“Carrier activation” and glucose transport in Chinese hamster fibroblasts

Down-regulation ("curb") of hexose transport in Chinese hamster lung fibroblasts has been studied in a metabolic mutant highly defective in phosphoglucose isomerase (PGI; glucosephosphate isomerase; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9). In the parental strain (PGI+) glucose as well as glucosamine and mannose were able to elicit a curb of the hexose transport system. In the PGI mutant, only glucose was able to mediate a transport curb. The inability of glucosamine and mannose to promote a transport curb in the PGV strain must be ascribed to the fact that the 6-esters of these aldohexoses are converted by their own specific deaminase and isomerase to fructose 6-phosphate, which initiates the pyruvate-tricarboxylate energyyielding pathway but cannot be converted to glucose 6-phosphate in the mutant. The latter ester can be metabolized, but its metabolism in the mutant is confined to the pentose shunt. It is shown that inhibitors such as 2,4-dinitrophenol and malonate exert only slight inhibition of the pentose shunt yet release the glucose-mediated curb elicited by glucose and glucosamine in the parental PGI+ strain and also the glucose transport curb persisting in the PGI mutant.It has recently been observed in cultured hamster fibroblasts that glucose-mediated down-regulation of the hexose transport system (abbreviated "transport curb") was released by addition of 2,4-dinitrophenol (DNP), oligomycin, and malonate (1, 2). Because these inhibitors also interfere with oxidative phosphorylation and the tricarboxylate cycle, one is led to believe that oxidative energy metabolism may be crucial for the preservation of the transport curb (2).The role of lactic acid generation for the establishment ofthe transport curb was considered dubious, especially after it was disclosed that the amino sugar D-glucosamine exerts a marked transport curb of the hexose transport system, yet only traces of lactic acid are generated (1, 2).Fructose, a furanoid ketohexose, is recognized neither by the hexose transporter from erythrocytes (3) nor by that of hamster fibroblasts (see table 1 of ref. 2). Yet the catabolism offructose, via fructose 6-phosphate (Fru-6-P), is presumably much like that of D-glucosamine.The glucose-mediated transport curb and its release by glucose starvation has recently been studied on a tumor line, 023, from Chinese hamster lung fibroblasts (4). In contrast to other fibroblast lines, in which the presence of cycloheximide at the onset of glucose starvation interferes with the release of the transport curb (5, 6), no interference was observed in 023 (4).In 1980, Pouyssegur et al. (7) isolated and characterized a metabolic mutant called DS-7. It was shown (7) to be highly defective in the enzyme phosphoglucose isomerase (PGI; glucosephosphate isomerase; D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9).This strain and the parental strain 023 are tumorigenic lines, although DS-7 is nonglycolytic (8). For the sake of clarity, we shall call the DS-7 strain with the defect in phosphoglucose is...

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confidence: 99%

Down-regulation of the hexose transport system: metabolic basis studied with a fibroblast mutant lacking phosphoglucose isomerase.

Ullrey¹,

Franchi²,

Pouysségur³

et al. 1982

“…the Chinese hamster fibroblast line 0 23, the increase in transport is mainly due to a mechanism of "carrier activation" (16). DS (Table 1).…”

mentioning

confidence: 99%

Isolation of a Chinese hamster fibroblast mutant defective in hexose transport and aerobic glycolysis: its use to dissect the malignant phenotype.

Pouysségur¹,

Franchi²,

Salomon³

et al. 1980

A procedure is described for the selection of glucose uptake mutants based upon radiation suicide of Chinese hamster fibroblasts by 2-deoxy[3H]glucose. In one of these mutants, DS 7, the ability to transport either 2-deoxyglucose or 3-O-methylglucose was decreased to one-fifth to one-fourth. Besides this defect, DS 7 produces l/,4th the lactic acid produced by the parent when grown on 5 mM glucose. This block in aerobic glycolysis is due to a mutation that affects the expression of the phosphoglucose isomerase gene because no isomerase activity is detected in cell extracts of DS 7. This glycolytic block makes that cell line dependent exclusively on respiration for its energy requirement. Consequently, DS 7 survives well after removal of glucose but dies quickly in the presence of oligomycin. The parental line 0 23 (subclone of CCI 39) grows at low serum concentration, is anchorage-independent, and is tumorigenic in nude mice. The derived glycolytic mutant DS 7 has retained both the in vitro transformed phenotype (low serum dependence and loss of anchorage dependence) and the tumor-forming capability. The tumor cells derived from the injection of DS 7 cells have kept the original glycolytic defect. This finding suggests that the transformed properties (high hexose transport and aerobic glycolysis) that can be uncoupled from abnormal growth control are not necessary for the expression of the malignant phenotype in fibroblasts.So far, among the various properties that accompany viral or spontaneous transformation (for review, see refs. 1-4), it has been difficult to assign to any particular transformed property a primary role in the establishment and maintenance of the abnormal growth control. A set of surface and membrane changes-including low adhesion to substratum, rounded shape, increased agglutinability by plant lectins, altered cell locomotion, decreased cell alignment, and loss of microfilament bundles-has been dissociated from the mechanism that alters the control of division (5-9).Increases in hexose transport and in aerobic glycolysis are two other characteristics that one finds closely linked to transformation and increased proliferation in cells (10)(11)(12)(13)(14) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

“…The course of the deregulation was reminiscent of the starvation response of hamster fibroblast cultures (7)(8)(9). CHX was permitting some deregulation of the transport system (data not shown).…”

Section: Resultsmentioning

confidence: 92%

“…The release of the transport curb by glucose starvation of the PGI mutant seems not to be as consistently unaffected by the presence of cycloheximide (CHX) as described in the case of the parental line (7).…”

Section: )mentioning

confidence: 88%

Further clues concerning the vectors essential to regulation of hexose transport, as studied in fibroblast cultures from a metabolic mutant.

Kalckar¹,

Ullrey²

1984

A close study of the metabolic regulation of hexose transport in a hamster fibroblast mutant, highly defective in the enzyme phosphoglucose isomerase (PGI mutant), reveals the requirement for at least three vectors for transport regulation. The downward regulation of the hexose transport system, called the "transport curb," requires (i) a ligand for the transport system, (it) oxidative energy metabolism, and (iiW) some specific enzymes of the glucose-6-phosphate metabolism. Deprivation of glucose was shown to deprive the PGI mutant of UDP hexose, whereas the glucose-fed mutant contained high levels. The parental strain preserved the UDP hexose with or without glucose feeding. Cycloheximide added to the mutant showed two different types of effects. If added at the onset of glucose starvation, the up-regulation of the transport system was scarcely affected. If cycloheximide was added to the mutant at the onset of glucose refeeding, it prevented the development of the glucose-mediated transport curb. In the mutant, the glucose-mediated curb is not derived from energy metabolism but is solely dependent on certain enzymes of glucose-6-phosphate metabolism. The interference of this curb by cycloheximide requires evidently a reassessment, including that of the role of the UDP hexose pathway in regulation of the hexose transport system.From our previous studies on the mediated hexose transport down-regulation in cultured mammalian fibroblasts, we have been able to disclose a number of essential vectors for the onset of this type of regulation. This search has been greatly facilitated by maintaining the hamster fibroblast cultures for 24 hr in media (with or without serum) devoid of L-glutamine and pyruvate and varying the hexoses (1, 2). The mediated down-regulation of the hexose transport system shall be named the transport "curb." We have found the following requirements for mediating a transport curb of the hexose transport system.(i) One ligand of the hexose transport system, be it glucose, galactose, mannose, or some of the nonmetabolizable analogues of glucose, must be present to initiate a transport curb. Fructose is not a ligand and is unable to initiate a curb (3).(ii) Metabolites able to generate oxidative energy metabolism are needed. Inhibitors of oxidative energy metabolism (2,4-dinitrophenol, malonate) release the transport curb (3-5).(iii) Glucose and glucose-6-phosphate metabolism, different from general energy metabolism [as disclosed from observations on a mutant DS7-defective phosphoglucose isomerase, PGI mutant (6)], generate vectors essential for the transport curb (2, 5).The release of the transport curb by glucose starvation of the PGI mutant seems not to be as consistently unaffected by the presence of cycloheximide (CHX) as described in the case of the parental line (7).The restoration of the transport curb by refeeding glucose to starved cultures of the PGI mutant turns out to be abolished in the presence of CHX, reminiscent of the response observed in NIL fibroblasts (8,9).In this arti...