Chronic respiratory failure, which is often caused by chronic obstructive pulmonary disease, chronic lower respiratory tract infection, or interstitial pneumonia, often leads to cachexia with disease progression. Patients who have chronic respiratory failure with cachexia exhibit increased morbidity. Although cachectic status is an important clinical problem, there are no effective therapies for cachexia. Ghrelin has various effects, including increasing food intake, attenuating sympathetic nerve activity, inhibiting inflammation, increasing cardiac output, and controlling fat utilization. These effects of ghrelin are ideal targets for the treatment of severely wasting chronic respiratory disease. In a few clinical studies, including a small randomized controlled trial, ghrelin administration to cachectic patients with chronic respiratory failure improved exercise tolerance, dyspnea, and appetite. The patients in these studies gained muscle mass and weight. In another study of chronic lower respiratory tract infection with cachexia, ghrelin suppressed airway inflammation by decreasing neutrophil accumulation in the airway, resulting in improvements in oxygenation and exercise tolerance. Although further clinical investigations are needed to clarify its usefulness, ghrelin is expected to become a novel therapy for cachectic patients with chronic respiratory failure.
Ghrelin has been identified in vertebrates from fish to mammals, and it has multiple biological activities including gastrointestinal (GI) motor-stimulating action. In some non-mammalian vertebrates, we examined the effects of ghrelin on contractility of the isolated GI tract as well as the mRNA expression of growth hormone secretagogue-receptor 1a (GHS-R1a) to determine whether the motor-stimulating action of ghrelin is common in vertebrates. The expression level of GHS-R1a mRNA differed depending on the species and on the GI region (stomach, small intestine, and colon). GI region-dependent expression of GHS-R1a mRNA was remarkable in chickens, and the expression levels changed depending on age. In a functional study, ghrelin did not cause contraction of unstimulated GI strips in fish (goldfish and rainbow trout) or amphibians (bullfrog and Japanese fire belly newts) even using their homologous ghrelin. In avian species, ghrelin caused contraction of the unstimulated GI tract of the chicken but not of the Japanese quail, and the responses to ghrelin in the chicken GI tract decreased with aging. Our in vitro studies show that the motor-stimulating action of ghrelin is not conserved across vertebrates and that the chicken is a unique animal species for evaluation of the GI-stimulating action of ghrelin of different age.
Ghrelin, a peptide hormone produced in the stomach, has been known to be involved in the regulation of gastric contraction in humans and rodents. To elucidate the detailed mechanisms of ghrelin on gastric contractions, we used Suncus murinus, a recently established small animal model for gastrointestinal motility. S. murinus produces motilin, a family peptide of ghrelin, and its stomach anatomy and physiological patterns of gastric contractions, in fed and fasted states, are closely similar to humans. Ghrelin administration in phase II, and latter half of phase I, of the migrating motor contractions (MMC) enhanced gastric motility in S. murinus. In addition, we showed that ghrelin and motilin coordinately stimulated strong gastric contractions in vitro and in vivo. We also demonstrated that a pretreatment with a ghrelin antagonist, D-Lys3-GHRP6, inhibited the effects of motilin-induced gastric contractions, and a γ-aminobutyric acid (GABA) antagonist reversed this inhibition. Our results suggest that ghrelin is essential for motilin-induced gastric contractions and that ghrelin-mediated GABAergic neurons are involved in this neural pathway.
We are exploring physiological importance of the ghrelin system in vertebrates. This review summarizes current knowledge of the ghrelin system in amphibians. Our study on ghrelin precursor in various amphibians revealed that the third amino acid with acyl modification has changed to threonine (Thr-3) instead of serine (Ser-3) only in the genus, Rana. Functional analyses of the ghrelin receptor in three species of amphibians, Japanese fire belly newt, American bullfrog and Japanese tree frog revealed that ghrelin and GHS-R1a agonists increase intracellular Ca2+ concentration in HEK293 cells expressing each receptor, and that ligand selectivity of ghrelin with Ser-3 and Thr-3 that expected to see in the bullfrog receptor was not found in the two frog receptors, but in the newt receptor. The brain, gastrointestinal tract, kidney and gonad highly express GHS-R1a mRNA. In frogs and newt, fasting did not increase GHS-R1a mRNA expression in the brain, but in the stomach. However, intraperitoneal (IP) injection of ghrelin did not affect food intake. A dehydration treatment increased GHS-R1a mRNA expression in the brain, stomach and ventral skin in the tree frog. However, intracerebroventricular (ICV) injection of ghrelin did not affect water absorption. Ghrelin did not influence gastrointestinal motility in in vitro studies using smooth muscle strips of the bullfrog and newt in vitro. These results suggest that the ghrelin system is present in various amphibians, but little is known about the physiological functions except hypophyseal hormone secretion.
The octanoyl modification of ghrelin by ghrelin O-acyltransferase (GOAT) is essential for exerting its physiologic actions. Since exogenous acylated-ghrelin has shown to stimulate food intake in humans and rodents, GOAT has been regarded as a promising target for modulating appetite, thereby treating obesity and diabetes. However, GOAT-knockout (KO) mice have been reported to show no meaningful body weight reduction, when fed a high-fat diet. In this study, we sought to determine whether GOAT has a role in the regulation of body weight and food intake when fed a dietary sucrose. We found that GOAT KO mice showed significantly reduced food intake and marked resistance to obesity, when fed a high-fat + high-sucrose diet. In addition, GOAT KO mice fed a medium-chain triglyceride (MCT) + high-sucrose diet showed a marked resistance to obesity and reduced feed efficiency. These results suggest that blockade of acylated-ghrelin production offers therapeutic potential for obesity caused by overconsumption of palatable food.
In the current study, we examined the effects of LPS and inflammatory cytokines including IL-1β, TNF-α, and IL-6 on the expression of ghrelin in MGN3-1 cells. We found that IL-1β, and TNF-α with lesser extent, significantly suppressed ghrelin mRNA expression in the cells. MGN3-1 cells expressed IL-1β receptor and IL-1β significantly stimulated NF-κB, p38, JNK, and ERK pathways. Knockdown of IKK2 by siRNA significantly attenuated the suppression of ghrelin mRNA by IL-1β. These results indicate that IL-1β directly suppressed ghrelin mRNA via NF-κB pathway at least partially, which may have a role in the regulation of appetite during inflammation.
Ghrelin is a peptide hormone with a unique structure comprising a medium chain fatty acid modification. Ghrelin cells are known to be abundantly localized in the gastric mucosa and are released into the blood stream to exert their multifunctional physiological effects. To elucidate the regulatory mechanisms of ghrelin secretion and acyl-modification, we developed novel ghrelin-producing cell lines. Using ghrelinoma cell lines, we focused on the mechanisms of ghrelin secretion and found that several GPCRs were highly expressed in ghrelin cells. Then, we showed that noradrenaline treatment stimulated ghrelin secretion via β1-adrenergic receptor, and fasting-induced ghrelin elevation was completely inhibited by the β1-adrenergic receptor antagonist in mice. In addition, we demonstrated that long chain fatty acids, glucose, and L-glutamate significantly inhibited ghrelin secretion. Furthermore, we recently revealed that the genes involved in fatty acid synthesis and long chain fatty acid metabolism were expressed in ghrelin cells, and that CPT-1 inhibitor treatment dramatically decreased the levels of acyl-modified ghrelin. Here, we introduce the current knowledge of the mechanisms involving ghrelin secretion and its acyl-modification.
To elucidate the clinical implication of ghrelin, we have been trying to generate variable models of transgenic (Tg) mice overexpressing ghrelin. We generated Tg mice overexpressing des-acyl ghrelin in a wide variety of tissues under the control of β-actin promoter. While plasma des-acyl ghrelin level in the Tg mice was 44-fold greater than that of control mice, there was no differences in the plasma ghrelin level between des-acyl ghrelin Tg and the control mice. The des-acyl ghrelin Tg mice exhibited the lower body weight and the shorter body length due to modulation of GH-IGF-1 axis. We tried to generate Tg mice expressing a ghrelin analog, which possessed ghrelin-like activity (Trp3-ghrelin Tg mice). The plasma Trp3-ghrelin concentration in Trp3-ghrelin Tg mice was approximately 85-fold higher than plasma ghrelin (acylated ghrelin) concentration seen in the control mice. Because Trp3-ghrelin is approximately 24-fold less potent than ghrelin, the plasma Trp3-ghrelin concentration in Trp3-ghrelin Tg mice was calculated to have approximately 3.5-fold biological activity greater than that of ghrelin (acylated ghrelin) in the control mice. Trp3-ghrelin Tg mice did not show any phenotypes except for reduced insulin sensitivity in 1-year old. After the identification of ghrelin O-acyltransferase (GOAT), we generated doubly Tg mice overexpressing both mouse des-acyl ghrelin and mouse GOAT in the liver by cross-mating the two kinds of Tg mice. The plasma ghrelin concentration of doubly Tg mice was approximately 2-fold higher than that of the control mice. No apparent phenotypic changes in body weight and food intake were observed in doubly Tg mice. Further studies are ongoing in our laboratory to generate Tg mice with the increased plasma ghrelin level to a greater extent. The better understanding of physiological and pathophysiological significance of ghrelin from experiments using an excellent animal model may provide a new therapeutic approach for human diseases.
Cancer was considered an incurable disease for many years; however, with the development of anticancer drugs and state-of-the art technologies, it has become curable. Cardiovascular diseases in patients with cancer or induced by cancer chemotherapy have recently become a great concern. Certain anticancer drugs and molecular targeted therapies cause cardiotoxicity, which limit the widespread implementation of cancer treatment and decrease the quality of life in cancer patients significantly. The anthracycline doxorubicin (DOX) causes cardiotoxicity. The cellular mechanism underlying DOX-induced cardiotoxicity include free-radical damage to cardiac myocytes, leading to mitochondrial injury and subsequent death of myocytes. Recently, circulating orexigenic hormones, ghrelin and des-acyl ghrelin, have been reported to inhibit DOX-induced cardiotoxicity. However, little is known about the molecular mechanisms underlying their preventive effects. In the present study, we show the possible mechanisms underlying the effects of ghrelin and des-acyl ghrelin against DOX-induced cardiotoxicity through in vitro and in vivo researches.
Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor (GHSR), is produced in the human stomach. Although ghrelin has therapeutic potential for cancer cachexia, ghrelin treatment may have a concern about accelerating cancer progression. Here, using the human lung adenocarcinoma cell line HLC-1, we investigated the effects of ghrelin on molecular mechanisms linked to cancer progression, including cell viability, proliferation, resistance to apoptosis, and mitochondrial activity. Both types of mouse alveolar epithelial cells (types I and II) expressed the GHSR, as did the human normal airway cell lines BEAS-2B and HLC-1. Treatment with ghrelin (10-2, 10-1, 1, 10 μM) did not affect cell viability or proliferation. Pretreatment of HLC-1 cells with ghrelin (10 μM) did not affect resistance to paclitaxel-induced apoptosis. The parameters of mitochondrial respiration, including basal respiration, proton leak, ATP production, maximal respiration, spare respiratory capacity, and non-mitochondrial respiration, of the HLC-1 cells pretreated with or without ghrelin were unchanged. Taken together, ghrelin does not influence cancer progression in lung adenocarcinoma cells.
Chronic kidney disease (CKD) impairs physical performance in humans, which leads to a risk of all-cause mortality. In our previous study, we demonstrated that a reduction in muscle mitochondria rather than muscle mass was a major cause of physical decline in 5/6 nephrectomized CKD model mice. Because ghrelin administration has been reported to enhance oxygen utilization in skeletal muscle, we examined the usefulness of ghrelin for a recovery of physical decline in 5/6 nephrectomized C57Bl/6 mice, focusing on the epigenetic modification of peroxisome proliferator activated receptor gamma coactivator-1α (PGC-1α), a master regulator of mitochondrial biogenesis. The mice were intraperitoneally administered acylated ghrelin (0.1 nmol/gBW; three times per week) for a month. Muscle strength and exercise endurance were measured by using a dynamometer and treadmill, respectively. Mitochondrial DNA copy number was determined by quantitative PCR. The methylation levels of the cytosine residue at 260 base pairs upstream of the translation initiation point (C-260) of PGC-1α, which has been demonstrated to decrease the expression, was evaluated by methylation-specific PCR and bisulfite genomic sequencing methods after the ghrelin administration. Ghrelin administration improved both muscle strength and exercise endurance in the mice and was associated with an increase in muscle mass and muscle mitochondrial content. Ghrelin administration decreased the methylation ratio of C-260 of PGC-1α in the skeletal muscle and increased the expression. Therefore, ghrelin administration effectively reduced the physical decline in 5/6 nephrectomized mice and was accompanied with an increased mitochondrial content through de-methylation of the promoter region of PGC-1α in the muscle.
Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes, and its progression significantly worsens the patient’s quality of life. Although several drugs are available for DPN, all of these provide only symptomatic relief. We investigated the therapeutic effects of ghrelin for DPN, based on its various physiological functions. Seven patients with type 2 diabetes with typical clinical signs and symptoms of DPN were hospitalized. Synthetic human ghrelin (1.0 μg/kg) was administered intravenously for 14 days. Motor nerve conduction velocity (MCV) of the posterior tibial nerve improved significantly after the treatment, compared to that at baseline (35.1 ± 1.8 to 38.6 ± 1.8 m/s, p < 0.0001), while the MCV in six untreated patients did not change throughout hospitalization. The subjective symptoms assessed based on the total symptom score also significantly improved (15.6 ± 3.1 to 11.1 ± 2.2, p = 0.047). Although sensory nerve conduction velocity (SCV) of the sural nerve could not be detected in three patients at baseline, it was detected in two of the three patients after 14 days of ghrelin administration. Overall, SCV did not change significantly. Plasma glucose, but not serum C peptide, levels during a liquid meal tolerance test significantly improved after treatment. These results suggest that ghrelin may be a novel therapeutic option for DPN; however, a double-blind, placebo-controlled trial is needed in the future.