RESUMO
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.
Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Encéfalo/metabolismo , Corantes Fluorescentes/química , Grelina/metabolismo , Rim/metabolismo , Animais , Células Cultivadas , Ingestão de Alimentos , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Domínios Proteicos , Transdução de SinaisRESUMO
The growth hormone secretagogue receptor (GHSR) is a G protein-coupled receptor that is highly expressed in the central nervous system. GHSR acts as a receptor for ghrelin and for liver-expressed antimicrobial peptide 2 (LEAP2), which blocks ghrelin-evoked activity. GHSR also displays ligand-independent activity, including a high constitutive activity that signals in the absence of ghrelin and is reduced by LEAP2. GHSR activity modulates a variety of food intake-related behaviours, including binge eating. Previously, we reported that GHSR-deficient mice daily and time-limited exposed to a high-fat (HF) diet display an attenuated binge-like HF intake compared to wild-type mice. In the present study, we aimed to determine whether ligand-independent GHSR activity affects binge-like HF intake in a 4-day binge-like eating protocol. We found that plasma levels of ghrelin and LEAP2 were not modified in mice exposed to this binge-like eating protocol. Moreover, systemic administration of ghrelin or LEAP2 did not alter HF intake in our experimental conditions. Interestingly, we found that central administration of LEAP2 or K-(D-1-Nal)-FwLL-NH2 , which are both blockers of constitutive GHSR activity, reduced binge-like HF intake, whereas central administration of ghrelin or the ghrelin-evoked GHSR activity blockers [D-Lys3]-GHRP-6 and JMV2959 did not modify binge-like HF intake. Taken together, current data indicate that GHSR activity in the brain affects binge-like HF intake in mice independently of plasma levels of ghrelin and LEAP2.
Assuntos
Peptídeos Catiônicos Antimicrobianos/fisiologia , Bulimia/fisiopatologia , Grelina/fisiologia , Receptores de Grelina/agonistas , Receptores de Grelina/fisiologia , Animais , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/sangue , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bulimia/prevenção & controle , Dieta Hiperlipídica , Grelina/administração & dosagem , Grelina/sangue , Grelina/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Infusões Intraventriculares , Masculino , Camundongos , Oligopeptídeos/farmacologia , Receptores de Grelina/antagonistas & inibidores , Fatores de Tempo , Triazóis/farmacologiaRESUMO
Ghrelin is a potent orexigenic peptide hormone that acts through the growth hormone secretagogue receptor (GHSR), a G protein-coupled receptor highly expressed in the hypothalamus. In vitro studies have shown that GHSR displays a high constitutive activity, whose physiological relevance is uncertain. As GHSR gene expression in the hypothalamus is known to increase in fasting conditions, we tested the hypothesis that constitutive GHSR activity at the hypothalamic level drives the fasting-induced hyperphagia. We found that refed wild-type (WT) mice displayed a robust hyperphagia that continued for 5 days after refeeding and changed their food intake daily pattern. Fasted WT mice showed an increase in plasma ghrelin levels, as well as in GHSR expression levels and ghrelin binding sites in the hypothalamic arcuate nucleus. When fasting-refeeding responses were evaluated in ghrelin- or GHSR-deficient mice, only the latter displayed an â¼15% smaller hyperphagia, compared with WT mice. Finally, fasting-induced hyperphagia of WT mice was significantly smaller in mice centrally treated with the GHSR inverse agonist K-(D-1-Nal)-FwLL-NH2, compared with mice treated with vehicle, whereas it was unaffected in mice centrally treated with the GHSR antagonists D-Lys3-growth hormone-releasing peptide 6 or JMV2959. Taken together, genetic models and pharmacological results support the notion that constitutive GHSR activity modulates the magnitude of the compensatory hyperphagia triggered by fasting. Thus, the hypothalamic GHSR signaling system could affect the set point of daily food intake, independently of plasma ghrelin levels, in situations of negative energy balance.