Genetic variants of LEAP2 are associated with anthropometric traits and circulating insulin-like growth factor-1 concentration: A UK Biobank study.

AIM: To test the hypothesis that liver-expressed antimicrobial peptide 2 (LEAP2) genetic variants might influence the susceptibility to human obesity. METHODS: Using data from the UK Biobank, we identified independent LEAP2 gene single nucleotide polymorphisms (SNPs) and examined their associations with obesity traits and serum insulin-like growth factor-1 (IGF-1) concentration. These associations were evaluated for both individual SNPs and after combining them into a genetic risk score (GRSLEAP2) using linear and logistic regression models. Sex-stratified analyses were also conducted. RESULTS: Five SNPs showed positive associations with obesity-related traits. rs57880964 was associated with body mass index (BMI) and waist-to-hip ratio adjusted for BMI (WHRadjBMI), in the total population and among women. Four independent SNPs were positively associated with higher serum IGF-1 concentrations in both men and women. GRSLEAP2 was associated with BMI and WHRadjBMI only in women and with serum IGF-1 concentration in both sexes. CONCLUSIONS: These findings reveal sex-specific associations between key LEAP2 gene variants and several obesity traits, while also indicating a strong independent association of LEAP2 variants with serum IGF-1 concentration.

Pre-prandial plasma liver-expressed antimicrobial peptide 2 (LEAP2) concentration in humans is inversely associated with hunger sensation in a ghrelin independent manner.

PURPOSE: The liver-expressed antimicrobial peptide 2 (LEAP2) is a newly recognized peptide hormone that acts via the growth hormone secretagogue receptor (GHSR) blunting the effects of ghrelin and displaying ghrelin-independent actions. Since the implications of LEAP2 are beginning to be elucidated, we investigated if plasma LEAP2 concentration varies with feeding status or sex and whether it is associated with glucose metabolism and appetite sensations. METHODS: We performed a single test meal study, in which plasma concentrations of LEAP2, ghrelin, insulin and glucose as well as visual analogue scales for hunger, desire to eat, prospective food consumption, fullness were assessed before and 60 min after breakfast in 44 participants (n = 21 females) with normal weight (NW) or overweight/obesity (OW/OB). RESULTS: Pre-prandial plasma LEAP2 concentration was ~ 1.6-fold higher whereas ghrelin was ~ 2.0-fold lower in individuals with OW/OB (p < 0.001) independently of sex. After adjusting for body mass index (BMI) and sex, pre-prandial plasma LEAP2 concentration displayed a direct relationship with BMI (ß: 0.09; 95%CI: 0.05, 0.13; p < 0.001), fat mass (ß: 0.05; 95%CI: 0.01, 0.09; p = 0.010) and glycemia (ß: 0.24; 95%CI: 0.05, 0.43; p = 0.021), whereas plasma ghrelin concentration displayed an inverse relationship with BMI and fat mass but not with glycemia. Postprandial plasma LEAP2 concentration increased ~ 58% in females with OW/OB (p = 0.045) but not in females with NW or in males. Pre-prandial plasma LEAP2 concentration displayed an inverse relationship with hunger score (ß: - 11.16; 95% CI: - 18.52, - 3.79; p = 0.004), in a BMI-, sex- and ghrelin-independent manner. CONCLUSIONS: LEAP2 emerges as a key hormone implicated in the regulation of metabolism and appetite in humans. TRIAL REGISTRATION: The study was retrospectively registered in clinicaltrials.gov (April 2023). CLINICALTRIALS: gov Identifier: NCT05815641.

Systemic Ghrelin Treatment Induces Rapid, Transient, and Asymmetric Changes in the Metabolic Activity of the Mouse Brain.

INTRODUCTION: Ghrelin regulates a variety of functions by acting in the brain. The targets of ghrelin in the mouse brain have been mainly mapped using immunolabeling against c-Fos, a transcription factor used as a marker of cellular activation, but such analysis has several limitations. Here, we used positron emission tomography in mice to investigate the brain areas responsive to ghrelin. METHODS: We analyzed in male mice the brain areas responsive to systemically injected ghrelin using positron emission tomography imaging of 18F-fluoro-2-deoxyglucose (18F-FDG) uptake, an indicator of metabolic rate. Additionally, we studied if systemic administration of fluorescent ghrelin or native ghrelin displays symmetric accessibility or induction of c-Fos, respectively, in the brain of male mice. RESULTS: Ghrelin increased 18F-FDG uptake in few specific areas of the isocortex, striatum, pallidum, thalamus, and midbrain at 0-10-min posttreatment. At the 10-20 and 20-30 min posttreatment, ghrelin induced mixed changes in 18F-FDG uptake in specific areas of the isocortex, striatum, pallidum, thalamus, and midbrain, as well as in areas of the olfactory areas, hippocampal and retrohippocampal regions, hypothalamus, pons, medulla, and even the cerebellum. Ghrelin-induced changes in 18F-FDG uptake were transient and asymmetric. Systemically administrated fluorescent-ghrelin-labeled midline brain areas known to contain fenestrated capillaries and the hypothalamic arcuate nucleus, where a symmetric labeling was observed. Ghrelin treatment also induced a symmetric increased c-Fos labeling in the arcuate nucleus. DISCUSSION/CONCLUSION: Systemically injected ghrelin transiently and asymmetrically affects the metabolic activity of the brain of male mice in a wide range of areas, in a food intake-independent manner. The neurobiological bases of such asymmetry seem to be independent of the accessibility of ghrelin into the brain.

GHSR controls food deprivation-induced activation of CRF neurons of the hypothalamic paraventricular nucleus in a LEAP2-dependent manner.

OBJECTIVE: Prolonged fasting is a major challenge for living organisms. An appropriate metabolic response to food deprivation requires the activation of the corticotropin-releasing factor-producing neurons of the hypothalamic paraventricular nucleus (PVHCRF neurons), which are a part of the hypothalamic-pituitary-adrenal axis (HPA), as well as the growth hormone secretagogue receptor (GHSR) signaling, whose activity is up- or down-regulated, respectively, by the hormones ghrelin and the liver-expressed antimicrobial peptide 2 (LEAP2). Since ghrelin treatment potently up-regulates the HPA axis, we studied the role of GHSR in mediating food deprivation-induced activation of the PVHCRF neurons in mice. METHODS: We estimated the activation of the PVHCRF neurons, using immuno-staining against CRF and the marker of neuronal activation c-Fos in brain sections, and assessed plasma levels of corticosterone and glucose in different pharmacologically or genetically manipulated mouse models exposed, or not, to a 2-day food deprivation protocol. In particular, we investigated ad libitum fed or food-deprived male mice that: (1) lacked GHSR gene expression, (2) had genetic deletion of the ghrelin gene, (3) displayed neurotoxic ablation of the hypothalamic arcuate nucleus, (4) were centrally treated with an anti-ghrelin antibody to block central ghrelin action, (5) were centrally treated with a GHSR ligand that blocks ghrelin-evoked and constitutive GHSR activities, or (6) received a continuous systemic infusion of LEAP2(1-12). RESULTS: We found that food deprivation results in the activation of the PVHCRF neurons and in a rise of the ghrelin/LEAP2 molar ratio. Food deprivation-induced activation of PVHCRF neurons required the presence and the signaling of GHSR at hypothalamic level, but not of ghrelin. Finally, we found that preventing the food deprivation-induced fall of LEAP2 reverses the activation of the PVHCRF neurons in food-deprived mice, although it has no effect on body weight or blood glucose. CONCLUSION: Food deprivation-induced activation of the PVHCRF neurons involves ghrelin-independent actions of GHSR at hypothalamic level and requires a decrease of plasma LEAP2 levels. We propose that the up-regulation of the actions of GHSR associated to the fall of plasma LEAP2 level are physiologically relevant neuroendocrine signals during a prolonged fasting.

Ghrelin treatment induces rapid and delayed increments of food intake: a heuristic model to explain ghrelin's orexigenic effects.

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.

A simple strategy for culturing morphologically-conserved rat hypothalamic tanycytes.

Hypothalamic tanycytes are specialized bipolar ependymal cells that line the floor of the third ventricle. Given their strategic location, tanycytes are believed to play several key functions including being a selective barrier and controlling the amount of hypothalamic-derived factors reaching the anterior pituitary. The in vitro culture of these cells has proved to be difficult. Here, we report an improved method for the generation of primary cultures of rat hypothalamic tanycytes. Ependymal cultures were derived from tissue dissected out of the median eminence region of 10-day-old rats and cultured in a chemically defined medium containing DMEM:F12, serum albumin, insulin, transferrin and the antibiotic gentamycin. After 7 days in vitro, ∼30% of the cultured cells exhibited morphological features of tanycytes as observed by phase contrast or scanning electron microscopy. Tanycyte-like cells were strongly immuno-reactive for vimentin and dopamine-cAMP-regulated phospho-protein (DARPP-32) and weakly immune-reactive for glial fibrillary acidic protein. Tanycyte-like cells displayed a stable negative resting plasma membrane potential and failed to show spiking properties in response to current injections. When exposed to fluorescent beads in the culture medium, tanycyte-like cells exhibited a robust endocytosis. Thus, the present method effectively yields cultures containing tanycyte-like cells that resemble in vivo tanycytes in terms of morphologic features and molecular markers as well as electrical and endocytic activity. To our knowledge, this is the first protocol that allows the culturing of tanycyte-like cells that can be individually identified and that conserve the morphology of tanycytes in their natural physiological environment.

Is Ghrelin Synthesized in the Central Nervous System?

Ghrelin is an octanoylated peptide that acts via its specific receptor, the growth hormone secretagogue receptor type 1a (GHSR-1a), and regulates a vast variety of physiological functions. It is well established that ghrelin is predominantly synthesized by a distinct population of endocrine cells located within the gastric oxyntic mucosa. In addition, some studies have reported that ghrelin could also be synthesized in some brain regions, such as the hypothalamus. However, evidences of neuronal production of ghrelin have been inconsistent and, as a consequence, it is still as a matter of debate if ghrelin can be centrally produced. Here, we provide a comprehensive review and discussion of the data supporting, or not, the notion that the mammalian central nervous system can synthetize ghrelin. We conclude that no irrefutable and reproducible evidence exists supporting the notion that ghrelin is synthetized, at physiologically relevant levels, in the central nervous system of adult mammals.

Obesity induces hypothalamic endoplasmic reticulum stress and impairs proopiomelanocortin (POMC) post-translational processing.

It was shown previously that abnormal prohormone processing or inactive proconverting enzymes that are responsible for this processing cause profound obesity. Our laboratory demonstrated earlier that in the diet-induced obesity (DIO) state, the appetite-suppressing neuropeptide α-melanocyte-stimulating hormone (α-MSH) is reduced, yet the mRNA of its precursor protein proopiomelanocortin (POMC) remained unaltered. It was also shown that the DIO condition promotes the development of endoplasmic reticulum (ER) stress and leptin resistance. In the current study, using an in vivo model combined with in vitro experiments, we demonstrate that obesity-induced ER stress obstructs the post-translational processing of POMC by decreasing proconverting enzyme 2, which catalyzes the conversion of adrenocorticotropin to α-MSH, thereby decreasing α-MSH peptide production. This novel mechanism of ER stress affecting POMC processing in DIO highlights the importance of ER stress in regulating central energy balance in obesity.

Melanocortin 4 receptor activation inhibits presynaptic N-type calcium channels in amygdaloid complex neurons.

The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor involved in food intake and energy expenditure regulation. MC4R activation modifies neuronal activity but the molecular mechanisms by which this regulation occurs remain unclear. Here, we tested the hypothesis that MC4R activation regulates the activity of voltage-gated calcium channels and, as a consequence, synaptic activity. We also tested whether the proposed effect occurs in the amygdala, a brain area known to mediate the anorexigenic actions of MC4R signaling. Using the patch-clamp technique, we found that the activation of MC4R with its agonist melanotan II specifically inhibited 34.5 ± 1.5% of N-type calcium currents in transiently transfected HEK293 cells. This inhibition was concentration-dependent, voltage-independent and occluded by the Gαs pathway inhibitor cholera toxin. Moreover, we found that melanotan II specifically inhibited 25.9 ± 2.0% of native N-type calcium currents and 55.4 ± 14.4% of evoked inhibitory postsynaptic currents in mouse cultured amygdala neurons. In vivo, we found that the MC4R agonist RO27-3225 increased the marker of cellular activity c-Fos in several components of the amygdala, whereas the N-type channel blocker ω conotoxin GVIA increased c-Fos expression exclusively in the central subdivision of the amygdala. Thus, MC4R specifically inhibited the presynaptic N-type channel subtype, and this inhibition may be important for the effects of melanocortin in the central subdivision of the amygdala.

Cell-to-cell transmitted alpha-synuclein recapitulates experimental Parkinson's disease.

Parkinson's disease is characterized by a progressive accumulation of alpha-Synuclein (αSyn) neuronal inclusions called Lewy bodies in the nervous system. Lewy bodies can arise from the cell-to-cell propagation of αSyn, which can occur via sequential steps of secretion and uptake. Here, by fusing a removable short signal peptide to the N-terminus of αSyn, we developed a novel mouse model with enhanced αSyn secretion and cell-to-cell transmission. Expression of the secreted αSyn in the mouse brain was under the control of a novel hybrid promoter in combination with adeno-associated virus serotype 9 (AAV9). This combination of promoter and viral vector induced a robust expression in neurons but not in the glia of injected mice. Biochemical characterization of the secreted αSyn revealed that, in cultured cells, this protein is released to the extracellular milieu via conventional secretion. The released αSyn is then internalized and processed by acceptor cells via the endosome-lysosome pathway indicating that the secreted αSyn is cell-to-cell transmitted. The secreted αSyn is aggregation-prone and amyloidogenic, and when expressed in the brain of wild-type non-transgenic mice, it induces a Parkinson's disease-like phenotype that includes a robust αSyn pathology in the substantia nigra, neuronal loss, neuroinflammation, and motor deficits, all the key features of experimental animal models of Parkinson's disease. In summary, a novel animal model of Parkinson's disease based on enhanced cell-to-cell transmission of αSyn was developed. The neuron-produced cell-to-cell transmitted αSyn triggers all phenotypic features of experimental Parkinson's disease in mice.

GHSR in a Subset of GABA Neurons Controls Food Deprivation-Induced Hyperphagia in Male Mice.

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 Pattern of GH Action in the Mouse Brain.

GH acts in numerous organs expressing the GH receptor (GHR), including the brain. However, the mechanisms behind the brain's permeability to GH and how this hormone accesses different brain regions remain unclear. It is well-known that an acute GH administration induces phosphorylation of the signal transducer and activator of transcription 5 (pSTAT5) in the mouse brain. Thus, the pattern of pSTAT5 immunoreactive cells was analyzed at different time points after IP or intracerebroventricular GH injections. After a systemic GH injection, the first cells expressing pSTAT5 were those near circumventricular organs, such as arcuate nucleus neurons adjacent to the median eminence. Both systemic and central GH injections induced a medial-to-lateral pattern of pSTAT5 immunoreactivity over time because GH-responsive cells were initially observed in periventricular areas and were progressively detected in lateral brain structures. Very few choroid plexus cells exhibited GH-induced pSTAT5. Additionally, Ghr mRNA was poorly expressed in the mouse choroid plexus. In contrast, some tanycytes lining the floor of the third ventricle expressed Ghr mRNA and exhibited GH-induced pSTAT5. The transport of radiolabeled GH into the hypothalamus did not differ between wild-type and dwarf Ghr knockout mice, indicating that GH transport into the mouse brain is GHR independent. Also, single-photon emission computed tomography confirmed that radiolabeled GH rapidly reaches the ventral part of the tuberal hypothalamus. In conclusion, our study provides novel and valuable information about the pattern and mechanisms behind GH transport into the mouse brain.

Genetic or pharmacological GHSR blockade has sexually dimorphic effects in rodents on a high-fat diet.

The stomach-derived hormone ghrelin regulates essential physiological functions. The ghrelin receptor (GHSR) has ligand-independent actions; therefore, GHSR gene deletion may be a reasonable approach to investigate the role of this system in feeding behaviors and diet-induced obesity (DIO). Here, we investigate the effects of a long-term (12-month) high-fat (HFD) versus regular diet on obesity-related measures in global GHSR-KO and wild-type (WT) Wistar male and female rats. Our main findings are that the GHSR gene deletion protects against DIO and decreases food intake during HFD in male but not in female rats. GHSR gene deletion increases thermogenesis and brain glucose uptake in male rats and modifies the effects of HFD on brain glucose metabolism in a sex-specific manner, as assessed with small animal positron emission tomography. We use RNA-sequencing to show that GHSR-KO rats have upregulated expression of genes responsible for fat oxidation in brown adipose tissue. Central administration of a novel GHSR inverse agonist, PF-5190457, attenuates ghrelin-induced food intake, but only in male, not in female mice. HFD-induced binge-like eating is reduced by inverse agonism in both sexes. Our results support GHSR as a promising target for new pharmacotherapies for obesity.

Mechanisms by which the orexigen NPY regulates anorexigenic α-MSH and TRH.

Protein posttranslational processing is a cellular mechanism fundamental to the generation of bioactive peptides, including the anorectic α-melanocyte-stimulating hormone (α-MSH) and thyrotropin-releasing hormone (TRH) peptides produced in the hypothalamic arcuate (ARC) and paraventricular (PVN) nuclei, respectively. Neuropeptide Y (NPY) promotes positive energy balance in part by suppressing α-MSH and TRH. The mechanism by which NPY regulates α-MSH output, however, is not well understood. Our results reveal that NPY inhibited the posttranslational processing of α-MSH's inactive precursor proopiomelanocortin (POMC) by decreasing the prohormone convertase-2 (PC2). We also found that early growth response protein-1 (Egr-1) and NPY-Y1 receptors mediated the NPY-induced decrease in PC2. NPY given intra-PVN also decreased PC2 in PVN samples, suggesting a reduction in PC2-mediated pro-TRH processing. In addition, NPY attenuated the α-MSH-induced increase in TRH production by two mechanisms. First, NPY decreased α-MSH-induced CREB phosphorylation, which normally enhances TRH transcription. Second, NPY decreased the amount of α-MSH in the PVN. Collectively, these results underscore the significance of the interaction between NPY and α-MSH in the central regulation of energy balance and indicate that posttranslational processing is a mechanism that plays a specific role in this interaction.

Toward a consensus nomenclature for ghrelin, its non-acylated form, liver expressed antimicrobial peptide 2 and growth hormone secretagogue receptor.

The stomach-derived octanoylated peptide ghrelin was discovered in 1999 and recognized as an endogenous agonist of the growth hormone secretagogue receptor (GHSR). Subsequently, ghrelin has been shown to play key roles in controlling not only growth hormone secretion, but also a variety of other physiological functions including, but not limited to, food intake, reward-related behaviors, glucose homeostasis and gastrointestinal tract motility. Importantly, a non-acylated form of ghrelin, desacyl-ghrelin, can also be detected in biological samples. Desacyl-ghrelin, however, does not bind to GHSR at physiological levels, and its physiological role has remained less well-characterized than that of ghrelin. Ghrelin and desacyl-ghrelin are currently referred to in the literature using many different terms, highlighting the need for a consistent nomenclature. The variability of terms used to designate ghrelin can lead not only to confusion, but also to miscommunication, especially for those who are less familiar with the ghrelin literature. Thus, we conducted a survey among experts who have contributed to the ghrelin literature aiming to identify whether a consensus may be reached. Based on the results of this consensus, we propose using the terms "ghrelin" and "desacyl-ghrelin" to refer to the hormone itself and its non-acylated form, respectively. Based on the results of this consensus, we further propose using the terms "GHSR" for the receptor, and "LEAP2" for liver-expressed antimicrobial peptide 2, a recently recognized endogenous GHSR antagonist/inverse agonist.

Chlorpromazine, an Inverse Agonist of D1R-Like, Differentially Targets Voltage-Gated Calcium Channel (CaV) Subtypes in mPFC Neurons.

The dopamine receptor type 1 (D1R) and the dopamine receptor type 5 (D5R), which are often grouped as D1R-like due to their sequence and signaling similarities, exhibit high levels of constitutive activity. The molecular basis for this agonist-independent activation has been well characterized through biochemical and mutagenesis in vitro studies. In this regard, it was reported that many antipsychotic drugs act as inverse agonists of D1R-like constitutive activity. On the other hand, D1R is highly expressed in the medial prefrontal cortex (mPFC), a brain area with important functions such as working memory. Here, we studied the impact of D1R-like constitutive activity and chlorpromazine (CPZ), an antipsychotic drug and D1R-like inverse agonist, on various neuronal CaV conductances, and we explored its effect on calcium-dependent neuronal functions in the mouse medial mPFC. Using ex vivo brain slices containing the mPFC and transfected HEK293T cells, we found that CPZ reduces CaV2.2 currents by occluding D1R-like constitutive activity, in agreement with a mechanism previously reported by our lab, whereas CPZ directly inhibits CaV1 currents in a D1R-like activity independent manner. In contrast, CPZ and D1R constitutive activity did not affect CaV2.1, CaV2.3, or CaV3 currents. Finally, we found that CPZ reduces excitatory postsynaptic responses in mPFC neurons. Our results contribute to understanding CPZ molecular targets in neurons and describe a novel physiological consequence of CPZ non-canonical action as a D1R-like inverse agonist in the mouse brain.

Ghrelin's orexigenic action in the lateral hypothalamic area involves indirect recruitment of orexin neurons and arcuate nucleus activation.

OBJECTIVE: Ghrelin is a potent orexigenic hormone, and the lateral hypothalamic area (LHA) has been suggested as a putative target mediating ghrelin's effects on food intake. Here, we aimed to investigate the presence of neurons expressing ghrelin receptor (a.k.a. growth hormone secretagogue receptor, GHSR) in the mouse LHA (LHAGHSR neurons), its physiological implications and the neuronal circuit recruited by local ghrelin action. METHODS: We investigated the distribution of LHAGHSR neurons using different histologic strategies, including the use of a reporter mice expressing enhanced green fluorescent protein under the control of the GHSR promoter. Also, we investigated the physiological implications of local injections of ghrelin within the LHA, and the extent to which the orexigenic effect of intra-LHA-injected ghrelin involves the arcuate nucleus (ARH) and orexin neurons of the LHA (LHAorexin neurons) RESULTS: We found that: 1) LHAGHSR neurons are homogeneously distributed throughout the entire LHA; 2) intra-LHA injections of ghrelin transiently increase food intake and locomotor activity; 3) ghrelin's orexigenic effect in the LHA involves the indirect recruitment of LHAorexin neurons and the activation of ARH neurons; and 4) LHAGHSR neurons are not targeted by plasma ghrelin. CONCLUSIONS: We provide a compelling neuroanatomical and functional characterization of LHAGHSR neurons in male mice that indicates that LHAGHSR cells are part of a hypothalamic neuronal circuit that potently induces food intake.

Ghrelin proteolysis increases in plasma of men, but not women, with obesity.

AIMS: Since plasma ghrelin can undergo des-acylation and proteolysis, the aim of this study was to investigate the extent to which an enhancement of these reactions is associated to the decrease of ghrelin in plasma after food intake or in individuals with obesity. MAIN METHODS: we performed an intervention cross-sectional study, in which levels of ghrelin, desacyl-ghrelin (DAG), glucose, insulin, ghrelin des-acylation and ghrelin proteolysis were assessed in plasma before and after a test meal in 40 people (n = 21 males) with normal weight (NW, n = 20) or overweight/obesity (OW/OB, n = 20). KEY FINDINGS: Preprandial ghrelin and DAG levels were lower, whereas preprandial ghrelin proteolysis was ∼4.6-fold higher in plasma of males with OW/OB. In males, ghrelin proteolysis positively correlated with glycemia. Ghrelin and DAG levels were also lower in females with OW/OB, but preprandial ghrelin proteolysis was not different between females with NW or OW/OB. Ghrelin and DAG levels decreased postprandially in males and females, independently of BMI, and ghrelin proteolysis increased postprandially ∼2 folds only in individuals with NW. Ghrelin des-acylation remained unaffected by BMI or feeding status in both sexes. SIGNIFICANCE: Current study shows that ghrelin proteolysis increases in males with obesity as well as after meal in lean individuals. Therefore, ghrelin proteolysis may be an important checkpoint and, consequently, a putative pharmacological target to control circulating ghrelin levels in humans.

Ghrelin Action in the PVH of Male Mice: Accessibility, Neuronal Targets, and CRH Neurons Activation.

The hormone ghrelin displays several well-characterized functions, including some with pharmaceutical interest. The receptor for ghrelin, the growth hormone secretagogue receptor (GHSR), is expressed in the hypothalamic paraventricular nucleus (PVH), a critical hub for the integration of metabolic, neuroendocrine, autonomic, and behavioral functions. Here, we performed a neuroanatomical and functional characterization of the neuronal types mediating ghrelin actions in the PVH of male mice. We found that fluorescent ghrelin mainly labels PVH neurons immunoreactive for nitric oxide synthase 1 (NOS1), which catalyze the production of nitric oxide [NO]). Centrally injected ghrelin increases c-Fos in NOS1 PVH neurons and NOS1 phosphorylation in the PVH. We also found that a high dose of systemically injected ghrelin increases the ghrelin level in the cerebrospinal fluid and in the periventricular PVH, and induces c-Fos in NOS1 PVH neurons. Such a high dose of systemically injected ghrelin activates a subset of NOS1 PVH neurons, which do not express oxytocin, via an arcuate nucleus-independent mechanism. Finally, we found that pharmacological inhibition of NO production fully abrogates ghrelin-induced increase of calcium concentration in corticotropin-releasing hormone neurons of the PVH whereas it partially impairs ghrelin-induced increase of plasma glucocorticoid levels. Thus, plasma ghrelin can directly target a subset of NO-producing neurons of the PVH that is involved in ghrelin-induced activation of the hypothalamic-pituitary-adrenal neuroendocrine axis.

Genetic or pharmacological GHSR blockade has sexually dimorphic effects in rodents on a high-fat diet.

The stomach-derived hormone ghrelin regulates essential physiological functions. The ghrelin receptor (GHSR) has ligand-independent actions, therefore, GHSR gene deletion may be a reasonable approach to investigate the role of this system in feeding behaviors and diet-induced obesity (DIO). Here we investigated the effects of a long-term (12 month) high-fat (HFD) versus regular diet on obesity-related measures in global GHSR-KO and wild type (WT) Wistar male and female rats. Our main findings were that the GHSR gene deletion protects against DIO and decreases food intake during HFD in male but not in female rats. GHSR gene deletion increased thermogenesis and brain glucose uptake in male rats and modified the effects of HFD on brain glucose metabolism in a sex-specific manner, as assessed with small animal positron emission tomography. RNA-sequencing was also used to show that GHSR-KO rats had upregulated expression of genes responsible for fat oxidation in brown adipose tissue. Central administration of a novel GHSR inverse agonist, PF-5190457, attenuated ghrelin-induced food intake, but only in male, not in female mice. HFD-induced binge-like eating was reduced by inverse agonism in both sexes. Our results support GHSR as a promising target for new pharmacotherapies for obesity.