| The morbidity of type 2 diabetes mellitus (T2DM) increased rapidly in recent years. Insulin resistance is the most important pathological basic of T2DM and is closely correlated with the development of hypertension, hyperlipidemia, obesity, cardiovascular diseases, and so on. It was called the"common soil"of these diseases. Insulin resistance is one state that the insulin-stimulated glucose uptake and clearance decreased in the peripheral tissues.Genetic and environmental factors play major roles in the development of insulin resistance. It characterized by the defects in multiple levels, the defects at pre-receptor, receptor, post-receptor levels all relate to the development of insulin resistance. Insulin integrates with its receptors at first, and then induces a series of signal transductions, and realizes its physiological effects at last.In the past, the studies about IR focused on the insulin target tissues and the insulin receptors. With the development of molecular biological technology, many specialists discovered that defects in the insulin signaling pathway, abnormal secretion of adipokines and the inflammatory mediators all contributed to the development of insulin resistance.Protein kinase B (PKB) and glucose transporter 4 (GLUT4) are two important molecules in the insulin signaling pathway. They can modulate the insulin-stimulated glucose uptake. Their genetic deficits or activity changes may contribute to the development of insulin resistance.In recent years, more and more people realized that adipose tissue was an endocrine organ. Many cytokines secreted by adipocyte such as leptin, adiponectin, tumor necrosis factor alpha (TNF-α) are all related with the insulin resistant state. The present studies demonstrated that these adipokines performed different actions in IR; adiponectin can improve the insulin sensitivity, while the increase of other cytokines can impair the insulin sensitivity.At present, thiazolidinediones (TZDs) are used widely in the treatment of IR-linked diseases. They can specifically bind with the peroxisome proliferator-activated receptor-gamma (PPARγ), and activate the PPARγ, and then induce a special signaling pathway. The pharmacological effects of TZDs include promoting the adipocyte differentiation, improving the glucose and lipid metabolism; its clinical effects are significant. Last year the argument about the safety of rosiglitazone limited the use of these kinds of drugs.The characteristics of Traditional Chinese Medicine (TCM) treatment are multiple positions, multiple links and multiple targets. There are much superiority in the prevention and treatment on Diabetes Mellitus. And now, to search the traditional medicine drugs or the extract of these drugs is important in the prevention and treatment of IR. If we can explore the possible mechanisms of these drugs, it will benefit the TCM modernization, the treatment of IR, Diabetes Mellitus and the cardiovascular disease.EGB is used widely and has remarkable curative effect in dyslipidemia, senile dementia, cardiovascular and cerebrovascular disease. Now it was also used in the treatment of diabetic complications, chronic obstructive pulmonary disease, hepatic fibrosis, and had affirmative effects. Its international standard about the production provides the safety of its clinical usage.EGB has many pharmacological effects; it can modulate the blood lipid, improve the glucose metabolism, and ameliorate the function of endothelio- cyte. It also has the anti-oxadative effect and can remove the free radical. These links are all related with the improvement of insulin resistance. EGB may improve the insulin sensitivity. To verify this hypothesis, we used EGB to intervene the insulin resistance rats induced by high-fat-fed, and wanted to know the effects and the targets of EGB on improving insulin sensitivity. ObjectivesThe aim was to study the effect of EGB on the insulin sensitivity in IR rats induced by high-fat diets and its relative mechanisms. The research included the duplication and evaluation of the insulin resistant model; the improvement of insulin sensitivity after intervened by EGB; the influences on the blood glucose and lipid metabolism by EGB intervention; the regulation on adipocytokine secretion; and the influence on expression of PKB and GLUT4 in skeletal muscle and expression of PPARγin adipose tissue.MethodsTotal 80 clean male Wistar rats weighed 280～320 grams were randomly divided into two groups, one was normal control group (NC), there were 20 rats in this group; the other was high-fat-fed group (HF), there were 60 rats in this group. The rats in NC group were fed with standard rat chow containing 10.3% fat, 24.2% protein, and 65.5% carbohydrate as percentage of total calories. The rats in HF group were fed with isocaloric high fat diet containing 59.8% fat, 20.1% protein and 20.1% carbohydrate as a percentage of total calories. Body weight was observed every week. Four weeks later, 10 rats were chosen randomly from each group. After fasted for 10 hours, the rats were taken blood sample from endocanthion venous plexus; the serum was separated for the detection of lipid, insulin, leptin, adiponectin, tumor necrosis factor alpha and free fatty acid. The hyperinsulinaemic-euglycaemic clamp was performed to confirm that the insulin resistant rat model was duplicated successfully. After this procedure, the rest 50 rats in HF group were divided randomly into high fat control group (HC), the first EGB group (EGB1), the second EGB group (EGB2), the third EGB group (EGB3), rosiglitazone group (RSG), there were 10 rats in each group. The rats were intervened by different drugs and were maintained original diets. Among the total, the rats in EGB1, EGB2, and EGB3 group were given different dose of SHUXUENING injecta by intraperitoneal injection. The rats in EGB1 group were given 4 mg·kg-1·d-1; in EGB2 group were given 8 mg·kg-1·d-1; and in EGB3 group were given 12 mg·kg-1·d-1. The rats in RSG group were given Rosiglitazone 3 mg·kg-1·d-1 orally. The rats in HC and HC group were given sodium chloride by intraperitoneal injection, and the volume was the same with the EGB2 group. Once per day, the intervention lasted for 4 weeks. Body weight was observed once a week, and the dosage was regulated according to the body weight. After four weeks of intervention, the blood sample was taken from endocanthion venous plexus; serum was separated for the detection of lipid, insulin, leptin, adiponectin, tumor necrosis factor alpha and free fatty acid once again. The concentration of leptin, adiponectin, TNF-αwas determined by ELISA, the serum FFA level was measured by copper-chromogenic technology. The oral glucose tolerance test was performed to know the metabolic ability under the glucose-loaded condition. The hyperinsulinaemic- euglycaemic clamp was carried out to detect the change of GIR in different groups. After the clamp, the rats were sacrificed by depletion, the adipose pad around kidneys and epididymides was weighed exactly, and the relative content of visceral adipose was calculated. Quadriceps femoris and the renal or epididymal adipose pad were removed respectively and flash-frozen in liquid nitrogen, then stored at -70°C for further detections. The protein level of the muscle PKB, GLUT4 and the adipose tissue PPARγwas assayed by Western blotting. The expression of muscle PKB mRNA, GLUT4 mRNA and adipose tissue PPARγmRNA was assessed by real time RT-PCR. Data was collected and analyzed through t test or analysis of variance. The statistic software was SPSS. Differences with P values <0.05 were considered significant.Results1 The parameters of the rats after four-week high fat feeding The body weight of HF group rats was higher than that of NC group, but there was no statistic difference between the two groups (P>0.05). The serum total cholesterol, triacylglycerol, fasting insulin in HF group rats increased remarkably, there were significant differences compared with the NC group (P<0.05). The fasting blood glucose, serum free fatty acid and tumor necrosis factor alpha in HF group rats increased notablely too, the difference between NC and HF group was significant (P<0.01). While the serum adiponectin level and the glucose infusion rate decreased dramatically, there were remarkable differences when compared with the NC group (P<0.01 or P<0.05). The serum leptin level increased, but there was no difference compared with NC group (P>0.05). The rats in HF group also showed increase in the relative content of visceral adipose tissue (P<0.01). The protein level of skeletal muscle PKB in HF group had a down-regulated tendency, while compared with NC group, there was no significant difference (P=0.071). The protein level of skeletal muscle GLUT4 and adipose tissue PPARγin HF group decreased obviously (P<0.05).2 The changes of above-mentioned parameters after intervened by rosiglitazone or EGB.2.1 The changes of blood glucose, blood lipid, fasting insulin and glucose infusion rate. At the end of the 8th week, the serum TC,TG,FBG,FINS in HC group rats all increased, while the GIR decreased remarkably, there were significant differences between the HC group and the NC group (P<0.01). After the intervetion, rat serum TC, TG, FBG, FINS in RSG group decreased and the GIR increased, there was significant difference when compared with HC group (P<0.01). After intervened by different dosages of EGB, serum TC, TG, FBG, FINS decreased in different degrees. In EGB1 group, the serum TC, TG decreased, the difference was remarkable when compared with HC group (P<0.05), But the FBG, FINS, GIR in EGB1 group had no change compared with HC group (P>0.05). In EGB2 and EGB3 groups, these parameters decreased obviously, while the GIR increased meanwhile compared with HC group, there were significant differences (P<0.01). There were no statistic differences among the EGB2, EGB3 and RSG groups (P>0.05). The serum TC, FINS levels in EGB2, EGB3 group were lower and the GIR was higher than that of EGB1 group (P<0.01).2.2 The results of oral glucose tolerance test. The glucose level of the rats in HC group was much higher than that of NC group at the time of 0′and 60′, 120′after glucose-loaded. AUC of glucose increased obviously in HC group (P<0.01). The blood glucose level at the time of 0′, 120′and AUC in EGB2, EGB3 and RSG group decreased, and the difference was remarkable compared with that in HC group (P<0.01). The blood glucose level in EGB2 group decreased at 60′when compared with RSG group and EGB1 group (P<0.05 or P<0.01). The blood glucose of 120′in EGB3 group was much lower than that in EGB1 group (P<0.05). The rats in HC group also showed a delayed glucose peak time after glucose loaded, the glucose peak time was at 120′, and the glucose peak time in other groups was at 30′or 60′after the glucose loaded.2.3 The serum concentration of leptin, adiponectin, tumor necrosis factorαand free fatty acids at the end of the eighth week. The FFA, TNF-αlevels were higher and the adiponectin concentration in HC group was lower than those of NC group (P<0.01). The serum leptin level had an increased tendency, but the difference was not significant when compared with the NC group (P=0.054). RSG or EGB decreased the level of serum FFA and TNF-α, increased the concentration of serum adiponectin at different degrees. The serum FFA concentration in EGB3 group rats was the lowest one among the intervened groups, and there was significant difference compared with the EGB1 group (P<0.05), there were no statistic differences among the EGB2, EGB3 and RSG groups (P>0.05). The serum leptin level in EGB3 group rats was lower than that of EGB1, EGB2 and RSG groups (P<0.05). The serum adiponectin level in EGB3 group rats was much higher than that of other groups, the difference was significant when compared with EGB1 group (P<0.05). The TNF-αlevel was no difference among the rats in EGB1, EGB3 and RSG groups (P>0.05), there was statistic difference when compared with EGB2 group (P<0.05). The relative content of visceral adipose tissue in HC group rats was much more than that of NC group (P<0.01). Intervened by EGB or rosiglitazone reduced the relative content of visceral adipose tissue, there was statistic differences when compared with the HC group (P<0.01). There was no difference between the EGB2, EGB3 group (P>0.05), but there were significant differences when these two groups compared with EGB1 and RSG group (P<0.01).2.4 The correlation analysis between the serum leptin, adiponectin, TNFα, FFA, the relative content of adipose tissue and GIR. The serum TNFα, FFA, relative content of adipose tissue were negatively correlated with GIR (P<0.01 or P<0.05), while the serum adiponetin was positively correlated with GIR (P<0.01). The serum leptin had no correlation with GIR (P=0.13).2.5 The expression of muscle PKB, GLUT4 and adipose tissue PPARγafter intervened by rosiglitazone or EGB.2.5.1 The PKB expression in rat skeletal muscle. The PKB protein level decreased in HC group, but there was no statistic difference when compared with NC group (P=0.063). In HC group rats, the expression of PKB mRNA was down-regulated remarkably compared with that of NC group (P<0.05). The skeletal muscle PKB protein level and mRNA expression increased in different degrees after intervened by middle and high dose of EGB or rosiglitazone (P<0.05 or P<0.01). And in EGB1 group, the PKB expression has no changes. The PKB expression level had no differences among the EGB2, EGB3 and RSG group (P>0.05).2.5.2 The GLUT4 expression in rat skeletal muscle. The GLUT4 protein level and mRNA expression decreased in HC group, and there were significant differences when compared with NC group (P<0.05 or P<0.01). The expression of skeletal muscle GLUT4 increased in different degrees after intervened by EGB or rosiglitazone (P<0.05 or P<0.01). In EGB2, EGB3 and RSG groups, the skeletal muscle GLUT4 protein level increased obviously, the difference was significant when compared with HC group (P<0.01). The skeletal muscle GLUT4 protein level in EGB1 group increased too (P<0.05). The GLUT4 mRNA expression in EGB2, EGB3 groups increased significantly compared with HC group (P<0.05 or P<0.01), and the difference was significant when the two groups compared with EGB1 group (P<0.05 or P<0.01). The change of GLUT4 mRNA expression in EGB1, RSG groups was not remarkable (P>0.05).2.5.3 The PPARγexpression in rat adipose tissue. The PPARγprotein level had a decreased tendency in HC group, but there was no statistic difference when compared with NC group (P=0.055). The adipose tissue PPARγprotein level increased in different degrees after intervened by EGB or rosiglitazone (P<0.05 or P<0.01), among these groups, the PPARγprotein level in EGB2, EGB3, RSG groups was no difference , but it was much higher than that of EGB1 group. The PPARγprotein level in EGB1 group had no change compared with the HC group (P>0.05). As for the PPARγmRNA expression, the adipose tissue PPARγmRNA expression in HC group decreased significantly when compared with the NC group (P<0.05). The adipose tissue PPARγmRNA expression in EGB2, EGB3 and RSG groups all increased remarkably compared with that in HC group (P<0.01 or P<0.05), and there were no significant differences among these three groups(P>0.05). There was statistic difference between the EGB1 and EGB3 group (P<0.05).Conclusions1 The high-fat-fed rats had higher level of serum TC, TG, FFA, FBG and FINS. The GIR was decreased significant in high-fat-fed group. The oral glucose tolerance test demonstrated that the function of dealing with glucose loading was impaied by high-fat diets.2 The EGB decreased the serum TC, TG, FFA level in high-fat-fed rats, and improved the glucose tolerance, increased the GIR. These results suggested that the EGB could ameliorate the insulin resistant state in high-fat-fed rats.3 The high-fat-fed rats also showed increase in serum TNF-αand leptin level, decrease in serum adiponectin concentration. The relative content of visceral adipose tissue increased in high-fat-fed rats. The correlation analysis demonstrated that the GIR was negatively correlated with serum FFA, TNF-αconcentration and the relative content of visceral adipose tissue, and was positively correlated with the serum adiponectin level.4 The protein levels of skeletal muscle PKB and adipose tissue PPARγhad a down-regulated tendency. The expression of skeletal GLUT4 protein and mRNA, adipose tissue PPARγmRNA all decreased in high-fat diets rats.5 EGB could modulate the secretion of adipokines. It can reduce the TNF-α, Lep secretion, and increase the APN secretion. It can regulate the expression of skeletal muscle PKB, GLUT4 and adipose tissue PPARγ.6 The above-mentioned effects of EGB showed a dose-dependent feature, the middle and high dosage of EGB intervention could improve insulin sensitivity more remarkably than the low dosage of EGB.
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