TOFA (5-(tetradecycloxy)-2-furoic acid) was from Abcam (Toronto; ON, Canada). Sodium acetate was from Sigma-Aldrich (Oakville;ON, Canada). (3S, 5S)-atorvastatin sodium salt was from My BioSource (San Diego; CA, USA). Oleic acid-albumen, BSA and α-amanitin were from Sigma (Oakville;ON, Canada). Dulbecco’s Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), fetal bovine serum (FBS), L-glutamine (200 mM), penicillin/streptomycin (10,000 Units/mL and 10,000 μg/mL, respectively), and 0.5% trypsin-EDTA-10X were from Gibco Thermofisher Scientific (Ottawa; ON, Canada). Hu-LPDS was from Millipore (Temecula-California). Anti-ApoA-I and anti-mouse IgG HRP- linked antibodies were from Cell Signaling technology (CST). Anti-beta actin antibodies were from Novus Biologicals (Centennial; CO, USA). Protease inhibitor cocktail and PMSF were from Roche, ethanol 100% was from Greenfield, Inc. (Ontario, Canada), trypan blue was from Thermofisher Scientific (Ottawa; ON, Canada).
Human hepatocellular carcinoma cells (HepG2) were freshly obtained from the ATCC (Manassas, VA). Cells were cultured in 10-cm2 culture dishes containing 1 mL of culture medium per cm2. Unless stated otherwise the standard medium was Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), penicillin and streptomycin (10,000 units /mL and 10,000 μg /mL respectively). One week before the start of experiments, cells were split at a ratio of 1:6 and seeded into 6-well plates at a density of about 105 cells/well in 2 mL standard medium. The medium was replaced after 3 days.
Human colorectal adenocarcinoma cells, (CaCo-2) were kindly provided by Dr. Ali Ahmed. Cells were cultured in Eagle’s minimal essential medium (EMEM) containing 10% FBS, L-glutamine, and penicillin/streptomycin (10,000 units/mL and 10,000 μg/mL respectively). HepG2 and Caco-2 cells were maintained at 37 °C in a saturating humidity atmosphere containing 95% air and 5% CO2. At the start of the incubations the cells were grown to confluence.
Experiments with test compounds were carried out in DMEM plus 10% FBS or 3 mg/mL of human lipoprotein deficient serum (LPDS) from Millipore (Etobicoke, ON, Canada). In some experiments the medium was DMEM (Gibco), with 1000 mg glucose/L supplemented with penicillin/streptomycin, non-essential amino acids and bovine acid oleic-albumin (BAOA). Single compounds were added from concentrated stock solutions in water or DMSO as appropriate.
HepG2 and Caco-2 incubation with ethanol or sodium acetate
HepG2 or Caco-2 cells were cultured in 6-well plates and incubated for varying times in the presence of final media concentrations of 0, 10, 25, 50 or 100 mM ethanol. The culture medium in each plate was changed every 24 h in induction experiments of longer duration. For the experiments in which ApoA1 RNA was measured, HepG2 and Caco-2 cells were incubated with varying concentration of ethanol (0–500 mM) for 24 h. For the experiments in which ABCA1 protein was measured, HepG2 cells were incubated with ethanol (50 or 100 mM) for 24 h. For the acetate experiments, HepG2 cells were incubated with medium containing sodium acetate (0, 5 or 10 mM) for 24 h. Cell death was assessed using trypan blue exclusion and was minimal in all experiments except in the presence of 500 mM ethanol or 10 mM sodium acetate.
HepG2 incubation with ethanol plus α-amanitin, atorvastatin or TOFA
HepG2 cells of approximately 70–80% confluence were grown for 24 h in standard medium media prior to experiments. At the initiation of incubations, cells were washed twice with phosphate buffered saline (PBS). Then the experimental medium plus DMEM with 10% FBS, or 3 mg/mL of LPDS, was added, containing variously either α-amanitin (20 uM), atorvastatin (20 uM) or TOFA (20 uM) alone, or in combination with ethanol, at varying concentrations as indicated in the relevant figure legends. In some experiments the experimental medium was DMEM (Gibco), also containing 1000 mg glucose/L) supplemented with penicillin/streptomycin, non-essential amino acid and BSA (Sigma), and different concentrations of TOFA and ethanol, or TOFA together with ethanol were added. Atorvastatin sodium salt and TOFA stock solutions were made with DMSO as solvent. An equivalent volume of DMSO was always added to control cells. Unless stated otherwise, cells were incubated at 37 °C for 24 h.
ApoA1 and ABCA1 protein measurement
ApoA1 protein levels were measured by Western blot. Cells were lysed by RIPA buffer (25 mM Tris-HCl pH 7.5, 5 mM NaCl, 0.5 mM EDTA, 0.1% SDS, 1% Triton X-100 (BioShop; Burlington, ON; Canada), and a protease/PMSF inhibitor cocktail. Lysates were centrifuged at 14,000 × rpm for 10 min at 4 °C in a fixed angle rotor FA-45-24-11, and the supernatant was collected. For measuring ApoA1 protein in conditioned medium, 1 mL of medium was precipitated with trichloroacetic acid (TCA) according to the manufacturer’s protocol (Sigma). Protein concentration was measured with a Quick Start™ BSA kit (Bio-Rad; Mississauga, ON, Canada). Cell lysates and protein precipitates with TCA were dissolved for electrophoresis in Laemmli sample buffer and incubated for 5 min at 95 °C, pH adjusted as needed with Tris-HCl. Samples (30 μg of total protein per well) were fractionated by electrophoresis in a 10% (wt/vol) SDS-polyacrylamide gel and transferred to a nitrocellulose membrane 0.45 μm (Bio-Rad). The membrane was blocked for 1 h with 5% (wt/vol) skim milk powder in TBS-Tween (T-TBS, Tween from BioShop). Membranes were incubated overnight at 4 °C with monoclonal primary antibody (anti-ApoA1 at 1:1000 dilution, cat. SAB1410670; anti-ABCA1 at 1:2000 dilution, cat NB100–2068; or anti-B-Actin at 1:5000 dilution; cat NB600–501 (Novus Biologicals), in 2.5% (vol/vol) blocking solution in T-TBS. After three washes with T-TBS, detection was performed by using goat anti-rabbit IgG H&L (cat. no ab 6721; Abcam), goat anti-mouse IgG (cat HAF007; Novus Biologicals), or alpha-mouse (cat. No ab 7075; Abcam) at dilutions of 1:3000, 1:5000, or 1:5000 respectively. Quantification was by the enhanced chemiluminescence reagent system (Pierce ECL, Thermofisher Scientific), using CCD camera-based imaging (GE Healthcare; Bio Science). For measurements of cytoplasmic ApoA1 protein, results were normalized to beta-actin in each lane and analyzed by ImageJ software. Due to the very large difference in concentration of ApoA1 or ABCA1 and actin, the same blots were probed and photographed separately with antibodies against the two proteins. For measurements of secreted ApoA1 protein, equivalent volumes of cell culture medium were TCA-precipitated as there is no consensus on an internal standard for secreted protein samples.
Total RNA extraction and real time (RT)-PCR
At the indicated time after incubation with medium plus ethanol or other reagents, total cell RNA was extracted using TRIzol (Invitrogen,Carlsbad, CA), according to the manufacturer’s instructions. RNA was electrophoresed on a 2100 Bioanalyzer using a Nano RNA chip to verify its integrity. Total RNA was treated with DNase and reverse transcribed using the Maxima First Strand cDNA synthesis kit with dsDNase (Thermo Scientific). Primer Assay Design Centre (https://lifescience.roche.com/en_ca/brands/universal-probe-library.html#assay-design-centre) was applied to design the primers and probes of human APOA1, APOC3, APOA4, APOB, HMGCR, LDLR, SREBF2, TBP and GAPDH (the sequences of primers and probes are shown in Table 1. RNA was determined by quantitative RT-PCR (qPCR) using assays designed with the Universal Probe Library from Roche. For each qPCR assay, a standard curve was performed to validate the dynamic range of the assay. qPCR reactions were performed using Perfecta QPCR FastMIX II (Quanta), 2 μM of each primer and 1 μM of the corresponding UPL probe. The Viia7 qPCR instrument (Life Technologies) was used to detect the amplification level and was programmed with an initial step of 20 s at 95 °C, followed by 40 cycles of: 1 s at 95 °C and 20 s at 60 °C. Relative expression (RQ = 2-ΔΔCT) was calculated using the Expression Suite software (Life Technologies), and normalization was done using both GAPDH and TBP as internal control housekeeping genes. Formally, this analysis measures total not cytoplasmic mRNA for the assayed genes.
Measurement of lipid synthesis and secretion
Lipid synthesis and secretion were assayed as previously described [32, 33]. Briefly, radiolabeled [14C]-oleic acid (specific activity 59.0 mCi/mmol; PerkinElmer, Boston, US) was added to unlabeled oleic acid-BSA (Sigma). The final oleic acid concentration was 0.7 mM (0.45 μCi)/well. Cells were first washed with PBS, and the [14C]-oleic acid-containing medium was added to the upper compartment. At the end of a 24-h incubation period, cells were washed, and then scraped with a rubber policeman in RIPA buffer containing anti-proteases (pepstatin, leupeptin, PMSF all at a final concentration of 1 mM). An aliquot was taken for lipid extraction by standard methods in the presence of unlabeled carrier (phospholipids, triglyceride and cholesteryl esters).
The various lipid classes synthesized from [14C]-oleic acid were separated by thin-layer chromatography (TLC) using the solvent mixture of hexane, ether, and acetic acid (80:20:3, vol:vol:vol), as previously described [32, 33]. The area corresponding to each lipid was scraped off the TLC plates, and the silica powder was placed in a scintillation vial with Ecolite (+) liquid scintillation cocktail (MP Biomedicals, CA). Radioactivity was then measured by scintillation counting (Hidex 300 SL). Cell protein was quantified as described above, and results were expressed as disintegrations per min (dpm) per milligram of cell protein. Lipid secreted in the basolateral compartment was analyzed and quantified, as described above, after centrifugation (2000 rpm for 30 min at 4 °C) to remove cell debris.
Blood was drawn 3 h after the oral intake of a fatty meal by a human volunteer, and postprandial plasma was prepared to serve as a carrier for the lipoproteins synthesized by HepG2 cells. The TG-enriched plasma was incubated at 56 °C for 1 h to inactivate enzymatic activity in the presence of anti-proteases.
Isolation of lipoproteins
For the determination of secreted lipoproteins, HepG2 cells were incubated with the lipid substrate as described above. The medium supplemented with anti-proteases (as described above) was first mixed with a plasma lipid carrier (4:1, vol:vol) to efficiently isolate de novo lipoproteins synthesized. The lipoproteins were isolated by sequential ultracentrifugation using a TL-100 ultracentrifuge (Beckman). Briefly, chylomicrons were isolated after ultracentrifugation (25,000 rpm for 40 min). Very low-density lipoprotein (1.006 g/mL) and low-density lipoprotein (LDL, 1.063 g/mL) were separated at 90,000 rpm for 2 h and 47 min in a tabletop ultracentrifuge 100.4 rotor at 4 °C. The high-density lipoprotein fraction was obtained by adjusting the LDL infranatant to a density of 1.21 g/mL by adding Kbr followed by centrifugation for 7 h and 15 min at 90,000 rpm. Each lipoprotein fraction was exhaustively dialyzed against 0.15 M NaCl and 0.001 M EDTA, pH 7.0, at 4 °C for 24 h.
Statistical analyses and software
Results are presented as means ± standard deviation. All experiments were repeated at least three times or five times with independent biological replicates. For assessing cell vitality individual biological experiments were performed as duplicates, respectively. Group analyzes were performed using one-way two-way analysis of variance (ANOVA) by GraphPad Prism 6.04 (GraphPad, La Jolla, CA, USA). P-alues < 0.05 were considered statistically significant.