| Objective: Diabetic cardiomyopathy is a severe complication of diabetes mellitus. It is characterized by myocardial hypertrophy, fibrosis, apoptosis, cadiocyte dysfunction, conveying discrepancy of calcium and metabolic abnormality of fatty acid in cellular level. It can lead to heart failure, arrhythmias, angina, cardiac shock and sudden death. The pathogenesis of diabetic cardiomyopathy remains uncertain. Glucagon is a peptide hormone secreted by pancreatic alpha cell. Its major physiological functions include promoting glycogenolysis, gluconeogenesis and inhibiting glycogenesis. Glucagon has been increasingly recognized in the past few years. Its role on the pathogenesy of type 2 diabetes mellitus has been emphasized. The aims of this study are to verify whether high glucagon has non-glucose-heightening and growth-promoting effect on cardiac myocytes of neonatal rats, and leads to lipid metabolic abnormalities, further to investigate the effect of hyperglucagonaemia on diabetes mellitus and diabetic cardiomyopathy, and also explain the theory foundations for pathogenesis and new therapy of diabetes mellitus and complications. Methods: Cardiac myocytes from neonatal rats were isolated by collagenase sequential digestion method and different speed adherence method and cultured in vitro. After seventy-two hours of culture, the culture solution was changed into Dulbecco's modified Eagle's medium (DMEM) without blood serum. The cells cultured in vitro were assigned into seven groups. Each group included five wells. Administration while the medium changed. The first group was the normal control group, no administration. The second was the high glucagon group administrated by 10-10mol/L glucagon. The third was the high glucose group administrated by 25.5mmol/L glucose. The fourth was the high insulin group administrated by 10-7mol/L insulin. The fifth was the high glucose-high glucagon group administrated by 25.5mmol/L glucose and 10-10mol/L glucagon. The sixth was the high glucose-high insulin group administrated by 25.5mmol/L glucose and 10-7mol/L insulin. The seventh is the high glucose-high insulin-high glucagon group administrated by 25.5mmol/L glucose, 10-7mol/L insulin and 10-10mol/L glucagon. The morphology and beating of cardiac myocytes were observed with inverted phase contrast microscope every day after administration and the following indexes were detected after forty-eight hours of administration:(1) The cell numbers of each group were counted by trypanblau staining method.(2) The apoptosis of cardiac myocytes was detected by terminal deoxynucleotidyl transferas (TdT) mediated dUTP nick end labeling(TUNEL), and the cardiac myocyte apoptotic index (CMAI%) was calculated.(3) The content of intracellular free fatty acid and lipoprotein lipase was detected by ultraviolet spectrophotometry. The levels of lactate dehydrogenase (LDH),creatine kinase (CK) and MB isoenzyme of creatine kinase (CK-MB) of cell culture fluid were detected by biochemistry.Results: (1) We cultured cardiac myocytes of neonatal rats successfully.(2) The cardiac myocyte numbers of each experimental group were more than those of the normal control group after administration. Cell counting: 8.0×105/ml of the normal control group, 9.0×105/ml of the high glucagon group, 9.1×105/ml of the high glucose group, 8.8×105/ml of the high insulin group, 9.7×105/ml of the high glucose-high glucagon group, 9.0×105/ml of the high glucose-high insulin group, and 10.0×105/ml of the high glucose-high insulin-high glucagon group.(3) The apoptosis of cardiac myocytes was detected after administration. There were cells with dark brown nucleus in every group. The CMAI% of the high glucose group was 32.80±2.64%, and the apoptosis cells were obviously more than those of the normal control group (t=20.941, P=0.000). There was no obviously difference between the high glucagon group and the normal control group (t=1.796, P=0.110), the high insulin group and the normal control group (t=0.460, P=0.658). There was no difference between the three combination groups and the normal control group respectively (p>0.05).(4) Compared with the normal control group, the intracellular FFA levels of the high glucagon group (t=26.422, P=0.000), the high glucose group (t=39.269, P=0.000), the high insulin group (t=4.615, P=0.002), and the high glucose-high glucagon group (t=8.415, P=0.001) were significantly higher. The intracellular LPL levels of the high glucagon group (t=6.295, P=0.002), the high glucose group (t=13.591, P=0.000), the high insulin group (t=4.913, P=0.001), and the high glucose-high glucagon group(t=14.052, P=0.000) were also significantly higher. The intracellular LPL of the high glucose-high glucagon-high insulin group was significant higher than that of the normal control group (t=2.768, P=0.024), but there was no significant difference of the intracellular FFA (t=-1.948, P=0.087). There were no significant differences of the intracellular FFA (t=-0.146, P=0.887) and LPL (t=0.948, P=0.371) between the high glucose -high insulin group and the normal control group. (5) Compared with the normal control group, the LDH levels in the cell culture fluid of the high glucagon group (t=5.090, P=0.001), the high glucose group (t=2.601, P=0.032), and the high glucose-high glucagon group (t=3.228, P=0.012) were higher. But there were no differences between the other experimental group and the normal control group (P>0.05). There were no significant differences of the CK and CK-MB level in the cell culture fluid of each experimental group compared with the normal control group (P>0.05). Conclusion: (1) High glucagon has non-glucose- heightening and growth-promoting effect on cardiac myocytes of neonatal rats. It may cause hyperplasia of cardiac myocytes as high glucose and high insulin do. (2) High glucose may facilitate apoptosis of cardiac myocytes, but high glucagon may not have this effect. (3) High glucagon may promote myocardium lipoclasis, and induce lipid metabolic abnormalities. High glucagon may play an important role on the pathogenesis of diabetic cardiomyopathy. |
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