Establishment of a Cell Line Stably Expressing the Growth Hormone Secretagogue Receptor to Identify Crocin as a Ghrelin Agonist.

The growth hormone secretagogue receptor-1a (GHSR1a) is the endogenous receptor for ghrelin. Activation of GHSR1a participates in many physiological processes including energy homeostasis and eating behavior. Due to its transitory half-life, the efficacy of ghrelin treatment in patients is restricted; hence the development of new adjuvant therapy is an urgent need. This study aimed to establish a cell line stably expressing GHSR1a, which could be employed to screen potential ghrelin agonists from natural compounds. First, by means of lentiviral transduction, the genome of a human HEK293T cell was modified, and a cell platform stably overexpressing GHSR1a was successfully established. In this platform, GHSR1a was expressed as a fusion protein tagged with mCherry, which allowed the monitoring of the dynamic cellular distribution of GHSR1a by fluorescent microscopy. Subsequently, the authenticity of the GHSR1a mediated signaling was further characterized by using ghrelin and teaghrelin, two molecules known to stimulate GHSR1a. The results indicated that both ghrelin and teaghrelin readily activated GHSR1a mediated signaling pathways, presumably via increasing phosphorylation levels of ERK. The specific GHSR1a signaling was further validated by using SP-analog, an antagonist of GHSR1a as well as using a cell model with the knockdown expression of GHSR1a. Molecular modeling predicted that crocin might be a potential ghrelin agonist, and this prediction was further confirmed by the established platform.

Teaghrelin Protects SH-SY5Y Cells against MPP+-Induced Neurotoxicity through Activation of AMPK/SIRT1/PGC-1α and ERK1/2 Pathways.

The prevalence and incidence of Parkinson's disease (PD), an age-related neurodegenerative disease, are higher among elderly people. Independent of etiology, dysfunction and loss of dopaminergic neurons are common pathophysiological changes in PD patients with impaired motor and non-motor function. Currently, preventive or therapeutic treatment for combating PD is limited. The ghrelin axis and ghrelin receptor have been implicated in the preservation of dopaminergic neurons and have potential implications in PD treatment. Teaghrelin, a compound originating from Chin-Shin Oolong tea, exhibits ghrelin agonist activity. In this study, the neuroprotective potential of teaghrelin against PD was explored in a cell model in which human neuroblastoma SH-SY5Y cells were treated with the mitochondrial toxin 1-methyl-4-phenylpyridinium (MPP+). Upon MPP+ exposure, SH-SY5Y cells exhibited decreased mitochondrial complex I activity and apoptotic cell death. Teaghrelin activated AMP-activated protein kinase (AMPK)/sirtuin 1(SIRT1)/peroxisome proliferator-activated receptor gamma (PPARγ) coactivator-1α (PGC-1α) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathways to antagonize MPP+-induced cell death. Herein, we propose that teaghrelin is a potential candidate for the therapeutic treatment of PD.

Ghrelin agonist HM01 attenuates chemotherapy-induced neurotoxicity in rodent models.

Chemotherapy-Induced Peripheral Neurotoxicity (CIPN) is often dose-limiting and impacts life quality and survival of cancer patients. Ghrelin agonists have neuroprotectant effects and may have a role in treating or preventing CIPN. We evaluated the CNS-penetrant ghrelin agonist HM01 in three experimental models of CIPN at doses of 3-30 mg/kg p.o. daily monitoring orexigenic properties, nerve conduction, mechanical allodynia, and intra-epidermal nerve fiber density (IENFD). In a cisplatin-based study, rats were dosed daily for 3 days (0.5 mg/kg i.p.) + HM01. Cisplatin treatment induced mechanical hypersensitivity which was significantly reduced by HM01. In a second study, oxaliplatin was administered to mice (6 mg/kg i.p. 3 times/week for 4 weeks) resulting in significant digital nerve conduction velocity (NCV) deficits and reduction of IENFD. Concurrent HM01 dose dependently prevented the decline in NCV and attenuated the reduction in IENFD. Pharmacokinetic studies showed HM01 accumulation in the dorsal root ganglia and sciatic nerves which reached concentrations > 10 fold that of plasma. In a third model, HM01 was tested in preventive and therapeutic paradigms in a bortezomib-based rat model (0.2 mg/kg i.v., 3 times/week for 8 weeks). In the preventive setting, HM01 blocked bortezomib-induced hyperalgesia and IENFD reduction at all doses tested. In the therapeutic setting, significant effect was observed, but only at the highest dose. Altogether, the robust peripheral nervous system penetration of HM01 and its ability to improve multiple oxaliplatin-, cisplatin-, and bortezomib-induced neurotoxicities suggest that HM01 may be a useful neuroprotective adjuvant for CIPN.

Sustained-release multiparticulates for oral delivery of a novel peptidic ghrelin agonist: Formulation design and in vitro characterization.

There is an impetus to provide appropriate sustained release oral delivery vehicles to protect biofunctional peptide loads from gastric degradation in vivo. This study describes the generation of a high load capacity pellet formulation for sustained release of a freely water-soluble dairy-derived hydrolysate, FHI-2571. The activity of this novel peptidic ghrelin receptor agonist is reported using in vitro calcium mobilization assays. Conventional extrusion spheronization was then used to prepare peptide-loaded pellets which were subsequently coated with ethylcellulose (EC) film coats using a fluid bed coating system in bottom spray (Wurster) mode. Aqueous-based EC coating dispersions produced mechanically brittle coats which fractured due to osmotic pressure build-up within pellets in simulated media. In contrast, an ethanolic-based EC coating solution provided robust, near zero-order release in both USP Type 1 and Type 4 dissolution studies. Interestingly, the functionality of aqueous-based EC film coats was restored by first layering pellets with a methacrylic acid copolymer (MA) subcoat, thereby hindering pellet core swelling in acidic media. Broadband Acoustic Resonance Dissolution Spectroscopy (BARDS) was utilised as a complementary technique to confirm the results seen in USP dissolution studies. Retention of activity of the ghrelinergic peptide hydrolysate in the final encapsulated product was confirmed as being greater than 80%. The described pellet formulation is amenable to oral dosing in small animal studies in order to assess in vivo efficacy of the whey-derived ghrelinergic hydrolysate. In more general terms, it is also suitable as a delivery vehicle for peptide-based bioactives to special population groups e.g paediatric and geriatric.

Cinnamomum osmophloeum Kanehira ethanol extracts prevents human liver-derived HepG2 cell death from oxidation stress by induction of ghrelin gene expression.

Diabetes patients associated with liver disease carry a significant risk of morbidity and mortality. Cinnamon has been reported to reduce fructose-induced oxidative stress in the rat liver. However, the mechanism by which cinnamon protects the liver in a high-saccharide environment remains to be investigated. HepG2 cells were cultured with 30 mM D-ribose to mimic the high-oxidative-stress environment, typical of a liver in a diabetic patient. Three different chemical types of C. osmophloeum ethanol extracts (CEEs) were added in HepG2 culture media and the administration of all three CEEs protected HepG2 cells from D-ribose damage and increased cell survival by approximately 20 percent. Exclusively, the transcript variant 1 of the ghrelin gene, but not variant 3, was 2-3 times induced by the addition of these CEEs. Moreover, the mRNAs of ghrelin processing enzyme, furin, and mboat4 were detected in HepG2 cells. The ghrelin hormones in the culture media were increased 4-9 times by the addition of CEEs. The protective effects of ghrelin on HepG2 cells in D-ribose environment were further confirmed by recombinant ghrelin transfection. We conclude that the CEEs induce ghrelin gene expression and protect HepG2 cells from D-ribose-induced oxidative damage through ghrelin signalling.

Hypothalamic Leptin and Ghrelin Signaling as Targets for Improvement in Metabolic Control.

Metabolic homeostasis requires a tight balance between energy intake and energy expenditure; hence, the physiological circuits implicated in the regulation of energy metabolism must be able to quickly adjust to changes in either side of the equation. Circulating orexigenic and anorexigenic factors, including ghrelin and leptin, are produced in the gastrointestinal tract and adipose tissue, respectively, in relation to an individual's nutritional status. These signals interact with central metabolic circuits to regulate the production and secretion of neuropeptides implicated in the control of appetite and energy expenditure. However, this physiological equilibrium can be perturbed by diverse processes, with weight gain occurring due to a positive energy balance and weight loss taking place if there is a negative energy balance. If a situation of positive energy balance continues for an extended period of time, excess weight is accumulated and this can eventually result in obesity. Obesity has become one of the most important health problems facing the industrialized world, indicating that metabolic equilibrium is frequently disrupted. Understanding how and why this occurs will allow new therapeutical targets to be identified.

Frailty, sarcopenia, and hormones.

Frailty is now a definable clinical syndrome with a simple screening test. Age-related changes in hormones play a major role in the development of frailty by reducing muscle mass and strength (sarcopenia). Selective Androgen Receptor Molecules and ghrelin agonists are being developed to treat sarcopenia. The role of Activin Type IIB soluble receptors and Follistatin-like 3 mimetics is less certain because of side effects. Exercise (resistance and aerobic), vitamin D and protein supplementation, and reduction of polypharmacy are keys to the treatment of frailty.

Ghrelin agonist JMV 1843 increases food intake, body weight and expression of orexigenic neuropeptides in mice.

Ghrelin and agonists of its receptor GHS-R1a are potential substances for the treatment of cachexia. In the present study, we investigated the acute and long term effects of the GHS R1a agonist JMV 1843 (H Aib-DTrp-D-gTrp-CHO) on food intake, body weight and metabolic parameters in lean C57BL/6 male mice. Additionally, we examined stability of JMV 1843 in mouse blood serum. A single subcutaneous injection of JMV 1843 (0.01-10 mg/kg) increased food intake in fed mice in a dose-dependent manner, up to 5-times relative to the saline-treated group (ED(50)=1.94 mg/kg at 250 min). JMV 1843 was stable in mouse serum in vitro for 24 h, but was mostly eliminated from mouse blood after 2 h in vivo. Ten days of treatment with JMV 1843 (subcutaneous administration, 10 or 20 mg/kg/day) significantly increased food intake, body weight and mRNA expression of the orexigenic neuropeptide Y and agouti-related peptide in the medial basal hypothalamus and decreased the expression of uncoupling protein 1 in brown adipose tissue. Our data suggest that JMV 1843 could have possible future uses in the treatment of cachexia.

Cisplatin-induced anorexia and ghrelin.

Cisplatin, a formidable anticancer treatment, is used for several varieties of cancer. There are, however, many cases in which treatment must be abandoned due to a decrease in the patient's quality of life from loss of appetite associated with vomiting and nausea. There is a moderate degree of improvement in prevention of cisplatin-induced nausea and vomiting when serotonin (5-HT) 3 receptor antagonists, neurokinin 1 receptor antagonists, and steroids-either alone or in combination-are administered. The mechanism of action for anorexia, which continues during or after treatment, is, however, still unclear. This anorexia is, similar to the onset of vomiting and nausea, caused by the action of large amounts of 5-HT released as a result of cisplatin administration on tissue 5-HT receptors. Among the 5-HT receptors, the activation of 5-HT2b and 5-HT2c receptors, in particular, seems to play a major role in cisplatin-induced anorexia. Following activation of these two receptors, there is reduced gastric and hypothalamic secretion of the appetite-stimulating hormone ghrelin. There is ample evidence of the usefulness of exogenous ghrelin, synthetic ghrelin agonists, and the endogenous ghrelin signal-enhancer rikkunshito, which are expected to play significant roles in the clinical treatment and prevention of anorexia in future.

Ghrelin and motilin in the gastrointestinal system.

Human ghrelin and human motilin, belonging to the ghrelin/motilin-related peptide family, share 36% amino acid sequence identity, while the human ghrelin receptor exhibits a remarkable 50% overall identity with the human motilin receptor. In addition to their structural resemblance, ghrelin and motilin are the only two mammalian hormones known to decrease in the postprandial period. Ghrelin and motilin participate in initiating the migrating motor complex in the stomach, and stimulate gastrointestinal motility, accelerate gastric emptying, and induce "gastric hunger". In addition to modulating the release of growth hormone and gut motility, ghrelin plays a crucial role in the secretion and protection of the stomach and colon. Ghrelin mimetics and motilin agonists are currently being developed to reverse gastrointestinal hypomotility disorders. With additional appetite-enhancing, adiposity-promoting, and anti-inflammatory effects, ghrelin and rikkunshito (a traditional Japanese herb enhancing acyl ghrelin signaling) are superior to motilin in the treatment of cancer-related anorexia and cachexia, post-chemotherapy symptoms, rheumatological diseases, age-related frailty, as well as post-operative, septic, and post-burn gut ileus.

Rikkunshito and ghrelin secretion.

Rikkunshito is a kampo herbal medicine which is widely used in Japan for the treatment of the upper gastrointestinal symptoms of patients with functional dyspepsia (FD), gastroesophageal reflux disease (GERD), dyspeptic symptoms of postgastrointestinal surgery patients, and chemotherapy-induced dyspepsia in cancer patients. Recently, very unique characteristics of rikkunshito have been unveiled; oral administration of rikkunshito potentiates orexigenic action of ghrelin through several different mechanisms. In addition, several lines of evidence obtained from both animal and human studies indicate that rikkunshito can be an attractive and promising therapeutic option for the anorectic conditions including cisplatin-induced dyspepsia, anorexia of aging, stress-induced hypophagia, cancer cachexia-anorexia syndrome. In this review, we will highlight what is known about the orexigenic effect of rikkunshito with a special focus on an interaction with ghrelin signaling system.

Neurohormones, rikkunshito and hypothalamic neurons interactively control appetite and anorexia.

Ghrelin is the orexigenic peptide produced in the periphery, and its plasma level shows remarkable pre/postprandial changes. Ghrelin is considered a pivotal signal to the brain to stimulate feeding. Hence, characterizing the target neurons for ghrelin in the hypothalamic feeding center and the signaling cascade in the target neurons are essential for understanding the mechanisms regulating appetite. Anorexia and cachexia associated with gastric surgery, stress-related diseases, and use of anti-cancer drugs cause the health problems, markedly deteriorating the quality of life. The anorexia involves several neurotransmitters and neuropeptides in the hypothalamic feeding center, in which corticotropin-releasing hormone (CRH), urocortine, serotonin (5HT) and brain-derived neurotrophic factor (BDNF) play a pivotal role. A Japanese herbal medicine, rikkunshito, has been reported to ameliorate the anorexia by promoting the appetite. This review describes 1) the interaction of ghrelin with the orexigenic neuropeptide Y (NPY) neurons in the hypothalamic arcuate nucleus (ARC) and underlying signaling cascade in NPY neurons, 2) the anorectic pathway driven by BDNF-CRH/urocortine and 5HTCRH/ urocortine pathways, 3) the effect of rikkunshito on the interaction of ghrelin and NPY neurons in ARC, and 4) the effect of rikkunshito on the interaction of 5HT on CRH neurons in paraventricular nucleus (PVN).

A review of traditional Japanese medicines and their potential mechanism of action.

Traditional Japanese herbal, or Kampo medicine was developed and modified from Chinese herbal medicine. After the Japanese government approved Kampo for clinical use, much attention has been paid to establishing scientific evidence for the effectiveness of these medicines. Recent progress has been made in elucidating the mechanisms of action of some types of Kampo medicine, including rikkunshito (RKT), daikenchuto, and yokukansan. In this review, we focused on identifying the target molecules and the active ingredients of RKT. Thus far, many target molecules have been implicated in the mechanism of action of Kampo medicines, such as ion channels, enzymes, and receptors. In particular, G protein-coupled receptors are attractive candidates for explaining herbal medicine activity. This is particularly true of RKT, which is composed of 8 independent, crude drug extracts. Recent reports have shown that RKT elicits its effects through dual action to the G protein-coupled receptors: inhibition of serotonergic 5-HT2C and 5-HT2B receptors and activation of ghrelin receptors via specific ingredients of RKT. In addition, we suggest that the identification of the effective ingredients from Kampo medicines could contribute to the discovery and development of new drugs by means of modern high-throughput drug screening technology.

Cachexia and herbal medicine: perspective.

Kampo, a form of traditional herbal medical practice, has become a substance of interest for scientific research. Although earlier clinical reports concerning Kampo are abundant, the scientific investigation of Kampo has a very short history. However, the process of acquiring quantifiable clinical trial evidence on herbal medicine is now clearly underway. The development of multi-component herbal medicines capable of targeting multiple sites could be useful both for future drug discovery and for the potential management of complex diseases. Additionally, mechanistic studies and the identification of active compounds could lead to new discoveries in the biological and biomedical sciences. Modern translational research on herbal medicines beyond basic science and clinical perspectives will contribute to the development of new medicines. This review covers the translational aspects of herbal medicine with a focus on cancer anorexiacachexia. The review gives perspective on a new horizon for herbal medicine from a scientific point of view and a basis for the further development of complementary and alternative medicine (CAM) for patients.