Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT), an Enzyme Essential for NAD+ Biosynthesis,Leads to Altered Carbohydrate Metabolism in Cancer Cells

Nicotinamide phosphoribosyltransferase (NAMPT) has been extensively studied due to its essential role in NAD⁺ biosynthesis in cancer cells and the prospect of developing novel therapeutics. To understand how NAMPT regulates cellular metabolism, we have shown that the treatment with FK866, a specific NAMPT inhibitor, leads to attenuation of glycolysis by blocking the glyceraldehyde 3-phosphate dehydrogenase step (Tan, B., Young, D. A., Lu, Z. H., Wang, T., Meier, T. I., Shepard, R. L., Roth, K., Zhai, Y., Huss, K., Kuo, M. S., Gillig, J., Parthasarathy, S., Burkholder, T. P., Smith, M. C., Geeganage, S., and Zhao, G. (2013) Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), an enzyme essential for NAD⁺ biosynthesis, in human cancer cells: metabolic basis and potential clinical implications. J. Biol. Chem. 288, 3500–3511). Due to technical limitations, we failed to separate isotopomers of phosphorylated sugars. In this study, we developed an enabling LC-MS methodology. Using this, we confirmed the previous findings and also showed that NAMPT inhibition led to accumulation of fructose 1-phosphate and sedoheptulose 1-phosphate but not glucose 6-phosphate, fructose 6-phosphate, and sedoheptulose 7-phosphate as previously thought. To investigate the metabolic basis of the metabolite formation, we carried out biochemical and cellular studies and established the following. First, glucose-labeling studies indicated that fructose 1-phosphate was derived from dihydroxyacetone phosphate and glyceraldehyde, and sedoheptulose 1-phosphate was derived from dihydroxyacetone phosphate and erythrose via an aldolase reaction. Second, biochemical studies showed that aldolase indeed catalyzed these reactions. Third, glyceraldehyde- and erythrose-labeling studies showed increased incorporation of corresponding labels into fructose 1-phosphate and sedoheptulose 1-phosphate in FK866-treated cells. Fourth, NAMPT inhibition led to increased glyceraldehyde and erythrose levels in the cell. Finally, glucose-labeling studies showed accumulated fructose 1,6-bisphosphate in FK866-treated cells mainly derived from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Taken together, this study shows that NAMPT inhibition leads to attenuation of glycolysis, resulting in further perturbation of carbohydrate metabolism in cancer cells. The potential clinical implications of these findings are also discussed.     

Metabolic profile of wound-induced changes in primary carbon metabolism in sugarbeet root

Injury to plant products by harvest and postharvest operations induces respiration rate and increases the demand for respiratory substrates. Alterations in primary carbon metabolism are likely to support the elevated demand for respiratory substrates, although the nature of these alterations is unknown. To gain insight into the metabolic changes that occur to provide substrates for wound-induced increases in respiration, changes in the concentrations of compounds that are substrates, intermediates or cofactors in the respiratory pathway were determined in sugarbeet (Beta vulgaris L.) roots in the 4 days following injury. Both wounded and unwounded tissues of wounded roots were analyzed to provide information about localized and systemic changes that occur after wounding. In wounded tissue, respiration increased an average of 186%, fructose, glucose 6-phosphate, ADP and UDP concentrations increased, and fructose 1,6-bisphosphate, triose phosphate, citrate, isocitrate, succinate, ATP, UTP and NAD⁺ concentrations decreased. In the non-wounded tissue of wounded roots, respiration rate increased an average of 21%, glucose 6-phosphate, fructose 6-phosphate, glucose 1-phosphate and ADP concentrations increased, and isocitrate, UTP, NAD⁺, NADP⁺, and NADPH concentrations declined. Changes in respiration rate and metabolite concentrations indicated that localized and systemic changes in primary carbon metabolism occurred in response to injury. In wounded tissue, metabolite concentration changes suggested that activities of the early glycolytic enzymes, fructokinase, phosphofructokinase, phosphoglucose isomerase, and phosphoglucomutase were limiting carbon flow through glycolysis. These restrictions in the respiratory pathway, however, were likely overcome by use of metabolic bypasses that allowed carbon compounds to enter the pathway at glycolytic and tricarboxylic acid (TCA) cycle downstream locations. In non-wounded tissue of wounded roots, metabolic concentration changes suggested that glycolysis and the TCA cycle were generally capable of supporting the small systemic elevation in respiration rate. Although the mechanism by which respiration is regulated in wounded sugarbeet roots is unknown, localized and systemic elevations in respiration were positively associated with one or more indicators of cellular redox status.     

Interrelationships between carbohydrate metabolism and nitrogen assimilation in cultured plant cells

In sycamore cells grown on nitrate as opposed to glutamate there is a higher pentose phosphate pathway carbon flux relative to glycolysis in the early stages of cell growth when nitrate assimilation is most active. The high pentose phosphate pathway activity compared with glycolysis in nitrate grown cells is accompanied by enhanced levels of hexokinase, pyruvate kinase, glucose-6-phosphate de-hydrogenase, 6-phosphogluconate dehydrogenase and transketolase. There is no significant increase in activity of the solely glycolytic enzyme, phosphofructokinase. It is suggested that the increased pentose phosphate pathway activity in nitrate grown cells is correlated with a demand by nitrite assimilation for NADPH.II=Jessup and Fowler, 1976 b     

CONTROL OF AEROBIC GLYCOLYSIS AND PYRUVATE KINASE ACTIVITY IN CEREBRAL CORTEX SLICES

Abstract— —The concentrations of glycolytic intermediates were measured in aerobically incubated guinea pig cerebral cortex slices. Increasing the concentration of potassium in the medium increased fructose diphosphate ten-fold and triose phosphates three-fold. Omitting calcium increased all the glycolytic intermediates except pyruvate; triose phosphates were increased most. The changed patterns of the glycolytic intermediate profile in the slices are consistent with the previously proposed hypothesis that the phosphofructokinase is a main regulatory step in glycolysis. Glycolysis is also limited at the step of pyruvate kinase, which is inhibited in cerebral cortex slices. Calcium in the tissue and cellular organization of the slices were shown to be responsible for this inhibition. It was concluded that the effects of potassium and calcium on aerobic glycolysis in cerebral cortex slices are direct—on the pyruvate kinase—and also indirect. Calcium was shown to be inhibitive also to the activities of hexokinase, phosphoglucoisomerase, phosphofructokinase, glyceraldehyde 3-phosphate dehydrogenase and enolase of guinea pig cerebral tissue.     

Mode of action of alpha-chlorohydrin as a male anti-fertility agent. Inhibition of the metabolism of ram spermatozoa by alpha-chlorohydrin and location of block in glycolysis

1. The effect of alpha-chlorohydrin on the metabolism of glycolytic and tricarboxylate-cycle substrates by ram spermatozoa was investigated. The utilization and oxidation of fructose and triose phosphate were much more sensitive to inhibition by alpha-chlorohydrin (0.1-1.0mm) than lactate or pyruvate. Inhibition of glycolysis by alpha-chlorohydrin is concluded to be between triose phosphate and pyruvate formation. Oxidation of glycerol was not as severely inhibited as that of the triose phosphate. This unexpected finding can be explained in terms of competition between glycerol and alpha-chlorohydrin. A second, much less sensitive site, of alpha-chlorohydrin inhibition appears to be associated with production of acetyl-CoA from exogenous and endogenous fatty acids. 2. Measurement of the glycolytic intermediates after incubation of spermatozoal suspensions with 15mm-fructose in the presence of 3mm-alpha-chlorohydrin showed a ;block' in the conversion of glyceraldehyde 3-phosphate into 3-phosphoglycerate. alpha-Chlorohydrin also caused conversion of most of the ATP in spermatozoa into AMP. After incubation with 3mm-alpha-chlorohydrin, glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase activities were decreased by approx. 90% and 80% respectively, and in some experiments aldolase was also inhibited. Other glycolytic enzymes were not affected by a low concentration (0.3mm) of alpha-chlorohydrin. Loss of motility of spermatozoa paralleled the decrease in glyceraldehyde 3-phosphate dehydrogenase activity. alpha-Chlorohydrin, however, did not inhibit glyceraldehyde 3-phosphate dehydrogenase or triose phosphate isomerase in sonicated enzyme preparations when added to the assay cuvette. 3. Measurement of intermediates and glycolytic enzymes in ejaculated spermatozoa before, during and after injection of rams with alpha-chlorohydrin (25mg/kg body wt.) confirmed a severe block in glycolysis in vivo at the site of triose phosphate conversion into 3-phosphoglycerate within 24h of the first injection. Glyceraldehyde 3-phosphate dehydrogenase activity was no longer detectable and both aldolase and triose phosphate isomerase were severely inhibited. Spermatozoal ATP decreased by 92% at this time, being quantitatively converted into AMP. At 1 month after injection of alpha-chlorohydrin glycolytic intermediate concentrations returned to normal in the spermatozoa but ATP was still only 38% of the pre-injection concentration. Motility of spermatozoa was, however, as good as during the pre-injection period. The activity of the inhibited enzymes also returned to normal during the recovery period and 26 days after injection were close to pre-injection values. 4. An unknown metabolic product of alpha-chlorohydrin is suggested to inhibit glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase of spermatozoa. This results in a lower ATP content, motility and fertility of the spermatozoa. Glycidol was shown not to be an active intermediate of alpha-chlorohydrin in vitro.     

Some effects of glucose concentration and anoxia on glycolysis and metabolite concentrations in the perfused liver of fetal guinea pig

Effects of glucose concentration and anoxia upon the metabolite concentrations and rates of glycolysis and respiration have been investigated in the perfused liver of the fetal guinea pig. In most cases the metabolite concentrations in the perfused liver were similar to those observed in vivo. Between 50 days and term there was a fall in the respiratory rate and in the concentration of ATP and fructose 1,6-diphosphate and an increase in the concentration of glutamate, glycogen and glucose. Reducing the medium glucose concentration from 10 mM to 1 mM or 0.1 mM depressed lactate production and the concentration of most of the phosphorylated intermediates (except 6-phosphogluconate) in the liver of the 50-day fetus. This indicates a fall in glycolytic rate which is not in accord with the known kinetic properties of hexokinase in the fetal liver. Anoxia increased lactate production by, and the concentrations of, the hexose phosphates ADP and AMP in the 50-day to term fetal liver, while the concentration of ribulose 5-phosphate, ATP and some triose phosphates fell. These results are consistent with an activation of glycolysis, particularly at phosphofructokinase and of a reduction in pentose phosphate pathway activity, particularly at 6-phosphogluconate dehydrogenase.The calculated cytosolic NAD⁺/NADH ratio for the perfused liver was similar to that measured in vivo and evidence is presented to suggest that the dihydroxyacetone phosphate/glycerol 3-phosphate ratio gives a better indication of cytosolic redox than the lactate/pyruvate ratio. The present observations indicate that phosphofructokinase and hexokinase and possibly pyruvate kinase control the glycolytic rate and that glyceraldehyde-3-phosphate dehydrogenase is at equilibrium in the perfused liver of the fetal guinea pig.     

The pentose phosphate pathway of glucose metabolism. Enzyme profiles and transient and steady-state content of intermediates of alternative pathways of glucose metabolism in Krebs ascites cells

1. The pentose phosphate pathway in Krebs ascites cells was investigated for regulatory reactions. For comparison, the glycolytic pathway was studied simultaneously. 2. Activities of the pentose phosphate pathway enzymes were low in contrast with those of the enzymes of glycolysis. The K(m) values of glucose 6-phosphate dehydrogenase for both substrate and cofactor were about four times the reported upper limit for the enzyme from normal tissues. Fructose 1,6-diphosphate and NADPH competitively inhibited 6-phosphogluconate dehydrogenase. 3. About 28% of the hexokinase activity was in the particulate fraction of the cells. The soluble enzyme was inhibited by fructose 1,6-diphosphate and ribose 5-phosphate, but not by 3-phosphoglycerate. The behaviour of the partially purified soluble enzyme in vitro in a system simulating the concentrations of ATP, glucose 6-phosphate and P(i) found in vivo is reported. 4. Kinetics of metabolite accumulation during the transient state after the addition of glucose to the cells indicated two phases of glucose phosphorylation, an initial rapid phase followed abruptly by a slow phase extending into the steady state. 5. Of the pentose phosphate pathway intermediates, accumulation of 6-phosphogluconate, sedoheptulose 7-phosphate and fructose 6-phosphate paralleled the accumulation of glucose 6-phosphate. Erythrose 4-phosphate reached the steady-state concentration by 2min., whereas the pentose phosphates accumulated linearly. 6. The mass-action ratios of the pentose phosphate pathway reactions were calculated. The transketolase reaction was at equilibrium by 30sec. and then progressively shifted away from equilibrium towards the steady-state ratio. The glucose 6-phosphate dehydrogenase was far from equilibrium at all times. 7. Investigation of the flux of [(14)C]glucose carbon confirmed the existence of an operative pentose phosphate pathway in ascites cells, contributing 1% of the total flux in control cells and 10% in cells treated with phenazine methosulphate. 8. The pentose phosphate formed by way of the direct oxidative route and estimated from the (14)CO(2) yields represented 20% of the total accumulated pentose phosphate, the other 80% being formed by the non-oxidative reactions of the pentose phosphate pathway. 9. The pentose phosphate pathway appears to function as two separate pathways, both operating towards pentose phosphate formation. Control of the two pathways is discussed.     

Deficiency of Nicotinamide Mononucleotide Adenylyltransferase 3 (Nmnat3) Causes Hemolytic Anemia by Altering the Glycolytic Flow in Mature Erythrocytes

NAD biosynthesis is of substantial interest because of its important roles in regulating various biological processes. Nicotinamide mononucleotide adenylyltransferase 3 (Nmnat3) is considered a mitochondria-localized NAD synthesis enzyme involved in de novo and salvage pathways. Although the biochemical properties of Nmnat3 are well documented, its physiological function in vivo remains unclear. In this study, we demonstrated that Nmnat3 was localized in the cytoplasm of mature erythrocytes and critically regulated their NAD pool. Deficiency of Nmnat3 in mice caused splenomegaly and hemolytic anemia, which was associated with the findings that Nmnat3-deficient erythrocytes had markedly lower ATP levels and shortened lifespans. However, the NAD level in other tissues were not apparently affected by the deficiency of Nmnat3. LC-MS/MS-based metabolomics revealed that the glycolysis pathway in Nmnat3-deficient erythrocytes was blocked at a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) step because of the shortage of the coenzyme NAD. Stable isotope tracer analysis further demonstrated that deficiency of Nmnat3 resulted in glycolysis stall and a shift to the pentose phosphate pathway. Our findings indicate the critical roles of Nmnat3 in maintenance of the NAD pool in mature erythrocytes and the physiological impacts at its absence in mice.     

Effect of transketolase modifications on carbon flow to the purine-nucleotide pathway in Corynebacterium ammoniagenes

Transketolase, one of the enzymes in the nonoxidative branch of the pentose phosphate pathway, operates to shuttle ribose 5-phosphate and glycolytic intermediates together with transaldolase, and might be involved in the availability of ribose 5-phosphate, a precursor of nucleotide biosynthesis. The tkt and tal genes encoding transketolase and transaldolase, respectively, were cloned from the typical nucleotide- and nucleoside-producing organism Corynebacterium ammoniagenes by a PCR approach using oligonucleotide primers derived from conserved regions of each amino acid sequence from other organisms. Enzymatic and molecular analyses revealed that the two genes were clustered on the genome together with the glucose 6-phosphate dehydrogenase gene (zwf). The effect of transketolase modifications on the production of inosine and 5'-xanthylic acid was investigated in industrial strains of C. ammoniagenes. Multiple copies of plasmid-borne tkt caused about tenfold increases in transketolase activity and resulted in 10-20% decreased yields of products relative to the parents. In contrast, site-specific disruption of tkt enabled both producers to accumulate 10-30% more products concurrently with a complete loss of transketolase activity and the expected phenotype of shikimate auxotrophy. These results indicate that transketolase normally shunts ribose 5-phosphate back into glycolysis in these biosynthetic processes and interception of this shunt allows cells to redirect carbon flux through the oxidative pentose pathway from the intermediate towards the purine-nucleotide pathway.     

Isotopomer profiling of Leishmania mexicana promastigotes reveals important roles for succinate fermentation and aspartate uptake in tricarboxylic acid cycle (TCA) anaplerosis, glutamate synthesis, and growth

Leishmania parasites proliferate within nutritionally complex niches in their sandfly vector and mammalian hosts. However, the extent to which these parasites utilize different carbon sources remains poorly defined. In this study, we have followed the incorporation of various (13)C-labeled carbon sources into the intracellular and secreted metabolites of Leishmania mexicana promastigotes using gas chromatography-mass spectrometry and (13)C NMR. [U-(13)C]Glucose was rapidly incorporated into intermediates in glycolysis, the pentose phosphate pathway, and the cytoplasmic carbohydrate reserve material, mannogen. Enzymes involved in the upper glycolytic pathway are sequestered within glycosomes, and the ATP and NAD(+) consumed by these reactions were primarily regenerated by the fermentation of phosphoenolpyruvate to succinate (glycosomal succinate fermentation). The initiating enzyme in this pathway, phosphoenolpyruvate carboxykinase, was exclusively localized to the glycosome. Although some of the glycosomal succinate was secreted, most of the C4 dicarboxylic acids generated during succinate fermentation were further catabolized in the TCA cycle. A high rate of TCA cycle anaplerosis was further suggested by measurement of [U-(13)C]aspartate and [U-(13)C]alanine uptake and catabolism. TCA cycle anaplerosis is apparently needed to sustain glutamate production under standard culture conditions. Specifically, inhibition of mitochondrial aconitase with sodium fluoroacetate resulted in the rapid depletion of intracellular glutamate pools and growth arrest. Addition of high concentrations of exogenous glutamate alleviated this growth arrest. These findings suggest that glycosomal and mitochondrial metabolism in Leishmania promastigotes is tightly coupled and that, in contrast to the situation in some other trypanosomatid parasites, the TCA cycle has crucial anabolic functions.     

An analysis of intermediary metabolism and its control in a fat-synthesizing yeast (Candida 107) growing on glucose or alkanes.

Enzymes of glycolysis, pentose phosphate pathway, gluconeogenesis, tricarboxylate acid cycle, glyoxylate by-pass and fatty-acid biosynthesis were assayed in extracts from Candida 107 grown continuously on glucose under carbon limitation, nitrogen limitation and on n-alkanes. The yeast was therefore either in a lipogenic or lipolytic state. Phosphofructokinase was absent under all conditions whereas enzymes of gluconeogenesis, including fructose 1,6-bisphosphatase and the pentose phosphate cycle, were all present. Glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were specific for NADP+ and were inhibited in a non-competitive manner by NADPH and NADH. Phosphoenolpyruvate, citrate, ATP and acetyl CoA had no inhibitory effects. Thus glucose metabolism appears to be by the pentose phosphate pathway which will rapidly produce NADPH. This can readily be consumed during fatty-acid biosynthesis and, as there appears to be no inhibition of the flow of carbon from glucose to acetyl CoA, fatty-acid synthesis can continue for as long as there is a supply of glucose. These results help to explain the probable causes of fat build-up to high concentrations (about 40% of the cell dry weight) in this and other organisms. In alkane-grown cells, lipogenesis is repressed and carbon is able to flow from the alkanes via acetyl CoA, oxaloacetate and pyruvate into pentoses and hexoses in a unidirectional manner, because of the strong repression of pyruvate kinase and the increased activities of phosphoenolpyruvate kinase and fructose 1,6-biosphosphatase under these conditions. Although there was little change in the total activity of the TCA cycle enzymes under the various growth conditions, isocitrate lyase was induced under lipolytic conditions.     

Nicotinamide Phosphoribosyltransferase Promotes Epithelial-to-Mesenchymal Transition as a Soluble Factor Independent of Its Enzymatic Activity

Boosting NAD⁺ biosynthesis with NAD⁺ intermediates has been proposed as a strategy for preventing and treating age-associated diseases, including cancer. However, concerns in this area were raised by observations that nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in mammalian NAD⁺ biosynthesis, is frequently up-regulated in human malignancies, including breast cancer, suggesting possible protumorigenic effects for this protein. We addressed this issue by studying NAMPT expression and function in human breast cancer in vivo and in vitro. Our data indicate that high NAMPT levels are associated with aggressive pathological and molecular features, such as estrogen receptor negativity as well as HER2-enriched and basal-like PAM50 phenotypes. Consistent with these findings, we found that NAMPT overexpression in mammary epithelial cells induced epithelial-to-mesenchymal transition, a morphological and functional switch that confers cancer cells an increased metastatic potential. However, importantly, NAMPT-induced epithelial-to-mesenchymal transition was found to be independent of NAMPT enzymatic activity and of the NAMPT product nicotinamide mononucleotide. Instead, it was mediated by secreted NAMPT through its ability to activate the TGFβ signaling pathway via increased TGFβ1 production. These findings have implications for the design of therapeutic strategies exploiting NAD⁺ biosynthesis via NAMPT in aging and cancer and also suggest the potential of anticancer agents designed to specifically neutralize extracellular NAMPT. Notably, because high levels of circulating NAMPT are found in obese and diabetic patients, our data could also explain the increased predisposition to cancer of these subjects.     

The enzymes of the classical pentose phosphate pathway display differential activities in procyclic and bloodstream forms of Trypanosoma brucei

The specific activities of each of the enzymes of the classical pentose phosphate pathway have been determined in both cultured procyclic and bloodstream forms of Trypanosoma brucei. Both forms contained glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconolactonase (EC 3.1.1.31), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), ribose-5-phosphate isomerase (EC 5.3.1.6) and transaldolase (EC 2.2.1.2). However, ribulose-5-phosphate 3'-epimerase (EC 5.1.3.1) and transketolase (EC 2.2.1.1) activities were detectable only in procyclic forms. These results clearly demonstrate that both forms of T. brucei can metabolize glucose via the oxidative segment of the classical pentose phosphate pathway in order to produce D-ribose-5-phosphate for the synthesis of nucleic acids and reduced NADP for other synthetic reactions. However, only procyclic forms are capable of using the non-oxidative segment of the classical pentose phosphate pathway to cycle carbon between pentose and hexose phosphates in order to produce D-glyceraldehyde 3-phosphate as a net product of the pathway. Both forms lack the key gluconeogenic enzyme, fructose-bisphosphatase (EC 3.1.3.11). Consequently, neither form should be able to engage in gluconeogenesis nor should procyclic forms be able to return any of the glyceraldehyde 3-phosphate produced in the pentose phosphate pathway to glucose 6-phosphate. This last specific metabolic arrangement and the restriction of all but the terminal steps of glycolysis to the glycosome may be the observations required to explain the presence of distinct cytosolic and glycosomal isoenzymes of glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. These same observations also may provide the basis for explaining the presence of cytosolic hexokinase and phosphoglucose isomerase without the presence of any cytosolic phosphofructokinase activity. The key enzymes of the Entner-Doudoroff pathway, 6-phosphogluconate dehydratase (EC 4.2.1.12) and 2-keto-3-deoxy-6-phosphogluconate aldolase (EC 4.1.2.14) were not detected in either procyclic or bloodstream forms of T. brucei.     

Stimulation of glycolysis by imidazole

Imidazole stimulated glycolysis by rat diaphragm homogenates; this effect increased as the concentration of imidazole was raised. The simultaneous changes in concentration of some of the glycolytic intermediates suggested an action of imidazole at a step between triose phosphate and pyruvate. Addition of glyceraldehyde phosphate dehydrogenase increased glycolysis and abolished the stimulating effect of imidazole, thus leading to the conclusion that in the homogenate the activity of this enzyme was rate-limiting for glycolysis and was increased by imidazole.     

Correlation between the rate of protein and nucleic acid synthesis and the intensity of various glucose metabolic pathways in rat liver cells

A sharp and strong suppression of protein synthesis by cycloheximide in liver cells of starving rats is paralleled with activation of RNA synthesis and glucose-6-phosphate dehydrogenase production. Subsequent reconstitution and stimulation of protein synthesis (6-12 hrs after cycloheximide injection) result in activation of hexokinase. Upon stimulation of DNA synthesis (48-60 hrs after cycloheximide injection) the activity of both enzymes is very low. Since glucose-6-phosphate dehydrogenase appears to be the limiting step of glucose decay via the pentose phosphate pathway, and hexokinase is the limiting step of glycolysis, it was assumed that RNA synthesis predominantly occurs via the pentose phosphate pathway, while that of proteins via glycolysis.     

Nicotinamide phosphoribosyltransferase can affect metastatic activity and cell adhesive functions by regulating integrins in breast cancer

NAD⁺ metabolism is an essential regulator of cellular redox reactions, energy pathways, and a substrate provider for NAD⁺ consuming enzymes. We recently demonstrated that enhancement of NAD⁺/NADH levels in breast cancer cells with impaired mitochondrial NADH dehydrogenase activity, through augmentation of complex I or by supplementing tumor cell nutrients with NAD⁺ precursors, inhibits tumorigenicity and metastasis. To more fully understand how aberrantly low NAD⁺ levels promote tumor cell dissemination, we here asked whether inhibition of NAD⁺ salvage pathway activity by reduction in nicotinamide phosphoribosyltransferase (NAMPT) expression can impact metastasis and tumor cell adhesive functions. We show that knockdown of NAMPT, the enzyme catalyzing the rate-limiting step of the NAD⁺ salvage pathway, enhances metastatic aggressiveness in human breast cancer cells and involves modulation of integrin expression and function. Reduction in NAMPT expression is associated with upregulation of select adhesion receptors, particularly αvβ3 and β1 integrins, and results in increased breast cancer cell attachment to extracellular matrix proteins, a key function in tumor cell dissemination. Interestingly, NAMPT downregulation prompts expression of integrin αvβ3 in a high affinity conformation, known to promote tumor cell adhesive interactions during hematogenous metastasis. NAMPT has been selected as a therapeutic target for cancer therapy based on the essential functions of this enzyme in NAD⁺ metabolism, cellular redox, DNA repair and energy pathways. Notably, our results indicate that incomplete inhibition of NAMPT, which impedes NAD⁺ metabolism but does not kill a tumor cell can alter its phenotype to be more aggressive and metastatic. This phenomenon could promote cancer recurrence, even if NAMPT inhibition initially reduces tumor growth.     

Comparison of dynamic responses of cellular metabolites in <Emphasis Type="Italic">Escherichia coli</Emphasis> to pulse addition of substrates

We conducted an integrated study of cell growth parameters, product formation, and the dynamics of intracellular metabolite concentrations using Escherichia coli with genes knocked out in the glycolytic and oxidative pentose phosphate pathway (PPP) for glucose catabolism. We investigated the same characteristics in the wild-type strain, using acetate or pyruvate as the sole carbon source. Dramatic effects on growth parameters and extracellular and intracellular metabolite concentrations were observed after blocking either glycolytic breakdown of glucose by inactivation of phosphoglucose isomerase (disruption of pgi gene) or pentose phosphate breakdown of glucose by inactivation of glucose-6-phosphate dehydrogenase (disruption of zwf gene). Reducing power (NADPH) was mainly produced through PPP when the pgi gene was knocked out, while NADPH was produced through the tricarboxylic acid (TCA) cycle by isocitrate dehydrogenase or NADP-linked malic enzyme when the zwf gene was knocked out. As expected, when the pgi gene was knocked out, intracellular concentrations of PPP metabolites were high and glycolytic and concentrations of TCA cycle pathway metabolites were low. In the zwf gene knockout, concentrations of PPP metabolites were low and concentrations of intracellular glycolytic and TCA cycle metabolites were high.     

Riboneogenesis in yeast

Glucose is catabolized in yeast via two fundamental routes, glycolysis and the oxidative pentose phosphate pathway, which produces NADPH and the essential nucleotide component ribose-5-phosphate. Here, we describe riboneogenesis, a thermodynamically driven pathway that converts glycolytic intermediates into ribose-5-phosphate without production of NADPH. Riboneogenesis begins with synthesis, by the combined action of transketolase and aldolase, of the seven-carbon bisphosphorylated sugar sedoheptulose-1,7-bisphosphate. In the pathway's committed step, sedoheptulose bisphosphate is hydrolyzed to sedoheptulose-7-phosphate by the enzyme sedoheptulose-1,7-bisphosphatase (SHB17), whose activity we identified based on metabolomic analysis of the corresponding knockout strain. The crystal structure of Shb17 in complex with sedoheptulose-1,7-bisphosphate reveals that the substrate binds in the closed furan form in the active site. Sedoheptulose-7-phosphate is ultimately converted by known enzymes of the nonoxidative pentose phosphate pathway to ribose-5-phosphate. Flux through SHB17 increases when ribose demand is high relative to demand for NADPH, including during ribosome biogenesis in metabolically synchronized yeast cells.     

Formation of a pentose phosphate cycle metabolite, erythrose-4-phosphate, from initial compounds of glycolysis by transketolase from the rat liver

Using ion-exchange chromatography of sucrose phosphates on Dowex-1, it was demonstrated that the highly purified rat liver transketolase (specific activity 1.7 mumol/min.mg protein) is capable of catalyzing the synthesis of erythrose-4-phosphate, a metabolite of the pentose phosphate pathway non-oxidizing step, from the initial participants of glycolysis, i. e., glucose-6-phosphate and fructose-6-phosphate. As can be evidenced from the reaction course, the second product of this synthesis is octulose-8-phosphate. The reaction was assayed by accumulation of erythrose-4-phosphate. The soluble fraction from rat liver catalyzes under identical conditions the synthesis of heptulose-7-phosphate (but not erythrose-4-phosphate), which points to the utilization of the erythrose-4-phosphate formed in the course of the transketolase reaction by transaldolase which is also present in the soluble fraction. The role of the transketolase reaction reversal from the synthesis of pentose phosphate derivatives to glycolytic products is discussed. The transketolase reaction provides for the relationship between glycolysis and the anaerobic step of the pentose phosphate pathway which share common metabolites, i. e. glucose-6-phosphate and fructose-6-phosphate.