RESUMEN
Alcohol-related liver disease (ALD) is characterized by accumulation of hepatic free fatty acids (FFAs) and liver injury. The present study aimed to investigate if mechanistic target of rapamycin complex 1 (mTORC1) plays a role in FFA-induced organelle dysfunction, thereby contributing to the development of ALD. Cell studies were conducted to define the causal role and underlying mechanism of FFA-activated mTORC1 signaling in hepatocellular cell injury. C57BL/6J wild-type mice were subjected to chronic alcohol feeding with or without rapamycin to inhibit mTORC1 activation. We revealed that palmitic acid (PA)-induced ER stress and suppression of LAMP2 and autophagy flux were mTORC1-dependent as rapamycin reversed such deleterious effects. C/EBP homologous protein (CHOP) was downstream of ATF4 which partially modulated LAMP2. Supplementation with rapamycin to alcohol-fed mice attenuated mTORC1 activation and ER stress, restored LAMP2 protein, and improved autophagy, leading to amelioration of alcohol-induced liver injury. Induction of mTORC1 signaling and CHOP were also detected in the liver of patients with severe alcoholic hepatitis. This study demonstrates that hepatic FFAs play a crucial role in the pathogenesis of ALD by activating mTORC1 signaling, thereby inducing ER stress and suppressing LAMP2-autophagy flux pathway, which represents an important mechanism of FFA-induced hepatocellular injury.
Asunto(s)
Autofagia , Estrés del Retículo Endoplásmico , Etanol/efectos adversos , Ácidos Grasos no Esterificados/farmacología , Hepatopatías/patología , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Factor de Transcripción Activador 4/metabolismo , Animales , Autofagia/efectos de los fármacos , Línea Celular Tumoral , Suplementos Dietéticos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Hepatitis Alcohólica/metabolismo , Hepatitis Alcohólica/patología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Ratones Endogámicos C57BL , Ácido Palmítico/farmacología , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Factor de Transcripción CHOP/metabolismoRESUMEN
Metabolite accumulation in lysosomal storage disorders (LSDs) results in impaired cell function and multi-systemic disease. Although substrate reduction and lysosomal overload-decreasing therapies can ameliorate disease progression, the significance of lysosomal overload-independent mechanisms in the development of cellular dysfunction is unknown for most LSDs. Here, we identify a mechanism of impaired chaperone-mediated autophagy (CMA) in cystinosis, a LSD caused by defects in the cystine transporter cystinosin (CTNS) and characterized by cystine lysosomal accumulation. We show that, different from other LSDs, autophagosome number is increased, but macroautophagic flux is not impaired in cystinosis while mTOR activity is not affected. Conversely, the expression and localization of the CMA receptor LAMP2A are abnormal in CTNS-deficient cells and degradation of the CMA substrate GAPDH is defective in Ctns(-/-) mice. Importantly, cysteamine treatment, despite decreasing lysosomal overload, did not correct defective CMA in Ctns(-/-) mice or LAMP2A mislocalization in cystinotic cells, which was rescued by CTNS expression instead, suggesting that cystinosin is important for CMA activity. In conclusion, CMA impairment contributes to cell malfunction in cystinosis, highlighting the need for treatments complementary to current therapies that are based on decreasing lysosomal overload.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Autofagia , Cistinosis/metabolismo , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Lisosomas/metabolismo , Chaperonas Moleculares/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Animales , Cistina/metabolismo , Cistinosis/genética , Cistinosis/fisiopatología , Humanos , Proteína 2 de la Membrana Asociada a los Lisosomas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Chaperonas Moleculares/genéticaRESUMEN
Puerarin, an active component of Pueraria montana var. lobata (Willd.) Sanjappa & Pradeep is well-known for its anti-oxidative and neuroprotective activities. Although anti-Parkinson's disease activity of puerarin was reported in both of in vivo and in vitro model, detailed mechanisms are not clarified. In this study, we addressed that puerarin attenuated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced behavioral deficits, dopaminergic neuronal degeneration and dopamine depletion. Puerarin administration enhanced glutathione (GSH) activity, glial cell line-derived neurotrophic factor (GDNF) expression and PI3K/Akt pathway activation, which might ameliorate MPTP injection-induced progressive elevation of reactive oxygen species (ROS) formation in mice. In addition to the effect on ROS, puerarin ameliorated MPTP-reduced lysosome-associated membrane protein type 2A (Lamp 2A) expression. Taken together, our data demonstrate that puerarin attenuates MPTP-induced dopaminergic neuronal degeneration via modulating GDNF expression, PI3K/Akt pathway and GSH activation, which subsequently ameliorate MPTP-induced ROS formation and decrease of Lamp 2A expression.
Asunto(s)
Isoflavonas/farmacología , Degeneración Nerviosa/inducido químicamente , Fármacos Neuroprotectores/farmacología , Enfermedad de Parkinson/tratamiento farmacológico , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/efectos adversos , Animales , Modelos Animales de Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/patología , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Glutatión/metabolismo , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalAsunto(s)
Autofagia/fisiología , Ferritinas/fisiología , Quelantes del Hierro/metabolismo , Hierro/metabolismo , Animales , Terapia por Quelación/métodos , Ferritinas/metabolismo , Humanos , Quelantes del Hierro/síntesis química , Quelantes del Hierro/farmacología , Quelantes del Hierro/uso terapéutico , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Proteína 2 de la Membrana Asociada a los Lisosomas/fisiología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Zinc is a trace element that is essential for the function of many enzymes and transcription factors. Zinc deficiency results in defects in innate and acquired immune responses. However, little is known about the mechanism(s) by which zinc affects immune cell function. Here we show that stimulation with the Toll-like receptor 4 agonist lipopolysaccharide (LPS) altered the expression of zinc transporters in dendritic cells and thereby decreased intracellular free zinc. A zinc chelator mimicked the effects of LPS, whereas zinc supplementation or overexpression of the gene encoding Zip6, a zinc transporter whose expression was reduced by LPS, inhibited LPS-induced upregulation of major histocompatibility complex class II and costimulatory molecules. These results establish a link between Toll-like receptor signaling and zinc homeostasis.