Lower ghrelin levels does not impact the metabolic benefit induced by Roux-en-Y gastric bypass.

Objective: Roux-en-Y gastric bypass is an effective intervention for metabolic disorder. We aim to elucidate whether ghrelin contributes to weight reduction, and glycemic and lipid control after Roux-en-Y gastric bypass (RYGB). Design: Four-week-old WT and Ghrl-TSC1-/- mice were fed high fat diet for 12 weeks before surgery, and continued to be on the same diet for 3 weeks after surgery. Body weight, food intake, glycemic and lipid metabolism were analyzed before and after surgery. Results: Gastric and circulating ghrelin was significantly increased in mice with RYGB surgery. Hypoghrelinemia elicited by deletion of TSC1 to activate mTOR signaling in gastric X/A like cells demonstrated no effect on weight reduction, glycemic and lipid control induced by Roux-en-Y gastric bypass surgery. Conclusion: Lower ghrelin levels does not impact the metabolic benefit induced by Roux-en-Y gastric bypass.

Traceable Peptidic Ligands that Target Ghrelin Receptors.

Ghrelin is a 28-amino acid octanoylated peptide hormone that is implicated in many physiological and pathophysiological processes. Specific visualization of ghrelin and its cognate receptor using traceable ligands is crucial in elucidating the localization, functions, and expression pattern of the peptide's signaling pathway. Here 12 representative radio- and fluorescently-labeled peptide-based ligands are reviewed for in vitro and in vivo imaging studies. In particular, the focus is on their structural features, pharmacological properties, and applications in further biochemical research.

Three-dimensional growth of breast cancer cells potentiates the anti-tumor effects of unacylated ghrelin and AZP-531.

Breast cancer is the most common type of cancer in women and notwithstanding important therapeutic advances, remains the second leading cause of cancer-related death. Despite extensive research relating to the hormone ghrelin, responsible for the stimulation of growth hormone release and appetite, little is known of the effects of its unacylated form, especially in cancer. The present study aimed to characterize effects of unacylated ghrelin on breast cancer cells, define its mechanism of action, and explore the therapeutic potential of unacylated ghrelin or analog AZP-531. We report potent anti-tumor effects of unacylated ghrelin, dependent on cells being cultured in 3D in a biologically-relevant extracellular matrix. The mechanism of unacylated ghrelin-mediated growth inhibition involves activation of Gαi and suppression of MAPK signaling. AZP-531 also suppresses the growth of breast cancer cells in vitro and in xenografts, and may be a novel approach for the safe and effective treatment of breast cancer.

LC-MS/MS assay coupled with carboxylic acid magnetic bead affinity capture to quantitatively measure cationic host defense peptides (HDPs) in complex matrices with application to preclinical pharmacokinetic studies.

Synthetic host defense peptides (HDP) are a new class of promising therapeutic agents with potential application in a variety of diseases. RP-182 is a 10mer synthetic HDP design, which selectively reduces M2-like tumor associated macrophages via engagement with the cell surface lectin receptor MRC1/CD206 and is currently being developed as an innate immune defense regulator to improve anti-tumor immunity in immunologically cold tumors. Herein, we describe a sensitive and specific liquid chromatography (LC) coupled to quadrupole electron spray tandem mass spectrometry method to measure positively charged HDPs and HDP peptide fragments in complex biological matrices. Carboxylic acid magnetic beads were used as an affinity-capturing agent to extract the positively charged RP-182 from both mouse plasma and tissue homogenates. Beads were eluted with 0.1% (v/v) formic acid and chromatographic separation was achieved on a Waters 2.1 × 100 mm, 3.5 µm XSelect Peptide CSH C18 column with a Vanguard pre-column of the same phase. MS/MS was performed on a Thermo TSQ Quantiva triple quadrupole mass spectrometer operating in Selected Reaction Monitoring (SRM) mode fragmenting the plus three parent ion 458.9+3 and monitoring ions 624.0+2, 550.5+2, and 597.3+1 for RP-182 and 462.4+3 > 629.1+2, 555.5+2, and 607.3+1 for isotopic RP-182 standard. The assay had good linearity ranging from 1 ng to 1000 ng in mouse plasma with the lower limit of detection for RP-182 at 1 ng in mouse plasma with good intra- and inter-sample precision and accuracy. Recovery ranged from 66% to 77% with minimum matrix effects. The method was successfully applied to an abbreviated pharmacokinetic study in mice after single IP injection of RP-182. The method was successfully tested on a second HDP, the 17mer D4E1, and the cationic human peptide hormone ghrelin suggesting that it might be a general sensitive method applicable to quantifying HDP peptides that are difficult to extract.

Current and emerging therapies for managing hyperphagia and obesity in Prader-Willi syndrome: A narrative review.

In early childhood, individuals with Prader-Willi syndrome (PWS) experience excess weight gain and severe hyperphagia with food compulsivity, which often leads to early onset morbid obesity. Effective treatments for appetite suppression and weight control are currently unavailable for PWS. Our aim to further understand the pathogenesis of PWS led us to carry out a comprehensive search of the current and emerging therapies for managing hyperphagia and extreme weight gain in PWS. A literature search was performed using PubMed and the following keywords: "PWS" AND "therapy" OR "[drug name]"; reference lists, pharmaceutical websites, and the ClinicalTrials.gov registry were also reviewed. Articles presenting data from current standard treatments in PWS and also clinical trials of pharmacological agents in the pipeline were selected. Current standard treatments include dietary restriction/modifications, exercise, and growth hormone replacement, which appear to have limited efficacy for appetite and weight control in patients with PWS. The long-term safety and effectiveness of bariatric surgery in PWS remains unknown. However, many promising pharmacotherapies are in development and, if approved, will bring much needed choices into the PWS pharmacological armamentarium. With the progress that is currently being made in our understanding of PWS, an effective treatment may not be far off.

Role of ghrelin isoforms in the mitigation of hepatic inflammation, mitochondrial dysfunction, and endoplasmic reticulum stress after bariatric surgery in rats.

BACKGROUND/OBJECTIVES: Bariatric surgery improves nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), but the underlying mechanisms remain elusive. We evaluated the potential role of ghrelin isoforms in the amelioration of hepatic inflammation after sleeve gastrectomy and Roux-en-Y gastric bypass (RYGB). SUBJECTS/METHODS: Plasma ghrelin isoforms were measured in male Wistar rats (n = 129) subjected to surgical (sham operation, sleeve gastrectomy, or RYGB) or dietary interventions [fed ad libitum a normal (ND) or a high-fat diet (HFD) or pair-fed diet]. The effect of acylated and desacyl ghrelin on markers of inflammation, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress in primary rat hepatocytes under palmitate-induced lipotoxic conditions was assessed. RESULTS: Plasma desacyl ghrelin was decreased after sleeve gastrectomy and RYGB, whereas the acylated/desacyl ghrelin ratio was augmented. Both surgeries diminished obesity-associated hepatic steatosis, CD68+- and apoptotic cells, proinflammatory JNK activation, and Crp, Tnf, and Il6 transcripts. Moreover, a postsurgical amelioration in the mitochondrial DNA content, oxidative phosphorylation (OXPHOS) complexes I and II, and ER stress markers was observed. Specifically, following bariatric surgery GRP78, spliced XBP-1, ATF4, and CHOP levels were reduced, as were phosphorylated eIF2α. Interestingly, acylated and desacyl ghrelin inhibited steatosis and inflammation of palmitate-treated hepatocytes in parallel to an upregulation of OXPHOS complexes II, III, and V, and a downregulation of ER stress transducers IRE1α, PERK, ATF6, their downstream effectors, ATF4 and CHOP, as well as chaperone GRP78. CONCLUSIONS: Our data suggest that the increased relative acylated ghrelin levels after bariatric surgery might contribute to mitigate obesity-associated hepatic inflammation, mitochondrial dysfunction, and ER stress.

Ghrelin effects on mitochondrial fitness modulates macrophage function.

Over the past years, systemic derived cues that regulate cellular metabolism have been implicated in the regulation of immune responses. Ghrelin is an orexigenic hormone produced by enteroendocrine cells in the gastric mucosa with known immunoregulatory roles. The mechanism behind the function of ghrelin in immune cells, such as macrophages, is still poorly understood. Here, we explored the hypothesis that ghrelin leads to alterations in macrophage metabolism thus modulating macrophage function. We demonstrated that ghrelin exerts an immunomodulatory effect over LPS-activated peritoneal macrophages, as evidenced by inhibition of TNF-α and IL-1ß secretion and increased IL-12 production. Concomitantly, ghrelin increased mitochondrial membrane potential and increased respiratory rate. In agreement, ghrelin prevented LPS-induced ultrastructural damage in the mitochondria. Ghrelin also blunted LPS-induced glycolysis. In LPS-activated macrophages, glucose deprivation did not affect ghrelin-induced IL-12 secretion, whereas the inhibition of pyruvate transport and mitochondria-derived ATP abolished ghrelin-induced IL-12 secretion, indicating a dependence on mitochondrial function. Ghrelin pre-treatment of metabolic activated macrophages inhibited the secretion of TNF-α and enhanced IL-12 levels. Moreover, ghrelin effects on IL-12, and not on TNF-α, are dependent on mitochondria elongation, since ghrelin did not enhance IL-12 secretion in metabolic activated mitofusin-2 deficient macrophages. Thus, ghrelin affects macrophage mitochondrial metabolism and the subsequent macrophage function.

Biochemical Assays for Ghrelin Acylation and Inhibition of Ghrelin O-Acyltransferase.

Ghrelin O-acyltransferase (GOAT) is an enzyme responsible for octanoylating and activating ghrelin, a peptide hormone that plays a key role in energy regulation and hunger signaling. Due to its nature as an integral membrane protein, GOAT has yet to be purified in active form which has complicated biochemical and structural studies of GOAT-catalyzed ghrelin acylation. In this chapter, we describe protocols for efficient expression and enrichment of GOAT in insect cell-derived microsomal fraction, HPLC-based assays for GOAT acylation activity employing fluorescently labeled peptides, and assessment of inhibitor potency against GOAT.

Development and Characterization of an 18F-labeled Ghrelin Peptidomimetic for Imaging the Cardiac Growth Hormone Secretagogue Receptor.

One-third of patients with heart disease develop heart failure, which is diagnosed through imaging and detection of circulating biomarkers. Imaging strategies reveal morphologic and functional changes but fall short of detecting molecular abnormalities that can lead to heart failure, and circulating biomarkers are not cardiac specific. Thus, there is critical need for biomarkers that are endogenous to myocardial tissues. The cardiac growth hormone secretagogue receptor 1a (GHSR1a), which binds the hormone ghrelin, is a potential biomarker for heart failure. We have synthesized and characterized a novel ghrelin peptidomimetic tracer, an 18F-labeled analogue of G-7039, for positron emission tomography (PET) imaging of cardiac GHSR1a. In vitro analysis showed enhanced serum stability compared to natural ghrelin and significantly increased cellular uptake in GHSR1a-expressing OVCAR cells. Biodistribution studies in mice showed that tissue uptake of the tracer was independent of circulating ghrelin levels, and there was negligible cardiac uptake and high uptake in the liver, intestines, and kidneys. Specificity of tracer uptake was assessed using ghsr -/- mice; both static and dynamic PET imaging revealed no difference in cardiac uptake, and there was no significant correlation between cardiac standardized uptake values and GHSR1a expression. Our study lays the groundwork for further refinement of peptidomimetic PET tracers targeting cardiac GHSR1a.

Effect of HTST and Holder Pasteurization on the Concentration of Immunoglobulins, Growth Factors, and Hormones in Donor Human Milk.

Donor human milk (DHM) is submitted to Holder pasteurization (HoP) to ensure its microbiological safety in human milk banks but this treatment affects some of its bioactive compounds. The objective of this work was to compare the effects of HoP and high temperature short time (HTST) treatments on some bioactive compounds found in DHM. A total of 24 DHM batches were processed in a continuous HTST system (70, 72, and 75°C for 5-25 s) and by HoP (62.5°C for 30 min). The concentrations of immunoglobulins (Igs) A, G, and M, transforming growth factor-beta 2 (TGF-ß2), adiponectine, ghrelin, and leptin were measured using a multiplex system, whereas the concentration of epidermal growth factor (EGF) was determined by ELISA. In relation to Igs, IgG showed the highest preservation rates (87-101%) after HTST treatments, followed by IgA (54-88%) and IgM (25-73%). Ig retention after any of the HTST treatments was higher than after HoP (p < 0.001). Treatment times required to reduce the concentration of IgM by 90% (D-value) were 130, 88, and 49 s at 70, 72, and 75°C, while the number of degrees Celsius required to change the D-value by one factor of 10 (z-value) was 11.79°C. None of the heat treatments had a significant effect on the concentrations of TGF-ß2, EGF, adiponectin, and ghrelin. In contrast, leptin was detected only in 4 of the samples submitted to HoP, whereas it was present in all samples after the different HTST treatments, with retention rates ranging between 34 and 68%. Globally, the concentration of IgA, IgG, IgM, and leptin in DHM was significantly higher after HTST pasteurization performed in a continuous system designed to be used in human milk banks than after the HoP procedure that is routinely applied at present.

[DTPA-(PABn)-Leu5]-des-acyl ghrelin(1-5) as a new carrier of radionuclides and potential precursor of radiopharmaceuticals.

BACKGROUND: Ghrelin is a peptide consisting of 28 aminoacids and an octadecyl side chain (acyl group) binding to the growth hormone secretagogue receptor type 1a (GHS-R1a). Its des-acylated form, des-acyl ghrelin (DAG) binds to the corticotropin releasing factor receptor type 2a (CRF2a) located on endocrine cancer cells such as the prostate carcinoma cell line DU 145. AIM: The aim of this study is to develop a new DAG-based carrier of radionuclides with potential application in therapy. MATERIALS AND METHODS: Trunctated C-terminal five aminoacids chain of the DAG peptide (H2N-Gly-Ser-Ser-Phe-Leu-COOH) was linked to DTPA to obtain [DTPA-(PABn)-Leu5]-DAG(1-5). For therapeutic application the lutetium-177 (177Lu) radionuclide was coordinated to the peptide. To determine biological and chemical properties of newly synthesized radiopharmaceutical, two iodine-131 (131I)-labelled compounds were used: [131I]-Tyr4-DAG(1-5) and full length [131I]-DAG(1-28) together with their nonradioactive forms: DAG(1-28) and DAG(1-5). RESULTS: Identical HPLC elution profiles of [177Lu-DTPA-(PABn)-Leu5]-DAG(1-5) before and after incubation with human serum proved its stability. The lipophilicity profile of [177Lu-DTPA-(PABn)-Leu5]-DAG(1-5) was log DO/W=-2.68±0.05, pH 7.4. Receptor affinity of the nonradioactive conjugate [Lu-DTPA-(PABn)-Leu5]-DAG(1-5) was IC50 (21.06 nmol/l), as shown against the [131I]-DAG(1-28) used as a competitor. The 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide assay indicated the significant cytotoxicity of the newly synthesized compounds, similar to that of [131I]-Tyr4-DAG(1-5). CONCLUSION: The results obtained suggest the potency of the [DTPA-(PABn)-Leu5]-DAG(1-5) as a new carrier of radionuclides in radiopharmacy.

Acylated and unacylated ghrelin inhibit apoptosis in myoblasts cocultured with colon carcinoma cells.

Cancer cachexia is a life­threatening syndrome associated with myofiber damage. Tumor factors impair muscle regeneration by promoting myoblast apoptosis. Ghrelin is a multifunctional hormone with an anti­apoptotic effect, but its mechanism of action is not fully understood. In the present study, we investigated whether the coculturing of C2C12 myoblasts with CT26 colon carcinoma cells may induce myoblast apoptosis, and whether acylated ghrelin (AG) and unacylated ghrelin (UnAG) may exert anti­apoptotic effects. We found that the coculture induced myoblast apoptosis and increased tumor necrosis factor (TNF)­α concentrations in the culture medium. Moreover, the coculture increased c­Jun N­terminal kinase (JNK) activity, suppressed Akt activity, increased the mitochondrial Bax/Bcl­2 ratio, impaired mitochondrial membrane potential (Δψm), increased the cytosolic cytochrome c levels, and activated the caspase­3/poly (ADP­ribose) polymerase (PARP) cascade in myoblasts. We also found that either AG or UnAG inhibited these changes. The present study describes a novel in vitro model that can be employed to investigate cancer­induced myoblast apoptosis, and our findings suggest a possible use for AG and UnAG in treating cancer cachexia.

Development of coated liposomes loaded with ghrelin for nose-to-brain delivery for the treatment of cachexia.

The aim of the present study was to develop a ghrelin-containing formulation based on liposomes coated with chitosan intended for nose-brain delivery for the treatment of cachexia. Among the three types of liposomes developed, anionic liposomes provided the best results in terms of encapsulation efficiency (56%) and enzymatic protection against trypsin (20.6% vs 0% for ghrelin alone) and carboxylesterase (81.6% vs 17.2% for ghrelin alone). Ghrelin presented both electrostatic and hydrophobic interactions with the anionic lipid bilayer, as demonstrated by isothermal titration calorimetry. Then, anionic liposomes were coated with N-(2-hydroxy) propyl-3-trimethyl ammonium chitosan chloride. The coating involved a size increment from 146.9±2.7 to 194±6.1 nm, for uncoated and coated liposomes, respectively. The ζ-potential was similarly increased from -0.3±1.2 mV to 6±0.4 mV before and after coating, respectively. Chitosan provided mucoadhesion, with an increase in mucin adsorption of 22.9%. Enhancement of permeation through the Calu3 epithelial monolayer was also observed with 10.8% of ghrelin recovered in the basal compartment in comparison to 0% for ghrelin alone. Finally, aerosols generated from two nasal devices (VP3 and SP270) intended for aqueous dispersion were characterized with either coated or uncoated liposomes. Contrarily to the SP270 device, VP3 device showed minor changes between coated and uncoated liposome aerosols, as shown by their median volume diameters of 38.4±5.76 and 37.6±5.74 µm, respectively. Overall, the results obtained in this study show that the developed formulation delivered by the VP3 device can be considered as a potential candidate for nose-brain delivery of ghrelin.

Acute effects of intravenous cocaine administration on serum concentrations of ghrelin, amylin, glucagon-like peptide-1, insulin, leptin and peptide YY and relationships with cardiorespiratory and subjective responses.

BACKGROUND: Food intake and use of drugs of abuse like cocaine share common central and peripheral physiological pathways. Appetitive hormones play a major role in regulating food intake; however, little is known about the effects of acute cocaine administration on the blood concentrations of these hormones in cocaine users. METHODS: We evaluated serum concentrations of six appetitive hormones: ghrelin (total and acyl-ghrelin), amylin, glucagon-like peptide-1 (GLP-1), insulin, leptin and peptide YY (PYY), as well as acute cardiorespiratory and subjective responses of 8 experienced cocaine users who received 25mg intravenous (IV) cocaine. RESULTS: Serum concentrations of GLP-1 (p=0.014) and PYY (p=0.036) were significantly decreased one hour following IV cocaine administration; there was a trend towards a decrease for insulin (p=0.055) and amylin (p=0.063) concentrations, while no significant IV cocaine effect was observed for ghrelin (total or acyl-ghrelin) or leptin concentrations (p's≫>0.5). We also observed associations between hormone concentrations acutely affected by IV cocaine (GLP-1, PYY, insulin, amylin) and some cocaine-related cardiorespiratory and subjective responses (e.g., increased heart and respiratory rates; feeling high and anxious). DISCUSSION: These findings show a significant effect of acute IV cocaine administration on some appetitive hormones and suggest potential associations between these hormones and cocaine-related cardiorespiratory and subjective responses. Additional research is needed to further investigate the potential mechanisms underlining these associations.

The oncogenic role of the In1-ghrelin splicing variant in prostate cancer aggressiveness.

BACKGROUND: The Ghrelin-system is a complex, pleiotropic family composed of several peptides, including native-ghrelin and its In1-ghrelin splicing variant, and receptors (GHSR 1a/b), which are dysregulated in various endocrine-related tumors, where they associate to pathophysiological features, but the presence, functional role, and mechanisms of actions of In1-ghrelin splicing variant in prostate-cancer (PCa), is completely unexplored. Herein, we aimed to determine the presence of key ghrelin-system components (native-ghrelin, In1-ghrelin, GHSR1a/1b) and their potential pathophysiological role in prostate cancer (PCa). METHODS: In1-ghrelin and native-ghrelin expression was evaluated by qPCR in prostate tissues from patients with high PCa-risk (n = 52; fresh-tumoral biopsies), and healthy-prostates (n = 12; from cystoprostatectomies) and correlated with clinical parameters using Spearman-test. In addition, In1-ghrelin and native-ghrelin was measured in plasma from an additional cohort of PCa-patients with different risk levels (n = 30) and control-healthy patients (n = 20). In vivo functional (proliferation/migration) and mechanistic (gene expression/signaling-pathways) assays were performed in PCa-cell lines in response to In1-ghrelin and native-ghrelin treatment, overexpression and/or silencing. Finally, tumor progression was monitored in nude-mice injected with PCa-cells overexpressing In1-ghrelin, native-ghrelin and empty vector (control). RESULTS: In1-ghrelin, but not native-ghrelin, was overexpressed in high-risk PCa-samples compared to normal-prostate (NP), and this expression correlated with that of PSA. Conversely, GHSR1a/1b expression was virtually absent. Remarkably, plasmatic In1-ghrelin, but not native-ghrelin, levels were also higher in PCa-patients compared to healthy-controls. Furthermore, In1-ghrelin treatment/overexpression, and to a much lesser extent native-ghrelin, increased aggressiveness features (cell-proliferation, migration and PSA secretion) of NP and PCa cells. Consistently, nude-mice injected with PC-3-cells stably-transfected with In1-ghrelin, but not native-ghrelin, presented larger tumors. These effects were likely mediated by ERK1/2-signaling activation and involved altered expression of key oncogenes/tumor suppressor genes. Finally, In1-ghrelin silencing reduced cell-proliferation and PSA secretion from PCa cells. CONCLUSIONS: Altogether, our results indicate that In1-ghrelin levels (in tissue) and circulating levels (in plasma) are increased in PCa where it can regulate key pathophysiological processes, thus suggesting that In1-ghrelin may represent a novel biomarker and a new therapeutic target in PCa.

Metabolic responses to exogenous ghrelin in obesity and early after Roux-en-Y gastric bypass in humans.

AIMS: Ghrelin is a gastric-derived hormone that stimulates growth hormone (GH) secretion and has a multi-faceted role in the regulation of energy homeostasis, including glucose metabolism. Circulating ghrelin concentrations are modulated in response to nutritional status, but responses to ghrelin in altered metabolic states are poorly understood. We investigated the metabolic effects of ghrelin in obesity and early after Roux-en-Y gastric bypass (RYGB). MATERIALS AND METHODS: We assessed central and peripheral metabolic responses to acyl ghrelin infusion (1 pmol kg-1 min-1 ) in healthy, lean subjects (n = 9) and non-diabetic, obese subjects (n = 9) before and 2 weeks after RYGB. Central responses were assessed by GH and pancreatic polypeptide (surrogate for vagal activity) secretion. Peripheral responses were assessed by hepatic and skeletal muscle insulin sensitivity during a hyperinsulinaemic-euglycaemic clamp. RESULTS: Ghrelin-stimulated GH secretion was attenuated in obese subjects, but was restored by RYGB to a response similar to that of lean subjects. The heightened pancreatic polypeptide response to ghrelin infusion in the obese was attenuated after RYGB. Hepatic glucose production and hepatic insulin sensitivity were not altered by ghrelin infusion in RYGB subjects. Skeletal muscle insulin sensitivity was impaired to a similar degree in lean, obese and post-RYGB individuals in response to ghrelin infusion. CONCLUSIONS: These data suggest that obesity is characterized by abnormal central, but not peripheral, responsiveness to ghrelin that can be restored early after RYGB before significant weight loss. Further work is necessary to fully elucidate the role of ghrelin in the metabolic changes that occur in obesity and following RYGB.

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.

Ghrelin and the Cardiovascular System.

Ghrelin is a small peptide released primarily from the stomach. It is a potent stimulator of growth hormone secretion from the pituitary gland and is well known for its regulation of metabolism and appetite. There is also a strong relationship between ghrelin and the cardiovascular system. Ghrelin receptors are present throughout the heart and vasculature and have been linked with molecular pathways, including, but not limited to, the regulation of intracellular calcium concentration, inhibition of proapoptotic cascades, and protection against oxidative damage. Ghrelin shows robust cardioprotective effects including enhancing endothelial and vascular function, preventing atherosclerosis, inhibiting sympathetic drive, and decreasing blood pressure. After myocardial infarction, exogenous administration of ghrelin preserves cardiac function, reduces the incidence of fatal arrhythmias, and attenuates apoptosis and ventricular remodeling, leading to improvements in heart failure. It ameliorates cachexia in end-stage congestive heart failure patients and has shown clinical benefit in pulmonary hypertension. Nonetheless, since ghrelin's discovery is relatively recent, there remains a substantial amount of research needed to fully understand its clinical significance in cardiovascular disease.

Ghrelin Inhibits Post-Operative Adhesions via Blockage of the TGF-ß Signaling Pathway.

Post-operative adhesions are a critical problem in pelvic and abdominal surgery despite a multitude of studies dedicated to finding modalities to prevent their occurrence. Ghrelin administration promotes an anti-fibrotic response in a surgical mouse model of adhesion-induction, but the mechanisms mediating this effect have not been established. In the current study, the molecular mechanisms that underlie the anti-adhesion effect of ghrelin were investigated. Post-surgical adhesions were experimentally created in C57BL/6 wild-type mice via a combination of ischemic peritoneal buttons and cecal multiple abrasions. Ghrelin or saline intraperitoneal injections were given twice daily from two days before surgery to selected time points post-surgically to assess the phenotypic and molecular effects of treatment (1 day (n = 20), 4 days (n = 20) and 20 days (n = 40) after surgery). Endpoints included the scoring of adhesions and gene and protein expression analysis of pro-fibrogenic factors conducted on peritoneal ischemic tissue by quantitative PCR and Western blot. Ghrelin administration significantly reduced post-surgical adhesions and down-regulated pro-inflammatory gene and protein expression, including Tgfb3 and Tgfbr2. The up-regulation of inhibitory proteins Smad6 and Smad7 confirmed the ghrelin-induced blockage of TGF-ß signaling. Ghrelin is a candidate therapeutic drug for post-operative adhesion prevention, inhibiting inflammatory responses via blockage of the TGF-ß signaling pathway at the onset of surgery before the occurrence of the granulation-remodeling phase.

Ghrelin O-acyltransferase knockout mice show resistance to obesity when fed high-sucrose diet.

Ghrelin is an appetite-stimulating hormone secreted from stomach. Since the discovery that acylation of the serine-3 residue by ghrelin O-acyltransferase (GOAT) is essential for exerting its functions, GOAT has been regarded as an therapeutic target for attenuating appetite, and thus for the treatment of obesity and diabetes. However, contrary to the expectations, GOAT-knockout (KO) mice have not shown meaningful body weight reduction, under high-fat diet. Here, in this study, we sought to determine whether GOAT has a role in body weight regulation and glucose metabolism with a focus on dietary sucrose, because macronutrient composition of diet is important for appetite regulation. We found that peripherally administered acylated-ghrelin, but not unacylated one, stimulated sucrose consumption in a two-bottle-drinking test. The role of acylated-ghrelin in sucrose preference was further supported by the finding that GOAT KO mice consumed less sucrose solution compared with WT littermates. Then, we investigated the effect of dietary composition of sucrose on food intake and body weight in GOAT KO and WT mice. As a result, when fed on high-fat diet, food intake and body weight were similar between GOAT KO and WT mice. However, when fed on high-fat, high-sucrose diet, GOAT KO mice showed significantly reduced food intake and marked resistance to obesity, leading to amelioration of glucose metabolism. These results suggest that blockade of acylated-ghrelin production offers therapeutic potential for obesity and metabolic disorders caused by overeating of palatable food.