Stimulation of hexose transport in L6 rat myoblasts by antibody and by glucose starvation

Abstract

Treatment of glucose-grown L6 rat myoblasts with rabbit or sheep anti-(L6-rat myoblast) antibody for 35 min or glucose starvation for at least 8 h results in a 2-fold increase in the Vmax. of 2-deoxy-D-glucose (dGlc) and 3-O-methyl-D-glucose uptake. In both cases, apparent transport affinities were not affected. Furthermore, once stimulation has occurred, further increases in hexose uptake could not be produced. Assays of antibody binding to whole cells suggested that the antibody is not internalized but remains bound on the cell surface. To elucidate the site and mechanism of antibody action, plasma-membrane vesicles from L6 cells were prepared. Anti-L6 antibody was found to cause a time- and dosage-dependent stimulation of dGlc transport in these vesicles. Maximum activation was achieved after 30 min exposure. This antibody-mediated activation could be inhibited by treatment of vesicles with various proteinase inhibitors. Treatment of vesicles with trypsin was also found to activate dGlc transport to levels observed with antibody. These results are virtually identical with those obtained with whole cells and suggest that antibody-mediated activation of hexose transport results from interaction of antibody with a specific membrane component(s).

Use of transport mutants to examine the identity and expression of GLUT isoforms in rat cardiac myoblasts

“…Analysis of data by non-linear regression fit to the Michaelis-Menten hyperbola revealed that the transport capacities of glucose-grown and starved cells were 263.91 and 264.62 nmoles/l0 s cells/min, respectively. Thus unlike rat L6 myoblasts [16,27], the glucose transport process in rat cardiac myoblasts was not activated by glucose starvation. GLUT transcript levels were determined by Northern blot analysis.…”

“…Clone EZ-4 is a spontaneous mutant selected by growth of RCM myoblast in fructose medium containing 4 mM dGlc. All three cell lines were maintained in Alpha medium (Flow Laboratories) supplemented with 10% (v/v) horse serum (Flow Laboratories) and gentamycin (50 lag/ml; Gibco) [27]. The D-glucose concentration in Alpha medium was 25 raM.…”

“…Preparation of plasmid cDNAs and poly (A) + RNAs, and northern blot studies were carried out by previously described procedures [ 15,16,34,35]. Immunoblotting studies using membrane preparations --Plasma membrane and microsome were fractionated and purified from glucose-grown and starved subconfluent day-2 cultures of H9C2, RCM, and EZ-4 myoblasts by differential centrifugation and followed by discontinuous sucrose gradient centrifugation [27,[36][37][38]. Immunoblotting studies were carried out by previously described procedures [15,39,40].…”

“…Glucose starvation has been shown to stimulate GLUT 1 expression, dGlc transport and cytochalasin B binding activities in a number of cell types [2,16,18,27,[49][50][51][52]. To examine its effect on the expression of rat cardiac GLUT isoforms, poly (A) § RNAs were prepared from day 2 culture of H9C2 myoblasts grown after several passages in media containing no glucose (fructose medium), low glucose (5.6 mM), or 25 mM D-glucose.…”

A possible role for a mammalian facilitative hexose transporter in the development of resistance to drugs.

“…Initial experiments indicated that the uptake of 0.1 mM 2-deoxy-D-glucose by both cells increased linearly with time for at least 5 min. Using short incubation periods, less than a twofold increase in uptake of 2-deoxy-D-glucose was observed in drug-resistant DC-3F/AC X cells (70 pmol (12,34). In addition, it has been shown that exposure of drug-sensitive cell lines to phorbol esters rapidly induces a drug-resistant phenotype that is sensitive to verapamil, which is -accompanied by a net decrease in intracellular drug accumulation (vincristine and doxorubicin) (20).…”