Ines C. M. Simoes

P66Shc is an adaptor protein and one of the cellular fate regulators since it modulates mitogenic signaling pathways, mitochondrial function, and reactive oxygen species (ROS) production. p66Shc is localized mostly in the cytosol and endoplasmic reticulum (ER); however, under oxidative stress, p66Shc is post-translationally modified and relocates to mitochondria. p66Shc was found in the intermembrane space, where it interacts with cytochrome c, contributing to the hydrogen peroxide generation by the mitochondrial respiratory chain. Our previous studies suggested that p66Shc is localized also in mitochondria-associated membranes (MAM). MAM fraction consists of mitochondria and mostly ER membranes. Contact sites between ER and mitochondria host proteins involved in multiple processes including calcium homeostasis, apoptosis, and autophagy regulation. Thus, p66Shc in MAM could participate in processes related to cell fate determination. Due to reports on various and conditional p66Shc intracellular localization, in the present paper, we describe the allocation of p66Shc pools in different subcellular compartments in mouse liver tissue and HepG2 cell culture. We provide additional evidence for p66Shc localization in MAM. In the present study, we use precisely purified subcellular fraction isolated by differential centrifugation-based protocol from control mouse liver tissue and HepG2 cells and from cells treated with hydrogen peroxide to promote mitochondrial p66Shc translocation. We performed controlled digestion of crude mitochondrial fraction, in which the degradation patterns of p66Shc and MAM fraction marker proteins were comparable. Moreover, we assessed the distribution of the individual ShcA isoforms (p46Shc, p52Shc, and p66Shc) in the subcellular fractions and their contribution to the total ShcA in control mice livers and HepG2 cells. In conclusion, we showed that a substantial pool of p66Shc protein resides in MAM in control conditions and after oxidative stress induction.

Despite decades of research, the pathogenesis of metabolic dysfunction‐associated steatotic liver disease (MASLD) is still not completely understood. Based on the evidence from preclinical models, one of the factors proposed as a main driver of disease development is oxidative stress. This study aimed to search for the resemblance between the profiles of oxidative stress and antioxidant defense in the animal model of MASLD and the group of MASLD patients. C57BL/6J mice were fed with the Western diet for up to 24 weeks and served as the animal model of MASLD. The antioxidant profile of mice hepatic tissue was determined by liquid chromatography‐MS3 spectrometry (LC–MS/MS). The human cohort consisted of 20 patients, who underwent bariatric surgery, and 6 controls. Based on histological analysis, 4 bariatric patients did not have liver steatosis and as such were also classified as controls. Total antioxidant activity was measured in sera and liver biopsy samples. The hepatic levels of antioxidant enzymes and oxidative damage were determined by Western Blot. The levels of antioxidant enzymes were significantly altered in the hepatic tissue of mice with MASLD. In contrast, there were no significant changes in the antioxidant profile of hepatic tissue of MASLD patients, except for the decreased level of carbonylated proteins. Decreased protein carbonylation together with significant correlations between the thioredoxin system and parameters describing metabolic health suggest alterations in the thiol‐redox signaling. Altogether, these data show that even though the phenotype of mice closely resembles human MASLD, the animal‐to‐human translation of cellular and molecular processes such as oxidative stress may be more challenging.

The clinical picture of autoimmune hepatitis (AIH) varies markedly between patients, potentially due to genetic modifiers. The aim of this study was to evaluate genetic variants previously associated with fatty liver as potential modulators of the AIH phenotype. The study cohort comprised 313 non-transplanted adults with AIH. In all patients, the MARC1 (rs2642438), HSD17B13 (rs72613567), PNPLA3 (rs738409), TM6SF2 (rs58542926), and MBOAT7 (rs641738) variants were genotyped using TaqMan assays. Mitochondrial damage markers in serum were analyzed in relation to the MARC1 variant. Carriers of the protective MARC1 allele had lower ALT and AST (both P < 0.05). In patients treated for AIH for ≥ 6 months, MARC1 correlated with reduced AST, ALP, GGT (all P ≤ 0.01), and lower APRI (P = 0.02). Patients carrying the protective MARC1 genotype had higher total antioxidant activity (P < 0.01) and catalase levels (P = 0.02) in serum. The PNPLA3 risk variant was associated with higher MELD (P = 0.02) in treated patients, whereas MBOAT7 increased the odds for liver cancer (OR = 3.71). None of the variants modulated the risk of death or transplantation. In conclusion, the MARC1 polymorphism has protective effects in AIH. Genotyping of MARC1, PNPLA3, and MBOAT7 polymorphisms might help to stratify patients with AIH.

The progression of non-alcoholic fatty liver (NAFL) into non-alcoholic steatohepatitis implicates multiple mechanisms, chief of which is mitochondrial dysfunction. However, the sequence of events underlying mitochondrial failure are still poorly clarified. In this work, male C57BL/6J mice were fed with a high-fat plus high-sucrose diet for 16, 20, 22, and 24 weeks to induce NAFL. Up to the 20th week, an early mitochondrial remodeling with increased OXPHOS subunits levels and higher mitochondrial respiration occurred. Interestingly, a progressive loss of mitochondrial respiration along “Western diet” feeding was identified, accompanied by higher susceptibility to mitochondrial permeability transition pore opening. Importantly, our findings prove that mitochondrial alterations and subsequent impairment are independent of an excessive mitochondrial reactive oxygen species (ROS) generation, which was found to be progressively diminished along with disease progression. Instead, increased peroxisomal abundance and peroxisomal fatty acid oxidation-related pathway suggest that peroxisomes may contribute to hepatic ROS generation and oxidative damage, which may accelerate hepatic injury and disease progression. We show here for the first time the sequential events of mitochondrial alterations involved in non-alcoholic fatty liver disease (NAFLD) progression and demonstrate that mitochondrial ROS are not one of the first hits that cause NAFLD progression.

According to the ‘multiple‐hit’ hypothesis, several factors can act simultaneously in nonalcoholic fatty liver disease (NAFLD) progression. Increased nitro‐oxidative (nitroso‐oxidative) stress may be considered one of the main contributors involved in the development and risk of NAFLD progression to nonalcoholic steatohepatitis (NASH) characterized by inflammation and fibrosis. Moreover, it has been repeatedly postulated that mitochondrial abnormalities are closely related to the development and progression of liver steatosis and NAFLD pathogenesis. However, it is difficult to determine with certainty whether mitochondrial dysfunction or oxidative stress are primary events or a simple consequence of NAFLD development. On the one hand, increasing lipid accumulation in hepatocytes could cause a wide range of effects from mild to severe mitochondrial damage with a negative impact on cell fate. This can start the cascade of events, including an increase of cellular reactive nitrogen species (RNS) and reactive oxygen species (ROS) production that promotes disease progression from simple steatosis to more severe NAFLD stages. On the other hand, progressing mitochondrial bioenergetic catastrophe and oxidative stress manifestation could be considered accompanying events in the vast spectrum of abnormalities observed during the transition from NAFL to NASH and cirrhosis. This review updates our current understanding of NAFLD pathogenesis and clarifies whether mitochondrial dysfunction and ROS/RNS are culprits or bystanders of NAFLD progression.

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. In the current work, we used an exploratory data analysis to 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 up-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. In fact, 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.

OBJECTIVE A key event in atherogenesis is the formation of lipid-loaded macrophages, lipidotic cells, which exhibit irreversible accumulation of undigested modified low-density lipoproteins in lysosomes. This event culminates with the loss of cell homeostasis, inflammation and cell death. In this study we propose to identify the chemical etiological factors and understanding the molecular and cellular mechanisms responsible for the impairment of lysosome function in macrophages. APPROACH AND RESULTS Using shotgun lipidomics we have discovered that a family of oxidized lipids (cholesteryl hemiesters, ChE), end products of oxidation of polyunsaturated cholesteryl esters, occurs at higher concentrations in the plasma of two cohorts of cardiovascular disease patients than in the plasma of a control cohort. Macrophages exposed to the most prevalent ChE, cholesteryl hemiazelate (ChA) exhibit lysosome enlargement, peripheral lysosomal positioning, lysosome dysfunction and lipidosis which are irreversible. The transcriptomic profile of macrophages exposed to ChA indicates that the lysosome pathway is deeply affected and is well correlated with lysosome phenotypic and functional changes. Interestingly, the dysfunctional peripheral lysosomes are more prone to fuse with the plasma membrane, secreting their undigested luminal content into the extracellular milieu with potential consequences to the pathology. CONCLUSION We identify ChA not only as one of the molecules involved in the etiology of irreversible lysosome dysfunction culminating with lipidosis but also as a promoter of exocytosis of the dysfunctional lysosomes. The latter event is a new mechanism that may be important in the pathogenesis of atherosclerosis.

Nonalcoholic fatty liver disease (NAFLD) is characterized by the development of steatosis, which can ultimately compromise liver function. Mitochondria are key players in obesity-induced metabolic disorders; however, the distinct role of hypercaloric diet constituents in hepatic cellular oxidative stress and metabolism is unknown. Male mice were fed either a high-fat (HF) diet, a high-sucrose (HS) diet or a combined HF plus HS (HFHS) diet for 16 weeks. This study shows that hypercaloric diets caused steatosis; however, the HFHS diet induced severe fibrotic phenotype. At the mitochondrial level, lipidomic analysis showed an increased cardiolipin content for all tested diets. Despite this, no alterations were found in the coupling efficiency of oxidative phosphorylation and neither in mitochondrial fatty acid oxidation (FAO). Consistent with unchanged mitochondrial function, no alterations in mitochondrial-induced reactive oxygen species (ROS) and antioxidant capacity were found. In contrast, the HF and HS diets caused lipid peroxidation and provoked altered antioxidant enzyme levels/activities in liver tissue. Our work provides evidence that hepatic oxidative damage may be caused by augmented levels of peroxisomes and consequently higher peroxisomal FAO-induced ROS in the early NAFLD stage. Hepatic damage is also associated with autophagic flux impairment, which was demonstrated to be diet-type dependent. The HS diet induced a reduction in autophagosomal formation, while the HF diet reduced levels of cathepsins. The accumulation of damaged organelles could instigate hepatocyte injuries and NAFLD progression.

Nonalcoholic fatty liver disease (NAFLD) is a common disease in Western society and ranges from steatosis to steatohepatitis to end-stage liver disease such as cirrhosis and hepatocellular carcinoma. The molecular mechanisms that are involved in the progression of steatosis to more severe liver damage in patients are not fully understood. A deeper investigation of NAFLD pathogenesis is possible due to the many different animal models developed recently. In this review, we present a comparative overview of the most common dietary NAFLD rodent models with respect to their metabolic phenotype and morphological manifestation. Moreover, we describe similarities and controversies concerning the effect of NAFLD-inducing diets on mitochondria as well as mitochondria-derived oxidative stress in the progression of NAFLD.