All experiments involving animals were approved by the Animal Care and Use Committee of Pusan National University (Miryang, Korea; PNU-2012-0056). Male, 6-week-old, Institute of Cancer Research (ICR) mice (Japan SLC Inc., Shizuoka, Japan) were acclimated for 7 days prior to assessment of health status. The study was approved by the ethics committee of Pusan National University (Miryang, Korea). The mice were individually housed with sawdust bedding and were maintained in environmentally controlled rooms (ambient temperature, 22±2°C; relative humidity, 50±10%) under a 12/12 h light-dark cycle (lights on 8:00 am–8:00 pm). Food and water were available ad libitum. The repeated dose (4 weeks) investigation was performed in 32 mice, which were divided into four groups of eight animals: Control (7% soybean oil), EA (6.5% soybean oil + 0.5% EA), TVA (6.5% soybean oil + 0.5% TVA) and CLA (6.5% soybean oil + 0.5% cis-9, trans-11 CLA), as shown in Table I. The dosages of each fatty acid were selected on the basis of the worldwide consumption report (12). The fatty acids were obtained from Matreya LLC (Pleasant Gap, PA, USA). The experimental diets were manufactured by Feedlab (Kyung-ki, Seoul, Korea), and no deterioration of the fatty acids in the diets was observed following storage for 4 weeks (data not shown).

The total fatty acid compositions of the diets were analyzed and are described in Table I. Briefly, the total lipids were extracted from 4 g of feed using 20 ml chloroform/methanol (2:1; v/v; Burdick & Jackson, Muskegon, MI, USA). The extracted lipids were subsequently converted into fatty acid methyl esters using 14% (w/v) boron trifluoride-methanol (cat. no. B1252; Sigma-Aldrich, St. Louis, MO, USA), according to a previously published method (13). The fatty acid methyl esters were then subjected to gas chromatography (Agilent 7890A GC system; Agilent Technologies, Inc., Santa Clara, CA, USA) using a system equipped with a 7863 series auto-sampler, 7683B series injector and flame ionization detector. An SP™-2560 fused silica capillary column (L × I.D. 100 m × 0.25 mm, df 0.20 μm) was used (Supelco Inc., Bellefonte, PA, USA) to analyze the fatty acid compositions of the samples. The peaks were routinely identified by comparing the duration of retention with those of the standards, including Supelco 37 Component Fatty acid methyl esters mix (47885-U), trans-11-octadecenoicmethyl ester (46905-U; Sigma-Aldrich) and cis-9, trans-11 CLA (1255; Matreya LLC, Pleasant Gap, PA, USA). The percentages of the individual fatty acids were calculated as the ratio of each area compared with that of the total identified fatty acids, as described previously (14). The four experimental diets contained a similar concentration of fatty acids. The concentrations of the three assessed fatty acids decreased to 0.25% from an initial concentration of 0.5%, which may have been due to losses acquired during production (Table I). The body weights and food intake of each mice were measured every 2 days. In addition, the general appearance, behavior and signs of morbidity and mortality were observed every 2 days throughout the experiment.

Following 4 weeks of dietary intervention, the mice were sacrificed by CO₂ asphyxiation and blood samples were collected (5 ml) from the external jugular veins. The blood was centrifuged at 400 × g at 4°C for 15 min and the supernatant was collected and stored at −80°C until subsequent analysis.

The blood metabolites were analyzed using a Toshiba Accute Biochemical Analyzer-TBA-40FR (Toshiba Medical Instruments, Tochigi-ken, Japan), according to the manufacturer’s instructions. A total of 10 metabolites, including blood urea nitrogen, calcium, magnesium, triglyceride, glucose, non-esterified fatty acid, albumin, total cholesterol, LDL cholesterol and HDL cholesterol were analyzed. All the reagents required for this procedure were purchased from Wako Pure Chemical Industries, Ltd. (Chuo-ku, Osaka, Japan).

Samples of the small intestine and liver (n=80/group) were collected and fixed in formaldehyde solution (Sigma-Aldrich). The samples for histopathological examination were processed, embedded in paraffin (Sigma-Aldrich), cut to a thickness of ~2–4 μm and stained using hematoxylin and eosin (Sigma-Aldrich). The length of the villi were then quantified (n=80/group) by measuring the distance between the crypt necks.

The relative protein quantities of cholesterol 7α-hydroxylase (CYP7α1) in the liver were determined using western blotting. Briefly, the proteins (15 μg protein/lane) were separated using 10% sodium dodecyl sulfate polyacryl-amide gel electrophoresis (SDS-PAGE; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and were subsequently transferred onto a polyvinylidenedifluoride membrane (GE Healthcare Life Sciences., Piscataway, NJ, USA). The membrane was blocked with phosphate-buffered saline (PBS)-T, containing 10% 10X PBS (pH 7.6), 0.2 M Tris base, 1.37 M NaCl and 10% Tween-20, with 5% skim milk (BD Biosciences, San Jose, CA, USA), for 1 h at room temperature. The membrane was incubated overnight with specific primary antibodies against rabbit monoclonal β-actin (1:1,000; cat. no. ab32575; Abcam, Cambridge, MA, USA) and mouse monoclonal CYP7α1 (1:1,000; cat. no. ab77157; Abcam) at 4°C. Following washing three times with TBS-T, the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (1:1,000; cat. no. ab6721; Abcam) for 30 min at room temperature. The membrane was then washed with TBS-T and antibody binding was visualized using an enhanced chemiluminescence system and detection kit (GE Healthcare Life Sciences, Pittsburgh, PA, USA), according to the manufacturer’s instructions. The membrane was then exposed to X-ray film (Fujifilm Corporation, Minato-ku, Tokyo, Japan) for 1 min (β-actin) or 3 min (CYP7α1). The film was scanned and bands were quantified using the Image J^® 1.43 software (NIH, Bethesda, MD, USA). The protein levels were normalized against that of β-actin on the same membrane.

The concentrations of tumor necrosis factor-α (TNF-α) in the plasma were determined using an ELISA kit (cat. no. 88-7324; eBioscience, Inc., San Diego, CA, USA) at the end of the experimental period, according to the manufacturer’s instructions. The absorbance was then measured at 450 nm using a Multiskan EX Microplate Reader (Thermo Fisher Scientific Inc., Waltham, MA, USA).

All data are expressed as the mean ± standard error of the mean and were analyzed using one-way analysis of variance with SPSS 11.5 software (SPSS Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Changes in the general condition and external appearance of the rodents were observed in mice fed with EA or TVA. The animals of these two groups were small and weak in appearance, and their fur, eyes and oral cavity conditions were poor (data not shown). Although all the mice had a similar daily food intake (Fig. 1A), the body weights of the animals in the EA, TVA and CLA groups were all gradually decreased following 10 days administration of the assessed fatty acids (Fig. 1B). However, no significant change was observed in the lengths of the intestine or colon (Fig. 1C). In addition, the weights of the mediastinal adipose tissue (MAT) of the animals in these three groups were decreased significantly, compared with the control group (P<0.05; Fig. 1C).

A previous study reported that TVA exerted no effect on either the body weight or food intake in obese or lean rats during experimental periods (15). In another previous study, no significant differences in body weight or body weight gain were observed between groups treated with enriched dairy fat compared with the control throughout the experimental period (16). However, in the present study, the body weight decreased gradually following treatment with TVA, the reason for this difference remains to be elucidated and requires further investigation.

Significant effects of the three assessed fatty acids on organ weights was observed (Table II). The weights of the liver and testis of mice in the EA, TVA and CLA groups were all lower compared with those in the control groups (P<0.05). The weights of the spleen, kidney and heart of mice in the EA and TVA groups were also decreased (P<0.05; Table II). However, a previous study reported no gross pathological changes or differences in organ weight following 4 weeks of treatment with TVA (16).

A metabolic profile test (MPT) was initially designed as a pre-symptomatic diagnostic aid, based on the statistical analyses of blood metabolites, to provide an early warning of certain types of metabolic imbalances (17). Previously, it was used to measure physiological characteristics for selection programs designed to improve animal production traits (18). In the present study, 10 blood metabolites were analyzed to determine if any of these factors were correlated with the consumption of pure EA, TVA or CLA. No significant changes were observed in the TVA or CLA group, compared with the control. The EA group exhibited lower levels of Mg and triglycerides compared with the other groups (P<0.05; Table III). The levels of Mg, triglycerides and glucose were significantly decreased in the EA group compared with those in the control group (P<0.05).

Neither the total cholesterol concentrations or the HDL:total cholesterol ratio in the plasma were significantly different between the groups (Fig. 2A–C). However, the presence of TVA in the diet reduced the levels of plasma LDL cholesterol, compared with the other groups (P<0.05; Fig. 2A–C). In addition, although no significant difference was observed, the expression of CYP7α1 was increased in the TVA and CLA groups (Fig. 2D). EA is the predominant trans fat in hydrogenated vegetable oils and it has been confirmed that the consumption of EA exerts negative effects on health (5,19,20). In particular, EA increases the activity of cholesterylester transfer protein, which increases the level of VLDL cholesterol and reduces the level of HDL cholesterol (21). CYP7α1 is a rate-limiting enzyme in the synthesis of bile acid from cholesterol via the classical pathway (22). A previous study reported primarily neutral effects of TVA/CLA-enriched butter on atherogenic risk factors and reduced aorta fatty streak development (5,9). However, the ratios of atherogenic to anti-atherogenic lipoproteins, VLDL+LDL to HDL, are significantly lower in TVA/CLA-enriched butter groups compared with those of trans-10-C18:1-enriched groups (9).

No significant liver injury was observed in any of the groups following 4 weeks of dietary intervention with EA, TVA or CLA (Fig. 3A). However, morphological analyses demonstrated that EA and TVA intervention structurally shortened the length of villi, whereas CLA increased the villi length compared with that of the control (Fig. 3B and C). Villi are finger-like projections in the small intestine, which are important for the digestion and absorption of food (23). These results suggested that EA and TVA had negative effects (P<0.05) on villi morphogenesis in the small intestines of the mice, where CLA exerted a positive effect (P<0.05; Fig. 3D).

Cytokines are fundamental in modulating inflammation, phagocytosis, tissue injury and cell death. Accordingly, the present study measured the plasma levels of circulating TNF-α. The CLA group exhibited higher levels of TNF-α (P<0.05), whereas the TVA group exhibited lower levels, compared with the control group (Fig. 3D). It has been confirmed that the plasma levels of TNF-α are significantly lower in rats fed CLA for 6 weeks compared with control-fed rats 2 h following LPS challenge, whereas the production of TNF-α in PBS-injection control mice remains unchanged (24). Additionally, a previous report indicated that dietary CLA inhibits the production of TNF-α in weaned pigs challenged with LPS at the protein and mRNA expression levels (25). Previous studies have also indicated that CLA inhibits LPS-stimulated production of TNF-α and expression, partially due to inhibition of the binding activity of nuclear factor-κB. However, the present study did investigate the effects of LPS challenge, therefore, the increased expression of TNF-α by CLA may offer potential in immune-modulation. A pro-inflammatory response can be beneficial for bacterial clearance, however, overactivation of the inflammatory response can lead to cell injury and shock. Therefore, consideration of the appropriate dietary concentrations of CLA is required.

In conclusion, the administration of EA, TVA or CLA in the present study reduced the body weights of the mice, specifically the weights of the liver, testis and MAT. Treatment with EA decreased the levels of magnesium and triglycerides, and the length of the villi, whereas treatment with CLA increased the length of the villi and the expression of TNF-α. TVA also reduced the length of the villi, and the levels of LDL and TNF-α. Overall, the mice exhibited different responses to EA, TVA and CLA, however, the underlying mechanism remains to be elucidated and requires further investigation.