RESUMEN
The coatomer protein complex 1 (COPI) is a multisubunit complex that coats intracellular vesicles and is involved in intracellular protein trafficking. Recently we and others found that depletion of COPI complex subunits zeta (COPZ1) and delta (ARCN1) preferentially kills tumor cells relative to normal cells. Here we delineate the specific cellular effects and sequence of events of COPI complex depletion in tumor cells. We find that this depletion leads to the inhibition of mitochondrial oxidative phosphorylation and the elevation of reactive oxygen species (ROS) production, followed by accumulation of lipid droplets (LDs) and autophagy-associated proteins LC3-II and SQSTM1/p62 and, finally, apoptosis of the tumor cells. Inactivation of ROS in COPI-depleted cells with the mitochondrial-specific quencher, mitoquinone mesylate, attenuated apoptosis and markedly decreased both the size and the number of LDs. COPI depletion caused ROS-dependent accumulation of LC3-II and SQSTM1 which colocalizes with LDs. Lack of double-membrane autophagosomes and insensitivity to Atg5 deletion suggested an accumulation of a microlipophagy complex on the surface of LDs induced by depletion of the COPI complex. Our findings suggest a sequence of cellular events triggered by COPI depletion, starting with inhibition of oxidative phosphorylation, followed by ROS activation and accumulation of LDs and apoptosis.
Asunto(s)
Autofagia , Neoplasias , Especies Reactivas de Oxígeno , Apoptosis , Proteína Coat de Complejo I/metabolismo , Lípidos , Neoplasias/metabolismoRESUMEN
Cortisol has wide-ranging actions, namely in gluconeogenesis and glycogenesis and exerts its effects through the glucocorticoid receptor. In the present study, we examined effects of glucocorticoid receptor blockade on type 2 diabetes control using the antagonist, RU486. Obese diabetic mice received daily injections of vehicle or RU486 over 28 days. Food intake, body weight, and plasma glucose were measured frequently. At 28 days, glucose tolerance, insulin sensitivity, and plasma triglycerides were assessed. Epididymal white adipose tissue and liver were excised for measurement of gene expression. Daily administration of RU486 had no effect on body weight or food intake, but plasma glucose concentrations were significantly lowered (1.4-1.6-fold; p<0.05 to p<0.001). Glucose concentrations were also significantly reduced (2.2-fold; p<0.001) following a glucose challenge. Similarly, exogenous insulin evoked a significantly greater reduction in plasma glucose (3.6-fold; p<0.01). Gene expression analysis revealed a significant reduction in hepatic mRNA of key enzymes, namely PEPCK-C (25%; p<0.01) and G6 Pase (32%; p<0.01) and also 11beta-HSD1 (18%; p<0.05). Investigation of adipose tissue gene expression also demonstrated reduced expression in 11beta-HSD1 (47%; p<0.05) and LPL (47%; p<0.001). These data demonstrate wide-ranging effects of glucocorticoid receptor antagonism on gene expression and metabolism, illustrating the therapeutic potential of specific glucocorticoid receptor antagonists in obesity-related diabetes.
Asunto(s)
Adipocitos/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/prevención & control , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/metabolismo , Mifepristona/farmacología , Obesidad/complicaciones , Adipocitos/efectos de los fármacos , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/metabolismo , Animales , Glucemia/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Diabetes Mellitus Tipo 2/genética , Esquema de Medicación , Epidídimo/efectos de los fármacos , Epidídimo/metabolismo , Ayuno/sangre , Conducta Alimentaria/efectos de los fármacos , Prueba de Tolerancia a la Glucosa , Insulina/metabolismo , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Obesos , Mifepristona/administración & dosificación , Obesidad/genética , Obesidad/prevención & control , Páncreas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Triglicéridos/metabolismoRESUMEN
AIMS/HYPOTHESIS: Premature death of retinal pericytes is a pathophysiological hallmark of diabetic retinopathy. Among the mechanisms proposed for pericyte death is exposure to AGE, which accumulate during diabetes. The current study used an in vitro model, whereby retinal pericytes were exposed to AGE-modified substrate and the mechanisms underlying pericyte death explored. METHODS: Pericytes were isolated from bovine retinal capillaries and propagated on AGE-modified basement membrane (BM) extract or non-modified native BM. The extent of AGE modification was analysed. Proliferative responses of retinal pericytes propagated on AGE-modified BM were investigated using a 5-bromo-2-deoxy-uridine-based assay. The effect of extrinsically added platelet-derived growth factor (PDGF) isoforms on these proliferative responses was also analysed alongside mRNA expression of the PDGF receptors. Apoptotic death of retinal pericytes grown on AGE-modified BM was investigated using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling labelling, mitochondrial membrane depolarisation and by morphological assessment. We also measured both the ability of PDGF to reverse Akt dephosphorylation that was mediated by AGE-modified BM, and increased pericyte apoptosis. RESULTS: Retinal pericytes exposed to AGE-modified BM showed reduced proliferative responses in comparison to controls (p<0.05-0.01), although this effect was reversed at low-AGE modifications. PDGF mRNA levels were differentially altered by exposure to low and high AGE levels, and AGE-modified BM caused significantly increased apoptosis in retinal pericytes. Pre-treatment of AGE-modified BM with PDGF-AA and -BB reversed the apoptosis (p<0.05-0.001) and restored Akt phosphorylation in retinal pericytes. CONCLUSIONS/INTERPRETATION: Evidence suggests that substrate-derived AGE such as those that occur during diabetes could have a major influence on retinal pericyte survival. During diabetic retinopathy, AGE modification of vascular BM may reduce bioavailability of pro-survival factors for retinal pericytes.
Asunto(s)
Supervivencia Celular/efectos de los fármacos , Productos Finales de Glicación Avanzada/metabolismo , Pericitos/fisiología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Retina/fisiología , Animales , Membrana Basal/fisiología , Becaplermina , Bovinos , Proteínas Proto-Oncogénicas c-sis , Retina/citología , Vasos Retinianos/citología , Vasos Retinianos/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
AIMS/HYPOTHESIS: This study was designed to determine whether inhibition of formation of AGE and advanced lipoxidation end-products (ALE) is a mechanism of action common to a diverse group of therapeutic agents that limit the progress of diabetic nephropathy. We compared the effects of the ACE inhibitor enalapril, the antioxidant vitamin E, the thiol compound lipoic acid, and the AGE/ALE inhibitor pyridoxamine on the formation of AGE/ALE and protection against nephropathy in streptozotocin diabetic rats. METHODS: Renal function and AGE/ALE formation were evaluated in rats treated with the agents listed above. Plasma was monitored monthly for triglycerides, cholesterol, creatinine and TNF-alpha, and 24-h urine samples were collected for measurement of albumin and total protein excretion. After 29 weeks, renal expression of mRNA for extracellular matrix proteins was measured, and AGE/ALE were quantified in skin and glomerular and tubular collagen. RESULTS: Diabetic animals were both hyperglycaemic and dyslipidaemic, and showed evidence of early nephropathy (albuminuria, creatinaemia). All interventions limited the progression of nephropathy, without affecting glycaemia. The order of efficacy was: pyridoxamine (650 mg.kg(-1).day(-1)) > vitamin E (200 mg.kg(-1).day(-1)) > lipoic acid (93 mg.kg(-1).day(-1)) approximately enalapril (35 mg.kg(-1).day(-1)). Pyridoxamine also significantly inhibited AGE/ALE accumulation in tissues; effects of other agents were mixed, but the degree of renoprotection was consistent with their effects on AGE/ALE formation. CONCLUSIONS/INTERPRETATION: All interventions inhibited the progression of nephropathy at the doses studied, but the maximal benefit was achieved with pyridoxamine, which also limited dyslipidaemia and AGE/ALE formation. These experiments indicate that the more effective the renoprotection, the greater the inhibition of AGE/ALE formation. For optimal protection of renal function, it would be beneficial to select drugs whose mechanism of action includes inhibition of AGE/ALE formation.