Measurements of basal d-glucose transport through GLUT1 across the intact plasma membrane of isolated segments from single fast- and slow-twitch skeletal muscle fibres ofrat.

Abstract

AimTo develop a method for direct measurement of the fluorescent d‐glucose analogue 2‐NBDG transport across the plasma membrane of single skeletal muscle fibres and derive the theoretical framework for determining the kinetic parameters for d‐glucose transport under basal conditions.MethodsA novel method is described for measuring free 2‐NBDG transport across plasma membrane of single rat muscle fibres at rest. The 2‐NBDG uptake was >90% suppressed by 100 µM cytochalasin B in both fast‐twitch and slow‐twitch fibres, indicating that the 2‐NBDG transport is GLUT‐mediated. Fibres were identified as fast‐twitch or slow‐twitch based on the differential sensitivity of their contractile apparatus to Sr2+.ResultsThe time course of 2‐NBDG uptake in the presence of 50 µM 2‐NBDG follows a one‐phase exponential plateau curve and is faster in fast‐twitch (rate constant 0.053 ± 0.0024 s‐1) than in slow‐twitch fibres (rate constant 0.031 ± 0.0021 s‐1). The rate constants were markedly reduced in the presence of 20 mM d‐glucose to 0.0082 ± 0.0004 s‐1 and 0.0056 ± 0.0002 s‐1 in fast‐twitch and slow‐twitch fibres respectively. 2‐NBDG transport was asymmetric, consistent with GLUT1 being the major functional GLUT isoform transporting 2‐NBDG in muscle fibres at rest. The parameters describing the transport kinetics for both 2‐NBDG and d‐glucose (dissociation constants, Michaelis–Menten constants, maximal rates of uptake and outflow) were calculated from the measurements made with 2‐NBDG.ConclusionFree 2‐NBDG and d‐glucose transport across the plasma membrane of single rat muscle fibres at rest is fast, conclusively showing that the rate‐limiting step in d‐glucose uptake in skeletal muscle is not necessarily the GLUT‐mediated transport of d‐glucose.