Changes in cholesterol homeostasis modify the response of F1B hamsters to dietary very long chain n-3 and n-6 polyunsaturated fatty acids

Abstract

Background: The plasma lipoprotein response of F1B Golden-Syrian hamsters fed diets high in very long chain (VLC) n-3 polyunsaturated fatty acids (PUFA) is paradoxical to that observed in humans. This anomaly is attributed, in part, to low lipoprotein lipase activity and is dependent on cholesterol status. To further elucidate the mechanism(s) for these responses, hamsters were fed diets containing supplemental fish oil (VLC n-3 PUFA) or safflower oil (n-6 PUFA) (both 10% [w/w]) and either cholesterol-supplemented (0.1% cholesterol [w/w]) or cholesterol-depleted (0.01% cholesterol [w/w] and 10 days prior to killing fed 0.15% lovastatin+2% cholestyramine [w/w]).

Results: Cholesterol-supplemented hamsters fed fish oil, relative to safflower oil, had higher non-high density lipoprotein (HDL) cholesterol and triglyceride concentrations (P < 0.001) which were associated with lower hepatic low density lipoprotein (LDL) receptor, sterol regulatory element binding protein (SREBP)-1c and acyl-CoA: cholesterol acyl transferase-2 (ACAT) mRNA and protein (p < 0.05), and higher hepatic apolipoprotein (apo) B-100 and apo E protein levels. In contrast, cholesterol-depleted hamsters fed fish oil, relative to safflower oil, had lower non-HDL cholesterol and triglyceride concentrations (P < 0.001) which were associated with lower hepatic SREBP-1c (p < 0.05) but not apo B-100, apo E or ACAT-2 mRNA or protein levels. Independent of cholesterol status, fish oil fed hamsters had lower HDL cholesterol concentrations (p < 0.001), which were associated with lower hepatic apoA-I protein levels (p < 0.05).

Conclusion: These data suggest disturbing cholesterol homeostasis in F1B hamsters alters their response to dietary fatty acids, which is reflected in altered plasma lipoprotein patterns and regulation of genes associated with their metabolism.

Figure 1

Effect of dietary n-3 (fish oil) and n-6 PUFA (safflower oil) on fasting plasma lipid and lipoprotein cholesterol concentrations in cholesterol-supplemented (A) and cholesterol-depleted (B) hamsters. Retro-orbital blood was collected into EDTA coated tubes from fasted hamsters. Plasma cholesterol and triglyceride concentrations were determined enzymatically. Bars represent means ± SEM, n = 15-16 animals per group. Appropriate transformations of the data (log HDL; square root total cholesterol, non-HDL cholesterol; inverse triglyceride) were made before statistical analysis. Asterisks indicate significant differences between safflower and fish oil within cholesterol-supplemented (+C) or depleted (-C) hamsters, P ≤ 0.05.

Figure 2

Effect of dietary n-3 (fish oil) and n-6 PUFA (safflower oil) on fast protein liquid chromatography (FPLC) cholesterol profiles of plasma from cholesterol-supplemented (+C) or depleted (-C) hamsters. Plasma (200 μL) was pooled from 4 hamsters and lipoprotein fractions were separated by FPLC. Cholesterol concentrations were measured in odd numbered fractions. Data represent the mean of 4-pooled groups.

Figure 3

Effect of dietary n-3 (fish oil) and n-6 PUFA (safflower oil) on hepatic mRNA levels of genes associated with cholesterol and lipoprotein metabolism in cholesterol-supplemented (A) and cholesterol-depleted (B) hamsters. Real time PCR was used to measure gene expression in the liver. A standard curve was run on all plates for each mRNA of interest to calculate relative levels. Values were normalized using beta-actin as an endogenous control. Bars represent means ± SEM, n = 14-16 animals per group. Appropriate transformations of the data (log SREBP-2, CYP7A1, MTP, apo B-100, ABCA1, HMG-CoA reductase; square root SR-B1, SREBP-1c, apo A-I) were made before statistical analysis. Asterisks indicate significant differences between safflower oil and fish oil within cholesterol-supplemented (+C) or depleted (-C) hamsters, P ≤ 0.05.

Figure 4

Effect of dietary n-3 (fish oil) and n-6 PUFA (safflower oil) on hepatic protein levels of genes associated with cholesterol and lipoprotein metabolism in cholesterol-supplemented (A) and cholesterol-depleted (B) hamsters. Proteins were extracted from the liver, separated by SDS-PAGE and detected by immunoblotting. Relative protein levels were normalized to the density of beta-actin. Bars represent means ± SEM, n = 14-16 animals per group. Appropriate transformations of the data (log apo A-I, apo E, LDL receptor, SREBP-1c, SR-B1; square root apo B-100) were made before statistical analysis. Asterisks indicate significant differences between safflower oil and fish oil within cholesterol-supplemented (+C) or depleted (-C) hamsters, P ≤ 0.05.

Figure 5

Effect of dietary n-3 (fish oil) and n-6 PUFA (safflower oil) on mRNA levels of small intestinal sterol transporters in cholesterol-supplemented (A) and cholesterol-depleted (B) hamsters. Real time PCR was used to measure gene expression in the small intestine. A standard curve was run on all plates for each mRNA of interest to calculate relative levels. Values were normalized using beta-actin as an endogenous control. Bars represent means ± SEM, n = 14-16 animals per group. Appropriate transformations of the data (log ABCG5) were made before statistical analysis. Asterisks indicate significant differences between safflower oil and fish oil within cholesterol supplemented (+C) or depleted (-C) hamsters, P ≤ 0.05.