A semi-quantitative RT-PCR method to measure the in vivo effect of dietary conjugated linoleic acid on porcine muscle PPAR gene expression.

Conjugated linoleic acid (CLA) can activate (in vitro) the nuclear transcription factors known as the peroxisome proliferators activated receptors (PPAR). CLA was fed at 11 g CLA/kg of feed for 45d to castrated male pigs (barrows) to better understand long term effects of PPAR activation in vivo. The barrows fed CLA had lean muscle increased by 3.5% and overall fat reduced by 9.2% but intramuscular fat (IMF %) was increased by 14% (P < 0.05). To measure the effect of long term feeding of CLA on porcine muscle gene expression, a semi-quantitative RT-PCR method was developed using cDNA normalized against the housekeeping genes cyclophilin and beta-actin. This method does not require radioactivity or expensive PCR instruments with real-time fluorescent detection. PPARgamma and the PPAR responsive gene AFABP but not PPARalpha were significantly increased (P < 0.05) in the CLA fed pig's muscle. PPARalpha and PPARgamma were also quantitatively tested for large differences in gene expression by western blot analysis but no significant difference was detected at this level. Although large differences in gene expression of the PPAR transcriptional factors could not be confirmed by western blotting techniques. The increased expression of AFABP gene, which is responsive to PPAR transcriptional factors, confirmed that dietary CLA can induce a detectable increase in basal PPAR transcriptional activity in the live animal.

Introduction

In 1997, Dugan and coworkers (1) reported that Conjugated Linoleic acid (CLA) acted as effective fat to lean “repartitioning” agent in swine. Adding CLA to 2% of the feed, increased lean muscle by 2.3% while reducing overall subcutaneous fat 6.8% in genetically lean, Large White pigs. In vitro studies using cell cultures have demonstrated that CLA acts as an agonist of the peroxisome proliferator activated receptor family (PPAR). PPARs are DNA binding transcription factors that bind the peroxisome proliferator repeat element (PPRE) (2, 3, 4) as a heterodimer with the retinoic acid receptor (RxR), which is activated by 9-cis-retinoic acid (vitamin A) (5, 6). The PPAR family is currently divided into three subgroups, α, β and γ. PPARβ is expressed throughout the body and is involved in embryo development (7, 8). PPARα has a 1000-fold higher affinity for CLA than PPARγ and both are expressed in hepatic, muscle, pancreatic, and adipose tissue.

Total RNA was prepared from pig muscle samples to examine if the CLA induced physiological changes were by differences in gene expression. The mRNA amount of a transcriptional factor such as PPARγ is often relatively very low and difficult to detect using standard Northern blot procedures. As a result, Northern blot methods usually require additional time consuming selective chromatography to isolate poly A+ mRNA and radioactive labeled probes to generate a measurable signal to quantify. Therefore, in order to reduce labor, a semi-quantitative RT-PCR method was developed. RT-PCR is the most sensitive method for detecting gene transcription products; however, it can be highly variable and may not accurately reflect gene activity (9). Subsequently, western blotting was used to quantitatively measure total PPAR protein. Ultimately however, even protein levels may not reflect actual gene activity due to post-translational modifications; therefore, the PPRE responsive genes, adipocyte fatty acid binding protein (AFBAP) and acyl Co A oxidase (ACO), were measured to confirm PPAR activity with its transcription rate.

Often a direct link cannot be made between a physiological response and a genetic marker due to missing biochemical information. Some studies in swine have found that dietary CLA increased fasting levels of serum insulin (10). Insulin increases muscle nutrient absorption. Glutamine:Fructose aminotransferase (GFAT) is the rate-limiting enzyme in the hexosamine pathway, which is involved in producing glycosylation substrates (11). GFAT mRNA expression is increased when intercellular concentrations of glucose or fatty acids are high (12). Therefore the expression of GFAT was measured to examine if the increase lean mass in pigs fed CLA was due to more available energy inside the muscle cells.

Materials and Methods

Animal treatments

Muscle samples from 20 castrated male Landrace X Large White pigs (barrows) were used in this study (1). The pigs were fed either a CLA supplemented diet containing 2%-conjugated linoleic acid (n = 10) or a Control diet containing 2% sunflower oil (n = 10). The isomeric composition of the CLA feed has been previously reported (13). The diets were formulated to meet nutrient requirements as outlined by the National Research Council (14). The feeding trial commenced when the pigs reached an average weight of 60 kg and lasted for 45 days. The pigs were slaughtered at an average weight of 105 kg, as outlined in Dugan (1). The animals were slaughtered at the Lacombe Research Center in accordance with the principles and guidelines set by the Canadian Council on Animal Care (15). Animal carcass evaluations for lean muscle (Lean) and subcutaneous fat (SQ) levels were calculated from in-depth dissections according to the procedures described by Martin (16). Intramuscular fat % (IMF %) was estimated from dried ground longissimus thoracis (LT) muscle (Method 39.1.05: AOAC 1995) (17).

RNA Isolation

Total RNA was isolated from 100 mg of longissimus thoracis (LT) muscle cores using the guanidine isothiocyanate based TRIzol solution (GIBCO-BRL, Burlington, ON, Canada) according to the manufacturer’s specifications (18). The RNA samples were resuspended in 100% formamide and quantified spectrophotometrically at 260nm. All RNA isolates had an OD260:OD280 between 1.8 and 2.0, indicating clean RNA isolates. The RNA quality was also checked by 1.0% agarose gel electrophoresis, stained with 1 ug/ml ethidium bromide. Genomic DNA was isolated from the meat samples using the guanidium based DNAzol solution (GIBCO-BRL, Burlington, ON, Canada) as outlined in Meadus and MacInnis (19).

Relative Reverse Transcriptase - Polymerase Chain Reaction (RT-PCR) analysis

A two-step semi-quantitative RT-PCR method was used to measure gene expression in the LT muscle samples at the time of slaughter. Oligo-(dT)10n (InVitrogen, Burlington ON) was used as primer in the first step of cDNA synthesis. Total RNA (5 μg) was combined with 0.5 μg oligo-dT, 200 μM dNTPs and H20 and preheated at 65oC for 2 min to denature secondary structures. The mixture was then cooled rapidly to 20oC and then 10 μl 5X RT Buffer, 10 mM DTT and 200 U MMLV Reverse Transcriptase (Sigma-Aldrich, Oakville, ON, Canada) was added for a total volume of 50 μl. The RT mix was incubated at 37oC for 90 min. then stopped by heating at 95oC for 5 min. The cDNA stock was stored at -20oC. The yield of cDNA was measured according to the PCR signal generated from the internal standard house-keeping gene cyclophilin [Genebank accession #AY008846] or β-actin amplified from 18 to 24 cycles starting with 0.1 μl of the cDNA solution. The volume of each cDNA pool was adjusted to give the same exponential phase PCR signal strength for β-actin after 20 cycles.

Relative RT-PCR (20) was performed to measure gene expression of PPARα, PPARγ, AFABP, GFAT, ACO, and m-calpain mRNAs. Primer sequences and optimal PCR annealing temperatures (ta) and are listed in Table 1. To insure that no false positive PCR fragments would be generated from pseudogenes in contaminating genomic DNA; primer sequences were designed to span intron regions, when genomic sequence data was available. In addition, all PCR primer combinations were tested using porcine genomic DNA as a negative control. Polymerase chain reactions were performed on a PTC-200 PCR machine (MJ Research Inc, MA, USA) using ~100 ng of cDNA, 5 pmoles each oligonucleotide primer, 200 μM of each dNTP, 1 unit of REDTaq Polymerase (Sigma-Aldrich, Oakville, ON, Canada) and 1X REDTaq polymerase buffer in a 20 μl volume. The PCR program initially started with a 95oC denaturation for 5 min, followed by 28 to 38 cycles of 95oC/1 min, ta oC /1 min, 72oC/1 min. Linear amplification range for each gene was tested on the adjusted cDNA. The less expressed transcripts of PPARα, and PPARγ required >32 cycles of PCR for detection. B-actin primers were added when 20 cycles were remaining in the specified gene’s linear amplification range. The PCR samples were electrophoresed on 8% polyacrylamide gels (8 X 10 cm) or 2.5% agarose gels in TBE buffer [89mM Tris-base pH 7.6, 89mM boric acid, 2mM EDTA]. The gels were stained with ethidium bromide [10 μg/ml] and photographed on top of a 280 nm UV light box. The gel images were digitally captured with a CCD camera and analyzed with the NIH Imager beta version 2 program. The quantity and base pair size of the PCR generated DNA fragments were estimated relative to DNA ladder standards. Densitometry values were measured at each cycle sampling using the One-Dscan software (Scanalytics, Fairfax, VA). RT-PCR values are presented as a ratio of the specified gene’s signal in the selected linear amplification cycle divided by the β-actin signal (Fig. 1).

Table 1

Sequence, expected fragment size and annealing temperature (ta) of primers used in the semi-quantitative RT-PCR analysis of mRNA levels of the genes expressed in porcine LT muscle.

Gene	Sequences	Expected fragment size (bp)	ta (oC)	GenBank accession number
PPARα	Sense: 5’-tta cag act acc agt act tag g-3’Antisense: 5’-agg ctt tgt ccc cac ag-3’	176	58	AF175309
PPARγ	Sense: 5’-tga cca tgg ttg aca ccg-3’Antisense: 5’-aag cat gaa ctc cat agt gg-3’	381	58	SSC6756
AFABP (aP2)Exon 1 → 2	Sense: 5’-tgg aaa ctt gtc tcc agt g-3’Antisense: 5’-ggt act ttc tga cta atg gtg-3’	147	56	SSY16039
Cyclophilin	Sense: 5’-acc gtc ttc ttc gac atc g-3’Antisense: 5’-caa cca ctc agt ctt ggc-3’	330	62	AY008846
Calpain 1Exon 1 → 2	Sense: 5’-tgg aag gac tgg agc ttt gt-3’Antisense: 5’-gac tcc agg gca tca gct g-3’	152	60	SSU23954
ACO	Sense: 5’-ccg gag ctg ctt aca cac at-3’Antisense: 5’-ggt cat acg tgg ctg tgg tt-3’	427	60	AF185048
GFAT	Sense 5’-aac cca gtc ctg tca ata gcc-3’Antisense 5’-acg aga gag att gca gct tcc-3’	420	60	AF441244
β-actin	Sense: 5’-gga ctt cga gca gga gat gg-3’Antisense: 5’-gca ccg tgt tgg cgt aga gg-3’	233	61	SSU07786

Fig. 1

Gel image of relative RT-PCR for A) PPARγ (381bp) at 34 cycles with β-actin (233bp) added at cycle 14 to act as the internal control. B) GFAT (420bp) at 34 cycles with β-actin added at cycle 14. C) AFABP (147bp) at cycle 26 with β-actin added at cycle 4. C; control fed animals. T: CLA fed animals.

Protein isolation and analysis

Protein extracts were prepared from the thawed muscle samples by homogenizing 100 mg of muscle in 1 ml of extract buffer A [100 mM Tris-HCl pH 7.5, 0.5 mM dithiothreitol, 2 mM phenymethylsulfonyl fluoride (PMSF), 5 mM ethylenediaminetetraacetic acid (EDTA), 500 mM NaCl, and 0.1% Triton X-100. The homogenate was then centrifuged at 1000 xg for 5 min to pellet large cellular debris. Protein concentrations were measured using the BCA method (Pierce, Rockford, IL). Samples were diluted to 1 ug/ul with extract buffer and then 20 ug were mixed with an equal volume of SDS-PAGE loading buffer [50 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.002% bromophenol blue], heated at 90oC for 5 min then loaded onto 10% SDS-PAGE for electrophoresis in TBE. To perform the Western analysis, the separated proteins were electroblotted to Protran nitrocellulose (Schleicher, Schuell, Keene, NH) and then probed with rabbit anti-rat m-calpain (Sigma, St. Louis, MO), rabbit anti-synthetic PPAR (Sigma, St. Louis, MO), and mouse anti-rabbit GAPDH (Chemicon Intl. Inc, Temecula, CA) which acts as an internal standard between samples. The primary antibodies were detected with secondary anti-rabbit or anti-mouse IgG conjugated with alkaline phosphatase to generate a NBT/BCIP signal (Roche Diagnostics, Laval, PQ). To help in identification, prestained SDS-PAGE protein broad range standards (BioRad, Hercules, CA) were included on the 10% SDS-PAGE. The protein standards were visible on the Protran western blots. The antibody labeled m-calpain (58kDa), PPARα (~54 kDa), PPARγ (~55 kDa) or GADPH (~35kDa) polypeptide bands on the developed blots were digitally captured with a HP Scanjet 5100C scanner (Helwett Packard Co., Greeley, CO) and band intensities were measured using the One-D Scan program (Scanalytics, Fairfax, VA). Western blotting gives a more direct quantitative of a gene’s expression level but depends on the availability of specific antibodies and is less sensitive than RT-PCR. Protein quantity also doesn’t necessarily reflect gene product activity, since the protein or enzyme may still undergo post-translational modifications.

Statistical analysis

RT-PCR signals were averaged from at least 3 replicates using 2 quantities of cDNA from 10 CLA and 10 Control samples. The RT-PCR ratio values were analyzed using GLM Frequency and Correlation procedures (21). Western blot data was generated from 3 replicate runs using 7 CLA samples and 7 Control samples. Significance was calculated using Student’s t-test.

Results and Discussion

In this study, barrows fed 2% CLA enriched oil for 45d had a significant reduction in subcutaneous fat (SQ) compared to pigs fed diets containing 2% sunflower oil. However, 2% CLA also significantly increased IMF%. The changes in physiology are believed to be due to CLA activating the nuclear transcription factor PPAR (2). The relationship between IMF and dietary CLA are explained in greater detail in previously published work of Meadus et al. (2002) (22). The relative gene expression level in LT muscle from pigs fed either the control diet or CLA diet is shown in Fig. F%[2]%. In the LT muscle of animals fed CLA; GFAT, AFABP, and PPARγ mRNAs were significantly (P < 0.05) increased 36.1%, 20.4% and 26.0% respectively. However, Western blotting analysis (Fig. F%[3]%) could not detect a difference in LT muscle PPARγ protein content between Control and CLA treatments.

Western blot of muscle calpain, PPARa (53 kDa) and GAPDH (35kDa) polypeptides labeled with conjugated antibodies from pigs fed control and CLA supplemented diets. The proteins were isolated from muscle homogenates, separated on 10% SDS-PAGE and electroblotted to nitrocellulose membrane as mentioned in the material and methods section. Blots were colormetrically labeled using alkaline phosphatase to generate a NBT/BCIP signal. Western blot A) labeled PPARα and GADPH signal B) m-calpain signal. Lanes 1-7 control diet, Lanes 8-14 CLA diet, std, SDS-PAGE prestained protein standard.

The adipocyte fatty acid binding protein (AFABP) is known to contain PPRE binding sites in its upstream promoter region (23, 24) and 2% CLA did increase AFABP gene expression (Fig. 2) in this trial by 26% (P < 0.05). Acyl-CoA oxidase (ACO) also contains a PPRE in it promoter and is expressed in both muscle and liver cells. However in rat studies (25), ACO mRNA is increased only in liver but not in muscle, by a CLA diet. The pig muscle ACO also did not show a significant response to dietary CLA. Since IMF adipose was not dissected from the lean muscle tissue for individual testing of RNA, it was possible that the observed increase in PPARγ expression was an artifact of the increased IMF% in the LT muscle from the CLA fed group. To investigate the possibility of sampling variance, a muscle to fat ratio was calculated using the gene markers, m-calpain (muscle), and AFABP (adipocytes). There was still a significant increase in GFAT and PPARγ mRNA expression in the 2% CLA fed animals after adjusting for the relative level of fat in each muscle sample.

Fig. 2

Adult muscle gene expression in barrows fed CLA (n = 10) relative to the Control sunflower diet (n = 10). Gene transcript quantity was measured by relative RT-PCR using the internal standard β-actin RT-PCR signal as the denominator as described in the materials and methods section. Error bars represent standard deviation. Significance is represented as (*) P<0.05, (**) P<0.01.

Recent pig studies have shown that prolonged dietary treatment with 3% CLA increases the plasma fasted levels of both free fatty acids and insulin (10, 26). One of the effects from increased insulin sensitivity is increased protein retention due to reduced skeletal muscle catabolism (27). The protease m-calpain mRNA was measured to determine if dietary CLA reduced protein catabolism. The mRNA and protein level of m-calpain was not significantly reduced by CLA in this trial. Increased insulin activity also increases in the number of insulin receptors and glucose transporters (GLUT) on the plasma membrane of skeletal muscle (28). The net effect is increased intercellular glucose and reduced protein catabolism needed to supply glycogenic amino acids (29). Expression of GFAT mRNA in the hexosamine pathway is increased when an excess amount of intercellular glucose is available. GFAT mRNA expression was significantly increased in the CLA fed pigs. Antibodies for porcine GFAT protein were not available for comparison and plasma insulin levels were not measured in this trial. However, the increased GFAT mRNA expression did indicate that intercellular glucose and possibly plasma insulin was increased by the dietary CLA. This supports the hypothesis that feeding CLA to the pigs increased muscle insulin sensitivity causing an increase in muscle intercellular glucose, GFAT expression, and the hexosamine pathway (12).

To conclude, the semi-quantitative RT-PCR results show that prolonged dietary CLA increased gene transcription of PPARγ, GFAT and AFABP in LT muscle core samples. The increased rate of gene transcription was not detected using western blotting techniques for these same genes but since the PPAR inducible AFABP gene was increased by CLA, it demonstrates that PPAR functional activity was increased in the muscle. Lean muscle mass and GFAT mRNA levels were also increased indicating that the intercellular supply of glucose was improved in the muscle of pigs fed CLA.