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Institute of Biomedicine, Pharmacology, University of Helsinki, Finland and * Department of Pathology, HUS Maria Hospital, Helsinki, Finland
3To whom correspondence should be addressed. E-mail: timo.vaskonen{at}helsinki.fi.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • atherosclerosis • hypertension • cholesterol • plant sterols • obese Zucker rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–4
). These disorders of industrialized societies are sensitive to various dietary factors. High intakes of saturated fat and cholesterol increase serum LDL cholesterol, probably by decreasing the amount and/or activity of LDL receptors in the liver (5
,6
). Elevated and modified LDL is one of the principal factors in the development of atherosclerosis. Accumulating in the vascular wall, it induces inflammation and endothelial dysfunction, leading eventually to permanent lesions (7
,8
). High intake of salt (sodium chloride), another feature of the typical Western diet, elevates blood pressure, aggravates end organ damage and reduces the effect of many antihypertensive drugs (9
–11
). Hypertension has proinflammatory effects as well by increasing the formation of hydrogen peroxide and free radicals (7
,12
). These substances impair the function of the endothelium and increase leukocyte adhesion, thus contributing to the process of atherosclerosis (7
).
On the other hand, certain changes in dietary habits have a favorable effect on cardiovascular health; therefore, recent recommendations include reduced intake of salt (sodium chloride), cholesterol and saturated fatty acids, and increased intake of potassium, magnesium and calcium (1
,4
,13
–16
). Diets supplemented with these minerals lower elevated blood pressure and also have other beneficial effects in both animal models and hypertensive patients (17
–22
). For prevention and treatment of lipid disorders, food products containing plant sterols or stanols have recently been introduced and proven to be useful (23
–25
).
The obese Zucker rat is an animal model of the metabolic syndrome; it carries a mutation in the leptin receptor and exhibits genetic obesity, hyperlipidemia, insulin resistance and renal injury as recessive traits (26
). Like other rodents and unlike humans, it normally has more HDL than LDL in circulation, which would make it unsuitable for studies of atherosclerosis. However, hypercholesterolemia can be induced in obese Zucker rats by increasing the dietary intake of saturated fat and cholesterol, as we have shown recently (27
). Both the fat and the cholesterol supplementation appeared to be necessary to induce a "human-like" serum lipid profile, i.e., a marked increase in LDL cholesterol, in these rats (27
). We also showed that supplementation with plant sterols counteracted in part this diet-induced hypercholesterolemia, and that calcium and magnesium, but not sodium and potassium, enhanced the effect of plant sterols (27
).
The purpose of the present study was to investigate the effects of a Western-type high fat, high cholesterol and high salt diet on blood pressure, endothelial function, and life expectancy in obese Zucker rats. We also tested the cardiovascular effects of dietary salt reduction and supplementation of plant sterols and mineral nutrients in this new model.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Female obese Zucker rats (n = 47; Harlan, Oxon, UK) were used. Rats, matched for blood pressure and serum cholesterol, were divided into three groups to receive different diets. The control group consumed basic dry rat food (Altromin, Lage, Germany). The atherogenic diet group consumed the same food into which was incorporated 18% butter (Valio, Helsinki, Finland), 1% cholesterol (Sigma, St. Louis, MO) and 6% sodium chloride (University Pharmacy, Helsinki, Finland). The plant sterol and mineral nutrient (PS + MN)–supplemented diet group consumed the same food containing 18% butter, 1% cholesterol, 1% plant sterols (see below), 5% potassium- and magnesium-enriched, sodium-reduced mineral salt (Pansalt, Oriola, Espoo, Finland), 0.5% calcium carbonate and 0.5% calcium chloride (Sigma).
The different components were mixed with powdered nonpurified diet and moistened with water using an industrial dough mixer. The prepared food was packed in amounts sufficient for 1 d and stored at -20°C. The final concentrations of the nutrients in the diets are given in Table 1
. The plant sterol mixture, purified from pine oil, was kindly provided by Mr. Matti Hautala of UPM-Kymmene, Lappeenranta, Finland. The chemical composition of the powder was as follows: sitosterol, 79.2%; sitostanol, 11.4%; campesterol, 7.5%; campestanol, 1.2%; cycloartenolol, 0.5%; and 
-7-avenasterol, 0.2%. The rats had free access to the food and tap water at all times. The procedures and protocols of the study were in accord to our institutional guidelines and approved by the Animal Experimentation Committee of the Institute of Biomedicine, University of Helsinki, Finland.
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Eighteen rats were used to study the hemodynamic effects of the different diets in a 4-wk experiment. The Dataquest IV telemetry system (Data Sciences International, St. Paul, MN) was used for measurements of systolic, diastolic and mean arterial pressure, heart rate and motor activity, as described previously (28
). At 12 wk of age, rats were anesthetized intraperitoneally with midazolam (Dormicum, 5 mg/kg) and fentanyl-fluanisone (Hypnorm, 1 mL/kg), and the flexible catheter of the transmitter was surgically secured in the abdominal aorta just below the renal arteries and pointing upstream (against the flow). The transmitter was sutured to the abdominal wall. The rats were given buprenorfin subcutaneously (Temgesic, 0.1 mg/kg) for two postoperative days. They were housed in individual cages after the operation and were allowed to stabilize for 2 wk. The rats were unrestrained and free to move within their cages at all times. The data were sampled every 5 min for 10 s for each rat. The pressure, heart rate and activity values were calculated by the Dataquest software. Mean values were calculated for intervals of 60 min for observation of diurnal patterns and for intervals of 12 h to obtain day- and nighttime curves for the entire 4-wk period.
During wk 4 of the study, the rats were housed individually in metabolic cages. Food intake was measured and urine collected for 24 h. At the end of the experiment, the rats were killed by decapitation. Blood samples were placed in two chilled tubes on ice. After centrifugation (4000 rpm, 15 min, -4°C), the serum samples were stored at -80°C. The left tibia was excised, carefully cleaned of adherent tissue and measured using a micrometer; the length of the tibia was used to estimate the skeletal body weight of the obese rats, as suggested by Zucker (29
).
Experiment 2.
Fifteen rats fed the atherogenic diet and 15 rats fed the PS + MN diet were followed in a long-term survival experiment. The rats had free access to food and tap water at all times. They were examined daily for survival and signs of stroke or other illness throughout the study. Rats that were either paralyzed or otherwise clearly ill, i.e., stopped moving and eating, were killed by decapitation and coded as dead on the same day, according to the ethical guidelines of the Animal Experimentation Committee. From these few rats, we were able to take tissue samples of heart and kidney. After excision, the tissue samples were fixed in phosphate buffered formalin and then prepared following standard laboratory procedures, embedded in paraffin and cut into 3-µm sections, stained with trichrome-stain, and examined and photographed under light microscopy.
Mesenteric arterial responses in vitro.
Three groups of six rats each were fed the same diets as in the telemetry experiment but did not undergo surgery so as not to interfere with the mesenteric artery. After the experiment, these rats were killed by decapitation, and 3-mm standard sections of the mesenteric artery, 3 mm distal from the artery-aorta junction, were cut. The rings were placed between stainless steel hooks and mounted in an organ bath chamber in physiological salt solution (pH 7.4) of the following composition (mmol/L): NaCl, 119.0; NaHCO3, 25.0; glucose, 11.1; CaCl2, 1.6; KCl, 4.7; KH2PO4, 1.2; MgSO4, 1.2; and aerated with 95% O2 and 5% CO2. The ring was equilibrated for 20 min at 37°C with a resting tension of 1.0 g. The force of contraction was measured with an isometric force-displacement transducer and registered on a polygraph (FTO3C transducer, Model 7C8 Polygraph, Grass Instrument, Quincy, MA). The contractile concentration curves to noradrenaline and potassium chloride, and the relaxation concentration curves to acetylcholine (to test endothelium-dependent relaxation) and sodium nitroprusside (for endothelium-independent relaxation) were determined as described previously (22
).
Biochemical determinations.
Serum cholesterol was measured by an accredited laboratory (United Laboratories, Helsinki, Finland; Hitachi 912 Automatic Analyzer, Hitachi, Tokyo, Japan) The concentrations of the mineral elements and albumin and creatinine in urine were analyzed by the same laboratory (for Na, K and Ca, BM/Hitachi 912 ion selective electrode, Boehringer-Mannheim, Germany; for Mg, Hitachi 180–80 Polarized Zeeman Atomic absorption spectrophotometer). Urinary excretion of the end products of nitric oxide metabolism, nitrate and nitrite (NOx), was measured using a colorimetric assay kit (Cayman Chemical, Ann Arbor, MI).
Statistics.
Statistical analysis was carried out by one-way ANOVA followed by Tukey’s test. Data for multiple observations over time were analyzed by two-way ANOVA with repeated measures for overall treatment effect, and Tukey’s test was used for multiple pair-wise comparisons of treatment groups at different times. Survival data were analyzed using the Kaplan-Meier test. Differences between means that had P < 0.05 were considered significant. The data were analyzed with SYSTAT Statistical Software (SYSTAT, Evanston, IL). The results are expressed as means ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The atherogenic diet induced a twofold increase in serum cholesterol compared with the control diet (Table 2
), and this was almost completely prevented by the PS + MN diet. The atherogenic diet produced a 10-fold increase in serum LDL cholesterol concentration, which the PS + MN diet decreased to about one third. Both the atherogenic and the PS + MN diets tended to decrease HDL cholesterol (P = 0.06) and increase triglyceride (P = 0.07) concentration. The HDL cholesterol/LDL cholesterol ratio was improved slightly but significantly by the PS + MN diet.
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The blood pressure of the rats fed the atherogenic diet increased steadily during the 4-wk study (Fig. 1
). The effect was most pronounced in nighttime systolic pressure, but even daytime and mean arterial pressures were significantly greater than in the other groups. The PS + MN diet completely prevented this hypertensive effect of the atherogenic diet. The pressure readings of the PS + MN diet group remained at the same low level as the control group, with no significant increase over time.
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. All of these variables had higher readings at night when rats are active and eat most of their food. There were no significant changes in the heart rate or motor activity levels over time during the 4-wk experimental period. However, heart rate was consistently lower at all times in the PS + MN diet group compared with the other groups.
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The endothelium-dependent relaxation responses to acetylcholine were significantly improved in the PS + MN diet group compared with the atherogenic diet and control groups (Fig. 3
). The endothelium-independent relaxation response to sodium nitroprusside was not significantly affected by diet. There were no significant differences among groups in the contractile responses to noradrenaline or potassium chloride (data not shown).
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The average life span of the rats was 306 d in the atherogenic diet group and 485 d in the PS + MN diet group (P < 0.001; Fig. 4
). Most of the deaths in both diet groups were sudden, and only two rats fed the atherogenic diet suffered from symptoms that required euthanasia according to preset criteria. In these two rats, atherosclerotic lesions, thromboses in coronary arteries and myocardial infarctions were observed (Fig. 5
). In the kidneys, intimal thickening and occlusion of arteries were seen, and most glomeruli were destroyed.
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Body weight gain was steadily slower in the PS + MN diet group than in the other groups (data not shown), resulting in lower final weight, although food and energy intakes in this group were greater (Table 3
). The skeletal weight and urine creatinine levels did not differ among groups. Urine volume was greater in the atherogenic and PS + MN diet groups, as were the excretions of sodium and potassium, approximately in the same proportions as they were added to the diets. The urinary excretions of calcium and magnesium were increased not only in the PS + MN diet group, but also in the atherogenic diet group. Urine albumin excretion was markedly increased in the atherogenic diet group, but not in the PS + MN diet group compared with the control group. The urinary excretion rate of the end-products of nitric oxide metabolism, nitrate and nitrite was greater (P < 0.001) in the PS + MN group (640 ± 136 µmol/24 h) than in the control group (51 ± 36 µmol/24 h) or the atherogenic diet group (60 ± 44 µmol/24 h).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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,30
). Although our data are preliminary, it is possible that this could be a suitable model for future studies of the mechanisms and prevention of human disease. The present study focused on the use of a dietary regimen consistent with current public health recommendations to reduce risk of chronic disease, i.e., increased intake of plant sterols and the minerals potassium, magnesium and calcium (15
,16
,24
,25
).
The PS + MN-supplemented diet effectively prevented the rise of serum total and LDL cholesterol induced by the atherogenic diet and markedly extended the life span of the rats. Part of this effect was due to the added plant sterols in the diet. Previous studies in both animals and humans have shown that natural plant sterols, or their chemically modified derivatives, lower serum cholesterol by inhibiting the intestinal absorption of cholesterol (23
–25
,31
). However, a part of the effect can also be attributed to the increased content of calcium and magnesium in the diet. We previously tested the components of this diet separately and found that supplementation with the divalent cations calcium and magnesium, but not the monovalent sodium and potassium, enhanced the cholesterol-lowering effect of plant sterols in obese Zucker rats consuming a high fat and high cholesterol diet (27
). Calcium decreases the intestinal absorption of fat (32
,33
), and a decrease in the intake of dietary saturated fatty acids lowers serum lipoprotein and cholesterol concentrations (6
,34
). Previous studies have also shown a lowering of serum cholesterol by calcium supplementation of humans (32
,33
). Orlistat, a drug that inhibits the intestinal absorption of fat, lowers serum cholesterol as well (35
). Thus, the remarkable decrease in serum cholesterol in the PS + MN diet group in the present study may have resulted from simultaneous decreases in the absorption of both cholesterol and fatty acids.
PS + MN supplementation did not prevent the decrease in HDL cholesterol induced by the atherogenic diet. HDL mediates reverse cholesterol transport and also has antioxidant properties (36
) that protect against atherogenesis. However, due to the reduction in LDL cholesterol, the HDL cholesterol/LDL cholesterol ratio was slightly improved in the PS + MN group compared with the atherogenic diet group, which may contribute to its overall beneficial effect.
In the present study, the atherogenic high salt diet markedly increased systolic and mean blood pressures. This is consistent with some previous studies in obese Zucker rats (37
,38
), although it contradicts some others (39
). This may be due to methodological differences. We used radiotelemetry, which is a continuous, stress-free and highly accurate method for measurement of blood pressure, and it showed a clear difference between the groups from the very beginning of the experiment. We also found previously that a mere fat and/or cholesterol supplementation did not raise the blood pressure (T. Vaskonen, unpublished data). Therefore, our results strongly suggest that the obese Zucker rat is a salt-sensitive animal model for hypertension. The PS + MN diet, using a potassium- and magnesium-enriched, sodium-reduced mineral salt instead of common salt, and calcium, completely prevented the development of hypertension in this model. The same mineral salt has a similar effect in another salt-sensitive model, the spontaneously hypertensive rat (21
), and it has been shown to lower blood pressure in humans as well (17
,19
,20
). The antihypertensive effect of potassium was also confirmed in a recent meta-analysis of human studies (40
). Similar evidence exists for calcium and magnesium (15
).
The PS + MN diet markedly increased the relaxation response to acetylcholine in a resistance artery in vitro with no significant effect on either the relaxation response to nitroprusside or the contraction response to noradrenaline or potassium chloride. This means that the function of the endothelium in obese Zucker rats was improved. Interestingly, it was even better than in the control rats. The poor relaxation response in the control diet group is, although exceptionally pronounced here, consistent with previous studies and probably related to obesity and hyperlipidemia (41
,42
).
Improvement in endothelial function of rats consuming the PS + MN diet is also suggested by our finding that the systemic production of nitric oxide, measured as urinary excretion rate of nitrate and nitrite, was considerably increased. Dietary nitrates and nitrites may have affected this measurement; however, because food and protein intakes did not differ among groups, this is not likely to explain the difference. Thus, restoration of the endothelial function may, at least in part, explain the blood pressure–lowering effect of the PS + MN diet. Previous studies have also demonstrated that calcium (43
) as well as potassium- and magnesium-enriched diets (22
,44
,45
) improve endothelial function and lower blood pressure in different rat models. Furthermore, because hyperlipidemia per se induces endothelial dysfunction (7
,8
), the effect of the PS + MN diet may also be related to the lipid-lowering effect of the plant sterols.
Previously, the development of hypertension in obese Zucker rats has been associated with renal damage in aging animals (26
). In the present study, increased albuminuria in the 16-wk-old rats consuming the atherogenic diet indicated an early development of renal damage, whereas there was no sign of such damage in the PS + MN diet group. Apparently, this diet prevented or at least delayed the development of the renal injuries and, consequently, the eventual development of hypertension. This view is supported by the results of the second study, in which the rats fed the PS + MN diet lived much longer than those fed the atherogenic diet, and severe damage was observed in the kidneys of the rats fed the atherogenic diet. Renal damage is also strongly linked to serum lipid profile, in both humans and several animal models (26
,46
). It has been shown that cholesterol feeding induces renal damage (47
), and treatment with cholesterol-lowering drugs improves renal functions (48
). In the present study, the rise of serum cholesterol was completely blocked by the PS + MN diet, which could explain in part the protection against renal damage.
In agreement with previous reports (29
,49
) Zucker rats fed the control and atherogenic diets developed marked obesity. However, enrichment of the high fat diet with mineral nutrients and plant sterols resulted in a considerably lower end weight of the rats. The slower increase of body weight in this group appeared to be due to reduced accumulation of fat tissue because the skeletal size of the rats was not affected, and because urinary creatinine excretion, as an indicator of muscle catabolism, was not increased. The estimated food and energy intakes of the PS + MN group were even greater than in the other groups. Therefore, the decline in the amount of fat tissue appeared to be caused by increased loss rather than decreased intake of energy. This is best explained by a blockade of fatty acid absorption by calcium and magnesium so that part of the ingested energy was lost into stools (32
,33
). Increased intakes of potassium and magnesium were demonstrated also to improve carbohydrate metabolism (50
–54
). However, the favorable metabolic changes may well be secondary to decreases in fat absorption and obesity, as suggested by studies with orlistat, a pancreatic lipase inhibitor that decreases intestinal absorption of fatty acids and results in weight loss and improvements in serum lipid, glucose and insulin profiles (35
).
In conclusion, the administration of a diet high in saturated fat, cholesterol and salt to obese Zucker rats resulted in hypercholesterolemia, hypertension, endothelial dysfunction and death in 12 mo, apparently due to renal damage and heart infarctions. Supplementation of this atherogenic diet with plant sterols together with the minerals calcium, magnesium and potassium provided effective protection against the harmful effects of the diet and extended the average life span of the rats by 60%. Hypertension and hyperlipidemia seem to be sensitive to diet composition changes that could easily be applied to human diets as well. In fact, the first clinical trials have already confirmed the cholesterol-lowering effect of food items prepared according to the principles described in the present study (55
).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by grants from the National Technology Agency Tekes, Finland; the Finnish Medical Foundation (T.V.), the Research and Science Foundation of Farmos (T.V.), the Ida Montin Foundation (T.V.), the Academy of Finland (E.M.), the Finnish Foundation for Cardiovascular Research (E.M.) and the Sigrid Juselius Foundation (E.M.).
Manuscript received 26 June 2001. Initial review completed 3 August 2001. Revision accepted 19 October 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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control group; 
atherogenic diet; 
PS + MN-supplemented diet. Repeated-measures ANOVA for daytime pressures: between-subjects effects P = 0.096, within-subjects effects P < 0.001, interaction P = 0.019; for nighttime pressures: between-subjects effects P = 0.029, within-subjects effects P < 0.001, interaction P = 0.004; the atherogenic diet group differed from the others.
9 mo. (A) A thrombosed coronary artery with atherosclerotic lesions in the intima (trichrome staining, original magnification X10). (B) The kidney, intimal thickening and occlusion of arteries, a destroyed glomerulus (trichrome staining, original magnification X20). (C) A destroyed glomerulus (trichrome staining, original magnification X40).