Effect of intracellular lipid accumulation in a new model of non-alcoholic fatty liver disease

Cell culture medium Dulbecco's modified Eagle's high glucose medium (DMEM) (ECB7501L), L- Glutamine (ECB3000D), Penicillin/Streptomycin (ECB3001D), were obtained from Euro-clone (Milan, Italy). Bicinconinc acid solution-kit (B9643); bovine albumin Cohn V fraction (A4503); dimethyl sulphoxide (DMSO) (D2438); fetal bovine serum (F7524); Hoechst 33258 (B1155); hydrogen peroxide (H1009); 3-(4,5 dimethylthiazol-yl-)-2,5-dipheniltetrazoliumbromide (MTT) (M2128); N-acetyl-L-cysteine (NAC) (A9165); Nile Red (N3013); oleic acid (C18:1) (O1008); palmitic acid (C16:0) (P0500); paraformaldehyde (P6148), phosphate-buffered saline (PBS) (D5652); propidium iodide (PI)(P4170) and Tri-Reagent (T9424) were from Sigma Chemical (St.Louis, MO, USA). iScript^™ cDNA Synthesis kit (170-8890) and iQ SYBR Green Supermix (170-8860) were purchased from Bio-Rad Laboratories (Hercules, CA, USA). 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) (D399) was obtained from Molecular Probes (Milan, Italy). Human annexin V-FITC Kit (BMS306FICE); Instant ELISA kit interleukin (IL)-6, IL-8 and tumor necrosis factor (TNF)-alpha (BMS213INSTCE; BMS204/3INSTCE; BMS223INSTCE respectively) were purchased to Bender MedSystems GmbH (Vienna, Austria)

Hepatoma derived cell line HuH7 (JHSRRB, Cat #JCRB0403) were obtained from the Health Science Research Resources Bank (Osaka, Japan). Cells were grown in DMEM high glucose medium supplemented with 10% v/v fetal bovine serum, 2 mM L-Glutamine, 10,000 U/mL penicillin and 10 mg/mL streptomycine at 37°C under 5% CO₂, in a 95% humidified atmosphere. For the treatment, palmitic and oleic acid 0.1 M stock solutions were prepare by dissolving FFA in DMSO. The cells were exposed for 24 h to increasing concentrations of a fresh mixture of exogenous FFA (100; 200; 400; 600 and 1200 μM) in molar ratio 1:2 palmitic:oleic respectively. Since albumin concentration is an important factor in determining the concentration of available FFA, the FFA were complexed with bovine serum albumin at a 4:1 molar ratio taking into consideration the albumin concentration already present in the medium due to the FBS supplementation. The experimental doses were determined in advance by performing dose curves and by assessing cell viability which was always higher than 85% as compared to control cells (100% viability) treated with the equivalent concentration v/v of the vehicle (DMSO).

Cell viability was assessed using the MTT colorimetric assay. When taken up by living cells, MTT is converted from a yellow to a water insoluble blue-colored precipitate by cellular dehydrogenases [6]. Briefly, 4 × 10⁴ cells/cm² were plated in 24 well dish and allowed to adhere overnight. The following day the cells were treated as described above. After 24 h of treatment, the medium was removed and the treatment was followed by addition of 0.5 mg/mL of MTT and incubation at 37°C for 1 h. The medium was then removed, the cells were lysed and the resulting blue formazan crystals were solved in DMSO. The absorbance of each well was read on a microplate reader (Beckman Coulter LD 400 C Luminescence detector) at 570 nm. The absorbance of the untreated controls was taken as 100% survival. Data are expressed as mean ± SD of three independent experiments.

Intracellular fat content was determined fluorimetrically based on Nile Red staining, a vital lipophilic dye used to label fat accumulation in the cytosol [7, 8]. After 24 h of FFA exposure, adherent monolayer cells were washed twice with PBS and detached by tripsinization. After a 5 min centrifugation at 1500 rpm, the cell pellet was resuspended in 3 mL of PBS and incubated with 0.75 μg/mL Nile red dye for 15 min at room temperature.

Nile red intracellular fluorescence was determined by flow cytofluorometry using a Becton Dickinson FACSCalibur System on the FL2 emission channel through a 585 ± 21 nm band pass filter, following excitation with an argon ion laser source at 488 nm [9]. Data were collected in 10,000 cells and analyzed using Cellquest software from BD Biosciences (San Jose, CA, USA).

HuH7 cells were seeded in a coverslip and exposed to FFA for 24 h as previously described. Cells were then washed with PBS twice, and fixed with 3% paraformaldehide for 15 min. Intracellular neutral lipids were stained with Nile Red (3.3 μg/mL) for 15 min. Cell nuclei were stained with Hoescht 33258 dye for 15 min. All the staining procedure was carried out at room temperature by protecting the samples from the direct light. Images were acquired with an inverted fluorescence microscope (Leica DM2000, Wetzlar Germany)

After the treatment, the medium was removed, cells were washed twice with PBS and after centrifugation, total RNA isolated using Tri-Reagent kit according to manufacturer's instructions. Briefly, cells were lysed with the reagent, chloroform was added and cellular RNA was precipitated by isopropyl alcohol. After washing with 75% ethanol, the RNA pellet was dissolved in nuclease-free water and stored at -80°C until further analysis. RNA was quantified spectrophotometrically at 260 nm in a Beckman Coulter DU^®730 spectrophotometer (Fullertone, CA, USA). The RNA purity was evaluated by measuring the ratio A260/A280, considering RNA with appropriate purity those showing values between 1.8 and 2.0; its integrity was evaluated by gel electrophoresis. The integrity of RNA was assessed on standard 1% agarose/formaldehyde gel. Isolated RNA was resuspended in RNAse free water and stored at -80°C until analysis. Total RNA (1 μg) was reverse transcribed using iScript^™ cDNA Synthesis kit BioRad according to manufacturer's instructions. Retrotranscription was performed in a Thermal Cycler (Gene Amp PCR System 2400, Perkin Elmer, Boston, MA, USA) in agreement with the reaction protocol proposed by the manufacturer's: 5 min at 25°C (annealing), 45 min at 42°C (cDNA synthesis), and 5 min at 85°C (enzyme denaturation).

Real Time quantitative PCR was performed in i-Cycler IQ; 18S and β-actin were used as housekeeping genes. All primer pairs were synthesized by Sigma Genosys Ltd. (London Road, UK) and were designed using the software Beacon Designer 7.51 (PREMIER Biosoft International, Palo Alto, CA, USA). Primer sequences and references are specified in Table 1. PCR amplification was carried out in 25 μL reaction volume containing 25 ng of cDNA, 1x iQ SYBR Green Supermix [100 mM KCl; 40 mM Tris-HCl, pH 8.4; 0.4 mM each dNTP; 50 U/mL iTaq DNA polymerase; 6 mM MgCl2; SYBR Green I; 20 nM fluorescein; and stabilizers] and 250 nM gene specific sense and anti-sense primers and 100 nM primers for 18S. Standard curves using a "calibrator" cDNA (chosen among the cDNA samples) were prepared for each target and reference gene. In order to verify the specificity of the amplification, a melt-curve analysis was performed, immediately after the amplification protocol. Non-specific products of PCR were not found in any case. The relative quantification was made using the Pfaffl modification of the ΔΔCt equation, taking into account the efficiencies of individual genes. The results were normalized to 18S and β-actin, the initial amount of the template of each sample was determined as relative expression versus one of the samples chosen as reference (in this case the control sample) which is considered the 1x sample. Results reported are the mean ± SD expression of at least 3 different determinations for each gene.

The cytokine released in the culture media was determined by Instant ELISA (Bender MedSystems GmbH; Vienna Austria) according to manufacturer's instructions. Cells were cultured and treated as previously described. The culture media was collected after 24 h, possible contamination of cellular fractions was eliminated by centrifugation and the test performed in cell-free supernatant. According to the manufacturer the lowest detection limits were: 0.92 pg/mL for IL-6, 1.3 pg/mL for IL-8 and 1.65 pg/mL for TNF-alpha.

Intracellular ROS generation was measured by the use of the cell permeable fluorogenic substrate H₂DCFDA. This non fluorescent probe is easily taken up by cells and, after intracellular cleavage of the acetyl groups, is trapped and may be oxidized to the fluorescent compound 2',7'-dichlorofluorescin (DCF; the monitored fluorophore) by intracellular ROS [10]. Cells were exposed to FFA for 24 h and after the treatment, adherent cells were washed with PBS, and loaded with 10 μM H₂DCFDA for 15 min at 37°C. The fluorescence was measured by using a Spectrofluorometer Jasco FP-770 (Excitation wavelength 505 nm and Emission 525 nm). Fluorescence was normalized by the μg of protein assessed by bicinconinic acid protein assay [11] and expressed as AU/μg of protein. The intracellular ROS production was also assessed by flow cytometric analysis by using Becton Dickinson FACSCalibur System. Briefly, attached cells were harvested by tripsinization, washed in PBS by centrifugation, and incubated with 5 μM H₂DCFDA for 30 min at 37°C in PBS, to exclude hydrogen peroxide generation in phenol red containing media. The cells were washed in PBS and the pellet was suspended in 500 μL of PBS. PI staining was performed to assess non viable cells. Cells within a negative control gate for PI fluorescence were back-gated (FL2) and the fluorescence histogram for H₂DCFDA (FL1) was applied for the living cells [12]. Considering the potential anti-oxidant effect of albumin [13], the albumin concentration was kept the same in the controls and in the treated samples. Cells exposed to 400 μM hydrogen peroxide were considered as positive control. In order establish if the ROS production induced by FFA could be reversed by an antioxidant agent, cells were co-treated with FFA and 300 μM n-acetylcysteine (NAC). The optimal NAC concentration was established by performing dose curve analysis by MTT, at the used concentration cell viability was unaltered (data not shown). Data were collected for 10,000 cells and analyzed using CellQuest software from BD Biosciences (San Jose, CA).

During apoptosis, phosphatidyl serine is exposed from the inner to the outer portion of the membrane and becomes available to bind to the Annexin-V/FITC conjugate. Accordingly, only cells prone to apoptosis will stain positive for Annexin-V/FITC [14]. The cells were harvested by gentle tripsinization which was inactivated by adding medium with 10% FBS. Cells were washed, the pellet suspended in the incubation buffer (10 mM Hepes/NAOH, pH 7.40, 140 mM NaCl, 5 mM CaCl₂) adjusting cell density to 5 × 10⁶ cell/mL, and 1 × 10⁶ cells were incubated with 5 μL Annexin V-FITC and 5 μL PI for 10 min at room temperature. The cells were diluted with the incubation buffer to a final volume of 600 μL and analyzed by flow cytometer using 488 nm excitation and 515 nm band-pass filter for fluorescein detection (FL1) and a filter > 600 nm for PI detection (FL2). Data were collected for 10,000 cells and analyzed using CellQuest software from BD Biosciences (San Jose, CA).

The addition of increasing concentrations of FFA (palmitic/oleic 1:2 molar ratio) did not affect the cell viability measured by MTT Test (data not shown). To assess the effect of the vehicle in which FFA were solubilized, cells were also treated with the equivalent concentration of DMSO of each FFA concentration. In both cases viability was not significantly reduced (10-15%) after 24-h incubation even at the DMSO concentration used with the highest FFA concentrations used (1200 μm).

The content of intracellular lipid droplets was determined by Nile Red staining. Cell exposure to 600 and 1200 μM FFA for 24 h induced a dose-dependent fat accumulation. Microscopic images (Figure 1A) showed the presence of cytoplasmic lipid droplets. This pattern was particularly evident after the exposure at 1200 μM FFA where the quantity and size of the droplets increased as compared with that seen with the lower dose. This qualitative data was confirmed by flow cytometry where the presence of cytoplasmatic lipids induced a shift in the median of the fluorescence peak (Figure 1B). After the treatment with FFA, a clear increase of events was observed both in M1 and M2 regions, where M1 represents the percentage of events above the maximum value and M2 the percentage of events above the median value of fluorescence. The total mean fluorescence shows a dose dependent accumulation of FFA (Table 2).

The role of inflammatory cytokines in the response of liver cells to several injuries has been described [15]. Figure 2 shows the gene expression of IL-6, IL-8 and TNF-alpha after 24 h of FFA-exposure. All the 3 cytokines were significantly over-expressed as compared to cells treated with the vehicle. However while IL-6 showed a 3-fold increase with no difference between the two doses used, IL-8 showed a clear dose-dependency with a 3-fold difference moving from 600 μM to 1200 μM. Conversely, no increment was observed for TNF-alpha at 600 μM while exposure to 1200 μM resulted in a 5-fold up-regulation in the gene expression.

To investigate if the up-regulation in the mRNA expression was associated to an increased cytokine cell production, the cytokine release into the culture media was also assessed. In line with the data obtained in the mRNA expression (Figure 2), the production of IL-8 was increased in a dose-dependent fashion at both 600 μM (1484 ± 14 vs. 900 ± 246 pg/mL, p < 0.05) and 1200 μM (2613 ± 354 vs. 900 ± 246 pg/mL, p < 0.05) of FFA (Figure 3). On the contrary and differently from the gene expression, no significant changes were observed for TNF-alpha at any of the experimental concentrations. In line with previous studies [16], IL-6 level was lower than the sensitivity of the method.

The involvement of hepatocytes on fibrogenesis has been previously described, and the first phase of fibrosis seems to be mediated by many cytokines [2, 16]. To analyze the effect of FFA in our in vitro model, a series of cytokines reported as marker of fibrosis in NAFLD [17] and in other liver diseases [18] were assessed. As shown in Figure 4, the intracellular lipid accumulation was associated with a significant up-regulation in TGFβ1, A2M, VEGFA, CTGF, THBS and IGF-2 mRNA. Among these cytokines, the highest increment in gene expression was observed for TGFβ1 (1.99 ± 0.23 and 2.33 ± 0.19 folds of expression vs. control for 600 and 1200 μM, respectively). The same pattern of expression was also observed for all the other cytokines with the exception of a non significant increase for A2M and IGF2 at the lower FFA concentration.

Cells treated with 1200 μM FFA showed a 1.38 ± 0.12 (p < 0.05) folds of increase in ROS generation vs. untreated control (Additional file 1: Figure S1 upper panel). The ROS generation was similar to that observed in positive control cells exposed to 400 μM hydrogen peroxide for 3 h (1.48 ± 0.22 folds of increase, p < 0.05). When cells treated wither with FFA or hydrogen peroxide were concomitantly exposed to 300 μM of NAC, the ROS level decreased to 1.22 ± 0.12 and 1.07 ± 0.16 (P = NS) fold respectively, indicating that the addition of NAC was able to reverse the alteration in redox state induced by both FFA treatment and hydrogen peroxide. The increment in ROS in FFA treated cells was also confirmed by flow cytometry analysis (333 vs.375 AU respectively) (Additional file 2: Figure S2 lower panel).

HuH7 cells were stained with Annexin V and PI to detect cells with disrupted membrane. According to the cell staining, cells were categorized as: 1) live cells negative for both annexin V-FITC and PI; 2) early apoptotic cells, positive for annexin V-FITC and negative for PI; and 3) late apoptotic cells, positive both for annexin-V-FITCH and PI; cells positive only for PI were considered as necrotic. As shown in Figure 5A 1200 μM of FFA induce an increase in the percentage of cells in early apoptosis (6.2%), late apoptosis (2.1%) and a 4.23% increase in the necrotic population (Figure 5B).