Human Ghrelin Research Articles

Glucose Regulatory Hormones in Camel, Cow, Goat, and Human Milk (P06-034-19)

ObjectivesDiabetes Mellitus is globally predicted to affect 592 million people by 2035 with the greatest rates of increase in low and middle income countries. Various plant and animal foods are being tested as alternatives to medication to control blood glucose. Regular consumption of milk from camels has had a positive anti-diabetic effect in both type 1 and type 2 diabetics. Epidemiological studies, research in diabetic rat and mouse model systems, and clinical studies in diabetic patients support the anti-diabetic effects of camel milk, but the mechanism of action is unclear.This study investigated hormones that impact glucose metabolism in humans from milks from different species and their stability with pasteurization. We tested hypotheses in the literature that camel milk antidiabetic properties are due to a high content of insulin or insulinotropic hormones. MethodsFive milk samples: Human, Bovine, Goat, and Camel from two different farms were analyzed with a multiplex panel for hormones that impact glucose metabolism in humans. Immunoreactivity to human Insulin, C-peptide, Glucagon, Ghrelin, Leptin, GIP, GLP-1, Resistin, Visfatin, and PAI-1 (total) were determined with a multiplex assay (Biorad Bioplex diabetes panel) using a Magpix analyzer. ResultsInsulin immunoreactivity was present in both human and goat milk at approximately 200 pg/mL. Both camel samples were below the range of detection with this anti-human insulin antibody; but 5 pg/mL of insulin was detected in bovine milk. GIP was detected in all milks but was highest in human milk. Camel milk had higher immunoreactivity to resistin, visfatin, and ghrelin than goat or bovine milk. GLP-1 and leptin were only detected in human milk. Neither HTST nor Holder pasteurization significantly reduced the immunoreactivity of these hormones in milk. ConclusionsAlthough literature suggests camel milk is an effective medical food for diabetics, we did not find high insulin concentration in camel milk. The hypoglycemic effect of camel milk might be due to other components of camel milk. Assays using different antibodies or insulin receptors might show different results. The insulin concentration in goat milk suggests that this product should be tested as a medical food for glucose control. Funding SourcesNIFA; NCSU; Saudi Cultural Mission.

Anti-inflammatory Effect of Ghrelin in Lymphoblastoid Cell Lines From Children With Autism Spectrum Disorder.

The gut hormone ghrelin has been implicated in a variety of functional roles in the central nervous system through the brain-gut axis, one of which is an anti-inflammatory effect. An aberrant brain-gut axis producing immune dysfunction has been implicated in the pathobiology of autism spectrum disorder (ASD), and elevated expression of inflammatory markers has been shown in blood and brain tissue from subjects with ASD. We hypothesized that ghrelin may mitigate this effect. Lymphoblastoid cell lines from typically developed children (TD-C) (N = 20) and children with ASD (ASD-C) (N = 20) were cultured with PBS or human ghrelin (0.01 μM) for 24 h, and mRNA expression levels of the inflammation-related molecules interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and nuclear factor kappa B (NF-κB) were measured to examine the effects of ghrelin as an anti-inflammatory agent. Expression levels of TNF-α and NF-κB mRNA, but not IL-1β or IL-6, were significantly elevated in ASD-C compared to TD-C. Ghrelin showed a tendency to reduce the expression of TNF-α and NF-κB, but this was not statistically significant. Considering the heterogenous pathobiology of ASD, we examined the effects of ghrelin on TD-C and ASD-C with expression levels of TNF-α and NF-κB in the highest and lowest quartiles. We found that ghrelin markedly reduced mRNA expression of TNF-α and NF-κB s in ASD-C with highest-quartile expression, but there were no effects in ASD-C with lowest-quartile expression, TD-C with highest quartile expression, or TD-C with lowest quartile expression. Together, these findings suggest that ghrelin has potential as a novel therapeutic agent for ASD with inflammation and/or immune dysfunction.

ASB20123: A novel C-type natriuretic peptide derivative for treatment of growth failure and dwarfism

C-type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor B (NPR-B) are physiological potent positive regulators of endochondral bone growth; therefore, the CNP/NPR-B signaling pathway is one of the most promising therapeutic targets for treating growth failure and dwarfism. In this article, we summarized the pharmacological properties of a novel CNP analog peptide ASB20123 as a therapeutic agent for short stature. ASB20123, one of the CNP/ghrelin chimeric peptides, is composed of CNP(1–22) and human ghrelin(12–28, E17D). Compared to CNP(1–22), ASB20123 showed similar agonist activity for NPR-B and improved biokinetics with a longer plasma half-life in rats. In addition, the distribution of ASB20123 to the cartilage was higher than that of CNP(1–22) after single subcutaneous (sc) injection to mice. These results suggested that the C-terminal part of ghrelin, which has clusters of basic amino acid residues and a BX7B motif, might contribute to the retention of ASB20123 in the extracellular matrix of the growth plate. Multiple sc doses of ASB20123 potently stimulated skeletal growth in rats in a dose-dependent manner, and sc infusion was more effective than bolus injection at the same dose. Our data indicated that high plasma levels of ASB20123 would not necessarily be required for bone growth acceleration. Thus, pharmaceutical formulation approaches for sustained-release dosage forms to allow chronic exposure to ASB20123 might be suitable to ensure drug effectiveness and safety.

Dopamine neuronal protection in the mouse Substantia nigra by GHSR is independent of electric activity

ObjectiveDopamine neurons in the Substantia nigra (SN) play crucial roles in control of voluntary movement. Extensive degeneration of this neuronal population is the cause of Parkinson's disease (PD). Many factors have been linked to SN DA neuronal survival, including neuronal pacemaker activity (responsible for maintaining basal firing and DA tone) and mitochondrial function. Dln-101, a naturally occurring splice variant of the human ghrelin gene, targets the ghrelin receptor (GHSR) present in the SN DA cells. Ghrelin activation of GHSR has been shown to protect SN DA neurons against 1-methyl-4-phenyl-1,2,5,6 tetrahydropyridine (MPTP) treatment. We decided to compare the actions of Dln-101 with ghrelin and identify the mechanisms associated with neuronal survival. MethodsHistologial, biochemical, and behavioral parameters were used to evaluate neuroprotection. Inflammation and redox balance of SN DA cells were evaluated using histologial and real-time PCR analysis. Designer Receptors Exclusively Activated by Designer Drugs (DREADD) technology was used to modulate SN DA neuron electrical activity and associated survival. Mitochondrial dynamics in SN DA cells was evaluated using electron microscopy data. ResultsHere, we report that the human isoform displays an equivalent neuroprotective factor. However, while exogenous administration of mouse ghrelin electrically activates SN DA neurons increasing dopamine output, as well as locomotion, the human isoform significantly suppressed dopamine output, with an associated decrease in animal motor behavior. Investigating the mechanisms by which GHSR mediates neuroprotection, we found that dopamine cell-selective control of electrical activity is neither sufficient nor necessary to promote SN DA neuron survival, including that associated with GHSR activation. We found that Dln101 pre-treatment diminished MPTP-induced mitochondrial aberrations in SN DA neurons and that the effect of Dln101 to protect dopamine cells was dependent on mitofusin 2, a protein involved in the process of mitochondrial fusion and tethering of the mitochondria to the endoplasmic reticulum. ConclusionsTaken together, these observations unmasked a complex role of GHSR in dopamine neuronal protection independent on electric activity of these cells and revealed a crucial role for mitochondrial dynamics in some aspects of this process.

Ghrelin transport across the blood–brain barrier can occur independently of the growth hormone secretagogue receptor

ObjectiveThe blood–brain barrier (BBB) regulates the entry of substrates and peptides into the brain. Ghrelin is mainly produced in the stomach but exerts its actions in the central nervous system (CNS) by crossing the BBB. Once present in the CNS, ghrelin can act in the hypothalamus to regulate food intake, in the hippocampus to regulate neurogenesis, and in the olfactory bulb to regulate food-seeking behavior. The goal of this study was to determine whether the primary signaling receptor for ghrelin, the growth hormone secretagogue receptor (GHSR), mediates the transport of ghrelin from blood to brain. MethodsWe utilized the sensitive and quantitative multiple-time regression analysis technique to determine the transport rate of mouse and human acyl ghrelin (AG) and desacyl ghrelin (DAG) in wildtype and Ghsr null mice. We also measured the regional distribution of these ghrelin peptides throughout the brain. Lastly, we characterized the transport characteristics of human DAG by measuring the stability in serum and brain, saturability of transport, and the complete transfer across the brain endothelial cell. ResultsWe found the transport rate across the BBB of both forms of ghrelin, AG, and DAG, were not affected by the loss of GHSR. We did find differences in the transport rate between the two isoforms, with DAG being faster than AG; this was dependent on the species of ghrelin, human being faster than mouse. Lastly, based on the ubiquitous properties of ghrelin throughout the CNS, we looked at regional distribution of ghrelin uptake and found the highest levels of uptake in the olfactory bulb. ConclusionsThe data presented here suggest that ghrelin transport can occur independently of the GHSR, and ghrelin uptake varies regionally throughout the brain. These findings better our understanding of the gut-brain communication and may lead to new understandings of ghrelin physiology.

The protective role of human ghrelin in sepsis: Restoration of CD4 T cell proliferation

Decrease of CD4 T cell numbers causes immunosuppression in sepsis. We previously showed the beneficial role of ghrelin in sepsis. We hypothesize that the protective outcome of ghrelin in sepsis is mediated partially through the restoration of CD4 T cells’ proliferation. Sepsis was induced in mice by cecal ligation and puncture (CLP). The percentage of CD4 T cells in spleen was assessed by flow cytometry and their proliferation was determined by carboxyfluorescein succinimidyl ester (CSFE). Compared to sham mice, the percentages of splenic CD4 T cells were reduced by 20%, 21%, and 29% at day 1, 2 and 3 after CLP, respectively. Human ghrelin was given to 3 day septic mice by s.c. injection at 5 and 24 h after CLP. Treatment with ghrelin restored the loss of CD4 T cells by increasing their proliferation in septic mice. The expression of cyclin D1 and B1 was significantly increased, while the expression of p57 was decreased in ghrelin-treated mice compared to vehicle-treated mice in sepsis. Treatment with human ghrelin significantly increased the p-AKT levels in the spleen compared to vehicle-treated septic mice. Human ghrelin plays an important role in reestablishing the proliferation of CD4 T cells and serves as a promising therapeutic agent in sepsis.

A Ketone Ester Drink Lowers Human Ghrelin and Appetite.

ObjectiveThe ketones d‐β‐hydroxybutyrate (BHB) and acetoacetate are elevated during prolonged fasting or during a “ketogenic” diet. Although weight loss on a ketogenic diet may be associated with decreased appetite and altered gut hormone levels, it is unknown whether such changes are caused by elevated blood ketones. This study investigated the effects of an exogenous ketone ester (KE) on appetite.MethodsFollowing an overnight fast, subjects with normal weight (n = 15) consumed 1.9 kcal/kg of KE, or isocaloric dextrose (DEXT), in drinks matched for volume, taste, tonicity, and color. Blood samples were analyzed for BHB, glucose, insulin, ghrelin, glucagon‐like peptide 1 (GLP‐1), and peptide tyrosine tyrosine (PYY), and a three‐measure visual analogue scale was used to measure hunger, fullness, and desire to eat.ResultsKE consumption increased blood BHB levels from 0.2 to 3.3 mM after 60 minutes. DEXT consumption increased plasma glucose levels between 30 and 60 minutes. Postprandial plasma insulin, ghrelin, GLP‐1, and PYY levels were significantly lower 2 to 4 hours after KE consumption, compared with DEXT consumption. Temporally related to the observed suppression of ghrelin, reported hunger and desire to eat were also significantly suppressed 1.5 hours after consumption of KE, compared with consumption of DEXT.ConclusionsIncreased blood ketone levels may directly suppress appetite, as KE drinks lowered plasma ghrelin levels, perceived hunger, and desire to eat.

Is there a hormonal regulation of phagocytosis at unicellular and multicellular levels? A critical review.

Phagocytosis is an ancient cell function, which is similar at unicellular and multicellular levels. Unicells synthesize, store, and secrete multicellular (mammalian) hormones, which influence their phagocytosis. Amino acid hormones, such as histamine, serotonin, epinephrine, and melatonin stimulate phagocytosis, whereas peptide hormones, such as adrenocorticotropic hormone (ACTH), insulin, opioids, arginine vasopressin, and atrial natriuretic peptide decreased it, independently on their chemical structure or function in multicellulars. Macrophage phagocytosis of multicellulars is also stimulated by amino acid hormones, such as histamine, epinephrine, melatonin, and thyroid hormones, however, the effect of peptide hormones is not uniform: prolactin, insulin, glucagon, somatostatin, and leptin have positive effects, whereas ACTH, human chorionic gonadotropin, opioids, and ghrelin have negative ones. Steroid hormones, such as estrogen, hydrocortisone, and dexamethasone are stimulating macrophage phagocytosis, whereas progesterone, aldosterone, and testosterone are depressing it. Considering the data and observations there is not a specific phagocytosis hormone, or a hormonal regulation of phagocytosis neither unicellular, nor multicellular level, however, hormones having specific functions in multicellulars also influence phagocytosis at both levels universally (in unicellulars) or individually (in macrophages). Nevertheless, the hormonal influence cannot be neglected, as phagocytosis (as a function) is rather sensitive to minute dose of hormones and endocrine disruptors. The hormonal influence of phagocytosis by macrophages can be deduced to the events at unicellular level.

Assessment of Serum Level of Ghrelin in Patients with Chronic Obstructive Pulmonary Disease

Objectives: to study the relationship between serum level of ghrelin and cachexia in patients with COPD. Background: Chronic obstructive pulmonary disease is a major cause of chronic morbidity and mortality throughout the world. COPD involves several systemic features. The link between ghrelin, cachexia and COPD needs to be clarified.Materials and methods: Serum levels of ghrelin were measured in 60 COPD patients (divided into 3 groups according to their BMI) and 30 control subjects using human ghrelin ELISA kits. Results: There was a significant increase in ghrelin level in COPD patients compared to control subjects. There was significant increase in serum ghrelin level in underweight COPD subgroup compared to normal weight and overweight subgroups. Serum levels of ghrelin in COPD patients were affected by the level of pulmonary obstruction. Conclusions: Serum level of ghrelin is increased in COPD patients, and is positively correlated with BMI and disease severity.

Z-505 hydrochloride, an orally active ghrelin agonist, attenuates the progression of cancer cachexia via anabolic hormones in Colon 26 tumor-bearing mice

Cancer cachexia is a progressive wasting syndrome characterized by anorexia and weight loss, specifically muscle wasting and fat depletion. There is no therapeutic agent for treatment of this syndrome. We investigated the anti-cachexia effects of Z-505 hydrochloride (Z-505), a new oral growth hormone secretagogue receptor 1a (GHSR1a) agonist, using a mouse model of cancer cachexia. We performed a calcium flux assay in Chinese hamster ovary (CHO-K1) cells stably expressing human GHSR1a to quantify the agonistic activity of Z-505. In Colon 26 tumor-bearing mice, Z-505 (300mg/kg, p.o., twice daily) was administered for 7 days to assess its anti-cachexia effects. Body weight and food intake were monitored during the period, and the skeletal muscle and epididymal fat weights were measured. Serum levels of insulin, insulin-like growth factor 1 (IGF-1), interleukin-6 (IL-6), and corticosterone were measured to confirm the mechanism of the anti-cachexia action of Z-505. Z-505 showed strong agonistic activity similar to that of human ghrelin, with a half maximal effective concentration (EC50) value of 0.45nM. Z-505 treatment significantly increased food intake and inhibited the progression of weight loss. Z-505 also significantly attenuated muscle wasting and fat loss, and increased circulating levels of anabolic factors such as insulin and IGF-1, but not catabolic factors such as IL-6 and corticosterone. These findings suggest that Z-505 might be effective in the treatment of cachexia via the increased anabolic hormone levels stimulated by the activation of the ghrelin receptor, GHSR1a.

MicroRNA in hormonal mechanisms of regulation of NK cell function

We investigated the effect of human chorionic gonadotropin, estriol, leptin, ghrelin, and kisspeptin on the microRNA expression in separated NK cells. All of these hormones are able to effectively modulate the expression of microRNAs both stimulating and suppressing the cytotoxic potential of NK cells and thereby indirectly regulate the functions of these lymphocytes.

Synthetic Triterpenoid Inhibition of Human Ghrelin O-Acyltransferase: The Involvement of a Functionally Required Cysteine Provides Mechanistic Insight into Ghrelin Acylation.

The peptide hormone ghrelin plays a key role in regulating hunger and energy balance within the body. Ghrelin signaling presents a promising and unexploited target for development of small molecule therapeutics for treatment of obesity, diabetes, and other health conditions. Inhibition of ghrelin O-acyltransferase (GOAT), which catalyzes an essential octanoylation step in ghrelin maturation, offers a potential avenue for controlling ghrelin signaling. Through screening a small molecule library, we have identified a class of synthetic triterpenoids that efficiently inhibit ghrelin acylation by the human isoform of GOAT (hGOAT). These compounds function as covalent reversible inhibitors of hGOAT, providing the first evidence of the involvement of a nucleophilic cysteine residue in substrate acylation by a MBOAT family acyltransferase. Surprisingly, the mouse form of GOAT does not exhibit susceptibility to cysteine-modifying electrophiles, revealing an important distinction in the activity and behavior between these closely related GOAT isoforms. This study establishes these compounds as potent small molecule inhibitors of ghrelin acylation and provides a foundation for the development of novel hGOAT inhibitors as therapeutics targeting diabetes and obesity.

Therapeutic effect of human ghrelin and growth hormone: Attenuation of immunosuppression in septic aged rats

Sepsis is a leading cause of mortality in intensive care units, and is more common in the geriatric population. The control of hyperinflammation has been suggested as a therapeutic approach in sepsis, but to date clinical trials utilizing this strategy have not lead to an effective treatment. In addition to hyperinflammation, patients with sepsis often experience a state of immunosuppression, which serves as an important determinant for increased morbidity and mortality. We previously used aged animals to demonstrate the effectiveness of combined treatment with human ghrelin (Ghr) and human growth hormone (GH) in improving organ injury and survival in septic animals. Here, we hypothesized that combined treatment with Ghr and GH could improve immune function in septic aged animals. Male 24-month-old rats were subjected to cecal ligation and puncture (CLP) for sepsis induction. Human Ghr (80nmol/kg BW) plus GH (50μg/kg BW) or vehicle (normal saline) was administrated subcutaneously at 5h after CLP. The ex vivo production of TNF-α, IL-6 and IL-10 to LPS-stimulation, as well as TNF-α, IL-6, IL-10 and IFN-γ production to anti-CD3/anti-CD28 antibody-stimulation, in splenocytes isolated 20h after CLP, was significantly decreased compared to production of these cytokines in splenocytes from sham animals. The production of cytokines from splenocytes isolated from septic animals that received the combined treatment, however, was significantly higher than from those isolated from vehicle-treated septic animals. Combined treatment prevented the loss of splenic CD4+ and CD8+ T cells in septic aged rats, and reduced lymphocyte apoptosis. Combined treatment also inhibited an increase in the regulatory T cell (Treg) population and expression of the immune co-inhibitory molecule PD-1 in the spleens of septic aged rats. In contrast, expression of HLA-DR was increased after combined treatment with Ghr and GH. Based on these findings, we conclude that co-administration of Ghr and GH is a promising therapeutic tool for reversing immunosuppression caused by sepsis in the geriatric population. This article is part of a Special Issue entitled: Immune and Metabolic Alterations in Trauma and Sepsis edited by Dr. Raghavan Raju.

Clinical application of ghrelin for diabetic peripheral neuropathy.

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.

Influence of ghrelin on rat pituitary GH3 cell line proliferation

Introduction. Human ghrelin is the endogenous ligand of the growth hormone secretagogue receptor type 1a (GHSR1a). It is suggested that ghrelin is involved in pituitary adenomas pathogenesis. There are inconsistent data regarding the effect of ghrelin on cell proliferation. In this study the outcome of ghrelin in the rat pituitary adenoma GH3 cell line on morphology and proliferation ratio was evaluated. The ghrelin receptor (Ghsr) mRNA expression in GH3 cell line was established as well, because it was found that heterogeneous expression pattern characterized physiological and pathological conditions of tissues of different origin.Material and Methods. Suitable experimental model pituitary tumor (rat GH3 cell line) was stimulated with ghrelin in the final concentrations 10–12 M, 10–9 M and 10–6 M. Reverse transcription followed by real time polymerase chain reaction was used for ghrelin receptor gene transcript detection. The morphology as well as cell cycle of those cells were analyzed using Axio Vert.A1 Microscope (Zeiss) and BD FACSCalibur™ flow cytometer (Beckton Dickinson), respectively. The percentages of cells in the G0/G1, S, G2/M cycle phases were evaluated using the ModFit™ software (Verity Software, Inc., USA). Results. Ghsr mRNA presence was confirmed in GH3 cells. Ghrelin did not affect conspicuously GH3 cells morphology, however the ghrelin‑induced proliferation index increase was caused by both decline of G0/G1 phases cells count and increase those being in S+G2/M (p &lt; 0.05). Conclusions. In conclusion, this study indicates that ghrelin stimulates GH3 cells proliferation and may play role in pituitary tumorigenesis via an autocrine/paracrine pathway.

Influence of ghrelin on rat pituitary GH3 cell line proliferation

Introduction. Human ghrelin is the endogenous ligand of the growth hormone secretagogue receptor type 1a (GHSR1a). It is suggested that ghrelin is involved in pituitary adenomas pathogenesis. There are inconsistent data regarding the effect of ghrelin on cell proliferation. In this study the outcome of ghrelin in the rat pituitary adenoma GH3 cell line on morphology and proliferation ratio was evaluated. The ghrelin receptor (Ghsr) mRNA expression in GH3 cell line was established as well, because it was found that heterogeneous expression pattern characterized physiological and pathological conditions of tissues of different origin.Material and Methods. Suitable experimental model pituitary tumor (rat GH3 cell line) was stimulated with ghrelin in the final concentrations 10–12 M, 10–9 M and 10–6 M. Reverse transcription followed by real time polymerase chain reaction was used for ghrelin receptor gene transcript detection. The morphology as well as cell cycle of those cells were analyzed using Axio Vert.A1 Microscope (Zeiss) and BD FACSCalibur™ flow cytometer (Beckton Dickinson), respectively. The percentages of cells in the G0/G1, S, G2/M cycle phases were evaluated using the ModFit™ software (Verity Software, Inc., USA). Results. Ghsr mRNA presence was confirmed in GH3 cells. Ghrelin did not affect conspicuously GH3 cells morphology, however the ghrelin-induced proliferation index increase was caused by both decline of G0/G1 phases cells count and increase those being in S+G2/M (p &lt; 0.05). Conclusions. In conclusion, this study indicates that ghrelin stimulates GH3 cells proliferation and may play role in pituitary tumorigenesis via an autocrine/paracrine pathway.

Effects of ghrelin receptor agonist, relamorelin, on gastric motor functions and satiation in healthy volunteers.

Synthetic human ghrelin accelerates gastric emptying, reduces gastric accommodation, and results in numerical increases in postprandial symptom scores. The ghrelin receptor agonist, relamorelin, accelerates gastric emptying in patients with diabetic gastroparesis. To measure pharmacological effects of relamorelin on gastric accommodation, distal antral motility, and satiation in healthy volunteers. In a placebo-controlled, double-blind, randomized study of 16 healthy volunteers, we compared effects of 30 μg subcutaneous (s.c.) relamorelin to placebo on: (i) gastric volumes measured by single photon emission computed tomography, (ii) 1-h postprandial distal antral motility index (MI) by 15-lumen perfusion gastroduodenal manometry, and (iii) satiation tested by Ensure nutrient drink test. Primary endpoints were: fasting and postprandial gastric volumes, distal antral phasic pressure activity (number of contractions, mean amplitude, and MI), and maximum tolerated volume. Results were normally distributed and the two treatment groups were compared using t-test. Relamorelin, 30 μg s.c., significantly increased the number of contractions in the distal antrum during 0-60 min postmeal when compared to placebo (p = 0.022); this was also observed in the first two 15-min periods (p = 0.005 and 0.015 for number of contractions 0-15 and 16-30). There was borderline increase in MI0-15 (p = 0.055) and numerically increased MI0-60 (p = 0.139) and MI16-30 (p = 0.116). The amplitude of contractions was not significantly increased. Relamorelin did not significantly alter fasting or postprandial gastric volumes, gastric accommodation, or satiation volumes and symptoms. Relamorelin increases frequency of distal antral motility contractions without significant effects on amplitude of contractions. The lack of inhibition of accommodation and absence of increase in satiation symptoms support relamorelin for the treatment of symptomatic gastroparesis (ClinicalTrials.gov NCT02466711).

Endothelial Dysfunction in Resistance Arteries of Hypertensive Humans: Old and New Conspirators.

A large body of homogeneous reports documented that endothelial dysfunction, which characterizes human essential hypertension is largely dependent on an impaired endothelial nitric oxide availability and an increased production of endothelium-derived contracting factors. These factors include endothelin, prostanoids, and reactive oxygen species (ROS). In particular, it was evidenced that acute intraarterial administration of indomethacin, a nonselective cyclooxygenase (COX)-inhibitor, and ascorbic acid, an antioxidant, normalized the blunted endothelial dysfunction by restoring nitric oxide availability at the level of peripheral microcirculation, thus demonstrating that COX-derived endothelium-derived contracting factors are one of the major sources of ROS. Recent studies put in evidence new lights on the mechanisms involved in endothelial dysfunction in human hypertension, identified as "new conspirators." In particular, functional and immunohistochemical experiments with selective COX inhibitors identified the isoform COX-2 as the main source of intravascular ROS generation in isolated small vessels from essential hypertensive patients. In addition, important vascular protective properties by human ghrelin have been demonstrated in human hypertension, in terms that its systemic reduction is involved in the pathophysiology of endothelial dysfunction, while a normalization of its levels may restore vascular homeostasis.

Effects of ghrelin in energy balance and body weight homeostasis.

Ghrelin is a gut peptide composed of 28 amino acids mostly secreted in the gastric fundus mucosa. It was isolated and described in 1999 by Kojima et al. and only three years later its specific receptor, GHSR1a, was also identified. Ghrelin, the endogenous ligand for the GH secretagogue receptor, is the only peripheral orexigenic hormone that activates the receptors to be found especially in the appetite center (hypothalamus and pituitary gland). Ghrelin is present in human plasma in two forms: an inactive form known as deacylated ghrelin, and an active form called acylated ghrelin synthesized under the action of ghrelin O-acyltransferase enzyme (GOAT). The literature even mentions an extremely complex ghrelin/GOAT/GHSR system involved in the regulation of human energy, metabolism and adaptation of energy homeostasis to environmental changes. In humans, there is a preprandial rise and a postprandial fall in plasma ghrelin levels, which strongly suggest that the peptide plays a physiological role in meal initiation and may be employed in determining the amount and quality of ingested food. Besides the stimulation of food intake, ghrelin determines a decrease in energy expenditure and promotes the storage of fatty acids in adipocytes. Thus, in the human body ghrelin induces a positive energy balance, an increased adiposity gain, as well as an increase in caloric storage, seen as an adaptive mechanism to caloric restriction conditions. In the current world context, when we are witnessing an increasing availability of food and a reduction of energy expenditure to a minimum level, these mechanisms have become pathogenic. As a consequence, the hypothesis that ghrelin is involved in the current obesity epidemic has been embraced by many scholars and researchers.

Mole ghrelin: cDNA cloning, gene expression, and diverse molecular forms in Mogera imaizumii

Here, we describe cDNA cloning and purification of the ghrelin gene sequences and ghrelin peptides from the Japanese true mole, Mogera imaizumii. The gene spans >2.9kbp, has four exons and three introns, and shares structural similarity with those of terrestrial animals. Mature mole ghrelin peptide was predicted to be 28 amino acids long (GSSFLSPEHQKVQQRKESKKPPSKPQPR) and processed from a prepropeptide of 116 amino acids. To further elucidate molecular characteristics, we purified ghrelin peptides from mole stomach. By mass spectrometry, we found that the mole ghrelin peptides had higher ratios of the odd-number fatty acids (C9 and C11 as much as C8) attached to the third serine residue than other vertebrate ghrelin. Truncated forms of ghrelins such as [1–27], [1–19], [1–16] and [1–15], and that lacked the 14th glutamine residue (des-Gln14 ghrelin) were produced in the stomach. Marked expression of ghrelin mRNA in lung was observed as in stomach and brain. Phylogenetic analysis indicated that the branch of M. imaizumii has slightly higher dN/dS ratios (the nucleotide substitution rates at non-synonymous and synonymous sites) than did other eulipotyphlans. Peptide length was positively correlated with human ghrelin receptor activation, whereas the length of fatty-acyl chains showed no obvious functional correlation. The basal higher luciferase activities of the 5′-proximal promoter region of mole ghrelin were detected in ghrelin-negative C2C12 cells and hypoxic culture conditions impaired transcriptional activity. These results indicated that moles have acquired diverse species of ghrelin probably through distinctive fatty acid metabolism because of their food preferences. The results provide a gateway to understanding ghrelin metabolism in fossorial animals.