The research conducted adhered to the Guidelines for the Care and Use of Laboratory Animals. The ethics committee of Kasetsart University Research and Development Institute, Kasetsart University, Thailand, approved this study (Approval No. ACKU61-VET-088).
Crocodile oil preparation
Crocodile oil extraction was performed according to the method described by Santativongchai et al. . Abdominal fat samples were collected from slaughtered C. siamensis (age: 3–5 years) obtained from a crocodile farm in Nakhon Pathom province, Thailand.
The samples were pressed through two layers of filter cloth with distilled water at the proportion of 1:1 (w/v). Subsequently, the solution was left undisturbed until separation of the mixture was observed. The upper clear oil fraction was then collected, evaporated, and stored in a sealed container at room temperature.
Animal care, diets and experimental design
Briefly, 21 Sprague–Dawley male rats (age: 7 weeks) were obtained from Nomura Siam International Co. Ltd., Samutprakan province, Thailand. The animals were individually housed under controlled environmental conditions (25 ± 2°C on a 12-h light/12-h dark cycle). Rats had ad libitum access to food and drinking water throughout the study. Rats were randomly divided into three groups (n = 7/group). Rats in group 1 were treated with sterile water (RO), those in group 2 were treated with crocodile oil (3% v/w) (CO), and those in group 3 were treated with commercial palm oil (3% v/w) (PO). The animals were orally administered sterile water, CO, and PO once daily for 7 weeks.
Measurement of body weight, food intake, and energy intake of animals
Food consumption was measured daily between 11:00 and 11:30 A.M. by weighting the rats. The food intake of each rat was measured by weighting the remaining chow. Food intake was also monitored daily to calculate the energy intake. The body weight was measured weekly throughout the experimental duration.
At the end of experiment, all animals were sacrificed by a lethal dose of pentobarbital sodium. Blood samples were collected by cardiac puncture and were centrifuged at 2,200 g for 15 min at 4°C. The serum was stored at -20°C until further analyses. The serum lipid profile included triglycerides, cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) and were determined by Hitachi 7080 analyzer (Hitachi, Japan).
Liver specimens were immediately collected and weighed. The liver specimens were stored on ice-cold homogenate buffer (0.32 M sucrose, 1 mM EDTA, and 10 mM Tris-HCL; pH 7.4) when performing a standard protocol for mitochondrial extraction. Some of the liver tissue was also collected and separated for histopathology analysis and preserved in 10% neutral buffer formalin. Liver samples were collected and homogenized in ice-cold PBS (20% w/v) and centrifuged at 2,000 g for 20 min at 4°C. The supernatants were stored at -80°C until further analysis.
Energy metabolism-related intermediates analysis
The frozen supernatants were mixed with methanol in a ratio of 2:8 (v/v). After centrifugation (20,000 g for 20 min, 4°C), the supernatants were taken out and evaporated using a freeze dyer at -80°C. The metabolites were then re-dissolved in 500 µL HPLC buffer. Each 5 µL sample was subjected to HPLC analysis. Chromatography was performed as follows: the injection volume was set to 5 µL and the column was kept at 40°C. An InertSustain C18 (150×4.6 mm) measuring 5 µm was used to achieve separation of the mobile phase consisting of 8% 1 N sulfuric acid. The gradient elution was used at a flow rate of 1 mL/min.
Hepatic histopathology and fat accumulation analysis
Fixed livers were stored in 10% formalin for 24-h at room temperature. The sample were embedded in paraffin and sectioned at 5-µm thickness, and stained with hematoxylin and eosin (H&E). Hepatic fat accumulation was determined by Oil Red O (ORO) staining. Liver cryosections (5 µm thick) were fixed with 10% NBF in PBS for 20 min and incubated in freshly prepared ORO solution for 10 min and then counterstained with hematoxylin for 20 sec. The sections were examined under a light microscope with a magnification 200×. The number and total surface area of lipid particles were analyzed.
Liver mitochondrial extraction
The liver mitochondrial extraction was performed within 1–2 h to prevent cellular damage. Liver samples from each group were pooled, weighed, homogenized, and washed in homogenate buffer. Liver specimens were homogenized in a glass Potter–Elvehjem tissue grinder with an appropriate volume of the homogenate buffer (4 mL homogenate buffer/1 g of liver specimen). During this step, several up and down strokes were performed using a motor-driven Teflon pestle at 600 rpm. Next, homogenates were centrifuged at 1,000 g at 4°C for 5 min. The supernatants were collected and centrifuged at 15,000 g at 4°C for 2 min. The mitochondrial pellets were collected and washed several times in homogenate buffer. The pellets were resuspended in ice-cold final equilibrated buffer (250 mM sucrose, 5 mM KH2PO4, 10 mM Tris-HCl, and 2 mg/mL bovine serum albumin [BSA]; pH 7.2), and 200 µL of the resuspended pellet was then fixed in 2.5% glutaraldehyde in 0.1 M sucrose phosphate buffer (SPB) for electron microscopy analysis. The mitochondrial protein content was measured by protein assay (Bio-Rad®) using a spectrophotometer (NanoDrop-1000, Thermo Scientific).
Conventional electron microscopy
Electron microscopy was used to examine the ultrastructure of liver mitochondria. Fixation of the liver specimens from each group was performed using 1% osmium tetroxide, dehydrated in graded ethanol, infiltrated in a series of LR white resin (EMS®, USA), embedded in pure LR white (EMS®, USA), polymerized at 60°C for 48 h, cut into 100-nm–thick sections, and stained with lead citrate and uranyl acetate. The liver ultrastructure was examined under a transmission electron microscope (model HT7700, Hitashi, Japan). Intact mitochondria in hepatocytes were counted (50 cells per group) and compared with other treatment groups.
Metabolite-protein and protein-protein interaction
We utilized STITCH V. 5.0 (http://stitch.embl.de/) to establish the interaction prediction between the expected metabolites and proteins that interact with the targeted mitochondrial energy-maintenance protein (haloacid dehalogenase-like hydrolase domain containing 3: HDHD3), such as the energy-related proteins (PRKAA, PRKAB, PRKAG, AKT, PPAR-α, PPAR-γ) and energy-related metabolites (lactate, pyruvate, oxaloacetate, citrate), with a required confidence threshold (score) of 0.40.
Immunogold labelling technique
The immunogold labelling technique was used to compare the expression of HDHD3 (mitochondrial energy marker), and its localization on mitochondria among the groups. Rabbit polyclonal anti-HDHD3 was used as the primary antibody marker (MyBioSource, USA).
The mitochondrial pellet from the pooled liver extract in each group was secondary fixed, and tissue processing was performed as described previously. The tissue sections were blocked using 50 mM glycine in phosphate-buffered saline (PBS) followed by 5% BSA (EMS, USA) in PBS for 30 min each. Then, they were incubated with 1:50 diluted primary antibodies for 1 h prior to applying goat anti-rabbit IgG conjugated with 10-nm gold particles (EMS®, USA). Between each step, sections were washed several times using 0.1% BSA in PBS. To improve contrast of the gold particle labelling, a silver enhancement kit (Aurion R-Gent SE-EM kit, EMS, USA) was used after rigorously washing the tissue sections with distilled water. Finally, the sections were stained with lead citrate and uranyl acetate prior to transmission electron microscopy. The number of labelled gold particles was counted for the intact stage of liver mitochondria (50 mitochondria/group were evaluated).
Data analysis and statistical methods
The data are expressed as means ± SD. Statistical analysis was performed by analysis of variance (one-way ANOVA) followed by Tukey’s post-hoc test in R project statistical computing package (R core team, 2019). For all analyses, p < 0.05 was considered to indicate statistical significance.