mammalian development is not well understood. Marsupials deliver highly altricial young that weigh less than 1g so they must feed and digest milk at a comparatively immature stage of development. Since they complete their growth and differentiation while in the pouch, they are accessible models in which to determine the time course of ghrelin production during development. We examined the distribution of gastric ghrelin cells, plasma ghrelin concentrations and pituitary expression of the ghrelin receptor (ghsr-1alpha) and GH in the tammar wallaby, Macropus eugenii. There were ghrelin immunopositive cells in the developing mesenchyme of the stomach from day 10 post partum (pp) to day 150pp. Subsequently ghrelin protein in the fore-stomach declined and was absent by day 250pp but remained in the gastric cells of the hind-stomach. Ghrelin was detected in the developing pancreas from day 10pp but was absent by day 150pp and in the adult. Pituitary ghsr-1alpha expression and plasma concentrations of ghrelin increased significantly up to day 70-120pp while GH expression was also elevated, declining with GH to reach adult levels by day 180pp. These results demonstrate an early onset of gastric ghrelin expression in the tammar in concert with a functional stomach at a relatively earlier stage than that of developmentally more mature eutherian young.

Gastric ghrelin cell development is hampered and plasma ghrelin is reduced by delayed weaning in rats

The duration of breastfeeding has attracted much interest, as a prolonged period of breastfeeding has been shown to reduce the risk of developing obesity. The mechanism behind the reduced risk is, however, poorly understood. The novel hormone ghrelin augments appetite, promotes body weight increase and increases adiposity. The majority of circulating ghrelin emanates from endocrine cells in the oxyntic mucosa of the stomach. In newborn humans and rodents, the number of ghrelin cells is low after birth until weaning, when the cell population is greatly expanded. To date, information about the influence of weaning perturbations on ghrelin cell development is scarce. Therefore, we studied the effect of delayed weaning on gastric ghrelin expression and plasma ghrelin concentration. To this end, special food separator cages were used to prevent the pups from eating solid food, forcing them to drink milk up to 21 days of age. Gastric ghrelin expression was examined by immunocytochemistry and in situ hybridisation, and plasma concentrations were assessed by RIA. Our data showed that gastric ghrelin expression and plasma ghrelin concentration are maintained at a lower level by delayed weaning. We also found that the relation between gastric ghrelin expression and body weight was altered by delayed weaning. Thus, control rats displayed a positive correlation between ghrelin expression and body weight, while no such correlation was evident in animals with delayed weaning. We conclude that delayed weaning exerts a negative influence on ghrelin expression, and that the onset of solid food intake may trigger normal ghrelin expression. Therefore, we suggest that ghrelin may constitute a hormonal link between the duration of breastfeeding and body weight development.

Gastric estrogen directly induces ghrelin expression and production in the rat stomach

Ghrelin, an endogenous ligand for the GH secretagogue receptor, is predominantly produced in the stomach. Little is known about the regulation mechanism of gastric ghrelin. Here, we report that estrogen synthesized in the stomach induces rat gastric ghrelin gene expression and production. We established a gastric ghrelin cell enrichment method using Percoll centrifugation and then studied the effect of estrogen and/or its antagonist on ghrelin expression and production. Treatment with estrogen for 8 h significantly increased the level of ghrelin expression, and ICI-182 780, an estrogen receptor (ER) antagonist, completely reversed this effect. Reverse transcriptase-PCR analysis clearly showed that ERalpha and aromatase are expressed in the female rat stomach. Moreover, treatment with an aromatase inhibitor, 4-hydro-xyandrostenedione (formestane), significantly decreased the level of ghrelin mRNA expression in minced stomach tissue. In vivo studies revealed that the ghrelin mRNA expression and production did not change in gonadectomized rat 3 weeks after surgery. These results strongly suggest that estrogen produced in the stomach directly induces ghrelin expression and production in both female and male rat stomachs.

Gastric immunolocalization and plasma profiles of acyl-ghrelin in fasted and fasted-refed prepuberal gilts

Ghrelin is a peripheral circulating hormone, mainly released from the stomach, which can stimulate food intake. We studied fed, fasted and fasted-refed prepuberal gilts in order to outline possible changes in gastric mucosal ghrelin cells and in plasma ghrelin profiles in response to food deprivation. Acyl-ghrelin-immunoreactive cells were numerous in oxyntic glands, less abundant in cardiac glands and least frequent in pyloric glands, with the addition of a minor population of labelled cells in the gastric pit mucosa. When fed and fasted animals were compared (72-h fast versus fed; n = 4 each), no clear-cut differences were revealed in labelled cell numbers, nor in their staining intensity. An RIA for plasma porcine acyl-ghrelin (n-octanoylated at Ser-3), not recognizing des-acyl-ghrelin, was validated. Plasma acyl-ghrelin progressively increased upon fasting (over 6, 12, 24 and 48 h); ghrelin levels significantly (P<0.05) higher than those prefast were reached at 72 h. After refeeding, plasma ghrelin was rapidly restored to basal values by 6 h. In the same animals, plasma insulin was significantly reduced throughout the fasting period (6-72 h), while rapidly increasing after refeeding. Non-esterified fatty acid levels increased during fasting (12-72 h) and rapidly returned to low values after refeeding. In conclusion, the present study demonstrates that starvation and refeeding influence ghrelin plasma level in prepuberal gilts. The absence of detectable changes in ghrelin cells, as seen in immunohistochemistry, could be due to a large intracellular storage of potentially releasable acylghrelin.