Use of hexose transport mutants to examine the expression and properties of the rat myoblast GLUT 1 transport process

Abstract

Rat L6 myoblasts were recently shown to possess the GLUT 1, 3 and 4 transporters, and not the GLUT 2 isoform [1]. This investigation examined the expression and properties of the GLUT 1 isoform. GLUT 1 transcript level was significantly reduced in cells grown at high densities and during myogenic differentiation. A comparison of the GLUT 1 and 4 transcript levels in myogenesis-competent and impaired cells revealed an inverse relationship between these two isoforms. This relationship was confirmed by studies using two independent spontaneous GLUT 3- GLUT 4- mutants, M1 and M3. These mutants possessed very high level of the GLUT 1 isoform, but negligible amount of the GLUT 3 and 4 isoforms. GLUT 1 expression was also subject to positive regulation. Glucose starvation was found to increase not only the levels of the GLUT 1 transcript and transporter, but also the intrinsic activity of the GLUT 1 transporter. Studies with M1 and M3 mutants revealed that the GLUT 1 transporter was not functional in glucose-grown cells, even though it was present at a very high level in the plasma membrane. This transporter became functional when cells were starved for glucose. The functional GLUT 1 transporter had an apparent Km value of around 0.9 mM, and was sensitive to cytochalasin B, phloretin, phlorizin and pCMBS.

Effects of 2‐deoxy‐D‐glucose on the functional state of the rat myoblast GLUT 1 transporter

“…BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL importantly, the GLUT 1 transporter was not functional in glucose-grown cells, however, it was activated when cells were grown in the absence of glucose [ 1].…”

“…Recent studies using fructose-grown rat myoblast GLUT 3-GLUT 4-mutants revealed that the apparent Km and Vmax values for the uptake of dGlc by the GLUT 1 transporter were 0.9 mM and 220 pmol/ 10 s cells per min, respectively [1,12].…”

“…These mutants also enabled one to determine the expression and identity of the GLUT I transcript and transporter, free of interference by other GLUT isoforms [ 1]. More…”

“…Mutants harbouring only one GLUT isoform allow a detailed examination of the in situ expression, intrinsic property and functional states of a particular isoform under different growth conditions. For example, rat myoblast GLUT 3-GLUT 4-mutants have been used to examine the properties of the GLUT 1 isoform [1]. These studies revealed that the GLUT 1 transport kinetic properties, substrate specificity and response to cytochalasin B were distinctly different from those of the GLUT 5 transporter, which is responsible for fructose transport [1,4].…”

“…In view of the presence of different GLUT isoforms in rat myoblasts, glucose transport mutants are invaluable in establishing the association of GLUT transcripts, transporters, and cytochalasin B binding sites with specific glucose transport processes [1,9]. Mutants harbouring only one GLUT isoform allow a detailed examination of the in situ expression, intrinsic property and functional states of a particular isoform under different growth conditions.…”