Animals and model of obesity
This was an experimental, cross-sectional and analytical study. The murine model that was used consisted of a population of Sprague Dawley rats strain that were obtained from an inbred colony in the bioterium of the Specialty Hospital of the National Medical Center, Mexican Social Security Institute (Mexico City, Mexico). A total of 20 males weighing 200-250 g, were used.
Animals were randomly allocated and divided into two groups: the control (C) group (n=12) was fed with the standard diet (Formulab 5008 Diet; PMI Nutrition International, Brentwood, MO, USA), and the obese (Ob) group (n=8) was fed with a high fat-fructose diet (standard diet supplemented with 10% lard and 30% fructose mixed and dissolved in drinking water) during 16 weeks’ ad libitum; both groups received this diet until reaching six months of age. Food and water intake, as well as, body weight, were recorded daily during this period. We have worked with this experimental model in other projects [5]. In this murine model the ingestion of a high fat-fructose diet is clearly associated with the development of insulin resistance, disturbed glucose homeostasis and endothelial dysfunction [4-6]. The hypercaloric diet was used to induce obesity in the Ob group.
All cages contained wood shavings, bedding and a cardboard tube for environmental enrichment. All rats of each group had ad libitum access to their pellet diet and drinking water and were housed in a hygienically controlled room in groups of 4 rats, in conventional cages at room temperature (22-25°C), under a light cycle of 12 hours’ light/12 hours’ dark.
Ethical statement
All experiments were performed in accordance with the relevant guidelines and regulations of the bioterium of the Specialty Hospital of National Center Medical, of the Mexican Social Security Institute (CMN SXXI-IMSS), in accordance with the Official Mexican Standard (NOM-062-ZOO-1999, revised 2001) for the care and use of laboratory animals. Additionally, we obtained written informed consent to use the animals in our study. This study was approved by the Ethical Committee and the Local Research and Health Committee of the Mexican Social Security Institute (registration number 3601-2015-95). The animals were treated according to the Official Mexican Standard for the care, use and sacrifice of laboratory animals. (NOM-062-ZOO-1999, revised 2001). In this work, the obese group had a hypercaloric diet for 4 months, and both groups, C and Ob, were studied at 6 months of age. However, to facilitate other tissue and organ analyses, the feeding continued in both groups until the animals reached 12 months of age.
Metabolic parameters and body weight
Body weight. The weight in both groups, (group C (n=12) and Ob (n=8)), was recorded from the beginning to the end of the study. Food and water intake were recorded each week.
Blood samples were taken at 8 am (during 7 hours fasting at 6 months of age) by a longitudinal cut on the end of the tail to measure biochemical blood parameters and inflammatory cytokines. The blood samples were transferred into anticoagulant containing tubes for the measurement of biochemical blood parameters and inflammatory cytokines, respectively. The samples were centrifuged at 5200 g for 15 minutes. Plasma was separated and stored at -70 °C until use for bioassay analyses.
Blood glucose levels were determined by placing one drop of blood on blood glucose test strips of the FreeStyle Optium Xceed glucometer (Abbot Diabetes Care Ltd. OYL, UK). Each animal was twice analyzed.
Determinations of the levels of total cholesterol, triglyceride (TG), high-density lipoprotein (HDL) and low-density lipoprotein (LDL) were made on a Cardiocheck apparatus after placing one drop of blood (see the previous section on blood samples) on a reactive strip, according to the manufacturer’s instructions.
Quantification of insulin and index HOMA-IR, insulin was measured by chemiluminiscence, using an insulin IMMULITE kit (LKIN1 insulin IMMULITE).
Insulin resistance was calculated using the HOMA-IR index with the following formula for the homeostasis model assessment-insulin resistance (HOMA-IR) index: insulin (μU/mL) × glucose (mg/dL)/405.
TNF-α, IL-6, and the adhesion molecules ICAM-1 and VCAM-1 were analyzed using enzyme immunoassay kits with ELISA R&D kit (R&D Systems, Inc., Minneapolis, MN, USA).
Ultrasound analysis
In this study, the rat was placed in the left lateral decubitus position, with a slight inclination of the head and the transducer was placed in the different acoustic windows. The equipment used was a Philips Affiniti 70 device with a linear transducer of 9 mHz, where the results were obtained in millimeters. During the echocardiographic examination, two cuts were used to evaluate the heart: the left longitudinal parasternal section and the right longitudinal parasternal section. In the left longitudinal sternal section, it is possible to visualize the right ventricle, aorta with aortic valve, left atrium, mitral valve and LV [11].
With rotation of the transducer the right longitudinal sternal section that appears on the left side and the base (atria) that appears on the right side reveal the left ventricular outlet, aortic valve, aortic root and proximal ascending aorta [11,12].
The measurement of the LV in diastole was performed from the left septal endocardium to the posterior wall endocardium, measuring below the level of the mitral valve. The interventricular septum is located between the mitral ring in its posterior part and the endocardial surface of the high septum in its anterior part. The diastolic thickness of the free wall of the LV was measured, and its value was approximately equal to the diameter of the interventricular (IV).
Statistical analysis
The data are presented as the means ± standard deviation (SD) of each group. The study groups were statistically analyzed for the difference in means between group C and group Ob with Student´s t test, and a value of p<0.05 was considered to indicate statistical significance. For each study variable (glucose, insulin, lipid profile, HOMA-IR index, proinflammatory cytokines, TNF-α, IL-6, adhesion molecules ICAM-1 and VCAM-1), a statistical analysis was carried out using GraphPad Prism (GraphPad Prism 8 for Windows, San Diego, CA); p<0.05 was considered significant.