Dietary Fat–Induced Alterations in Atherosclerosis Are Abolished by ACAT2-Deficiency in ApoB100 Only, LDLr−/− Mice
Objectives— The enzyme acyl-coenzymeA (CoA):cholesterol O-acyltransferase 2 (ACAT2) in the liver synthesizes cholesteryl esters (CE) from cholesterol and fatty acyl-CoA, which get incorporated into apoB-containing lipoproteins that are secreted into the bloodstream. Dietary fatty acid composition influences the amount and fatty acid composition of CE within apoB-containing lipoproteins. We hypothesized that when ACAT2 activity is removed by gene deletion, hepatic CE synthesis and secretion would be minimal and, as a result, dietary fat-related differences in atherosclerosis would be eliminated.
Methods and Results— Groups of female apoB100 only, LDLr−/− mice with and without ACAT2 were fed diets enriched in either ω-3 or ω-6 polyunsaturated fat, saturated fat, and cis or trans monounsaturated fat. After 20 weeks on diet, mice fed diets enriched in monounsaturated or saturated fat exhibited significantly higher amounts of plasma cholesterol, larger LDL particles enriched in monounsaturated CE, and more atherosclerosis than mice fed polyunsaturated fat. The dietary fat-induced shifts in plasma cholesterol, LDL size, LDL CE composition, and atherosclerosis were not observed in ACAT2−/− mice. Regardless of the diet fed, the ACAT2−/− mice were protected from atherosclerosis.
Conclusions— The results indicate that in apoB100 only, LDLr−/− mice, ACAT2 plays an essential role in facilitating dietary fat type–specific atherosclerosis through its various effects on plasma lipoprotein concentration and composition.
Plasma cholesterol has been established as a predictor of coronary heart disease (CHD) risk. The seminal epidemiological studies conducted in Framingham, Mass found that, beyond total plasma cholesterol measurements, the LDL and HDL cholesterol concentrations provide a more accurate assessment of CHD risk.1 The incidence of CHD is increased in subjects with elevated amounts of LDL cholesterol or lower quantities of HDL cholesterol. The numerous observations associating elevated LDL with increased CHD risk has prompted studies to unlock the qualities of LDL that give it this proatherogenic behavior.
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A growing body of evidence from atherosclerosis studies conducted in animal models suggests the fatty acid composition of the cholesteryl ester of LDL plays a critical role in determining the atherogenic potential of LDL. Studies using nonhuman primates fed diets containing fat and cholesterol found a positive correlation between LDL size and coronary artery atherosclerosis.2 Analysis of the lipoproteins found the increase in LDL size was attributable to an enrichment of the neutral lipid core with cholesteryl ester (CE), primarily as cholesteryl oleate.3 Cholesteryl oleate is the primary product of the enzyme acyl CoA: cholesterol acyltransferase (ACAT). ACAT2 has been identified as the isoform of ACAT responsible for the synthesis of cholesteryl esters incorporated into apo-B–containing lipoproteins secreted from the liver and intestine.4,5
The relationship between ACAT2 and LDL atherogenicity has been further examined in studies in which African Green monkeys were fed cholesterol-containing diets enriched in either saturated, monounsaturated or polyunsaturated fat.6,7 Subsequent studies identified that the majority of hepatic ACAT activity can be attributed to the ACAT2 isoform.8 The consequence of increased hepatic ACAT2 activity was increased secretion of cholesteryl ester from the liver and subsequent enrichment of LDL with cholesteryl oleate.6,9 The enrichment of LDL with cholesteryl oleate increased mean LDL size which was highly correlated with coronary artery atherosclerosis in the monkeys fed saturated and monounsaturated fat, and higher than in the monkeys fed polyunsaturated fat.6,7
Using the apoB100 only, LDLr−/− mouse model,10 preliminary studies showed similar effects of dietary fat on LDL composition and atherosclerosis. We hypothesized that when ACAT2 activity is negated by gene deletion, relationships between LDL composition and atherosclerosis would be lost. ApoB100-only, LDLr−/− mice and apoB100-only, LDLr−/−, ACAT2−/− mice were fed diets enriched in several different fats including polyunsaturated, saturated, and monounsaturated fats. Mice lacking ACAT2 were protected from atherosclerosis regardless of the type of dietary fat fed. Overall, our results show that ACAT2 is a potent mediator of dietary fat-induced atherosclerosis.
Mice, Diets, and Study Outline
The apoB100 only, LDLr−/− and apoB100 only, LDLr−/−, ACAT2−/− female mice were created as described in the expanded Materials and Methods (see online supplement, available at http://atvb.ahajournals.org). These mice were fed 1 of 6 diets consisting of 10% calories from fat and 0.02% cholesterol by weight for 20 weeks. The diets were enriched in specific types of dietary fat, either fish- or plant-derived ω-3 polyunsaturated fat, ω-6 polyunsaturated fat, saturated fat, and cis or trans monounsaturated fat. The complete details of dietary ingredients and fatty acid composition are summarized in supplemental Tables I through III (available online at http://atvb.ahajournals.org). The time course for lipid measurement and the tissue collection techniques used at termination are described in the online supplement.
Plasma Lipid and Lipoprotein Measurements
Measurement of plasma lipid concentrations were performed using enzymatic assays according to established methods as described in detail in the online supplement.11,12 Plasma VLDL, LDL, and HDL were isolated for compositional analyses as described previously.13,14 These techniques are described in detail in the online supplement.
Quantification of Atherosclerosis
Atherosclerosis was evaluated by biochemical methods according to techniques previously described.15 These methods are described in detail in the expanded Materials and Methods section.
The type of fat fed as well as the presence of ACAT2 had demonstrable effects on total plasma cholesterol (TPC) concentration during the treatment period as shown in the data summarized in Figure 1. As early as 2 weeks on diet, control (ACAT2+/+) mice segregated themselves into 3 response groups by cholesterol level (Figure 1A). Control mice fed the diet containing fish-oil consistently had the lowest concentrations of TPC, which remained around 200 mg/dL for the duration of the study. The n-6 polyunsaturated fat fed mice had the next lowest concentrations of TPC, which started around 330 mg/dL and increased to approximately 470 mg/dL after 20 weeks on diet. The saturated and monounsaturated fat fed control mice had the highest concentrations of TPC, which at 2 weeks was slightly less than 500 mg/dL. Control mice fed saturated fat maintained TPC levels around 500 mg/dL for much of the treatment period with an unexpected drop-off to 460 mg/dL at 20 weeks. The TPC concentration for control mice fed cis-monounsaturated fat continued to increase throughout the treatment period and reached 625 mg/dL at the termination of the study, whereas the mice fed trans-Mono achieved the highest average TPC near 725 mg/dL at 20 weeks. These dietary fat effects on TPC were lost in ACAT2−/− mice (Figure 1B). All but 1 of the ACAT2−/− diet groups had TPC values around 200 mg/dL after 2 weeks which increased to approximately 400 mg/dL by the end of the treatment period. The exception was ACAT2−/− mice fed the fish oil diet, which started with a lower TPC that had increased to about 400 mg/dL by 20 weeks.
A detailed analysis of lipid measurements from the terminal plasma sample is displayed in Table 1. In the control mice, the concentration of plasma free cholesterol (FC) increased as the type of dietary fat fed was changed from polyunsaturated to saturated and monounsaturated. Control mice fed fish oil had the lowest concentration of plasma FC, at 76 mg/dL, and control mice fed the trans-Mono diet had the highest concentration of plasma FC, at 209 mg/dL. In the ACAT2−/− mice, the different diets did not provide similar shifts in plasma FC and no differences in plasma FC were found.
Dietary fat type and ACAT2 activity had a greater impact on plasma cholesteryl ester (CE) concentrations (Table 1). In control mice, dietary fat type had a significant effect on plasma CE. Control mice fed fish oil had significantly lower concentrations (249 mg/dL) when compared with the other diet groups except for the saturated fat group. The other control diet groups had plasma CE concentrations over 500 mg/dL, with mice fed trans-Mono having the highest concentration of CE at 858 mg/dL. The differences in plasma CE concentrations between dietary fat groups of ACAT2−/− mice were not significantly different.
Surprisingly, the most dramatic effect of ACAT2 deletion on plasma lipid values was on triglycerides (TG) as shown in Table 1. With the exception of the fish oil– and n-6 Poly–fed mice, ACAT2−/− mice exhibited a significant increase in plasma TG when compared with control mice fed the same diet. No discernable trends could be established between the type of dietary fat fed and plasma TG concentration in either genotype. The only significant difference found between 2 diet groups of the same genotype was between ACAT2−/− mice fed fish oil and trans-Mono fat, which had plasma TG concentrations of 230 and 495 mg/dL, respectively.
In Table 2, the effects of dietary fat and ACAT2 activity on plasma lipoprotein CE distribution in subsets of mice at 20 weeks from each diet group are shown. In control mice, the different dietary fat enriched diets apparently altered VLDL-CE concentration although not in a statistically significant way, and higher averages for VLDL-CE concentrations were observed when the saturated and monounsaturated diets were fed. Except for ACAT2−/− mice fed fish oil, a lower average VLDL-CE concentration was seen in all ACAT2−/− diet groups when compared with control mice fed the same diet. In the ACAT2−/− mice, the different diets did not appear to have a similar effect on mean VLDL-CE concentration to what was observed in the control mice.
In control animals, the average LDL-CE concentration was lowest in the fish oil group and was highest in the cis-Mono group; for these small subsets of animals, the value in the latter group was only significantly higher than for the fish oil group. With the exception of the fish oil diet groups, LDL-CE values tended to be lower in the ACAT2−/− mice when compared with the control groups. The largest difference among the ACAT2−/− animals was between the LDL-CE of fish oil and flaxseed oil groups, averaging 208 and 494 mg/dL, respectively.
Among diet groups of control animals, the lowest HDL-CE concentrations were seen in the fish oil and the trans-Mono groups, whereas the highest value was seen in the cis-Mono group. With the exception of the cis-Mono diet groups, HDL-CE values tended to higher in ACAT2−/− mice when compared with the control mice. In the fish oil and the trans-Mono groups, HDL-CE was almost 300% higher in ACAT2−/− mice, a finding suggesting that the absence of ACAT2 can markedly increase HDL concentrations, at least in some dietary fat groups.
Although dietary fats and presence or absence of ACAT2 activity did not produce large shifts in LDL-CE concentrations, a significant alteration in LDL-CE fatty acid composition occurred, as shown in Figure 2. In control mice, the percentage of monounsaturated CE was higher in mice fed flaxseed oil, saturated, or monounsaturated fat when compared with mice fed fish oil or the ω-6 polyunsaturated diet. The higher percentages of saturated and monounsaturated CE were no longer present in the ACAT2−/− mice, and Figure 2A and 2B show the inverse effects on polyunsaturated fatty acid CE percentages when compared with the saturated plus monounsaturated CE percentages.
Figure 3 shows the data on effects of dietary fat and genotype on LDL size and atherosclerosis. In control mice the different dietary fats were able to alter mean LDL size, becoming larger as the fatty acid composition of the diet shifted from polyunsaturated to saturated and monounsaturated (Figure 3A). Fish oil–fed mice had the smallest mean LDL at 4.1 g/μmole, and the largest LDL particles were found in control mice fed cis-Mono at 5.9 g/μmole. Any effects of dietary fat on LDL size were lost when ACAT2 activity was absent and LDL size was equivalent in all dietary fat groups with means near 4.0 g/μmole for each group (Figure 3B).
A parallel relationship occurred in the extent of atherosclerosis quantified by chemical measurement of aortic CE and FC accumulation (Figure 3C and 3E). In control mice, aortic CE accumulation increased as the fatty acid composition of the diet shifted from polyunsaturated to saturated and monounsaturated. Mice fed fish oil had the lowest average aortic CE at 9.0 mg/g protein, and mice fed trans-Mono had the highest average at 38.3 mg/g protein. In the control mice, with exception of the mice fed cis-Mono, the concentration of aortic FC also increased with the type of dietary fat fed although not to the same extent as CE. Control mice fed the n-6 Poly diet had the lowest concentration of aortic FC at 19.5 mg/g protein, and mice fed trans-Mono had the highest concentration at 47.1 mg/g protein. The ACAT2−/− mice did not have a similar trend of diet-induced atherosclerosis among dietary fat groups; markedly lower aortic CE concentrations were found for all ACAT2−/− diet groups averaging 6.9 mg/g protein, a value lower than in any of the control diet groups. The ACAT2−/− mice also did not show an effect of dietary fat on aortic FC concentration, and the bulk of the FC in these groups likely represents membrane cholesterol. In Figure 4, LDL size and atherosclerosis extent measured as aortic CE were correlated. A strong relationship was found between these 2 variables with a correlation coefficient of r=0.87. Significant correlations of atherosclerosis with other plasma lipid parameters were also found (for example, a correlation of r=0.78 with TPC as shown in supplemental Figure I) but none were as strong as the correlation between LDL size and atherosclerosis.
In pilot studies in the apoB100 only, LDLr−/− mouse a relationship between dietary fat type, LDL size, and atherosclerosis was seen, similar to what had been found in studies of coronary artery atherosclerosis in monkeys6,7,9 where ACAT2 activity was implicated in the diet-induced difference in atherosclerosis. In the present study, ACAT2 activity was removed by gene deletion so that the role of this enzyme could be examined. Remarkably, dietary fat–related differences in atherosclerosis were all lost and atherosclerosis was minimal in ACAT2−/− mice (Figure 3). This is the first demonstration of a liver enzyme that can control the differential atherosclerosis responses to individual dietary fats. The striking association with LDL size (Figure 4) suggests that the prevention of dietary fat–related shifts in types and amounts of CE in LDL is a major contributor to the effect. The association with LDL size was higher even than with TPC (see supplemental Figure I) and although it was only determined for the blood samples collected at 20 weeks, there is little room for the strength of the association to increase with more data such as from additional time points.
Earlier results from studies in nonhuman primates showed that the increase in LDL size that occurred when saturated and monounsaturated fat were fed was attributable to an enrichment of the lipoprotein particle core with monounsaturated CE, primarily cholesteryl oleate.3 A similar relationship between dietary fat and LDL-CE fatty acid composition occurred in the apoB100 only, LDLr−/− mice as seen in the data of Figure 2. An increase in the percentage of monounsaturated CE in LDL occurred as the type of fat shifted from polyunsaturated to saturated and monounsaturated fat (Figure 2A). When this shift in LDL-CE composition is coupled with the change in LDL size shown in Figure 3A, the net effect is a marked increase in the amount of monounsaturated CE within the LDL particles of mice fed saturated and monounsaturated fat enriched diets. In the monkey studies and in our current work, enrichment of the LDL core with monounsaturated CE is highly associated with an increase in atherosclerosis.
The ACAT2−/− mice in these studies allowed for an assessment of the role of ACAT2 derived CE in the pathogenesis of atherosclerosis. Figure 2B shows that LDL-CE fatty acid composition is dramatically altered when ACAT2 activity is removed, resulting in a shift in LDL-CE fatty acid composition to be predominantly polyunsaturated with a lower CE content per particle. The change in LDL-CE fatty acid composition is believed to be attributable to a compensatory action of lecithin:cholesterol acyltransferase (LCAT) to provide CE for the LDL particle core.13,15 LCAT in the plasma compartment produces polyunsaturated CE by using phospholipid sn-2 fatty acids as substrates. LCAT in the ACAT2−/− mice provided LDL-CE concentrations nearly comparable to those observed in the control mice. Further, LCAT can potentially contribute to the increased HDL-CE found in most of the ACAT2−/− diet groups (Table 2). Because the control and ACAT2−/− mice had similar concentrations of LDL-CE, the differences in atherosclerosis observed between these two genotypes (Figure 3C and 3D) appear to be importantly related to the shift in LDL-CE fatty acid content and composition. This result strengthens the conclusion that ACAT2-derived CE determines the atherogenicity of LDL. The higher the percentage of saturated and monounsaturated CE in LDL particles, especially as they become larger in size, will result in an increase in the number of these higher melting cholesteryl esters per LDL particle. This shift is thought to result in cholesteryl ester accumulation in atherosclerotic lesions, possibly as a result of a decreased rate of CE clearance from foam cells.16,17
The apoB100 only mice were originally developed to compare the atherogenic potential of LDL-sized particles to larger VLDL remnant–sized particles that were free of apoB48.18 To do so, the apoB100 only mice were crossed onto both the LDLr−/− and apoE−/− mouse models of atherosclerosis. Despite having similar levels of plasma cholesterol, the apoB100 only, LDLr−/− mice had significantly higher amounts of atherosclerosis when compared with the apoB100 only, apoE−/− mice. The difference in atherosclerosis between the 2 groups of mice was suggested to be attributable to the significantly increased particle number in the apoB100 only, LDLr−/− mice. Our current studies in the apoB100 only, LDLr−/− mice, allow a further refinement to the assessment of particle size/atherosclerosis associations. By using different dietary fats, we have shifted mean apoB100-LDL sizes while keeping the majority of the particles within the LDL size range. The results from our studies indicate in the apoB100 only, LDLr−/− mice, an increase in LDL size is attributable to an increase in ACAT2-derived cholesteryl oleate in LDL; the effect to increase LDL size enhances atherosclerosis.
LDL size as an indicator of CHD risk has been examined in detail in clinical studies.19,20 Both large and small LDL have been positively associated with CHD in humans. The data indicate that the utility of LDL size as a predictor of atherosclerosis in the absence of other measurements such as LDL particle concentration and composition is limited. In earlier studies in nonhuman primates2,7,9 and in our current work in mice, an increase in LDL size is associated with an enrichment of LDL with ACAT2-derived monounsaturated CE. Additionally, a recent study in humans found a positive relationship between monounsaturated CE in plasma and carotid artery intimal thickness.21 The results from these studies suggest that assessing LDL size alone is not sufficient and that beyond LDL size, measurements of LDL-CE fatty acid composition including the content of cholesteryl oleate in LDL, may provide a more accurate indication of CHD risk.
When ACAT2 was present, the effects the different dietary fats had on TPC were rapid and specific to the type of fat fed (Figure 1A). In nonhuman primates, dietary saturated and monounsaturated fat enhanced CE production and secretion from the liver when compared with polyunsaturated fat.6,9 The current results suggest that the effects the different fatty acid enriched diets had on hepatic ACAT2 activity and hepatic CE secretion may have paralleled the earlier observations from nonhuman primates and the absence of ACAT2 blocked this response. Using liver perfusion experiments in mice with and without ACAT2, Lee et al directly assessed the function of ACAT2 in lipoprotein CE secretion from the liver.22 Liver perfusate from ACAT2−/−, LDLr−/− mice had an 87% decrease in VLDL-CE accumulation when compared with LDLr−/− mice, indicating ACAT2 was vital in supplying newly synthesized apoB-lipoproteins with CE.
Regardless of the diet fed, the apoB100 only, LDLr−/−, ACAT2−/− mice had elevated plasma TG (Table 1). This observation is typical of those made in other studies conducted in ACAT2 knockout mice.15,22 Based on the results from isolated liver perfusion, ACAT2−/−, LDLr−/− mice showed an increased hepatic secretion of VLDL measured as a higher accumulation rate of TG in perfusate when compared with LDLr−/− mice.22 The increased incorporation of TG into newly formed lipoproteins in the liver of ACAT2 knockout mice apparently compensates in some way for the decreased CE production in the hepatocyte. Exactly why this phenomenon occurs is not clear and needs further study. Nevertheless, the hypertriglyceridemia identified in ACAT2 knockout mice appears to be benign because atherosclerosis was much attenuated at the same time that the elevation in triglyceride concentrations occurred.
Three different polyunsaturated fats were compared in these studies, including 1 ω-6 fatty acid enriched diet and 2 ω-3 polyunsaturated fat–containing diets. One diet used fish oil and another used flaxseed oil as the ω-3 fatty acid source. The key difference between the flax and fish oil diets is that the primary ω-3 fatty acid in the flax diet is α-linolenic acid, a precursor to the eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids of the fish oil diet. The flax diet did not decrease plasma cholesterol, VLDL-CE, LDL size, or the percentage of monounsaturated CE in LDL in an analogous fashion to what was observed in the fish oil fed mice. The lack of equivalent effects on plasma lipoprotein measurements apparently led to higher amounts of atherosclerosis in mice fed the flax versus fish oil diets (Figure 3C). The bulk of the ω-3 fatty acids found in plasma and liver lipids in mice fed the flax diet were EPA and DHA, not α-linolenic acid (data not shown). However, the percentage of EPA and DHA in plasma and liver cholesteryl esters and triglycerides of mice fed the flax diet was only about half as much as was found in mice fed the fish oil diet (data not shown). The results suggest that α-linolenic acid is not as effective as EPA and DHA in atheroprotection. Others have shown that the Δ6-desaturase enzyme required for the conversion of α-linolenic acid to EPA can be limiting, resulting in poor conversion of α-linolenic acid to more potent ω-3 fatty acids.23
Interestingly, mice fed the ω-6 polyunsaturated fat and fish oil diets presented with a similar reduction in aortic CE (Figure 3C), despite the ω-6 fatty acid fed mice having plasma CE and LDL-CE concentrations more similar to what was found in mice fed saturated or monounsaturated fat. The smaller LDL size (Figure 3A) and reduced percentage of monounsaturated CE in LDL (Figure 2A) appears to have protected the ω-6 fatty acid fed mice from atherosclerosis. These data in mice fed ω-6 fatty acids highlight how measurements of plasma lipoprotein cholesterol distribution are not always sufficient in and of themselves to accurately predict atherosclerosis.
The relative impact that different dietary fats have on CHD risk has been studied and debated for quite some time, and yet a consensus as to which types of fatty acids promote or prevent atherosclerosis has not been reached. The data from the present study are consistent with other observations in showing that monounsaturated fatty acids do not protect against atherosclerosis.7,24,25 In humans, the LDL to HDL cholesterol ratio is the biomarker commonly used to support beneficial effects of monounsaturated fat.26 However, usefulness of this ratio is believed to be confounded by the effects of monounsaturated fat to promote LDL cholesteryl oleate enrichment.27 Several studies in humans have indicated that a decrease in the proportion of cholesteryl linoleate together with an increase in cholesteryl oleate in plasma can be detrimental.24 The results from our studies in the apoB100 only, LDLr−/− mice show that ACAT2 derived CE are atherogenic and when ACAT2 activity is removed, less atherosclerosis occurs and dietary fat differences are eliminated. The implication is that ACAT2 might also be important in humans, although this has yet to be directly tested.
Sources of Funding
This work was supported by NIH Grants AT-002782 and HL-49373.
Original received January 9, 2007; final version accepted March 30, 2007.
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