RESUMEN
We have recently found that GPR120, which is abundantly expressed in intestine, functions as a receptor for unsaturated long-chain free fatty acids (FFAs) and that GPR120 stimulation promotes the secretion of glucagons-like peptide-1 (GLP-1) in the mouse (Hirasawa et al., Nat Med 11:90-94, 2005). In this study, we cloned and characterized rat GPR120 (rGPR120), and then we examined the in vivo effects of acute and long-term administration of the natural ligand alpha-linolenic acid (alpha-LA). The cloned rat GPR120 complimentary DNA had a seven transmembrane structure, and a homology comparison of human, mouse, and rat GPR120 revealed that the rat GPR120 (rGPR120) shares 85 and 98% sequence identity with the human and mouse GPR120 proteins, respectively. The tissue distribution and ligand properties of rGPR120 were similar to those of mouse GPR120. In addition, alpha-LA provoked a transient increase in [Ca2+]i levels in HEK293 cells expressing rGPR120. Furthermore, administration of alpha-LA to the rat increased plasma GLP-1 levels, and long-term administration of alpha-LA led to proliferation of pancreatic beta cells, probably because of the enhanced GLP-1 secretion. These results show that rat GPR120 is a G-protein-coupled receptor whose ligand is a free fatty acid, and it may play an important role in the FFA-associated physiological responses.
Asunto(s)
Péptido 1 Similar al Glucagón/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ácido alfa-Linolénico/farmacología , Secuencia de Aminoácidos , Animales , Proliferación Celular/efectos de los fármacos , Clonación Molecular , ADN Complementario/metabolismo , Péptido 1 Similar al Glucagón/efectos de los fármacos , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiología , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Ácido alfa-Linolénico/administración & dosificaciónRESUMEN
Connective tissue growth factor (CTGF) is induced by transforming growth factor-beta (TGF-beta) via Smad activation in mesangial cells. We recently reported that sphingosine 1-phosphate (S1P) induces CTGF expression in rat cultured mesangial cells. However, the mechanism by which S1P induces CTGF expression is unknown. The present study revealed that S1P-induced CTGF expression is mediated via pertussis toxin-insensitive pathways, which are involved in the activation of small GTPases of the Rho family and protein kinase C. We also showed by luciferase reporter assays and chromatin immunoprecipitation that S1P induces CTGF expression via Smad activation as TGF-beta does.
Asunto(s)
Mesangio Glomerular/citología , Mesangio Glomerular/efectos de los fármacos , Proteínas Inmediatas-Precoces/efectos de los fármacos , Lisofosfolípidos/farmacología , Esfingosina/análogos & derivados , Esfingosina/farmacología , Transcripción Genética , Animales , Western Blotting , Línea Celular Transformada , Transformación Celular Viral , Inmunoprecipitación de Cromatina , Factor de Crecimiento del Tejido Conjuntivo , Genes Reporteros , Vectores Genéticos , Péptidos y Proteínas de Señalización Intercelular , Luciferasas , MicroARNs/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Free fatty acids (FFAs) provide an important energy source and also act as signaling molecules. FFAs are known to exert a variety of physiological responses via their G protein-coupled receptors (GPCRs), such as the GPR40 family. Recently, we identified a novel FFA receptor, GPR120, that promotes secretion of glucagon-like peptide-1 (Hirasawa, A., Tsumaya, K., Awaji, T., Katsuma, S., Adachi, T., Yamada, M., Sugimoto, Y., Miyazaki, S., and Tsujimoto, G. (2005) Nat. Med. 11, 90-94). Here we showed that FFAs inhibit serum deprivation-induced apoptosis of murine enteroendocrine STC-1 cells, which express two types of GPCRs, GPR120 and GPR40, for unsaturated long chain FFA. We first found that linolenic acid potently activated ERK and Akt/protein kinase B (Akt) in STC-1 cells. ERK kinase inhibitors significantly reduced the anti-apoptotic effects of linolenic acid. Inhibitors for phosphatidylinositol 3-kinase (PI3K), a major target of which is Akt, significantly reduced the anti-apoptotic effects. Transfection of STC-1 cells with the dominant-negative form of Akt also inhibited the anti-apoptotic effect. These results suggested that the activation of ERK and PI3K-Akt pathways is required for FFA-induced anti-apoptotic effects on STC-1 cells. Transient transfection of STC-1 cells with GPR120 cDNA, but not GPR40 cDNA, enhanced inhibition of caspase-3 activation. RNA interference experiments showed that reduced expression of GPR120, but not GPR40, resulted in reduced ERK activation and reduced effects of FFAs on caspase-3 inhibition. Collectively, these results demonstrated that FFAs promote the activation of ERK and PI3K-Akt pathways mainly via GPR120, leading to the anti-apoptotic effect of STC-1 cells.