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

  • ️Fri Jan 01 2021

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

Ricardo Amorim et al. Nutrients. 2021.

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.

Keywords: Hepg2 cells; exploratory data analysis; in vitro cell model; lipid accumulation; mitochondria dys(function); non-alcoholic fatty liver disease (NAFLD).

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Conflict of interest statement

The authors state that they have no conflicts of interest. The funding agencies had no role in the decision to publish or in the contents of the manuscript.

Figures

Figure 1
Figure 1

Effect of supra-physiological concentrations of FA on the accumulation of lipid and mitochondrial phospholipids content. (A) Lipid droplet content in HepG2 cells treated with palmitic acid (PA, 0.5 mM) or a mix of free fatty acids (FFA, 0.25 mM) in the presence or absence of fructose (F, 10 mM) for 1, 6, and 24 h. (B) A typical chromatogram showing the mitochondrial phospholipids profile (CL, cardiolipin; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PC, phosphatidylcholine; SM, sphingomyelin; LPC, lysophosphatidylcholine) of HepG2 cells treated with PA or FFA in the presence or absence of F for 24 h. This image was inverted and contrast-optimized for visualization purposes. Quantification of the bands was performed using the original images. (C) Quantification of phospholipid content (CL, PE, PI, PC, SM, and LPC) in multiple experiments. (D) PC/PE ratios obtained in all conditions. Data are the mean ± SEM of four independent experiments, and the results normalized on the control condition (CTL = 100%, marked by a dotted line). Significance was accepted with * p < 0.05, ** p < 0.01, *** p < 0.0005, **** p < 0.0001 for comparations between treatment vs. CTL (BSA 0.01 g/mL) and # p < 0.05, ## p < 0.01, ### p < 0.0005 for comparations during time in the same group (24 and 6 h vs. 1 h).

Figure 2
Figure 2

Time-dependent effect of supra-physiological concentrations of FA on the levels of CM-H2DCFDA-oxidizing ROS. Average cellular CM-H2DCFDA oxidation signal in cells treated with palmitic acid (PA, 0.5 mM) or a mix of free fatty acids (FFA, 0.25 mM) in the presence or absence of fructose (F, 10 mM) for 1, 6 and 24 h. Data are the mean ± SEM of four independent experiments, and the results normalized on the control condition (CTL = 100%, marked by a dotted line). Significance was accepted with * p < 0.05, ** p < 0.01, **** p < 0.0001 for comparisons between treatment vs. CTL (BSA 0.01 g/mL) and ## p < 0.01, ### p < 0.0005 for comparisons during time in the same group (24 and 6 h vs. 1 h). Significance for additional fructose effect as accepted with $$$ p < 0.0005.

Figure 3
Figure 3

Effect of supra-physiological concentrations of FA on mitochondrial morphology and mtDNA copy number. (A) Typical background-corrected (COR) image of HepG2 cells stained with the fluorescent cation TMRM and Hoechst 33,342 after treatment with palmitic acid (PA, 0.5 mM) or a mix of free fatty acids (FFA, 0.25 mM) in the presence or absence of fructose (F, 10 mM) for 6 and 24 h. The TMRM and Hoechst fluorescence intensity was color-coded to red and blue, respectively. (B) Average mitochondrial TMRM fluorescence intensity calculated from the images. (C) Index of mitochondrial interconnectivity calculated from the images (D) mtDNA copy number in HepG2 cells treated with PA or FFA in the presence or absence F for 6 and 24 h. mtDNA copy number was based on the amplification of cytochrome B (encoded on the mitochondrial genome) and β-2-microglobulin (encoded on the nuclear genome) ratio. Data are the mean ± SEM of three independent experiments, and the results normalized on the control condition (CTL = 100%, marked by a dotted line). Significance was accepted with * p < 0.05, ** p < 0.01, *** p < 0.0005 for comparisons between treatment vs. CTL (BSA 0.01 g/mL) and ## p < 0.01 for comparisons during time in the same group (24 and 6 h vs. 1 h).

Figure 4
Figure 4

Effect of supra-physiological concentrations of FA on mitochondrial OXPHOS protein levels. (A) Typical Western blot result of whole cell homogenates showing the protein level of NDUFB8 (complex I), SDHB (complex II), UQCRC2 (complex III), COXII (complex IV), ATP5A (complex V) subunits, and β-actin (cytosolic marker) in cells treated with palmitic acid (PA, 0.5 mM) or a mix of free fatty acids (FFA, 0.25 mM) in the presence or absence of fructose (F, 10 mM) for 6 and 24 h. This blot was inverted and contrast-optimized for visualization purposes. Quantification of the bands was performed using the original blots. (B) Quantification of OXPHOS proteins levels in multiple experiments normalized to β-actin levels and for the control group (100% marked by a dotted line). (C) Typical BN-PAGE in-gel activity result of mitochondrial-enriched fraction homogenates depicting the protein activity of mitochondrial complex I (NADH:ubiquinone oxidoreductase) in cells treated with palmitic acid (PA; 0.5 mM) or a mix of free fatty acids (FFA; 0.25 mM) in the presence or absence of fructose (F; 10 mM) for 6 and 24h. This image was inverted and contrast-optimized for visualization purposes. Quantification of the bands was performed using the original images. Data are the mean ± SEM of four independent experiments, and the results normalized on the control condition (CTL = 100%, marked by a dotted line). Significance was accepted with * p < 0.05, ** p <0.01, *** p < 0.0005 for comparisons between treatment vs. CTL (BSA 0.01 g/mL) and # p < 0.05, ## p < 0.01 for comparisons during time in the same group (24 and 6 h vs. 1 h). Significance for additional fructose effect as accepted with $ p < 0.05, $$ p < 0.01, $$$ p < 0.0005.

Figure 5
Figure 5

Time-dependent effect of supra-physiological concentration of FA on mitochondrial oxygen consumption. (A) Typical representation of oxygen consumption rate (OCR) measurement in HepG2 cells treated with palmitic acid (PA, 0.5 mM) or a mix of free fatty acids (FFA, 0.25 mM) in the presence or absence of fructose (F, 10 mM) for 1, 6 and 24 h. Several respiratory parameters were evaluated: (B) cellular basal respiration; (C) maximal respiration; (D) ATP production-linked respiration; and (E) proton leak. Data are mean ± SEM (expressed as pmol O2/min/cell mass) of four independent experiments, and the results normalized on the control condition (CT = 100%, marked by a dotted line. The data obtained for the different treatments was compared with the control group. Significance was accepted with * p < 0.05, ** p < 0.01, *** p < 0.0005, **** p < 0.0001 for comparisons between treatment vs. CTL (BSA 0.01 g/mL) and # p < 0.05, ## p < 0.01, ### p < 0.0005, #### p < 0.0001 for comparisons during time in the same group (24 and 6 h vs. 1 h). Significance for additional fructose effect as accepted with $ p < 0.05. Legend: FCCP—carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, ROT—rotenone, AA—antimycin A.

Figure 6
Figure 6

Time-dependent effect of fatty acid excess on caspase activity and cell viability. (A) Caspase 8-like activity in HepG2 cells treated with palmitic acid (PA, 0.5 mM) or a mix of free fatty acids (FFA, 0.25 mM) in the presence or absence of fructose (F, 10 mM) for 6 and 24 h. (B) Same as panel A but now for caspase 9-like activity. (C) Same as panel A but now for caspase 3/7. (D) Cell viability of HepG2 cells cultured in the presence of palmitic acid (PA, 0.5 mM) or a mix of free fatty acids (FFA, 0.25 mM) in the presence or absence of fructose (F, 10 mM) for 6 and 24 h. Data are the mean ± SEM of four independent experiments, and the results normalized on the control condition (CT = 100%, marked by a dotted line). Significance was accepted with * p < 0.05, ** p < 0.01, *** p < 0.0005, **** p < 0.0001 for comparisons between treatment vs. CTL (BSA 0.01 g/mL) and ## p < 0.01, ### p < 0.0005, ####p < 0.0001 for comparisons during time in the same group (24 and 6 h vs. 1 h). Significance for additional fructose effect as accepted with $ p < 0.05, $$ p < 0.01.

Figure 7
Figure 7

Computational data analysis of all experimental endpoint measures analyzed in different lipotoxicity models on human hepatocytes. (A) Correlation matrices of HepG2 cells treated with palmitic acid (PA, 0.5 mM) or a mix of free fatty acids (FFA, 0.25 mM) in the presence or absence of fructose (F, 10 mM). (B) Mutual information gain of each individual experimental endpoint (24 h), regarding the existence of 3 or 6 experimental groups. (C) K-means clustering results, for k = 3, using a subset of selected experimental endpoints.

Figure 8
Figure 8

Proposed mechanism for fatty acid overload and mitochondrial dysfunction on human hepatocytes. FA overload are toxic and trigger a time-dependent caspase apoptotic cell death. The progressive increase in neutral lipids content and phospholipid modifications in mitochondria, followed by a large increase in ROS levels compromise mitochondrial function (decrease in ΔΨm, O2 consumption, and OXPHOS protein levels). Mitochondrial impairment can potentiate even more ROS production. ROS itself, or in conjugation with mitochondrial dysfunction, can lead to activation of caspase-dependent apoptotic cell death pathways. Thus, fatty acid overload led to a time-dependent decrease in cell viability.

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