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The effects of conjugated linoleic acid isomers on the morphological changes in adipose tissue and adipogenic genes expressions on primary adipose tissue

Abstract Previous studies carried out in mouse 3T3-L1 cell culture have shown that conjugated linoleic acid (CLA) inhibited adipocyte differentiation. The present study was undertaken to investigate the effect of cis-9, trans-11 and trans-10, cis-12 CLA isomers on morphological changes and on mRNA expressions in the in vitro adipocyte isolated from specific pathogen-free (SPF) chicken. The adipocytes were isolated from SPF chicken and cultured in differentiation-induction medium with different concentrations of cis-9, trans-11 and trans-10, cis-12 CLA. After day 7, the adipocyte differentiations were monitored morphologically and mRNA expressions of lipoprotein lipase (LPL) and acyl-coenzyme A binding domain containing 5 (ACBD 5) were quantified by real-time polymerase chain reaction (PCR) analysis. Our data suggested that cis-9, trans-11 CLA downregulated the expression of LPL and ACBD 5 genes, which was concurrently observed with decrease in the adipocyte area size and cell number compared to the control and trans-10, cis-12 treated groups. Based on this finding, we concluded that dietary CLA modulate fat reduction in chicken via alteration of transcription of key adipogenic genes and adipose cellularity.


Introduction
Conjugated linoleic acid (CLA) is a family of linoleic acid isomers with various conjugated double-bond arrangements. CLA dienes are fatty acids predominantly found naturally in meat and dairy products from ruminant. They have been receiving much attention for their putative healthful benefits, such as anticancer (Belury et al., 1996), atherosclerosis (McLeod et al., 2004), decreased body fat in various animal species and humans (Park et al., 1997;Ramiah et al., 2014). On the other hand, the trans-10, cis-12 isomers accumulate 10% in dairy or meat products (Kennedy et al., 2010). Chemically synthesised dietary supplement of CLA contains equal mixtures of cis-9, trans-11 and trans-10, cis-12 CLA isomers.
Accumulation of adipose tissue is a major problem in modern broiler industries (Claire et al., 2013). Fat deposition in chicken occurs due to the increased percentage of body fat with a concomitant increase in the mass of abdominal and visceral fat (Zhou et al., 2007).
Supplementation with CLA has been recorded with positive effect on fat deposition in animal models (Zhao et al., 2011). Both in vivo and in vitro studies suggested that CLA decreases adipocyte cellularity via decreasing adipocyte proliferation or adipocyte size in cell cultures in rodents (Azain et al., 2000). Several reports have demonstrated that CLA inhibits differentiation and represses adipocyte gene expression during the in vitro differentiation of mouse 3T3-L1 preadipocytes into adipocytes (Evans et al., 2000).
Adipose tissue acquired its ability to respond to insulin, and expressed fat metabolism-related genes such as lipoprotein lipase (LPL), adiposin and glucose transporter 4 (GLUT4) during their differentiation into mature adipocytes (Botelho et al., 2009). Study by Zhou et al. (2007) demonstrated LPL mRNA in cultures treated with CLA remained unchanged. In contrast, Botelho et al. (2009) found that the LPL activity was reduced. Similar changes were also observed in the in vivo studies when the mice were treated with CLA (Park et al., 1997). These findings indicated that inhibition of LPL activity played an important role in body fat reduction. Benefit of CLA supplementation on chicken fat metabolism has been reported by Zhang et al. (2007), where supplementation of CLA reduced abdominal fat percentage associated with decreasing LPL level in chicken plasma.
There is little information available about isomerspecific effect of CLA on the adipose tissue cellularity and adipogenic genes, particularly in chickens because CLA mixtures were used in most studies (Shin et al., 2011). To date, only studies that mainly focus on mammary tissue of rodents and humans have reported that trans-10, cis-12 CLA alone can act as antiadipogenic agents (Warren et al., 2003;Wang & Jones, 2004). Therefore, it is important to compare the regulation of both CLA isomers in chickens' adipocyte differentiation. Nevertheless, detailed analysis on the effects of cis-9, trans-11 and trans-10, cis-12 CLA individually, particularly focussing on the fat reduction capabilities of these CLA isomers remain to be investigated further. Thus, the purpose of this study was to compare the effects of cis-9, trans-11 and trans-10, cis-12 CLA on morphological changes and mRNA expression of fat metabolism-related genes of adipose tissue isolated from chickens.

Experimental birds
Nine-day-old specific pathogen-free (SPF) eggs were obtained from the Veterinary Research Institute, Ipoh, Perak, and hatched at the Laboratory of Vaccines and Immunotherapeutics, Institute of Bioscience, Universiti Putra Malaysia (UPM). Birds were fed with standard commercial diet obtained from Gold Coin Feed Mill (Malaysia).

Preparation of CLAs
Both isomers of CLAs were mixed to fatty acid-free (!98%) bovine serum albumin (BSA) at a 4:1 molar ratio using 1 mmol/L bovine serum albumin (BSA) stocks, as previously defined by Brown et al. (2001).

Animal welfare
This study was conducted following the guidelines approved by the Universiti Putra Malaysia Institutional Animal Care and Use Committee. The animals were cared, and their health and welfare were monitored by a qualified veterinarian who is a member of the research team.
Isolation of adipose tissue from primary culture of chicken adipocytes Chicken preadipocytes were cultured according to the method of Ramsay and Rosebrough (2003), with several modifications. One gram of adipose tissue was removed from the abdominal cavity of four SPF layer chickens. The tissues were washed with phosphate buffer saline (PBS) (Sigma-Aldrich, USA) for three times. Then, the adipose tissue was minced into fine sections with scissors, added with 10 mL of digestion buffer; DMEM:HEM's (50:50) (Sigma-Aldrich, St. Louis, MO), 1.5% BSA, 100 mM HEPES (Sigma-Aldrich, USA), 2mg/ml collagenease type I (Sigma-Aldrich, USA) and 1% penicillin/streptomycin (Sigma-Aldrich, USA), and was incubated in water bath shaker at 37 C for 45 minutes. After the incubation, the digested tissue was filtered through a wire mesh (80lm) and the collected filtrate was pelleted (2000 rpm, 10 minutes) and washed twice with PBS. Erythrocytes were removed using erythrocytes lysis buffer (0.154M NH 4 Cl, 10 mM KHCO 3, 0.1mM EDTA) (Sigma-Aldrich, USA) and centrifuged at 500 Â g for 10 minutes. After that, the cell were added with 10 mL of DMEM/HAM's supplemented with 10% foetal bovine serum (FBS), counted by haemacytometer, seeded at cell density of 1 million cells in T25 cell culture flask coated with collagen and incubated at 37 C with 5% CO 2 . After the cell was confluent on day 5, preadipocytes were trypsinized by TrypLE TM (Life Technology, Carlsbad, CA), seeded in 6-well plate precoated with collagen at 1 Â 104 cell/mL in differentiation media (1:1 ratio of DMEM/HEM's; 10% FBS (Gibco, Carlsbad, CA); 20 lg/mL bovine insulin (Sigma-Aldrich, USA); 1% L-glutamine (Gibco, USA); 1% penicillin/ streptomycin). The media were replaced every day for 7 days to allow the differentiation of preadipocytes. Both isomers of CLA were added to the differentiation-induction medium for 7 days. The final concentration of CLA isomers in the cell culture added to medium as follows: (C1) 1.51% and (C2) 2.56% for cis-9, trans-11 (C) and (T1) 1.51% and (T2) 2.56% for trans-10, cis-12 (T) with a control group (CON) (without CLA isomers). These concentrations of the CLA isomers were based on the concentration of CLA isomers deposited in the adipose tissue of chicken in an in vivo study done by Ramiah et al. (2014). After the incubation period, all samples were subjected to lipid staining and quantitative real-time PCR (qRT-PCR) studies.

Lipid staining
The adipocyte cells were fixed in 4% formaldehyde solution for 1 hour. The cells were rinsed with deionised water and stained with 0.3% Oil Red O (ORO) (Merck, Germany) (0.12% ORO in 60% isopropanol) for 1 hour at room temperature. Finally, the cells were washed twice with 60% isopropanol to remove undissolved stain. Aliquots of suspended adipocytes were placed in a haemacytometer and observed under an Olympus microscope BX51 (Olympus, Tokyo, Japan). Adipocytes were then enumerated and their diameters were measured using ImageJ software (National Institute of Health, Rockville, MD). The mean adipocyte diameter was calculated as an average of the diameters of 200 cells.
Trizol RNA extraction and qRT-PCR analysis of LPL and ACBD 5 genes Total RNA was extracted from cultured cells using Trizol Reagent (Life Science Technologies, Ontario, Canada) as per instructions. Briefly, 600 lL for Trizol was added into 6-well plates each. Each sample was pipetted up and down to detach the cells. 200 lL of chloroform was added into each well and shaken vigorously for $15 seconds to mix. The samples were incubated at room temperature for 2-3 minutes and centrifuged at 12,000 Â g in a cold chamber centrifuge (Eppendorf, Hamburg, Germany) for 20 minutes. Total RNA purity was determined by the 260/280 nm ratio of absorbance readings using NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE).

Quantitative real-time polymerase chain reaction (RT-PCR)
The adipocytes cells were cultured at a density of 5 Â 10 3 cells on a 6-well plate for a week with changing mediums every day. Total RNA was extracted from cultured cells using Zymo research Direct-zol RNA MiniPrepKit as per instructions. Total RNA concentration was then quantified by measuring the optical density (ratio of absorption 260/280 nm) of all preparations between 1.8 and 2.0. Five milligrams of RNA from each sample was reverse-transcribed using the QuantiTect Rev Transcription Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The gene expression of ACBD 5 and LPL was conducted using QuantiTect Primer Assays (Qiagen, Hilden, Germany) according to the manufacturer's instruction. Beta-actin gene (GenBank accession number: L08165) was used as the reference gene for normalisation. Beta-actin primers (Forward: 5 0 -ATGAAG CCCAGAGCAAAAGA-3 0 and Reverse: 5 0 -GGGGTGTT GAAGGTCTCAAA-3 0 ) were synthesised by Integrated DNA Technologies (IDT), Singapore. The amplification procedure was performed in a 20 lL reaction volume with final concentration of 1Â QuantiFast SYBR green PCR kit (Qiagen, Hilden, Germany),1 lg cDNA, 400 nM of each forward and reverse primers and 8.5 lL of RNase-free water. The following thermal-cycling conditions were used: PCR initial activation (10min at 95 C), and 40 cycles of denaturation (15 s at 95 C), annealing (20 s at 60 C for ACBD 5 and LCL primers, 62 C for beta-actin primers) and extension (20 s at 72 C). The efficiency of each target and reference genes were validated by titrating respective cDNA template at six serial dilutions in the PCR experiment with optimum efficiencies between 94% and 110% for respective primer pairs. Each sample was run in triplicate, and averaged triplicates were used to assign Cq (quantification cycle) values. Template control was included in each run. The gene expression levels were calculated as described by Ramiah et al. (2014). All mRNA expressions of the samples were normalised to the values of beta-actin that served as the reference gene in this study. The gene expression levels of LPL and ACBD 5 in the treated groups were compared with control groups and expressed as normalised fold-change.

Statistical analysis
Adipocyte cellularity and transcription were analysed using the analysis of variance (ANOVA) procedure of the SAS software package, version 9.1 (SAS Institute Inc., Cary, NC). Significantly different mean values were further elucidated using Duncan's test. The results were expressed as mean and pooled standard error of mean (SEM). 5% or lower p-value is considered to be statistically significant.

Adipose cellularity changes by CLA
The area size, diameter and number of abdominal adipocytes are shown in Figures 1 and 2 respectively. Adipose cellularity data presented in this work revealed that the cis-9, trans-11 significantly decreased adipocyte area size following a reduction in the number of adipocytes. The data clearly indicated that different CLA isomers and concentration had pronounced effect on fat cell cellularity. Figure 1 shows that the cis-9, trans-11-treated groups have significant lower adipocytes area size and diameter compared to the trans-10, cis-12-treated groups and control. As the concentration of cis-9, trans-11 increased, the adipocytes area size and diameter decreased. Trans-10, cis-12 CLA-treated groups had no observable effect on the chicken adipocytes area size and diameter distribution compared to the control group, as shown in Figure 1. C1 has the lowest adipocyte cell count compared to the other treatment group from cis-9, trans-11. Cis-9, trans-11 CLA at a concentration of 1.51% treatment resulted in lower cell numbers compared to the other concentration of cis-9, trans-11. Regardless of the concentration, confluent cells treated with trans-10, cis-12 were unaffected (un-induced cells), as shown in Figure 2.

Transcriptional expression level of LPL and ACBD 5 from CLA isomers treated adipocytes
The expression of the mRNAs of the known adiposespecific genes, LPL and ACBD 5 are shown in Figures 3  and 4, respectively. LPL was downregulated at C2, which was 5-fold lower than the control. No significant differences (p > .05) in LPL expression from other treatment groups were noted (Figure 3). In Figure 4, ACBD 5 was found to be almost 3-fold lower in C2 compared to the control. At the concentration of T1, ACBD 5 was upregulated almost 2-fold higher than C2. No significant changes of ACBD 5 transcription were observed from C1 and T2 compared to the control.   Values were normalized with reference genes, b-actin. Treated samples were expressed relative to transcriptional level of CON group. CON: without CLA isomers; C: (C1) 1.51% and (C2) 2.56% for cis-9, trans-11 (C) and (T1) 1.51% and (T2) 2.56% for trans-10, cis-12 (T). Values are means ± 1 standard error (n ¼ 10). Different superscripts between bars denote significant differences at p < .05. Figure 1. Effects of conjugated linoleic acid (CLA) isomers at different concentrations on the adipocyte area size and diameter of chicken adipocytes compared to CON (control). The data represent the mean cell diameter. Data are presented as means ± SEM. CON: without CLA isomers; (CI) 1.51% and (C2) 2.56% for cis-9, trans-11 (C) and (T1) 1.51% and (T2) 2.56% for trans-10, cis-12 (T). Different superscripts among bars denote significant differences at p < .05.

Discussion
This study has found that the cis-9, trans-11 significantly decreased the adipocyte area size. On the other hand, the primary adipose tissue treated with trans-10, cis-12 concentrations imparted negligible changes on the adipose cellularity. Cartwright (1991) reported that excessive fat deposition is highly correlated with increased adipocyte size. Other studies have demonstrated a similar phenomenon, where feeding with 0.5% and 5% of CLA isomers mixture decreased the size of adipocytes in Zucker rats (Sisk et al., 2001) and chicken (Ramiah et al., 2014), respectively. Besides adipocytes size, our data also suggested that CLA could reduce the number of adipocytes, which may subsequently control the deposition of adipose tissue. Evans et al. (2000) suggested that CLA stimulated preadipocytes differentiation, decreased cell size and decreased triacylglycerol content in the adipocytes cultured, resulted in a decrease in body fat.
In an in vivo trial, broiler chickens fed with cis-9, trans-11 CLA isomer diet showed a smaller number of adipocytes (Ramiah et al., 2014), which is similar with this study. Although these reports utilised the mixture of CLA-isomers, many other reports (Evans et al., 2000;Brown et al., 2001) have suggested that trans-10, cis-12 CLA induced reduction in fat deposition in 3T3-L1 adipocytes and rats. However, our study indicated that cis-9, trans-11 CLA also could reduce fat deposition more effectively. This may be due to different biological effects produced by cis-9, trans-11 and trans-10, cis-12 CLA (Pariza et al., 2001). Thus, the use of CLA preparations containing different amounts and ratios of CLA isomers should be considered when utilised in different biological subjects.
To understand the regulation of these CLA isomers on fat metabolism of adipocytes during differentiation, expression of LPL and ACBD genes were evaluated using the quantitative real-time PCR. These target genes were selected as they play central roles in fat deposition where LPL is involved in the regulation of lipid metabolism (Hermier, 1997). ACBD families act as an intracellular acyl-CoA pool formers and transporters that play critical roles in lipid metabolism and energy homeostasis (Stewart et al., 2014).
In the current study, LPL expression was downregulated by cis-9, trans-11 CLA at 2.56%. Similar to our result, Park et al. (1997) have reported that the LPL expression in the CLA-treated 3T3-L1 adipocytes was reduced compared to the control cells. In contrast, porcine stromal-vascular cells (preadipocytes) containing CLA was found with increasing LPL mRNA expression at days 5 and 7 (McNeel & Mersmann, 2003). Differentiation of 3T3-L1 pre-adipocytes to full adipocytes resulted in a 6-fold increase in LPL mRNA expression (Gonzales & Orlando, 2007). In the same study, reduction in adipocyte LPL expression resulted in a parallel decrease in lipid storage of $80% (Gonzales & Orlando, 2007). Taken together, downregulation of LPL gene in the current study is in parallel with the reduction of adipocyte size which reduced lipid accumulation.
There are several families of lipid-binding proteins that regulate homeostasis and function of fatty acids, such as ACBDs and fatty acid-binding protein (FABP) (Chen et al., 2012). ACBD 5 was involved in triglyceride synthesis and b-oxidation (Ji et al., 2012) as well as in the distribution of long-chain acyl-coenzyme A (Cheng et al., 2015). The current study shed light on the role of ACBD 5 in CLA-mediated adiposity defects. It seems that ACBD 5 was upregulated at lower level of trans-10, cis-12 CLA, but inhibited by higher levels of cis-9, trans-11 CLA. This showed that the effect of isomerspecific action of CLA on ACBD 5 expression levels. In view of the roles of ACBD 5 on the mobilisation and b-oxidation of fats, it is postulated that higher level of cis-9, trans-11 CLA isomers coupled with decreased LPL genes expression levels.
It is apparent that this results in lower net deposition in the adipose tissue. This is also consistent with the view of those long-chain fatty acids, available in lesser amount when ABCD 5 expression is downregulated. It is important to extend further studies on ACBDs regarding its role in lipid haemostasis and therapeutic potential for diseases attributed to the imbalance of lipid metabolism, such as obesity, diabetic and cancer (Chen et al., 2012).

Conclusions
Our study has demonstrated the efficacy of cis-9, trans-11 CLA to out-perform trans-10, cis-12 CLA in reducing adiposity in chicken primary adipocytes, as opposed to previous claims that trans-10, cis-12 CLA played a bigger role. Understanding the isomer-specific action of CLA on the adipocyte will help us to devise designer CLA mixtures that, when fed to chicken, will result in higher quality chicken products that are healthier to the consumer. Therefore, the effect of feeding pure CLA isomers needs to be addressed in depth if the fatty acids is to be used to alter the accumulation of fats chicken and also in other species. research was supported by the Malaysian Government Fundamental Research Grant Scheme (FRGS) under grant number 5450632.

Disclosure statement
The authors declare no conflict of interest regarding the publication of this paper.