Immunochemical identification of the mammalian peptide hormone obestatin from tea plant (Camellia sinensis).

This study determines obestatin-like substances from the young shoots of the tea plant [Camellia sinensis (L.) O. Kuntze (Theaceae)]. Proteins were extracted from the vegetative tea leaves using the QB (Quick Buffer) buffer as an extraction buffer. Obestatin-like substances in tea extract were investigated using an indirect home-made enzyme-linked immunosorbent assay (ELISA). Human obestatin-like immunoreactive substances from tea extract were isolated and characterized by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (tricine-SDS-PAGE) and immunoblotting techniques. Immunochemical results showed that there are strong human obestatin-like immunoreactive substances (0.048±0.0064ng/mg protein) in vegetative tea leaves. This finding was completely unexpected since this hormone was considered to be present solely in animals. Furthermore, a single obestatin-like immunoreactive protein band of 13kDa was identified by tricine-SDS-PAGE and Western blotting of extract of vegetative tea leaf proteins. Present investigation is the first report of presence of obestatin-like immunoreactive substances in plants. It is concluded that obestatin-like bioactive peptides derived from plants can affect gastrointestinal tract structures as endogenous obestatin does and hence play a role in appetite regulation and body weight gain.

Determination of ghrelin and desacyl ghrelin in human plasma and urine by means of LC-MS/MS for doping controls.

The hunger hormone ghrelin (G) is classified as prohibited substance in professional sport by the World Anti-Doping Agency (WADA), due to its known growth hormone releasing properties. The endogenous bioactive peptide consists of 28 amino acids with a caprylic acid attached to serine at position 3. Within this study, it was aimed to develop methods to determine G and desacyl ghrelin (DAG) in plasma and urine by means of LC-MS/MS. Two strategies were applied with a bottom-up approach for plasma and top-down analyses for urine. Both sample preparation procedures were based on solid-phase extraction for enrichment and sample clean-up. Method validation showed good results for plasma and urine with limits of detection (LODs) for G and DAG between 30 and 50 pg/ml, recoveries between 45-50%, and imprecisions (intra- and inter-day) between 3% and 24%. Plasma analysis was also valid for quantification with accuracies determined with ~100% for G and ~106% for DAG. The minimum required performance level for doping control laboratories is set to 2 ng/ml in urine, and the herein established method yielded acceptable results even at 5% of this level. As proof-of-concept, plasma levels (G and DAG) of healthy volunteers were determined and ranged between 30 and 100 pg/ml for G and 100-1200 pg/ml for DAG. In contrast to earlier reported studies using ligand binding assays for urinary G and DAG, in this mass spectrometry-based study, no endogenous urinary G and DAG were found, although the LODs should enable this.

Gastric ghrelin cells in obese patients are hyperactive.

BACKGROUND/OBJECTIVES: Distribution and activity of ghrelin cells in the stomach of obese subjects are controversial. SUBJECTS/METHODS: We examined samples from stomachs removed by sleeve gastrectomy in 49 obese subjects (normoglycemic, hyperglycemic and diabetic) and quantified the density of ghrelin/chromogranin endocrine cells by immunohistochemistry. Data were compared with those from 13 lean subjects evaluated by gastroscopy. In 44 cases (11 controls and 33 obese patients) a gene expression analysis of ghrelin and its activating enzyme ghrelin O-acyl transferase (GOAT) was performed. In 21 cases (4 controls and 17 obese patients) the protein levels of unacylated and acylated-ghrelin were measured by ELISA tests. In 18 cases (4 controls and 14 obese patients) the morphology of ghrelin-producing cells was evaluated by electron microscopy. RESULTS: The obese group, either considered as total population or divided into subgroups, did not show any significant difference in ghrelin cell density when compared with control subjects. Inter-glandular smooth muscle fibres were increased in obese patients. In line with a positive trend of the desacylated form found by ELISA, Ghrelin and GOAT mRNA expression in obese patients was significantly increased. The unique ghrelin cell ultrastructure was maintained in all obese groups. In the hyperglycemic obese patients, the higher ghrelin expression matched with ultrastructural signs of endocrine hyperactivity, including expanded rough endoplasmic reticulum and reduced density, size and electron-density of endocrine granules. A positive correlation between ghrelin gene expression and glycemic values, body mass index and GOAT was also found. All obese patients with type 2 diabetes recovered from diabetes at follow-up after 5 months with a 16.5% of weight loss. CONCLUSIONS: Given the known inhibitory role on insulin secretion of ghrelin, these results suggest a possible role for gastric ghrelin overproduction in the complex architecture that takes part in the pathogenesis of type 2 diabetes.

The distribution of ghrelin cells in the human and animal gastrointestinal tract: a review of the evidence.

The growth hormone and appetite are regulated by a 28-peptide hormone called ghrelin, which is produced in the stomach, pituitary gland, and other body tissues. The physiological roles fulfilled by ghrelin include regulation of food intake, cardiac output, reproductive system, proliferation of cells, and formation of osteoblasts, as well as action against inflammation/fibrosis. The ghrelin present in the body can be distinguished as acylated ghrelin and deacylated ghrelin. Furthermore, both in humans and other animals, the entirety of the gastrointestinal tract comprises ghrelin cells, which are classified as open-type and closed-type cells. The present study reviews the evidence about how ghrelin cells are distributed in the human and the animal body.

Developmental stage-dependent relationships between ghrelin levels and hippocampal white matter connections in low-weight anorexia nervosa and atypical anorexia nervosa.

INTRODUCTION: Disruptions in homeostatic and hedonic food motivation are proposed to underlie anorexia nervosa (AN) and atypical AN, restrictive eating disorders which commonly onset in puberty. Ghrelin, a neuroprotective hormone that drives hedonic eating is increased in AN and is expressed in the hippocampus. White matter (WM) undergoes significant change during puberty in regions involved in food motivation, particularly WM tracts connected with the hippocampus. The association between ghrelin and WM region of interest (ROI) with hippocampal connections in restrictive eating disorders, particularly in adolescence during key neurodevelopmental growth, is unknown. METHODS: We evaluated fasting plasma ghrelin and WM microstructure (measured by free-water corrected fractional anisotropy (FA-t)) in WM ROIs with hippocampal connections - the fornix and the hippocampal portion of the cingulum - in 56 adolescent females (age range: 11.9 - 22.1 y; mean: 19.0 y) with low-weight eating disorders including AN and atypical AN (N = 36) and healthy controls (N = 20). RESULTS: FA-t in the fornix or hippocampal portion of the fornix did not differ between groups. Ghrelin was higher in AN/atypical AN vs. HC and was positively correlated with puberty stage in the AN/atypical AN group, but not the HC group. The correlation between ghrelin and FA-t in the fornix was significantly different in females with AN/atypical AN compared to controls. In AN/atypical AN, pubertal stage moderated the relation between fasting plasma ghrelin and FA-t in the fornix: higher fasting ghrelin was associated with lower FA-t in the fornix in late-post-puberty, but was not associated with FA-t in the early to mid stages of puberty. CONCLUSIONS: In post-pubertal females with low-weight AN/atypical AN, higher levels of ghrelin are associated with lower FA-t in the fornix. This relationship is not evident in the early to mid stages of puberty in AN/atypical AN or in HC, and may reflect a lack of possible neuroprotective effects of ghrelin in late-post puberty only. Understanding the effects of ghrelin on WM microstructure longitudinally and following recovery from AN/Atypical AN and how this differs across pubertal stages will be an important next step. These findings could ultimately inform treatment staging and aid in diagnosis and detection of AN/atypical AN.

Localization and distribution of CCK-8, NPY, Leu-ENK-, and Ghrelin- in the digestive tract of Prochilodus lineatus (Valenciennes, 1836).

This study describes the histological characteristics and distribution of gastrointestinal tract endocrine cells (ECs) of Prochilodus lineatus (detritivorous fish) using immunohistochemical procedures. The digestive tract of P. lineatus was divided into seven portions: stomach (cardial and pyloric), pyloric caeca, and intestine (anterior, glandular, middle and posterior). A pool of specific antisera against cholecystokinin (CCK-8), -neuropeptide Y (NPY), -ghrelin (Ghre) and -leu-enkephalin (Leu-ENK) to identify ECs were used. According to the morphological characteristics of ECs, two different types were identified and classified as open or closed-type. The number of ECs varied throughout the gastrointestinal tract, though a high abundance was found in the anterior intestine and pyloric caeca. A large number of ECs immunoreactive to CCK-8 and NPY were recorded in the anterior, glandular and middle intestine. ECs immunopositive to Leu-ENK were distributed in the stomach and pyloric caeca. For Ghre, immunopositive ECs were restricted to the glandular intestine. The results of the present study indicate that P. lineatus presents an ECs distribution pattern with species-specific particularities. However, CCK showed a distribution similar to that of omnivores, which is possibly related to local signaling functions in order to achieve the correct digestion of the various organisms found in the detritus.

Ghrelin suppresses migration of macrophages via inhibition of ROCK2 under chronic intermittent hypoxia.

OBJECTIVES: Migration of macrophages and atherosclerosis result in various diseases, including coronary heart disease. This study aimed to clarify the roles that ghrelin and Rho-associated coiled-coil-containing protein kinase 2 (ROCK2) play in migration of macrophages under chronic intermittent hypoxia (CIH). METHODS: A rat model of CIH was constructed and changes in ghrelin and ROCK2 protein expression were measured by western blot assay. The migratory ability of macrophages was determined by the transwell assay. Hematoxylin and eosin staining was applied to detect the changes in intima-media thickness. RESULTS: We found that CIH enhanced migration of macrophages, and this effect was attenuated by exogenous ghrelin. Additionally, the facilitative effect of CIH on migration of macrophages was strengthened or decreased by upregulation or downregulation of ROCK2, respectively. This phenomenon indicated that ROCK2 was involved in CIH-induced migration in macrophages. Furthermore, western blot and transwell assays showed that ghrelin inhibited CIH-induced migration via ROCK2 suppression in macrophages. CONCLUSIONS: In summary, the present study shows that ghrelin inhibits CIH-induced migration via ROCK2 suppression in macrophages. Our research may help lead to identifying a new molecular mechanism for targeted therapy of atherosclerosis and its associated coronary artery diseases under intermittent hypoxia.

Expression of ghrelin and leptin in the chemosensory system of adult zebrafish.

Numerous data show that the chemosensory system seems to be modulated by changes in the circulating levels of different molecules such as ghrelin, orexin, leptin, NPY, CCK. The chemosensory system of the zebrafish is represented by the taste buds (skin, oral and oropharyngeal), the olfactory rosette and the solitary chemosensorial cells (SCCs). The purpose of our study was to analyze the distribution of two peripheral hormones such as ghrelin and leptin in the chemosensory organs of the zebrafish. Our results demonstrated the presence of immunoreaction for all antibodies used in the zebrafish chemosensory organs even if with different distribution. In particular, IR was observed for ghrelin in the olfactory rosette while IR for leptin was found in the olfactory rosette, in the skin and oropharyngeal taste buds and in the gills. Both these hormones were detected in the intestine, used as a control.

Changes in appetite-regulating hormones following food intake are associated with changes in reported appetite and a measure of hedonic eating in girls and young women with anorexia nervosa.

BACKGROUND: Females with anorexia nervosa (AN) have higher ghrelin and peptide YY (PYY) and lower brain-derived neurotropic factor (BDNF) levels than controls, and differ in their perception of hunger cues. Studies have not examined appetite-regulating hormones in the context of homeostatic and hedonic appetite in AN. OBJECTIVE: To examine whether alterations in appetite-regulating hormones following a standardized meal are associated with homeostatic and hedonic appetite in young females with AN vs. controls. METHODS: 68 females (36 AN, 32 controls) 10-22 years old were enrolled. Ghrelin, PYY and BDNF levels were assessed before, and 30, 60 and 120 min following a 400-kilocalorie standardized breakfast. Visual Analog Scales (VAS) assessing prospective food consumption, hunger, satiety, and hedonic appetite were administered before and 20 min after breakfast. A Cookie Taste Test (CTT) was conducted after a snack as a measure of hedonic eating behavior ∼3 h after breakfast. RESULTS: AN had higher fasting ghrelin and PYY, and lower fasting BDNF (p = 0.001, 0.002 and 0.044 respectively) than controls. Following breakfast (over 120 min), ghrelin and PYY area under the curve (AUC) were higher, while BDNF AUC was lower in AN vs. controls (p = 0.007, 0.017 and 0.020 respectively). Among AN (but not controls), reductions in ghrelin and increases in PYY in the first 30-minutes following breakfast were associated with reductions in VAS scores for prospective food consumption. AN consumed fewer calories during the CTT vs. controls (p < 0.0001). In AN (particularly AN-restrictive subtype), BDNF AUC was positively associated with kilocalories consumed during the CTT CONCLUSIONS: In young females with AN, changes in ghrelin and PYY following food intake are associated with reductions in a prospective measure of food consumption, while reductions in BDNF are associated with reduced hedonic food intake. Further studies are necessary to better understand the complex interplay between appetite signals and eating behaviors in AN.

Concentration of ghrelin and leptin in serum and human milk in nursing mothers according to the type of feeding.

INTRODUCTION: Objective: we assessed the relationship between serum and human foremilk and hindmilk concentrations of ghrelin and leptin in nursing mothers according to the type of feeding. Methods: this cohort design was carried out on 131 mother-newborn dyads admitted to a physiological puerperium ward. The independent variables were the type of feeding, full breastfeeding (FBF, 56.5%) and partial breastfeeding (PBF, 43.5%). The dependent variables were the concentration of total ghrelin (pg/ml) and leptin (ng/ml) in serum, foremilk and hindmilk at eight and 16 weeks. Fasting blood samples were obtained from the nursing mothers at four months for serum assays. Unpaired Student's t-test, Mann-Whitney U test, Pearson's correlation tests, coefficient of determination and linear regression were used. Results: the concentration of ghrelin and leptin in hindmilk was higher than that of foremilk in both groups at eight and 16 weeks. The concentration of ghrelin and leptin was higher in serum than in foremilk in both groups. These values showed a direct and significant linear correlation with the exception of ghrelin in the FBF group. The serum concentration of leptin in mothers explained 32% of the variance of its concentration in foremilk in the FBF and 13% in the PBF groups. Conclusion: the hindmilk/foremilk gradient suggests an intake regulating mechanism during the fed. The concentration of ghrelin and leptin was higher in the serum than in foremilk and its correlation and determination coefficients could suggest plasma-milk transfer in addition to synthesis regulation by the mammary gland, adipose tissue or other organs.

INTRODUCCIÓN: Objetivo: evaluar la relación entre la concentración de suero y la leche materna y la concentración de grelina y leptina en leche materna en madres lactantes según el tipo de alimentación. Métodos: diseño de cohorte realizado en 131 diadas madre-lactante que ingresaron en una sala de puerperio fisiológico. Variables independientes: tipo de alimentación, lactancia materna completa (LMC, 56,5%) y lactancia materna parcial (LMP, 43,5%). Variables dependientes: concentración sérica de grelina total (pg/ml) y leptina (ng/ml), leche humana pre-tetada y pos-tetada a las ocho y 16 semanas. Se utilizaron pruebas no pareadas t de Student, U de Mann-Whitney, correlación de Pearson, coeficiente de determinación y regresión linear. Resultados: la concentración de grelina y leptina en leche humana pre-tetada fue mayor que en leche humana pos-tetada en ambos grupos a las ocho y 16 semanas. La concentración de grelina y leptina fue mayor en suero que en leche humana en ambos grupos; estos valores mostraron una correlación lineal directa y significativa con la excepción de la grelina en el grupo de LMC. La concentración sérica de leptina en las madres explicó el 32% de la varianza de su concentración en leche humana en LMC y el 13% en madres en LMP. Conclusión: el gradiente de leche humana pre-tetada/pos-tetada sugiere un mecanismo de regulación e ingestión de leche. La concentración de grelina y leptina fue mayor en suero que en leche humana y los coeficientes de determinación sugieren una transferencia de suero-leche, además de una regulación de la síntesis por la glándula mamaria, el tejido adiposo u otros órganos.

Influence of subliminal intragastric fatty acid infusion on subjective and physiological responses to positive emotion induction in healthy women: A randomized trial.

BACKGROUND: Subliminal intragastric fatty acid infusion attenuates subjective and brain responses to negative emotion induction. However, the underlying gut-brain signaling mechanisms remain unclear, and it is unknown whether such effect equally applies to positive emotion. OBJECTIVE: We aimed to investigate the interaction between fatty acid-induced gut-brain signaling and subjective responses to positive emotion, and the potential mediational role of gastrointestinal (GI) hormones. DESIGN: Twelve fasting healthy women underwent intragastric infusion of 2.5 g lauric acid or saline, after which either positive or neutral emotion was induced for 30 min, in 4 separate visits. Appetite-related sensations, subjective emotional state, and GI hormones were measured at baseline and every 10 min after infusion. Heart rate variability was measured at baseline and at t = 20-30 min to quantify vagal tone (root mean square of successive differences, RMSSD), and sympathovagal balance (low frequency to high frequency ratio, LF/HF). RESULTS: Fatty acid infusion did not influence appetite-related sensations (as expected), nor emotional state ratings (contrary to expectations). As anticipated, fatty acid stimulated release of CCK at t = 20-40 min (p < 0.001), and GLP1 at t = 30-40 min (p < 0.001), but not PYY. Interestingly, positive emotion induction suppressed plasma octanoylated ghrelin at t = 20-40 min (p = 0.020). Further, both positive emotion and fatty acid attenuated RMSSD (p = 0.012 & 0.0073, respectively). Positive emotion attenuated LF/HF after fatty acid (p = 0.0006), but raised LF/HF after saline (p = 0.004). CONCLUSIONS: Subliminal fatty acid did not influence subjective responses to positive emotion induction. However, positive emotion induction suppressed octanoylated ghrelin release. Moreover, both positive emotion and subliminal fatty acid decreased cardiac vagal tone. Further, the fatty acid reversed the effect of positive emotion on sympathovagal balance.

Supplementation with eicosapentaenoic and docosahexaenoic acids in late gestation in ewes changes adipose tissue gene expression in the ewe and growth and plasma concentration of ghrelin in the offspring1.

Omega-3 long chain fatty acids have a positive impact on production. When consumed during late gestation, it might have fetal programming effects on the fetus, which will have lifelong impacts on development and production. The objectives of the present study were to evaluate the effect of increasing doses of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the diet of ewes in the last third of gestation on their body weight (BW), subcutaneous adipose tissue relative mRNA abundance of genes associated with adipose tissue metabolism, and growth performance and plasma metabolites and hormones of their offspring during the finishing phase. Ewes (n = 72) were blocked by BW and allotted to pens (8 per treatment) with 3 ewes per pen. Ewes were supplemented with an EPA and DHA source (Strata G113) at concentrations of 0, 1, or 2% of dry matter intake during the last 50 d of gestation. At lambing, all ewes were penned together and offered the same diet. After weaning at 60 d of age, lambs were blocked by BW and sex and fed for 56 d. All lambs were fed the same pellet diet (61.09% ground corn, 24.08% soy hulls, 11.09% soybean meal, 1.48% Ca salt of palm oil, and 2.26% mixed mineral vitamin), and were weighed every 14 d until the end of the trial. Blood samples were collected on the weight sampling days. Dry matter intake and refusals were weighed daily. Data were analyzed as a randomized complete block design with repeated measurements (SAS 9.4). Polynomial contrast (linear-L and quadratic-Q) was used for mean separation. There were no differences in ewe body condition score, milk production, milk fat, or milk protein, but there was a trend for increased (L, P = 0.06) lactose concentration, and also differences in DGAT1 (L, P = 0.04), Δ5-desaturase (Q, P = 0.06) and Δ6-desaturase (Q, P = 0.07), PPARα (Q, P = 0.03), ELOVL2 and 5 (Q, P < 0.07), FABP4 (Q, P = 0.04), FATP1 (Q, P = 0.06), leptin (Q, P = 0.02), and resistin (L, P = 0.05). Feeding pregnant ewes an increased amount of EPA and DHA in late gestation increased final BW (L, P = 0.01), ADG (L, P = 0.04; Q, P = 0.01), DMI (Q, P ≤ 0.01), plasma glucose concentration (L, P = 0.04), and trended to decrease ghrelin concentrations (L, P = 0.07) in offspring during the finishing period. Dam supplementation did not affect G:F, nor plasma NEFA concentration (P ≥ 0.53) of lambs. Therefore, increasing supplementation of EPA and DHA in pregnant ewes has an impact on offspring performance, increasing DMI, ADG, and BW.

Therapeutic Effects of Sleeve Gastrectomy and Ileal Transposition on Type 2 Diabetes in a Non-Obese Rat Model by Regulating Blood Glucose and Reducing Ghrelin Levels.

BACKGROUND Nowadays, more than 170 million patients suffer from diabetes mellitus worldwide. This study aimed to investigate the effects of sleeve gastrectomy (SG) and ileal transposition (IT) surgery on the control of diabetes. MATERIAL AND METHODS Goto-Kakizaki rats were used to establish type 2 diabetes models and undergo SG or IT surgery. At 2 months post-surgery, insulin, glucose, triglycerides (TG), total cholesterol (TC), glucose tolerance, glucagon-like peptide-1 (GLP-1) levels, and insulin sensitivity were evaluated. RESULTS SG significantly shortened operative time and post-operative recovery time compared to IT surgery (P<0.05). SG and IT surgery resulted in significantly induced weight loss, significantly decreased levels of glucose, and significantly enhanced levels of Ghrelin compared the Sham surgery group (P<0.001). SG and IT surgery resulted in significantly increased GLP-1 levels compared to Sham surgery (P<0.001). SG resulted in better reduction of oral glucose tolerance test (OGTT) glucose compared to IT surgery (P<0.05). SG and IT surgery significantly upregulated insulin tolerance test (ITT) levels compared to Sham surgery (P<0.001). SG induced better reductions in TC and TG compared to IT surgery (P<0.05). CONCLUSIONS In non-obese rats with spontaneous diabetes, both SG and IT surgery were found to control diabetes by regulating body weight and levels of glucose, Ghrelin, GLP-1, OGTT glucose, insulin, TC, and TG. Moreover, SG demonstrated advantages of shorter operative time, shorter post-operative recovery time, and better control of diabetes compared to IT surgery.

Enhancement of the chondrogenic differentiation of mesenchymal stem cells and cartilage repair by ghrelin.

Transforming growth factor beta (TGF-ß) is commonly utilized in chondrogenic differentiation protocols, but this often results in incomplete maturation of the derived chondrocytes. Gene expression analysis, quantitation of sulfated glycosaminoglycan and collagen, and histological staining were performed to assess the effects of ghrelin. The signaling pathways involved were investigated with inhibitors or targeted by shRNAs. Joint cavity delivery of TGF-ß with or without ghrelin, within a rat cartilage defect model was performed to evaluate the in vivo effects of ghrelin. Ghrelin dramatically enhanced gene expression levels of SOX9, ACAN, and COL II and resulted in increased synthesis of sulfated glycosaminoglycan (sGAG) and collagen in vitro. Combined treatment with TGF-ß and ghrelin synergistically enhanced the phosphorylation of ERK1/2 and DMNT3A, which accounted for increased expression of chondrogenic genes. Delivery of ghrelin in combination with TGF-ß after MSC implantation within a rat osteochondral defect model significantly enhanced de novo cartilage regeneration, as compared to delivery with TGF-ß alone. In conclusion, ghrelin could significantly enhance MSC chondrogenic differentiation in vitro and can also enhance cartilage regeneration in vivo when used in combination with TGF-ß. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1387-1397, 2019.

Development and initial characterization of a novel ghrelin receptor CRISPR/Cas9 knockout wistar rat model.

BACKGROUND/OBJECTIVES: Ghrelin, a stomach-derived hormone implicated in numerous behaviors including feeding, reward, stress, and addictive behaviors, acts by binding to the growth hormone secretagogue receptor (GHSR). Here, we present the development, verification, and initial characterization of a novel GHSR knockout (KO) Wistar rat model created with CRISPR genome editing. METHODS: Using CRISPR/Cas9, we developed a GHSR KO in a Wistar background. Loss of GHSR mRNA expression was histologically verified using RNAscope in wild-type (WT; n = 2) and KO (n = 2) rats. We tested the effects of intraperitoneal acyl-ghrelin administration on food consumption and plasma growth hormone (GH) concentrations in WT (n = 8) and KO (n = 8) rats. We also analyzed locomotion, food consumption, and body fat composition in these animals. Body weight was monitored from early development to adulthood. RESULTS: The RNAscope analysis revealed an abundance of GHSR mRNA expression in the hypothalamus, midbrain, and hippocampus in WTs, and no observed probe binding in KOs. Ghrelin administration increased plasma GH levels (p = 0.0067) and food consumption (p = 0.0448) in WT rats but not KOs. KO rats consumed less food overall at basal conditions and weighed significantly less compared with WTs throughout development (p = 0.0001). Compared with WTs, KOs presented higher concentrations of brown adipose tissue (BAT; p = 0.0322). CONCLUSIONS: We have verified GHSR deletion in our KO model using histological, physiological, neuroendocrinological, and behavioral measures. Our findings indicate that GHSR deletion in rats is not only associated with a lack of response to ghrelin, but also associated with decreases in daily food consumption and body growth, and increases in BAT. This GHSR KO Wistar rat model provides a novel tool for studying the role of the ghrelin system in obesity and in a wide range of medical and neuropsychiatric disorders.

Ghrelin and NUCB2/Nesfatin-1 Co-Localization With Digestive Enzymes in the Intestine of Pejerrey (Odontesthes bonariensis).

Ghrelin (orexigenic) and nesfatin-1 (anorexigenic) are two peptides with opposing actions on food intake regulation and are mainly expressed in the hypothalamus and gut of mammals and fish. Both are involved in the regulation of a wide range of physiological processes in vertebrates, including metabolism, growth, and reproduction. However, the anatomical relationship between these peptides and the nutrient assimilation processes are not well understood. Thus, the aim of this work was to determine the localization of ghrelin, nesfatin-1, and several enzymes involved in the digestive process (lipoprotein lipase, aminopeptidase A, trypsin, and sucrase-isomaltase) in the intestine of pejerrey (Odontesthes bonariensis), a species with commercial importance in South America. We observed co-localization of ghrelin and nesfatin-1 in enteroendocrine cells, absorptive cells, and in cells of the lamina propia. Approximately half of the cells displaying ghrelin-like immunoreactivity co-localized the NUCB2/nesfatin-1-like signal. In addition, both peptides showed co-localization with lipoprotein lipase, aminopeptidase A, trypsin, or sucrase-isomaltase. All digestive enzymes except for aminopeptidase A and trypsin, showed high co-localization (68-88%) with both ghrelin-like and NUCB2/nesfatin-1-like signals in absorptive, enteroendocrine, and lamina propria cells. Together, our results provide immunohistochemical evidence supporting a role for both ghrelin and NUCB2/nesfatin-1 in the regulation of nutrient assimilation in fish. Anat Rec, 302:973-982, 2019. © 2018 Wiley Periodicals, Inc.

Relationship between irritable bowel syndrome and plasma and tissue ghrelin levels.

BACKGROUND/AIMS: This study aimed to evaluate the relationship between irritable bowel syndrome (IBS) and plasma and tissue ghrelin levels. MATERIALS AND METHODS: Patients who had undergone gastroscopy procedure for any reason previously were enrolled in the study. Among these, patients with IBS symptoms were evaluated according to the Roma III criteria. The healthy control group comprised patients with no IBS symptom and had undergone gastroscopy procedure for another reason. The plasma ghrelin level and tissue ghrelin level obtained by immunohistochemical examination of biopsy specimens taken from the gastric antrum and corpus were evaluated in all participants. RESULTS: The mean age of 90 participants was 43.64}12.64 years. The median value of the plasma ghrelin level was 3.29 (1.2-12.7) in the diarrhea group (IBS-D), 1.49 (0.82-7.08) in the constipation group (IBS-C), and 1.5 (0.2-3.7) in the control group. The plasma ghrelin levels between the groups were found to be significantly higher in IBS-D than in IBS-C and the control groups (p=0.001 and p=0.001, respectively). On comparing antral mucosal gland biopsy outcomes among the groups, staining intensity score was found to be significantly high in IBS-C as compared with the control group, whereas no significant difference was observed between IBS-D and the control groups (p=0.020 and p=0.429, respectively). CONCLUSION: The plasma ghrelin level in IBS-D and the staining intensity in the antral mucosal gland in IBS-C were found to be significantly higher. In addition, there was no difference between the groups in terms of ghrelin staining intensity in the gastric corpus.

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.

Alcohol consumption is positively associated with fasting serum ghrelin in non-dependent adults: Results from the population-based LIFE-Adult-Study.

BACKGROUND: Animal experiments and studies in alcohol dependent patients indicate that ghrelin signaling in the brain is causally involved in the regulation of alcohol reward and intake. Increasing ghrelin levels enhances alcohol craving and intake, blocking ghrelin receptors abolishes these effects. If ghrelin is also involved in non-dependent alcohol consumption in humans, though, remains unknown. The aim was therefore to investigate the relationship between ghrelin serum levels and alcohol consumption in a large population-based sample. METHODS: Total ghrelin was determined after an overnight fast in 1666 subjects participating in a population-based cross-sectional study ('LIFE') including 10,000 adults. 1521 subjects were included in this analysis. Alcohol consumption was assessed using a food frequency questionnaire (FFQ). Multiple linear regression analyses and extreme group comparisons testing for statistical differences of alcohol consumption between the highest and lowest quartile according to ghrelin levels were performed. RESULTS: Alcohol consumption was positively associated with serum ghrelin; total sample: ß = 0.003, p = 0.002; men: ß = 0.005, p = 0.023; women: ß = 0.002, p = 0.007, adjusted for age, BMI and smoking status. Mean alcohol consumption in men/women belonging to the highest quartile of serum ghrelin levels (men: 21.5 (21.1) g/day; women: 7.5 (11.4) g/day) was considerably higher than in those belonging to the lowest quartile (men: 16.5 (19.3) g/day p < 0.002; women: 4.59 (10.7) g/day p = 0.0001). CONCLUSION: This is the first study showing that alcohol consumption is positively associated with serum ghrelin in a population-based sample. The study provides an initial indication that ghrelin is also involved in the regulation of alcohol consumption in non-dependent subjects.

[Association of leptin, adiponectin, and ghrelin in breast milk with the growth of infants with exclusive breastfeeding].

OBJECTIVE: To investigate the association of leptin, adiponectin, and ghrelin in breast milk with the weight growth velocity of infants with exclusive breastfeeding. METHODS: A total of 67 full-term singleton infants who received regular child care and exclusive breastfeeding and their mothers were enrolled. The nutritional status was evaluated based on the measurements of body weight and body length (underweight, growth retardation, emaciation, overweight, and obesity). Z score was used to calculate growth velocity, and according to the ΔZ score, the infants were divided into poor growth group, low growth velocity group, and normal growth velocity group. Mature breast milk samples were collected from their mothers, and ELISA was used to measure the levels of leptin, adiponectin, and ghrelin. RESULTS: The emaciation group had a significantly lower level of leptin in breast milk than the non-emaciation group (P<0.05), and the overweight/obesity group had a significantly lower level of adiponectin than the non-overweight/obesity group (P<0.05). The correlation analysis showed that the level of ghrelin in breast milk was positively correlated with Z score of current body weight and ΔZ score compared with birth weight (rs=0.280 and 0.290 respectively; P<0.05). The regression analysis showed that the level of ghrelin in breast milk was an important influencing factor for the Z score of body weight (ß=0.161, P<0.05). CONCLUSIONS: Various active constituents in breast milk, including leptin, adiponectin, and ghrelin, may regulate the growth and development of infants to a certain degree, but long-term studies and observation are needed to investigate their association with offspring growth and development and the health-promoting effect of breast milk on offspring.