Previous studies revealed an inverse relationship between GLUT 1 and GLUT 4 expression in rat myoblasts [L. Xia, Z. Lu, T.C.Y. Lo, J. Biol. Chem., 268 (1993) 23258-23266]. It was not clear whether these were coincidental or causal occurrences. To examine the regulatory roles of the GLUT 4 isoform, rat L6 myoblasts were transfected with full length GLUT 4 cDNAs (2.5 kb) in the sense or antisense orientation. L6 myoblasts transfected with the GLUT 4 sense cDNA (L6/G4S transfectants) possessed much elevated levels of both endogenous GLUT 4 transcripts (1.4 kb and 2.8 kb). Transport and immunofluorescence studies showed that this GLUT 4 sense cDNA was responsible for a functional GLUT 4 transporter. L6 cells transfected with the GLUT 4 antisense cDNA (L6/G4A transfectants) possessed only 6% of the L6 level in day 6 cultures. These antisense transfectants were essentially devoid of any functional GLUT 4 transporter. The activation of transcription of the endogenous GLUT 4 gene in L6/G4S myoblasts suggested auto-regulation of GLUT 4 expression. GLUT 3 expression and activity were not altered in both sense and antisense GLUT 4 transfectants. More interestingly, GLUT 1 expression was reduced in L6/G4S myoblasts, whereas it was elevated in L6/G4A myoblasts. This was the first direct evidence indicating GLUT 4 might play an important role in suppressing GLUT 1 expression.
We have recently demonstrated a close relationship between the GLUT 3 transporter and the myogenic ability of rat skeletal L6 myoblasts [1]. This investigation examined the effects of over‐and under‐expression of the GLUT 3 transporter on both biochemical and morphological differentiation. L6 transfectants expressing two to five times the normal L6 GLUT 3 transcript level were impaired in the expression of myogenesis‐associated genes, such as myogenin, MLC, MHC and TnT, and in myotube formation. Similar defects were also observed in myoblast mutants expressing less than 20% of the normal GLUT 3 level. Forced expression of an exogenous GLUT 3 cDNA could partially rescue the myogenic defect of these GLUT 3‐ mutants. However, such myogenic defects were not observed in L6 GLUT 3 antisense transfectants expressing 39% of the normal L6 GLUT 3 level. These data suggest that myogenic differentiation will proceed only within a critical level of the GLUT 3 transporter. The optimal GLUT 3 content for myogenesis ranges from around 2 × 105 to 5 × 105 molecules per cell in day 2 cultures; GLUT 3 levels outside this range have a negative effect on myogenesis. Our data suggest that GLUT 3 may regulate myogenesis by modulating the levels of signal transducers required for expression of myogenin and muscle‐specific contractile protein genes.
Myogenesis is a complex process characterized by both biochemical and morphological differentiation. Recent transfection studies suggested a close relationship between the GLUT 3 transporter and the myogenic ability of rat skeletal L6 myoblast. In this study, the myogenic properties of GLUT 3- mutants were examined. Studies using three different GLUT 3- mutants (D2, D9 and D23) revealed that these mutants were defective not only in the GLUT 3 transporter, but also in the expression of myogenesis-associated genes. The properties of mutant D23 were further characterized. Overexpression of an exogenous functional GLUT 3 transporter was unable to restore the myogenic defects of this mutant. This mutant was subsequently found to be altered in components acting on at least two different sites of the L6 myogenic pathway. Transfection studies revealed that mutant D23 was deficient in component(s) essential for the myogenin promoter activity. The second component was required for the transcription of muscle-specific protein genes, as overexpression of myogenin was unable to rescue the inability of this mutant to express muscle-specific genes and to form myotubes. Mutant D23 was therefore thought to be deficient in a regulatory component which controlled the expression of GLUT 3, myogenin and muscle-specific genes.
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