Establishing mammalian GLUT kinetics and lipid preferences in a reconstituted-liposome system

Abstract

Glucose transporters (GLUTs) are essential for organism-wide glucose homeostasis in mammals. Due to their critical role in cellular growth and metabolism, GLUT dysfunction is associated with numerous diseases, such as diabetes and cancer. Despite structural advances, transport assays using purified GLUT have proven to be difficult to implement, hampering deeper mechanistic studies. Indeed, our understanding of the role of GLUT transporters to whole-body glucose homeostasis is largely derived from the kinetics of 2-deoxy-glucose and not D-glucose, as this sugar is rapidly phosphorylated in the cell. Here, we have optimized a transport assay in liposomes for the fructose-specific isoform GLUT5, and demonstrate how inaccuracies can be generated when assessing the impact of mutations and inhibitors with suboptimal activity. By combining lipidomic analysis with native MS and thermal-shift assays, we replicate the GLUT5 transport activities seen in crude lipids with a small number of synthetic lipids. Subsequently, we show how GLUT5 is only active under a specific range of membrane fluidity, and that human GLUT1-4 prefers a similar lipid composition. Although GLUT3 is designated as the high-affinity glucose transporter, in vitro D-glucose kinetics demonstrates that GLUT1 and GLUT3 actually have a similar KM, but GLUT3 has a higher turnover. Interestingly, GLUT4 has a high KM for D-glucose and yet a very slow turnover, which might be to ensure D-glucose uptake is regulated by insulin-dependent trafficking. Overall, our study provides a much-needed transport assay for analyzing in vitro GLUT activities, and implies that high-levels of free fatty acids in membranes, as found in those suffering from metabolic disorders, are a likely contributor to the impairment of glucose transport.

Determinants of sugar-induced influx in the mammalian fructose transporter GLUT5

“…Consistently, the H386F and S391G variants that were constructed to match the most common residue in the other GLUT transporters ( Deng et al, 2015 ), abolished D -fructose transport ( Figure 4B ). As shown recently ( Saudea et al, 2022 ), the A395W variant is also incapable of D -fructose transport ( Figure 4B ). All GLUT5 variants were unable to transport D-glucose ( Figure 4—figure supplement 1C ).…”

“…Interestingly, the combined variant is now re-able to transport D -fructose transport with ~40% wildtype activity ( Figure 4B , Figure 4—figure supplement 1B ), although it is still unable to transport D-glucose ( Figure 4—figure supplement 1C ). Given the already low affinity (10 mM) of D -fructose for wildtype GLUT5, we are unable to measure kinetics of this combined variant under the current experimental setup ( Saudea et al, 2022 ). Nonetheless, the much higher accumulation observed above background indicates multiple turnover events.…”

“…To strengthen the proposed MD model for D -fructose coordination, we evaluated 14 C- D -fructose transport of purified GLUT5 variants reconstituted into liposomes using our recently developed transport assay ( Saudea et al, 2022 ) (see Methods for details). We focused the mutagenesis on GLUT residues that are different in GLUT5 from GLUT transporters unable to transport D -fructose ( Li et al, 2004 ).…”

“…Firstly, the energy barriers are estimated along a path that describes structural transitions in the extra- and intracellular gates, rather than all conformational changes across the entire protein, that is, whilst the CVs allowed us to explore the entire transport pathway, different CVs that more accurately separate metastable states are required, which we have explored in an accompanying work using co-evolution analysis ( Mitrovic et al, 2022 ). Secondly, our models consider a membrane bilayer made from POPC lipids, whereas it is well established that transport by GLUT proteins requires the presence of anionic lipids ( Saudea et al, 2022 ; Hresko et al, 2016 ). The fact that the activation barrier for GLUT1 has been shown to increase from 10 to 16 kcal/mol in liposomes made from lipids with longer fatty acids highlights just how sensitive GLUT proteins are to the lipid composition ( Carruthers and Melchior, 1984 ).…”

“…rGLUT5 protein was incorporated into proteoliposomes and radiolabeled sugar uptake measured as recently described ( Saudea et al, 2022 ). Briefly, liposomes were prepared in 500 µl batches by mixing total bovine brain lipid extract (Sigma-Aldrich, final 30 mg/ml) and cholesteryl-hemisuccinate powder (Sigma-Aldrich, final 6 mg/ml) were mixed in a buffer of 10 mM Tris–HCl (pH 7.6) and 2 mM MgSO 4 .…”