Biomedical subjects
Publications and source records attributed to H M Kalckar.
The all-cis hexose D-allose, fed to hamster fibroblast cultures over 14-20 hr, brings about a striking down-regulation of hexose transport. This down-regulation by allose is inhibited by 2,4-dinitrophenol. By using D-[14C]allose and thin-layer chromatography, two types of products accumulated that have been identified as allose and allose phosphate. We suspect that allose phosphate might be the down-regulator.
In 1930 adenosine triphosphate appeared in the literature from W. A. Engelhardt's work on avian erythrocytes. This was an early example of oxidative phosphorylation in intact cells, and it required methylene blue and oxygen. Both Belitser and I realized that the use of Warburg manometers for aeration was critical in order to generate oxidative phosphorylation of glucose in tissue preparations. Test tube techniques did not work. In 1956 we were able to describe a human type of diabetes called "galactose diabetes," in which consumption of human or cows' milk provokes mental retardation. Replacement of human or cows' milk products with "vegetable milk" formula in early infancy can prevent retardation. We determined that the disease results from a defect of galactose-one-phosphate uridylyl-transferase, a hereditary enzyme. This type of enzyme defect, if discovered and treated in early infancy, is a benign molecular disease. Regulation of transport systems in mammalian cell cultures are frequently complex energized systems. Perhaps my greatest surprise in this regard was the mere fact that an all-cis "odd" hexose-D-allose turned out to be a highly intense down-regulator of the hexose transport system. Additions of inhibitors of oxidative phosphorylation (such as oligomycin or di-nitrophenol) arrested the allose-mediated down-regulation. We have reason to suspect that the strong down-regulator is a phosphorylated form of D-allose. Thus ends my story about oxidative energized biological phosphorylation systems.
The hexose transport in a hamster fibroblast mutant (DS7), unable to use glucose for generation of energy, is nevertheless subject to a marked down-regulation ("curb") after prolonged incubation of monolayer cultures with glucose; fructose is unable to exert any curb. D-Allose, an all-cis hexose, mediates a vigorous curb of the transport system. Moreover, prolonged coincubation of glucose or allose with tunicamycin (TM) brings about an additional effect that is not an inhibition of the transport system, which we shall call the "concerted" transport curb. This type of concerted transport curb requires L-glutamine in the maintenance medium; moreover, addition of cycloheximide prevents the development of this TM effect. Apparently, cellular protein synthesis or protein turnover or both are required for the development of the TM-concerted transport curb. The concerted transport curb can be reversed in sugar-free or in fructose-containing medium, even upon readdition of TM. In contrast, the sole readdition of glucose or D-allose renders the concerted curb irreversible. This raises the question of whether the cells under the condition of the concerted curb somehow have internalized the TM into the membrane.
The hexose transport system of a fibroblast mutant, DS7, unable to convert glucose 6-phosphate to fructose 6-phosphate ("the phosphoglucose isomerase mutant"), is subject to a specific down-regulation ("curb") evoked by only glucose or D-allose. Neither fructose nor mannose has a curbing effect on this mutant. Further addition of tunicamycin intensified the transport curb on the mutant mediated by glucose or allose. Mannose added to the parental cell line 023 seems able to mimic a glucose-mediated transport curb. In this line, but not the mutant, tunicamycin also intensifies a mannose-mediated curb. It seems that the tightening of the allose-mediated curb is a function of a specific type of transport regulation and perhaps too of interference with glycosylation of the hexose transporter. Furthermore, this type of curb can be strikingly reversed by shifting the cultures to medium containing fructose.
Endogenous oligonucleotides were found in trichloroacetic acid extracts of hamster lung fibroblasts and Tetrahymena cells. Peaks of radioactivity that eluted with retention times similar to oligonucleotide markers (5- to 50-mer) were found by HPLC in cells labeled briefly with 32Pi. Only minute amounts of UV-absorbing material were detected, consistent with a rapid turnover of phosphate groups. The 32P-labeled material also migrated as oligonucleotides on 20% polyacrylamide gels; it was not hydrolyzed by alkaline phosphatase but was digested by snake venom phosphodiesterase, S1 nuclease, and pancreatic RNase and was phosphorylated by T4 polynucleotide kinase. The 32P-labeled material isolated by HPLC was alkali labile and the hydrolyzate ran as nucleotides on paper chromatography. It is concluded that the oligonucleotides are mainly oligoribonucleotides, but it is possible that oligodeoxynucleotides are also present.
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The interrelationships of hexose feeding and insulin action were studied in the Chinese hamster fibroblast cell lines 023 and DS-7. The latter, derived from 023 and deficient in phosphoglucose isomerase, has been used to map the metabolic requirements for aldohexose-mediated down-regulation or "curbing" of hexose transport. We have characterized insulin binding and the response to insulin in both cell lines to determine if the insulin-mediated stimulation of transport is similarly dependent on hexose metabolism. DS-7 cells exhibited 5-6 times as many high-affinity insulin binding sites as the parental strain. Apart from this difference, 023 and DS-7 cells showed comparable insulin binding characteristics, which are similar to those observed in other cell types. Insulin at a concentration of 1 microgram/ml (167 nM) was found to stimulate 3-O-methylglucose uptake by approximately equal to 50% in glucose-fed cells of both lines. In neither line did glucose starving significantly alter insulin binding or the insulin-induced stimulation of transport. Feeding with mannose or fructose was found to increase both parameters in 023 cells but had no effect on DS-7 cells. The increase in hexose uptake with the administration of insulin or with glucose starving was shown to be due to an increase in Vmax. Our studies suggest that insulin binding and effect are not regulated by hexose metabolism in the same manner as in the curbing process and insulin induces the recruitment of a transporter pool that is insensitive to hexose curbing.
By studying the energy-requiring control of the hexose transport system (the transport "curb") in a lung fibroblast mutant called the phosphoglucose isomerase mutant (because it is devoid of the enzyme phosphoglucose isomerase) the following features were noted. The aldohexose D-allose, if added over 20 hr to a culture of the mutant, promotes the development of an intense curb of the hexose transport system, greatly surpassing that brought about by incubation with glucose. The allose-mediated curb can be circumvented by various metabolic inhibitors as well as by the presence of other aldohexoses such as mannose.
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The generation of ATP in a hamster fibroblast mutant devoid of the enzyme phosphoglucose isomerase (PGI) has been studied and compared with that in the parental line, which is PGI+. Both cell lines could be maintained for 24 hr in hexose media devoid of L-glutamine. Under these conditions in mannose medium, both the parental line and the PGI mutant maintained high intracellular ATP levels. With glucose under the same conditions, the parental line was able to keep the ATP level high. In contrast, the mutant line lost most of its ATP pool after incubation with glucose; the ATP/ADP ratio fell about 80% after incubation in glucose medium. Addition of pyruvate, with or without glucose, preserved the ATP pool at high levels even in the mutant, as did the presence of L-glutamine. When the PGI mutant was maintained for 3-4 days in growth medium, containing 4 mM L-glutamine and 10% dialyzed calf serum, in which glucose was replaced by mannose, the UDP-glucose pool dwindled and mediated control of the hexose uptake system did not ensue, in contrast to results of the same exposure to glucose-containing medium.
A protracted type of down-regulation of the hexose transport system in cultured fibroblasts that depends on one main factor in their nutritional state, the presence or absence of metabolizable D- aldohexoses in the culture fluid, is discussed. Fructose feeding is unable to elicit a down-regulation, whereas mannose and D-glucosamine, regulation of the transport system. This down-regulation or transport curb depends on oxidative energy metabolism, because inhibitors of this type of metabolism bring about a striking release of the transport curb. Studies with a fibroblast mutant that lacks the enzyme glucosephosphate isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9, abbreviated phosphoglucose isomerase) (pgi-) have indicated that two types of metabolism are needed: 1) oxidative energy metabolism, which in the pgi- mutant can still be generated effectively from L-glutamine or, in its absence, from mannose or D-glucosamine; 2) glucose-6-phosphate metabolism, either its catabolism through the pentose shunt or through the anabolic pathway to UDP glucose and UDP galactose. The schism in carbohydrate metabolism in the pgi- fibroblasts is clearly reflected through the development of the metabolically mediated curb of the hexose transport or uptake system.
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, (ii) oxidative energy metabolism, and (iii) 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.
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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 PGI 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 energy-yielding 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.
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Regulation of hexose transport in NIL hamster fibroblasts has been studied in confluent cultures preconditioned for 24 hr in media deprived of glutamine or of serum or of both. Cultures maintained in media containing dialyzed fetal calf serum and 4 mM glutamine accumulated up to 72 nmol of glutamine per mg of cell protein; in contrast, cells deprived of glutamine contained less than 1 nmol/mg of cell protein. Glutamine elicited a general enhancement of hexose transport compared with transport in glutamine-deprived cultures. This enhancement was particularly pronounced in glucose-fed cultures which in the absence of glutamine showed conspicuously low transport activity. When maintained in glucose media, cultures deprived of serum also showed a marked loss of hexose transport which, in this case, was not compensated for by addition of glutamine. However, regardless of the presence or absence of glutamine, these cultures were able to develop the usual transport enhancement response to glucose starvation. Moreover, 2,4-dinitrophenol was also able to elicit a pronounced enhancement of hexose transport in the glucose-fed cultures; this effect surpassed even the transport derepression observed in the glucose-starved cultures. In polyoma-transformed cultures maintained in serum-free media, hexose transport remained relatively high, even in the presence of glucose. However, addition of glutamine brought about an enhancement in both the presence and absence of serum. The various phenomena are discussed in regard to protein turnover in general and more specifically the turnover of hexose transport carriers.