| ObjectiveTo investigate the effects of three Chinese herb-extracts:Ku Gua (Momordica Charantia L., MC) extract, Di Gu Pi (Lycium Chinense Mill.,LCM) extract, and Fan Bai Cao(Potentilla Discolor bunge,PDB) extract on glucose and lipid metabolism in insulin resistance and type 2 diabetic animal models. There are also further studies on liver and skeletal muscle of insulin signaling pathway:the PI3K/Akt signaling pathway and insulin independent signaling pathway:the AMPK signaling pathway by evaluating several related protein expressions to explore the molecular mechanism of the three Chinese herb extracts on the improvement of insulin resistance and type 2 diabetes.Methods1. Eighty male Wistar rats,3 weeks old, are divided randomly into two groups according to random data table:control group (n=10 with rat standard diet) and high-fat diet group (HFD) (n=70 rats). At the end 16 a week feeding period, in order to induce insulin resistance and type 2 diabetes, the 70 HFD group of Wistar rats are given an intraperitoneal-injection of small doses of STZ (35mg·kg-1·bw-1). Then,56 rats are randomly selected from the successful induced insulin resistance and type 2 diabetic model (the sum of successful induced-model is a total of 63) and are divided into 8 groups which are the three Chinese herb-extract high and low dose groups, respectively, as well as western medicine compared group. There are 7 rats in each group with the remaining 7 successfully induced model rats set as a model group. Another 7 rats are randomly selected from the normal diet group. The Thiazolidine (TZDs) insulin sensitizer Pioglitazone is used as western medicine control group (4.05mg/kg). The three Chinese herb extracts are obtained by high-dose group (400mg·kg-1·bw-1) and low dose group (100mg·kg-1·bw-1) with intragastric administration, respectively. The normal control group and model group fed with distilled water at 10ml·kg-1·bw-1. After 4 weeks of drug intervention, to evaluate the effect of insulin resistance improvement by three Chinese herb extracts, the Hyperinsulinemic-Euglycemic Clamp (HEC) experiments are examined. The fasting blood glucose (FBG), oral glucose tolerance (OGTT), fasting serum insulin (FINS), insulin sensitivity index (ISI), serum triglyceride (TG), serum total cholesterol (TC), serum free fatty acid (FFA), serum low-density lipoprotein (LDL-C) and serum high density lipoprotein (HDL-C) are also examined. The liver AMPK, Akt protein, and phosphorylation levels are detected by Western Blot. The liver AMPKα1-mRNA/AMPKa2-mRNA gene transcription are detected by RT-PCR.2. Forty-eight male BKS.Cg-m +/+ Leprdb/J db/db mice,6 weeks old, are divided randomly into 8 groups according to random data table:the three Chinese herb-extract high and low dose group, respectively, as well as western medicine compared group. There are 6 mice in each group, and the remaining 6 mice are set as model group. Another 6 mice were randomly selected from the normal C57BL/KsJ (+/-) group. The Thiazolidine (TZDs) insulin sensitizer Pioglitazone is used as western medicine control group (1.2g·kg-1·bw-1), three Chinese herb-extracts are obtained by high-dose group (4g·kg-1·bw-1) and low dose group (1g·kg-1·bw-1) with intragastric administration, respectively. The normal control group and model group are fed with distilled water at 1ml·kg-1·bw-1. After 4 weeks of drug intervention; fasting blood glucose (FBG), fasting serum insulin (FINS), insulin sensitivity index (ISI), serum triglyceride (TG), serum total cholesterol (TC), serum free fatty acid (FFA), serum low-density lipoprotein (LDL-C) and serum high density lipoprotein (HDL-C) are examined. The skeletal muscle AMPK, Akt, AS 160 protein, and phosphorylation levels are detected by Western Blot. The skeletal muscle GLUT4-mRNA gene transcription is detected by RT-PCR.Results1 The effects on IR by the three Chinese herb-extracts in type 2 diabetic IR rats1.1 Hyperinsulinemic-Euglycemic Clamp (HEC) experiment shows that:after 4 week treatments (compared with the MO group, the KG group, the DGP group, and the FBC group with high dose significantly increase the glucose infusion rate [GIR] (P<0.05) the low-dose group with the three Chinese herb extracts have an increased GIR intendancy which indicates that the insulin sensitivity is improved after treatment.1.2 Blood biochemical results show that after 4 week treatments (compared with the model group) the three Chinese herb extracts significantly improve OGTT, increased serum FINS and HDL-C levels level, and decreased serum FBG, TC, TG, LDL -C level (P<0.05).1.3 HE staining shows under a light microscope that (compared with the NO group) the MO group shows mild fatty degeneration of liver cells, fatty degeneration of liver cells along the central vein showed concentric distribution, many small visible lipid droplet vacuoles around the cytoplasm, no significant change in nuclear location, and the structure of hepatic lobule is integrated. After 4 week treatments, the three Chinese herb extracts significantly reduce fat distortion of the rat liver cells with occasional small lipid droplets cavity around the cytoplasm in focal liver cell, no significant change in nuclear location, and the structure of hepatic lobule is integrated.1.4 Western Blotting results of rat liver shows that (compared with the Model group) all of the three Chinese herb-extracts increase P-AMPK, P-Akt (Ser 473) protein expression (P<0.05) in liver. There is no significant difference among Chinese herb extracts and Pioglitazone group.1.5 RT-PCR results of liver and skeletal muscle shows that (compared with the MO group) all of the three Chinese herb extracts increase liver AMPKα1-mRNA and AMPKa2-mRNA's expression level (P<0.05).2. The effects on insulin resistance by the three Chinese herb-extracts in BKS.Cg-m +/+ Leprdb/J db/db mice2.1 Blood biochemical results show that after 4 week treatments (compared to with the model group), the Chinese herb extracts significantly increase serum FINS and HDL-C levels, and decrease serum FBG,TC,TG, LDL -C level (P<0.05).2.2 Western Blotting results of skeletal muscle shows that (compared with the model group) all of the three Chinese herb-extracts increase P-AMPK, P-Akt (Ser 473), P-AS160 protein expression (P<0.05) in skeletal muscle tissue. There is no significant difference among Chinese herb extracts and Pioglitazone group.2.3 RT-PCR results of skeletal muscle shows that (compared with the model group) all of the three Chinese herb extracts increase skeletal muscle GLUT 4-mRNA expression levels (P<0.05).ConclusionThe three Chinese herb-extracts:Ku Gua (Momordica Charantia L.,MC) extract, Di Gu Pi (Lycium Chinense Mill.,LCM) extract, and Fan Bai Cao (Potentilla Discolor Bunge,PDB) extract can improve insulin resistance in both long-term high-fat-diet with the low-dose STZ-induced Wistar IR rat model, and the leptin-receptor-deficient BKS.Cg-m+/+Leprdb/J db/db IR mice model. The improvement mechanism of insulin resistance by the three Chinese herb extracts is through the activation of AMPK which can increase GLUT4 translocation, so that glucose utilization is accelerated. Meanwhile, the three Chinese herb-extracts also active PI3k/Akt signaling pathway by the activation of Akt which leads to further activation of AS 160, so as to strengthen GLUT4 translocation. The three Chinese herb extracts regulate the AMPK signaling pathway through increasing the genetic transcription of AMPKal-mRNA and AMPKa2-mRNA levels in the liver and promoting the GLUT4-mRNA level in the skeletal muscle tissue, thereby the overall insulin resistance can be ameliorated. |
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