Effects And Mechanism Studies Of Linderane And Coronarin A On Hepatic Glucose Metabolism

Diabetes is a severe worldwide epidemic,which is characterized by hyperglycemia.Hepatic glucose metabolism plays key role in the regulation of whole-body glucose homeostasis.Dysregulation in hepatic glucose metabolism is the predominant reason for hyperglycemia.We found that two natural compounds linderane and coronarin A significantly inhibited gluconeogenesis in rat primary hepatocytes.In addition,coronarin A could also enhance glycogen synthesis in rat primary hepatocytes.Therefore,the present study will thoroughly investigate the molecular mechanisms of linderane and coronarin A to modulate hepatic glucose metabolism,and will provide experimental evidences and theoretical basis for the development of linderane and coronarin A as anti-diabetic lead compounds.Part one:Effects and mechanism studies of the natural compound linderane on hepatic gluconeogenesis.Aims:Inhibiting hepatic gluconeogenesis was thought to be effective in ameliorating fast blood glucose in type 2 diabetes.Our study explored the effect of linderane on gluconeogenesis and key gluconeogenic gene expression.We further investigated the mechanism of linderane in inhibiting gluconeogenesis,and evaluated its effect on ameliorating diabetic syndromes in vivo.Methods:The effect of linderane on gluconeogenesis was examined under both basal and forskolin-induced conditions in rat primary hepatocytes.The expression of PEPCK and G6Pase was detected by real-time PCR.cAMP ELISA kit was used to determine the cAMP content in rat primary hepatocytes.Phosphorylation of CREB,ERK and STAT3 was detected in western blot analysis.We examined the indirect and direct activity of total PDE and PDE3 using the PDE activity assay kit.Small molecular inhibitors were applied to explore the mechanism for linderane to inhibit gluconeogenesis.Our study evaluated the effect of single administration of linderane on hepatic PDE in C57BL/6 mice,as well as the chronic administration of linderane on diabetic syndromes in ob/ob mice.Results:Linderane could significantly suppress gluconeogenesis under both basal and forskolin-induced conditions in rat primary hepatocytes.It also reduced the mRNA expression of PEPCK and G6Pase.Linderane reduced cAMP content and CREB phosphorylation in rat primary hepatocytes.Linderane indirectly activated PDE3through ERK/STAT3 pathway,which led the increase of cAMP degradation,and both pan-PDE and PDE3 specific inhibitors blocked the effect of linderane on cAMP/CREB pathway,as well as gluconeogenesis.For in vivo study,a single oral administration of linderane elevated the phosphorylation of ERK and STAT3,and activity of PDE in the liver,which inhibited hepatic cAMP/CREB pathway in C57BL/6 mice.Linderane ameliorated random-fed and fast blood glucose,as well as HbA_1c level during the chronic treatment in ob/ob mice.In addition,linderane could reduce the serum and hepatic triglycerides content.Conclusions:Linderane indirectly activated PDE3 through ERK/STAT3,and inhibited cAMP/CREB pathway,leading to the suppression of gluconeogenesis.Linderane ameliorated random-fed and fast blood glucose,as well as lipid metabolism in ob/ob mice.These results highlighted the role of PDE3 in regulating hepatic gluconeogenesis,which provided experimental evidences and theoretical basis for the development of linderane as anti-diabetic lead compounds.Part two:Effects and mechanism studies of the natural compound coronarin A on hepatic glycogen synthesis and gluconeogenesis.Aims:Dual regulation of hepatic glycogen synthesis and gluconeogenesis might be effective in alleviating diabetic syndromes.We found that the natural compound coronarin A could promote glycogen synthesis and inhibit gluconeogenesis in rat primary hepatocytes.The present study will explore the mechanism of coronarin A in regulating hepatic glucose metabolism.In addition,the anti-diabetic effect of coronarin A was evaluated in ob/ob mice.Methods:The effect of coronarin A on glycogen synthesis was examined in rat primary hepatocytes.Detect the phosphorylation of Akt and GSK3?using western blot analysis.Pharmacological inhibitors for PI3K and Akt were applied to explore the manner coronarin A enhanced glycogen synthesis.Phosphorylation of mTOR,S6K,S6 and IRS1 Ser1101 was also detected.The effect of coronarin A on gluconeogenesis was determined in rat primary hepatocytes,as well as the mRNA expression of PEPCK and G6Pase using real-time PCR.We also detected the expression of TCF7L2 and its downstream genes,and phosphorylation of ERK and?-catenin.The effect of coronarin A in improving glucose and lipid metabolism was evaluated in ob/ob mice.Results:Coronarin A enhanced the glycogen synthesis in rat primary hepatocytes in a time-and dose-dependent manner.Meanwhile,PI3K and Akt inhibitors blocked this effect.Coronarin A inhibited mTORC1,reduced the inhibition of IRS1 by S6K,and subsequently activated PI3K/Akt pathway.Coronarin A suppressed gluconeogenesis and the mRNA expression of PEPCK and G6Pase under both basal and forskolin-induced conditions.This inhibitory effect was dependent on?-catenin/TCF7L2 pathway via ERK.Intraperitoneal injection of coronarin A ameliorated fast and random-fed blood glucose,HbA_1c,glucose tolerance and overall insulin sensitivity,as well as lipid metabolism in ob/ob mice.Oral administration of coronarin A improved glucose and lipid metabolism,and increased hepatic glycogen content in chronic treatment in ob/ob mice.Conclusions:Coronarin A modulated hepatic glucose metabolism in a dual-regulation way.Coronarin A activated PI3K/Akt pathway through mTORC1,leading to the increase of glycogen synthesis.Coronarin A activated ERK/?-catenin pathway and inhibited gluconeogenesis.Intraperitoneal or oral administration of coronarin A ameliorated glucose and lipid metabolism in ob/ob mice.These results provided experimental evidences and theoretical basis for the development of coronarin A as anti-diabetic lead compounds.