A White Grape Juice Extract Reduces Fat Accumulation through the Modulation of Ghrelin and Leptin Expression in an In Vivo Model of Overfed Zebrafish.

A caloric surplus and a sedentary lifestyle are undoubtedly known to be the leading causes of obesity. Natural products represent valuable allies to face this problematic issue. This study was planned to assess the effect of a white grape (Vitis vinifera) juice extract (WGJe) in diet-induced obese zebrafish (Danio rerio). Fish were divided into four different diet groups: (i) normally fed (NF); (ii) overfed (OF); (iii) WGJe-supplemented NF (5 mL/L in fish water); (iv) WGJe-supplemented OF. Body mass index (BMI) was extrapolated each week. After the fourth week, euthanized zebrafish were processed for both microscopic evaluations and gene expression analyses. OF zebrafish showed higher BMI values with respect to NF counterparts, an effect that was hindered by WGJe treatment. Moreover, histological analyses showed that the area of the adipose tissue, as well as the number, size, and density of adipocytes was significantly higher in OF fish. On the other hand, WGJe was able to avoid these outcomes both at the subcutaneous and visceral levels, albeit to different extents. At the gene level, WGJe restored the altered levels of ghrelin and leptin of OF fish both in gut and brain. Overall, our results support the anti-obesity property of WGJe, suggesting its potential role in weight management.

Ghrelin Affects Gastric Cancer Progression by Activating AMPK Signaling Pathway.

As the endogenous ligand for the GH secretagogue receptor (GHSR), Ghrelin is aberrant expressed in multiple malignant carcinoma, and involved in regulating a number of progression of cancer, especially in metastasis and proliferation. However, the precise role of Ghrelin in tumorigenesis of gastric cancer (GC) is still poorly understood. In this study, we extensively investigated the roles and mechanisms of Ghrelin in human gastric cancer. Ghrelin levels in cancer tissues and cell lines were analyzed by immunohistochemistry, qRT-PCR, and Western blot. Functional studies were performed after Ghrelin overexpressed or knockdown in AGS cell line. Cell proliferation was evaluated in by MTT and clone formation assays. The wound healing and Transwell system were used to assess the cell migration and invasive ability of GC cells. Cell apoptosis was detected by flow cytometry, and metabolic assays were performed to reveal the function of Warburg effect in the process. Ghrelin was lowly expressed in gastric cancer tissues and cell lines. Overexpression of Ghrelin inhibited gastric cancer cell proliferation, migration, invasion, and promoted apoptosis by activating the AMPK pathway, while D-[lys3]-GHRP-6 (a GHSR agonist) treatment relieved the effect, promoting tumorigenesis. Ghrelin knockdown increased the glucose uptake and lactic acid release, suggesting that Ghrelin elicited an anti-Warburg effect via AMPK pathway to inhibit gastric tumorigenesis. Ghrelin inhibits cell proliferation, migration, and invasion by eliciting an anti-Warburg effect via AMPK signaling pathway in gastric cancer cells.

The Role of Malnutrition and Muscle Wasting in Advanced Lung Cancer.

PURPOSE OF REVIEW: Malnutrition, cancer cachexia, and sarcopenia often co-occur in patients with advanced cancer and are associated with poorer response to chemotherapy and reduced survival. Here, we evaluate the current literature regarding the role of nutrition and these associated conditions in patients with advanced lung cancer. RECENT FINDINGS: While rates of malnutrition are high, nutritional intervention studies have generally been limited by small sample sizes. Novel strategies such as home-based meal delivery may have promise. While no therapy is approved for cancer cachexia, ghrelin agonists and other targeted therapies have yielded promising data in clinical trials. Recent data also suggest that obesity may improve immunotherapy responsiveness. Malnutrition and associated muscle wasting are clearly negative prognostic markers in advanced lung cancer. Patients with malnutrition should be urgently referred for dietary counseling and guidelines for nutritional support should be followed. Optimal treatment of these syndromes will likely include nutrition and anti-cachexia interventions used in combination.

Estradiol Replacement Improves High-Fat Diet-Induced Obesity by Suppressing the Action of Ghrelin in Ovariectomized Rats.

This study aims to investigate the effects of estradiol replacement on the orexigenic action of ghrelin in ovariectomized (OVX) obese rats fed with a high-fat diet (HFD). Four weeks after OVX at 9 weeks of age, Wistar rats were subcutaneously implanted with either 17ß-estradiol (E2) or placebo (Pla) pellets and started on HFD feeding. After 4 weeks, growth hormone-releasing peptide (GHRP)-6, a growth hormone secretagogue receptor (GHSR) agonist injected intraperitoneally, induced changes in HFD intake, and c-Fos-positive neurons in the hypothalamic arcuate nucleus (ARC) were measured in both groups. The ghrelin protein and mRNA levels, as well as GHSR protein in stomach, were analyzed by Western blotting and real-time PCR. HFD increased energy intake and body weight in the Pla group, while it temporarily reduced these in the E2 group. GHRP-6 enhanced HFD intake and activated neurons in the ARC only in the Pla group. Furthermore, gastric ghrelin and GHSR protein levels were lower in the E2 group than in the Pla group, but plasma acyl ghrelin levels were similar in both groups. Our results suggest that E2 replacement improves obesity by inhibiting the orexigenic action of ghrelin via downregulation of ghrelin and its receptor in stomach in HFD-fed OVX rats.

Ghrelin Fights Against Titanium Particle-Induced Inflammatory Osteolysis Through Activation of ß-Catenin Signaling Pathway.

Aseptic loosening is a major complication of prosthetic joint surgery, in which exaggerated inflammation and impaired osteoblastogenesis are detected. Ghrelin is a recently discovered neuropeptide that is closely associated with inflammatory conditions and bone regeneration. Here, we report that titanium particles inhibited ghrelin expression in MC3T3-E1 cells. Furthermore, exogenous ghrelin effectively inhibited titanium particle-induced inflammation in vitro by interacting with its receptor GHSR1a; as an inhibitor of GHSR1a, Dlys repressed the function of ghrelin. Moreover, ghrelin attenuated the impairment of osteoblastogenesis and the exaggeration of osteolysis induced by titanium particles. Furthermore, the protective role of ghrelin in aseptic loosening might be associated with the Wnt/ß-catenin signaling pathway. Collectively, these findings suggest that ghrelin might be a potential therapeutic target for wear-debris-induced inflammation and osteolysis.

Prokinetics and ghrelin for the management of cancer cachexia syndrome.

Cancer cachexia (CC) is one of the most distressing syndromes for both patients and their families. CC can have an impact on patient reported quality of life and overall survival. It is often associated with symptoms such as fatigue, depressed mood, early satiety, and anorexia. Prokinetic agents have been found to improve chronic nausea and early satiety associated with CC. Among the prokinetic agents, metoclopramide is one of the best studied medications. The role of the other prokinetic agents, such as domperidone, erythromycin, haloperidol, levosulpiride, tegaserod, cisapride, mosapride, renzapride, and prucalopride is unclear for use in cachectic cancer patients due to their side effect profile and limited efficacy studies in cancer patients. There has been an increased interest in the use of ghrelin-receptor agonists for the treatment of CC. Anamorelin HCl is a highly selective, novel ghrelin receptor agonist. A meta-analysis was conducted of the recent randomized trials using anamorelin (daily dose of 50 and 100 mg daily). Results show that both total body weight and lean body mass were significantly increased from baseline in the anamorelin group. Anamorelin did not improve overall survival or hand grip strength, and there were no significant differences between groups for frequency or severity of any adverse events. In this review, the authors discuss the available evidence for the use of prokinetics such as metoclopramide and ghrelin receptor agonists for the treatment of CC.

Peptide mimetic of N-terminal ghrelin enhances ghrelin-induced growth hormone secretion and c-Fos expression in mice.

Orexigenic peptide ghrelin and its receptor have been extensively investigated as potential therapeutic targets, primarily because of their role in feeding initiation and growth hormone (GH) release. However, no specific ghrelin targeting anti-obesity or cachexia therapeutics are available for clinical use thus far and further efforts in this direction are warranted. The present study aimed to find new peptide drug leads modulating ghrelin signal transduction. By targeting neutralising antibodies against ghrelin with phage display libraries, we aimed to identify peptides binding to the cognate receptor. Four synthetic peptides were selected and tested using calcium screening assays. The most effective competitive antagonist FSFLPPE was further tested in vivo. Administration of the peptide produced no significant effect on either food intake or GH release. Surprisingly, when co-administered with ghrelin, the peptide significantly enhanced GH secretion and c-Fos expression. The evidence obtained in the present study indicates that FSFLPPE might act as an ago-allosteric modulator.

Hydrogen sulfide suppresses ghrelin secretion in vitro and delays postprandial ghrelin secretion while reducing appetite in mice.

Ghrelin is a stomach-derived hormone that regulates several metabolic functions including growth hormone release, appetite, adiposity, and gastric motility. Nutrients, the autonomic nervous system, and other metabolic hormones have all been implicated in the regulation of ghrelin secretion. Despite this, ongoing efforts to develop modulators of ghrelin secretion in human diseases are still underway. Hydrogen sulfide (H2 S) is a gaseous signaling molecule that is produced both endogenously in many tissues and by the gut microbiome. H2 S has established roles in cardiovascular and immune health, however, more recently H2 S has been implicated in the regulation of metabolic hormone secretion. We hypothesized that H2 S is able to directly regulate ghrelin secretion and in turn, regulate appetite. We first demonstrated that GYY4137 (an H2 S donor molecule) directly suppresses ghrelin secretion in rat primary gastric culture, in part through the activation of the protein kinase B (AKT) pathway. We then demonstrated the colocalization of ghrelin-positive gastric cells with the H2 S producing enzyme cystathionine-γ-lyase (CSE). While GYY4137 suppressed ghrelin secretion, inhibition of CSE caused a stimulation in ghrelin secretion in primary gastric culture. In mice, GYY4137 treatment prolonged the postprandial drop of circulating ghrelin and caused reduced food consumption up to 4 h after treatment. These results demonstrate for the first time a role for H2 S in the regulation of ghrelin and appetite. Modulating H2 S levels may be a novel approach to regulate ghrelin secretion in the treatment of metabolic diseases.

TNF-α inhibits SCF, ghrelin, and substance P expressions through the NF-κB pathway activation in interstitial cells of Cajal.

Ulcerative colitis is a chronic inflammatory disease of the colon where intestinal motility is disturbed. Interstitial cells of Cajal (ICC) are required to maintain normal intestinal motility. In the present study, we assessed the effect of tumor necrosis factor-alpha (TNF-α) on viability and apoptosis of ICC, as well as on the expression of stem cell factor (SCF), ghrelin, and substance P. ICC were derived from the small intestines of Swiss albino mice. Cell viability and apoptosis were measured using CCK-8 assay and flow cytometry, respectively. ELISA was used to measure the concentrations of IL-1ß, IL-6, ghrelin, substance P, and endothelin-1. Quantitative RT-PCR was used to measure the expression of SCF. Western blotting was used to measure the expression of apoptosis-related proteins, interleukins, SCF, and NF-κB signaling pathway proteins. TNF-α induced inflammatory injury in ICC by decreasing cell viability and increasing apoptosis and levels of IL-1ß and IL-6. TNF-α decreased the levels of SCF, ghrelin, and substance P, but had no effect on endothelin-1. TNF-α down-regulated expressions of SCF, ghrelin, and substance P by activating the NF-κB pathway in ICC. In conclusion, TNF-α down-regulated the expressions of SCF, ghrelin, and substance P via the activation of the NF-κB pathway in ICC.

LEAP2 Is an Endogenous Antagonist of the Ghrelin Receptor.

Ghrelin, an appetite-stimulatory hormone secreted by the stomach, was discovered as a ligand for the growth hormone secretagogue receptor (GHSR). Through GHSR, ghrelin stimulates growth hormone (GH) secretion, a function that evolved to protect against starvation-induced hypoglycemia. Though the biology mediated by ghrelin has been described in great detail, regulation of ghrelin action is poorly understood. Here, we report the discovery of liver-expressed antimicrobial peptide 2 (LEAP2) as an endogenous antagonist of GHSR. LEAP2 is produced in the liver and small intestine, and its secretion is suppressed by fasting. LEAP2 fully inhibits GHSR activation by ghrelin and blocks the major effects of ghrelin in vivo, including food intake, GH release, and maintenance of viable glucose levels during chronic caloric restriction. In contrast, neutralizing antibodies that block endogenous LEAP2 function enhance ghrelin action in vivo. Our findings reveal a mechanism for fine-tuning ghrelin action in response to changing environmental conditions.

Effect of vertical sleeve gastrectomy on alcohol consumption and preferences in dietary obese rats and mice: A plausible role for altered ghrelin signaling.

Vertical sleeve gastrectomy (VSG) and Roux-en-Y gastric bypass (RYGB) are the most common surgical options for the treatment of obesity and metabolic disorder. Whereas RYGB may result in greater and more durable weight loss, recent clinical and pre-clinical studies in rats have raised concerns that RYGB surgery may increase risk for alcohol use disorder (AUD). In contrast, recent clinical reports suggest a lesser risk for AUD following VSG, although no preclinical studies have been done to confirm that. Therefore, the present study sought to determine the effects of VSG on ethanol intake and preferences in rodent models using protocols similar to those previously used in animal studies for RYGB. Male Sprague Dawley rats and male C57B6 mice were made obese on a high fat diet (60%kcal from fat) and received VSG or no surgery (controls). All animals then were given access to increasing concentrations of ethanol (2%, 4%, 6%, and 8%), presented for few days each. Compared to controls, VSG rats consumed significantly less of 2, 6 and 8% ethanol and showed significantly reduced preferences to 6 and 8% ethanol over water. VSG mice also displayed reduced intake and preference for 6 and 8% ethanol solutions. After a two-week period of forced abstinence, 8% ethanol was reintroduced and the VSG rats and mice continued to exhibit reduced consumption and less preference for ethanol. Regarding the underlying mechanism, we hypothesized that the removal of the ghrelin producing part of the stomach in the VSG surgery is a possible contributor to the observed reduced ethanol preference. To test for functional changes at the ghrelin receptors, the VSG and control rats were given IP injections of acyl-ghrelin (2.5nmol and 5nmol) prior to ethanol access. Neither concentration of ghrelin resulted in a significant increase in 8% ethanol consumption of VSG or control subjects. Next, the rats were given IP injections of the ghrelin receptor antagonist, JMV (2.5mg/kg body weight). This dose induced a significant reduction in 8% ethanol consumption in the VSG group, but no effect on ethanol intake in the controls. While ghrelin injection was uninformative, increased sensitivity to subthreshold doses of the ghrelin receptor antagonist may indicate reduced ghrelin signaling following VSG. Overall, these findings suggest that bariatric patients with increased susceptibility to AUD may benefit from receiving VSG instead of RYGB surgery, and that changes in ghrelin signaling, at least in part, may play a role in the differential AUD risks between the two most commonly performed bariatric surgical procedures.

Ghrelin ameliorates acute lung injury induced by oleic acid via inhibition of endoplasmic reticulum stress.

AIMS: Acute lung injury (ALI) is associated with excessive mortality and lacks appropriate therapy. Ghrelin is a novel peptide that protects the lung against ALI. This study aimed to investigate whether endoplasmic reticulum stress (ERS) mediates the protective effect of ghrelin on ALI. MAIN METHODS: We used a rat oleic acid (OA)-induced ALI model. Pulmonary impairment was detected by hematoxylin and eosin (HE) staining, lung mechanics, wet/dry weight ratio, and arterial blood gas analysis. Plasma and lung content of ghrelin was examined by ELISA, and mRNA expression was measured by quantitative real-time PCR. Protein levels were detected by western blot. KEY FINDINGS: Rats with OA treatment showed significant pulmonary injury, edema, inflammatory cellular infiltration, cytokine release, hypoxia and CO2 retention as compared with controls. Plasma and pulmonary content of ghrelin was reduced in rats with ALI, and mRNA expression was downregulated. Ghrelin (10nmol/kg) treatment ameliorated the above symptoms, but treatment with the ghrelin antagonists D-Lys3 GHRP-6 (1µmol/kg) and JMV 2959 (6mg/kg) exacerbated the symptoms. ERS induced by OA was prevented by ghrelin and augmented by ghrelin antagonist treatment. The ERS inducer, tunicamycin (Tm) prevented the ameliorative effect of ghrelin on ALI. The decreased ratio of p-Akt and Akt induced by OA was improved by ghrelin treatment, and was further exacerbated by ghrelin antagonists. SIGNIFICANCE: Ghrelin protects against ALI by inhibiting ERS. These results provide a new target for prevention and therapy of ALI.

TNF-α inhibits SCF, ghrelin, and substance P expressions through the NF-κB pathway activation in interstitial cells of Cajal

Ulcerative colitis is a chronic inflammatory disease of the colon where intestinal motility is disturbed. Interstitial cells of Cajal (ICC) are required to maintain normal intestinal motility. In the present study, we assessed the effect of tumor necrosis factor-alpha (TNF-α) on viability and apoptosis of ICC, as well as on the expression of stem cell factor (SCF), ghrelin, and substance P. ICC were derived from the small intestines of Swiss albino mice. Cell viability and apoptosis were measured using CCK-8 assay and flow cytometry, respectively. ELISA was used to measure the concentrations of IL-1β, IL-6, ghrelin, substance P, and endothelin-1. Quantitative RT-PCR was used to measure the expression of SCF. Western blotting was used to measure the expression of apoptosis-related proteins, interleukins, SCF, and NF-κB signaling pathway proteins. TNF-α induced inflammatory injury in ICC by decreasing cell viability and increasing apoptosis and levels of IL-1β and IL-6. TNF-α decreased the levels of SCF, ghrelin, and substance P, but had no effect on endothelin-1. TNF-α down-regulated expressions of SCF, ghrelin, and substance P by activating the NF-κB pathway in ICC. In conclusion, TNF-α down-regulated the expressions of SCF, ghrelin, and substance P via the activation of the NF-κB pathway in ICC.

Ghrelin and Obesity: Identifying Gaps and Dispelling Myths. A Reappraisal.

The etiology of obesity is complex. Environmental and genetic causes have been implicated in the development of this disease. Ghrelin is a hormone known to stimulate appetite. There are numerous possible actions through which ghrelin exerts its effect in the body: a) Overproduction of ghrelin, b) reduced ghrelin following meals, and c) increased receptor sensitivity to ghrelin action. Sleeve gastrectomy, a bariatric procedure, leads to reduction of ghrelin levels and subsequently to weight loss. However, there are many limitations to measurement of the fasting plasma level of the active form of ghrelin. The establishment of the exact correlation between ghrelin, appetite and obesity could be vital for the fight against obesity.

Butyrylcholinesterase gene transfer in obese mice prevents postdieting body weight rebound by suppressing ghrelin signaling.

The worldwide prevalence of obesity is increasing at an alarming rate but treatment options remain limited. Despite initial success, weight loss by calorie restriction (CR) often fails because of rebound weight gain. Postdieting hyperphagia along with altered hypothalamic neuro-architecture appears to be one direct cause of this undesirable outcome. In response to calorie deficiency the circulating levels of the appetite-promoting hormone, acyl-ghrelin, rise sharply. We hypothesize that proper modulation of acyl-ghrelin and its receptor's sensitivity will favorably impact energy intake and reprogram the body weight set point. Here we applied viral gene transfer of the acyl-ghrelin hydrolyzing enzyme, butyrylcholinesterase (BChE), in a mouse model of diet-induced obesity. Our results confirmed that BChE overexpression decreased circulating acyl-ghrelin levels, suppressed CR-provoked ghrelin signaling, and restored central ghrelin sensitivity. In addition to maintaining healthy body weights, BChE treated mice had modest postdieting food intake and showed normal glucose homeostasis. Spontaneous activity and energy expenditure did not differ significantly between treated and untreated mice after body weight rebound, suggesting that BChE gene transfer did not alter energy expenditure in the long term. These findings indicate that combining BChE treatment with CR could be an effective approach in treating human obesity and aiding lifelong weight management.

Ghrelin fibers from lateral hypothalamus project to nucleus tractus solitaries and are involved in gastric motility regulation in cisplatin-treated rats.

Ghrelin can alleviate cancer chemotherapy-induced dyspepsia in rodents, though the neural mechanisms involved are not known. Therefore, ghrelin projections from the lateral hypothalamus (LH) and its involvement in the regulation of gastric motility in cisplatin-treated rats were investigated with a multi-disciplined approach. Retrograde tracing combined with fluoro-immunohistochemical staining were used to investigate ghrelin fiber projections arising from LH and projecting to nucleus tractus solitaries (NTS). Results revealed that ghrelin fibers originating in LH project to NTS. Expression of ghrelin and its receptor growth hormone secretagogue receptor (GHS-R1a) in LH and NTS were detected by Western Blot. 2days after cisplatin dosing, expression of ghrelin in LH decreased while GHS-R1a in both LH and NTS increased. In electrophysiological experiments, the effects of N-methyl-d-aspartate (NMDA) microinjection in LH on neuronal discharge of gastric distension-responsive neurons in NTS and gastric motility were assessed. NMDA in LH excited most of ghrelin-responsive gastric distension (GD)-sensitive neurons in NTS and promoted gastric motility. This effect was partially blocked by ghrelin antibody in NTS. Furthermore, the excitatory effects of NMDA in cisplatin-treated rats were weaker than those in saline-treated rats. Behaviorally, cisplatin induced a significant increase of kaolin consumption and decrease of food intake. These studies reveal a decreased expression of ghrelin in LH and up-regulation of GHS-R1a in LH and NTS, which are involved in the regulation of GD neuronal discharge in NTS and gastric motility.

The Neurobiological Impact of Ghrelin Suppression after Oesophagectomy.

Ghrelin, discovered in 1999, is a 28-amino-acid hormone, best recognized as a stimulator of growth hormone secretion, but with pleiotropic functions in the area of energy homeostasis, such as appetite stimulation and energy expenditure regulation. As the intrinsic ligand of the growth hormone secretagogue receptor (GHS-R), ghrelin appears to have a broad array of effects, but its primary role is still an area of debate. Produced mainly from oxyntic glands in the stomach, but with a multitude of extra-metabolic roles, ghrelin is implicated in complex neurobiological processes. Comprehensive studies within the areas of obesity and metabolic surgery have clarified the mechanism of these operations. As a stimulator of growth hormone (GH), and an apparent inducer of positive energy balance, other areas of interest include its impact on carcinogenesis and tumour proliferation and its role in the cancer cachexia syndrome. This has led several authors to study the hormone in the cancer setting. Ghrelin levels are acutely reduced following an oesophagectomy, a primary treatment modality for oesophageal cancer. We sought to investigate the nature of this postoperative ghrelin suppression, and its neurobiological implications.

Intracerebroventricular urocortin 3 counteracts central acyl ghrelin-induced hyperphagic and gastroprokinetic effects via CRF receptor 2 in rats.

PURPOSE: Urocortin 3 is a key neuromodulator in the regulation of stress, anxiety, food intake, gut motility, and energy homeostasis, while ghrelin elicits feeding behavior and enhances gastric emptying, adiposity, and positive energy balance. However, the interplays between urocortin 3 and ghrelin on food intake and gastric emptying remain uninvestigated. METHODS: We examined the differential effects of central O-n-octanoylated ghrelin, des-Gln14-ghrelin, and urocortin 3 on food intake, as well as on charcoal nonnutrient semiliquid gastric emptying in conscious rats that were chronically implanted with intracerebroventricular (ICV) catheters. The functional importance of corticotropin-releasing factor (CRF) receptor 2 in urocortin 3-induced responses was examined by ICV injection of the selective CRF receptor 2 antagonist, astressin2-B. RESULTS: ICV infusion of urocortin 3 opposed central acyl ghrelin-elicited hyperphagia via CRF receptor 2 in satiated rats. ICV injection of O-n-octanoylated ghrelin and des-Gln14-ghrelin were equally potent in accelerating gastric emptying in fasted rats, whereas ICV administration of urocortin 3 delayed gastric emptying. In addition, ICV infusion of urocortin 3 counteracted central acyl ghrelin-induced gastroprokinetic effects via CRF receptor 2 pathway. CONCLUSION: ICV-infused urocortin 3 counteracts central acyl ghrelin-induced hyperphagic and gastroprokinetic effects via CRF receptor 2 in rats. Our results clearly showed that enhancing ghrelin and blocking CRF receptor 2 signaling in the brain accelerated gastric emptying, which provided important clues for a new therapeutic avenue in ameliorating anorexia and gastric ileus found in various chronic wasting disorders.

Ghrelin treatment prevents development of activity based anorexia in mice.

Stimulation of feeding is necessary for treatment of pathological conditions of chronic malnutrition due to anorexia. Ghrelin, a hunger hormone, is one of the candidate for pharmacological treatments of anorexia, but because of its instability in plasma has limited efficacy. We previously showed that plasmatic IgG protect ghrelin from degradation and that IgG from obese subjects and mice may increase ghrelin׳s orexigenic effect. In this study we tested if ghrelin alone or combined with IgG may improve feeding in chronically food-restricted mice with or without physical activity-based anorexia (ABA) induced by free access to a running wheel. Mice received a single daily intraperitoneal injection of ghrelin (1nM) together or not with total IgG (1nM) from obese ob/ob or lean mice before access to food during 8 days of 3h/day feeding time. We found that both ghrelin and ghrelin combined with IgG from obese, but not lean mice, prevented ABA, however, they were not able to diminish body weight loss. Physical activity was lower during the feeding period and was increased shortly after feeding in mice receiving ghrelin together with IgG from obese mice. In food-restricted mice without ABA, ghrelin treatments did not have significant effects on food intake. Thus, this study supports pharmacological use of ghrelin or ghrelin combined with IgG from obese animals for treatment of anorexia accompanied by elevated physical activity. The utility of combining ghrelin with protective IgG should be further determined in animal models of anorexia with unrestricted access to food.

RNAi-mediated Ghrelin affects gastric H(+)-K(+)-ATPase activity and expression of GOAT-Ghrelin system in vitro.

Ghrelin has been implicated in the regulation of gastric functional development, and its physiological functions are mediated by Ghrelin-O-acyltransferase (GOAT) which is capable of generating the active form of this polypeptide hormone. However, whether and how ghrelin gene silencing may modify gastric acid secretion and GOAT-Ghrelin system is yet to be explored. The study was performed in gastric mucosal cells from weanling piglets in vitro. We evaluated the effect of ghrelin on gastric acid secretion, gene expression of GOAT and ghrelin as well as ghrelin levels by RNA interference assay. shGhrelin triggered the down-regulation of ghrelin mRNA expression (P<0.05) via an RNAi mechanism, as observed by real-time RT-PCR. In addition, shGhrelin showed reduced total ghrelin production and secretion (P<0.05) using ELISA in vitro. We also detected that GOAT mRNA expression was reduced in shGhrelin group (P<0.05), compared with control groups. In accordance with the GOAT expression, acylated ghrelin production and secretion were reduced in gastric mucosal cells and culture medium (P<0.05). Silencing of ghrelin gene achieved by RNAi-mediation inhibited the activity of H(+)-K(+)-ATPase and pepsin (P<0.05) in gastric mucosal cells. These results indicated that RNAi of Ghrelin gene inhibited the gastric acid secretion with decreased GOAT mRNA and acylated Ghrelin in gastric mucosal cells.