François Mifsud

Les neurones hypothalamiques exprimant le récepteur aux mélanocortines de type 4 (MC4R) sont des acteurs clés de la régulation de la balance énergétique à long terme. Ils intègrent les différents signaux neuro-hormonaux dépendants de la concentration de leptine circulante pour contrôler l'appétit et la dépense énergétique au long cours. Bien que le locus MC4R soit fortement associé à l'obésité commune dans les GWAS (Genome Wide Association Studies), la majorité des polymorphismes associés à l'obésité dans ces études se situent dans des régions non-codantes, dont le lien avec la prédisposition à l'obésité est encore inconnu. Notre équipe a récemment démontré que MC4R est localisé à la membrane du cil primaire dans les neurones hypothalamiques. De plus, des travaux récents ont montré que la localisation ciliaire de MC4R est nécessaire pour le maintien de l'homéostasie énergétique. Or il existe d'autres voies de signalisation connues pour passer par des récepteurs ciliaires, comme la voie Hedgehog. Dans ces voies ciliaires, on sait que la localisation au cil primaire du récepteur varie en fonction de l'activation de la voie de signalisation, et que l'activation du récepteur déclenche in fine des modifications transcriptionnelles dans la cellule. Par analogie, cette thèse repose sur l'hypothèse que la régulation de l'homéostasie énergétique par les neurones MC4R passe par un contrôle dynamique de la localisation du récepteur au cil primaire, et que l'activation ou non du récepteur au cil déclenche des mécanismes transcriptionnels dans le neurone. La seconde hypothèse est que certains variants génétiques associés à l'obésité dans les GWAS et exprimés dans les neurones pourraient en réalité perturber l'expression de gènes impliqués dans ce programme transcriptionnel en aval de MC4R. Dans la première partie de ce travail nous rapportons l'élaboration d'un algorithme basé sur l'intelligence artificielle pour quantifier la localisation de MC4R au cil primaire sur des images du noyau paraventriculaire chez la souris, dans différentes conditions physiologiques et pharmacologiques. Cette approche permet de comparer des dizaines d'images contenant des milliers de cils primaires dans ces différentes conditions, de façon entièrement autonome et objective. Grâce à cette méthode, nous montrons que la localisation de MC4R au cil est dynamique, et dépend de la concentration circulante de leptine. Nous démontrons que la localisation de MC4R au cil primaire est dépendante d'un de ses ligands, l'agoniste inverse AgRP, et que l'échelle de temps de cette dynamique est de plusieurs jours à semaines. La deuxième partie est consacrée à la mise au point d'une technique expérimentale pour étudier le transcriptome et les régions régulatrices actives dans les neurones MC4R chez la souris. Nous démontrons l'efficacité du protocole pour isoler les noyaux des neurones MC4R par cytométrie en flux, étudier leur transcriptome par RNA-seq, et identifier les régions régulatrices actives dans ces neurones par ATAC-seq. Cela nous permet d'établir le paysage transcriptionnel et épigénétique des neurones MC4R dans l'hypothalamus, et de prouver l'importance de ces neurones dans la prédisposition à l'obésité commune en s'appuyant sur les résultats de GWAS chez l'homme. Enfin, nous caractérisons l'hétérogénéité des neurones MC4R dans l'hypothalamus en construisant leur cartographie génomique par séquençage sur noyau unique. Dans la dernière partie, nous étudions les changements transcriptionnels et épigénétiques dans les neurones MC4R dans différentes conditions d'activation du système leptine-mélanocortine. En comparant les variations transcriptionnelles chez des souris soumises à une restriction calorique prolongée, à un régime gras, à un déficit en leptine ou à une infusion intra-cérébrale d'AgRP, nous tentons de mettre en évidence les cibles en aval de la signalisation de MC4R, et d'identifier de nouveaux gènes candidats impliqués dans l'homéostasie énergétique.

Objective: The melanocortin‐4 receptor agonist setmelanotide has demonstrated effectiveness in phase 3 clinical trials for patients with monogenic obesity caused by biallelic variants in the leptin receptor (LEPR) and pro‐opiomelanocortin (POMC), as well as for individuals with Bardet–Biedl syndrome (BBS). However, real‐world evidence remains limited. This study evaluates the long‐term effectiveness and safety of setmelanotide in patients who received treatment under a pre‐marketing early‐access authorization.Methods: This ongoing prospective monocentric cohort includes 17 patients with obesity due to BBS (n = 11) or biallelic variants in LEPR (n = 4) or POMC (n = 2) who either started setmelanotide in routine care between 2022 and 2024 or continued therapy after participating in the RM‐493‐022 clinical trial. The average follow‐up time was 14.4 months.Results: Patients experienced a clinically significant weight reduction of 20% from their highest pre‐treatment weight within the first year. Those previously treated in a clinical trial maintained their weight loss over time. Additionally, eating behavior improved, with significant reductions in hunger (−62%), food craving (−41%), and external eating evaluated by DEBQ. The overall safety profile was consistent with phase 3 trials data, without any new safety signals.Conclusions: These findings confirm the drug's long‐term clinical benefit and safety profile in a routine‐care setting.

Glucagon-like peptide 1 (GLP-1) stimulates insulin secretion and holds significant pharmacological potential. Nevertheless, the regulation of energy homeostasis by centrally-produced GLP-1 remains partially understood. Preproglucagon cells, known to release GLP-1, are found in the olfactory bulb (OB). We show that activating GLP-1 receptors (GLP-1R) in the OB stimulates insulin secretion in response to oral glucose in lean and diet-induced obese male mice. This is associated with reduced noradrenaline content in the pancreas and blocked by an α₂-adrenergic receptor agonist, implicating functional involvement of the sympathetic nervous system (SNS). Inhibiting GABA_A receptors in the paraventricular nucleus of the hypothalamus (PVN), the control centre of the SNS, abolishes the enhancing effect on insulin secretion induced by OB GLP-1R. Therefore, OB GLP-1-dependent regulation of insulin secretion relies on a relay within the PVN. This study provides evidence that OB GLP-1 signalling engages a top-down neural mechanism to control insulin secretion via the SNS.

The G protein–coupled receptor melanocortin-4 receptor (MC4R) and its associated protein melanocortin receptor–associated protein 2 (MRAP2) are essential for the regulation of food intake and body weight in humans. MC4R localizes and functions at the neuronal primary cilium, a microtubule-based organelle that senses and relays extracellular signals. Here, we demonstrate that MRAP2 is critical for the weight-regulating function of MC4R neurons and the ciliary localization of MC4R. More generally, our study also reveals that GPCR localization to primary cilia can require specific accessory proteins that may not be present in heterologous cell culture systems. Our findings further demonstrate that targeting of MC4R to neuronal primary cilia is essential for the control of long-term energy homeostasis and suggest that genetic disruption of MC4R ciliary localization may frequently underlie inherited forms of obesity.

Rheumatoid arthritis (RA) remains a cause of morbidity and mortality in many patients while new treatments have changed the face of the disease. Despite the emergence of these new drugs, cardiovascular (CV) diseases remain more frequent in RA patients compared with the general population. However, predictive biomarkers of RA severity and precise guidelines to manage the CV risk in these patients are still lacking. Pro-inflammatory cytokines contribute both to RA and CV pathogenesis. Focusing on IL-17A, high levels of bioactive IL-17A were associated with destruction in RA but also during myocardial infarction. The study aimed to assess the relationship between bioactive IL-17A, destruction and the occurrence of CV events (CVE) in RA patients with a very long follow-up. Thirty-six RA patients were followed between 1970 and 2012 in Lyon, France. They were tested for bioactive IL-17A and clinical and biological characteristics were recorded at baseline. Then, the occurrence of CVE was registered during the follow-up. To study the bioactive fraction of IL-17A, the bioassay used the ability of human umbilical vein endothelial cells to produce IL-8 in presence of RA plasma samples with or without an anti-IL-17A antibody. Bioactive IL-17A level at baseline was higher in RA patients who later experienced a CVE compared to those without (0.77 vs 0.21 ng/ml, p-value = 0.0095, Mann-Whitney test) and synergized with joint destruction (p-value = 0.020, Kruskal-Wallis test). Through its effects on vessels and thrombosis, high levels of bioactive IL-17A could represent a long-term marker of CV risk.

Monogenic diabetes (MgD) accounts for 1–2% of all diabetes cases. In adults, MgD is difficult to distinguish from common diabetes causes. We assessed the diagnosis rate and genetic spectrum of MgD using next-generation sequencing in patients with late adolescence/adult-onset diabetes referred for a clinical suspicion of MgD. This cross-sectional study was performed in 1564 probands recruited in 116 Endocrinology departments. Inclusion criteria were the absence of diabetes autoantibodies, and at least two of the three following criteria: an age ≤ 40 years and a body mass index (BMI) < 30 kg/m2 at diagnosis in the proband or in at least two relatives with diabetes, and a family history of diabetes in ≥ 2 generations. Seven genes (GCK, HNF1A, HNF4A, HNF1B, ABCC8, KCNJ11, and INS) were analyzed. Variant pathogenicity was assessed using current guidelines. Pathogenic variants were identified in 254 patients (16.2%) and in 23.2% of EuroCaucasian patients. Using more stringent selection criteria (family history of diabetes in ≥ 3 generations, age at diabetes ≤ 40 years and BMI < 30 kg/m2 in the proband, EuroCaucasian origin) increased the diagnosis rate to 43%, but with 70% of the identified cases being missed. GCK (44%), HNF1A (33%), and HNF4A (10%) accounted for the majority of the cases. HNF1B (6%), ABCC8/KCNJ11 (4.4%), and INS (2.8%) variants accounted for 13% of the cases. As compared to non-monogenic cases, a younger age, a lower BMI and the absence of diabetes symptoms at diagnosis, a EuroCaucasian origin, and a family history of diabetes in ≥ 3 generations were associated with MgD, but with wide phenotype overlaps between the two groups. In the total population, two clusters were identified, that mainly differed by the severity of diabetes at onset. MgDs were more prevalent in the milder phenotypic cluster. The phenotypes of the 59 patients (3.8%) with variants of uncertain significance were different from that of patients with pathogenic variants, but not from that of non-monogenic patients. Variants of HNF1B and the K-ATP channel genes were more frequently involved in MgD than previously reported. Phenotype overlapping makes the diagnosis of MgD difficult in adolescents/adults and underlies the benefit of NGS in clinically selected patients.