The use of animals was approved by the Research Ethics (Animal) Committee, Universiti Sains Malaysia (Penang, Malaysia). Male Sprague Dawley rats (weight, 80–100 g; age, 4 weeks) were obtained from the Animal House of the Universiti Sains Malaysia (Kubang Kerian, Kelantan, Malaysia). The rats were randomly assigned to two groups: Six control rats were fed a normal intake of food (5 g pellet/100 g body weight per day) and six experimental rats were fed an excessive intake of food (10 g pellet/100 g body weight per day). The animal feed was obtained from Gold Coin Feedmills Sdn. Bhd (Penang, Malaysia). The rats were provided pre-weighed quantities of food each day and the quantity remaining in the food dish was weighed every 24 h. The body weight of each rat was measured weekly to determine growth. A total of four groups were used, with six rats in the 8 week group (three control rats; three experimental rats) and six in the 16 week group (three control rats; three experimental rats). The experiment was repeated twice. Following a feeding period of 8 weeks, the waist circumferences of the three rats in each group were measured prior to sacrifice via brief exposure to 10% (v/v) diethyl ether in a chamber. The same measurement was peformed prior to sacrifice in the three rats in each of the two 16 week feeding period groups. Blood was also collected from the rats in each group following the feeding periods of 8 and 16 weeks by cardiac puncture, left to clot and centrifuged at 3,000 × g for 15 mins to obtain serum for ELISA. For long-term storage, the serum samples were maintained at −80°C. The adipose tissues (visceral and subcutaneous) were collected and weighed. The visceral adipose tissues were used for total RNA extraction or rinsed in TRI Reagent^® (Molecular Research Center, Inc., Cincinnati, OH, USA), frozen in liquid nitrogen and stored at −80°C until use.

Total RNA extraction was performed using the TRI Reagent^®, according to the manufacturer’s instructions. For this, total RNA was extracted from the aqueous phase (colourless top layer) of the homogenised tissue samples and the remaining organic phase was stored at −80°C for subsequent protein extraction. The integrity of the total RNA extracted was confirmed using agarose gel electrophoresis, while the purity and yield of the RNA were measured using a nanophotometer (Implen, München, Germany). Subsequently, 1 μg total RNA was reverse-transcribed to cDNA using the RevertAid™ First Strand cDNA Synthesis kit (Fermentas, Waltham, MA, USA), according to the manufacturer’s instructions. The success of cDNA synthesis was validated by conventional PCR using rat GAPDH primers. The sequences of the GAPDH and PPAR primers used in the present study are listed in Table I.

Following cDNA synthesis, the PPAR expression levels were determined using 2X Power SYBR Green PCR master mix (Applied Biosystems, Inc., Foster City, CA, USA) in a Rotor-Gene 6000 PCR machine (Qiagen, Hilden, Germany). The final concentrations of cDNA and each primer used were 5 ng/μl and 0.4 μM, respectively. The GAPDH gene served as a reference gene, to compare the expression levels amongst different samples. All primers were designed with an annealing temperature of 55°C using the Primer Express 2.0 software (Applied Biosystems, Inc.). All PCR reactions were performed at 94°C for 10 min followed by 40 cycles of denaturation at 94°C for 20 sec, primer annealing at 55°C for 20 sec and extension at 72°C for 30 sec. A dissociation curve program was added following the completion of the reaction in order to generate the derivative dissociation curve. The derivative dissociation curve was created to confirm the primer-dimer artifact and to ensure the RT-qPCR specificity. The dissociation curve program was conducted at 95°C for 90 sec and 72°C to 95°C at 1°C/5 sec (1°C increase every 5 sec between 72 and 95°C) using the same PCR machine. Each PCR reaction was performed as three replicates in two independent experiments. The PPAR gene expression levels were determined using the relative quantification approach. The 2^(−ΔΔCt) method was used to analyse relative change in gene expression level (12). The value for fold-change in gene expression level was calculated as determined by the target gene expression level versus the reference gene expression level.

The total protein from visceral adipose tissue was extracted from the organic phase of the samples as described above. The protein concentration was determined using the DC Protein Assay kit (Bio-Rad, Hercules, CA, USA)and then used for Western blot analysis.

The protein samples (30 μg per well) were separated on a 10% SDS-polyacrylamide gel. The samples were then electrophoretically transferred to a nitrocellulose membrane, then blocked using 5% skimmed milk powder for 60 min at room temperature with constant agitation. The membrane was washed three times for 10 min each using 1X Tris-buffered saline and Tween-20, and then incubated with 1:250 responsive primary rabbit polyclonal antibody for PPARα (H-98: sc-9000), PPARδ (H-74: sc-7197) and PPARγ (H-100: sc-7196) at 4°C, overnight. All antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Subsequent to washing, the membrane was incubated at room temperature for 1 h with 1:5,000 horseradish peroxidase-conjugated rat anti-rabbit antibody (Santa Cruz Biotechnology, Inc.). Subsequent to another washing step, the blot was incubated with the SuperSignal West Pico Chemiluminescent Substrate (Pierce Biotechnology, Inc., Rockford, IL, USA). The blot was then developed on film in a dark room. Anti-rabbit GAPDH antibody (Santa Cruz Biotechnology, Inc.) was used as a control, to verify equal sample loading.

The expression levels of the CCL2, TNF-α, adiponectin, leptin, resistin, IL-6 adipokines and C-reactive protein (CRP; control) in the serum samples of control and experimental rats, were determined using commercially available ELISA kits (Abnova, Taipei, Taiwan). In these kits, antibodies specific for the adipokines had been pre-coated onto 96-well microtitre plates. The serum samples were then added to the wells and allowed to react with the bound antibody for 2.5 h at room temperature. The unbound antibodies were washed away with a Wash Solution, according to the manufacturer’s instructions. Subsequently, an enzyme-linked antibody specific to the adipokine was added to the wells. The antibody was incubated with the targeted protein for 1 h. Following another washing step, a substrate solution was added to the wells for colour development. The colour developed was proportional to the quantity of adipokine present in the samples. The colour intensity was then measured using an ELISA reader (Tecan Group, Ltd., Männedorf, Switzerland) at a wavelength of 450 nm and the level of each adipokine in the serum samples was calculated. The ELISAs were performed in triplicate and repeated in two independent experiments.

All graphs and statistical analyses were performed using the GraphPad Prism5 software (GraphPad Software Inc., La Jolla, CA, USA). All values are expressed as the mean ± standard deviation. Student’s t-test was used to compare the means of two groups and one way analysis of variance (ANOVA) was used to compare the means between the groups. P<0.05 was considered to indicate a statistically significant difference.

The results demonstrated that weight gain increased with time and that the rats in the experimental group, which consumed more food, gained more weight compared with the rats in the control group. The weight gain reached a plateau phase at a feeding period of 14 weeks, whereby the weights of the control and experimental rats were ~325 and 400 g, respectively. However, following feeding periods of 14 weeks, no more weight gain was observed in either the control or the experimental rats. In the experimental rats (fed with excessive food intake), the accumulation of adipose tissue mass was first observed at 8 weeks.

In the experimental rats (fed with excessive food intake), the accumulation of adipose tissue mass was first observed at 8 weeks. The subcutaneous adipose tissue mass collected was higher than the visceral adipose tissue mass (Fig. 1). At 16 weeks, the quantity of subcutaneous adipose tissue mass in the experimental rats was unchanged as compared with the fat mass at 8 weeks. However, the accumulation of visceral adipose tissue mass was significantly increased (P<0.05) in the experimental rats at 16 weeks, when compared with the fat mass at 8 weeks. The visceral adipose tissue mass in the experimental rats was also significantly higher than the mass of the subcutaneous adipose tissue (P<0.05) at 16 weeks.

The PCR results detected no significant difference (P>0.05) in the PPARα and PPARδ mRNA expression levels in the visceral adipose tissue between samples obtained at 8 and 16 weeks (Fig. 2). Conversely, at 16 weeks, the experimental rats exhibited a significant increase (P<0.05; ~2-fold) in the mRNA expression level of PPARγ relative to the result obtained at 8 weeks. A similar pattern in the protein expression levels of all PPARs, as assessed by Western blotting, was observed in the visceral adipose tissue of experimental rats at 8 and 16 weeks (Fig. 3).

As shown in Fig. 4, no significant differences (P>0.05) were observed in the levels of CCL2 and IL-6 in serum samples between experimental and control rats at 8 weeks. However, at 16 weeks, significantly higher levels of CCL2 (P<0.05) were detected in the serum samples of the experimental rats (1,465 ng/ml), compared with those of the control rats (939.8 ng/ml; Fig. 4B). In addition, at 16 weeks, the levels of IL-6 in the experimental rats (179.1 pg/ml) were significantly higher (P<0.05), when compared with those at 8 weeks (84.3 pg/ml; Fig. 4C). The levels of leptin at 8 weeks were found to be significantly increased (P<0.05) in the serum samples of experimental rats (2,341 pg/ml), compared with those of the control rats (526.7 pg/ml). This phenomenon was also observed at 16 weeks, with 2,971 pg/ml in experimental rats and 928.9 pg/ml in control rats (P<0.05; Fig. 4D). Conversely, significantly reduced levels of adiponectin (P<0.05) were observed in the serum samples of experimental rats compared with the control rats at 8 and 16 weeks (Fig. 4E). No significant differences were identified in the levels of the resistin and TNF-α between experimental and control rats in all serum samples (Fig. 4F and G).