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ORIGINAL ARTICLE |
From the Departments of Psychology (J.A.G., A.S., J.P., C.C., M.M.L., N.S., P.M.M), Medicine (A.M., R.B.G.), and Pathology (J.Z.), University of Miami, Coral Gables, and the University of Miami School of Medicine, Miami, FL; and the Department of Neurobiology and Physiology (J.E.L.), Northwestern University, Evanston, IL.
Address correspondence and reprint requests to Philip M. McCabe, PhD, Department of Psychology, University of Miami, P.O. Box 248185, Coral Gables, FL 33124. E-mail: pmccabe{at}miami.edu
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ABSTRACT |
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Methods: Forty-two WHHLs were assigned to a dietary, exercise, or control condition, and 12 NZWs were used as a comparison control group. The intervention ran from 3 to 7 months of age, and all animals received an intravenous glucose tolerance test at the beginning and end of the intervention.
Results: WHHLs were insulin-resistant relative to NZWs at 3 months of age. Whereas the dietary intervention was effective in controlling insulin resistance, WHHLs in the exercise group without dietary restriction and the control group exhibited significant increases in insulin resistance. No intervention significantly influenced the progression of atherosclerosis.
Conclusions: Young WHHLs are insulin-resistant during an early period when atherosclerosis is developing rapidly. Dietary restriction, but not exercise without weight control, is effective in controlling insulin metabolic variables in the WHHL model. Although dietary intervention can reduce cardiovascular risk factors such as insulin resistance, it is not effective in slowing the development of atherosclerosis in these genetically dyslipidemic animals. Similarly, exercise training, without dietary control, does not influence the progression of disease in WHHLs.
Key Words: diet • exercise • insulin resistance • WHHL rabbit • atherosclerosis
Abbreviations: WHHL = Watanabe heritable hyperlipidemic rabbit; VLDL = very-low-density lipoprotein; LDL = low-density lipoprotein; HDL = high-density lipoprotein; NZW = New Zealand white rabbit; IVGTT = intravenous glucose tolerance test; HOMA = Homeostasis Model Assessment; CV = coefficient of variation; SBP = systolic blood pressure; MAP = mean arterial pressure; DBP = diastolic blood pressure; HR = heart rate; BMI = body mass index; CNS = central nervous system.
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INTRODUCTION |
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It has been proposed that insulin resistance is associated with a clustering of metabolic disorders, including obesity, hyperinsulinemia, lipid abnormalities, atherosclerotic disease, and hypertension (5). In humans, insulin resistance is associated with an atherogenic lipid profile, such that resistance to the action of insulin leads to enhanced very-low-density lipoprotein (VLDL) synthesis, resulting in hypertriglyceridemia, small, dense low-density lipoprotein (LDL) and reduced high-density lipoprotein (HDL) levels (5). Insulin resistance, independent of its effects on plasma lipids and blood pressure, has direct effects on vascular tissue, inhibiting endothelial vasodilatation through reduced nitric oxide and increased endothelin activity (6). This promotes a procoagulant state by increasing the production of PAI-1, enhancing the action of angiotensin II, which is both a vasoconstrictor and a proproliferative hormone in the vascular wall, and raising levels of oxidative stress, thereby inducing a proinflammatory state. All of these actions would be expected to contribute to an acceleration of processes known to be important in atherogenesis.
Schneiderman and Skyler (7) proposed a pathway for atherogenesis that is based on an interactive relation among insulin resistance, hyperinsulinemia, and sympathetic tone. According to this notion, social and emotional factors can interact with insulin metabolic variables to promote the development of cardiovascular disease. Importantly, it has been shown in humans that behavioral interventions such as caloric restriction in overweight patients or regular physical exercise can improve tissue sensitivity to insulin and reduce blood pressure (5). In the recent Diabetes Prevention Program, it was reported that a lifestyle intervention, including weight loss and increased physical activity, improved glycemic control and reduced the incidence of type 2 diabetes in persons at high risk for the disease (8).
Given the accumulating evidence that insulin metabolic variables may play an important role in human disease, and that lifestyle interventions are effective in treating these risk factors, the present study sought to examine these issues in the WHHL model. Specifically, the purpose of this study was to: 1) determine if young, individually caged WHHLs are insulin-resistant relative to a control strain of rabbits during the early period of disease progression, and 2) assess whether dietary or exercise interventions in young individually caged WHHLs can improve insulin metabolic variables and slow the progression of atherosclerosis in this animal model of disease.
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METHODS |
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The rabbits were housed in individual cages (6 sq ft) under a 12-hour light/dark cycle (lights on at 7:00 am). The animals were given water ad libitum and were fed a standard rabbit chow (Purina, 2.5% total fat, 0% cholesterol) according to their experimental group (see "Experimental Design"). Animals were fed at the same time each day, and any uneaten food from the previous day was weighed and subtracted from the total food provided to calculate the food consumed per day. During the course of the experiment, 2 WHHLs died from causes other than heart disease or aortic atherosclerosis, and the data from these animals were eliminated from the study.
Experimental Design
All rabbits were acclimated to the experimental procedures for 2 weeks. The experiment began when the rabbits were 3 months of age and was continued until the animals were 7 months old. At the beginning of the study, the WHHLs were assigned to 1 of 3 experimental groups. The WHHL control group (n = 14) was fed ad libitum and received no exercise training during the study. The exercise group (n = 14) was fed ad libitum but was exercised using a rabbit treadmill. Using a modified version of a protocol developed previously for rabbits (9), the exercise group was trained to run 12.6 m/min for 60 minutes per day 5 days per week. Exercise training consisted of gradually increasing the treadmill speed and the duration of training over the first 4 weeks of the study until the rabbits were running at 12.6 m/min for 60 minutes per day. It has been demonstrated that this intensity and duration of exercise enhances aerobic fitness in rabbits (9). The diet group (n = 12) was not exercised but was fed a restricted quantity of rabbit chow. These WHHLs received 4 oz (80 g) of rabbit chow for the first 4 weeks of the study and 5 oz (100 g) for the remainder of the study. These are age-appropriate amounts of food (10), and over the course of the study, the diet group consumed approximately 25% less food than the other groups on a daily basis. Using this design, we were able to assess independently the effects of diet or exercise on insulin metabolic variables and atherosclerosis in WHHLs. A fourth group, the NZW control group (n = 12), consisted of NZW rabbits that were fed ad libitum and not exercised (ie, comparable to the WHHL control group).
Blood Sampling, Intravenous Glucose Tolerance Test and Biochemical Assays
Blood was drawn from the marginal ear vein in loosely restrained animals at 3 and 7 months of age for the measurement of plasma corticosterone and cortisol, as well as fasted lipids, leptin, glucose, and insulin. Because rabbit glucocorticoids follow a circadian rhythm that is 6 to 12 hours out of phase with the human glucocorticoid cycle (11); blood was drawn for cortisol and corticosterone measurement between 1:00 and 3:00 pm. Fasted samples were drawn at 7:00 am after an 8-hour overnight fast.
Intravenous glucose tolerance tests (IVGTT) were performed for all animals at 3 and 7 months of age. For the IVGTT, a solution of 0.6 g glucose per kilogram body weight was injected into the marginal ear vein, and blood was drawn at 0, 15, 30, 45, 60, 75, and 90 minutes after the injection. At the end of the IVGTT, the animals were rehydrated with a bolus of sterile saline injected subcutaneously. Because 2 mL of blood was sampled at each time point, more frequent sampling of blood was not possible without potential harm to the rabbit. The number and frequency of IVGTT samples did not permit the use of the Bergman Minimal Model (12) to assess insulin sensitivity. Instead, insulin sensitivity was determined through the Homeostasis Model Assessment (HOMA (13)) and by fasting baseline insulin values. Both of these measures have been shown to provide reliable estimates of insulin sensitivity (14–18). For the calculation of HOMA, glucose values were converted into millimolar concentrations, and thus the formula used was: fasting glucose (mmoles/L) x fasting insulin (µIU/mL)/22.5.
The plasma was separated by centrifugation at 4°C, drawn off, and stored in aliquots at –80°C until assay. Insulin and leptin were measured by radioimmunoassay using a rat insulin assay (Linco Research #RI-13K, using rat insulin standards) and multispecies leptin assay (Linco Research, #XL-85K, using human leptin standards), respectively. The intra- and interassay coefficients of variation (CV) for the insulin assay were 4.3% and 9.1%, respectively, and limit of detection was 0.1 ng/mL. For the leptin assay, the intra- and interassay CV were 3.6% and 8.7%, respectively, and the limit of detection was 1.0 ng/mL. Cholesterol, triglycerides, and glucose were measured using an enzymatic assay (Roche Diagnostics). HDL cholesterol was measured through the same cholesterol assay after precipitation of apoB-containing lipoproteins. The intra- and interassay CVs for cholesterol, HDL cholesterol, and triglycerides were 1.8% and 2.8%, 3.2% and 5.6%, and 1.1% and 3.3%, respectively. The limit of detection was 5 mg/dL for cholesterol, 2 mg/dL for HDL cholesterol, and 10 mg/dL for triglycerides. For glucose, the limit of detection was 5.0 mg/dL, and the intra- and interassay CVs were 1.6% and 3.6%, respectively. The assays for corticosterone and cortisol were performed using commercially available radioimmunoassay kits (ICN Biomedicals, Costa Mesa, CA). The intra- and interassay CVs for corticosterone were 4.6% and 9.6%, respectively, and for cortisol, they were 3.8% and 10.5%, respectively. The limit of detection for corticosterone was 5.7 ng/mL, and for cortisol, it was 0.2 µg/dL.
Urine Sampling and Biochemical Assays
Twenty-four-hour urine samples were collected in all animals at 3 and 7 months of age for the measurement of epinephrine and norepinephrine. Animals were placed in a metabolic cage and urine samples were collected on ice and acidified with HCl to a final concentration of 5.0 mmol/L as a preservative, drawn off in aliquots, and stored at –80°C until assay. Urinary catecholamines were assayed using a radioimmunoassay kit (ICN Pharmaceuticals, Inc.) following the manufacturer’s instructions for extraction and assay. The intra- and interassay CVs for epinephrine were 4.6% and 6.1%, respectively, and for norepinephrine, they were 4.6% and 6.1%, respectively. Urinary creatinine was measured with the Jaffe reagent (Roche Diagnostics). This analysis was performed using an automated clinical chemistry analyzer (Roche Cobas-Mira). The limit of detection for the creatinine assay was 3.0 mg/dL, and intra- and interassay CVs were 1.4% and 2.1%, respectively.
Cardiovascular Measures
At 3 and 7 months of age, resting systolic blood pressure (SBP), mean arterial pressure (MAP), diastolic blood pressure (DBP), and heart rate (HR) were measured in loosely restrained animals by an automated tail-cuff system (model 29SSP; IITC, Inc., Woodland Hills, CA). Three separate measures of blood pressure and HR were taken, and the daily mean for each measure was computed.
Morphometric Measures
Body weight was measured weekly to the nearest 0.1 kg. The length (cm) of the rabbit from the tip of the snout to the base of the tail was used as a measure of height to calculate body mass index (BMI; h/wt2). BMI measures were obtained at 3 and 7 months of age.
Histomorphometic Methods and Measures
After euthanasia, the entire aorta was removed through a midline thoracic and abdominal incision and the tissue was placed in a 10% solution of buffered formalin. In addition, all visceral and mesenteric fat, including the retroperitoneal fat pads, were removed and weighed (g). Fixed tissue samples were coded, and all histomorphometric procedures were performed in a blind fashion. Under gross examination, the 2-dimensional area of each aortic lesion from each animal was calculated by multiplying the length and width of the lesion in millimeters. For the entire extent of the aorta, each lesion that was identified by gross examination was sectioned at the point of maximum height or at midlesion. For sectioning, the tissue was paraffin-embedded, sectioned at 5 µM, stained with hematoxylin & eosin, and observed under light microscopy. In the absence of a gross lesion, the aorta was sectioned at predetermined locations for histopathology.
Statistical Analyses
To address the major research questions, 2 separate analyses were performed. First, the WHHL control group was compared with the NZW control group at 3 months of age and again at 7 months to determine whether WHHLs are insulin-resistant relative to NZWs at an early stage of disease progression (analysis 1). Second, the 3 WHHL groups (exercise, diet, control) were compared at 3 months and at 7 months to determine if behavioral interventions influence insulin metabolic variables and atherosclerosis in this genetically predisposed animal model of disease (analysis 2). Because of the small sample size and the large variability in many of the dependent measures, as well as the unreliability of measuring change over time, we did not use a repeated-measures design. Instead, baseline measures were compared across groups at 3 months of age, and then we focused on the treatment outcome at 7 months of age. For both analysis 1 and analysis 2, insulin and glucose data could not be obtained from 1 WHHL, and thus there was 1 less total number of subjects for these analyses.
For both sets of analyses, analysis of variance with treatment as the between-groups factor was used to examine group differences in morphometric, hormonal, glucose, lipid, and cardiovascular measures, as well as atherosclerosis. Significant treatment effects were followed with interactions were followed with Tukey’s post hoc tests (p <.05 for significance).
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RESULTS |
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By 7 months of age, the NZWs were still significantly heavier (F [1,24] = 65.3, p <.001) and consumed more food that the WHHLs (F [1,23] = 53.8, p <.001). In contrast to the baseline measures, at 7 months, there were no significant group differences in visceral/mesenteric fat (F [1,24] = 3.8, p >.05) or in plasma leptin (F [1,24] = 1.7, p >.05). There were no significant group differences at 7 months of age in insulin (F [1,24] = 1.2, p >.05), HOMA (F [1,24] = 0.8, p >.05), or plasma glucose (F [1,24] = 0.2, p >.05), which was presumably the result of the large weight gain in both groups of rabbits (Table 1, Fig. 1). SBP remained elevated in the WHHLs relative to the NZWs (F [1,24] = 4.8, p <.05), but there were no group differences in catecholamines or glucocorticoids at 7 months of age. Finally, WHHLs exhibited significant aortic atherosclerosis, whereas disease was virtually absent in the NZWs (F [1,24] = 38.8, p <.001).
Analysis 2
In this analysis, the 3 WHHL groups (exercise, diet, control) were examined at 3 months and 7 months of age to determine if common behavioral interventions can influence insulin metabolic variables and the progression of atherosclerosis in this model of disease. Before the interventions were begun (ie, at 3 months of age), the groups were similar on morphometric variables, lipids, leptin, food consumption, cardiovascular measures, and glucocorticoids (Table 2). At this preintervention time point, the WHHL control group did exhibit significantly greater urinary epinephrine values compared with the exercise group (F [2,37] = 4.3, p <.05) and greater norepinephrine values relative to the diet group (F [2,37] = 5.1, p <.05). For the preintervention IVGTT, there were no significant differences among the groups for insulin or glucose at any of the time points (Fig. 2), nor were there any group differences in the indices of insulin sensitivity (Table 2) (insulin, [F{2,36} = 0.9, p >.05]; HOMA, [F{2,36} = 0.3, p >.05]).
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After the 4-month behavioral intervention (ie, at 7 months of age), the diet group weighed less (F [2,37] = 10.5, p <.001), had lower BMI (F [2,37] = 8.7, p <.001), and less visceral fat (F [2,37] = 25.9, p <.001) than the other 2 freely feeding groups (Table 2). The average weekly food consumption for the diet group was less than the other groups (F [2,37] = 8.4, p <.001), and plasma leptin was also significantly lower in this group (F [2,37] = 11.0, p <.001). After 4 months of dietary restriction, the plasma total cholesterol was significantly higher in the diet group than the other groups (F [2,37] = 14.6, p <.001). Plasma HDL and triglycerides were not significantly different among the groups postintervention. The dietary invention also significantly reduced heart rate relative to heart rate in WHHL control group (Tukey, p <.05); however, there were no significant differences in the resting blood pressures among the 3 groups. Moreover, there were no significant group differences in plasma glucocorticoids or urinary catecholamines at the end of the study.
The exercise group also exhibited a significant decrease in heart rate compared with the WHHL control group (Table 2; Tukey, p <.05), suggesting that the daily exercise produced an aerobic training effect. Although these animals exercised for 60 minutes per day 5 days per week for 4 months, their body weight, BMI, and visceral fat measures were not significantly different from the WHHL control group. Similarly, the weekly food consumption and plasma leptin levels at the end of the study were not significantly different in the exercise group relative to the controls.
After the 4-month behavioral intervention, the diet group exhibited significantly lower insulin resistance relative to the other 2 groups (Table 2), as illustrated by the HOMA index (F [2,37] = 5.4, p <.01) and insulin values (F [2,37] = 5.8, p <.01). In addition, the insulin values for the diet group were significantly lower at each time point during the postintervention IVGTT (Fig. 2). The exercise group and the WHHL control group did not differ on measures of insulin sensitivity.
Finally, although the diet intervention had significant effects on morphometric, cardiovascular, and insulin sensitivity measurements, and the exercise intervention influenced heart rate, all 3 groups developed aortic atherosclerosis (Table 2). Importantly, there were no significant differences in the total area of aortic atherosclerosis among the 3 groups (F [2,37] = 0.1, p >.05). Similarly, there were no significant differences in the amount of disease within the aortic arch (F [2,37] = 0.3, p >.05), thoracic aorta (F [2,37] = 0.2, p >.05), abdominal aorta (F [2,37] = 0.02, p >.05), or iliac arteries (F [2,37] = 0.7, p >.05).
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DISCUSSION |
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By 7 months of age, when atherosclerosis is well-established in WHHLs, many of the metabolic differences between strains had disappeared. These results differ from earlier reports in which older, food-restricted WHHLs were found to be insulin-resistant relative to healthy comparison rabbits (3,4). In the current study, when allowed unlimited access to food for 4 months, NZWs doubled their body weight and insulin resistance, as evidenced by a 2-fold increase in the HOMA index. Although insulin resistance increased in the WHHLs over the course of the study, these animals gained less weight, possibly as a result of fact that their food consumption was significantly less than the NZWs. The decreased food consumption may be related to sickness behavior (21), because WHHLs have significant atherosclerosis by 7 months of age. The data suggest that food consumption and weight gain are important variables to monitor in this model, especially in the control group, because NZWs with free access to food do a poor job of regulating their food intake and body weight, becoming just as insulin-resistant as WHHL rabbits.
Analysis 2
Given the finding that WHHLs exhibit significant insulin metabolic dysregulation early in life when atherosclerosis is developing rapidly (1), the second aim of this study was to assess the effectiveness of common behavioral interventions in treating metabolic variables and the progression of atherosclerosis in this disease model. Dietary restriction, independent of exercise, improved insulin sensitivity, controlled body weight and visceral fat, and lowered plasma leptin levels and heart rate relative to the control group. Interestingly, total serum cholesterol increased dramatically in the diet group compared with the other 2 groups. Although dietary restriction and weight loss in humans typically leads to a reduction in serum cholesterol, it has been reported that diet-restricted, cholesterol-fed NZW rabbits exhibit hypercholesterolemia, including elevations in LDL and VLDL cholesterol (22,23).
In the current study, exercise training without dietary restriction did not prevent the increase in insulin resistance, body weight, or plasma leptin, as was seen in the diet group, nor did exercise training attenuate the feeding behavior of these animals. The exercise group did exhibit lower resting heart rate compared with the control group, suggesting that the 4 months of exercise did produce a training effect. In addition, these animals did have less visceral fat and a smaller abdominal girth than control animals, although these effects were statistically nonsignificant. After the 4-month intervention, there were no significant group differences in plasma glucocorticoids or urinary catecholamines; however, urinary norepinephrine was slightly elevated compared with the other 2 groups, which is consistent with chronic exercise training (24).
Although dietary restriction was very successful in controlling insulin resistance, body weight, visceral fat, and heart rate, this intervention did not slow the progression of atherosclerosis in the WHHL model. Similarly, chronic exercise training did not influence disease progression relative to the control group, which is a result similar to that reported in the cynomolgus monkey model of atherosclerosis (25). The design of the current study allowed us to assess the effects of diet and exercise independently; however, it did not address whether a combination of diet and exercise would be more effective in slowing the development of atherosclerosis. It is likely that in the WHHL model, genetic influences and profound dyslipidemia are primary factors in disease progression. Although WHHLs are insulin-resistant relative to other rabbit strains, the current study suggests that behavioral manipulation of insulin metabolic variables does not alter the course of disease in this particular model. Interestingly, however, it has been reported that a combination pharmacologic treatment with an insulin action enhancer (troglitazone) and pravastatin decreased atherosclerosis in WHHLs more than that of monotherapy with either drug (26). The fact that an insulin sensitizer such as troglitazone reduced atherosclerosis in this model, whereas diet and exercise sufficient to significantly improve surrogate measures of insulin sensitivity did not, is intriguing and may have broader consequences for cardiovascular disease prevention. Because it is thought that much of the effect that diet and exercise has on improved insulin signaling occurs through restriction of weight gain, although troglitazone enhances insulin signaling by activating PPAR gamma receptors, it is conceivable that the latter approach may better target pathways to atherogenesis in subjects with advanced atherosclerosis than "lifestyle" changes do.
Although the measure of disease used in the current study was the extent of atherosclerosis, other measures of lesion status (eg, stability, inflammatory status) may provide different information regarding cardiovascular risk. It is possible that the interventions used in this study could have altered plaque stability or inflammation without affecting the size of the lesion. Future studies will examine other features of these atherosclerotic lesions and their potential contribution to cardiovascular event risk.
Despite the observation that manipulation of insulin metabolic variables does not affect atherosclerosis in WHHLs, these findings lend added importance to the role of social environment on disease progression. A previous study from our laboratory found that stable social environment, characterized by increased affiliative social behavior and relatively less agonistic behavior, slowed the progression of atherosclerosis in WHHLs by approximately 50% (2). Future work will focus on the central nervous system, autonomic and endocrine mechanisms by which social environment and emotional behavior may influence the course of disease in this model.
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NOTES |
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Received for publication August 10, 2004; revision received October 18, 2004.
DOI:10.1097/01.psy.0000155674.95497.ab
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