Mitobullet: antioxidant targeting of mitochondria to prevent nonalcoholic fatty liver disease-induced oxidative stress

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a public health concern affecting 24% of the population worldwide. Non-alcoholic steatohepatitis (NASH), one of the deleterious stages of non-alcoholic fatty liver disease, remains a significant cause of liver-related morbidity and mortality worldwide. NAFLD is a multifactorial disease and is considered the hepatic component of metabolic syndrome. Although mechanisms underlying disease pathophysiology are not fully clarified, mitochondrial dysfunction and oxidative stress (OxS) are potential key players. Mitochondria are key organelles involved in cellular survival, differentiation, and death induction. In this regard, mitochondrial morphology and/or function alterations are involved in stress-induced adaptive pathways, priming mitochondria for mitophagy or apoptosis induction. In this context and driven by the lack of effective pharmacological therapies, we propose a new approach using a mitochondria-targeted antioxidant (AntiOxCIN4) to prevent non-alcoholic fatty liver (NAFL) development. Previously studies shown that the mitochondriotropic antioxidant AntiOxCIN4 (100 μM; 48 h) presented significant cytoprotective effect without affecting the viability of human hepatoma-derived (HepG2) cells. Moreover, AntiOxCIN4 (12.5 μM; 72 h) caused a mild increase of reactive oxygen species (ROS) levels without toxicity to primary human skin fibroblasts (PHSF). As Nrf2 is a master regulator of the OxS response inducing antioxidant-encoding gene expression, we hypothesized that AntiOxCIN4 could increase the resistance of human hepatoma-derived HepG2 to oxidative by Nrf2-dependent mechanisms, in a process mediated by mitochondrial ROS (mtROS). In fact, in chapter 7, we showed that after an initial decrease in oxygen consumption paralleled by a moderate increase in superoxide anion levels, AntiOxCIN4 led to a time-dependent Nrf2 translocation to the nucleus. This was followed later by an increase in basal respiration (150 %) and extracellular acidification (120 %). AntiOxCIN4 treatment enhanced mitochondrial quality by triggering the clearance of defective organelles by autophagy and/or mitophagy, coupled with increased mitochondrial biogenesis. Anti-OxCIN4 also upregulated the cellular antioxidant defense system. AntiOxCIN4 seems to have the ability to maintain hepatocyte redox homeostasis, regulating the electrophilic/nucleophilic tone, and preserve cellular physiological functions. The obtained data in chapter 7 opened a new avenue to explore the effects of AntiOxCIN4 in the context of preserving hepatic mitochondrial function in disorders, such as NASH/NAFLD and type II diabetes. In agreement with that hypothesis, we next characterized the human HepG2 cells as an in vitro model for steatosis (chapter 8), in order to screen mitochondriotropic antioxidants lipid-lowering ability. Using an exploratory data analysis, we investigate time-dependent cellular and mitochondrial effects of different supra-physiological fatty acids (FA) overload strategies, in the presence or absence of fructose (F), on human hepatoma-derived HepG2 cells. We measured intracellular neutral lipid content and reactive oxygen species (ROS) levels, mitochondrial respiration and morphology, and caspases activity and cell death. FA-treatments induced a time-dependent increase in neutral lipid content, which was paralleled by an increase in ROS. Fructose, by itself, did not increase intracellular lipid content nor aggravated the effects of palmitic acid (PA) or free fatty acids mixture (FFA), although it led to an increased expression of hepatic fructokinase. Instead, F decreased mitochondrial phospholipid content, as well as OXPHOS subunits levels. Increased lipid accumulation and ROS in FA-treatments preceded mitochondrial dysfunction, comprising altered mitochondrial membrane potential (ΔΨm) and morphology, and decreased oxygen consumption rates, especially with PA. Consequently, supra-physiological PA alone or combined with F prompted the activation of caspase pathways leading to a time-dependent decrease in cell viability. Exploratory data analysis methods support this conclusion by clearly identifying the effects of FA treatments. Unsupervised learning algorithms created homogeneous and cohesive clusters, with a clear separation between PA and FFA treated samples to identify a minimal subset of critical mitochondrial markers in order to attain a feasible model to predict cell death in NAFLD or for high throughput screening of possible therapeutic agents, with particular focus in measuring mitochondrial function. Finally, to validate the beneficial effects of hydroxycinnamic-derived mitochondriotropic antioxidant AntiOxCIN4, C57BL/6J mice daily supplemented with 2.5 mg An-tiOxCIN4 were fed with a standard diet (SD) or Western diet (WD) (30% high-fat, 30% high-sucrose) for 16 weeks to induce simple steatosis in vivo. Additionally, we treated HepG2 cells with AntiOxCIN4 (100 μM, 48 h) before the exposure to bovine serum albumin (BSA) alone or free fatty acids (FFAs) mixture (250 µM, 24 h) to induce lipid accumulation in vitro (chapter 9). In WD-fed mice, AntiOxCIN4 supplementation de-creased body (by 43 %), liver weight (by 39 %) and plasma hepatocyte damage markers. The improvement in hepatic-related parameters was associated with a reduction of fat liver accumulation (600 %) and the remodeling of fatty acyl chain composition compared with the WD-fed group. Data on human HepG2 cells confirmed a lower lipid accumulation by the evident reduction of lipid droplets size and number in AntiOxCIN4-treated cells. AntiOxCIN4 supplementation induced mitochondrial metabolism remodeling by upregulating oxidative phosphorylation (OXPHOS) subunits, mediated by the PGC-1α-SIRT3. Accordingly, human HepG2 data corroborated that AntiOxCIN4 pre-treatment stimulated fatty acid oxidation-linked oxygen consumption rates and OXPHOS gene expression remodeling. AntiOxCIN4 also upregulated hepatic antioxidant defense system in WD-fed mice and counteracted cellular ROS exacerbation in FFA-treated human HepG2 cells. Finally, AntiOxCIN4 supplementation prevented lipid ac-cumulation-driven autophagic flux impairment, by increasing lysosomal proteolytic capacity as observed both in vivo and in vitro Overall, the results obtained in this dissertation added novel and relevant knowledge by showing that AntiOxCIN4 improved NAFL in vivo, via three main mechanisms: a) increasing mitochondrial function (fatty acid oxidation); b) regulating antioxidant capacity (enzymatic and non-enzymatic) and; c) preventing the impairment in autophagy. Together, the findings support the potential use of AntiOxCIN4 in the prevention/ treatment of NAFLD.