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Contents The objective of this study was to describe testosterone (T) response to GnRH challenge in antagonist-treated dogs over a 30-day period. Eight mongrel dogs were randomly assigned to either the GnRH antagonist acyline 330 μg/kg sc (ACY; n = 4) or a placebo group (PLA; n = 4). The dogs were serially challenged with the GnRH agonist, buserelin 0.2 μg/kg sc on days −1, 1, 3, 7, 10, 14, 21 and 30. On these days, blood samples for T determinations were collected before (−30 min) and 60, 120 and 180 min after the agonist injection. Basal (−30 min) and post-GnRH agonist stimulation T values were compared by anova for repeated measures. Before treatments (day −1), there were no differences in basal T serum concentrations between groups (p > 0.1). After treatments, basal T showed a significant interaction between treatment and day (p < 0.05). Furthermore, when both groups were analysed independently, basal T varied in the ACY (p < 0.01) but not in the PLA group (p > 0.1). On day −1, before treatments, the stimulation tests had only a time effect (p = 0.05) although on days 1 (p < 0.01), 3 (p < 0.01), 7 (p < 0.01), 10 (p < 0.01) and 14 (p < 0.05), the response to the agonist differed between groups, becoming similar on days 21 (p > 0.05) and 30 (p > 0.05). It was concluded that, in dogs, a single administration of the GnRH antagonist prevented canine gonadal axis to physiologically respond to agonistic challenge during 14 days. [ABSTRACT FROM AUTHOR]

The growth hormone secretagogue receptor (GHSR), primarily known as the receptor for the hunger hormone ghrelin, potently controls food intake, yet the specific Ghsr-expressing cells mediating the orexigenic effects of this receptor remain incompletely characterized. Since Ghsr is expressed in gamma-aminobutyric acid (GABA)-producing neurons, we sought to investigate whether the selective expression of Ghsr in a subset of GABA neurons is sufficient to mediate GHSR's effects on feeding. First, we crossed mice that express a tamoxifen-dependent Cre recombinase in the subset of GABA neurons that express glutamic acid decarboxylase 2 (Gad2) enzyme (Gad2-CreER mice) with reporter mice, and found that ghrelin mainly targets a subset of Gad2-expressing neurons located in the hypothalamic arcuate nucleus (ARH) and that is predominantly segregated from Agouti-related protein (AgRP)-expressing neurons. Analysis of various single-cell RNA-sequencing datasets further corroborated that the primary subset of cells coexpressing Gad2 and Ghsr in the mouse brain are non-AgRP ARH neurons. Next, we crossed Gad2-CreER mice with reactivable GHSR-deficient mice to generate mice expressing Ghsr only in Gad2-expressing neurons (Gad2-GHSR mice). We found that ghrelin treatment induced the expression of the marker of transcriptional activation c-Fos in the ARH of Gad2-GHSR mice, yet failed to induce food intake. In contrast, food deprivation-induced refeeding was higher in Gad2-GHSR mice than in GHSR-deficient mice and similar to wild-type mice, suggesting that ghrelin-independent roles of GHSR in a subset of GABA neurons is sufficient for eliciting full compensatory hyperphagia in mice., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. See the journal About page for additional terms.)

Ghrelin is a stomach-derived hormone that acts via the growth hormone secretagogue receptor (GHSR). Recent evidence suggests that some of ghrelin's actions may be mediated via the supramammillary nucleus (SuM). Not only does ghrelin bind to cells within the mouse SuM, but ghrelin also activates SuM cells and intra-SuM ghrelin administration induces feeding in rats. In the current study, we aimed to further characterize ghrelin action in the SuM. We first investigated a mouse model expressing enhanced green fluorescent protein (eGFP) under the promoter of GHSR (GHSR-eGFP mice). We found that the SuM of GHSR-eGFP mice contains a significant amount of eGFP cells, some of which express neuronal nitric oxide synthase. Centrally-, but not systemically-, injected ghrelin reached the SuM, where it induced c-Fos expression. Furthermore, a 5-day 40% calorie restriction protocol, but not a 2-day fast, increased c-Fos expression in non-eGFP+ cells of the SuM of GHSR-eGFP mice, whereas c-Fos induction by calorie restriction was not observed in GHSR-deficient mice. Exposure of satiated mice to a binge-like eating protocol also increased c-Fos expression in non-eGFP+ cells of the SuM of GHSR-eGFP mice in a GHSR-dependent manner. Finally, intra-SuM-injected ghrelin did not acutely affect food intake, locomotor activity, behavioral arousal or spatial memory but increased recognition memory. Thus, we provide a compelling neuroanatomical characterization of GHSR SuM neurons and its behavioral implications in mice., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Ghrelin is a stomach-derived peptide hormone with salient roles in the regulation of energy balance and metabolism. Notably, ghrelin is recognized as the most powerful known circulating orexigenic hormone. Here, we systematically investigated the effects of ghrelin on energy homeostasis and found that ghrelin primarily induces a biphasic effect on food intake that has indirect consequences on energy expenditure and nutrient partitioning. We also found that ghrelin-induced biphasic effect on food intake requires the integrity of Agouti-related peptide/neuropeptide Y-producing neurons of the hypothalamic arcuate nucleus, which seem to display a long-lasting activation after a single systemic injection of ghrelin. Finally, we found that different autonomic, hormonal and metabolic satiation signals transiently counteract ghrelin-induced food intake. Based on our observations, we propose a heuristic model to describe how the orexigenic effect of ghrelin and the anorectic food intake-induced rebound sculpt a timely constrain feeding response to ghrelin., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Growth hormone secretagogue receptor (GHSR), the receptor for ghrelin, is expressed in key brain nuclei that regulate food intake. The dopamine (DA) pathways have long been recognized to play key roles mediating GHSR effects on feeding behaviors. Here, we aimed to determine the role of GHSR in DA neurons controlling appetitive and consummatory behaviors towards high fat (HF) diet. For this purpose, we crossed reactivable GHSR-deficient mice with DA transporter (DAT)-Cre mice, which express Cre recombinase under the DAT promoter that is active exclusively in DA neurons, to generate mice with GHSR expression limited to DA neurons (DAT-GHSR mice). We found that DAT-GHSR mice show an increase of c-Fos levels in brain areas containing DA neurons after ghrelin treatment, in a similar fashion as seen in wild-type mice; however, they did not increase food intake or locomotor activity in response to systemically- or centrally-administered ghrelin. In addition, we found that satiated DAT-GHSR mice displayed both anticipatory activity to scheduled HF diet exposure and HF intake in a binge-like eating protocol similar to those in wild-type mice, whereas GHSR-deficient mice displayed impaired responses. We conclude that GHSR expression in DA neurons is sufficient to both mediate increased anticipatory activity to a scheduled HF diet exposure and fully orchestrate binge-like HF intake, but it is insufficient to restore the acute orexigenic or locomotor effects of ghrelin treatment. Thus, GHSR in DA neurons affects appetitive and consummatory behaviors towards HF diet that take place in the absence of caloric needs., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Liver-expressed antimicrobial peptide 2 (LEAP2) was recently recognized as an endogenous ligand for the growth hormone secretagogue receptor (GHSR), which also is a receptor for the hormone ghrelin. LEAP2 blocks ghrelin-induced activation of GHSR and inhibits GHSR constitutive activity. Since fluorescence-based imaging and pharmacological analyses to investigate the biology of GHSR require reliable probes, we developed a novel fluorescent GHSR ligand based on the N-terminal LEAP2 sequence, hereafter named F-LEAP2. In vitro, F-LEAP2 displayed binding affinity and inverse agonism to GHSR similar to LEAP2. In a heterologous expression system, F-LEAP2 labeling was specifically observed in the surface of GHSR-expressing cells, in contrast to fluorescent ghrelin labeling that was mainly observed inside the GHSR-expressing cells. In mice, centrally-injected F-LEAP2 reduced ghrelin-induced food intake, in a similar fashion to LEAP2, and specifically labeled cells in GHSR-expressing brain areas. Thus, F-LEAP2 represents a valuable tool to study the biology of GHSR in vitro and in vivo., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Since inbred C57BL/6 mice are known to show inter-individual phenotypic variability for some traits, we tested the hypothesis that inbred C57BL/6 mice display a different tendency to consume a high fat (HF) diet. For this purpose, we used a compilation of HF intake data from an experimental protocol in which satiated mice were exposed to a HF pellet every morning for 2-h over 4 consecutive days. We found that mice displayed a large degree of variability in HF intake. Since day 1 HF intake significantly correlated with HF intake in successive days, we applied a hierarchical clustering algorithm on HF intake measurements in days 2, 3, and 4 in order to classify mice into "low" or "high" HF intake groups. "Low" HF intake group showed a day 1 HF intake similar to that seen in mice exposed to regular chow, while "high" HF intake group showed a higher day 1 HF intake as compared to "low" HF intake group. Both groups of mice increased HF consumption over the successive days, but "high" HF intake group always displayed a higher HF consumption than the "low" HF intake group. As compared to "low" HF intake group, "high" HF intake group showed a higher number of dopamine neurons positive for c-Fos in the VTA after the last event of HF intake. Thus, inbred C57BL/6 mice show inter-individual variability for HF intake and such feature may be linked to a different response to the rewarding properties of the HF diet.

Ghrelin is a stomach-derived hormone that regulates a variety of biological functions such as food intake, gastrointestinal function and blood glucose metabolism, among others. Ghrelin acts via the growth hormone secretagogue receptor (GHSR), a G-protein-coupled receptor located in key brain areas that mediate specific actions of the hormone. GHSR is highly expressed in the nucleus of the solitary tract (NTS), which is located in the medulla oblongata and controls essential functions, including orofacial, autonomic, neuroendocrine and behavioral responses. Here, we used a mouse model, in which the expression of enhanced green fluorescent protein (eGFP) is controlled by the promoter of GHSR (GHSR-eGFP mice), to gain neuroanatomical and functional insights of the GHSR-expressing neurons of the NTS. We found that GHSR-expressing neurons of the NTS are segregated in clusters that were symmetrically distributed to the midline: (1) a pair of rostral clusters, and (2) a caudal and medially located cluster. We also identified that a subset of GHSR neurons of the caudal NTS are GABAergic. Finally, we found that rostral NTS GHSR neurons increase the levels of the marker of neuronal activation c-Fos in mice exposed to fasting/refeeding or high-fat diet bingeing protocols, while caudal NTS GHSR neurons increase the levels of c-Fos in mice exposed to gastric distension or LiCl-induced malaise protocols. Thus, current data provide evidence that ghrelin receptor signaling seems to target segregated clusters of neurons within the NTS that, in turn, may be activated by different stimuli.

Various combinations of gonadotropin-releasing hormone (GnRH) antagonists and long-acting GnRH agonists have been assessed in several species to prevent the "flare-up" effect that agonists cause on the pituitary-gonadal axis. To determine the effect of a single administration of the GnRH antagonist acyline in anestrous GnRH agonist-implanted domestic bitches, 19 dogs (canis familiaris) were randomly assigned to receive either 10mg sc deslorelin acetate (DA; n=6) or DA combined with 330 microg/kg sc acyline within the first 48 h (DA & ACY; n=13). These bitches were examined daily for detection of posttreatment flare-up, manifested as an estrous response during the month after treatment. In the DA and DA&ACY groups, an estrous response was detected in 6 of 6 and 9 of 12 (P<0.5) of the bitches, starting 5.3+/-1.3 and 10.1+/-1.8 d (mean+/-SEM, P=0.5), respectively, after treatment. Based on serum progesterone concentrations, ovulation occurred in 6 of 6 and 5 of 9 of these bitches (P=0.1). None of the dogs had any local or systemic side effects related to the treatments. In five DA and six DA&ACY bitches that could be followed up after the trial, interestrus intervals were 385+/-22.5 and 330+/-69.1 d, respectively (P>0.1). It was concluded that the current antagonist protocol prevented initial ovarian stimulation in one quarter of the treated dogs, whereas the stimulation period was postponed and ovulation was inhibited in approximately half of the remainder.

The polycystic ovary syndrome (PCOS) and hyperandrogenism are some of the most common endocrine disorders in women of fertile age. Insulin resistance is present in a significant proportion of hyperandrogenic patients, yet also, impaired beta-cell function, even in absence of clinically evident glucose intolerance, is a frequent finding, especially in patients with familial history of type 2 diabetes mellitus. Therefore, it is not surprising that hyperandrogenism, PCOS, and disorders of carbohydrate metabolism are associated frequently. This association was first reported 75 years ago and, although the mechanisms responsible are not precisely understood, insulin resistance plays an important role in the development of both disorders. PCOS patients develop type 2 diabetes mellitus more frequently than non-hyperandrogenic women and, conversely, women with type 2 diabetes have a greater risk of having PCOS compared with the normal population. Although type 1 diabetes mellitus is a disease characterized by complete abolition of endogenous insulin secretion, a certain degree of hyperinsulinism may exist, resulting from the relatively excessive insulin doses needed to maintain a strict metabolic control. This exogenous hyperinsulinism may increase ovarian androgen secretion, and it has been reported that there is an increased prevalence of hyperandrogenic disorders in type 1 diabetic women. Considering that insulin resistance, hyperinsulinemia and androgen excess may collaborate in increasing the risk for CVD in these women, the identification of hyperandrogenic symptoms in diabetic women, and the identification of disorders of glucose tolerance in hyperandrogenic patients, may have important consequences for the correct management of these women.