Glucocorticoid action on rat thymic lymphocytes. Experiments utilizing adenosine to support cellular metabolism lead to a reassessment of catabolic hormone actions.

Inhibition of glucose uptake has been proposed as a primary cause of many of the subsequent inhibitory effects of glucocorticoids. This hypothesis has been tested in experiments where adenosine is substituted for glucose. Like glucose, adenosine maximally supports glycolytic and oxidative ATP generation, and by its use the hormonal inhibition of glucose uptake is circumvented. With adenosine, inhibition by cortisol is seen at at least one other metabolic site, respiratory ATP synthesis. This action can be observed by hormone-induced increases in levels of lactate, pyruvate, and AMP that accompany a lowering of ATP. Evidence for this metabolic action is also seen when cells are provided with a limiting amount of glucose; despite inhibition of glucose uptake, a cortisol-induced increase in lactate accompanies the reduction in levels of ATP. Decreased respiratory ATP synthesis is also suggested by a hormonal reduction in the metabolism of labeled exogenous pyruvate to 14CO2. Several experimental approaches suggest that inhibition of oxidative ATP production, rather than of glucose uptake, is the event most responsible for glucocorticoid-induced changes in the balance of adenine nucleotides, which in turn contribute to effects on protein synthesis and uridine uptake. First, the characteristic inhibitory cortisol effects on adenine nucleotides and protein synthesis are undiminished when adenosine is substituted for glucose. Second, in adenosine-supported cells the onset of the hormone-induced increase in levels of lactate corresponds closely to the appearance of measurable reductions in ATP. In contrast, when cells are supported by glucose, the hormonal inhibition of glucose uptake is maximal by 30 to 35 min, nearly an hour before effects on levels of ATP are detectable. Third, when cells are made strongly dependent upon glucose for ATP production by deprivation of exogenous substrate and cortisol is added at 90 min, a characteristic inhibition of the uptake of glucose added 40 min later is seen; nevertheless, this is insufficient to prevent added glucose from immediately and fully restoring ATP, rates of protein synthesis, and uridine uptake. Inhibitory effects on ATP, protein synthesis, and uridine do appear after an additional hour or so, a time commensurate with the development of an inhibition of oxidative metabolism. Fourth, limiting added glucose can reduce uptake more than cortisol, without reducing levels of ATP.     

Decreased glycolytic metabolism contributes to but is not the inducer of apoptosis following IL-3-starvation

IL-3 regulates the glycolytic pathway. In Baf-3 cells IL-3 starvation leads to a decrease in glucose uptake and in lactate production. To determine if there is a link between the decreased metabolism induced by growth factor-starvation and the induction of cell death, we have compared the cell death characteristics and the metabolic modifications induced by IL-3-deprivation or glucose-deprivation in Baf-3 cells. We show that in both conditions cells die by an apoptotic process which involves the activation of similar Caspases. Different metabolic parameters (i.e. intracellular ATP levels and lactate accumulation in the culture medium) were measured. We show that IL-3 deprivation leads to a partial decrease in lactate production in contrast to glucose deprivation that completely inhibits lactate production. Similarly following IL-3-starvation a significant drop in the intracellular ATP levels in live cells is observed only after 16 h when a large fraction, more than 50 per cent of cells, is already apoptotic. On the contrary, glucose deprivation is followed by an abrupt decrease in ATP levels in the first 2 h of treatment. However, in the presence of IL-3, cells are able to survive for an extended time in these conditions since 70% of cells survived with low ATP levels for up to 16 h. This was not due to partial inhibition of the apoptotic process by the low level of ATP as glucose-deprivation in the absence of IL-3 led to faster death kinetics of Baf-3 cells compared with IL-3 starvation only. These results indicate that the drop in ATP levels and the triggering of apoptosis can be dissociated in time and that when the glycolytic pathway is strongly inhibited, cells are able to survive with relatively low ATP levels if IL-3 is present. Finally we show that induction of bcl-x by IL-3 protects cells from glucose-deprivation induced cell death.     

31P-NMR studies of the metabolisms of the parasitic helminths Ascaris suum and Fasciola hepatica

31P-NMR has been applied to the study of the metabolisms of the intact parasitic helminths Ascaris suum (the intestinal roundworm) and Fasciola hepatica (the liver fluke). After calibration of the chemical shift of Pi in muscle extracts the internal pH of adult Ascaris worms and the effect of the pH of the external medium on the organism's internal pH were measured. Assignments of nearly all of the observable 31P resonances could be made. A large resonance from glycerophosphorylcholine whose function is unclear was observed but no signals from energy storage compounds such as creatine phosphate were detected. The profiles of the phosphorus-containing metabolites in both organisms were monitored as a function of time. Changes in sugar phosphate distributions but not ATP/ADP were observed. Studies of the drug closantel on Fasciola hepatica were performed. Initial effects of the drug were a decrease in glucose 6-phosphate and an increase in Pi with no substantial change in ATP levels as observed by 31P-NMR. Studies involving treatment with closantel followed by rapid freezing, extraction, and analytical determination of glycolytic intermediates confirmed NMR observations. This NMR method can serve as a simple noninvasive procedure to study parasite metabolism and drug effects on metabolism.     

ER stress modulates cellular metabolism

Changes in metabolic processes play a critical role in the survival or death of cells subjected to various stresses. In the present study, we have investigated the effects of ER (endoplasmic reticulum) stress on cellular metabolism. A major difficulty in studying metabolic responses to ER stress is that ER stress normally leads to apoptosis and metabolic changes observed in dying cells may be misleading. Therefore we have used IL-3 (interleukin 3)-dependent Bak-/-Bax-/- haemopoietic cells which do not die in the presence of the ER-stress-inducing drug tunicamycin. Tunicamycin-treated Bak-/-Bax-/- cells remain viable, but cease growth, arresting in G1-phase and undergoing autophagy in the absence of apoptosis. In these cells, we used NMR-based SIRM (stable isotope-resolved metabolomics) to determine the metabolic effects of tunicamycin. Glucose was found to be the major carbon source for energy production and anabolic metabolism. Following tunicamycin exposure, glucose uptake and lactate production are greatly reduced. Decreased 13C labelling in several cellular metabolites suggests that mitochondrial function in cells undergoing ER stress is compromised. Consistent with this, mitochondrial membrane potential, oxygen consumption and cellular ATP levels are much lower compared with untreated cells. Importantly, the effects of tunicamycin on cellular metabolic processes may be related to a reduction in cell-surface GLUT1 (glucose transporter 1) levels which, in turn, may reflect decreased Akt signalling. These results suggest that ER stress exerts profound effects on several central metabolic processes which may help to explain cell death arising from ER stress in normal cells.     

MAPK14/p38α-dependent modulation of glucose metabolism affects ROS levels and autophagy during starvation

Increased glycolytic flux is a common feature of many cancer cells, which have adapted their metabolism to maximize glucose incorporation and catabolism to generate ATP and substrates for biosynthetic reactions. Indeed, glycolysis allows a rapid production of ATP and provides metabolic intermediates required for cancer cells growth. Moreover, it makes cancer cells less sensitive to fluctuations of oxygen tension, a condition usually occurring in a newly established tumor environment. Here, we provide evidence for a dual role of MAPK14 in driving a rearrangement of glucose metabolism that contributes to limiting reactive oxygen species (ROS) production and autophagy activation in condition of nutrient deprivation. We demonstrate that MAPK14 is phosphoactivated during nutrient deprivation and affects glucose metabolism at 2 different levels: on the one hand, it increases SLC2A3 mRNA and protein levels, resulting in a higher incorporation of glucose within the cell. This event involves the MAPK14-mediated enhancement of HIF1A protein stability. On the other hand, MAPK14 mediates a metabolic shift from glycolysis to the pentose phosphate pathway (PPP) through the modulation of PFKFB3 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase 3) degradation by the proteasome. This event requires the presence of 2 distinct degradation sequences, KEN box and DSG motif Ser273, which are recognized by 2 different E3 ligase complexes. The mutation of either motif increases PFKFB3 resistance to starvation-induced degradation. The MAPK14-driven metabolic reprogramming sustains the production of NADPH, an important cofactor for many reduction reactions and for the maintenance of the proper intracellular redox environment, resulting in reduced levels of ROS. The final effect is a reduced activation of autophagy and an increased resistance to nutrient deprivation.     

Effects of glucose and insulin on HepG2‐C3A cell metabolism

HepG2, hepatocellular carcinoma cells, are used in drug toxicity studies and have also been explored for bioartificial livers. For these applications, the cells are under variable levels of nutrients and hormones, the effects of which on metabolism are poorly understood. In this study, HepG2‐C3A cells were cultured under varying levels of glucose (high, low, and glucose‐free) and insulin (without and with physiological levels of insulin) for 5 days. Cell growth was found to be comparable between high and low glucose media and lowest for glucose‐free medium. Several features of central metabolism were affected profoundly by the medium glucose levels. Glucose consumption was greater for low glucose medium compared to high glucose medium, consistent with known glucose feedback regulation mechanisms. Urea productivity was highest in glucose‐free medium. Further, it was seen that lactate acted as an alternative carbon source in the absence of glucose, whereas it acted as a sink for the high and low glucose media. Using a metabolic network flexibility analysis (MNFA) framework with stoichiometric and thermodynamic constraints, intracellular fluxes under varying levels of glucose and insulin were evaluated. The analysis indicates that urea production in HepG2‐C3A cells arises via the arginase II pathway rather than from ammonia detoxification. Further, involvement of the putrescine metabolism with glutamine metabolism caused higher urea production in glucose‐free medium consistent with higher glutamine uptake. MNFA indicated that in high and low glucose media, glycolysis, glutaminolysis, and oxidative phosphorylation were the main sources of energy (NADH, NADPH, and ATP). In the glucose‐free medium, due to very low glycolytic flux, higher malate to pyruvate glutaminolytic flux and TCA cycle contributed more significantly to energy metabolism. The presence of insulin lowered glycerol uptake and corresponding fluxes involved in lipid metabolism for all glucose levels but otherwise exerted negligible effect on metabolism. HepG2‐C3A cells thus show distinct differences from primary hepatocytes in terms of energy metabolism and urea production. This knowledge can be used to design media supplements and metabolically engineer cells to restore necessary hepatic functions to HepG2‐C3A cells for a range of applications. Biotechnol. Bioeng. 2010;107: 347–356. © 2010 Wiley Periodicals, Inc.     

Pyridine nucleotide analog interference with metabolic processes in mitogen-stimulated human T lymphocytes

The differential metabolic effects of three nicotinamide analogs, 6-aminonicotinamide, 3-aminobenzamide, and 5-methylnicotinamide, were analyzed in mitogen-stimulated preparations of human T lymphocytes. Mitogen stimulation with the phorbol ester TPA and a monoclonal antibody to the T3 cell surface antigen caused an increase in cellular NAD and ATP levels and a marked increase in glucose metabolism as demonstrated by an increase in cellular levels of glucose 6-phosphate and a sevenfold increase in radioactive CO2 formation from [l-14C]glucose. 6-Aminonicotinamide had drastic inhibitory effects on the mitogen-stimulated increases in NAD and ATP levels as well as on the metabolism of glucose. Treatment of the mitogen-stimulated cells with 6-aminonicotinamide also caused a marked increase in cellular levels of 6-phosphogluconate, suggesting inhibition of the hexose monophosphate shunt at 6-phosphogluconate dehydrogenase. Radioactive CO2 formation from [6-14C]glucose showed that metabolism through the tricarboxylic acid cycle was not used to compensate for the inhibition of the hexose monophosphate shunt pathway. Treatment of cells with 3-aminobenzamide had the opposite effect of 6-aminonicotinamide in that cellular NAD levels increased, presumable due to inhibition of poly(ADP-ribose) polymerase. 3-Aminobenzamide did not interfere with ATP or glucose 6-phosphate levels and did not cause significant elevations of 6-phosphogluconate. Thus, 6-aminonicotinamide appears to have direct inhibitory effects on the synthesis of both pyridine nucleotides and poly(ADP-ribose), whereas 3-aminobenzamide has its major inhibitory effect on poly(ADP-ribose) synthesis. 5-Methylnicotinamide also interferes with the mitogen-stimulated increase in NAD levels but not as effectively as 6-aminonicotinamide. The alterations in pyridine nucleotide metabolism resulting from treatment with these nicotinamide analogs can produce drastic and diverse alterations in pathways of glucose utilization and energy generation.     

Brain glucose-sensing mechanisms: ubiquitous silencing by aglycemia vs. hypothalamic neuroendocrine responses

Interest in brain glucose-sensing mechanisms is motivated by two distinct neuronal responses to changes in glucose concentrations. One mechanism is global and ubiquitous in response to profound hypoglycemia, whereas the other mechanism is largely confined to specific hypothalamic neurons that respond to changes in glucose concentrations in the physiological range. Although both mechanisms use intracellular metabolism as an indicator of extracellular glucose concentration, the two mechanisms differ in key respects. Global hyperpolarization (inhibition) in response to 0 mM glucose can be reversed by pyruvate, implying that the reduction in ATP levels acting through ATP-dependent potassium (K-ATP) channels is the key metabolic signal for the global silencing in response to 0 mM glucose. In contrast, neuroendocrine hypothalamic responses in glucoresponsive and glucose-sensitive neurons (either excitation or inhibition, respectively) to physiological changes in glucose concentration appear to depend on glucokinase; neuroendocrine responses also depend on K-ATP channels, although the role of ATP itself is less clear. Lactate can substitute for glucose to produce these neuroendocrine effects, but pyruvate cannot, implying that NADH (possibly leading to anaplerotic production of malonyl-CoA) is a key metabolic signal for effects of glucose on glucoresponsive and glucose-sensitive hypothalamic neurons.     

BAD-dependent regulation of fuel metabolism and K(ATP) channel activity confers resistance to epileptic seizures

Neuronal excitation can be substantially modulated by alterations in metabolism, as evident from the anticonvulsant effect of diets that reduce glucose utilization and promote ketone body metabolism. We provide genetic evidence that BAD, a protein with dual functions in apoptosis and glucose metabolism, imparts reciprocal effects on metabolism of glucose and ketone bodies in brain cells. These effects involve phosphoregulation of BAD and are independent of its apoptotic function. BAD modifications that reduce glucose metabolism produce a marked increase in the activity of metabolically sensitive K(ATP) channels in neurons, as well as resistance to behavioral and electrographic seizures in vivo. Seizure resistance is reversed by genetic ablation of the K(ATP) channel, implicating the BAD-K(ATP) axis in metabolic control of neuronal excitation and seizure responses.     

Depletion of mitochondrial DNA alters glucose metabolism in SK-Hep1 cells

Maternally inherited mitochondrial DNA (mtDNA) has been suggested to be a genetic factor for diabetes. Reports have shown a decrease of mtDNA content in tissues of diabetic patients. We investigated the effects of mtDNA depletion on glucose metabolism by use of rho(0) SK-Hep1 human hepatoma cells, whose mtDNA was depleted by long-term exposure to ethidium bromide. The rho(0) cells failed to hyperpolarize mitochondrial membrane potential in response to glucose stimulation. Intracellular ATP content, glucose-stimulated ATP production, glucose uptake, steady-state mRNA and protein levels of glucose transporters, and cellular activities of glucose-metabolizing enzymes were decreased in rho(0) cells compared with parental rho(+) cells. Our results suggest that the quantitative reduction of mtDNA may suppress the expression of nuclear DNA-encoded glucose transporters and enzymes of glucose metabolism. Thus this may lead to diabetic status, such as decreased ATP production and glucose utilization.     

Energetic effects of adenosine on vascular smooth muscle

Adenosine (Ado) is a naturally occurring compound that has several important cardiovascular actions, including activation of ATP-sensitive K(+) channels in vascular smooth muscle, vasorelaxation, and an effect to alter glucose metabolism of cardiac muscle. The metabolic effects of Ado on vascular smooth muscle have not been defined and were examined in this study. Porcine carotid artery strips were incubated in the presence and absence of 0.5 mM Ado. Compared with the control, Ado had no effect on glucose uptake, glucose oxidation, or fatty acid (octanoate) oxidation. Ado suppressed glycolysis but enhanced glycogen synthesis. Relative to the rate of glycolysis, Ado increased lactate production. Ado stimulated O(2) consumption by 52 +/- 10%, altered the activities of the tricarboxylic acid cycle and malate-aspartate shuttle, and increased the content of ATP, ADP, AMP, and phosphocreatine. Alteration in the metabolic variables by Ado could not be attributed to diminished energy requirements of reduced resting muscle tone of the arterial strips. Relaxation of the arterial strips in response to Ado were abolished in arteries incubated under hypoxic conditions (95% N(2)-5% CO(2)). Hypoxia was associated with increased ADP content. It is concluded that Ado affected glucose metabolism indirectly. The metabolic and energetic effects of 0.5 mM Ado are mediated by alterations in the concentrations of AMP, ATP, and phosphorylation potential (ATP/ADP).     

Lactate-Mediated Protection of Retinal Ganglion Cells

Loss of retinal ganglion cells (RGCs) is a leading cause of blinding conditions. The purpose of this study was to evaluate the effect of extracellular l-lactate on RGC survival facilitated through lactate metabolism and ATP production. We identified lactate as a preferred energy substrate over glucose in murine RGCs and showed that lactate metabolism and consequently increased ATP production are crucial components in promoting RGC survival during energetic crisis. Lactate was released to the extracellular environment in the presence of glucose and detained intracellularly during glucose deprivation. Lactate uptake and metabolism was unaltered in the presence and absence of glucose. However, the ATP production declined significantly for 24 h of glucose deprivation and increased significantly in the presence of lactate. Finally, lactate exposure for 2 and 24 h resulted in increased RGC survival during glucose deprivation. In conclusion, the metabolic pathway of lactate in RGCs may be of great future interest to unravel potential pharmaceutical targets, ultimately leading to novel therapies in the prevention of blinding neurodegenerative diseases, for example, glaucoma.     

HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia

Activation of glycolytic genes by HIF-1 is considered critical for metabolic adaptation to hypoxia through increased conversion of glucose to pyruvate and subsequently to lactate. We found that HIF-1 also actively suppresses metabolism through the tricarboxylic acid cycle (TCA) by directly trans-activating the gene encoding pyruvate dehydrogenase kinase 1 (PDK1). PDK1 inactivates the TCA cycle enzyme, pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA. Forced PDK1 expression in hypoxic HIF-1alpha null cells increases ATP levels, attenuates hypoxic ROS generation, and rescues these cells from hypoxia-induced apoptosis. These studies reveal a hypoxia-induced metabolic switch that shunts glucose metabolites from the mitochondria to glycolysis to maintain ATP production and to prevent toxic ROS production.     

Organic mercurial diuresis: inhibition of glutamine utilization in the acidotic rat.

Organic mercurials inhibit mitochondrial glutamine metabolism in vitro while metabolic acidosis, a condition in which the predominant renal fuel is glutamine, potentiates mercurial diuresis. The following studies were undertaken to determine whether potentiation of diuresis reflects mercurial inhibition of glutamine utilization. (1) All three mercurials employed (mersalyl, chlormerodrin, and p-chloromercuribenzoate) are diuretics in the rat and this effect was potentiated by NH4Cl. (2) Despite reabsorbing less sodium, mercurial-treated rats had lower kidney ATP content (4.35 +/- 0.26 and 3.84 +/- 0.43 mumol/g dry weight (mercurial plus NH4Cl) than did controls (4.95 +/- 0.31 and 4.87 +/- 0.39 mumol/g dry weight (NH4Cl). (3) Isolated kidneys from NH4Cl and NH4Cl plus mercurial treated rats were perfused with 1 mM L-[U-14C]glutamine to determine rates of extraction and oxidation. Mercurial-treated acidotic rat kidneys had a reduced rate of glutamine uptake (40.8 +/- 7.4 vs. 64.8 +/- 5.8 mumol/h per kidney), a diminished rate of glutamine conversion to CO2 (14.8 +/- 3.6 vs. 26.4 +/- 5.2 mumol/h per kidney), and a reduction in glucose production (16 +/- 5 vs. 27 +/- 4 mumol/h per kidney). These results are consistent with an effect of organic mercurials upon glutamine utilization, limiting ATP availability, and thereby reducing tubular active sodium reabsorption.     

Carbon and energy balances of glucose fermentation with hydrogenproducing bacterium Citrobacter amalonaticus Y19

For the newly isolated H2-producing chemoheterotrophic bacterium Citrobacter amalonaticus Y19, anaerobic glucose metabolism was studied in batch cultivation at varying initial glucose concentrations (3.5- 9.5 g/l). The carbon-mass and energy balances were determined and utilized to analyze the carbon metabolic-pathways network. The analyses revealed (a) variable production of major metabolites (H2, ethanol, acetate, lactate, CO2, and cell mass) depending on initial glucose levels; (b) influence of NADH regeneration on the production of acetate, lactate, and ethanol; and (c) influence of the molar production of ATP on the production of biomass. The results reported in this paper suggest how the carbon metabolic pathway(s) should be designed for optimal H2 production, especially at high glucose concentrations, such as by blocking the carbon flux via lactate dehydrogenase from the pyruvate node.     

Inhibition of oxidative metabolism by nitric oxide restricts EMCV replication selectively in pancreatic beta-cells

Environmental factors, such as viral infection, are proposed to play a role in the initiation of autoimmune diabetes. In response to encephalomyocarditis virus (EMCV) infection, resident islet macrophages release the pro-inflammatory cytokine IL-1β, to levels that are sufficient to stimulate inducible nitric oxide synthase (iNOS) expression and production of micromolar levels of the free radical nitric oxide in neighboring β-cells. We have recently shown that nitric oxide inhibits EMCV replication and EMCV-mediated β-cell lysis and that this protection is associated with an inhibition of mitochondrial oxidative metabolism. Here we show that the protective actions of nitric oxide against EMCV infection are selective for β-cells and associated with the metabolic coupling of glycolysis and mitochondrial oxidation that is necessary for insulin secretion. Inhibitors of mitochondrial respiration attenuate EMCV replication in β-cells, and this inhibition is associated with a decrease in ATP levels. In mouse embryonic fibroblasts (MEFs), inhibition of mitochondrial metabolism does not modify EMCV replication or decrease ATP levels. Like most cell types, MEFs have the capacity to uncouple the glycolytic utilization of glucose from mitochondrial respiration, allowing for the maintenance of ATP levels under conditions of impaired mitochondrial respiration. It is only when MEFs are forced to use mitochondrial oxidative metabolism for ATP generation that mitochondrial inhibitors attenuate viral replication. In a β-cell selective manner, these findings indicate that nitric oxide targets the same metabolic pathways necessary for glucose stimulated insulin secretion for protection from viral lysis.     

Leptin regulates energy metabolism in MCF-7 breast cancer cells

Obesity is known to be a poorer prognosis factor for breast cancer in postmenopausal women. Among the diverse endocrine factors associated to obesity, leptin has received special attention since it promotes breast cancer cell growth and invasiveness, processes which force cells to adapt their metabolism to satisfy the increased demands of energy and biosynthetic intermediates. Taking this into account, our aim was to explore the effects of leptin in the metabolism of MCF-7 breast cancer cells. Polarographic analysis revealed that leptin increased oxygen consumption rate and cellular ATP levels were more dependent on mitochondrial oxidative metabolism in leptin-treated cells compared to the more glycolytic control cells. Experiments with selective inhibitors of glycolysis (2-DG), fatty acid oxidation (etomoxir) or aminoacid deprivation showed that ATP levels were more reliant on fatty acid oxidation. In agreement, levels of key proteins involved in lipid catabolism (FAT/CD36, CPT1, PPARα) and phosphorylation of the energy sensor AMPK were increased by leptin. Regarding glucose, cellular uptake was not affected by leptin, but lactate release was deeply repressed. Analysis of pyruvate dehydrogenase (PDH), lactate dehydrogenase (LDH) and pyruvate carboxylase (PC) together with the pentose-phosphate pathway enzyme glucose-6 phoshate dehydrogenase (G6PDH) revealed that leptin favors the use of glucose for biosynthesis. These results point towards a role of leptin in metabolic reprogramming, consisting of an enhanced use of glucose for biosynthesis and lipids for energy production. This metabolic adaptations induced by leptin may provide benefits for MCF-7 growth and give support to the reverse Warburg effect described in breast cancer.     

Overexpression of catalase or Bcl-2 alters glucose and energy metabolism concomitant with dexamethasone resistance

Glucocorticoids induce apoptosis in lymphocytes by causing the release of cytochrome c into the cytosol; however, the events in the signaling phase between translocation of the steroid-receptor complex to the nucleus and the release of cytochrome c have not been elucidated. Previously, we found that, in response to steroid treatment, WEHI7.2 mouse thymic lymphoma cells overexpressing catalase (CAT38) show delayed apoptosis (delayed cytochrome c release) compared to the parental cells, while Bcl-2 overexpressing cells (Hb12) are protected from steroid-induced apoptosis. In lymphocytes, glucocorticoid treatment decreases glucose uptake. Both glucose deprivation and the attendant ATP drop are known inducers of apoptosis. Therefore, we used (31)P and (1)H NMR spectroscopy to compare metabolic profiles of WEHI7.2, CAT38 and Hb12 cells in the presence and absence of dexamethasone to determine: (1) whether glucocorticoid effects on glucose metabolism contribute to the mechanism of steroid-induced apoptosis; and (2) whether catalase or Bcl-2 overexpression altered metabolism thereby providing a mechanism of steroid resistance. Loss of mitochondrial hexokinase activity was correlated to the induction of apoptosis in WEHI7.2 and CAT38 cells. CAT38 and Hb12 cells have an altered basal metabolism which includes increases in hexokinase activity, lactate production when subcultured into new medium, use of mitochondria for ATP production and potentially increased glutaminolysis. These data suggest that: (1) glucocorticoid effects on glucose metabolism may contribute to the mechanism of steroid-induced lymphocyte apoptosis; and (2) the altered metabolism seen in catalase and Bcl-2 overexpressing cells may contribute to both the steroid resistance and increased tumorigenicity of these variants.     

Haemopoietic cell growth factor mediates cell survival via its action on glucose transport

A number of haematopoietic precursor cell lines have been established which exhibit an absolute dependence on haematopoietic cell growth factor (HCGF) which is secreted by WEHI-3 myelomonocytic leukaemia cells. In the presence of HCGF, ATP levels are maintained in these factor-dependent cells (FDC-P cells); in the absence of HCGF, intracellular ATP levels undergo a steady depletion. The cell death that follows this ATP depletion can be prevented by supplying exogenous ATP suggesting that HCGF maintains these cells via its effects on energy metabolism. We have investigated the effect of HCGF on FDC-P cells further and found that: (i) HCGF markedly and rapidly increases lactate production; (ii) high extracellular glucose or glycolytic intermediate concentrations can maintain FDC-P cell viability to some extent whilst stimulating lactate production; (iii) the uptake of 2-deoxyglucose by FDC-P2 cells is stimulated by HCGF in a dose-dependent fashion. This uptake is inhibited by cytochalasin B; (iv) HCGF does not stimulate L-glucose uptake by FDC-P cells. These results suggest that HCGF acts to maintain FDC-P cells via its action on glucose transport. The significance of these results to haemopoiesis and leukaemogenesis is discussed.     

Metabolic effects of interleukin 3 on 32D cl23 cells analyzed by NMR

31P NMR of living 32D cl23 cells and 1H NMR of cell extracts were used to study the metabolic effects of interleukin 3 (IL3). When IL3 was removed from 32D cl23 for 9-10 hours 31P spectra showed a decrease in sugar phosphate, gamma ATP/ADP, alpha ATP/ADP/NAD, and beta ATP resonances which declined progressively over a time period of up to 16 hours. By comparison, ATP measurements using the luciferin/luciferase method resulted in the decline of ATP levels from 12 hours in the absence of IL3. At this time, viability of the cells was unaffected. For 1H NMR experiments cells were grown in the presence and absence of IL3 for 4 and 24 hours, after which acid cell extracts were prepared. These spectra revealed a four-fold decrease in lactate 4 hours post-IL3 removal. Alanine levels were unchanged but glycine was elevated 1.5-fold whilst various other amino acids were elevated slightly. After 24 hours without IL3, only 22% of cells were viable which was reflected in a general decline of most resonance intensities. These findings suggest that IL3 exerts its effect primarily on glucose metabolism and has a delayed secondary effect on maintenance of ATP levels in the cell. We have demonstrated the applicability of high resolution 1H and 31P NMR to the study of cellular metabolism in hemopoietic cells.