RESUMEN
The link between obesity and diabetes is not fully understood but there is evidence to suggest that hypothalamic signalling pathways may be involved. The hypothalamic neuropeptides, pro-opiomelanocortin (POMC), neuropeptide Y (NPY) and agouti-related protein (AGRP) are central to the regulation of food intake and have been implicated in glucose homeostasis. Therefore, the expression of these genes was quantified in hypothalami from diabetic Zucker fatty (ZDF) rats and nondiabetic Zucker fatty (ZF) rats at 6, 8, 10 and 14 weeks of age. Although both strains are obese, only ZDF rats develop pancreatic degeneration and diabetes over this time period. In both ZF and ZDF rats, POMC gene expression was decreased in obese versus lean rats at all ages. By contrast, although there was the expected increase in both NPY and AGRP expression in obese 14-week-old ZF rats, the expression of NPY and AGRP was decreased in 6-week-old obese ZDF rats with hyperinsulinaemia and in 14-week-old rats with the additional hyperglycaemia. Therefore, candidate genes involved in glucose, and insulin signalling pathways were examined in obese ZDF rats over this age range. We found that expression of the ATP-sensitive potassium (K(ATP)) channel component, Kir6.2, was decreased in obese ZDF rats and was lower compared to ZF rats in each age group tested. Furthermore, immunofluorescence analysis showed that Kir6.2 protein expression was reduced in the dorsomedial and ventromedial hypothalamic nuclei of 6-week-old prediabetic ZDF rats compared to ZF rats. The Kir6.2 immunofluorescence colocalised with NPY throughout the hypothalamus. The differences in Kir6.2 expression in ZF and ZDF rats mimic those of NPY and AGRP, which could infer that the changes occur in the same neurones. Overall, these data suggest that chronic changes in hypothalamic Kir6.2 expression may be associated with the development of hyperinsulinaemia and hyperglycaemia in ZDF rats.
Asunto(s)
Proteína Relacionada con Agouti/biosíntesis , Diabetes Mellitus/metabolismo , Hipotálamo/metabolismo , Neuropéptido Y/biosíntesis , Obesidad/metabolismo , Canales de Potasio de Rectificación Interna/biosíntesis , Animales , Diabetes Mellitus/genética , Diabetes Mellitus/patología , Expresión Génica/fisiología , Glucosa/fisiología , Hiperglucemia/sangre , Hiperglucemia/genética , Hiperinsulinismo/sangre , Hiperinsulinismo/genética , Hipotálamo/crecimiento & desarrollo , Hipotálamo/patología , Inmunohistoquímica , Inflamación/patología , Insulina/fisiología , Leptina/fisiología , Masculino , Neuropéptido Y/genética , Obesidad/genética , Páncreas/patología , Canales de Potasio de Rectificación Interna/genética , Proopiomelanocortina/biosíntesis , Ratas , Ratas Wistar , Ratas Zucker , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Interactions between pro-opiomelanocortin (POMC)-derived peptides, agouti-related protein (AGRP) and the melanocortin-4 receptor (MC4-R) are central to energy homeostasis. In this study we have undertaken comprehensive pharmacological analysis of these interactions using a CHOK1 cell line stably transfected with human MC4-R. Our main objectives were (1) to compare the relative affinities and potencies of POMC-derived peptides endogenously secreted within the hypothalamus, (2) to investigate the potency of AGRP(83-132) antagonism with respect to each POMC-derived peptide and (3) to determine whether AGRP(83-132) and POMC-derived peptides act allosterically or orthosterically. We have found that beta melanocyte-stimulating hormone (betaMSH), desacetyl alpha MSH (da-alphaMSH) and adrenocorticotrophic hormone all have very similar affinities and potencies at the MC4-R compared with the presumed natural ligand, alphaMSH. Moreover, even MSH precursors, such as beta lipotrophic hormone, showed significant binding and functional activity. Therefore, many POMC-derived peptides could have important roles in appetite regulation and it seems unlikely that alphaMSH is the sole physiological ligand. We have shown that AGRP(83-132) acts as a competitive antagonist. There was no significant difference in the potency of inhibition by AGRP(83-132) or agouti(87-132) at the MC4-R, regardless of which POMC peptide was used as an agonist. Furthermore, we have found that AGRP(83-132) has no effect on the dissociation kinetics of radiolabelled Nle4,D-Phe7 MSH from the MC4-R, indicating an absence of allosteric effects. This provides strong pharmacological evidence that AGRP(83-132) acts orthosterically at the MC4-R to inhibit Gs-coupled accumulation of intracellular cAMP.
Asunto(s)
Regulación del Apetito , Fragmentos de Péptidos/farmacología , Proopiomelanocortina/metabolismo , Receptor de Melanocortina Tipo 4/antagonistas & inhibidores , alfa-MSH/análogos & derivados , Proteína Relacionada con Agouti , Animales , Unión Competitiva , Células CHO , Cricetinae , AMP Cíclico/metabolismo , Humanos , Unión Proteica , Receptor de Melanocortina Tipo 4/genética , Transfección , alfa-MSH/metabolismoRESUMEN
The signalling pathways used by the human endothelin A receptor to activate phospholipase A2 in Chinese hamster ovary cells were investigated. Pertussis toxin caused a partial but significant reduction in endothelin-1-induced arachidonic acid release although cAMP-dependent kinase inhibitors did not mimic its action. Extracellular calcium and its entry into the cell was essential for activation of phospholipase A2 as its removal from media or incubation with an intracellular calcium chelator-reduced activation. Nifedipine had no effect on endothelin-1-induced arachidonic acid release while divalent cations caused a significant reduction indicating the possible role of CRAC. Thapsigargin caused an increase in arachidonic acid release which was completely inhibited by pertussis toxin treatment. This further supports the involvement of CRAC in calcium influx and activation of phospholipase A2 by the human endothelin A receptor.
Asunto(s)
Calcio/metabolismo , Proteínas de Unión al GTP/metabolismo , Fosfolipasas A/metabolismo , Receptores de Endotelina/metabolismo , Animales , Ácido Araquidónico/metabolismo , Transporte Biológico Activo , Células CHO , Calcio/farmacología , Canales de Calcio/metabolismo , Cricetinae , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Endotelinas/farmacología , Activación Enzimática/efectos de los fármacos , Espacio Extracelular/metabolismo , Humanos , Nifedipino/farmacología , Toxina del Pertussis , Fosfolipasas A2 , Proteínas Recombinantes/metabolismo , Transducción de Señal , Terpenos/farmacología , Tapsigargina , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
We have developed a cell based assay for the functional screening of chemical libraries for novel chemical entities active at the human endothelin A (ET(A)) receptor. The assay is relatively inexpensive, suitable for dealing with large number of samples, and simple to operate; it generates results quickly. We achieved this by expressing the cDNA for the receptor in mammalian cell lines and determining whether coupling to pIA(A) occurred through the quantification of released radiolabeled arachidonic acid (AA) into the culture medium. Significant coupling was observed only when the receptor was expressed in the Chinese hamster ovary (CHO) line DG44. The assay could distinguish between ET(A)r agonists and antagonists, and the IC50 (the concentration that inhibits maximum response by 50%) values obtained were similar to those from other sources of receptor. The ET(B) receptor-selective agonist BQ3020 did not stimulate AA release, indicating that the assay can also discriminate between receptor subtypes.