Fig. 1: S1P loading of murine RBC by sphingosine reduces glucose uptake, and S1P unloading by albumin or a S1P neutralizing antibody restores glucose uptake.
A C57Bl6 RBC S1P levels and (B) S1P efflux after incubation with 1 µM sphingosine (Sph) or vehicle (EtOH) for 30 min followed by incubation with or without 1% BSA for 20 min (n = 6 each). C Glucose uptake rate in RBC loaded or not with sphingosine and incubated subsequently with or without 1% BSA (n = 8). D S1P efflux from S1P or vehicle loaded RBC induced by incubation with 800 pmol/ml of the S1P neutralizing antibody Sphingomab or an isotype-matched control IgG1 (n = 6). E Glucose uptake rate in S1P-loaded RBC in the presence of Sphingomab or control IgG (n = 7/7/9/9). F GLUT4 cell surface localization without and with sphingosine as determined by flow cytometry using the LM608 antibody that recognizes a strictly extracellular GLUT4 domain (n = 4). G 13C3 lactate/13C2 lactate ratio, and (H) 13C2 PRPP relative exchange rate in metabolic flux experiments using 1,2,3-13C3-D-glucose (n = 6 each). 1 pmol S1P/Mio RBC equals a concentration of 21 µMol/L S1P as calculated based on a mean MCV of 47.5 fl. The ‘n’ in the A-E refers to individual mice. Data are presented as mean ± sd and tested with paired one-way ANOVA followed by a Tukey’s multiple comparison test (A–E), a paired two-tailed t test (F) or stack matched two-way ANOVA (G, H); ns= not significant; p** < 0.01; p*** < 0.001; p**** < 0.0001.
