The objectives of our study were:
- to compare the severity of myocardial damage caused by transient ischemia ex vivo in animals with IGT, experimental type 2 diabetes and in animals without disorders of glucose metabolism.
- to study of the potential cardioprotective effect of metformin (MET) and a glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide (LIRA) in animals with IGT and to compare the prominence of this effect in transient myocardial ischemia ex vivo.
We chose MET as it is the first-line drug for DM therapy [18], which is often used for IGT and has shown its effectiveness in patients with IGT in terms of preventing the development of DM. For GLP-1 receptor agonists, numerous pleotropic effects have been described in recent years, among which the cardioprotective effect occupies one of the leading positions. Thus, LIRA therapy, according to the LEADER study, demonstrated the ability to reduce overall mortality, mainly by reducing cardiovascular mortality [19]. Currently, GLP-1 receptor agonists are recommended for both primary and secondary prevention of cardio-vascular diseases.
Animals
The study was carried out in male Wistar rats weighing 150-250 g (n = 32).
The animals were maintained in fixed light mode, 12.00: 12.00 h (light: dark), no more than 5 animals per cage with free access to food and water. The temperature was maintained within the range of 22-25 ° C, the relative humidity - 50-70%.
The duration of quarantine (acclimatization period) for all animals was 14 days. During the quarantine, every animal was examined daily. The color of the skin and visible mucous membranes, behavior, the motor activity, the presence of seizures, changes in the respiratory movements, and tail position were assessed. Weighing was carried out upon arrival of the animals and during the quarantine period - at least 1 time per week. Animals with deviations in weight, general condition or behavior were not included in the experiment.
Study groups
After the acclimatization period, the following experimental groups were formed:
- «CRL» (n=6) - control group – rats were fed with standard chow for 16 weeks
- «HFD» (n=8) - high-fat diet group – rats were fed with high-fat diet for 16 weeks
- «DM» (n=4) - type 2 diabetes mellitus group – rats with experimental type 2 diabetes mellitus were kept without treatment for 12 weeks
- «IGT» (n=4) - impaired glucose tolerance group - rats with impaired glucose tolerance were kept without treatment for 12 weeks
- «IGT+MET» (n=4) - impaired glucose tolerance + metformin group – in 4 weeks after the induction of impaired glucose tolerance started 8-weeks metformin therapy
- «IGT+LIRA» (n=4) - impaired glucose tolerance + liraglutide group – in 4 weeks after the induction of impaired glucose tolerance started 8-weeks liraglutide therapy
The number of animals in these groups was determined during the experiment, depending on which variant of the carbohydrate metabolism disorder developed after streptozotocin and nicotinamide injection.
The study design is shown in Figure 1.
Induction of type 2 diabetes mellitus and impaired glucose tolerance
Animals were kept on the diet with increased amount of saturated fat (25%) (further: high-fat diet, HFD) during all the experiment.
After first 4 weeks of high-fat diet a solution of nicotinamide (Nicotimamide, Sigma-Aldrich, St. Louis, MO, USA) 230 mg/kg was injected intraperitoneally as a pancreatic protector, after 15 minutes - a solution of streptozotocin (Streptozocin, Sigma-Aldrich, St. Louis, MO, USA) 60 mg/kg intraperitoneally as a pancreatic toxin [20].
On the second and third days after the administration of nicotinamide and streptozotocin, glycemia was determined. For this, a tail vein puncture was performed, after which the glucose content in the obtained venous blood drop was determined using an Accu-Chek Performa glucometer (Roshe, Germany). Glycemic values of 3.3 to 7.8 mmol/L were considered normal, since the measurement was made during the day (not fasting). DM was diagnosed when two measurements performed in different days showed glycemia elevation more than 11.1 mmol/L [21, 22]. If lower glycemic values were found in at least one of the measurements, an oral glucose tolerance test (OGTT) was performed. Glycemia was measured initially (fasting), as well as 15, 30, and 60 minutes after the gastric administration of a 40% glucose solution 3 g/kg of animal body weight. If we detected glycemia more than 11.1 mmol/L at any of the measurement points during OGTT we diagnosed the presence of DM. If glycemia was in the interval of 7.8-11.0 mmol/L we diagnosed IGT [23]. Animals in which the glycemic parameters at all measurement points did not exceed 7.8 mmol/L were regarded as having no disturbances in glucose metabolism and were excluded from further experiment.
Glycemic measurements for making the diagnosis of DM and IGT are shown in Table 1.
Table 1. Blood glucose measurement for DM and IGT diagnostics.
Group
|
animal №
|
Blood glucose level, mmol/L
|
1st measurement
|
2nd measurement
|
ORAL GLUCOSE TOLERANCE TEST
|
baseline
|
15 min
|
30 min
|
60 min
|
90 min
|
DM
|
1
|
16.0
|
12.1
|
|
|
|
|
|
2
|
20.5
|
21.6
|
|
|
|
|
|
3
|
19.2
|
16.0
|
|
|
|
|
|
4
|
9.7
|
8.6
|
3.8
|
10.5
|
17.6
|
18.0
|
15.3
|
IGT
|
5
|
9.9
|
7.7
|
4.2
|
7.0
|
9.3
|
10.2
|
10.9
|
6
|
6.5
|
6.4
|
4.6
|
8.9
|
8.7
|
10.2
|
9.2
|
7
|
6.1
|
6.3
|
3.8
|
5.7
|
7.1
|
9.5
|
8.0
|
8
|
7.7
|
7.0
|
5.3
|
5.9
|
7.9
|
8.8
|
9.6
|
IGT+MET
|
9
|
6.8
|
5.7
|
4.2
|
10.1
|
10.4
|
9.3
|
9.9
|
10
|
6.7
|
6.9
|
4.5
|
9.6
|
10.4
|
10.6
|
9.9
|
11
|
5.6
|
5.8
|
5.4
|
7.7
|
9.2
|
9.7
|
9.2
|
12
|
6.7
|
5.3
|
5.3
|
6.6
|
9.8
|
10.7
|
10.8
|
IGT+LIRA
|
13
|
8.6
|
8.5
|
4.6
|
6.9
|
8.1
|
7.3
|
9.0
|
14
|
5.7
|
6.3
|
4.7
|
8.2
|
8.6
|
9.5
|
9.0
|
15
|
5.4
|
8.0
|
4.5
|
7.4
|
7.7
|
9.8
|
8.5
|
16
|
5.3
|
7.7
|
4.8
|
7.3
|
8.8
|
10.5
|
9.6
|
Study drugs
Metformin powder (Metformin hydrochloride Sigma-Aldrich, St. Louis, MO, USA) was dissolved in distilled water and given per os by gastric tube 200 mg/kg of body weight once daily for 8 weeks (56 days).
Liraglutide (Victoza, NovoNordisk, Denmark) was administered subcutaneously 0.06 mg/kg of body weight once daily for 8 weeks (56 days).
Body weight and food consumption measurement
Once every two days during the entire experiment, the animals were weighed, and the weight of the chow consumed in 2 days was determined.
Glucose measurement
Glucose measurement was performed with the help of Accu-Chek Performa glucometer (Roshe, Germany).
In CRL and HFD groups glucose assessment was performed at the end of the 4th week (28th day), 8th week (56th day), 10th week (70th day), 12th week (84th day), 14th week (98th day) and 16th week (112th day) of experiment at the same daytime (not fasting measurement).
In DM and IGT groups glucose assessment was performed after 4 weeks of experiment on the 2nd and 3rd day after streptozotocin injection, at the end of the 8th week (56th day), 10th week (70th day), 12th week (84th day), 14th week (98th day) and 16th week (112th day) of experiment at the same daytime (not fasting measurement).
In groups IGT+MET and IGT+LIRA groups glucose assessment was performed after 4 weeks of experiment on the 2nd and 3rd day after streptozotocin injection, at the end of the 8th week (56th day), then every third day during the 8 weeks of treatment at the same daytime (not fasting measurement), 5 hours after the certain treatment.
Isolated heart perfusion according to Langendorff
Anesthesia was performed by Zoletil (tiletamine hydrochloride 30 mg/kg and zolazepam hydrochloride 30 mg/kg) intramuscularly and xylazine hydrochloride 6 mg/kg intramuscularly. After reaching the surgical stage of anesthesia (Zoletil + Xylazine IM), a wide thoracolaparotomy was performed, the organs of the chest cavity were exposed and the heart was removed, after which it was connected to the modified Langendorff apparatus. Perfusion was performed retrogradely through the ascending aorta, while venous outflow of perfusate occurred from the right chambers of the heart. A polyethylene balloon was inserted into the left ventricular cavity, connected to a pressure transducer to register intra-left ventricular pressure and create an adequate preload. Coronary perfusion volumetric rate was also recorded by measuring venous outflow. Perfusion was performed with modified Krebs-Henseleit buffer solution (consisting of the following [in mmol/L]: NaCl, 118.5; KCl, 4.7; NaHCO3, 25; KH2PO4, 1.2; MgSO4, 1.2; glucose, 11; and CaCl2, 1.5) at a constant pressure of 80 mm Hg and temperature +37 С [24]. In this case, the left ventricle contracted in isovolumetric mode due to the fact that the volume of the balloon introduced into its cavity was constant and provided preload at the physiological level (no more than 12 mm Hg). Intra-ventricular pressure was recorded using PhysExp software (Cardioprotect Ltd., Saint Petersburg, Russian Federation).
After the end of the stabilization period lasting 5 minutes, the functional parameters of the heart were recorded. Left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP) were measured isovolumetrically using a nonelastic polyethylene balloon introduced into the left ventricle via the left atrium. Left ventricular developed pressure (LVDP) was calculated as the difference between LVSP and LVEDP. Intensity of coronary perfusion (coronary flow rate (CFR) was determined by measuring the time for the collection of perfusate outflow.
Total 30-minute normothermic myocardial ischemia and subsequent 90-minute reperfusion were induced by reversible shutdown of perfusion. During the period of ischemia, the value of intra-left ventricular pressure was recorded every 5 minutes in order to assess the severity of ischemic contracture. During the reperfusion period, functional parameters (coronary blood flow and intra-ventricular pressure) were recorded every 15 minutes.
Infarction size measurement
At the end of reperfusion, the volume of the irreversibly damaged myocardium was measured by the method of histochemical staining of transverse sections of the heart with 1% triphenyltetrazolium chloride solution. The sections were incubated in the indicated solution for 15 minutes and the viable myocardium was stained bright red. Areas of irreversibly damaged myocardium remained unstained. Then the photographs of the sections were made with a stereomicroscope (SMZ18; Nikon, Tokyo, Japan) coupled to a digital camera (DS-Fi2, Nikon, Tokyo, Japan) and computer processing of the images was performed. Infarction size was expressed as a percentage of total ventricular area minus the cavities.
Statistical analysis
Statistical data processing was performed using the software package IBM SPSS Statistics-22 (IBM, USA) and Statistica-10 (Statsoft, USA).
The significance of differences between groups was assessed using the nonparametric Kruskal-Wallace and Mann-Whitney test for independent samples, using a nonparametric analysis of variance (a posteriori pairwise comparison of groups using the Dunn test). The significance of differences within one group was assessed using the nonparametric Friedman and Wilcoxon tests for dependent variables with the introduction of the Bonferroni correction with false discovery rate. All indicators are presented as "median (25%; 75%)". P values less than 0.05 were considered significant.