| Objective:Fla-CN is a flavonoid derivative with significant anti-diabetic activity discovered previously in our research group.This compound can significantly promote the glucose consumption in insulin resistant HepG2 cells,regulate glucose metabolism and lipid metabolism in insulin resistant obese mice,and improve insulin resistance.This article investigated the molecular mechanism of Fla-CN in regulating glucose and lipid metabolism in vitro,focusing on the molecular mechanism of the activation of adenosine-dependent protein kinase?AMPK?signal pathway of the compound.Method:1.Western blot was used to study the effect of Fla-CN on the protein expression and phosphorylation of AMP-dependent protein kinase?AMPK?in HepG2 cells.2.The insulin resistant model of HepG2 cells was established and Western blot was used to examine the effect of Fla-CN on the expression of AMPK protein and phosphorylation of insulin resistant HepG2 cells?IR-HepG2?.3.Western blot was used to study the effect of Fla-CN on the expression of AMPK protein and phosphorylation in mature 3T3-L1 adipocytes.4.Reversed-phase ion-pair high performance liquid chromatography?RPIP-HPLC?was used to measure adenine nucleotide?AMP?and adenosine triphosphate?ATP?content in cells.5.The AMP/ATP ratio in HepG2 cells was determined by RPIP-HPLC.6.HepG2 cell insulin resistance model was established and RPIP-HPLC was used to determine the ratio of AMP/ATP in IR-HepG2 cells.7.The AMP/ATP ratio in mature 3T3-L1 adipocytes was determined by RPIP-HPLC.Result:1.Western blot results showed that Fla-CN up-regulated the phosphorylation level of AMPK in HepG2 cells dose-dependently,while had no effect on the expression level of AMPK protein.2.Western blot results showed that the phosphorylation of AMPK and pAMPK/AMPK were decreased in insulin-resistant HepG2 cells.Fla-CN could upregulate AMPK phosphorylation in IR-HepG2 cells dose-dependently,but had no effect on AMPK protein expression.3.Western blot results showed that Fla-CN up-regulated the phosphorylation level of AMPK in mature 3T3-L1 adipocytes in a dose dependent manner,while had no effect on the expression level of AMPK protein.4.RP-IP-HPLC analysis of AMP and ATP content in cells was established.The mobile phase was consisted of buffer?containing100 mM potassium dihydrogen phosphate and 1mM tetrabutylammonium dihydrogen phosphate,pH6.0?and methanol?9:1,v/v?.The mobile phase was pumped through a SUPELCOSIL LC-18T?25cm×4.6?m,5?m?at the column temperature of 35°C at the flow rate of1 m L/min.The injection volume was 10?L while the detection wavelength was set at 258nm.The methodological validation showed that the linear range of AMP and ATP was 0.625-25?g·mL-1?r=0.9999?,and 0.5-25?g·mL-1,?r=0.9999?respectively,and the RSD was less than 2%.Sample repeatability RSD was less than 3%and the stability RSD was less than 4%in 12 hours.The sample recovery rate was 92%-106%.Therefore,the chromatographic adaptability of the method was good,with the precision,reproducibility,stability and accuracy meeting the requirements and therefore,can be used to analyze AMP and ATP content in cell samples.5.The content of AMP and ATP in HepG2 cells was determined using RP-IP-HPLC and the results showed that Fla-CN can increase the ratio of AMP/ATP in HepG2cells in a concentration-dependent manner,which in turn activated AMPK.6.The content of AMP and ATP in IR-HepG2 cells was determined using RP-IP-HPLC and results showed that Fla-CN can increase the ratio of AMP/ATP in HepG2 cells in a concentration-dependent manner,which in turn activated AMPK.7.The content of AMP and ATP in mature 3T3-L1 adipocytes was determined using RP-IP-HPLC and the results showed that Fla-CN can increase the ratio of AMP/ATP in HepG2 cells at the maximum concentration and activate AMPK. |
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