Discovery Of Glucokinase Activator, The Anti-diabetic Effects Of LGK-01 And Initial Mechanism Approach

Glucokinase(GK) is a 50 KDa protein which reside in mammalian liver and pancreas.It was also called hexokinaseⅣ.It is the key enzyme in glycolysis to restrict the reaction rate.The GK has dual role in liver and pancreas.The effect of GK in liver is increasing glucose phosphorylation and glycogen synthesis.In pancreasβ-cells,GK is as the glucose sensor to increase glucose stimulated insulin release in high concentration of glucose.The disfunction of GK will directly induce the derangement of glucose metabolism,in order to produce abnormality of glucose tolerance and pancreatic cell function.Activation of GK leads to an increased of glucose phosphorylation,glycogen synthesis and release of insulin as well as improvement of glucose tolerance and pancreatic cell function.Glucokinase regulatory protein(GKRP) is a 68 KDa protein which only reside in mammalian liver.The GK has no physiological effects in the combination complex of GKRP and GK.So,activation of GK and inhibition of GKRP represent a new therapeutic approach to treatment of type 2 diabetes.The activators of GK and the inhibitors of GKRP are in development all over the world.In this paper,human GK gene(cDNA) was obtained from human liver tissue and coding sequence with the length of 1.4 kb was cloned into the yeast expression vector pPIC9K.The recombinant plasmid pPIC9K-GK was introduced into Pichia pastoris GS115 strain by electroporation.The cultural filtrate was partially purified and the activity of recombinant protein stock solution was determined.After the optimization of substrates and recombinant enzyme,the screening system for the GK activators was set up.Meanwhile,human GKRP gene(cDNA) was obtained from human liver tissue and coding sequence with the length about 1.9 kb was cloned into the E.coli expression vector pET32a(+).The recombinant plasmid pET32a -GKRP was introduced into BL21trx(DE3) strain to obtain activity protein solution.In addition,human GKRP gene was cloned into the yeast expression vector pPIC9K.The recombinant plasmid pPIC9K-GKRP was introduced into Pichia pastoris GS115 strain to obtain protein solution.This protein solution will be used in the inhibition effect of GK activators on GKRP.Using this screening system,orient and non-orient compounds were screened in vitro and lead compound LGK-01 was identified which had GK activity.We studied the effects of LGK-01 on enzyme analysis,cell biology,in vivo study,molecular biology and so on.Results followed below.1.LGK-01 increased the maximum velocity of glucose phosphorylation,decreased the affinity of substrate glucose and depressed the inhibition of GKRP.2.LGK-01 increased the glucose comsumption on HepG2 cells and 3T3-L1 cells,promoted the glucose stimulated insulin release on NIT-1βcells.3.LGK-01 promoted the GK from nuclear to cytoplasm in HepG2 cells.4.LGK-01 decreased fasting blood glucose on ICR mice and improved glucose tolerance on obesity MSG mice.5.LGK-01 improved glucose tolerance and insulin tolerance,decreased fasting blood glucose and suppressed gluconeogenesis on HFD-C57BL/6J mice.6.LGK-01 increased phaseⅠinsulin releasing,increasing liver glycogen synthesis and increased liver GK activity in HFD-C57BL/6J mice.7.LGK-01 improved glucose tolerance and insulin resistance in spontaneous type 2 diabetes KKAy mice.8.LGK-01 increased PPARγactivity partly.Conclusions 1.Activity recombinant proteins were obtained by heterologous expression.2.Set up GK screening system and to screen GK activators by High Throughput Screening.3.A lead compound LGK-01 was identified.It was a novel GK activator and increased liver glucose uptake,glycogen synthesis and glucose stimulated insulin release in pancreas.4.The first time to introduce the ideas that unify the activity of GK and PPARγ,but the activity effect of LGK-01 on PPARγwas warranted to make sure.