Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. IV. Role of energy metabolism

The role of energy metabolism on tumor cell killing by in vitro activated macrophages was studied. Depletion of extracellular glucose had little effect on the cytotoxic capacity of mediator-activated macrophages. Respiratory antagonists did not inhibit cytotoxicity regardless of whether or not the assays were carried out in low-glucose-containing medium. Sodium fluoride, a known inhibitor of glycolysis, inhibited the killing of tumor cells by activated macrophages. 2-Deoxyglucose, an analog of glucose, was found to be an effective inhibitor of cytotoxicity. Three other analogs, 5-thio-d-glucose, 3-O-methylglucose, and 2-deoxy-d-galactose, were without effect. The concentrations of 2-DG that inhibited cytotoxicity did not lower cellular ATP levels to an appreciable extent. The combined addition of inhibitors of glycolysis and respiration resulted in a marked reduction in ATP levels. Under these experimental conditions, macrophage-mediated cytotoxicity was also significantly inhibited.     

Glucose transport and its inhibition by short-chain n-alkanes in Cladosporium resinae.

Glucose transport in Cladosporium resinae was studies with the aid of the non-metabolizable glucose analogue 3-O-methyl-D-glucose (3-O-MG). 3-O-MG, transported as a free sugar without phosphorylation, was found to inhibit glucose uptake competitively. Conversely, glucose was a competitive inhibitor of 3-O-MG uptake. Moreover, both glucose and 3-O-MG were able to bring about rapid counterflow intracellular 3-O-MG. Thus, glucose and 3-O-MG share the same entry and exit systems. The transport of 3-O-MG is carrier mediated and energy dependent as shown by saturation kinetics, strong temperature dependence, accumulation of unaltered 3-O-MG against a concentration gradient, and inhibition of uptake by NaN3, NaCN, and 2,4-dinitrophenol. The glucose transport system appeared to be constitutive for glucose transport in cells grown on fructose, galactose, mannose, xylose, or glucose. There was no derepressible low-Km glucose transport system in C. resinae. n-Hexane and n-heptane were found to inhibit 3-O-MG uptake rapidly at temperatures above 20 C. Over 50% inhibition of the uptake rate occurred after only 10 min of incubation with n-hexane at 30 C. The percentage of inhibition in the presence of n-hexane, compared to controls in the absence of n-hexane, was found to increase with increasing temperature. Longer-chain n-alkanes (C8 to C18) had no significant effect on uptake. The efflux of intracellular 3-O-MG, which appeared to occur by facilitated diffusion, was not affected by any of the n-alkanes tested including n-hexane.     

Net movements of monovalent and bivalent cations, and their relation to energy metabolism, in slices of hepatoma 3924A and of a mammary tumour

1. During incubation at 1° in saline medium buffered either with phosphate or bicarbonate, slices of Morris hepatoma 3924A, and of a chemically induced tumour of rat mammary gland, lost K⁺ and gained Na⁺, Ca²⁺ and water.

2. Upon subsequent incubation at 38° in oxygenated medium, these changes were partially reversed. In the hepatoma, the reaccumulation of K⁺ was equally efficient in phosphate or bicarbonate medium, and in the presence and absence of glucose. Ca²⁺ was extruded in bicarbonate, but not in phosphate medium, and its extrusion was reduced in the presence of glucose.

3. When respiration was inhibited in the presence of glucose, K⁺ transport by the hepatoma continued to an extent which varied with the glycolytic activity of the slices, suggesting that the rate of ATP synthesis was a limiting factor under these conditions.

4. In the absence of glucose, the transport of Na⁺ and K⁺ was completely stopped by respiratory inhibition. However, more than 50% of the O₂ uptake had to be inhibited before any effect on transport was observed, suggesting that the rate of synthesis of ATP from endogenous respiration is in excess of that required to maintain transport.

5. Inhibition of transport by ouabain was accompanied by a 30% fall in the rate of endogenous respiration, and by a fall of 33% in the rate of glycolysis in the presence of cyanide plus glucose.

6. Comparison of the minimum rates of respiration and of glycolysis (in the presence of glucose plus cyanide) required to maintain the maximal extent of K⁺ transport in the hepatoma slices, suggests that ATP derived from oxidative phosphorylation or from anaerobic glycolysis is equally efficient as a source of energy for ion transport.     

Diverse effects of insulin-induced hyperpolarization on 3-O-methyl-D-glucose (3-O-MG) transport in frog skeletal muscles

It has been suggested that the insulin-induced hyperpolarization might be a mediator of the stimulatory action of insulin on glucose transport. The purpose of the present study was to investigate the relationship between the insulin-induced hyperpolarization and the stimulatory action of insulin on glucose transport in skeletal muscle. Satorius muscles dissected from bullfrogs (Rana catesbeiana) were used. Insulin induced a hyperpolarization of the membrane and an increase in the 3-O-Methyl-D-glucose (3-O-MG) uptake and extrusion. In the presence of valinomycin, insulin had no significant effect on the membrane potential. Insulin still had the stimulatory action on both the 3-O-MG uptake and extrusion even in the presence of valinomycin, under whose condition insulin had no significant effect on the membrane potential. The magnitude of the stimulatory action of insulin on the 3-O-MG uptake in the presence of valinomycin was smaller than that in the absence of valinomycin. The magnitude of the stimulatory action of insulin on the 3-O-MG extrusion was, on the contrary, larger than that in the absence of valinomycin. The abolishment of the insulin-induced hyperpolarization decreased the 3-O-MG uptake and increased the 3-O-MG extrusion. The observation in the present study concludes that insulin has two different actions on glucose transport. One of them is developed through the insulin-induced hyperpolarization, which increases the 3-O-MG uptake and decreases the 3-O-MG extrusion. The other action is irrelevant of the insulin-induced hyperpolarization and stimulates both the 3-O-MG uptake and extrusion.     

Overexpression of Mitochondrial Sirtuins Alters Glycolysis and Mitochondrial Function in HEK293 Cells

SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose) all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.     

Uptake and Metabolism of d-Glucose by Neocosmospora vasinfecta E. F. Smith

Freshly harvested, nongrowing mycelium of Neocosmospora vasinfecta E. F. Smith rapidly absorbed exogenous glucose but converted a greater proportion to trehalose and glucan than to respiratory CO(2). This effect was accentuated in mycelium preincubated for 3.5 hours in water before exposure to glucose. Glucose was absorbed via two uptake systems, both apparently constitutive, with apparent Km values for glucose of 0.02 mm (high affinity) and 2 mm (low affinity). The glucose derivative 3-O-methylglucose (3-O-MG) was also absorbed by two apparently constitutive systems with apparent Km values for 3-O-MG of 0.065 mm and 1.9 mm. Absorption of 3-O-MG by both freshly harvested and preincubated mycelium led to its accumulation. Freshly harvested mycelium lost accumulated 3-O-MG rapidly to water, whereas preincubated mycelium showed reduced or no leakage. The reduction in leakage due to preincubation was prevented by 5 mug/ml cycloheximide in the preincubation medium. Glucose competitively inhibited 3-O-MG uptake via the high affinity system and induced loss of previously accumulated 3-O-MG from preincubated mycelium. The uptake of both glucose and 3-O-MG was associated with a transient alkalinization of the uptake medium. It is concluded that uptake of both glucose and 3-O-MG by at least the high affinity system is energy-linked and probably mediated by proton cotransport.     

Structural evaluation of anorectic action induced by 1,5-anhydro-D-glucitol

In previous studies, 1,5-anhydro-D-glucitol (1-DG), an endogenous glucose analog, was found to significantly influence physiological feeding behavior. The relationships between the hydroxyl group positions on the pyranose ring carbons and the anorectic action induced by 1-DG and its analogs are discussed. To investigate the effects of these glucose analogs on ingestive behavior, 24 mumole of test solution was injected into the rat third cerebral ventricle immediately before normal eating time, which starts at the beginning of the dark. After initial transient hyperphagia, 1-DG suppressed feeding during the first 12-hr dark period. It prolonged postprandial intermeal interval beginning shortly after injection, but eating rate was not affected and meal size did not decrease until near the end of the normal feeding period. The incidence of drinking episodes decreased concomitant with feeding suppression. Feeding and drinking suppression were also produced by 1,2-dideoxy-D-glucose, 1,3-dideoxy-D-glucose, and 1,4-dideoxy-D-glucose, although they were less potent than 1-DG. They suppressed feeding by prolonging the postprandial intermeal interval, but did not change meal size or eating rate. The anorectic effects of 1-DG were abolished by removal of the hydroxyl group at carbon 6 and by epimerization at carbons 2, 3, and 4. These findings indicate that feeding suppression induced by 1-DG and its analogs is induced mainly by prolongation of the postprandial intermeal interval, and the presence or absence of a hydroxyl group on each carbon of 1-DG is important for its feeding suppression.     

ATP-independent glucose stimulation of sphingosine kinase in rat pancreatic islets

Sphingosine kinase (SPHK) catalyzes sphingosine 1-phosphate production, promoting cell survival and reducing apoptosis in isolated rat pancreatic islets. Glucose, the primary islet β-cell growth factor and insulin secretagogue, increased islet SPHK activity by 3- to 5-fold following acute (1 h) or prolonged (7 days) stimulation. Prolonged stimulation of islets with glucose induced SPHK1a and SPHK2 mRNA levels; there were no changes in SPHK protein expression. To isolate the metabolic effects of glucose on SPHK activation, islets were stimulated with glucose analogs or metabolites. 2-deoxy-D-glucose (2-DG), an analog phosphorylated by glucokinase but not an effective energy source, activated SPHK similarly to glucose. In contrast, 3-o-methylglucose (3-oMeG), which is transported but neither phosphorylated nor metabolized, did not increase islet SPHK activity. Glyceraldehyde and α-ketoisocaproic acid (KIC), metabolites that stimulate glycolysis and the citric acid cycle, respectively, did not activate islet SPHK. Moreover, inorganic phosphate blocked glucose-induced SPHK activation. A role for SPHK activity in β-cell growth was confirmed when small interfering (si)SPHK2 RNA transfection reduced rat insulinoma INS-1e cell SPHK levels and activity and cell growth. Glucose induced an early and sustained increase in islet SPHK activity that was dependent on glucose phosphorylation, but independent of ATP generation or new protein biosynthesis. Glucose-supported β-cell growth appears to be in part mediated by SPHK activity.     

Persistent Overexpression of Phosphoglycerate Mutase,a Glycolytic Enzyme,Modifies Energy Metabolism and Reduces Stress Resistance of Heart in Mice

Background

Heart failure is associated with changes in cardiac energy metabolism. Glucose metabolism in particular is thought to be important in the pathogenesis of heart failure. We examined the effects of persistent overexpression of phosphoglycerate mutase 2 (Pgam2), a glycolytic enzyme, on cardiac energy metabolism and function.

Methods and Results

Transgenic mice constitutively overexpressing Pgam2 in a heart-specific manner were generated, and cardiac energy metabolism and function were analyzed. Cardiac function at rest was normal. The uptake of analogs of glucose or fatty acids and the phosphocreatine/βATP ratio at rest were normal. A comprehensive metabolomic analysis revealed an increase in the levels of a few metabolites immediately upstream and downstream of Pgam2 in the glycolytic pathway, whereas the levels of metabolites in the initial few steps of glycolysis and lactate remained unchanged. The levels of metabolites in the tricarboxylic acid (TCA) cycle were altered. The capacity for respiration by isolated mitochondria in vitro was decreased, and that for the generation of reactive oxygen species (ROS) in vitro was increased. Impaired cardiac function was observed in response to dobutamine. Mice developed systolic dysfunction upon pressure overload.

Conclusions

Constitutive overexpression of Pgam2 modified energy metabolism and reduced stress resistance of heart in mice.     

Ileal resection and dietary content of sucrose influence the intestinal uptake of monosaccharides

The intestinal uptake of 0.5 and 40 mM glucose, galactose, and 3-O-methyl glucose (3-O-MG) was examined in vitro in rabbits fed a high (HS) or a low (LS) sucrose diet. In animals with an intact intestinal tract, the jejunal uptake of 0.5 mM 3-O-MG was unaffected by the dietary content of sucrose, whereas the uptake of 40 mM 3-O-MG was lower in LS than HS. The uptake of 40 mM galactose was higher in LS than HS and the uptake of 0.5 mM galactose was similar in HS and LS, whereas the uptake of 0.5 mM but not 40 mM glucose was lower in LS than HS. In animals subjected 6 weeks previously to an ileal resection, the adaptive changes in the jejunal uptake of the hexoses in response to alterations in the dietary content of sucrose differed from the changes observed in rabbits with an intact intestinal tract. For example, feeding HS to ileal resected animals was associated with increased jejunal uptake of 40 mM galactose, decreased uptake of 40 mM glucose, and unchanged uptake of 40 mM 3-O-MG; whereas in control animals with an intact intestinal tract, feeding HS resulted in increased uptake of 40 mM 3-O-MG, decreased uptake of 40 mM galactose, and no change in the uptake of 40 mM glucose. A similar adaptive pattern was noted in the jejunum and ileum for the effect of dietary sucrose on the uptake of 0.5 and 40 mM glucose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of leptin secretion from white adipocytes by insulin, glycolytic substrates, and amino acids

The aim of the present study was to determine the respective roles of energy substrates and insulin on leptin secretion from white adipocytes. Cells secreted leptin in the absence of glucose or other substrates, and addition of glucose (5 mM) increased this secretion. Insulin doubled leptin secretion in the presence of glucose (5 mM), but not in its absence. High concentrations of glucose (up to 25 mM) did not significantly enhance leptin secretion over that elicited by 5 mM glucose. Similar results were obtained when glucose was replaced by pyruvate or fructose (both 5 mM). L-Glycine or L-alanine mimicked the effect of glucose on basal leptin secretion but completely prevented stimulation by insulin. On the other hand, insulin stimulated leptin secretion when glucose was replaced by L-aspartate, L-valine, L-methionine, or L-phenylalanine, but not by L-leucine (all 5 mM). Interestingly, these five amino acids potently increased basal and insulin-stimulated leptin secretion in the presence of glucose. Unexpectedly, L-glutamate acutely stimulated leptin secretion in the absence of glucose or insulin. Finally, nonmetabolizable analogs of glucose or amino acids were without effects on leptin secretion. These results suggest that 1) energy substrates are necessary to maintain basal leptin secretion constant, 2) high availability of glycolysis substrates is not sufficient to enhance leptin secretion but is necessary for its stimulation by insulin, 3) amino acid precursors of tricarboxylic acid cycle intermediates potently stimulate basal leptin secretion per se, with insulin having an additive effect, and 4) substrates need to be metabolized to increase leptin secretion.     

Transient knockdown and overexpression reveal a developmental role for the zebrafish enosf1b gene

Background

Cancer cells possess unique metabolic phenotypes that are determined by their underlying oncogenic pathways. Activation of the PI3K/Akt/mTOR signaling cascade promotes glycolysis and leads to glucose-dependence in tumors. In particular, cells with constitutive mTORC1 activity secondary to the loss of TSC1/TSC2 function are prone to undergo apoptosis upon glucose withdrawal in vitro, but this concept has not been tested in vivo. This study examines the effects of restricting glucose metabolism by pharmacologic and dietary means in a tuberous sclerosis complex (TSC) tumor xenograft model.

Results

Tumor-bearing mice were randomly assigned to receive unrestricted carbohydrate-free ("Carb-free") or Western-style diet in the absence or presence of 2-deoxyglucose (2-DG) in one of four treatment groups. After 14 weeks, tumor sizes were significantly different among the four treatment groups with those receiving 2-DG having the smallest tumors. Unexpectedly, the "Carb-free" diet was associated with the largest tumors but they remained responsive to 2-DG. PET imaging showed significant treatment-related changes in tumor ¹⁸fluorodeoxyglucose-uptake but the standard uptake values did not correlate with tumor size. Alternative energy substrates such as ketone bodies and monounsaturated oleic acid supported the growth of the Tsc2-/- cells in vitro, whereas saturated palmitic acid was toxic. Correspondingly, tumors in the high-fat, "Carb-free" group showed greater necrosis and liquefaction that contributed to their larger sizes. In contrast, 2-DG treatment significantly reduced tumor cell proliferation, increased metabolic stress (i.e., ketonemia) and AMPK activity, whereas rapamycin primarily reduced cell size.

Conclusions

Our data support the concept of glycolytic inhibition as a therapeutic approach in TSC whereas dietary withdrawal of carbohydrates was not effective.     

Effects of 2-deoxy-D-glucose on the immune system of seabream (Sparus aurata L.)

Stressful situations are a major problem in aquaculture because they affect the immune system. 2-Deoxy-d-glucose (2-DG) is a derivative of a glucose analogue that reduces the availability of energy, thereby inhibiting cell metabolism so that it is unable to enter the glycolysis pathway. In this paper, 2-DG has been administered in order to study if the immune function is compromised during metabolic stress. Blood glucose level was measured as an indicator of the inhibition of glycolysis, and the effects of intraperitoneal administration of 2-DG on the main parameters of the humoral (complement, IgM levels and peroxidase activity in blood plasma) and cellular (respiratory burst, intracellular peroxidase level and phagocytosis activity) immune parameters of gilthead seabream (Sparus aurata, L) were evaluated. Furthermore, the expression levels of immune-associated genes (CSF-1R, NCCRP-1, Hep, TCR-β, IgM_H, MHC-IIα, C3 and IL-1β) were analyzed by real-time PCR in head-kidney. A total of 5 intraperitoneal injections were performed at 48 h intervals. Three experimental groups were established: a control group injected with phosphate buffer saline, group 2-DG 500 and group 2-DG 750 injected with 500 mg kg^?1 and 750 mg kg^?1 2-DG, respectively (N = 15). After the third and fourth injection, some specimens of both DG-treated groups died. Following the first and third injection, the blood glucose levels of both 2-DG treated groups increased to a statistically significant extent with respect to the control group. While the humoral immune parameters were not significantly affected as a consequence of 2-DG administration, the cellular activities of leucocytes were. The injection of 500 mg kg^?1 2-DG provoked up- or down-regulation of the immune-relevant genes analyzed, while the injection of 750 mg kg^?1 always caused down-regulation of these genes. The results suggest that 2-DG provokes metabolic stress, which reduces the activities carried out by immune cells (leucocytes) and induces down-regulation of the immune-relevant genes analyzed when the energy available to the cell decreases.     

In vivo effect of 2-deoxy-D-glucose on adenine nucleotide levels in the liver and skeletal muscle of rats

The present report indicates that 2-deoxy-D-glucose (2-DG) at a single dose causing reduction of Tre has no influence on liver and skeletal muscle content of ATP, ADP and AMP, the ATP/ADP ratio, energy charge potential (ECP) and total adenine nucleotides (TAN). After administration of 2-DG for 3) successive days, the level of ATP, ATP/ADP ratio, the values of ECP and TAN are decreased both in the liver and skeletal muscle. However, 72 hours after the last injection of 2-DG adenine nucleotide contents returned to the values observed in control group, indicating that the in vivo effect of this glucose analogue is fully reversible.     

MuRF1-dependent regulation of systemic carbohydrate metabolism as revealed from transgenic mouse studies

Under various pathophysiological muscle-wasting conditions, such as diabetes and starvation, a family of ubiquitin ligases, including muscle-specific RING-finger protein 1 (MuRF1), are induced to target muscle proteins for degradation via ubiquitination. We have generated transgenic mouse lines over-expressing MuRF1 in a skeletal muscle-specific fashion (MuRF1-TG mice) in an attempt to identify the in vivo targets of MuRF1. MuRF1-TG lines were viable, had normal fertility and normal muscle weights at eight weeks of age. Comparison of quadriceps from MuRF1-TG and wild type mice did not reveal elevated multi-ubiquitination of myosin as observed in human patients with muscle wasting. Instead, MuRF1-TG mice expressed lower levels of pyruvate dehydrogenase (PDH), a mitochondrial key enzyme in charge of glycolysis, and of its regulator PDK2. Furthermore, yeast two-hybrid interaction studies demonstrated the interaction of MuRF1 with PDH, PDK2, PDK4, PKM2 (all participating in glycolysis) and with phosphorylase β (PYGM) and glycogenin (both regulating glycogen metabolism). Consistent with the idea that MuRF1 may regulate carbohydrate metabolism, MuRF1-TG mice had twofold elevated insulin blood levels and lower hepatic glycogen contents. To further examine MuRF1's role for systemic carbohydrate regulation, we performed glucose tolerance tests (GTT) in wild type and MuRF1-TG mice. During GTT, MuRF1-TG mice developed striking hyperinsulinaemia and hepatic glycogen stores, that were depleted at basal levels, became rapidly replenished. Taken together, our data demonstrate that MuRF1 expression in skeletal muscle re-directs glycogen synthesis to the liver and stimulates pancreatic insulin secretion, thereby providing a regulatory feedback loop that connects skeletal muscle metabolism with the liver and the pancreas during metabolic stress.     

Properties of the 3-o-methyl-D-glucose transport system in Acholeplasma laidlawii.

Transport of 3-O-methyl-D-glucose (3-O-MG) by Acholeplasma laidlawii cells was studied. The 3-O-MG transport system appeared to be constitutive in cells grown on 3-O-MG and glucose; the transport process depended on the concentration of substrate used and exhibited typical saturation kinetics, with an apparent Km of 4.6 muM. 3-O-MG was transported as a free carbohydrate and was not metabolized further in the cell. Dependence on pH and temperature and the results of efflux and "counterflow" experiments demonstrated the carrier nature of the transport system. 6-Deoxyglucose and glucose competitively inhibited 3-O-MG transport, whereas maltose inhibited in non-competitively. p-Chloromercuribenzoate, p-chloromercuribenzene sulfonate, N-ethylmaleimide, and iodoacetate inhibited transport of 3-O-MG. Cells were able to accumulate 3-O-MG against a concentration gradient. Some electron transfer inhibitors (rotenone and amytal), arsenate, dicyclohexylcarbodiimide, and proton conductors such as 2,4-dinitrophenol, carbonylcyanide, m-chlorophenylhydrazone, pentachlorophenol, and tetrachlorotrifluoromethylbenzimidazole inhibited this process.     

The tricarboxylic acid cycle and glycolysis in relation to ion transport by the ciliary body

1. The respiration and aerobic glycolysis of pig ciliary processes in oxygenated phosphate and bicarbonate buffers have been investigated. 2. Significant amounts of lactic acid are produced only in the presence of added glucose, but this does not change the endogenous respiration rate. 3. Succinate and citrate increase the oxygen uptake considerably, but pyruvate has almost no effect; oxaloacetate and fumarate stimulate slightly in the presence of glucose. Aspartate and fumarate together stimulate pyruvate utilization and are oxidized as fast as citrate. 4. Ouabain inhibits the oxidation of glucose and other substrates by limiting the ADP supply from the sodium transport system. Cyanide and azide inhibit respiration and stimulate glycolysis. 5. The transport mechanism depends largely on ATP from oxidative phosphorylation and regulates the rate of respiration and glycolysis by controlling ADP production from the Na(+)-K(+)-activated adenosine triphosphatase.     

Critical Role of H2O2 Generated by NOX4 during Cellular Response under Glucose Deprivation

Glucose is the most efficient energy source, and various cancer cells depend on glycolysis for energy production. For maintenance of survival and proliferation, glucose sensing and adaptation to poor nutritional circumstances must be well organized in cancer cells. While the glucose sensing machinery has been well studied in yeasts, the molecular mechanism of glucose sensing in mammalian cells remains to be elucidated. We have reported glucose deprivation rapidly induces AKT phosphorylation through PI3K activation. We assumed that regulation of AKT is relevant to glucose sensing and further investigated the underlying mechanisms. In this study, AKT phosphorylation under glucose deprivation was inhibited by galactose and fructose, but induced by 2-deoxyglucose (2-DG). Both 2-DG treatment and glucose deprivation were found to induce AKT phosphorylation in HepG2 cells. These findings suggested that glucose transporter may not be involved in the sensing of glucose and induction of AKT phosphorylation, and that downstream metabolic events may have important roles. A variety of metabolic stresses reportedly induce the production of reactive oxygen species (ROS). In the present study, glucose deprivation was found to induce intracellular hydrogen peroxide (H₂O₂) production in HepG2 cells. N-acetylcysteine (NAC), an antioxidant reagent, reduced both the increase in cellular H₂O₂ levels and AKT phosphorylation induced by glucose deprivation. These results strongly suggest that the glucose deprivation-induced increase of H₂O₂ in the cells mediated the AKT phosphorylation. RNA interference of NOX4, but not of NOX5, completely suppressed the glucose deprivation-induced AKT phosphorylation as well as increase of the intracellular levels of ROS, whereas exogenous H₂O₂ could still induce AKT phosphorylation in the NOX4-knockdown cells. In this study, we demonstrated that the ROS generated by NOX4 are involved in the intracellular adaptive responses by recognizing metabolic flux.     

Effects of NO-Generating Compounds on Synaptosomal Energy Metabolism

Abstract: The effects of nitroprusside and S -nitrosocysteine, compounds that generate nitric oxide (NO), on synaptosomal energy-producing pathways and energy level were investigated. The decrease in respiration was much faster and more pronounced with S -nitrosocysteine than with nitroprusside. S -Nitrosocysteine, at 10 µ M , inhibited by 80% respiration with glucose and succinate (plus rotenone) in intact synaptosomes and with ascorbate/cytochrome c in broken preparations. Oxygenated hemoglobin reversed and/or prevented the inhibition, whereas glutathione (GSH) prolonged it. Under aerobic conditions, the synaptosomal energy level (creatine phosphate/creatine and ATP/ADP ratios) was reduced by the presence of S -nitrosocysteine, whereas lactate generation was enhanced. The effects on energy parameters were greater at 5 min than at 15 min of incubation and were more pronounced in the presence of GSH. Under strictly anaerobic conditions, lactate production was reduced by the NO-generating compounds in a concentration-dependent manner. It is concluded that (a) inhibition of oxidative phosphorylation by NO leads to a fall in the synaptosomal energy level, which in turn stimulates glycolysis; (b) glycolysis can be inhibited by higher concentrations of the radical; and (c) inhibitory effects on the energy-generating pathway and ATP level could contribute to NO toxicity under some in vivo situations.