Correlation Between TXNIP And Geniposide Regulating GLUT2 Expression And Distribution In INS-1 Cells And Its Mechanism

Type 2 Diabetes Mellitus(T2DM)is characterized by impaired insulin secretion from pancreatic β‐cells.Our previous studies suggested that geniposide isolated from Gardenia jasminoide was a novel agonist for glucagon-like peptide 1(GLP-1)receptor,which can rapidly regulate insulin secretion in INS-1 cells.It was found that Adenosine 5-Monophosphate(AMP)-activated Protein Kinase(AMPK)inhibitor and AMPK siRNA could decrease glucose uptake,ATP production,and attenuate Glucose-Stimulated Insulin Secretion test(GSIS)which regulated by geniposide,proved that AMPK pathway plays an important role in the regulation of GSIS by geniposide.Thioredoxin Interacting Protein(TXNIP),a regulator of cellular oxidative stress,which plays an important role in regulating insulin secretion.During the pathogenesis of T2 DM,elevated TXNIP levels induce β-cell apoptosis and insulin sensitivity decrease,which in turn inhibit glucose uptake and utilization.In pancreatic β cells,Glucose Transporter 2(GLUT2)is required for glucose-stimulated insulin secretion.It has been suggested TXNIP regulates GLUT1 through endocytosis in Hep G2 cells.So is there a link between TXNIP and GLUT2 in pancreatic beta cells? Previous studies have shown that geniposide can affect pancreatic β-cell function by regulating GLUT2 endocytosis,so is this process related to the regulation of TXNIP levels by geniposide? Does AMPK also have an important role in the regulation of TXNIP levels by geniposide? These are the the central questions in the present study.Based on present research and our preliminary work,we propose the following hypothesis: in INS-1 cells,geniposide may reduce high-glucose-induced TXNIP level via the AMPK signaling pathway,affect the interactions between TXNIP and GLUT2,influence the distribution of GLUT2 levels between cytosolic and plasma membrane,and ultimately affects glucose uptake,glucose metabolism and insulin secretion.In this study,we first investigated the effect of the high glucose on the TXNIP protein levels,and the regulatory effect and mechanisms of geniposide in this process.It is showed that the high glucose will induce the increase of TXNIP level in INS-1 cells in a timedependent manner,but TXNIP mRNA levels were not significantly affected.The proteasome inhibitor MG132 could significantly block the regulation of geniposide on TXNIP,indicated that geniposide may regulate TXNIP degradation through the proteasome pathway under high glucose state.Then,we used AMPK agonists and inhibitors and RNA interference methods to analyze the role of AMPK signaling in the above process.The study confirmed that the AMPK activator AICAR can not only downregulate the level of TXNIP protein but also significantly enhance the regulatory effect of geniposide on TXNIP,while the AMPK antagonist Compound C blocks the effect of geniposide.After using AMPK siRNA to interfere with the AMPK gene,the effect of geniposide to degrade TXNIP was also inhibited,which further clarified the important role of AMPK in the regulation of high glucose-induced TXNIP levels by geniposide.Subsequently,co-immunoprecipitation(CO-IP)combined with immunofluorescence(IF)experiments were used to investigate the interaction and co-localization of TXNIP and GLUT2 in INS-1 cells.The results showed an interaction between TXNIP and GLUT2 in INS-1 cells and a colocalization of TXNIP and GLUT2 which is mainly concentrated on the cell membrane.Finally,gene interference and overexpression techniques were used to study the correlation between TXNIP and geniposide regulating the distribution of GLUT2 levels between cytosolic and plasma membrane,and the effect of TXNIP on geniposide regulating the function of INS-1 cells.The experimental results showed that under high glucose(25 mM glucose)state,both TXNIP gene interference and geniposide could significantly reduce the level of GLUT2 protein in the cell membrane and increase the level of GLUT2 protein in the cytoplasm;Under normal glucose(11 mM glucose)state,after TXNIP overexpression,the cytoplasmic GLUT2 protein level in INS-1 cells was significantly increased,and the cytomembranic GLUT2 protein level decreased.Geniposide could inhibit the effect of TXNIP overexpression that influence the distribution of GLUT2 levels between cytosolic and plasma membrane.Both geniposide and TXNIP siRNA could significantly inhibit the absorption of glucose,the production of ATP and the secretion of insulin under high glucose state,and the above effects of geniposide were further enhanced after TXNIP gene interference.The results of this study confirmed the interaction and colocalization of GLUT2 and TXNIP in INS-1 cells.Under high glucose state,geniposide may degrade TXNIP protein through AMPK pathway,and then regulate the interaction between TXNIP and GLUT2,indirectly affecting distribution of GLUT2 levels between cytosolic and plasma membrane and finally affecting the pancreas β Cell function.The relationship between TXNIP and geniposide regulating the expression and distribution of GLUT2 in INS-1 cells was elucidated,which provides the experimental basis for the development of Geniposide as a drug for T2 DM.