Prolactin Suppression of Gonadotropin-Releasing Hormone Initiation of Mammary Gland Involution in Female Rats.

It has been demonstrated that mammary gland involution after lactation is initiated by accumulation of milk in alveoli after weaning. Here, we report that involution is also dependent on mammary GnRH expression that is suppressed by PRL during lactation. Reduction of plasma prolactin (PRL) by the withdrawal of suckling stimuli increased GnRH and annexin A5 (ANXA5) expression in the mammary tissues after lactation with augmentation of epithelial apoptosis. Intramammary injection of a GnRH antagonist suppressed ANXA5 expression and apoptosis of epithelial cells after forcible weaning at midlactation, whereas local administration of GnRH agonist (GnRHa) caused apoptosis of epithelial cells with ANXA5 augmentation in lactating rats. The latter treatment also decreased mammary weight, milk production, and casein accumulation. Mammary mast cells were strongly immunopositive for GnRH and the number increased in the mammary tissues after weaning. GnRHa was shown to be a chemoattractant for mast cells by mammary local administration of GnRHa and Boyden chamber assay. PRL suppressed the mammary expression of both ANXA5 and GnRH mRNA. It also decreased mast cell numbers in the gland after lactation. These results are the first to demonstrate that GnRH, synthesized locally in the mammary tissues, is required for mammary involution after lactation. GnRH is also suggested to introduce mast cells into the regressing mammary gland and would be in favor of tissue remodeling. The suppression of these processes by PRL is a novel physiological function of PRL.

Expression of insulin-like factor 3 hormone-receptor system in the reproductive organs of male goats.

Relaxin-like factor (RLF), generally known as insulin-like factor 3 (INSL3), is essential for testis descent during fetal development. However, its role in adult males is not fully understood. We investigate the function of INSL3 in male Saanen goats by identifying cell types expressing its receptor, relaxin/insulin-like family peptide receptor (RXFP)2 and by characterizing the developmental expression pattern of INSL3 and RXFP2 and the binding of INSL3 to target cells in the male reproductive system. A highly specific RXFP2 antibody that co-localizes with an anti-FLAG antibody in HEK-293 cells recognizes RXFP2-transcript-expressing cells in the testis. INSL3 and RXFP2 mRNA expression is upregulated in the testis, starting from puberty. INSL3 mRNA and protein expression has been detected in Leydig cells, whereas RXFP2 mRNA and protein localize to Leydig cells, to meiotic and post-meiotic germ cells and to the epithelium and smooth muscle of the cauda epididymis and vas deferens. INSL3 binds to all of these tissues and cell types, with the exception of Leydig cells, in a hormone-specific and saturable manner. These results provide evidence for a functional intra- and extra-testicular INSL3 ligand-receptor system in adult male goats.

The insulin-like factor 3 (INSL3)-receptor (RXFP2) network functions as a germ cell survival/anti-apoptotic factor in boar testes.

Relaxin-like factor, commonly known as insulin-like factor (INSL3), is essential for testis descent during fetal development; however, its function in the adult testis is still being elucidated. The study aimed to identify a relaxin family peptide receptor 2 (RXFP2)-specific antibody suitable for immunological approaches, analyze which testicular germ cell types express RXFP2, and clarify its expression dynamics in the boar testis. In addition, the function of INSL3-RXFP2 signaling on the germ cells was explored by neutralizing INSL3 using long-term active immunization. Samples were collected from Duroc boars, and a commercially available RXFP2-specific antibody directed against the human RXFP2 endodomain was identified by characterizing its specificity in HEK-293 cells expressing mouse RXFP2, and by demonstrating the suitability for analyzing RXFP2 expression in porcine tissues. RXFP2 mRNA and protein were both localized mainly in meiotic and post-meiotic germ cells, but not in Leydig cells. Functional RXFP2, which enables INSL3 to bind, was detected as an ∼85-kDa band, which increased in intensity from the pubertal stage onward. Interestingly, INSL3 immunization significantly reduced testis weight and induced a 4-fold increase in the frequency of apoptotic germ cells, which was associated with the up-regulation of pro-apoptotic caspase-3 (CASP3) and BAX, and the down-regulation of anti-apoptotic XIAP and BCL2, and a substantial reduction in sperm concentration. These results revealed that RXFP2 was expressed in boar meiotic and post-meiotic germ cells, where INSL3 neutralization led to increased germ cell apoptosis and reduced sperm output, suggesting that INSL3 acts as a survival/anti-apoptotic factor in maintaining sperm production.

A new pathway mediating social effects on the endocrine system: female presence acting via norepinephrine release stimulates gonadotropin-inhibitory hormone in the paraventricular nucleus and suppresses luteinizing hormone in quail.

Rapid effects of social interactions on transient changes in hormonal levels are known in a wide variety of vertebrate taxa, ranging from fish to humans. Although these responses are mediated by the brain, neurochemical pathways that translate social signals into reproductive physiological changes are unclear. In this study, we analyzed how a female presence modifies synthesis and/or release of various neurochemicals, such as monoamines and neuropeptides, in the brain and downstream reproductive hormones in sexually active male Japanese quail. By viewing a female bird, sexually active males rapidly increased norepinephrine (NE) release in the paraventricular nucleus (PVN) of the hypothalamus, in which gonadotropin-inhibitory hormone (GnIH) neuronal cell bodies exist, increased GnIH precursor mRNA expression in the PVN, and decreased luteinizing hormone (LH) concentration in the plasma. GnIH is a hypothalamic neuropeptide that inhibits gonadotropin secretion from the pituitary. It was further shown that GnIH can rapidly suppress LH release after intravenous administration in this study. Centrally administered NE decreased plasma LH concentration in vivo. It was also shown that NE stimulated the release of GnIH from diencephalic tissue blocks in vitro. Fluorescence double-label immunohistochemistry indicated that GnIH neurons received noradrenergic innervations, and immunohistochemistry combined with in situ hybridization have further shown that GnIH neurons expressed α2A-adrenergic receptor mRNA. These results indicate that a female presence increases NE release in the PVN and stimulates GnIH release, resulting in the suppression of LH release in sexually active male quail.

Follistatin, an activin antagonist, ameliorates renal interstitial fibrosis in a rat model of unilateral ureteral obstruction.

Activin, a member of the TGF-ß superfamily, regulates cell growth and differentiation in various cell types. Activin A acts as a negative regulator of renal development as well as tubular regeneration after renal injury. However, it remains unknown whether activin A is involved in renal fibrosis. To clarify this issue, we utilized a rat model of unilateral ureteral obstruction (UUO). The expression of activin A was significantly increased in the UUO kidneys compared to that in contralateral kidneys. Activin A was detected in glomerular mesangial cells and interstitial fibroblasts in normal kidneys. In UUO kidneys, activin A was abundantly expressed by interstitial α-SMA-positive myofibroblasts. Administration of recombinant follistatin, an activin antagonist, reduced the fibrotic area in the UUO kidneys. The number of proliferating cells in the interstitium, but not in the tubules, was significantly lower in the follistatin-treated kidneys. Expression of α-SMA, deposition of type I collagen and fibronectin, and CD68-positive macrophage infiltration were significantly suppressed in the follistatin-treated kidneys. These data suggest that activin A produced by interstitial fibroblasts acts as a potent profibrotic factor during renal fibrosis. Blockade of activin A action may be a novel approach for the prevention of renal fibrosis progression.

The influence of feeding pattern on changes in plasma ghrelin in the Holstein cow.

We measured the plasma ghrelin and cortisol concentrations in non-lactating cows under fixed-time feeding conditions followed by an acute or gradual fasting treatment. During the 4 days before fasting, animals in Group 1 were fed a fixed amount of rations at 0800 and 1600 hr, and those in Group 2 were fed a gradually reduced amount. Thereafter, the plasma ghrelin concentrations of each animal were measured for 40 hr. The plasma ghrelin concentrations, which were low at the onset of fasting, increased before and after 0800 during fasting in Group 1, but not in Group 2. There were no significant differences in the plasma cortisol concentration within or between the groups. It was demonstrated that acute fasting induces elevation of the plasma ghrelin concentration, but that gradual fasting does not. This result suggests that fixed-time and fixed-quantity feeding caused a daily ghrelin rhythm in the cow and that this rhythm influenced changes in plasma ghrelin.

Dynamics of insulin-like factor 3 and its receptor expression in boar testes.

Relaxin-like factor (RLF), now mainly known as insulin-like factor 3 (INSL3), is essential for testis descent during fetal development; however, its function in the adult testis is still being elucidated. As a major step toward understanding the as-yet-unknown function of INSL3 in boars, this study aimed to develop a time-resolved fluoroimmunoassay for boar INSL3, characterize the dynamics of INSL3 expression during development, and demonstrate the expression of the INSL3 hormone-receptor system in the testis. All samples were collected from Duroc boars. The sensitivity of the assay system established was 8.2 pg/well (164 pg/ml), and no cross-reactivity with other hormones, such as porcine relaxin, was observed. Circulating INSL3 was shown to increase progressively during development. INSL3 secreted from the Leydig cells was released not only into the blood circulation but also into the interstitial and seminiferous compartments in sufficient concentrations. A testicular fractionation study revealed that its receptor RXFP2 transcripts were expressed mainly in testicular germ cells. In addition, INSL3 bound to the germ cell membranes in a hormone-specific and saturable manner. These results reveal that INSL3 secreted into the interstitial compartment from the Leydig cells is transported into the seminiferous compartments, where its receptor RXFP2 is expressed mainly in the germ cells to which INSL3 binds, suggesting that INSL3 functions as a paracrine factor on seminiferous germ cells.

Plasma cortisol and prolactin secretion rhythms in cattle under varying external environments and management techniques.

The secretion rhythms of plasma cortisol (CORT) and prolactin (PRL), hormones related to stress responsiveness and biological rhythm and controlled by light and temperature, were investigated under varying external environments and different management techniques. Serial blood samples were collected from female cattle reared in free-stall and freely fed (FF) conditions (n = 4) or in tie-stall and restricted feeding (RF) conditions (hay and concentrate twice daily, n = 4). Plasma CORT and PRL concentrations, eating behavior, and environmental parameters were analyzed. Cyclic patterns for each parameter were examined using spectral analysis, and correlations between CORT, PRL and other parameters were investigated using cross-spectral analysis. Under FF conditions, CORT secretion was not related to the lighting intensity and eating behavior. However, under RF conditions, the CORT secretion rhythm showed a distinct correlation with lighting intensity and eating behavior. Under FF conditions, the PRL secretion rhythm was similar in all seasons. However, under RF conditions, the PRL rhythm oscillated with high frequency in summer and low frequency in winter, indicating a seasonal change in rhythm. The present study demonstrates that hormone secretion rhythms change under different environments and management techniques.

Biological rhythms related to metabolism in Japanese Shorthorn cattle under varying environments and management techniques.

Plasma insulin (INS), thyroxin (T4 ), glucose (GLU), non-esterified fatty acid (NEFA), blood urea nitrogen (BUN), rectal temperature (RT) and eating behavior were evaluated in Japanese Shorthorn cattle under varying external environments and management techniques. Serial blood collection and assessments of RT and eating behavior were performed over 48 h in the spring, summer, autumn and winter in four female cattle reared under either free-stall and ad libitum feeding (FA) conditions or tie-stall and restricted feeding (TR) conditions. Cycle patterns for each parameter were examined using spectral analysis, and correlations between parameters were investigated using cross-spectral analysis. Rhythms for all parameters, except eating behavior and T4 , did not differ significantly among the varied external environments and between management techniques, although seasonal differences in the concentration or value of parameters were observed. An approximate 3- or 4-h rhythm cycle detected in T4 , GLU, NEFA, BUN, and RT might be the common metabolic rhythm. Under both conditions, the metabolite levels showed strong correlations with eating behavior. Moreover, GLU positively correlated with INS at lag time of 0 h, as did eating behavior and RT.

Effects of intravenous ghrelin injection on plasma growth hormone, insulin and glucose concentrations in calves at weaning.

Ghrelin action, which stimulates growth hormone (GH) secretion, may alter during the weaning period in calves. Our objective was to compare the effects of intravenous ghrelin injection on plasma GH, insulin and glucose concentrations in calves around the weaning period. Four Holstein bull calves were fed whole milk and allowed free access to solid feeds, and weaned at 7 weeks of age. Measurements were performed at weeks 1, 2, 4, 6, 7, 9, 11 and 13, when calves were intravenously injected with ghrelin (1.0 µg/kg body weight (BW)) through a catheter, and jugular blood samples were obtained temporally relative to the injection time. Estimated digestible energy intake per metabolic BW transiently decreased at week 7 because of low solid intake immediately after weaning, and thereafter gradually increased. Plasma insulin and glucose concentrations were not affected by ghrelin injection at all ages. In contrast, plasma GH concentrations increased with ghrelin injection at all ages. The incremental area of GH at week 7 was greatest and significantly higher compared with weeks 2, 4, 6 and 9. This result suggests that nutrient insufficiency immediately after weaning enhances GH responsiveness to ghrelin.

Plasma melatonin secretion rhythms in cattle under varying seasonal conditions.

The purpose of the present study was to investigate the secretion cycles of melatonin (MEL) in cattle over the course of four seasons. Four female Japanese Shorthorn cattle under free-stall and ad libitum feeding conditions were used, and plasma MEL concentrations were measured over a 48 h period at 1 h intervals. The time-series data were analyzed by spectral analysis, and the cycle hour was determined. Data indicated that the secretion cycle for MEL was approximately 23.5 h for all four seasons. The area under the curve of MEL from start to end of experiment for 48 h did not differ significantly among the four seasons. However, the duration of high MEL secretion which defined the duration time of the values were more than 10 pg/mL and differed significantly among the four seasons. In conclusion, this study, which was the first to use spectral analysis to evaluate the cyclic rhythm of MEL in cattle, revealed that MEL secretion cycles did not differ among the seasons. These findings are inconsistent with previous study results in that previous reports suggested that the MEL secretion cycle differed under different lighting conditions.

Role of (18)F-fluorodeoxyglucose positron emission tomography/computed tomography in predicting the pathologic response to preoperative chemoradiation therapy in patients with resectable T3 pancreatic cancer.

BACKGROUND: The purpose of this study was to evaluate whether (18)F-fluorodeoxyglucose positron emission tomography in combination with computed tomography (FDG-PET/CT) could correctly predict the pathologic response to preoperative chemoradiation therapy (CRT) for resectable pancreatic cancer. METHODS: Each of the 40 patients underwent FDG-PET/CT before and after preoperative CRT. The maximum standard uptake value (SUV) was measured for the primary tumor before and after preoperative CRT, defined as pre-CRT SUV and post-CRT SUV, respectively. The proportional alteration of the SUV decline (regression index) between post-CRT SUV and pre-CRT SUV was also calculated. These three indicators were associated with the pathologic response. RESULTS: Patients were classified as 21 responders and 19 nonresponders according to the histologic features. A pre-CRT SUV ≥ 4.7 was seen in 15 (71 %) of 21 responders and in 6 (32 %) of 19 nonresponders (p = 0.03). A regression index ≥ 0.46 was seen in 15 (71 %) responders and 5 (26 %) nonresponders (p = 0.01). CONCLUSIONS: A better pathological response can be expected for pancreatic cancer patients who have a high regression index (≥ 0.46) and a high pre-CRT SUV (≥ 4.7). The SUV measurement using FDG-PET/CT is a useful tool for predicting the pathologic response to preoperative CRT.

Expression and localization of RLF/ INSL3 receptor RXFP2 in boar testes.

This study investigated the possibility of the presence of specific receptor for relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) in boar testes. While RLF/INSL3 was produced by Leydig cells in the boar testis, its own receptor RXFP2 was expressed mainly in meiotic and post-meiotic germ cells, but not in Leydig cells, suggesting the existence of RLF/INSL3-RXFP2 signaling in germ cells of boars.

Loss of maternal annexin A5 increases the likelihood of placental platelet thrombosis and foetal loss.

Antiphospholipid syndrome is associated with an increased risk of thrombosis and pregnancy loss. Annexin A5 (Anxa5) is a candidate autoantigen. It is not known, however, whether endogenous Anxa5 prevents foetal loss during normal pregnancy. We found significant reductions in litter size and foetal weight in Anxa5-null mice (Anxa5-KO). These changes occurred even when only the mother was Anxa5-KO. A small amount of placental fibrin deposition was observed in the decidual tissues, but did not noticeably differ between wild-type and Anxa5-KO mice. However, immunoreactivity for integrin beta 3/CD61, a platelet marker, was demonstrated within thrombi in the arterial canals only in Anxa5-KO mothers. Subcutaneous administration of the anticoagulant heparin to pregnant Anxa5-KO mice significantly reduced pregnancy loss, suggesting that maternal Anxa5 is crucial for maintaining intact placental circulation. Hence, the presence of maternal Anxa5 minimises the risk of thrombosis in the placental circulation and reduces the risk of foetal loss.

Identification and characterization of canine growth differentiation factor-9 and its splicing variant.

Growth differentiation factor-9 (GDF-9), a member of the transforming growth factor-ß (TGF-ß) superfamily, is expressed exclusively in the oocyte within the ovary and plays essential roles in the ovarian function in mammals. However, a possible involvement of GDF-9 in canine ovarian physiology that has a unique ovulation process among mammals has not been studied. Interestingly, we have isolated two types of cDNA clones generated by an alternative splicing from a canine ovarian total RNA. The predominant long form cDNA shares a common precursor structure with GDF-9s in other species whereas the minor short form cDNA has a 172 amino acid truncation in the proregion. Using a transient expression system, we found that the long form cDNA has a defect in mature protein production whereas the short form cDNA readily produces mature protein. However, mutations at one or two N-glycosylation sites in the mature domain of the short form GDF-9 caused a loss in mature protein production. These results suggest that the prodomain and N-linked glycosylation of the mature domain regulate proper processing and secretion of canine GDF-9. Based on the biological functions of GDF-9, these characteristics of canine GDF-9 could be causatively linked to the unique ovulation process in the Canidae.

Effects of amino acids infused into the vein on ghrelin-induced GH, insulin and glucagon secretion in lactating cows.

To investigate the effects of amino acids on ghrelin-induced growth hormone (GH), insulin and glucagon secretion in lactating dairy cattle, six Holstein cows were randomly assigned to two infusion treatments in a cross-over design. Mixture solution of amino acids (AMI) or saline (CON) was continuously infused into the left side jugular vein via catheter for 4 h. At 2 h after the start of infusion, synthetic bovine ghrelin was single injected into the right side jugular vein through the catheter. Ghrelin injection immediately increased plasma GH, glucose and non-esterified fatty acids (P<0.05) with no difference between both treatments. Additionally, plasma insulin and glucagon concentrations were increased by ghrelin injection in both treatments. The peak value of plasma insulin concentration was greater in AMI compared with CON (P<0.05). Plasma glucagon concentration showed no difference in the peak value reached at 5 min between both treatments, and then the plasma levels in AMI compared with CON showed sustained higher values (P<0.05). After plasma glucose concentration reached the peak, the decline was greater in AMI compared with CON (P<0.05). These results showed that the increased plasma amino acids may enhance ghrelin action which in turn enhances insulin and glucagon secretions in lactating cows.

Occult mediastinal lymph node metastasis in NSCLC patients diagnosed as clinical N0-1 by preoperative integrated FDG-PET/CT and CT: Risk factors, pattern, and histopathological study.

BACKGROUND: Integrated F18-fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) is widely used for mediastinal lymph node (MLN) staging in patients with non-small cell lung cancer (NSCLC). However, FDG-PET/CT has certain limitations. Prediction of occult MLN metastasis could allow selection of candidates for preoperative cervical mediastinoscopy or endobronchial ultrasound-guided transbronchial needle aspiration. This study defined risk factors for occult MLN metastasis in patients with NSCLC patients who were diagnosed as clinical N0-1 by preoperative integrated FDG-PET/CT and CT. METHODS: Consecutive patients with NSCLC who underwent staging using integrated FDG-PET/CT as an adjunct to CT prior to lung resection from October 2006 to September 2009 were evaluated retrospectively. The prevalence of MLN metastasis in patients diagnosed as clinical N0-1 was analyzed according to clinicopathological factors such as tumor location, tumor size, histology, and FDG uptake by the primary tumor. Risk factors for occult MLN metastasis were defined by multivariate analysis. Patterns of occult MLN metastasis were also analyzed and the involved MLNs were further examined histopathologically. RESULTS: The incidence of MLN metastasis was 11% (24 patients of 224). Multivariate analysis identified adenocarcinoma (P=0.04), tumors located in upper or middle lobe (P=0.02), tumor size >3 cm (P=0.01), and SUV(max) of primary tumor >4.0 g/ml (P=0.04) as significant risk factors for MLN metastasis. The pattern of occult MLN metastasis was typical for NSCLC cases. The size of metastatic foci were small, with 68% of foci smaller than 4.0mm. CONCLUSIONS: The present study demonstrated that adenocarcinoma, tumors located in the upper or middle lobe, tumor size >3 cm, and SUV(max) of primary tumor >4.0 g/ml are risk factors for occult MLN metastasis in patients with NSCLC who were diagnosed as clinical N0-1 by preoperative integrated FDG-PET/CT and CT. Patients with tumors located in the right upper or middle lobe are considered candidates for cervical mediastinoscopy because the involved metastatic mediastinal lymph nodes are easily accessible by these modalities.

Molecular cloning and expression of canine prolactin gene.

We isolated canine prolactin cDNA from a dog pituitary cDNA library. The 930-bp nucleotide sequence covered the entire open reading frame encoding the putative 229 amino acids. It was located in chromosome 35, and had five exons. The amino acid sequence was highly homologous to the feline and porcine sequences. To generate recombinant canine prolactin, plasmids for full-length canine prolactin were constructed and transfected into the mammalian HEK293 cell line. The recombinant prolactin was secreted into the medium as an N-linked glycosylated (31 kDa) or non-glycosylated (27 kDa) protein. Western blotting revealed both of these bands were canine pituitary protein extracts.

Transforming growth factor beta family expression at the bovine feto-maternal interface.

BACKGROUND: Endometrial remodelling is necessary for implantation in all mammalian species. The TGF beta super-family plays a crucial role in this event in humans and mice. However, the role of TGF beta super-family members during implantation is still unclear in ruminants. In the present study, the spacio-temporal expression of TGF beta super-family members including activin was explored in bovine trophoblasts and endometrial tissue during the peri-implantation period in order to elucidate whether it is essential for promoting cell proliferation at the implantation site. METHODS: Gene expression in the fetal membrane and endometrium of the gravid and non-gravid horn around Day 35 of gestation were analyzed with a custom-made oligo-microarray in cattle. The expression of activin and its related genes was also analyzed with quantitative RT-PCR. Activin-like activity in trophoblastic tissue and BT-1 cells was examined using a fibroblast cell proliferation test and Western blotting. RESULTS: The expression of various TGF beta super-family related genes including activin was detected in trophoblasts and the endometrium in cattle. The most intensive activin expression was found in the gravid horn endometrium, and rather intense expression was detected in the non-gravid trophoblastic tissue. Extracts from the fetal membrane including trophoblasts and purified activin both stimulated fibroblast proliferation effectively, and activin was immunologically detected in BT-1 cells, which have trophoblastic features. CONCLUSIONS: Specific expression of the activin gene (gene name: inhibin beta A) was found in the gravid horn endometrium during peri-implantation. An activin-like molecule, which was derived from the endometrium and trophoblasts, stimulated the proliferation of fibroblast cells. These results suggested that as in other species, the activity of TGF beta super-family members including activin-like molecules plays a pivotal role in endometrial remodelling, which is an essential process in implantation and placentogenesis during the peri-implantation period in cattle.

Effects of glucose and amino acids on ghrelin secretion in sheep.

Two experiments were conducted to elucidate the effects of post-ruminal administration of starch and casein (Exp. 1), plasma amino acids concentrations (Exp. 2), and plasma glucose and insulin concentrations (Exp. 2) on plasma ghrelin concentrations in sheep. In Exp. 1, plasma ghrelin concentrations were determined by four infusion treatments (water, cornstarch, casein and cornstarch plus casein) in four wethers. Abomasal infusion of casein increased plasma alpha-amino N (AAN) concentrations. Infusion of starch or casein alone did not affect plasma ghrelin concentrations, but starch plus casein infusion increased plasma levels of ghrelin, glucose and AAN. In Exp 2, we investigated the effects of saline or amino acids on ghrelin secretion in four wethers. Two hours after the initiation of saline or amino acid infusion into the jugular vein, glucose was also continuously infused to investigate the effects of blood glucose and insulin by hyper-glycemic clump on plasma ghrelin concentrations. Infusion of amino acids alone raised plasma levels of ghrelin, but the higher plasma glucose and insulin concentrations had no effect on plasma ghrelin concentrations. These results suggest that high plasma levels of amino acids can stimulate ghrelin secretion, but glucose and insulin do not affect ghrelin secretion in sheep.