HepG2 cells (Banco de Células do Rio de Janeiro – BCRJ, code: 0103) were cultivated in Dulbecco's Modified Eagle Medium (DMEM; Gibco, Life-technologies, USA), containing fetal bovine serum (FBS, 10 %; Gibco, Life-technologies, USA) and 1 % penicillin/streptomycin antibiotics (ATB; Gibco, Life-technologies, USA). The cells were incubated in a humidified atmosphere at 37 °C with 5 % CO₂. The OG stock solutions were prepared in dimethylsulfoxide (DMSO; Neon, Brazil) and the dilution was prepared in DMEM containing 10 % FBS. Oleic acid (OA; Synth, Brazil) was used as a positive control to induce lipid droplet (LD) accumulation in HepG2 cells (Cui et al., 2010[6]). The OA stock solutions were prepared in isopropanol and the dilution was prepared in DMEM containing 10 % FBS. The control cells were maintained in DMEM, 10 % FBS and DMSO 0.2 % (vehicle). The treated cells received OG at 40 µM and the positive control cells received OA at 100 µM. The HepG2 cells were incubated and the analyses were performed 24 h after treatment.

HepG2 cells (12 x 10⁴ cells/well) were seeded into 24-well plates, cultured, and treated as aforementioned. Cellular morphological changes were evaluated after 24 h of treatment using a phase contrast inverted microscope (INV100, BEL Engineering, Italy) at 400× magnification and photographed using a Bel Photonics camera.

Cells were seeded as described for the morphological analysis and treated as aforementioned. After the treatment period, the culture medium was removed and HepG2 cells were washed twice with a phosphate-buffered saline (PBS) solution. Afterwards, HepG2 cells were fixed for 30 min with 4 % paraformaldehyde solution, then washed thrice with PBS, and subsequently rinsed with propylene glycol. Then, cells were stained with freshly prepared propylene glycol solution containing 0.5 % Oil Red (Sigma-Aldrich, USA), previously filtered with a 0.45 µm filter, for 15 min. Thus, cells were rinsed with 60 % propylene glycol and washed with distilled water. Images of cells with the stained lipid droplets were acquired using a phase contrast inverted microscope (INV100; BEL Engineering, Italy) at 400× magnification and photographed using a Bel Photonics camera.

After the Oil Red (Sigma-Aldrich, USA) stained lipid droplet images were acquired, 200 µL of isopropanol was added to each plate well for the lipid dissolution. Optical density (OD) was monitored with a VICTOR® microplate reader (PerkinElmer) at a wavelength of 490nm. Cell viability was evaluated using the Trypan blue (Sigma-Aldrich, USA) exclusion assay, as previously described (Lima et al., 2018[22]), in order to correct the optical density lipid reading by the number of cells. Results were expressed as Lipid OD/100,000 cells.

Cells were seeded into 60.8 cm² plates (5 x 10⁶ cells/well), cultured and treated as aforementioned. Then, HepG2 cells were transferred to 1.5 mL tubes and all samples were centrifuged at 3000rpm for 5min. The pellet was washed once using PBS, resuspended with 400 μL of PBS solution, and submitted to ultrasonication. The cellular triglyceride (TG) concentration was measured through TG Color assay kit (Wiener lab, Argentina). All measurements were normalized by protein concentration, following the protocol recommended by the manufacturer. Absorbance was measured at 505 nm using CT300i Chemistry Analyzer (Wiener lab, Argentina).

Initially, a trypsinization was performed for the collection of semi-confluent HepG2 cells. Then, the cells were prepared as previously described (Lima et al., 2018[22]). For the ultrastructural analysis in both control (DMSO 0.2 %) and treated cells (OG 40 µM), a transmission electron microscopy (FEI Tecnai T20 G2) and the Image Pro Plus software (Image Pro Plus 6.1, Media Cybernetics, Silver Spring, EUA) were used. All the analyses were performed by two blinded specialists in imaging analysis. The TEM images were used for morphological evaluation (lipid droplet density and area).

In order to estimate lipid droplet density, a quantitative analysis was performed. For that, randomized squares (3.0 μm²), named as areas of interest (AOIs), were overlaid on each image captured (4500×) and the lipid droplet located inside each square, as well as in the edges (upper and/or left), were counted. On the other hand, lipid droplet located in the lower and/or right edges of the squares were not counted (de Senna et al., 2017[8]). In average, 7 AOIs were evaluated in each cell and a total of 10 cells were evaluated per group.

The lipid droplet area was calculated using a stereological tool and the point counting method (Weibel 1979[32]; Zacharová and Kubínová, 1995[34]; Ilha et al., 2008[15]; Lazzarotto Rucatti et al., 2015[19]; Fernandes et al., 2016[13]). A grid mask containing an area/point value of 6518.5 nm² was placed over the lipid droplet image (13500×). Each point of the grid has a cross shape, thus delimiting four quadrants. The lipid droplets that were superimposed on the upper right quadrant of the grid points were counted. The areas were obtained through the following equation: Â = ∑p.a/p (Â is the area and ∑p is the sum of points counted, and a/p is the area/point value). In average, 5 lipid droplets were evaluated in each cell and a total of 6 cells were evaluated per group.

HepG2 cells (5 x 10⁵ cells/well) were seeded in 6-well plates, cultured and treated as aforementioned. The preparation of the cells was performed as previously described (Lima et al., 2018[22]). Protein samples were separated with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the nitrocellulose membrane. Next, the blot was blocked for 1 hour with Tris-buffered saline containing 0.05 % Tween-20 and 5 % non-fat dry milk. Then, the blot was incubated overnight at 4 °C with the following primary antibodies: mTOR (1:500) (2972, Cell Signaling) and GAPDH (1:1000) (39-8600, Invitrogen). After incubation with primary antibodies, the blot was incubated again for 1 hour with the secondary antibody conjugated with horseradish peroxidase (HRP) (1:1000). The chemiluminescence and digital images were taken in a Carestream Gel Logic 2200 PRO Imaging System. The protein concentration was measured using the NanoDrop Lite (Thermo Fisher Scientific®). GAPDH was used to normalize protein quantification (de Mesquita et al., 2017[7]).

HepG2 cells (2.5 x 10⁵ cells/well) were seeded in 6-well plates, cultured and treated as aforementioned. The RNA of all cells was extracted using TRIzol® Reagent (Invitrogen), according to the manufacturer’s recommendations. Then, RNA was converted to complementary deoxyribonucleic acid (cDNA) using Superscript III First-Strand Synthesis SuperMix (Invitrogen), according to the manufacturer’s instructions. Primer sequences used were synthesized by Integrated DNA Technologies (Iowa, USA) and the sequences are described in Table 1(Tab. 1). The quantity of cDNA was evaluated using the NanoDrop 2000 (Thermo Fisher Scientific). The gene expression of SREBP1c, PPAR-α, PPAR-γ, and β-actin were quantified using StepOneTM (Applied Biosystems). The reaction was catalyzed using the SYBR Green I (Applied Biosystems - Thermo Fisher Scientific), according to the manufacturer’s recommendation. The β-actin was used as an endogenous control gene.

The results from three independent experiments are presented as mean ± standard deviation (SD). The Student’s t-test was used for comparison between groups in the Ultrastructural analysis (TEM), Western blotting, and quantitative PCR assays. For the comparisons between groups in the Lipid Measurement and TG assays, a one-way analysis of variance (ANOVA), followed by Tukey post hoc test, was used. In all cases the significance level was set at p<0.05. Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA).

Treatment with OG induced vesicle accumulation in the cell cytoplasm, as observed in the phase contrast inverted microscope (Figure 1B(Fig. 1)). The positive control for the induction of lipid droplet accumulation (treatment with oleic acid) has also led to cytoplasmic vesicle accumulation, although with a greater intensity, as shown in Figure 1C(Fig. 1). Oil Red staining assay revealed that treatment with OG significantly increased lipid accumulation, as shown in Figure 1G(Fig. 1) (representative image shown in Figure 1E(Fig. 1)). As expected, the positive control increased lipid accumulation in a greater intensity (Figure 1F and Figure 1G(Fig. 1)). The results also showed an increase in the TG concentration after OG treatment (Figure 1H(Fig. 1)), corroborating the accumulation of lipids demonstrated in the Oil Red assay.

In order to confirm the effects of OG on lipid accumulation and to evaluate lipid droplet area and density, an ultrastructural analysis of HepG2 cells was performed. Representative images are shown in Figure 2A(Fig. 2) (control) and Figure 2B(Fig. 2) (40 µM OG). Transmission electron microscopy images revealed that treatment with 40 µM of OG increased the lipid droplet area (p<0.05; Figure 2C(Fig. 2)) without changing lipid droplet density (p=0.97; Figure 2D(Fig. 2)).

In order to investigate whether increased mammalian target of rapamycin (mTOR) expression could be related to lipid accumulation caused by OG, we have evaluated its expression using Western blot. However, results have shown that treatment with OG decreased mTOR expression (p<0.05; Figure 3A and B(Fig. 3)).

Therefore, the involvement of mTOR/ SREBP-1c signaling in lipid accumulation was investigated by measuring the SREBP-1c gene expression. Results have shown that treatment with OG increased SREBP-1c gene expression (p<0.05; Figure 3C(Fig. 3)), although a reduction in the mTOR protein expression was seen.

Finally, we have investigated whether the modification of PPAR-γ and PPAR-α gene expression could be related to lipid accumulation induced by OG, by evaluating the expression of its genes. Results have shown that OG increased PPAR-γ gene expression (p<0.05; Figure 3D(Fig. 3)) without modifying PPAR-α (Figure 3E(Fig. 3)).