Echium oil reduces plasma lipids and hepatic lipogenic gene expression in apoB100-only LDL receptor knockout mice

Abstract

We tested the hypothesis that dietary supplementation with echium oil (EO), which is enriched in stearidonic acid (SDA; 18:4 n-3), the product of Delta-6 desaturation of 18:3 n-3, will decrease plasma triglyceride (TG) concentrations and result in conversion of SDA to eicosapentaenoic acid (EPA) in the liver. Mildly hypertriglyceridemic mice (apoB100-only LDLrKO) were fed a basal diet containing 10% calories as palm oil (PO) and 0.2% cholesterol for 4 weeks, after which they were randomly assigned to experimental diets consisting of the basal diet plus supplementation of 10% of calories as PO, EO or fish oil (FO) for 8 weeks. The EO and FO experimental diets decreased plasma TG and VLDL lipid concentration, and hepatic TG content compared to PO, and there was a significant correlation between hepatic TG content and plasma TG concentration among diet groups. EO fed mice had plasma and liver lipid EPA enrichment that was greater than PO-fed mice but less than FO-fed mice. Down-regulation of several genes involved in hepatic TG biosynthesis was similar for mice fed EO and FO and significantly lower compared to those fed PO. In conclusion, EO may provide a botanical alternative to FO for reduction of plasma TG concentrations.

Fig. 1. Plasma lipid response to dietary supplementation with PO, EO, or FO

Eight week old chow-fed apoB100-only LDLrKO mice were fed a basal 10% PO diet for four weeks, after which they were randomly assigned to an experimental diet consisting of the basal diet plus an additional 10% of calories as PO (20% PO; n=8), EO (10% PO + 10% EO; n=8) or FO (10% PO + 10% FO; n=9) for an additional 8 weeks. Mice were bled at the end of the basal diet period (0 wk of experimental phase) and every two weeks during the experimental diet period for measurement of total plasma cholesterol (TPC), triglyceride (TG), esterified cholesterol (EC), free cholesterol (FC), and phospholipid (PL) concentrations. Values are mean ± SEM.

Fig. 2. Plasma lipoprotein lipid concentration

Three groups of apoB100-only LDLr KO mice were fed diets containing PO, EO and FO as described in the Methods. At the end of the 8 week experimental diet period, plasma was fractionated by FPLC and lipoprotein elution was monitored by enzymatic cholesterol assay. Fractions corresponding to the elution position of VLDL, LDL, and HDL were pooled and assayed for TC, FC, PL and CE using enzymatic assays. Cholesterol ester content was calculated as total cholesterol – free cholesterol) × 1.67. Data represent mean ± SEM for 6 mice in the PO and EO groups and 8 mice in the FO group. Bars with different letters denote significant differences among diet groups (p<0.05).

Fig. 3. Liver lipid content

Mice were fed experimental diets containing PO, EO, or FO for 8 weeks before livers were harvested for extraction and measurement of lipid content by enzymatic assays. Bars represent mean ± SEM; n=8 for PO and EO, n=9 for FO. Values with different letters are significantly different for one another (p<0.05).

Fig. 4. Association between plasma TG and hepatic TG content

Mice were fed experimental diets containing PO, EO, or FO for 8 weeks before plasma and livers were harvested for TG mass measurement. The plot of plasma TG concentration vs. hepatic TG content shows the line of best fit (r²=0.56) determined by linear regression analysis with GraphPad Prizm software. Each data point represents an individual animal in one of the three designated diet groups.

Fig. 5. Liver and plasma lipid percentage fatty acid composition

Mice were fed experimental diets containing PO, EO, or FO for 8 weeks before plasma and livers were harvested for measurement of PL, TG and CE fatty composition. Lipids from plasma and liver were extracted and separated by TLC into PL, TG and CE bands that were visualized with premulin and scraped for fatty acid analysis as described in the Methods section. Data represent mean ± SEM; n=5–8 for each group. Values with different letters are significantly different (p<0.05) by ANOVA; data bars not marked with letters were not significantly different. Only the polyunsaturated fatty acids (≥ 2 double bonds) are shown in the figure.

Fig. 6. Hepatic gene expression

Mice were fed experimental diets containing PO, EO, or FO for 8 weeks before livers were harvested for measurement of gene expression by quantitative real time PCR. Liver RNA was isolated using TRIzol from individual mice and quantified for the indicated genes. Values represent mean ± SEM; n=7–9 per diet group. Values with different letters are significantly different for one another (p<0.05) for each gene. SREBP1c= sterol regulatory element binding protein 1c; ACC=acetyl CoA carboxylase; SCD-1=stearoyl CoA desaturase-1; FAS=fatty acid synthase; HMGCoA syn= hydroxymethylglutaryl CoA syntase; LDLr=low density lipoprotein receptor; LXR=liver X receptor α; PGC1α=peroxisome proliferator-activated receptor γ, coactivator 1 and PPARα=peroxisome proliferator-activated receptor α.