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 [23
], we observed a significant reduction in plasma TG values for EO-fed mice compared with PO-fed mice that was similar to the TG reduction obtained with FO. Plasma TC, EC, FC and PL were also reduced with EO compared to PO supplementation. Of the three lipoprotein classes, EO had the greatest effect on VLDL lipid concentration, with significant reductions in plasma concentration of all lipid classes, suggesting the hepatic secretion of VLDL lipid might be reduced with EO consumption. Supporting this notion, the average content of hepatic TG and TC was lower in EO vs. PO-fed mice and several hepatic genes involved in fatty acid and TG biosynthesis were significantly down regulated with EO and FO consumption. In addition, there was a significant positive association between hepatic TG content and plasma TG concentration, suggesting that down regulation of hepatic fatty acid and TG synthetic genes was responsible, in part, for the reduced plasma TG concentrations of EO and FO-fed mice through reduced hepatic TG synthesis and secretion in VLDL particles. Finally, EO supplementation resulted in significant enrichment of plasma and liver neutral lipid fractions with EPA and plasma and liver TG with DHA, though not to the extent achieved with FO. Thus, dietary EO resulted in a reduction of plasma lipids that was similar to that of FO with only half the enrichment of plasma and liver lipid fractions with EPA and DHA. These results suggest that dietary supplementation with EO may represent a botanical alternative to FO to reduce the risk of premature atherosclerosis.
Consumption of fatty fish or FO supplements is associated with many cardioprotective effects [11
]. The cardioprotective ingredient in FO is believed to be highly unsaturated fatty acids (≥ 5 double bonds) with carbon chains ≥ 20, such as EPA and DHA. Although the benefits of EPA and DHA are well-recognized, dietary consumption of these PUFAs in the US population is far below that needed for cardiovascular benefit [30
]. As a result, attempts have been made to find dietary botanical sources enriched in n-3 PUFA that might function to increase cellular EPA and DHA content and provide cardioprotective benefit. There was initial hope that dietary botanical oils enriched in ALA might result in enrichment of tissues with EPA and DHA through fatty acid elongation and desaturation pathways. However, this approach proved to be disappointing because the Δ-6 desaturase reaction is rate limiting and results in only a small fraction of dietary ALA being converted to EPA (4 to 15 %, refs [17
]). Several groups have used dietary supplementation with SDA or EO, which is relatively enriched in SDA, as an alternative to FO because SDA is the immediate product of Δ-6 desaturation of ALA and SDA supplementation results in significant enrichment of plasma and blood cells with EPA. In one study with mildly hypertriglyceridemic subjects, EO supplementation was shown to lower plasma TG concentrations, which is a consistently observed effect of FO, EPA and/or DHA supplementation in humans [21
]. However, no study to date has examined the role of EO supplementation on hepatic lipid phenotype or gene expression. Given the central role of the liver in TG production and energy partitioning in the body, this represents a fundamental gap in knowledge.
Several lines of evidence support the role of n-3 PUFAs in decreasing hepatic TG synthesis and secretion in VLDL. Several kinetic studies in humans demonstrated that dietary n-3 PUFAs decreased VLDL TG synthesis and secretion (reviewed in [29
]). Liver perfusion studies in rats and non-human primates demonstrated that hepatic TG secretion, but not apoB secretion in the non-human primate study, was decreased with n-3 PUFA consumption compared to the control fat [32
]. Several mechanisms for the decrease in hepatic TG synthesis and secretion are supported by experimental data. N-3 PUFA decreases expression of lipogenic genes, such as fatty acid synthase (FAS) and acetyl CoA carboxylase (ACC), through the decreased expression of the nuclear form of sterol regulatory element binding protein-1c (SREBP-1c) [34
]. Part of the decreased expression appears related to accelerated SREBP-1c mRNA decay [39
]. Another reported mechanism for decreased TG secretion with n-3 PUFA consumption is increased fatty acid oxidation, likely mediated by peroxisome proliferator activated receptor α (PPARα), a nuclear transcription factor that is activated by fatty acid binding [34
]. However, the latter mechanism is not rate limiting in vivo since FO reduced plasma TG concentrations in PPARα knockout mice [40
]. N-3 PUFA also downregulates fatty acid desaturation pathways in the liver (i.e., Δ5, Δ6, SCD-1) via SREBP-1c mediated transcriptional con- trol [41
]. Taken together, these studies suggest a multifaceted control of TG synthesis and secretion by n-3 PUFA in the liver and that dietary n-3 PUFA lowers plasma TG concentrations, in part, by decreased hepatic VLDL TG synthesis and secretion. Whether these mechanisms are responsible for the lower plasma TG concentrations in mice fed EO will require additional studies.
Another potential mechanism for plasma TG reduction by n-3 PUFA is increased TG-enriched lipoprotein catabolism. VLDL turnover studies in human subjects suggest a trend towards faster catabolic rates in those given n-3 PUFA [29
]. Chylomicron lipid removal from plasma was also faster in n-3 PUFA-fed subjects compared with olive oil [43
]. Post-heparin lipase activity, consisting of LPL and HL, in human subjects has not been particularly responsive to dietary n-3 PUFA [45
], although increased LPL activity was observed several studies [47
]. However, the rate of hydrolysis of TG-enriched lipoproteins may be greater when TG is enriched with n-3 PUFA [49
]. Another study demonstrated increased hepatic clearance of IDL/LDL in LDLrKO mice with a hepatic specific deletion of the LDL related protein (LRP) receptor, suggesting an increased removal of apoB lipoproteins by the liver by an LDLr and LRP-independent pathway [51
]. The results from theses studies collectively suggest that part of the plasma TG lowering effect of n-3 PUFA is related to increased apoB lipoprotein catabolism and this may also be the case with EO supplementation.
In summary, we have identified EO a dietary botanical oil that may function to reduce plasma TG concentrations and the development of cardiovascular disease by providing an enriched source of SDA that can be converted to EPA and DHA, providing similar health benefits as FO, but presumably without the side effects and low compliance of FO. A combination of EO with other sources of n-3 PUFAs, such as fatty fish, would aid in improving the intake of n-3 relative to n-6 PUFA in the general population. The results from this study suggest a relatively simple dietary modification that may have a high impact on reduction of premature cardiovascular disease in the American population.