Research articleEchium oil reduces plasma lipids and hepatic lipogenic gene expression in apoB100-only LDL receptor knockout mice☆
Introduction
Cardiovascular disease (CVD) is the leading cause of death in the United States and other Westernized societies. Elevated plasma LDL and low HDL concentrations have long been known as important risk factors for development of premature CVD in humans and animal models [1]. Recently, elevated plasma TG concentrations have also been recognized as an independent risk factor for CVD, suggesting that attention should be given to reducing elevated levels of plasma TG in addition to treatment of elevated plasma total cholesterol (TC) and LDL cholesterol [2].
Long-chain (≥20 carbons) n-3 polyunsaturated (≥4 double bonds) fatty acids (PUFA) in fish oil (FO) are among the most effective and widely documented dietary components to reduce CVD risk in human and animal models. A wealth of evidence has accumulated for the efficacy of dietary FO in reducing CVD risk since the initial observation that Greenland Eskimos consuming a high fat diet, containing large amounts of FO, had lower death rates from CVD compared with their counterparts in Denmark [3]. Various animal models have produced similar results including pigs, monkeys, rats, dogs and mice [4], [5], [6], [7], [8], [9]. Multiple mechanisms have been proposed for the cardioprotective benefits of FO including reduced blood pressure, decreased thrombosis, decreased arrhythmias, decreased inflammation, decreased endothelial activation and decreased plasma TG concentration [10]. Indeed, one of the most consistent observations associated with the consumption of FO or purified n-3 PUFA is a reduction in plasma TG levels [11], [12]. Thus, increased consumption of n-3 PUFA in FO seems to be an effective way to reduce plasma TG and, presumably, the risk of developing premature CVD.
Despite the well-documented health benefits of n-3 PUFA, the intake of n-6 to n-3 PUFA in the United States is approximately 10:1, with the principal n-3 PUFA consumed being ALA (90% of n-3 PUFA intake) [13]. The recommended intake ratio is 2.3:1 [13]. There are multiple barriers to achieving the recommended n-6 to n-3 PUFA ratio in the American diet. To achieve this by fish consumption alone would require a fourfold increased intake of fatty fish [13]. Given the relatively higher cost of fish to other sources of meat in the American diet and personal preferences in food, this option seems very unlikely. Supplementation of the diet with FO would be another means of increasing n-3 PUFA intake, but this option is unlikely to succeed due to the organoleptic aversion (i.e., fishy aftertaste) to FO supplements. Yet another possibility is to increase the consumption of foods and oils containing ALA, which can go through elongation and desaturation to EPA. However, studies have shown that ALA is poorly converted to EPA in humans and rodents (i.e., 4–15% conversion efficiency) and the degree of conversion depends on the amount of 18:2 in the diet, since 18:2 competes with ALA for Δ6-desaturation and diminishes the conversion of ALA to EPA [14], [15], [16], [17], [18]. One suggested way to circumvent these problems is to enrich existing foods with EPA, DHA and ALA using biotechnology, but the introduction of genetically engineered food sources has met stiff social resistance. Finally, an approach that holds promise is to use a botanical oil that is enriched in SDA (18:4 n-3), which is the immediate product of Δ6-desaturation of ALA. Since Δ6 desaturase is the rate limiting step in the formation of EPA from ALA, dietary supplementation with SDA can enrich cellular membranes and plasma lipoproteins with EPA and may result in the beneficial cardiovascular effects of FO without the side effects mentioned above.
Echium oil (EO) from the seeds of Echium plantagineum has been identified as a natural source of SDA, which accounts for approximately 13% of total fatty acids in the oil [19], [20]. In a recent human study, dietary supplementation of mild-to-moderate hypertriglyceridemic subjects with 15 g EO per day for 4 weeks resulted in 21% reduction in serum TG concentrations compared with baseline values. Decreased serum TG levels were associated with a significant increase in long chain n-3 PUFA, including EPA, in plasma and neutrophils in these subjects [19]. Similar enrichment of EPA in plasma and red cells by SDA supplementation has also been reported in another human study [18]. However, the mechanisms for the plasma TG-lowering effects of EO are unknown. Considering the central role of the liver in TG synthesis and secretion, it is likely that dietary EO lowers plasma TG through effects on hepatic gene expression. However, how EO supplementation affects liver fatty acid composition, lipid content and gene expression is not known and can only be examined in a suitable animal model.
To address the gaps in knowledge on EO supplementation, we performed supplementation experiments using apoB100-only LDLrKO mice. The B100-only LDLrKO mouse is an animal model of atherosclerosis that exhibits a mild elevation in plasma TG concentrations [21]. The goal of this study was to determine whether EO supplementation would reduce plasma TG concentrations, enrich hepatic lipid fractions with EPA and alter hepatic expression of genes involved in TG synthesis to a similar extent as that observed with FO supplementation.
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Animals and diets
The apoB100-only LDLrKO mice in the C57BL/6 background (∼93%) were housed in a specific pathogen-free facility at Wake Forest University School of Medicine. The mice were generated by crossing the apoB100-only mice, originally generated by Dr. Steve Young at the Gladstone Institute (San Francisco, CA, USA) [21], with LDLrKO mice in the C57BL/6 background. All protocols and procedures were approved by the Institutional Animal Care and Use committee.
Mice were maintained on a chow diet from
Plasma lipid response to EO supplementation
Our initial studies were designed to determine how much EO supplementation was necessary to result in a 30–40% reduction in plasma TG in apoB100-only LDLrKO mice as a prelude to future atherosclerosis studies. We started with supplementing the basal diet (10% Cal from PO, 0.2% cholesterol) with an additional 10% Cal as PO, EO or FO for a total of 20% Cal from fat. Body weight and liver weight of mice fed the three experimental diets did not differ significantly at the end of the 8-week
Discussion
The purpose of this study was to determine whether EO can serve as a botanical source of n-3 dietary PUFA to enrich tissues in EPA and result in some of the cardioprotective effects that have been documented for FO. One of the better documented effects of dietary FO is a reduction in plasma TG concentrations. Indeed, in our study using a mouse model of atherosclerosis with mildly elevated plasma TG concentrations [21], [25], we observed a significant reduction in plasma TG values for EO-fed
Acknowledgments
The authors gratefully acknowledge Bioriginal Food and Science Corporation for supplying echium oil for the study.
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This study was supported by NIH grant P50 AT0027820. No conflicts of interest to report.