The effects of feeding conjugated linoleic acid on pig ...

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The effects of feeding conjugated linoleic acid on pig liver vitamin A and retinol binding protein mRNA

Can. J. Anim. Sci. Downloaded from pubs.aic.ca by Agriculture and Agri-food Canada on 01/19/15 For personal use only.

M. E. R. Dugan, D. C. Rolland, D. R. Best, and W. J. Meadus Meat Research Section, Agriculture and Agri-Food Canada, Lacombe Research Centre, 6000 C&E Trail, Lacombe, Alberta, Canada T4L 1W1 (e-mail: [email protected]). Received 21 September 2001, accepted 22 May 2002. Dugan, M. E. R., Rolland, D. C., Best, D. R. and Meadus, W. J. 2002. The effects of feeding conjugated linoleic acid on pig liver vitamin A and retinol binding protein mRNA. Can. J. Anim. Sci. 82: 461–463. Banni et al. (1999) indicated feeding conjugated linoleic acid (CLA) increases rat liver retinol and retinyl-ester levels. We wanted to determine if feeding CLA would affect pig vitamin A status. Feeding 0.5% CLA did not increase pig liver retinyl-palmitate but did increase retinol from 1.56 to 2.56 µg g–1 (P < 0.05) and the level of retinol binding protien mRNA relative to actin mRNA (P < 0.05). Key words: Conjugated linoleic acid, pig, vitamin A, retinol, retinol binding protein Dugan, M. E. R., Rolland, D. C., Best, D. R. et Meadus, W. J. 2002. Incidence de l’addition d’acide linoléique conjugué à la ration sur la concentration de vitamine A et d’ARNm codant la protéine RBP dans le foie du porc. Can. J. Anim. Sci. 82: 461–463. Selon Banni et ses collaborateurs (1999), l’addition d’acide linoléique conjugué (ALC) à la ration augmente la concentration de rétinol et d’ester de rétinyle dans le foie du rat. Les auteurs voulaient savoir si l’ingestion d’ALC par le porc modifie le bilan de la vitamine A. L’addition de 0,05 % d’ALC à la ration des porcs n’augmente pas la concentration de palmitate de rétinyle dans le foie du porc, mais celle de rétinol passe de 1,56 à 2,56 µg g–1 (P < 0,05) et on observe une hausse de la quantité d’ARNm codant la protéine RBP par rapport à celle d’ARNm codant l’actine (P < 0,05). Mots clés: Acide linoléique conjugué, porc, vitamine A, rétinol, protéine RBP

total of 36 animals per diet. We, however, arbitrarily limited our vitamin A analyses to 10 pigs from each of the 2% total oil diets and these originated from four blocks of pigs. Diet compositions were reported previously (Dugan et al. 2001) and met or exceeded National Research Council (1998) nutrient requirements. Diets were typical commercial grain (barley and wheat) based grow/finish rations (16.6% CP, 14.2 MJ kg–1 DE, 1% lysine, 0.65% threonine, 2% total added oil) and had 8000 IU vitamin A added per kg [4.39 mg retinyl-palmitate (RP) per kg of diet]. The CLA oil fed contained 65% CLA (33% cis-9, trans-11-CLA and 32% trans-10, cis-12-CLA). The CLA was a gift from Conlinco Inc. (Detroit Lakes, MN) and was synthesized using proprietary methods. The stock oil used to prepare the CLA was first methylated and then further refined by distillation and collection of fatty acid methyl esters, and this, incidentally, left any vitamin A/pro-vitamin A in the residual. Diets were fed from 37 to 116 kg liveweight, and at 116

Conjugated linoleic acid (CLA) has been shown to have anti-cancer (Scimeca 1999) and fat-to-lean repartitioning effects in a number of animal models (Jahreis et al. 2000). CLA has also been shown to protect against atherosclerosis (Lee et al. 1994; Nicolosi et al. 1997) and prevent growth depression induced by immune simulation (Cook et al. 1993; Miller et al. 1994). The mechanism(s) of CLA action are not, however, clearly understood. Recently, Banni et al. (1999) found feeding CLA to rats increased liver retinol (R) and retinyl-ester levels and indicated some CLA effects might be linked to vitamin A because both have been shown to inhibit mammary gland development and tumorogenisis. We, therefore, wanted to determine if feeding CLA would affect pig liver vitamin A levels and potentially vitamin A requirements for pigs. Pigs used for this study were raised in compliance with CCAC (1993) guidelines. Livers for analyses had been collected prior to inception of this study. Growth performance, average daily gain, feed conversion and carcass composition data for the original study have previously been reported (Dugan et al. 2001). In the original study, three levels of dietary CLA oil (0, 0.25 and 0.5%) were fed to barrows in combination with canola oil to make up 2 and 5% total oil. The original experiment comprised 12 blocks of six pens (i.e., one pen per diet per block) and three pigs per pen for a

Abbreviations: CLA, conjugated linoleic acid; CP, crude protein; DE, digestible energy; mRNA, messenger ribonucleic acid; PPAR, peroxisomal proliferator activated receptor; R, retinol; RP, retinyl-palmitate; RBP, retinol binding protein 461

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CANADIAN JOURNAL OF ANIMAL SCIENCE

Table 1. The effects of feeding CLA to barrows from 37 to 116 kg on growth performance, liver weight, liver vitamin A status and relative level of liver retinol binding protein (RBP) mRNAz Dietary CLA (%) Days on feed (d) Avg. daily gain (kg.d–1) Liver weight (kg)


Retinol (µg g–1) Retinyl-palmitate (µg g–1) Retinol to retinyl palmitate ratio Relative RBP mRNA zValues are means. yStandard error. xNot determined.

0%

0.25%

0.5%

SEy

83.4 0.907 1.94

83.5 0.992 1.97

84.2 0.947 1.99

0.76 0.016 0.03

1.56a 599 0.0026a 1.30a

1.92a 628 0.0031a NDx

2.56b 589 0.0045b 1.52b

0.11 21.6 0.0002 0.04

For each diet, n = 10.

a, b Means within row with different letters are different (P < 0.05).

kg pigs were stunned (400 V for 3 s) and slaughtered at the Lacombe Research Centre abattoir and liver samples were frozen and stored at –80°C until analyses. All extractions and analyses for vitamin A were conducted under subdued lighting. For each liver sample, 1 g was pulverized at dry ice temperature (Kramer and Hulan 1978). Total lipids were then extracted using the method described by Bligh and Dyer (1959), and then dried under vacuum and resuspended in 10 mL of chloroform/methanol (2:1, vol/vol containing 1 mg mL–1 butylated hydroxytoluene). The final concentration of liver immediately prior to HPLC analysis was 37.5 mg mL–1 of pre-extraction weight and retinylacetate was added as an internal standard (2.4 µg mL–1). Vitamin A was analyzed using a Varian HPLC system (Varian, 2700 Mitchell Drive, Walnut Creek, CA 945981675, USA) including a Prostar 230 HPLC, Prostar 410 Autosampler and Prostar 360 fluorescence detector set at 300 nm excitation and 480 nm emission. Twenty µL of sample or standard was injected and R and retinyl-esters were separated on a 15 cm × 4.6 mm Supelcosil LC-18 column (3 µm particle size) protected with a Pelligard column (4 cm × 4.6 mm with 40 µm particle size LC-18 packing) using two 1.1 mL per min isocractic elutions (6 min of 96:4 methanol:water followed by 18 min of 80:20 methanol: ethanol). Retention times for R, retinyl-acetate and RP were 3.7, 5.1 and 19 min, respectively. These retention times compare favorably to those indicated by Banni et al. (1999); however, our solvent composition was adjusted to compensate for our larger column diameter (4.6 mm vs. 3 mm) and smaller diameter packing material (3 µm vs. 5 µm). Relative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was also performed on liver samples from pigs fed 0 and 0.5% CLA to determine the relative level of retinol binding protein (RBP) mRNA. Total RNA was isolated from 100 mg of liver tissue using the guanidine isothiocyanate based TRIzol solution (InVitrogen, Scarborough, ON) according to the manufacturers specifications (Chomczynski and Sacchi 1987). Oligo-(dT)10 was used as primer in the first step of cDNA synthesis using total RNA and MMLV Reverse Transcriptase (Sigma-Aldrich, Oakville, ON). RNA expression was measured using a rela-

tive RT-PCR method (Spencer and Christensen 1999). To measure the RBP mRNA levels (GenBank accession #M68860), new primers designated RBP+221for (5’-ttt ctg cag gac aac atc gtc g-3) and RBP + 565rev (5’-ttt ctg cac ttc tgg gga gaa gc-3’) were used to generate a 344 bp PCR fragment. Polymerase chain reactions were performed on a PTC-200 PCR machine (MJ Research Inc, MA) using ~100 ng of cDNA, and REDTaq Polymerase (Sigma, Oakville, ON). The PCR program initially started with a 95°C denaturation for 5 min, followed by 95°C for 1 min, 60°C for 1 min, 72°C for 1 min for 26, 28, and 30 cycles, which was predetermined to be in the exponential amplification range of the RBP signal. The PCR reactions were completed with a 72°C for 10 min final elongation step then cooled to 4°C. No PCR fragments were generated when tested on porcine genomic DNA. As an internal standard, β-actin mRNA (GenBank accession #SSU23954) was co-amplified using the primers β-actin for (5’-gga ctt cga gga gat gg-3’) and βactin rev (5’-gca ccg tgt tgg cgt aga gg-3’) to generate a 233 bp PCR fragment. Primers for the β-actin sequence were added at cycle number 10 to generate an endpoint signal from the predetermined exponential amplification range of 16 to 20 cycles. The PCR samples were electrophoresed on 8% polyacrylamide gels (8 × 10 cm) or 2.5% agarose TBE gels and stained with ethidium bromide (10 µg per mL) and photographed on 280 nm UV light source. 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 a 20 bp DNA ladder standard (BioRad, Mississauga, ON). Changes in RBP mRNA expression were determined relative to β-actin mRNA levels. The General Linear Model procedure of the SAS Institute, Inc. (1990) was used for statistical analysis. The model included block, diet and diet by block interaction as factors and the Student-Newman-Keuls’ multiple comparison test was used to determine if diet means were different (P < 0.05)(Steel and Torrie 1980). Feeding CLA did not affect liver weight, days on feed or average daily gain (Table 1) and, as previously reported, feed intake did not differ between diets (Dugan et al. 2001).


DUGAN ET AL. — CONJUGATED LINOLEIC ACID EFFECTS ON LIVER VITAMIN A

Feeding 0.5% CLA increased liver R (P < 0.05) and the R/RP ratio (P < 0.05; Table 1) compared to controls. Standards for retinyl-esters other than RP were not available, but if the RP response factor was used for other tentatively identified retinyl-esters, RP comprised 82.2% of retinylesters and this value did not change for control versus CLA added diets (data not shown). In rats, Banni et al. (1999) found that feeding 0.5% CLA increased liver R by 74% and retinyl-esters by 49% and also increased mammary R levels by 115%. Banni et al. (1999) speculated that higher levels of R and retinyl-esters may in part have resulted from CLA increasing intestinal mucosal RBP and an increased absorption of R. In addition, the higher liver R and R/RP ratio may have resulted from higher liver-RBP (i.e., more protein bound retinol versus esterified retinol). Conjugated linoleic acid is a known ligand for the peroxisome proliferator-activated receptor-α (PPAR-α)(Bleury et al. 1997) and PPAR-α has recently been demonstrated to be involved in RBP expression in intestinal cells (Suzuki et al. 1998). The potential for CLA to increase levels of liver RBP is thus further supported by our finding that CLA increases the relative level of RBP mRNA in liver (P < 0.05; Table 1). Feeding pigs CLA at the 0.5% level, therefore, altered vitamin A status in a second animal model by increasing the level of R, the R/RP ratio and the relative level of RBP mRNA in pig liver. Vitamin A in pigs is mainly stored in the liver as RP, and because the level of liver RP was unaltered, feeding CLA will likely not change pig vitamin A requirements. The active forms of vitamin A (retinol, retinal and retinoic acid) are derived from RP via hydrolysis and progressive oxidation. Feeding CLA may still, therefore, induce some of its physiological effects by changing the balance between protein bound and esterified retinol. Further work is needed to determine to what extent CLA’s physiological effects may be attributed to its effects on tissue PPAR receptors, RBP and vitamin A levels. The CLA used for this study was a gift from Conlinco Inc. The authors wish to thank Sigrid Windsor and the staff of the Lacombe Research Centre swine unit for their assistance in the procurement, feeding, weighing and delivery of pigs and the staff at the Lacombe Research Centre abattoir for the slaughter and dressing of pigs. The technical assistance provided by F. Costello, I. Larsen, R. Thacker, and Robin McInnis is also greatly appreciated. Banni, S., Angioni, E., Casu, V., Melis, M. P., Scrugli, S., Carta, G., Corongiu, F. P. and Ip, C. 1999. An increase in vitamin A status by feeding conjugated linoleic acid. Nutr. Cancer 33: 53–57. Bleury, M. A., Moya-Camarena, S. Y., Liu, K. L. and Vanden Heuvel, J. P. 1997. Dietary conjugated linoleic acid induces peroxisome-specific enzyme accumulation and ornithine decarboxylase activity in mouse liver. J. Nutr. Biochem. 8: 579–584.

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Bligh, E. G. and Dyer, W. J. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37: 911–917. Canadian Council on Animal Care. 1993. A guide to the care and use of experimental animals. Vol.1 2nd ed. E. D. Olfert, B. M. Cross, and A. A. McWilliams, eds. CCAC, Ottawa, ON. Chomczynski, P. and Sacchi, N. 1987. Single step method of RNA isolation by guanidium thiocyanate phenol chloroform extraction. Anal. Biochem. 161: 156–159. Cook, M. E., Miller, C. C., Park, Y. and Pariza, M. 1993. Immune modulation by altered nutrient metabolism – nutritional control of immune-induced growth depression. Poultry Sci. 72: 1301–1305. Dugan, M. E. R., Aalhus, J. L., Lien, K. A., Schaefer, A. L. and Kramer, J. K. G. 2001. Effects of feeding different levels of conjugated linoleic acid and total oil to pigs on live animal performance and carcass composition. Can. J. Anim. Sci. 81: 505–510 Jahreis, G., Kraft, J., Tischendorf, F., Schone, F. and von Loeffelholz, C. 2000. Conjugated linoleic acids: Physiological effects in animal and man with special regard to body composition Eur. J. Lipid Sci. Technol. 102: 695–703. Kramer, J. K. G. and Hulan, H. W. 1978. A comparison of procedures to determine free fatty acids in heart. J. Lipid Res. 19: 103–106 Lee, K. N., Kritchevsky, D. and Pariza, M. W. 1994. Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 108: 19–25. Miller, C. C., Park, Y., Pariza, M. W. and Cook, M. E. 1994. Feeding conjugated linoleic acid to animals partially overcomes catabolic responses due to endotoxin injection. Biochem. Biophys. Res. Commun. 198: 1107–1112. National Research Council. 1998. Nutrient requirements of swine. 10th ed. National Academy Press, Washington, DC. Nicolosi, R. J., Rogers, E. J., Kretchevsky, D., Scimeca, J. A. and Huth, P. J. 1997. Dietary conjugated linoleic acid reduces plasma lipoproteins and early aortic atherosclerosis in hypercholesterolemic hamsters. Artery 22: 266–277. SAS Institute, Inc. 1990. SAS/STAT® user’s guide. Release 6.08. SAS Institute, Inc., Cary, NC. Scimeca, J. A. 1999. Cancer inhibition in animals. Pages 420–443 in M. P. Yurawecz et al., eds. Advances in conjugated linoleic acid research. Vol 1. AOCS Press, Champain, IL. Spencer, W. E. and Christensen, M. J. 1999. Multiplex relative RT-PCR method for verification of differential gene expression. BioTechniques 27: 1044–1052. Steel, R. G. D. and Torrie, J. H. 1980. Principles and procedures of statistics a biometric approach. McGraw-Hill, New York, NY. Suzuki, R., Suruga, K., Goda, T. and Takase, S. 1998. Peroxixsome proliferator enhances gene expression of cellular retinol-binding protein, type II in Caco-2 cells. Life Sci. 62: 861–871.
