Showing papers on "Glycolysis published in 2002"

Mitochondria, calcium regulation, and acute glutamate excitotoxicity in cultured cerebellar granule cells.

Abstract: Exposure of cultured cerebellar granule cells to 100 μM glutamate plus glycine in the absence of Mg 2+ causes calcium loading of the in situ mitochondria and is excitotoxic, as demonstrated by a collapse of the cellular ATP/ADP ratio, cytoplasmic Ca 2+ deregulation (the failure of the cell to maintain a stable cytoplasmic free Ca 2+ concentration), and extensive cell death. Glutamate-evoked Ca 2+ deregulation is exacerbated by the mitochondrial respiratory chain inhibitor rotenone. Cells maintained by glycolytic ATP, i.e., in the presence of the mitochondrial ATP synthase inhibitor oligomycin, remain viable for several hours but are still susceptible to glutamate; thus, disruption of mitochondrial ATP synthesis is not a necessary step in glutamate excitotoxicity. In contrast, the combination of rotenone (or antimycin A) plus oligomycin, which collapses the mitochondrial membrane potential, therefore preventing mitochondrial Ca 2+ transport, allows glutamate-exposed cells to maintain a high ATP/ADP ratio while accumulating little 45 Ca 2+ and maintaining a low bulk cytoplasmic free Ca 2+ concentration determined by fura-2. It is concluded that mitochondrial Ca 2+ accumulation is a necessary intermediate in glutamate excitotoxicity, whereas the decreased Ca 2+ flux into cells with depolarized mitochondria may reflect a feedback inhibition of the NMDA receptor mediated by localized Ca 2+ accumulation in a microdomain accessible to the mitochondria.

Acid Production in Glycolysis-impaired Tumors Provides New Insights into Tumor Metabolism

Abstract: Purpose: Low extracellular pH is a hallmark of solid tumors. It has long been thought that this acidity is mainly attributable to the production of lactic acid. In this study, we tested the hypothesis that lactate is not the only source of acidification in solid tumors and explored the potential mechanisms underlying these often-observed high rates of acid production. Experimental Design: We compared the metabolic profiles of glycolysis-impaired (phosphoglucose isomerase-deficient) and parental cells in both in vitro and two in vivo models (dorsal skinfold chamber and Gullino chamber). Results: We demonstrated that CO 2 , in addition to lactic acid, was a significant source of acidity in tumors. We also found evidence supporting the hypothesis that tumor cells rely on glutaminolysis for energy production and that the pentose phosphate pathway is highly active within tumor cells. Our results also suggest that the tricarboxylic acid cycle is saturable and that different metabolic pathways are activated to provide for energy production and biosynthesis. Conclusions: These results are consistent with the paradigm that tumor metabolism is determined mainly by substrate availability and not by the metabolic demand of tumor cells per se . In particular, it appears that the local glucose and oxygen availabilities each independently affect tumor acidity. These findings have significant implications for cancer treatment.

The glycolytic flux in Escherichia coli is controlled by the demand for ATP.

Abstract: The nature of the control of glycolytic flux is one of the central, as-yet-uncharacterized issues in cellular metabolism. We developed a molecular genetic tool that specifically induces ATP hydrolysis in living cells without interfering with other aspects of metabolism. Genes encoding the F1 part of the membrane-bound (F1F0) H+-ATP synthase were expressed in steadily growing Escherichia coli cells, which lowered the intracellular [ATP]/[ADP] ratio. This resulted in a strong stimulation of the specific glycolytic flux concomitant with a smaller decrease in the growth rate of the cells. By optimizing additional ATP hydrolysis, we increased the flux through glycolysis to 1.7 times that of the wild-type flux. The results demonstrate why attempts in the past to increase the glycolytic flux through overexpression of glycolytic enzymes have been unsuccessful: the majority of flux control (>75%) resides not inside but outside the pathway, i.e., with the enzymes that hydrolyze ATP. These data further allowed us to answer the question of whether catabolic or anabolic reactions control the growth of E. coli. We show that the majority of the control of growth rate resides in the anabolic reactions, i.e., the cells are mostly “carbon” limited. Ways to increase the efficiency and productivity of industrial fermentation processes are discussed.

High expression of inducible 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (iPFK-2; PFKFB3) in human cancers.

Abstract: Tumor cells maintain an especially high glycolytic rate to supply the anabolic precursors essential for de novo nucleotide synthesis. We recently cloned an inducible isozyme of 6-phosphofructo-2 kinase (iPFK-2) that bears an oncogene-like regulatory element in its mRNA and functions to produce fructose-2,6-bisphosphate, which is a powerful allosteric activator of glycolysis. Rapidly proliferating cancer cells constitutively express iPFK-2 in vitro, and inhibition of iPFK-2 expression decreases tumor growth in experimental animal models. We report herein that the expression of iPFK-2 mRNA and protein, as assessed by in situ hybridization and immunohistochemistry, is increased in many human cancers when compared with corresponding normal tissues. In particular, iPFK-2 expression was found to be markedly elevated in multiple aggressive primary neoplasms, including colon, breast, ovarian, and thyroid carcinomas. iPFK-2 mRNA and protein expression were induced by hypoxia in cultured human colon adenocarcinoma cells, and an examination of normal lung fibroblasts showed that iPFK-2 and fructose-2,6-bisphosphate levels increased specifically during the S phase of the cell cycle. These data indicate that iPFK-2 is abundantly expressed in human tumors in situ and may serve as an essential regulator of glycolysis during cell cycle progression and growth in an hypoxic microenvironment.

Metabolic Flux Responses to Pyruvate Kinase Knockout in Escherichia coli

Abstract: The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient Escherichia coli was estimated using biosynthetically directed fractional 13C labeling experiments with [U-13C6]glucose in glucose- or ammonia-limited chemostats, two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, and a comprehensive isotopomer model. The general response to disruption of both pyruvate kinase isoenzymes in E. coli was a local flux rerouting via the combined reactions of phosphoenolpyruvate (PEP) carboxylase and malic enzyme. Responses in the pentose phosphate pathway and the tricarboxylic acid cycle were strongly dependent on the environmental conditions. In addition, high futile cycling activity via the gluconeogenic PEP carboxykinase was identified at a low dilution rate in glucose-limited chemostat culture of pyruvate kinase-deficient E. coli, with a turnover that is comparable to the specific glucose uptake rate. Furthermore, flux analysis in mutant cultures indicates that glucose uptake in E. coli is not catalyzed exclusively by the phosphotransferase system in glucose-limited cultures at a low dilution rate. Reliability of the flux estimates thus obtained was verified by statistical error analysis and by comparison to intracellular carbon flux ratios that were independently calculated from the same NMR data by metabolic flux ratio analysis.

Contribution by different fuels and metabolic pathways to the total ATP turnover of proliferating MCF-7 breast cancer cells.

Abstract: For the past 70 years the dominant perception of cancer metabolism has been that it is fuelled mainly by glucose (via aerobic glycolysis) and glutamine. Consequently, investigations into the diagnosis, treatment and the basic metabolism of cancer cells have been directed by this perception. However, the data on cancer metabolism are equivocal, and in this study we have sought to clarify the issue. Using an innovative system we have measured the total ATP turnover of the MCF-7 breast cancer cell line, the contributions to this turnover by oxidative and glycolytic ATP production and the contributions to the oxidative component by glucose, lactate, glutamine, palmitate and oleate. The total ATP turnover over approx. 5 days was 26.8 micromol of ATP.10(7) cells(-1).h(-1). ATP production was 80% oxidative and 20% glycolytic. Contributions to the oxidative component were approx. 10% glucose, 14% glutamine, 7% palmitate, 4% oleate and 65% from unidentified sources. The contribution by glucose (glycolysis and oxidation) to total ATP turnover was 28.8%, glutamine contributed 10.7% and glucose and glutamine combined contributed 40%. Glucose and glutamine are significant fuels, but they account for less than half of the total ATP turnover. The contribution of aerobic glycolysis is not different from that in a variety of other non-transformed cell types.

Activity of key enzymes involved in glucose and triglyceride catabolism during bovine oocyte maturation in vitro.

Abstract: Little is known about the metabolic profile of cumulus-oocyte complexes (COCs) during maturation. The aim of this study was to determine the differential participation of enzymatic activity in cumulus cells and the oocyte during in vitro maturation of bovine oocytes, by measuring the activity of key enzymes involved in the regulation of glycolysis (phosphofructokinase), the pentose phosphate pathway (glucose-6-phosphate dehydrogenase) and lipolysis (lipase). COCs were matured in medium 199 plus 10% (v/v) steer serum for 22-24 h at 39 degrees C in 5% CO(2):95% humidified air. Phosphofructokinase, glucose-6-phosphate dehydrogenase and lipase activities were measured in immature and in vitro matured COCs, denuded oocytes and cumulus cells, respectively. Phosphofructokinase and glucose-6-phosphate dehydrogenase activities (enzymatic units) remained constant during in vitro maturation of COCs, but there was a significant decrease in lipase activity (units) (P < 0.05), as activity in cumulus cells decreased significantly (P < 0.05). For the three enzymes studied, enzyme activity (units) remained unchanged in the oocyte during in vitro maturation. Specific activity increased in the oocyte (P < 0.05) and decreased in cumulus cells as a result of maturation (P < 0.05). In cumulus cells, phosphofructokinase was the most abundant of the three enzymes followed by glucose-6-phosphate dehydrogenase and then lipase (P < 0.05), whereas in the denuded oocyte this order was reversed (P < 0.05). Thus, the metabolism of cumulus cells is adapted to control the flow of metabolites toward the oocyte, which maintains its enzymatic activity even when dissociated from cumulus cells during maturation. The high activity of phosphofructokinase in cumulus cells indicates that glucose is metabolized mainly via the glycolytic pathway in these cells. The greater relative activity of glucose-6-phosphate dehydrogenase recorded in the oocyte indicates that glucose uptake could be directed mainly toward the pentose phosphate pathway. The marked lipolytic activity concentrated in the oocyte indicates an active participation in lipid catabolism during maturation.

Energy metabolism in the hypertrophied heart.

Abstract: In response to a prolonged pressure- or volume-overload, alterations occur in myocardial fatty acid, glucose, and glycogen metabolism. Oxidation of long chain fatty acids has been found to be reduced in hypertrophied hearts compared to non-hypertrophied hearts. However, this observation depends upon the degree of cardiac hypertrophy, the severity of carnitine deficiency, the concentration of fatty acid in blood or perfusate, and the myocardial workload. Glycolysis of exogenous glucose is accelerated in hypertrophied hearts. Despite the acceleration of glycolysis, glucose oxidation is not correspondingly increased leading to lower coupling between glycolysis and glucose oxidation and greater H(+) production than in non-hypertrophied hearts. Although glycogen metabolism does not differ in the absence of ischemia, synthesis and degradation of glycogen are accelerated in severely ischemic hypertrophied hearts. These alterations in carbohydrate metabolism may contribute to the increased susceptibility of hypertrophied hearts to injury during ischemia and reperfusion by causing disturbances in ion homeostasis that reduce contractile function and efficiency to a greater extent than normal. As in non-hypertrophied hearts, pharmacologic enhancement of coupling between glycolysis and glucose oxidation (e.g., by directly stimulating glucose oxidation) improves recovery of function of hypertrophied hearts after ischemia. This observation provides strong support for the concept that modulation of energy metabolism in the hypertrophied heart is a useful approach to improve function of the hypertrophied heart during ischemia and reperfusion. Future investigations are necessary to determine if alternative approaches, such as glucose-insulin-potassium infusion and inhibitors of fatty acid oxidation (e.g., ranolazine, trimetazidine), also produce beneficial effects in ischemic and reperfused hypertrophied hearts.

Pyruvate kinase type M2: a crossroad in the tumor metabolome

Abstract: Cell proliferation is a process that consumes large amounts of energy. A reduction in the nutrient supply can lead to cell death by ATP depletion, if cell proliferation is not limited. A key sensor for this regulation is the glycolytic enzyme pyruvate kinase, which determines whether glucose carbons are channelled to synthetic processes or used for glycolytic energy production. In unicellular organisms pyruvate kinase is regulated by ATP, ADP and AMP, by ribose 5-P, the precursor of the nucleic acid synthesis, and by the glycolytic intermediate fructose 1,6-P2 (FBP), thereby adapting cell proliferation to nutrient supply. The mammalian pyruvate kinase isoenzyme type M2 (M2-PK) displays the same kinetic properties as the pyruvate kinase enzyme from unicellular organisms. The mammalian M2-PK isoenzyme can switch between a less active dimeric form and a highly active tetrameric form which regulates the channeling of glucose carbons either to synthetic processes (dimeric form) or to glycolytic energy production (tetrameric form). Tumor cells are usually characterized by a high amount of the dimeric form leading to a strong accumulation of all glycolytic phosphometabolites above pyruvate kinase. The tetramer-dimer ratio is regulated by ATP, FBP and serine and by direct interactions with different oncoproteins (pp60v-src, HPV-16 E7). In solid tumors with sufficient oxygen supply pyruvate is supplied by glutaminolysis. Pyruvate produced in glycolysis and glutaminolysis is used for the synthesis of lactate, glutamate and fatty acids thereby releasing the hydrogen produced in the glycolytic glyceraldehyde 3-phosphate dehydrogenase reaction.

Metabolic engineering of glycerol production in Saccharomyces cerevisiae.

Abstract: Inactivation of TPI1, the Saccharomyces cerevisiae structural gene encoding triose phosphate isomerase, completely eliminates growth on glucose as the sole carbon source. In tpi1-null mutants, intracellular accumulation of dihydroxyacetone phosphate might be prevented if the cytosolic NADH generated in glycolysis by glyceraldehyde-3-phosphate dehydrogenase were quantitatively used to reduce dihydroxyacetone phosphate to glycerol. We hypothesize that the growth defect of tpi1-null mutants is caused by mitochondrial reoxidation of cytosolic NADH, thus rendering it unavailable for dihydroxyacetone-phosphate reduction. To test this hypothesis, a tpi1delta nde1delta nde2delta gut2delta quadruple mutant was constructed. NDE1 and NDE2 encode isoenzymes of mitochondrial external NADH dehydrogenase; GUT2 encodes a key enzyme of the glycerol-3-phosphate shuttle. It has recently been demonstrated that these two systems are primarily responsible for mitochondrial oxidation of cytosolic NADH in S. cerevisiae. Consistent with the hypothesis, the quadruple mutant grew on glucose as the sole carbon source. The growth on glucose, which was accompanied by glycerol production, was inhibited at high-glucose concentrations. This inhibition was attributed to glucose repression of respiratory enzymes as, in the quadruple mutant, respiratory pyruvate dissimilation is essential for ATP synthesis and growth. Serial transfer of the quadruple mutant on high-glucose media yielded a spontaneous mutant with much higher specific growth rates in high-glucose media (up to 0.10 h(-1) at 100 g of glucose. liter(-1)). In aerated batch cultures grown on 400 g of glucose. liter(-1), this engineered S. cerevisiae strain produced over 200 g of glycerol. liter(-1), corresponding to a molar yield of glycerol on glucose close to unity.

Suppression of Mitochondrial Succinate Dehydrogenase, a Primary Target of β‐Amyloid, and Its Derivative Racemized at Ser Residue

Abstract: beta-Amyloid cores contain considerable amounts of D-Ser and D-Asp residues in Alzheimer's disease. We investigated the cytotoxic effects of various synthetic beta-amyloids, including D-Ser-substituted derivatives, on primary cultured neurons and nonneuronal HeLa cells. beta 25-35, its D-Ser26-substituted derivative, and beta 1-40 in 10-100 nM specifically suppressed mitochondrial succinate dehydrogenase activity [MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] reduction] in HeLa cells, which are dependent on ATP production mainly from glycolysis, but did not exert detectable cytotoxicity, assessed by dye exclusion test, NADH levels, and uptake of [3H]Leu and [3H]Tdr. The beta-amyloids, on the other hand, did exert neurodegenerative effects on rat hippocampal cultured neurons in which ATP is mostly synthesized by the mitochondrion. The activities of beta 25-35 and [D-Ser26] beta 25-35 are dependent on their having beta-structures and not random forms. Although beta 25-35 was degraded rapidly by proteinase(s) in brain extract or leucine aminopeptidase, [D-Ser26] beta 25-35 is fairly resistant. These results indicate that one of the primary targets of beta-amyloids is suppression of mitochondrial succinate dehydrogenase, and the vulnerability of the brain of beta-amyloids can be explained by its large dependence on mitochondrial energy production. Moreover, racemization of serine residues of beta-amyloids may be involved in neurodegeneration and formation of senile plaques through escaping from the degradation process by brain proteinases.

Analysis of gluconeogenic and anaplerotic enzymes in Campylobacter jejuni: an essential role for phosphoenolpyruvate carboxykinase

Abstract: Campylobacter jejuni is unable to utilize glucose as a carbon source due to the absence of the key glycolytic enzyme 6-phosphofructokinase. The genome sequence of C. jejuni NCTC 11168 indicates that homologues of all the appropriate enzymes for gluconeogenesis from phosphoenolpyruvate (PEP) are present, in addition to the anaplerotic enzymes pyruvate carboxylase (PYC), phosphoenolpyruvate carboxykinase (PCK) and malic enzyme (MEZ). Surprisingly, a pyruvate kinase (PYK) homologue is also present. To ascertain the role of these enzymes, insertion mutants in pycA, pycB, pyk and mez were generated. However, this could not be achieved for pckA, indicating that PCK is an essential enzyme in C. jejuni. The lack of PEP synthase and pyruvate orthophosphate dikinase activities confirmed a unique role for PCK in PEP synthesis. The pycA mutant was unable to grow in defined medium with pyruvate or lactate as the major carbon source, thus indicating an important role for PYC in anaplerosis. Sequence and biochemical data indicate that the PYC of C. jejuni is a member of the alpha4beta4, acetyl-CoA-independent class of PYCs, with a 65.8 kDa subunit containing the biotin moiety. Whereas growth of the mez mutant was comparable to that of the wild-type, the pyk mutant displayed a decreased growth rate in complex medium. Nevertheless, the mez and pyk mutants were able to grow with pyruvate, lactate or malate as carbon sources in defined medium. PYK was present in cell extracts at a much higher specific activity [>800 nmol x min(-1) x (mg protein)(-1)] than PYC or PCK [<65 nmol x min(-1) x (mg protein)(-1)], was activated by fructose 1,6-bisphosphate and displayed other regulatory properties strongly indicative of a catabolic role. It is concluded that PYK may function in the catabolism of unidentified substrates which are metabolized through PEP. In view of the high K(m) of MEZ for malate (approximately 9 mM) and the lack of a growth phenotype of the mez mutant, MEZ seems to have only a minor anaplerotic role in C. jejuni.

The mechanism of inhibition of Ran-dependent nuclear transport by cellular ATP depletion

Abstract: Rran-dependent nuclear transport requires a nuclear pool of RanGTP both for the assembly of export complexes and the disassembly of import complexes. Accordingly, in order for these processes to proceed, Ran-dependent nuclear import and export assays in vitro require the addition of GTP to produce RanGTP. Notably, no ATP requirement can be detected for these transport processes in vitro. But in vivo, when cells are depleted of ATP by the addition of sodium azide and 2-deoxyglucose to block ATP production by oxidative phosphorylation and glycolysis, respectively, Ran-dependent nuclear import and export are rapidly inhibited. This raised the question of whether there is an ATP requirement for these nuclear transport pathways in an intact cell that has remained undetected in vitro. Here we report that the free (but not total) GTP concentration rapidly drops to an undetectable level upon ATP depletion as does the availability of RanGTP. Our conclusion is that the inhibition of Ran-dependent nuclear transport observed upon ATP depletion in vivo results from a shortage of RanGTP rather than the inhibition of some ATP-dependent process.

Adenosine, Inosine, and Guanosine Protect Glial Cells During Glucose Deprivation and Mitochondrial Inhibition: Correlation Between Protection and ATP Preservation

Abstract: The purpose of this study was to determine the mechanism by which adenosine, inosine, and guanosine delay cell death in glial cells (ROC-1) that are subjected to glucose deprivation and mitochondrial respiratory chain inhibition with amobarbital (GDMI). ROC-1 cells are hybrid cells formed by fusion of a rat oligodendrocyte and a rat C6 glioma cell. Under GDMI, ATP was depleted rapidly from ROC-1 cells, followed on a much larger time scale by a loss of cell viability. Restoration of ATP synthesis during this interlude between ATP depletion and cell death prevented further loss of viability. Moreover, the addition of adenosine, inosine, or guanosine immediately before the amobarbital retarded the decline in ATP and preserved cell viability. The protective effects on ATP and viability were dependent on nucleoside concentration between 50 and 1,500 microM. Furthermore, protection required nucleoside transport into the cell and the continued presence of nucleoside during GDMI. A significant positive correlation between ATP content at 16 min and cell viability at 350 min after the onset of GDMI was established (r = 0.98). Modest increases in cellular lactate levels were observed during GDMI (1.2 nmol/mg/min lactate produced); however, incubation with 1,500 microM inosine or guanosine increased lactate accumulation sixfold. The protective effects of inosine and guanosine on cell viability and ATP were >90% blocked after treatment with 50 microM BCX-34, a nucleoside phosphorylase inhibitor. Accordingly, lactate levels also were lower in BCX-34-treated cells incubated with inosine or guanosine. We conclude that under GDMI, the ribose moiety of inosine and guanosine is converted to phosphorylated glycolytic intermediates via the pentose phosphate pathway, and its subsequent catabolism in glycolysis provides the ATP necessary for maintaining plasmalemmal integrity.

Generalized sensory stimulation of conscious rats increases labeling of oxidative pathways of glucose metabolism when the brain glucose-oxygen uptake ratio rises.

Abstract: Interpretation of functional metabolic brain images requires understanding of metabolic shifts in working brain. Because the disproportionately higher uptake of glucose compared with oxygen ("aerobic glycolysis") during sensory stimulation is not fully explained by changes in levels of lactate or glycogen, metabolic labeling by [6-14C]glucose was used to evaluate utilization of glucose during brief brain activation. Increased labeling of tricarboxylic acid cycle-derived amino acids, mainly glutamate but also gamma-aminobutyric acid, reflects a rise in oxidative metabolism during aerobic glycolysis. The size of the glutamate, lactate, alanine, and aspartate pools changed during stimulation. Brain lactate was derived from blood-borne glucose and its specific activity was twice that of alanine, revealing pyruvate compartmentation. Glycogen labeling doubled during recovery compared with rest and activation; only 4% to 8% of the total 14C was recovered in lactate plus glycogen. Restoration of glycogen levels was slow, and diversion of glucose from oxidative pathways to restore its level could cause a prolonged reduction of the global O2/glucose uptake ratio. The rise in the brain glucose-oxygen uptake ratio during activation does not simply reflect an upward shift of glycolysis under aerobic conditions; instead, it involves altered fluxes into various (oxidative and biosynthetic) pathways with different time courses.

ATP and the Heart

Abstract: List of Topics. Prologue. Part I: ATP: The Molecule. 1. ATP and the heart: An overview. 2. The basics. 3. The chemistry of the ATPase reaction. 4. Ways to measure ATP and related metabolites. Part II: ATP: Degradation Pathway and De Novo Synthesis. 5. Degradation and synthesis of ATP. Part III: ATP Utilizing Pathways. 6. The work of contraction: Myosin ATPase. 7. The work of ion movements. 8. The work of macromolecular synthesis and degradation. Part IV: ATP Synthesizing Pathways. 9. ATP synthesis pathways: glycolysis. 10. ATP synthesis pathways: oxidative phosphorylation. 11. ATP synthesis pathways: phosphotransferase reactions. Part V: ATP and the Heart. 12. Integration of ATP synthesis and ATP utilization pathways. Index.

Metabolic changes detected by in vivo magnetic resonance studies of HEPA-1 wild-type tumors and tumors deficient in hypoxia-inducible factor-1beta (HIF-1beta): evidence of an anabolic role for the HIF-1 pathway.

Abstract: Hypoxia-inducible factor-1 (HIF-1) regulates many pathways potentially important for tumor growth, including angiogenesis and glycolysis. Most attention has focused on its role in the response to hypoxia, but HIF-1 is also constitutively expressed in many tumors. To analyze the role of this pathway in vivo, we used magnetic resonance (MR) methods and complementary techniques to monitor metabolic changes in tumors derived from HEPA-1 mouse hepatoma lines that were either wild type (WT) or deficient in hypoxia-inducible transcription factor HIF-1beta (c4). The c4 tumors grew significantly more slowly than the WT tumors (P < 0.05), but were examined at a similar size (0.4-0.6 g). At the tumor size used in these studies, no differences in vascularity were observed, and MR parameters measured that related to tumor blood flow, vascularity, and oxygenation demonstrated no significant differences between the two tumor types. Unexpectedly, the ATP content of the c4 tumor was approximately 5 times less than in the WT tumor [measured in tumor extracts (P < 0.001) and by metabolic imaging (P < 0.05)]. Noninvasive (31)P MR spectroscopy showed that the nucleoside triphosphate/P(i) ratio of the two tumor types was similar, so the low ATP content of the c4 tumors was not caused by (or a cause of) impaired cellular bioenergetics. Rather, glycine, an essential precursor for de novo purine formation, was significantly lower in the c4 tumors (P < 0.05), suggesting that ATP synthesis was impaired in the mutant tumor cells. Supporting evidence for this hypothesis came from the significantly lower concentrations of betaine, phosphocholine, and choline in the c4 tumors (P < 0.05); these are intermediates in an alternative pathway for glycine synthesis. No significant differences were seen in lactate or glucose content. MR resonances from phosphodiesters, which relate to the metabolic turnover of phospholipid membranes, were significantly lower in the WT tumors than in the c4 tumors, both in vivo (P < 0.05) and in extracts (P < 0.01). We propose that loss of up-regulation of expression of the genes for glucose transporters and glycolytic enzymes in the c4 tumors decreased formation of glycine, an essential precursor of ATP synthesis, and thus caused the low ATP content of the c4 tumors. In summary, these data suggest that disruption of the HIF-1 pathway in these tumor cells impairs the supply of anabolic precursors required for cell synthesis. They suggest potential biochemical targets that may be modified by therapy blocking HIF-1 function.

Lactate induces insulin resistance in skeletal muscle by suppressing glycolysis and impairing insulin signaling.

Abstract: Elevation of plasma lactate levels induces peripheral insulin resistance, but the underlying mechanisms are unclear. We examined whether lactate infusion in rats suppresses glycolysis preceding ins...