The Role Of Glucokinase In Liver Tissue Is Regulated By Glucokinase Activation Agent HMS5552 In Rats Of Type 2 Diabetes

OBJECTIVE To observe the regulating effect of and possible mechanism of action that glucose kinase activator(HMS5552) to the glucosekinase in the liver tissue of type 2 diabetic rats. METHODS The animal models of type 2 diabetes is induced that using high fat and sugar and urea with intraperitoneal injection of one-time chain cephalosporins streptozotocin(STZ) with 40 mg/Kg.From 60 healthy SD rats randomly selected 10 as normal group, the rest were randomly divided into diabetic group, low dose treatment group and high dose treatment group.Normal group is feed with normal fodder and the other three groups are feed with high fat and sugar fodder.Take heart blood in each of rats to measure the cholesterol, triglyceride, blood glucose, insulin and glucagon indicators before the treatment of HMS5552. During the treatment of HMS5552 and at 8 o ’clock, the normal group with normal saline lavage, diabetes group with dissolved HMS5552 buffer to fill the stomach, and low and high dose treatment group respectively with 10 and 30 mg/Kg HMS5552 lavage. During the treatment of HMS5552,detect fasting plasma glucose and oral glucose tolerance test(OGTT) and oral drug tolerance test(ODTT) of each group of rats through the tail vein blood. Kill the all rats and take their blood to detect the cholesterol, triglyceride, blood glucose, insulin and glucagon indicators after a month.Take the liver, pancreas and small intestine paraffin section for the fresh tissue: HE staining to observe the histopathological changes, immunohistochemical staining to observe the tissue glucose kinase(GK), insulin(INS), glucagon(GC) and pancreatic blood sugar peptide 1(GLP-1) expression.Take fresh liver tissue, using glucose 6- phosphate dehydrogenase coupling colorimetric method of kinase of glucose in the liver tissue of Km.Using Western Blotting and RT-PCR method respectively from the level of protein and nucleic acid level detection of GK expression changes. RESULTS(1) The model of type 2 diabetic rats is successful that the fasting blood glucose ≥ 8.0 mmol/L.(2) According to the results of fasting glucose compared with normal group, diabetes group fasting glucose continue to rise, and the treatment group fasting glucose have varying degrees of decline in the diabetic group. Compared with diabetes group that low dose treatment group fasting glucose down 28.57%(P < 0.05) and high dose treatment group fasting glucose down 42.45%(P < 0.05).(3) According to the results of the the OGTT, normal group blood sugar spikes at about 15 minutes and after the meal 2h blood glucose levels rise to normal levels. Compared with normal group that the peak of diabetic group moves to the right and the peak is at 90 minutes and 2h blood sugar after meals can’t return to baseline levels.One month after treatment, the low dose treatment group and high dose group of fasting plasma glucose values were obviously improved, blood glucose peak compared with diabetes group, peak shift to the left.The area under the concentration time curve(AUC) measurement showed that low dose treatment group and high dose treatment group Δ AUC0-240 compared with diabetes group, with significant difference statistically significant(P < 0.01), low dose treatment group and high dose treatment group Δ AUC0-240, there was no statistically significant difference(P = 0.57).(4) According to the results of giving drugs HMS5552 in ODTT that lavage after 30, 60, 90, 120, 180, 120 min, blood sugar levels gradually decline, 240 min compared with on an empty stomach, low dose treatment group and high dose treatment group blood sugar was reduced by 25.73% and 26.89% respectively, 60 min after meal blood sugar levels are lower in the diabetic group.After medication for a month, the treatment group significantly lower fasting blood sugar levels, giving drugs HMS5552 lavage after 240 min compared with on an empty stomach, low dose treatment group and high dose treatment group blood sugar was reduced by 16.67% and 16.67% respectively.(5) According to the results of glucose 6- phosphate dehydrogenase coupling colorimetric that the Km of diabetes group kinase increased obviously in liver tissue and the Km of the treatment group decreased significantly(P < 0.01) compared with normal group.(6) According to the results of fasting insulin that diabetes group of fasting insulin level rise compared with normal group and the treatment group fasting insulin have varying degrees of decline in the diabetic group, compared with diabetes group that low dose treatment group fasting insulin down 19.71%(P < 0.05) and high dose treatment group fasting insulin down 47.02%(P < 0.05).(7) HE staining results showed that form the normal liver of normal group, liver cells arrange compact and tidy, radiate out in around the central vein.Arrange osteoporosis diabetes group of liver cells, the serious fat cavitation can be seen infiltrating through the.The diabetes treatment group liver tissue form have different degrees of improvement.The pancreatic tissue of normal group is normal.Diabetes group of visible partislet cell degeneration atrophy.The diabetes treatment group in the pancreas form have different degrees of improvement.(8) Immunohistochemical results showed that the expression of GK in the liver, INS in the pancreas and GLP-1 in the small intestine in diabetes group were significantly lower compared with normal group. The GK, INS and GLP-1 in the treatment group has different degrees of improvement compared with diabetes group. Compared with normal group that the pancreas GC increases in diabetes group and the GC in the treatment group with varying degrees of decline compared with the diabetes group.(9) According to the results of RT-PCR that the expression of GK m RNA in the liver tissue reduced in diabetes group compared with normal group. The expression of GK mRNA in the treatment group increased to different extent compared with the diabetic group and the high dose treatment group increased significantly(P < 0.01).(10) According to the results of Western blotting that the amount of GK protein in liver tissue lower in diabetes group compared with normal group. The content of GK protein in the treatment group increased to different degree compared with the diabetic group and the high dose treatment group increased significantly(P < 0.01). CONCLUSION(1) The type 2 diabetic rats model is copied successfully through the method that high fat and sugar feed induction combined with one-off chain urea with cephalosporins injection. The physiological and pathological changes of the rats cconform the clinical symptoms of type 2 diabetes.(2) The kinase activity of glucose in the liver tissue of rats in type 2 diabetes related the change of blood glucose levels closely.(3) The glucose kinase activator HMS5552 can make rat liver tissue GK expression.(4) The glucose kinase activator HMS5552 can lower the blood sugar levels through the regulation of the concentration and activity of GK in rat liver.