Mitochondria and liver disease.

Resumen

Motivation:Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of pathologies, from simple steatosis tosteatohepatitis with fibrosis and inflammation. Steatosis is characterized by a hepatic fat content. Efficient drugtreatments are still lacking [1]. Hepatic lipid accumulation is associated with mitochondria dysfunction, which mayfavor the progression of the disease. Tricarboxylic acid cycle (TCA) and oxidative phosphorylation are key metabolicpathways of mitochondria. Succinate dehydrogenase (SDH), that consists of four subunits (A-B-C-D), is involved inthe TCA cycle (oxidises succinate to fumarate) and in the electron transport chain (provides electrons to complex IIIand enables the reduction of ubiquinone to ubiquinol) [2]. Previous work in the laboratory showed that NAFLD ischaracterized by decreased levels of the subunit D of SDH (SDHD).The aim of this Master's Thesis (TFM) is to analyse the impact of SDHD levels in the progression of NAFLD in anexperimental mouse model.Methods:For this purpose, male C57BL/6J mice were fed a methionine-choline deficient diet (MCDD), a NASH-inducing diet [3],and control diet (CD) for 4 and 6 weeks. Two weeks after the start of the diets, adeno-associated virus (AAV)encoding SDHD (AAV-SDHD) or control (AAV-renila) were injected intravenously. Mice were sacrificed and serum,livers and pancreas were obtained. Serum alanine transaminase (ALT) and aspartate transaminase (AST) weredetermined. Livers and pancreas were dissected out and fixed in formalin for hematoxylin & eosin and Sirius redstaining (for liver fibrosis). For cryosections, livers were frozen in OCT compound to perform Oil Red staining (forintracellular lipids) and SDH activity. Mouse livers were flash-frozen in liquid nitrogen, and homogenized in RIPAbuffer for proteins and RNAzol for RNA. Western blots were achieved to analyze protein levels and mRNA wasreverse transcribed into cDNA. Quantitative real-time PCR was performed using SYBR green PCR master mix.Relative RNA expression for pro-inflammatory cytokines, macrophages, fibrosis and mitochondrial genes werenormalized using two different housekeeping genes (cyclophilin and Rlpo).