Exploratory Data Analysis of Cell and Mitochondrial High-Fat, High-Sugar Toxicity on Human HepG2 Cells.

Ricardo Amorim,Rui Simões,Inês Simões,Amália Jurado,Andrea Normann,José Teixeira,Paulo Oliveira,Caroline Veloso,Catarina Morais,Adriana Carvalho,Mariusz Wieckowski,Theresa Thiel,Francisco Pereira

doi:10.3390/nu13051723

Abstract

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.

Highlights

Non-alcoholic fatty liver disease (NAFLD) is a worldwide public health concern
These results demonstrate that the increase in intracellular lipid content is mainly due to the fatty acids (FA) added, as the presence of sugar has a residual role on the different degrees of hepatic steatosis
Our work showed that palmitic acid (PA) or PA + F treatments resulted in mitochondrial network fragmentation and decreased mitochondrial membrane potential (∆Ψm) The data suggests that the free fatty acids mixture (FFA)-treatment regimens and PA-treatment led to different oxidative damage status, being the later more severe, and mitochondrial dysfunction, probably due to alterations in mitochondrial structure and integrity

Summary

Introduction

Non-alcoholic fatty liver disease (NAFLD) is a worldwide public health concern. NAFLD can progress from simple steatosis to non-alcoholic steatohepatitis (NASH), a pathological stage characterized by inflammation and hepatocellular ballooning. Further progression of this condition can result in a fibrotic phenotype (cirrhosis) and, in hepatocellular carcinoma (HCC). (more than 5%) in the absence of excessive alcohol consumption [2] or other liver disease etiology. Mitochondria are responsible, among other functions, for the oxidation of short and median chain fatty acids through β-oxidation. The deleterious sequence of cellular events includes fatty acid and/or carbohydrates overload followed by an increased reactive oxygen species (ROS)

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