Study Of Type 2 Diabetic Rat Model And Its Application In The Valuation Of Hypoglycemic Functional Food

BackgroudsWith the socio-economic development and people's diet and lifestyle changes, diabetes (DM) has become a serious hazard to human health. Diet and nutrition therapy is an important part of diabetes treatment. Preventing and controlling diabetes by diet and functional foods become a trend. Hypoglycemic functional foods mainly originate from natural plant foods or their extracts, their wide variety of sources and different mechanisms of hypoglycemic make a great challenge to the evaluation of functional foods.Current standards of health food evaluation only given a similar type 1 DM chemically induced animal model, clearly can not meet the requirements of the multiform development of health food. Therefor we initiated this study with the objective of developing a suitable type 2 diabetic rat model that would on the one hand closely mimic the natural history of the disease events (from insulin resistance to beta cell dysfunction) as well as metabolic features of human type 2 diabetes and useful for the investigation as well as preclinical testing of various functional foods viz. insulin sensitizers and insulinotropics for the treatment of type 2 diabetes.Objective1. To develop a rat model that replicates the natural history and metabolic characteristics of human type 2 diabetes.2. To indentify the reproducibility and applicability of the animal models3. To verify the applicability of the established animal models by drug and fuctional foods assessment.4. To study the preventive effects and possible mechanisms of Pueraria and EGCG on the regulating blood glucose.Materials and methods1. The establishment of type 2 diabetic rat model1.1 Effects of high-fat diet ratio on the modeling 55 healthy Wistar rats were randomly assigned to normal group and high-fat-sugar feeding group (HFS) based on initial body weight. According to the ratio of lard (F) and sucrose (S) in feeds, HFS feeding group diets were repartition to 10F10S (representing containing 10% lard and 10% sucrose in feeds, same below),10F20S, 20F10S. Body weight was weekly recorded. At the 8th week, fasting blood glucose and 2h postload glucose after oral glucose torlerence test (OGTT) were tested. Blood lipid level were detected. The influence of fat and sugar in diets on body weight gain, glucose response and lipid was analyzed.Then, each dietary group was injected intraperitoneally (i.p.) with low dose of STZ(25 mg/kg). Fast plasma glucose and OGTT were carried out every week. The influence of dietary regimens on the animal model was envaluated based on integration of injury intensity, blood glucose levels and mortality.1.2 Effects of STZ injection dose on the modelingThe rats were allocated into two dietary regimens consisting of 20 and 55 rats by feeding either common or hight-fat diet. Base on body weight, blood glucose and lipid level,55 HFS-fed rats were randomly divided into 4 groups:STZO, STZ20, STZ25 and STZ30. After fasted for overnight, each groups were respectively intraperitoneally injected with 0,20,25,30 mg/kg.bw of streptozotocin (STZ). The basis of observation on the relationship between dose depended blood glucose response and death, the appropriate dose of STZ used in model made was indentified. Then,5 weeks of blood glucose monitor was preceeded in each groups. The influence of STZ injection dose on the modeling was evaluated based on integration of injury intensity, blood glucose levels, mortality and islet morphology.2. The replication and characteristics of type 2 diabetes rat modelThe rats were allocated into two dietary regimens consisting of 10 and 85 rats by feeding either common or high-fat diet. Eight weeks later, blood lipid was measured and OGTT was carried out in each group and insulin sensitivity index (ISI) was calculated and compared.Then 75 high-fat diet rats were injected intraperitoneally (i.p.) with low dose of STZ(25 mg/kg). Fast plasma glucose and OGTT were carried out 2 weeks later to make sure that type 2 diabetes was induced in rats.10 rats with type 2 diabetes and 10 common or high-fat diet rats were chosen and continued on their original diets for 5 weeks. The body weight and ALT, AST, CR, LDL, HDL, TG, TC, GLU and INS were carried out. The pancreatic, kidney, liver, heart tissues were stained with hemotoxylin and erosin. The expressions of InsR, IRS, PPARy were examined using Western blot method.3. The validation of Type 2 diabetic rat model10 normal control rats (N) and 27 rats with type 2 diabetes were selected and then assign to insulin secretion agent repaglinide (1mg/kg), insulin sensitizer rosiglitazone (0.5mg/kg) or distilled water.Each group continued on their original diets for 5 weeks. Five weeks later, the body weight and ALT, AST, CR, LDL, HDL, TG, TC, GLU and INS were carried out. The pancreatic, kidney, liver and heart tissues were stained with hemotoxylin and erosin. The expressions of InsR, IRS and PPARy were examined using Western blot method.4. The applicability of Type 2 diabetic rat model for the assessment of fuctional food4.1 Effects of Puerarin on rats with type 2 diabetes10 normal control rats (N) and 36 rats with type 2 diabetes were selected and then assigned to low dose of puerarin (50mg/kg), middle dose of puerarin (100mg/kg) high dose of puerarin (200mg/kg), or distilled water.Each group continued on their original diets for 5 weeks. Five weeks later, the body weight and ALT, AST, CR, LDL, HDL, TG, TC, GLU and INS were carried out. The pancreatic, kidney, liver and heart tissues were stained with hemotoxylin and erosin. The expressions of InsR, IRS, PPARy, and NF-κB were examined using Western blot method.4.2 Effects of EGCG on the prevention of type 2 diabetesSelected 10 normal control rats (N); 40 rats with insulin resistant were randomly divided into 4 groups:H, E1, E2 and E3 group.One time hypoglycemic effect of EGCG:single intragastric administration were given to N, H, E1, E2 and E3 group with distilled water group, distilled water, EGCG low dose (200mg/kg), EGCG middle dose (700mg/kg) or EGCG high dose (1000mg/kg). OGTT were carried out.Short-term hypoglycemic effect of EGCG:after one time experiment, group N, H, E1, E2 and E3 group were given distilled water, distilled water, EGCG low dose (20mg/kg), EGCG middle dose (50mg/kg), EGCG high dose (200mg/kg) orally. After EGCG intervention 10 days, OGTT were carried out.Long-term hypoglycemic effect of EGCG:After EGCG interference 5 weeks. The body weight, ALT, AST, CR, LDL, HDL, TG, TC, GLU and INS were carried out. The pancreatic, kidney, liver and heart tissues were stained with hemotoxylin and erosin.Results1. The establishment of type 2 diabetic rat model1.1 Effects of high-fat diet ratio on the modelingInsulin resistance was induced in high fat diet feeding rats 8 weeks later. The weight of HFS groups has exceeded 10% of N group, but no significant difference amog HFS groups (P<0.05). The PBG of HFS groups increased also (P<0.05), insulin sensitivity index (ISI) was significantly lower than the control group (P<0.05). After STZ injection, body weight decreased rapidly in the first week.2 weeks later, body weight begun to stabilize. The level of blood glucose in 20F10S increased significantly (p<0.05). The level of PBG in HFS groups significantly increased compared with the pre-modeling (p<0.05).20F10S had the highest level of blood glucose, there were 4 rats'PBG over 30mmol/L. The level of FBG in 10F20S group was not obvious increased, but the PBG increased significantly (p<0.05), range of 11.1-20mmol/L. The achievement ratio in 10F20S and 20F10S were 73.3%,10F10S was 66.6%.1.2 Effects of STZ injection dose on the modelingThe 20mg/kg of STZ injection group had low successful rate(30%); The 25mg/kg of STZ injection group had the highest rate (73.3%) of establishing model successfully, and stable moderate hyperglycemia. Islet structural integrity, but the number ofβ-cell and cytoplasmic brown granules decreased; The 30mg/kg of STZ injection group had the highest mortality and highest blood glucose, the number of islets decreased obviously, small islets more common, islet structure is not complete.2. The replication and characteristics of type 2 diabetes rat modelInsulin resistance was induced in high fat diet feeding rats 8 weeks later. The insulin resistant rats had higher body weight and levels of FBG and PBG compared to control group(P<0.05). Islets mass enlarged in HFS groups,but the expression of insulin was lower than N group. The expressions of IRS-2 and PPARy decreased significantly.After low-dose intraperitoneal injection of STZ, rats had the following changes:1) FBG (8.14±5.87mmol/L) and PBG (17.23±8.98mmol/L) were moderately elevated. ISI was less than N group (p<0.05), liver glycogen increased 5 times above the N group. The achievement ratio was 71.6%.2) histological detection:Islet structural integrity, but the number ofβ-cell and cytoplasmic brown granules decreased; the liver were small bubble-like steatosis 3) The expressions of IRS-2 and PPARγdecreased significantly in the Peripheral tissue.3. The validation of Type 2 diabetic rat modelAfter 5 weeks of insulin promoter (repaglinide) and insulin sensitizer (rosiglitazone) treatment, FBG and PBG levels in rats were significantly improved (p<0.05); Insulin immunohistochemistry:rosiglitazone and repaglinide could improveβ-cell volume and cytoplasmic brown granular level, repaglinide stimulateβ-cell insulin secretion was slightly stronger than rosiglitazone; rosiglitazone and repaglinide could increase the expressions of IRS-2 and PPARγ.4. The applicability of Type 2 diabetic rat model for fuctional foods assessment.4.1 Effects of Puerarin on rats with type 2 diabetesPuerarin could significantly improve the indicator level of DM rats by 5 weeks intervention.1) Blood biochemical indicators:compared with the diabetic control group, puerarin could reduce the levels of FBG and PBG in diabetic rats, improve the ISI, improve liver and kidney function, and had dose-response relationship (p<0.05).2) Histological detection:with the dosage increased, the number of pancreas islets gradually increased, vacuolar degeneration decreased, the number ofβcells and insulin content was increased, but not yet reached the level of the normal group.3) Oxidative stress:puerarin could increase the antioxidant capacity of diabetic rats, increase the level of SOD and reduce the level of 8-OHdG and MDA (p<0.05).4) Insulin signaling pathway:puerarin inhibited the expression of NF-κB in peripheral tissue (liver, muscle) and increased the expressions of IRS-2 and PPARy.4.2 Effects of EGCG on the prevention of type 2 diabetesThe hypoglycemic effect of EGCG was concerned with the length of action time. In this study, we analyzed the effects of EGCG on blood sugar in three differern stages. There was no obvious effect in one time intervention. After 10 consecutive days of intervention, EGCG's middle and high dose group had a trend to improve glucose tolerance and reduce the level of postprandial blood (P<0.05). After 5 weeks intervention, EGCG lowered blood glucose (P<0.05), improved liver steatosis and isletβcell function.Conclusions1. Through study the effects of high-fat diet ratio and STZ injection dose on the modeling, we established the type 2 diabetic rat model with 10% lard and 10% sucrose high-fat diet and low dose of STZ(25 mg/kg) injection.2. The hyperglycemia models have strong consistency in duplication checks, and are suitable for long-term observation in prevention and intervention studies.3. These fat-fed/STZ-treated rats simulate natural disease progression and metabolic characteristics typical of individuals at increased risk of developing type 2 diabetes because of insulin resistance.4. Insulin secretion agent repaglinide and insulin sensitizer rosiglitazone, which could reduce blood glucose, confirmed from another aspect that the model still had adequate number ofβ-cell with function of contolling blood glucose level, and insulin resistance was one of the reasons leading to high blood sugar.5. The model had a good application in different kinds of functional foods and could reflect the different mechanisms (antioxidant, anti-inflammatory, insulin resistance).6. Puerarin could improve insulin signal transduction, its hypoglycemic effect might be related to raising the level of antioxidant, as well as inhibition the expression of NF-κB in inflammatory signaling pathway.7. The hypoglycemic effect of EGCG was concerned with the length of action time.The mechanism might be related to increasing the body's antioxidant capacity and its a-glucosidase inhibitor effects.