Phenethylamine in chlorella alleviates high-fat diet-induced mouse liver damage by regulating generation of methylglyoxal

Materials and reagents

WEC was prepared and supplied by Sun Chlorella (Kyoto, Japan). WEC contained ~10 μg/g PHA in dry matter　and 4.4 mg/g half cystine (information from supplier). PHA, proteinase inhibitor cocktail, butylated hydroxytoluene (BHA), phosphate-buffered saline (PBS, pH 7.4), diethylenetriamine pentaacetic acid (DTPA), 1,1,3,3-tetraethoxypropane (TEP), thiobarbituric acid (TBA), 2,3-diaminonaphthalene (DAN), enhanced chemiluminescence (ECL) reagent, acetonitrile (high-performance liquid chromatography grade), and 4-vinylpyridine (4-VP) were purchased from Nacalai Tesque (Kyoto, Japan). Toronto Research Chemicals (Toronto, ON, Canada) provided 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ). Heparin sodium was obtained from Nipro (Osaka, Japan). Cell lysis reagent was obtained from Sigma-Aldrich (St. Louis, MO, USA). Tween 20 was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Rabbit immunoglobulin G (IgG) against mouse SOD-1 and GPX-1 were obtained from Abcam (Cambridge, UK). Mouse anti-GAPDH and β-actin monoclonal antibodies, were obtained from Proteintech (Chicago, IL, USA) and Santa Cruz Biotechnology. Goat horseradish peroxidase (HRP)-conjugated rabbit and mouse IgGs were obtained from Cell Signaling Technology (Danvers, MA, USA). Stable isotope-labeled L-cysteine (L-cysteine-¹³C₃,¹⁵N) was purchased from Taiyo Nippon Sanso (Tokyo, Japan).

Animal experiments

All animal experiments were performed according to the guidelines of the National Institutes of Health for the use of experimental animals. The experimental procedures were performed at the Louis Pasteur Center for Medical Research (Kyoto, Japan) and were approved by its Animal Care Committee (No. 20192).

Male C57BL/6 J mice (aged 7 weeks; bodyweight, 21–23 g) were purchased from Japan SLC (Shizuoka, Japan). The mice, which were housed in cages (three mice/cage), were maintained at 22 °C under a 12-h light/dark cycle with free access to rodent chow (Certified Diet MF, Oriental Yeast, Osaka, Japan) and tap water for 3 days. Subsequently, the mice were randomly divided into the following four groups (n = 6): ND group, fed on a standard diet; HFD group, fed on HFD; WEC group, fed on HFD and orally administered with WEC (100 mg/kg bodyweight) through drinking water; PHA group, fed on HFD and orally administered with PHA (10 μg/kg bodyweight) through drinking water. The proximate composition of animal diets is shown in Table 2. HFD (high-fat diet 32, CLEA Japan, Tokyo, Japan) comprises 32% crude fat with 60% of the calories derived from the fat. The amounts of WEC and PHA in drinking water were calculated based on the consumption of drinking water by mice in the previous 3 days. All mice received experimental diets for 12 weeks. The mice were then euthanized under isoflurane in the morning without fasting. The blood samples were collected from the inferior vena cava using a heparin-treated syringe. The plasma was separated by centrifuging the blood samples at 410 g for 5 min. The liver was excised and the blood in the liver was purged by infusing cold PBS into the portal vein. The plasma and liver samples were stored at −30 °C.

Biochemical analyses

The analysis of plasma AST and ALT activities and the plasma levels of TG, TC, HDL-C, and LDL-C contents were outsourced to Oriental Yeast (Osaka, Japan). To determine the TG levels in the liver, liver samples (20–30 mg) were homogenized in 200 μL of isopropanol using a BioMasher II (Nippi, Tokyo, Japan). The homogenates were centrifuged at 1100 × g for 10 min. The supernatant was subjected to a TG assay using the TG E-test Wako kit (Wako, Osaka, Japan).

TBARS assay

The working solutions of DTPA (2 mM) and BHA (10%) were prepared in 1 M sodium acetate buffer (pH 3.5) and methanol, respectively. To prepare the TBA solution, 0.1 g TBA was mixed with 5 mL of DTPA solution, 45 mL of hot distilled water, and 100 μL of BHA solution. The stock solution (2 mM) of TEP (a precursor of malondialdehyde) was prepared by adding 4.8 μL TEP to 10 mL of methanol. The standards (2–50 μM) were prepared from the stock solution of TEP. The liver tissues were homogenized in nine volumes (v/w) of 1.15% KCl solution. The homogenates were centrifuged at 2000 × g for 1 min. The supernatant or standard solution (25 μL) was mixed with TBA solution (100 μL), vortexed, and heated at 95 °C for 60 min. The reaction was terminated by cooling the reaction mixture on ice for 5 min. The reactants were centrifuged at 14,200 × g for 10 min and the absorbance at 515 nm of the supernatant was measured.

Evaluation of hepatic SOD-like and GPX-like activities

Liver samples were homogenized in five volumes (v/w) of 10 mM Tris-HCl buffer (pH 7.4) containing 0.25 M sucrose and 1 mM ethylenediaminetetraacetic acid (EDTA) using a BioMasher II. The homogenates were centrifuged at 10000 × g for 60 min and the total SOD-like activity in the supernatant was assayed using a WST-1 SOD assay kit (Dojindo, Kumamoto, Japan). To examine the SOD-like activity in the low molecular weight compounds in the extract, the liver tissues were homogenized with one volume (v/w) of PBS using a BioMasher II. The homogenate was mixed with six volumes (v/w) of ethanol. The resultant suspension was centrifuged at 14200 × g for 10 min. The SOD-like activity in the supernatant was assayed. The liver extract used for the TBARS assay was also used for assaying GPX-like activity. The GPX-like activity in the supernatant was assayed using a glutathione peroxidase activity colorimetric assay kit (BioVision, Milpitas, CA, USA).

Determination of hepatic methylglyoxal levels

The methylglyoxal level was determined using the liquid chromatography-tandem mass spectrometry system (LC-MS/MS) after derivatization with DAN following the methods of Han et al. with minor modifications⁴⁶. Liver samples were homogenized with one volume (v/w) of PBS using a BioMasher II. The homogenate was mixed with six volumes (v/w) of ethanol and the suspension was centrifuged at 14200 × g and 4 °C for 10 min. The supernatant (10 µL) was incubated with 50 μL of 0.1 % (w/v) DAN solution at 50 °C for 1 h. The reactants were mixed with 500 μL ethyl acetate and vortexed. The ethyl acetate layer (400 μL) was collected and evaporated. The residue was dissolved in a 30% aqueous acetonitrile solution. Aliquots of the solution were clarified by passing it through a Cosmonice filter W (Nacalai Tesque). DAN derivatives of methylglyoxal were quantified using an LC-MS/MS system equipped with an ODS-3 column in multiple reaction monitoring (MRM) mode. The MRM condition for the DAN derivatives were optimized using LabSolutions Version 5.65. A binary linear gradient, with 0.1% formic acid (solvent A) and 0.1% formic acid containing 80% acetonitrile (solvent B), was used at a flow rate of 0.2 mL/min. The gradient program was as follows: 0–15 min, 0–100% B; 15–20 min, 100% B; 20–20.1 min, 100–0% B; 20.1–30 min, 0% B. The column temperature was maintained at 40 °C.

Western blotting

The liver samples (~50 mg) were homogenized with 300 μL of cell lysis reagent containing 1% proteinase inhibitor using a BioMasher II. The homogenate was centrifuged at 12000 × g and 4 °C for 15 min. The protein concentration in the supernatant was measured using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA) and the concentration was adjusted to 10 μg/15 μL with the same buffer. All samples were mixed with the same volume of pre-stained marker (Prestained XL-Ladder, Integrale, Tokushima, Japan) to ensure the consistency of loading volume and transfer efficiency. The proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a 12.5% gel. The resolved proteins were transferred to a polyvinylidene difluoride membrane (0.45 μm; GE Healthcare Life Sciences, Chicago, IL, USA) using a semi-dry blotting apparatus (WSE 4020, Atto, Tokyo, Japan). The band intensity of pre-stained markers was quantified using ImageJ 1.52a. The membranes were then blocked with 4% Block Ace solution (Megmilk Snow Brand, Sapporo, Japan) at room temperature (25 °C) for 30 min and washed five times with 50 mM Tris-HCl buffer (pH 7.5) containing 2.68 mM KCl, 137 mM NaCl, and 0.05% (v/v) Tween 20 (TBST) for 5 min at room temperature. Primary antibodies against SOD-1, GPX-1, GAPDH, and β-actin were diluted to 1:5000, 1:5000, 1:8000 and 1:1000 with 0.4% Block Ace solution containing 0.05% (v/v) Tween 20, respectively. After overnight incubation with the primary antibody, the membranes were washed with TBST for 5 min (5 times). HRP-conjugated secondary antibodies against rabbit or mouse IgG were diluted to 1:10000 and 1:12500 with 0.4% Block Ace containing 0.05% (v/v) Tween 20, respectively. The membranes were incubated with secondary antibodies for 1 h, followed by washing with TBST for 5 min (5 times) at room temperature. The membranes were soaked with ECL reagent for 1 min. Immunoreactive bands were detected using a Lumino Graph I (Atto). To correct loading volume and transfer efficiency, chemiluminescence intensity of each　band was divided by the band intensity of pre-stained markers near the target protein　(35 kDa for GAPDH and β-actin, 30 kD for SOD-1 and GPX-1). The ratio was used as band intensity. To compare band intensity of specific protein in different gels, the band intensity of each gel was normalized by the band intensity of common sample in HFD group, which was analyzed by all gels.

Determination of hepatic levels of reduced and oxidative forms of cysteine

The same sample used for the determination of methylglyoxal level was used for the determination of cysteine level. The supernatant (37.5 μL) was mixed with same volume of an internal standard (0.1 mM of L-cysteine-¹³C₃,¹⁵N) and 22.5 μL of 75% ethanol or 2 mM DTT in 75% ethanol solution for 1 min to detect the levels of reduced cysteine and total cysteine, respectively. Next, the sample was incubated with 4-VP (2.5 μL) at 37 °C for 2 h. Ethyl acetate (500 μL) and distilled water (100 μL) were added to the reactants and vortexed. The water layer (50 μL) was collected and evaporated under vacuum. Sodium borate buffer (50 mM; pH 8.8; 80 μL) and 0.3% AccQ acetonitrile solution (20 μL) were added to the residue. The resultant solution was kept at 50 °C for 10 min. The reactant was clarified by passing it through a Cosmonice filter W. The resultant cysteine derivatives with 4-VP and AccQ were quantified using an LC-MS/MS system equipped with an ODS-3 column in MRM mode. The MRM conditions for the cysteine and cysteine-¹³C₃,¹⁵N derivatives were optimized using LabSolutions Version 5.65. A binary linear gradient, with 0.1% formic acid (solvent A) and 0.1% formic acid containing 80% acetonitrile (solvent B), was used at a flow rate of 0.2 mL/min. The gradient program was as follows: 0–15 min, 0–50% B; 15–20 min, 50–100% B; 20–25 min, 100% B; 25–25.1 min, 100–0% B; 25.1–35 min, 0% B. The column temperature was maintained at 40 °C. The levels of cysteine in the extract were estimated using the following equation: [(peak area of cysteine derivative in the sample/peak area of the　internal standard) × concentration of the internal standard].

Statistical analyses

The results are presented as mean ± standard deviation. The means was analyzed using one-way analysis of variance, followed by Dunnett’s test for multiple comparisons vs. HFD group. The differences were considered significant at p < 0.05 and to have a certain tendency at 0.05 <p < 0.1. GraphPad Prism 7 software (GraphPad Software, San Diego, CA, USA) was used for statistical analyses.