GLUT2 proteins are regulated by the AKT pathway under diabetic conditions in intestinal epithelial cells

Abstract

BackgroundInsulin secretion and nutrient transport are regulated by Glucose dependent Insulinotropic Peptide (GIP). Excessive GIP secretion after a meal contributes to diabetes. GIP also increases the transport of glucose, which stimulates additional GIP secretion. High blood glucose levels decrease apical Na+‐glucose cotransporter‐1 (SGLT1) mediated glucose absorption and increase apical glucose transporter‐2 (GLUT2) mediated glucose absorption. In contrast, normal blood glucose levels maintain glucose absorption through SGLT1, while GLUT2 is not expressed on the apical membrane. Signaling mechanisms of how GIP regulates glucose transporters in the presence of normal and high extracellular glucose concentrations is not known. Earlier results indicate that the cAMP pathway through EPAC partially regulates GLUT2 expression and function.HypothesisExtracellular glucose concentration alters the glucose transporters on the apical membrane of differentiated epithelial cells through the AKT pathway.MethodsIEC6 cells were grown 3–5 days post confluent on snap‐wells using standard DMEM media with 5% FBS and either normal (0.45 g/L) or high (4.5 g/L) glucose. Mucosal to serosal (m‐s) and serosal to mucosal (s‐m) glucose fluxes were measured across IEC6 monolayers mounted under voltage clamp conditions in Ussing chambers. Short circuit currents (Isc) were measured to distinguish SGLT1 (electrogenic) and GLUT2 (electroneutral) mediated glucose absorption. Apical membranes were isolated from IEC6 cell monolayers and the apical proteins were isolated using biotinylation techniques. Western blot analyses were then performed using SGLT1 and GLUT2 antibodies.ResultsGlucose absorption is increased, while electrogenic (i.e. Isc) and GIP‐stimulated glucose absorption are reduced in IEC6 monolayers grown in high glucose media. In contrast, higher Isc and lower level of glucose absorption in IEC6 monolayers grown in normal glucose medium. The absorption of GIP stimulated glucose absorption is inhibited by AKT inhibitors Wortmannin and an AKT specific inhibitor that inhibits glucose absorption in IEC6 monolayers grown in only high glucose medium. Although GIP stimulates glucose absorption in IE6 monolayers grown in both normal and high glucose medium, the GIP‐stimulated glucose absorption (flux) is higher in IEC6 monolayers grown in high glucose medium. Western blot analyses revealed that GLUT2 expression increased, but not SGLT1 on the apical membrane of IEC6 monolayers grown in high glucose medium. AKT inhibitors and Wortmanin (PI3K inhibitor) decreased GLUT2 protein levels in the presence of GIP when compared to untreated GIP stimulated cells.Conclusions1) In addition to EPAC pathway, the AKT pathway also regulates GLUT2, but not SGLT1 in high glucose media (diabetes) rather than in low glucose media (normal). 2) SGLT1is the major glucose transporter present in IEC6 monolayers grown in normal extracellular glucose environment. Speculation: It is likely that GIP increases glucose absorption through trafficking of GLUT2 to the apical membrane.Support or Funding InformationSupported by Montana IMBRE