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ANIMAL NUTRITION |
Department of Animal Nutrition and Feed Science, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, P. R. China
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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(PPAR), IL-1β0, IL-6, and tumor necrosis factor-
(TNF-); and plasma concentrations of the 3 proinflammatory cytokines were measured and analyzed. Average daily feed intake did not differ among treatment groups (P > 0.05), but ADG (P < 0.05) and G:F (P < 0.01) responded quadratically to the duration of linseed diet feeding, and pigs in the 60-d treatment group had the greatest ADG and G:F. The mRNA expression of PPAR in loin muscle and spleen increased linearly (P < 0.01) with the duration of linseed diet feeding, whereas its expression in adipose tissue was not affected (P = 0.095). Tumor necrosis factor- and IL-6 mRNA expression in muscle, adipose, and spleen, as well as serum concentration of TNF-, decreased linearly (P < 0.01) with the duration of linseed diet feeding. Peroxisome proliferator-activated receptor- mRNA abundance was negatively correlated with IL-1β, IL-6, and TNF- mRNA abundance both in muscle (R2 = 0.63, P < 0.001) and in spleen (R2 = 0.69, P < 0.001), and PPAR mRNA expression in spleen (R2 = 0.59, P < 0.01) and muscle (R2 = 0.52, P < 0.05) was negatively correlated with serum TNF- concentration. There were also significant quadratic relations between ADG and expression of PPAR (P < 0.05) and splenic TNF- (P < 0.05). These data suggest that intake of n-3 PUFA from the linseed diet led to significant decreases in the expression of proinflammatory cytokine genes, which may stimulate growth in growing-finishing barrows, at least in part, through a PPAR-dependent mechanism.
Key Words: growth • linseed • n-3 polyunsaturated fatty acid • peroxisome proliferator-activated receptor- • pig • proinflammatory cytokine
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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(TNF-), have been reported to have profound behavioral, neuroendocrine, and metabolic effects (Johnson, 1997
). It is well documented that proinflammatory cytokines increase muscle protein degradation, reduce muscle protein synthesis, and divert nutrients to the synthesis of components in the immune system, such as acute-phase proteins (Spurlock, 1997; Mitchell et al., 2001).
Interestingly, n-3 PUFA consumption could attenuate the growth-inhibitory effect by reducing the production of proinflammatory cytokines in several species (Korver and Klasing, 1997; Xi et al., 2001; Gaines et al., 2003). However, most of the n-3 PUFA effects mentioned above have been observed during inflammation or immune stress. Reports on the effects of dietary n-3 PUFA on the expression of inflammatory cytokines under physiological conditions are scarce. Although the antiinflammation effects of n-3 PUFA might come partly from their eicosanoid derivatives, which are able to regulate expression of inflammatory genes (Calder 2002), evidence is emerging that n-3 PUFA may exert some of their effects through activation of 1 or more transcription factors, such as peroxisome proliferator-activated receptor- (PPAR; Olsen and Haldosén, 2006).
A potential commercial source of n-3 PUFA for pigs is whole linseed, which contains approximately one-third oil, of which more than 50% is -linolenic acid (ALA, C18:3n-3) (Matthews et al., 2000). In this study, we used linseed as the source of n-3 PUFA and tested the hypothesis that dietary consumption of n-3 PUFA might activate the expression of nuclear transcription factor PPAR; might downregulate the expression of the proinflammatory cytokines IL-1β, IL-6, and TNF-; and consequently might stimulate the growth of growing-finishing barrows.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Animals and Diets
Two isoenergetic and isonitrogenous diets were formulated, a basal diet and a linseed diet containing 10% linseed. Diet composition is presented in Table 1, and the fatty acid composition of the diets was analyzed and is listed in Table 2. Twenty-four Landrace x Yorkshire barrows weighing 35 ± 3.7 kg were randomly assigned to 1 of 4 treatment groups, with 6 pigs per group. Throughout the experimental period of 90 d, pigs in treatment groups 1, 2, 3, and 4 were first fed the control diet for 90, 60, 30, and 0 d and then fed the linseed diet for 0, 30, 60, and 90 d, respectively. Pigs were housed individually and fed ad libitum. Pigs were weighed individually on the beginning day and at the end of the trial. Feed consumption was recorded daily per pen. Average daily gain, ADFI, and G:F were calculated. At the end of the trial, the pigs were humanely slaughtered at 170 d of age by electrical stunning, coupled with exsanguination. Tissue samples were rapidly removed, wrapped in foil, and frozen in liquid nitrogen to be stored at –70°C. Skeletal muscle samples were removed from LM between the 10th and the last rib, adipose tissue samples containing the upper and middle layers were removed from the dorsal subcutaneous fat in the neck region, and a spleen sample was collected for subsequent RNA extraction. Another set of LM samples was also collected from each pig and stored at –20°C for fatty acid analysis.
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concentration. Fatty Acid Analysis
Fatty acid analysis was conducted on diet and backfat samples. Lipids were extracted by the choloroform:methanol procedure (Folch et al., 1957). The samples were analyzed by using a CP-3800 gas chromatograph (Varian Inc., Walnut Creek, CA) equipped with an 1177 injector, a flame-ionization detector, and a CPSil88 capillary chromatographic column for fatty acid methyl esters (50 m x 0.25 mm i.d. x 0.20 µm). The injector temperature was 250°C, the detector temperature was 270°C, column flux was 1.0 mL/min, and the split ratio was 1:100. The temperature program was as follows: increased from 100 to 200°C at 5°C/min and held 5 min, then increased to 225°C at 2°C/min and held 2 min. The total analysis time was 39.5 min. Peaks were identified by comparing their retention times with individual reference standard fatty acids.
RNA Isolation
Total RNA was extracted by using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the specifications of the manufacturer. The RNA samples were quantified spectrophotometrically at 260 and 280 nm. The ratio of light absorbance at 260 nm to that at 280 nm was between 1.8 and 2.0, indicating that they were pure and clean. The quality of RNA was also checked by 1.0% agarose gel electrophoresis and staining with 1 µg/mL of ethidium bromide.
Semiquantitative Reverse Transcription-PCR
A 2-step semiquantitative reverse transcription-PCR (RT-PCR) method (Spencer and Christensen, 1999) was used to measure gene expression at the time of slaughter. Oligo-(dT)20n was used as a primer in the first step of complementary DNA (cDNA) synthesis. The reverse transcription reaction solution (20 µL) consisted of 4 µg of total RNA, 100 U of Moloney murine leukemia virus reverse transcriptase (Toyobo, Osaka, Japan), 20 U of an RNase inhibitor (Toyobo), 0.5 mmol/L of deoxyribonucleotide triphosphate, and 0.5 µL of oligo-(dT)20n primers. The cDNA stock was stored at –20°C. The yield of cDNA was measured by the PCR signal generated for the internal standard housekeeping gene β-actin, which was amplified for 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 intensity for β-actin after 20 cycles (Meadus et al., 2002).
Primer sequences and optimal PCR annealing temperatures for amplifying PPAR, IL-1β, IL-6, and TNF- messenger RNA (mRNA) are listed in Table 3. To ensure that no false-positive PCR fragments would be generated from pseudogenes in genomic DNA, primer sequences were designed to span intron regions when genomic sequence data were available. In addition, all PCR primer combinations were tested by using porcine genomic DNA as a negative control. The PCR were performed with a 2720 PCR instrument (Applied Bio-systems, Foster City, CA) by using 100 ng of cDNA, 5 pmol of each oligonucleotide primer, 200 µM each dNTP, 1 U of Taq DNA Polymerase (Fermentas/Life Sciences, Vilnius, Lithuania), and 20 µL of 10x reaction buffer. The PCR program began with a 95°C denaturation for 5 min, continued with 28 to 32 cycles at 94°C for 1 min, optimal PCR annealing temperature for 1 min, 72°C for 1 min, and then a final extension at 72°C for 10 min. The linear amplification range for each gene was tested on the adjusted cDNA. The optimal cycle number was then considered to be 2 cycles less than the greatest cycle of linearity. The PCR samples were electrophoresed on a 2% agarose gel and stained with ethidium bromide. Pictures of the gels were digitally captured by using the BioImaging System GeneGenius (Syngene, Cambridge, UK), and densitometry values were measured by using Gene Tool software (Syngene). The RT-PCR values were presented as a ratio of the specified gene signal in the selected linear amplification cycle divided by the β-actin signal. Data for each gene represented the mean of 3 individual RT-PCR.
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Serum IL-1β, IL-6, and TNF- concentrations were measured by using commercially available porcine ELI-SA kits (Quantikine PLB00, P6000, and PTA00, respectively, R&D Systems, Minneapolis, MN) according to the instructions of the manufacturer (http://www.rndsystems.com/pdf/plb00.pdf, http://www.rndsystems.com/pdf/p6000.pdf, and http://www.rndsystems.com/pdf/pta00.pdf, respectively). The intraassay and interassay precision, sensitivity, recovery, and linearity of the assays were described in the instructions of the manufacturer. Each sample was assayed in triplicate and the absorbance values at 450 nm were recorded. The minimum detectable concentration was 15 pg/ mL for IL-1β, 10 pg/mL for IL-6, and 20 pg/mL for TNF-.
Statistical Analyses
Experimental animals were assigned to different treatments in a completely randomized design. The basic variable was linseed addition time (d). Linear and quadratic relationships with day were determined for parameters collected from treatment groups. The data variances were analyzed by using PROC GLM (SAS Inst. Inc., Cary, NC). The equations that describe the relations between gene expression and ADG were generated by iterative nonlinear least squares regression. The correlation between ADG and expressions of inflammation-related genes was also assessed. Data are presented as least squares means. Means were considered statistically different at P < 0.05.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Average daily feed intake was not affected (P = 0.87) by treatment. However, ADG (P < 0.05) and G:F (P < 0.05) exhibited a quadratic response; treatment group 3 had the greatest ADG value among the 4 groups (Table 4).
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Expression data of PPAR and the 3 proinflammatory cytokine genes (IL-1β, IL-6, and TNF-) are presented in Figure 1 and Table 5. The expression of PPAR in muscle and in spleen increased (P < 0.01; Figure 1A and 1C, respectively) linearly with increasing duration of linseed diet feeding, but its expression in adipose tissue was not affected (P = 0.095; Figure 1B). In contrast, TNF- and IL-6 expression in muscle, adipose, and spleen decreased (P < 0.01) linearly with duration on the linseed diet, and the expression of IL-1β decreased quadratically in adipose tissue (P < 0.01; Figure 1E) and spleen (P < 0.01; Figure 1F).
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Serum concentration of TNF- decreased (P < 0.01) linearly with increasing duration of feeding the linseed diet, but the concentrations of IL-1β and IL-6 in serum were not significantly different (P > 0.05) between different diet treatments (Table 6).
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and Proinflammatory Cytokine Genes
Significant negative linear correlations were found between expression of PPAR and expression of the 3 proinflammatory cytokines (IL-1β, IL-6, and TNF-) both in muscle (R2 = 0.75, P < 0.001; R2 = 0.63, P = 0.001; and R2 = 0.70, P < 0.001, respectively) and in spleen (R2 = 0.73, P < 0.001; R2 = 0.70, P < 0.001; and R2 = 0.77, P < 0.001, respectively), as well as in adipose tissue for IL-1β (R2 = 0.54, P = 0.007), but not in adipose tissue for IL-6 (R2 = 0.30, P = 0.166) and TNF- (R2 = 0.32, P < 0.138).
Correlation Between PPAR Expression and Plasma Cytokine Concentrations
Significant negative linear correlations were found between splenic PPAR expression and serum TNF- concentration (R2 = 0.59, P < 0.01), but not between splenic PPAR expression and serum IL-1β (R2 = 0.15, P = 0.52) or IL-6 (R2 = 0.25, P = 0.28) concentration, respectively. Similarly, significant negative linear correlations were detected between PPAR expression and serum TNF- concentration (R2 = 0.52, P = 0.02) in muscle, but not between PPAR and IL-1β (R2 = 0.12, P = 0.62) or IL-6 (R2 = 0.33, P = 0.16). In adipose tissue, there were no significant linear negative correlations between PPAR expression and serum proinflammatory cytokine concentrations (IL-1β: R2 = 0.15, P = 0.52; IL-6: R2 = 0.18, P = 0.44; and TNF-: R2 = 0.33, P = 0.16).
Relation Between Gene Expression and Growth Performance
The mRNA expressions for PPAR and proinflammatory cytokines in treatment groups 1, 2, 3, and 4 ranged from 0.72 to 2.79. These mRNA expression data were pooled and compared with ADG to evaluate a possible correlation between expression of inflammation-related genes and growth performance.
There were statistically significant (P < 0.05) quadratic relations between expression of muscular PPAR or splenic TNF- mRNA and ADG (Figure 2 and 3, respectively). Average daily gain increased as the expression of PPAR mRNA in muscle increased (Figure 2), whereas the optimal expression level of TNF- (relative to β-actin) in spleen ranged from 1.4 to 1.8, beyond or below which the growth performance of pigs declined (Figure 3).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, b) reported that feeding pigs with up to 15% linseed in the diet for 25 d did not significantly influence feed intake. Other researchers also found that the differences in feed intake among pigs fed linseed oil diets were not significant (Fontanillas et al., 1998; Kouba et al., 2003). However, the results for ADG and G:F in the present study were in contrast to the reports of Fontanillas et al. (1998) and Kouba et al. (2003), who showed no statistically significant differences of ADG and G:F by feeding linseed. Nguyen et al. (2004) reported that feeding linseed appeared to affect the ADG of pigs, but this failed to reach statistical significance (P = 0.077). Indeed, the stimulatory effect of linseed on growth may not be significant when the amount added is less than 5% or the feeding duration is shorter than 60 d (Romans et al., 1995a,b; Fontanillas et al., 1998; Kouba et al., 2003; Nguyen et al., 2003, 2004). However, additional intake of ALA may stimulate growth in growing-finishing pigs (Nguyen et al., 2004). In the current study, linseed supplementation in the diet increased the growth of growing-finishing barrows, which was consistent with a previous report by Liu et al. (2003), who showed that pigs fed fish oil (rich in n-3 PUFA) had greater BW gain independent of a lipopolysaccharide challenge.
It seems unlikely that the stimulatory effect of linseed oil on growth is due to an ALA deficiency in the control diet because LM, backfat lipids, and liver, in all cases, contained a moderate concentration (at least more than 0.5, 0.9, and 2.1%, respectively; data for backfat, muscle, and liver ALA content not presented) of ALA at the end of the experiment. However, the duration of feeding the linseed diet did affect the expression of inflammation-related genes in the current study. Previous studies in humans and pigs have demonstrated that consumption of n-3 PUFA from linseed oil or fish oil inhibits IL-1β and TNF- production (Caughey et al., 1996; Gaines et al., 2003). Therefore, feeding the 10% linseed diet to pigs might improve growth performance partially by modulating the production of proinflammatory cytokines.
Cytokines play important roles in immunoregulation. The 3 cytokines IL-1β, IL-6, and TNF- could induce great metabolic changes (Melchior et al., 2004). Although they are mostly derived from lymphocytes and macrophages, it is now clear that cytokines are also produced by cells not traditionally considered part of the immune system, such as adipocytes and myofibers, which are effective sources and targets of cytokines (Pié et al., 2004; Jacobi et al., 2006). Production of appropriate amounts of proinflammatory cytokines is beneficial in response to infection, but insufficient production or overproduction of these cytokines can be dangerous (Simopoulos, 2002). Proinflammatory cytokines mediate "reprogramming" of metabolism and shift the partitioning of dietary nutrients away from skeletal muscle accretion toward metabolic responses that support the immune system (Spurlock, 1997; Melchior et al., 2004). However, the majority of previous results were observed under conditions of inflammation or immune stress. Interestingly, the present study showed that during normal physiological processes, feeding the linseed diet suppressed the expression of proinflammatory cytokines and decreased the circulatory TNF- concentration of growing-finishing barrows. However, the total circulatory TNF- did not show a significant correlation with ADG. The reason for this requires further study.
Both human and marine studies have demonstrated that dietary n-3 PUFA are able to regulate expression of inflammatory genes. The n-3 PUFA might exert their effects through direct actions on intracellular signaling pathways, which lead to activation of 1 or more transcription factors, including PPAR (Xi et al., 2001; Calder, 2002). Peroxisome proliferator-activated receptor- is a member of the nuclear receptor superfamily of PPAR, and its main function was originally thought to be regulation of adipocyte differentiation (Hammad et al., 2004; Pegorier et al., 2004). Recently, growing evidence points to their involvement in regulating the immune response, particularly in inflammation control (Soko
owska et al., 2005). In the present study, significant negative linear correlations were found between expression of PPAR and expression of proinflammatory cytokines in muscle and spleen, and a significant negative linear correlation was also observed between PPAR and IL-1β expression in adipose tissue. These suggest that feeding linseed might decrease the expression of proinflammatory cytokines by activating PPAR.
The increased expression of PPAR in skeletal muscle indicated that feeding linseed induced the development of preadipocytes from stromal-vascular stem cells to promote intramuscular fat content (Meadus et al., 2002). Surprisingly, the stimulatory effect of the linseed diet on PPAR expression was weaker in subcutaneous fat than in skeletal muscle. This conflict might be due to prolonged PPAR stimulation by dietary n-3 PUFA increasing the apparent intramuscular fat content, because more stem cells are available for intramuscular fat locations than for subcutaneous fat locations (May et al., 1994; Meadus et al., 2002).
Recently, PPAR has also attracted attention as a key factor in insulin sensitivity (Sokoowska et al., 2005). Furthermore, IL-1 has been shown to inhibit the anabolic effects of insulin on skeletal muscle (Klasing and Johnstone, 1991). Meadus et al. (2002) have demonstrated that the glucose supply of muscle cells could be increased with CLA. This finding suggests that the growth-enhancing effect of ALA may also be related to an increase in muscle glucose uptake by a mechanism similar to that of CLA.
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Footnotes |
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2 Corresponding author: naturevoice{at}hotmail.com
Received for publication March 19, 2007. Accepted for publication August 11, 2008.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and interleukin 1β production of diets enriched in n-3 fatty acids from vegetable oil or fish oil. Am. J. Clin. Nutr. 63:116–122. in dendritic cells inhibits the development of eosinophilic airway inflammation in a mouse model of asthma. Am. J. Pathol. 164:263–271. and glutamine-fructose aminotransferase gene expression in vivo. J. Mol. Endocrinol. 28:79–86.[Abstract]owska, M., M. L. Kowalski, and R. Pawliczak. 2005. Peroxisome proliferator-activated receptors- (PPAR-) and their role in immunoregulation and inflammation control. Post
py Hig. Med. Do
w. 59:472–484. http://www.phmd.pl/fulltxt.php?ICID=429272 Accessed Oct. 29, 2005.This article has been cited by other articles:
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
), 30 (
), 60 (
), or 90 (