RESUMEN
Human diploid fibroblasts metabolize up to 13% of the glutamine in tissue culture medium to lactate. Four microCi of glutamine-U-14C were added to media containing 5 mM or 65 microM glucose or medium containing no added glucose, but supplemented with purine and pyrimidine nucleosides (HGTU). Aliquots of the media were taken at daily intervals and were assayed for glucose, lactate, pyruvate, malate, citrate, aspartate, glutamine, and glutamate. The label incorporation into these compounds was determined, except for glutamine and glucose. The distribution of label from glutamine-U14C in 5 mM glucose medium by day 4 was lactate (10.2%), glutamate (15.2%), citrate (1.9%), pyruvate (2.0%), malate (1.1%), and aspartate (< 0.1%). The accumulation of label in lactate and glutamate occurred continuously during the growth cycle. Malate, citrate, and aspartate accumulation occurred primarily in confluent cultures. The label in aspartate was seen only in stationary phase cells or when the glucose concentration was decreased to 65 microM or less; net aspartate accumulation was increased twofold in low glucose media. These data demonstrate an actively functioning pathway for the conversion of 4-carbon TCA-cycle intermediates to 3-carbon glycolytic intermediates in human diploid fibroblasts.
Asunto(s)
Fibroblastos/metabolismo , Glutamina/metabolismo , Lactatos/biosíntesis , Ácidos Carboxílicos/metabolismo , División Celular , Células Cultivadas , Metabolismo Energético , HumanosRESUMEN
The activity of glutaminase per milligram of protein increased threefold after cultured human diploid fibroblasts were subcultured in fresh medium. The maximum activity was reached after 2 days of growth and decreased once the cells reached confluency. The increase of glutaminase activity was independent of the glutamine concentration between 0.2 and 2.0 mmol/l. In contrast, the specific activity of glutamate dehydrogenase was independent both of the glutamine concentration and the growth phase of the cultured cells. These results indicate that glutaminase, the first enzyme involved in the ultilization of glutamine as an energy source, is elevated in rapidly dividing human diploid fibroblasts.
Asunto(s)
Fibroblastos/enzimología , Glutaminasa/metabolismo , Ciclo Celular , Células Cultivadas , Diploidia , Fibroblastos/citología , Glutamina/metabolismo , HumanosRESUMEN
The activities of orotate phosphoribosyl transferase (OPRT) and orotidine monophosphate decarboxylase (ODC) were significantly elevated (P less than 0.001) in erythrocytes (RBC) from five patients with prednisone-responsive congenital hypoplastic anaemia (CHA). (OPRT: patients - 10.1--64.2 nmol/h/10(9) RBC; controls - 2.8 +/- 0.3 (mean +/- SEM, n = 37); ODC: patients = 30--124 nmol/h/10(9) RBC; controls = 10.2 +/- 0.7 (mean SEM, n = 37).) Two patients had a less pronounced, but significant, increase of aspartate transcarbamylase activity and three patients had marginal increases of dihydroorotase activity. Dihydroorotate dehydrogenase activity was not detected in any CHA patient or control. In one patient prior to prednisone therapy, the OPRT and ODT activities were elevated 10-fold and remained elevated 3-fold after 16 months of therapy. An elevated enzyme pattern similar to that of RBC from CHA patients was observed in three parents of three CHA patients, but not in three parents of two other CHA patients. The activities of all five pyrimidine enzymes were normal for one patient with transient erythroblastopenia of childhood. In contrast, the activities of all the pyrimidine biosynthetic enzymes were elevated in blood from patients with a young RBC population: sickle cell anaemia, sickle-beta-thalassaemia, hereditary spherocytosis, and DiGuglielmo syndrome and from the newborn. It is postulated that factors which affect the activities of pyrimidine enzymes in CHA may also result in diminished erythropoiesis.
Asunto(s)
Anemia Aplásica/congénito , Carboxiliasas/sangre , Eritrocitos/enzimología , Orotato Fosforribosiltransferasa/sangre , Orotidina-5'-Fosfato Descarboxilasa/sangre , Pentosiltransferasa/sangre , Adolescente , Adulto , Anemia Aplásica/enzimología , Anemia Aplásica/genética , Aspartato Carbamoiltransferasa/sangre , Niño , Preescolar , Dihidroorotasa/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Prednisona/uso terapéuticoRESUMEN
Human diploid fibroblasts deplete 50% of their cellular glycogen by day 4 after subcultivation in 100 mg% glucose medium. The glycogen content increases again as the cells approach confluency. Growth of cells in low glucose medium results in rapid glycogen depletion and indicates that stored glycogen has a limited potential as an energy source.
Asunto(s)
Fibroblastos/metabolismo , Glucógeno/metabolismo , Células Cultivadas , Medios de Cultivo , Fibroblastos/efectos de los fármacos , Glucosa/farmacología , Humanos , Proteínas/metabolismo , Factores de TiempoRESUMEN
Human diploid fibroblasts utilize both glucose and glutamine as energy sources. The utilization of glutamine by fibroblasts is regulated by glucose, and vice versa. This conclusion is supported by the following observations: (1) essentially identical growth rates were observed in Eagle's minimum essential medium (MEM)3 in which the glucose concentration was either 5.5 mM or was maintained between 25 and 40 micrometer, (2) the total glutamine utilization by fibroblasts increase at least 30% in medium with 25 micrometer to 70 micrometer glucose compared to medium with 5.5 mM glucose, while the rate of glutamine-1 or 5-14C oxidation to CO2 increased 5-fold as the glucose concentration was decreased to zero, (3) 2 mM glutamine inhibited glucose-6-14C oxidation by 88% and stimulated glucose-1-14C by 77% in log phase cells and (4) glutamine oxidation in normal medium contributed approximately 30% of the energy requirement of human diploid fibroblasts.
Asunto(s)
Fibroblastos/metabolismo , Glucosa/metabolismo , Glutamina/metabolismo , División Celular , Células Cultivadas , Diploidia , Fibroblastos/citología , Glutamatos/biosíntesis , Humanos , Oxidación-ReducciónRESUMEN
Normal human diploid fibroblasts were able to undergo one to two cell divisions without glucose utilization in Eagle's minimum essential medium plus 10% dialyzed fetal calf serum if the medium was supplemented with hypoxanthine, thymidine, and uridine (supplemented medium termed HTU-MEM). Under these conditions, the added purine and pyrimidines were required for nucleic acid synthesis, as shown by the inability of Lesch-Nyhan fibroblasts to grow in HTU-MEM. Normal human diploid fibroblasts continued to produce lactate in HTU-MEM, but at a greatly reduced rate. Since cells grew in HTU-MEM without glucose utilization, the probable energy and carbon source was glutamine, which is present in relatively high concentration. Furthermore, the rate of glutamine utilization per cell division was 2-fold greater in HTU-MEM than in medium with 5.5 mM glucose. These results suggest that glutamine can be a major energy source for cells grown in vitro.