Two-compartment model for determination of glycolytic rates of solid tumors by in vivo 13C NMR spectroscopy

Carbon-13 NMR spectroscopy of 13C enriched substrates is useful for non-invasively determining metabolic fluxes of cells and tissues. Our study demonstrates that for RIF-1 tumor cells, examined under monolayer culture with continuous perfusion and also grown as solid subcutaneously (sc) implanted tumors in vivo, the levels of intracellular glucose and intermediates of the glycolytic pathway are below the level of detection by NMR spectroscopy. For these tumors, glucose transport into the cell is the most probable rate limiting step of the glycolytic pathway. Under these limiting conditions a simple two-compartment model of glycolysis applies. This model yields two parameters: the average rate of glycolysis and the rate of lactate clearance through the vasculature. For the RIF-1 tumor these parameters were 0.022 +/- 0.01 and 0.034 +/- 0.006 min(-1), respectively.     

RETRACTED: Penfluridol triggers mitochondrial-mediated apoptosis and suppresses glycolysis in colorectal cancer cells through down-regulating hexokinase-2

Penfluridol, a commonly used antipsychotic agent in a clinical setting, exhibits potential anticancer properties against various human malignancies. Here, we investigated the effect of penfluridol on the biological behavior of colorectal cancer (CRC) cells. Cell viability and clonogenic potential were detected by the cell counting kit-8 and colony formation assay. The cell apoptosis and cell cycle distribution were quantified through flow cytometry. Caspase-3 activity, glucose consumption, lactate production, and intracellular ATP levels were evaluated using the corresponding commercial detection kits. The protein levels of related genes were detected through western blotting. Mitochondrial membrane potential was detected using JC-1 staining. A CRC xenograft tumor model was used to validate the antitumor activity of penfluridol in vivo. Penfluridol reduced cell survival and promoted apoptotic cell death effectively through the mitochondria-mediated intrinsic pathway in a dose-dependent manner. Furthermore, the process of glycolysis in HCT-116 and HT-29 cells was inhibited upon penfluridol treatment, as evidenced by the decrease in glucose consumption, lactate production, and intracellular ATP levels. Further mechanistic studies revealed that penfluridol influenced cell apoptosis and glycolysis in CRC cells by downregulating hexokinase-2 (HK-2). The proapoptotic effect and glycolytic inhibition-induced by penfluridol were effectively reversed by HK-2 overexpression. Consistent with in vitro results, penfluridol could also suppress tumor growth and trigger apoptosis in vivo. Penfluridol triggers mitochondrial-mediated apoptosis and induces glycolysis inhibition via modulating HK-2 in CRC and provides a theoretical basis to support penfluridol as a repurposed drug for CRC patients.     

Glycolytic genes in cancer cells are more than glucose metabolic regulators

Nearly a hundred years of scientific research has revealed a notable preference of cancer cells to utilize aerobic glycolysis rather than mitochondrial oxidative phosphorylation for glucose-dependent ATP production, which is thought to be the root of tumor formation and growth. Glycolysis is a complex biochemical process that is mediated by multiple glycolytic genes. Besides regulating glucose metabolism, these genes are also suggested to possess various other functions related to cancer, including roles in cancer development and promotion, inhibition of apoptosis, cell cycle progression, and tumor metastasis. This article highlights the biological functions of glycolytic genes beyond their role in regulation of glycolysis and discusses their clinical implications, especially in regard to the use of glycolytic genes as biomarkers for early detection of cancer or as targets for novel anticancer treatments.     

13C high-resolution-magic angle spinning MRS reveals differences in glucose metabolism between two breast cancer xenograft models with different gene expression patterns

Tumor cells have increased glycolytic activity, and glucose is mainly used to form lactate and alanine, even when high concentrations of oxygen are present (Warburg effect). The purpose of the present study was to investigate glucose metabolism in two xenograft models representing basal-like and luminal-like breast cancer using (13) C high-resolution-magic angle spinning (HR-MAS) MRS and gene expression analysis. Tumor tissue was collected from two groups for each model: untreated mice (n=19) and a group of mice (n=16) that received an injection of [1-(13) C]-glucose 10 or 15 min before harvesting the tissue. (13) C HR-MAS MRS was performed on the tumor samples and differences in the glucose/alanine (Glc/Ala), glucose/lactate (Glc/Lac) and alanine/lactate (Ala/Lac) ratios between the models were studied. The expression of glycolytic genes was studied using tumor tissue from the same models. In the natural abundance MR spectra, a significantly lower Glc/Ala and Glc/Lac ratio (p<0.001) was observed in the luminal-like model compared with the basal-like model. In the labeled samples, the predominant glucose metabolites were lactate and alanine. Significantly lower Glc/Ala and Glc/Lac ratios were observed in the luminal-like model (p<0.05). Most genes contributing to glycolysis were expressed at higher levels in the luminal-like model (fdr<0.001). The lower Glc/Ala and Glc/Lac ratios and higher glycolytic gene expression observed in the luminal-like model indicates that the transformation of glucose to lactate and alanine occurred faster in this model than in the basal-like model, which has a growth rate several times faster than that of the luminal-like model. The results from the present study suggest that the tumor growth rate is not necessarily a determinant of glycolytic activity.     

Clotrimazole disrupts glycolysis in human breast cancer without affecting non-tumoral tissues

Human breast cancer tissues, as well as normal tissues from the same patients, were treated with clotrimazole (CTZ) and have their capacities for glucose consumption and lactate production evaluated. This treatment strongly decreased the lactate production rate by tumor tissues (85% inhibition) without affecting the other measurements made, i.e. lactate production by control tissues or glucose consumption by both, control and tumor tissues. This result directly correlates with the inhibition promoted by CTZ on the activity of the major regulatory glycolytic enzyme 6-phosphofructo-1-kinase (PFK) that was observed in tumor tissues (84% inhibition) but not in control tissues. Fractionation of the tissues revealed that this inhibition does not occur in the soluble fraction of the enzyme, but is exclusive of a particulate fraction. It has been previously shown that the particulate fraction of PFK activity in tumors is associated to actin filaments (f-actin). Thus, we investigated whether CTZ would affect the association between PFK and f-actin and we found that the drug directly induces the dissociation of the two proteins in the same extent that it inhibits lactate production, total PFK activity and the particulate PFK activity. We concluded that CTZ disrupts glycolysis on human breast tumor tissues, inhibiting PFK activity by dissociating the enzyme from f-actin.     

Robust glycogen shunt activity in astrocytes: Effects of glutamatergic and adrenergic agents

The significance and functional roles of glycogen shunt activity in the brain are largely unknown. It represents the fraction of metabolized glucose that passes through glycogen molecules prior to entering the glycolytic pathway. The present study was aimed at elucidating this pathway in cultured astrocytes from mouse exposed to agents such as a high [K⁺], d-aspartate and norepinephrine (NE) known to affect energy metabolism in response to neurotransmission. Glycogen shunt activity was assessed employing [1,6-¹³C]glucose, and the glycogen phosphorylase inhibitor 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) to block glycogen degradation. The label intensity in lactate, reflecting glycolytic activity, was determined by mass spectrometry. In the presence of NE a substantial glycogen shunt activity was observed, accounting for almost 40% of overall glucose metabolism. Moreover, when no metabolic stimulant was applied, a compensatory increase in glycolytic activity was seen when the shunt was inhibited by DAB. Actually the labeling in lactate exceeded that obtained when glycolysis and glycogen shunt both were operational, i.e. supercompensation. A similar phenomenon was seen when astrocytes were exposed to d-aspartate. In addition to glycolysis, tricarboxylic acid (TCA) cycle activity was monitored, analyzing labeling by mass spectrometry in glutamate which equilibrates with α-ketoglutarate. Both an elevated [K⁺] and d-aspartate induced an increased TCA cycle activity, which was altered when glycogen degradation was inhibited. Thus, the present study provides evidence that manipulation of glycogen metabolism affects both glycolysis and TCA cycle metabolism. Altogether, the results reveal a highly complex interaction between glycogenolysis and glycolysis, with the glycogen shunt playing a significant role in astrocytic energy metabolism.     

Stimulation of Glucose Metabolism in Rat Mast Cells by Antigen,Dextran and Compound 48/80, Used as Histamine Releasing Agents

Histamine release in aerobic medium from rat peritoneal mast cells by anaphylactic reaction, dextran and compound 48/80 was associated with a stimulation of exogenous glucose metabolism as determined by carbon dioxide and lactate production. There was generally a correlation between the amount of histamine released and the degree of metabolic stimulation. In anaerobic medium a stimulation of lactate production was observed in most of the experiments when histamine was released by anaphylactic reaction and dextran. Compound 48/80-induced histamine release in presence of oligomycin was not, however, associated with any appreciable change in lactate production. Galactose and fructose were metabolized by rat mast cells to CO2 and lactate, but the rate of CO2 production was only 18 % and 29 %, respectively, and that of lactate production (aerobic) 5 % and 3.6 %, respectively, as compared to the metabolism of glucose to these products under the same conditions. 80 mM galactose could partially reverse the cyanide inhibition of histamine release while the same concentration of fructose was ineffective.     

Effects of exposure to a simulated altitude of 5500 m on energy metabolic pathways in rats

We examined the effect of exposure to 5500 m on three closely related metabolic pathways: anaerobic glycolysis, the pentose phosphate shunt (PPS), and fatty acid metabolism. Rats were exposed to simulated altitude of 5500 m for up to 3 months. The maximal rate of lactate production in tissue homogenates, tissue lactic acid dehydrogenase and blood lactate levels were measured to evaluate the capacity for anaerobic glycolysis. The uptake of 14C-1-palmitate, oxidation of 14C-1-palmitate to 14CO2, incorporation of 14C-1-palmitate into tissue lipids, plasma and tissue free fatty acids (FFA) levels and total lipid contents were measured to assess the magnitude of lipid metabolism. Activities of glucose-6-phosphate dehydrogenase (G-6-PD) and 6-phophogluconate dehydrogenase (6-PGD) in the PPS pathway were measured to assess the capacity to generate reducing power. Acute and chronic hypoxia did not affect most of the measurements of anaerobic glycolysis, but depressed lactate production in liver and kidney. Chronic hypoxia enhanced all aspects of lipid metabolism in liver and enhanced the uptake and oxidation to CO2 of palmitate in skeletal muscle. Chronic hypoxia did not alter the activity of the G-6-PD in any tissue studied, but the activity of 6-PGD was depressed in heart, kidney, thymus and adrenal gland. The lack of major changes in the capacities of anaerobic glycolytic pathways and the activities of the PPS dehydrogenases is consistent with the maintenance of normal aerobic metabolism in rats at 5500 m. We found no evidence that anaerobic metabolic processes were upregulated to sustain energy consumption during chronic hypoxia. On the other hand, enhanced fatty acid metabolism may spare carbohydrate for metabolic fuel under conditions of extreme hypoxic limitation.     

Modulation of M2-type pyruvate kinase activity by the cytoplasmic PML tumor suppressor protein

The promyelocytic leukemia (PML) tumor suppressor protein accumulates in PML nuclear bodies (PML-NBs), and can induce growth arrest, cellular senescence and apoptosis. PML has also been localized in the cytoplasm, although its function in this localization remains elusive. A general property of primary cancers is their high glycolytic rate which results from increased glucose consumption. However, the mechanism by which cancer cells up-regulate glycolysis is not well understood. Here, we have shown that cytoplasmic PML (cPML) directly interacts with M2-type pyruvate kinase (PKM2), a key regulator of carbon fate. PKM2 determines the proportion of carbons derived from glucose that are used for glycolytic energy production. Over-expression of PML-2KA mutant in the cytoplasm, which was generated by mutagenesis of the nuclear localization signals of PML, in MCF-7 breast cancer cells suppressed PKM2 activity and the accumulation of lactate. PKM2 exists in either an active tetrameric form which has high affinity for its substrate phosphoenolpyruvate (PEP) or a less active dimeric form which has low affinity for its substrate. Over-expression of PML-2KA suppressed the activity of the tetrameric form of PKM2, but not the dimeric form. Our findings suggest that cPML plays a role in tumor metabolism through its interaction with PKM2.     

The Metabolism of Monosaccharides in Isolated Rat Mast Cells and its Influence on Histamine Release Induced by Adenosine-5‘-Triphosphate.

Histamine release from isolated rat mast cells induced by extracellularly applied ATP in the presence of calcium was enhanced by D(+)glucose and D(+)mannose, but not by D(-)-fructose or D(+) galactose (0.6 mM of each). Only those monosaccharides which enhanced the histamine release were metabolized by the mast cells through the Embden-Meyerhof pathway as judged from the accumulation of lactate in samples incubated with the sugars. Lactate (1 mM) as well as pyruvate (1 mM) also enhanced the histamine release induced by ATP. Antimycin A (10^-7M) completely inhibited ATP-induced histamine release irrespectively of the presence of glucose, pyruvate or lactate. Iodoacetate (10^-4M) blocked the enhancing effect of glucose, but not that of pyruvate or lactate. In the presence of glucose (0.6 mM) or mannose (0.6 mM) the accumulation of lactate in suspensions of rat mast cells was enhanced by antimycin A (10^-7M) and blocked by iodoacetate (10^-4M). In the absence of glycolytic substrates and metabolic inhibitors no accumulation of lactate was noted whereas in the presence of antimycin A (10^-7M) a slight but statistically significant accumulation occurred.     

Nonoxidative pentose phosphate pathways and their direct role in ribose synthesis in tumors: is cancer a disease of cellular glucose metabolism?

Pentose phosphate pathways (PPP) are considered important in tumor proliferation processes because of their role in supplying tumor cells with reduced NADP and carbons for intracellular anabolic processe. Direct involvement of PPP in tumor DNA/RNA synthesis is not considered as significant as in lipid and protein syntheses. Currently, PPP activity in tumor cells is measured by lactate production, which shows a moderate activity: about 4% to 7% compared with glycolysis. Recent data generated in our laboratory indicate that PPP are directly involved in ribose synthesis in pancreatic adenocarcinoma cells, through oxidative steps (< 31 %) and transketolase reactions (69%). These findings raise serious questions about the adequacy of lactate in measuring PPP activity in tumors. We hypothesize that ribose, not lactate, is the major product of PPP in tumor cells. Control of both oxidative and nonoxidative PPP may be critical in the treatment of cancer. PPP are substantially involved in the proliferation of human tumors, which raises the prospect of new treatment strategies targeting specific biochemical reactions of PPP by hormones related to glucose metabolism, controlling thiamine intake, the cofactor of the nonoxidative transketolase PPP reaction, or treating cancer patients with antithiamine analogues.     

Effect of lonidamine on the utilization of 14C-labeled glucose by human astrocytoma cells

The effect of lonidamine (LND), 1-(2,4-dichlorobenzyl)-1H-indazol-3 carboxylic acid, on the utilization of carbon from 14C-labeled glucose by cell cultures of the permanent strain LI derived from a human glioblastoma multiforme (astrocytoma) has been investigated. The results may be summarized as follows. Aerobic glycolysis is the main energy-yielding process as shown by the fact that the greatest part of glucose carbon atoms is incorporated into lactate. Nevertheless, the amount of glucose converted accounts for only 63% of the lactate produced, indicating the presence of an elevated endogenous aerobic glycolysis. The amount of glucose carbon atoms incorporated into CO2, lipids, nucleic acid, and supporting structures is low. LND decreased the incorporation of 14C activity in all the above mentioned isolated compounds because of its ability to inhibit glucose phosphorylation. Consequently, there is a lower concentration of glucose-6-phosphate which, in turn, affects the rate of formation of several metabolites in glycolytic and pentose phosphate pathways. Experiments with [1-14C]-2-deoxy-D-glucose further substantiate the idea of glucose phosphorylation as a main target of LND and strongly suggest the presence of a mitochondrially bound hexokinase. The higher inhibition of glucose phosphorylation in exponentially growing cells indicates a further shift of the enzyme toward mitochondria-bound form and confirms the importance of the energy status of the cell in eliciting the response to LND. The reduced capacity of LND-treated cells to synthetize ATP and glucose-6-phosphate reflects the decreased synthesis of proteins and nucleic acids, which affects cell growth and duplication.     

Announcements

During red cell storage the pH of the medium and that of the intracellular fluid decreases. The decrease in pH is due to glycolysis and is a likely contributor to red cell storage lesion. The trisodium salt of 3-phosphoglycerate (PGA) would be an ideal non-acid producing fuel for stored red cells as it would enter the glycolytic pathway below the acid-producing steps (glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase), and in comparison with glucose, would enable the production of equivalent amounts of ATP per carbon of the fuel. A medium containing PGA (PGA) was evaluated for 21 days in comparison with a standard dextrose-phosphate-citrate additive solution (DCP) and a medium containing minimal fuel (NF). Fuel utilisation, lactate and pyruvate production, pH, adenylates, Hb, GSH, Na⁺ and K⁺ were measured, and in vitro incubations were used to assess the potential for flux through glycolysis and the pentose pathway. DCP was the superior medium, although this is not evident for the first 7 days. 3-Phosphoglycerate did enter the cells, but was metabolised too slowly to be a useful fuel. The cells in PGA were similar to those in DCP in one respect in that they maintained their pentose pathway potential. Apart from flux through the pentose pathway, PGA and NF were identical in that all tests indicated substantial storage lesion.     

Pim-2 Modulates Aerobic Glycolysis and Energy Production during the Development of Colorectal Tumors

Tumor cells have higher rates of glucose uptake and aerobic glycolysis to meet energy demands for proliferation and metastasis. The characteristics of increased glucose uptake, accompanied with aerobic glycolysis, has been exploited for the diagnosis of cancers. Although much progress has been made, the mechanisms regulating tumor aerobic glycolysis and energy production are still not fully understood. Here, we demonstrate that Pim-2 is required for glycolysis and energy production in colorectal tumor cells. Our results show that Pim-2 is highly expressed in colorectal tumor cells, and may be induced by nutrient stimulation. Activation of Pim-2 in colorectal cells led to increase glucose utilization and aerobic glycolysis, as well as energy production. While knockdown of Pim-2 decreased energy production in colorectal tumor cells and increased their susceptibility to apoptosis. Moreover, the effects of Pim-2 kinase on aerobic glycolysis seem to be partly dependent on mTORC1 signaling, because inhibition of mTORC1 activity reversed the aerobic glycolysis mediated by Pim-2. Our findings suggest that Pim-2-mediated aerobic glycolysis is critical for monitoring Warburg effect in colorectal tumor cells, highlighting Pim-2 as a potential metabolic target for colorectal tumor therapy.     

The effect of glucose metabolism on murine follicle development and steroidogenesis in vitro

Previous studies have shown that mouse ovarian follicles producelarge amounts of lactate during growth and maturation in vitro,suggesting a metabolic preference for glycolysis. Further investigationswere therefore undertaken to determine firstly the effect ofgonadotrophins on glucose uptake and secondly the role of glucosein follicle lactate production, development and steroidogenesis.Pre-antral mouse follicles were cultured individually in mediacontaining different concentrations of glucose (1–5.5mM) using a system which supported development to the pre-ovulatorystage within 5 days. Samples of media were removed every 24h and analysed for oestradiol using an enzyme-linked immunosorbentassay technique and for glucose uptake and lactate productionusing a fluorimetric assay. Results showed that all the glucoseconsumed was converted to lactate, irrespective of the glucoseconcentration. Growth and steroidogenesis were also dependenton glucose concentration, and at concentrations below 2 mM,follicle development was significantly retarded. Results confirmthat follicles adopt a predominantly glycolytic mode of energyproduction to sustain growth and steroidogenesis, and that theglycolytic rate is stimulated by gonadotrophins.     

The 1H NMR visibility of intracellular lactate in Streptococcus faecalis

1H NMR studies of glycolysis in washed cell suspensions of Streptococcus faecalis indicated that intracellular lactate is not 1H NMR visible. Evidence for this was gained from time course studies of glycolysis at increasing concentrations of glucose. A close correlation existed between the relative increase in the lactate integral and the enzymatically determined extracellular lactate concentration [Lo]. When ionophores which cause the collapse of the positive intracellular/extracellular lactate gradient were added to cell suspensions following fermentation of 5, 10 and 50 mM glucose, the increase in the lactate integral was proportional to the respective increase in [Lo]. A more direct method for determining the origin of the lactate signal involved centrifugation of a cell suspension after fermentation of 50 mM glucose and measurement of lactate in the extracellular and intracellular fluid. 1H spectra of the cell suspension, supernatant and sonicated pellet revealed that the lactate observed in the cell suspension was equivalent to the lactate in the supernatant alone. The intracellular lactate contained in the pellet represented 42% of the total lactate, indicating that only 58% of lactate is detected by in vivo 1H MRS of S. faecalis. This result is in contrast with the high percentage (70-90%) of in vitro lactate which is detected by in vivo 1H MRS of mammalian brain tissue (Williams S. R. et al. Magn. Res. Med. 7, 425-431, 1988). This may be due to a higher proportion of extracellular lactate in mammalian tissue or differences in the intracellular environments of bacterial and mammalian cells.     

Hydrogen peroxide excretion by oral streptococci and effect of lactoperoxidase-thiocyanate-hydrogen peroxide

Approved type strains of Streptococcus sanguis, S. mitis, S. mutans, and S. salivarius were grown under aerobic and anaerobic conditions. The rate of hydrogen peroxide excretion, oxygen uptake, and acid production from glucose by washed-cell suspensions of these strains were studied, and the levels of enzymes in cell-free extracts which reduced oxygen, hydrogen peroxide, or hypothiocyanite (OSCN-) in the presence of NADH or NADPH were assayed. The effects of lactoperoxidase-thiocyanate-hydrogen peroxide on the rate of acid production and oxygen uptake by intact cells, the activity of glycolytic enzymes in cell-free extracts, and the levels of intracellular glycolytic intermediates were also studied. All strains consumed oxygen in the presence of glucose. S. sanguis, S. mitis, and anaerobically grown S. mutans excreted hydrogen peroxide. There was higher NADH oxidase and NADH peroxidase activity in aerobically grown cells than in anaerobically grown cells. NADPH oxidase activity was low in all species. Acid production, oxygen uptake, and, consequently, hydrogen peroxide excretion were inhibited in all the strains by lactoperoxidase-thiocyanate-hydrogen peroxide. S. sanguis and S. mitis had a higher capacity than S. mutans and S. salivarius to recover from this inhibition. Higher activity in the former strains of an NADH-OSCN oxidoreductase, which converted OSCN- into thiocyanate, explained this difference. The change in levels of intracellular glycolytic intermediates after inhibition of glycolysis by OSCN- and the actual activity of glycolytic enzymes in cell-free extracts in the presence of OSCN- indicated that the primary target of OSCN- in the glycolytic pathway was glyceraldehyde 3-phosphate dehydrogenase.