Chemical Basis And Mechanism Of The Novel Matrine Derivative 9a In Promoting Glucose Metabolism And Anti-diabetic Effect

Diabetes mellitus is a metabolic disease caused by genetic and environmental factors.It has become one of the major health concerns all over the world with complex pathogenic factors and a large number of patients.Now,there are various therapeutic approaches for the management of diabetes which include a series of glucose-lowering drugs or insulin injection,and so on.These therapeutic agents can ameliorate diabetes to some extent.However,due to inadequate efficacy or adverse effects,it is of scientific significance to develop novel anti-diabetic drugs with high safety profile,superior glucose-lowering efficacy,or novel mechanisms of action.It is reported that many natural products may have glucose-lowering activities.Therefore,chemical modification of natural products and screening of glucoselowering activities are reasonable strategies for the development of novel anti-diabetic drugs.Recently,we found that 12N-p-trifluoromethylbenzenesulfonyl matrinane(compound 1)displayed promising effects on lowering blood glucose,glucosylated haemoglobin and AGE levels,as well as improving glucose tolerance and insulin resistance in KK-Ay mice,with the advantages of high safety profile and ameliorating diabetic nephropathy.Therefore,thirty-five tricyclic matrinic derivatives are continuously synthesized and determined for their stimulatory effects on glucose consumption in L6 myotubes,taking 1 as the lead.Among them,we found compound 9a greatly promoted glucose consumption in L6 cells and gave a superior activity to the lead 1.So we conducted a more in-depth study about 9a.Firstly,the safety of 9a was determined in vitro and in vivo,in HepG2 cells and Kunming mice,respectively.Compound 9a gave the half maximal inhibitory concentration(IC50)values of 859 μM in HepG2 cells.In Kunming mice,compound 9a gave the median lethal dose(LD50)values of over 1000 mg/kg via oral route and caused no significant toxicity to liver and kidney of mice.Taken together,compound 9a owned a high safety profile.Secondly,the glucose-lowering effects of 9a were explored both in vitro and in vivo.In the in vitro experiments,compound 9a was given to three kinds of glucose metabolic cells,which include L6 myotubes,HepG2 cells and 3T3-L1 adipocytes,taking MET(5 mM)as a positive control.The data suggest that compound 9a effectively stimulates the glucose consumption in these cells,and the efficacies of 9a at 40 μM were close to those of MET at 5 mM.To explore the anti-diabetic effects of 9a in vivo,compound 9a was given to KK-Ay diabetic mice or HFD+STZ induced diabetic mice,taking RGZ(5 mg/kg)or MET(300 mg/kg)as the positive control,respectively.The data suggest that compound 9a effectively lowers blood glucose and improves glucose tolerance in these animal model.It also increases insulin sensitivity,repairs islet damage,ameliorates diabetic nephropathy,improves lipid metabolism,and ameliorates fatty liver.The anti-diabetic and anti-diabetic nephropathy efficacies of 9a were similar to those of RGZ and MET in our experiments.Finally,we explored the possible hypoglycemic mechanisms of compound 9a in L6 myotubes.Through experiments like glucose uptake,immunofluorescence,and western blot,we find that compound 9a stimulates GLUT4 membrane translocation and 2-DG uptake in L6 myotubes through activating AMPK.Through examing AMPK up-stream kinases like LKB1 and CaMKK,and ATP/AMP contents,we find that 9a may activate AMPK through increasing AMP/ATP ratio or activating LKB1 and CaMKK.Through determination of mitochondrial complex I activity,cellular oxygen consumption,and lactate release,we find that 9a inhibits complex I activity,reduces oxygen consumption,and stimulates glucose consumption of L6 myotubes through promoting glycolysis.As downstream proteins of AMPK,the Src/Cbl pathway is also critical for GLUT4 translocation stimulated by compound 9a.In summary,compound 9a has favorable hypoglycemic efficacies and good safety profile both in cells and in animal models.It targets mitochondrial complex I to increase AMP/ATP ratio,and affects LKB1 and CaMKK to activate AMPK,thereby stimulating GLUT4 membrane translocation and glucose uptake.It also stimulates cellular glucose consumption and thereby lowers glucose levels.Our results suggest that compound 9a may be a potential candidate for the development of novel anti-diabetic or anti-diabetic nephropathy drugs in the future.