Cardiometabolic Alterations in Wistar Rats on a Six-Week Hyperlipidic, Hypercholesterolemic Diet

Mailing Address: Dora Maria Grassi Kassisse Rua Monteiro Lobato, n:255. Postal Code: 13083-862, Barão Geraldo, Campinas, SP – Brazil E-mail: doramgk@unicamp.br; doramgk@gmail.com Cardiometabolic Alterations in Wistar Rats on a Six-Week Hyperlipidic, Hypercholesterolemic Diet Danilo Roberto Xavier de Oliveira Crege, Alexandre Marcucci Miotto, Filipy Borghi, Valéria Wolf-Nunes, Dora Maria Grassi Kassisse Universidade Estadual de Campinas (UNICAMP), Campinas, SP – Brazil


Introduction
Obesity, atherosclerosis and hypertension, when associated in one patient, constitute a series of metabolic risk factors known as metabolic syndrome.[6] The beginning of this complex syndrome is not yet completely clear, and an important question to be answered is: What is the triggering factor for this disease?Studies show that there is a close relation between this syndrome and insulin resistance 7,8 and high-fat diets. 1,4unique characteristic of this study is that, most studies found in literature that involve the administration of hyperlipidic diets and cardiovascular alterations are done in mice.Published studies that were done in Wistar Cardiometabolic profile of dyslipidemic rats Int J Cardiovasc Sci.2016;29 (5):362-369 Original Article rats present long treatment protocols such as 20 9,10 or 30 11 weeks, generating a high cost to maintain the rats.Thus, in this study, we attempted to evaluate the efficacy of the short-period dietary treatment to trigger cardiovascular and metabolic disorders in Wistar rats.

Animals
We used male Wistar rats that weighed 150 g (4 weeks old) at the beginning of the study.The animals were kept in a room with temperature control (22±2ºC), and 12-hour dark light cycle (lights on at 6:30 am).The experiments were performed in accordance with the principles adopted by COBEA (Brazilian College of Animal Experimentation) for the use of animals in research and education, under number 226-1,2561-1, according to the Declaration of Helsinki of 1975, revised in 2008.

Catheterization of blood vessels
The rats were anaesthetized with xylazine (50 mg/Kg, i.m.) and ketamine hydrochloride (0.01 mg/Kg, i.m.), and the left carotid artery was catheterized with a siliconized polyethylene cannula (PE 20) filled with 5 mM sodium citrate.The catheter was connected to a PE 50 tube (with 25 cm of length, filled with 0.9% NaCL solution), exteriorized in the interscapular region, fixed on the animal's skin. 14This method allowed us to collect blood samples from non-anaesthetized rats with no movement restriction.Before the experiment, all rats had a 48-hour surgery recovery time in individual cages.

Blood pressure measurement
After recovering from surgery, the catheterized rats were kept in the cages, conscious, and with no movement restriction in a quiet environment.Blood pressure was registered at every beat, with a pressure transducer (Gould-Strain Gauge) connected to a pre-amplifier linked to an intra-arterial catheter.The measurements were recorded in a computer to be later analysed by WINDAQ-PRO Data Acquisition software (DI220 AT CODAS data acquisition system, Data Instruments Co).Systolic, diastolic and mean blood pressures and heart rate in basal conditions were calculated with the software Advanced CODAS.

Blood samples
After 16 hours of fasting, blood samples were collected with a catheter.Total cholesterol, HDL cholesterol, triglycerides (TAG) and glucose concentrations were determined in the serum by a colorimetric method with diagnostic kits (laborlab, Barueru, SP, Brazil).An indirect method was used to determine the concentrations of LDL and VLDL cholesterol. 15Free fatty acids (FFA) were also determined by the colorimetric method with a specific diagnostic kit (Wako Chemicals GmbH; Neuss, Germany).Insulin concentrations were determined by radioimmunoassay (RIA), as described in the literature. 16he atherogenic index (AI), a measurement that indicates the organism's tendency to develop atherosclerosis, was determined by the calculation: AI = [(Total cholesterol -HDL)/HDL].

Histologic analysis
The rats were euthanized by anaesthetic overdose, and cardiac tissue samples were taken for the histologic analysis and verification of ventricular hypertrophy.The ventricle was washed in a physiological solution, weighed and immediately placed in a container with formaldehyde.Ventricle slices were dehydrated with alcohol in gradual concentrations (70% to absolute), followed by diaphonization in xylene, immersion in Bouin solution and paraffin embedding (58°C -60°C).Blocks were sectioned 5 µm thick and stained with Trichrome Masson.Chamber diameter, and ventricular area and wall thickness were determined with the software Scion Image (NIH Image Software).

Statistical analysis
Calculation of the sample number of experiments for each of the different conditions was done according to the Lenth, 17 with the software Statistica 7.0 (Stat Soft, Inc. 2004: version 7. www.statsoft.com).This calculation was done to ensure the following parameters: minimum

Serum concentrations of total, LDL, VLDL and TAG cholesterol
The rats that were on the hyperlipidic and hypercholesterolemic diet during six weeks (from their 4 th to their 10 th week of life) presented significant increases in serum concentrations of total, LDL, VLDL cholesterol and triglycerides (Table 1).No significant differences were observed for HDL cholesterol concentrations.Moreover, rats in the hyperlipidic, hypercholesterolemic diet presented higher AI than those in the control group (Table 1).

Plasma concentrations of glucose
Plasma concentrations of glucose were not altered in rats on the hyperlipidic, hypercholesterolemic diet.On the other hand, insulin concentrations were significantly higher, in comparison to rats that were on a standard feed diet (Table 2).

Blood pressure
Mean, systolic and diastolic blood pressures and heart rate were significantly higher in rats on the six-week hyperlipidic, hypercholesterolemic diet (Table 3).

Histologic analysis of the heart
Left ventricular mass was significantly higher in rats on the hyperlipidic, hypercholesterolemic diet.Similar results were found for the ventricular mass index and for the left ventricular wall thickness (p < 0.0001), internal diameter (p < 0.0001) and ratio of the wall thickness to the internal diameter (p < 0.0001).The area of the left ventricular chamber, on the other hand, showed no alterations (Table 4).Morphological changes are depicted in Figure 1

Discussion
9][20] After six weeks of a hyperlipidic, hypercholesterolemic diet, the rats presented dyslipidemia associated to hyperinsulinemia and moderate hypertension.Elevations in serum concentration of triglycerides, total, LDL and VLDL cholesterol characterize atherogenic dyslipidemia.AI was higher in rats on the hyperlipidic, hypercholesterolemic diet.Serum concentrations of HDL and FFA were not affected by the diet.
High concentrations of lipids may be a triggering factor to other metabolic alterations.[24] Excessive nutrient intake, especially glucose and lipids, may cause insulin resistance in the adipose tissue and muscles, just as it compromises endogenous glucose production.Experimental models with animals on hyperlipidic diets have shown a reduction in glucose tolerance associated to a lower basal glucose uptake, stimulated by insulin. 25The results seen here showed that rats on the hyperlipicid, hypercholesterolemic diet were hyperinsulinemic and euglycemic, which likely suggests a reduction of glucose tolerance.2-deoxyglucose uptake by adipocytes, as the euglycemic-hyperinsulinemic clamp, is being evaluated in our laboratories to confirm this condition in the experimental model.
A compromise in insulin sensitivity and/or in glucose transport were related to changes in the composition of fatty acids of the plasma membrane induced by a lipid based diet.Saturated fats, present in hyperlipidic and hypercholesterolemic diets, seem to be more deleterious than monounsaturated or polyunsaturated fats [26][27][28] because lipids induce a reduction of insulin sensitivity.Studies with rabbits have demonstrated that a diet-induced hypercholesterolemia resulted in depressed systolic and diastolic functions as a consequence of the increased amount of cholesterol in the cardiac sarcolemma, alterations in calcium permeability, and/or SERCA-2 activity. 18Therefore, animal fat intake, associated to a sedentary life-style, predisposes atherogenic lipoprotein elevation and metabolic syndrome. 1,4ts on the hyperlipidic, hypercholesterolemic diet, in comparison to the control group, also showed a significant increase in HR and MBP, with a moderate, but significant, increase in SP and DP.HR increase is an indicator of sympathetic nervous system (SNS) hyperactivity, 29,30 and this SNS activity increases with calorie intake. 22,31Elevated concentrations of insulin appear to be the link between nutrient absorption and sympathetic activity.HR increase, associated to dyslipidemia, is a factor involved in the genesis of hypertension and atherosclerosis.
Insulin resistance and hyperinsulinemia have also been described as important mechanisms in the pathophysiology of dyslipidemia and atherosclerosis. 32,33t has been suggested that insulin regulates the peripheral vascular resistance, leading to vasoconstriction through the activation of the SNS.In a situation of insulin resistance, nerve endings of the sympathetic system are not significantly affected, thus, the sympathetic stimulus is continuous. 33On the other hand, insulin increases nitric oxide (NO) synthesis, 34,35 reducing catecholamine-mediated vasoconstriction and potentiating acetylcholine-mediated vasodilatation in Cardiometabolic profile of dyslipidemic rats Int J Cardiovasc Sci.2016;29(5):362-369 Original Article the blood vessels.These actions seem protective against the development of arterial hypertension.However, in the condition of insulin resistance, such effects are reduced, 32,36 leading to hypertension.Moreover, the stimulation of growth and migration of smooth muscle cells in the vessel wall is directly associated to the beginning and progression of this process, 37 in which the NO synthesis is reduced.Evidence that hyperinsulinemia precedes the development of vascular disorders has been previously described. 38 has been demonstrated that nitric oxide is an effective anti-hypertrophic and cardiac remodeling inhibitor. 39However, the elevated plasma concentrations of LDL observed in rats fed with the hyperlipidic diet can promote an increase of this oxidative stress through lipid peroxidation, reducing NO availability, 40 and increasing concentrations of nitrite and nitrate. 41Moreover, molecules such as tumor necrosis factor (TNF-α), which is secreted by the adipose tissue and is a factor that contributes to insulin resistance and dyslipidemia, 42 are also additional inducers of apoptosis of myocytes.Even though we did not verify apoptosis in the evaluated hearts, this condition is associated to the progressive reduction of ventricular function or myocardial lesion, 43 and concentric hypertrophy due to the pressure overload, as shown in our results.
Rosen et al., 44 in a study with chronic heart failure and hypertrophic cardiomyopathy patients, confirmed the normal activation of the SNS with density reduction of beta-adrenoceptor of the myocardium.In preliminary studies, we identified that rats on the hyperlipidic diet, for six weeks, presented subsensitivity to b-adrenergic agonists in the right atrium. 45e main limitation of this study is the lack of investigation of mechanisms involved in the presented disorders.However, considering this is a model of dyslipidemia induction by a diet in only six weeks, further studies are necessary to unravel the mechanisms related to this syndrome.

Conclusion
Results show that a six-week hyperlipidic, hypercholesterolemic diet induced dyslipidemia associated to hyperinsulinemia, hypertension and left ventricular hypertrophy.This condition is associated to cardiometabolic diseases.Therefore, this could be a short-term experimental model to induce cardiometabolic disorders through a diet in Wistar rats, which can contribute to elucidate the mechanisms of this complex pathology.

Figure 1
Figure 1 Representative figures of the histologic analysis of the rats treated during six weeks with standard (S) or hyperlipidic, hypercholesterolemic (H) diets.Tissues were stained with Trichrome Masson.Column A: images of the cross-section of half the heart.Left ventricular wall thickness was significantly increased in rats on the hyperlipidic, hypercholesterolemic diet; Column B: surface area of cardiomyocytes (scale = 7µmm); Column C: longitudinal view of cardiomyocytes; Column D: myocardial fibrosis; and Column E: perivascular fibrosis (Column B-E -400x increase).

Table 2 Serum concentrations of glucose and plasma concentrations of insulin in rats on the standard or hyperlipidic, hypercholesterolemic diets for six weeks
of the test of 0.80 and alpha preset at 0.05.The animals were removed from the vivarium Biotério Central da Universidade Estadual de Campinas and, upon arriving at the department of functional and structural biology, four animals were randomly separated to form the normolipidemic group.The five remaining animals constituted the dyslipidemic group.After indication of normal distribution of data through Kolmogorov Smirnov analysis, results were analysed by unpaired Student's t-test as stated in the legends.Statistical significance level was set at 5%.Results were expressed as mean ± standard error of the mean (SEM).Statistical study was done with the software Prisma.
* Blood samples were taken after 16 hours of fasting, from conscious rats, with no movement restriction.Results are expressed as mean ± SEM.The number of animals is stated between parenthesis; * significantly different mean from the control group; (p < 0.05; unpaired Student's t-test).power

Table 4 Body weight, left ventricular mass, ventricular mass index, left ventricular wall thickness, internal diameter, chamber area and ratio of the left ventricular thickness and the internal diameter of the left ventricle of rats on standard or hyperlipidic and hypercholesterolemic diets for six weeks
BW: Body weight; VM: Left ventricular mass; VMI: Ventricular mass index; VT: Left ventricle was thickness; ID: Internal diameter; CA: Chamber area; VT/ID: Ratio of the ventricular thickness and internal diameter of the left ventricle.Results are expressed as mean ± SEM.Ventricular mass index = ventricle weight / body weight.The number of animals is stated between parenthesis; * significantly different mean from the control group; (p < 0.05; unpaired Student's t-test).