Periodic Reporting for period 1 - FEEDING (Finding new target to modulate food intake)
Periodo di rendicontazione: 2023-10-01 al 2025-09-30
Obesity is recognized as a major burden to public health as it increases the risk of several chronic diseases, like diabetes, cardiovascular diseases or some cancers. Therefore, there is substantial interest in developing safe, effective anti-obesity medications. Typically, obesity develops when energy from food intake chronically exceeds energy expenditure. Energy homeostasis is centrally regulated by the hypothalamus; a brain region filled with several interdependent nuclei. The hypothalamus regulates an impressive number of vital functions such as body temperature, appetite control, sleep-wake cycles/circadian rhythm and somatic growth. The arcuate nucleus (ARC) located at the base of the hypothalamus, is thought to orchestrate the regulation of feeding behaviors and energy balance. Two neuronal populations of the ARC are considered as the first-order neurons mediating the effects of various signals (such as leptin and insulin), the orexigenic neuropeptide (NP)Y/Agouti-related peptide (AgRP) neurons and the anorexigenic pro-opiomelanocortin (POMC)/Amphetamine-regulated transcript (CART) neurons. Leptin is an adipocyte-secreted peptidic hormone that inhibits food intake and regulates body weight. Within the ARC, leptin binds to the isoform b of the leptin receptor (LepR) which signals mainly by activating kinases of the janus family to phosphorylate signal transducers and activators of transcription 3 (STAT3). Phospho(p)-STAT3 promotes the release of POMC and CART while inhibiting the secretion of other neuropeptides such as NPY and AgRP. Noteworthy, POMC is cleaved to give rise to multiple peptides such as alpha melanocyte stimulating hormone (αMSH). αMSH mainly acts on neuron expressing melanocortin 4 receptors (MC4R) in the paraventricular nucleus (PVN) to suppress appetite. Loss-of-function mutations in the genes that encode LepR, POMC or MC4R lead to persistent hunger and obesity in both humans and rodents. In nearly all forms of obesity, circulating levels of leptin increase but fail to suppress food intake and body weight. This condition has been identified as “leptin resistance”, and one of the identified mechanisms arises from the impaired LepR signaling (through SOCS3 notably) by reducing both p-STAT3 levels and αMSH production and release upon leptin administration. Despite our knowledge of the principal circuits regulating feeding, satiety, hunger and energy expenditure, we have been unable to deliver effective drugs targeting directly these systems. Most of the pharmacotherapeutic interventions to induce weight loss have so far proven dangerous or ineffective. However, Glucagon-like peptide (GLP)-1 receptor agonists, Semaglutide and Liraglutide, have recently proven that safe pharmacological reduction of body weight was a practicable therapeutic option. The failure in targeting the melanocortin pathway comes out partly from an incomplete understanding of leptin action, especially leptin resistance, at the central level.
GPCRs are the largest family of membrane proteins and a notoriously successful source of drug targets. However, many GPCRs are understudied or even orphans (without known ligands). Most GPCRs share a common activation scheme that comprises activation of intracellular G proteins. Once bound, the G protein dissociates into Gα and Gβγ subunits and propagates the signal through various pathways depending on the G protein family. Following G protein binding, ß-arrestins contribute to signaling and promote internalization to endosomes. GPR101 is an orphan GPCR highly enriched in the hypothalamus. Hence, we aim to study the role of GPR101 in this brain region, to investigate the interplay between GPR101 and leptin receptor in leptin sensitive cells of the arcuate nucleus and to address the impact of G protein signaling on the leptin axis.
In conclusion, the discovery of new regulators of food intake like GPCRs offers the chance of improving the quality of life of patients suffering from diseases that affect weight and metabolism. This can allow the development of better treatment protocols and diagnosis in these disorders.
GPCRs are the largest family of membrane proteins and a notoriously successful source of drug targets. However, many GPCRs are understudied or even orphans (without known ligands). Most GPCRs share a common activation scheme that comprises activation of intracellular G proteins. Once bound, the G protein dissociates into Gα and Gβγ subunits and propagates the signal through various pathways depending on the G protein family. Following G protein binding, ß-arrestins contribute to signaling and promote internalization to endosomes. GPR101 is an orphan GPCR highly enriched in the hypothalamus. Hence, we aim to study the role of GPR101 in this brain region, to investigate the interplay between GPR101 and leptin receptor in leptin sensitive cells of the arcuate nucleus and to address the impact of G protein signaling on the leptin axis.
In conclusion, the discovery of new regulators of food intake like GPCRs offers the chance of improving the quality of life of patients suffering from diseases that affect weight and metabolism. This can allow the development of better treatment protocols and diagnosis in these disorders.
The technological approaches that we used to answer our research question is multidisciplinary and combine molecular & cellular biology techniques. We designed and we used several transgenic mouse models to follow the expression of the GPR101 receptor, as well as full and cell-specific knockout mouse lines in order to understand how GPR101 affects body weight and feeding. We performed metabolic phenotyping, cell profiling of hypothalamic neurons such as RNAscope and RNA analysis by transcriptomics. We used also several techniques such as cell culture, flow cytometry, microscopy (confocal and Light-Sheet), patch clamp and histology to determine the nature of neurons expressing GPR101. in vitro, we used different pharmacological approaches such as the discovery, the design and the use of pharmacological modulators to characterize the signaling pathway implicated in the molecular mechanism of leptin signaling and regulation of food intake. Finally in silico, we determined the amino acids that are present in the pocket and involved in the activation of the receptor. We confirmed our virtual model by testing several peptides derived from the extracellular loop 2 activating the receptor.
Here we deciphered novel signaling pathways and characterized a new cell population (Gpr101+) involved in metabolism and food intake. The outcome of the work goes beyond the state of the art as its goal is to unveil an unknown circuit regulating food intake. The description of a new cell population, signaling pathway, receptor or ligand able to modulate this fundamental process would lead to a groundbreaking and disruptive new insights highly relevant to health and diseases. In addition, it would open novel avenues to develop new therapies. Drug targets able to mitigate food intake in obese patients represent an unmet medical need. Given their successes in drug discovery, GPCR constitute ideal candidates. Therefore, this project could lead to the validation of GPR101 as an innovative drug target to modulate feeding behaviors in humans and could expand our horizon toward the discovery of efficient medicines against obesity and metabolic disorders.