Elaidic acid (EA) generates dysfunctional high-density lipoproteins and consumption of EA exacerbates hyperlipidemia and fatty liver change in zebrafish

2.1 Materials

SA (18:0, cat no. S4751), OA (18:1, cis-9-Octadecenoic acid, O1008), and EA (18:1 trans-9-Octadecenoic acid, E4637) were purchased from Sigma (St. Louis, MO, USA).

2.2 Purification of plasma apoA-I

ApoA-I was purified from human plasma using ultracentrifugation and column chromatography following the method described previously 19.

2.3 Synthesis of rHDL containing FA

Discoidal rHDL containing the FA was prepared by the sodium cholate dialysis method 20, 21 using initial molar ratios of palmitoyloleoyl phosphatidylcholine (POPC):free cholesterol:apoA-I:FA:sodium cholate of 95:5:1:x:150, where x represents 1, 5, or 10. For example, the SA-rHDL (1:10) indicates the molar ratio of 95:5:1:10:150 for POPC:free cholesterol:apoA-I:SA:sodium cholate. The rHDL particles were used without further purification because they showed high homogeneity. Their sizes were determined using 8–25% native polyacrylamide gradient gel electrophoresis (PAGGE; Pharmacia Phast system, GE Healthcare, Uppsala, Sweden) to compare them with standard globular proteins (AmershamPharmacia, Uppsala, Sweden). Particle sizes of rHDLs were determined by native 8–25% PAGGE comparison with standard globular proteins (cat no. 17–0445–01, AmershamPharmacia). The relative migrations were compared via densitometric scanning analysis using a Gel Doc® XR (Bio-Rad, Hercules, CA, USA) with Quantity One software, version 4.5.2. The number of apoA-I molecules per rHDL particle, as well as the self-association properties of lipid-free proteins, was determined by cross-linking with BS₃ using the method described previously 22 and then analyzing the products of the reaction by SDS-PAGGE on precasted 8–25% gradient gels (AmershamPharmacia).

2.4 Circular dichroism

The average α-helix content of proteins in lipid-free and lipid-bound states was measured by circular dichroism spectroscopy using a J-715 Spectropolarimeter (Jasco, Tokyo, Japan) as described in our previous report 16 and the α-helical content was calculated from the molar ellipticity at 222 nm 23.

2.5 Characterization of tryptophan fluorescence

The wavelengths of the maximum fluorescence of tryptophan residues in apoA-I protein was determined from uncorrected spectra obtained using an LS55 spectrofluorometer (Perkin-Elmer, Norwalk, CT, USA) in conjunction with the WinLab software package 4.00 (Perkin-Elmer) and a 1-cm path-length Suprasil quartz cuvette (Fisher Scientific, Pittsburgh, PA, USA) as in our previous report 16.

2.6 Inhibition of LDL oxidation

Fresh LDL was incubated with lipid-free or rHDL-associated apoA-I at a concentration of 2 μM in the presence of 10 μM CuSO₄ for up to 3 h. During incubation, the quantity of conjugated dienes formed was monitored by following the absorbance at 234 nm (Abs₂₃₄) and 37°C 24 using a Beckman DU 800 spectrophotometer (Fullerton, CA, USA) equipped with a MultiTemp III thermocirculator (Amersham, Uppsala, Sweden).

2.7 Acetylation of LDL

The acetylation of LDL (acLDL) was performed using saturated sodium acetate and acetic anhydride according to a previously described method 25. After acetylation and subsequent dialysis, the acLDL protein content was determined and filtered through a 0.22-μm filter (Millex; Millipore, Bedford, MA, USA). To visualize phagocytosis of acLDL, a fluorescent cholesterol derivative (22-(N-7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-23,24-bisnor-5-cholen-3beta-ol (NBD-cholesterol), Molecular Probes N-1148; 70 μg of NBD-cholesterol/mg of LDL) was added to acLDL particles.

2.8 Cholesteryl ester transfer assay

An rHDL containing apoA-I and cholesteryl oleate was synthesized and the CETP reaction was measured as previously described method 26 using trace amounts of [³H]-cholesteryl oleate (TRK886, 3.5 μCi/mg of apoA-I; GE Healthcare).

2.9 LDL phagocytosis assay

Differentiated and adherent macrophages from THP-1 cells were incubated with 400 μL of fresh RPMI-1640 medium containing 1% FBS, 50 μL of the acetylated LDL (acLDL, 1 mg of protein/mL in PBS), and 50 μL of PBS or each rHDL (final 2 μM of apoA-I in PBS) for 48 h at 37°C in a humidified incubator. In prior to treatment, apoA-I protein concentration in the rHDL and lipid-free states was determined by Lowry–Markwell assay 27 and our group 28 with BSA as a standard. The uptaken LDL was visualized and THP-1 macrophage-derived foam cells were then observed and photographed using a Nikon Eclipse TE2000 microscope (Tokyo, Japan). The NBD fluorescence (Ex = 485 nm, Em = 535 nm) and dihydroethidium (DHE) fluorescence (Ex = 485 nm, Em = 616 nm) were quantified by Victor X4 multimode plate reader (Perkin-Elmer Inc., Waltham, MA, USA).

2.10 Cellular senescence assay

Primary human dermal fibroblasts (HDFs) were cultured in DMEM (Life Technologies, Gaithersburg, MD, USA). HDFs were plated in DMEM at 1 × 10⁵ cells per 100-mm culture plate and cultured at 37°C in a 5% CO₂ humidified incubator as described in our previous report 17. HDFs were exposed at passage 11 (approximately 40% confluence) to the indicated concentrations of trans-fat (0.1, 1, 10, or 100 μM) for 30 days with subculture to passage 18. The extent of aging and cellular SA-β-gal activity was compared as previously described 29 by comparing intensity and area of stained extent.

2.11 Western blotting

In order to compare delivery efficiency and stability of each rHDL containing apoA-I and FA, equal amounts (22 μg of protein) of cell lysate were loaded and electrophoresed on 15% SDS-PAGE gels and detected by anti-human full-length apoA-I goat antibody (ab7613; Abcam, Cambridge, UK) and donkey anti-goat IgG-horseradish peroxidase (SC2020; Santa Cruz Biotechnology, Santa Cruz, CA, USA) as secondary antibody (1:2000 diluted). Anti-human GAPDH antibody (AAS79585C; Antibody Verify, Las Vegas, NV, USA) and secondary antibody was goat anti-rabbit IgG-horseradish peroxidase (SC2004; Santa Cruz, Dallas, TX, USA).

In order to normalize the expressional level, anti GAPDH antibody was employed as internal standard. The same amount of protein (25 μg/lane) from each lysate was loaded after protein quantification using the Bradford reagent (Bio-Rad). The relative band intensity was compared via band scanning with a Gel Doc XR (Bio-Rad) using Quantity One software (version 4.5.2) after normalization to the band intensity of the GAPDH.

2.12 Zebrafish and embryos

Wildtype zebrafish (Linebrass, AB strain) and embryos were maintained according to standard protocols 30. Maintenance and experimental procedures for zebrafish were approved (YUHS 01–13–004) by the Committee of Animal Care and Use of Yeungnam University (Gyeongsan, Korea). The fish were maintained in a system cage at 28°C during treatment under a 14:10 h light:dark cycle.

2.13 Microinjection of zebrafish embryo

Embryos at 2 days postfertilization were individually microinjected using a pneumatic picopump (PV820; World Precision Instruments, Sarasota, FL, USA) equipped with a magnetic manipulator (MM33; Kantec, Bensenville, IL, USA) with a pulled microcapillary pipette (PC-10; Narishigen, Tokyo, Japan). To minimize bias, injections were performed at the same position on the yolk abdomen. Just prior to injection, each rHDL containing FA (1.4 ng of FA) was mixed with 50 nL of PBS in the presence of 13 ng of oxLDL. Following injection, live embryos were observed at 48 h under a stereomicroscope (Motic SMZ 168; Hong Kong) and imaged using a Motic Cam2300 CCD camera 31.

2.14 Consumption of TFA

High-cholesterol diets (HCD) containing cholesterol (final 4%, wt/wt) and each FA (final 0.58%, wt/wt in tetrabit) was prepared as our previous report 32. Each diet was made by soaking Tetrabit (47.5% crude protein; 6.5% crude fat; 2.0% crude fiber; 10.5% crude ash; vitamin A, 29 770 IU/kg; vitamin D₃, 1860 IU/kg; vitamin E, 200 mg/kg; and vitamin C, 137 mg/kg; Tetrabit Gmbh D49304, Melle, Germany) in a diethyl ether solution of cholesterol (Sigma #C-3045) and each FA. We calculated the amount of FA in the diet (0.53 cal per 0.058 mg of FA) should be 2% of calorie in the total calorie of diet (25.65 cal/10 mg of tetrabit) in according to the formula.

Each group (n = 80) consumed the designated diet (20 mg of diet/day/fish), as shown in Table 1. The zebrafish were maintained at 28 ± 1°C under a 14:10 h light:dark cycle. After feeding for 20 weeks, blood (2 μL) was drawn from the hearts of adult fish and combined with 5 μL of PBS-EDTA (final concentration, 1 mM), then collected in EDTA-treated tubes. Plasma and histologic analysis was carried out as described in Supporting Information—supplemental methods.

2.15 Statistical analysis

All data are expressed as the mean ± SD of at least three independent experiments with duplicate samples. Comparisons between results were made by Student's t-test using the SPSS program (version 12.0; SPSS, Inc., Chicago, IL, USA). Statistical significance was defined as p < 0.05.

3.1 Synthesis of rHDL

All rHDLs containing FA were synthesized well without precipitation with particle size of 89–94 Å as shown in Fig. 1. Incorporation of each FA in the particle size induced decreased size of rHDL and alpha-helix content of apoA-I with dosage-dependent manner as shown in Supporting Information Table 1. With increasing molar ratio of FA, rHDL containing EA (EA-rHDL) showed the highest increase of smaller band (around 78 Å) as indicated by arrow head. The FA incorporation induced smaller particle size with increasing content of FA especially EA. Furthermore, cross-linking ability of apoA-I in rHDL by the BS₃ cross-linker was much impaired under the highest molar ratio of apoA-I:FA, 1:10 (Fig. 1 and Supporting Information Table 1).

3.2 LDL oxidation and more uptake of oxLDL by EA-rHDL

SA-rHDL and OA-rHDL showed potent inhibition abilities against cupric ion-mediated LDL oxidation until 80 min of incubation, whereas EA-rHDL exacerbated LDL oxidation induced by cupric ion after 60 min (Supporting Information Fig. 1).

Native rHDL could inhibit uptake of oxLDL into macrophages from observation of NBD fluorescence as previously described (Supporting Information Fig. 2). EA-rHDL caused the most extent of oxLDL uptake with increasing FA content, dosage-dependent manner, while OA-rHDL showed the least uptake of oxLDL (Supporting Information Fig. 2A). This result suggests that SA-rHDL and OA-rHDL could inhibit uptake of oxLDL with the least production of reactive oxygen species (ROS).

DHE staining, in order to visualize ROS in the cell as red fluorescence, revealed that EA-rHDL-treated cell showed the strongest red intensity with dosage-dependent manner, while OA-rHDL-treated cell showed the least red intensity. Merged image showed co-incidental existence of uptaken NBD-cholesterol and ROS in the cell. These results suggest that EA caused more atherogenic and proinflammatory response, while OA showed antiatherogenic and anti-inflammatory properties (Supporting Information Fig. 2A and B).

3.3 EA-rHDL suppressed cellular uptake of apoA-I in the rHDL

After 48-h incubation with rHDL, immunodetection of apoA-I in the treated cell revealed that EA-rHDL-treated cell showed the least detection of apoA-I detection in all dosage as shown in Supporting Information Fig. 2C. Under 1:1 ratio, the apoA-I detection level of EA-rHDL treatment was decreased only 70% level in comparison with native rHDL treatment (lane N), while SA-rHDL- and OA-rHDL-treated cell showed 120 and 140% based on densitometric quantification. OA-rHDL- and SA-rHDL-treated cell showed higher level of apoA-I in cell lysate than EA-rHDL, indicating that EA might interfere uptake of HDL into the cell. Since PBS-treated cell showed no band of apoA-I, the detected apoA-I band was appeared from exogenously treated rHDL containing each FA.

3.4 EA-rHDL caused more ROS generation and cellular senescence

Fructose treatment caused more ROS production in THP-1 cell (photos in Fig. 2) via inflammatory response as our previous report, however, native rHDL suppressed it (graph in Fig. 2). Among the rHDL containing FA, OA-rHDL-treated cell showed the least ROS production, while OA-rHDL-treated cell showed the least ROS. This result indicates that anti-inflammatory activity of rHDL could be impaired by trans-fat (EA), while unsaturated FA (OA) did not.

As shown in Fig. 3, EA-rHDL (final 2 μM of apoA-I and 17 μM of FA) caused the highest cellular senescence in the HDF cells at passage 13, while OA-rHDL caused the lowest senescence from SA-β-gal staining.

3.5 Survivability of zebrafish embryo

As shown in Fig. 4, injection of oxLDL caused the most severe embryo death around 47% survivability as similar with our previous report 31. Among rHDL, EA-rHDL showed the least survivability around 52%, while OA-rHDL-injected embryo showed 65% of survivability. As a control, native rHDL-treated embryo showed the highest survivability around 69%. There was no significant difference of survivability between OA-rHDL and SA-rHDL.

3.6 Serum profiles of zebrafish

After 20 weeks feeding of each FA (0.58% in diet, wt/wt), SA-fed group and EA-fed group showed 8 and 10% more body weight than control under normal diet (ND) supplementation, while OA-fed group showed 6% more body weight than control (Table 1). Under HCD supplementation, SA-fed and EA-fed groups showed 15 and 10% more gaining of body weight, respectively. Interestingly, OA-fed group showed even 4% smaller body weight than that of control (HCD alone diet), indicating that OA might exert antiobese effect.

Serum cholesterol and triglycerides (TG) were elevated by the HCD feeding around 4.0- and 1.8-fold more than ND (tetrabit) control as shown in Table 1. Under ND, SA-fed group showed the highest total cholesterol (TC) and TG levels with 33 and 45% more increase than control, while EA-fed group showed 14 and 25% more increase of TC and TG control. Under HCD, EA-fed group showed the highest increase of TC and TG levels although extent of body weight gaining was less than SA-fed group. The EA group showed 41 and 42% more increased serum cholesterol and TG level, respectively, compared with HCD control. SA-fed group showed the most increase of body weight and second highest TC level around 30% more than HCD control. Interestingly, OA-fed group exhibited the lowest level of serum TC level under both ND and HCD, indicating that OA exerted hypolipidemic effect. The OA-fed group showed 23 and 17% lower TC and TG levels, respectively, than control under HCD, while SA-fed group showed 1.3- and 1.2-fold higher serum TC and TG levels, respectively.

In hepatic inflammation parameters, EA-fed group showed the highest serum glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels, around twofold higher than control under either ND or HCD. Under HCD, all group showed similar GOT level around 150 Karmen/mL except EA group around 340 Karmen/mL, while GPT level was similar in around 65–67 Karmen/mL.

Under ND, serum CETP activity was around threefold increased by consumption of FA especially in EA-fed group, around 47% of CE-transfer activity. OA-fed group showed second highest CETP activity around 36%. Under HCD, control and SA-fed group showed higher CETP activity than OA-fed and EA-fed group, indicating that the serum CETP activity might be influenced by different FA consumption.

3.7 Fatty liver change

As shown in Fig. 5, histological analysis with hepatic tissue revealed that EA-fed group showed severe fatty liver change by oil-red O and hematoxylin staining under both ND and HCD, while OA-fed group showed the least oil-stained area. Among HCD group, OA-fed group showed the lowest oil-red O stained area, indicating that OA consumption can ameliorate the fatty liver change by the high cholesterol consumption.

Malondialdehyde determination with the extract of hepatic tissue revealed that EA-fed group showed the highest production of MDA 39 and 32 μmol/mg of protein under ND and HCD consumption, respectively. However, OA-fed group showed the lowest MDA production among the FA group with 11 and 19 μmol/mg of protein (Supporting Information Fig. 3). These results suggest that EA consumption caused the most impairment of antioxidant activity in hepatic tissue with fatty liver change.