RESUMEN
Leptin is an appetite-controlling peptide secreted from adipose tissue. Previously, we showed that the gene expression of acetoacetyl-CoA synthetase (AACS), the ketone body-utilizing enzyme for lipid synthesis, was suppressed by leptin deficiency-induced obesity in white adipose tissue. In this study, to clarify the effects of leptin on ketone body utilization in the central nervous system, we examined the effects of leptin signaling on AACS expression. In situ hybridization analysis of ob/ob and db/db mice revealed that AACS mRNA level was reduced by leptin deficiency in the arcuate nucleus (Arc) and ventromedial hypothalamic nucleus (VMH) in hypothalamus but not in other brain regions. Moreover, AACS mRNA level was increased by leptin treatment both in primary cultured neural cells and in N41 neural-like cells. In N41 cells, AACS level was decreased by AMPK inducer but increased by AMPK inhibitor. These results suggest that the up-regulation of AACS expression by leptin is due to the suppression of AMPK activity via neural leptin signaling and that the deficiency of this regulation may be responsible for neurological disorders in central appetite control.
Asunto(s)
Hipotálamo/citología , Cuerpos Cetónicos/metabolismo , Leptina/farmacología , Neuronas/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Animales , Células Cultivadas , Coenzima A Ligasas/genética , Coenzima A Ligasas/metabolismo , Coenzima A Transferasas/genética , Coenzima A Transferasas/metabolismo , Inhibidores Enzimáticos/farmacología , Hipoglucemiantes/farmacología , Masculino , Ratones , Ratones Obesos , Neuronas/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacología , ARN Mensajero/metabolismo , Receptores de Leptina/genética , Receptores de Leptina/metabolismo , Ribonucleótidos/farmacología , Factores de TiempoRESUMEN
Medullary thyroid carcinoma (MTC) is a rare endocrine tumor that frequently metastasizes, and treatment with irinotecan (CPT-11) is limited because of side effects. Mutations in the Rearranged during transfection (RET) proto-oncogene are considered the causative event of MTC. The objective of this study was to examine whether small interfering RNA (siRNA) and its combined treatment with CPT-11 could inhibit MTC cell growth in vitro and in vivo. The transfection of RET siRNA suppressed RET expression, reduced proliferation, and increased caspase-3/7 activity via the down-regulation of Bcl-2 expression. Combined treatments with CPT-11 or SN-38 significantly increased caspase 3/7 activity compared with RET siRNA, CPT-11 or SN-38 treatment alone. Importantly, intratumoral injection of RET siRNA along with intravenous injection of CPT-11 significantly inhibited the tumor growth of MTC xenografts via an increased apoptotic effect. These findings that RET siRNA enhanced sensitivity for CPT-11 will provide a novel strategy for the treatment of MTC with RET mutation.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Apoptosis/efectos de los fármacos , Camptotecina/análogos & derivados , Carcinoma Medular/metabolismo , Carcinoma Medular/terapia , Proteínas Proto-Oncogénicas c-ret/genética , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/terapia , Animales , Apoptosis/genética , Camptotecina/farmacología , Camptotecina/uso terapéutico , Carcinoma Medular/genética , Carcinoma Medular/patología , Caspasa 3/genética , Caspasa 3/metabolismo , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Regulación hacia Abajo , Femenino , Humanos , Irinotecán , Ratones , Ratones Endogámicos ICR , Ratones Desnudos , Proto-Oncogenes Mas , Proto-Oncogenes , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/farmacología , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The hydrodynamic method by rapid intravenous injection of a large volume of plasmid DNA is known to be an efficient and liver-specific method of in vivo gene delivery and achieves high levels of foreign gene expression, particularly in hepatocytes. Low transgene activities have also been observed in other organs such as the spleen and lung; however, the expression profiles of mRNA and protein are still unknown. Therefore, we investigated the localization of luciferase mRNA by in situ hybridization and luciferase activity in mice after transfection of pCMV-luc encoding the luciferase gene under the control of cytomegalo virus (CMV) promoter. We found that hydrodynamic injection effectively induced mRNA expression of the transgene only in the liver although transgene activities were observed in other organs. The transgene activity observed in other organs may be due to leakage from hepatocyte gene expression by transient increase in the permeability of the hepatocyte cellular membrane caused by increased pressure by hydrodynamic injection.
Asunto(s)
Técnicas de Transferencia de Gen , ARN Mensajero/biosíntesis , Transgenes/genética , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Excipientes , Femenino , Expresión Génica , Hibridación in Situ , Inyecciones Intravenosas , Hígado/metabolismo , Luciferasas/genética , Ratones , Ratones Endogámicos BALB C , Plásmidos/genética , ARN Mensajero/genética , Distribución Tisular , beta-Galactosidasa/genéticaRESUMEN
Obesity causes various physiological disorders between the central nervous system and peripheral tissues. Ketone bodies have a neuro-protective role and are strongly affected by obesity-related metabolic disorders. To clarify the effects of obesity on ketone body utilization in brain, we examined the mRNA localization of acetoacetyl-CoA synthetase (AACS), which activates ketone bodies for the synthesis of fatty acid and cholesterol, in various brain regions of Zucker fatty rats by in situ hybridization. The AACS mRNA level was increased in the paraventricular thalamic nucleus (PVT) but not affected in the cerebrum and hippocampus in Zucker fatty rats. In contrast, the AACS mRNA level was reduced in the arcuate hypothalamic nucleus (Arc) and ventromedial hypothalamic nucleus (VMH) in the hypothalamus. Succinyl-CoA:3-oxoacid CoA-transferase (SCOT) mRNA level was decreased only in the PVT but not affected in the Arc and VMH. These data raise the possibility that AACS is regulated by the leptin signaling pathway in the hypothalamus but not in the PVT. As AACS was expressed in neural-like cells, ketone bodies are assumed to be utilized for the synthesis of lipidic substances and to cause metabolic disorders in the nervous system.
