Isolation and characterization of hexose transport mutants in L6 rat myoblasts

A method for the selection and isolation of hexose transport mutants in undifferentiated rat myoblast L6 cells is reported; 2-deoxy-D-glucose (2-DOG)-and 2-deoxy-2-fluoro-D-glucose (2FG)-resistant mutants were selected after mutagenization of L6 cells with ethyl methanesulfonate. Of these, D18 and D23 (selected with 0.1 mM 2-DOG) and F72 and F76 (selected with 0.1 mM 2FG) exhibited the lowest hexose transport activity. Uptake of 0.06 mM 2-DOG, 2FG, or 3-O-methyl-D-glucose (3-OMG) by mutants grown in fructose medium supplemented with 0.05 mM 2FG was about four- to five-fold lower than the parental L6 cells. These mutants contain normal levels of ATP and glycolytic enzyme activities. They also exhibit normal transport activities for alpha-aminoisobutyric acid and fructose. Furthermore, hexose transport was observed to be decreased in plasma membrane vesicles prepared from these mutants. Kinetic analysis of 2-DOG and 3-OMG transport in mutant F72 demonstrated that the Vmax for 2-DOG uptake was significantly reduced, whereas the Vmax for 3-OMG transport was not affected. In all cases, the affinity for these hexose analogues was unaffected. In addition mutant F72 was found to be only slightly affected by treatment with various energy inhibitors and sulfhydryl reagents. The results suggest that this mutant is defective in, or has low levels of, a plasma membrane component(s) involved in the high-affinity hexose transport system.     

Approaches used to examine the mechanism and regulation of hexose transport in rat myoblasts

This review discusses some of the approaches and general criteria that we have used to examine the properties of the hexose transport system in undifferentiated L6 rat myoblasts. These approaches include studying the kinetics of hexose transport in whole cells and plasma membrane vesicles, the effects of various inhibitors on hexose transport, the isolation and characterization of hexose transport mutants, and the use of cytochalasin B (CB) to identify the transport component(s). Transport kinetics indicated that two transport systems are present in these cells. 2-Deoxy-D-glucose is transported primarily by the high affinity system, whereas 3-O-methyl-D-glucose is transported by the low affinity system. Furthermore, these two transport systems are inactivated to different extents by CB. CB has a higher binding affinity for the low affinity hexose transport system. The inhibitory effect of various hexose analogues also revealed the presence of two hexose transport systems. The effects of various ionophores and energy uncouplers on hexose transport suggest that the high affinity system is an active transport process, whereas the low affinity system is of the facilitated diffusion type. The high affinity system is also sensitive to sulfhydryl reagents, whereas the low affinity system is not. Further evidence for the presence of two transport systems comes from the characterization of hexose transport mutants. Two of the mutants isolated are shown to be defective in the high affinity transport system, but not in the low affinity transport system. These mutants are also defective in the CB low affinity binding site. Based on our results a tentative working model for hexose transport in L6 rat myoblasts is presented.     

Coordinate regulation of histone mRNAs during growth and differentiation of rat myoblasts

To determine whether histone genes are coordinately regulated, histone mRNA concentrations were measured in exponentially growing L6 myoblasts, S-phase synchronized myoblasts and in differentiating myoblasts. The levels of various histone mRNA subspecies declined rapidly and coordinately once myoblasts were given the signal to differentiate. mRNA levels were reduced on average to 1-5% of the amount observed in exponentially growing cells by 48 h after the signal to differentiate. The reductions occurred in concert with the cessation of DNA synthesis as the cells differentiated. Inhibition of DNA synthesis by treating myoblasts with Ara-C or hydroxyurea resulted in a histone mRNA half-life of 10-13 min for each of the histones examined. One example of non-coordinate regulation was observed however among the H4 mRNA subspecies in S-phase synchronized cells. The levels of two major subspecies of H4 mRNA increased coordinately in S-phase compared to levels observed in cells growing exponentially. A third subspecies of H4 mRNA on the other hand was found to decline by 50%. These studies suggest that the majority of histone mRNA subspecies are under coordinate control, although one exception has been noted among the subspecies of histone H4.     

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

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).     

Involvement of hexose transport in myogenic differentiation

A high (HAHT) and a low (LAHT) affinity hexose transport system are present in undifferentiated rat L6 myoblasts; however, only the latter can be detected in multinucleated myotubes. This suggests that HAHT is either down-regulated or modified as a result of myogenesis. The present investigation examined the relationship between HAHT and myogenic differentiation. While myogenesis could be inhibited by the potent hexose transport inhibitor phloretin, it was not affected by phlorizin which had no effect on hexose transport. This relationship was further explored using six different HAHT-defective mutants. All six mutants, altered in either the HAHT transport affinity (Type I mutants) or capacity (Type II mutants), were impaired in myogenesis. Since these mutants were selected from both mutagenized and non-mutagenized cells with different reagents, or with different concentrations of the same reagent, the deficiency in myogenesis was likely due to changes in HAHT properties. This notion was confirmed by the observation that growth of Type I mutants in high D-glucose concentrations could rectify the defect in myogenesis. D-glucose was unlikely to rectify the defect in myogenesis, if this defect was due to a second unrelated mutation that may have arisen during isolation of the mutants. Since both types of mutants were not altered in LAHT, D-glucose should still be taken up into the cells. The fact that the glucose-mediated increase in fusion could not be observed in Type II mutants (deficient in the HAHT transporter) suggested that myogenesis was dependent on the presence of D-glucose or its metabolites in specific HAHT-accessible compartments. It is tempting to speculate that trans-acting regulators involved in myogenesis may be synthesized from the glucose metabolites in these specialized HAHT-accessible compartments.     

Recombinant platelet-derived growth factor-BB stimulates growth and inhibits differentiation of rat L6 myoblasts.

We previously found that L6 myoblasts and skeletal muscle isolated from developing rats express the platelet-derived growth factor (PDGF) beta-receptor gene (Jin, P., Rahm, M., Claesson-Welsh, L., Heldin, C.-H., and Sejersen, T. (1990) J. Cell Biol. 110, 1665-1672). We now report that recombinant human PDGF-BB is a mitogen for L6 myoblasts and also a potent inhibitor of myogenic differentiation. Treatment of L6J1 myoblasts with PDGF-BB increased the rate of DNA synthesis and stimulated cell proliferation. In differentiation medium (Dulbecco's modified Eagle's medium/0.5% fetal calf serum or Dulbecco's modified Eagle's medium/insulin), PDGF-BB prevented fusion of confluent myoblasts and suppressed biochemical differentiation in L6J1 cells. Inhibition of myoblast differentiation was, however, reversible. Withdrawal of PDGF-BB from the medium allowed myoblast fusion to occur. Northern blot hybridization showed that the PDGF beta-receptor mRNA was down-regulated to an undetectable level when confluent cultures of L6J1 myoblasts in growth medium (Dulbecco's modified Eagle's medium/5% fetal calf serum) were shifted to differentiation medium. Receptor binding assays further indicated that binding of PDGF-BB to its receptors on L6J1 myoblasts declined rapidly before creatine kinase activity rose. Our results provide the first demonstration that PDGF-BB is a potent regulator of myogenesis of L6 rat myoblasts and suggest that it may regulate muscle differentiation in vivo.     

Regulation of hexose transport in respiration deficient hamster lung fibroblasts

The transport of [3H]2-deoxy-D-glucose (2DG) and [3H]3-O-methyl-D-glucose (3-OMG) was elevated in a respiration deficient (NADH coenzyme Q [Co Q] reductase deficient) Chinese hamster lung fibroblast cell line (G14). This sugar transport increase was related to an increased Vmax for 2DG transport, 26.9 +/- 4.2 nmoles 2DG/mg protein/30 sec in the G14 cell line vs 9.5 +/- 0.6 nmoles 2DG/mg protein/30 sec in the parental V79 cell line. No differences were observed in their respective Km values for 2DG transport (3.9 +/- .6 vs. 3.0 +/- .13 mM). Factors which increase sugar transport (e.g., glucose deprivation, serum or insulin exposure) or decrease sugar transport (e.g., serum deprivation) in the parental V79 cell line had little effect on sugar transport in the G14 respiration deficient cell lines. Amino acid transport, specific 125I-insulin binding to cells, and insulin-stimulated DNA synthesis, however, were similar in both cell lines. Exposure of both cell lines to varying concentrations of cycloheximide (0.1-50 micrograms/ml) for 4 h resulted in differential effects on 2DG transport. In the parental cell line (V79) low cycloheximide concentrations resulted in decreased 2DG transport, while higher concentrations (greater than or equal to 1 microgram/ml) resulted in elevated 2DG transport. In the G14 cell line, 2DG transport decreased at all concentrations of cycloheximide (up to 50 micrograms/ml). The data indicate that the G14 mutant has been significantly and specifically affected in the expression of sugar transport activity and in the regulatory controls affecting sugar transport activity.     

Alterations of purine salvage pathways during differentiation of rat heart myoblasts towards myocytes

Enzyme activities of purine catabolism and salvage, the concentrations of high-energy phosphates and the reutilisation of purine bases and purine nucleosides were studied in rat heart myoblasts and myocytes. Rat heart myoblasts H9c2(2-1) were grown in Dulbecco's modified Eagle's minimum essential medium supplemented with 10% fetal calf serum. Reduction of fetal calf serum to 2% for 1 week resulted in a differentiation into myocytes with respect to their morphological features and their enzyme pattern. In differentiated myocytes, activity of 5'-nucleotidase was increased more than 2-fold, and AMP deaminase and creatine kinase activities were more than 10-fold elevated. The concentration of creatine phosphate in differentiated myocytes was doubled compared to that in myoblasts. The uptake into myoblasts and myocytes and the incorporation into adenine nucleotides was highest using adenosine, inosine and adenine uptake rates were intermediate, and hypoxanthine was utilised least. Differentiation of myoblasts into myocytes resulted in a slightly lower overall uptake of adenosine and adenine, whereas about 40% more inosine and hypoxanthine were utilised by myocytes. Increasing the phosphate concentration in the incubation medium up to 50 mmol/l resulted in a stimulation of uptake of all purine compounds tested. This stimulation was more pronounced in myoblasts.     

Evidence against necessary phosphorylation during hexose transport in Aspergillus nidulans

The transport of 2-deoxy-d-glucose, a nonmetabolizable glucose analogue, into Aspergillus nidulans against a concentration gradient does not appear to require phosphorylation, despite the high levels of sugar phosphates accumulated rapidly within the cell. Two other deoxy analogues of d-glucose, 6-deoxy-d-glucose and 1,5-anhydro-d-glucitol (1-deoxy-d-glucose), although they lack the C-6 and the C-1 hydroxyl groups, respectively, and thus cannot be phosphorylated in those positions, still competitively inhibit the entry of 2-deoxy-d-glucose. Moreover, 6-deoxy-d-glucose can be concentrated against a gradient within the cell without the accumulation of 6-deoxy-d-glucose-phosphate. d-Galactose shows an intracellular ratio of free to phosphorylated sugar similar to that found for 2-deoxy-d-glucose in cells that have galactokinase, but no sugar phosphates are found in a galactokinaseless mutant strain. These data suggest that intracellular kinases are responsible for the sugar phosphate pool; and indeed, a kinase capable of phosphorylating 2-deoxy-d-glucose has been demonstrated. Finally, experiments on the kinetics of labeling of intracellular free sugar and sugar phosphate pools with (14)C-2-deoxy-d-glucose show that radioactivity appears first in the free sugar pool and after a delay enters the sugar phosphate pool.     

Filament-directed intercellular contacts during differentiation of cultured chick myoblasts

Detergent-extracted, critical point dried chicken myoblasts at early stages of development in tissue culture were observed by electron microscopy. It was found that the organization of filaments within these cells changes significantly during development. A particular specialization of the cellular filament framework is the formation of microprocesses; long extensions of the cellular filament system. These microprocesses appear to be involved in cell-to-cell contact. The filaments of these processes appear to integrate with the filament system of a contacted cell, and possibly transmit tension from one cell to another. The role of these structures in effecting muscle differentiation by forming cytoplasmic connections and the implications for muscle gene expression are discussed.     

Regulation of polyamine synthesis in physarum polycephalum during growth and differentiation

The levels and synthesis of polyamines were investigated in Physarum polycephalum to obtain information about their regulation during growth and differentiation in a lower eukaryote. Putrescine pools rapidly increased 4–5 fold during the change from dormant spherules to growing plasmodia. The activity of ornithine decarboxylase (EC 4.1.1.17), which converts ornithine to putrescine, reflected this rapid change in the level of putrescine. Spermidine levels were closely correlated with protein concentrations during differentiation due to variations in the activity of S-adenosyl-l-methionine decarboxylase which is involved in the conversion of putrescine to spermidine This enzyme was not stimulated by putrescine, unlike the similar enzyme in other eukaryotes, thereby permitting independent regulation of putrescine and spermidine levels. The high levels of both putrescine and spermidine suggest separate functions for these polyamines in Physarum.The half-lives of ornithine decarboxylase and S-adenosyl-l-methionine decarboxylase were 14 and 21.5 min, respectively. These short half-lives keep the polyamine metabolism under a very tight control as illustrated by the rapid fluctuations in enzyme activity during differentiation and the synchronous mitotic cycle. The step patterns of these unstable enzymes during the mitotic cycle suggest that these enzyme levels are limited by gene dosage.     

Embryonic and fetal rat myoblasts express different phenotypes following differentiation in vitro

Myosin heavy chain (MHC) is encoded by a multigene family containing members which are expressed in developmental and fiber type-specific patterns. In developing rats, primary (1°) and secondary (2°) myotjbes can be disfinguished by differences in MHC expression: 1° myotubes coexpress embryonic and slow MHC, while 2° myotubes initially express only embryonic MHC. We have used monoclonal antibodies which recognize the embryonic, slow, neonatal, and adult fast IIB/IIX MHCs to examine MHC accumulation in myoblasts obtained from hindlimbs of embryonic day (ED) 14 and ED 20 Sprague-Dawley rats during differentiation in vitro. Embryonic myoblasts (ED 14), which develop into 1° myotubes in vivo, differentiate as myocytes or small myotubes (i.e., 1–4 nuclei) which express both embryonic and slow MHC. They do not accumulate detectable levels of neonatal or adult fast IIB/IIX MHC. Fetal myoblasts, which develop into secondary myotubes in vivo, fuse to form large myotubes (i.e., 10–50 nuclei) and express predominantly embryonic MHC at 3 days in culture. These myotubes accumulate neonatal and adult fast IIB/IIX isoforms of MHC and eventually contract spontaneously. In contrast to embryonic myotubes, they do not accumulate slow MHC. Our results demonstrate that embryonic and fetal rat myoblasts express different phenotypes in vitro and suggest that they represent distinct myoblast lineages similar to those previously described in chickens and mice. These two lineages may be responsible for the generation of distinct populations of 1° and 2° myotubes in vivo. © 1993Wiley-Liss, Inc.     

The inhibition of hexose transport by permeant and impermeant sulfhydryl agents in rat adipocytes

The importance of exofacial sulfhydryl groups for hexose transport and its regulation was studied by comparing the effects of plasma membrane-permeant maleimide (N-ethylmaleimide) to an impermeant maleimide (glutathione-maleimide I) on 3-O-methylglucose transport into isolated rat adipocytes. The impermeant nature of glutathione-maleimide was confirmed by the finding that after a 15-min incubation, concentrations as high as 10 mM had no effect on intracellular glutathione content, while 1.7 mM N-ethylmaleimide decreased intracellular glutathione by 61%. Although N-ethylmaleimide appeared to be a more potent inhibitor of transport below 5 mM and at incubation times of less than 5 min, neither agent at concentrations which did not cause significant cell breakage inhibited basal transport rates more than 60-70%. The inhibition of transport by both agents was unaffected by extensive washing, suggesting a possible covalent interaction with the carrier. Preincubation with p-chloromercuribenzenesulfonic acid protected against the transport inhibition induced by both agents. However, only the transport inhibition induced by glutathione-maleimide was prevented by preincubation with D-glucose (50 mM) and maltose (50 mM). Transport in cells pretreated with insulin was inhibited by both agents to a similar extent as basal transport. However, treatment of cells with the maleimides before insulin caused a greater degree of inhibition. Thus, the insulin-induced increase in transport was inhibited half-maximally by 1 mM glutathione-maleimide. These results show that exofacial sulfhydryl groups, perhaps on the hexose-binding site of the carrier, are important for both the function and regulation of hexose transport.     

Changes in ganglioside metabolism during in vitro differentiation of quail embryo myoblasts

The metabolism of gangliosides was studied during the in vitro differentiation of both normal quail myoblasts and myoblasts which have been transformed by a temperature-sensitive mutant of Rous sarcoma virus (RSV). These transformed cells can be maintained undifferentiated if incubated at 35 degrees C, but they will differentiate when shifted to 41 degrees C. (D. Montarras and M. Y. Fiszman (1983) J. Biol. Chem. 258, 3882-3888). The analysis of [14C]Glucosamine-labeled gangliosides by two-dimensional thin-layer chromatography reveals variations in the metabolism of the gangliosides during the process of differentiation. During the formation of myotubes, it was observed that the accumulation of GD1a is reduced, while the accumulation of GD3 is increased. Therefore, this results in the variation of the ratio GD3/GD1a which increases from 1.8 to 25 in the case of clones of transformed myoblasts, and from 0.5 to 1.7 in the case of uninfected myoblasts. These variations which have been observed seem to be specific of the myogenic differentiation since they cannot be reproduced when differentiation is inhibited by BUdR treatment or when fibroblasts reach confluency and are blocked in the G1 phase of cell cycle. Furthermore, the transformed myoblasts in vitro are shown to be a good model system since their gangliosides composition is very similar to that of muscle cells in vivo.     

Regulation of hexose transport in L8 myocytes by glucose: possible sites of interaction

Previous work demonstrated that glucose controls its own transport rate in rat skeletal muscle: exposure to high glucose levels down-regulates muscle hexose transport, while glucose withdrawal results in elevated transport rates (J. Biol. Chem. 261:16827-16833, 1986). The present study investigates the mechanism of this autoregulatory system. Preincubation of L8 myocytes at 16 mM glucose reduced subsequent 2-deoxy-D-glucose (dGlc) uptake by 40% within 3 h. Cycloheximide (1 microM) mimicked the action of glucose; the effects of glucose and cycloheximide were not additive. At 50 microM, cycloheximide prevented the modulations of glucose transport induced by exposure of muscle cells to high or low glucose concentrations. Inhibition of glycosylation with tunicamycin A1 reduced the basal dGlc uptake, but did not prevent its up-regulation following glucose withdrawal. Inhibition of RNA synthesis by actinomycin D prevented the down-regulatory effect of glucose. These results indicate that continuous protein synthesis and protein glycosylation are required for the maintenance of the steady-state dGlc uptake. We suggest that glucose exerts its autoregulatory effect on hexose transport by modifying the incorporation of active glucose transporters into the plasma membrane rather than changing their rate of degradation. It is hypothesized that this effect is mediated by a non-glycosylated protein involved in the translocation or activation of glucose transporters.     

RIP2 regulates growth and differentiation of normal myoblasts and of rhabdomyosarcoma cells

RIP2 is an important regulator of myoblast proliferation and differentiation. We have previously demonstrated that in the myoblast cell line C2C12 and in primary myoblasts, downregulation of rip2 gene expression is a prerequisite for differentiation. To further study the role of rip genes in myogenesis, we compared expression patterns of rip1–4 in two myoblast cell lines, C2C12 and C2F3, after the induction of differentiation. These two cell lines are derived from the same clonal origin, but differ with respect to their differentiation behaviour: specifically, the differentiation process is slower and more incomplete in C2F3 cells. When analyzing cells up to 4 days after the induction of differentiation, we found no downregulation of rip2 gene expression in C2F3 cells, which might be linked to the low differentiation potential of these cells. In addition, in contrast to C2C12 cells, the rip3 gene was not expressed in C2F3 cells. To further study the role of rip genes in the regulation of myoblast growth and differentiation, we analyzed expression patterns of rip1–4 in rhabdomyosarcoma cell lines. We found that in these cells, rip2 expression was not downregulated after the induction of differentiation. Furthermore, in contrast to normal myoblasts, they did not express the rip3 and rip4 genes. Thus, we focused on the functional role of RIP2 in rhabdomyosarcoma cells. Inhibition of rip2 gene expression in C2C12 and in rhabdomyosarcoma cells using specific siRNAs led to decreased proliferation and promoted the differentiation process of these cells. These data indicate that differential expression of rip genes can be associated with abnormal growth and differentiation behaviour of skeletal myoblasts.     

Mechanism of murine leukemia virus-induced fusion in rat myoblasts defective in differentiation.

fu-1 cells, a line of rat myoblasts defective in differentiation, can be fused into multinucleate syncytia by Moloney murine leukemia virus. The effects of treating the virus with specific antibody, UV irradiation, and elevated temperature and the requirements for cellular RNA and protein synthesis have been studied as they relate to this virus-induced fusion. The results indicate that intact, but not necessarily infectious, virions are required to promote fusion of fu-1 cells. Neither actinomycin D nor cycloheximide altered the formation of syncytia; thus, neither viral nor cellular RNA or protein synthesis is required for fusion. fu-1 cells infected with the ts3 temperature-sensitive mutant of Moloney murine leukemia virus accumlate large amounts of budding virus on their cell membrane; however, this membrane-associated virus failed to induce syncytia. Upon release of the virus at the permissive temperature, fusion did occur. We conclude that contact or attachment of the immature virus to the cell membrane is not sufficient to promote murine leukemia virus-induced cell fusion; complete virions are required. From these data, we propose that adsorption and penetration of the virus may induce a change in the cell membrane that subsequently promotes the fusion of susceptible cells.