Showing papers on "Glycolysis published in 2006"

p53 Regulates Mitochondrial Respiration

Abstract: The energy that sustains cancer cells is derived preferentially from glycolysis. This metabolic change, the Warburg effect, was one of the first alterations in cancer cells recognized as conferring a survival advantage. Here, we show that p53, one of the most frequently mutated genes in cancers, modulates the balance between the utilization of respiratory and glycolytic pathways. We identify Synthesis of Cytochrome c Oxidase 2 (SCO2) as the downstream mediator of this effect in mice and human cancer cell lines. SCO2 is critical for regulating the cytochrome c oxidase (COX) complex, the major site of oxygen utilization in the eukaryotic cell. Disruption of the SCO2 gene in human cancer cells with wild-type p53 recapitulated the metabolic switch toward glycolysis that is exhibited by p53-deficient cells. That SCO2 couples p53 to mitochondrial respiration provides a possible explanation for the Warburg effect and offers new clues as to how p53 might affect aging and metabolism.

Glycolysis inhibition for anticancer treatment

Abstract: Most cancer cells exhibit increased glycolysis and use this metabolic pathway for generation of ATP as a main source of their energy supply. This phenomenon is known as the Warburg effect and is considered as one of the most fundamental metabolic alterations during malignant transformation. In recent years, there are significant progresses in our understanding of the underlying mechanisms and the potential therapeutic implications. Biochemical and molecular studies suggest several possible mechanisms by which this metabolic alteration may evolve during cancer development. These mechanisms include mitochondrial defects and malfunction, adaptation to hypoxic tumor microenvironment, oncogenic signaling, and abnormal expression of metabolic enzymes. Importantly, the increased dependence of cancer cells on glycolytic pathway for ATP generation provides a biochemical basis for the design of therapeutic strategies to preferentially kill cancer cells by pharmacological inhibition of glycolysis. Several small molecules have emerged that exhibit promising anticancer activity in vitro and in vivo, as single agent or in combination with other therapeutic modalities. The glycolytic inhibitors are particularly effective against cancer cells with mitochondrial defects or under hypoxic conditions, which are frequently associated with cellular resistance to conventional anticancer drugs and radiation therapy. Because increased aerobic glycolysis is commonly seen in a wide spectrum of human cancers and hypoxia is present in most tumor microenvironment, development of novel glycolytic inhibitors as a new class of anticancer agents is likely to have broad therapeutic applications.

Hexokinase II: Cancer's double-edged sword acting as both facilitator and gatekeeper of malignancy when bound to mitochondria

Abstract: A key hallmark of many cancers, particularly the most aggressive, is the capacity to metabolize glucose at an elevated rate, a phenotype detected clinically using positron emission tomography (PET). This phenotype provides cancer cells, including those that participate in metastasis, a distinct competitive edge over normal cells. Specifically, after rapid entry of glucose into cancer cells on the glucose transporter, the highly glycolytic phenotype is supported by hexokinase (primarily HK II) that is overexpressed and bound to the outer mitochondrial membrane via the porin-like protein voltage-dependent anion channel (VDAC). This protein and the adenine nucleotide transporter move ATP, newly synthesized by the inner membrane located ATP synthase, to active sites on HK II. The abundant amounts of HK II bind both the ATP and the incoming glucose producing the product glucose-6-phosphate, also at an elevated rate. This critical metabolite then serves both as a biosynthetic precursor to support cell proliferation and as a precursor for lactic acid, the latter exiting cancer cells causing an unfavorable environment for normal cells. Although helping facilitate this chemical warfare, HK II via its mitochondrial location also suppresses the death of cancer cells, thus increasing their possibility for metastasis and the ultimate death of the human host. For these reasons, targeting this key enzyme is currently being investigated in several laboratories in a strategy to develop novel therapies that may turn the tide on the continuing struggle to find effective cures for cancer. One such candidate is 3-bromopyruvate that has been shown recently to eradicate advanced stage, PET positive hepatocellular carcinomas in an animal model without apparent harm to the animals.

Metabolic imaging by hyperpolarized 13C magnetic resonance imaging for in vivo tumor diagnosis.

Abstract: The “Warburg effect,” an elevation in aerobic glycolysis, may be a fundamental property of cancer cells. For cancer diagnosis and treatment, it would be valuable if elevated glycolytic metabolism could be quantified in an image in animals and humans. The pyruvate molecule is at the metabolic crossroad for energy delivery inside the cell, and with a noninvasive measurement of the relative transformation of pyruvate into lactate and alanine within a biologically relevant time frame (seconds), it may be possible to quantify the glycolytic status of the cells. We have examined the metabolism after i.v. injection of hyperpolarized 13 C-pyruvate in rats with implanted P22 tumors. The strongly enhanced nuclear magnetic resonance signal generated by the hyperpolarization techniques allows mapping of pyruvate, lactate, and alanine in a 5 × 5 × 10 mm 3 imaging voxel using a 1.5 T magnetic resonance scanner. The magnetic resonance scanning (chemical shift imaging) was initiated 24 seconds after the pyruvate injection and had a duration of 14 seconds. All implanted tumors showed significantly higher lactate content than the normal tissue. The results indicate that noninvasive quantification of localized Warburg effect may be possible. (Cancer Res 2006; 66(22): 10855-60)

5'-AMP-activated protein kinase (AMPK) is induced by low-oxygen and glucose deprivation conditions found in solid-tumor microenvironments.

Abstract: We have been studying the relationship between the activity of hypoxia-inducible factor 1 (HIF-1), the primary transcriptional regulator of the response of mammalian cells to oxygen deprivation (e.g., see references 21, 43, and 50) and the regulation of c-Jun/AP-1 transcription factors (31, 32). We determined that c-Jun N-terminal phosphorylation is induced by low-oxygen conditions (hypoxia or anoxia; called hypoxia hereafter) in an HIF-1-dependent manner (31) and showed that this HIF-1-dependent c-Jun phosphorylation absolutely requires extracellular glucose utilization (32). Together, these findings suggest that enhanced glucose absorption and/or glycolytic activity mediated by HIF-1 in response to hypoxia activates c-Jun/AP-1, as well as other targets of c-Jun N-terminal kinases. To further investigate this potential mechanism, we focused on determining the contribution of bioenergetics—ATP depletion—to hypoxia-inducible c-Jun phosphorylation in wild-type (WT) and HIF-1-null mouse embryo fibroblasts (MEFs). While exploring cellular mechanisms of ATP regulation, we observed that 5′-AMP-activated protein kinase (AMPK) activity was induced in both cell types, particularly under conditions of hypoxia and glucose deprivation. This observation suggested the hypothesis that AMPK is important for the adaptive responses of energetically stressed cells in the hypoxic and glucose-deprived microenvironments present in solid tumors (e.g., reviewed in references 35 and 59). AMPK activity is defined by a class of evolutionarily conserved serine/threonine kinases that are sensitive to various environmental stresses, especially those that perturb cellular energy status (reviewed in references 9, 19, and 47). Different members of the AMPK catalytic subunit subfamily have been characterized; the α subunits (collectively, AMPKα1 and -α2) are the most widely expressed in mammalian cells (36). AMPK is a heterotrimeric complex consisting of an α subunit and β and γ regulatory subunits, each of which is encoded by distinct genes (α1 and α2; β1 and β2; γ1, γ2, and γ3) (19). In terms of a role in ATP regulation, decreased cellular ATP levels promote AMPK activation through the allosteric binding of AMP, which in effect enables AMPK to sense increases in the cellular [AMP]/[ATP] ratio. Full activation of AMPK also requires specific phosphorylation within the activation loop of the catalytic domain of the α subunit (at Thr172 in humans and mice) by LKB1, a serine/threonine protein kinase and tumor suppressor (36, 37, 52). LKB1 is thus an AMPK kinase. Recently, mammalian Ca2+/calmodulin-dependent kinase kinases have also been identified as AMPK kinases (reviewed in reference 6). Activated AMPK phosphorylates diverse targets, including many that are directly involved in controlling cellular energy metabolism (22, 34). In cells exposed to an energy-depleting stress, AMPK is believed to function as an energy sensor that inhibits ATP-consuming processes and stimulates ATP-producing processes to optimize total cellular ATP levels for maintaining critical physiological functions (or for survival in response to extreme stress) (19). For example, in cells exposed to hypoxic or ischemic conditions that significantly deplete total ATP, activated AMPK can stimulate ATP generation by increasing both glucose absorption and glycolysis (e.g., see references 2, 19, and 22). AMPK can also generate ATP by phosphorylating and inhibiting the metabolic enzymes acetyl coenzyme A (acetyl-CoA) carboxylases 1 and 2 (ACC1/2), which synthesize malonyl-CoA (19). Malonyl-CoA synthesized by ACC1 is necessary for de novo fatty acid synthesis, whereas that synthesized by ACC2 inhibits fatty acid transport into the mitochondrion, the site of ATP production by the process of fatty acid β oxidation (18). Thus, AMPK-dependent inhibition of ACC1/2 can divert cellular metabolism from consuming ATP during fatty acid biosynthesis to producing ATP by oxidizing fatty acid stores. In the present study, we found that the combination of hypoxia and glucose deprivation decreased total cellular ATP levels to the same extent in both WT and HIF-1α-null cells. This finding supports our previous conclusion that increased intracellular glucose, rather than decreased ATP levels, is responsible for the stimulation of c-Jun N-terminal kinase activity in WT cells exposed to hypoxia. AMPK activity, conventionally defined by phosphorylation of AMPK target sites on the metabolic enzymes ACC1/2 (19), was strongly activated in both WT and HIF-1α-null cells under the same conditions of hypoxia and glucose deprivation, which is consistent with its function as a sensor of ATP depletion. However, AMPK activity was also rapidly induced in both cell types following exposure to hypoxia in the presence of glucose, even though total cellular ATP was not significantly depleted. By using genetically manipulated MEFs nullizygous for AMPK expression, we directly demonstrated that AMPK activity is sensitive to a wide range of low-oxygen conditions, at least in mesenchymal cells. To determine whether these hypoxia-inducible responses of AMPK also occur in vivo, we prepared tumor xenografts from transformed derivatives of the same WT and HIF-1α-null cells, and exposed tumor-bearing mice to the hypoxia probe pimonidazole (3, 10, 44, 45). Immunohistochemical analysis of these tumors indicated that AMPK activity was prevalent in hypoxic regions of both tumor types, especially in viable areas near necrosis. By using tumor xenografts prepared from identically transformed WT and AMPKα-null cells, we determined that the absence of AMPK activity greatly inhibited the growth of this experimental tumor type. We propose that activation of AMPK in hypoxic or ischemic microenvironments may be critical for cell survival and thus would represent a novel protective mechanism for metabolically depressed or ATP-deficient cells.

Fatty acid oxidation is a dominant bioenergetic pathway in prostate cancer.

Abstract: Most malignancies have increased glycolysis for energy requirement of rapid cell proliferation, which is the basis for tumor imaging through glucose analog FDG (2-deoxy-2-fluoro-D-glucose) with positron emission tomography. One of significant characteristics of prostate cancer is slow glycolysis and low FDG avidity. Recent studies showed that prostate cancer is associated with changes of fatty acid metabolism. Several enzymes involved in the metabolism of fatty acids have been determined to be altered in prostate cancer relative to normal prostate, which is indicative of an enhanced beta-oxidation pathway in prostate cancer. Increased fatty acid utilization in prostate cancer provides both ATP and acetyl-coenzyme A (CoA); subsequently, increased availability of acetyl-CoA makes acceleration of citrate oxidation possible, which is an important energy source as well. Dominant fatty acid metabolism rather than glycolysis has the potential to be the basis for imaging diagnosis and targeted treatment of prostate cancer.

Neuronal–Glial Glucose Oxidation and Glutamatergic–GABAergic Function

Abstract: Prior 13C magnetic resonance spectroscopy (MRS) experiments, which simultaneously measured in vivo rates of total glutamate-glutamine cycling (V(cyc(tot))) and neuronal glucose oxidation (CMR(glc(ox), N)), revealed a linear relationship between these fluxes above isoelectricity, with a slope of approximately 1. In vitro glial culture studies examining glutamate uptake indicated that glutamate, which is cotransported with Na+, stimulated glial uptake of glucose and release of lactate. These in vivo and in vitro results were consolidated into a model: recycling of one molecule of neurotransmitter between glia and neurons was associated with oxidation of one glucose molecule in neurons; however, the glucose was taken up only by glia and all the lactate (pyruvate) generated by glial glycolysis was transferred to neurons for oxidation. The model was consistent with the 1:1 relationship between DeltaCMR(glc(ox), N) and DeltaV(cyc(tot)) measured by 13C MRS. However, the model could not specify the energetics of glia and gamma-amino butyric acid (GABA) neurons because quantitative values for these pathways were not available. Here, we review recent 13C and 14C tracer studies that enable us to include these fluxes in a more comprehensive model. The revised model shows that glia produce at least 8% of total oxidative ATP and GABAergic neurons generate approximately 18% of total oxidative ATP in neurons. Neurons produce at least 88% of total oxidative ATP, and take up approximately 26% of the total glucose oxidized. Glial lactate (pyruvate) still makes the major contribution to neuronal oxidation, but approximately 30% less than predicted by the prior model. The relationship observed between DeltaCMR(glc(ox), N) and DeltaV(cyc(tot)) is determined by glial glycolytic ATP as before. Quantitative aspects of the model, which can be tested by experimentation, are discussed.

Dynamics of the Cellular Metabolome during Human Cytomegalovirus Infection

Abstract: Viral replication requires energy and macromolecular precursors derived from the metabolic network of the host cell. Despite this reliance, the effect of viral infection on host cell metabolic composition remains poorly understood. Here we applied liquid chromatography-tandem mass spectrometry to measure the levels of 63 different intracellular metabolites at multiple times after human cytomegalovirus (HCMV) infection of human fibroblasts. Parallel microarray analysis provided complementary data on transcriptional regulation of metabolic pathways. As the infection progressed, the levels of metabolites involved in glycolysis, the citric acid cycle, and pyrimidine nucleotide biosynthesis markedly increased. HCMV-induced transcriptional upregulation of specific glycolytic and citric acid cycle enzymes mirrored the increases in metabolite levels. The peak levels of numerous metabolites during infection far exceeded those observed during normal fibroblast growth or quiescence, demonstrating that HCMV markedly disrupts cellular metabolic homeostasis and institutes its own specific metabolic program.

Mitochondrial respiration defects in cancer cells cause activation of Akt survival pathway through a redox-mediated mechanism

Abstract: Cancer cells exhibit increased glycolysis for ATP production due, in part, to respiration injury (the Warburg effect). Because ATP generation through glycolysis is less efficient than through mitochondrial respiration, how cancer cells with this metabolic disadvantage can survive the competition with other cells and eventually develop drug resistance is a long-standing paradox. We report that mitochondrial respiration defects lead to activation of the Akt survival pathway through a novel mechanism mediated by NADH. Respiration-deficient cells (ρ-) harboring mitochondrial DNA deletion exhibit dependency on glycolysis, increased NADH, and activation of Akt, leading to drug resistance and survival advantage in hypoxia. Similarly, chemical inhibition of mitochondrial respiration and hypoxia also activates Akt. The increase in NADH caused by respiratory deficiency inactivates PTEN through a redox modification mechanism, leading to Akt activation. These findings provide a novel mechanistic insight into the Warburg effect and explain how metabolic alteration in cancer cells may gain a survival advantage and withstand therapeutic agents.

Glycolysis and sperm motility: does a spoonful of sugar help the flagellum go round?

Abstract: It is doubtful that diffusion can deliver sufficient ATP from the mitochondria to sustain activity at the distal end of the sperm flagellum. Glycolytic enzymes bound to the fibrous sheath could provide energy along the flagellum at the point it is required. An obligatory role for glycolysis is supported by the lack of progressive motility in sperm from mice where the gene for sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDHs) had been ‘knocked out’. Here, I review some evidence against this idea. First, pure diffusion from the mitochondrion is likely to be adequate in species with smaller sperm, and it is possible that rapid ATP delivery required in larger sperm could be achieved by an adenylate kinase shuttle. Second, experience with -chlorohydrin demonstrates that sperm can remain motile with normal ATP concentrations despite inhibition of GAPDHs; adverse effects only occur if glucose is added and high levels of glycolytic intermediates accumulate. These observations undermine the GAPDHs knockout mouse as evidence for an essential role of local glycolysis. Third, sperm from many species can remain motile for long periods in sugarfree media and excepting dog sperm, evidence that gluconeogenesis is a possible explanation, is weak. In most species, it is unlikely that local glycolysis is the only way that ATP can be supplied to the distal flagellum.

Regulation of fat metabolism in skeletal muscle.

Abstract: Regulation of carbohydrate and fat utilization by skeletal muscle at rest and during exercise has been the subject of investigation since the early 1960s when Randle et al. proposed the so-called glucose-fatty acid cycle to explain the reciprocal relationship between carbohydrate and fat metabolism. The suggested mechanisms were based on the premise that an increase in fatty acid (FA) availability would result in increased fat metabolism and inhibition of carbohydrate metabolism. Briefly, accumulation of acetyl-CoA would result in inhibition of pyruvate dehydrogenase (PDH), accumulation of citrate would inhibit phosphofructokinase (PFK), and accumulation of glucose-6-phosphate (G6P) would reduce hexokinase (HK) activity. Ultimately, this would inhibit carbohydrate metabolism with increasing availability and oxidation of FA. Although there is some evidence for the existence of the glucose-FA cycle at rest and during low-intensity exercise, it cannot explain substrate use at moderate to high exercise intensities. More recently, evidence has accumulated that increases in glycolytic flux may decrease fat metabolism. Potential sites of regulation are the transport of FA into the sarcoplasma, lipolysis of intramuscular triacylglycerol (IMTG) by hormone-sensitive lipase (HSL), and transport of FA across the mitochondrial membrane. There are several potential regulators of fat oxidation: first, malonyl-CoA concentration, which is formed from acetyl-CoA, catalyzed by the enzyme acetyl-CoA carboxylase (ACC), which in turn will inhibit carnitine palmitoyl transferase I (CPT I). Another possible mechanism is accumulation of acetyl-CoA that will result in acetylation of the carnitine pool, reducing the free carnitine concentration. This could theoretically reduce FA transport into the mitochondria. There is also some recent evidence that CPT I is inhibited by small reductions in pH that might be observed during exercise at high intensities. It is also possible that FA entry into the sarcolemma is regulated by translocation of FAT/CD36 in a similar manner to glucose transport by GLUT-4. Studies suggest that the regulatory mechanisms may be different at rest and during exercise and may change as the exercise intensity increases. Regulation of skeletal muscle fat metabolism is clearly multifactorial, and different mechanisms may dominate in different conditions.

Expression of transketolase TKTL1 predicts colon and urothelial cancer patient survival: Warburg effect reinterpreted.

Abstract: Tumours ferment glucose to lactate even in the presence of oxygen (aerobic glycolysis; Warburg effect). The pentose phosphate pathway (PPP) allows glucose conversion to ribose for nucleic acid synthesis and glucose degradation to lactate. The nonoxidative part of the PPP is controlled by transketolase enzyme reactions. We have detected upregulation of a mutated transketolase transcript (TKTL1) in human malignancies, whereas transketolase (TKT) and transketolase-like-2 (TKTL2) transcripts were not upregulated. Strong TKTL1 protein expression was correlated to invasive colon and urothelial tumours and to poor patients outcome. TKTL1 encodes a transketolase with unusual enzymatic properties, which are likely to be caused by the internal deletion of conserved residues. We propose that TKTL1 upregulation in tumours leads to enhanced, oxygen-independent glucose usage and a lactate-based matrix degradation. As inhibition of transketolase enzyme reactions suppresses tumour growth and metastasis, TKTL1 could be the relevant target for novel anti-transketolase cancer therapies. We suggest an individualised cancer therapy based on the determination of metabolic changes in tumours that might enable the targeted inhibition of invasion and metastasis.

Inhibition of glycogenolysis in astrocytes interrupts memory consolidation in young chickens

Abstract: Glycolysis and glycogenolysis are involved in memory processing in day-old chickens and, aside from the provision of energy for neuronal and astrocytic energy metabolism these pathways enable astrocytes to supply neurones with precursor for transmitter glutamate by glucose-based de novo synthesis. We have previously shown that memory processing for bead discrimination learning is dependent on glycolysis; however, the metabolic inhibitor used, iodoacetate, inhibits pyruvate formation from both glucose and glycogen. At specific time points after training transient reductions in brain glycogen content occur, mirrored by increases in glutamate/glutamine content. In the present study, we used intracerebral injection of a glycogen phosphorylase inhibitor, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), which does not affect glucose breakdown, to evaluate the role of glycogen metabolism in memory consolidation. Dose-dependent inhibition of learning occurred when DAB was administered at specific time periods in relation to training: (i) 5 min before training, (ii) around 30 min posttraining, and (iii) 55 min posttraining. After injection at either of the two earlier periods, memory disappeared after consolidation 30 min postlearning, and after injection 55 min after learning memory was absent at 70 min. The memory loss caused by early administration could be prevented after training by central injection of the glutamate precursor glutamine or the astrocyte-specific substrate acetate together with aspartate, substituting for pyruvate carboxylation. Thus, glycogenolysis is essential for learning in this paradigm and, aside from energy supply considerations, we suggest that an important role for glycogenolysis is to provide neurones with glutamine as the precursor for neuronal glutamate and GABA. © 2006 Wiley-Liss, Inc.

Mitochondria in cancer.

Abstract: Prominent features of cancer cells include metabolic imbalances and enhanced resistance to mitochondrial apoptosis. The fact that tumors rely heavily on glycolysis to meet their metabolic demands has been recognized since the beginning of the twentieth century, yet a complete elucidation of the so-called Warburg effect has not been achieved. Several mechanisms have been proposed to explain this phenomenon, including the upregulation of rate-limiting steps of glycolysis, the accumulation of mutations in the mitochondrial genome, the hypoxia-induced switch from mitochondrial respiration to glycolysis or the metabolic reprogramming resulting from the loss-of-function of enzymes like fumarate and succinate dehydrogenases. How aerobic glycolysis and apoptosis resistance are linked remains to be elucidated. On the one hand, these alterations may be acquired independently by cancer cells during multistep oncogenesis. On the other hand, the suppression of the intrinsic apoptotic program may be achieved through mechanisms that directly lead to the Warburg phenotype. Cancer-specific mitochondrial alterations and bioenergetics may be taken advantage for the development of two novel classes of antineoplastic agents. A first approach would target glycolysis and/or revert the Warburg phenomenon, whereas a second approach would aim at inducing apoptosis by targeting mitochondrial proteins and membranes. In both instances, encouraging pre-clinical results have been obtained.

Determining and understanding the control of glycolysis in fast-growth tumor cells. Flux control by an over-expressed but strongly product-inhibited hexokinase.

Abstract: Control analysis of the glycolytic flux was carried out in two fast-growth tumor cell types of human and rodent origin (HeLa and AS-30D, respectively). Determination of the maximal velocity (V(max)) of the 10 glycolytic enzymes from hexokinase to lactate dehydrogenase revealed that hexokinase (153-306 times) and phosphofructokinase-1 (PFK-1) (22-56 times) had higher over-expression in rat AS-30D hepatoma cells than in normal freshly isolated rat hepatocytes. Moreover, the steady-state concentrations of the glycolytic metabolites, particularly those of the products of hexokinase and PFK-1, were increased compared with hepatocytes. In HeLa cells, V(max) values and metabolite concentrations for the 10 glycolytic enzyme were also significantly increased, but to a much lesser extent (6-9 times for both hexokinase and PFK-1). Elasticity-based analysis of the glycolytic flux in AS-30D cells showed that the block of enzymes producing Fru(1,6)P2 (i.e. glucose transporter, hexokinase, hexosephosphate isomerase, PFK-1, and the Glc6P branches) exerted most of the flux control (70-75%), whereas the consuming block (from aldolase to lactate dehydrogenase) exhibited the remaining control. The Glc6P-producing block (glucose transporter and hexokinase) also showed high flux control (70%), which indicated low flux control by PFK-1. Kinetic analysis of PFK-1 showed low sensitivity towards its allosteric inhibitors citrate and ATP, at physiological concentrations of the activator Fru(2,6)P2. On the other hand, hexokinase activity was strongly inhibited by high, but physiological, concentrations of Glc6P. Therefore, the enhanced glycolytic flux in fast-growth tumor cells was still controlled by an over-produced, but Glc6P-inhibited hexokinase.

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside and metformin inhibit hepatic glucose phosphorylation by an AMP-activated protein kinase-independent effect on glucokinase translocation.

Abstract: AMP-activated protein kinase (AMPK) controls glucose uptake and glycolysis in muscle. Little is known about its role in liver glucose uptake, which is controlled by glucokinase. We report here that 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), metformin, and oligomycin activated AMPK and inhibited glucose phosphorylation and glycolysis in rat hepatocytes. In vitro experiments demonstrated that this inhibition was not due to direct phosphorylation of glucokinase or its regulatory protein by AMPK. By contrast, AMPK phosphorylated liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase without affecting activity. Inhibitors of the endothelial nitric oxide synthase, stress kinases, and phosphatidylinositol 3-kinase pathways did not counteract the effects of AICAR, metformin, or oligomycin, suggesting that these signaling pathways were not involved. Interestingly, the inhibitory effect on glucose phosphorylation of these well-known AMPK activators persisted in primary cultured hepatocytes from newly engineered mice lacking both liver alpha1 and alpha2 AMPK catalytic subunits, demonstrating that this effect was clearly not mediated by AMPK. Finally, AICAR, metformin, and oligomycin were found to inhibit the glucose-induced translocation of glucokinase from the nucleus to the cytosol by a mechanism that could be related to the decrease in intracellular ATP concentrations observed in these conditions.

Mitochondrial UCP4 mediates an adaptive shift in energy metabolism and increases the resistance of neurons to metabolic and oxidative stress.

Abstract: The high-metabolic demand of neurons and their reliance on glucose as an energy source places them atrisk for dysfunction and death under conditions of metabolic and oxidative stress. Uncoupling proteins (UCPs) are mitochodrial inner membrane proteins implicated in the regulation of mitochondrial membrane potential (ΔΨm) and cellular energy metabolism. The authors cloned UCP4 cDNA from mouse and rat brain, and demonstrate that UCP4 mRNA is expressed abundantly in brain and at particularly high levels in populations of neurons believed to have high-energy requirements. Neural cells with increased levels of UCP4 exhibit decreased ΔΨm, reduced reactive oxygen species (ROS) production and decreased mitochondrial calcium accumulation. UCP4 expressing cells also exhibited changes of oxygen-consumption rate, GDP sensitivity, and response of ΔΨm to oligomycin that were consistent with mitochondrial uncoupling. UCP4 modulates neuronal energy metabolism by increasing glucose uptake and shifting the mode of ATP production from mitochodnrial respiration to glycolysis, thereby maintaining cellular ATP levels. The UCP4-mediated shift in energy metabolism reduces ROS production and increases the resistance of neurons to oxidative and mitochondrial stress. Knockdown of UCP4 expression by RNA interference in primary hippocampal neurons results in mitochondrial calcium overload and cell death. UCP4-mRNA expression is increased in neurons exposed to cold temperatures and in brain cells of rats maintained on caloric restriction, suggesting a role for UCP4 in the previously reported antiageing and neuroprotective effects of caloric restriction. By shifting energy metabolism to reduce ROS production and cellular reliance on mitochondrial respiration, UCP4 can protect neurons against oxidative stress and calcium overload.

Ras transformation requires metabolic control by 6-phosphofructo-2-kinase.

Abstract: Neoplastic cells transport large amounts of glucose in order to produce anabolic precursors and energy within the inhospitable environment of a tumor. The ras signaling pathway is activated in several cancers and has been found to stimulate glycolytic flux to lactate. Glycolysis is regulated by ras via the activity of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFK2/FBPase), which modulate the intracellular concentration of the allosteric glycolytic activator, fructose-2,6-bisphosphate (F2,6BP). We report herein that sequential immortalization and ras-transformation of mouse fibroblasts or human bronchial epithelial cells paradoxically decreases the intracellular concentration of F2,6BP. This marked reduction in the intracellular concentration of F2,6BP sensitizes transformed cells to the antimetabolic effects of PFK2/FBPase inhibition. Moreover, despite co-expression of all four mRNA species (PFKFB1-4), heterozygotic genomic deletion of the inducible PFKFB3 gene in ras-transformed mouse lung fibroblasts suppresses F2,6BP production, glycolytic flux to lactate, and growth as soft agar colonies or tumors in athymic mice. These data indicate that the PFKFB3 protein product may serve as an essential downstream metabolic mediator of oncogenic ras, and we propose that pharmacologic inhibition of this enzyme should selectively suppress the high rate of glycolysis and growth by cancer cells.

Exercise affects energy metabolism and neural plasticity-related proteins in the hippocampus as revealed by proteomic analysis.

Abstract: Studies were conducted to evaluate the effect of a brief voluntary exercise period on the expression pattern and post-translational modification of multiple protein classes in the rat hippocampus using proteomics. An analysis of 80 protein spots of relative high abundance on two-dimensional gels revealed that approximately 90% of the proteins identified were associated with energy metabolism and synaptic plasticity. Exercise up-regulated proteins involved in four aspects of energy metabolism, i.e. glycolysis, ATP synthesis, ATP transduction and glutamate turnover. Specifically, we found increases in fructose-bisphosphate aldolase C, phosphoglycerate kinase 1, mitochondrial ATP synthase, ubiquitous mitochondrial creatine kinase and glutamate dehydrogenase 1. Exercise also up-regulated specific synaptic-plasticity-related proteins, the cytoskeletal protein alpha-internexin and molecular chaperones (chaperonin-containing TCP-1, neuronal protein 22, heat shock 60-kDa protein 1 and heat shock protein 8). Western blot was used to confirm the direction and magnitude of change in ubiquitous mitochondrial creatine kinase, an enzyme essential for transducing mitochondrial-derived ATP to sites of high-energy demand such as the synapse. Protein phosphorylation visualized by Pro-Q Diamond fluorescent staining showed that neurofilament light polypeptide, glial fibrillary acidic protein, heat shock protein 8 and transcriptional activator protein pur-alpha were more intensely phosphorylated with exercise as compared with sedentary control levels. Our results, together with the fact that most of the proteins that we found to be up-regulated have been implicated in cognitive function, support a mechanism by which exercise uses processes of energy metabolism and synaptic plasticity to promote brain health.