Direct observation of the autophosphorylation of insulin receptor kinase by mass spectrometry

The catalytic and signaling activities of insulin receptor kinase （IRK） are regulated by the autophosphorylation of three tyrosine residues in a cytoplasmic protein-tyrosine kinase domain at Tyro 1158, Tyro 1162 and Tyro 1163. In this study, time-course of the auphosphorylation of the core kinase （residues 978-1283） from IRK was directly investigated by online electrospray ionization mass spectrometry. It is found that two tyrosine residues were phosphorylated in reaction time range of 30 min. This study implies that mass spectrometric technique must be a powerful tool to directly monitor the biological macromolecular modification and will also provide the information of the order and the mechanism of autophosphorylation at the tyrosine sites coupled with tandem mass spectrometric technique.

Clean-DM1, a Korean Polyherbal Formula, Improves High Fat Diet-Induced Diabetic Symptoms in Mice by Regulating IRS/PI3K/AKT and AMPK Expressionsin Pancreas and Liver Tissues

Objective:To investigate the effects of Clean-DM1(C-DM1),a polyherbal formulation of Radix Scrophulariae,Radix Astragali,Rhizoma Atractylodis,and Radix Salviae Miltiorrhizae,on high-fat diet(HFD)-induced diabetes mice.Methods:The information about active components of C-DM1 extract and molecular mechanism was obtained from network pharmacology analysis.Main compounds of C-DM1 extract by high performance liquid chromatography-mass spectrometry(HPLC-MS)analysis were conducted for quality control.For in vivo study,mice were induced diabetes by HFD for 12 weeks.The mice in the normal group(Nor)were maintained with a regular diet and treated with saline by gavage.The HFD model mice were randomly divided into 3 groups,including a HFD diabetic model group,a C-DM1 extract-administered group(C-DM1,500 mg/kg),and metformin-administered groups(Met,500 mg/kg),8 mice in each group.Food intake,body weight(BW),and fasting blood glucose(FBG)levels were recorded weekly for 4 weeks.After 4 weeks of treatment,alanine aminotransferase(ALT),aspartate aminotransferase(AST),blood glucose,low-density lipoprotein cholesterol(LDL-C)were determined using an automated clinical chemistry analyzer,and homeostatic model for assessing insulin resistance(HOMA-IR)levels and oral glucose tolerance test(OGTT)were detected.The histopathological changes of liver and pancreatic tissues were observed by hematoxylin-eosin staining.Insulin receptor substrate(IRS)/phosphatidylinositol 3 kinase(PI3K)/protein kinase B(AKT)and adenosine 5'-monophosphate-activated protein kinase(AMPK)expressions in liver and pancreas tissues were detected by Western blot analysis.Results:HPLC-MS identified dihydroisotanshinone,dihydroisotanshinone I,cryptotanshinone,harpagoside,and atractyloside A in C-DM1 extract.The administration of C-DM1 extract significantly decreased body weight,calorie intake,and the levels of blood glucose and insulin in the diabetic mice(P<0.05 or P<0.01).The C-DM1 extract administration improved the impaired glucose tolerance and insulin resistance in the diabetic mice and significantly decreased the levels of LDL-C,ALT and AST(P<0.01).The C-DM1 extract inhibited the histopathological changes of fatty liver and hyperplasia of pancreatic islets in the diabetic mice.The C-DM1 extract significantly increased the phosphorylation of IRS,AKT,and AMPK and the expression of PI3K in pancreas and liver tissues(P<0.05 or P<0.01),which was consistent with the analysis results of network pharmacology.Conclusion:C-DM1 extract improved diabetes symptoms in longterm HFD-induced mice by regulation of IRS/PI3K/AKT and AMPK expressions in pancreas and liver tissues,suggesting that C-DM1 formulation may help prevent the progression of T2DM.