INTRODUCTION

Cardiovascular diseases (CVD) are the major cause of death in adults in several countries.[1] The development of CVD is multifactorial, with risk factors that are modifiable, such as diet, exercise, and smoking, and others that are not, such as gender, age, and genetics. The behavioral factors might modify a number of biochemical markers involved in dyslipidemia, hypertension, inflammation, and insulin resistance.[2, 3]

Alterations in lipid and lipoprotein metabolisms have been investigated as important CVD risk factors. High triglycerides (TG) and low high-density lipoprotein (HDL) concentration are strong predictable factors of cardiac events.[1, 4] Either environmental or genetic factors can affect the lipoproteins concentration, which may affect the risk of developing CVD. Thus, key metabolism regulators such as the peroxisome proliferator-activated receptor (PPAR) family are the candidate genes to investigate genetic predisposition of this complex disease.[1]

PPAR family members are nuclear receptors involved in lipid and carbohydrate metabolisms, adipogenesis, and insulin sensitivity.[5] PPARα is highly expressed in tissues with high fatty acid catabolism and integrates several metabolic pathways, including fatty acid β-oxidation, lipoprotein synthesis, and amino acid catabolism.[5] PPARα is located on 22q13.3, and it consists of eight exons. Single nucleotide polymorphisms (SNPs) were described in this gene and were found to be associated with dyslipidemia, insulin resistance, diabetes, and CVD.[1, 5, 6] Hypertriglyceridemia (HTG) is a common and heterogeneous metabolic disorder that represents a risk factor for premature coronary heart disease. HTG can be caused by various interactions between environmental and genetic factors.[6]

In humans, several variants in PPARα have been identified. One variant in exon 5, Leu162Val, has been studied extensively and has been associated with body mass index (BMI), fasting concentration of total cholesterol (TC), HDL-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C), apolipoprotein B (ApoB), apolipoprotein A-I (ApoA-I), and the progression of atherosclerosis.[7, 8, 9, 10, 11, 12] Furthermore, this variant has been shown to have an effect on transactivation efficiency in vitro.[8, 9, 10, 11, 12, 13] In addition to its effect on fasting lipid parameters, PPARα variants may exert their effect on atherosclerosis progression via the modulation of postprandial lipid metabolism.[12]

L162V polymorphism represents a C to G substitution leading to a leucine to valine amino acid exchange in codon 162.[8] Studies evaluating the association between this polymorphism and lipid profile reported controversial results.[5] The intron7 G/C polymorphism leads to a G to C substitution on intron7. An association between this SNP and lower TG levels was observed in diabetic patients. Conversely, the C allele of intron7 G/C was associated with atherosclerosis progression[14] and left ventricular growth induced by exercise.[15] The V162 allele was also associated with higher levels of TC,[8, 11] LDL-C,[11] and ApoB levels[11, 16] in other studies. Previous studies demonstrated that although the V162 allele was associated with lipid abnormalities, carriers of the less common allele were not at increased risk of developing cardiovascular disease or diabetes.[8, 11, 12, 17]

Activation of PPARα by agonists leads to reduced adiposity and lowered triglyceride levels by reduced food intake.[18]

Prospective cohort studies and secondary prevention trials indicate that n-3 polyunsaturated fatty acids (PUFAs) from fish or plant sources lower the risk of CVD.[19, 20, 21, 22, 23, 24] Furthermore, since fatty acids are ligands for PPARα, we conducted this study to determine the lipid lowering effects of eicosapentaenoic acid (EPA) supplementation in PPARα polymorphism.

METHODS

RESULTS

The baseline characteristics of subjects were described previously.[25, 27] For comparison of groups with different PPARα genotypes, the data obtained from Val162/Val and Leu162/Val subjects were combined. EPA supplementation decreased the levels of serum lipid and lipoprotein in the two study groups. No interaction was observed between PPARα genotypes and the degree of changes in plasma lipids and lipoproteins after EPA consumption [Table 1]. EPA supplementation decreased levels of serum lipid and lipoprotein in the two study groups. No interaction was observed between CC and GC carriers and the degree of changes in plasma lipids and lipoproteins after EPA consumption [Table 2]. Although EPA consumption lowered the lipid and lipoprotein concentrations in Leu162 and Val162 carriers and CC and GC carriers, the observed difference between the studied groups was not statistically significant [Tables 1 and 2]. EPA supplementation increased the plasma EPA in both Leu162 and Val162 carriers [Table 3] (P = 0.01 and P = 0.05, respectively), but it was observed that it has been significantly increased more in Val162 carriers than Leu162 (P = 0.001) [Table 3].

Table 1. Changes in serum lipids and lipoproteins of hypertriglyceridemic subjects at baseline and after 8-weeks eicosapentaenoic acid supplementation in the studied groups

Table 2. Changes in serum lipids and lipoproteins of hypertriglyceridemic subjects at baseline and after 8-weeks eicosapentaenoic acid supplementation in the studied groups

Table 3. Changes in plasma eicosapentaenoic acid (EPA) levels in hypertriglyceridemic subjects after 8-week EPA supplementation stratified by Leu162 and Val162 peroxisome proliferator-activated receptor alpha genotypes*

Supplementation increased plasma EPA in GG and GC carriers (P = 0.001 and P =0.001) [Table 4], but it was observed that it has been significantly increased more in GC carriers than in GG carriers (P = 0.001) [Table 4].

Table 4. Changes in plasma eicosapentaenoic acid levels in hypertriglyceridemic subjects after 8-weeks EPA supplementation stratified by GG and GC polymorphism of peroxisome proliferator-activated receptor alpha genotypes*

DISCUSSION

As PPARα has a role in lipid homeostasis, we were therefore interested in their variable plasma lipid levels that often account for the independent increased risk of atherosclerosis[28, 16] and cardiovascular risk factors.[29]

The major aim of this study was to identify the role that PPARα Leu162Val polymorphism may play in the variability of serum lipid levels, and changes in lipids and lipoproteins level in response to EPA supplementation.

Among the reported polymorphisms for the PPARα gene, Leu162Val polymorphism is the most prevalent with a frequency of 10.45% in diabetic and non-diabetic patients.[13] It is also associated with alterations in lipoprotein concentrations in both diabetic and non-diabetic subjects.[16] We found a prevalence of 21.8% for the Leu162/Val polymorphism in these Iranian hypertriglyceridemic subjects.[27] There was no difference in the frequency of Leu162/Val between men and women, and there were no significant differences between lipid and lipoprotein in Leu162 and Val162 carriers except for TC. In our study, a comparison between Val162 carries and Leu carriers has indicated a significant association between PPARα Leu162Val polymorphism and blood lipids, where the Val162 carriers showed higher concentrations of TC.[27]

This polymorphism has been previously found to be associated with greater blood TC,[16] LDL-C,[8, 11, 12] TG levels,[9, 30] and lower HDL-C concentration[8] depending upon the population studied. Most studies that have found an association between the Leu162Val polymorphism and greater blood lipid concentrations were conducted in diabetic subjects; few studies have found a positive association in healthy subjects.[11, 31]

The intron7 allele has been shown to be associated with increased progression of atherosclerosis. The Val162 allele and intron7 allele are in strong allelic association, such that 78% of Val162 carriers are found in combination with the intron7.[14] In our study the frequency of the intron7 polymorphism was 55.3%, which is much greater than the frequency of Leu162Val polymorphism in the studied subjects, and all of the subjects who were Leu162/Val carriers also had an intron7 allele. Except for blood TC level, there were no significant associations between intron7 allele and blood lipid, ApoB, or apolipoprotein CIII (ApoCIII) concentrations. Similar results have been observed in Foucher's study in which intron7 polymorphism was only associated with blood TC levels.[32]

Frequency of intron7 polymorphism is greater than Leu162Val and all hypertriglyceridemic subjects with Leu162 polymorphism might be carriers of the intron7 polymorphism.[31]

The expected changes in post-supplementation plasma lipids and lipoproteins were observed with EPA intervention. Results of the present study clearly show that two carriers of PPARα genotypes can influence the lipid-lowering effects of EPA supplementation in hypertriglyceridemic subjects.

Although EPA supplementation decreased serum lipids and lipoproteins and increased serum HDL-C in Leu162, Val162 carriers and GG, GC carriers, there was no significant interaction between the two carriers after EPA consumption.

We believe that these data provide some insight into the pathways that could be involved in generating the association between LDL cholesterol concentration and the Leu162Val polymorphism at the PPARα locus.[11] There was no association between the Leu162Val polymorphism at the PPARα locus and TG, HDL-C, very low-density lipoprotein, ApoB, and ApoCIII concentrations. Several possible reasons exist for these results. Intrer-individual variation in the plasma lipid and lipoprotein. This phenomenon will reduce the chance of finding an association between a genetic variant and plasma lipid and lipoprotein concentrations, especially when the changes are small, as in this case. Alternatively, additional gene–gene or gene–diet interactions may exist that modulates the effect of the L162V polymorphism, and this will need to be addressed in future studies. Potential gene–environment interactions may be particularly relevant to this polymorphism. The level of exposure to known endogenous (such as non-esterified fatty acids) or exogenous ligands (such as dietary PUFAs) may therefore be important in determining the biochemical phenotype observed with this polymorphism.[11]

Previous studies have shown the PPARα L162V SNP to be associated with multiple lipid and lipoprotein measures, with one study finding the effect of the L162V SNP on triacylglycerol and ApoCIII concentrations to be dependent on PUFA intake.[9, 10, 11, 12, 33]

In the current study, we did not observe significant associations between the PPARα L162V SNP and multiple lipid and lipoprotein measures. However, we did not observe a significant interaction between the Leu162, Val162 and EPA consumption with regards to lipid and lipoprotein concentrations in hypertriglyceridemic subjects. But we could observe a significant difference between EPA supplementation and Leu162 and Val162, GG and GC polymorphism. This finding suggests that genetic variation within the PPARα Leu162/Val cannot modulate the association of EPA intakes with lipid and lipoprotein profiles. It is possible that the sample of study is not high enough to affect the association between PPARα genotype and lipid after EPA consumption However, previous studies of genetic variation in the PPARα gene have found gene-by-diet interactions specific to different polymorphisms in different racial-ethnic groups (i.e., L162V in whites and V227A in Asians).[33]

The role of dietary fatty acids in regulating plasma lipoprotein and lipid concentrations is well documented.[34] Compared with SFA intake, PUFA intake has been shown to lower LDL-C and HDL-C concentrations. However, the great inter-individual lipoprotein and lipid response to such dietary changes is not well understood. Genetic factors are likely to play a significant role, because polymorphisms in several genes seem to modulate lipoprotein and lipid responses to dietary modifications.[35] This study is the first in hypertriglyceridemic subjects to examine whether plasma lipid and lipoprotein responsiveness to EPA supplementation are influenced by the PPARα Leu162Val gene polymorphism or not.

In vitro studies have shown that the Val162 allele has a greater transactivation activity when treated with a PPARα agonist.[8] Previous studies showed the increasing effect of the Leu162Val polymorphism on TC, LDL-C, HDL-C,[8] ApoB,[11, 12, 16] apo A-1,[16] and ApoCIII.[11] This polymorphism was also found to be associated with decreased concentrations of fasting serum triacylglycerol among white subjects with normal glucose tolerance.[36, 37] Their investigation of the gene-by-EPA consumption interaction effects on the metabolic profile led us to several interesting observations. They found that EPA consumption interacts with the Leu162/Leu and Leu162Val polymorphism to modulate the TC concentrations These gene-by-EPA supplementation interaction effects may then modulate plasma lipid concentrations and need further replication in different intervention studies and later in a meta- analysis.[37] Although we cannot interaction between EPA consumption and PPARα genotypes. One study of the gene-by-diet interaction effects on the metabolic profile led to several interesting observations. They found that diet interacts with the Leu162Val polymorphism to modulate TC concentrations, plasma ApoA-1 concentrations, and cholesterol in small LDL particles. This gene-by diet interaction effects may then modulate plasma lipid concentrations and need further replication in different intervention studies and later in a meta-analysis. In addition, independent of the genotype, diet had an effect on HDL.[36, 38]

CONCLUSIONS

EPA consumption has lipid-lowering effect in hypertriglyceridemic subjects in both Leu162 and Val162, GG, and GC carriers. But there was no significant interaction between EPA supplementation and PPARα genotypes. Thus, genetic variation within the PPARα Leu162/Val cannot modulate the association of EPA intakes with lipid and lipoprotein profiles. However, we must note the sample size in this study.