The aim of the present study was to compare the indices (product/precursor ratios) estimating desaturases and elongases participating in the formation of MUFAs, and n-6 and n-3 LCPUFAs with the expression of genes involved in lipid metabolism, and with fat percent in breast muscle of chickens fed diets with or without linseed oil and high and low selenium. Desaturases and elongases are important regulators in metabolism of fatty acids and they are expressed in many tissues, e.g. in skeletal muscle[26, 27]. The expression of genes involved in fat metabolism are regulated by nutrients such as minerals, vitamins and fatty acids, hormones and other environmental factors[26, 27]. To our knowledge, there is little, if any, information of comparison of the expression of genes in fat metabolism with indices of elongases and desaturases and with individual fatty acid profiles and fatty concentrations in muscle from chickens fed diets enriched in ALA and Se.
In the present study, the diets with high and low Se supplementation showed that high Se supplementation did not cause large differences in fatty acid profiles in chicken breast muscle.
Linseed oil, on the contrary, resulted in large differences in fatty acid profiles in chicken muscles; significantly increased levels of ALA, EPA, DPA and DHA, and reduced content of LA and AA, compared to muscle from chickens without LO in the feed. The product/precursor indices for fatty acids were highly significantly different for desaturation and elongation of ALA to EPA, DPA and DHA, and LA to AA between the diet groups without or with LO. The differences in indices of product/precursor fatty acid were not a result of changes in gene expression since the gene expression of enzymes participating in desaturation and in general lipid metabolism was similar in the diet groups without and with LO.
The decreased Delta-6 and Delta-5 desaturase activity in the LO supplemented diet, estimated by product/precursor indices are in line with studies on fatty acid desaturase activity in erythrocytes that were decreased due to low n-6/n-3 ratios[28, 29]. In the present study, the animals that were given a diet with LO had an n-6/n-3 ratio on 1.4 in breast meat and the ratio was 6.4 in breast meat from the ‘without LO’ group. Since the product/precursor estimate for the two elongase reactions 20:3n-6/18:3n-6 and 22:5n-3/20:5n-3 showed opposite responses to LO supplementation, it could be suggested that two different elongases (Elovl2 and Elovl5) might be the main enzymes in these two conversions. But this suggestion has to be verified by direct enzyme measurements.
The sum of EPA + DPA + DHA in the chicken breast muscle was 0.3 mg/g in the group without LO, and 0.7 mg/g in the LO supplemented group. The AA content was 0.6 mg/g and 0.3 mg/g in the two groups, respectively, thus the sum of n-3 + n-6 LCPUFAs were close to identical in the groups without and with LO supplementation. The sum of LA + ALA was 2.3 mg/g and 1.6 mg/g muscle tissue in group without and with LO supplementation, respectively. Since the animals did not have any n-3 and n-6 LCPUFAs in the diet during their growth from 40 g at hatching till 1.3 kg at slaughtering, it must be anticipated that AA, EPA, DPA and DHA was produced by endogenous conversion of ALA to n-3 LCPUFAs and LA to AA.
The n-3 and n-6 fatty acids compete for the same desaturase and elongase enzymes in the fatty acid pathway from LA to AA and from ALA to EPA + DPA + DHA. Since the sum of n-3 + n-6 PUFAs in breast muscle was lower in muscle of the chickens fed with LO compared to without LO, the total need for elongases and desaturases could be anticipated to be less in the group ‘With LO’ compared to ‘Without LO’. However, the gene expression of Fads1 and 2 were the same in breast meat from chickens fed diets with or without LO (Table 4). These findings are in line with the study by Tu et al. in rats that were fed diets containing between 0.2 and 2.9 energy percent ALA, showing no differences in expression levels of desaturases and elongases between the diet groups (in the present study the energy percent from ALA was 0.7% in the feed without LO, and 2.5% in the feed with LO).
The expression of Fads9 in chicken breast muscle was not affected by supplementation of Se or LO to the feed. Delta-9 desaturase indices estimated by the ratios between 16:1n-7/16:0 and 18:1n-9/18:0 in breast muscle showed that the indices were not changed by Se supplementation, but the 16:1n-7/16:0 index was slightly higher in the ‘With LO’ group, compared to ‘Without LO’. Delta-9 desaturase is known to be inhibited by PUFA. The sum of total PUFAs in the breast muscle in the ‘With LO’ group was 2.8 mg/g, and it was 3.4 mg/g in the ‘Without LO’ muscle. It might be speculated that this difference in PUFA caused the higher 16:1n-7/16:0 Delta-9 desaturase index in the group supplemented with LO.
The Se concentration was either 0.13 mg/kg or 1.1 mg/kg feed, which is at the lower and higher range of Se intake for chickens. The birds having 0.13 mg Se/kg feed or 1.1 mg Se/kg had a normal growth, and had not higher mortality than normal, indicating that the intake was acceptable. The recommended lowest intake is 0.15 mg/kg feed for poultry, and highest recommended intake is 0.5 mg/kg.
Comparison of desaturase indices in muscle from the animals fed with low Se without LO to high Se with LO, (Table 6, right panel), with animals fed without and with LO (Table 6 middle panel), shows that LO had major effects on the indices, and Se supplementation had only minor effect on the indices.
It has been shown that a high intake of ALA resulted in a relatively lower conversion of ALA to n-3 LCPUFAs compared to a diet with less ALA[12, 31], and this is confirmed in the present study: The indices for desaturation and elongation of ALA to the sum EPA + DPA + DHA, and ALA to DPA and DHA in the LO fed animals showed a decrease compared to the group fed without LO (Table 6), indicating that the LO supplementation slowed down the formation to n-3 LCPUFAs compared to a diet containing less ALA. The fatty acid pathway from LA to AA; the index AA/LA was reduced in the breast muscle from chickens supplemented with LO (Table 6), showing that the formation of AA was also reduced by the supplementation of ALA. LO supplementation resulted in more than a doubling of n-3 LCPUFAs in breast muscle. Since the expression of the measured genes in lipid and antioxidant metabolism was not affected by the LO supplementation in the present study, this indicates that the profile of n-3 LCPUFAs in chicken muscle may be controlled through substrate competition, feed-back control and esterification rather than by alterations in gene expression of enzymes in lipid metabolism such as Fads1, Fads2, Fads9, HMGCoA reductase, Acox and Cpt1. This is consistent with findings in rats.
Breast muscle Se content, Gpx activity in whole blood and Gpx4 expression were all increased by high Se intake. The increased expression of the Gpx4 gene with high Se intake indicates an up-regulation when Se intake is increased from 0.13 to 1.1 mg/kg feed. An increase in Gpx4 expression following Se supplementation in chicken muscle has also been shown by others.
Associations between fatty acid indices, fat percent and expression of genes in lipid metabolism in breast muscle from the birds fed low Se and without LO
Noteworthy, as shown in Table 7, there is a strong positive correlation between muscle fat content and synthesis of MUFA as estimated by 16:1n-7/16:0 and 18:1n-9/18:0 indices, and a strong negative correlation between muscle fat and synthesis of AA, EPA, DPA, DHA and DGLA. The fat percent in muscle has been shown to be a determinant for metabolic disease, inflammation and non-chronic diseases. Indeed, the present results confirm that there are strong correlations between fat percent and indices for desaturases and elongases, apparent supporting the role of muscle fat percent as a marker in relation to chronic diseases in man. However, studies in humans are required to substantiate this hypothesis.
The lack of correlations between muscle fat percent and expression of genes in lipid metabolism is surprising. There were no correlations between gene expression of Fads1 and the index estimate of Delta-5 desaturase 18:3n-6/18:2n-6, and no correlation between expression of Fads2 and the estimate of Delta-6 desaturase 20:4n-6/20:3n-6, as shown in Table 7 in the ‘low Se-without LO’ group, but also the same was shown when calculated on the other animals in the study. A positive association between Fads9 and the estimates of Delta-9 desaturase indices 16:1n-7/16:0 and 18:1n-9/18:0 were expected, but as shown in Table 7, the association did not reach level of significance. The lack of association between the gene expression for the fatty acid desaturases and the corresponding product/precursors ratios confirms the role of fatty acid concentrations, and not gene expressions, as the main regulators of the enzyme activities.
As shown in Table 7, the fat percent of the muscle was positively associated with percent of most fatty acids with 14 to 18 C-atoms (not 18:0), and negatively associated with 18:0 and all the C20 and C22 unsaturated fatty acids. Thus, the amount of fat in the muscle is fundamental for the fatty acid profile of meat; much fat in muscle results in lower percentage of LCPUFAs. This is also shown and discussed by others[33, 34].
The positive correlation between the expression of the genes for Fads1, HMGCoA reductase, Acox, Cpt1 and Sod indicates that there may be a link between these genes, possibly via a central regulation of the gene expression. It is known that many genes related to lipid metabolism are controlled by the same transcription factors such as peroxisome proliferator-activated receptor (PPAR) and sterol response element binding protein (SREBP)[20, 35]. Many reactive oxygen species are continuously produced as byproducts of aerobic metabolism. Sod is connected to β-oxidation of fatty acids by catalyzing the conversion of reactive oxygen species produced during β-oxidation to hydrogen peroxide.
The expression of HMGCoA reductase was positively correlated to the Acox and Gpx4 expression. HMGCoA reductase is involved in cholesterol, coenzyme Q10 and dolichole synthesis. Coenzyme Q10 has a double function, it is an essential part of the electron transport system in the mitochondrial respiratory chain but it is also an important endogenous antioxidant, especially in its reduced form (ubiquinol). It has a rapid turnover in mammals, presumably because of rapid peroxidative degradation of its side chain with numerous double bonds. With higher dietary intake of LO rich in ALA, it could be suggested that the synthesis of the antioxidant ubiquinol was up-regulated, but no effect was observed on expression of HMGCoA reductase in the present study (Table 4).
The positive association between Acox and the expression of Cpt1 and Gpx4 may indicate a link to mitochondrial β-oxidation and antioxidant status, but the lack of effect on gene expression of Acox by Se and LO, and the lack of association between Acox and other fatty acids and fatty acid ratios indicates that other factors than the Se content and the LO intake affected Acox.