Changes in Circulating Satiety Hormones in Obese Children: A Randomized Controlled Physical Activity‐Based Intervention Study

The aims of this study are to examine in children: (i) obesity‐related alterations in satiety factors such as leptin, ghrelin, and obestatin; (ii) the link between satiety factors and cardiometabolic risk factors; and (iii) the impact of a physical activity‐based lifestyle intervention on the levels of these satiety factors in the obese. We studied a total of 21 adolescents (BMI percentile, 99.0 ± 0.6 for 15 obese and 56.2 ± 1.1 for 6 lean). The obese subjects underwent a 3‐month randomized controlled physical activity‐based lifestyle intervention. Leptin, soluble leptin receptor (sOB‐R), ghrelin, and obestatin levels were determined as the primary outcome measures. Other markers of cardiometabolic disease such as inflammation and insulin resistance were also determined. Body composition was measured by dual‐energy X‐ray absorptiometry. The concentrations of ghrelin, obestatin, and sOB‐R were significantly lower in the obese children compared to the lean controls, whereas that of leptin was higher (all P < 0.05). Although intervention led to a net increase in obestatin (P < 0.01) and no change in ghrelin levels, the balance between ghrelin and obestatin (ratio of ghrelin to obestatin, G/O) decreased (P < 0.02). Intervention reduced leptin and increased sOB‐R (P < 0.01 for both). Significant associations between satiety factors and other cardiometabolic risk factors were also observed. Taken together, alterations in the levels of satiety factors are evident early in the clinical course of obesity, but physical activity‐based lifestyle intervention either prevented their continued increase or normalized their levels. These beneficial effects appear to aid in the maintenance of body weight and reduction in cardiovascular risk.     

Distribution of obestatin and ghrelin in human tissues: immunoreactive cells in the gastrointestinal tract, pancreas, and mammary glands.

Obestatin and ghrelin are two peptides derived from the same prohormone. It is well established that ghrelin is produced by endocrine cells in the gastric mucosa. However, the distribution of human obestatin immunoreactive cells is not thoroughly characterized. A polyclonal antibody that specifically recognizes human obestatin was produced. Using this antibody and a commercial antibody vs ghrelin, the distribution of obestatin and ghrelin immunoreactive cells was determined in a panel of human tissues using immunohistochemistry. The two peptides were detected in the mucosa of the gastrointestinal tract, from cardia to ileum, and in the pancreatic islets. Interestingly, epithelial cells in the ducts of mammary glands showed distinct immunoreactivity for both ghrelin and obestatin. By double immunofluorescence microscopy, it was shown that all detected cells were immunoreactive for both peptides. Furthermore, the subcellular localization of obestatin and ghrelin was essentially identical, indicating that obestatin and ghrelin are stored in the same secretory vesicles.     

Ghrelin and obestatin: different role in fetal lung development?

Ghrelin and obestatin are two proteins that originate from post-translational processing of the preproghrelin peptide. Various authors claim an opposed role of ghrelin and obestatin in several systems. Preproghrelin mRNA is significantly expressed in airway epithelium throughout lung development, predominantly during the earliest stages. The aim of this study was to evaluate the role of ghrelin and obestatin in fetal lung development in vitro. Immunohistochemistry studies were performed at different gestational ages in order to clarify the expression pattern of ghrelin, GHS-R1a, obestatin and GPR39 during fetal lung development. Fetal rat lung explants were harvested at 13.5 days post-conception (dpc) and cultured during 4 days with increasing doses of total ghrelin, acylated ghrelin, desacyl-ghrelin, ghrelin antagonist (D-Lys(3)-GHRP-6) or obestatin. Immunohistochemistry studies demonstrated that ghrelin, GHS-R1a, obestatin and GPR39 proteins were expressed in primitive rat lung epithelium throughout all studied gestational ages. Total and acylated ghrelin supplementation significantly increased the total number of peripheral airway buds, whereas desacyl-ghrelin induced no effect. Moreover, GHS-R1a antagonist significantly decreased lung branching. Finally, obestatin supplementation induced no significant effect in the measured parameters. The present study showed that ghrelin has a positive effect in fetal lung development through its GHS-R1a receptor, whereas obestatin has no effect on lung branching.     

Obestatin is present in saliva: alterations in obestatin and ghrelin levels of saliva and serum in ischemic heart disease

Ghrelin and obestatin are a single gene products and are a multiple functional peptides that regulates energy homeostasis, and food intake. In the present work, we studied the secretion of ghrelin and its co-secreted peptide obestatin in 44 patients with ischemic heart disease with that of 27 healthy matched controls. Here we first conducted using an immunohistochemistry assay to screen whether human salivary glands have any obestatin immunoreactivity. Then, serum and saliva obestatin and acylated ghrelin levels were determined by using Radioimmunoassay. Our immunohistochemical analysis demonstrated that obestatin was localized in the striated and excretory duct of human salivary gland. We also report for the first time that obestatin, like ghrelin, is present in human salivary gland and saliva. No evidence of the role of obestatin or ghrelin saliva levels in the context of ischemic heart disease was found. Salivary ghrelin and obestatin levels are correlated in controls with the blood levels. Determination of salivary values could represent a non-invasive alternative to serum ones that can be useful in clinical practice.     

Different responses of circulating ghrelin, obestatin levels to fasting, re-feeding and different food compositions, and their local expressions in rats

Obestatin, a sibling of ghrelin derived from preproghrelin, opposes several physiological actions of ghrelin. Our previous study has demonstrated that both plasma ghrelin and obestatin levels were decreased significantly 2h after food intake in human. To further expand current knowledge, we investigated the temporal profiles of their levels in ad libitum fed rats, 48h fasted rats and 48h fasted rats refed 2h with a standard chow, crude fiber, 50% glucose or water, and their expressions in stomach, liver and pancreatic islets immunohistochemically. Plasma ghrelin and obestatin levels were measured by EIA. Plasma leptin, insulin and glucose levels were also evaluated. Both plasma ghrelin and obestatin levels increased significantly in fasted rats compared with ad libitum fed rats. The ingestion of standard chow produced a profound and sustained suppression of ghrelin levels, whereas plasma obestatin levels decreased significantly but recovered quickly. Intake of crude fiber or 50% glucose, however, produced a more profound and sustained suppression of obestatin levels, though they had relatively less impact on ghrelin levels. Plasma glucose was the only independent predictor of ghrelin levels, obestatin levels, and ghrelin to obestatin ratios. Obestatin immunoreactivity was detected in the fundus of stomach, liver and pancreatic islets, with roughly similar patterns of distribution to ghrelin. These data show quantitative and qualitative differences in circulating ghrelin and obestatin responses to the short-term feeding status and nutrient composition, and may support a role for obestatin in regulating metabolism and energy homeostasis.     

Proghrelin-derived peptides influence the secretion of insulin, glucagon, pancreatic polypeptide and somatostatin: a study on isolated islets from mouse and rat pancreas

Proghrelin, the precursor of the orexigenic and adipogenic peptide hormone ghrelin, is synthetized in endocrine (A-like) cells in the gastric mucosa. During its cellular processing, proghrelin gives rise to the 28-amino acid peptide desacyl ghrelin, which after octanoylation becomes active acyl ghrelin, and to the 23-amino acid peptide obestatin, claimed to be a physiological opponent of acyl ghrelin. This study examines the effects of the proghrelin products, alone and in combinations, on the secretion of insulin, glucagon, pancreatic polypeptide (PP) and somatostatin from isolated islets of mice and rats. Surprisingly, acyl ghrelin and obestatin had almost identical effects in that they stimulated the secretion of glucagon and inhibited that of PP and somatostatin from both mouse and rat islets. Obestatin inhibited insulin secretion more effectively than acyl ghrelin. In mouse islets, acyl ghrelin inhibited insulin secretion at low doses and stimulated at high. In rat islets, acyl ghrelin inhibited insulin secretion in a dose-dependent manner but the IC(50) for the acyl ghrelin-induced inhibition of insulin release was 7.5 x 10(-8) M, while the EC(50) and IC(50) values, with respect to stimulation of glucagon release and to inhibition of PP and somatostatin release, were in the 3 x 10(-12)-15 x 10(-12) M range. The corresponding EC(50) and IC(50) values for obestatin ranged from 5 x 10(-12) to 20 x 10(-12) M. Desacyl ghrelin per se did not affect islet hormone secretion. However, at a ten times higher concentration than acyl ghrelin (corresponding to the ratio of the two peptides in circulation), desacyl ghrelin abolished the effects of acyl ghrelin but not those of obestatin. Acyl ghrelin and obestatin affected the secretion of glucagon, PP and somatostatin at physiologically relevant concentrations; with obestatin this was the case also for insulin secretion. The combination of obestatin, acyl ghrelin and desacyl ghrelin in concentrations and proportions similar to those found in plasma resulted in effects that were indistinguishable from those induced by obestatin alone. From the data it seems that the effects of endogenous, circulating acyl ghrelin may be overshadowed by obestatin or blunted by desacyl ghrelin.     

Plasma obestatin levels in men with chronic atrophic gastritis

Obestatin is a recently discovered active peptide isolated from the stomach. The purpose of the present study was to investigate the modification of plasma obestatin levels in men with chronic atrophic gastritis. Men older than 65 years undergoing upper gastrointestinal endoscopy were included. All patients with chronic atrophic gastritis underwent multiple biopsies. Fasting plasma obestatin and ghrelin levels were examined in 50 men with chronic atrophic gastritis and 50 healthy men. Plasma obestatin levels were significantly lower in patients with chronic atrophic gastritis than in healthy subjects. Plasma ghrelin levels and ghrelin to obestatin ratio was decreased in men with chronic atrophic gastritis. There was a significant relationship between atrophy and decreased obestatin. A negative correlation was found between circulating obestatin levels and body mass index (BMI) in healthy subjects, but not in patients with chronic atrophic gastritis. The data indicated that chronic atrophic gastritis influenced plasma obestatin levels as well as ghrelin to obestatin ratio in elderly men.     

Neither intravenous nor intracerebroventricular administration of obestatin affects the secretion of GH, PRL, TSH and ACTH in rats

To examine the effect of obestatin, a recently identified peptide derived from preproghrelin, on pituitary hormone secretion, obestatin was administered in anesthetized male rats. Intravenous administration of obestatin did not show any effect on plasma GH, PRL, ACTH and TSH levels. Since obestatin has been reported to have opposite effects of ghrelin in regulating food intake, gastric emptying and intestinal contractility, GH suppressive effect, which is opposite effect of ghrelin, was tested. Intravenous administration of GHRH or GHRP-2, a ghrelin receptor ligand, resulted in a marked plasma GH elevation. However obestatin did not show any effect on GHRH- or GHRP-2-induced GH rise. Furthermore intracerebroventricular administration of obestatin also did not influence plasma GH, PRL, ACTH and TSH levels. These findings suggest that obestatin has no effect on pituitary hormone secretions despite the presence of GPR39, a receptor for obestatin, in the pituitary.     

Ghrelin and obestatin expression in oral squamous cell carcinoma: an immunohistochemical and biochemical study

The underlying molecular mechanism of carcinogenesis in oral squamous cell carcinoma (OSCC) is poorly understood and appears to be controlled on many genetic, environmental, and hormonal factors. Obestatin and ghrelin, two recently discovered hormones, are co-expressed in endocrine cells. The purpose of this investigation was to examine the immunohistochemical features of OSCCs in relation to the tissue concentration of ghrelin and obestatin. The association between OSCC and Epstein Barr Virus (EBV) status was also explored. The expression of ghrelin and obestatin was examined by immunohistochemistry and immunoassay in oral biopsy specimens: 10 benign squamous epithelial cell samples, 10 microinvasive squamous cell carcinomas, and seven well-differentiated and seven poorly differentiated OSCCs. The presence of EBV was evaluated in these samples using immunohistochemistry. The concentrations of ghrelin and obestatin in tissue homogenates were measured by RIA and ELISA, respectively. Squamous cell carcinomas and benign tissue samples were positive for anti-EBV antibody, and obestatin and ghrelin were shown to be co-expressed in all stratified squamous epithelium samples. Expression of ghrelin and obestatin was decreased or absent in OSCCs in relation to the invasiveness of the carcinoma; ghrelin and obestatin levels in cancerous tissue homogenates were lower than in benign tissue homogenates. These results indicate that the concentrations and distribution of immunoreactive obestatin and ghrelin might be helpful in distinguishing OSCC from benign tumors. Maintaining normal levels of these hormones might be required for regulation of normal cell division. However, detailed studies will be required for better understanding of the complex mechanism of carcinogenesis relating to OSCCs.     

Obestatin, acylated and total ghrelin concentrations in the perinatal rat pancreas

BACKGROUND: Ghrelin and obestatin are encoded by the preproghrelin gene and originate from posttranslational processing of the preproghrelin peptide. The fetal rat pancreas contains acylated and desacylated ghrelin peptides, as well as growth hormone secretagogue receptor -1a mRNA. Acylated ghrelin inhibits insulin secretion. We investigated the plasma and tissue ontogeny of ghrelin and obestatin in the rat. METHODS: We measured obestatin and acylated and total ghrelin concentrations in plasma, pancreas and stomach from rat fetuses (F20) and neonates at postnatal day (PN) 1, 6, 12 and 21). RESULTS: Overall, obestatin concentrations were markedly lower than total ghrelin concentrations. In plasma, total ghrelin concentrations decreased abruptly after birth (p < 0.05), contrasting with a 3 times increase in the concentration of acylated ghrelin between F20 and PN1 (p < 0.05). In pancreas, total ghrelin and obestatin concentrations decreased progressively from PN1 to PN21 but acylated ghrelin concentrations increased 6-7 times from F20 (18 [6] pg/ml) to PN6 (122 [59] pg/ml). The percent of acylated ghrelin increased from 1.8 (0.6) at F20 to 39.7 (13.0) % of total ghrelin immunoreactivity at PN12 (p < 0.05). There were significant positive correlations between postnatal obestatin, acylated or total ghrelin and insulin concentrations in the pancreas (all p < 0.02, r(2) > 0.21) and between postnatal total ghrelin and obestatin (in pancreas, r(2) = 0.37) or acylated ghrelin (in stomach, r(2) = 0.27) (p < 0.001). CONCLUSION: Ghrelin and obestatin are present in the perinatal pancreas where they could potentially affect insulin secretion.     

Plasma obestatin levels in normal weight, obese and anorectic women

Obestatin is a recently discovered peptide produced in the stomach, which was originally described to suppress food intake and decrease body weight in experimental animals. We investigated fasting plasma obestatin levels in normal weight, obese and anorectic women and associations of plasma obestatin levels with anthropometric and hormonal parameters. Hormonal (obestatin, ghrelin, leptin, insulin) and anthropometric parameters and body composition were examined in 15 normal weight, 21 obese and 15 anorectic women. Fasting obestatin levels were significantly lower in obese than in normal weight and anorectic women, whereas ghrelin to obestatin ratio was increased in anorectic women. Compared to leptin, only minor differences in plasma obestatin levels were observed in women who greatly differed in the amount of fat stores. However, a negative correlation of fasting obestatin level with body fat indexes might suggest a certain role of obestatin in the regulation of energy homeostasis. A significant relationship between plasma obestatin and ghrelin levels, independent of anthropometric parameters, supports simultaneous secretion of both hormones from the common precursor. Lower plasma obestatin levels in obese women compared to normal weight and anorectic women as well as increased ghrelin to obestatin ratio in anorectic women might play a role in body weight regulation in these pathologies.     

Changes of plasma obestatin, ghrelin and NPY in anorexia and bulimia nervosa patients before and after a high-carbohydrate breakfast

Peptides ghrelin, obestatin and neuropeptide Y (NPY) play an important role in regulation of energy homeostasis, the imbalance of which is associated with eating disorders anorexia (AN) and bulimia nervosa (BN). The changes in ghrelin, obestatin and NPY plasma levels were investigated in AN and BN patients after administration of a high-carbohydrate breakfast (1604 kJ). Eight AN women (aged 25.4+/-1.9, BMI: 15.8+/-0.5), thirteen BN women (aged 22.0+/-1.05, BMI: 20.1+/-0.41) and eleven healthy women (aged 25.1+/-1.16, BMI: 20.9+/-0.40) were recruited for the study. We demonstrated increased fasting ghrelin in AN, but not in BN patients, while fasting obestatin and NPY were increased in both AN and BN patients compared to the controls. Administration of high-carbohydrate breakfast induced a similar relative decrease in ghrelin and obestatin plasma levels in all groups, while NPY remained increased in postprandial period in both patient groups. Ghrelin/obestatin ratio was lower in AN and BN compared to the controls. In conclusions, increased plasma levels of fasting NPY and its unchanged levels after breakfast indicate that NPY is an important marker of eating disorders AN and BN. Different fasting ghrelin and obestatin levels in AN and BN could demonstrate their diverse functions in appetite and eating suppression.     

Differential BBB interactions of three ingestive peptides: obestatin, ghrelin, and adiponectin

Endogenous compounds, including ingestive peptides, can interact with the blood-brain barrier (BBB) in different ways. Here we used in vivo and in vitro techniques to examine the BBB permeation of the newly described satiety peptide obestatin. The fate of obestatin in blood and at the BBB was contrasted with that of adiponectin. By the sensitive multiple time-regression method, obestatin appeared to have an extremely fast influx rate to the brain whereas adiponectin did not cross the BBB. HPLC analysis, however, showed the obestatin result to be spurious, reflecting rapid degradation. Absence of BBB permeation by obestatin and adiponectin was in contrast to the saturable transport of human ghrelin reported previously. As a positive control, ghrelin showed saturable binding and endocytosis in RBE4 cerebral microvessel endothelial cells. By comparison, obestatin lacked specific binding and endocytosis, and the small amount internalized showed rapid intracellular degradation before the radioactivity was released by exocytosis. The differential interactions of obestatin, adiponectin, and ghrelin with the BBB illustrate their distinctive physiological interactions with the CNS.     

Expression of obestatin and ghrelin in papillary thyroid carcinoma

Ghrelin and obestatin are two peptide hormones with opposing roles in the control of appetite: orexigenic and anorexigenic, respectively. Loss of appetite is a common, serious complication of many forms of malignancy. The goals of this study were to investigate: (i) whether there are differences in ghrelin and obestatin peptide expression in thyroid tissues from a series of papillary carcinoma cases and normal controls, and (ii) whether there are correlations between tissue ghrelin and obestatin levels in series of papillary carcinoma cases and normal controls. Immunohistochemical analysis showed that in sections of benign human thyroid tissue, anti-ghrelin antibody reacted with intense staining in colloid-filled follicles. In benign thyroid tissues, colloids displayed plentiful dispersion in comparison with papillary microcarcinomas, whereas colloids in malignant thyroid tissues were uncommon. We found markedly lower tissue ghrelin levels in thyroid tissue of patients with papillary carcinomas, compared with normal thyroid tissues (P = 0.001). Immunohistochemical analysis also showed that obestatin in papillary carcinoma stained positively to various degrees. Obestatin tissue levels in papillary carcinomas tended to be slightly higher than those in normal thyroid tissue, but this was not statistically significant (P = 0.29). We also report that thyroid tissue of patients with Hashimoto’s thyroiditis produced ghrelin and obestatin at similar levels as in normal thyroid tissue, even though colloid in Hashimoto’s disease is scarce. We conclude that depressed expression of ghrelin, but not obestatin, is specific to papillary carcinoma, and this difference might constitute a diagnostic tool to differentiate papillary carcinoma from normal thyroid tissue. We currently do not know how these peptides are regulated and what factors are involved in papillary carcinoma, which inhibit the expression of ghrelin but not obestatin. This issue warrants further studies.     

Simultaneous decrease of plasma obestatin and ghrelin levels after a high-carbohydrate breakfast in healthy women

Ghrelin is a gut peptide produced mainly by stomach, well known to induce appetite stimulatory actions. Obestatin, a recently identified peptide derived from preproghrelin, was initially described to antagonize stimulatory effect of ghrelin on food intake. The postprandial response of obestatin and its relationship with ghrelin in humans remains unknown. We therefore investigated the postprandial response of obestatin and total ghrelin, acyl and desacyl ghrelin and neuropeptide Y (NPY) to a high-carbohydrate breakfast (1 604 kJ) in eight healthy women (age: 24.2+/-0.82 years; BMI 21.6+/-0.61 kg/m(2)). Blood samples were collected before the meal, and 30, 60, 90, 120 and 150 min after the breakfast consumption. Postprandial plasma obestatin concentrations significantly decreased compared with preprandial levels as well as total ghrelin concentrations and reached the lowest values 90 and 120 min after the meal consumption, respectively (p<0.05). Plasma acyl and desacyl ghrelin concentrations decreased after the breakfast and reached lowest values in 30 and 60 min, respectively (p<0.05). Plasma NPY concentrations were lower than preprandial levels 90 and 150 min after consuming breakfast (p<0.05). In conclusion, we demonstrated in healthy young women that plasma obestatin concentrations decrease similarly to ghrelin after a high-carbohydrate breakfast.     

Obestatin对血浆ghrelin和nesfatin-1水平及胃排空的影响

目的:探讨侧脑室注射obestatin对大鼠血浆酰基化ghrelin、去酰基化ghrelin、nesfatin-1水平的影响以及对胃排空的调控。方法:侧脑室注射obestatin,采用酶免疫测定(EIA)法检测血浆酰基化ghrelin、去酰基化ghrelin、nesfatin-1水平以及胃排空率的变化。结果:侧脑室分别注射0.1、0.3或1.0 nmol obestatin,大鼠血浆酰基化ghrelin、去酰基化ghrelin以及nesfatin-1水平无显著改变(P0.05),且酰基化ghrelin与去酰基化ghrelin比率无显著改变(P0.05);侧脑室注射obestatin,大鼠摄食量无显著改变,但胃排空率明显增加(P0.05);胃排空率明显延迟(P0.05)。与侧脑室注射1.0 nmol Obestatin组相比,注射1.0 nmol Obestatin+CRF,大鼠摄食量无显著改变,胃排空率明显延迟(P0.05)。各组摄食量及进入十二指肠内食物量无明显差异(P0.05)。结论:中枢obestatin促进大鼠的胃排空,可能与h/r CRF通路有关。     

EFFECT OF EXERCISE ON APPETITE-REGULATING HORMONES IN OVERWEIGHT WOMEN

Over the past decade, our knowledge of how homeostatic systems regulate food intake and body weight has increased with the discovery of circulating peptides such as leptin, acyl ghrelin, des-acyl ghrelin and obestatin. These hormones regulate the appetite and food intake by sending signals to the brain regarding the body''s nutritional status. The purpose of this study was to investigate the response of appetite-regulating hormones to exercise. Nine overweight women undertook two 2 h trials in a randomized crossover design. In the exercise trial, subjects ran for 60 min at 50% of maximal oxygen uptake followed by a 60 min rest period. In the control trial, subjects rested for 2 h. Obestatin, acyl ghrelin, des-acyl ghrelin and leptin concentrations were measured at baseline and at 20, 40, 60, 90 and 120 min after baseline. A two-way ANOVA revealed a significant (P < 0.05) interaction effect for leptin and acyl ghrelin. However, changes in obestatin and des-acyl ghrelin concentration were statistically insignificant (P > 0.05). The data indicated that although acute treadmill exercise resulted in a significant change in acyl ghrelin and leptin levels, it had no effect on plasma obestatin and des-acyl ghrelin levels.     

Ghrelin and obestatin can promote human ovarian granulosa cell functions and FSH effects

The aim of the present in-vitro experiments was to examine the direct influence of ghrelin and obestatin on viability, proliferation and progesterone release by human ovarian granulosa cells and their response to FSH administration. Human granulosa cells were cultured in presence of ghrelin or obestatin (both at 0, 1, 10 or 100 ng/ml) alone or in the presence of FSH (10 ng/ml). Cell viability, accumulation of proliferation markers PCNA and cyclin B1 and release of progesterone were analyzed by Trypan blue extrusion test, quantitative immunocytochemistry and ELISA. Ghrelin, obestatin and FSH up-regulated all the measured ovarian cell parameters. Moreover, both ghrelin and obestatin promoted all the stimulatory effects of FSH. The obtained results demonstrate the direct stimulatory action of ghrelin, obestatin and FSH on basic ovarian cell functions, as well as the ability of metabolic hormones to improve FSH action on human ovarian cells.     

A Natural Variant of Obestatin,Q90L,Inhibits Ghrelin's Action on Food Intake and GH Secretion and Targets NPY and GHRH Neurons in Mice

Background

Ghrelin and obestatin are two gut-derived peptides originating from the same ghrelin/obestatin prepropeptide gene (GHRL). While ghrelin stimulates growth hormone (GH) secretion and food intake and inhibits γ-aminobutyric-acid synaptic transmission onto GHRH (Growth Hormone Releasing Hormone) neurons, obestatin blocks these effects. In Humans, GHRL gene polymorphisms have been associated with pathologies linked to an unbalanced energy homeostasis. We hypothesized that one polymorphism located in the obestatin sequence (Q to L substitution in position 90 of the ghrelin/obestatin prepropeptide, rs4684677) may impact on the function of obestatin. In the present study, we tested the activity of native and Q90L obestatin to modulate ghrelin-induced food intake, GH secretion, cFos activity in GHRH and Neuropeptide Y (NPY) neurons and γ-aminobutyric-acid activity onto GHRH neurons.

Methodology/Principal findings

Food intake, GH secretion and electrophysiological recordings were assessed in C57BL/6 mice. cFos activity was measured in NPY-Renilla-GFP and GHRH-eGFP mice. Mice received saline, ghrelin or ghrelin combined to native or Q90L obestatin (30 nmol each) in the early light phase. Ghrelin stimulation of food intake and GH secretion varied considerably among individual mice with 59–77% eliciting a robust response. In these high-responders, ghrelin-induced food intake and GH secretion were reduced equally by native and Q90L obestatin. In contrast to in vivo observations, Q90L was slightly more efficient than native obestatin in inhibiting ghrelin-induced cFos activation within the hypothalamic arcuate nucleus and the nucleus tractus solitarius of the brainstem. After ghrelin injection, 26% of NPY neurons in the arcuate nucleus expressed cFos protein and this number was significantly reduced by co-administration of Q90L obestatin. Q90L was also more potent that native obestatin in reducing ghrelin-induced inhibition of γ-aminobutyric-acid synaptic transmission onto GHRH neurons.

Conclusions/Significance

These data support the hypothesis that Q90L obestatin partially blocks ghrelin-induced food intake and GH secretion by acting through NPY and GHRH neurons.     

Obestatin induction of early-response gene expression in gastrointestinal and adipose tissues and the mediatory role of G protein-coupled receptor, GPR39

Obestatin was identified as a brain/gut peptide hormone encoded by the ghrelin gene and found to interact with the G protein-coupled receptor, GPR39. We investigated target cells for obestatin based on induction of an early-response gene c-fos in different tissues. After ip injection of obestatin, c-fos staining was found in the nuclei of gastric mucosa, intestinal villi, white adipose tissues, hepatic cords, and kidney tubules. Immunohistochemical analyses using GPR39 antibodies further revealed cytoplasmic staining in these tissues. In cultured 3T3-L1 cells, treatment with obestatin, but not motilin, induced c-fos expression. In these preadipocytes, treatment with obestatin also stimulated ERK1/2 phosphorylation. Because phenotypes of GPR39 null mice are partially consistent with a role of GPR39 in mediating obestatin actions, we hypothesized that inconsistencies on the binding of iodinated obestatin to GPR39 are due to variations in the bioactivity of iodinated obestatin. We obtained monoiodoobestatin after HPLC purification and demonstrated its binding to jejunum, stomach, ileum, pituitary, and white adipose tissue. Furthermore, human embryonic kidney 293T cells transfected with plasmids encoding human or mouse GPR39 or a human GPR39 isoform, but not the ghrelin receptor, exhibited high-affinity binding to monoiodoobestatin. Binding studies using jejunum homogenates and recombinant GPR39 revealed obestatin-specific displacement curves. Furthermore, treatment with obestatin induced c-fos expression in gastric mucosa of wild-type, but not GPR39 null, mice, underscoring a mediating role of this receptor in obestatin actions. The present findings indicate that obestatin is a metabolic hormone capable of binding to GPR39 to regulate the functions of diverse gastrointestinal and adipose tissues.