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Am J Physiol Endocrinol Metab 294: E496-E505, 2008. First published December 26, 2007; doi:10.1152/ajpendo.00509.2007
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Generation and characterization of two novel mouse models exhibiting the phenotypes of the metabolic syndrome: Apob48–/–Lepob/ob mice devoid of ApoE or Ldlr

David J. Lloyd,1 Jocelyn McCormick,1 Joan Helmering,1 Ki Won Kim,1 Minghan Wang,1 Preston Fordstrom,1 Stephen A. Kaufman,2 Richard A. Lindberg,1 and Murielle M. Véniant1

Departments of 1Metabolic Disorders and 2Pathology, Amgen Incorporated, Thousand Oaks, California

Submitted 3 August 2007 ; accepted in final form 20 December 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The metabolic syndrome is a group of disorders including obesity, insulin resistance, atherogenic dyslipidemia, hyperglycemia, and hypertension. To date, few animal models have been described to recapitulate the phenotypes of the syndrome. In this study, we generated and characterized two lines of triple-knockout mice that are deficient in either apolipoprotein E (Apoe–/–) or low-density lipoprotein receptor (Ldlr–/–) and express no leptin (Lepob/ob) or apolipoprotein B-48 but exclusively apolipoprotein B-100 (Apob100/100). These two lines are referred to as Apoe triple-knockout-Apoe 3KO (Apoe–/–Apob100/100Lepob/ob) and Ldlr triple-knockout-Ldlr 3KO (Ldlr–/–Apob100/100Lepob/ob) mice. Both lines develop obesity, hyperinsulinemia, hyperlipidemia, hypertension, and atherosclerosis. However, only Apoe 3KO mice are hyperglycemic and glucose intolerant and are more obese than Ldlr 3KO mice. To evaluate the utility of these lines as pharmacological models, we treated both with leptin and found that leptin therapy ameliorated most metabolic derangements. Leptin was more effective in improving glucose tolerance in Ldlr 3KO than Apoe 3KO animals. The reduction of plasma cholesterol by leptin in Ldlr 3KO mice can be accounted for by its suppressive effect on food intake. However, in Apoe 3KO mice, leptin further reduced plasma cholesterol independently of its effect on food intake, and this improvement correlated with a smaller plaque lesion area. These effects suggest a direct role of leptin in modulating VLDL levels and, likewise, the lesion areas in VLDL-enriched animals. These two lines of mice represent new models with features of the metabolic syndrome and will be useful in testing therapies targeted for combating the human condition.

atherosclerosis; leptin; hypercholesterolemia; diabetes; obesity

IN RECENT YEARS, THE METABOLIC SYNDROME has been increasingly recognized by clinicians as a major risk factor for cardiovascular disease and diabetes (28, 32, 36). Although the definition varies, it generally involves three of the following abnormal metabolic parameters (13, 41): central obesity (with excessive fat tissue in and around the abdomen), atherogenic dyslipidemia [mainly high triglycerides and low high-density lipoprotein (HDL)], insulin resistance or glucose intolerance, hyperglycemia, and elevated blood pressure. Since coronary heart disease and diabetes are the leading causes of death in the United States, the pharmaceutical industry has now focused on discovering drugs that can address multiple arms of the metabolic syndrome (23).

Mouse models are widely used to study lipid metabolism, atherosclerosis, and diabetes, but, to date, animal models that encompass all of these characteristics remain limited. Furthermore, a model that develops hypertension in addition to all of the above aspects of metabolic disorders would be desirable. Extensively used models in the lipid and atherosclerosis fields are the apolipoprotein E (Apoe–/–) (27, 43) and low-density lipoprotein receptor knockout (Ldlr–/–) mice (18). These two models have distinct lipoprotein profiles, with Apoe–/– mice containing high levels of very-low-density lipoprotein (VLDL) and Ldlr–/– mice exhibiting high levels of low-density lipoprotein (LDL). Both models (15, 24) have been used extensively in understanding the mechanisms of lipoprotein metabolism and the development of atherosclerosis. However, certain features of these models have limited their use. For instance, cholesterol diet-fed Ldlr–/– mice develop only a minimal amount of atherosclerotic lesions, even at 9–12 mo of age (33). In addition, the plasma cholesterol in this model is carried in the ApoB-48-containing VLDL particles and chylomicron remnants, making it less relevant to human conditions because most humans with atherosclerosis have high plasma levels of ApoB-100-containing LDL and do not produce ApoB-48 in the liver (20, 31). Most importantly, both Apoe–/– and Ldlr–/– mice are models for hyperlipidemia and atherosclerosis, and insulin resistance cannot be addressed along with lipid disorders in these models. To develop mouse models that exhibit hyperglycemia, insulin resistance, and hyperlipidemia with a lipoprotein profile close to that in humans, we obtained mice that synthesize exclusively ApoB-100 backcrossed on an ApoE-deficient or LDL receptor-deficient background (Apoe–/–Apob100/100 or Ldlr–/–Apob100/100) (8, 34) and bred them to Lepob/ob mice to generate mice that develop symptoms corresponding to multiple arms of the metabolic syndrome.

In this report, we characterized the degree of obesity, diabetes, dyslipidemia, atherosclerosis, and hypertension in Ldlr 3KO (Ldlr–/–Apob100/100Lepob/ob) and Apoe 3KO (Apoe–/–Apob100/100Lepob/ob) mice. To assess their potential for developing new therapies, we treated both models with leptin for 11 wk. To further assess the benefits, if any, of leptin therapy beyond its potential to reduce body weight, we included a group of mice that had been food restricted to match the body weights of the leptin-treated animals. In addition, a group of mice fed ad libitum was used as a control group. Our data demonstrate that these two mouse models develop not only obesity, insulin resistance, and hyperlipidemia but also hypertension and atherosclerosis. Although Ldlr 3KO mice had more atherosclerotic lesions than Apoe 3KO mice, Apoe 3KO mice were significantly more obese and more diabetic than Ldlr 3KO mice. Treatment of both lines with leptin or food restriction improved many of these metabolic perturbations. In general, food restriction was effective in mimicking the effects of leptin. However, leptin treatment was more potent in lowering cholesterol levels and aortic lesion areas in Apoe 3KO mice.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of Ldlr 3KO and Apoe 3KO mice. Three breeding pairs of Apoe–/–Apob100/100, Ldlr–/–Apob100/100, and Lepob/+ mice were purchased from the Jackson Laboratory (34) and bred to produce multiple breeding pairs of Apoe–/–Apob100/100Lepob/+ mice. These mice were then intercrossed to generate Apoe 3KO and Ldlr 3KO mice. The background of each of the lines was mainly C57BL/6. Mice were weaned at 21 days of age and fed a chow diet containing 22.0% crude protein, 5% crude fat, and 4.5% crude fiber (22/5 Rodent Diet; Harlan Teklad, Indianapolis, IN). All offspring were genotyped by polymerase chain reaction (34). Only male mice were included in this study. Blood samples were collected by retroorbital bleed on nonanesthetized mice either in the fed state or following a 4-h fast (0900-1300) using EDTA microtainer tubes. Animals were singly housed and kept under constant day-night (lights on 0630-1830) conditions. All experiments were approved by Amgen's Institutional Animal Use and Care Committee.

Blood chemistry measurements. Total plasma cholesterol, triglyceride, and nonesterified fatty acid (NEFA) levels were measured from frozen plasma using the Hitachi 717 clinical chemistry analyzer. Blood glucose levels were measured immediately following blood collection on a glucose monitor (One Touch Profile Meter; Lifescan, Milpitas, CA). Reagents for cholesterol and triglyceride measurements were purchased from Boehringer Mannheim (Indianapolis, IN). Reagents for NEFA measurement were obtained from Wako Diagnostics (Richmond, VA).

Cholesterol lipid profiles. Plasma samples were collected and stored on ice, vortexed, and cleared by brief centrifugation. Plasma (100 µl) was diluted to 400 µl in PBS and fractionated by AKTA FPLC gel filtration using two serial Superose 6 (10/300) GL columns run at a flow rate of 0.5 ml/min. Fractions were assayed by enzymatic colorimetric methods (test kit from Wako Diagnostics).

Insulin measurements. Insulin levels were determined in plasma samples using Mouse Endocrine Lincoplex 96-well plate assay kit (MENDO-75K; Linco Research, St. Charles, MO). Samples were run in duplicate according to the manufacturer's instructions and analyzed using Upstate Beadview (Temecula, CA) software.

Glucose tolerance test. Glucose tolerance tests were carried out in mice fasted for 12 h (2100-0900) for the characterization of the two lines and 8 h (2300-0700) for the experiments with leptin treatment. Fasted blood glucose was measured prior to an intraperitoneal injection of glucose (0.5 g/ml dextrose) at 1 g/kg body wt. Blood samples were collected from the retroorbital sinuses of nonanesthetized mice. To minimize stress levels in the tested animals, only 30- and 90-min blood glucose levels were measured using the One Touch Profile Meter.

Analysis of atherosclerotic lesions by morphometry. Aortas were dissected for quantification of atherosclerosis. Pentobarbital-anesthetized mice were perfused with phosphate-buffered saline followed by fixative (4% paraformaldehyde, 5% sucrose, 20 mM EDTA, pH 7.4). With the major branching vessels attached, the aorta was opened longitudinally from the iliac arteries to the aortic root. All branching vessels were removed, including the great vessels in the neck, and most of the aorta (from the iliac bifurcation to a point equidistant between the aortic valve and the bracheocephalic artery) was removed and pinned flat onto a paraffin wax board. Sudan IV stain solution 0.5% (wt/vol) was dissolved in equal parts 70% ethanol and 100% acetone. The mounted aorta was stained in the following manner: the fixative was rinsed out for 3 min in PBS followed by 5 min in 70% ethanol and then stained in freshly filtered Sudan IV for 6 min with occasional agitation. The stained aorta was then differentiated in 80% ethanol for 3 min and finally washed in PBS for 3 min. After staining, the aorta was removed from the board and cut into four 1-cm segments, beginning at the proximal end. The segments were individually positioned on a glass plate and mounted under a glass coverslip using PBS, taking care to eliminate all folds and air bubbles. Images of the aortas were captured with a Nikon DXM 1200 digital camera utilizing a Nikon SMZ-U dissecting microscope (Nikon USA, Melville, NY) and the Automatic Camera Tamer imaging software. Each image was analyzed with Metamorph Imaging System software version 6.1 (Molecular Devices, Sunnyvale, CA) red-green-blue thresholding to define lesion areas. Atherosclerotic lesions were quantified by a trained observer blinded to mouse genotype and treatment. Data are reported as the percent of the aortic surface covered by lesions. The total length of the aorta was also measured, making it possible to determine the extent of atherosclerotic lesions in different segments of the aorta.

Blood pressure measurements. Blood pressure was measured indirectly using the BP-200 Blood Pressure system for mice (Visitech Systems, Apex, NC) (37). Briefly, mice were placed on a heated platform for precisely 5 min prior to initiating the measurements to facilitate the dilation of the tail vessels (platform heated to 98–100°F). Restrainers were custom made to allow as much space around the obese mice as the restrainers used for the C57BL/6 mice. As part of the acclimation period, 10 preliminary measurements were followed by 10 experimental measurements. Ten-week-old Apoe 3KO, Ldlr 3KO, and two groups of C57BL/6 mice were measured for 5 consecutive days. Measurements from the 4th and 5th days were averaged for data analysis. After the 5th day, drinking water bottles were replaced with new drinking water bottles containing enalapril (catalog no. E6888–5G; Sigma-Aldrich, St. Louis, MO) for three of four groups at a dose of 10 mg/kg body wt. Two concentrations of enalapril were prepared to account for the differences in water consumption in 3KO mice (5.8 ml/day) compared with C57BL/6 mice (5 ml/day), as determined in preliminary experiments (data not shown). Mice were allowed to recover for 2 days, after which blood pressure monitoring resumed for another 5 days. For posttreatment data analysis, measurements from the 4th and 5th days were averaged. C57BL/6 mice receiving enalapril and mice not receiving treatment but exposed to the same experimental regime were used as controls.

Recombinant leptin and food restriction treatments. At the beginning of the study (mice at 10 wk of age), three treatment groups were randomized from each mouse line on the basis of their blood glucose levels. Mice were treated daily with saline or leptin, and food was restricted (with daily saline injection). Saline-treated mice received intraperitoneal saline injections 5 times/wk (shown to be similar to saline injections 7 times/wk; data not shown). The leptin-treated mice were injected with recombinant murine leptin every day for 86 days. Recombinant murine leptin was generated as previously described (26). Solutions for injection were freshly diluted each day with saline. The leptin doses were adjusted throughout the experiment to maintain minimal changes in body weight. Recombinant leptin doses ranged from 20 to 80 µg·kg–1·day–1 for Apoe 3KO mice and 10 to 120 µg·kg–1·day–1 for Ldlr 3KO mice. Food intake was measured daily for all mice. The food-restricted groups received a portion of the amount of food consumed by the leptin-treated mice. Food amount was adjusted daily for every food-restricted mouse with the aim to mirror the average body weight of groups treated with leptin. Nevertheless, mice from the food-restricted groups changed their feeding behavior; these mice immediately (within 30 min after administration) ate their food and underwent a fast for the rest of the day. To minimize an extensive fasting period meals were provided at two different times of the day, 66% of the meal at 1530 and 33% of the meal the next morning at 0830.

Statistical analyses. All statistical analyses were carried out using a one-way ANOVA incorporating a Tukey posttest to compare all pairs of data unless otherwise stated.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ldlr 3KO and Apoe 3KO mice exhibit many characteristics of the metabolic syndrome. The body weights of the Ldlr 3KO and Apoe 3KO mice were significantly greater than that of the C57BL/6 mice (P < 0.0001 for both groups; Fig. 1A). Furthermore, Apoe 3KO mice grew heavier than Ldlr 3KO mice. At 15–16 wk of age, Apoe 3KO mice weighed 63 ± 1 g, whereas Ldlr 3KO mice weighed 58 ± 1 g. Interestingly, only Apoe 3KO mice were hyperglycemic (Fig. 1B), and glycemic control in these mice fluctuated with age in a trend similar to the glucose levels of Lepob/ob mice (26). The glucose levels of Ldlr 3KO mice were indistinguishable from normal C57BL/6 mice. Despite this blood glucose variation, both mouse lines exhibited marked hyperinsulinemia. Plasma insulin levels of both lines of triple-knockout mice were significantly higher than that of C57BL/6 mice (Fig. 1C). Again, Ldlr 3KO mice were less severely affected than Apoe 3KO mice; on average, Ldlr 3KO mice had 34.6% lower plasma insulin concentrations. Consistent with worsened glucose homeostasis, we observed glucose intolerance in Apoe 3KO mice (Fig. 1D). Glucose intolerance was evident in these mice at 7–8 wk of age and sustained until 15–16 wk (data not shown). Ldlr 3KO mice, although hyperinsulinemic, showed no deterioration in glucose tolerance when compared with C57BL/6 mice.


Figure 1
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  		Fig. 1. Body weight and glucose homeostasis in LDL receptor (Ldlr) triple-knockout (3KO) and apoplipoprotein E (Apoe) 3KO mice. Body weight and glucose homeostasis were profiled in cohorts of mice 7–16 wk of age. A: body weight of C57BL/6 (n = 8–18), Ldlr 3KO (n = 22–52), and Apoe 3KO (n = 18–52). Body weight was significantly increased in Ldlr 3KO and Apoe 3KO mice compared with C57BL/6 mice. Apoe 3KO mice were significantly heavier than Ldlr 3KO mice. B: fed blood glucose levels of C57BL/6 (n = 8–18), Ldlr 3KO (n = 5–17), and Apoe 3KO (n = 10–24) mice. Fed blood glucose levels were significantly increased in Apoe 3KO only compared with C57BL/6 mice. C: fed insulin levels of C57BL/6 (n = 16–28), Ldlr 3KO (n = 16–32), and Apoe 3KO (n = 18–46) mice. Insulin levels were significantly higher in both Ldlr 3KO and Apoe 3KO mice compared with C57BL/6 mice and were significantly higher in Apoe 3KO mice than in Ldlr 3KO mice. D: glucose tolerance test performed in 13- to 14-wk-old mice. Glucose tolerance test in C57BL/6 (n = 8), Ldlr 3KO (n = 11), and Apoe 3KO (n = 10) mice showed glucose intolerance in Apoe 3KO mice compared with C57BL/6 mice. All data represent means ± SE. *P < 0.05, **P < 0.01, ***P < 0.0001 vs. C57BL/6 mice; ++P < 0.01, +++P < 0.0001 vs. Ldlr 3KO mice.

 
Both Ldlr 3KO and Apoe 3KO mice were hypercholesterolemic, and their cholesterol levels were elevated almost 10-fold when compared with that of C57BL/6 mice (Fig. 2A). There was no difference in cholesterol level between the two lines of triple-knockout mice. However, when the plasma lipoproteins were fractionated, the distributions of VLDL, LDL, and HDL were distinctive between the two mouse knockout lines and the C57BL/6 mice. In keeping with published data (3), HDL is the dominant lipoprotein species in C57BL/6 mice, with very low levels of LDL and VLDL (Fig. 2B). In contrast, the cholesterol distribution among VLDL, LDL, and HDL was very different between the two mouse lines (Fig. 2B). Ldlr 3KO mice had more LDL than VLDL, with lower HDL levels, whereas Apoe 3KO mice had higher VLDL than LDL and again lower HDL.


Figure 2
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  		Fig. 2. Plasma lipid profiles in Ldlr 3KO and Apoe 3KO mice. A: cholesterol levels were measured in cohorts of 15- to 16-wk-old C57BL/6 (n = 8–18), Ldlr 3KO (n = 7–18), and Apoe 3KO (n = 7–16) mice. Ldlr 3KO and Apoe 3KO mouse cholesterol levels are similar and both significantly higher than those of C57BL/6 mice. B: cholesterol plasma lipoprotein profiles of C57BL/6, Ldlr 3KO, and Apoe 3KO mice. Ldlr 3KO mice were enriched with LDL cholesterol, whereas Apoe 3KO mice were enriched with VLDL cholesterol. C: triglyceride levels were measured in the same mice described in A and found to be significantly higher in both Ldlr 3KO and Apoe 3KO mice compared with C57BL/6 mice. D: nonesterified fatty acid (NEFA) levels were measured in the same mice described in A and found to be significantly higher in both Ldlr 3KO and Apoe 3KO mice compared with C57BL/6 mice. All data represent means ± SE. ***P < 0.0001 vs. C57BL/6 mice.

 
At 15–16 wk of age, plasma triglyceride levels of the Ldlr 3KO and Apoe 3KO mice were similar and were three- and fourfold higher than that observed in C57BL/6 mice, respectively (Fig. 2C). The plasma NEFA levels of the Ldlr 3KO and Apoe 3KO mice were also significantly greater than those of the C57BL/6 animals (Fig. 2D).

We investigated the extent of lesion formation in both lines of mice. At 12 wk of age, atherosclerotic lesions were found mainly in the aortic region of the whole aorta in both lines, and, as expected, no lesions were found in the aortas of C57BL/6 mice (Fig. 3).


Figure 3
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  		Fig. 3. Aortic plaques and blood pressure in Ldlr 3KO and Apoe 3KO mice. A: representative images of dissected aorta from 11- to 12-wk-old mice stained with Sudan IV. Atherosclerotic lesions are evident at this young age in both Ldlr 3KO and Apoe 3KO animals. C57BL/6 mice served as a negative control group. B: indirect blood pressure measurement was assessed in 2 groups of C57BL/6 (n = 17 for each group), Apoe 3KO (n = 16), and Ldlr 3KO mice (n = 22). Systolic pressure values are averages of the last 2 days of measurements obtained from 5-day trained animals (for both pre- and posttreatment); see METHODS. Enalapril (10 mg·kg–1·day–1 given to 3 of 4 groups of mice) was administered in the drinking water for 7 days. All data represent means ± SE. *P < 0.05 vs. C57BL/6 mice pretreatment. ns, No significant difference vs. C57BL/6 mice posttreatment with enalapril.

 
To further establish these two lines of mice as representative models of the metabolic syndrome, we measured indirect systolic blood pressure. Following a training period of 3 days, we recorded blood pressure in 10-wk-old mice from each line and from C57BL/6 mice (in the 4th and 5th days). Both Ldlr 3KO and Apoe 3KO mice had elevated blood pressure (Fig. 3B). To investigate whether this was true hypertension and not a consequence of poor acclimation, we treated both strains with enalapril in drinking water (1 group of C57BL/6 mice was maintained on drinking water without enalapril). The hypertension observed in Ldlr 3KO and Apoe 3KO mice was corrected by enalapril, and the blood pressure was normalized to the level in C57BL/6. Blood pressure was also slightly decreased in C57BL/6 mice. This decrease is unlikely to be a consequence of the blood pressure-lowering effects of enalapril, as it was also observed in C57BL/6 mice drinking water only. Thus, the decrease in blood pressure is more likely associated with prolonged exposure of the animals to the apparatus. Therefore, enalapril does not lower blood pressure in all mice but rather only in hypertensive mice, confirming the phenotype in the Ldlr 3KO and Apoe 3KO animals.

Ldlr 3KO and Apoe 3KO serve as pharmacological models. We sought to determine the potential of these two mouse lines as tools for in vivo pharmacological studies. Additionally, we were interested in understanding the role of leptin in lipid metabolism and considered the contrasting characteristics of these two lines to be useful toward this goal. We formed three groups for each line: 1) control mice injected with saline and fed ad libitum (saline ad libitum), 2) leptin-treated mice to attain a stable body weight but with free access to food (leptin ad libitum), and 3) mice injected with saline but food restricted to match the body weight for the leptin-treated group (saline food restricted). The last group was included as a control for the leptin-treated mice and intended to identify the direct role of leptin in glucose and lipid metabolism independent of its weight-lowering effects.

At the beginning of the study and prior to the treatments, Apoe 3KO mice had significantly higher body weights than Ldlr 3KO mice (58.5 ± 1.9 vs. 50.7 ± 1.3 g, respectively, P = 0.0036). The body weights of Ldlr 3KO and Apoe 3KO mice (saline ad libitum) increased during the study (Fig. 4, A and B) and diverged from those of leptin-treated and food-restricted mice. The mice in the leptin treatment group maintained a constant body weight throughout the 86 days of the study period. As intended, there was no difference in body weight between the food-restricted and leptin-treated mice for both Apoe 3KO and Ldlr 3KO lines.


Figure 4
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  		Fig. 4. Body weights and glucose tolerance in Ldlr 3KO and Apoe 3KO mice injected with leptin or food restricted. A and B: the body weights of Ldlr 3KO (A) and Apoe 3KO (B) mice were monitored over a period of 85 days from the age of 10 wk. Each line was separated into 3 groups. The first group was injected with saline (Ldlr 3KO n = 9, Apoe 3KO n = 7). The second group was injected with leptin (Ldlr 3KO 10–120 µg·kg–1·day–1, n = 12; Apoe 3KO 20–80 µg·kg–1·day–1, n = 7). The third group was food restricted to attain the same weight as the leptin-treated group of animals (Ldlr 3KO n = 13, Apoe 3KO n = 8). C and D: glucose tolerance was assessed in the Ldlr 3KO (C) and Apoe 3KO (D) mice (described above) at the end of the study (day 86). Mice were fasted for 8 h and ip injected with 1 g/kg of dextrose. Glucose tolerance was improved in the leptin-treated animals in both lines. All data represent means ± SE. **P < 0.01, ***P < 0.0001 vs. saline ad libitum (strain matched) mice; +P < 0.05, ++P < 0.01, +++P < 0.0001 vs. saline food-restricted (strain matched) mice.

 
Consistent with the differences in body weight, Apoe 3KO mice ate more than Ldlr 3KO mice at the start of the study (1.7 g more; Table 1). Administration of leptin significantly lowered food consumption in both lines of mice compared with the saline-treated animals (beginning on day 7 for Apoe 3KO mice, P = 0.0294, beginning on day 9 for Ldlr 3KO mice, P = 0.0047; data not shown). Consistent with the extra-satiety effects of leptin, the food-restricted groups in both lines required significantly less food than the leptin-treated groups to maintain similar body weights.


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  		Table 1. Effect of leptin treatment vs. food restriction on metabolic parameters in Ldlr 3KO and Apoe 3KO mice after 76 days of regimen

 
Glucose homeostasis was investigated in both lines of mice (Table 1 and Fig. 4, C and D). Neither leptin nor food restriction decreased glucose in Ldlr 3KO mice, although these mice were initially euglycemic compared with C57BL/6 mice (Fig. 1B). Nonetheless, a drastic reduction was observed in fasted insulin levels in both treatments compared with the saline control group. Furthermore, leptin treatment significantly lowered insulin more than food restriction in these mice. Apoe 3KO mice, on the other hand, exhibited an improvement in blood glucose with food restriction but not with leptin treatment, and there was no reduction in insulin levels with either treatment. The effect of leptin on glucose tolerance was evident during the glucose tolerance test in Ldlr 3KO mice only (Fig. 4, C and D). Similar observations were made at different ages (data not shown). Leptin-treated Ldlr 3KO mice had lower glucose levels than the saline-treated controls at the 30- (40% reduced) and 90-min (21% reduced) time points, whereas the Apoe 3KO leptin group had only a 20% reduction in blood glucose levels at the 90-min time point compared with the saline-treated group. These data suggest that Ldlr 3KO mice are less diabetic and more glucose tolerant than Apoe 3KO mice, and these differences were seen when mice were treated with leptin to restore insulin sensitivity. Interestingly, no improvements were seen in glucose tolerance in the food-restricted mice for both lines despite their improved glucose and insulin levels following a 4-h fast (Table 1). The glucose levels of these mice at the beginning of the glucose tolerance test were significantly elevated compared with the saline- and leptin-treated mice. These higher levels were observed only when the animals had been fasted for 8 h (for 4-h fast, see Table 1) and may be a long-term adaptation to fasting as a consequence of 86 days of food restriction. Indeed, when the glucose tolerance test data were analyzed as a percentage change from baseline (T0), glucose tolerance in the food-restricted groups was significantly improved (vs. saline-treated mice of the same strain, P < 0.01 for Apoe 3KO mice and P < 0.001 for Ldlr 3KO mice, as assessed by area under the curve; data not shown). Altogether, these results indicate that leptin and food restriction are similar in their ability to improve glucose and insulin following a short fasting period but dissimilar in their effect on improving glucose tolerance after a prolonged fast unless baseline glucose values are taken into account.

Leptin treatment improves lipid profile and plaque progression in Apoe 3KO mice. Circulating lipid levels were investigated in both lines of mice (Table 1). In Ldlr 3KO mice, both leptin treatment and food restriction resulted in pronounced reductions in cholesterol levels. Leptin treatment resulted in a 39.2% reduction in cholesterol and food restriction in a 30.0% reduction. Apoe 3KO mice exhibited similar changes with leptin treatment and food restriction. However, the effects of food restriction were more subtle than in Ldlr 3KO mice. Leptin-treated Apoe 3KO mice had 52.6% lower cholesterol levels than the saline-treated controls after 76 days of treatment, whereas food-restricted Apoe 3KO mice had a nonsignificant 18.5% reduction. The cholesterol levels of the leptin-treated mice were significantly lower than the food-restricted mice in the Apoe 3KO line only. Consistent with a reduction in cholesterol levels in the Ldlr 3KO mice, both leptin treatment and food restriction resulted in reduction of circulating triglyceride levels (Table 1). In this case, food restriction had a greater effect on reducing triglyceride levels than leptin treatment (due to a lower calorie intake, which was even lower for the Ldlr 3KO than Apoe 3KO mice). After 76 days of food restriction, a 58% reduction in triglyceride levels was observed compared with control Ldlr 3KO mice, whereas only a 32% reduction was seen in the leptin-treated animals. As described above, Apoe 3KO mice had {approx}300 mg/dl plasma triglycerides, and neither food restriction nor leptin administration resulted in any detectable differences. These data demonstrate that food restriction significantly improves triglyceride levels in LDL-enriched mice; however, leptin is much more potent in lowering cholesterol levels in VLDL-enriched mice.

The more robust effect of leptin in lowering cholesterol in the Apoe 3KO mice than those in the Ldlr 3KO mice led us to investigate the extent of atherosclerotic lesion progression in the two lines. Total plaque sizes, which were measured after 86 days of treatment, were larger in saline-treated Ldlr 3KO mice than in Apoe 3KO mice treated with saline (P = 0.012; Fig. 5, A and B). As expected, most of the lesions were found in the proximal region of the aorta. The lesion sizes in the Ldlr 3KO mice treated with leptin were significantly lower than those in the saline-treated group; the same reduction was seen in the food-restricted mice. Similarly, lesion sizes of the proximal region in the Apoe 3KO mice treated with leptin were significantly decreased compared with the same region in the saline-treated group. In contrast, there were no improvements in lesion sizes in the weight-matched, food-restricted group; lesion area in leptin-treated Apoe 3KO mice was only 36% of the area of food-restricted mice (P < 0.0001).


Figure 5
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  		Fig. 5. Proximal plaque size in Ldlr 3KO and Apoe 3KO mice injected with leptin or food restricted. A: proximal plaque size was measured on day 86 in Ldlr 3KO and Apoe 3KO mice treated with saline, treated with leptin, or food restricted, as described above. Images were collected from pinned-out aortas stained with Sudan IV. B: the %area occupied by the lesions as indicated by Sudan IV staining was quantified from the aortas of several mice. Ldlr 3KO mice injected with leptin (n = 10), injected with saline (n = 13), or food restricted (n = 9). Apoe 3KO mice injected with leptin (n = 7), injected with saline (n = 7), or food restricted (n = 6). All data represent means ± SE. *P < 0.05 vs. saline ad libitum (strain matched) mice; +++P < 0.0001 vs. saline food restricted (strain matched). Aortic lesion area was significantly lower in Apoe 3KO mice than in Ldlr 3KO mice (open bars, saline ad libitum groups; P < 0.05) as determined by a separate analysis using an unpaired 2-tailed Student's t-test assuming unequal variances.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The metabolic syndrome is a cluster of metabolic abnormalities, including central obesity, insulin resistance, atherogenic dyslipidemia (high triglycerides and low HDL cholesterol), hyperglycemia, and hypertension. It has been recognized as a major cause of type 2 diabetes and cardiovascular diseases. Previous animal models present some but not all features of the syndrome. For example, Hasty et al. (14) and Wu et al. (39) have described mouse models of Ldlr–/– crossed with Lepob/ob mice and Apoe–/– crossed with leptin receptor null (Leprdb/db) mice, respectively. Both lines have increased lipid levels and atherosclerotic lesions, but there has been no report on blood pressure with either line (14, 39). In addition, these models contain ApoB-48 in their lipoproteins, which does not fully recapitulate human lipoprotein metabolism because ApoB-48 mRNA is edited from ApoB-100 in the livers of mice but not humans (11). Although this issue was addressed previously in Apoe–/–Apob100/100 and Ldlr–/–Apob100/100 mice (32), these double-knockout mice are intended for studies of lipoprotein metabolism and atherosclerosis and do not exhibit insulin resistance. In this study, our goal was to develop mouse models that exhibit hyperglycemia, insulin resistance, and hyperlipidemia with a lipoprotein profile close to that in humans for use in testing new therapies that target multiple arms of the metabolic syndrome. Given that 1) Lepob/ob mice are one of the most commonly used models for type 2 diabetes research, 2) Apoe–/– and Ldlr–/– mice are the commonly used model for atherosclerosis, and 3) Apob100/100 mice do not make ApoB-48 in their liver, establishing the triple-knockout mice was a logical approach to match our goal. Here we report Ldlr 3KO and Apoe 3KO mice, which recapitulate most characteristics of the metabolic syndrome. However, a direct comparison to the human disorder cannot be made, since these mice are leptin deficient and most humans with metabolic syndrome are not. Furthermore, the species-related differences in lipoprotein biology and dissimilar underlying pathogenic mechanisms between these mice and human metabolic syndrome limit the interpretation of the Apoe and Ldlr 3KO mice as true metabolic syndrome models. Nonetheless, both lines are present with atherosclerotic lesions in their aortas when animals are put on a regular chow diet. Additionally, the Apoe 3KO were diabetic.

Our Ldlr 3KO and Apoe 3KO mouse models share some similarities with those reported by Hasty et al. (14) and Wu et al. (39) with respect to glucose and lipid levels but also have some differences. Our models have lipid profiles much more similar to human lipid profiles. This could be due to the fact that the 3KO lines synthesize lipoproteins with only ApoB-100 and thus more closely resemble human hepatic lipoproteins than other mouse metabolic syndrome models. The advantage of this modification is important in addressing the role of lipoprotein metabolism in the development of atherosclerosis in humans. High-fat feeding mouse models of lipoprotein abnormalities (Ldlr–/– or Apoe–/–) also result in the development of a metabolic syndrome (40). Indeed, a recent publication (6) describing the generation of a leptin-resistant (induced by ectopic expression of the agouti protein) Ldlr-null mouse offers an alternative method for deriving a mouse model with multiple metabolic defects. These mice developed many metabolically relevant phenotypes and perhaps more closely recapitulate the biology of human metabolic syndrome (as evidenced by the elevation of leptin); on the other hand, these mice are neither diabetic nor similar to humans in ApoB-containing lipoproteins. Nonetheless, high-fat-fed agouti-Ldlr–/–, Ldlr 3KO, and Apoe 3KO mice will be complimentary in understanding the metabolic syndrome and providing pharmacological models to assess agents targeting metabolic syndrome.

Although both lines of mice presented here were obese, body weight and appetite were greater in Apoe 3KO mice. Furthermore, these mice were hyperglycemic, more insulin resistant, and less glucose tolerant when compared with Ldlr 3KO mice. Because these two lines of mice were very different in lipoprotein profiles, it is possible that the lipid profile of the Apoe 3KO mice (elevated VLDL) leads to worsened obesity and more severe diabetes. In keeping with this hypothesis, VLDL receptor null mice are protected from diet-induced obesity and hyperinsulinemia (10). The interpretation of this study implicated VLDL in the transport of fatty acids from the circulation to peripheral tissues, and thus it is possible that VLDL excess in the case of the Apoe 3KO mice resulted in increased obesity and insulin resistance. Furthermore, it has recently been shown (21) that increased VLDL secretion precedes the development of diabetes in California mice.

Another possible explanation for the difference between the two strains is that VLDL excess also has deleterious effects on the survival and function of the pancreatic β-cells, as seen by injection of VLDL into mice (29), and may explain the hyperglycemia in the Apoe 3KO animals. Each of these explanations relies on the deficiency in leptin signaling for the manifestation of diabetes and obesity in the Apoe 3KO mice, since these phenotypes do not develop in the single-knockout Apoe–/– mice. It is also possible that these two lines of mice are unequally matched for genetic background, and this may be responsible for the observed effects. Although we attempted to achieve genetic homogeneity in the backcrossing of the double-knockout mice (already backcrossed 5 times to C57BL/6) by one generation to Lepob/+ mice, we discovered that the backgrounds were not similar as expected. Ldlr 3KO mice and Apoe 3KO mice are 94.7 and 74.6% C57BL/6, respectively, as determined by single-nucleotide polymorphism analysis (data not shown). To rule out the possibility of genetic background influences on these parameters, future congenic breeding will be required. As previously described (34), total cholesterol levels of the double-knockout mice Apoe–/–Apob100/100 and Ldlr–/–Apob100/100 were very similar ({approx}250 mg/dl). In these two lines of 3KOs, we also saw a similarity in the total cholesterol levels, but at much higher levels (>900 mg/dl). The ob mutation did not alter the lipoprotein characteristics of the Apoe–/–Apob100/100 (preponderance of VLDL) and Ldlr–/–Apob100/100 (preponderance of LDL); however, HDL was additionally elevated.

Even at a young age of 11–12 wk on a normal chow diet, both lines of mice developed lesions in their aortic regions, with fewer lesions found in the proximal and distal regions of the aorta. Also, we believe that there are significantly more lesions in both lines than in the Apoe–/–Apob100/100 and Ldlr–/–Apob100/100 in our previous report (34). Schreyer et al. (30) showed that Ldlr–/– mice were susceptible to diet-induced obesity (diet rich in saturated fat and sucrose), dyslipidemia, and accelerated atherosclerosis. In contrast, fat- and sucrose-fed Apoe–/– mice exhibited no increased propensity for dyslipidemia and atherosclerosis beyond what was observed for chow-fed Apoe–/– mice. In the study conducted by Hasty et al. (14), Ldlr–/–Lepob/ob mice displayed signs of lesions in the aortic sinus and the aortic arch, whereas the Ldlr+/–Lepob/ob mice were completely lesion free. Our findings of worsened lesion areas in Ldlr 3KO mice are consistent with these studies.

Both Ldlr 3KO and Apoe 3KO mice were hypertensive at 11–12 wk of age. Enalapril treatment normalized blood pressure in these two lines. It has been reported (42) that Apoe–/– mice exhibit hypertension at 7.5 mo, whereas no hypertension was found at a younger age. In addition, Ldlr–/–Lepob/ob mice have been found to be hypertensive when blood pressure was measured directly using a telemetric system (35). The indirect tail-cuff method of monitoring blood pressure has limitations because the animals must be trained for repeated handling when placed on a heated pad and restrained. To adjust for these confounding factors we performed our blood pressure measurements in a very controlled manner (time on the heated pad, size of the restrainers, and no. of measurement cycles). Also, direct vs. indirect blood pressure comparisons in mice have been reported to be, for the most part, correlative (38). Our data, generated from modified equipment and training acclimated mice, demonstrated that both lines of 3KO are hypertensive and responsive to the angiotensin-converting enzyme inhibitor enalapril treatment.

A second part of this study focused on the effects of low-dose leptin therapy in these two lines of 3KO mice to understand the role of leptin in modulating lipid homeostasis and atherosclerotic plaques. Low doses of leptin were chosen to maintain a constant body weight in the Ldlr 3KO and Apoe 3KO mice, which allowed us to make the assumption that leptin was administered at physiological levels. Improvement in glucose homeostasis was apparent in both lines of mice when treated with leptin. Apoe 3KO mice treated with leptin became normoglycemic but remained hyperinsulinemic, whereas leptin-treated Ldlr 3KO mice maintained normal glucose levels and did not become as hyperinsulinemic as the Ldlr 3KO saline ad libitum controls. We found that the improvement in glucose levels (Apoe 3KO mice) and insulin levels (Ldlr 3KO mice) were most likely attributable to the reduction in body weight, as evidenced by similar changes in the weight-matched, food-restricted mice. A reduction in caloric intake is known to reduce the severity of diabetes in humans (19) as well as several animal models of obesity and diabetes (1, 7, 22) and likely explains the observations seen in the food-restricted Ldlr 3KO and Apoe 3KO mice. Although the effects of food restriction and leptin treatment were similar on blood glucose and insulin, there was a clear difference in glucose tolerance. Only Ldlr 3KO mice treated with leptin were more glucose tolerant than the saline-treated controls. In fact, in both lines the food-restricted groups were initially hyperglycemic after the 8-h fast. This phenomenon could be explained by a physiological adaptation to the 86 days of food restriction. Indeed, chronic food restriction has been shown to increase gluconeogenesis under fasting conditions (9).

In both lines of mice, leptin significantly decreased cholesterol levels. Interestingly, it has been reported that leptin treatment reduces HDL in Lepob/ob mice (16) and improves bile acid elimination (17). The reduction in cholesterol levels observed with leptin might be related to an improvement in plasma HDL cholesterol elimination. It is also possible that leptin exerts its effects by reducing the VLDL or LDL levels in both lines of mice. A notable difference in this study was the potent effect of leptin on lowering cholesterol levels in the Apoe 3KO mice to a far greater extent than food restriction alone or in Ldlr 3KO mice. These data suggest that leptin may exert direct effects on the metabolism of VLDL in the setting of VLDL enrichment and leptin deficiency. Additionally, impaired VLDL clearance has recently been described in Lepob/obLdlr–/– mice, exacerbated by the absence of leptin compared with Ldlr–/– mice (5).

Consistent with the reduction in plasma cholesterol, the extent of atherosclerotic lesion formation was also decreased in leptin-treated animals. In both Apoe 3KO and Ldlr 3KO mice, leptin resulted in a greater reduction in atherosclerosis than food restriction. This effect was far more pronounced in the Apoe 3KO mice than in the Ldlr 3KO mice and correlated closely with the more potent effect of leptin on plasma cholesterol. It is possible that leptin not only reduces plaques by decreasing VLDL but also directly modulates lesion progression. However, this mechanism may be unlikely, since leptin has been suggested to cause inflammation-induced atherogenic events and has been found to be a risk factor in clinical trials (2).

Recently, it has been reported (4) that leptin deficiency suppresses atherosclerosis in Lepob/obApoe–/– and leptin treatment in Apoe–/– mice increases lesion area. Our data do not agree with this report but confirm the findings of numerous other studies carried out by Coenen et al. (5), Gruen et al. (12), and others (25, 35) where Lepob/ob mice were bred with either Ldlr–/– or Apoe–/– animals. Furthermore, leptin receptor-null mice lacking Apoe or Ldlr also develop more lesions (5, 39), indicating that defective leptin signaling results in increased lesion formation, and high concentrations of leptin (as expected in a Lepdb/db animal) do not protect from atherosclerosis. It is noteworthy to mention that, in the study by Chiba et al. (4), there was no effect of leptin in Lepob/obApoe–/– mice.

In summary, we have shown the implementation of two new mouse models with the phenotypes of the metabolic syndrome combining obesity, hyperglycemia, hyperinsulinemia, hyperlipidemia, and hypertension. The two lines have different lipid profiles and thus can be used for different purposes, e.g., VLDL vs. LDL cholesterol-lowering studies. We also showed that leptin is capable of reducing many of the metabolic disturbances created in these two models. Furthermore, we identified a potential role of leptin in cholesterol metabolism that may be elicited through VLDL modulation. Altogether, these data highlight the potential of both Apoe 3KO and Ldlr 3KO mice not only for testing newly emerging therapies for the metabolic syndrome but also in exploring biological mechanisms of glucose and lipid homeostasis.


   ACKNOWLEDGMENTS
 
We thank Dr. George Carlson for help on the genetic single nucleotide polymorphism analysis and Tom Graves for statistical analyses.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. M. Véniant, Amgen, Inc., One Amgen Centre Dr., Thousand Oaks, CA 91320 (e-mail: mveniant{at}amgen.com)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Barzilai N, Banerjee S, Hawkins M, Chen W, Rossetti L. Caloric restriction reverses hepatic insulin resistance in aging rats by decreasing visceral fat. J Clin Invest 101: 1353–1361, 1998.[Web of Science][Medline]
  2. Beltowski J. Leptin and atherosclerosis. Atherosclerosis 189: 47–60, 2006.[CrossRef][Web of Science][Medline]
  3. Camus MC, Chapman MJ, Forgez P, Laplaud PM. Distribution and characterization of the serum lipoproteins and apoproteins in the mouse, Mus musculus. J Lipid Res 24: 1210–1228, 1983.[Abstract]
  4. Chiba T, Shinozaki S, Nakazawa T, Kawakami A, Ai M, Kaneko E, Kitagawa M, Kondo K, Chait A, Shimokado K. Leptin deficiency suppresses progression of atherosclerosis in apoE-deficient mice. Atherosclerosis 196: 68–75, 2008.[CrossRef][Medline]
  5. Coenen KR, Gruen ML, Hasty AH. Obesity causes very low density lipoprotein clearance defects in low-density lipoprotein receptor-deficient mice. J Nutr Biochem 18: 727–735, 2007.[CrossRef][Web of Science][Medline]
  6. Coenen KR, Hasty AH. Obesity potentiates development of fatty liver and insulin resistance, but not atherosclerosis, in high-fat diet-fed agouti LDLR-deficient mice. Am J Physiol Endocrinol Metab 293: E492–E499, 2007.[Abstract/Free Full Text]
  7. Dhahbi JM, Mote PL, Wingo J, Rowley BC, Cao SX, Walford RL, Spindler SR. Caloric restriction alters the feeding response of key metabolic enzyme genes. Mech Ageing Dev 122: 1033–1048, 2001.[CrossRef][Web of Science][Medline]
  8. Farese RV Jr, Véniant MM, Cham CM, Flynn LM, Pierotti V, Loring JF, Traber M, Ruland S, Stokowski RS, Huszar D, Young SG. Phenotypic analysis of mice expressing exclusively apolipoprotein B48 or apolipoprotein B100. Proc Natl Acad Sci USA 93: 6393–6398, 1996.[Abstract/Free Full Text]
  9. Feuers RJ, Duffy PH, Leakey JA, Turturro A, Mittelstaedt RA, Hart RW. Effect of chronic caloric restriction on hepatic enzymes of intermediary metabolism in the male Fischer 344 rat. Mech Ageing Dev 48: 179–189, 1989.[CrossRef][Web of Science][Medline]
  10. Goudriaan JR, Tacken PJ, Dahlmans VE, Gijbels MJ, van Dijk KW, Havekes LM, Jong MC. Protection from obesity in mice lacking the VLDL receptor. Arterioscler Thromb Vasc Biol 21: 1488–1493, 2001.[Abstract/Free Full Text]
  11. Greeve J, Altkemper I, Dieterich JH, Greten H, Windler E. Apolipoprotein B mRNA editing in 12 different mammalian species: hepatic expression is reflected in low concentrations of apoB-containing plasma lipoproteins. J Lipid Res 34: 1367–1383, 1993.[Abstract]
  12. Gruen ML, Saraswathi V, Nuotio-Antar AM, Plummer MR, Coenen KR, Hasty AH. Plasma insulin levels predict atherosclerotic lesion burden in obese hyperlipidemic mice. Atherosclerosis 186: 54–64, 2006.[CrossRef][Web of Science][Medline]
  13. Hanley AJ, Karter AJ, Williams K, Festa A, D'Agostino RB Jr, Wagenknecht LE, Haffner SM. Prediction of type 2 diabetes mellitus with alternative definitions of the metabolic syndrome: the Insulin Resistance Atherosclerosis Study. Circulation 112: 3713–3721, 2005.[Abstract/Free Full Text]
  14. Hasty AH, Shimano H, Osuga J, Namatame I, Takahashi A, Yahagi N, Perrey S, Iizuka Y, Tamura Y, Amemiya-Kudo M, Yoshikawa T, Okazaki H, Ohashi K, Harada K, Matsuzaka T, Sone H, Gotoda T, Nagai R, Ishibashi S, Yamada N. Severe hypercholesterolemia, hypertriglyceridemia, and atherosclerosis in mice lacking both leptin and the low density lipoprotein receptor. J Biol Chem 276: 37402–37408, 2001.[Abstract/Free Full Text]
  15. Horton JD, Shimano H, Hamilton RL, Brown MS, Goldstein JL. Disruption of LDL receptor gene in transgenic SREBP-1a mice unmasks hyperlipidemia resulting from production of lipid-rich VLDL. J Clin Invest 103: 1067–1076, 1999.[Web of Science][Medline]
  16. Hyogo H, Roy S, Cohen DE. Restoration of gallstone susceptibility by leptin in C57BL/6J ob/ob mice. J Lipid Res 44: 1232–1240, 2003.[Abstract/Free Full Text]
  17. Hyogo H, Roy S, Paigen B, Cohen DE. Leptin promotes biliary cholesterol elimination during weight loss in ob/ob mice by regulating the enterohepatic circulation of bile salts. J Biol Chem 277: 34117–34124, 2002.[Abstract/Free Full Text]
  18. Ishibashi S, Brown MS, Goldstein JL, Gerard RD, Hammer RE, Herz J. Hypercholesterolemia in low density lipoprotein receptor knockout mice and its reversal by adenovirus-mediated gene delivery. J Clin Invest 92: 883–893, 1993.[Web of Science][Medline]
  19. Kelley DE, Wing R, Buonocore C, Sturis J, Polonsky K, Fitzsimmons M. Relative effects of calorie restriction and weight loss in noninsulin-dependent diabetes mellitus. J Clin Endocrinol Metab 77: 1287–1293, 1993.[Abstract]
  20. Krauss RM. Atherogenicity of triglyceride-rich lipoproteins. Am J Cardiol 81: 13B–17B, 1998.[CrossRef][Web of Science][Medline]
  21. Krugner-Higby L, Shelness GS, Holler A. Heritable, diet-induced hyperlipidemia in California mice (Peromyscus californicus) is due to increased hepatic secretion of very low density lipoprotein triacylglycerol. Comp Med 56: 468–475, 2006.[Web of Science][Medline]
  22. Lane MA, Tilmont EM, De Angelis H, Handy A, Ingram DK, Kemnitz JW, Roth GS. Short-term calorie restriction improves disease-related markers in older male rhesus monkeys (Macaca mulatta). Mech Ageing Dev 112: 185–196, 2000.[CrossRef][Web of Science][Medline]
  23. Massi-Benedetti M. Changing targets in the treatment of type 2 diabetes. Curr Med Res Opin 22, Suppl 2: 5–13, 2006.
  24. Masucci-Magoulas L, Goldberg IJ, Bisgaier CL, Serajuddin H, Francone OL, Breslow JL, Tall AR. A mouse model with features of familial combined hyperlipidemia. Science 275: 391–394, 1997.[Abstract/Free Full Text]
  25. Mertens A, Verhamme P, Bielicki JK, Phillips MC, Quarck R, Verreth W, Stengel D, Ninio E, Navab M, Mackness B, Mackness M, Holvoet P. Increased low-density lipoprotein oxidation and impaired high-density lipoprotein antioxidant defense are associated with increased macrophage homing and atherosclerosis in dyslipidemic obese mice: LCAT gene transfer decreases atherosclerosis. Circulation 107: 1640–1646, 2003.[Abstract/Free Full Text]
  26. Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, Collins F. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269: 540–543, 1995.[Abstract/Free Full Text]
  27. Plump AS, Smith JD, Hayek T, Aalto-Setala K, Walsh A, Verstuyft JG, Rubin EM, Breslow JL. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell 71: 343–353, 1992.[CrossRef][Web of Science][Medline]
  28. Pollex RL, Hegele RA. Genetic determinants of the metabolic syndrome. Nat Clin Pract Cardiovasc Med 3: 482–489, 2006.[CrossRef][Web of Science][Medline]
  29. Roehrich ME, Mooser V, Lenain V, Herz J, Nimpf J, Azhar S, Bideau M, Capponi A, Nicod P, Haefliger JA, Waeber G. Insulin-secreting beta-cell dysfunction induced by human lipoproteins. J Biol Chem 278: 18368–18375, 2003.[Abstract/Free Full Text]
  30. Schreyer SA, Lystig TC, Vick CM, LeBoeuf RC. Mice deficient in apolipoprotein E but not LDL receptors are resistant to accelerated atherosclerosis associated with obesity. Atherosclerosis 171: 49–55, 2003.[CrossRef][Medline]
  31. Segrest JP. The role of non-LDL:non-HDL particles in atherosclerosis. Curr Diab Rep 2: 282–288, 2002.[Medline]
  32. Smith SR. Importance of diagnosing and treating the metabolic syndrome in reducing cardiovascular risk. Obesity (Silver Spring) 14, Suppl 3: 128S–134S, 2006.
  33. Tangirala RK, Rubin EM, Palinski W. Quantitation of atherosclerosis in murine models: correlation between lesions in the aortic origin and in the entire aorta, and differences in the extent of lesions between sexes in LDL receptor-deficient and apolipoprotein E-deficient mice. J Lipid Res 36: 2320–2328, 1995.[Abstract]
  34. Véniant MM, Sullivan MA, Kim SK, Ambroziak P, Chu A, Wilson MD, Hellerstein MK, Rudel LL, Walzem RL, Young SG. Defining the atherogenicity of large and small lipoproteins containing apolipoprotein B100. J Clin Invest 106: 1501–1510, 2000.[Web of Science][Medline]
  35. Verreth W, De Keyzer D, Pelat M, Verhamme P, Ganame J, Bielicki JK, Mertens A, Quarck R, Benhabiles N, Marguerie G, Mackness B, Mackness M, Ninio E, Herregods MC, Balligand JL, Holvoet P. Weight-loss-associated induction of peroxisome proliferator-activated receptor-alpha and peroxisome proliferator-activated receptor-gamma correlate with reduced atherosclerosis and improved cardiovascular function in obese insulin-resistant mice. Circulation 110: 3259–3269, 2004.[Abstract/Free Full Text]
  36. Wagh A, Stone NJ. Treatment of metabolic syndrome. Expert Rev Cardiovasc Ther 2: 213–228, 2004.[CrossRef][Medline]
  37. Wassmann S, Czech T, van Eickels M, Fleming I, Bohm M, Nickenig G. Inhibition of diet-induced atherosclerosis and endothelial dysfunction in apolipoprotein E/angiotensin II type 1A receptor double-knockout mice. Circulation 110: 3062–3067, 2004.[Abstract/Free Full Text]
  38. Whitesall SE, Hoff JB, Vollmer AP, D'Alecy LG. Comparison of simultaneous measurement of mouse systolic arterial blood pressure by radiotelemetry and tail-cuff methods. Am J Physiol Heart Circ Physiol 286: H2408–H2415, 2004.[Abstract/Free Full Text]
  39. Wu KK, Wu TJ, Chin J, Mitnaul LJ, Hernandez M, Cai TQ, Ren N, Waters MG, Wright SD, Cheng K. Increased hypercholesterolemia and atherosclerosis in mice lacking both ApoE and leptin receptor. Atherosclerosis 181: 251–259, 2005.[CrossRef][Medline]
  40. Wu L, Vikramadithyan R, Yu S, Pau C, Hu Y, Goldberg IJ, Dansky HM. Addition of dietary fat to cholesterol in the diets of LDL receptor knockout mice: effects on plasma insulin, lipoproteins, and atherosclerosis. J Lipid Res 47: 2215–2222, 2006.[Abstract/Free Full Text]
  41. Wubben DP, Adams AK. Metabolic syndrome: what's in a name? WMJ 105: 17–20, 2006.[Medline]
  42. Yang R, Powell-Braxton L, Ogaoawara AK, Dybdal N, Bunting S, Ohneda O, Jin H. Hypertension and endothelial dysfunction in apolipoprotein E knockout mice. Arterioscler Thromb Vasc Biol 19: 2762–2768, 1999.[Abstract/Free Full Text]
  43. Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science 258: 468–471, 1992.[Abstract/Free Full Text]




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