AMPK involvement in endoplasmic reticulum stress and autophagy modulation after fatty liver graft preservation: a role for melatonin and trimetazidine cocktail

Ischemia/reperfusion injury (IRI) associated with liver transplantation plays an important role in the induction of graft injury. Prolonged cold storage remains a risk factor for liver graft outcome, especially when steatosis is present. Steatotic livers exhibit exacerbated endoplasmic reticulum (ER) stress that occurs in response to cold IRI. In addition, a defective liver autophagy correlates well with liver damage. Here, we evaluated the combined effect of melatonin and trimetazidine as additives to IGL-1 solution in the modulation of ER stress and autophagy in steatotic liver grafts through activation of AMPK. Steatotic livers were preserved for 24 hr (4°C) in UW or IGL-1 solutions with or without MEL + TMZ and subjected to 2-hr reperfusion (37°C). We assessed hepatic injury (ALT and AST) and function (bile production). We evaluated ER stress (GRP78, PERK, and CHOP) and autophagy (beclin-1, ATG7, LC3B, and P62). Steatotic livers preserved in IGL-1 + MEL + TMZ showed lower injury and better function as compared to those preserved in IGL-1 alone. IGL-1 + MEL + TMZ induced a significant decrease in GRP78, pPERK, and CHOP activation after reperfusion. This was consistent with a major activation of autophagic parameters (beclin-1, ATG7, and LC3B) and AMPK phosphorylation. The inhibition of AMPK induced an increase in ER stress and a significant reduction in autophagy. These data confirm the close relationship between AMPK activation and ER stress and autophagy after cold IRI. The addition of melatonin and TMZ to IGL-1 solution improved steatotic liver graft preservation through AMPK activation, which reduces ER stress and increases autophagy

Mecanismos de protección del precondicionamiento isquémico en el trasplante hepático de injertos esteatósicos

[spa] Actualmente la principal limitación del trasplante hepático es la falta de donantes. Para suplir la carencia de órganos en muchas ocasiones se recurre al trasplante de órganos que no son óptimos para este fin, los denominados hígados subóptimos. Entre estos injertos destacan, por su prevalencia, los hígados esteatósicos. Los hígados esteatósicos son más susceptibles a la lesión de isquemia/reperfusión (I/R) asociada al trasplante hepático que los hígados no grasos, y por ello presentan un mayor riesgo de disfunción tras ser trasplantados. Estudios llevados a cabo en modelos de isquemia normotérmica señalan hacia una mayor alteración en la microcirculación hepática, un aumento en la acumulación de neutrófilos, una mayor liberación de mediadores inflamatorios o una mayor lesión por estrés oxidativo como posibles causas de esta mayor vulnerabilidad a la lesión de I/R. El Precondicionamiento (PC) isquémico, una estrategia quirúrgica basada en la aplicación de breves periodos de isquemia-reperfusión antes de someter al órgano a una I/R prolongada, ha resultado eficaz en la protección de los hígados grasos frente a la lesión de I/R en modelos de isquemia normotérmica, donde se sugiere que la preservación de los niveles de ATP ejercida por el PC isquémico tendría un papel determinante en la protección del PC isquémico en este tipo de hígados. Diversos autores sugieren que el NO y la AMPK están implicados en la protección del PC isquémico frente a la lesión de I/R en modelos experimentales de isquemia normotérmica. Sobre estas bases, en esta tesis se plantearon los siguientes objetivos: (1) estudiar si el PC isquémico, modulando los mecanismos implicados en la mayor vulnerabilidad de los hígados grasos a la lesión de I/R, protege frente a la lesión hepática y pulmonar asociada al trasplante de hígados grasos. Así como la evaluación de la implicación del NO en los efectos protectores del PC isquémico; (2) evaluar el papel de la AMPK y el NO en los efectos del PC isquémico en el metabolismo energético de los hígados grasos sometidos a trasplante. En las condiciones evaluadas en la presente tesis las alteraciones en la microcirculación, la acumulación de neutrófilos o la producción de TNF-α no parecen ser los mecanismos responsables de la mayor susceptibilidad de los hígados grasos a la lesión de I/R; por contra, el estrés oxidativo tiene un papel fundamental en la mayor susceptibilidad que presentan este tipo de hígados a la lesión de I/R asociada al trasplante de hígado. El PC isquémico protegió a los hígados esteatósicos frente a la lesión de I/R asociada al trasplante hepático. El PC isquémico, modulando el sistema xantina/XOD, redujo la lesión hepática y pulmonar asociadas al trasplante de injertos esteatósicos. El NO está implicado en la protección del PC isquémico sobre la lesión hepática y pulmonar asociada al trasplante de hígados esteatósicos. Este mediador vasoactivo al modular el sistema xantina/XOD y los efectos nocivos de los RLO, protegió frente a los desórdenes locales y sistémicos asociados al trasplante hepático. La síntesis de NO durante el PC isquémico es inducida por la activación de la AMPK. Estrategias farmacológicas basadas en la administración de activadores de la AMPK o donadores de NO simulan los efectos beneficiosos del PC isquémico sobre la lesión de I/R asociada al trasplante de injertos no esteatósicos. Si bien los activadores de la AMPK son también beneficiosos en el caso de los injertos esteatósicos, los donadores de NO resultaron ineficaces en este tipo de injertos. En estas condiciones, el NO se combinó con el exceso de RLO presentes en este tipo de injertos generando peroxinitritro, agravando aún más la vulnerabilidad de este tipo de hígados a la lesión de I/R.[eng] Despite the significant improvement during the last decade, the dramatic shortage of organs for transplantation obliges us to consider use of steatotic grafts, which are more vulnerable to hepatic ischemia-reperfusion (I/R) injury than non-steatotic livers. The use of steatotic livers is associated with an increased risk of primary non-function or dysfunction after transplantation. Several hypotheses have been put forward to explain why steatotic livers are more liable to I/R injury than normal livers. These include increases in: oxidative stress, neutrophil infiltration, microcirculatory alterations and release of tumor necrosis factor ; (TNF) from Kupffer cell activation. Recently, it has been reported that ischemic preconditioning, based on brief episodes of ischemia and reperfusion, protects steatotic livers undergoing normothermic ischemia. Results obtained suggest that ATP preservation induced by ischemic preconditioning may be central to the protection of steatotic liver against the hepatic I/R injury. NO and AMPK are candidates for mediating ischemic preconditioning. In the present work we examined: (1) whether preconditioning protects against hepatic and lung damage associated with steatotic liver transplantation and the effect of preconditioning on the mechanisms involved in the increased vulnerability of fatty livers to hepatic I/R. Evaluate the role of NO in ischemic preconditioning; (2) Whether AMPK and NO are involved in the benefits of ischemic preconditiong on energy metabolism in steatotic liver transplantation. Steatotic and non-steatotic liver grafts were similar in their blood flow, neutrophil accumulation, and TNF release after transplantation. Neither the increased vulnerability of fatty livers of Zucker rats to hepatic I/R injury associated with liver transplantation nor the protection conferred by preconditioning could be explained by changes in hepatic blood flow, neutrophil accumulation, and TNF release after transplantation under the conditions evaluated. However, in the presence of steatosis, lipid peroxidation injury increased. Preconditioning reduced the xanthine accumulation and percentage of xanthine oxidase seen in steatotic liver grafts during cold ischemia, and conferred protection against liver and lung damage following transplantation. The benefits of preconditioning could be mediated by nitric oxide. NO reduce the deleterious effects of ROS and modulate XDH/XOD activity, thus protecting steatotic livers against hepatic I/R injury. Preconditioning, through AMPK activation, induce NO synthesis. Pharmacological strategies based in pretreatment with AMPK activators or NO donors could protect against I/R injury associated with non-steatotic liver transplantation. However, the effective dose of exogenous NO may differ in both steatotic and non-steatotic livers.The injurious effects of exogenous donors on hepatic injury and oxidative stress in steatotic grafts were associated with increase nitrotyrosine levels. The results of nitrotyrosines suggest that peroxynitrite is a predominant form of ROS in steatotic grafts. This could explain, at least partially, the increased vulnerability of steatotic grafts to I/R injury and the injurious effects of exogenous NO on steatotic liver transplantation

Adenosine monophosphate-activated protein kinase and nitric oxide in rat steatotic liver transplantation

Background/Aims: Hepatic steatosis is a risk factor for transplantation. We examined the role of AMP-activated protein kinase (AMPK) and nitric oxide (NO) in the benefits of preconditioning in steatotic liver transplantation. Methods: Steatotic liver transplantation with or without preconditioning was induced in Zucker rats. The activities of AMPK and NO synthase (NOS) were measured and altered pharmacologically. Results: Preconditioning or AMPK activation with aminoimidazole-4-carboxamide ribonucleoside (AICAR) increased AMPK and constitutive NOS activities and protected against lipid peroxidation, nitrotyrosine formation and hepatic injury in both grafts. Inhibition of AMPK activity removed the benefits of preconditioning. NO synthesis inhibition abolished the benefits of preconditioning or AICAR. Therefore, preconditioning or AICAR, through AMPK activation, may induce NO synthesis, thus protecting against hepatic injury in both steatotic and non-steatotic liver transplantation. In non-steatotic grafts, NO donors simulated the benefits of preconditioning. However, in steatotic grafts, NO supplementation was ineffective. Conclusions: These results indicate (a) a potential relationship between AMPK and NO in the benefits of preconditioning in steatotic liver transplantation, (b) AICAR as a new phamacological strategy in steatotic liver transplantation and (c) a differential effect of NO supplementation in both grafts. © 2005 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.Supported by The Ministerio de Ciencia y Tecnología (project grant no. BFI 2002-00704 and BFI-2003-00912 and Ramón y Cajal research contract to Carmen Peralta) and the Ministerio de Sanidad y Consumo (project grant no. V2003-REDC03G-O) (Madrid, Spain)Peer Reviewe