Development of a sensitive and quantitative HPLC-FLD method for the determination of obestatin in human plasma.

Obestatin is a gastrointestinal system peptide. The quantification of this peptide is conventionally performed using immunological techniques. In this study, a selective and sensitive HPLC method coupled with fluorescence detection for the quantitation of obestatin in human plasma was developed and validated. The separation was obtained on a C18 (4.6 × 100 mm, 3.5-µm particles) column using a mobile phase composed of acetonitrile and water, both including 0.1% trifluoroacetic acid. The developed method was found to be linear in the concentration range of 20 to 1000 ng/mL, with a coefficient of determination of 0.9982. The precision results were less than 10%, and the accuracy results were between 92% and 107%. The detection and quantification limit values were obtained as 2.8 and 9.4 ng/mL, respectively. Analyte solutions were found stable for 24 h at room temperature, three freeze-thaw cycles, and 2 weeks at -20°C. The developed method was successfully used for the quantification of obestatin in human plasma samples. In conclusion, the developed method is sensitive and specific for measuring the plasma concentrations of obestatin.

Concurrent Akt, ERK1/2 and AMPK Activation by Obestatin Inhibits Apoptotic Signaling Cascades on Nutrient-Deprived PC12 Cells.

Targeting apoptosis in the ischemic penumbra is a rational therapeutic approach for restricting cerebral infarct volume after clinical stroke. The present work explored the capability of the obestatin peptide, as a novel approach to inhibit apoptotic signaling cascades on PC12 cells. According to the results, obestatin treatment significantly reduced nutrient deprivation-induced apoptotic cell death. The protective effects were related to the regulation of the anti-apoptotic protein, BCL-2, and the apoptotic protein caspase-3. This encompasses the control of apoptosis by the interplay between Akt, ERK1/2 and AMPK signaling pathways. The activation of Akt and AMPK was concomitant with the phosphorylation of their downstream targets, GSK3 and ACC, respectively. Besides, obestatin also causes FoxO1 nuclear export supporting the prevention of the apoptosome formation. The concurrent activation of Akt and AMPK by obestatin via the GPR39 receptor, supports a role for this system in the balance concerning the catabolic and the anabolic signaling to sustain cellular function and viability. Furthermore, these results provide both an insight into how the obestatin/GPR39 system regulates anti-apoptotic pathways, and a framework for ascertaining how this system can be optimally targeted in treatment of brain cell death after stroke.

Human Milk Metabolic Hormones: Analytical Methods and Current Understanding.

Human milk (HM) contains a wide array of peptide hormones including leptin and adiponectin, which are involved in the regulation of infant growth and development. These essential hormones might play an important role in the regulation of metabolic reprogramming of the new-born infant. However, HM hormone studies are sparse and heterogeneous in regard to the study design, sample collection, preparation and analysis methods. This review discussed the limitations of HM hormone analysis highlighting the gaps in pre-analytical and analytical stages. The methods used to quantify HM metabolic hormones (leptin, adiponectin, ghrelin, insulin, obestatin, resistin and apelin) can be classified as immunoassay, immunosensor and chromatography. Immunoassay methods (ELISA and RIA) have been predominantly used in the measurement of these HM hormones. The relative validity parameters of HM hormones analysis are often overlooked in publications, despite the complexity and differences of HM matrix when compared to that of plasma and urine. Therefore, appropriate reports of validation parameters of methodology and instrumentation are crucial for accurate measurements and therefore better understanding of the HM metabolic hormones and their influences on infant outcomes.

Lower leptin level at discharge in acute anorexia nervosa is associated with early weight-loss.

OBJECTIVE: Predictive values of acute phase metabolic abnormalities of anorexia nervosa (AN) have seldom been studied. As early postrestoration weight loss is associated with poor outcome, discharge biologic parameters were assessed to detect an association with 2-month follow-up weight loss as a proxy to poor outcome. METHOD: Fasting plasma levels of leptin, acyl-ghrelin, obestatin, PYY, oxytocin and BDNF were measured in 26 inpatients, at inclusion, at discharge and 2 months later. A body mass index less than 18 2-month postdischarge was considered a poor outcome. RESULTS: Nineteen patients (73%) had a fair outcome and seven (27%) had a poor one with a mean loss of 0.69 versus 4.54 kg, respectively. Only discharge leptin levels were significantly higher in fair versus poor outcome patients (14.1 vs. 7.0 ng/ml, p = 0.006). The logistic regression model using discharge leptin, acyl-ghrelin, obestatin, oxytocin, PYY and BDNF levels as predictors of outcome disclosed a nearly significant effect of leptin (p < 0.10). Receiver operating characteristic analysis showed 11.9 ng/ml was the best value of threshold. Neither clinical variables differed according to outcome. CONCLUSION: Leptin level may be a biomarker of early weight relapse after acute inpatient treatment of AN. Its clinical usefulness in monitoring care in AN should further be determined.

Potential Therapeutic Effects of Gut Hormones, Ghrelin and Obestatin in Oral Mucositis.

Chemotherapy and/or head and neck radiotherapy are frequently associated with oral mucositis. Oral pain, odynophagia and dysphagia, opioid use, weight loss, dehydration, systemic infection, hospitalization and introduction of a feeding tube should be mentioned as the main determinated effect of oral mucositis. Oral mucositis leads to a decreased quality of life and an increase in treatment costs. Moreover, oral mucositis is a life-threatening disease. In addition to its own direct life-threatening consequences, it can also lead to a reduced survival due to the discontinuation or dose reduction of anti-neoplasm therapy. There are numerous strategies for the prevention or treatment of oral mucositis; however, their effectiveness is limited and does not correspond to expectations. This review is focused on the ghrelin and obestatin as potentially useful candidates for the prevention and treatment of chemo- or/and radiotherapy-induced oral mucositis.

Serum ghrelin and obestatin levels in patients with acne vulgaris: are they important for the severity?

INTRODUCTION: Acne vulgaris (AV) is a chronic, inflammatory disease of the pilosebaceous unit. Recently, three peptide-structured hormones, products of a single gene, have been discovered. These hormones are acylated ghrelin, des-acyl ghrelin and obestatin. AIM: To demonstrate the association of serum acylated ghrelin, des-acyl ghrelin, and obestatin levels with acne severity. MATERIAL AND METHODS: A total of 63 patients grouped as mild (n = 22), moderate (n = 21) and severe (n = 20) acne according to the Global Acne Grading System and 20 medically healthy volunteers were included in the study. Serum ghrelin and obestatin levels obtained from the participants were examined. RESULTS: When mean ghrelin, des-acyl-ghrelin and obestatin values of the acne-group (AG) were compared with the control group (CG), they were found be lower in the AG, but were not statistically significant. Among the patient groups, while acylated ghrelin values were highest in the severe AG, des-acyl ghrelin values were highest in mild severe AG and mean obestatin values were highest in moderate severe AG (p > 0.05). When the groups were compared for obestatin values; the highest average value was detected in the CG. However, it was not significant when the groups were compared. CONCLUSIONS: It has been suggested that there may be a link between acne and the levels of acylated ghrelin, des-acyl ghrelin and obestatin which are decreased in the serum of acne patients. Because of the decrease observed in the levels of these hormones which have antimicrobial features, we suggest that inflammation in acne cannot be suppressed and the reproduction of the microorganisms that play a role in the aetiology of the disease cannot be prevented. The replacement of these hormones at physiologic concentrations may contribute to the acne treatment.

Obestatin can potentially differentiate Wharton's jelly mesenchymal stem cells into insulin-producing cells.

In vitro-generation of ß-cells from Wharton's jelly mesenchymal stem cells (WJ-MSCs) could provide a potential basis for diabetes mellitus cell therapy. However, the generation of functional insulin-producing cells (IPCs) from WJ-MSCs remains a challenge. Recently, obestatin, a gut hormone, was found to promote ß-cell generation from pancreatic precursor cells. Accordingly, we hypothesize that obestatin can induce the differentiation of WJ-MSCs into IPCs. Therefore, the purpose of the current study is to examine the ability of obestatin to generate IPCs in comparison to well-known extrinsic factors that are commonly used in IPCs differentiation protocols from MSCs, namely exendin-4 and glucagon-like peptide-1 (GLP-1). To achieve our aims, WJ-MSCs were isolated, cultured and characterized by immunophenotyping and adipocytes differentiation. Afterwards, WJ-MSCs were induced to differentiate into IPCs using two differentiation protocols incorporating either exendin-4, GLP-1 or obestatin. The pancreatic progenitor marker, nestin and ß-cell differentiation markers were assessed by qRT-PCR, while the functionality of the generated IPCs was assessed by glucose-stimulated insulin secretion (GSIS). Our results showed that WJ-MSCs exhibit all the characteristics of MSCs. Interestingly, using obestatin in both the short and long differentiation protocols managed to induce the expression of ß-cell markers, similar to exendin-4. In GSIS, IPCs generated using either GLP-1 or obestatin showed higher secretion of insulin as compared to those generated using exendin-4 under low-glucose conditions but failed to show a significant response to increased glucose. These results indicate obestatin can be considered as a novel potential factor to consider for generation of IPCs from WJ-MSCs.

Serum Obestatin: A Biomarker of Cardiovascular and All-Cause Mortality in Hemodialysis Patients.

BACKGROUND: Recent experimental studies have suggested that obestatin, a proposed anorexigenic gut hormone and a physiological opponent of acyl-ghrelin, has protective cardiovascular effects. We tested the hypothesis that obestatin is independent of inflammatory mediators and/or acyl-ghrelin in predicting outcomes of the maintenance hemodialysis (MHD) population. METHODS: It was a 6-year cohort study on 261 MHD patients. Obestatin, acyl-ghrelin, adipokines (leptin and adiponectin), markers of inflammation and nutrition, prospective all-cause and cardiovascular mortality were studied. RESULTS: During the follow-up, 160 patients died in total, with 74 deaths due to cardiovascular causes. For each ng/mL increase in baseline obestatin level in fully adjusted models (including malnutrition-inflammation score, Interleukin-6 [IL-6], adipokines and acyl-ghrelin), the hazard for death from all causes was 0.90 (95% CI 0.81-0.99) and for cardiovascular death 0.85 (95% CI 0.73-0.99). However, these associations were more robust in the subgroup of patients aged above 71 years: 0.85 (95% CI 0.73-0.98) for all-cause death and 0.66 (95% CI 0.52-0.85) for cardiovascular death. An interaction between high IL-6 (above median) and low obestatin (below median) levels for increased risk of all-cause mortality (synergy index [SI] 5.14, p = 0.001) and cardiovascular mortality (SI 4.81, p = 0.02) emerged in the development of multivariable adjusted models. Interactions were also observed between obestatin, Tumor necrosis factor-alpha, adipokines and acyl-ghrelin, which were associated with mortality risk. CONCLUSION: Serum obestatin behaves as a biomarker for cardiovascular and all-cause mortality in MHD patients. The prognostic ability of obestatin in this regard is independent of inflammation, nutritional status, acyl-ghrelin's and adipokines' activity and is modified by age being very prominent in patients older than 71 years.

Unacylated ghrelin and obestatin: promising biomarkers of protein energy wasting in children with chronic kidney disease.

BACKGROUND: Impairment in orexigenic/anorexigenic hormone balance may be key in the pathogenesis of protein energy wasting in children with chronic kidney disease (CKD). Measurement of ghrelin and obestatin concentrations in children with CKD would help assess the potential contribution of these hormones to uremic protein energy wasting. METHODS: This was a cross-sectional case-control study. Acylated and unacylated ghrelin and obestatin were measured in 42 children on conservative treatment (CT), 20 children on hemodialysis, 48 pediatric renal transplant (RTx) recipients and 43 controls (CTR) (mean age 11.9, range 5-20 years). Weight, height and bicipital, tricipital, subscapular and suprailiac folds were measured, and the body mass index-standard deviation score (BMI-SDS), percentage of fat mass and fat-free mass were calculated. Urea and creatinine were measured and the glomerular filtration rate (GFR) calculated. RESULTS: Unacylated ghrelin level was higher in patients than controls (p = 0.0001), with the highest levels found in hemodialysis patients (p = 0.001 vs. CKD-CT, p = 0.0001 vs. RTx, p < 0.0001 vs. CTR). Obestatin level was significantly higher in patients on hemodialysis than those on conservative treatment, RTx recipients and controls (p < 0.0001 in each case). Unacylated ghrelin negatively correlated with weight-SDS (p < 0.0001), BMI-SDS (p = 0.0005) and percentage fat mass (p = 0.004) and positively correlated with percentage fat-free mass (p = 0.004). Obestatin concentration negatively correlated with weight-SDS (p = 0.007). Unacylated ghrelin and obestatin concentrations positively correlated with creatinine and urea and inversely with eGFR, even after adjustments for gender, age, puberty and BMI-SDS (p < 0.0001 for each model). CONCLUSIONS: Unacylated ghrelin and obestatin, negatively related to renal function, seem to be promising inverse indicators of nutritional status in children with CKD. Potential therapeutic implications in terms of optimization of their removal in patients on hemodialysis could be hypothesized.

Serum obestatin level strongly correlates with lipoprotein subfractions in non-diabetic obese patients.

BACKGROUND: Obestatin is a ghrelin-associated peptide, derived from preproghrelin. Although many of its effects are unclear, accumulating evidence supports positive actions on both metabolism and cardiovascular function. To date, level of obestatin and its correlations to the lipid subfractions in non-diabetic obese (NDO) patients have not been investigated. METHODS: Fifty NDO patients (BMI: 41.96 ± 8.6 kg/m2) and thirty-two normal-weight, age- and gender-matched healthy controls (BMI: 24.16 ± 3.3 kg/m2) were enrolled into our study. Obestatin level was measured by ELISA. Low-density lipoprotein (LDL) and high-density lipoprotein (HDL) subfractions, intermediate density lipoprotein (IDL) and very low-density lipoprotein (VLDL) levels and mean LDL size were detected by nongradient polyacrylamide gel electrophoresis (Lipoprint). RESULTS: Serum level of obestatin was significantly lower in NDO patients compared to controls (3.01 ± 0.5 vs. 3.29 ± 0.6 µg/ml, p < 0.05). We found significant negative correlations between the level of obestatin and BMI (r = - 0.33; p < 0.001), level of serum glucose (r = - 0.27, p < 0.05), HbA1c (r = - 0.38; p < 0.001) and insulin (r = - 0.34; p < 0.05). Significant positive correlation was found between obestatin level and the levels of ApoA1 (r = 0.25; p < 0.05), large HDL subfraction ratio and level (r = 0.23; p < 0.05 and r = 0.24; p < 0.05), IDL (r = 0.25 p < 0.05) and mean LDL size (r = 0.25; p < 0.05). Serum VLDL ratio and level negatively correlated with obestatin (r = - 0.32; p < 0.01 and r = - 0.21; p = 0.05). In multiple regression analysis obestatin was predicted only by VLDL level. CONCLUSIONS: Based on our data, measurement of obestatin level in obesity may contribute to understand the interplay between gastrointestinal hormone secretion and metabolic alterations in obesity.

Alteration of ghrelin/obestatin ratio in adolescence with polycystic ovarian syndrome.

Ghrelin, an endoggenous for the growth hormone secretagogue receptor, has been shown to participate in the regulation of energy homeostasis and pituitary hormone secretion. Obestatin, encoded by the same gene as ghrelin, is described as a physiological opponent of ghrelin. Ghrelin and obestatin are altered in polycystic ovary syndrome (PCOS), which is characterized by insulin resistance and pituitary hormone secretion disorder. The aim of this study was to evaluate ghrelin/obestatin imbalance in relation to insulin resistance and pituitary hormone in adolescence with PCOS. This restrospective case-control study included 33 adolescence with PCOS and 38 control adolescence. Ghrelin and obestatin concentrations in serum were determined by RIA, and the serum fasting glucose and Insulin were determined by the glucose oxidase color method and INS-EASIA. The serum LH and FSH were measured by highly specific hemiluminescence immunoassays. We found that the serum ghrelin levels and ghrelin/obestatin ratio were significant lower in PCOS group than in control group, and the serum obestatin levels were significant higher in PCOS group than in control group. The ghrelin/obestatin ratios were negatively correlation with LH/FSH ratio and insulin resistant index in PCOS group. The findings of this study suggest that ghrelin/obestatin imbalance may play a role in pathogenesis of adolescent PCOS.

Treatment with Obestatin-A Ghrelin Gene-Encoded Peptide-Reduces the Severity of Experimental Colitis Evoked by Trinitrobenzene Sulfonic Acid.

Obestatin is a 23-amino acid peptide derived from proghrelin, a common prohormone for ghrelin and obestatin. Previous studies showed that obestatin exhibited some protective and therapeutic effects in the gut. The aim of our presented study was to examine the effect of treatment with obestatin on trinitrobenzene sulfonic acid (TNBS)-induced colitis. In rats anesthetized with ketamine, colitis was induced through intrarectal administration of 25 mg of 2,4,6-trinitrobenzene sulfonic acid (TNBS). Obestatin was administered intraperitoneally at doses of 4, 8, or 16 nmol/kg, twice per day for four consecutive days. The first dose of obestatin was given one day before the induction of colitis, and the last one was given two days after administration of TNBS. Fourteen days after the induction of colitis, rats were anesthetized again with ketamine, and the severity of colitis was determined. The administration of obestatin had no effect on the parameters tested in rats without the induction of colitis. In rats with colitis, administration of obestatin at doses of 8 or 16 nmol/kg reduced the area of colonic damage, and improved mucosal blood flow in the colon. These effects were accompanied by a reduction in the colitis-evoked increase in the level of blood leukocytes, and mucosal concentration of pro-inflammatory interleukin-1ß. Moreover, obestatin administered at doses of 8 or 16 nmol/kg reduced histological signs of colonic damage. The administration of obestatin at a dose of 4 nmol/kg failed to significantly affect the parameters tested. Overall, treatment with obestatin reduced the severity of TNBS-induced colitis in rats. This effect was associated with an improvement in mucosal blood flow in the colon, and a decrease in local and systemic inflammatory processes.

Does obestatin modulate the hypothalamic appetite-regulating network in peripubertal sheep?

The participation of peripheral peptides in the processes regulating the food intake (energy homeostasis) at the central nervous system level remains unclear. This study focuses on the role of obestatin in neuronal activity within the hypothalamic appetite-regulating network in ruminants. The animals (n = 28) were randomly divided into two groups. The sheep in the control group received intracerebroventricular infusions of the Ringer-Locke solution, and the sheep in obestatin group were infused with obestatin (diluted in the Ringer-Locke solution) at 25 µg per 120 µl/hr. The series of four 1-hr infusions on 3 consecutive days were performed, and immediately after the experiment, the sheep were decapitated. Selected brain regions were fixed in situ for further immunohistochemical analysis, while the remaining ones were frozen for real-time RT-qPCR analysis. Obestatin infusion elicited changes in the neuropeptide Y (NPY) neuronal network in the hypothalamus. The results obtained show that exogenous obestatin evoked an increase in npy and agrpmRNA expression in the mediobasal hypothalamus (MBH), while the immunoreactivity for NPY was decreased in the arcuate and periventricular nuclei. The increase in cart and pomcmRNA expression in the MBH was also observed. Moreover, increased levels of gpr39 receptor and npy receptor 1 mRNA expression were evident in obestatin-infused sheep. Based on these results, it can be concluded that obestatin plays a role in the modulation of appetite-regulating network at the central level in sheep. The results obtained suggest that the underlying mechanism may involve the modification of the activity of NPY/AgRP and CART/α-MSH neurons in the arcuate nucleus.

The effect of orlistat and weight loss diet on plasma ghrelin and obestatin.

BACKGROUND: The objective of this study was to evaluate the effect of weight loss with hypocaloric diet and orlistat treatment in addition to hypocaloric diet on gut-derived hormones ghrelin and obestatin. MATERIALS AND METHODS: A total of 52, euglycemic and euthyroid, obese female patients were involved in the study. The patients were assigned to two groups: Group 1 (n = 26) received hypocaloric diet alone and Group 2 (n = 26) received orlistat in addition to hypocaloric diet for 12 weeks. Anthropometric measurements, serum lipid, insulin levels, and obestatin and ghrelin values were assessed at the beginning of the study and after 12 weeks of therapy. RESULTS: Baseline clinical characteristics and laboratory parameters including serum ghrelin and obestatin concentrations and ghrelin/obestatin ratio were similar between the two groups. After 12 weeks, mean change in BMI, fat mass, and fat-free mass (FFM) were -1.97 ± 1.56 kg/m2 (P = 0.003), -2.63% ±2.11% (P = 0.003), and -1.06 ± 0.82 kg (P = 0.003), respectively, in Group 1. In Group 2, mean change in BMI was -2.11 ± 1.24 kg/m2 (P = 0.001), fat mass was -3.09% ±2.28% (P = 0.002), and FFM was -1.26 ± 0.54 kg (P = 0.001). However, fasting glucose, lipid, and insulin levels did not change in Group 1. Furthermore, except serum high-density lipoprotein cholesterol and triglyceride levels, no significant change was observed in Group 2. Although serum ghrelin and obestatin concentrations increased significantly in both groups (Group 1: pGhrelin: 0.047, pobestatin: 0.001 and Group 2: pGhrelin: 0.028, pobestatin: 0.006), ghrelin/obestatin ratio did not change significantly. When the changes in anthropometric assessments and laboratory parameters were compared, no significant difference was observed between the two groups. Furthermore, no correlation was observed between ghrelin or obestatin and any other hormonal and metabolic parameters. CONCLUSION: Weight loss with diet and diet plus orlistat is both associated with increased ghrelin and obestatin concentrations.

Obestatin regulates cardiovascular function and promotes cardioprotection through the nitric oxide pathway.

Patients with ischaemic heart disease or chronic heart failure show altered levels of obestatin, suggesting a role for this peptide in human heart function. We have previously demonstrated that GH secretagogues and the ghrelin gene-derived peptides, including obestatin, exert cardiovascular effects by modulating cardiac inotropism and vascular tone, and reducing cell death and contractile dysfunction in hearts subjected to ischaemia/reperfusion (I/R), through the Akt/nitric oxide (NO) pathway. However, the mechanisms underlying the cardiac actions of obestatin remain largely unknown. Thus, we suggested that obestatin-induced activation of PI3K/Akt/NO and PKG signalling is implicated in protection of the myocardium when challenged by adrenergic, endothelinergic or I/R stress. We show that obestatin exerts an inhibitory tone on the performance of rat papillary muscle in both basal conditions and under ß-adrenergic overstimulation, through endothelial-dependent NO/cGMP/PKG signalling. This pathway was also involved in the vasodilator effect of the peptide, used both alone and under stress induced by endothelin-1. Moreover, when infused during early reperfusion, obestatin reduced infarct size in isolated I/R rat hearts, through an NO/PKG pathway, comprising ROS/PKC signalling, and converging on mitochondrial ATP-sensitive potassium [mitoK(ATP)] channels. Overall, our results suggest that obestatin regulates cardiovascular function in stress conditions and induces cardioprotection by mechanisms dependent on activation of an NO/soluble guanylate cyclase (sGC)/PKG pathway. In fact, obestatin counteracts exaggerated ß-adrenergic and endothelin-1 activity, relevant factors in heart failure, suggesting multiple positive effects of the peptide, including the lowering of cardiac afterload, thus representing a potential candidate in pharmacological post-conditioning.

Endogenous peptide profile for elucidating biosynthetic processing of the ghrelin precursor.

Ghrelin is an orexigenic peptide primarily produced by gastric endocrine cells. The biosynthetic cleavage site of ghrelin has been well documented, but how its downstream region undergoes proteolytic processing remains poorly explored. Here, we provide the first snapshot of endogenous peptides from the ghrelin precursor by profiling the secretopeptidome of cultured mouse ghrelin-producing cells during exocytosis. Mapping of MS/MS sequenced peptides to the precursor highlighted three atypical monobasic processing sites, including the established C-terminus of ghrelin and the N-terminal cleavage site for obestatin, a putative 23-amino-acid C-terminally amidated peptide. However, we found that mouse obestatin does not occur in the form originally reported, but that a different amidation site is used to generate a shorter peptide. These data can be extended to study and characterize the precursor-derived peptides located downstream of ghrelin in different biological contexts.

The influence of enteral obestatin administration to suckling rats on intestinal contractility.

This study investigated the effect of enteral administration of obestatin on the contractility of whole-thickness preparations of duodenum and middle jejunum, as well as on the morphology of the enteric nervous system (ENS). Suckling rats were assigned to 3 groups (n=12) treated with: C-saline solution; LO-obestatin (125nmol/kgb.wt); HO-obestatin (250nmol/kgb.wt). Saline solution or obestatin were administered twice daily, from the 14th to the 21st day of life. Sections were studied in an organ bath, for isometric recording in the presence of acetylocholine (ACh), atropine (ATR) and tetradotoxin (TTX). Thickness of intestinal muscularis layer, the number of interstitial cells of Cajal (ICC) were measured in the paraffin sections. The immunodetection of Muscarinic Acetylocholine Receptor 2 (M2 receptor) was performed in the intestinal segments. In both intestinal segments HO treatment decreased the amplitude of spontaneous contraction compared to that observed in the C group. In the middle jejunum, the LO treatment also decreased the amplitude. TTX and ATR had no effect on amplitude of spontaneous contraction in the jejunum of LO and HO-treated animals. Compared to the C group, duodenal sections from HO animals and middle jejunum sections from LO and HO groups displayed a lower amplitude in response to ACh and EFS evoked contraction. An increase in the thickness of the muscularis layer was observed in the duodenum of LO and HO groups whereas the number ICC did not change significantly after treatment with obestatin. Moreover, the enteral administration of obestatin did not effect significantly on the cytoplasmic expression of M2 receptor in the jejunum. Our study demonstrated that enteral administration of obestatin to suckling rats influences small intestine contractility in the segment specific manner.

ß-Arrestin scaffolds and signaling elements essential for the obestatin/GPR39 system that determine the myogenic program in human myoblast cells.

Obestatin/GPR39 signaling stimulates skeletal muscle repair by inducing the expansion of satellite stem cells as well as myofiber hypertrophy. Here, we describe that the obestatin/GPR39 system acts as autocrine/paracrine factor on human myogenesis. Obestatin regulated multiple steps of myogenesis: myoblast proliferation, cell cycle exit, differentiation and recruitment to fuse and form multinucleated hypertrophic myotubes. Obestatin-induced mitogenic action was mediated by ERK1/2 and JunD activity, being orchestrated by a G-dependent mechanism. At a later stage of myogenesis, scaffolding proteins ß-arrestin 1 and 2 were essential for the activation of cell cycle exit and differentiation through the transactivation of the epidermal growth factor receptor (EGFR). Upon obestatin stimulus, ß-arrestins are recruited to the membrane, where they functionally interact with GPR39 leading to Src activation and signalplex formation to EGFR transactivation by matrix metalloproteinases. This signalplex regulated the mitotic arrest by p21 and p57 expression and the mid- to late stages of differentiation through JNK/c-Jun, CAMKII, Akt and p38 pathways. This finding not only provides the first functional activity for ß-arrestins in myogenesis but also identify potential targets for therapeutic approaches by triggering specific signaling arms of the GPR39 signaling involved in myogenesis.

Ghrelin and Motilin Control Systems in GI Physiology and Therapeutics.

Ghrelin and motilin are released from gastrointestinal endocrine cells during hunger, to act through G protein-coupled receptors that have closely related amino acid sequences. The actions of ghrelin are more complex than motilin because ghrelin also exists outside the GI tract, it is processed to des-acyl ghrelin which has activity, ghrelin can exist in truncated forms and retain activity, the ghrelin receptor can have constitutive activity and is subject to biased agonism and finally additional ghrelin-like and des-acyl ghrelin receptors are proposed. Both ghrelin and motilin can stimulate gastric emptying, acting via different pathways, perhaps influenced by biased agonism at the receptors, but research is revealing additional pathways of activity. For example, it is becoming apparent that reduction of nausea may be a key therapeutic target for ghrelin receptor agonists and perhaps for compounds that modulate the constitutive activity of the ghrelin receptor. Reduction of nausea may be the mechanism through which gastroparesis symptoms are reduced. Intriguingly, a potential ability of motilin to influence nausea is also becoming apparent. Ghrelin interacts with digestive function through its effects on appetite, and ghrelin antagonists may have a place in treating Prader-Willi syndrome. Unlike motilin, ghrelin receptor agonists also have the potential to treat constipation by acting at the lumbosacral defecation centres. In conclusion, agonists of both ghrelin and motilin receptors hold potential as treatments for specific subsets of digestive system disorders.

Effect of Chronic Administration of Obestatin and Its Fragment FNAP-NH2 on Behavioral Activity and Nociceptive Threshold in Rats with Normal and Excess Body Weight.

The effects of chronic intranasal administration of 300 nmol/kg obestatin and its fragment FNAP-NH2 on behavioral activity and nociceptive threshold were examined in male Wistar rats with normal body weight or alimentary obesity. In normal rats, obestatin produced no effect on behavior and nociception, whereas FNAP-NH2 fragment enhanced risk-taking behavior. Rats with excess body weight demonstrated less pronounced risk-taking behavior and elevated nociceptive threshold in comparison with normal animals, but these differences were abolished by chronic administration of FNAP-NH2.