Ghrelin Administration Research Articles

Ghrelin as a Novel Pharmacological Treatment for Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the leading cause of mortality from gastrointestinal (GI) disease in preterm neonates. Its precise pathogenesis is unknown, but early stages of NEC are thought to be characterized by damage to the intestinal mucosa; while full intestinal necrosis and perforation occur during the later stages.The high‐frequency power spectra of heart rate variability (HF‐HRV) is a non‐invasive measure of vagal tone, our laboratory has shown that decreases in HF‐HRV may predict which neonates are at increased risk for NEC before the onset of clinical signs (Doheny et al. Neurogastroenterol. Motil., 2014). Furthermore, experimental NEC in newborn rat pups reduces HF‐HRV and increases NOS immunoreactivity in the myenteric neurons of the lower GI tract (Meister et al., Neurogastroenterol. Motil., 2018). Ghrelin is a GI hormone known to increase gastric motility via actions at several sites, including an excitation of dorsal vagal motoneurons upon brainstem microinjection, hence leading to an increase in vagal output to the GI tract.The aim of the present study was to test the hypotheses that ghrelin administration in a rodent model of NEC will: i) increase HF‐HRV values, and ii) restore the neurochemical phenotype of myenteric neurons of the lower GI tract.Newborn Sprague‐Dawley rats were randomly separated in the following groups: i) control, ii) mild NEC (<grade 2) induced via exposure to cold stress and hypoxia twice daily, and iii) mild NEC and anterior subdiaphragmatic vagotomy (grade >2). All groups underwent twice daily intraperitoneal injections of saline or ghrelin (0.05mg/kg) on postnatal days (PD)‐5 to PD‐10. Electrocardiogram recordings using bilateral electrodes to measure HF‐HRV were conducted on PD‐1, ‐5, and ‐10, prior to sacrifice on PD12. Upon sacrifice, GI tissues were collected to confirm NEC grade via H&E staining, and to assess the neurochemical phenotype of myenteric neurons through fluorescence immunohistochemistry.Mild NEC was confirmed via grade >2 histological examination; these pups did not display typical developmental increases in HF‐HRV. Ghrelin, but not saline, treatment reduced the NEC histological scale, increased HF‐HRV values and restored NOS‐immunoreactivity (NOS‐IR) in neurons of the myenteric plexus to levels similar to controls. Data are summarized in the Table below.Our data indicate that ghrelin administration in rodent model of NEC: i) decreased the histological severity of NEC, ii) increased HF‐HRV values, and iii) restored the neurochemical phenotype of myenteric neurons of the lower GI tract. We suggest that ghrelin may be a promising treatment to attenuate, or even reverse, the impaired HF‐HRV and altered neurochemical phenotype observed after NEC induction.Support or Funding InformationAmerican Physiological Society grant to C.R.B. & NIH grant DK99350 to R.A.T. & K.K.D.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of dorsal raphe nucleus GHS-R1a receptors in the regulation of inhibitory avoidance and escape behaviors in rats.

Ghrelin is a recently discovered peptide, mainly produced in the stomach and involved in body's energy-maintenance processes. Ghrelin exerts its actions by activating the growth hormone secretagogue receptor (GHS-R). Recent analyses indicate that ghrelin targets the brain to regulate a wealth of functions, including behavioral responses that have been associated with stress and anxiety mechanisms. In this context, evidence shows the presence of GHS-R receptors in the dorsal raphe nucleus (DRN), the main source of serotonergic neurons that innervate encephalic structures involved in emotional control. Our study aims to evaluate the effects of the pharmacological manipulation of ghrelin receptors located in the DRN on the expression of the behavioral responses of Wistar rats. Such responses were assessed in the elevated T maze (ETM), an experimental model that allows the measurement, in the same animal, of two defensive tasks, inhibitory avoidance and escape. Our results showed that the intra-DRN infusion of ghrelin impaired the acquisition of inhibitory avoidance, an anxiolytic-like effect, and facilitated the expression of escape response in the ETM, indicating a panicogenic-like effect. The intra-DRN administration of the ghrelin receptor (GHS-R1a) antagonist PF-04628935 did not alter the behavioral tasks assessed in the ETM. Finally, our results revealed that intra-DRN infusions of PF-04628935 prior to the administration of ghrelin into this area neutralized the behavioral effects obtained in the ETM. Taken together, our data reveal the involvement of DRN GHS-R1a receptors in the regulation of defensive tasks that have been associated with generalized anxiety and panic disorders.

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.

Glucagon-Like Peptide-1 (GLP-1) and 5-Hydroxytryptamine 2c (5-HT2c) Receptor Agonists in the Ventral Tegmental Area (VTA) Inhibit Ghrelin-Stimulated Appetitive Reward.

Current literature indicates that the orexigenic peptide ghrelin increases appetitive motivation via signaling in the mesolimbic reward system. Another gastric peptide, glucagon-like peptide-1 (GLP-1), and the neurotransmitter 5-hydroxytryptamine (5-HT), are both known to suppress operant responding for food by acting on key mesolimbic nuclei, including the ventral tegmental area (VTA). In order to investigate the interaction effects of ghrelin, GLP-1, and 5-HT within the VTA, we measured operant responding for sucrose pellets after the administration of ghrelin, the GLP-1 receptor agonist exendin-4 (Ex-4), and the 5-HT2c receptor agonist Ro60-0175 in male Sprague-Dawley rats. Following training on a progressive ratio 3 (PR3) schedule, animals were first injected with ghrelin into the VTA at doses of 3 to 300 pmol. In subsequent testing, separate rats were administered intraperitoneal (IP) Ex-4 (0.1–1.0 µg/kg) or VTA Ex-4 (0.01–0.1 µg) paired with 300 pmol ghrelin. In a final group of rats, the 5-HT2c agonist Ro60-0175 was injected IP (0.25–1.0 mg/kg) or into the VTA (1.5–3.0 µg), and under both conditions paired with 300 pmol ghrelin delivered into the VTA. Our results indicated that ghrelin administration increased operant responding for food reward and that this effect was attenuated by IP and VTA Ex-4 pretreatment as well as pre-administration of IP or VTA Ro60-0175. These data provide compelling evidence that mesolimbic GLP-1 and serotonergic circuitry interact with the ghrelinergic system to suppress ghrelin’s effects on the mediation of food reinforcement.

New Metabolic Influencer on Oxytocin Release: The Ghrelin

Background: The hypothalamic–pituitary axis by secreting neuropeptides plays a key role in metabolic homeostasis. In light of the metabolic regulation, oxytocin is a potential neuropeptide for therapies against obesity and related disorders. The aim of our study is to measure ghrelin-induced oxytocin secretion in rats and to detect the changes after administration of ghrelin antagonist. Methods: Ghrelin was administrated centrally (intracerebroventricular, i.c.v., 1.0, 10.0, and 100.0 pmol) or systemically (intravenous, i.v., 1.0, and 10.0 nmol). [d-Lys3]-GHRP-6 ghrelin antagonist was injected 15 min before ghrelin injection in a dose of 10.0 pmol i.c.v. and 10.0 nmol i.v. Results: Either i.c.v. or i.v. administration of ghrelin dose-dependently increased the plasma oxytocin concentration. Following pretreatment with the ghrelin antagonist [d-Lys3]-GHRP-6, the high plasma oxytocin level induced by ghrelin was significantly reduced. Conclusion: The results indicate that the release of oxytocin is influenced directly by the ghrelin system. Examination of the mechanism of ghrelin-induced oxytocin secretion is a new horizon for potential therapeutic options.

Brain accessibility delineates the central effects of circulating ghrelin.

Ghrelin is a hormone produced in the gastrointestinal tract that acts via the growth hormone secretagogue receptor. In the central nervous system, ghrelin signalling is able to recruit different neuronal targets that regulate the behavioural, neuroendocrine, metabolic and autonomic effects of the hormone. Notably, several studies using radioactive or fluorescent variants of ghrelin have found that the accessibility of circulating ghrelin into the mouse brain is both strikingly low and restricted to some specific brain areas. A variety of studies addressing central effects of systemically injected ghrelin in mice have also provided indirect evidence that the accessibility of plasma ghrelin into the brain is limited. Here, we review these previous observations and discuss the putative pathways that would allow plasma ghrelin to gain access into the brain together with their physiological implications. Additionally, we discuss some potential features regarding the accessibility of plasma ghrelin into the human brain based on the observations reported by studies that investigate the consequences of ghrelin administration to humans.

Activation of the rat hypothalamic supramammillary nucleus by food anticipation, food restriction or ghrelin administration.

The circulating orexigenic hormone ghrelin targets many brain areas involved in feeding control and signals via a dedicated receptor, the growth hormone secretagogue receptor 1A. One unexplored target area for ghrelin is the supramammillary nucleus (SuM), a hypothalamic area involved in motivation and reinforcement and also recently linked to metabolic control. Given that ghrelin binds to the SuM, we explored whether SuM cells respond to ghrelin and/or are activated when endogenous ghrelin levels are elevated. We found that peripheral ghrelin injection activates SuM cells in rats, reflected by an increase in the number of cells expressing c-Fos protein in this area, as welll as by the predominantly excitatory response of single SuM cells recorded in in vivo electrophysiological studies. Further c-Fos mapping studies reveal that this area is also activated in rats in situations when circulating ghrelin levels are known to be elevated: in food-restricted rats anticipating the consumption of food and in fed rats anticipating the consumption of an energy-dense food. We also show that intra-SuM injection of ghrelin induces a feeding response in rats suggesting that, if peripheral ghrelin is able to access the SuM, it may have direct effects on this brain region. Collectively, our data demonstrate that the SuM is activated when peripheral ghrelin levels are high, further supporting the emerging role for this brain area in metabolic and feeding control.

Ghrelin protects against contact dermatitis and psoriasiform skin inflammation by antagonizing TNF-\u03b1/NF-\u03baB signaling pathways

Contact dermatitis and psoriasis are skin disorders caused by immune dysregulation, yet much remains unknown about their underlying mechanisms. Ghrelin, a recently discovered novel peptide and potential endogenous anti-inflammatory factor expressed in the epidermis, is involved in skin repair and disease. In this study, we investigated the expression pattern and therapeutic effect of ghrelin in both contact dermatitis and psoriasis mouse models induced by oxazolone (OXA) and imiquimod (IMQ), respectively, and in TNF-α-stimulated RAW264.7 macrophages, NHEKs and skin fibroblasts. Ghrelin expression was reduced in both the OXA-induced contact dermatitis and IMQ-induced psoriasis mouse models. Furthermore, treatment with ghrelin attenuated skin inflammation in both the contact dermatitis and psoriasis mouse models. Mice administered PBS after OXA- or IMQ-induced model generation exhibited typical skin inflammation, whereas ghrelin treatment in these mouse models substantially decreased the dermatitis phenotype. In addition, exogenous ghrelin attenuated the inflammatory reaction induced by TNF-α in RAW264.7 cells. Moreover, ghrelin administration limited activation of NF-κB signaling. In summary, ghrelin may represent a potential molecular target for the prevention and treatment of inflammatory skin diseases, including contact dermatitis and psoriasis.

Attenuated levels of ghrelin in synovial fluid is related to the disease severity of ankle post-traumatic osteoarthritis.

Post-traumatic osteoarthritis (PTOA) of ankle joints results in pain and reduced joint function. Ghrelin, a 28-amino-acid polypeptide, has been previously identified as the first cognate natural ligand that binds to the growth hormone secretagogue receptor. In the present study, ghrelin has been validated to exert cartilage-protective and anti-inflammatory effects. The current study was aimed at investigating the potential role of the levels of serum and synovial fluid (SF) ghrelin on the severity of disease in patients suffering from ankle PTOA. Ninety-seven patients with ankle osteoarthritis who received an arthroscopical examination and debridement or replacement of the ankle joint were included in the study cohort. Meanwhile, 95 healthy individuals (whose age and sex were matched) who received periodic body checkups were enrolled as healthy controls. Enzyme-linked immunosorbent assay (ELISA) was used to analyze the ghrelin levels in serum and SF. SF was also probed for cartilage degradation enzyme matrix metalloproteinases-3 (MMP-3) and tumor necrosis factor alpha (TNF-α), which is a known pro-inflammatory cytokine. The clinical evaluation was carried out using the American Orthopedic Foot and Ankle Society (AOFAS) ankle-hindfoot rating scale and visual analogue scale (VAS). The radiographic severity was evaluated using the modified Kellgren-Lawrence (K-L) grading system. We scored for the modified Mankin's score to depict histopathological changes due to cartilage lesions. The diagnostic relevance of the ghrelin concentrations in the prediction of the radiographic grading (in comparison with MMP-3 and TNF-α) was evaluated by calculating the area under the curve of the receiver operating characteristic (ROC) curve. The serum abundance of ghrelin was not significantly altered between ankle PTOA patients and healthy controls. SF ghrelin was negatively correlated with radiographic progression determined by modified ankle K-L grades. In addition SF ghrelin concentrations were negatively related to VAS scores, and positively associated with AOFAS ankle-hindfoot rating. Moreover, SF ghrelin was inversely proportional to the expressions of MMP-3 and TNF-α. ROC analysis curve demonstrated that ghrelin serves as a favorable marker for the diagnosis of radiographic severity by modified ankle K-L grade. The ghrelin concentration in SF is negatively proportional to disease progression in patients suffering from ankle PTOA. Local administration of ghrelin may function as a decent adjuvant therapy to delay the progress of ankle PTOA. © 2019 BioFactors, 45(3):463-470, 2019.

Exogenous ghrelin ameliorates acute lung injury by modulating the nuclear factor κB inhibitor kinase/nuclear factor κB inhibitor/nuclear factor κB pathway after hemorrhagic shock

Previous studies have shown that ghrelin, a peptide produced in the stomach, attenuates acute lung injury (ALI) in various animal models, and that some of these effects are associated with inhibition of the nuclear factor κB signaling pathway. This study investigated whether ghrelin exerts beneficial effects on hemorrhagic shock (HS)-induced ALI by modulating nuclear factor κB inhibitor kinase/nuclear factor κB inhibitor/nuclear factor κB (IKK/IκBα/NF-κB) pathway activity. HS was induced in male SD rats by withdrawing blood to a mean arterial pressure (MAP) of 40 mm Hg for 1 h; rats then received ghrelin (10 nmol/kg) or vehicle intravenously and were resuscitated with the shed blood and an equal volume of Ringer lactate solution followed by observation for 2 h. After resuscitation, samples were collected and analyzed for lung histopathology, wet to dry weight ratio (W/D), bronchoalveolar lavage fluid (BALF) protein, neutrophil infiltration, plasma inflammatory cytokines (TNF-α and IL-6), and cytoplasmic phosphorylated IKKβ, IκBα, phosphorylated IκBα and nuclear NF-κB expression. Compared to those in the two sham groups, lung injury, W/D, BALF protein, neutrophil infiltration, plasma TNF-α and IL-6 levels, and IKK/IκBα/NF-κB pathway activation were significantly increased in HS rats. After ghrelin administration, all parameters analyzed were decreased compared to those without ghrelin in HS rats. Moreover, ghrelin alleviated the decreased MAP after resuscitation compared to that in HS rats. Exogenous ghrelin attenuates the inflammatory response and acute lung injury after HS. These beneficial effects appear to be mediated through inhibition of IKK/IκBα/NF-κB signaling.

MTOR Signaling in X/A-Like Cells Contributes to Lipid Homeostasis in Mice.

Gastric mechanistic target of rapamycin (mTOR) signaling is inversely associated with the expression and secretion of ghrelin, a 28-aa peptide hormone produced by gastric X/A-like cells. Ghrelin contributes to obesity and hepatic steatosis. We sought to control global lipid metabolism via the manipulation of gastric mTOR signaling in X/A-like cells. We established a ghrl-cre transgene in which the Cre enzyme is expressed in X/A-like cells under the control of the ghrelin-promoter. mTORflox/flox and tuberous sclerosis 1 (TSC1)flox/flox mice were separately bred with ghrl-cre mice to generate mTOR-ghrl-cre or TSC1-ghrl-cre mice, within which mTOR signaling was suppressed or activated, respectively. Lipid metabolism in liver and adipose depots was analyzed. Under the control of the ghrelin-promoter, the Cre enzyme was exclusively expressed in stomach X/A-like cells in adult animals. Knockout of mTOR in X/A-like cells increased circulating acyl-ghrelin and promoted hepatic lipogenesis with effects on adipose depots. Activation of mTOR signaling by deletion of its upstream inhibitor, TSC1, decreased ghrelin expression and secretion, altering lipid metabolism as evidenced by resistance to high-fat diet-induced obesity and hepatic steatosis. Both ghrelin administration and injection of rapamycin, an inhibitor of mTOR, altered the phenotypes of TSC1-ghrl-cre mice. Conclusion: Gastric mTOR signaling in X/A-like cells contributes to organism lipid homeostasis by regulating hepatic and adipose lipid metabolism. Gastric mTOR signaling may provide an alternative strategy for intervention in lipid disorders.

No effect of unacylated ghrelin administration on subcutaneous PC3 xenograft growth or metabolic parameters in a Rag1-/- mouse model of metabolic dysfunction.

Ghrelin is a peptide hormone which, when acylated, regulates appetite, energy balance and a range of other biological processes. Ghrelin predominately circulates in its unacylated form (unacylated ghrelin; UAG). UAG has a number of functions independent of acylated ghrelin, including modulation of metabolic parameters and cancer progression. UAG has also been postulated to antagonise some of the metabolic effects of acyl-ghrelin, including its effects on glucose and insulin regulation. In this study, Rag1-/- mice with high-fat diet-induced obesity and hyperinsulinaemia were subcutaneously implanted with PC3 prostate cancer xenografts to investigate the effect of UAG treatment on metabolic parameters and xenograft growth. Daily intraperitoneal injection of 100 μg/kg UAG had no effect on xenograft tumour growth in mice fed normal rodent chow or 23% high-fat diet. UAG significantly improved glucose tolerance in host Rag1-/- mice on a high-fat diet, but did not significantly improve other metabolic parameters. We propose that UAG is not likely to be an effective treatment for prostate cancer, with or without associated metabolic syndrome.

Does a ghrelin stimulus during zebrafish embryonic stage modulate its performance on the long-term?

Metabolic programming refers to the induction, deletion, or impaired development of a somatic structure or “setting” of a physiological system by an early life stimulus operated at a critical period during development. Ghrelin is the only known orexigenic gut hormone, is an acylated peptide that acts as an endogenous ligand specific for growth-hormone secretagogue-receptor. The aim of the present work was to evaluate if an in ovo ghrelin administration could positively influence the zebrafish performance in the long-term and to gain insight on the mechanisms associated to ghrelin regulation of food intake during the larval phase. Food intake, growth potential, protein metabolism, expression of target genes involved in ghrelin, feeding behaviour regulation and locomotor activity were assessed in zebrafish (Danio rerio) larvae at 25 days post-fertilization. Elevated levels of acylated ghrelin in zebrafish eggs did not result in increased growth or food intake. Differences in mRNA expression between larvae fasted for 16 h before and 1 h after feeding were found for igf1ra, gh1 and pomca. Moreover, ghrelin treated larvae showed higher swimming activity, indicating that the peptide may have an important role on foraging activity. The present study addressed for the first time the effects of an early stimulus of ghrelin during the embryonic stage of zebrafish, however, further studies are needed to clarify the metabolic pathways affected by the early stimulus as well as focus on the effects on metabolic regulation of energy balance through lipid and carbohydrate metabolism.

Effects of Ghrelin on the Oxidative Stress and Healing of the Colonic Anastomosis in Rats

BackgroundAnastomotic leakage is the deadliest complication of colonic procedures. Ghrelin is an orexigenic hormone with potent actions on growth hormone release and functions in the processes of growth, tissue inflammation, repair, and oxidative stress. We evaluated the hypothesis that the exogenous administration of ghrelin causes beneficial effects on the healing of colonic anastomosis. Materials and methodsSixty-four male Wistar rats were randomly assigned to eight subgroups receiving postoperative intraperitoneal administration of ghrelin (23 μg/kg/d) or saline after a colonic anastomosis. The anastomotic tissue was evaluated on the third, seventh, and 14th postoperative days. Anastomotic bursting pressure, histological parameters, hydroxyproline content, and tissue oxidative stress markers were compared. ResultsThere was a significant increase in the mean anastomotic bursting pressure in the ghrelin subgroup on the seventh postoperative day (P = 0.035). Histological evaluation demonstrated a significant difference in the neutrophilic infiltrate (P = 0.035) on the third and 14th d and in apoptosis (P = 0.004), granulation tissue (P = 0.011) and peritoneal inflammation (P = 0.014) on the 14th postoperative day. There was a statistically significant increase in the hydroxyproline content in the ghrelin subgroup on the 14th postoperative day (P = 0.043). There were significant differences in the nitrite tissue levels (P = 0.021) on day 3 and in reactive oxygen species (P = 0.012) on day 14. ConclusionsThe administration of ghrelin had beneficial anti-inflammatory and antioxidant effects, increasing the resistance of the anastomosis and the hydroxyproline tissue content in the postoperative period.

Evidence Supporting a Role for the Blood-Cerebrospinal Fluid Barrier Transporting Circulating Ghrelin into the Brain.

The stomach-derived hormone ghrelin mainly acts in the brain. Studies in mice have shown that the accessibility of ghrelin into the brain is limited and that it mainly takes place in some circumventricular organs, such as the median eminence. Notably, some known brain targets of ghrelin are distantly located from the circumventricular organs. Thus, we hypothesized that ghrelin could also access the brain via the blood-cerebrospinal fluid (CSF) barrier, which consists of the choroid plexus and the hypothalamic tanycytes. Using systemic injection of ghrelin or fluorescent-ghrelin in mice, we found that cells of the blood-CSF barrier internalize these molecules. In time-response studies, we found that peripherally injected fluorescent-ghrelin quickly reaches hypothalamic regions located in apposition to the median eminence and more slowly reaches the periventricular hypothalamic parenchyma, adjacent to the dorsal part of the third ventricle. Additionally, we found that CSF ghrelin levels increase after the systemic administration of ghrelin, and that central infusions of either an anti-ghrelin antibody, which immuno-neutralizes CSF ghrelin, or a scrambled version of ghrelin, which is also internalized by cells of the blood-CSF barrier, partially impair the orexigenic effect of peripherally injected ghrelin. Thus, current evidence suggests that the blood-CSF barrier can transport circulating ghrelin into the brain, and that the access of ghrelin into the CSF is required for its full orexigenic effect.

Unacylated ghrelin stimulates steroidogenesis in lean rats and reverses reproductive dysfunction in high fat diet-fed rats

ABSTRACTThis study investigated the effect of sub-chronic administration of unacylated ghrelin (UAG) on steroidogenesis, sperm parameter, and reproductive function in lean and high fat diet (HFD)-induced obese male rats. Rats were divided into 4 groups (n = 12 each) as 1) Control-fed standard diets (STD): (10 kcal%), 2) STD + UAG (200 ng/kg, i.p.), 3) HFD obese: fed HFD (45 kcal%), and 4) HFD + UAG. Diet regimen was continued for 16 weeks after which normal saline as a vehicle or UAG was administered to desired groups for the next 4 weeks. In vitro, testicular slices were incubated with increasing concentrations of UAG (10−8–10−6 M) in the presence or absence of GSH-R1a antagonist, [D-Lys-3]-GHRP-6 (10−6 M). UAG significantly increased the circulatory levels of FSH, LH and testosterone, increased testicular testosterone levels and sperm count and motility in lean and obese rats and reduced sperm morphological abnormalities and increased pregnancy rate and number of pups at birth in HFD-obese rats. Associated with the reduction in the final body and fat masses weights and independent of food intake, UAG post-therapy to both lean and HFD-fed rats significantly lowered fasting blood glucose and insulin levels, lowered HOMA-IR value, enhanced OGTT and ITT, lowered circulatory leptin levels, downregulated aromatase expression in adipose and testicular tissue and inhibited HFD-induced testicular oxidative stress and activation of cleaved caspase-3. Dysregulation of testicular levels of StAR, SF-1, CYP11A1 in the testis of both groups as well as in the in vitro preparation, in a dose-dependent manner, independent of GSH-R1a and not associated with activation of STAT3, a mediator of leptin signaling was apparent. In conclusion, administration of UAG can enhance reproductive function in lean rats and reverses HFD-induced reproductive dysfunction in obese rats.Abbreviations: AG: acylated ghrelin; BMI: body mass index; CHOL: cholesterol;FSH: follicular stimulating hormone; GHS: growth hormone secretagogues; GSH: reduced glutathione; HFD: high fat diet; HOMA-IR: homeostasis model assessment of insulin resistance: IR: insulin resistance; OGTT: oral glucose tolerance test; ITT: insulin tolerance test; LH: luteinizing hormone; MDA: malondialdehyde; STAT3: signal transducer and activator of transcription; SOD: superoxide dismutase; STD: standard diet; SF-1: steroidogenic factor-1; StAR: steroidogenic acute regulatory protein; CYP11A1: cholesterol side-chain cleavage enzyme (or P450scc); TGs: triglycerides; UAG: unacylated ghrelin.

Ghrelin Alleviates Angiotensin II-Induced H9c2 Apoptosis: Impact of the miR-208 Family.

BackgroundGhrelin is a novel peptide with abundant cardioprotective effects. The miR-208 family, consisting of cardiac-specifically expressed microRNAs, are not only involved in hypertrophy and fibrosis, but are also closely related with myocyte apoptosis. This study explored the role of the miR-208 family in the protective effect of ghrelin on angiotensin II (Ang II)-induced apoptosis.Material/MethodsH9c2 cells were exposed to Ang II with or without ghrelin. Cell viability was detected by MTT assay and the percentage of apoptotic cells was confirmed by flow cytometry. miRNAs expression levels were measured by qRT-PCR. Then, cells transfected with miR-208 negative control, mimics, and inhibitors were treated with Ang II and ghrelin, followed by flow cytometry. PCR array was performed to explore the pathways affected by miR-208.ResultsThe miR-208 level was reduced in Ang II-treated H9c2 cells, accompanied with increased cell apoptosis, which were both reversed by ghrelin administration. Flow cytometry revealed that miR-208 inhibitors clearly upregulated the apoptotic percentage, whereas miR-208 mimics showed the opposite effects in the Ang II group. miR-208a further alleviated apoptosis when treated with ghrelin. miR-208 mainly affected caspase, inflammatory-related genes, and several signaling pathways.ConclusionsWe provide new evidence that the miR-208 family is regulated by Ang II and ghrelin. Overexpression of miR-208 family alleviated Ang II-induced cell apoptosis and miR-208a assisted in the protective effect of ghrelin. Several apoptosis pathways affected by miR-208 family were found. These findings suggest the pathogenesis of cardiomyocyte apoptosis and the protective mechanism of ghrelin.

Glucose Availability Predicts the Feeding Response to Ghrelin in Male Mice, an Effect Dependent on AMPK in AgRP Neurons.

Metabolic feedback from the periphery to the brain results from a dynamic physiologic fluctuation of nutrients and hormones, including glucose and fatty acids, ghrelin, leptin, and insulin. The specific interactions between humoral factors and how they influence feeding is largely unknown. We hypothesized that acute glucose availability may alter how the brain responds to ghrelin, a hormonal signal of energy availability. Acute glucose administration suppressed a range of ghrelin-induced behaviors as well as gene expression changes in hypothalamic neuropeptide Y (NPY) and agouti-related peptide (AgRP) neurons after ghrelin administration. Knockdown of the energy-sensing molecule AMP-activated protein kinase (AMPK) in AgRP neurons resulted in loss of the glucose effect, and mice responded as though pretreated with saline. Conversely, 2-deoxyglucose (2-DG), which decreases glucose availability, potentiated ghrelin-induced feeding and increased hypothalamic NPY mRNA levels. AMPK knockdown did not alter the additive effect of 2-DG and ghrelin on feeding. Our findings support the idea that computation of energy status is dynamic, is informed by multiple signals, and responds to acute fluctuations in metabolic state. These observations are broadly relevant to the investigation of neuroendocrine control of feeding and highlight the underappreciated complexity of control within these systems.

Chronic ghrelin administration restores hippocampal long-term potentiation and ameliorates memory impairment in rat model of Alzheimer's disease.

Alzheimer's disease (AD), as a common age-related dementia, is a progressive manifestation of cognitive decline following synaptic failure resulted majorly by senile plaques composed of deposits of amyloid beta (Aβ). Ghrelin is a multifunctional peptide hormone with receptors present in various brain tissues including hippocampus and has been associated with neuroprotection, neuromodulation, and memory processing. Here, we investigated the neuroprotective and therapeutic effects of intracerebroventricular (icv) ghrelin infusion for 2 weeks on passive avoidance learning (PAL), memory retention, and synaptic plasticity in the hippocampal dentate gyrus (DG) and CA1 of both normal rats and Aβ1-42-induced neurotoxicity in AD model. Male Wistar rats were evaluated for their passive memory performance using a shuttle box while some groups had already received Aβ1-42 and/or chronic ghrelin. Using field potential recording, the induction of short- and long-term potentiation (STP and LTP) was studied in DG granule cells along with the LTP changes in CA1 pyramidal neurons through stimulation of the medial perforant path (mPP) and Schaffer collaterals (SCs), respectively. Our results demonstrated that chronic ghrelin treatment not only improved memory processing and retrieval in normal rats during the PAL task, but also promoted memory retention and alleviated memory loss by amelioration of Aβ1-42-induced synaptic plasticity impairment in AD subjects through augmentation of field excitatory postsynaptic potential (fEPSP) slope that led to LTP restitution in both the mPP-DG and the CA3-CA1 synapses. Meanwhile, STP was not significantly changed, meaning that although ghrelin enhanced postsynaptic excitability in DG, it did not change presynaptic transmitter release significantly. This suggests the involvement of postsynaptic mechanisms in long-term ghrelin-enhanced memory. In conclusion, it can be inferred that chronic ghrelin administration has an auspicious therapeutic value for impaired cognitive performance and memory deficits in AD-like neuropathology.

Electrophysiological Effects of Ghrelin in the Hypothalamic Paraventricular Nucleus Neurons.

The paraventricular nucleus (PVN) is involved in the control of sympathetic tone and the secretion of hormones, both functions known to be influenced by ghrelin, suggesting direct effect of ghrelin in this nucleus. However, the effects of ghrelin on the excitability of different PVN neuronal populations have not been demonstrated. This study assessed the effects of ghrelin on the activity of PVN neurons, correlating the responses to subpopulations of PVN neurons. We used a 64 multielectrode array to examine the effects of ghrelin administration on extracellular spike frequency in PVN neurons recorded in brain slices obtained from male Sprague-Dawley rats. Bath administration of 10 nM ghrelin increased (29/97, 30%) or decreased (37/97, 38%) spike frequency in PVN neurons. The GABAA and glutamate receptors antagonists abolish the decrease in spike frequency, without changes in the proportion of increases in spike frequency (23/53, 43%) induced by ghrelin. The results indicate a direct effect of ghrelin increasing PVN neurons activity and a synaptic dependent effect decreasing PVN neurons activity. The patch clamp recordings showed similar proportions of PVN neurons influenced by 10 nM ghrelin (33/95, 35% depolarized; 29/95, 30% hyperpolarized). Using electrophysiological fingerprints to identify specific subpopulations of PVN neurons we observed that the majority of pre-autonomic neurons (11/18 -61%) were depolarized by ghrelin, while both neuroendocrine (29% depolarizations, 40% hyperpolarizations), and magnocellular neurons (29% depolarizations, 21% hyperpolarizations) showed mixed responses. Finally, to correlate the electrophysiological response and the neurochemical phenotype of PVN neurons, cell cytoplasm was collected after recordings and RT-PCR performed to assess the presence of mRNA for vasopressin, oxytocin, thyrotropin (TRH) and corticotropin (CRH) releasing hormones. The single-cell RT-PCR showed that most TRH-expressing (4/5) and CRH-expressing (3/4) neurons are hyperpolarized in response to ghrelin. In conclusion, ghrelin either directly increases or indirectly decreases the activity of PVN neurons, this suggests that ghrelin acts on inhibitory PVN neurons that, in turn, decrease the activity of TRH-expressing and CRH-expressing neurons in the PVN.