31485 Glucose-dependent Glycolytic Enzyme Induction and Its Effects on Glucose M

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31485 Glucose-dependent Glycolytic Enzyme Induction and Its Effects on Glucose Metabolism TABLE I Effects of PMS and 2,4-DNP on axidative glucose metabolism of resting und proliferating rat thymocytes Proliferation of cultured rat thymocytes was achieved by concanavalin A (10 ug ml-1) and interleukin 2 (10 units-ml-1). Glucose concentration in all incubations was 4 mM. For details see Experimental Procedures. Mean values ±S E. are given in ?molh(10' cells) from eight separate experiments. WCO, production from Lactate production Conditions consumption 13,4-"CIGIe [6- CIGlo Resting thymocytes 2.4-DNP (0.1 mM) PMS (0.050 mM) Proliferating thymocytes 42.6+ 1.23 142 3.10 96.7 + 1.65 37.2 2.51 279 ± 13.7 81.83.22 5.100.51 3.22+ 0.31 36.2 2.61 21.3 t 2.18 20.2 ± 2.08 34.3+1.89 1.98 0.19 3.44+ 0.38 2.01 ± 0.20 38.6 ± 2.16 32.6 ± 2.37 96.6 5.64 2.4-DNP (0.1 mM) PMS (0.010 mM) 740 + 5.09 1240 : 21.1 644 5.29 1320 t 88.6 1940 t 86.1 571 t 19.0 11.6 t 1.01 13.5 1.15 207 14.1 20.1 2.15 33.5 3.90 34.93.54 0.93 ± 0.06 3.02 0.28 11.7 ± 0.83 36.1 t 3.24 60.1 t 4.35 266+14.7 Since the observed Crabtree effect might result from the ments fetal calf serum was dialyzed against phosphate-buffered saline. 8-10-fold increase in glycolytic enzyme activities (Guppy et al. The dialyzed serum contained less than 5 p M glucose Cultured cells 1993), we have investigated and quantified the pathways of were harvested by centrifugation, washed with phosphate-buffered sa- line, pH 7.4, and resuspended in the same buffer at the indicated time. Viability of cultured cells was at least 90% as judged by trypan blue and capacities of oxidative glucose metaboliam were analyzed exclusion. Proliferation of the rat thymocytes was confirmed by a dou in the context of aerobic glycolysis. In an attempt to investigate bling of the cell numbers and by analyzing the DNA synthesis, which glucose metabolism and potential limiting processes. Activities mechanisms leading to glycolytic enzyme induction and conse- was determined by the incorporation of 0.5 uCi of PHjthymidine (2 quently to proliferation, stimulated rat thymocytes were cul- Ci/mmol) into 1-3 × 10° cells for time intervals of 3 h (Schöbitz el al. tured with glucose, with glutamine or with a mixture of gluta- 1990) mine and ribose or uridine, respectively. Based on these data Incubations and Metabolite Assays-Measurements on proliferating cells cultured in RPMI 1640 containing 12 mM glucose and all the supplements listed above were done 48 h after stimulation by ConA and interleukin 2. Incubations, "CO2 collection, scintillation counting, and calculations were done as described by Brand (1985). Glucose was meas- ured by the coupled hexokinase/glucose-6-phosphate dehydrogenase Materials -Female outbred Wistar rats (6-9 weeks old) were used for method described by Bergmeyer et al. (1974), lactate by the method of . [U-14C)glucose, [1Holglucose, 13,4 ?)glucose, Gawehn and Bergmeyer (1974), and pyruvate by the method of Lam -uClalucose, and [U."Clglutamine were obtained from Amersham precht and Heinz (1984). The contribution of the pentose phosphate (Braunschweig, Germany (FRG)). Concanavalin A (ConA), pathway to glucose metabolism was calculated according to Katz et al. 2 mercantoethanol. EDTA, and Hepes buffer were pur (1966), while the rates of the pyruvate dehydrogenase reaction and the cells we discuss the mechanisms of the aerobic to anaerobic transi- tion of glucose metabolism in the proliferating thymocyte EXPERIMENTAL PROCEDURES all experiments I6

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