Effects of ghrelin on feeding regulation and interdigestive migrating complex in rats

OBJECTIVE: Ghrelin is an orexigenic peptide with remarkable prokinetic effects. However, its mechanisms in regulating feeding and gastrointestinal migrating myoelectrical complex (MMC) are not fully understood. The aim of this study was to examine the effects of ghrelin on feeding regulation and duodenal motility in rats. MATERIAL AND METHODS: Feeding regulation was investigated at different times after intravenous injection of ghrelin and its receptor antagonist (D-Lys3)GHRP-6 in fasted rats. Rats were supplied with a pair of silver bipolar electrodes embedded in the duodenal serosa for electromyography. Ghrelin was injected intravenously into the rats during the fed state or fasted state. Some rats were pretreated with atropine, phentolamine, propranolol, L-arginine, the 5-hydroxytryptamine3 receptor antagonist ondansetron, and (D-Lys3)GHRP-6. RESULTS: Ghrelin had little effect on food intake in the first 30 min after administration, but was found to increase feeding during the subsequent hours. (D-Lys3)GHRP-6 decreased feeding and antagonized the effect of ghrelin. Ghrelin induced duodenal MMC after administration in the fed state, and shortened the duodenal MMC cycle length and the duration of phase III during fasting. The amplitude and frequency of phase III were increased without affecting the percentage of phase III in the MMC cycle. Pretreatment with atropine, L-arginine, ondansetron, and (D-Lys3)GHRP-6 inhibited the effects of ghrelin. Propranolol and phentolamine had little influence on these effects. CONCLUSIONS: Ghrelin appears to be closely related to feeding and intestinal motility. The excitatory effects of ghrelin are dependent on the cholinergic pathway, and it has a close relationship with the NOS-NO or 5-HT pathway. The ghrelin receptor is involved in its activities.     

Ghrelin stimulates phagocytosis and superoxide production in fish leukocytes

To clarify the role of ghrelin in the fish immune system, the in vitro effect of ghrelin was examined in phagocytic leukocytes of rainbow trout (Oncorhynchus mykiss). Administration of trout ghrelin and des-VRQ-trout ghrelin, in which three amino acids are deleted from trout ghrelin, increased superoxide production in zymosan-stimulated phagocytic leukocytes from the head kidney. Gene expression of growth hormone (GH) secretagogue-receptor (GHS-R) was detected by RT-PCR in leukocytes. Pretreatment of phagocytic leukocytes with a GHS-R antagonist, [D-Lys3]-GHRP-6, abolished the stimulatory effects of trout ghrelin and des-VRQ-trout ghrelin on superoxide production. Ghrelin increased mRNA levels of superoxide dismutase and GH expressed in trout phagocytic leukocytes. Immunoneutralization of GH by addition of anti-salmon GH serum to the medium blocked the stimulatory effect of ghrelin on superoxide production. These results suggest that ghrelin stimulates phagocytosis in fish leukocytes through a GHS-R-dependent pathway, and also that the effect of ghrelin is mediated, at least in part, by GH secreted by leukocytes.     

Effects of ghrelin on feeding regulation and interdigestive migrating complex in rats

Objective. Ghrelin is an orexigenic peptide with remarkable prokinetic effects. However, its mechanisms in regulating feeding and gastrointestinal migrating myoelectrical complex (MMC) are not fully understood. The aim of this study was to examine the effects of ghrelin on feeding regulation and duodenal motility in rats. Material and methods. Feeding regulation was investigated at different times after intravenous injection of ghrelin and its receptor antagonist (D-Lys3)GHRP-6 in fasted rats. Rats were supplied with a pair of silver bipolar electrodes embedded in the duodenal serosa for electromyography. Ghrelin was injected intravenously into the rats during the fed state or fasted state. Some rats were pretreated with atropine, phentolamine, propranolol, L-arginine, the 5-hydroxytryptamine3 receptor antagonist ondansetron, and (D-Lys3)GHRP-6. Results. Ghrelin had little effect on food intake in the first 30 min after administration, but was found to increase feeding during the subsequent hours. (D-Lys3)GHRP-6 decreased feeding and antagonized the effect of ghrelin. Ghrelin induced duodenal MMC after administration in the fed state, and shortened the duodenal MMC cycle length and the duration of phase III during fasting. The amplitude and frequency of phase III were increased without affecting the percentage of phase III in the MMC cycle. Pretreatment with atropine, L-arginine, ondansetron, and (D-Lys3)GHRP-6 inhibited the effects of ghrelin. Propranolol and phentolamine had little influence on these effects. Conclusions. Ghrelin appears to be closely related to feeding and intestinal motility. The excitatory effects of ghrelin are dependent on the cholinergic pathway, and it has a close relationship with the NOS-NO or 5-HT pathway. The grehlin receptor is involved in its activities.     

Role of endogenous ghrelin in the hyperphagia of mice with streptozotocin-induced diabetes

Ghrelin is an orexigenic peptide involved in the regulation of energy homeostasis. To investigate the role of ghrelin in the hyperphagia associated with uncontrolled streptozotocin-induced diabetes, food intake was followed in diabetic ghrelin knockout (ghrelin(-/-)) and control wild-type (ghrelin(+/+)) mice and diabetic Naval Medical Research Institute noninbred Swiss mice treated with either saline or the ghrelin receptor antagonist, D-Lys3-GH-releasing peptide-6 (D-Lys3-GHRP-6) for 5 d. In diabetic ghrelin(-/-) mice, hyperphagia was attenuated, and the maximal increase in food intake was 50% lower in mutant than in wild-type mice. The increased food intake observed during the light period (1000-1200 h) in ghrelin(+/+) mice was abolished in mutant mice. Diabetic ghrelin(-/-) mice lost 12.4% more body weight than ghrelin(+/+) mice. In diabetic ghrelin(+/+) mice, but not in ghrelin(-/-) mice, the number of neuropeptide Y (NPY)-immunoreactive neurons was significantly increased. Diabetic Naval Medical Research Institute noninbred Swiss mice were hyperphagic and had increased plasma ghrelin levels. Treatment with D-Lys3-GHRP-6 reduced daily food intake by 23% and reversed the increased food intake observed during the light period. The change in the number of NPY- (2.4-fold increase) and alpha-MSH (1.7-fold decrease)-immunoreactive hypothalamic neurons induced by diabetes was normalized by D-Lys3-GHRP-6 treatment. Our results suggest that enhanced NPY and reduced alpha-MSH expression are secondary to the release of ghrelin, which should be considered the underlying trigger of hyperphagia associated with uncontrolled diabetes.     

Effects of the thyroid hormone derivatives 3-iodothyronamine and thyronamine on rat liver oxidative capacity

It was demonstrated that estrogen deficiency and consuming high fat (HF) diet enhanced orexigenic activity of ghrelin. Therefore, we hypothesized that antagonizing of ghrelin action would attenuate food intake and body weight in mice obese both from ovariectomy (OVX) and feeding a HF diet. Ghrelin receptor antagonist [D-Lys(3)]GHRP-6 after seven days of subcutaneous treatment markedly decreased food intake in OVX mice fed both HF and standard diets; furthermore, it reduced body weight and blood glucose, insulin and leptin, and increased β-hydroxybutyrate level and uncoupling-protein-1 mRNA in brown adipose tissue. Pair-feeding revealed that effect of [D-Lys(3)]GHRP-6 was primary anorexigenic. Estrogen supplementation reduced anorexigenic effects of [D-Lys(3)]GHRP-6. OVX [D-Lys(3)]GHRP-6 treatment in mice on HF diet resulted in markedly increased circulating level and liver expression of a major metabolic regulator, fibroblast growth factor 21. Our data suggest that ghrelin antagonists could be especially beneficial in individuals with common obesity combined with estrogen deficiency.     

Effects of ghrelin on gastric distension sensitive neurons and gastric motility in the lateral septum and arcuate nucleus regulation

Background

Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R) and a peptide hormone that promotes food intake and gastric motility. Our aims are to explore the effects of ghrelin on gastric distension (GD) sensitive neurons in the lateral septum, and the possible regulation of gastric motility by ghrelin through the hypothalamic arcuate nucleus (ARC).

Methods

Single-unit discharges were recorded, extracellularly, and the gastric motility was monitored by the administration of ghrelin in the lateral septum. The projection of nerve fiber and expression of ghrelin were observed by retrograde tracer and fluo-immunohistochemistry staining. The expression of GHS-R and ghrelin was determined by real-time polymerase chain reaction and western blotting analysis.

Results

There were GD neurons in the lateral septum. The administration of ghrelin could excite both GD-excitatory (GD-E) and GD-inhibitory (GD-I) neurons in the lateral septum. Gastric motility was significantly enhanced by the administration of ghrelin in the lateral septum in a dose-dependent manner. Pretreatment with [d-Lys-3]-GHRP-6, however, could completely abolish the ghrelin-induced effects. Electrical stimulation of the ARC could significantly excite the response of GD neurons to ghrelin, increase ghrelin protein expression in the lateral septum and promote gastric motility. Nevertheless, these effects could be mitigated by pretreatment of [d-Lys-3]-GHRP-6. Electrical lesion of the lateral septum resulted in decreased gastric motility. The GHS-R and Ghrelin/FG-double labeled neurons were observed in the lateral septum and ARC, respectively.

Conclusions

It is suggested that the lateral septum may receive afferent information from the gastrointestinal tract and promote gastric motility. Ghrelin plays an important role in promoting gastric motility in the lateral septum. The ARC may be involved in the regulation of the lateral septum’s influence on gastric motility.     

Effect of inhibitor and activator of ghrelin receptor (GHS-R1a) on porcine ovarian granulosa cell functions

It was previously shown, that ghrelin and its agonistic analogue, ghrelin 1-18, can be a stimulator of ovarian cell functions (promoter of proliferation, inhibitor of apoptosis and stimulator of hormones release). The aim of our studies was to compare the action of two ghrelin analogues - ghrelin 1-18, activator of ghrelin receptors (GHS-R1a), and (d-Lys3)-GHRP-6, its inhibitor, on porcine ovarian granulosa cell functions. Effects of (d-Lys3)-GHRP-6 added at doses of 0, 1, 10 or 100 ng/ml on the expression of markers of proliferation (PCNA, cyclin B1, MAPK/ERK1,2), apoptosis (bax, p53, caspase 3) and release of steroid hormones (progesterone, testosterone, estradiol) were examined. In addition, some effect of ghrelin 1-8 on some of these parameters (expression of MAPK/ERK1,2, bax, p53) were verified. It was shown, that (d-Lys3)-GHRP-6 promotes all markers of granulosa cell proliferation, inhibits all markers of apoptosis and stimulates the release of all three steroid hormones. Similar effects of (d-Lys3)-GHRP-6 (inhibitor of GHS-R1a) and ghrelin 1-18 (its stimulator) suggest that the examined effects of these substances on porcine ovaries are not mediated by GHS-R1a. Both chemical analogues could be potentially useful for stimulation of reproductive processes, at least in in vitro conditions.     

Expression and homologous regulation of GH secretagogue receptor mRNA in rat adrenal gland

OBJECTIVE: GH secretagogues (GHSs) elicit a variety of biological effects in several endocrine and non-endocrine target tIssues, including activation of the hypothalamic-pituitary-adrenal axis. The latter is mainly carried out through a central hypothalamic action; yet the possibility of additional effects directly at the adrenal level cannot be ruled out. The aims of this study were to evaluate the expression and homologous regulation of the GHS-receptor (GHS-R) gene in rat adrenal and to assess the effects of synthetic (GH releasing peptide-6 - GHRP-6) and natural (ghrelin) ligands of GHS-R upon basal and ACTH-stimulated corticosterone secretion in vitro. DESIGN AND METHODS: Analysis of adrenal expression of target mRNAs (GHS-R, GHS-R1a, ghrelin, and several steroidogenic factors) was conducted by means of primer-specific, semi-quantitative RT-PCR. Evaluation of corticosterone secretion by incubated adrenal tIssue was carried out by specific RIA. RESULTS: RT-PCR analysis demonstrated expression of the GHS-R gene, but not of the gene encoding the cognate ligand ghrelin, in rat adrenal. Moreover, expression of the mRNA coding for the type 1a GHS-R (GHS-R1a), i.e. the biologically active receptor form, was demonstrated. The adrenal expression of the GHS-R message appeared under the regulation of homologous signals in vitro, as short-term incubation of adrenal samples in serum-free medium induced a significant increase in GHS-R mRNA levels that was inhibited by exposure to different doses of GHRP-6 (10(-9)-10(-5) mol/l) or ghrelin (10(-7) mol/l). Notably, an opposite pattern of homologous regulation of GHS-R gene expression was observed at the pituitary. Finally, short-term stimulation with increasing concentrations of GHRP-6 (10(-9)-10(-5) mol/l) or ghrelin (10(-7) mol/l) failed to alter basal and ACTH-stimulated corticosterone secretion in vitro, neither did it modify ACTH-stimulated mRNA expression levels of several upstream elements in the steroidogenic route: the steroidogenic acute regulatory (StAR) protein, and the enzymes P450 cholesterol side-chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). CONCLUSIONS: Our study provides novel evidence for the expression and homologous regulation of the GHS-R gene in rat adrenal. However, our results cast doubts on the possibility of direct adrenal actions of ligands of the GHS-R in the regulation of corticosterone secretion in the rat.     

Antagonism of ghrelin receptor reduces food intake and body weight gain in mice

BACKGROUND AND AIMS: Ghrelin, an endogenous ligand for growth hormone secretagogue receptor (GHS-R), is an appetite stimulatory signal from the stomach with structural resemblance to motilin. We examined the effects of the gastric peptide ghrelin and GHS-R antagonists on energy balance and glycaemic control in mice. MATERIALS AND METHODS: Body weight, fat mass, glucose, insulin, and gene expression of leptin, adiponectin, and resistin in white adipose tissue (WAT) were measured after repeated administrations of ghrelin under a high fat diet. Gastric ghrelin gene expression was assessed by northern blot analysis. Energy intake and gastric emptying were measured after administration of GHS-R antagonists. Repeated administration of GHS-R antagonist was continued for six days in ob/ob obese mice. RESULTS: Ghrelin induced remarkable adiposity and worsened glycaemic control under a high fat diet. Pair feeding inhibited this effect. Ghrelin elevated leptin mRNA expression and reduced resistin mRNA expression. Gastric ghrelin mRNA expression during fasting was increased by a high fat diet. GHS-R antagonists decreased energy intake in lean mice, in mice with diet induced obesity, and in ob/ob obese mice; it also reduced the rate of gastric emptying. Repeated administration of GHS-R antagonist decreased body weight gain and improved glycaemic control in ob/ob obese mice. CONCLUSIONS: Ghrelin appears to be closely related to excess weight gain, adiposity, and insulin resistance, particularly under a high fat diet and in the dynamic stage. Gastric peptide ghrelin and GHS-R may be promising therapeutic targets not only for anorexia-cachexia but also for obesity and type 2 diabetes, which are becoming increasingly prevalent worldwide.     

Toward office-based measurement of gastric emptying in symptomatic diabetics using [13C]octanoic acid breath test

OBJECTIVE: Current methods for measuring gastric emptying by breath test require sampling over several hours and are too inaccurate for clinical use. The aim of this study was to develop an office-based method for measuring gastric emptying of solids in patients with diabetes using a [13C]octanoic acid breath test. METHODS: In 22 symptomatic diabetic patients (17 insulin-dependent diabetes, 5 non-insulin-dependent diabetes) and 6 controls, we simultaneously measured gastric emptying of an egg meal (420 kcal) by scintigraphy and [13C]octanoic acid breath test. Conventional (nonlinear) methods for scintigraphic and [13C]octanoic acid breath test emptying and generalized linear regression method to predict scintigraphic half-life (t(1/2)) using four breath samples obtained during the first 3 h. RESULTS: Despite 8 h of breath sampling, the t(1/2) estimate using the conventional method was markedly different from the scintigraphic value (delta t(1/2): median, 113 min; range, 19-282 min). The generalized linear model (using samples at baseline, 30, and 120 or 150 min) yielded predicted scintigraphic tLAG and t(1/2) that were more accurate than the conventional method; mean standard deviations of differences were 16 and 27 min, respectively. Breath test correctly assessed normal or prolonged emptying in 21 of 22 patients. CONCLUSIONS: The [13C]octanoic acid breath test can be simplified to measure gastric tLAG and t(1/2) and can be expected to correctly identify normal t(1/2) in symptomatic diabetics. Further refinement of the model will need to include studies of patients with markedly delayed t(1/2).     

Decreased GH secretion and enhanced ACTH and cortisol release after ghrelin administration in Cushing’s disease: Comparison with GH-releasing peptide-6 (GHRP-6) and GHRH

GH responsiveness to GH secretagogues (GHS) is blunted in Cushing’s disease (CD), while ACTH/cortisol responses are enhanced, by mechanisms still unclear. Ghrelin, the endogenous ligand for GHS-receptors (GHS-R), increases GH, ACTH, cortisol and glucose levels in humans. This study evaluated the GH, ACTH, cortisol and glucose-releasing effects of ghrelin in CD in comparison with GHRP-6. GHRH-induced GH release was also studied. Ten patients with CD (BMI 26.9 ± 1.0 kg/m²) and ten controls (BMI 24.4 ± 1.1 kg/m²) received ghrelin (1 μg/kg), GHRP-6 (1 μg/kg) and GHRH (100 μg) separately. GH, ACTH, cortisol and glucose levels were measured. In CD ghrelin-induced GH (μg/L; mean ± SE) release (peak: 7.2 ± 3.0) was higher than seen with GHRP-6 (2.7 ± 1.0) and GHRH (0.7 ± 0.2), but lower than in controls (ghrelin: 58.3 ± 12.1; GHRP-6: 22.9 ± 4.8; GHRH: 11.3 ± 3.7). In controls ACTH (pg/mL) release after ghrelin (79.2 ± 26.8) was higher than after GHRP-6 (23.6 ± 5.7). In CD these responses (ghrelin: 192 ± 43; GHRP-6: 185 ± 56) were similar, and enhanced compared to controls. The same was observed with cortisol. Glucose levels failed to increase after ghrelin in CD, differently than in controls. Our data suggests that hypothalamic and pituitary pathways of GH release activated by ghrelin, GHRP-6 and GHRH are deranged in chronic hypercortisolism. The increased ACTH/cortisol responses to ghrelin and GHRP-6 in CD could be mediated by overexpression of GHS-R in ACTH-secreting adenomas. Hypercortisolism apparently impairs the ability of ghrelin to increase glucose levels.     

Pharmacological modulation of gastric emptying rate of solids as measured by the carbon labelled octanoic acid breath test: influence of erythromycin and propantheline.

The *C (13C or 14C) labelled octanoic acid breath test was recently developed to measure the gastric emptying rate of solids. This study aimed to investigate whether it is sensitive enough to detect pharmacologically induced changes in the gastric emptying rate. Nine healthy volunteers were studied in basal condition, after intravenous administration of 200 mg erythromycin, and after peroral administration of 30 mg propantheline. Erythromycin significantly enhanced gastric emptying in all subjects, with an increase of the gastric emptying coefficient (p = 0.0043) in eight of nine and a fall in both the gastric half emptying time (p = 0.0020) and the lag phase (p = 0.0044) in all nine. Propantheline significantly reduced the gastric emptying rate, with a decreased gastric emptying coefficient (p = 0.0007) and an increased gastric half emptying time (p = 0.0168) in all subjects, but no change in the lag phase (p = 0.1214). Further mathematical analysis showed that breath sampling at 15 minutes intervals over a four hour period is recommended to guarantee accuracy and the discriminative value of the breath test in various gastric emptying patterns. In conclusion the *C labelled octanoic acid breath test is sufficiently sensitive to show pharmacologically induced changes of gastric emptying rates of solids.     

Prokinetic effects of a ghrelin receptor agonist GHRP-6 in diabetic mice

AIM： To investigate the effects of a ghrelin receptor agonist GHRP-6 on delayed gastrointestinal transit in alloxan-induced diabetic mice. METHODS： A diabetic mouse model was established by intraperitoneal injection with alloxan. Mice were randomized into two main groups： normal mice and diabetic mice treated with GHRP-6 at doses of 0, 20, 50, 100 and 200 μg/kg ip. Gastric emptying （GE）, intestinal transit （IT）, and colonic transit （CT） were studied in mice after they had a phenol red meal following injection of GHRP-6. Based on the most effective GHRP-6 dosage, atropine was given at 1 mg/kg for 15 rain before the GHRP-6 injection for each measurement. The mice in each group were sacrificed 20 min later and the percentages of GE, IT, and CT were calculated. RESULTS： Percentages of GE, IT, and CT were significantly decreased in diabetic mice as compared to control mice. In the diabetic mice, GHRP-6 improved both GE and IT, but not CT. The most effective dose of GHRP-6 was 200 μg/kg and atropine blocked the prokinetic effects of GHRP-6 on GE and IT. CONCLUSION： GHRP-6 accelerates delayed GE and IT, but has no effect on CT in diabetic mice. GHRP-6 may exert its prokinetic effects via the cholinergic pathway in the enteric nervous system, and therefore, has therapeutic potential for diabetic patients with delayed upper gastrointestinal transit.     

Stimulatory effect of ghrelin on isolated porcine somatotropes

Research on the mechanism for growth hormone secretagogue (GHS) induction of growth hormone secretion led to the discovery of the GHS receptor (GHS-R) and later to ghrelin, an endogenous ligand for GHS-R. The ability of ghrelin to induce an increase in the intracellular Ca(2+) concentration - [Ca(2+)](i) - in somatotropes was examined in dispersed porcine pituitary cells using a calcium imaging system. Somatotropes were functionally identified by application of human growth hormone releasing hormone. Ghrelin increased the [Ca(2+)](i) in a dose-dependent manner in 98% of the cells that responded to human growth hormone releasing hormone. In the presence of (D-Lys(3))-GHRP-6, a specific receptor antagonist of GHS-R, the increase in [Ca(2+)](i) evoked by ghrelin was decreased. Pretreatment of cultures with somatostatin or neuropeptide Y reduced the ghrelin-induced increase of [Ca(2+)](i). The stimulatory effect of ghrelin on somatotropes was greatly attenuated in low-calcium saline and blocked by nifedipine, an L-type calcium channel blocker, suggesting involvement of calcium channels. In a zero Na(+) solution, the stimulatory effect of ghrelin on somatotropes was decreased, suggesting that besides calcium channels, sodium channels are also involved in ghrelin-induced calcium transients. Either SQ-22536, an adenylyl cyclase inhibitor, or U73122, a phospholipase C inhibitor, decreased the stimulatory effects of ghrelin on [Ca(2+)](i) transiently, indicating the involvement of adenylyl cyclase-cyclic adenosine monophosphate and phospholipase C inositol 1,4,5-trisphosphate pathways. The nonpeptidyl GHS, L-692,585 (L-585), induced changes in [Ca(2+)](i) similar to those observed with ghrelin. Application of L-585 after ghrelin did not have additive effects on [Ca(2+)](i). Preapplication of L-585 blocked the stimulatory effect of ghrelin on somatotropes. Simultaneous application of ghrelin and L-585 did not cause an additive increase in [Ca(2+)](i). Our results suggest that the actions of ghrelin and synthetic GHS closely parallel each other, in a manner that is consistent with an increase of hormone secretion.     

Ghrelin在离体大鼠胰岛中抑制胰岛素分泌和上调Kir6．2表达

目的明确ghrelin在离体大鼠胰岛中对胰岛素分泌和内向整流钾通道（Kit6．2）表达的影响,并讨论两者的关系。方法离体大鼠胰岛以高浓度葡萄糖及不同浓度ghrelin和（或）其受体拮抗剂ED-lys^3]-GHRP-6孵育1h,采用放免法测定上清液的胰岛素,采用RT-PCR检测Kir6．2、磺酰脲受体1（SUR-1）、解偶联蛋白2（UCP-2）、葡萄糖转运子2（GluT-2）、胰十二指肠同源盒1（PDX-1）等基因的表达。结果10。～10“mol／Lghre｜in呈剂量依赖性抑制离体大鼠胰岛高浓度葡萄糖刺激的胰岛素释放,且剂量依赖性增加Kir6．2mRNA表达,但对SUR-1、UCP-2、GluT-2及PDX-1 mRNA表达则无显著影响。ED-lys^3]-GHRP-6可消除ghrelin对Kir6．2 mRNA表达的上调作用。结论在离体大鼠胰岛中,ghrelin通过作用于其受体,促进ATP敏感性钾通道组成成分Kir6．2的表达,改变钾通道功能状态。这可能是ghrelin抑制葡萄糖刺激的胰岛素分泌的机制之一。     

Small-molecule ghrelin receptor antagonists improve glucose tolerance, suppress appetite, and promote weight loss

Ghrelin, through action on its receptor, GH secretagogue receptor type 1a (GHS-R1a), exerts a variety of metabolic functions including stimulation of appetite and weight gain and suppression of insulin secretion. In the present study, we examined the effects of novel small-molecule GHS-R1a antagonists on insulin secretion, glucose tolerance, and weight loss. Ghrelin dose-dependently suppressed insulin secretion from dispersed rat islets. This effect was fully blocked by a GHS-R1a antagonist. Consistent with this observation, a single oral dose of a GHS-R1a antagonist improved glucose homeostasis in an ip glucose tolerance test in rat. Improvement in glucose tolerance was attributed to increased insulin secretion. Daily oral administration of a GHS-R1a antagonist to diet-induced obese mice led to reduced food intake and weight loss (up to 15%) due to selective loss of fat mass. Pair-feeding experiments indicated that weight loss was largely a consequence of reduced food intake. The impact of a GHS-R1a antagonist on gastric emptying was also examined. Although the GHS-R1a antagonist modestly delayed gastric emptying at the highest dose tested (10 mg/kg), delayed gastric emptying does not appear to be a requirement for weight loss because lower doses produced weight loss without an effect on gastric emptying. Consistent with the hypothesis that ghrelin regulates feeding centrally, the anorexigenic effects of potent GHS-R1a antagonists in mice appeared to correspond with their brain exposure. These observations demonstrate that GHS-R1a antagonists have the potential to improve the diabetic condition by promoting glucose-dependent insulin secretion and promoting weight loss.     

Gastric emptying in diabetic patients by the 13C-octanoic acid breath test: role of insulin in gastric motility

Background Impairment of gastric emptying is well recognized in patients with diabetes mellitus (DM), especially long-standing insulin-dependent diabetes mellitus (IDDM). The aim of this study was to evaluate the cause of delayed gastric emptying in DM patients. Methods In 16 controls, 16 non-insulin-dependent diabetes mellitus (NIDDM) patients and 23 IDDM patients, gastric emptying was studied using the ¹³C octanoic acid breath test. Breath samples were taken before a test meal labeled with 100 mg of ¹³C octanoic acid, and at 15-min intervals over a 300-min period postprandially. Results In all DM patients, the gastric emptying coefficient was lower than that in the controls (P < 0.05), and lag time and half-emptying time were significantly longer (P < 0.05). Both NIDDM and IDDM patients showed delayed ¹³CO₂ excretion compared with the controls, but IDDM patients showed more delayed gastric emptying than NIDDM patients (P < 0.05). There were no significant differences in sex, HbA1c level, or the rate of neuropathy between the two groups. Conclusions IDDM patients showed delayed gastric emptying compared with NIDDM patients, and the ¹³C octanoic acid breath test is useful for evaluating DM patients with delayed gastric emptying.     

Role of ghrelin in the control of growth hormone secretion in prepubertal rats: interactions with excitatory amino acids

Ghrelin is a 28-amino-acid peptide, with an essential n-octanoyl modification at Ser3, that elicits growth-hormone (GH) secretion in rats and humans. At present, the mechanisms of ghrelin action and its interactions with other systems controlling GH secretion remain poorly characterized. In this context, the present study was undertaken to obtain information about ontogeny and possible gender differences in the GH-releasing activity of ghrelin, and to delineate its primary site(s) of action at the hypothalamus and/or pituitary. In addition, the interactions between ghrelin and other relevant signals in the control of GH secretion, such as excitatory amino acids (EAAs), nitric oxide (NO) and serotonin, were assessed. Experiments were carried out in infantile-prepubertal animals, when GH pulsatility is not yet established. Systemic administration of ghrelin (25 nmol/rat, i.p.) to 5-, 10- and 23-day-old male and female rats increased plasma GH levels from day 10 onwards. This action was NO dependent, since it disappeared in 23-day-old males after pretreatment with an inhibitor of NO synthase (NAME). Similarly, central infusion of ghrelin (3 nmol/rat, i.c.v.) elicited GH responses in 10- and 23-day-old animals significantly higher than after systemic administration. By contrast, in vitro challenge of pituitary tissue with increasing doses of ghrelin (10(-9)-10(-7) M) failed to enhance GH release into the incubation medium, whereas stimulation with GH-releasing hormone (GHRH; 10(-7) M) or GHRP-6 (10(-7) M) was effective. Finally, effects of ghrelin were blocked by pretreatment with MK-801 and NBQX antagonists of EAA ionotropic receptors and after manipulation of endogenous serotoninergic tone. In addition, the potent releasing activity of EAA agonists NMDA and AMPA was blunted by pretreatment with D-Lys3-GHRP-6, a selective antagonist of the cognate ghrelin receptor, i.e. the GH-secretagogue receptor. In conclusion, our results demonstrate that GH-releasing activity of ghrelin appears early in the infantile period, is NO dependent and involves a primary hypothalamic site of action. The data also demonstrate for the first time the existence of a cross-talk between ghrelin and other neurotransmitter systems, such as EAAs and serotonin, in precise control of GH secretion.     

Effect of atropine sulfate on gastric emptying and gastrocecal transit time evaluated by using the [1-(13)C]acetic acid and lactose-[(13)C]ureide breath test in conscious rats

Breath tests have been used to investigate many physiological functions. Recently, we have developed a breath test system by using a non-invasive technique in conscious rats. In this study, we investigated the effect of atropine sulfate on the gastric emptying and gastrocecal transit time using [1-(13)C]acetic acid and lactose-[(13)C]ureide, respectively. Gastric emptying was significantly delayed by atropine sulfate in a dose-dependent manner (0.03-0.3 mg kg(-1)), but the effects of 0.1 and 0.3 mg kg(-1) were almost equal. C(max) and T(max) values were also significantly and dose-dependently decreased and delayed, respectively. However, AUC(120 min) values were not significantly different for the three doses of atropine sulfate. This suggests that although atropine sulfate slows the emptying of the gastric content, it is excreted eventually to the same content as the control. The gastrocecal transit time was significantly delayed by atropine sulfate at a dose of 1 mg kg(-1), but not at a dose of 0.1 mg kg(-1). These findings demonstrate that the breath tests can evaluate the effect of atropine sulfate on the gastric emptying and gastrocecal transit time. The effective dose of atropine sulfate may be different for gastric emptying and gastrocecal transit time.