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【摘要】
Objective- Western-type high-fat/high-cholesterol diets used to induce atherogenesis in low-density lipoprotein (LDL) receptor-deficient mice also lead to obesity with concomitant metabolic complications, eg, hypertriglyceridemia, hyperinsulinemia, and insulin resistance. Our aim was to design a diet inducing atherosclerosis through moderate hypercholesterolemia without associated parameters of the metabolic syndrome.
Methods and Results- Male LDL receptor-deficient mice were fed regular chow (RC; 0.01% cholesterol/4.4% fat), cholesterol-enriched regular chow (HC; 1% cholesterol/4.4% fat), or Western diet (WD 0.06% cholesterol/21% milk fat) for 28 weeks. HC-feeding led to elevated plasma ( 20.7 mmol/L [800 mg/dL]) and LDL cholesterol and accelerated atherosclerosis. Plasma triglycerides were unaffected. Compared with RC-fed controls, HC-fed mice had normal body weight gain and normal fasting levels of glucose, free fatty acids, and insulin. In contrast, WD-fed mice were extremely hypercholesterolemic 41.4 mmol/L), obese, hypertriglyceridemic, hyperinsulinemic, insulin resistant, and showed adverse health such as skin/fur abnormalities and hepatic steatosis. Although atherosclerotic surface areas in the entire aorta were similar in HC-fed and WD-fed mice, lesions in aortic origin cross sections were significantly larger in WD-fed mice. However, morphology was similar in lesions of equal size.
Conclusions- The HC diet induced moderate hypercholesterolemia and extensive atherosclerosis and should be useful to study specific aspects of atherogenesis in the absence of confounding effects of the metabolic syndrome.
High-fat/high-cholesterol diets commonly used to induce atherogenesis in LDLR -/- mice also induce the metabolic syndrome. This study demonstrates that cholesterol enrichment of regular chow provides a simple atherogenic diet that moderately elevates LDL cholesterol generating a human-like lipoprotein profile and avoids confounding effects of the metabolic syndrome.
【关键词】 atherosclerosis insulin resistance lipoproteins metabolic syndrome mouse models
Introduction
Atherosclerosis is a complex disorder that displays many of the characteristics of a chronic inflammatory process. 1 To accelerate atherogenesis in mice, plasma cholesterol levels must be raised extensively. 2 With extreme degrees of hypercholesterolemia, multiple pathways that can independently sustain lesion formation appear to be activated. 3 In this setting, for instance induced by the administration of a so-called Western diet (WD), the extreme hypercholesterolemia and associated features of the metabolic syndrome such as obesity, hypertriglyceridemia, and hyperinsulinemia can overcome the modulating impact of immune functions that can be often demonstrated at more moderate levels of hypercholesterolemia. 4-7 Thus, a diet that increases low-density lipoprotein (LDL) cholesterol levels modestly, without associated parameters of the metabolic syndrome, would be preferable for many studies of atherogenesis.
The LDL receptor-deficient (LDLR -/- ) mouse on the C57BL/6 background fed a high-fat/high-cholesterol WD is a commonly used murine model of atherosclerosis. 2 Young LDLR -/- mice on regular chow (RC) have twice the plasma cholesterol levels of C57BL/6 mice, ie, 5.2 mmol/L (200 mg/dL), 8 which slowly increases to 9.0 mmol/L (350 mg/dL) with age. 9,10 The increased plasma cholesterol is found primarily in atherogenic LDL with only small amounts in very low-density lipoprotein (VLDL). 8 This lipoprotein profile is more comparable to the human profile, for instance, than that of RC-fed apolipoprotein E-deficient (ApoE -/- ) mice, which have elevated cholesterol-enriched VLDL-like particles as the major lipoprotein. 11
Because RC-fed LDLR -/- mice develop little atherosclerosis, 2,11 various high-fat/high-cholesterol WDs have been used. However, they not only induce extreme hypercholesterolemia but also lead to marked weight gain with concomitant components of the metabolic syndrome, eg, hypertriglyceridemia caused by elevated VLDL levels, decreased high-density lipoprotein (HDL) levels, hyperinsulinemia, and insulin resistance. 12-15 In addition, the WD generates pathologies independent of atherosclerosis, eg, changes in fur and skin integrity that lead to ulceration and infection and decreases in body weight and plasma lipids, caused in part by hepatic steatosis 16 and gastritis. 17 The associated infections and metabolic disturbances may independently impact atherogenesis and complicate interpretation of atherosclerosis data. Thus, alternative diets that produce atherosclerosis without these confounding effects would be helpful.
For these reasons, we developed a simple cholesterol-enriched regular chow diet (high-cholesterol diet, 1% cholesterol plus 4.4% fat) that induces more moderate hypercholesterolemia ( 20.7 mmol/L [800 mg/dL]), predominately increases LDL cholesterol, and leads to atherosclerosis without concomitant obesity and other health and metabolic consequences.
Methods
Animals and Diets
All mice were tenth-generation male LDLR -/- mice on the C57BL/6J background. Mice were weaned at 21 days of age and fed RC consisting of 0.01% cholesterol and 4.4% fat (TD8604; Harlan-Teklad, Madison, Wis) until initiation of the diet interventions. In 3 separate studies, groups of mice were matched for age, litter, body weight, and plasma cholesterol and triglycerides, and housed in cages equipped with rodent enrichments (igloo and gnawing bone; Bio-Serv, Frenchtown, NJ) in a facility with a 12-hour light cycle. Animals had ad libitum access to water and food. All animal experiments were performed according to NIH guidelines and were approved by the University of California, San Diego Animal Subjects Committee.
In addition to the RC diet, 2 experimental diets were used. One diet was a widely used WD consisting of 0.06% cholesterol and 21% milk fat (TD98338; Harlan-Teklad). The second diet consisted of regular chow that was only enriched with cholesterol and designated as HC diet. This HC diet was prepared as follows: 1.0% (wt/wt chow) cholesterol (Sigma-Aldrich, St. Louis, Mo) was dissolved in diethyl ether and sprayed over RC pellets in a fume hood. The diet was stored at -20°C after the diethyl ether had evaporated. The HC diet was changed twice per week, whereas the milk fat-containing WD was changed 3 times per week because of the visual discoloration (white to yellow), suggestive of accelerated lipid peroxidation. The compositions of the diets are listed in Table I (see http://atvb.ahajournals.org).
Intervention Design
Study 1 included 2 age groups of LDLR -/- mice, designated "younger" (age 14.1±0.2 weeks; n=23) and "older" (age 44.4±0.2 weeks; n=36), and each was divided into 3 diet intervention groups and fed either RC, HC diet, or WD for 28 weeks. Mice were bled after 0 and 14 weeks, subjected to an oral glucose tolerance test (OGTT) after 19 to 22 weeks, and euthanized after 28 weeks of feeding for atherosclerosis analysis.
Study 2 included LDLR -/- mice (age 16.5±0.2 weeks; n=44) that were divided into 5 diet intervention groups and fed commercial pelleted custom-made diets (Harlan-Teklad) that had been enriched with 0.00% (TD8604; n=12), 0.15% (TD04409; n=8), 0.25% (TD99260; n=8), 0.50% (TD97234; n=8), and 1.00% (TD97131; n=8) cholesterol, using the TD8604 RC as the base. Plasma was obtained after 0 and 4 weeks of cholesterol feeding and analyzed for lipid levels.
Study 3 included LDLR -/- mice (age 34.1±0.4 weeks; n=25) that were fed the HC diet. Mice were bled after 0, 4, and 15 weeks of HC feeding to monitor lipid levels and euthanized in groups of 4 to 5 mice after 8, 12, 18, 24, 28, and 40 weeks of HC feeding for atherosclerosis analysis.
Atherosclerosis, Hepatic, and Plasma Analyses
Atherosclerosis lesion areas were measured in cross-sections of the aortic origin and as surface area in the en face preparation of the entire aorta. Trichrome-stained cross-sections were used to assess lesion morphology. Plasma and blood collected after a 3-hour fast or an OGTT were analyzed for levels of cholesterol, triglycerides, glucose, insulin, and free fatty acids. Cholesterol and triglyceride distribution in plasma lipoproteins separated by fast protein liquid chromatography (FPLC) were determined. Hepatic lipid content was measured. All experimental data are expressed as mean±SEM. See http://atvb.ahajournals.org for full description of methods and statistics.
Results
Impact of Diets on Body and Organ Weights and Hepatic Lipid Content
The consequences of HC feeding were compared with those of RC and WD feeding in both younger and older male LDLR -/- mice (starting age 14 and 44 weeks, respectively). Figure 1 shows the body weight gain over 28 weeks of RC, HC, and WD feeding of the younger and older LDLR -/- mice. Independently of age, HC-fed mice gained weight equal to RC-fed mice, whereas WD-fed mice gained 25% to 35% more body weight ( P <0.01; Figure 1 ). All HC-fed and RC-fed mice appeared healthy and active throughout the 28-week intervention period (even though the "older" mice were 17 months old). Some WD-fed mice exhibited signs of adverse health, including a sudden decrease in plasma lipid levels and 10 grams over 7 days), and/or abnormal changes in fur and skin integrity (including ulcerations). In fact, 3 of the 12 older WD-fed mice had to be euthanized before 12 weeks of feeding and a fourth mouse after 18 weeks. All other mice were euthanized after 28 weeks of feeding and hearts, spleens, livers, and pancreases were weighed (supplemental Figure IA to ID). No statistical differences in organ weights were observed between the HC-fed and RC-fed mice, whereas WD-fed mice, independently of age, had 45% to 65% heavier livers ( P <0.01). Livers from HC-fed and RC-fed mice appeared normal, in contrast to the livers from WD-fed mice, which appeared yellow with patchy coloration indicative of hepatic steatosis. 16 In addition, the older WD-fed mice also had heavier spleens than older HC-fed and RC-fed mice ( P <0.05).
Figure 1. HC-fed LDLR -/- mice have normal weight gain compared with RC-fed mice. WD-fed mice have significantly higher body weights than age-matched RC-fed and HC-fed mice throughout the study ( P <0.01). Decreases in body weight (18 to 22 weeks) are attributable to OGTTs. Data are expressed as mean±SEM.
Livers were analyzed for cholesterol and triglyceride content. Compared with RC-fed and HC-fed mice, WD-fed mice were found to have highly significant and massive hepatic accumulation of both free and esterified cholesterol as well as triglycerides (supplemental Figure IIA, IIB). In fact, the levels of lipid accumulation are comparable to that seen in leptin-deficient C57BL/6 mice 18 consistent with hepatic steatosis. In contrast, HC-fed mice had only mild, although significant, elevations of hepatic free and esterified cholesterol as well as triglycerides compared with RC-fed mice.
Impact of Diets on Plasma Lipid and Lipoprotein Levels
Figure 2 shows 3-hour fasting plasma lipid levels and lipoprotein distributions in younger and older LDLR -/- mice after RC, HC, and WD feeding. Independently of age, HC-fed mice had significantly elevated plasma cholesterol compared with RC-fed mice ( 20.7 versus 7.8 mmol/L [800 versus 300 mg/dL]; Figure 2A ) without concomitant changes in plasma triglyceride levels ( 2.0 mmol/L [175 mg/dL]; Figure 2 B). This is in marked contrast to the effect of the WD, which induced extreme hypercholesterolemia 6.8 mmol/L [600 mg/dL]). Cholesterol and triglyceride lipoprotein distribution in male mice ( Figure 2C, 2 D, respectively) by FPLC after 28 weeks of feeding showed that the HC diet induced an almost exclusive elevation in LDL cholesterol with minimal changes in HDL levels and only trace increases in VLDL levels. The same lipoprotein profile was also observed in HC-fed male LDLR -/- mice after 14, 40, and 80 weeks of feeding. Together, this shows a reproducible cholesterol distribution in LDL, HDL, and VLDL of 83%, 14%, and 3%, respectively. In contrast, WD-fed mice had markedly different lipid profiles, as both LDL and VLDL particles were greatly enriched in cholesterol and triglyceride ( Figure 2C, 2 D), suggesting different phenotypes and perhaps atherogenicity. The lipoprotein profiles were confirmed by agarose gel electrophoresis (supplemental Figure III). Female LDLR -/- mice fed the same HC-diet had plasma cholesterol and triglyceride levels of 18.1 mmol/L (700 mg/dL) and 0.85 mmol/L (75 mg/dL), respectively, and a lipoprotein cholesterol distribution similar to HC-fed male mice (data not shown).
Figure 2. HC-fed LDLR -/- mice have isolated elevation of LDL cholesterol. Fasting plasma cholesterol (A) and triglyceride (B) levels in younger and older RC-fed, HC-fed, and WD-fed LDLR -/- mice. Representative cholesterol (C) and triglyceride (D) profiles of fast protein liquid chromatography separated lipoproteins from intra-grouped plasma of 28-week RC-fed, HC-fed, and WD-fed younger mice. Statistically significant differences between age-matched HC-fed mice and RC-fed or WD-fed mice at the same time point are indicated with ** P <0.01 and *** P <0.001. Data are given as mean±SEM.
To investigate if the increase in plasma cholesterol levels could be titrated according to the content of dietary cholesterol, we fed male LDLR -/- mice for 4 weeks with commercial custom-made TD8604 RC-based diets that had been enriched with 0.00% (RC), 0.15%, 0.25%, 0.50%, and 1.00% cholesterol (study 2). Figure 3 shows a dose-dependent plasma cholesterol response to the dietary cholesterol resulting in 1.1-, 1.8-, 2.5-, 2.9-, and 3.8-fold increase in plasma cholesterol levels, respectively. These increases in plasma cholesterol were significantly different for the cholesterol-enriched groups (all P <0.001), but not for the RC-fed mice. After 4 weeks of feeding, all 5 diet groups had significantly different mean plasma cholesterol levels of 8.2, 11.7, 17.8, 20.7, and 25.9 mmol/L (317, 453, 687, 800, and 1003 mg/dL, respectively). Body weights and plasma triglycerides were unaffected by the cholesterol feeding (data not shown).
Figure 3. Titration of plasma cholesterol levels in male LDLR -/- mice fed 0% (RC), 0.15%, 0.25%, 0.5%, and 1.0% cholesterol-enriched commercial pelleted custom-made regular chow (study 2). All cholesterol-enriched diet groups had significantly higher plasma cholesterol after 4 weeks of feeding ( $$$ P <0.001). Statistically significant differences between diet groups at the same time point are indicated with * P <0.05 and *** P <0.001. Data are expressed as mean±SEM.
Impact of Diets on Glucose and Insulin Metabolism
To assess the effect of these diets on nonlipid metabolic parameters, blood was collected at 0, 14, and 20 weeks of feeding after a 3-hour fast. HC-fed and RC-fed mice had nearly identical fasting levels of glucose ( Figure 4A, 4 B), insulin ( Figure 4C, 4 D), and unbound free fatty acid (supplemental Figure IV), as well as normal insulin-to-glucose ratios ( Figure 4E, 4 F) (all P 0.05). In contrast, WD-fed mice were hyperinsulinemic as early as week 14 ( P <0.003), tended to have lower glucose levels, and had progressively increased insulin-to-glucose ratios with continued feeding ( P <0.01) ( Figure 4A to 4 F).
Figure 4. HC-fed LDLR -/- mice have normal fasting levels of glucose (A, B) and insulin (C, D), as well as normal insulin-to-glucose ratios (E, F) as compared with RC-fed control mice (all data points; P 0.05). In contrast, WD-fed mice are relatively hyperinsulinemic and at least for the older mice have lower glucose levels. Statistically significant differences between age-matched RC-fed and HC-fed mice versus WD-fed mice at the same time point are indicated with ** P <0.01 and *** P <0.001 and differences between RC-fed mice versus WD-fed mice are indicated with $ P <0.05 and $$ P <0.01. Data are given as mean±SEM.
Mice were subjected to an OGTT after 19 to 22 weeks of feeding. Figure 5 A to 5D illustrates that HC-fed and RC-fed mice had similar responses to the glucose challenge (all time points: P 0.05). The blood glucose profiles ( Figure 5A, 5 B) show that despite higher glucose levels at 7.5 and 15 minutes after glucose administration for the WD-fed mice, the glucose dynamics were similar among all groups. In contrast, in both age groups, the WD-fed mice had very abnormal and highly variable insulin responses to the administered glucose, ie, 3-fold insulin elevations (WD, 0.94 to 1.3 nmol/L [5.4 to 7.3 ng/mL] versus HC and RC, 0.23 to 0.40 nmol/L [1.3 to 2.3 ng/mL]), 5-minute delayed insulin peak (WD, 7.5 to 15 minutes versus HC and RC, 7.5 minutes), and 30-minute delayed return to fasting insulin levels in addition to fasting hyperinsulinemia (WD versus HC and RC, both P <0.01) ( Figure 5C, 5 D). Direct comparison of insulin levels between diets at the different time points revealed significantly higher insulin levels in the WD-fed mice ( Figure 5C, 5 D). Thus, in contrast to RC-fed and HC-fed mice, WD-fed mice were hyperinsulinemic, and the glucose and insulin responses after OGTT suggested a degree of insulin resistance.
Figure 5. HC-fed male LDLR -/- mice show normal dynamics to an oral glucose challenge. Blood glucose and plasma insulin levels were determined at baseline (after 3-hour fast) and 7.5, 15, 30, 60, and 90 minutes after oral administration of 1 mg glucose/gram body weight. Statistically significant differences between age-matched HC-fed mice and RC-fed or WD-fed mice at the same time point are indicated with * P <0.05, ** P <0.01, and *** P <0.001. Data are expressed as mean±SEM.
Impact of Diets on Atherosclerosis
Figure 6 reports the extent of atherosclerosis in the entire aorta of WD-fed and HC-fed mice, as evaluated by the en face method and at the aortic origin by cross-sectional analysis. Independently of age and diet, no significant differences in en face aortic lesion areas were obtained ( Figure 6A, 6 C). There were no quantitative differences in the extent of lesion formation in arch, thoracic, and abdominal subsections of the aorta (data not shown). The en face method yields a 2-dimensional measurement and does not take into account lesion volume. To address this, we weighed the carefully cleaned aortas, as we have previously shown that aorta weight correlates well with extent of atherosclerosis, 19 but again we found no differences (results not shown). In contrast, cross-sectional analysis of the aortic origin showed that WD-fed mice had 35% to 45% larger lesions than HC-fed mice (younger mice, 0.377±0.048 versus 0.547±0.039 mm 2 /section; P =0.022; Figure 6 B; older mice, 0.423±0.037 versus 0.565±0.041 mm 2 /section; P =0.021; Figure 6 D).
Figure 6. A and C, Independently of age, HC-fed and WD-fed male LDLR -/- mice have equal extent of aortic atherosclerosis as evaluated by the en face Sudan IV-stained area after 28 weeks of feeding (all groups, P 0.05). B and D, Both younger and older HC-fed mice have significantly smaller lesion areas at the aortic origin than corresponding WD-fed mice ( P <0.05). Symbols and horizontal bars indicate individual mice and group means, respectively. E, The HC diet induces near-linear temporal lesion formation in the entire aorta of male LDLR -/- mice. Data are expressed as mean±SEM.
Lesion composition assessed in trichrome-stained cross-sections of the aortic origin showed that the majority of lesions in both groups of mice were quite advanced and contained areas of collagen-rich fibrous caps, necrotic cores, cholesterol clefts, and cellular enrichment adjacent to the lumen. To determine whether diets resulted in compositional differences, we compared similar-sized small, intermediate, and large cross-sectional lesions, as described in Methods. In general, no overall qualitative differences in cellular composition were noted, but the degree of core necrosis appeared to be increased in some lesions from the WD-fed mice. An example is shown in supplemental Figure V, which shows a series of cross-sections over a distance of 333 µm from one lesion under one valve leaflet from 2 representative HC-fed and WD-fed mice with equal lesion burden.
To delineate the temporal lesion progression of atherosclerosis in HC-fed LDLR -/- mice, a separate study was designed (study 3). Twenty-five 34-week-old male LDLR -/- mice were fed the HC diet and euthanized in 6 cohorts of 3 to 5 mice after 8, 12, 18, 24, 28, and 40 weeks of feeding. Again, these HC-fed mice appeared healthy despite their old age and the long diet intervention, ie, obesity and changes in fur and skin integrity were not observed. Steady plasma lipid levels were achieved within 4 weeks of initiation of diet (supplemental Figure VIA) and lipoprotein distributions (supplemental Figure VIB) were identical to those shown for HC-fed mice in Figure 2C and 2 D. The en face analysis of the aorta demonstrated progressive lesion formation throughout the entire aortic tree ( Figure 6 E).
Discussion
The present work demonstrates that male LDLR -/- mice fed 1% cholesterol-enriched regular chow, without other added fat or cholate, had predominant elevation of LDL cholesterol and extensive atherosclerosis, without concomitant alterations characteristic of the metabolic syndrome, typically seen with use of WDs, eg, increases in weight gain, VLDL triglycerides, VLDL cholesterol, glucose, insulin, and alterations in glucose/insulin dynamics. Moreover, HC-fed mice appeared as healthy as RC-fed mice and did not display features typically seen with WD-fed mice such as fatty infiltration of livers and/or changes in skin and fur integrity that often lead to ulceration. Thus, the HC diet minimized conditions that might independently impact atherogenesis. Also, compared with WD-fed mice, the degree of plasma cholesterol elevation with HC feeding was less. Because severe hypercholesterolemia may override effects that may otherwise impact atherogenesis, the development of atherosclerosis at more moderate levels is a distinct advantage for many studies, for example, those of the impact of immune deficiency. 4-7 Thus, we propose that the simple HC diet described here is advantageous for many atherosclerosis studies in mice.
The metabolic syndrome is defined as a cluster of factors that leads to a substantial increase in cardiovascular disease. Some of the known components are: (1) obesity; (2) dyslipidemia, ie, elevated VLDL with associated hypertriglyceridemia and low HDL; (3) hyperinsulinemia, insulin resistance, and glucose intolerance; (4) hypertension; (5) proinflammatory state, eg, high C-reactive protein in humans or corresponding SAA in mice; and (6) prothrombotic state. 15 Our work has demonstrated that atherosclerotic male LDLR -/- mice fed the high-cholesterol diet do not have the first 3 components, which are central features of the metabolic syndrome. Recently, Teupser et al, 20 feeding male LDLR -/- mice a semi-synthetic low-fat HC diet, demonstrated that dietary cholesterol did not induce hypertension in this setting. Moreover, Chait et al 21 5-fold elevated plasma SAA levels in WD-fed male LDLR -/- mice compared with RC-fed controls. In collaboration with Chait et al, we determined SAA levels in the mice from study 1 and confirmed the strong SAA-elevating effect of WD, which could not be demonstrated with HC feeding (unpublished data, 2005). Thus, male LDLR -/- mice fed low-fat, cholesterol-enriched chow do not have confounding effects of the metabolic syndrome.
The concept of a low-fat, cholesterol-enriched RC is not novel. 2 However, this is the first report to our knowledge that directly compares such a diet with RC and a WD. Teupser et al 20 fed male LDLR -/- mice low-fat, cholesterol-enriched RC diets, using the American Institute of Nutrition?s cholesterol-free, casein-containing, semi-synthetic AIN-76A diet (Table I). LDLR -/- mice fed 0% or 0.02% cholesterol-enriched diets had plasma cholesterol levels of 12.9 mmol/L (500 mg/dL), whereas mice fed 0.15%, 0.3%, or 0.5% cholesterol-enriched AIN-76A had cholesterol levels of 36.2 mmol/L (1400 mg/dL). 20 On the AIN-76A diet, only mice with cholesterol levels of 36.2 mmol/L developed atherosclerosis in the aorta, and in addition these mice developed a complex lipoprotein profile with high levels of unusual triglyceride-poor, cholesterol-rich VLDL. 20 In contrast, in our studies with regular murine chow enriched with 1% cholesterol only, the LDLR -/- mice had plasma cholesterol levels of 20.7 mmol/L (800 mg/dL) and developed reproducible aortic lesions. In fact, feeding male LDLR -/- mice of different ages the HC diet for a period of 28 weeks revealed a high degree of reproducibility of total aortic en face lesions: 20.2±2.0% (beginning age 14 weeks, n=7; Figure 6 A), 17.1±1.4% (beginning age 28 weeks, n=19; unpublished data from an unrelated study), 17.7±2.5% (beginning age 34 weeks, n=4; Figure 6 E), and 21.3±2.2% (beginning age 44 weeks, n=12; Figure 6 C).
To further investigate the uses of the cholesterol-enriched TD8604 RC, we attempted to titrate the plasma cholesterol levels in male LDLR -/- mice using commercial custom-made cholesterol-enriched diets with 0.15%, 0.25%, 0.5%, and 1% added cholesterol (study 2). We found that these diets significantly increased plasma cholesterol 1.8-, 2.5-, 2.9-, and 3.8-fold, respectively ( Figure 3 ), which demonstrated the feasibility of manipulating plasma cholesterol levels between 11.7 to 25.9 mmol/L (450 to 1000 mg/dL) using cholesterol-enriched TD8604 RC as base. Interestingly, such dose-response curve of dietary cholesterol using the AIN-76A as base was not observed by Teupser et al, who found 2 different cholesterol levels in response to various dietary cholesterol amounts, ie, 12.9 mmol/L at <0.02% dietary cholesterol and 0.15% dietary cholesterol. 20 Moreover, our study 2 revealed that the cholesterol-sprayed HC diet and the commercial custom-made 0.5% cholesterol-enriched diet, TD97234, resulted in equal plasma cholesterol levels. The explanation for the dose-dependent response using the TD8604 RC as base, but not the semi-synthetic AIN-76A, is most likely caused by the content of casein and fructose in AIN-76A (Table I). Compared with plant-derived proteins, casein has been reported to elevate levels of plasma and liver cholesterol and triglycerides, as well as atherosclerosis in rodents. 22 Likewise, sucrose and fructose induce hypertriglyceridemia and are more atherogenic compared with starch. 22 Thus, using the TD8604 RC as base for cholesterol-enriched low-fat diets may have advantages over the AIN-76A diet with respect to titration of plasma cholesterol, lipoprotein distribution, and liver lipid levels.
WD-fed male LDLR -/- mice on the C57BL/6J background are known to be susceptible to obesity. 12-14 However, we found that enriching RC with cholesterol (ie, the HC diet) did not induce the marked weight gain seen with WD feeding ( Figure 1 ). It is interesting to note that the intervention changed the weight trajectory of the mice. After 28 weeks of intervention, the younger RC-fed and HC-fed mice were 42 weeks old and weighed 33.5 grams, which was less than expected, because the older RC-fed and HC-fed mice weighed 37.5 grams at 44 weeks of age at the beginning of the diet intervention. We have consistently made this observation and speculate that procedures such as blood drawing and OGTT are the cause. This emphasizes the importance of internally controlled mouse interventions.
This report confirms that WD-fed male LDLR -/- mice developed obesity, hyperinsulinemia, and some degree of insulin resistance, as we and others have reported. 12-14 In contrast, female LDLR -/- mice respond differently to WD feeding 14; therefore, we focused the present study on male mice. Although insulin resistance and diabetes are known to accelerate atherosclerosis in humans, it has been difficult to show this in murine models. 23 Previously, we have shown that the WD-induced metabolic complications in the male LDLR -/- mice may be dramatically improved by PPAR ligands, and that these agents decrease the extent of atherosclerosis in these male mice. 14 However, it is not clear whether the anti-atherogenic impact of these agents is caused by improvement of the insulin resistance. In fact, it appears more likely that their anti-atherogenic impact is more related to a decrease in the inflammatory properties of macrophages and/or to advantageous alterations in intracellular cholesterol metabolism. 14,24,25 Thus, it is of considerable interest that in our current studies, despite the metabolic complications and the 2- to 3-fold higher cholesterol exposure in the WD-fed mice compared with HC-fed mice, the extent of aortic atherosclerosis was not significantly different ( Figure 6A, 6 C). In contrast, quantification of the extent of atherosclerosis at the aortic origin revealed that the WD-fed mice had 35% to 45% larger lesion burden compared with HC-fed mice ( Figure 6B, 6 D). Nevertheless, qualitative comparison of lesion composition in lesions of same size in the 2 diet groups did not reveal any gross differences (supplemental Figure V). It should be noted that the relatively long diet interventions (chosen chiefly with regards to the metabolic parameters) used in this study have resulted in very large and advanced lesions, and it is conceivable that shorter interventions would have yielded a better differentiation of the atherogenic properties of the tested diets.
Because WD feeding caused significantly greater hypercholesterolemia, the larger lesion burden in the aortic origin of WD-fed mice was expected. In contrast, the finding of equal aortic en face lesion burden with HC and WD feeding was unexpected. Thus, there appears to be site-specific responses in the arterial tree to the WD and HC diets. Such site-specificity in murine atherosclerosis models in response to different diets, gender, or immunologic interventions is now well-described. 26 In this regard, it is worth emphasizing that the lipoprotein cholesterol and triglyceride profiles from the HC-fed LDLR -/- mice are different from those of the WD-fed mice. Much of the elevated cholesterol found in the WD-fed mice is associated with larger VLDL-like particles ( Figure 2 C-D), and at least as compared with LDL particles, these are relatively less atherogenic. 27,28 Duff et al 29 originally commented on the failure of diabetes, which caused a marked enrichment of such large VLDL, to accelerate atherosclerosis in rabbits presumably for the same reasons. Also, the cholesterol and triglyceride profiles ( Figure 2C, 2 D) reveal that LDL particles from WD-fed mice, compared with HC-fed mice, have higher triglyceride content, suggesting LDL particles with different phenotype and potentially atherogenicity. 28 Of course, the failure to detect differences in aortic surface atherosclerosis using the en face method may be caused by the fact that it is a 2-dimensional measure and does not take into account the thickness and depth of the lesions, which potentially becomes increasingly problematic with the severity of lesion progression.
The HC diet provided an intermediate level of plasma cholesterol of 20.7 mmol/L (800 mg/dL), which distributed between VLDL, LDL, and HDL as 0.65 (3%), 17.1 (83%), and 2.8 (14%) mmol/L, respectively ( Figure 2 C). This cholesterol distribution is comparable to those obtained in 2 "apoB-100 only" mouse models of atherosclerosis; ie, LDLR -/- ApoB 100/100 and LDLR -/- Apobec1 -/- mice. 9,28,30 RC-fed LDLR -/- ApoB 100/100 and LDLR -/- Apobec1 -/- mice have plasma cholesterol levels of 7.8 mmol/L (300 mg/dL) and 14.2 mmol/L (550 mg/dL), respectively, and both develop aortic atherosclerotic lesions covering 14% of the surface of the entire aorta at age 34 to 40 weeks. 9,11,28 In our study, we found that LDLR -/- mice after 28 weeks of HC feeding develop total aortic en face lesions of 20%, which is consistent with the higher plasma cholesterol level ( 20.7 mmol/L [800 mg/dL]). The cholesterol distribution within the lipoproteins of these 3 murine models of atherosclerosis is much more comparable to the usual human profile than those of the commonly used LDLR -/- mice on high-fat/high-cholesterol diets or ApoE -/- mice, as well as LDLR -/- mice fed the cholesterol-enriched casein-containing AIN-76A diet.
Thus, the HC diet described here and the WD and the cholesterol-enriched AIN-76A diets described by others produce different biological consequences. Overall, at the level of the aorta, the HC diet appears to be as atherogenic as the WD, despite marked differences in total cholesterol content. In contrast, at the aortic origin, the WD was more atherogenic. These data emphasize the complexity of lesion formation at different sites in the aortic tree. 26
In summary, the present work describes an alternative and simple murine model of diet-induced atherogenesis, in which LDLR -/- mice are fed casein-free RC enriched with cholesterol without other added fat or cholate. We propose that this simple diet may be advantageous for many atherosclerosis studies in mice.
Addition Note
During the review process of our manuscript, Goldberg, Dansky, and colleagues published data relevant to this report (Wu et al, J Lipid Res 2006;47:2215- 2222). In 2 independent diet interventions using male LDLR -/- mice, the authors tested the effects of low-fat and high-fat diets in the setting of matched levels of moderate hypercholesterolemia. Their diets were based on the casein-containing AIN-76A diet, similar to that used by Teupser et al. 20 Compared with the mice fed the low-fat diet, the mice fed the high-fat diet became obese, hypertriglyceridemic, hyperglycemic, hyperinsulinemic, and insulin resistant, and thus showed a type 2 diabetic phenotype. Despite this, no differences in atherosclerotic lesion burden and composition could be demonstrated after 20 weeks of feeding. The authors did observe, however, that in one group of mice fed diets for 40 weeks, the mice fed the high-fat diet had larger lesions at the aortic origin but not in the entire aorta (en face method) and brachiocephalic artery, and also had a more atherogenic lipoprotein profile. Although, the authors did not compare their test diets to a baseline RC that does not induce hypercholesterolemia, their report supports our observations that low-fat atherogenic diets do not induce parameters of the metabolic syndrome and that the type 2 diabetic/insulin resistance phenotype has limited atherogenic potential in the male LDLR -/- mouse model.
Acknowledgments
We thank Florencia Casanada, Richard Elam, Jennifer Pattison, and Mercedes Silvestre for excellent technical assistance.
Sources of Funding
NIH-HL56989 (SCOR in Atherosclerosis and Molecular Medicine). K.H. was supported by a fellowship from the AHA (Western States Affiliates), a faculty start-up grant from The Sam and Rose Stein Institute for Research on Aging, and grants from the Novo-Nordisk/Danish-American Foundation, the Reinholdt W. Jorck & Hustrus Foundation, the Otto Mønsteds Foundation, the Arvid Nilssons Foundation, and the Villum Kann Rasmussen Foundation. C.J.B. was supported by a fellowship from the AHA (Western States Affiliates). A.C.L. was supported by a beginning grant-in-aid from the AHA (Western States Affiliates).
Disclosure
None.
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作者单位：Department of Medicine (K.H., C.J.B., L.F.H., A.R., J.J., S.H., D.S., W.P., J.L.W.) and Department of Cellular and Molecular Medicine (A.C.L.), University of California, San Diego, La Jolla, Calif.