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The regulation of hexose transport was studied in a human diploid fibroblast respiration-deficient cell strain (WG750). Transport of 2-deoxy-D-glucose (2-DG) was greater than sixfold higher compared with an in vivo age-matched normal cell strain (MCH55). In addition, 3-O-methylglucose transport and 14CO2 production were elevated in the mutant cell strain. Kinetic analysis revealed that the increased sugar transport in mutant cells was due to an average 5.7-fold increase in the 2-DG maximal transport rate, with no observed differences in the transport Michaelis constant for both normal and mutant cells. Also, the inhibitor constants for D-glucose inhibition of 2-DG transport were nearly identical for both cell types. Glucose deprivation led to a similar time-dependent increase in hexose transport in both cell strains. Serum refeeding of glucose-fed serum-deprived cultures led to a progressive increase in 2-DG transport in normal cells, whereas mutant cells displayed a time-delayed increase in 2-DG transport. Exposure to 67 and 670 nM insulin stimulated 2-DG transport on average 1.99 +/- 0.25- and 2.33 +/- 0.26-fold, respectively, over basal transport in the normal cells, whereas the mutant cells were significantly less sensitive to the stimulatory effects of the hormone. Insulin binding and amino acid transport (i.e., alpha-aminoisobutyric acid uptake) in the normal and mutant cells were not different. Data obtained using Western blot analysis showed that WG750 (mutant) cells expressed an increase (approximately 4-fold) in total cellular HepG2 (erythroid-brain) transporter protein compared with normal cells, thus reflecting the changes seen in hexose transport.(ABSTRACT TRUNCATED AT 250 WORDS)

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The base at the first anticodon ("wobble") position of certain eukaryotic tRNA species is either guanine or the hypermodified base queuine. These tRNA species are synthesized with guanine in the wobble position (tRNAG); this guanine can then be replaced with queuine by the action of the enzyme tRNA-guanine ribosyltransferase. In the present report, we show that tRNAG levels increased in response to the induction of erythroid differentiation of murine erythroleukemia (MEL) cells. We also found that tRNA-guanine ribosyltransferase was significantly inhibited by tetrahydrobiopterin. MEL cells showed a transient threefold increase in tetrahydrobiopterin levels 6 to 12 h after exposure of the cells to inducers such as DMSO or tetramethylurea. The increase in tetrahydrobiopterin preceded the increase in tRNAG which in turn preceded the appearance of phenotypic changes characteristic of differentiation. By contrast, a mutant MEL cell line unable to differentiate in response to inducers showed no change in the level of tetrahydrobiopterin or of tRNAG upon exposure to DMSO. N-acetylserotonin, a well-characterized inhibitor of tetrahydrobiopterin synthesis, prevented the DMSO-mediated increase in tetrahydrobiopterin in normal MEL cells. N-acetylserotonin also inhibited the increase in tRNAG levels and the appearance of phenotypic differentiation in these cells.

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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.

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