Incremental replacement of saturated fats by n-3 fatty acids in high-fat, high-cholesterol diets reduces elevated plasma lipid levels and arterial lipoprotein lipase, macrophages and atherosclerosis in LDLR-/- mice

Abstract

Objective: Effects of progressive substitution of dietary n-3 fatty acids (FA) for saturated FA (SAT) on modulating risk factors for atherosclerosis have not been fully defined. Our previous reports demonstrate that SAT increased, but n-3 FA decreased, arterial lipoprotein lipase (LpL) levels and arterial LDL-cholesterol deposition early in atherogenesis. We now questioned whether incremental increases in dietary n-3 FA can counteract SAT-induced pro-atherogenic effects in atherosclerosis-prone LDL-receptor knockout (LDLR-/-) mice and have identified contributing mechanisms.

Methods and results: Mice were fed chow or high-fat diets enriched in SAT, n-3, or a combination of both SAT and n-3 in ratios of 3:1 (S:n-3 3:1) or 1:1 (S:n-3 1:1). Each diet resulted in the expected changes in fatty acid composition in blood and aorta for each feeding group. SAT-fed mice became hyperlipidemic. By contrast, n-3 inclusion decreased plasma lipid levels, especially cholesterol. Arterial LpL and macrophage levels were increased over 2-fold in SAT-fed mice but these were decreased with incremental replacement with n-3 FA. n-3 FA partial inclusion markedly decreased expression of pro-inflammatory markers (CD68, IL-6, and VCAM-1) in aorta. SAT diets accelerated advanced atherosclerotic lesion development, whereas all n-3 FA-containing diets markedly slowed atherosclerotic progression.

Conclusion: Mechanisms whereby dietary n-3 FA may improve adverse cardiovascular effects of high-SAT, high-fat diets include improving plasma lipid profiles, increasing amounts of n-3 FA in plasma and the arterial wall. Even low levels of replacement of SAT by n-3 FA effectively reduce arterial lipid deposition by decreasing aortic LpL, macrophages and pro-inflammatory markers.

Keywords: Atherosclerosis; Inflammation; LDLR−/−; Lipoprotein lipase; n−3 fatty acids.

Figure 1

Effects of FA on plasma lipid profiles in LDLR−/− mice. Blood samples were determined for plasma free fatty acids (A, FFA), triglycerides (B, TG), and total cholesterol (C, Chol) at the end of 12-weeks of feeding with specific diets (n=20 from two separate but identical feeding studies, mean±SE). *, p<0.01, vs. chow; **, p<0.05, vs. SAT; #, vs. S:n-3 3:1; ¥ vs. S:n-3 1:1.

Figure 2

Effects of diet on LpL, macrophages and inflammatory markers in proximal aorta in LDLR−/− mice. (A). Representative images of proximal artery sections stained for endothelial cells (EC), LpL and macrophages (MΦ) in LDLR−/− mice that were fed the specific diets for 12 weeks. “L” indicates lumen. (B). Immunofluorescence of aortic LpL (open bars) and macrophages (filled bars) quantitated for each group. Mean±SE, n=3–4. (C) mRNA analyses of arterial pro-inflammatory markers. Proximal aorta homogenates of LDLR−/− mice (n=5) were analyzed for mRNA of CD68, VCAM-1, IL-6 and TLR-4 in each group. Data are expressed as mean±SE. *, SAT vs. chow; **, vs. SAT, p<0.05.

Figure 3

Effects of diet on LpL and macrophage localization and LpL/PPAR mRNA at the aortic origin in LDLR−/− mice. (A) Images shown are the colocalization of aortic EC, LpL, and macrophages (MΦ) in LDLR−/− mice fed a chow, SAT, or n-3 diet for 12 weeks. L=lumen. Aortic macrophages were collected by LCM and pooled (n=3–5)for measuring mRNA expression of LpL (B), PPARα (C), γ (D), and β/δ (E) with triplicate runs (mean±SE) in each group as described in Methods.

Figure 4

Atherosclerotic lesion development in LDLR−/− mice after feeding with different diets. At the end of each feeding period, aorta of LDLR−/− mice were collected as described in Methods. Representative images of the aortic origin stained with hematoxylin and Oil-Red-O (A). (B) Quantitation of atherosclerosis in the aortic origin in LDLR−/− mice (n=8–10 for each group). The results are expressed as mean lesion area (μm²) ±SE. *, vs. chow; **, S:n-3 3:1/1:1/n-3 vs. SAT, p<0.01.