Animal studies and diets
Male Wistar rats were obtained at weaning from the Primate Unit (South African Medical Research Council/ SAMRC, Tygerberg, South Africa). The rats were given standard rodent chow (low fat, LF) for two weeks during adaptation to the housing conditions. The animals were then bodyweight matched and divided into LF and HF groups. The rats were fed ad libitum on their specific diets with free access to fresh water for the duration of the study. The rats were singly caged in a temperature-controlled environment at 23 °C, and 12 h light/dark cycle (6:00 am/6:00 pm). The rats were weighed weekly after they started eating their respective diets. At termination, all animals were weighed and sacrificed. Various tissues were weighed, collected and stored at − 20 °C until further analysis. The use of animals was in line with the SAMRC and ARRIVE guidelines that recognize the NC3Rs initiative. All the procedures involving animals were approved by the South African Medical Research Council Ethics Committee for Research on Animals (SAMRC-ECRA) Ref: P04/10/021.
Induction of diet-induced obesity
Two independent studies were conducted. The first study was performed to determine the effects of three high-calorie diets on the development of obesity for 8 weeks, and the second study was to explore one diet that showed the highest rate of obesity induction for 42 weeks. The composition of the four diets was analyzed using Gas chromatography mass spectrometer (GC–MS).
28 rats were bodyweight matched into four groups (n = 7 rats/group). One group served as the control (LF) group and was fed chow (Epol, South Africa), and the remaining three groups were fed three different chow-based HF diets to induce obesity for 8 weeks. Dietary composition of the four diets is shown in Table 3.
14 rats were body weight matched into LF and HF groups (n = 7). HF1 diet was used to induce obesity in the HF group, whereas the LF group remained on chow. The rats were fed their respective diets for 42 weeks.
Collection of blood samples
Blood samples were collected at 0, 8 weeks for the first study, as well as 42 weeks for the second study. On the day of blood collection for the baseline samples (0 weeks), food was removed for 4 h in the morning, in order to obtain postprandial state. Rats were lightly anaesthetized with 5% Halothane (Safe Life Pharmaceuticals, Sri Lanka), and blood samples (0.5 ml) were collected into Vacutainer serum tubes (Becton Dickinson, South Africa) by slightly snipping off the tip of the tail. At 8 and 42 weeks, blood samples were collected through a hepatic vein during exsanguination. The blood samples were left at room temperature for 30 min, followed by centrifugation at 7 000 rpm for 10 min at 4 °C. The supernatant (serum) was collected into new tubes and stored at − 80 °C until further analysis.
Removal of high abundant proteins
The serum samples were precipitated to remove albumin following the TCA/acetone protocol described by Chen et al.,7 with some modifications. Four volumes of 10% TCA/acetone was added to the serum samples and incubated at − 20 °C for 90 min. The samples were centrifuged on an Eppendorf Refrigerated Microcentrifuge (Model 5417R, Sigma-Aldrich, MO, USA) at 14 000 rpm for 15 min at 4 °C. The supernatant was transferred into a new tube and kept on ice. The pellet was washed with 0.5 mL cold acetone and incubated for 15 min at − 20 °C, then centrifuged as before. The supernatants were combined, and 0.5 mL of fresh acetone was added and incubated at − 20 °C for 90 min and centrifuged as above. Both the TCA/acetone precipitated (albumin-depleted) and acetone precipitated (albumin-containing) pellets were air dried and resuspended in 0.2 mL of solubilization buffer (7 M urea, 2 M thiourea and 4% CHAPS; Sigma, USA). The protein concentrations were quantified by Bradford assay and the samples were stored at − 20 °C until further analysis.
Two-dimensional gel electrophoresis
Samples containing 100 µg protein were passively rehydrated onto 7 cm IPG strips (Bio-Rad, Hercules, CA, USA) with a pH range 4–7 for 12–16 h, followed by isoelectric focusing through the three-step protocol: the initial voltage was limited to 250 V for 10 min then increased to 3,500 V for 2,800 Vhr, then continued at 3,500 V to 3,700 Vhr. The current was limited to 50 µA per strip at 20 °C. After isoelectric focusing, the strips were first equilibrated in equilibration (EQ) buffer containing 2% 1,4-dithiothreitol (Bio-Rad, Hercules, CA, USA) for 15 min, followed by another 15 min equilibration in the EQ buffer containing 2.5% Iodoacetamide (Bio-Rad, Hercules, CA, USA) with shaking. The proteins were resolved on a 10% SDS-PAGE for the albumin-containing samples and 12% SDS-PAGE for the albumin-depleted samples. The gels were fixed in methanol/acetic acid/water solution (4:1:5) for 2 h, followed by 3 h of staining with 1X Flamingo fluorescent stain (Bio-Rad, Hercules, CA, USA).
Quantitative analysis of gel images
The gel images from both the LF and HF groups were scanned on a Molecular Imager Pharos FX system (Bio-Rad, Hercules, CA, USA). Image analysis including image editing, spot finding, quantitation, and matching, was carried out using Quantity One Software (Bio-Rad) and PD Quest version 8.0 2-D gel analysis software (Bio-Rad, Hercules, CA, USA). The protein spots were detected by following the PD Quest software instructions using the following parameters: horizontal and vertical streaking removal with a radius of 33, smoothing by Power Mean filter with kernel size 3 × 3, speckle removal at a sensitivity of 50. The densities of protein spots were normalized using the Local Regression Model. The spots were then quantitatively compared between LF and HF groups using the approach by Sun et al.43. Protein spots were considered to be differentially expressed if the difference between the average of spot densities between the two groups was twofold greater or lesser.
In gel proteolysis
The protein spots that were differentially expressed between the LF and HF groups were excised from the gels using the EX Quest spot cutter (Bio-Rad, Hercules, CA, USA). The protein spots were tryptically digested following the protocol adapted from Shevchenko42. The Proteomics Analyzer, Voyager DE PRO MALDI MS (Applied Biosystems, UK) was used to identify the obtained mass spectra that were searched through a Mascot search engine (https://www.matrixscience.com) then queried against the National Centre for Biotechnology Information (NCBI) protein database for protein identification.
Western blot analysis
The identities of the differentially expressed proteins were validated by western blotting. The protein samples, 20 µg for albumin-depleted sample and 10 µg for albumin-containing sample, were resolved on a 12% SDS-PAGE. The proteins were electroblotted onto Hybond-P nitrocellulose membrane (Amersham Biosciences, UK). The membranes were incubated overnight at 4 °C with 1:5,000 primary antibodies (Apo A-IV, CRP, Fetuin-A and transferrin (TFR); Santa Cruz Biotechnology, TX, USA), washed and incubated with 1:10 000 secondary antibodies conjugated to horseradish peroxidase for 1 h at room temperature. The membranes were developed with LumiGLO Chemiluminescent Substrate System (Whitehead Scientific) then exposed to X-ray film (Amersham Biosciences) for 5 min in a dark room. The X-ray films were processed using the X-ray processor (Agfa, South Africa), and the images were captured using a Digital Camera. Transferrin was used as a loading control. The X-ray films were scanned with a Molecular Imager Pharos FX scan. Image analysis including image editing and quantitation was carried out using ImageJ Software (National Institute of Health).
Interaction between selected proteins were analysed through String v10.5 (https://string-db.org) and GeneMANIA (https://genemania.org). The following gene codes: APOA4, AMBP, AHSG, CRP and HPX were used to determine how these proteins interact with each other at gene and protein levels.
The animal body weights were analyzed statistically using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc analysis performed using GraphPad Prism version 5.00 for Windows, GraphPad Software, La Jolla Carlifonia USA, www.graphpad.com. The results were presented as means ± SEM. Statistical analysis for 2D gel analysis, the comparison between LF and HF groups was assessed using Student’s t-test and Boolean analysis sets by the PD Quest software (Bio-Rad, South Africa). The differences were considered statistically significant at p < 0.05.