Discrimination of GLUTs by Fructose Isomers Enables Simultaneous Screening of GLUT5 and GLUT2 Activity in Live Cells

Abstract

Facilitative carbohydrate transporters (GLUTs, SLC2 gene family) are transmembrane proteins transporting hexoses and other sugars based on cellular metabolic demands. While a direct link between GLUTs and metabolic disorders has framed them as important biological and medicinal targets, targeting disease-relevant GLUTs remains challenging. In this study, we aimed to identify substrate–GLUT interactions that would discriminate between major fructose transporters. We examined the uptake distribution for conformational and configurational isomers of fructose using the corresponding conformationally locked fluorescently labeled mimetics as probes for assessing GLUT preferences in real time. Through comparative analysis of the uptake of the probes in the yeast-based single GLUT expression systems and the multi-GLUT mammalian cell environment, we established the ability of fructose transporters to discriminate between fructose conformers and epimers. We demonstrated that recreating the conformational and configurational mixture of fructose with molecular probes allows for the specific probe distribution, with fructofuranose mimetic being taken up preferentially through GLUT5 and β-d-fructopyranose mimetic passing through GLUT2. The uptake of α-d-fructopyranose mimetic was found to be independent of GLUT5 or GLUT2. The results of this study provide a new approach to analyzing GLUT5 and GLUT2 activity in live cells, and the findings can be used as a proof-of-concept for multi-GLUT activity screening in live cells. The research also provides new knowledge on substrate–GLUT interactions and new tools for monitoring alterations in GLUT activities.

Turn-on Rhodamine Glycoconjugates Enable Real-Time GLUT Activity Monitoring in Live Cells and In Vivo

“…The differences in competition outcomes appear to relate to the differences in the uptake specificities or the lack thereof. For ManRho-B, the lack of competition with fructose can be explained by the 270-fold higher affinity of the probe vs fructose (37 μM vs 10 mM) for GLUT5 and by the ability of fructose to pass through other fructose GLUTs in the presence of a GLUT5 binder . The increase in fructose uptake in the presence of ManRho-B appears to reflect the activation of GLUT5 in the high (100 mM) fructose environment.…”

“…For ManRho-B, the lack of competition with fructose can be explained by the 270-fold higher affinity of the probe vs fructose (37 μM vs 10 mM) for GLUT5 and by the ability of fructose to pass through other fructose GLUTs in the presence of a GLUT5 binder. 32 The increase in fructose uptake in the presence of ManRho-B appears to reflect the activation of GLUT5 in the high (100 mM) fructose environment. In contrast, potent inhibition of ManRho-Az uptake by fructose further highlights the nonspecific uptake of the probe, as it reflects the competition for GLUT5 and other fructose GLUTs.…”