Glucose Transporters are differentially regulated in rat intestinal epithelial crypt cells (IEC6) grown in high and low glucose medium

Abstract

BackgroundGlucose dependent insulinotropic peptide (GIP) is a hormone that regulates both insulin release and nutrient transport. When GIP is secreted in large amounts, it contributes to diabetes and obesity. It is a cyclic process in which GIP increases transport of glucose, which in turn stimulates additional GIP secretion. High blood glucose decreases Na+‐glucose cotransporter‐1 (SGLT‐1) mediated glucose absorption and increases apical glucose transporter‐2 (GLUT2) mediated glucose absorption. In contrast, normal blood glucose maintains glucose absorption through SGLT1, while GLUT2 is not expressed on the apical membranes. Although SGLT1 is the primary apical glucose absorptive process in normal, GLUT2 has been localized on the apical membranes of obese human and mouse intestine. However, signaling mechanisms of how GIP regulates glucose transporters in the presence of normal and high extracellular glucose concentrations is not known.HypothesisExtracellular glucose concentration alters the glucose transporters on the apical membrane of differentiated epithelial cells.MethodsIntestinal epithelial crypt cell‐6 (IEC6 cells) were grown 3–5 days post confluent on snap‐wells using standard DMEM media supplemented with 5% FBS and either normal (0.45 g/L) or high (4.5 g/L) glucose, respectively. Mucosal to serosal (m‐s) glucose fluxes were measured across IEC6 monolayers mounted under voltage clamp conditions in Ussing chambers. Short circuit currents (Isc) were measured to distinguish SGLT1 (i.e. electrogenic) and GLUT2 (i.e. 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 substantially increased, while electrogenic (i.e. Isc) and GIP‐stimulated glucose absorption are reduced in IEC6 monolayers grown in medium with high glucose. In contrast, significantly higher Isc and lower level of glucose absorption are present in IEC6 monolayers grown in normal glucose medium. The absorption of glucose is inhibited by a cAMP inhibitor inhibits glucose absorption in IEC6 monolayers grown in both normal and high glucose medium. In contrast, inhibition of EPAC inhibits the glucose absorption in IEC6 monolayers grown in high, but not in low glucose medium. Although GIP stimulated 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, but not SGLT1 specific protein is expressed on the apical membrane of IEC6 monolayers grown in high glucose medium.ConclusionsThe EPAC regulated GLUT2, but not SGLT1 is the major glucose transporter present in IEC6 monolayers grown in high extracellular glucose environment. The PKA regulated SGLT1 is the major glucose transporter present in IEC6 monolayers grown in normal extracellular glucose environment.Support or Funding InformationThis research is supported by Montana IMBREThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.