© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Low-Cholesterol and High-Fat Diets Reduce Atherosclerotic Lesion Development in ApoE-Knockout Mice
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Abstract |
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Abstract—We have investigated the effect of most common oils used in human nutrition on the development of atherosclerosis in apoE-knockout mice. Seven groups of animals, separated according to sex, were fed for 10 weeks either chow diet or the chow diet 10% (wt/wt) enriched with different oils (palm, coconut, 2 types of olive oil, and 2 types of sunflower oil) without addition of cholesterol. At the end of this period, plasma lipid parameters were measured and vascular lesions scored. None of the diets induced changes in plasma cholesterol concentrations, whereas plasma triglycerides were uniformly reduced in all diet groups. Some diets caused significant reductions in the size of atherosclerotic lesions in males and others in females; males responded most to sunflower oils and females to palm oil and one olive oil (II). The lesion reduction in males consuming sunflower oils was associated with the decrease of triglycerides in triglyceride-rich lipoproteins, whereas the decrease in females consuming olive oil II or palm oil was accompanied by an increase in plasma apoA-I. The increase in plasma apoA-I in the latter condition, is mainly due to overexpression of hepatic message elicited by a mechanism independent of apoE ligand. The data suggest that the different diets modulate lesion development in a gender specific manner and by different mechanisms and that the development of atherosclerosis, due to genetic deficiencies, may be modulated by nutritional maneuvers that may be implemented in human nutrition.
Key Words: cholesterol • apolipoprotein • atherosclerosis • dietary fat
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Introduction |
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ApoE is a 34-kDa glycoprotein that circulates in plasma as a component of several lipoproteins such as chylomicron remnants, IDL, VLDL, ß-migrating VLDL, and HDL.1 It is involved in the uptake and degradation of chylomicron and VLDL remnants by the LDL receptor and the LDL receptor related protein.1 2 Besides its role in lipoprotein metabolism, other biological functions have been attributed, such as macrophage differentiation and mobilization and utilization of lipids in the central nervous system.1 Genetic deficiency of apoE in humans has shown accumulation of plasma remnant lipoproteins and development of atherosclerosis.3 ApoE-deficient mice develop severe hypercholesterolemia and atherosclerosis on a regular low-fat/low-cholesterol diet. The progression and histopathology of lesions in this animal model show similar features to those observed in humans and other species, including fatty streaks, necrotic cores, and fibrous caps.4 5 6 7 8 9
Dietary fat is one of the most important environmental factors associated with the incidence of cardiovascular diseases. Diets high in cholesterol and saturated fat have shown to promote the development of atherosclerosis.9 10 11 Conversely, dietary polyunsaturated fats have shown to reduce the development of atherosclerosis in several species.11 12 Studies dealing with the effects of monounsaturated fatty acids on lesion development are scarce.10 This is surprising because traditional Mediterranean diets are followed in a geographical area with a low incidence of coronary heart disease, and they combine a relatively low cholesterol content and the use of olive oil as the main source of fat.13 Therefore, the hypothesis that a combination of high dietary oil consumption with reduced cholesterol is protective for atherosclerosis, needs to be tested in more detail.
Due to the above described properties, apoE-knockout mice are excellent animal models for evaluating the influence of pharmacological and nutritional agents on atherosclerosis development.14 The absence of the apoE, specific ligand to remove remnants, adds an additional value to this model to test the influence of different nutrients on the regulation of some apolipoproteins. In this study, we report the effect of the most common oils used in human nutrition, provided in diets of low cholesterol content, on the development of atherosclerotic lesions and the regulation of apoA-I gene expression in the apoE-deficient mice.
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Methods |
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Animals
Homozygous apoE-knockout mice, hybrids of C57BL/6J and 129 Ola strains, were progeny of those described by Piedrahita et al15 and bred in The Unidad Mixta de Investigación, Zaragoza, Spain. Seventy-seven males and sixty-three females, aged 2 months, were randomly allocated into 7 groups for each sex and housed in sterile filter-top cages. Animals had ad libitum access to food and water. Normal mouse chow was supplied by B & K Universal Ltd. Body weights were recorded throughout the experiment. None of the animals were lost during the study.
Diets
Regular mouse diet was supplemented with fat of different
origins to achieve isocaloric diets, differing only in the source of
fat. No extra cholesterol was provided. Experimental diets
were fed for 10 weeks, and all diets were well tolerated. A composition
of the different diets is shown in Table 1
. Hydrogenated coconut oil and palm oil
(42% of oleic acid) were supplied by Agra-Unilever. Olive oil I (70%
wt/wt of oleic acid) and II (81% of oleic acid) were prepared from
different cultivars in our pilot plant. Sunflower oil I (22% oleic
acid) and sunflower oil high in oleic acid (76.4% oleic acid),
described as II, were obtained from Koipe.
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Diet Analysis
Diets were analyzed chemically for total energy (bomb
calorimetry), protein (Kjeldahl), total fat (Soxhlet, and fatty acids
using gas-liquid chromatography.16 Vitamin
E was measured by high-pressure liquid chromatography
using a Lichorspher SI60 column and is expressed as
d-
-tocopherol.17 Cholesterol
was assayed using a kit for food analysis.
Lipids and Lipoprotein Analysis
Blood samples were obtained from animals fasted overnight by
retroorbital bleeding under light diethylether anesthesia
before the experimental diets. After the experimental period, animals
were killed by the Avertin (2,2,2-tribromoethanol) injection
(Aldrich Chemical Co) and blood was drawn from their hearts. Total
plasma cholesterol and triglyceride
concentrations (without free glycerol) were measured enzymatically in a
microtiter assay, using commercial kits from Sigma Chemical Co and
Boehringer Mannheim Gmbh. Cardiolipid (Sigma) was used as
quality control. Plasma apoA-I concentration was evaluated by a
chemiluminescent ELISPOT procedure (ECL kit; Amersham Corp), using
immunopurified rabbit IgG against rat apoA-I as primary antibody and
purified apoA-I as standard. For a more detailed analysis of
the plasma lipoprotein profiles, 200 µL of pooled plasma samples from
11 males and 9 females from each dietary group, were subjected to fast
protein liquid chromatography gel filtration using a
Superose 6B column (Pharmacia LKB Biotechnology), according to
conditions described by Vilella et al.18 Fractions (0.5
mL) were collected, and their total cholesterol and
triglyceride content were measured as described above.
RNA Preparation and Analysis
At the moment of killing, livers and small intestines were
obtained and quickly frozen in liquid nitrogen. RNA was isolated using
Trigent reagent MRC following the manufacturer's instructions. A pool
of 5 µg of each group was denatured by 2.2 mol/L formaldehyde and
50% formamide at 68°C for 15 minutes, run on a 1% agarose gel with
formaldehyde, transferred to a nylon membrane (Hybond-N, Amersham), and
hybridized to probes following current protocols.19 The
mouse Apoa1 probe was a 180bp EcorI/NheI fragment
corresponding to the fourth exon of the gene. A mouse G3PDH probe
obtained from Ambion was used to normalize the amount of RNA
loaded on the gel. Both probes were labeled using
[-32P]-dCTP and Rediprime (Amersham).
Filters were exposed to Biomax film (Kodak, Amersham) and
analyzed using Molecular Analysis (BioRad).
Evaluation of Atherosclerotic Lesions
The heart and the arterial tree were perfused with
phosphate-buffered formalin (4%, pH 7.4, Panreac) under
physiological pressure. Hearts and aortas were
dissected out, cleaned, and stored in neutral formaldehyde. The base of
the hearts and the aortic roots were taken for analysis and
transferred to liquid OCT (Bayer Diagnostic), where
they remained for 24 hours to eliminate bubbles. Hearts were placed in
new OCT on a cryostat chuck (Microm HM505E). Serial cryosections of the
proximal aorta and the aortic sinus were made and stained with Sudan IV
B (Sigma Chemical Co) and counter-stained with hematoxylin and eosin
(Sigma Chemical Co). Average lesion sizes of 4 sections, spanning the
region from the very proximal aorta to the point in the aortic sinus
that contains 3 complete valve leaflets, were used for morphometric
evaluations based on the method of Paigen et al.20 Images
were captured using a Leica microscope equipped with a video camera
connected to a computer. Quantification of atherosclerotic lesions was
performed using Win-Q500C software (Leica).
Statistical Analysis
Data were analyzed using Instat for Macintosh software
(GraphPad). Most of the parameters in this study did not
follow normal distribution according to Shapiro-Wilk test. Therefore,
analysis of statistically significant differences was carried
out using Wilcoxon test for paired data and Mann-Whitney U test
for unpaired data. Differences were considered nonsignificant when
P>0.05. Association between variables was assessed by
Spearman's rank-order correlation coefficient
(rs).
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Results |
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Dietary Characteristics
Table 1
summarizes the results of chemical analysis
of the diets. As shown in this table, chow and experimental diets were
almost isocaloric. Distinctive general features among them were lower
carbohydrate, higher percentage of fat, and a slight decrease in
cholesterol in the experimental diets. The vitamin E
content was highest in sunflower oils, at an intermediate level in
olive and palm oils, and at the lowest level in chow and coconut oil
diets. Chow diet presented a
monounsaturated fatty acid content of 35.2% and
almost equal amounts of polyunsaturated and saturated fatty acids (P/S
ratio 0.9). Coconut oil was a diet with a high content of short chain
saturated fatty acids, whereas palm oil contained saturated fatty acids
of long chain and monounsaturated fatty acids.
Olive oil I and II diets were high in
monounsaturated fatty acids, differing in their
percentages of saturated, monounsaturated, and
polyunsaturated fatty acids. Sunflower oil diets differed in their
content of monounsaturated (oleic acid) and
polyunsaturated fatty acids (linoleic acid).
Effects of Different Diets on Body Weight and Plasma
Lipoprotein Concentrations
During the 10 weeks of experimental period, both male and female
animals fed chow gained an average 6 g body weight (Tables 2 and 3).
In contrast, all fat-fed female groups gained significantly less weight
than animals consuming regular mouse chow (Table 2). This effect
was less evident in males, and only those groups consuming diets
supplemented with coconut oil, olive oil II, and sunflower oil I gained
significantly less weight than their littermates fed chow, olive oil I,
palm oil, or sunflower oil II diets (Table 3). No significant
variations in the amount of food consumption was found, with the
exception of the coconut oil diet (consumed less than other diets),
although the difference was not statistically significant (data not
shown).
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Plasma cholesterol, triglycerides, and apoA-I
were determined at the beginning and at the end of the experimental
period in both female and male animals, as summarized in Tables 2 and 3, respectively. In females, (Table 2)
plasma cholesterol levels did not change significantly,
with the exception of a significant decrease in animals fed sunflower
oil I. At the beginning, males in all groups had similar
cholesterol levels and, on average, 100 mg/dL higher than
females, but no significant change was observed with the dietary
intervention (Table 3). In contrast, all the high-fat diets
induced significant decreases in plasma levels of
triglycerides in both sexes compared with chow diet (Tables 2 and 3). Plasma apoA-I in the male of all groups (Table 3) showed uniform baseline levels, whereas there was a high
variability among the females in different groups (Table 2).
Diets supplemented with olive oil II or palm oil significantly induced
their plasma apoA-I concentrations in both sexes (Tables 2 and 3), and sunflower oil I diet reduced apoA-I levels only in
females (Table 2). The effect of sunflower oil was not
present when it was enriched in oleic acid (sunflower oil II)
(Table 2).
The distribution of cholesterol and
triglycerides among the different plasma lipoproteins was
analyzed by Superose 6B column chromatography
of pooled plasma from animals in each experimental group.
Cholesterol distribution after 10 weeks of feeding
different diets in females and males is shown in Figure 1. Minimal changes in females were
observed in cholesterol distribution of all lipoproteins.
However, cholesterol carried by
triglyceride-rich lipoproteins (TRL) increased in males fed
olive oil I, sunflower oil I, and sunflower oil II compared with chow
diet, whereas diets containing coconut, palm, or olive oil II decreased
cholesterol in TRL. No dramatic changes were observed in
other lipoprotein fractions in males. The distribution of
triglycerides among lipoproteins is also shown in Figure 1 for females and males. The triglycerides were
mainly carried in TRL. Olive oil II, sunflower oils I, and sunflower
oil II reduced the triglyceride content dramatically in
these particles in both sexes. Olive oil I increased
triglycerides in TRL in males but reduced them in females.
An opposite trend was found in animals fed palm oil.
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Quantification of Lesion Area
Figures 2 and 3 show atherosclerotic lesion area in
females and males killed at the end of the study. Lesions observed in
all animals killed at 18 weeks of age were foam cell infiltration into
the intima (data not shown). There was a trend to have smaller lesions
in control males than in females, but it did not reach statistical
significance due to the high variability in both groups (Figures 2 and 3). In either sex, enrichment of mouse diet with
fat did not increase atherosclerotic lesions in any studied conditions.
However, differences in size among dietary groups were found. In
females (Figure 2), diets supplemented with palm oil or olive
oil II induced a significant decrease in lesion size. Sunflower oil
also decreased lesion size, although it did not reach statistical
significance (P<0.08). Enrichment of sunflower oil with
oleic acid (sunflower oil II) did not have any additional effect
compared with sunflower oil I. Except the palm oil group, males (Figure 3) showed a trend of decreased lesion sizes due to the
supplement of fat compared with chow diet. However, this decrease did
not reach statistical significance. When compared with palm oil diet,
both sunflower oils with low or high oleic acids presented
statistically significant decreases of lesion (Figure 3). Taking
into consideration the responses to palm oil and olive oil II in both
sexes (Figures 2 and 3), there was significantly less
effects in males than in females, thus indicating that a complex
interaction with hormones may be involved.
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RNA Analysis
To test whether the expression of apoA-I can be regulated by the
different oils in absence of apoE ligand, and to verify whether mRNA
induction was involved in some changes in plasma apoA-I, we determined
apoA-I message levels in liver and intestine by triplicate Northern
blot analyses. Data in Figure 4
are expressed as arbitrary units and refer to the level of
glyceraldehyde 3-phosphate dehydrogenase. When mice
were fed chow diets, no differences were found between sexes in hepatic
or intestinal expression, and intestinal levels were slightly lower
than hepatic levels. In females, sunflower oil I diet induced a
significant decrease of intestinal expression of apoA-I compared with
chow (Figure 4A), whereas no significant changes by the effect
of other diets were observed. In the liver, no significant change in
the expression of apoA-I mRNA (Figure 4B) was seen in males.
Administration of high-fat diets induced significant increases (200%)
in the hepatic expression of this message in females consuming
saturated and polyunsaturated diets. All these data suggest a
differential response to fat in different organs in a sex-dependent
manner.
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Discussion |
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Although dietary fat is considered to be one of the risk factors for cardiovascular diseases, it is not well understood whether a high fat content in diet has an effect on lipid metabolism and on atherosclerotic lesion development when the amount of cholesterol in the diet is low. To address this issue and to study possible mechanisms by which dietary fat regulates such parameters, we have conducted a dietary trial in which apoE-knockout mice were given 7 different diets for 10 weeks. We found that dietary fat supplement without addition of cholesterol does not increase lesion sizes either in males or females. On the contrary, palm and olive II oils significantly reduced lesion size but only in female mice. Furthermore, different types of fat have different effects, and even a particular oil from different cultivars has different effects, as in the case of olive oil I and II. Reduction in lesion size was independent of plasma cholesterol levels because no change in the latter was observed in any of the studied conditions, and no correlation between total plasma cholesterol levels and lesion size was found (data not shown).
Overexpression of apoA-I has been shown to reduce atherosclerosis in apoE-deficient mice.21 We found that the females consuming olive oil II and palm oil (the groups with significant decreases in lesions) had a significant elevation of plasma concentration of apoA-I. The decreased lesion in these groups may therefore be explained by the increase in apoA-I. However, changes in total apoA-I levels do not appear to be responsible for the sizes of lesions in other diets because a constant reduction in lesion occurred in all fat-fed groups, but no such parallel increase in plasma apoA-I was observed. Furthermore, although increase in apoA-I was present in males fed olive oil II and palm oil, it did not have the same relevance as in females regarding lesion development.
The implication of hypertriglyceridemia in
atherosclerosis is a matter for open discussion because
it is not easy to delineate its role without considering other related
parameters. For example, in apoE-null mice made openly
chylomicronemic by crossing them with mice overexpressing
apoC-III,22 no further increase in lesion was observed.
This suggests that in a dramatic lesion forming condition, as in the
case of apoE knockout, additional increase in plasma
triglyceride levels is not a further risk factor. The
opposite situation appeared when mice lacking apoA-I were crossed with
apoB-transgenic mice, in which an increase in triglycerides
together with an increase in cholesterol was related to
accelerated lesion development.23 In apoB overexpressing
apoE-null mice,24 a negative correlation between
triglyceride and lesion development was seen, although this
effect was overlapped with the increase in cholesterol. A
clear correlation between triglyceride levels and
atherosclerotic lesion, independent of cholesterol, was
present in apoE mice treated with estrogens and fed chow
diet.25 In fact, their correlation coefficient values are
similar to those obtained by us (rs=0.24,
P<0.05 for females and rs=0.19,
P<0.05 for males), suggesting that a decrease in
triglyceride may be a direct cause of a key for the
reduction of vascular lesion formation, but this contribution would not
be higher than 20%. Clearly, a decrease in the amount of
triglycerides in TRL, when their cholesterol is
high (Figure 1), may explain the decrease in lesion observed in
males consuming sunflower oils (Figure 3). The influence of this
particular dietary condition and the complex genetics, as emerging from
the referred animal models, might explain the controversial results
obtained for triglyceride as risk factor in human
studies.26 27
Plasma triglycerides at the beginning of the present study were higher than those initially described for this mouse model.5 6 Although our colony was established from 1 pair of apoE-/- mice, some of the animals that were randomly allocated into the olive oil I group were openly hypertriglyceridemic. Different authors have found both more and less intense hypertriglyceridemia in different progenies of this animal.22 28
A remarkable and surprising finding in our study is the decrease in lesions in females fed palm oil. This oil was found to increase lesion in C57BL/6J females by Nishina et al.10 The main differences between their studies and ours are that we did not add cholesterol or cholate to the diet and no plasma cholesterol change was observed by the palm oil used. Another important aspect to be considered is that we used a special fraction of palm oil that is liquid at room temperature that had been refined to be used for human consumption. In the aforementioned study,10 there was a correlation between lesion and type of fatty acids provided in diets, which was not confirmed in our study. Additionally, the percentage of fat used (10%) in our study was lower than that used by Nishina et al10 (15%). Furthermore, we used genetically manipulated apoE-/- animals, whereas Nishina et al10 used C57BL/6J inbred mice. Lesion development in these 2 models may be influenced differently by other aspects apart from plasma lipids, such as monocyte adhesion29 and endothelium function,30 which can be modulated by different diets.
The decrease in body weight induced by these diets was sex-specific and more pronounced in females than in males. This sex difference was not attributable to differences in the amount of food consumed. Interestingly, we observed significant correlations (rs=0.35, P<0.0026 for males and rs=0.32, P<0.01 for females) between body weight and plasma triglyceride concentrations at the end of diet experiment. A fat-enriched diet with low cholesterol content therefore not only fails to induce obesity in this animal model but helps to reduce body weight. These data suggest a presence of important physiological aspects that are regulated in a gender-specific way and modulated by the dietary amount of fat.
The cellular response of hepatic apoA-I mRNA elicited by the different
diets points out that remnants are taken by the liver through an
apoE-independent mechanism, as suggested by Chang et
al31 32 and Quarfordt et al33 and poses an
exciting question regarding the ligand that facilitates the uptake in
these conditions. Induction of the hepatic transgene cholesterol ester
transfer proteins expression in apoE-null mice in response to
dietary cholesterol has been reported by Masucci-Magoulas
et al.34 Their results and ours clearly show that
transcription machinery in animals lacking apoE ligand is sensitive to
dietary manipulations. Compared with previous results in rats that
showed that a minimum threshold of 40% dietary fat was necessary to
obtain significant responses of apoA-I expression,35
apoE-/- mice responded more acutely with lesser percentage of fat
(10%, wt/wt). Our observation of the tissue expressions of apoA-I also
corroborate dietary expression studies carried out by Sorci-Thomas
et al,36 who showed that hepatic expression of apoA-I is
more adaptable to diet than its intestinal expression. A
low-cholesterol high-fat diet increases apoA-I expression
in female liver, and this effect is independent of the type of fat.
This particular behavior may be specific to mice, and the response to
different diets may differ among species. Indeed, a hypotrigliceridemic
response to high-fat diets, with or without
cholesterol,10 23 has only been shown in mice.
Our findings with the apoE-knockout mice may, therefore, not be
directly applicable to humans. Nevertheless, it is surprising that
diets rich in polyunsaturated fatty acid from sunflower oil did not
decrease hepatic expression (Figure 4). An enrichment of this
type of oil with oleic acid may be crucial to maintain levels of apoA-I
in plasma in both males and females. This effect was not observed
between olive oil I and II, perhaps because the difference of oleic
acid content between 2 olive oil diets is <10%, whereas the
difference in 2 sunflower oil diets is 43%. It is also possible that
other components of oils, such as sytosterols and polyphenols, may also
have influence. The sources of polyunsaturated (corn oil, sunflower
oil) or monounsaturated fatty acids (olive oil,
canola oil) are not often considered to be important. However, our
data, analyzing apoA-I concentration as well as lesion size, clearly
show that even olive oils from different cultivars are not all the same
in their biological properties. They also suggest that the previous
assumption of similar effects for different oils based on their fatty
acid contents may be too simplistic.
In conclusion, our results in apoE-knockout mice show that a dietary intervention is a useful tool to palliate the consequences of severe genetic backgrounds. The approach, although possibly not as effective as potential gene therapies,37 is more readily adaptable in populations for controlling incidence of atherosclerosis. Our data also show that some fat diets with low cholesterol content could reduce atherosclerotic lesions and help maintain body weight in a gender-specific fashion.
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Acknowledgments |
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This work was supported by grants FISS 95/703, CICYT OLI96-2204, FISS 98/0425, and by the Fundación Mapfre-Medicina. We thank Angel Beltrán, Jesús Cazo, Luis Mógica, Carmen Navarro, and Clara Tapia from Unidad Mixta de Investigación for their invaluable help in maintaining animals. We thank Dr Jesús Rodríguez Chaverría for supplying palm and coconut oils and Dr Manuel Sarasa for helpful hints in histological preparations. The support of María Iturralde and Carmen Trallero is also appreciated. Our thanks to Sandra Kennelly for help in correcting the text.
Received December 21, 1998; accepted February 23, 1999.
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