RESUMEN
The phosphotyrosine interacting domain-containing protein 1 (PID1) serves as a cytosolic adaptor protein of the LDL receptor-related protein 1 (LRP1). By regulating its intracellular trafficking, PID1 controls the hepatic, LRP1-dependent clearance of pro-atherogenic lipoproteins. In adipose and muscle tissues, LRP1 is present in endosomal storage vesicles containing the insulin-responsive glucose transporter 4 (GLUT4). This prompted us to investigate whether PID1 modulates GLUT4 translocation and function via its interaction with the LRP1 cytosolic domain. We initially evaluated this in primary brown adipocytes as we observed an inverse correlation between brown adipose tissue glucose uptake and expression of LRP1 and PID1. Insulin stimulation in wild type brown adipocytes induced LRP1 and GLUT4 translocation from endosomal storage vesicles to the cell surface. Loss of PID1 expression in brown adipocytes prompted LRP1 and GLUT4 sorting to the plasma membrane independent of insulin signaling. When placed on a diabetogenic high fat diet, systemic and adipocyte-specific PID1-deficient mice presented with improved hyperglycemia and glucose tolerance as well as reduced basal plasma insulin levels compared to wild type control mice. Moreover, the improvements in glucose parameters associated with increased glucose uptake in adipose and muscle tissues from PID1-deficient mice. The data provide evidence that PID1 serves as an insulin-regulated retention adaptor protein controlling translocation of LRP1 in conjunction with GLUT4 to the plasma membrane of adipocytes. Notably, loss of PID1 corrects for insulin resistance-associated hyperglycemia emphasizing its pivotal role and therapeutic potential in the regulation of glucose homeostasis.
Asunto(s)
Adipocitos Marrones/metabolismo , Proteínas Portadoras/genética , Transportador de Glucosa de Tipo 4/genética , Glucosa/metabolismo , Insulina/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Obesidad/genética , Adipocitos Marrones/patología , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Pardo/patología , Animales , Transporte Biológico , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Dieta Alta en Grasa/efectos adversos , Endosomas/metabolismo , Regulación de la Expresión Génica , Transportador de Glucosa de Tipo 4/metabolismo , Homeostasis/genética , Resistencia a la Insulina , Hígado/metabolismo , Hígado/patología , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Obesidad/etiología , Obesidad/metabolismo , Obesidad/patología , Cultivo Primario de Células , Transducción de SeñalRESUMEN
OBJECTIVE: Insulin resistance is associated with impaired receptor dependent hepatic uptake of triglyceride-rich lipoproteins (TRL), promoting hypertriglyceridemia and atherosclerosis. Next to low-density lipoprotein (LDL) receptor (LDLR) and syndecan-1, the LDLR-related protein 1 (LRP1) stimulated by insulin action contributes to the rapid clearance of TRL in the postprandial state. Here, we investigated the hypothesis that the adaptor protein phosphotyrosine interacting domain-containing protein 1 (PID1) regulates LRP1 function, thereby controlling hepatic endocytosis of postprandial lipoproteins. METHODS: Localization and interaction of PID1 and LRP1 in cultured hepatocytes was studied by confocal microscopy of fluorescent tagged proteins, by indirect immunohistochemistry of endogenous proteins, by GST-based pull down and by immunoprecipitation experiments. The in vivo relevance of PID1 was assessed using whole body as well as liver-specific Pid1-deficient mice on a wild type or Ldlr-deficient (Ldlr-/-) background. Intravital microscopy was used to study LRP1 translocation in the liver. Lipoprotein metabolism was investigated by lipoprotein profiling, gene and protein expression as well as organ-specific uptake of radiolabelled TRL. RESULTS: PID1 co-localized in perinuclear endosomes and was found associated with LRP1 under fasting conditions. We identified the distal NPxY motif of the intracellular C-terminal domain (ICD) of LRP1 as the site critical for the interaction with PID1. Insulin-mediated NPxY-phosphorylation caused the dissociation of PID1 from the ICD, causing LRP1 translocation to the plasma membrane. PID1 deletion resulted in higher LRP1 abundance at the cell surface, higher LDLR protein levels and, paradoxically, reduced total LRP1. The latter can be explained by higher receptor shedding, which we observed in cultured Pid1-deficient hepatocytes. Consistently, PID1 deficiency alone led to increased LDLR-dependent endocytosis of postprandial lipoproteins and lower plasma triglycerides. In contrast, hepatic PID1 deletion on an Ldlr-/- background reduced lipoprotein uptake into liver and caused plasma TRL accumulation. CONCLUSIONS: By acting as an insulin-dependent retention adaptor, PID1 serves as a regulator of LRP1 function controlling the disposal of postprandial lipoproteins. PID1 inhibition provides a novel approach to lower plasma levels of pro-atherogenic TRL remnants by stimulating endocytic function of both LRP1 and LDLR in the liver.
Asunto(s)
Proteínas Portadoras/metabolismo , Hipertrigliceridemia/metabolismo , Lipoproteínas/metabolismo , Triglicéridos/metabolismo , Animales , Carcinoma Hepatocelular , Proteínas Portadoras/genética , Línea Celular Tumoral , Endocitosis/fisiología , Hepatocitos/metabolismo , Humanos , Hipertrigliceridemia/genética , Insulina/metabolismo , Resistencia a la Insulina/fisiología , Lipoproteínas/fisiología , Hígado/metabolismo , Neoplasias Hepáticas , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Periodo Posprandial , Receptores de LDL/metabolismo , Sinapsinas/metabolismo , Sinapsinas/fisiología , Triglicéridos/fisiología , Proteínas Supresoras de Tumor/metabolismoRESUMEN
FGF21 decreases plasma triglycerides (TGs) in rodents and humans; however, the underlying mechanism or mechanisms are unclear. In the present study, we examined the role of FGF21 in production and disposal of TG-rich lipoproteins (TRLs) in mice. Treatment with pharmacological doses of FGF21 acutely reduced plasma non-esterified fatty acids (NEFAs), liver TG content, and VLDL-TG secretion. In addition, metabolic turnover studies revealed that FGF21 facilitated the catabolism of TRL in white adipose tissue (WAT) and brown adipose tissue (BAT). FGF21-dependent TRL processing was strongly attenuated in CD36-deficient mice and transgenic mice lacking lipoprotein lipase in adipose tissues. Insulin resistance in diet-induced obese and ob/ob mice shifted FGF21 responses from WAT toward energy-combusting BAT. In conclusion, FGF21 lowers plasma TGs through a dual mechanism: first, by reducing NEFA plasma levels and consequently hepatic VLDL lipidation and, second, by increasing CD36 and LPL-dependent TRL disposal in WAT and BAT.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Factores de Crecimiento de Fibroblastos/farmacología , Hipolipemiantes/farmacología , Lipoproteínas VLDL/metabolismo , Triglicéridos/sangre , Adipocitos Blancos/efectos de los fármacos , Adipocitos Blancos/metabolismo , Tejido Adiposo Pardo/efectos de los fármacos , Tejido Adiposo Blanco/efectos de los fármacos , Animales , Células Cultivadas , Evaluación Preclínica de Medicamentos , Femenino , Factores de Crecimiento de Fibroblastos/uso terapéutico , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hipolipemiantes/uso terapéutico , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
The endocannabinoids and their main receptor, cannabinoid type-1 (CB1), suppress intracellular cyclic AMP levels and have emerged as key players in the control of energy metabolism. CB1 agonists and blockers have been reported to influence the thermogenic function of white and brown adipose tissue (WAT and BAT), affecting body weight through the inhibition and stimulation of energy expenditure, respectively. The purpose of the current study was to investigate the regulation of the endocannabinoid system in WAT and BAT following exposure to either cold or specific agonism of ß3-adrenoceptors using CL316,243 (CL), conditions known to cause BAT activation and WAT browning. To address this question, we performed quantitative PCR-based mRNA profiling of genes important for endocannabinoid synthesis, degradation, and signaling, and determined endocannabinoid levels by LC-MS in WAT and BAT of control, cold-exposed, and CL-treated wild-type mice as well as primary brown adipocytes. Treatment with CL and exposure to cold caused an upregulation of endocannabinoid levels and biosynthetic enzymes in WAT. Acute ß3-adrenoceptor activation increased endocannabinoids and a subset of genes of biosynthesis in BAT and primary brown adipocytes. We suggest that the cold-mediated increase in endocannabinoid tone is part of autocrine negative feed-back mechanisms controlling ß3-adrenoceptor-induced BAT activation and WAT browning.