© 1998 American Heart Association, Inc.
Original Contributions |
Dietary Monounsaturated Fatty Acids Promote Aortic Atherosclerosis in LDL Receptor–Null, Human ApoB100–Overexpressing Transgenic Mice
From the Departments of Pathology (Comparative Medicine) (L.L.R., K.K., J.K.S., R.S., M.D.W.) and Biochemistry (L.L.R.), Wake Forest University School of Medicine, Winston-Salem, NC.
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Abstract |
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Abstract—In mice with genetically engineered high levels of plasma low density lipoprotein (LDL), we tested the hypothesis that an increase in the dietary content of monounsaturated fatty acids but not of polyunsaturated fatty acids would promote atherosclerosis. The mouse model used was an LDL receptor–null, human apoB100–overexpressing strain. Six experimental groups of 19 to 38 mice of both sexes were established when the animals had reached 8 weeks of age. For the next 16 weeks, individual groups were fed either a commercial diet or prepared diets including fat as 10% of energy, with 5 different fatty acid enrichment patterns including the following: saturated (sat), cis and trans monounsaturated (mono), and n-3 and n-6 polyunsaturated (poly). Highly significant differences (ANOVA, P<0.0001) in LDL cholesterol (in mg/dL) were found, with the rank order at 16 weeks being trans mono (mean, 1390)>sat (922) =cis mono (869)=n-6 poly (868)>n-3 poly (652)>commercial diet (526). Significant elevations in very low density lipoprotein cholesterol were also found in the trans and cis mono and sat groups, and triacylglycerol concentrations were also elevated in all groups. High density lipoprotein cholesterol concentrations were consistently low (20 to 50 mg/dL) in all groups. Highly significant differences (ANOVA, P<0.0001) in atherosclerosis, quantified by measurement of aortic cholesteryl ester concentration (mg/g protein) among dietary fatty acid groups were found, with the order being trans mono (mean, 50.4)>sat (35.6)=cis mono (34.6)>n-6 poly (18.3)=n-3 poly (9.7)=commercial diet (7.8). Therefore, in this mouse model of hypercholesterolemia, dietary cis or trans monounsaturated fat did not protect against atherosclerosis development, whereas aortic atherosclerosis in either of the polyunsaturated fat groups was significantly less than in the saturated fat group.
Key Words: cholesterol • lipoprotein metabolism • polyunsaturated fat • saturated fat • trans fatty acids
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Introduction |
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The effects of dietary cholesterol and type of fat on coronary heart disease have been frequently studied. Generally, recommendations from organizations such as the American Heart Association have been to limit the amount of total and saturated fat in the diet, replace saturated with unsaturated fat, and limit dietary cholesterol. Data indicate that we have been better at increasing the amount of vegetable oil in our diet than in decreasing the percentage of energy intake as fat.1 Decreased saturated fat and increased polyunsaturated fat intake may be a component of the overall 50% reduction in coronary heart disease rates that have occurred over the past 2 decades in the United States.2 A significant body of evidence demonstrates that for linoleic acid, accumulation in adipose tissue3 4 5 and enrichment in plasma cholesteryl esters6 7 8 9 10 are both factors associated with decreased rates of premature complications of coronary heart disease. In addition, several studies have demonstrated that linoleic acid–rich diets directly protect against atherosclerosis development.11 12 In an attempt to make linoleic acid–enriched fats more suitable for a variety of cooking needs, hydrogenation has been used to increase the melting temperature and reduce the oxidizability of these preparations, in the process introducing higher amounts of trans fatty acids, a by-product of hydrogenation, into our diet. Data have been presented that trans fatty acids may detrimentally affect coronary heart disease risk,13 14 though again, establishing a firm basis for these risk factor associations with direct observations of effects on atherosclerosis remains to be achieved.15
Interestingly, in our society at the present time, monounsaturated fatty acids in the form of olive oil and canola oil instead of linoleic acid–rich corn and soybean oils, for example, are appearing more frequently as the cooking oils available for sale in grocery stores. This shift is apparently based, at least in part, on recommendations that monounsaturated fatty acids provide similar or improved risk factor relationships compared with those seen for polyunsaturated fatty acids,16 17 although protection against atherosclerosis development in studies of experimental coronary artery atherosclerosis, per se, has not been found.18 Diets enriched in n-3 fatty acids derived from fish oils have been found to be beneficial in limiting the development of experimental atherosclerosis,19 20 although effects on risk factors have led to some skepticism in favoring their use in protection against coronary heart disease.21
Simultaneous evaluation of the variety of different unsaturated dietary fats with direct comparisons to the effects of saturated fat on experimental atherosclerosis has not been attempted. With the development of genetically modified mouse models in recent years, it has become possible to use mice to evaluate some of the important factors affecting atherosclerosis development. One difficulty with the mouse as a model of human atherosclerosis is that the typical plasma lipoprotein spectrum in mice is quite different from that in humans, with a pattern of HDL as the predominant lipoprotein with low concentrations of LDL and VLDL, and drastic diets have been needed to induce arterial changes consistent with early atherosclerosis development.22 23
The feasibility of using mice to evaluate dietary fatty acid effects on atherosclerosis was pioneered by Paigen and associates.24 A mouse model (LDL receptor–null with human apoB100 overexpression) with LDL as the predominant lipoprotein has recently been developed by Hobbs and associates.25 A mouse model of LDL-driven atherogenesis would appear to have advantages over other mouse models for evaluation of dietary fatty acid effects on atherosclerosis with extrapolation to humans, because at least a portion of the dietary effect in humans is on LDL particle composition. Therefore, we have used this mouse as a model to compare atherosclerosis outcomes in response to 5 different dietary fat types containing enrichments in saturated, cis and trans monounsaturated, and n-3 and n-6 polyunsaturated fatty acids, with all groups being compared with a standard commercial mouse diet (control) group. To the extent that dietary fatty acid–induced alterations in LDL cholesteryl ester composition participate in the overall atherogenicity of LDL, as has been hypothesized to be the case on the basis of outcomes in monkeys,12 18 a mouse model with LDL as the primary lipoprotein may permit the hypothesis to be tested.
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Methods |
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Animals and Diets
The mice used in these studies were reared in our medical school animal facility. The original breeding pair was provided by Dr Helen Hobbs at the University of Texas Southwestern Medical Center in Dallas, and this mouse has been described in a separate publication.25 In brief, this mouse strain is a hybrid cross between the LDL receptor -/- mouse described by Ishibashi et al,26 which itself is a hybrid of 129sv and C57BL/6 strains, and the human apoB100 transgenic mouse,27 itself a hybrid of the SJL and C57BL/6B strains. Breeding demonstrated the LDL receptor knockout and apoB overexpression traits to be homozygous, and all animals maintained grossly elevated LDL levels that contained principally apoB100. When the animals reached 8 to 9 weeks of age, they were randomly assigned to 1 of the 6 dietary fat groups. Groups of between 20 and 38 animals containing at least 10 members of each sex were established and entered into the study as they became of age. A total of 167 animals were studied, including 93 males and 74 females. All animals were housed in our American Association for the Accreditation of Laboratory Animal Care–approved animal facility, and their care was supervised by a veterinarian. All animal procedures were preapproved by the institutional animal care and use committee.
The diets used in the study were prepared on the basis of those
fed previously to monkeys,28 29 and we added a
group containing trans fatty acids in a fat blend provided
to us through the generous offices of Dr Pete Huth of Kraft Foods and
the Institute of Shortening and Edible Oils. Dietary ingredients are
shown in Table 1
. The diets contained
10% of energy as fat with the fatty acid compositions shown in Table 2. Cholesterol content in the
prepared diets was consistently low at 0.005%. The
cholesterol content of the commercial mouse food (Prolab
3000, PMI Feeds, Inc) used as a control was 0.06%, and the
measured fat content of this food was 4.6% by weight.
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At weeks 2, 8, and 16 of the experimental period, blood was taken from each mouse for measurement of lipids and lipoproteins. Ketamine HCl was administered (40 mg/kg) together with xylazine (8 mg/kg) to restrain the animals. Blood was taken via orbital bleeding and transferred to tubes containing 0.1% EDTA, 0.01% NaN3, aprotinin (1 µg/mL), and benzamidine (1 mmol/L).30 At the time of sacrifice, the animals were anesthetized with the ketamine (80 mg/kg) and xylazine (8 mg/kg) mixture, blood was drawn via heart puncture, and the mice were then euthanized in a CO2 chamber. Plasma was promptly separated from cells by centrifugation at 5000g at 4°C.
Lipid, Lipoprotein, and Atherosclerosis Measurements
Lipoprotein separations were promptly begun on aliquots of fresh
plasma isolated from each mouse. Whole plasma was injected onto a 30-cm
Superose 6 chromatography column, which was
subsequently run at 0.5 mL/min with 0.9% NaCl containing 0.05% EDTA,
pH 7.4, and 0.05% NaN3, as
described.31 The average LDL particle size was
measured from a standard curve constructed from known standards plotted
against elution time.31 Fractions were collected
and pooled according to the elution times for VLDL, LDL, and HDL, and
aliquots of isolated lipoprotein fractions were used for enzymatic
measurement of cholesterol32 and
triacylglycerols.33 Assays
were monitored for accuracy and precision with simultaneous
measurements in appropriate quality control serum samples.
In some cases for which purified lipoprotein fractions were needed for compositional analyses, plasma from mice in each diet group and of each sex was pooled and then ultracentrifuged for 24 hours at 50 000 rpm in a Ti 70.1 rotor (Beckman Instruments) at a density of 1.21 g/mL to isolate the combined lipoprotein fraction at the top of the tube. Fractions within a single lipoprotein class were then separated on agarose chromatography columns, and chemical compositions were determined for individual classes. For selected LDL samples, apolipoproteins were separated by polyacrylamide gel electrophoresis in the presence of SDS. In brief, aliquots of the lipoprotein sample containing 50 µg of protein were lyophilized and resolubilized by boiling for 5 minutes in a 0.05 mol/L barbital buffer, pH 8.6, containing 2.5% SDS, 3% glycerol, 5% 2-mercaptoethanol, and 0.001% bromophenol blue. Samples containing 10 µg of protein were then applied to a 4% to 30% polyacrylamide gel containing SDS, and electrophoresis was carried out as described previously.34 The gels were then fixed and stained with Coomassie blue dye.
At the end of the study after the animals had been euthanized, the
heart with attached aorta was removed from the body and placed in 10%
neutral buffered formalin for subsequent processing. The end points of
atherosclerosis quantified for each mouse in this study
were aortic free cholesterol and cholesteryl ester
concentrations, measured as mg/g protein. To obtain these end points,
the tissue was placed on the platform of a dissecting microscope, and
the adventitia was carefully and completely dissected and removed. The
aortic intimal-medial preparation was then detached at the base of the
heart and placed in a tube with 3 mL of chloroform-methanol, 2:1
vol/vol, containing 5
-cholestane as an internal standard, and
the lipids were extracted. The lipid extract was separated by
filtration from the delipidated protein, and extracts were dried under
N2 at 60°C and then dissolved in hexane.
Analysis of free and total cholesterol was carried
out with 2 injections per sample on a DB 17 (0.53-mm IDx15 mx1
µm) gas-liquid chromatography column (J&W Scientific)
at 250°C and installed in a Hewlett-Packard 5890 gas
chromatograph equipped with an HP 7673A automatic injector
using on-column injection and a flame ionization detector. Cholesteryl
ester (esterified cholesterolx1.67) was calculated as the
difference between free and total cholesterol, as measured
before and after saponification and reextraction of the nonsaponifiable
sterol into hexane. The delipidated tissue protein was then digested
and dissolved in 1N NaOH, and total protein was
determined.35
Statistical Evaluations
The data were first evaluated for main effects (sex and
dietary fat) by 2-way ANOVA. For post hoc analyses to identify
individual group differences (where they were found), 1-way ANOVA for
individual diets was then used with post hoc analyses by
Fisher's protected least significant difference test. Statistical
significance was considered at P
0.05. The outcomes for
post hoc analyses are indicated in the tables.
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Results |
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The body weights of the animals were measured when the animals were 8 to 9 weeks old, and at the time of sacrifice, 16 weeks into the experimental diet period, when all of the mice were 24 to 25 weeks of age. At the start of the study, the males weighed 28.2±2.0 g (mean±SD) and the females, 23.2±1.9 g. On average, the males weighed
5 g more than the females, and this was also true at the end of the
study, when the overall average weight for males was 35.8±2.6 g and
for females, 30.3±1.8 g. Thus, during the course of the study, both
males and females gained 7 g, and this increase was true in each of
the diet groups. Much of this weight gain appeared to be due to an
increase in adipose tissue, because the carcasses contained significant
amounts of fat at sacrifice. No apparent health problems were found in
any of the diet groups.
Time-related total plasma cholesterol (TPC) responses to
the different diets are shown in Figure 1. At 2 weeks into the study, TPC values
already had begun to show diet-specific differences, with the sat and
cis and trans mono groups already being higher
(near 800 mg/dL) than the n-6 poly (near 650 mg/dL) and n-3 poly and
commercial diet groups (near 550 mg/dL). This trend continued and
became even more marked at 8 weeks into the study, with TPC in the 2
poly groups being between 800 and 900 mg/dL, which was higher than the
TPC of 600 in the commercial diet group; even higher TPC values near
1300 mg/dL were found in the cis and trans mono
and sat groups. At 16 weeks, the TPC in the trans mono group
was clearly highest, at 2000 mg/dL, with the average TPC in the sat
and cis mono groups near 1300 mg/dL, and that in the n-6
poly group near 1100 mg/dL. The n-3 poly group remained lower, near 700
mg/dL, but the commercial diet group had not changed and remained near
600 mg/dL after 16 weeks of diet treatment. In all cases, the values
for males and females were averaged together after no statistically
significant differences between the sexes were found.
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After 16 weeks of diet treatment, the distribution of
cholesterol and triacylglycerol among
lipoprotein fractions was determined (Table 3). Plasma cholesterol
concentrations were high and dietary fat dependent, as discussed above.
The concentration of cholesterol in VLDL was highest in the
trans group, elevated in the sat and cis mono
group, and lower in both poly groups and in the commercial diet group.
LDL cholesterol was surprisingly dependent on the type of
dietary fat fed. It was lowest in the commercial diet and n-3 poly
groups; equivalently higher in the n-6 poly, cis mono, and
sat groups; and highest in the trans mono group. The
cholesterol concentrations in HDL were low in all groups,
although they were somewhat higher in the n-6 poly group.
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Plasma triacylglycerol concentrations were also
very high and diet dependent (Table 3
). The highest value was in the
trans mono group, and the lowest value was in the commercial
diet group. The other groups were intermediate and not different from
each other. The triacylglycerol values in VLDL were
highest in the trans and cis mono groups. The sat
group also had an elevated VLDL triacylglycerol
concentration, whereas the VLDL triacylglycerol in
the poly groups remained lower and similar to the commercial diet
value. Triacylglycerol concentrations in LDL were
high but not diet dependent. Triacylglycerol
concentrations in HDL were low and not diet dependent.
Plasma LDL particle size and composition have been shown to be diet
dependent in other species, and these characteristics were compared in
these mice. LDL particle size was estimated as molecular weight during
gel filtration chromatography31
of individual animal samples in each group, and the results are shown
in Table 4. In general, the LDL particles
were small, with the average in the commercial diet group being
2.43 g/µmol (or 2.43x106 Da). In most
of the groups fed the prepared diets, except for the n-3 poly group,
the particles were somewhat larger, with the average being between 2.5
and 2.7x106 Da. In the cis mono
group, the LDL particles were the largest and averaged
3.0x106 Da. The percentage compositions are also
shown in Table 4. LDLs used for this evaluation represent the
material from the LDL peaks isolated from the Superose column, which
were pooled from 3 to 4 animals into a total of 4 pools (2 for males
and 2 for females) for each diet group.
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The LDL particles of each pool were then floated at a density of 1.063
g/mL in the ultracentrifuge to remove extraneous proteins. The
compositions of LDL from each diet group were similar, and there were
not major differences among LDLs from the different diet groups. The
protein, phospholipid, and free cholesterol percentages
were quite similar in LDLs from each of the diet groups and together
made up 50% of the mass of the particle. The core lipid was
somewhat different in composition, with the
triacylglycerol percentage being higher in the
commercial diet and n-3 poly groups than in that of other groups,
wherein the cholesteryl ester made up close to 35% of the mass.
The apolipoprotein composition of the LDL particles was also examined,
and a gel showing some of the data is shown in Figure 2. The LDL peak was separated into large,
medium, and small fractions by pooling the material in the front,
middle, and back of the LDL peak from the column. The major
apolipoprotein in all fractions was apoB100, and lesser amounts of
apoB48 were apparent. Small amounts of apoE were visible in the
fraction containing larger LDL particles, and in 1 preparation, a small
amount of apoA-I was also seen in the fraction containing the smaller
LDL particles; for the most part, however, apoB100 was the principal
apolipoprotein on the LDL particles. The small amounts of apoE and
apoA-I could represent the presence of particles distinct from
the apoB-containing LDL particle.
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The cholesteryl ester composition of LDL was also examined, and the
data are shown in Table 5. The pattern
was characteristic of rodent species and different from that typical of
primates. The commercial diet group had 35% as cholesteryl linoleate,
15% as cholesteryl palmitoleate and oleate, and 10% as cholesteryl
palmitate and stearate. Approximately 25% was cholesteryl
arachidonate. A similar distribution was seen in the n-6
poly group, although the percentage of cholesteryl linoleate was
10% lower and the percentage of cholesteryl
arachidonate 10% higher. The n-3 poly group was
distinctive, in that >40% of the cholesteryl esters contained
long-chain, highly polyunsaturated fatty acids, the majority being n-3
polyunsaturated fatty acids. The remaining groups had only 12% to
13% cholesteryl linoleate, between 20% and 25% cholesteryl
arachidonate, and as the major cholesteryl ester,
cholesteryl oleate, with a significant amount of cholesteryl
palmitoleate as well. This pattern of
<\+aq;I>monounsaturated fatty acid enrichment of
cholesteryl esters is typical of the pattern in primates fed saturated
and monounsaturated fat, although the actual
percent is lower in mice due to the higher percentage of cholesteryl
arachidonate. The trans fatty acids appeared to
interfere to some extent with the accumulation of cis
monounsaturated fatty acids in cholesteryl esters,
although this outcome was not due to accumulation of trans
fatty acids in cholesteryl esters, since <1% of the LDL cholesteryl
esters in the mice of this group were identified as containing a
trans fatty acid.
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The variety of plasma lipoprotein responses to the different dietary
fatty acid challenges was somewhat unexpected, given the low energy
percent of fat in the diet. Nevertheless, the atherogenic response in
the aorta was considered the primary indicator of beneficial or
detrimental effects of any particular fatty acid on
atherosclerosis, and the lipoprotein responses
suggested that differences would be present, so
atherosclerosis quantification in each of the animals
in the study was performed. Whereas most studies in mice have used a
tissue histology–based system for atherosclerosis
evaluation, the method is labor intensive, the tissue available for
evaluation is small, and the accuracy of this type of evaluation for
determining the overall extent of aortic
atherosclerosis is unknown. Therefore, we measured the
accumulation of free cholesterol and cholesteryl ester in
the whole aorta as a chemical end point that quantitatively monitors
the extent of atherosclerosis. With a 2-way ANOVA, we
found no differences between males and females; thus, the following
analyses were done after combining the data from all of the
animals in the study. The group averages for aortic free
cholesterol (mg/g protein) are shown in Figure 3A. Aortic free cholesterol
concentration was significantly different among diet groups
(P<0.0001). It was the same, at about 20 mg/g, in the
commercial diet and the 2 poly groups, but this value was significantly
(P<0.008) elevated in the cis mono (29.8 mg/g)
and sat (30.5 mg/g) groups and was even higher in the trans
mono group (41.9 mg/g) (P<0.0001 compared with all other
groups).
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The atherogenic response as measured by aortic cholesteryl ester
concentration showed even more exaggerated differences among diet
groups (Figure 3B). The lowest values were in the commercial diet and
n-3 poly groups, 7.8 and 9.7 mg/g, respectively. The cholesteryl ester
concentration in the n-6 poly group was significantly higher
(P<0.05) than that in the commercial diet group, at 18.2
mg/g, and was barely higher (P=0.07) than the value in the
n-3 poly group, but the averages in the cis mono group, at
34.6 mg/g, and sat group, at 35.6 mg/g, were each higher
(P<0.001) than either of the poly groups. The highest value
was found in the trans mono group (50.4 mg/g), and this
value was significantly higher than either the sat or cis
mono group (P<0.002), as well as higher than that of any of
the other groups. Thus, the outcome found for both free and esterified
cholesterol concentrations, as indicators of
atherosclerosis extent, was similar. The greatest
accumulation of both forms of cholesterol occurred in the
trans mono group, the next highest occurred in the sat and
cis mono groups, and less accumulation occurred in the poly
groups, which were not much different from the group fed the commercial
diet. A very similar outcome was obtained when the analyses
were done on cholesterol and cholesteryl ester as measured
in mg/g of wet tissue (data not shown). The data expressed as mg/g
protein are thought to be more accurate, owing to the small amounts of
tissue (2 mg per aorta) and the potential weighing errors of wet
tissue that could occur.
The individual animal data for cholesteryl ester concentration are
plotted in rank order from least involved to most involved in Figure 4 for each of the diet groups to
illustrate the degree of difference among the groups. These data
indicate that the diet group differences identified by the statistical
analyses are present across the entire spectrum of
response, from low to high responders, and are not confined to a
particular segment of the response spectrum.
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Finally, the data shown in Figure 5
indicate that the data from 5 of the groups all described a similar
log-linear relationship, with a correlation coefficient of
r=0.7 between TPC and aortic cholesteryl ester
concentration. The data for individual groups are also indicated and
were shown to essentially fit the line in Figure 4 when each data set
was plotted separately. The data comparing plasma LDL and VLDL
cholesterol concentrations were also plotted (not shown),
and similar relationships were seen, although the correlation
coefficients were not as high as for TPC. We interpret this finding to
mean that both lipoprotein fractions are contributing to the
development of atherosclerosis, although LDL appears to
be the more important, presumably because it is present in much
higher concentrations. Interestingly, the data for the trans
mono group did not fit the relationship shown in Figure 5 (data not
shown). The scatter in the data from this group, if plotted as in
Figure 4, was large (r=0.24), and the regression line for
the data of this group was displaced, such that at any aortic
cholesteryl ester concentration, the TPC concentration was apparently
higher. This result is probably related to the distinct pattern of
increase in plasma cholesterol between 8 and 16 weeks for
this particular diet, as shown in Figure 1 for the trans
mono group. The pattern suggests that the extended increase induced by
trans monounsaturated fat in plasma
cholesterol during the 8- to 16-week interval may not have
been associated with an immediate and proportional effect on
atherosclerosis, although the study needs to be carried
out for a longer time to determine the real consequences for
atherosclerosis.
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Discussion |
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The data of this study illustrate the strongly positive effects of polyunsaturated fat compared with saturated fat to reduce the development of atherosclerosis in a mouse model that has LDL as the predominant lipoprotein in the circulation. This observation has also been made in several studies of coronary artery atherosclerosis in primates11 12 18 19 and is reproduced in the aortas of the mice of this study. The n-3 polyunsaturated fat appeared to be more effective than the n-6 polyunsaturated fat, although only 1 level of either polyunsaturated fat was studied here (7.2 energy% of n-6 versus 1.4 energy% of n-3 fatty acids), and therefore, the dose response is not known. Nevertheless, because almost 5 times more n-6 than n-3 polyunsaturated fatty acids were fed in the diet and because the n-3 polyunsaturated fatty acids kept aortic cholesteryl ester concentrations lower (Figures 3
and 4), the data suggest that n-3 fatty
acids are more potent in reducing atherosclerosis.
The reduction in aortic atherosclerosis was not found
when either cis or trans
monounsaturated fatty acids were fed. Rather, just
as much atherosclerosis was seen when cis
monounsaturated fat diets were fed (6.8 energy%)
as when saturated fat was fed, and significantly more
atherosclerosis was seen when the trans
monounsaturated fatty acids were fed as 2.8
energy% (Figures 3 and 4). The average plasma LDL
cholesterol concentration was very similar in the mice fed
n-6 polyunsaturated fat, saturated fat, and cis
monounsaturated fat, yet the amount of
atherosclerosis was greater in the cis
monounsaturated and saturated fat groups. The HDL
cholesterol concentrations were slightly higher in the
group fed n-6 polyunsaturated fat, but the LDL to HDL
cholesterol ratios were very high in all groups (in the
range of 15 to 30), and it is not clear that HDL at these ratios can
offer much protection in the face of such high plasma LDL
concentrations. The VLDL cholesterol levels were lower in
the n-6 polyunsaturated fat group, and even though these lipoproteins
were present in lower concentrations than LDL, this could account
for some of the difference in atherosclerosis.
The observation in these LDL receptor–null, human apoB–overexpressing mice that cis monounsaturated fatty acids in the diet promote as much atherosclerosis as saturated fatty acids and more atherosclerosis than polyunsaturated fatty acids is similar to an earlier observation on coronary artery atherosclerosis in primates.18 This is an important outcome when one considers that monounsaturated fats, often in the form of olive oil, are widely promoted as being healthful and effective for protection against heart disease.16 17 Although effects to modify lipoprotein risk factor profiles to be apparently less atherogenic were seen in response to dietary monounsaturated fat in monkeys and humans,17 18 effects to reduce atherosclerosis did not follow. In the present study in mice, the difference between the n-6 polyunsaturated fat group and the cis monounsaturated fat group in TPC and LDL cholesterol concentrations was not statistically significant, yet the former dietary fat still protected against atherosclerosis while the latter did not. Clearly, some other factor in response to cis monounsaturated fatty acids but not in response to polyunsaturated fatty acids appears to promote atherosclerosis, and as a result, the amount of atherosclerosis in animals fed monounsaturated fat was comparable to that when saturated fat was fed.
On the basis of data in monkeys, we speculated that another factor promoting atherogenesis could have been the remarkable enrichment of the lipoprotein particles with cholesteryl oleate.12 18 We examined the livers of monkeys in isolated liver perfusion studies and found a high correlation between cholesteryl ester accumulation in apoB-containing particles secreted during perfusion and the extent of coronary artery atherosclerosis in the liver donor animals, suggesting that the accumulation of cholesteryl esters in LDL and the associated increase in atherosclerosis might be in response to stimulated hepatic secretion.36 The stimulation of hepatic cholesteryl ester secretion was apparently due to a stimulation of hepatic acyl coenzyme A:cholesterol acyltransferase by oleic acid.37 38
In the mice of these studies, enrichment of LDL with cholesteryl oleate
was most pronounced in the cis mono and sat groups, although
the lipid milieu in the core of the LDL particles of these mice was
quite different from that found in monkeys and humans. A high
percentage of triacylglycerol was found in the
mouse LDL (Table 4), and significant quantities of cholesteryl
arachidonate (and n-3 fatty acid cholesteryl esters in the
n-3 poly group) were also found (Table 5). These lipids should modify
the proatherogenic effect of the cholesteryl oleate enrichment, if
effects on the physical state of the lipoprotein core are important in
atherosclerosis, as has been
proposed.39 Triacylglycerols
and polyunsaturated cholesteryl esters both have the effect of reducing
the transition temperature of the neutral lipids in the core of the LDL
particle.40 Perhaps this factor contributes to
the apparently modest rate of atherosclerosis
development in our mouse model, in which plasma cholesterol
concentrations were >500 mg/dL in the commercial diet–fed animals,
for example, whereas the degree of cholesteryl ester accumulation in
the aorta at 24 weeks of age was measurable but not remarkable (Figures 3 and 4). For example, in the African green monkey coronary
arteries of our earlier publication,18 average
cholesteryl ester concentrations in mg/g protein were 57, 179, and 137
in the polyunsaturated, monounsaturated, and
saturated fat groups, respectively, values higher than those found in
the mice of the present study (Figure 3). On the other hand, if
cholesteryl oleate enrichment of LDL were to promote cholesteryl ester
accumulation in the artery wall by forming a higher-melting-point lipid
"phase" during lysosomal processing of lipoprotein particles, for
example, then enrichment of LDL with cholesteryl oleate might still be
part of the effect to increase atherosclerosis through
delayed clearance of arterial cholesteryl
esters.41
Clearly, more work is needed on the mechanism(s) by which an atherosclerosis-promoting effect of dietary monounsaturated fat would occur. What is clear when comparing the present studies in mice with the earlier studies in monkeys18 is that a lack of protection against atherosclerosis development by monounsaturated fat occurred in 2 experimental animal models with quite different lipoprotein profiles and lipoprotein lipid compositions. It seems important to discern whether a similar outcome is present in humans before more recommendations are made to consume more fat enriched with monounsaturated fatty acids.
Animals fed polyunsaturated fat have LDL particles that are greatly
enriched in the polyunsaturated fatty acid content in their cholesteryl
esters42 and
phospholipids,43 and this result also appears to
be true in the present study in mice (Table 5). Numerous studies,
including our own,44 have demonstrated that LDL
particles enriched in polyunsaturated fatty acids are more easily
oxidized in vitro. To the extent that LDL oxidation promotes
atherosclerosis, data on dietary polyunsaturated fat
protection against atherosclerosis present a
paradox. It appears that other factors, possibly including LDL
cholesterol concentration and/or particle composition, are
more important in promoting atherosclerosis development
in this circumstance. In the present studies in mice, in which the
degree of atherosclerosis was less in the n-6 poly
group (Figures 3 and 4) even though LDL cholesterol
concentrations were not different among the n-6 poly, cis
mono, and sat groups (Table 3), it remains a distinct possibility that
LDL particle composition is an important factor in determining the
extent of atherogenesis. Perhaps the LDLs from mice fed either
cis mono or sat diets interact with the matrix within the
artery more effectively than do LDLs from the polyunsaturated fat
groups, as has been demonstrated in monkeys.45
Alterations in the metabolism of cholesteryl esters and
other lipids within the artery wall in response to dietary fatty acid
composition, such as an effect of monounsaturated
fatty acids to promote acyl coenzyme A:cholesterol
acyltransferase activity, might also be involved. Clearly, more data
are needed before we will fully understand the relative importance of
the various effects of individual dietary fatty acids on
atherosclerosis.
The effect of trans fatty acids to promote even more
atherosclerosis than do saturated fatty acids was not
anticipated on the basis on the literature.46 47 48 49
In the current study, trans fatty acids accounted for only
2.8% of energy, yet they dramatically promoted plasma
cholesterol concentration increases more than any other
type of fat (Figure 1 and Table 3). The mechanism by which plasma
cholesterol was increased is unknown, but it seems likely
that this increase was at least in part responsible for the worse
atherosclerosis (Figures 3 and 4). The fact that the
proportionality between TPC and aortic atherosclerosis
was not high among animals in the trans mono group may be
due to the fact that plasma cholesterol concentrations rose
to such high levels during the last 8 weeks of the study that the
impact of this increase was not fully registered in the
arterial response measured at 16 weeks. Perhaps if the diet
had been continued for a longer time, the extent of
atherosclerosis would have been more highly correlated
with the plasma cholesterol concentration. Alternatively,
it is possible that trans fatty acids have a unique effect
in promoting hypercholesterolemia but that the
proportionality of atherosclerotic lesion development to plasma
cholesterol concentration is different, due to an effect on
the lipoprotein profile or on the artery wall, which was not delineated
by the analyses performed in the present study.
Finally, these studies can be compared with those of Sanan et
al,25 wherein mice similar to those used in this
study were studied after 26 weeks on a commercial diet. The range of
cholesteryl ester concentrations observed for the commercial diet group
was between 2 and 20 mg/g protein (Figure 4), whereas the range of
values for the proportion of aortic surface area involved with lesion
in the comparable group of mice of the Sanan study was between 2% and
50%. This result indicates that significant lesion development
occurred during the 6-month study, even in mice fed the commercial
diet, and that this animal model is indeed an excellent one for
studying the factors affecting atherosclerosis in the
mouse. We have shown the effects on atherosclerosis of
individual dietary fatty acids in this study in mice that parallel
those of our studies of coronary artery
atherosclerosis in monkeys.18
This fact suggests that identification of factors important in the
development of atherosclerosis in this LDL
receptor–null, human apoB100–overexpressing mouse model could be
appropriate for understanding the relevant factors in this disease
process in humans.
|
Acknowledgments |
|---|
This work was supported by National Institutes of Health (Bethesda, Md) grants HL-49373 and HL-24736 (to L.L.R.). The assistance given to us by Dr Helen Hobbs from the University of Texas Southwestern Medical Center in Dallas in providing the transgenic/knockout mice used in these studies is greatly appreciated. We value greatly the assistance of Dr Pete Huth in obtaining and formulating the dietary fats, the contributions of dietary fats made by the members of Institute of Edible Fats and Oils, and the assistance of the US Marine Fisheries Institute, which provided the fish oil used as the source of the n-3 fatty acids in these studies. The assistance of Linda Odham in manuscript preparation is also appreciated.
|
Footnotes |
|---|
Reprint requests to Lawrence L. Rudel, Department of Comparative Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157.
Received May 4, 1998; accepted August 31, 1998.
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References |
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