t-10, c-12 CLA dietary supplementation inhibits atherosclerotic lesion development despite adverse cardiovascular and hepatic metabolic marker profiles

Abstract

Animal and human studies have indicated that fatty acids such as the conjugated linoleic acids (CLA) found in milk could potentially alter the risk of developing metabolic disorders including diabetes and cardiovascular disease (CVD). Using susceptible rodent models (apoE(-/-) and LDLr(-/-) mice) we investigated the interrelationship between mouse strain, dietary conjugated linoleic acids and metabolic markers of CVD. Despite an adverse metabolic risk profile, atherosclerosis (measured directly by lesion area), was significantly reduced with t-10, c-12 CLA and mixed isomer CLA (Mix) supplementation in both apoE(-/-) (p<0.05, n = 11) and LDLr(-/-) mice (p<0.01, n = 10). Principal component analysis was utilized to delineate the influence of multiple plasma and tissue metabolites on the development of atherosclerosis. Group clustering by dietary supplementation was evident, with the t-10, c-12 CLA supplemented animals having distinct patterns, suggestive of hepatic insulin resistance, regardless of mouse strain. The effect of CLA supplementation on hepatic lipid and fatty acid composition was explored in the LDLr(-/-) strain. Dietary supplementation with t-10, c-12 CLA significantly increased liver weight (p<0.05, n = 10), triglyceride (p<0.01, n = 10) and cholesterol ester content (p<0.01, n = 10). Furthermore, t-10, c-12 CLA also increased the ratio of 18∶1 to 18∶0 fatty acid in the liver suggesting an increase in the activity of stearoyl-CoA desaturase. Changes in plasma adiponectin and liver weight with t-10, c-12 CLA supplementation were evident within 3 weeks of initiation of the diet. These observations provide evidence that the individual CLA isomers have divergent mechanisms of action and that t-10, c-12 CLA rapidly changes plasma and liver markers of metabolic syndrome, despite evidence of reduction in atherosclerosis.

Conflict of interest statement

Competing Interests: The authors have the following interests. CLA was provided by Cognis Nutrition and Health and produced by Natural Lipids (Hovdebygda, Norway). There are no further patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Figure 1. Plasma lipoprotein cholesterol and triglyceride profiles for apoE−/− and LDLr−/− mice.

After 11 weeks on a HFC diet supplemented with 0.5% (w/w) of the indicated fatty acid, serum was obtained from apoE−/− (top) or LDLr−/− (bottom) mice by cardiac puncture. Plasma was separated from the blood cells and pooled plasma samples (n = 4–5 animals) were separated by density gradient ultracentrifugation. Cholesterol (TC, left) and triglyceride (TG, right) were measured in each fraction. ▪, HFC; □, LA; ▵, c-9, t-11 CLA; ▿ t-10, c-12 CLA; ⋄, CLA Mix.

Figure 2. Atherosclerotic lesion measurement in aortic en face preparations and aortic root cross sections.

The images show representative en face (left) and aortic root cross-section preparations (right) from apoE−/− (A) or LDLr−/− (B) mice. Mice were fed HFC diet with or without supplementation for 11 weeks as described in Figure 1. Quantification of percent lesion area in the en face preparations was calculated relative to total aorta area. Quantification of cross-sectional area occupied by lesion in the aortic root was determined relative to total cross-sectional area. * vs. HFC, p<0.05; ** vs. HFC, p<0.01.

Figure 3. Principal component scores plot of liver and plasma metabolites in apoE−/− mice fed fatty acid supplemented HFC diet (n = 56).

(A) The t-10, c-12 CLA diet exhibits a distinctly different response from the CLA mix and c-9, t-11 CLA; LA; HFC diets respectively, based on the variables shown in the loadings plot. (B) The loadings plot corresponding to the scores in (A) showing variables that distinguish diets into their respective groups as they appear in the scores plot. See text for details.

Figure 4. Principal component scores plot of liver and plasma metabolites in LDLr−/− mice fed fatty acid supplemented HFC diet (n = 50).

(A) There are three distinct clusters separated along the PC1, corresponding to: t-10, c-12 CLA; CLA mix; and c-9, t-11 CLA, LA, HFC diets respectively. (B) The loadings plot corresponding the scores plot shown in (A), depicting the variables that are significant for each cluster as they appear in (A).

Figure 5. Combined principal component analysis of apoE−/− and LDLr−/− mice fed fatty acid supplemented HFC diet (n = 106).

(A) and (B) are the scores and corresponding loadings plots respectively. See text for interpretation.