Study On The Glucose And Lipid Metabolism Regulation And Mechanism Of Kaempferol Derivative In Vitro

Objective:Fla-CN was a novel derivative of Potentilla flavonoid previously synthesized in our group with significant anti-obesity and anti-diabetic effects.Therefore,a series of flavonoid derivatives were synthesized using Fla-CN as leading compound.The insulin-resistant hepatic HepG2 cell model was used to select candidate compounds from the Kaempferol family of Fla-CN derivatives which promoted glucose depletion in insulin resistant cells to improve insulin resistance.We also evaluated the role of these candidate compounds in the regulation of glucose and lipid metabolism using mouse preadipocyte cell line 3T3-L1 and human hepatoma carcinoma cell line HepG2.Primary exploration was performed on their molecular mechanism,so as to provide experimental evidence for subsequent synthesis and discovery of novel drugs.Methods:1.The insulin resistant cell model was established by inducing HepG2 cells with high glucose and high insulin.The model group was then given different concentrations of Kaempferol derivatives.Glucose test kit was used to measure glucose consumption in supernatants and the effects of each derivative on glucose consumption in insulin resistant HepG2 cells were investigated.2.Using HepG2 cells as the research object,the intracellular glycogen content in each group was detected by the sulfuric acid-anthrone colorimetry method after intervention with different concentration of kaempferol derivatives.The lactic acid content,the final product of glycolysis pathway,was measured by the lactic acid test kit.2-deoxyglucose?2-NBDG?was used to determine glucose uptake in HepG2 cells.Glucose assay kit was used to analyze glucose production during gluconeogenesis and to investigate the effect of each derivative on glycogen synthesis,glycolysis,glucose uptake and glucose of in HepG2 cells.3.The mature and differentiated 3T3-L1 adipocytes were used as the research object.Different concentrations of Kaempferol derivatives were given and the intracellular triglyceride,extracellular glycerol and free fatty acid content were measured by kits to investigate the effect of these derivatives on lipid metabolism4.The effect of kaempferol derivatives on the phosphorylation levels of AMPK,and ACC in HepG2 cells and 3T3-L1 cells were determined using western blot.The effect of kaempferol derivatives on the expression of Glucose 6 phosphatase?G6Pase?and phosphoenolpyruvate carboxykinase?PEPCK?in HepG2 cells were measured.Results:1.The activity of kaempferol derivatives was screened using insulin resistant HepG2 cell model.The results showed that each kaempferol derivative can promote glucose consumption to some extent,and therefore improve insulin resistance.C6^C9,C11,C14,C21,C26,C27,C34,C37^C39,C41^C46 had stronger activity in enhancing glucose consumption in insulin-resistant cells,among which C7,C14,C26,C27,C41,C42,C43,C45 had the strongest activity in improving insulin resistance and EC₅₀ values were between 0.5 and 9.0nM.2.HepG2 cells was incubated with compound?Fla-CN,C7,C42 and C45?for24hrs before analysis.The glycogen synthesis experiment was performed.Fla-CN and C42 can dose-dependently increase HepG2 intracellular glycogen content.C7 can elevate glycogen content in HepG2 cells at its low concentration while the effect was decreased at higher concentration;At a concentration of 12.5?M,glycogen synthesis was inhibited.In the glycolysis experiment,Fla-CN,C7,C42,and C45 had no significant effect on the lactate content in HepG2 cells.In the gluconeogenesis test,Fla-CN,C7 can inhibit glucose production in a dose-dependent manner;C42,C45had no significant effect on gluconeogenesis.In the glucose uptake experiments,Fla-CN,C7,C42,and C45 all dose-dependently increased glucose uptake in HepG2cells.3.Differentiated and mature 3T3-L1 adipocytes were administrated with different concentrations of C7,C42,and C45.Compared with the normal control group,C7,C42,and C45 within certain concentrations had significant effects on fat cell lipolysis,in which C45 dose-dependently reduced the content of triglyceride and increased the content of extracellular glycerol and free fatty acids which was positively correlated with lipolysis.However,low concentrations of C7 and C42 can significantly reduce the content of triglyceride in adipocytes,increase the content of extracellular glycerol and free fatty acids,and inhibit the lipolysis of high concentrations of C7 and C42,suggesting that within a certain concentration range,their effect on lipolysis was negatively correlated with the concentration.4.C7 can dose-dependently up-regulate phosphorylation of AMPK and ACC in HepG2 cells and mature 3T3-L1 adipocytes.In addition,C7 can dose-dependently down-regulate gluconeogenesis-related G6Pase and PEPCK protein expression in HepG2 cells.Conclusion:Using the insulin-resistant HepG2 cell model,the anti-diabetic activity of kaempferol derivatives were screened and results showed that C7,C14,C26,C27,C41,C42,C43,and C45 had strong anti-diabetic activity with EC₅₀ values reaching the nM level.The strong active compound C7 can activate AMPK in HepG2 cells,up-regulate the phosphorylation of ACC,and down-regulate the protein expression of gluconeogenesis-related G6Pase and PEPCK,and therefore play a role in regulating glucose metabolism.In addition,C7 promoted fat hydrolysis of 3T3-L1 adipocytes by activating AMPK.AMPK was activated in the concentration range of 0.1-12.5?M of C7,but this effect was negatively correlated with the concentration.This effect of was weakened with high concentration,and the mechanism entails further investigation.