The aim of this study was to determine the effect of melatonin on activity of pyruvate kinase (PK) and lactate dehydrogenase (LDH) in the heart tissue and on the activity of succinate dehydrogenase (SDG) and H+-ATPase in mitochondria of cardiomyocytes of rats with dexamethasone diabetes. Methods. The research was performed in compliance with the Rules of the work with test animals (1977) and the European Convention on the Protection of Vertebrate Animals used in experiments and other scientific purposes (18 March 1986). The experiments were carried out on 36 sexually mature male albino rats with the body mass 0.18 – 0.20 kg. The animals were divided into 3 groups:1) control group; 2) group with diabetes mellitus DM (BG level ≥ 8.0 mmol/l); 3) diabetic animals which were injected with melatonin. Melatonin (“Sigma”, USA) was injected intraperitoneally in a dose of 10 mg/kg of body weight daily over 13 days. Dexamethasone diabetes was evoked by daily subcutaneous administration of dexamethasone (solution for injection 4 mg / ml, KRKA, Slovenia) at a dose of 0.125 mg / kg body weight for 13 days (O.V. Stefanov, 2001). The mathematical model of the insulin-glucose correlation by the Homeostasis Model Assessment (HOMA) was used to evaluate the insulin resistance in the animals (D.R.Matthews et al., 1985). The blood was taken from the tail vein to evaluate the BG level using “OneTouchUltra” (“LifeScan”, USA). The rats were sacrificed on the 14th day of the experiments in accordance with the ethical treatment of animals. 5% homogenate was prepared from cold-isolated rat hearts on chilled 50 mM Tris-HCl buffer (pH = 7.4) with sucrose. Mitochondria were isolated by differential centrifugation. Enzyme activities in the mitochondrial fraction was determined by: succinate dehydrogenase (SDG) according to the method of Eshchenko N.D. et al. (1992), H + -ATP-ase - for Gabibiv M.M. (1986). Serum insulin content was determined using an automatic immunochemiluminescent analyzer (Snibe Co., Ltd, PRC) using a Maglumi test kit. The insulin resistance index (HOMA-IR) was calculated by the formula: HOMA-IR = (glucose (mmol / l) • insulin (mcU / ml)) / 22.5. To determine the enzymes activities by standart methods, heart muscle tissue was quickly removed, rinsed in saline, blotted, weighed and homogenized. Then the homogenate (5% in ice-cold 0.25 mM tris-HCl-buffer) was ultracentrifugated (10 min at 1500r/min) and the supernatant was used for measurements. Statistical analysis was performed using Statistica 10 (StatSoft Inc). Prior to analysis, Shapiro-Wilk test was used to assess the normality of the group data. According to the criterion, the samples distributions differed from normal distribution. Given these, use of the Mann-Whitney test was considered sufficient for valid conclusions to be made. Differences were considered to be statistically significant when P ≤ 0.05. Results. In the blood of all diabetic rats on the 14th day the glucose content exceeded 8.9 mmol / l, and the HOMA-IR index increased 7 times. According to results, we have obtained the activity of PK in the heart tissue of diabetic animals was 55% less than in control group. It can be explained by low uptake of glucose from the blood by heart muscle tissue in conditions of insulin resistance. Reduced glucose uptake by peripheral tissues, in turn, leads to a reduced rate of glucose metabolism. That means now substrate for next catabolic changes in glycolysis. The activity of LDH increases by 32% in the heart muscles of diabetic rats compared with the control values. We have found out that the activity of PK and LDH has become normalized by the introducing of melatonin in dose of 10 mg/kg of body weight. In the mitochondria of the animals with dexamethasone diabetes, the activity of SDG and H+ -ATPase is significantly lower than that in intact animals by 45 and 32%, respectively. Probably, melatonin enhances the uptake of glucose by heart muscles. The blood glucose content of diabetic rats, which were administered is not significantly different from that of the intact animals, and the HOMA-IR was 6.3 times lower than that of diabetic rats. The activity of SDG and H+-ATPase in the mitochondria of rats receiving both melatonin and dexamethasone does not differ from that of intact rats. Conclusion. A 14-day introduction of melatonin leads to a marked improvement of the state of carbohydrate metabolism as well as the energy metabolism in heart tissue that is accompanied by the normalization of the indices studied.
THE EFFECT OF MELATONIN ON THE ENERGY METABOLISM IN HEART OF DIABETIC RATS