© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Western-Type Diets Induce Insulin Resistance and Hyperinsulinemia in LDL Receptor-Deficient Mice But Do Not Increase Aortic Atherosclerosis Compared With Normoinsulinemic Mice in Which Similar Plasma Cholesterol Levels Are Achieved by a Fructose-Rich Diet
From the Department of Medicine, University of California San Diego, La Jolla
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Abstract—The role of insulin resistance (IR) in atherogenesis is poorly understood, in part because of a lack of appropriate animal models. We assumed that fructose-fed LDL receptor-deficient (LDLR-/-) mice might be a model of IR and atherosclerosis because (1) fructose feeding induces hyperinsulinemia and IR in rats; (2) a preliminary experiment showed that fructose feeding markedly increases plasma cholesterol levels in LDLR-/- mice; and (3) hypercholesterolemic LDLR-/- mice develop extensive atherosclerosis. To test whether IR could be induced in LDLR-/- mice, 3 groups of male mice were fed a fructose-rich diet (60% of total calories; n=16), a fat-enriched (Western) diet intended to yield the same plasma cholesterol levels (n=18), or regular chow (n=7) for approximately 5.5 months. The average cholesterol levels of both hypercholesterolemic groups were similar (849±268 versus 964±234 mg/dL) and much higher than in the chow-fed group (249±21 mg/dL). Final body weights in the Western diet group were higher (39±6.2 g) than in the fructose- (27.8±2.7 g) or chow-fed (26.7±3.8 g) groups. Contrary to expectation, IR was induced in mice fed the Western diet, but not in fructose-fed mice. The Western diet group had higher average glucose levels (187±16 versus 159±12 mg/dL) and 4.5-fold higher plasma insulin levels. Surprisingly, the non–insulin-resistant, fructose-fed mice had significantly more atherosclerosis than the insulin-resistant mice fed Western diet (11.8±2.9% versus 7.8±2.5% of aortic surface; P<0.01). These results suggest that (1) fructose-enriched diets do not induce IR in LDLR-/- mice; (2) the Western diets commonly used in LDLR-/- mice may not only induce atherosclerosis, but also IR, potentially complicating the interpretation of results; and (3) IR and hyperinsulinemia do not enhance atherosclerosis in LDLR-/- mice, at least under conditions of very high plasma cholesterol levels. The fact that various levels of hypercholesterolemia can be induced in LDLR-/- mice by fat-enriched diets and that such diets induce IR and hyperinsulinemia suggest that LDLR-/- mice may be used as models to elucidate the effect of IR on atherosclerosis, eg, by feeding them Western diets with or without insulin-sensitizing agents.
Key Words: arteriosclerosis • diabetes • fructose • hypercholesterolemia • lipoproteins
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Introduction |
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Individuals with underlying insulin resistance (IR) and resulting impaired glucose tolerance (IGT) and non–insulin-dependent diabetes mellitus (NIDDM) have an increased prevalence of atherosclerosis and increased rates of coronary heart disease (CHD),1 2 3 4 5 but the mechanisms responsible are poorly understood. Hyperglycemia has been hypothesized to enhance atherosclerosis in NIDDM, but the specific contribution of hyperglycemia has been difficult to demonstrate in either population studies or animal models.5 6 7 8 Moreover, hyperglycemia per se is unlikely to play a role in the development of atherosclerosis in individuals with IGT who usually demonstrate only modest postprandial hyperglycemia. Insulin resistance is frequently associated with a number of metabolic abnormalities such as obesity, hypertriglyceridemia, low HDL, and hypertension. These risk factors explain some, but not all, of the increased risk for CHD.9 10 Thus, additional factors associated with IR are likely to contribute to the accelerated development of atherosclerosis. Hyperinsulinemia is frequently present in both IGT and NIDDM, and several lines of evidence suggest that hyperinsulinemia itself may be proatherogenic.11 12 For example, insulin has been shown to increase smooth muscle cell proliferation in vitro11 13 and to enhance accumulation of cholesterol ester in aortas of rats.14 Although several mechanisms have been proposed by which IR or hyperinsulinemia could enhance atherosclerosis, there is little direct evidence for a causal role of IR. This is in part due to the lack of animal models that develop both IR and atherosclerosis.
High-fructose diets have frequently been used to induce a hyperinsulinemic, euglycemic, insulin-resistant condition in rats.15 16 Fructose feeding enhances hepatic secretion of VLDL17 and may decrease its plasma clearance, which frequently results in modest hypercholesterolemia and hypertriglyceridemia.18 19 20 Although fructose-fed rats have been useful for studies of IR, rats (and mice) are generally resistant to diet-induced hypercholesterolemia21 22 and thus do not develop extensive atherosclerosis.
In contrast, the recently developed LDL receptor-deficient (LDLR-/-) mouse appears to be a very good model of atherosclerosis. As in humans with this condition, there is a marked increase in plasma cholesterol levels, particularly when fed Western diets rich in fat and cholesterol. When fed such diets, these mice develop extensive atherosclerosis in the vicinity of the aortic valve (as seen in other atherosclerosis-susceptible strains of mice), as well as in the arch, thoracic, and abdominal regions of the aorta.7 23 24 25 26 These aortic plaques show many morphological similarities to human atherosclerotic lesions and consist of both fatty streaks and more advanced lesions.7 25
Given the close phylogenetic relationship between the mouse and the rat, we presumed that a fructose-rich diet would also lead to IR in mice. Furthermore, we presumed that a fructose-rich diet would result in an increased production of VLDL in LDLR-/- mice (as in rats) and, because of the reduced clearance of lipoproteins caused by the lack of LDL receptors, to elevated plasma cholesterol levels, which could be matched in control LDLR-/- mice by feeding them a standard Western diet. Thus, we hypothesized that it should be possible to study the effect of IR on atherosclerosis in mice with similar plasma cholesterol levels. Because diets with a very high fat content can induce IR in rats27 28 29 30 and some strains of mice,31 32 it was conceivable that a Western diet moderately rich in fat could also induce some degree of IR in the control mice. Even though the potential occurrence of IR in LDLR-/- mice fed a Western diet could complicate the interpretation of this study, we thought such an observation would be of importance because of the common use of Western diets in murine atherosclerosis studies.
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Methods |
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Animals and Diets
A breeding colony was generated from homozygous LDLR-/- mice (C57BL/6x129 SV background mice; fifth generation bred back into C57BL/6 mice) obtained from Jackson Laboratories (Bar Harbor, ME). Two groups of male LDLR-/- mice were matched for age (2 to 4 months), plasma cholesterol levels, and litter. One group (n=16) was fed a fructose-enriched diet (TD 96130, Harlan-Teklad; 13% of calories from fat, 67% from carbohydrates, 20% from protein) for approximately 5.5 months. A preliminary experiment on 4 first-generation LDLR-/- mice (C57BL/6x129 SV background) had shown that this diet resulted in a marked elevation of plasma cholesterol levels. To achieve similar overall cholesterol exposure in both diet groups, the second group (n=18) was fed a Western-type diet (TD 96125, Harlan-Teklad; 42% of calories from fat, 43% from carbohydrates, 15% from protein), starting 1 month later. The plasma cholesterol levels achieved in the fructose group were used to select the Western diet fed to the second group to achieve similar plasma cholesterol levels. The composition of the diets is listed in Table 1
. In addition, a third group (n=7) of
age-matched mice was fed regular murine diet (TD 8604, Harlan-Teklad)
throughout the study period to provide a basis for comparison for the
plasma lipid and insulin levels achieved in the Western and fructose
diet groups. Monthly blood samples for determination of plasma glucose,
cholesterol, and triglyceride levels were
obtained by drawing blood from the retroorbital plexus of
anesthetized mice into heparinized capillary tubes. All animals
were fasted for 1 hour before drawing blood, which was
consistently performed at the same time of day (9
AM).
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In addition, to confirm the early onset of IR, 3 groups of 8 to 9 mice each were fed the same diets for 6 weeks. In these mice, blood glucose and plasma insulin levels were determined at baseline and after 2 and 4 weeks on the diet, and an oral glucose tolerance test (see below) was performed on a subset of mice after 6 weeks.
Glucose, Insulin, Lipid Levels, and Lipoprotein Profiles
Glucose levels were determined in whole blood using a One Touch
glucometer (Lifescan Inc). Plasma insulin was measured in 1-hour
fasting blood samples obtained at 9 AM by competitive
radioimmunoassay (Linco Research Laboratories). Plasma
cholesterol and triglyceride levels were
measured by enzymatic methods using an automated bichromatic
analyzer (Abbott Diagnostics). The
cholesterol content of lipoprotein fractions in the plasma
was determined by fast-performance liquid
chromatography (FPLC). One hundred microliters of each
mouse plasma sample was added to a Superose 6B column (0.7x50
cm), and 250-µL sample fractions were collected for
cholesterol analysis. Mouse lipoproteins isolated
by density gradient ultracentrifugation were run as
standards to facilitate lipoprotein peak identification.
Oral Glucose Tolerance Test
An oral glucose tolerance test (OGTT) was carried out on the
additional set of mice fed fructose, Western diet, or regular chow,
after 6 weeks on the diets. A review of the literature revealed a wide
range of "fasting" periods for rats and mice, from no fasting to up
to 12 hours of fasting.19 31 32 33 34 35 In view of the faster
metabolism of mice, we opted for a 4-hour fast. Zero time
(baseline) blood samples were obtained in heparinized capillary tubes
between 12 and 1 PM by tail bleed of restrained (but not
anesthetized) mice. A glucose load of 1.5 g/kg body weight was
then administered by oral gavage to mice that were manually restrained
for this purpose, using a 20% glucose solution. Tail vein blood
samples (25 µL) were obtained at 15, 30, and 60 minutes after the
glucose load. Blood glucose levels were determined immediately by the
method described above. Plasma was then prepared from the blood by
centrifugation (3000g for 15 minutes) and
stored at -70°C for subsequent insulin determination.
Morphometric Determination of Atherosclerosis
At the end of the study, mice were euthanized by an overdose of
anesthesia and the aorta was perfused with
phosphate-buffered saline containing 20 µmol/L butylated
hydroxytoluene and 2 mmol/L EDTA, pH 7.4. The aorta was then
dissected, opened longitudinally, and stained with Sudan
IV.36 Quantification of atherosclerosis
was performed by computer-assisted image analysis, as
previously described in detail.24 The extent of
atherosclerosis was expressed as percent of total
aortic surface area covered by Sudan-positive lesions.
Tissue Preparation and Immunocytochemistry
After determining the extent of atherosclerosis,
7-mm-long segments of the opened aorta containing prominent lesions
were paraffin-embedded, and 8-µm-thick serial sections were prepared
for immunocytochemical evaluation of advanced glycation end
products (AGE) and "oxidation-specific" epitopes. Sections were
rehydrated and immunostained using an
avidin-biotin-alkaline phosphatase system (Vector Labs), as previously
described.7 25 36 The following antibodies were used:
FLI-2 (1:700 dilution), an antiserum prepared by immunization of guinea
pigs with furoyl-furanyl-imidazole (FFI)-conjugated homologous LDL,
which recognizes FFI-lysine epitopes on FFI-LDL and other FFI-protein
adducts37 ; GPA-1 (1:700 dilution), a guinea-pig antiserum
generated with AGE-modified guinea pig albumin37 ;
MAL-2 (1:500 dilution), an oxidation-specific guinea pig antiserum to
MDA-lysine epitopes present on Ox-LDL38 39 ; and goat
anti-CD106 (vascular cell adhesion molecule-1 [VCAM-1]; 1:50
dilution; Research Diagnostics). Control slides were
incubated without primary antibody and were devoid of any specific
staining.
Immunocytochemistry using primary and secondary antibodies and avidin-biotin-enzyme complexes yields only semiquantitative results. To detect differences in the presence of AGE- and oxidation-specific epitopes, as well as VCAM-1, in lesions by comparative immunocytochemistry, staining of all sections with the same antibody was performed in a single assay, using rigidly controlled conditions (ie, reagent volumes, incubation times), and alternating slides from different groups. Furthermore, only large lesions of similar stage and composition from the 2 hypercholesterolemic groups were used for comparison. Results were quantified by the same investigator, using a scoring system for the different staining intensities.7 The scores were defined as 0=sections devoid of staining, 2=mild staining, 4=strong staining, 6=very strong staining (as well as 1, 3, and 5 for intermediate staining intensities). All sections were read twice, alternating between sections from animals in the fructose-fed and Western diet groups. A total of 30 sections per group were evaluated for each of the 3 antibodies.
Statistics
Groups were compared by ANOVA and unpaired t tests,
and correlations between variables were made by Pearson's
coefficient of correlation. All data shown are mean±SD. A
P<0.05 was defined as significant.
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Results |
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During the study period, 1 of the mice in the Western diet group died. All other animals were healthy with normal coat conditions. The weights of animals in all groups increased during the study period (Table 2
). However, this increase was
only significant in the Western diet group, perhaps due in part to the
greater caloric density of the Western diet (4.78 versus 3.88
cal/g).
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Extensive hypercholesterolemia developed in
both the fructose and Western diet groups (Table 2
and Figure 1). The average plasma
cholesterol and triglyceride levels throughout
the study in the fructose-fed and Western diet groups were not
significantly different from each other, but were significantly higher
than those in the chow-fed group (Table 2). Figure 1
shows the plasma cholesterol levels of the Western and
fructose diet groups throughout the intervention period. We had
attempted to match the plasma cholesterol levels in these 2
groups by selecting a Western diet not supplemented with additional
cholesterol. Nevertheless, the Western diet group
consistently had slightly higher plasma cholesterol
levels throughout the study. The differences between the 2 groups were
not significant at any given time point. However, the overall
cholesterol exposure, defined as the area under the curve
for cholesterol versus time, was significantly greater in
the Western than in the fructose diet group (184 013 versus 146 394
days · mg/dL respectively; P<0.05), even though we
had slightly shortened the feeding period in the Western diet group
(from 169 days for the fructose-fed to 158 days for the Western diet
group). Cholesterol distribution among plasma lipoproteins
was determined in individual samples (from 8 mice in the fructose-fed
and 5 mice in the Western diet group; Figure 2 and Table 3). The percent of total plasma
cholesterol carried in the VLDL fraction was higher in the
fructose-fed than in the Western diet group (20.8±5.4% versus
11.87±6.7%; P<0.05). There was no significant difference
in the percent of plasma cholesterol carried in the LDL
fraction between both groups. Typical cholesterol
distribution in the plasma of chow-fed LDLR-/-
mice was 65% in the LDL fraction and 35% in the HDL fraction. Plasma
triglyceride levels in both
hypercholesterolemic groups were significantly higher
than those in the chow-fed group (Table 2).
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Blood glucose levels (after 1-hour fasting) were slightly higher in the
Western diet group than in the fructose group (187±16 versus 159±12
mg/dL; P<0.01), but were not significantly different from
those of the chow-fed group (Table 2). Plasma insulin levels
were measured after 4 months, using pooled plasma samples (equal
aliquots of plasma from each animals) and in individual samples from
all mice at the end of the study. At the 4-month point, the plasma
insulin level in the Western diet group (1.23 ng/mL) was 2.5 times that
in the fructose- and chow-fed groups (0.49 and 0.46 ng/mL,
respectively). At the end of the study, the average plasma insulin
level in the Western diet group was more than 4.5 times that in the
fructose- or chow-fed groups (Table 2). This combination of
modestly higher glucose and markedly higher insulin levels is
indicative of IR. Thus, mice in the Western diet group developed IR and
hyperinsulinemia, whereas mice in the fructose
group did not. We also observed a strong correlation between the final
plasma insulin levels and the final body weight of animals, when data
of both groups were analyzed together (r=0.88;
P<0.01). This suggests that the development of IR in the
Western diet group may have resulted at least in part from the
substantially greater weight gain that occurred in this group.
At the end of the intervention period, the extent of
atherosclerosis in the aortic tree of the 2
hypercholesterolemic groups was evaluated by
computer-assisted image analysis. Atherosclerotic lesions were
found in the aortas of all hypercholesterolemic
animals. Lesions were similar in distribution and occurred at typical
predilection sites for lesions in LDLR-/- mice
exposed to prolonged
hypercholesterolemia.7 25 However,
the extent of atherosclerosis was significantly greater
in the fructose-fed group (11.84±2.86% of the aortic surface area)
than in the Western diet group (7.76±2.54%; P<0.01)
(Figure 3). One animal in the Western
diet group was excluded from the study, because the extent of
atherosclerosis in this animal (21.63%) was more than
3 SD greater than the group average. However, even when the lesion area
from this animal was included, the fructose-fed group still had a
significantly greater extent of atherosclerosis than
the Western diet group (P<0.05). As expected, chow-fed mice
did not develop measurable atherosclerosis in the
aorta.
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These results suggest that in LDLR-/- mice, IR
and hyperinsulinemia do not enhance
atherosclerosis, at least in the presence of very high
plasma cholesterol levels. This conclusion is based on the
assumption that significant IR was present in mice fed the Western
diet throughout the study. Although we had clearly established IR was
present after 4 months and at the end of the study, we needed to
demonstrate the early onset of IR to document the validity of this
assumption. The development of IR was therefore examined in a separate
set of mice of the same strain and age as the animals used in the
original experiment. These additional mice were fed the same Western
diet (n=8), fructose diet (n=9), and regular chow (n=8) as the mice in
the original study. Glucose and insulin levels were determined at
baseline and after 2 and 4 weeks (Table 4). Glucose levels were measured in each
individual animal, whereas insulin levels were measured in 1 or 2
pooled plasma samples from each group (with each pool containing an
equal volume of plasma from each animal). Compared with baseline,
glucose levels remained stable 1 month into the feeding period and were
not significantly different between the 3 diet groups. In contrast,
insulin levels in the Western diet group were already slightly elevated
at 2 weeks, compared with the levels in the chow- or fructose-fed
groups. After 4 weeks, insulin levels in the Western diet group were
more than 4 times higher than those of the other 2 groups (Table 4).
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An OGTT was performed 6 weeks after the start of the diet on mice
from these additional experimental groups. Figure 4 shows the blood glucose and plasma
insulin levels of 3 animals from each diet group during the OGTT. The
mice on Western diet had higher average fasting blood glucose and
insulin levels at baseline (before administration of the glucose load)
and at 15, 30, and 60 minutes after the glucose load than mice in the
other 2 groups. However, there was considerable variability in insulin
levels during the OGTT within the Western diet group.
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To ensure that the differences in plasma insulin levels between groups persisted throughout the day, we determined insulin levels on these additional groups of chow-, fructose-, and fat-fed mice during a 10-hour period, starting at 9 AM. The results showed marked hyperinsulinemia in the Western diet group at all points (0, 5, and 10 hours), compared with the fructose and chow groups (data not shown). Although elevations in fasting and post–glucose-challenge plasma insulin levels provide only an indirect measure of IR, these results taken together demonstrate rather convincingly that LDLR-/- mice fed a Western diet develop IR and do so within a short time.
We also investigated whether differences in AGE formation occurred in
the artery wall, which could have influenced
atherogenesis.40 41 42 43 We therefore performed a
semiquantitative determination of AGE epitopes in atherosclerotic
lesions. We also compared the presence of oxidation-specific epitopes,
because extensive in vitro and in vivo evidence suggests that lipid
peroxidation and glycation are mutually enhancing.37 41 44
However, when the intensity of the immunostaining with
antibodies to AGE- (FLI-2 and GPA-1) and oxidation-specific epitopes
(MAL-2) in atherosclerotic lesions of similar size was assessed by the
scoring system described in Methods, no significant differences between
the fructose and Western diet groups was detected (Table 5). This suggests that the extent of AGE
formation and oxidation was similar in both groups. Normal tissue (ie,
areas in the aorta without atherosclerotic lesions) showed minimal, if
any, staining. Finally, we immunostained for VCAM-1, an
adhesion molecule hypothesized to play important role in monocyte
recruitment into the intima.45 No significant differences
in VCAM-1 staining were seen between similar-size lesions from the
Western diet and fructose groups (Table 5).
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A potential mechanism by which fructose feeding could enhance
atherosclerosis is by inducing a shift in lipoprotein
composition that may be atherogenic. Indeed, our observation that the
VLDL cholesterol was increased in the fructose-fed group
(Table 3, Figure 2) supports this notion. The extent of
atherosclerosis correlated well with VLDL
cholesterol (r=0.68; P<0.01) when
data from both hypercholesterolemic groups were
analyzed together (Figure 5).
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Discussion |
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The aim of this study was to determine whether IR could be induced in LDLR-/- mice and to find out whether these mice are a suitable model to investigate whether IR enhances atherosclerosis. Our study yielded 3 main results. The first of these was the unexpected finding that a diet rich in fructose that consistently causes IR in rats resulted in marked hypercholesterolemia but failed to induce IR in LDLR-/- mice. The second result was the observation of marked and early onset IR in mice fed a standard, moderately high-fat, Western diet (42% of calories as fat). Diets with much higher fat content (55% to 65% of calories) have been used previously to induce IR in other animal models,27 28 29 30 46 and Surwit et al31 32 have shown that high-fat diets also induce IR in mice. Our study documents that Western-type diets also cause IR in LDLR-/- mice. This is important because it raises the possibility that IR may have occurred in many previous studies on atherogenesis in murine models that used similar diets to induce extensive lesion formation.7 23 24 25 26 47 Because in these studies IR may have influenced lipoprotein profiles, coagulation, blood pressure, and other atherogenic factors, interpretation of results may be more complex than previously assumed. Finally, the fact that the insulin-resistant, hyperinsulinemic mice fed a Western diet had equal or slightly higher plasma cholesterol levels but significantly less aortic atherosclerosis than fructose-fed normoinsulinemic mice would appear to suggest that IR does not enhance atherogenesis in LDLR-/- mice, at least under conditions of extensive hypercholesterolemia.
Mice fed the Western diet showed a 61% increase in body weight at the end of the study. Much of this weight gain is likely to be in the form of fat. Indeed, in rats it has been demonstrated that high-fat diets increase body weight mainly by increasing body fat.46 Extensive clinical and epidemiological evidence supports the correlation between body fat and IR in humans.48 49 50 51 52 Similar observations have also been made in animal models of obesity.53 54 It is therefore tempting to assume that IR in LDLR-/- mice was caused in part by an increase in body fat. The notion that in our study increased body fat was an important contributor to IR is also supported by the strong correlation between body weight and plasma insulin levels at the end of the intervention period.
The reasons why a diet rich in fructose did not yield significant IR in LDLR-/- mice are not clear. It is quite possible that the effects of fructose metabolism in LDLR-/- mice are different from those in rats. Although it is likely that fructose feeding results in increased hepatic synthesis and secretion of VLDL in mice, as has been shown in rats, we saw marked hypercholesterolemia only in LDLR-/- mice. Presumably, the very high level of hypercholesterolemia induced by the fructose-rich diet is mainly caused by the inability of the LDLR-/- mouse to clear VLDL and LDL from the plasma via the LDL receptor. However, we cannot rule out that there are other effects of fructose metabolism in the liver or peripheral tissues unique to rats that affect the sensitivity to insulin. Another potential explanation for the failure of fructose to induce IR could be that the fructose diet group did not gain nearly as much weight as did the Western diet group. Another long-term feeding study also reported that high fructose diets were associated with reduced weight gain, relative to control diets.55 This could in part be caused by a fructose-induced increase in thermogenesis or a higher metabolic cost of fat synthesis and storage from dietary carbohydrates compared with dietary fat.56 Finally, it is possible that minor genetic variability within the LDLR-/- strain may influence their susceptibility to develop some degree of IR on a fructose-rich diet. There are numerous examples of variations in the susceptibility to develop conditions such as IR, diabetes, and atherosclerosis resulting from differences in genetic background.8 57 58 59 60 Although we consistently failed to observe increased plasma insulin levels in the present study, a preliminary experiment on 4 LDLR-/- mice with the same C57BL/6 micex129 SV background that had not been back-bred into the C57BL/6 background yielded elevated insulin levels in some mice, compared with chow-fed controls (data not shown).
The association of diabetes and IR with atherosclerosis and CHD is well documented in humans.1 2 3 11 12 Therefore, it appears paradoxical that fat-fed, insulin-resistant, hyperinsulinemic mice developed less extensive atherosclerosis than their fructose-fed, normoinsulinemic counterparts, even though the latter had similar or even slightly lower plasma cholesterol levels throughout the study. Although we cannot rule out that IR may have altogether different effects on atherosclerosis in LDLR-/- mice than in humans, several other explanations exist that may reconcile this apparent contradiction.
The first of these is that the fructose-rich diet may have had atherogenic effects independent of hypercholesterolemia. In rats, fructose-enriched diets not only induce IR, but have also been reported to increase blood pressure and adherence of leukocytes to the artery wall.35 61 The latter was observed in the absence of significant elevations in plasma cholesterol and triglycerides.35 A diet-induced difference in blood pressure could have affected atherogenesis, particularly in association with hypercholesterolemia. To investigate this possibility, we subsequently determined blood pressure by the tail cuff method in mice fed the same diets for 10 months and indeed saw a slightly but significantly higher blood pressure in the fructose group than in the Western diet group. However, the absolute blood pressures of both groups were in the normal range published for mice, and the blood pressure of the fructose group was not significantly higher than that of the chow-fed control (data not shown). Therefore, the potential impact of diet-induced blood pressure changes on atherosclerosis will require further investigation.
We also found a slightly higher percentage of cholesterol in the VLDL fraction of the fructose-fed group compared with the Western diet group. Furthermore, there was a good correlation between the amount of cholesterol carried in VLDL and the extent of atherosclerosis in our study. Thus, we cannot rule out that a combination of decreased VLDL clearance19 20 and increased VLDL cholesterol was responsible for the accelerated atherosclerosis in the fructose group. Finally, a fructose-enriched diet might have increased AGE formation, which could have enhanced atherogenesis. Increased AGE formation has been described in many tissues of diabetic subjects, including the arterial wall, and AGE formation in the aorta is enhanced by both hyperglycemia and hyperlipidemia.40 41 42 43 44 However, the finding of similar intensity of immunostaining for AGE- and oxidation-specific epitopes in both groups indicated that this mechanism could not have been responsible for the differences in atherogenesis. Similarly, no evidence for different VCAM-1 expression between the 2 hypercholesterolemic groups was found.
A second explanation for the apparent lack of an atherogenic effect of IR would be that IR and hyperinsulinemia do not directly enhance lesion formation, but that the specific shifts in the lipoprotein profile (such as lower HDL or higher triglyceride concentrations) or an elevated blood pressure that are frequently found in insulin-resistant humans3 49 50 but were lacking in LDLR-/- mice are necessary for an accelerated atherogenesis. Indeed, some, but not all, multivariate analyses of human cohort studies indicated that after adjusting for other cardiovascular risk factors, insulin levels were not an independent predictor of CHD.62 63 64
Finally, it is conceivable that any effects of IR on atherosclerosis may have been dwarfed by the more potent atherogenic effect of high plasma cholesterol levels. In the present study, the extreme level of hypercholesterolemia in the Western diet group was dictated by the need to match levels achieved in the fructose group. To avoid such overriding effects of hypercholesterolemia, future studies on the role of IR in atherogenesis should be performed at lower cholesterol levels. A potential approach suggested by the present study would be to induce IR in LDLR-/- mice by feeding them a diet with a more moderate fat content, and to compare them with a control group receiving the same diet, but supplemented with insulin-sensitizing agents65 66 to reduce IR.
In conclusion, the present study demonstrates that Western diets commonly used to induce atherosclerosis in murine models also induce IR and hyperinsulinemia, potentially complicating the interpretation of previous atherosclerosis studies in mice. Our results also raise the possibility that IR and hyperinsulinemia may not enhance atherogenesis in the absence of metabolic changes commonly associated with IR in humans. The fat-fed LDLR-/- mouse may be a suitable model to investigate whether this is indeed the case.
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Acknowledgments |
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The authors thank Drs J.L. Witztum and G.M. Reaven for valuable discussions and Dr Y. Chen for assistance in determining plasma insulin levels. We also thank Jennifer Pattison, Joe Juliano, and Richard Elam for excellent technical assistance. These studies were supported by NHLBI grants HL14197 and HL56989 (La Jolla Specialized Center of Research in Molecular Medicine and Atherosclerosis; W.P., P.D.R.), 2 grants from the Whittier Institute for Diabetes (W.P., P.D.R.), and by a grant from the American Federation of Aging Research (P.D.R).
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Footnotes |
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Address correspondence to Wulf Palinski, MD, University of California, San Diego, Department of Medicine, 0682, 9500 Gilman Dr, BSB 1080, La Jolla, CA 92093-0682.
Received April 14, 1998; accepted August 31, 1998.
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