Study On The Effect Of Hyperlipidemia On Dysfunction Of β Cells Of Diabetic Rats And The Effect Of Insulin Therapy

Study on the mechanism of the dysfunction of β cell of diabetic rat induced by hyperlipidemia and the effect of insulin therapyObjective: To explore the effect of hyperlipidemia on β cell function of diabetic rat and the possible mechanism involved, and also to explore the effect of insulin therapy.Methods: DM rats were set up by small doze of streptozotocin (STZ, 25mg/kg body weight) and high fat diet. DM rats were randomly divided into three groups: DM high fat (DH) group, DM normal chow(DN) group and DM high fat diet plus insulin therapy(DHI) group, and normal Wistar rats was normal control (NC) group. The rats of DH group and DHI group were fed with high fat diet while the rats of DN group and NC group were fed with normal chow. The rats of DHI group were also received insulin therapy. 10 weeks later, all the rats were performed oral glucose tolerence test (OGTT) and the level of plasma glucose and insulin were tested. The ratio of the increment value of insulin and glucose after glucose charge(ΔI30/ΔG30) and the modified β cell secretion index (MBCI) were calculated according to the value of plasma glucose and insulin. The pancreas were resected, and the head part of pancreas were used to make paraffin slice while the tail part of pancreas were stored in the liquid nitrogen. The insulin protein, the average β cell area and the relative β cell mass were tested by SABC method. The expression of insulin gene, Caspase-3 gene and serine palmitoyl transferase (SPT) gene were tested by RT-PCR method and the insulin in pancreas was extracted by the method of acid-alcohol and tested. The expression of PCNA protein and Bcl-2 protein were tested by the method of immunofluorescence double mark. The expression of proliferating cell nuclear antigen (PCAN) protein was also tested by the immunestaining. The rate of apoptosis of β cell was tested by prodimum iodide (PI). The lipid in the plasma and pancreas was also tested.Conclusions: Compared with NC group, the FFA and TG in the plasma and pancreas of DH group and DN group were increased, while the expression of insulin gene and insulin protein, the insulin content, the curve of insulin secretion after glucose charge, ΔI30/ΔG30 and MBCI of DH group and DN group were lower than those of NC group, and the above changes were more to DH group(P<0.01～P<0.05).Compared with NC group, the relative β mass and the average of β cell area of DH group and DN group were decreased; the rat of β cell apoptosis, the expression of SPT gene and Caspase-3 gene were increased while the expression of Bcl-2 and PCNA protein were decreased, and the above changes were more to DH group(P<0.01～P<0.05).Compared with DH group, although the plasma FFA and TG of DHI group were not improved, the FFA and TG in pancreas were decreased dramatically. The expression of insulin gene and protein, the insulin content, the secretion function of insulin, ΔI30/ΔG30, MBCI, the relative β cell mass and the average β cell area of DHI group were improved dramatically; and the rate of β cell apoptosis, the expression of SPT and Caspase-3 gene and the expression of Bcl-2 and PCNA protein were also improved (P<0.01～P<0.05).Conclusions: Hyperlipidemia could impair the secretion function of insulin of diabetic rats induced by small doze of STZ and high fat diet. The dysfunction of insulin secretion was related with the decrement of insulin conten caused by the decrement of the expression of insulin gene and protein and the decrement of β cell mass caused by the decrement of replication and the increment of apoptosis of β cell. Insuin therapy had protective effect on the above changes caused by hyperlipidemia, and this effect correlated with the the decrement of FFA and TG content in pancreas. Chapter Ⅰ Study on the setting of the model of diabetic rat and the metabolic characters of itObjective: To explore the character of the plasma glucose, plasma lipid (FFA and TG), plasma insulin, insulin sensitivity index(ISI) and the insulin resistance index of the diabetic rat induced by small dose of STZ and high fat diet.Methods: Male Wistar rats were randomly divided into NC group and model group. The rats of model group were treated with STZ (25mg/kg body weight) ip, while the rats of NC group were treated with citric acid buffer. Two weeks later, all rats were received OGTT, and the rats bearing the character of impaired glucose tolerence were futher fed with high fat diet. The rats of NC group were still fed with normal chow. 8 weeks later, all rats were received OGTT again and plasma glucose was tested to make sure whether the DM rats were set up or not. And the plasma free fatty acid (FFA), plasma triglyceride (TG), plasma cholesterin (Chol) and plasma insulin of DM rats were tested. And ISI and insulin resistance index were calculated according to the plasma glucose and plasma insulin.Results: The plasma glucose at 0min, 30min, 60min 和 120min of DM rats were 1.5times, 1.4times, 1.5 times and 1.6 times those of NC gourp (P<0.01) . Compared with NC group , plasma FFA, TG and Chol of DM rats were also increased and the difference between them has the stastical meaning (P<0.01) . Compared with NC group, The fasting plasma insulin of DM rats was 1.2 times that of NC group (P<0.05), and ISI were decreased (P<0.01)while the insulin resistance index were increased (P<0.01).Conclusions: The diabetic model setted up by the small dose of STZ and high fat diet bear the character of hyperglycemia, dyslipidemia, insulin resistance(IR) and hyperinsulinemia. Chapter Ⅱ Study on the effect of hyperlipidemia on the expression of insulin gene and protein , the insulin content and the secretion function of β cell of the diabetic ratsObjective:To explore the effect of hyperlipidemia on the expression of insulin gene and protein , the insulin content and the secretion function of β cell of the diabetic rats.Methods: DM rats were divided into two groups randomly: DH group and DM group; and normal Wistar rats was NC group. The rats of DH group were fed with high fat diet and the rats of DN group and NC group were fed with normal chow. 10 weeks later, all the rats were performed OGTT and the value of plasma glucose and insulin at 0min, 30min, 60min and 120min were tested. ΔI30/ΔG30 and MBCI were calculated according to the value of plasma glucose and insulin. The pancreas were resected. The head part of pancreas were used to make paraffin slice and the insulin protein in β cell were tested by SABC method and the expression of insulin protein were tested. The tail part of pancreas were stored in the liquid nitrogen, the expression of insulin gene was tested by RT-PCR method and the insulin in pancreas were extracted by the method of acid-alcohol and tested. The lipid in the plasma and pancreas were also tested.Results: Compared with NC group, the lipid in the plasma and the pancrease were increased, while the curves of insulin secretion after glucose charge of DH group and DN group were lower, and the ΔI30/ΔG30 and MBCI were decreased also dramatically, especially to the rats of DH group (P<0.01～P<0.05) .The expression of insulin protein and gene and the insulin content in pancreas of DH and DN group were dramatically lower than those of NC group, and the above changes were more to DH group(P<0.05).Conclusions: Long-term hyperlipidemia could impair the expression of insulin gene and protein and the insulin content in the pancreas of DM , and the secretion function of insulin of β cells was also decreased dramatically. And those results of hyperlipidemia may correlate with the lipid content in the pancreas. Chapter Ⅲ Study on the effect of hyperlipidemia on beta cell mass of dibetic rat and the mechanism involvedObjective: To explore the effect of hyperlipidemia on β cell mass and the mechanism involvedMethods: After marking the insulin protein of β cell by SABC method, the average β cell area and the relative β cell mass were tested. The expression of PCNA protein was tested by the methods of immunofluorescence double mark and immunestaining. The expression of Bcl-2 protein was tested by the method of immunofluorescence double mark. The rate of apoptosis of β cell was tested by PI and the expression of SPT and Caspase-3 gene were also tested by RT-PCR.Results:The relative β cell mass and the average β cell area of DN group and DH group were less than those of NC group(P<0.05,P<0.01). Compared with NC group, the β cell apoptosis and the expression of SPT and Caspase-3 gene of DH group and DN group were higher, while the expression of PCNA and Bcl-2 were lower (P<0.05). And these indexes between the DH group and DN group also had stastical difference(P<0.01).Conclusions: Hyperlipidemia could cause the β cell loss in the diabetic rat, and the loss correlated with the increment of β apoptosis and the decrement of β cell replicaion. The increment of the expression of SPT and Caspase-3 gene and the decrement of the expression of Bcl-2 took part in β cell apoptosis. Chapter Ⅳ Effects of insulin therapy on the dysfunction of β cell of diabetic rats caused by hyperlipidemiaObjective: To explore the effect of insulin therapy on the dysfunction of diabetic rat caused by hyperlipidemia and the mechanism involved.Methods: DM rats were randomly divided into three groups: DH group, DHI group and DN group; and NC group. The rats of DH group and DHI group were fed with high fat diet while the rats of DN and NC group were fed with normal chow. The rats of DHI group were also received insulin therapy. 10 weeks after insulin therapy, all the rats were executed and the above indexes in the part Ⅱ and part Ⅲ were tested.Results: Compared with DH group, although the plasm FFA and TG were not improved, the TG and FFA content in pancreas of the rats of DHI group were decreased. The expression of insulin gene and protein and the insulin content were increased, were respectively 4.41 times(P<0.01), 2.11 times(P<0.01) and 1.47 times those of DH group. Compared with DH group, the secretion function of group was improved and ΔI30/ΔG30 , MBCI were higher than those of DH group(P<0.01～ P<0.05); The relative β cell mass and the average β cell area were also improved(P<0.01～P<0.05). And this is also true to the apoptosis of β cell, the expression of SPT and Caspase-3 gene and the expression of Bcl-2 and PCNA protein (P<0.01～P<0.05).Conclusions: Insulin therapy could improve the dysfunction of β cell caused by hyperlipidemia, and may partly relate to the decrement of FFA and TG content in pancreas.