FIG. 3.
The substrate dependence of SGLT1-mediated glucose uptake expressed in oocytes is different from the substrate dependence of SGLT1-mediated glucose uptake across small intestinal BBM. A and B: Transport measurements in oocytes. Oocytes were injected with 5 ng of mouse Sglt1 cRNA and incubated 2 days for expression. Uptake of different concentrations of [14C]AMG (A) or [3H]d-glucose (B) was measured in the absence and presence of 100 μmol/L phlorizin. Phlorizin-inhibitable uptake rates of individual experiments were normalized to the uptake rates measured at 2 mmol/L AMG or d-glucose. Mean values + SE of 25–30 oocytes from 3 independent experiments are shown. Similar apparent Km values of 0.17 ± 0.05 mmol/L and 0.13 ± 0.01 mmol/L were obtained for AMG and d-glucose, respectively. The Vmax values of SGLT1-mediated uptake of AMG and d-glucose were similar. In oocytes of the same batch, which were injected with 5 ng of Sglt1 cRNA per oocyte and incubated for 2 days, Vmax values (measured at monosaccharide concentrations of 2 mmol/L without and with 100 μmol/L phlorizin) of 5.0 ± 0.6 (AMG) and 4.8 ± 0.6 (d-glucose) pmol × oocyte−1 × min−1 were obtained (n = 25 each, not significant). C: Transport measurements in BBMVs. Wild-type mice fed with standard diet were starved for 18 h and killed at 3–4 p.m. BBMVs were prepared. BBMVs were incubated for 2 s at 37°C with different concentrations of [14C]AMG in the presence of an inwardly directed gradient of 100 mmol/L NaSCN or 100 mmol/L KSCN. The sodium-dependent uptake rates of individual experiments were normalized to the values obtained at 10 mmol/L AMG. Mean values ± SE of 12 measurements from three independent experiments are shown. A Km value of 1.20 ± 0.04 mmol/L (n = 5) was determined. D: Glucose-induced short circuit currents across small intestinal mucosa. Wild-type mice fed with standard diet were starved for 18 h and killed at 4 p.m. The jejunal small intestinal wall was then mounted to an Ussing chamber. The mucosal side was superfused with different concentrations of d-glucose, and d-glucose–induced short circuit currents (Isc) mediated by Na+ -d-glucose cotransport was measured. Mean values ± SE of 7 experiments are shown. The Michaelis Menten equation was fitted to the data. A Km value of 1.9 ± 0.6 mmol/L was determined.