Altered neurosecretion is a common feature of diverse pathophysiological conditions, including central nervous system disorders, hypertension, and tumorigenesis, where chromogranin A (CgA) is widely used as a biomarker, and both phosphatidic acid (PA) synthesis and catecholamine (CA) secretion are dysregulated. Here, we identify a direct interaction between CgA and PA at the plasma membrane of living cells using a newly developed synthetic PA fluorescent probe and Förster′s resonance energy transfer (FRET) combined with fluorescence lifetime imaging microscopy (FLIM). Confocal microscopy and transmission electron microscopy (TEM) further reveal that this interaction is spatially confined to exocytic sites. Using Total Internal Reflection Fluorescence microscopy (TIRF-M), we show that expression of a CgA variant lacking the PA-binding domain (PABD) in COS-7 cells increases the frequency of exocytic events and accelerates CgA release kinetics. In chromaffin cells, amperometry and live tracking of exocytosis-endocytosis demonstrate that CgA overexpression enhances granular CA content, extends fusion pore opening, and accelerates exocytosis-endocytosis coupling, effects that are abolished upon expression of CgA variant. Together, these findings unveil a new role of CgA/PA interaction in fine-tuning neurohormone secretion, suggesting unexplored avenues for restoring neurosecretion in disease-relevant contexts. ### Competing Interest Statement The authors have declared no competing interest. Fondation pour la Recherche Médicale, https://ror.org/04w6kn183, DEI20151234424 Agence Nationale de la Recherche, ANR-19-CE44-0019 Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-10-INBS-04

Graphical abstract: Abstract:The present study aims to investigate the impact of orexin deficiency on the regulation of energy and glucose metabolism using a mouse model depleted of the prepro-orexin gene. Our data reveal that, despite a decrease in food consumption (at least in males), orexin deficiency induces a significant increase in body weight that is associated with an alteration in the body composition, as male and female orexin-deficient mice display increased fat mass compared to wild-type littermates. Nevertheless, no significant differences of global energy expenditure and locomotor activity were observed in the mutant mice relative to the control. Glucose homeostasis is also impaired in the absence of orexins, since glucose tolerance and insulin secretion are diminished, and insulin sensitivity is slightly reduced. In addition, the livers of male orexin-KO mice are significantly larger and heavier with more adipose tissue than wild-type mice. Interestingly, orexin-deficient mice present an upregulation of liver enzymes involved in gluconeogenesis and a downregulation of GCK, an enzyme that promotes glycogen storage, which may participate to the altered glucose metabolism of orexin mutant mice. To conclude, the present study indicates that orexin deficiency induces profound alterations in the regulation of energy and glucose metabolism, which is more pronounced in males than in females. These findings support the idea that dysfunction of this orexin system may promote obesity and diabetes, and could represent an interesting therapeutic target in the context of 'diabesity'.

26RFa is a regulatory peptide initially isolated from the brain. 26RFa was found to be involved in the regulation of vital functions such as the regulation of energy and glucose metabolism. However, the whole distribution of 26RFa in the organs/tissues of the organism remains fragmentary although it represents a crucial step to discover novel physiological functions for this regulatory peptide. To this aim, we have generated a mouse line that expresses the fluorescent protein tdTomato in 26RFa-expressing cells, and visualization of tdTomato immunostaining in toto was performed using the tridimensional imaging approach. Our observations reveal that 26RFa is largely distributed among the organism of male and female mice. 26RFa-expressing cells were notably found in numerous regions of the central nervous system including the olfactory bulbs, the cortex, the hippocampus, the hypothalamus, the cerebellum, the brainstem and the spinal cord. At the periphery, 26RFa-expressing structures were detected all along the gastrointestinal tract, in the liver, the white adipose tissue, the kidney, the adrenal gland, the lungs, the male and female reproductive tracts, the striated muscles, the thymus and a number of exocrine glands such as the salivary glands, the prostate, the seminal vesicles. In conclusion, the present anatomical observations are in agreement with the main physiological functions previously reported for 26RFa such the regulation of energy and glucose metabolism or the control of the hypothalamo-pituitary-gonadal or adrenal axis. However, 26RFa is present in other organs/tissues in which its physiological relevance is totally unknown opening therefore a new field of research for this regulatory peptide.

BACKGROUND:Obesity and type 2 diabetes are strongly associated pathologies, currently considered as a worldwide epidemic problem. Understanding the mechanisms that drive the development of these diseases would enable to develop new therapeutic strategies for their prevention and treatment. Particularly, the role of the brain in energy and glucose homeostasis has been studied for 2 decades. In specific, the hypothalamus contains well-identified neural networks that regulate appetite and potentially also glucose homeostasis. A new concept has thus emerged, suggesting that obesity and diabetes could be due to a dysfunction of the same, still poorly understood, neural networks. SUMMARY:The neuropeptide 26RFa (also termed QRFP) belongs to the family of RFamide regulatory peptides and has been identified as the endogenous ligand of the human G protein-coupled receptor GPR103 (QRFPR). The primary structure of 26RFa is strongly conserved during vertebrate evolution, suggesting its crucial roles in the control of vital functions. Indeed, the 26RFa/GPR103 peptidergic system is reported to be involved in the control of various neuroendocrine functions, notably the control of energy metabolism in which it plays an important role, both centrally and peripherally, since 26RFa regulates feeding behavior, thermogenesis and lipogenesis. Moreover, 26RFa is reported to control glucose homeostasis both peripherally, where it acts as an incretin, and centrally, where the 26RFa/GPR103 system relays insulin signaling in the brain to control glucose metabolism. KEY MESSAGES:This review gives a comprehensive overview of the role of the 26RFa/GPR103 system as a key player in the control of energy and glucose metabolism. In a pathophysiological context, this neuropeptidergic system represents a prime therapeutic target whose mechanisms are highly relevant to decipher.

Aim: To investigate the evolution of the incretin-like peptide 26RFa in a prospective cohort of women living with obesity with or without type 2 diabetes (T2D) before and after sleeve gastrectomy (SG). Methods: In this study, a total of 61 women were divided into three groups: women living with severe obesity without T2D (WlwOB group), women living with severe obesity and T2D (WlwOB-T2D group) and lean healthy volunteers (control group). Serum 26RFa concentrations were measured using a 26RFa enzyme-linked immunosorbent assay developed specifically for this study during meal tests before SG, and 30 and 180 days after SG. Results: At baseline, serum 26RFa levels were reduced in the WlwOB (P < .05) and WlwOB-T2D (P < .01) groups compared with controls. In the WlwOB-T2D group, fasting 26RFa levels were found to increase throughout the entire follow-up period up to 6 months after the SG (P < .001). During the meal tests, serum 26RFa levels increased, especially in the WlwOB-T2D group at baseline. At the end of the follow-up, the profile of 26RFa concentrations obtained during the meal test in patients with severe obesity and T2D was similar to that of the controls. Conclusions: This prospective clinical study provides the first evidence that circulating 26RFa is altered mainly in WlwOB-T2D, and that these defects are partially reversed after SG.

INTRODUCTION:Immunoglobulins (Ig) reactive with α-melanocyte-stimulating hormone (α-MSH), an anorexigenic neuropeptide, are present in humans and were previously associated with eating disorders. In this longitudinal study involving patients with anorexia nervosa (AN), we determined whether α-MSH in serum is bound to IgG and analyzed long-term dynamics of both α-MSH peptide and α-MSH-reactive Ig in relation to changes in BMI and gut microbiota composition. METHODS:The study included 64 adolescents with a restrictive form of AN, whose serum samples were collected at hospital admission, discharge, and during a 1-year follow-up visit and 41 healthy controls, all females. RESULTS:We found that in both study groups, approximately 40% of serum α-MSH was reversibly bound to IgG and that levels of α-MSH-reactive IgG but not of α-MSH peptide in patients with AN were low at hospital admission but recovered 1 year later. Total IgG levels were also low at admission. Moreover, BMI-standard deviation score correlated positively with α-MSH IgG in both groups studied but negatively with α-MSH peptide only in controls. Significant correlations between the abundance of specific bacterial taxa in the gut microbiota and α-MSH peptide and IgG levels were found in both study groups, but they were more frequent in controls. CONCLUSION:We conclude that IgG in the blood plays a role as an α-MSH-binding protein, whose characteristics are associated with BMI in both patients with AN and controls. Furthermore, the study suggests that low production of α-MSH-reactive IgG during the starvation phase in patients with AN may be related to altered gut microbiota composition.

The regulatory peptide 26RFa (QRFP) is involved in the control of glucose homeostasis at the periphery by acting as an incretin, and in the brain by mediating the central antihyperglycemic effect of insulin, indicating the occurrence of a close relationship between 26RFa and insulin in the regulation of glucose metabolism. Here, we investigated the physiological interactions between 26RFa and insulin in two complementary models i.e. a model of obese/hyperglycemic mice deficient for 26RFa and a model of diabetic mice deficient for insulin. For this, transgenic 26RFa-deficient mice were made obese and chronically hyperglycemic by a 3-month high fat diet (HFD) and second group of mice was made diabetic by destruction of the β cells of the pancreatic islets using a single injection of streptozotocin. Our data reveal that 26RFa deficiency does not impact significantly the "glycemic" phenotype of the HFD mice. The pancreatic islets, liver, white adipose tissue masses are not altered by the lack of 26RFa production but the brown adipose tissue (BAT) weight is significantly increased in these animals. In diabetic insulin-deficient mice, the injection of 26RFa does not exhibit any beneficial effect on the impaired glucose homeostasis characterizing this model. Finally, we show that streptozotocin diabetic mice display lowered plasma 26RFa levels as compared to untreated mice, whereas the expression of the peptide in the duodenum is not affected. Taken together, the present results indicate that dysregulation of glucose homeostasis in obese/hyperglycemic mice is not aggravated by the absence of 26RFa that may be compensated by the increase of BAT mass. In diabetic insulin-deficient mice, the antihypergycemic effect of 26RFa is totally blunted probably as a result of the impaired insulin production characterizing this model, avoiding therefore the action of the peptide.

A commercial strain of Hafnia alvei (H. alvei) 4597 bacteria was shown to reduce food intake and promote weight loss, effects possibly induced by the bacterial protein ClpB, an antigen-mimetic of the anorexigenic α-melanocyte-stimulating hormone. A decrease in the basal plasma glucose levels was also observed in overweight fasted humans and mice receiving H. alvei. However, it is not known whether H. alvei influences sweet taste preference and whether its protein extract or ClpB are sufficient to increase glucose tolerance; these are the objectives tested in the present study. C57BL/6J male mice were kept under standard diet and were gavaged daily for 17 days with a suspension of H. alvei (4.5 × 107 CFU/animal) or with H. alvei total protein extract (5 μg/animal) or saline as a control. Sweet taste preference was analyzed via a brief-access licking test with sucrose solution. Glucose tolerance tests (GTT) were performed after the intraperitoneal (IP) or intragastric (IG) glucose administration at the 9th and 15th days of gavage, respectively. The expression of regulatory peptides' mRNA levels was assayed in the hypothalamus. In another experiment performed in non-treated C57BL/6J male mice, effects of acute IP administration of recombinant ClpB protein on glucose tolerance were studied by both IP- and IG-GTT. Mice treated with the H. alvei protein extract showed an improved glucose tolerance in IP-GTT but not in IG-GTT. Both groups treated with H. alvei bacteria or protein extract showed a reduction of pancreatic tissue weight but without significant changes to basal plasma insulin. No significant effects of H. alvei bacteria or its total protein extract administration were observed on the sweet taste preference, insulin tolerance and expression of regulatory peptides' mRNA in the hypothalamus. Acute administration of ClpB in non-treated mice increased glucose tolerance during the IP-GTT but not the IG-GTT, and reduced basal plasma glucose levels. We conclude that both the H. alvei protein extract introduced orally and the ClpB protein administered via IP improve glucose tolerance probably by acting at the glucose postabsorptive level. Moreover, H. alvei probiotic does not seem to influence the sweet taste preference. These results justify future testing of both the H. alvei protein extract and ClpB protein in animal models of diabetes.

Oxytocin is a neuropeptide produced mainly in the hypothalamus and secreted in the CNS and blood. In the brain, it plays a major role in promoting social interactions. Here we show that in human plasma about 60% of oxytocin is naturally bound to IgG which modulates oxytocin receptor signaling. Further, we found that IgG of violent aggressive inmates were characterized by lower affinity for oxytocin, causing decreased oxytocin carrier capacity and reduced receptor activation as compared to men from the general population. Moreover, peripheral administration of oxytocin together with human oxytocin-reactive IgG to resident mice in a resident-intruder test, reduced c-fos activation in several brain regions involved in the regulation of aggressive/defensive behavior correlating with the attack number and duration. We conclude that IgG is a natural oxytocin carrier protein modulating oxytocin receptor signaling which can be relevant to the biological mechanisms of aggressive behavior.

Narcolepsy with cataplexy is a sleep disorder caused by deficiency in the hypothalamic neuropeptide hypocretin/orexin (HCRT), unanimously believed to result from autoimmune destruction of hypocretin-producing neurons. HCRT deficiency can also occur in secondary forms of narcolepsy and be only temporary, suggesting it can occur without irreversible neuronal loss. The recent discovery that narcolepsy patients also show loss of hypothalamic (corticotropin-releasing hormone) CRH-producing neurons suggests that other mechanisms than cell-specific autoimmune attack, are involved. Here, we identify the HCRT cell-colocalized neuropeptide QRFP as the best marker of HCRT neurons. We show that if HCRT neurons are ablated in mice, in addition to Hcrt, Qrfp transcript is also lost in the lateral hypothalamus, while in mice where only the Hcrt gene is inactivated Qrfp is unchanged. Similarly, postmortem hypothalamic tissues of narcolepsy patients show preserved QRFP expression, suggesting the neurons are present but fail to actively produce HCRT. We show that the promoter of the HCRT gene of patients exhibits hypermethylation at a methylation-sensitive and evolutionary-conserved PAX5:ETS1 transcription factor-binding site, suggesting the gene is subject to transcriptional silencing. We show also that in addition to HCRT, CRH and Dynorphin ( PDYN ) gene promoters, exhibit hypermethylation in the hypothalamus of patients. Altogether, we propose that HCRT , PDYN , and CRH are epigenetically silenced by a hypothalamic assault (inflammation) in narcolepsy patients, without concurrent cell death. Since methylation is reversible, our findings open the prospect of reversing or curing narcolepsy.

26RFa, also referred to as QRFP, is a hypothalamic neuropeptide mainly known for its role in the regulation of appetite and glucose metabolism. Its possible relevance to emotional regulation is largely unexplored. To address this, in the present exploratory study, we analyzed the plasma concentrations of 26RFa in humans characterized by different levels of anxiety and aggressive behavior. For this purpose, the study included 13 prison inmates who have committed violent crimes and 19 age-matched healthy men from the general population as controls. Anxiety, depression and aggressive behavior were evaluated in both groups using standard questionnaires. The inmate group was characterized by increased aggression and anxiety compared to the controls. We found that the mean plasma levels of 26RFa did not significantly differ between the inmates and the controls. However, several high outliers were present only in the inmate group. The plasma levels of 26RFa correlated positively with the anxiety scores in all the studied subjects and controls. After removing the high outliers in the inmate group, positive correlations of 26RFa with anxiety and a subscale of hostility in the aggression scale were also recorded in this group. No significant correlations of 26RFa with depression scores or other parameters of aggressive behavior were found. Thus, the present results did not support an involvement of 26RFa in aggressive behavior in humans but pointed to a link between this neuropeptide and anxiety. Nevertheless, considering the exploratory nature of the present study, this conclusion should be verified in a larger cohort, including the clinical degree of anxiety.

The gut microbiota is increasingly recognized as a key environmental factor that shapes host development and physiology, including neural circuits formation and function. Concurrently, there has been growing concern that early-life antibiotic exposure may alter brain developmental trajectories, increasing the risk for neurodevelopmental disorders such as autism spectrum disorder (ASD). Here, we assessed whether perturbation of the maternal gut microbiota in mice during a narrow critical perinatal window (last week of pregnancy and first three postnatal days), induced by exposure to a commonly used broad-spectrum oral antibiotic (ampicillin), influences offspring neurobehavioral outcomes relevant to ASD. Our results demonstrate that neonatal offspring from antibiotic-treated dams display an altered pattern of ultrasonic communication, which was more pronounced in males. Moreover, juvenile male, but not female, offspring from antibiotic-treated dams showed reduced social motivation and social interaction, as well as context-dependent anxiety-like behavior. However, no changes were observed in locomotor or exploratory activity. This behavioral phenotype of exposed juvenile males was associated with reduced gene expression of the oxytocin receptor (OXTR) and several tight-junction proteins in the prefrontal cortex, a key region involved in the regulation of social and emotional behaviors, as well as a mild inflammatory response in the colon. Further, juvenile offspring from exposed dams also showed distinct alterations in several gut bacterial species, including, Lactobacillus murinus, and Parabacteroides goldsteinii. Overall, this study highlights the importance of the maternal microbiome in early-life, and how its perturbation by a widely used antibiotic could contribute to atypical social and emotional development of offspring in a sex-dependent manner.

Introduction: The aim of the study is to investigate whether acute or chronic central administration of the hypothalamic neuropeptide 26RFa may ameliorate the glycemic control of obese/diabetic mice. Methods: Mice were treated for 4 months with a high-fat (HF) diet and received a single i.c.v. injection of 26RFa (3 µg) or a chronic i.c.v. administration of the peptide during 28 days via osmotic minipumps (25 µg/day). i.p. and oral glucose (GLU) tolerance tests, insulin (INS) tolerance test, glucose-stimulated insulin secretion (GSIS), food/water intake, horizontal/vertical activity, energy expenditure, meal pattern, and whole-body composition were monitored. In addition, 26RFa and GPR103 mRNA expressions as well as plasma 26RFa levels were evaluated by RT-QPCR and radioimmunoassay. Results: Acute administration of 26RFa in HF mice induced a robust antihyperglycemic effect by enhancing INS secretion, whereas chronic administration of the neuropeptide is unable to improve glucose homeostasis in these obese/diabetogenic conditions. By contrast, chronic 26RFa treatment induced an increase of the body weight accompanied with an enhanced food intake and a decreased energy expenditure. Finally, we show that the HF diet does not alter the hypothalamic expression of the 26RFa/GPR103 neuropeptidergic system nor the levels of circulating 26RFa. Conclusion: Our data indicate that the central beneficial effect of 26RFa on glucose homeostasis, by potentiating GSIS, is preserved in HF mice. However, chronic administration of the neuropeptide is unable to balance glycemia in these pathophysiological conditions, suggesting that the hypothalamic 26RFa/GPR103 neuropeptidergic system mainly affects short-term regulation of glucose metabolism.

AIMS/HYPOTHESIS:26RFa (pyroglutamilated RFamide peptide [QRFP]) is a biologically active peptide that regulates glucose homeostasis by acting as an incretin and by increasing insulin sensitivity at the periphery. 26RFa is also produced by a neuronal population localised in the hypothalamus. In this study we investigated whether 26RFa neurons are involved in the hypothalamic regulation of glucose homeostasis.METHODS:26Rfa+/+, 26Rfa-/- and insulin-deficient male C57Bl/6J mice were used in this study. Mice received an acute intracerebroventricular (i.c.v.) injection of 26RFa, insulin or the 26RFa receptor (GPR103) antagonist 25e and were subjected to IPGTTs, insulin tolerance tests, acute glucose-stimulated insulin secretion tests and pyruvate tolerance tests (PTTs). Secretion of 26RFa by hypothalamic explants after incubation with glucose, leptin or insulin was assessed. Expression and quantification of the genes encoding 26RFa, agouti-related protein, the insulin receptor and GPR103 were evaluated by quantitative reverse transcription PCR and RNAscope in situ hybridisation.RESULTS:Our data indicate that i.c.v.-injected 26RFa induces a robust antihyperglycaemic effect associated with an increase in insulin production by the pancreatic islets. In addition, we found that insulin strongly stimulates 26Rfa expression and secretion by the hypothalamus. RNAscope experiments revealed that neurons expressing 26Rfa are mainly localised in the lateral hypothalamic area, that they co-express the gene encoding the insulin receptor and that insulin induces the expression of 26Rfa in these neurons. Concurrently, the central antihyperglycaemic effect of insulin is abolished in the presence of a GPR103 antagonist and in 26RFa-deficient mice. Finally, our data indicate that the hypothalamic 26RFa neurons are not involved in the central inhibitory effect of insulin on hepatic glucose production, but mediate the central effects of the hormone on its own peripheral production.CONCLUSION/INTERPRETATION:We have identified a novel mechanism in the hypothalamic regulation of glucose homeostasis, the 26RFa/GPR103 system, and we provide evidence that this neuronal peptidergic system is a key relay for the central regulation of glucose metabolism by insulin.

Le neuropeptide 26RFa, aussi dénommé QRFP (pour pyroglutamilatedRfamide peptide) et son récepteur, le GPR103, sont impliqués dans la régulation de la prise alimentaire et l’homéostasie du glucose. En complément de données déjà rapportées (Prévost G et al., 2017), qui ont permis de décrire une activité orexigène et incrétine du 26RFa, des résultats récents viennent renforcer l’implication de ce système peptidergique dans la régulation de la glycémie et, plus spécifiquement, au cours du diabète de type 2.

26RFa (QRFP) is a biologically active peptide that regulates glucose homeostasis by acting as an incretin and by increasing insulin sensitivity at the periphery. 26RFa is also produced by a neuronal population localized in the hypothalamus. In the present study, we have investigated whether the 26RFa neurons may be involved in the hypothalamic regulation of glucose homeostasis. Our data indicate that 26RFa, i.c.v. injected, induces a robust antihyperglycemic effect associated with an increase of insulin production by the pancreatic islets. In addition, we found that insulin strongly stimulates 26RFa expression and secretion by the hypothalamus. RNAscope experiments revealed that neurons expressing 26RFa in the lateral hypothalamic area and the ventromedial hypothalamic nucleus also express the insulin receptor and that insulin induces the expression of 26RFa in these neurons. Concurrently, we show that the central antihyperglycemic effect of insulin is abolished in presence of a 26RFa receptor (GPR103) antagonist as well as in mice deficient for 26RFa. Finally, our data indicate that the hypothalamic 26RFa neurons are not involved in the central inhibitory effect of insulin on hepatic glucose production, but mediate the central effects of the hormone on its own peripheral production. To conclude, in the present study we have identified a novel actor of the hypothalamic regulation of glucose homeostasis, the 26RFa/GPR103 system and we provide the evidence that this neuronal peptidergic system is a key relay for the central regulation of glucose metabolism by insulin.

26RFa is a neuropeptide that activates the rhodopsin-like G protein-coupled receptor QRFPR/GPR103. This peptidergic system is involved in the regulation of a wide array of physiological processes including feeding behavior and glucose homeostasis. Herein, the pharmacological profile of a homogenous library of QRFPR-targeting peptide derivatives was investigated in vitro on human QRFPR-transfected cells with the aim to provide possible insights into the structural determinants of the Phe residues to govern receptor activation. Our work advocates to include in next generations of 26RFa(20–26)-based QRFPR agonists effective substitutions for each Phe unit, i.e., replacement of the Phe22 residue by a constrained 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid moiety, and substitution of both Phe24 and Phe26 by their para-chloro counterpart. Taken as a whole, this study emphasizes that optimized modifications in the C-terminal part of 26RFa are mandatory to design selective and potent peptide agonists for human QRFPR.