Project 2- Echium oil, triglyceride metabolism, and atherosclerosis

Project Leader: John S. Parks, PhD
Perry L. Colvin, M.D., Co-Investigator
Floyd (Ski) H. Chilton, Ph.D., Co-Investigator
Ji-Young Lee, Ph.D., Co-Investigator
Lawrence L. Rudel, Ph.D., Co-Investigator
Amanda Wibley - predoctoral student
Lolita Forrest - predoctoral student

Cardiovascular disease (CVD) is the leading cause of death in the United States and other Westernized societies.  Consumption of fish oil (FO) or n-3 polyunsaturated fatty acid (PUFA) supplements has been shown to reduce the extent of atherosclerosis (i.e., hardening of the arteries), coronary heart disease (CHD), and stroke in epidemiological studies, in clinical trials, and in animal studies.  The cardioprotective benefits of fish oil have been largely attributed to two n-3 fatty acids, eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3), whose enrichment in cells and plasma lipoproteins results in decreased inflammation, thrombosis, blood pressure, arrhythmias, endothelial activation, and plasma triglyceride (TG) concentrations. 

In spite of the beneficial effects of FO, the consumption of n-3 fatty acids in the North American population is low.  Consumption of vegetable oils containing -linolenic acid (ALA, 18:3 n-3) has only limited impact on tissue concentrations of EPA and DHA, since the conversion of 18:3 n-3 to EPA (20:5 n-3) is limited by the inefficiency of the -6 desaturase-catalyzed step.  One strategy to enrich tissues in 20 and 22-carbon PUFAs is to use botanical oils that are enriched in fatty acids that bypass the -6 desaturase step of fatty acid elongation and desaturation.  Echium oil, derived from the seeds of Echium Plantagineum, is enriched in stearidonic acid (SDA; 18:4 n-3), which is the immediate product of -6 desaturation of 18:3 n-3.  Our preliminary clinical trials in mild to moderate hypertriglyceridemic human subjects have demonstrated that fatty acids in echium oil, presumably SDA, are metabolized to EPA and result in lower plasma TG concentrations that are similar to those observed with FO consumption.

The goals of this project are to determine the mechanisms by which echium oil reduces plasma TG concentrations and to determine whether echium oil supplementation will retard the development of atherosclerosis.  Our hypothesis is that echium oil supplementation decreases plasma TG concentrations by decreasing hepatic TG synthesis and secretion and by increasing the catabolism of plasma TG-enriched lipoproteins.  In addition, we hypothesize that echium oil supplementation will reduce atherosclerosis to an extent similar to that observed with FO supplementation, by decreasing the production of inflammatory mediators and through plasma lipoprotein lowing.  We will test our hypothesis using a moderately hypertriglyceridemic animal model, the apoB100 only-LDL receptor knockout (B100-LDLrKO) mouse that responds to FO with a significant decrease in plasma TG concentrations and very low density lipoprotein (VLDL) cholesterol concentrations, without a significant decrease in low density lipoprotein (LDL) or high density lipoprotein (HDL) cholesterol, compared to saturated (sat’d) fat fed controls.

In specific aim 1, we hypothesize that echium oil supplementation will reduce plasma TG concentrations to a degree similar to that observed with fish oil supplementation in B100-LDLrKO mice.  Mice will be fed atherogenic diets containing 10% calories as palm oil, 0.2% cholesterol, and supplemented with increasing amounts of palm oil (control), echium oil, or fish oil (control) up to 20% of total calories as fat.  After eight weeks of diet consumption, mice will be bled for quantification of plasma lipids, and whole plasma and liver cholesteryl ester (CE), phospholipid (PL), and TG fatty acid compositions.  On the basis of results in this specific aim, we will use an amount of echium oil supplementation that results in a 30-40% decrease in plasma TG concentrations relative to palm oil for specific aims 2-4.

In specific aim 2, we will test the hypothesis that echium oil supplementation decreases plasma TG concentrations by down regulation of genes involved in hepatic TG synthesis, resulting in less hepatic TG synthesis and reduced VLDL TG, but not apoB, secretion.  Using the experimental groups outlined in specific aim 1, the following will be determined:

1. plasma lipid and lipoprotein concentrations, and chemical and fatty acid compositions of plasma lipoproteins,
   
2. hepatic perfusate VLDL TG and apoB100 accumulation during recirculating liver perfusion and hepatic perfusate VLDL TG and apoB synthesis during non-recirculating liver perfusion,
   
3. hepatic mRNA abundance of genes involved in hepatic TG synthesis and sterol regulation.
   

In specific aim 3, we will test the hypothesis that echium oil supplementation of the experimental animals will increase the lipolysis and catabolism of plasma TG enriched lipoproteins.  Using the experimental groups outlined in specific aim 1, the following will be determined:

1. postprandial TG response to the supplemented oils during atherogenic diet consumption,
   
2. post heparin plasma lipoprotein lipase (LPL) and hepatic lipase (HL) activities with a standard TG substrate preparation,
   
3. rate of lipolysis of postprandial TG enriched particles by purified LPL in vitro,
   
4. rate of endogenous lipolysis after intravenous heparin injection and oral supplement oil load,
   
5. in vivo catabolism of plasma VLDL particles.
   

In specific aim 4, we will test the hypothesis that echium oil supplementation, relative to palm oil supplementation, will reduce aortic atherosclerosis and the reduction will be similar to that observed with FO supplementation.  Using the experimental groups, the following will be determined:

1. atherosclerosis extent by measuring aortic free (FC) and esterified cholesterol (EC) content,
   
2. aortic mRNA abundance for genes involved in endothelial activation and inflammation,
   
3. production of inflammatory mediators by elicited macrophages that have been activated in vitro with oxidized LDL (oxLDL) or lipopolysaccharide (LPS).
   

This project will determine whether echium oil supplementation results in beneficial lipid and lipoprotein changes and decreased atherosclerosis similar to that of fish oil supplementation and, if so, the mechanisms by which this occurs.  If echium oil supplementation proves to be atheroprotective, it could be used in conjunction with dietary guidelines to meet the American Heart Association suggested intake of n-3 fatty acids necessary for cardioprotection.