Life Sciences Vol . 20, pp .1599-1606, 1977 . Printed in the U .S .A .
FACTORS CONTROLLING THE METABOLIC RESPONSE TO GLUCOSE IN ISOLATED RAT LUNG CELLS Julio Parez -Diaz, Angeles Martin Requero Matilde S . Ayuso-Parrilla and Roberto Parrilla Department of Metabolism, Instituto G . Maran6n, C .S .I .C ., Velazquez 144, Madrid-6, SPAIN (Received in final form April 6, 1977)
SUMMARY Glucose decreases the oxygen utilization by isolated rat lung cells . Its effect displays saturation type kinetics with a "Ki" of 2 .2 mM . The similarity of this value with the reported "Km" of 2 .4 mM described for glucose uptake by these cells, suggests that both processes may be intimately related and both of them are under the control of the same rate limiting step . Several arguments point to glucose transport into these cells as the most important rate limiting step for its utilization : 1) Phloridzin prevented glucose inhibition of oxygen uptake while mannoheptulose did not ; 2) The activity of hexokinase which is the least active glycolytic enzyme in these cells far exceeded the observed rates of glucose utilization and a decrease of 45 per cent in its activity in starved animals did not affect the rate of glucose uptake ; 3) The "Km" of hexokinase for glucose is two orders of magnitude below the observed "Rm" for glucose uptake and the "Ki" for glucose inhibition of respiration . Glucose has been reported to inhibit oxygen utilization by isolated rat lung cells (1,2) . This effect of glucose depressing oxidative metabolism was originally described by Crabtree (3) . It~was thought to be restricted to tumoral cells (3), however, similar effects of glucose inhibiting oxygen utilization have been also described occasionally in normal tissues (4-6) . The mechanism of glucose action inhibiting oxygen utilization has not been elucidated yet although it is thought that glucose acts altering somehow the availability of ADP to the phosphorylation sites in the respiratory chain . In this work advantage has been taken of this peculiar response of the isolated rat lung cells oxidative metabolism to glucose to further explore some of their metabolic characteristics . MATERIAL AND METHODS Animals . Male Wistar rats, 200-250 g in body weight were used in all experiments . These rats were maintained in our institution under controlled conditions of light, temperature and food intake for at least two weeks prior to their use for experimental purposes . 1599
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Isolation of lung - cells . Rat lung cells were isolated by collagenase digestion of minced lung tissue, Anesthesized rats were infused with Krebs Ringer bicarbonate buffer (7) through the portal vein and the aorta and vena cava severed close to their bifurcation to allow a rapid evacuation of the blood . This procedure yields lungs virtually devoid of any contaminating blood cells . After mincing finely the tissue with scissors it was incubated for 20 min at 36 .5°C in Krebs Ringer bicarbonate buffer containing 1 mg/ml of collagenase and 1 mq_/ml of defatted (8) bovine serum albumin (9) . At the end of the incubation the digested tissue was filtered through a double layer cheese cloth and the collagenase was removed by repeated washing of the cells by centrifugation . For their experimental use the cells were resuspended in fresh oxygenated Krebs Ringer bicarbonate buffer containing 1 mg/ml of albumin . The number of cells in each preparation was determined by counting them in a hemocytometer chamber o .f 6Thoma . Routinely lungs from two rats yielded 8 ml of 2-4 x 10 cells . The viability of the cells preparation was assesed by the vital colorants exclusion test . 95$ of the cells excluded nigrosin and tripan blue indicating that their plasma membrane was intact . Analytical- procedures . Oxygen utilization was determined polarographycally using a Clark type oxygen electrode (Yellow Spring, Ohio, U .S .A .), by incubating the cells in an airtight microchamber . The isolated rat lung cells were able of maintaining constant rates of oxygen utilization for several hours . Hexokinase activity was assayed fluorimetrically by measuring the rate of reduction of NADP . 1 :10 homogenates were prepared in 50 mM phosphate buffer, pH 7 .2 . The composition of the assay buffer was : 20 mM triethanolamine hydrochloride, 50 mM potassium phosphate, 60 mM potassium acetate, 5 mM magnesium chloride, pH 7 .3 . The reaction mixture (1 .5-2 ml) contained 0 .02 mM NADP, 5 mM ATP, 10 mM glucose, 1 ug/ml of glucose-6-phosphate dehydrogenase (Boehringer Mannheim, C"ermany) and 1-10 ul of homogenate . Glucose and lactate were measured enzymically by methods already described (10) . RESULTS Increasing concentrations of glucose produced a progressive inhibition of oxygen utilization by isolated rat lung cells . This phenomenon displayed saturation type kinetics . Fig . 1 shows a reciprocal plot of the data obtained in a typical experiment indicating a "Ki" of 2 .2 mM . The "Km" for the process of glucose uptake was 2 .4 mM (il) . This similarity seems to suggest that both events, glucose utilization and glucose induced inhibition of oxygen uptake by isolated lung cells, share the same common step . As it is shown in Fig . 2 pretreatment of the cells with
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Glucose Utilization by Rat Ltmg Cella
"Ki " " 2.2mM
FIG . 1
Double reciprocal plot of the glucose induced inhibition of oxygen utilization by isolated rat lung cells . Cells were suspended in Rrebs Ringer bicarbonate buffer containing 1 mg/ml of defatted bovine serum albumin . 1 ml of cells suspensions were incubated in a thermostated airtight microchamber and rates of oxygen uptake measured with a Clark type platinum electrode .
FIG . 2
â â W
e ßLUCOSE 5mM
Effect of phloridzin pretreatment on the glucose induced inhibition of oxygen utilization by isolated rat lung cells . The experimental conditions were similar to those described in Fig .l . ( .- .- .-) 1 mM phloridzin added at zero time ; (-----) no phloridzin added .
Glucose Utilisation by Rat Lung Cells
Vol. 20, No . 9, 1977
â ~ v z t W W Y X O
FIG, 3 Effect of pretreatment with mannoheptulose on the glucose induced inhibition of oxygen utilization by isolated lung cells . The experimental conditions were similar to those in FIG . 1 . phloridzin completely abolished the effect of glucose, indicating that glucose has to be transported into the cells to exert its characteristic effect on respiration . On the other hand, mannoheptulose, inhibitor of sugar phosphorylation, does not affect the glucose inhibition of oxygen uptake (Fig .3) . This finding may be interpreted as the result of permeability restrictions to the passage of the inhibitor into the cell since mannoheptulose, in agreement with previous reports in other tissues (12,13), was found to inhibit glucose phosphorylation by 858 by a lung homogenate . These observations indicate that glucose does not diffuses freely into the lung cells but it has to be transported to exert its characteristic effects In order to explore whether it is the transport system or the phosphorylation of glucose what is rate limiting for its utiliza-
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Glucose Utilization by Rat Ltmg Celle
tion, rates of glucose uptaké by cells isolated from starved animals were studied . As it can be appreciated in Table 1 the rates of glucose utilization were not affected by starvation in spite of a significant decrease in the hexokinase activity which is the least active glycolytic enzyme in these cells . The "Km" of hexokinase for glucose was found to be 10 uM . These observations seem to support that the sugar phosphorylation is not the limiting step for glucose utilization .
TABLE 1 Effect of the nutritional state on the hexokinase activity, rates of glucose utilization and lactate production by isolated rat lung cells . Lung cells isolated from :
Hexokinase activity (umole /gxmin)
(nm les/10 6 cells x hr)
0 .537 + 0 .07
210 + 8
260 + 5
0 .298 + 0 .05
243 + 2
286 + 6
1 ml aliquots of the isolated lung cells suspension were placed in 25 ml Erlenmeyer flasks sealed with rubber caps . After equilibration with 95$ O2 : 5$ C0 2 gas mixture, the flasks were incubated in a rotary sh$ker water bath at 36 .5°C . The incubation was stopped by addition of perchloric acid up to a final concentration of 6$ . After neutralizing the samples with potassium carbonate, glucose and lactate were measured enzymically as described in Methods . Values are means of at least eight observations + error of the mean .
DISCUSSION Lung cells seem to share in common with other cells displaying glucose inhibition of respiration a high glycolytic capacity (14) . As much as 60 percent of the glucose utilized can be accounted for as lactate (Table 1) . The importance of glucose as a metabolic fuel for lung cells has been previously emphasized (15) but glucose is not only important for lung tissue as a fuel, its presence enhances, by mechanisms as yet unknown, the synthesis of protein (16) and lipids (17) . Nevertheless, in spite of all these observations, very little is known about the control of glucose utilization by lung cells . In this work advantage has been taken of the peculiar response of these cells inhibiting oxygen consumption in the presence of glucose, to explore the relative
Glucose Utilization by Rat Luag Cells
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importance of its transport and/or phosphorylation in the control of its utilization . According to the differential responses to glucose of the oxygen uptake in the presence of mannoheptuloae and phloridzin (Figs, 2 and 3) it may be concluded that glucose has to be transported into the cells in order to produce its characteristic effect . In agreement with this, the isomer L-glucose was found to be unable to induce this inhibitory effect (results not shown) suggesting that a stereospecific transport system is implicated in the phenomenon, Some of the herein reported findings seem to indicate that the process of glucose transport across the lung cells plasma membrane may be an important rate limiting step for its utilization . The observation that the activity of hexokinase, the least active glycolytic enzyme, far exceeds the rate of glucose utilization in conjuction with lack of effect of a decrease in hexokinase activity during starvation on the glucose to lactate flux (Table 1), suggests that the phosphorylation of glucose is not a limiting step for its utilization by lung cells, If sugar phosphorylation was the limiting step it should be expected a "Km" for glucose utilization much lower than actually it was found to be, since the apparent "Km" of hexokinase for glucose is two orders of magnitude lower that the "Km" for glucose utilization, On the other hand it is shown that inhibition of glycolysis with iodoacetate does not prevent the effect of glucose on respiration (18,19) ; however there is strong evidence suggesting that at least glucose has to be phosphorylated in order to exert its effect on oxidative metabolism (20) . If this is the case the finding of a "Ki" of 2,2 mM for glucose inhibition of respiration (Fig .l) also points to a step prior to phosphorylation as the rate limiting factor for the glucose effect . ACRNOWLEDGEMENTS This work was supported by grants from Fundacidn Rodriguez Pascual, Essex (Espana) S .A, and Comisißn Asesora para et Desarrollo de la Investigaci6n, J,P .D . and A,M .R . are recipients of research fellowships from the Spanish Secretary of Education and Science . Authors wish to thank Mr . Tomas Fontela and Mr, Fernando Martin for devoted and skilfull technical assistance . REFERENCES 1 . M, S . AYUSO-PARRILLA, A . B . FISHER, R . PARRILLA and J, R . WILLIAMSON, Am, J, Physiol . 2_25, 1153-1160 (1973), 2, J, PEREZ-DIAZ, B, CARBALLO, M S . AYUSO-PARRILLA and R, PARRILLA, Biochimie (In press) . 3 . H . G, CRABTREE, Biochem . J . 23, 536-545 (1929), 4, W . A . BELITZER, Biochem . Z, ~$3, 339-342 (1936) . 5, O . ROSENTHAL, M, A . HOWLE an~G . WAGONER, Science 92, 382-383 (1940) , 6 . L, H . COHEN, Federation Proc, 16, 165 (1957) .
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