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National Institute of Nutrition and Seafood Research, N-5817 Bergen, Norway;
* Institute for Marine Biosciences, Halifax, Nova Scotia, Canada B3H 3Z1; and
Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, Scotland, UK
3To whom correspondence should be addressed. E-mail: ann-elise.jordal{at}nifes.no.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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5 fatty acid desaturase gene expression was significantly greater in fish fed 75% RO than in fish fed the control diet; this was confirmed by quantitative real time PCR analysis. In addition, several genes, among these mitochondrial proteins, peroxisome proliferator-activated receptor 
, as well as other transcription factors, coactivators, and signal transducers, showed significant differential regulation. This partially validated microarray may be used for further gene expression profiling using other dietary comparisons, and for further characterization of selected genes.
KEY WORDS: • fatty acid • microarray • quantitative PCR • lipid metabolism • Atlantic salmon
Partial substitution of marine fish oils (FO)5 by vegetable oils in fish diets has become increasingly common in aquaculture, due to limited supplies of FO (1). These dietary changes are thought to influence lipid metabolic gene expression patterns in liver of Atlantic salmon, as in mammals, where changes in quantity or composition of dietary fat affect hepatic de novo lipogenesis, VLDL synthesis and secretion, fatty acid oxidation, and cholesterol metabolism (2). Several genes involved in these metabolic pathways are regulated in mammals through response elements for nuclear receptors. Hepatic nuclear factor-4
(HNF-4) positively regulates apolipoprotein (apo) C-II (2) and apo A-I gene expression (3). The (n-3) and (n-6) fatty acids and other peroxisome proliferator-activated receptor (PPAR) activators induce transcription of liver X receptor (LXR)- through DR1 elements (4) and regulate the expression of apo C-II (5) and sterol regulatory element binding protein (SREBP)-1c (6). In addition, 9-fatty acid desaturase (SCD) (2,7), 6-, and 5- (8) fatty acid desaturases (FADs) were induced by elevated SREBP-1c and PPAR ligands. PPAR is required for the induction of mitochondrial enzymes such as long-chain acyl CoA synthetase (long-chain ACS) (2) and peroxisomal bifunctional enzyme containing enoyl CoA hydratase (9).
The influences of dietary lipids on hepatic de novo lipogenesis, lipoprotein metabolism, fatty acid oxidation, and cholesterol metabolism (10–12) as well as nutritional effects on fatty acid desaturation and elongation activities (13,14) in Atlantic salmon (Salmo salar) have been thoroughly assayed. However, little is known about the expression of hepatic genes involved in lipid metabolism in salmonids. Fatty acids regulate the expression of genes involved in fatty acid desaturation and elongation (15). Leaver and co-workers (16) characterized PPARs from 3 fish species, including Atlantic salmon (unpublished results). All PPARs were detected in all tissues studied, and it was suggested that fish PPARs bind a wide range of fatty acids. PPAR, involved in the induction of mammalian FADs (2,7,8), was particularly responsive to unsaturated fatty acids.
Hepatic mitochondrial ß-oxidation exhibits substrate specificity in fish (17–19), and carnitine palmitoyltransferase (CPT)-II, which is induced by 20:5(n-3) and 22:6(n-3) in rat liver (20), has been used as a marker for mitochondrial ß-oxidation activity in the liver of brown trout (Salmo trutta L.) (21) and Atlantic salmon (22,23). However, to date, no one has studied mitochondrial ß-oxidation gene expression patterns in salmon.
In the present study, we examined whether nutrigenomics could be used as a tool for the analysis of nutrient-gene interactions in aquaculture nutrition. Because large-scale fish-specific arrays were not available at the time of the study, a small-scale lipid metabolism cDNA microarray was designed using amplicons from available sequenced cDNAs. This microarray was then used to determine the effects of dietary oil on hepatic gene expression in salmon fed either a 100% FO or a 75% rapeseed oil (RO) diet in a large-scale nutritional trial. Samples were pooled to ensure low variability and a normal distribution within the population. In accordance with this, an experimental design was chosen using a high number of technical replicates to validate the array itself for use in further studies. To further validate some of the result on the array quantitative real time PCR (Q-PCR) analyses were performed to confirm some of the results from the microarray profiling research.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The dietary experiment was performed at Gildeskaal Research Station, Inndyr, Norway as described in more detail in Torstensen et al. (24). In wk 21, 2001, 
600 postsmolt Atlantic salmon, mean body weight 0.142 kg, were randomly distributed to 7 net pens, of 125 m3 each, and fed a control diet containing 100% FO (2 replicates) or experimental diets in which FO was replaced by 0, 25, 50, 75, and 100% RO or 50% olive oil (OO) (one replicate for each oil concentration) in a linear regression design for 42 wk (Table 1). Diets, produced by Nutreco ARC, differed only in their oil composition. The chemical compositions of diets were (g/kg): lipid, 300; protein, 450; moisture, 60; ash, 70; and nitrogen-free extract, 120.
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Sampling procedure
Samples were collected during wk 22 and 42 for Q-PCR analysis, and during 42 wk for microarray analysis. The fish were deprived of food for 24 h before sampling. Randomly sampled fish from each tank, (i.e., dietary treatment) were anesthetized with methomidate (7 g/L; Norsk Medisinaldepot) before being killed with a blow to the head. Liver samples taken from 30 individual fish from each dietary group were pooled and preserved in RNAlater ® (Ambion) according to the manufacturer’s recommendations. For RT-PCR analysis, liver samples were immediately removed from 6 individual fish from each dietary group, snap-frozen in liquid nitrogen, and stored at –80°C. The protocol was approved by the local representatives for the Norwegian State Board of Biological Experiments with Living Animals.
Fatty acid composition
Lipids from livers and diets were extracted by homogenization in chloroform:methanol (2:1, v:v). After extraction of total lipid, an aliquot was saponified and methylated using 12% BF3 in methanol. The fatty acid composition of total lipids was analyzed using methods described previously by Lie and Lambertsen (26). Briefly, methyl esters were separated using a trace gas chromatograph 2000 ("cold on column" injection, 60°C for 20 s, 25°C/min; 160°C for 28 min, 25°C/min; 190°C for 17 min, 25°C/min; 220°C for 9 min), equipped with a 50-m CP-sil 88 (Chromopack) fused silica capillary column (i.d., 0.32 mm). The fatty acids were identified by retention time using standard mixtures of methyl esters (Nu-Chek-Prep), and quantified using Totalchrom software (version 6.2, Perkin Elmer). The amount of fatty acid per gram tissue was calculated using 19:0 methyl ester as an internal standard.
Microarray analysis
Minimum information about a microarray experiment (MIAME). MIAME standards (27) were used to define the content and the structure of the information for this microarray experiment. In accordance with these standards features used (Online Supplemental Table 1), the experimental conditions (Microarray hybridization protocol), the experiment (Experimental design), and array design (Microarray construction) as well as a description of samples, hybridizations, and normalizations controls (RNA isolation and preparation, Microarray hybridization protocol, and Normalization and statistical analysis) were reported.
Experimental design. Two arrays (i.e., technical replicates) were scanned using cohybridized labeled total RNA from control and experimental samples labeled with cyanine (Cy)3 and Cy5, respectively. Each of these experiments was replicated with Cy dyes switched to control for labeling bias. The same experimental design was used for cohybridized mRNA samples from fish fed the 2 diets.
Microarray construction. PCR amplicons from selected Atlantic salmon expressed sequence tags (ESTs) (28) were printed on bar-coded CMT-GAPS coated slides (Corning Microarray Technology) using a manual pin caster microarray printing system (Micro Caster 8 pin system, Schleicher & Schuell). cDNA clones were selected on the basis of their similarity [BLAST search of EST sequence (29)] to features involved in lipid metabolism. The Microarray Assistant Clone Organizer and Array Simulator (30) was used to organize clones and amplicons. A total of 73 features, mainly from Atlantic salmon, were spotted on the array in quadruplicate (4 subarrays with 96 probes on each subarray). Some features were spotted in duplicate on different areas of each subarray to control for changes in signal/hybridization in different parts of the slide. In total, there were 89 different amplicons, 2 empty spots and 5 controls consisting of a concentration gradient of an Arabidopsis thaliana chlorophyll synthetase G4 (GenBank Accession U19382). Several ribosomal proteins and ß-actin were included to monitor the expression of housekeeping genes. Controls were used only to ensure quality. Empty spots were included on the array for controlling the background intensity. All features were spotted using 1 spot length distance between spots to avoid interference of signals.
RNA isolation and preparation. Total RNA was purified from 30 pooled livers using the RNAwiz protocolTM (Ambion). RNA was subjected to DNase treatment (DNA-freeTM, Ambion) and quality and quantity determined by denaturing gel electrophoresis and spectrophotometry (A260/280). mRNA was isolated from total RNA using the Oligotex ® mRNA kit (Qiagen).
Microarray hybridization protocol. A total of 8 arrays were analyzed, 4 using total RNA and 4 using mRNA as templates for direct cDNA labeling. Both sources had an A260/280 ratio of 1.9:2.0 and were of sufficient quality for hybridization. Total RNA (50 µg) and mRNA (3 µg) were subjected to separate cDNA syntheses using a direct labeling protocol with Cy3- or Cy5-labeled dCTP (Perkin Elmer Life Sciences). Arabidsopsis thaliana chlorophyll synthetase G4 mRNA (80 pg) was added to the labeling reaction as an internal control; 5 µL concentrated and purified labeled cDNAs were combined, and 1 µL Poly (A)-DNA (20 g/L) (Amersham Pharmacia, Cat #27-7836-01) was added. The mixture was incubated at 95°C for 3 min, followed by quick cooling on ice for 2 min to block renaturation. Subsequently, 12 µL hybridization buffer (Amersham Pharmacia Cat#PRK0325) and 24 µL deionized formamide (Sigma, Cat #F9037) were added and the mixture was hybridized to preincubated arrays (80 µL hybridization buffer, 42°C) in a humid chamber at 42°C overnight. Scans were performed at a resolution of 10 mm using a microarray scanner (ScanArray ® 5000XL, Packard BioChip Technologies).
Normalization and statistical analysis. Background subtraction and normalization to total spots were performed using the QuantArray ® microarray analysis software (Packard BioChip Technologies), and low intensity spots (intensity < 1000 pixels) were eliminated from further analysis. All 8 scanned arrays were subjected to a significance analysis of microarrays (SAM) (31) using separate mRNA and total RNA data sets as well as the combined dataset. A simple gene specific t test analysis of the mean log ratio (32) was also used to analyze the separate mRNA and total RNA datasets (data not shown). Pooling of 30 individual samples was done in accordance with the central limit theorem (33,34) to remove sources of individual variation due to genetic differences and to ensure a low SD and a normal distribution within the population. Significance analysis was performed with a high number of technical replicates of normally distributed treatment groups.
RT-Q-PCR analysis
RNA isolation and preparation. Total RNA was purified using TRIZOL ® (Invitrogen) and subjected to DNase treatment (DNA-free, Ambion). RNA quality and quantity were determined as described above.
RT-PCR.
cDNA (125 ng) was synthesized following a modified protocol from the Taq Man Reverse Transcription Reagents kit (Applied Biosystems). Modifications included increasing the total reaction volume to 30 µL and accordingly increasing the volume of all reagents used. Oligo d(T)16 primers were used for 5 FAD and liver basic fatty acid binding protein (Lb-FABP), and random hexamers were used for 18S rRNA. The reactions were incubated at 25°C for 10 min and 48°C for 60 min, and the reverse transcriptase enzyme was inactivated at 95°C for 5 min followed by a decrease to 4°C. RT-PCR efficiency was monitored using a 4-step, 2-fold dilution curve of RNA.
Q-PCR.
BLAST searches (29) for Atlantic salmon 18S rRNA, 5 FAD, and Lb-FABP were performed to identify highly conserved regions for primer design. Selected sequences were translated using the Expasy translate tool (35) and intron-exon borders were identified from alignments of genomic and cDNA sequence using ClustalW software (36). Primer and probe sequences were constructed using the Assay by Design service (Applied Biosystems). The primer and probe sequences (5' to 3') for 18S rRNA (AJ427629) were: CCCCGTAATTGGAATGAGTACACTTT (F), ACGCTATTGGAGCTGGAATTACC (R), FAM-CACCAGACTTGCCCTCC-MGBNFQ (reverse probe), for 5 FAD (AF478472.1) GGAACCACAAACTGCACAAGT (F), GTGCTGGAAGTGACGATGGT (R) and FAM-CAGAGGCACCCTTTAGGTG-MGBNFQ (reverse probe), and for Lb-FABP (BG935057) CCGACATCACCACCATGGA (F), GCTTCCCTCCC TCCAGTTTG(R) and FAM-CAGTGCA CTTGAGCTT- MGBNFQ (reverse probe).
Q-PCR was performed using FAM fluorescent chemistry on an ABI prism 7000 (Applied Biosystems). The reaction mixture (25 µL) contained primers (900 nmol/L each), FAM probe (200 nmol/L), 1X TaqMan universal PCR master mix (Applied Biosystems art. no. 430 4437) and 5 µL cDNA. All samples were assayed in triplicate with nontemplate controls on the same plate. The reaction was incubated at 50°C for 2 min followed by 95°C for 10 min and 50 cycles of 95°C for 10 s and 60°C for 15 s. Liver RNA samples from 6 fish from each diet were tested.
Normalization and statistical analysis. 18S rRNA levels were used to normalize the results and to calculate relative expression levels using the Q gene method (37). The normalized relative expression data were analyzed using the nonparametric Kruskal-Wallis method (Statistica 6.1, Statsoft; n = 6 and n = 5) due to heterogeneity in variance within the 75% RO group. Differences were considered significant at P < 0.05.
Statistical analysis of dietary and liver fatty acid composition
For fatty acid composition in the experimental diets (Table 1, n = 2 analytical parallels) and in liver samples (Table 2, n = 3 and 6 pooled samples), means and medians (ranges) were calculated, respectively, using descriptive statistics (Statistica 6.1, Statsoft). Data were not analyzed for significant differences.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The concentration of 20:5(n-3) was 60% lower in the 75% RO diet compared with the control (FO) diet (Table 1). The concentrations of 22:5(n-3) and 22:6(n-3) were 56 and 50% lower, respectively. The total highly unsaturated fatty acid (HUFA) content, the sum of SFA, and the sum of (n-3) fatty acids were 58, 32, and 16% lower, respectively, whereas the sum of (n-6) fatty acids was 3-fold greater in the 75% RO diet compared with control. In addition, the amount of total (n-9) fatty acids was almost 2-fold greater in the 75% RO diet compared with the control diet; 18:1(n-9) was 3-fold greater. The concentrations of 18:1(n-9) (40%) and 18:2(n-6) (15%) fatty acids were high in the 75% RO diet. Those of 18:2(n-6) and 18(3n-3) were 4- and 6-fold greater, respectively, in the 75% RO diet. The (n-3):(n-6) ratio in the 75% RO diet was 79% less than the control.
Irrespective of diet, 22:6(n-3) dominated in the salmon liver total lipid (Table 2). The concentrations of 18:1(n-9) and 18:2(n-6) were 2-fold greater, and that of 18:3(n-3) > 6-fold greater (median values) compared with livers from salmon fed the control diet. In contrast, concentrations of 22:5(n-3), 20:5(n-3), and 22:6(n-3) in liver of salmon fed the 75% RO diet were 46, 34, and 31% lower, respectively, than those of controls. Further details on fatty acid composition in livers of salmon fed different RO diets were published (11).
Microarray analysis demonstrated that expression of hepatic fatty acid 5 desaturase was significantly greater in livers of 75% RO-fed salmon than FO-fed salmon, whether total RNA or mRNA was analyzed (Table 3). Expression of apo A1 precursor was upregulated in analyses using labeled total RNA, and the expression of apo C-II was upregulated in analyses using combined and mRNA datasets. Long-chain ACS3 was among several genes that were downregulated in livers of 75% RO-fed salmon in all analyses.
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, and catalase as downregulated genes in livers of 75% RO-fed salmon. Data from Q-PCR analysis confirmed results from the microarray screening showing an 2-fold increased gene expression of 5-FAD in livers of 75% RO-fed salmon after 42 wk of feeding (Fig. 1). Gene expression of 5-FAD in livers of 75% RO-fed salmon after 22 wk of feeding, was higher (P < 0.05, nonparametric statistics, n = 5) than in the control (n = 6).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Q-PCR analysis, performed to validate expression data from microarray analysis, using essays for Lb-FABP (results not shown) (Online Supplemental Table 1) confirmed the lack of gene specific variation between dietary treatments. However, both the microarray and Q-PCR results demonstrated that 5 FAD gene expression was higher in livers of 75% RO-fed salmon (Table 3 and Fig. 1). This is in agreement with Zheng and co-workers (15) who observed an increase in the expression of hepatic 5 desaturase and fatty acid elongase genes in salmon fed increased levels of dietary linseed oil for 20 wk. The values for expression of fatty acid elongase obtained in the present microarray study were too low to be deemed reliable. Bell and co-workers (13,14) suggested that the stimulation of hepatic desaturation and elongation by vegetable oils presumably reflects increased substrate availability [18:2(n-6) and/or 18:3(n-3)] coupled with reduced end product concentration [20:5(n-3) and 22:6(n-3)], with high concentrations of the latter inhibiting the pathway.
In contrast, SCD gene expression was reduced in livers of 75% RO-fed salmon compared with control salmon. Supporting these data, Berge et al. (41) demonstrated that SCD enzyme activity was reduced in salmon fed conjugated linoleic acid (CLA), and gene regulation of SCD was shown to differ from regulation of 5- and 6-FAD (42). However, SCD has not been cloned in salmon, and further studies are required.
Dietary 20:4(n-6), 20:5(n-3), and 22:6(n-3) were reduced in the 75% RO diet (Table 1) compared with control diet, and 20:4(n-6) and 22:6(n-3) were reported to affect hepatic gene expression (3). Therefore, this long-term dietary fatty acid change may reduce mRNA transcript synthesis in livers of 75% RO-fed salmon.
The HUFA, 20:5(n-3) and 22:6(n-3), were reported to be 2- to 4-fold more potent inducers of fatty acid ß-oxidation than 18:2(n-6) and 18:3(n-3) in mammals (43). Although there was an increase in the amount of 18:2(n-6) and 18:3(n-3), the reduced dietary 20:5(n-3) and 22:6(n-3) in the 75% RO diet compared with control correlated with a significant downregulation of several mitochondrial genes. These include long-chain ACS3, enoyl CoA hydratase, 2 mitochondrial outer membrane proteins, CPT-II, and mitochondrial acyl carrier protein (precursor). Furthermore, several additional mitochondrial genes (malate dehydrogenase, translocase of inner mitochondrial membrane, and mitochondrial solute carrier protein) were identified as significantly downregulated in fish fed 75% RO when gene-specific t tests, allowing genes with smaller fold changes to be detected, were used for statistical analysis (results not shown).
CPT-I and CPT-II activate and transport long-chain fatty acids into the mitochondrial matrix (44) and therefore occupy a key regulatory step in their catabolism (23,44). CPT-II was significantly downregulated in livers of 75% RO-fed salmon, possibly indicating lower ß-oxidation capacity in this group (23,45). Turchini and co-workers (21) showed that hepatic CPT-II in brown trout (Salmo trutta L.) had significantly higher activities in fish fed a FO compared with fish fed a canola (rapeseed) oil diet.
Two long-chain ACS features were present on the microarray, one from hagfish, Myxine glutinosa, which had the highest sequence similarity to the rat long-chain ACS3 (NM_057107) and one from Salmo salar, which had the highest similarity to the rat long-chain ACS1 gene (NM_012820). Only the former showed a significant change in mRNA levels due to diet, with a downregulation in livers from 75% RO-fed fish. Rat long-chain ACS3 and ASC1 are from different subfamilies with distinct functions, and share only 30% amino acid sequence identity (46). Rat long-chain ACS3 activates 20:5(n-3) and 20:4(n-6) most efficiently and has a 4-fold higher capacity for utilization of C20 HUFA than rat ASC1 (47). Long-chain ACS3 is thought to direct fatty acids to mitochondrial oxidation, whereas long-chain ACS1 may play a role in channeling fatty acids to triacylglycerol synthesis (46). Higher expression of the ACS3-like isoform could be expected in FO-fed salmon, but a fully characterized clone of salmon long-chain ACS3 is required to completely elucidate the effects of dietary fatty acids on ACS expression.
High dietary 20:5(n-3) (48) and (n-3):(n-6) ratio (49) both induce mitochondrial proliferation in rodents; thus, the reduced 20:5(n-3) and (n-3):(n-6) ratio in the 75% RO diet may also reduce mitochondrial proliferation in livers of 75% RO-fed salmon compared with the control. Mootha and co-workers (50) used proteomic and mRNA expression profiling to reveal several genes and transcriptional regulators exhibiting expression patterns that correlated with mitochondrial biogenesis. These included cellular nucleic acid binding protein and PPAR, which is consistent with the expression data in the present study. Furthermore, PPAR was downregulated by CLA and sunflower oil compared with FO in gilthead sea bream (Sparus aurata) (51), suggesting that vegetable oil inclusion may negatively affect PPAR expression. Whether PPAR is involved in mitochondrial biogenesis in salmon fed dietary RO remains to be elucidated.
The apo A-I precursor [sequence similarity to apo A-I-I (BE 518583)] and apo C-II were affected by the dietary oil source, showing upregulation in livers of 75% RO-fed salmon compared with control. Apo A-I–like protein was associated with HDL, and apo-C–like protein with LDL in rainbow trout (52). In contrast to the apolipoprotein expression results in the present study, the amount of HDL was significantly decreased in plasma of 75% RO-fed salmon, compared with fish fed FO (11). Torstensen and co-workers (11), however, measured the total lipoprotein levels in plasma, which is a relatively crude measurement compared with measuring the expression levels of a single apolipoprotein. Furthermore, the recirculation time of HDL particles in salmon plasma is not known, and increases in the expression of apo A-I-I in the 75% RO group may be a response to decreased plasma HDL levels as a result of increased HDL uptake in liver. Unraveling the complex gene and protein expression patterns of apolipoproteins in Atlantic salmon awaits further characterization of these individual physiological components.
The microarray data presented here as well as other recent data utilizing microarrays (53), confirm the effects of (n-3) HUFA on the regulation of gene expression, suggesting that cDNA microarray technology is a powerful tool for studying nutrient-gene interactions. Therefore, further work may be performed using the present array, or other high-density arrays developed recently using features of salmonid origin (39,54,55), as a complement to the specific microarray utilized in study.
In conclusion, the current thoroughly validated and quality controlled microarray study (technical replicates, internal controls, and PCR) showed differential, although relatively minor, regulation of several genes related to hepatic lipid metabolism in Atlantic salmon in response to changes in dietary fatty acids. These included 5 FAD, PPAR, and several genes whose gene products are involved in mitochondrial functions. Salmon have a partially tetraploedic genome, and genes may be present in more than one isotype, which may differ in expression patterns (56,57). Thus, there is an argued need for a more thorough examination of genes and gene expression patterns. However, the effects of nutrients are under strict homeostatic control (58), and microarray analysis is a valuable tool with which to screen for nutritional effects on a gene-expression level in Atlantic salmon, and should be further pursued.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supplemental Table 1 is available as Online Supporting Material with the online posting of this paper at www.nutrition.org.
4 Present address: Department of Obstetrics and Gynecology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72205.
5 Abbreviations used: ACS, acyl-CoA synthetase; apo, apolipoprotein; CLA, conjugated linoleic acid; CPT, carnitine palmitoyltransferase; Cy, cyanine; EST, expressed sequence tag; FAD, fatty acid desaturase; FO, fish oil; HNF-4, hepatic nuclear factor-4; HUFA, highly unsaturated fatty acid (carbon chain length 
C20 with 3 double bonds); Lb-FABP, liver basic fatty acid binding protein; LO, linseed oil; LXR, liver X receptor; MIAME, minimum information about a microarray experiment; OO, olive oil; PPAR, peroxisome proliferator-activated receptor; Q-PCR, quantitative real time-PCR; RO, rapeseed oil; SAM, significance analysis of microarrays; SCD, 9 fatty acid desaturase; SREBP, sterol regulatory element binding protein.
Manuscript received 25 February 2005. Initial review completed 14 April 2005. Revision accepted 21 July 2005.
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