The Regulation Of Phanginin A And Its Derivates On Glucose Metabolism Through SIK1 In Liver And Skeletal Muscle

Aims:As a member of AMP-activated protein kinase(AMPK)-related kinases,salt inducible kinase 1(SIK1)has emerged as a regulator of various metabolic pathways.However,the regulatory effect of SIK1 on glucose metabolism in liver and skeletal muscle has not been fully elucidated,and small-molecule compounds that can activate SIK1 are rarely reported.Previous studies in our laboratory have found that natural compound phanginin A could activate SIK1,then activate PDE4 to inhibit the c AMP/PKA/CREB signaling pathway,and further inhibit hepatic gluconeogenesis.Moreover,phanginin A also showed a profound therapeutic effect in ob/ob mice.Therefore,phanginin A is the first reported small-molecule compound that can activate SIK1,and it could also be a lead compound for the treatment of type 2 diabetes.In the first part of the present study,we investigated the inhibition of Fwo-143,a derivative of phanginin A synthesized by our chemistry cooperative group on hepatic gluconeogenesis and clarified the involvement of the activation of SIK1 by Fwo-143.In addition,we also investigated the hypoglycemic effect of Fwo-143 in type 2 diabetic mice to evaluate the potential of Fwo-143 as a new anti-type 2 diabetes drug candidate.In the second part of the present study,using phanginin A as a probe,we investigated the regulation of SIK1 activation on glucose uptake in skeletal muscle cells and its molecular mechanism to provide new clues for the deeper understanding of the function of SIK1 activation in the regulation of skeletal muscle glucose metabolism.Methods:The effect of Fwo-143 on gluconeogenesis was evaluated in primary mouse hepatocytes,and the effect of Fwo-143 on the content of cyclic adenosine monophosphate(c AMP)in primary mouse hepatocytes was measured by ELISA kit.The phosphorylation levels of c AMP-response element binding protein(CREB)and SIK1 were measured by western blot.A pan-SIK inhibitor was used to elucidate the correlation between SIK1 activation and Fwo-143 inhibition of gluconeogenesis.The anti-diabetic effects of Fwo-143 were evaluated in ob/ob mice after single and long-term oral administration,and the effects of Fwo-143 on hepatic SIK1 and CREB phosphorylation in ob/ob mice were analyzed by western blot.The regulation of phanginin A on glucose uptake in skeletal muscle was investigated in C2C12 myotubes.Real-time q PCR was used to detect the effect of phanginin A on the m RNA expression of glucose transporter 4(GLUT4)and junction plakoglobin(JUP).The effect of phanginin A on the phosphorylation of SIK1,histone deacetylase 4/5/7(HDAC4/5/7),protein kinase B(PKB/Akt),and Akt serine/threonine kinase(AS160),the total and membrane protein levels of GLUT4 were analyzed by western blot.PanSIK inhibitor and small interfering RNA(si RNA)-mediated SIK1 knockdown were used to elucidate the correlation between SIK1 activation and phanginin A promotion of skeletal muscle glucose uptake.Liver kinase B1(LKB1)si RNA was used to explore the correlation between phanginin A activation of SIK1 and the promotion of glucose uptake with LKB1.PI3 K inhibitor and Akt inhibitor were used to explore the correlation between PI3K/Akt activation and phanginin A promotion of skeletal muscle glucose uptake.JUP interference was used to explore the correlation between JUP and phanginin A activated Akt signaling and increased glucose uptake.Chronic oral treatment of phanginin A was performed in C57BL/6J mice to observe the regulation of phanginin A on glucose utilization and related signaling pathways in skeletal muscle.Results:Phanginin A derivative Fwo-143 significantly inhibited gluconeogenesis in primary mouse hepatocytes,promoted SIK1 phosphorylation,and consequently decreased the content of c AMP and the phosphorylation of CREB.In vivo experiments showed that long-term administration of Fwo-143 improved the hyperglycemia in ob/ob mice,increased hepatic SIK1 phosphorylation and inhibited CREB phosphorylation,suggesting that Fwo-143 has a significant therapeutic effect on type 2 diabetic ob/ob mice.Phanginin A significantly promoted glucose uptake and increased GLUT4 expression and translocation in C2C12 myotubes.Phanginin A significantly promoted SIK1 phosphorylation in C2C12 myotubes.Both pan-SIK inhibitor and interference with SIK1 could block the promotion of glucose uptake by phanginin A.Interfering with LKB1 expression completely blocked the promotion of glucose uptake and SIK1 phosphorylation by phanginin A.Phanginin A was able to promote HDAC4/5/7phosphorylation and the m RNA expression of MEF2 a and GLUT4.Interfering with SIK1 expression completely blocked the promotion of HDAC phosphorylation and MEF2 a and GLUT4 expression caused by phanginin A.Phanginin A was able to activate the Akt/AS160 signaling pathway by promoting HDAC7 phosphorylation to promote JUP expression.PI3 K and Akt inhibitors were able to reverse the promotion of glucose uptake by phanginin A.Interfering with JUP reverses the role of phanginin A in promoting Akt/AS160 phosphorylation and glucose uptake.Interfering with SIK1 reverses the role of phanginin A in promoting JUP expression and Akt phosphorylation.Phanginin A administration can increase the phosphorylation levels of SIK1 and HDAC4/5/7,increase the expression of MEF2 a,GLUT4 and JUP,activate the Akt/AS160 signaling pathway and promote the translocation of GLUT4,increase glycogen content and m RNA expression of glycolysis genes in skeletal muscle of C57BL/6J mice.Conclusion:Fwo-143,a derivative of phanginin A,could inhibit hepatic gluconeogenesis by activating SIK1 to inhibit c AMP/CREB signaling pathway.Fwo-143 also showed a profound anti-type 2 diabetes effect in ob/ob mice,which suggested Fwo-143 might be a potential drug candidate for the treatment of type 2 diabetes.Phanginin A promoted glucose uptake dependent on the activation of SIK1 in C2C12 myotubes.Activation of SIK1 by phanginin A promoted GLUT4 expression through inactivating HDAC4/5 to relieve the inhibition on MEF2 a.Meanwhile,activation of SIK1 by phanginin A inactivated HDAC7 to increase JUP expression and activated Akt/AS160 to promote the GLUT4 translocation.Using phanginin A as a probe,we elucidated that the activation of SIK1 could promote glucose uptake in C2C12 myotubes through a dual mechanism of increasing GLUT4 expression and promoting GLUT4 translocation,providing new clues and experimental basis for understanding the involvement of SIK1 in regulating skeletal muscle glucose uptake.