RESUMEN
Obesity is accompanied by a low-grade inflammation state, characterized by increased proinflammatory cytokines levels such as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1ß). In this regard, there exists a lack of studies in hepatic tissue about the role of TNFα receptor 1 (TNFR1) in the context of obesity and insulin resistance during the progression of nonalcoholic fatty liver disease (NAFLD). The aim of this work was to evaluate the effects of high-caloric feeding (HFD) (40% fat, for 16 weeks) on liver inflammation-induced apoptosis, insulin resistance, hepatic lipid accumulation and its progression toward nonalcoholic steatohepatitis (NASH) in TNFR1 knock-out and wild-type mice. Mechanisms involved in HFD-derived IL-1ß release and impairment of insulin signaling are still unknown, so we determined whether IL-1ß affects liver insulin sensitivity and apoptosis through TNFα receptor 1 (TNFR1)-dependent pathways. We showed that knocking out TNFR1 induces an enhanced IL-1ß plasmatic release upon HFD feed. This was correlated with higher hepatic and epididymal white adipose tissue mRNA levels. In vivo and in vitro assays confirmed an impairment in hepatic insulin signaling, in part due to IL-1ß-induced decrease of AKT activation and diminution of IRS1 levels, followed by an increase in inflammation, macrophage (resident and recruited) accumulation, hepatocyte apoptotic process and finally hepatic damage. In addition, TNFR1 KO mice displayed higher levels of pro-fibrogenic markers. TNFR1 signaling disruption upon an HFD leads to an accelerated progression from simple steatosis to a more severe phenotype with many NASH features, pointing out a key role of TNFR1 in NAFLD progression.
Asunto(s)
Dieta Alta en Grasa/efectos adversos , Enfermedad del Hígado Graso no Alcohólico/etiología , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Animales , Apoptosis/genética , Insulina/metabolismo , Resistencia a la Insulina , Interleucina-1beta/metabolismo , Hígado/metabolismo , Hígado/patología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/patología , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Transducción de SeñalRESUMEN
Diabetes mellitus is a risk factor for prognosis after liver resection. In previous work, we found a pro-apoptotic state in the diabetic rat liver. In this work, this was also observed 1 hour post-partial hepatectomy (PH) and resulted in a deficient regenerative response 24 hours post-PH. Treatment with insulin and/or Desferoxamine (DES) (iron chelator) or Tempol (TEM) (free radicals scavenger) was effective in preventing the liver reactive oxygen species (ROS) production induced by diabetic state. High levels of ROS play a role in hepatic lipid peroxidation in diabetes before and after PH, and lead to increased pro-apoptotic events, which contribute to a reduced regenerative response. This becomes of relevance for the potential use of antioxidants/free radical scavengers plus insulin for improvement of post-surgical recovery of diabetic patients subjected to a PH.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Regeneración Hepática/fisiología , Especies Reactivas de Oxígeno/metabolismo , Animales , Óxidos N-Cíclicos/farmacología , Deferoxamina/farmacología , Diabetes Mellitus Experimental/fisiopatología , Modelos Animales de Enfermedad , Hepatectomía , Insulina/farmacología , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/fisiopatología , Pronóstico , Ratas , Ratas Wistar , Marcadores de Spin , EstreptozocinaRESUMEN
We analyzed the contribution of TNF-α intracellular pathway in the development of apoptosis in the liver of streptozotocin-induced diabetic rats. In liver tissue, diabetes promoted a significant increase of TNF-α/TNF-R1, and led to the activation of caspase-8, of nuclear factor kappa B (NFκB), and JNK signaling pathways. The activation of NFκB led to an induction of iNOS and consequent increase in NO production. As a consequence of such changes a significant increase of caspase-3 activity and of apoptotic index were observed in the liver of diabetic animals. Importantly, the treatment in vivo of diabetic rats with etanercept (TNF-α blocking antibody) or aminoguanidine (selective iNOS inhibitor) significantly attenuated the induction of apoptosis by reduction of caspase-3 activity. Overall, we demonstrated that in the diabetes enhances TNF-α in the liver, which may be a fundamental key leading to apoptotic cell death, through activation of caspase-8, NFκB and JNK pathways.
Asunto(s)
Apoptosis , Diabetes Mellitus Tipo 1/metabolismo , Hígado/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Western Blotting , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Inhibidores de Caspasas , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1/patología , Espectroscopía de Resonancia por Spin del Electrón , Etanercept , Guanidinas/farmacología , Inmunoglobulina G/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Hígado/patología , Masculino , FN-kappa B/metabolismo , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo II/metabolismo , Ratas , Ratas Wistar , Receptores del Factor de Necrosis Tumoral , Transducción de Señal , EstreptozocinaRESUMEN
In this study, we analyzed the contribution of hydroxyl radical in the liver apoptosis mediated by hyperglycemia through the Bax-caspase pathway and the effects of insulin protection against the apoptosis induced by hyperglycemia. Male adult Wistar rats were randomized in three groups: control (C) (sodium citrate buffer, i.p.), streptozotocin (STZ)-induced diabetic (SID) (STZ 60 mg/kg body weight, i.p.), and insulin-treated SID (SID+I; 15 days post STZ injection, SID received insulin s.c., twice a day, 15 days). Rats were autopsied on day 30. In liver tissue, diabetes promoted a significant increase in hydroxyl radical production which correlated with lipid peroxidation (LPO) levels. Besides, hyperglycemia significantly increased mitochondrial BAX protein expression, cytosolic cytochrome c levels, and caspase-3 activity leading to an increase in apoptotic index. Interestingly, the treatment of diabetic rats with desferoxamine or tempol (antioxidants/hydroxyl radical scavengers) significantly attenuated the increase in both hydroxyl radical production and in LPO produced by hyperglycemia, preventing apoptosis by reduction of mitochondrial BAX and cytosolic cytochrome c levels. Insulin treatment showed similar results. The finding that co-administration of antioxidants/hydroxyl radical scavengers together with insulin did not provide any additional benefit compared with those obtained using either inhibitors or insulin alone shows that it is likely that insulin prevents oxidative stress by reducing the effects of hydroxyl radicals. Importantly, insulin significantly increased apoptosis inhibitor protein expression by induction of its mRNA. Taken together, our studies support that, at least in part, the hydroxyl radical acts as a reactive intermediate, which leads to liver apoptosis in a model of STZ-mediated hyperglycemia. A new anti-apoptosis signal for insulin is shown, given by an increase of apoptosis inhibitor protein.
Asunto(s)
Apoptosis , Radical Hidroxilo/metabolismo , Hiperglucemia/metabolismo , Insulina/metabolismo , Hígado/citología , Animales , Caspasa 3/genética , Caspasa 3/metabolismo , Citocromos c/metabolismo , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Humanos , Hiperglucemia/genética , Hiperglucemia/fisiopatología , Hígado/metabolismo , Masculino , Distribución Aleatoria , Ratas , Ratas WistarRESUMEN
We have already demonstrated that interferon alfa-2b (IFN-alpha2b) induces apoptosis in isolated hepatocytes from preneoplastic rat livers via the secretion of transforming growth factor beta(1) (TGF-beta(1)), and this process is accompanied by caspase-3 activation. The aim of this study was to further investigate the mechanism of this activation. Isolated hepatocytes from preneoplastic livers induced DNA fragmentation in response to IFN-alpha2b, which was completely blocked when anti-TGF-beta(1) was added to the culture media. IFN-alpha2b mediated radical oxygen species (ROS) production that preceded the loss of mitochondrial transmembrane potential (DeltaPsi), release of cytochrome c, and activation of caspase-3. Bax levels increased in a time-dependent fashion, and Bcl-x(L) was down-regulated in the early hours of IFN-alpha2b treatment. The delayed translocation of Bid into the mitochondria was in concordance with late caspase-8 activation. In conclusion, endogenous TGF-beta(1) secreted under IFN-alpha2b stimulus seems to induce cytochrome c release through a mechanism related to Bcl-2 family members and loss of mitochondrial DeltaPsi. Bax protein could be responsible of the release of cytochrome c during the initial hours of IFN-alpha2b-induced apoptosis via TGF-beta(1). Activated Bid by caspases could amplificate the mitochondrial events, enhancing the release of cytochrome c.
Asunto(s)
Apoptosis , Hepatocitos/citología , Interferón-alfa/farmacología , Neoplasias Hepáticas/metabolismo , Hígado/citología , Lesiones Precancerosas/metabolismo , Animales , Anexina A5/metabolismo , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Fragmentación del ADN , Hepatocitos/metabolismo , Interferón alfa-2 , Masculino , Potencial de la Membrana Mitocondrial , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
In previous work we showed that interferon alfa-2b (IFN-alpha2b) increases apoptosis on rat hepatic preneoplastic foci. The aim of this study was to determine if transforming growth factor beta1 (TGF-beta1) was involved in the programmed cell death on the foci. Animals were divided into 6 groups: subjected to a 2-phase model (diethylnitrosamine plus 2-acetylaminofluorene) of preneoplasia development (group 1); treated with IFN-alpha2b during the 2 phases (group 2); treated with IFN-alpha2b during initiation with diethylnitrosamine (group 3); treated with IFN-alpha2b during 2-acetylaminofluorene administration (group 4); subjected only to an initiation stage (group 5); and treated with IFN-alpha2b during the initiation period (group 6). Serum TGF-beta1 levels were increased in IFN-alpha2b-treated rats. Immunohistochemical studies showed that IFN-alpha2b significantly increased the quantity of TGF-beta1-positive hepatocytes in groups 2 to 4. Phosphorylated-Smads-2/3 (p-Smads-2/3) proteins in liver nuclear extracts were significantly elevated. To determine the source of TGF-beta1, isolated hepatocytes, Kupffer cells, and peritoneal macrophages from animals in groups 1 and 5 were cultured with or without IFN-alpha2b. IFN-alpha2b stimulus induced several-fold increases of TGF-beta1 secretion from hepatocytes. Neither Kupffer cells nor peritoneal macrophages secreted detectable TGF-beta1 levels when they were treated with IFN-alpha2b. IFN-alpha2b-stimulated cultured hepatocytes from preneoplastic livers showed enhanced apoptosis, measured by fluorescence microscopy and caspase-3 activity. They presented higher nuclear accumulation of p-Smads-2/3, indicating increased TGF-beta1 signaling. When anti-TGF-beta1 was added to the culture media, TGF-beta1 activation and apoptosis induced by IFN-alpha2b were blocked. In conclusion, IFN-alpha2b-induced production of TGF-beta1 by hepatocytes from preneoplastic liver is involved in the apoptotic elimination of altered hepatic foci.
Asunto(s)
Apoptosis , Interferón-alfa/farmacología , Neoplasias Hepáticas Experimentales/fisiopatología , Hígado/efectos de los fármacos , Hígado/fisiopatología , Lesiones Precancerosas/fisiopatología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Caspasa 3 , Caspasas/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Medios de Cultivo/metabolismo , Proteínas de Unión al ADN/metabolismo , Activación Enzimática , Hepatocitos/enzimología , Hepatocitos/metabolismo , Interferón alfa-2 , Hígado/metabolismo , Masculino , Fosforilación , Ratas , Ratas Wistar , Proteínas Recombinantes , Proteína Smad2 , Proteína smad3 , Transactivadores/metabolismo , Factor de Crecimiento Transformador beta/sangre , Factor de Crecimiento Transformador beta1RESUMEN
The expression and activity of the apical ileal sodium-dependent bile acid transporter (asbt) was examined in the small intestine of control, pregnant, and lactating postpartum rats 2, 12, and 21 days after delivery. Western blot analysis of brush border membrane vesicles (BBMV) prepared from different regions of the small intestine demonstrated that expression of asbt was maximal in the most distal segments for all experimental groups, was not substantially affected in pregnant and 2-day postpartum rats, and was significantly increased in 12- and 21-day postpartum rats. Analysis of mRNA suggested that asbt protein was regulated at the posttranscriptional level in postpartum rats. Increased expression of asbt protein postpartum was maximal (approximately 2-fold) in the proximal region of the ileum, consistent with a 60% increase in taurocholate (TC) transport in BBMV from the proximal ileum in 14- to 21-day postpartum rats relative to control rats. Absorption of TC, determined from the intact proximal ileum using an intestinal loop model, demonstrated a 30% increase in TC uptake per unit weight of tissue in 14- to 21-day postpartum rats relative to control rats. Together with the marked increase in intestinal mass observed at peak lactation, these data indicate a significant increase in asbt-mediated reclamation of bile acids in the intestine of lactating rats.
Asunto(s)
Ácidos y Sales Biliares/metabolismo , Proteínas Portadoras/genética , Íleon/metabolismo , Absorción Intestinal/fisiología , Transportadores de Anión Orgánico Sodio-Dependiente , Periodo Posparto/fisiología , Simportadores , Fosfatasa Alcalina/metabolismo , Animales , Grasas de la Dieta/farmacocinética , Circulación Enterohepática/fisiología , Femenino , Expresión Génica/fisiología , Péptidos Similares al Glucagón , Lactancia/fisiología , Hígado/fisiología , Ovariectomía , Péptidos/farmacología , Embarazo , Prolactina/farmacología , ARN Mensajero/análisis , Ratas , Ácido Taurocólico/farmacocinéticaRESUMEN
Para conocer si el IFN a previene la oncogenesis in vivo, en estadíos tempranos del desarrollo tumoral, evaluamos la acción del IFN a-2b sobre focos preneoplásicos en hígado de rata. Los animales se dividieron en los siguientes grupos: sujetos a un modelo de iniciación-promoción (G1), tratados con IFN a-2b durante: a) iniciación-promoción (G2), b) iniciación (G3), c) promoción (G4); sujetos solo al estadío de iniciación (G5) y tratados con IFNa-2b en este período (G6). El área y el número de los focos preneoplásicos rGST P-positivos se mostraron significativamente disminuidos y el Indice Apoptótico aumentado en los G2, 3 y 6. Los niveles de Bcl-2 y Bcl-xL están disminuidos en los grupos tratados con IFN a-2b y los de Bax mitocondrial aumentados en los G2, 3 y 6. En conclusión, los hepatocitos preneoplásicos de ratas que recibieron IFN a-2b sufren muerte celular programada como resultado de un aumento sustancial de Bax y de su translocación a la mitocondria. (AU)
Asunto(s)
Animales , Masculino , Ratas , RESEARCH SUPPORT, NON-U.S. GOVT , Apoptosis/efectos de los fármacos , Interferón-alfa/farmacología , Antineoplásicos/farmacología , Neoplasias Hepáticas/fisiopatología , Lesiones Precancerosas/fisiopatología , Apoptosis/fisiología , Hígado/patología , Hígado/enzimología , Proteínas Proto-Oncogénicas/análisis , Western Blotting , Ratas WistarRESUMEN
Para conocer si el IFN a previene la oncogenesis in vivo, en estadíos tempranos del desarrollo tumoral, evaluamos la acción del IFN a-2b sobre focos preneoplásicos en hígado de rata. Los animales se dividieron en los siguientes grupos: sujetos a un modelo de iniciación-promoción (G1), tratados con IFN a-2b durante: a) iniciación-promoción (G2), b) iniciación (G3), c) promoción (G4); sujetos solo al estadío de iniciación (G5) y tratados con IFNa-2b en este período (G6). El área y el número de los focos preneoplásicos rGST P-positivos se mostraron significativamente disminuidos y el Indice Apoptótico aumentado en los G2, 3 y 6. Los niveles de Bcl-2 y Bcl-xL están disminuidos en los grupos tratados con IFN a-2b y los de Bax mitocondrial aumentados en los G2, 3 y 6. En conclusión, los hepatocitos preneoplásicos de ratas que recibieron IFN a-2b sufren muerte celular programada como resultado de un aumento sustancial de Bax y de su translocación a la mitocondria.