278
Biochimica et Biophysica Acta, 496 (1977) 278--291
© Elsevier/North-Holland Biomedical Press
BBA 28155 METABOLIC EFFECTS OF 2-DEOXY-D-GLUCOSE IN ISOLATED FAT CELLS
VISVANATHAN CHANDRAMOULI and JAMES R. CARTER, Jr. Department of Medicine, Case Western Reserve University, and Endocrine Division, University Hospitals, Cleveland, Ohio 44106 (U.S.A.)
(Received July 30th, 1976)
Summary The uptake and phosphorylation of 2-deoxy-D-glucose by isolated adipocytes of the rat was determined by a method of rapid flotation through oil coupled with separation of sugar from sugar phosphate by chromatography on Dowex1-formate. Uptake of the sugar is rapid and linear over 5 min, with a gradual decline thereafter; by 1 h, no further uptake is observed. Initially only 2-deoxyglucose phosphate is observed within the cells; by 1 h, however, free 2-deoxyglucose accumulates to levels approximating those in the medium. Phosphorylation ceases when intracellular levels of 2-deoxyglucose phosphate are about 50 mM regardless of the medium concentration of 2-deoxyglucose; this does not represent feedback inhibition of hexokinase, since the enzyme in fat cell homogenates is n o t inhibited by 50 mM 2-deoxyglucose 6-phosphate. Accumulation of deoxyglucose 6-phosphate is associated with a marked decline in intracellular ATP levels. Fat cell respiration is also depressed b y approximately 50 per cent after a I h preincubation with 10 or 20 mM 2-deoxyglucose. Intracellular ATP levels and 02 uptake are only partially corrected b y the addition of pyruvate to the incubation medium. Since no glucose was present in the medium, and intracellular concentrations of glycogen are known to be small in adipose tissue, it is proposed that accumulation of 2-deoxyglucose 6-phosphate within fat cells has a direct inhibitory effect on cell respiration unrelated to inhibition of glycolysis. No increase in intracellular free fatty acids was observed to explain this, and under the conditions of the incubations it is unlikely that Pi availability was rate limiting. The exact locus of inhibition is unknown. 2-Deoxy-D-glucose is a glucose analogue which is a potent inhibitor of glycolysis in a variety of tissues [1--8]. This sugar is taken up in most cells by a saturable process which appears to be the same as that for glucose, and it is therefore a competitive inhibitor of glucose transport [9--12]. 2-Deoxyglucose
279 is phosphorylated by hexokinase [1,3,13--15] and in most tissues accumulates predominantly as 2-deoxyglucose 6-phosphate [8,9,11,15,16] ; in tissues which contain an active glucose-6-phosphatase, such as kidney, it may accumulate primarily as the free sugar [17,18]. Because of the lack of an oxygen on carbon 2, 2-deoxyglucose 6-phosphate is not a substrate for phosphohexose isomerase and is thus not metabolized further in the glycolytic pathway; it inhibits the metabolism of glucose 6-phosphate at this site [2,19--21]. Small amounts of 6phospho-2-deoxygluconate may be found intracellularly [2,21], indicating some metabolism by glucose-6-phosphate dehydrogenase. 2-Deoxyglucose 6phosphate has been claimed to inhibit glucose transport [8,9], although unlike glucose 6-phosphate it does not inhibit hexokinase [1,2,9,14]. In view of the multiple sites at which 2-deoxyglucose and 2-deoxyglucose 6-phosphate could inhibit glucose metabolism, there has been uncertainty as to the major locus of inhibition of glycolysis. Since glucose 6-phosphate does not accumulate in cells inhibited by 2-deoxyglucose [1,9,12], and since, at least with yeast, 2-deoxyglucose does not inhibit glycolysis in broken cell preparations [1], it seems likely that the major site of inhibition is at the level of glucose transport. This, however would not explain the apparent sensitivity of fructose metabolism to inhibition by 2-deoxyglucose [3,20]. Recently, in the course of studies on the relation of fat cell metabolism to insulin-stimulated glucose transport, we observed striking depression of ATP levels when fat cells were incubated with 2-deoxyglucose even in the absence of added glucose in the medium. Since relatively little is known of the effects of this glucose analogue in adipose as compared to other tissues such as muscle [9,15], we decided to investigate the effects of 2-deoxyglucose on adipose tissue metabolism. Materials and Methods
Materials. Crude bacterial collagenase was obtained from Worthington Biochemicals. Labeled sugars were products of New England Nuclear. Unlabeled sugars and firefly lanterns were obtained from Sigma. All other chemicals were reagent grade. Preparation of fat cells and incubation. Male Sprague-Dawley rats fed ad libitum, weighing 150--250 g, were used. The animals were stunned by a blow to the head, killed by cervical disolcation, and the epididymal fat pads rapidly removed. Isolated fat cells were prepared by collagenase digestion according to Rodbell [22] and washed several times at room temperature with Krebs-Ringer phosphate buffer containing 2% bovine albumin. Incubations of fat cells were carried out in plastic counting vials at 24 ° or 37°C as indicated. Cell numbers were determined by direct counting in a hemocytometer chamber. Determination of 2-deoxyglucose and 2-deoxyglucose 6-phosphate accumulation. Isolated fat cells in Krebs-Ringer-phosphate buffer containing bovine albumin were incubated in a shaker with equimolar concentrations of 2-deoxyD-[3H]glucose and [U-l~C]sucrose. At the end of the incubation, 0.3 ml aliquots of the cell suspension were rapidly transferred to 0.45 ml plastic microfuge tubes containing two drops of castor oil and centrifuged for 15 s at 10 000 rev./min in a Beckman microcentrifuge. The fat cell cake (above the oil)
280 was separated from the medium (below the oil) by slicing the tube through the o i l layer. The upper part of the tube containing the fat cells was then extracted three times with I ml of H20 in a boiling water bath and the extracts pooled. Preliminary experiments indicated that there was no residual radioactivity left in the fat cells or tube after extraction. The pooled extracts were then passed over a small Dowex-l-formate column in a pasteur pipette and the column washed with three 1.5 ml aliquots of water containing 1 mM unlabeled sucrose and 2-deoxyglucose. An aliquot of the combined water washes, containing the labeled 2-deoxyglucose and sucrose, was added to 10 ml of counting solution (aquasol, New England Nuclear). Radioactivity was determined in a Nuclear Chicago Isocap 300 under conditions in which essentially no 3H (
Biochimica et Biophysica Acta, 496 (1977) 278--291
© Elsevier/North-Holland Biomedical Press
BBA 28155 METABOLIC EFFECTS OF 2-DEOXY-D-GLUCOSE IN ISOLATED FAT CELLS
VISVANATHAN CHANDRAMOULI and JAMES R. CARTER, Jr. Department of Medicine, Case Western Reserve University, and Endocrine Division, University Hospitals, Cleveland, Ohio 44106 (U.S.A.)
(Received July 30th, 1976)
Summary The uptake and phosphorylation of 2-deoxy-D-glucose by isolated adipocytes of the rat was determined by a method of rapid flotation through oil coupled with separation of sugar from sugar phosphate by chromatography on Dowex1-formate. Uptake of the sugar is rapid and linear over 5 min, with a gradual decline thereafter; by 1 h, no further uptake is observed. Initially only 2-deoxyglucose phosphate is observed within the cells; by 1 h, however, free 2-deoxyglucose accumulates to levels approximating those in the medium. Phosphorylation ceases when intracellular levels of 2-deoxyglucose phosphate are about 50 mM regardless of the medium concentration of 2-deoxyglucose; this does not represent feedback inhibition of hexokinase, since the enzyme in fat cell homogenates is n o t inhibited by 50 mM 2-deoxyglucose 6-phosphate. Accumulation of deoxyglucose 6-phosphate is associated with a marked decline in intracellular ATP levels. Fat cell respiration is also depressed b y approximately 50 per cent after a I h preincubation with 10 or 20 mM 2-deoxyglucose. Intracellular ATP levels and 02 uptake are only partially corrected b y the addition of pyruvate to the incubation medium. Since no glucose was present in the medium, and intracellular concentrations of glycogen are known to be small in adipose tissue, it is proposed that accumulation of 2-deoxyglucose 6-phosphate within fat cells has a direct inhibitory effect on cell respiration unrelated to inhibition of glycolysis. No increase in intracellular free fatty acids was observed to explain this, and under the conditions of the incubations it is unlikely that Pi availability was rate limiting. The exact locus of inhibition is unknown. 2-Deoxy-D-glucose is a glucose analogue which is a potent inhibitor of glycolysis in a variety of tissues [1--8]. This sugar is taken up in most cells by a saturable process which appears to be the same as that for glucose, and it is therefore a competitive inhibitor of glucose transport [9--12]. 2-Deoxyglucose
279 is phosphorylated by hexokinase [1,3,13--15] and in most tissues accumulates predominantly as 2-deoxyglucose 6-phosphate [8,9,11,15,16] ; in tissues which contain an active glucose-6-phosphatase, such as kidney, it may accumulate primarily as the free sugar [17,18]. Because of the lack of an oxygen on carbon 2, 2-deoxyglucose 6-phosphate is not a substrate for phosphohexose isomerase and is thus not metabolized further in the glycolytic pathway; it inhibits the metabolism of glucose 6-phosphate at this site [2,19--21]. Small amounts of 6phospho-2-deoxygluconate may be found intracellularly [2,21], indicating some metabolism by glucose-6-phosphate dehydrogenase. 2-Deoxyglucose 6phosphate has been claimed to inhibit glucose transport [8,9], although unlike glucose 6-phosphate it does not inhibit hexokinase [1,2,9,14]. In view of the multiple sites at which 2-deoxyglucose and 2-deoxyglucose 6-phosphate could inhibit glucose metabolism, there has been uncertainty as to the major locus of inhibition of glycolysis. Since glucose 6-phosphate does not accumulate in cells inhibited by 2-deoxyglucose [1,9,12], and since, at least with yeast, 2-deoxyglucose does not inhibit glycolysis in broken cell preparations [1], it seems likely that the major site of inhibition is at the level of glucose transport. This, however would not explain the apparent sensitivity of fructose metabolism to inhibition by 2-deoxyglucose [3,20]. Recently, in the course of studies on the relation of fat cell metabolism to insulin-stimulated glucose transport, we observed striking depression of ATP levels when fat cells were incubated with 2-deoxyglucose even in the absence of added glucose in the medium. Since relatively little is known of the effects of this glucose analogue in adipose as compared to other tissues such as muscle [9,15], we decided to investigate the effects of 2-deoxyglucose on adipose tissue metabolism. Materials and Methods
Materials. Crude bacterial collagenase was obtained from Worthington Biochemicals. Labeled sugars were products of New England Nuclear. Unlabeled sugars and firefly lanterns were obtained from Sigma. All other chemicals were reagent grade. Preparation of fat cells and incubation. Male Sprague-Dawley rats fed ad libitum, weighing 150--250 g, were used. The animals were stunned by a blow to the head, killed by cervical disolcation, and the epididymal fat pads rapidly removed. Isolated fat cells were prepared by collagenase digestion according to Rodbell [22] and washed several times at room temperature with Krebs-Ringer phosphate buffer containing 2% bovine albumin. Incubations of fat cells were carried out in plastic counting vials at 24 ° or 37°C as indicated. Cell numbers were determined by direct counting in a hemocytometer chamber. Determination of 2-deoxyglucose and 2-deoxyglucose 6-phosphate accumulation. Isolated fat cells in Krebs-Ringer-phosphate buffer containing bovine albumin were incubated in a shaker with equimolar concentrations of 2-deoxyD-[3H]glucose and [U-l~C]sucrose. At the end of the incubation, 0.3 ml aliquots of the cell suspension were rapidly transferred to 0.45 ml plastic microfuge tubes containing two drops of castor oil and centrifuged for 15 s at 10 000 rev./min in a Beckman microcentrifuge. The fat cell cake (above the oil)
280 was separated from the medium (below the oil) by slicing the tube through the o i l layer. The upper part of the tube containing the fat cells was then extracted three times with I ml of H20 in a boiling water bath and the extracts pooled. Preliminary experiments indicated that there was no residual radioactivity left in the fat cells or tube after extraction. The pooled extracts were then passed over a small Dowex-l-formate column in a pasteur pipette and the column washed with three 1.5 ml aliquots of water containing 1 mM unlabeled sucrose and 2-deoxyglucose. An aliquot of the combined water washes, containing the labeled 2-deoxyglucose and sucrose, was added to 10 ml of counting solution (aquasol, New England Nuclear). Radioactivity was determined in a Nuclear Chicago Isocap 300 under conditions in which essentially no 3H (
