Hepatic cyclooxygenase-2 expression protects against diet-induced steatosis, obesity and insulin resistance

Resumen del póster presentado a la Conferencia: FASEB SRC: Liver Biology: Fundamental Mechanisms and Translational Applications, celebrada en Keystone-Colorado (US) del 6 al 11 de julio de 2014.[Background and Aims: Accumulation evidence links obesity-induced inflammation as an important contributor to the induction of insulin resistance. Moreover, insulin resistance plays a key role in the pathophysiology of obesity-related diseases such as type 2 diabetes and non alcoholic fatty liver disease. Cyclooxygenase-1 and -2 catalyze the first step in prostanoid biosynthesis. Since adult hepatocytes fail to induce COX-2 expression regardless of the pro-inflammatory factors used, we have evaluated whether this lack of expression under mild pro-inflammatory conditions might constitute a permissive condition for the onset of insulin resistance. [Methods]: We evaluated the role of COX-2 expression in hepatocytes in a model of insulin resistance and altered energy homeostasis induced by high fat diet by metabolic parameters in transgenic mice constitutively expressing human COX-2 in hepatocytes. [Results]: COX-2 expression in hepatocytes protects from high fat diet-induced hepatic steatosis, obesity and hence insulin resistance, as demonstrated by a decreased hepatic steatosis, adiposity and adipocyte area, an enhanced insulin sensitivity and glucose tolerance, decreased plasmatic and hepatic triglycerides and free fatty acids levels, increased adiponectin/leptin ratio and decreased levels of pro-inflammatory cytokines. COX-2 transgenic mice exhibited increased whole body energy expenditure and fatty acid oxidation. Moreover, when hepatic insulin signaling was analyzed, an increase in insulin receptor-mediated Akt phosphorylation was found in hCOX-2 transgenic mice. Similar results were obtained in human and murine hepatic cells expressing a COX-2 transgene. [Conclusion]: Constitutively expression of COX-2 in hepatocytes protects against adiposity, inflammation and hepatic insulin resistance in mice under high fat diet.Peer Reviewe

Disruption of tumor necrosis factor alpha receptor 1 signaling accelerates NAFLD progression in mice upon a high-fat diet

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-1β). 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-1β release and impairment of insulin signaling are still unknown, so we determined whether IL-1β affects liver insulin sensitivity and apoptosis through TNFα receptor 1 (TNFR1)-dependent pathways. We showed that knocking out TNFR1 induces an enhanced IL-1β 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-1β-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.This work was supported by research grants from Consejo Nacional de Investigaciones Científicas y Técnicas (PIP-CONICET 112201500508, to C.E.C.) and SAF2016-75004-R and SAF2015-70270-REDT (MINECO, Spain)to P.M.S. and O.M.Peer reviewe

Role of reactive oxygen species in the early stages of liver regeneration in streptozotocin-induced diabetic rats

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.Peer Reviewe

Hyperglycemia induces apoptosis in rat liver through the increase of hydroxy, radical: New insights into the insulin effect

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/kgbody weight, i.p.), and insulintreated 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. © 2010 Society tor Endocrinology.This work was supported by research grants from ANPCyT (PICT no. 32413, CEC) and from CONICET (PIP no. 5531, CEC).Peer Reviewe

Benznidazole, the trypanocidal drug used for Chagas disease, induces hepatic NRF2 activation and attenuates the inflammatory response in a murine model of sepsis

Molecular mechanisms on sepsis progression are linked to the imbalance between reactive oxygen species (ROS) production and cellular antioxidant capacity. Previous studies demonstrated that benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, has immunomodulatory effects, increasing survival in C57BL/6 mice in a model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). The mechanism by which BZL inhibits inflammatory response in sepsis is poorly understood. Also, our group recently reported that BZL is able to activate the nuclear factor erytroide-derived 2-Like 2 (NRF2) in vitro. The aim of the present work was to delineate the beneficial role of BZL during sepsis, analyzing its effects on the cellular redox status and the possible link to the innate immunity receptor TLR4. Specifically, we analyzed the effect of BZL on Nrf2 regulation and TLR4 expression in liver of mice 24 hours post-CLP. BZL was able to induce NRF2 nuclear protein localization in CLP mice. Also, we found that protein kinase C (PKC) is involved in the NRF2 nuclear accumulation and induction of its target genes. In addition, BZL prompted a reduction in hepatic CLP-induced TLR4 protein membrane localization, evidencing its immunomodulatory effects. Together, our results demonstrate that BZL induces hepatic NRF2 activation with the concomitant increase in the antioxidant defenses, and the attenuation of inflammatory response, in part, by inhibiting TLR4 expression in a murine model of sepsis.This work was supported by Research Grants PICT 2007-1730 from Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) and by Secretaría de Estado de Ciencia, Tecnología e Innovación del Gobierno de la Provincia de Santa Fe, Argentina (112/10) to M.T.R.; PICT 0360 from Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) to V.A.C.; PICT 2012-2383 from Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) to D.E.F. and SAF2012-33283 (MINECO, Spain), S2010/BMD-2423 (Comunidad de Madrid, CAM), SAF2013-43713-R (MINECO, Spain) to O.M. and P.M.S.Peer Reviewe

Cyclooxygenase-2 expression in hepatocytes attenuates non-alcoholic steatohepatitis and liver fibrosis in mice

Cyclooxygenase-2 (COX-2) is involved in different liver diseases but little is known about the significance of COX-2 in the development and progression of non-alcoholic steatohepatitis (NASH). This study was designed to elucidate the role of COX-2 expression in hepatocytes in the pathogenesis of steatohepatitis and hepatic fibrosis. In the present work, hepatocyte-specific COX-2 transgenic mice (hCOX-2-Tg) and their wild-type (Wt) littermates were either fed methionine-and-choline deficient (MCD) diet to establish an experimental non-alcoholic steatohepatitis (NASH) model or injected with carbon tetrachloride (CCl) to induce liver fibrosis. In our animal model, hCOX-2-Tg mice fed MCD diet showed lower grades of steatosis, ballooning and inflammation than Wt mice, in part by reduced recruitment and infiltration of hepatic macrophages, with a corresponding decrease in serum levels of pro-inflammatory cytokines. Furthermore, hCOX-2-Tg mice showed a significant attenuation of the MCD diet-induced increase in oxidative stress and hepatic apoptosis observed in Wt mice. Even more, hCOX-2-Tg mice treated with CCl had significantly lower stages of fibrosis and less hepatic content of collagen, hydroxyproline and pro-fibrogenic markers than Wt controls. Collectively, our data indicates that constitutive hepatocyte COX-2 expression ameliorates NASH and liver fibrosis development in mice by reducing inflammation, oxidative stress and apoptosis and by modulating activation of hepatic stellate cells, respectively, suggesting a possible protective role for COX-2 induction in NASH/NAFLD progression.This work was supported by Financing Program for short stays abroad for Assistant Researchers (CONICET-Argentina) (2618/13) and ANPCyT-PICT2383-2012 to D.F.; SAF2012-39732 (MINECO, Spain) and CIBERehd (ISCIII, Spain) (CB06/04/1069) to M.C.; SAF2014-52492 (MINECO, Spain) to L.B.; S2010/BMD-2378 (Comunidad de Madrid, CAM) to L.B. and P.M.S.; RD12/0042/0019 (ISCIII, Spain) and CIBERehd (ISCIII, Spain) to L.B. and P.M.S.; SAF-2015-65267-R (MINECO/FEDER), S2010/BMD-2423 (Comunidad de Madrid, CAM), EFSD and Amylin Paul Langerhans Grant and CIBERdem (ISCIII, Spain) to A.M.V.; PI13/01299 (ISCIII, Spain) to C.G-M.; SAF2013-43713-R (MINECO, Spain) to P.M.S.Peer Reviewe

La expresión hepática de la ciclooxigenasa-2 (COX-2) protege de la esteatosis, adiposidad y resistencia a la insulina inducida por dieta rica en grasa

Resumen del póster presentado al XXXVII Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Granada del 9 al 12 de septiembre de 2014.La resistencia a la insulina (RI) participa en la fisiopatología de enfermedades relacionadas con la obesidad como la diabetes mellitus tipo 2 y la enfermedad de hígado graso no alcohólica. Además, la RI está asociada con un estado de inflamación crónica leve que contribuye significativamente a su desarrollo. Las ciclooxigenasas 1 y 2 (COX-1 y COX-2) catalizan el primer paso en la biosíntesis de los prostanoides. COX-1 se expresa constitutivamente en diversos tejidos, mientras que la expresión de COX-2 es inducida por diferentes estímulos. Los hepatocitos adultos son incapaces de inducir la expresión de COX-2 independientemente de los factores proinflamatorios presentes. Se ha analizado el papel de la expresión hepática de COX-2 en ratones transgénicos (hCOX-2-Tg) en un modelo de RI y de alteración de la homeostasis energética inducido por una dieta rica en grasa. Nuestros resultados muestran que la expresión de COX-2 en el hepatocito protege frente a la RI, esteatosis hepática y obesidad, en parte debido a una mayor sensibilidad a la insulina, un aumento en la tolerancia a la glucosa, y de la relación adiponectina/leptina. Asimismo, se observó una disminución de la esteatosis hepática, adiposidad y en el área de los adipocitos, de los niveles de triglicéridos y ácidos grasos libres tanto hepáticos como plasmáticos, y de las citoquinas proinflamatorias. Los ratones hCOX-2-Tg exhibieron también un incremento en el gasto energético, inducción de la termogénesis y de los niveles de expresión de genes implicados en la oxidación de ácidos grasos, así como un aumento en la señalización de insulina hepática (fosforilación del receptor de insulina y AKT). Resultados similares se obtuvieron en líneas celulares hepáticas murinas y humanas que sobreexpresan COX-2. Nuestros datos demuestran que la expresión constitutiva de COX-2 en hepatocitos protege frente a la RI hepática, la adiposidad e inflamación en ratones hCOX-2-Tg sometidos a una dieta rica en grasa.Peer Reviewe