The Study Of Anti-diabetic,Anti-obesity Mechanism And Direct Targets Of Lead Compound Fla-CN

According to the“Global Report on Diabetes”published by World Health Organization in April 2016,there are around 422 million diabetic patients worldwide.In China,this figure is about 129 million.This report also pointed out that diabetes is closely correlated with obesity.Globally,more than one person in every ten developed obese in 2014.In China,the obesity rate has reached to 7.3%.Diabetes and obesity were the major chronic diseases that are detrimental to one’s health.Fla-CN,A flavonoid derivative with the function of regulating carbohydrate and lipid metabolism,was discovered through structural modification of an anti-diabetic compound tiliroside in previous research.In this paper,the anti-diabetic and anti-obesity role of Fla-CN and its molecular mechanism were further investigated using DIO mouse model.The activity based probes containing Fla-CN fragment were synthesized as an activity group and used for target fishing and localization.The direct targets of Fla-CN was identified using proteomics analysis and the biological validation.The animal experiments demonstrated that Fla-CN could reduce the body weight and visceral fat-pad weights of DIO mice with no effect on their food intake.Fla-CN could improve glucose tolerance and insulin tolerance,reduce the blood glucose level and ameliorate insulin resistance in DIO mice.The blood biochemical analysis showed that Fla-CN could increase the serum adiponectin level and decrease the leptin level which was abnormally induced by high fat diet.Fla-CN treatment could also rise the ratio of HDL/LDL,and reduce the total cholesterol,triglyceride and non-esterified fatty acid level.Fla-CN could reverse the gene expression changes associated with the gluconeogenesis,lipid metabolism,inflammation and oxidative stress in the liver and adiposity tissues of DIO mice（PEPCK,G6Pase,TGH,HSL,HIF-1α,PPARγ,C/EBPα,FAS SREBP-1c and S6K1）.The western blotting results showed that Fla-CN could increase the phosphorylation of AMPK and ACC in the livers and adiposity tissues of DIO mice in a dose dependent manner.The western blotting results also showed that Fla-CN could downregulate the expression of SREBP-1c and ratio of p S6K1/S6K1.It was suggested that Fla-CN could activate AMPK,andfurtherregulateAMPK/ACCpathwayand AMPK/m TOR/S6K1/SREBP-1c pathway.In a conclusion,the improvement of insulin sensitivity and inhibition of fat synthesis were associated with AMPK activation.The AMPK activation also reduced body weight and lipid accumulation in livers and adiposity tissues.Moreover,the epigenetic regulation of mi RNA-27 which contributed to lipid synthesis was researched.These studies implied that Fla-CN upregulated mi R-27expression in livers and adiposity tissues of DIO mice in a dose dependent manner.As a result of miR-27 upregulation,the target genes including PPARγ,C/EBPα,FAS and SREBP-1c were downregulated.The protein expression of C/EBPαand SREBP-1c also decreased dose-dependently.In part two,two kinds of affinity based probes containing Fla-CN fragment were synthesized as an activity group through multiple reactions.All of the probes were characterized by NMR and MS.The screening results showed that fluorescent probe3 and biotin probe 6 exhibited potential anti-diabetic effects.The confocal images showed that probe 6 could be quickly absorbed and localized with mitochondria both in Hep G2 cells and pre-adipocyte3T3-L1.The probe 6 was incubated with Hep G2cells or 3T3-L1 pre-adipocyte for target protein fishing.The target proteome was obtained through photo-crosslinking,protein enrichment,SDS-PAGE and biological mass spectrometry identification.The target proteome fished by probe 6 contained two kinds of targets of anti-diabetic drugs on the market as well as a number of key enzymes associated with glycolysis,TCA cycle,gluconeogenesis,pentose phosphate pathway,lipid synthesis,lipid oxidation and oxidative phosphorylation.Primary biological evaluation of these target proteins suggested that the pyruvate carboxylase,mitochondrial malate dehydrogenase,mitochondrial F₁F_O-ATPase and mitochondrial Complex I were the direct targets of Fla-CN with IC₅₀ value of 5.16±0.18μM,4.93±0.15μM,4.94±0.59μM和5.01±0.34μM respectively.The pyruvate kinase and DPP4 were not the direct targets of Fla-CN.In summary,the major mechanisms of Fla-CN’s anti-diabetic and anti-obesity effects were the AMPK activation and mi R-27 upregulation.After AMPK activation,downstream pathways including AMPK/ACC and AMPK/m TOR/S6K1/SREBP-1c pathways were regulated.Upon mi R-27 upregulation,their target genes including PPARγ,C/EBPα,FAS和SREBP-1c were downregulation.Target searching investigations showed that Fla-CN could regulate key enzymes in multiple biochemical processed including glycolysis,TCA cycle,gluconeogenesis,pentose phosphate pathway,lipid synthesis,lipid oxidation and oxidative phosphorylation.The pyruvate carboxylase,mitochondrial malate dehydrogenase,mitochondrial F₁F_O-ATPase and mitochondrial Complex I were the direct targets of Fla-CN.The direct inhibition of pyruvate carboxylase and mitochondrial malate dehydrogenase by Fla-CN could result in an inhibition of gluconeogenesis.The direct inhibition of mitochondrial malate dehydrogenase,mitochondrial F₁F_O-ATPase and mitochondrial Complex I by Fla-CN could result in decreased cellular ATP.The decrease of cellular ATP could active AMPK.Fla-CN’s target study results exhibited the molecular bases of the anti-diabetic and anti-obesity effects.