Author(s):
Amorim, Ricardo ; Simões, Inês C. M. ; Veloso, Caroline ; Carvalho, Adriana ; Simões, Rui F. ; Pereira, Francisco B. ; Thiel, Theresa ; Normann, Andrea ; Morais, Catarina M. ; Jurado, Maria Amália da Silva ; Wieckowski, Mariusz R. ; Teixeira, José ; Oliveira, Paulo J.
Date: 2021
Persistent ID: https://hdl.handle.net/10316/105302
Origin: Estudo Geral - Universidade de Coimbra
Project/scholarship:
info:eu-repo/grantAgreement/other/722619/EU/other;
Subject(s): non-alcoholic fatty liver disease (NAFLD); in vitro cell model; Hepg2 cells; lipid accumulation; mitochondria dys(function); exploratory data analysis; Carcinoma, Hepatocellular; Cell Death; Data Analysis; Diet, High-Fat; Dietary Carbohydrates; Fatty Acids; Fatty Acids, Nonesterified; Fructose; Hep G2 Cells; Hepatocytes; Humans; Lipid Metabolism; Liver; Liver Neoplasms; Mitochondria; Non-alcoholic Fatty Liver Disease; Oxidative Stress; Palmitic Acid; Reactive Oxygen Species; Sugars
Description
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.
This work was funded by FEDER funds through the Operational Programme Competitiveness Factors-COMPETE and national funds by FCT-Foundation for Science and Technology under research grants PTDC/BTM-SAL/29297/2017, POCI-01-0145-FEDER-029297, PTDC/DTPFTO/2433/2014, POCI-01-0145-FEDER-016659, PTDC/BIA-MOL/28607/2017, POCI-01-0145-FEDER028607, POCI-01-0145-FEDER-006980, NORTE-01-0145-FEDER-000028, PTDC/ASP-HOR/29152/2017, POCI-01-0145-FEDER-029152 and UIDB/04539/2020. R. Amorim (SFRH/BD/131070/2017), J. Teixeira (NORTE-01-0145-FEDER-000028) are supported by FCT, POPH and QREN. MRW was supported by the National Science Centre, Poland (UMO-2018/29/B/NZ1/00589). I. Simões, M. Wieckowski, T.Thiel, A. Normann, and P. Oliveira gratefully acknowledge funding from the European Union’s Horizon 2020 Research and Innovation programme under the Marie Skłodowska-Curie Grant Agreement No. 722619 (FOIE GRAS) and Grant Agreement No. 734719 (mtFOIE GRAS).
