| Type 2 diabetes mellitus is a complex metabolic syndrome with multiple genes involved,which is related to many factors such as heredity and environment.Its pathogenesis is generally considered to be related to defects in β-cell function and insulin resistance.Insulin resistance means that the effect of a unit concentration of insulin is weakened,that is,the sensitivity of tissues to insulin is reduced.It causes insulin-mediated cellular glucose uptake and metabolism impaired.Firstly,insulin resistance causes a compensatory increase in insulin secretion from pancreatic islet β cells.Then,it gradually leads to β-cell failure,insufficient insulin secretion and increased blood glucose and eventually.Type 2 diabetes mellitus occurs.Therefore,Insulin resistance is one of the major contributors to the pathogenesis of type 2 diabetes mellitus.One of the key mechanisms of insulin regulating glucose homeostasis is to activate glucose transporter 4(GLUT4)in target tissue such as muscle and adipocytes.Insulin triggers GLUT4 to translocate from cytoplasm to the surface of the cell membrane to take up glucose for metabolism.In patients with type 2 diabetes mellitus,decreased expression and abnormal translocation function of GLUT4 are often observed in organ tissues,indicating that decreased expression and abnormal function of GLUT4 are closely related to insulin resistance and type 2 diabetes.GLUT4 expression and function disorders are one of the important molecular mechanisms of type 2 diabetes mellitus.Therefore,GLUT4 is regarded as one of the important molecular targets for the control of insulin resistance and the treatment of type 2 diabetes mellitus.In our previous study,more than 300 kinds of natural compounds were screened by adopting luciferase reporter assay method.We have obtained natural compounds TG that can significantly promote GLUT4 promoter activity.Here in this study,we have conducted a series of in-depth studies on the role of TG in promoting GLUT4 expression and insulin resistance in vitro and in vivo.1.The experimental results in vitro are as follows:(1)TG promotes the expression of GLUT4 in muscle-derived cells.TG treated undifferentiated and differentiated mouse muscle cell C2C12 and rat muscle cell L6 cells,respectively.It was confirmed that TG can promote the expression of GLUT4 in these cells at the levels of m RNA and protein by RT-q PCR and Western Blot methods,respectively.(2)Activation of AMPK signaling pathway by TG is one of the mechanisms that promote the expression of GLUT4.For the AMPK signaling pathway,we measured the intracellular ATP and phosphorylated AMPK levels,respectively.The result shows that,after TG treatment of C2C12 cells at different time points,the intracellular ATP levels gradually decreased,which may be the upstream signal to activate AMPK signaling.Then,we found that TG did activate AMPK in undifferentiated and differentiated C2C12 cells to increase the level of phosphorylated AMPK by Western Blot.And AMPK inhibitor Compound C can inhibit AMPK activation and inhibit TG-induced GLUT4 expression as well.The above results indicate that TG promotes the expression of GLUT4 by decreasing intracellular ATP levels and subsequently activating AMPK signaling pathway.(3)TG promotes glucose uptake in muscle-derived cells.By measuring the residual glucose in the cell culture supernatant,it was found that after TG treatment of C2C12,the glucose content in the cell culture supernatant was significantly decreased compared to the control group,indicating that TG treated cells uptaked more glucose.It suggests that TG might enhance glucose uptake by promoting GLUT4 expression in muscle cells.(4)TG increases the sensitivity of insulin-resistant cells to insulin.High dose of insulin was used to continuously stimulate undifferentiated and differentiated C2C12 cells for 24 hours to induce insulin resistance,which showed that low-dose insulin stimulation could not effectively activate downstream AKT to make it fully phosphorylated.Using this model,we detected insulin-stimulated phosphorylated AKT levels by Western Blot.TG did not significantly affect phosphorylated AKT levels in normal and insulin-resistant cells derived from undifferentiated C2C12 cells.However,TG significantly increased phosphorylated AKT levels in insulin-resistant cells derived from differentiated mature C2C12 cells,suggesting that TG has the effect of increasing insulin sensitivity and against insulin resistance in vitro.2.The experimental results in vivo are as follows:(1)Construction of type 2 diabetes mellitus animal model and establishment of protocol to study the preventive effect of TG on type 2 diabetes mellitus.Feeding 45% high-fat diet for three months can establish a type 2 diabetes mellitus model with insulin resistance in mice.Thirty 4-week-old male mice were randomly divided into 5 groups: normal control group,diabetic group,10 mg/kg TG group,20 mg/kg TG group,40 mg/kg TG group.The mice in the normal control group were fed normally,and all other groups of mice were fed with high-fat diets.When the fasting blood glucose of the diabetic group reached ≥7.0mmol/L,implying the model of type 2 diabetes mellitus was successfully constructed.The following indicators were then examined.(2)Preventive administration of TG reduced blood glucose levels in high-fat diet fed mice,improved glucose intolerance and insulin intolerance and prevented type 2 diabetes mellitus.Compared with diabetic group,TG significantly reduce high fasting plasma glucose levels in high-fat diet induced mice,with the best effect at the dose of 40 mg/kg.Compared with diabetic group,glucose tolerance and insulin tolerance were significantly improved in three different doses of TG prevention group.This shows that TG has the effect of improving insulin resistance and preventing type 2 diabetes mellitus.(3)Preventive administration of TG prevented the rising of serum insulin levels and decreasing of serum C-peptide levels in high-fat diet fed mice.We measured the levels of serum insulin and serum C-peptide in mice by ELISA.Compared with diabetic group,40mg/kg TG preventive administration could effectively prevent the increase of serum insulin levels and the decrease of serum C-peptide levels.This suggests that TG inhibits hyperinsulinemia by improving insulin resistance and protects pancreatic β-cell function.(4)Preventive administration of TG inhibited the decrease of GLUT4 expression in muscle tissue of high-fat diet fed mice.We also used RT-q PCR,Western Blot and immunohistochemistry methods to measure the expression of GLUT4 in muscle tissues of all mice.The results showed that compared with diabetic group,different doses of TG can significantly increase GLUT4 expression in muscle tissues at both m RNA and protein levels.(5)Preventive administration of TG blocked pancreatic islet atrophy in high-fat diet fed mice.We performed HE staining and insulin immunohistochemistry on pancreatic tissue.We found that the pancreatic islets in mouse pancreas of high-fat diabetic control group were significantly reduced and the boundaries were blurred.Limited islet beta cells compensatoryly secrete increased insulin.Compared with diabetic group,three different doses of TG can significantly prevent pancreatic islet atrophy and maintain islet structural integrity.Compensatory insulin secretion has also been significantly improved.This indicates that TG can effectively protect the structure and function of pancreatic islet β cells in long-term high-fat diet fed mice.(6)Preventive administration of TG improved liver function but had no significant effects on lipid metabolism in high-fat diet fed mice.We found that compared with diabetic group,the increase of serum ALT and AST levels were remarkably reduced in the different doses of TG treated mice,indicating that liver damage caused by high-fat feeding has been improved.There was no statistical difference about serum Triglyceride levels,serum CHOL levels and serum LDL levels in all experimental groups.Only in 10 mg/kg TG group mice,the increase of serum HDL levels was prevented.It implies that TG may have no significant effect on lipid metabolism in mice fed with high fat.In conclusion,this study demonstrated that TG promotes the expression of GLUT4 and can improve insulin resistance both in vitro and in vivo.Therefore,it can effectively prevent the occurrence of type 2 diabetes mellitus.This provides theoretical and experimental evidence for the development of TG as a drug for the prevention and treatment of type 2diabetes mellitus in the future. |
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