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J. Anim Sci. 2009. 87:244-252. doi:10.2527/jas.2008-1005
© 2009 American Society of Animal Science
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ANIMAL NUTRITION

Effect of level and duration of dietary n-3 polyunsaturated fatty acid supplementation on the transcriptional regulation of {Delta}9-desaturase in muscle of beef cattle1

S. M. Waters*,2, J. P. Kelly{dagger}, P. O’Boyle*, A. P. Moloney* and D. A. Kenny{dagger}

* Teagasc, Animal Bioscience Centre, Grange, Dunsany, Co. Meath, Ireland; and {dagger} School of Agriculture, Food Science & Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland


   Abstract
Top
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
The objective of this study was to examine the effect of level and duration of feeding of an n-3 PUFA-enriched fish oil (FO) supplement in combination with soybean oil (SO) on the transcriptional regulation of {Delta}9-desaturase gene expression in bovine muscle. Beef bulls (n = 40) were assigned to 1 of 4 iso-lipid and isonitrogenous concentrate diets fed for ad libitum intake for a 100-d finishing period. Concentrates were supplemented with one of the following: 1) 6% SO (CON); 2) 6% SO + 1% FO (FO1); 3) 6% SO + 2% FO (FO2); or 4) 8% palmitic acid for the first 50 d and 6% SO + 2% FO for the second 50 d [FO2(50)]. Samples of LM were harvested and concentrations of fatty acids were measured. Total RNA was isolated and the gene expression of {Delta}9-desaturase was determined. The mRNA expression of putative regulators of {Delta}9-desaturase gene expression, sterol regulatory element binding protein-1c (SREBP-1c) and peroxisome proliferator activated receptor-{alpha} (PPAR-{alpha}), were also measured in the CON and FO2 groups. Expression of mRNA for {Delta}9-desaturase was decreased (P < 0.05) 2.6-, 4.4-, and 4.9-fold in FO1, FO2(50), and FO2 compared with CON, respectively. Expression of {Delta}9-desaturase mRNA tended to be reduced (P = 0.09) by increasing FO from 1 to 2%, but was not affected by duration of supplementation (P > 0.24). Expression of mRNA for SREBP-1c was decreased 2-fold in FO2 compared with CON (P < 0.05), whereas expression of PPAR-{alpha} was not affected (P > 0.30). There was a positive relationship between {Delta}9-desaturase and SREBP-1c gene expression (P < 0.01), but the expression of both genes was negatively related to tissue concentrations of n-3 PUFA (P < 0.05) and positively related to concentration of n-6 PUFA (P < 0.01). Simultaneous enhancement of tissue concentrations of CLA and n-3 PUFA concentrations in bovine muscle may be hindered by negative interactions between n-3 PUFA and {Delta}9-desaturase gene expression, possibly mediated through reduced expression of SREBP-1c.

Key Words: beef cattle • conjugated linoleic acid • {Delta}9-desaturase • fish oil • n-3 polyunsaturated fatty acids • sterol regulatory element binding protein-1c


   INTRODUCTION
Top
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Long-chain n-3 PUFA and the ruminant-derived CLA have potential human health benefits (Ip et al., 1999Go; Wahle et al., 2004Go). The cis-9, trans-11 isomer of CLA is most prevalent, comprising 80 to 90% of total CLA in food products from ruminants (Parodi, 2003Go). Thus, increasing the concentration of both n-3 PUFA and cis-9, trans-11 CLA in milk and meat is beneficial to human health. Supplementation of cattle diets with a blend of oils rich in n-3 PUFA and linoleic acid results in a synergistic accumulation of ruminal and tissue concentrations of vaccenic acid (VA; AbuGhazaleh et al., 2002Go; Kenny et al., 2007Go), the substrate for {Delta}9-desaturase–catalyzed de novo tissue synthesis of the cis-9, trans-11 isomer of CLA. However, despite increases in its substrate, muscle tissue concentrations of cis-9, trans-11 CLA have not increased by using this strategy (Kenny et al., 2007Go).

Studies have reported that the expression of the {Delta}9-desaturase gene is regulated by the transcription factors sterol regulatory element binding protein (SREBP)-1c and peroxisome proliferator activator receptor (PPAR)-{alpha} (Renaville et al., 2006Go; Sampath and Ntambi, 2006Go). The promoter region of the bovine {Delta}9-desaturase gene has been isolated and characterized (Keating et al., 2005Go) and apparently contains a conserved PUFA response region including a critical binding site for a SREBP transcription factor. Indeed, studies with mice (Tabor et al., 1999Go) and human intestinal cells (Renaville et al., 2006Go) have suggested that PUFA downregulate activity of the {Delta}9-desaturase gene by interfering with the SREBP-1c function. There is no published information, however, on the effect of n-3 PUFA on {Delta}9-desaturase gene expression or indeed its transcriptional regulators in bovine muscle. Thus, the objective of the current study was to investigate the effect of dietary level and duration of supplementation with n-3 PUFA on the expression of {Delta}9-desaturase, SREBP-1c, and PPAR-{alpha} genes in the muscle of beef cattle.


   MATERIALS AND METHODS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
All procedures were carried out under license in accordance with the European Community Directive, 86–609-EC. Animals were slaughtered in a licensed abattoir (Meadow Meats, Rathdowney, Co. Laois, Ireland).

Animals, Experimental Design, and Animal Measurements

Animals were maintained at University College Dublin Lyons research farm (Newcastle, Co. Dublin, Ireland). Forty continental cross young beef bulls were blocked on age, BW, and breed (Charolais and Limousin) and assigned to 1 of 4 isolipid and isonitrogenous (15% CP) dietary treatments over a 100-d finishing period as described by Kenny et al. (2007)Go. Briefly, animals were individually offered straw (10% of DMI) and barley-based concentrate rations (90% of DMI) for ad libitum intake. The concentrates contained 1) 6% soybean oil (SO, Redmills Animal Feeds, Goresbridge, Co. Kilkenny; control; CON diet); 2) 6% SO + 1% fish oil (FO, Nutreco, Boxmeer, the Netherlands; FO1 diet) for 100 d; 3) 6% SO + 2% FO (FO2 diet) for 100 d; or 4) 8% palmitic acid (Palmit 80, Trouw Nutrition, Belfast, UK) for the first 50 d and 6% SO + 2% FO (FO2 diet) for the second 50 d [FO2(50)]. Palmit 80 was added to CON and FO1 as appropriate to give 8% added lipid on a DM basis. The SO had a linoleic acid concentration of 53%, whereas the FO had concentrations of eicosopentaenoic acid (EPA) docosohexaenoic acid (DHA) of 39 and 24%, respectively. The ingredient and chemical composition of the experimental concentrates and straw is outlined in Table 1Go. All rations were prepared on the farm and fed within 30 d of manufacture. Feed intake data have been described by Kenny et al. (2007)Go. Briefly, the average DMI for CON, FO1, FO2, and FO2(50) were 8.78, 7.31, 6.50, and 6.84 kg/d, respectively. There was a linear decrease in DMI with increasing dietary inclusion of fish oil (P < 0.05). However, there was no effect of duration of supplementation [FO2 vs. FO2(50); P > 0.10].


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  		Table 1. Ingredients (g/kg; as fed) and chemical composition (g/kg of DM) of the experimental concentrates and straw
 
Tissue Collection and RNA Extraction and Purification

All surgical instruments used for tissue collection were sterilized and treated with RNA Zap (Ambion, Applera Ireland, Dublin, Ireland). Samples of LM were harvested, washed with sterile PBS, snap-frozen in liquid nitrogen, and stored immediately at –80°C. Muscle fatty acid concentrations were determined using GLC as described by Kenny et al. (2007)Go.

A {Delta}9-desaturase index to estimate the activity of the {Delta}9-desaturase enzyme at the tissue level was calculated as described by Kelsey et al. (2003)Go: [cis-9, trans-11 CLA]/[cis-9, trans-11 CLA + 18:1 trans-11]. Muscle lipid concentrations were measured and analyzed as described by Kenny et al. (2007)Go.

Total RNA was isolated from fragmented frozen muscle tissue using TRIzol reagent (Sigma-Aldrich Ireland Ltd., Dublin, Ireland) and chloroform and subsequently precipitated using isopropanol. Samples were treated with RQ1 RNase-free DNase (Promega UK Ltd., South-ampton, UK) and purified using the RNeasy mini kit (Qiagen Ltd., Crawley, UK). The RNA quantity was determined by absorbance at 260 nm using a Nanodrop spectrophotometer (Labtech International Ltd., East Sussex, UK). The RNA quality was also assessed using 18S/28S ratio and RNA integrity number on an Agilent Bioanalyser with the RNA 6000 Nano LabChip kit (Agilent Technologies Ireland Ltd., Dublin, Ireland).

Complementary DNA Synthesis

First-strand complementary (c)DNA was synthesized using the reverse transcription system according to the manufacturer’s instructions (Promega UK Ltd.). Total RNA (1 µg) from each sample was reverse transcribed into cDNA using random hexamers. The converted cDNA was quantified by absorbance at 260 nm, diluted to 50 ng/µL working stocks, and stored at –20°C for subsequent analyses.

Real-Time Quantitative Reverse Transcription PCR

Primers for real-time reverse transcription (RT)-PCR were commercially synthesized (Sigma-Aldrich Ireland Ltd.). Primers were first tested using end-point PCR to optimize amplification conditions. All amplified PCR products from this study were also sequenced to verify their identity. In the case of all genes examined in this study, DNA sequences were 100% identical to published sequences.

As the effect of various experimental treatments on the expression of traditional real-time RT-PCR reference or "housekeeping" genes is largely unknown, it was essential to identify a stable reference gene for the physiological conditions inherent in the current study. Analysis of putative reference genes was carried out using the geNorm version 3.4 Excel (Microsoft, Redmond, WA) add-in (Vandesompele et al., 2002Go). In this study, housekeeping genes analyzed included those for β-actin (Bahar, 2006Go), ubiquitin (Neuvians et al., 2003Go), glyceraldehyde phosphate dehydrogenase (Brunswig-Spickenheier and Mukhopadhyay, 2003Go), 18S ribosomal RNA (De Ketelaere et al., 2006Go), and peptidylprolyl isomerase A (O’Gorman et al., 2006Go). The gene stability measure M that geNorm determines is defined as the average pairwise variation V of a particular gene with all other potential reference genes (Vandesompele et al., 2002Go). This measure is based on the principle that the expression ratio of 2 ideal control genes should be identical in all samples; thus, genes with the least M value are the most stably expressed. The gene that exhibited the greatest stability during real-time RT-PCR of muscle mRNA samples analyzed was β-actin, with M values ranging from 0.10 to 0.22. GeNorm was used to determine the optimal number of reference genes for normalization and, based on a recommended cut-off V value of 0.15, additional reference genes would not have contributed to a more accurate normalization factor. Therefore, β-actin was used as a single standard reference gene for these experiments.

Details of primer sets used in this study to measure the gene expression of {Delta}9-desaturase, SREBP-1c, PPAR-{alpha}, and β-actin are listed in Table 2Go. Primer sequences for {Delta}9-desaturase and β-actin were as described by Bahar (2006)Go. Sequences for PPAR-{alpha} primers were designed using Primer3 software program based on GenBank accession number AF229356. All primers were commercially synthesized by Sigma Aldrich Ireland Ltd. There is no published sequence available for SREBP-1c in the bovine. The human SREBP-1c (National Center for Biotechnology Information gene ID NM_001005291) displays high sequence homology to the bovine cDNA sequence of National Center for Biotechnology Information gene ID XM-879234. The ClustalW web-based software program was used to align these sequences and to design bovine-specific primers for SREBP-1c in the bovine. The primers were subjected to BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and were 100% homologous to the bovine sequence.


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  		Table 2. Bovine oligonucleotide primers used for real-time reverse transcription-PCR
 
Each real-time PCR reaction was carried out in a total volume of 20 µL with 1 µL of cDNA (50 ng/µL), 10 µL of SYBR Green I master mix (Langanbach, Bray, Co. Wicklow, Ireland), 1 µL of forward and reverse primers (20 ng of each), and 8 µL of nuclease-free H2O. Dissociation curves were examined for the presence of a single PCR product. Real-time RT-PCR was performed using a Corbett Rotor-Gene 3000 quantitative PCR system (Corbett Life Sciences, Sydney, Australia) with the following cycling parameters: 95°C for 15 min followed by 40 cycles of 95°C for 30 s, 60°C for 30 s, 72°C for 30 s, followed by amplicon dissociation (95°C for 1 min, 50°C for 45 s, increasing at 0.5°C/cycle until 95°C was reached). Gene expression results were calculated using the delta delta cycle threshold (2–{Delta}{Delta}CT) method (Livak and Schmittgen, 2001Go). The 2–{Delta}{Delta}CT method was used to determine mean fold changes in gene expression between the control and each of FO1, FO2, and FO2(50). To test the effect of amount of dietary n-3 PUFA, the mean relative gene expression levels for FO1 and FO2 were compared. The effect of duration of feeding on the relative gene expression of {Delta}9-desaturase was examined by comparing the FO2 and FO2(50) groups. Differences in gene expression results between the CON and the FO2 treatment group for SREBP-1c and PPAR-{alpha} were also calculated using this method (Livak and Schmittgen, 2001Go).

Statistical Analysis

Data were analyzed using SAS (SAS Inst. Inc., Cary, NC). Data were examined for adherence to normality using PROC UNIVARIATE (SAS Inst. Inc.). Data were then analyzed to determine the effect of treatment on {Delta}9-desaturase, SREBP-1c, and PPAR-{alpha} expression level using a mixed model ANOVA (PROC MIXED). In all analyses the individual animal was denoted as the experimental unit, and animal within treatment was set as the error term. The statistical model included terms for treatment and block. The Tukey critical difference test was used to determine statistical difference between treatment means. Univariate and stepwise multiple regression analyses were carried out to establish relationships between tissue fatty acid concentrations and relative gene expression of {Delta}9-desaturase, SREBP-1c, and PPAR-{alpha} using PROC REG and PROC STEPWISE of SAS, respectively. Relationships were also determined between the relative gene expression of each of the genes measured.

Average daily feed intake was included as a covariate in the statistical model. Furthermore, {Delta}9-desaturase gene expression has previously been shown to be related to adiposity in bovine subcutaneous tissue (Martin et al., 1999Go); therefore, a relationship between fat content of muscle and gene expression of {Delta}9-desaturase was tested for and as no relationship was detected (P = 0.55), it was not necessary to correct gene expression data for tissue fat concentration.


   RESULTS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Muscle Fatty Acid Concentrations

The effect of treatment on the concentrations of 30 fatty acids in muscle has been described by Kenny et al. muscle concentrations of the n-3 PUFA, EPA, DHA, docosapentaenoic acid (DPA), and total n-3 PUFA as well as VA were greater in the 3 FO treatment groups compared with CON (P < 0.01); however, there was no effect (P = 0.18) of treatment on the concentration of cis-9, trans-11 CLA or total n-6 PUFA. Tissue concentrations of linoleic acid were similar for CON and FO2(50) but were greater for CON compared with either FO1 or FO2 (P < 0.05), whereas concentrations of linolenic acid were not affected by treatment (P = 0.12).

Effect of PUFA on mRNA Expression of {Delta}9-Desaturase

The effect of level and duration of n-3 PUFA supplementation is shown in Figures 1Go and 2Go. Compared with CON, {Delta}9-desaturase gene expression was decreased 2.6-fold in animals on FO1 (P < 0.05), 4.9-fold on FO2 (P < 0.01), and 4.4-fold on FO2(50) (P < 0.05; Figure 1Go). There was a tendency for the level of {Delta}9-desaturase gene expression to be decreased in animals on FO2 compared with those on FO1 (P = 0.09; Figure 2Go). There was no difference, however, in {Delta}9-desaturase gene expression between animals on FO2 compared with those on FO2(50) (P = 0.24; Figure 2Go).


Figure 1
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  		Figure 1. Effect of dietary n-3 PUFA supplementation on {Delta}9-desaturase mRNA in LM of cattle offered FO2, FO2(50), or FO1 diets compared with the control (fold change ± SEM; n = 10/treatment). Control = 6% soybean oil for 100 d; FO2 = 6% soybean oil + 2% fish oil for 100 d; FO2(50) = 8% palmitic acid for first 50 d and 6% soybean oil + 2% fish oil (FO2) for the last 50 d; FO1 = 6% soybean oil + 1% fish oil for 100 d. Units for fold change in gene expression units = 2–{Delta}{Delta}CT.

 

Figure 2
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  		Figure 2. Effect of level (FO1/FO2) and duration of feeding (FO2(50)/FO2) of n-3 PUFA on the expression of {Delta}9-desaturase mRNA in LM of cattle. FO1 = 6% soybean oil + 1% fish oil for 100 d; FO2 = 6% soybean oil + 2% fish oil for 100 d; FO2(50) = 8% palmitic acid for first 50 d and 6% soybean oil + 2% fish oil (FO2) for the last 50 d. Units for fold change in gene expression = 2–{Delta}{Delta}CT.

 
Effect of PUFA on mRNA Expression of SREBP-1c and PPAR-{alpha}

Expression of mRNA for SREBP-1c was decreased 2-fold in animals fed the FO2 diet compared with CON (P < 0.05; Figure 3Go). Dietary supplementation with FO2 did not alter the gene expression of PPAR-{alpha} (P = 0.3; Figure 3Go).


Figure 3
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  		Figure 3. Effect of 2% dietary fish oil supplementation (FO2) compared with control on mRNA expression of putative transcriptional regulators of {Delta}9-desaturase, sterol regulatory element binding protein-1c (SREBP-1c) and peroxisome proliferator activated receptor-{alpha} (PPAR-{alpha}), in LM of cattle (fold change ± SEM; n = 10/treatment). Control = 6% soybean oil for 100 d; FO2 = 6% soybean oil + 2% fish oil for 100 d. Units for fold change in gene expression = 2–{Delta}{Delta}CT.

 
Relationship Between Tissue Fatty Acid Concentrations and Gene Expression of {Delta}9-Desaturase, SREBP-1c, and PPAR-{alpha}

The relationships between concentration of several fatty acids (VA; cis-9, trans-11 CLA; cis-10, trans-12 CLA; oleic acid; linoleic acid; linolenic acid; EPA; DHA; DPA; total n-3 PUFA; total n-6 PUFA; and the ratio of n-6:n-3 PUFA) and gene expression for {Delta}9-desaturase is presented in Table 3Go. Similarly, relationships between muscle concentrations of these fatty acids and SREBP-1c gene expression are presented in Table 4Go. There was a negative relationship between {Delta}9-desaturase gene expression and the concentrations of EPA, DPA, DHA, and total n-3 PUFA (P < 0.05). Likewise, a tendency (P = 0.07) toward a negative relationship between linolenic acid and {Delta}9-desaturase gene expression was observed. A positive relationship was detected between {Delta}9-desaturase gene expression and concentrations of linoleic acid (P < 0.05), total n-6 PUFA (P < 0.05), and n-6:n-3 PUFA (P < 0.01). There was no relationship (P > 0.05) observed between gene expression for {Delta}9-desaturase and any other fatty acid measured.


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  		Table 3. Regression coefficients1 for the relationship between tissue fatty acid concentrations and {Delta}9-desaturase gene expression
 

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  		Table 4. Regression coefficients1 for the relationship between tissue fatty acid concentrations and sterol regulatory element binding protein (SREBP)-1c gene expression
 
A negative relationship was detected between SREBP-1c mRNA expression and both DHA (P < 0.05) and cis-9, trans-11 CLA (P < 0.05). Furthermore, the negative relationship observed between SREBP-1c gene expression and VA approached statistical significance (P = 0.08) as did that between SREBP-1c gene expression and total n-3 PUFA (P = 0.08). Similar to that observed for {Delta}9-desaturase, there was a positive relationship between SREBP-1c gene expression and linoleic acid (P < 0.01), total n-6 PUFA (P < 0.01), and n-6:n-3 PUFA (P < 0.01). No relationship was exhibited between EPA or any other fatty acid and SREBP-1c gene expression (P > 0.10). No statistically significant relationship existed between PPAR-{alpha} gene expression and any of the fatty acids measured (P > 0.10).

A significant positive relationship was found between {Delta}9-desaturase and SREBP-1c gene expression (R2 = 0.55; P < 0.01); however, no relationship existed between PPAR-{alpha} mRNA abundance and gene expression of either {Delta}9-desaturase or SREBP-1c (P > 0.10; Table 5Go).


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  		Table 5. Regression coefficients1 for the relationships between concentrations of {Delta}9-desaturase, sterol regulatory element binding protein (SREBP)-1c, and peroxisome proliferator activated receptor (PPAR)-{alpha} gene expression
 
Following stepwise multiple regression analysis, SREBP-1c gene expression and EPA tissue concentrations explained most of the variation observed in {Delta}9-desaturase gene expression (R2 = 0.65); thus, there was no advantage in including further variables in the regression model. There was no relationship between the calculated desaturase index and either {Delta}9-desaturase or SREBP-1c gene expression (P > 0.10).


   DISCUSSION
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
To our knowledge, this is the first study to examine the effect of level and duration of n-3 PUFA supplementation on {Delta}9-desaturase mRNA expression in the muscle of beef cattle. This study demonstrates, for the first time, that dietary n-3 PUFA inhibits expression of the gene that codes for the critical enzyme required to desaturate VA to CLA in bovine muscle tissue. Furthermore, there is evidence from the current study that the degree of inhibition of transcription for this gene is related to the level of dietary n-3 PUFA intake. Although the minimum time required to evoke a significant reduction in {Delta}9-desaturase mRNA expression was not established in the current study, 50 d was found to be sufficient, and extending the supplementation period beyond this did not further depress {Delta}9-desaturase expression. This study also investigated the effect of n-3 PUFA supplementation on the expression of mRNA for putative regulators of {Delta}9-desaturase, SREBP-1c, and PPAR-{alpha}. This is the first published study to measure gene expression of SREBP-1c in the bovine, and we found transcription of the gene coding for this transcription factor to be significantly decreased in muscle tissue of cattle fed a diet high in n-3 PUFA compared with the control animals. However, supplementation with n-3 PUFA did not alter the gene expression of the other putative regulator of {Delta}9-desaturase expression investigated, PPAR-{alpha}.

The expression of {Delta}9-desaturase is known to be strongly modulated by several nutrients (PUFA and fructose), drugs (sterculic acid), hormones such as insulin and leptin (Ntambi and Miyazaki, 2004Go), and cholesterol (Kim et al., 2002Go). Results of the current study showed that although there was evidence of a negative relationship between VA (the substrate for de novo tissue synthesis of cis-9, trans-11 CLA) and SREBP-1c gene expression, there was no relationship between VA and {Delta}9-desaturase gene expression and hence, no evidence of substrate inhibition. This is contrary to the report of Lin et al. (2004)Go, who found that {Delta}9-desaturase mRNA abundance was decreased because of the accumulation of VA in mammary gland tissue of lactating mice. Although a negative relationship was displayed between cis-9, trans-11 CLA and SREBP-1c gene expression, there was no evidence from the current study of a CLA-mediated inhibition of {Delta}9-desaturase gene expression, which is in contrast to that postulated by other researchers (Lin et al., 2004Go; Keating et al., 2005Go). However, the lack of a relationship between CLA and {Delta}9-desaturase gene expression in the current study may have been the result of a lack of sufficient variation in tissue CLA concentrations between treatments.

In the current study there was no relationship between calculated {Delta}9-desaturase activity index (Kelsey et al., 2003Go) and {Delta}9-desaturase gene expression. These results concur with the findings of Archibeque et al. (2005)Go, who reported that a calculated {Delta}9-desaturase index did not reflect actual {Delta}9-desaturase enzyme activity in adipose tissue of beef steers.

Daniel et al. (2004)Go reported that increased oleic acid content of sheep tissue in response to concentrate-rich diets is associated with an increase in {Delta}9-desaturase gene expression. There was a positive but not statistically significant relationship between oleic acid concentrations and {Delta}9-desaturase gene expression in the current study. This is contrary to another report in the literature by Keating et al. (2006)Go, who found that the bovine {Delta}9-desaturase gene promoter was downregulated by oleic acid.

The current study established a positive relationship between n-6 PUFA, linoleic acid, total n-6 PUFA, and n-6:n-3 PUFA; and both {Delta}9-desaturase and SREBP-1c gene expression. Contrary to this report, the in vitro work of Sessler et al. (1996)Go showed that mRNA expression for {Delta}9-desaturase was decreased in a dose-dependent manner by addition of n-6 PUFA, linoleic acid, and arachidonic acid to a murine cell line. Furthermore, in that study it was found that the half-life of {Delta}9-desaturase mRNA could be shortened by arachidonic acid, which led the authors to suggest that the repressed gene expression was due to a reduction in the stability of its mRNA (Sessler et al., 1996Go).

There is evidence in the current study of a negative relationship between the parent n-3 PUFA, linolenic acid, and {Delta}9-desaturase gene expression. Furthermore, a significant negative relationship was observed between linolenic acid concentration and SREBP-1c gene expression. Similarly, the results of a study with sheep showed a reduction in {Delta}9-desaturase gene expression in adipose and liver tissues of lambs fed forage compared with a concentrate-based diet (Daniel et al., 2004Go). The authors attributed this repression in mRNA levels to the greater concentrations of linolenic acid in the forage compared with the concentrate diet. Furthermore, there is some evidence from the work of McGettrick et al. (2007)Go that grazing cattle supplemented with FO had lesser relative quantities of {Delta}9-desaturase mRNA in muscle and adipose tissue. In agreement, it has been demonstrated using a mouse adipocyte cell line that linolenic acid inhibited {Delta}9-desaturase gene expression (Sessler et al., 1996Go). Given that {alpha}-linolenic acid is the predominant PUFA in grass (Dewhurst et al., 2003Go), these results may have implications for strategies to further augment concentrations of CLA in the tissue of cattle reared on pasture.

In the current study, multiple regression analysis demonstrated that, of the variables measured, EPA and SREBP-1c gene expression accounted for most of the variation in expression of the {Delta}9-desaturase gene. Indeed, we found a negative relationship between {Delta}9-desaturase gene expression and n-3 PUFA, EPA (C20:5n-3), DHA (C22:6n-3), and linolenic acid (C18:3n-3). These findings are in agreement with those of a previous report showing that feeding a mixture of n-3 PUFA, EPA, DHA, and linolenic acid resulted in a 50% suppression of {Delta}9-desaturase mRNA in rat liver (Bellinger et al., 2004Go). Similarly, Renaville et al. (2006)Go showed that a decrease in the conversion rate of VA to CLA in a human intestinal cell line following addition of EPA was attributed to a negative effect of this PUFA on mRNA expression of {Delta}9-desaturase.

Nutrients, in particular fatty acids, have been shown to regulate {Delta}9-desaturase at both a transcriptional (Renaville et al., 2006Go) and enzyme activity (Sessler et al., 1996Go) level. In human cell lines the transcription of {Delta}9-desaturase has been shown to be under the control of 2 transcription factors, SREBP-1c and PPAR-{alpha} (Renaville et al., 2006Go). The current study also examined the gene expression of these 2 transcription factors following n-3 PUFA dietary supplementation. Similar to {Delta}9-desaturase gene expression, SREBP-1c mRNA abundance was significantly decreased, whereas PPAR-{alpha} mRNA levels remained unchanged.

The SREBP-1c is a key regulator of {Delta}9-desaturase, which mediates its transcriptional activation (Nakamura and Nara, 2002Go). The present study found that a negative relationship existed between SREBP-1c gene expression and both DHA and cis-9, trans-11 CLA, whereas the relationship between the expression of this gene and EPA concentrations, although negative in direction, did not reach statistical significance. Similar to {Delta}9-desaturase gene expression, a positive relationship was displayed between linoleic acid, total n-6 PUFA, and n-6:n-3 PUFA; and SREBP-1c gene expression. Importantly, there was a highly significant positive relationship displayed between the gene expression of {Delta}9-desaturase and its putative transcription factor SREBP-1c, indicating that these 2 genes are co-regulated. As SREBP-1c appears to regulate {Delta}9-desaturase gene transcription, we suggest that in the bovine the effect of n-3 PUFA on {Delta}9-desaturase mRNA levels is mediated by reduced SREBP-1c gene expression. Furthermore, there is evidence from the current study that the balance between n-6 and n-3 PUFA tissue concentrations is important in the regulation of {Delta}9-desaturase, acting through SREBP-1c.

This is the first report of a reduction of SREBP-1c gene expression in muscle tissue following n-3 PUFA supplementation in the bovine species and is consistent with results of others (Renaville et al., 2006Go) who found that reduced SREBP-1c mRNA levels were associated with decreased gene expression of {Delta}9-desaturase in human intestinal cell cultures and proposed that decreased levels of SREBP-1c would decrease {Delta}9-desaturase gene transcription and expression, which would in turn decrease {Delta}9-desaturase activity. In rat hepatic tissue, dietary PUFA were shown to exert their effects by reducing both mRNA and proteolytic activation of SREBP-1c (Xu et al., 2001Go, 2002Go). In support of this theory, dietary PUFA have been shown to be associated with a decrease in the formation of mature cleaved SREBP-1c protein in the liver tissue of mice (Yahagi et al., 1999Go). Although {Delta}9-desaturase gene expression may also be reduced by leptin, this mechanism has been shown to be independent of SREBP-1c (Biddinger et al., 2006Go).

The PPAR-{alpha} has been shown to induce {Delta}9-desaturase expression in rats and in pigs (Forman et al., 1997Go; Cheon et al., 2005Go). The PPAR can directly induce {Delta}9-desaturase in cell culture through the peroxisome proliferator response element present in the promoter region (Miller and Ntambi, 1996Go). As a regulatory relationship has been reported between {Delta}9-desaturase and PPAR-{alpha} (Nakamura and Nara, 2002Go), the present study investigated whether reduced expression of {Delta}9-desaturase could be associated with alterations in PPAR-{alpha} gene expression. However, supplementation of cattle diets with FO had no effect on PPAR-{alpha} mRNA levels. Furthermore, unlike {Delta}9-desaturase, there was no significant relationship between PPAR-{alpha} gene expression and any of the fatty acids measured. In addition, the current study found that no relationship existed between gene expression of {Delta}9-desaturase and PPAR-{alpha}. Other investigators using human intestinal cell lines also reported that, although treatment with EPA did reduce {Delta}9-desaturase and SREBP-1c gene expression, PPAR-{alpha} mRNA levels remained unaltered. Moreover, a study in mice reported that a reduction in {Delta}9-desaturase enzyme activity may be partly dependent on SREBP-1c but most likely independent of PPAR-{alpha} (Sampath and Ntambi, 2006Go). The PPAR-{alpha} may have an indirect role to play in the regulation of {Delta}9-desaturase gene expression (Nakamura and Nara, 2002Go).

Increased human consumption of CLA and n-3 PUFA is strongly recommended by nutritionists (Mac-Rae et al., 2004Go). The CLA in human tissues may be synthesized through the tissue desaturation of VA by {Delta}9-desaturase (Turpeinen et al., 2002Go) and thus may be increased by VA in the human diet. Data presented in the current study has important implications for those ingesting n-3 PUFA, as n-3 PUFA may have negative effects on de novo synthesis of CLA in human muscle through potential reductions in {Delta}9-desaturase gene expression. However, because a positive relationship of n-6 PUFA, and particularly the ratio of n-6 to n-3 PUFA concentration, with {Delta}9-desaturase gene expression was observed, the correct balance of n-6 to n-3 PUFA concentrations ingested in the diet appears to be of critical importance to achieve optimal {Delta}9-desaturase gene expression levels and, in turn, CLA production in muscle tissue.

Beef that is naturally enriched with CLA and n-3 PUFA could be a good regular source of these important fatty acids and an alternative to expensive nutritional supplements. As such the research presented herein is important in the context of any potential human health benefits of food sources of these fatty acids. It also illustrates the potential interactions between nutrients and gene transcription at the tissue level. This has important implications for the development of dietary strategies to augment the concentration of both CLA and n-3 PUFA in ruminant meat. Hence, further work is required to elucidate the molecular and biochemical mechanisms controlling the synthesis and deposition of n-3 PUFA and CLA in muscle to minimize the negative effects of n-3 PUFA supplementation on the {Delta}9-desaturase enzyme. This will ultimately provide improved strategies to consistently produce nutritionally enhanced beef.


   Footnotes
 
1 The technical assistance of A. McArthur (Grange Beef Research Centre) and P. Duffy (University College Dublin) is gratefully acknowledged. The co-operation of staff at Meadow Meats (Rathdowney, Co. Laois, Ireland) is also gratefully acknowledged. Back

2 Corresponding author: Sinead.Waters{at}teagasc.ie

Received for publication March 5, 2008. Accepted for publication September 5, 2008.


   LITERATURE CITED
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 


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