Showing papers on "Glycolysis published in 2005"

Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1.

Abstract: Homeostatic mechanisms in mammals respond to hormones and nutrients to maintain blood glucose levels within a narrow range. Caloric restriction causes many changes in glucose metabolism and extends lifespan; however, how this metabolism is connected to the ageing process is largely unknown. We show here that the Sir2 homologue, SIRT1--which modulates ageing in several species--controls the gluconeogenic/glycolytic pathways in liver in response to fasting signals through the transcriptional coactivator PGC-1alpha. A nutrient signalling response that is mediated by pyruvate induces SIRT1 protein in liver during fasting. We find that once SIRT1 is induced, it interacts with and deacetylates PGC-1alpha at specific lysine residues in an NAD(+)-dependent manner. SIRT1 induces gluconeogenic genes and hepatic glucose output through PGC-1alpha, but does not regulate the effects of PGC-1alpha on mitochondrial genes. In addition, SIRT1 modulates the effects of PGC-1alpha repression of glycolytic genes in response to fasting and pyruvate. Thus, we have identified a molecular mechanism whereby SIRT1 functions in glucose homeostasis as a modulator of PGC-1alpha. These findings have strong implications for the basic pathways of energy homeostasis, diabetes and lifespan.

Inhibition of glycolysis in cancer cells: a novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia.

Abstract: Cancer cells generally exhibit increased glycolysis for ATP generation (the Warburg effect) due in part to mitochondrial respiration injury and hypoxia, which are frequently associated with resistance to therapeutic agents. Here, we report that inhibition of glycolysis severely depletes ATP in cancer cells, especially in clones of cancer cells with mitochondrial respiration defects, and leads to rapid dephosphorylation of the glycolysis-apoptosis integrating molecule BAD at Ser 112 , relocalization of BAX to mitochondria, and massive cell death. Importantly, inhibition of glycolysis effectively kills colon cancer cells and lymphoma cells in a hypoxic environment in which the cancer cells exhibit high glycolytic activity and decreased sensitivity to common anticancer agents. Depletion of ATP by glycolytic inhibition also potently induced apoptosis in multidrug-resistant cells, suggesting that deprivation of cellular energy supply may be an effective way to overcome multidrug resistance. Our study shows a promising therapeutic strategy to effectively kill cancer cells and overcome drug resistance. Because the Warburg effect and hypoxia are frequently seen in human cancers, these findings may have broad clinical implications. (Cancer Res 2005; 65(2): 613-21)

ATP citrate lyase is an important component of cell growth and transformation

Abstract: Cell proliferation requires a constant supply of lipids and lipid precursors to fuel membrane biogenesis and protein modification. Cytokine stimulation of hematopoietic cells directly stimulates glucose utilization in excess of bioenergetic demand, resulting in a shift from oxidative to glycolytic metabolism. A potential benefit of this form of metabolism is the channeling of glucose into biosynthetic pathways. Here we report that glucose supports de novo lipid synthesis in growing hematopoietic cells in a manner regulated by cytokine availability and the PI 3 K/Akt signaling pathway. The net conversion of glucose to lipid is dependent on the ability of cells to produce cytosolic acetyl CoA from mitochondria-derived citrate through the action of ATP citrate lyase (ACL). Stable knockdown of ACL leads to a significant impairment of glucose-dependent lipid synthesis and an elevation of mitochondrial membrane potential. Cells with ACL knockdown display decreased cytokine-stimulated cell proliferation. In contrast, these cells resist cell death induced by either cytokine or glucose withdrawal. However, ACL knockdown significantly impairs Akt-mediated tumorigenesis in vivo. These data suggest that enzymes involved in the conversion of glucose to lipid may be targets for the treatment of pathologic cell growth.

Short-Term Overexpression of a Constitutively Active Form of AMP-Activated Protein Kinase in the Liver Leads to Mild Hypoglycemia and Fatty Liver

Abstract: AMP-activated protein kinase (AMPK) is a major therapeutic target for the treatment of diabetes. We investigated the effect of a short-term overexpression of AMPK specifically in the liver by adenovirus-mediated transfer of a gene encoding a constitutively active form of AMPKalpha2 (AMPKalpha2-CA). Hepatic AMPKalpha2-CA expression significantly decreased blood glucose levels and gluconeogenic gene expression. Hepatic expression of AMPKalpha2-CA in streptozotocin-induced and ob/ob diabetic mice abolished hyperglycemia and decreased gluconeogenic gene expression. In normal mouse liver, AMPKalpha2-CA considerably decreased the refeeding-induced transcriptional activation of genes encoding proteins involved in glycolysis and lipogenesis and their upstream regulators, SREBP-1 (sterol regulatory element-binding protein-1) and ChREBP (carbohydrate response element-binding protein). This resulted in decreases in hepatic glycogen synthesis and circulating lipid levels. Surprisingly, despite the inhibition of hepatic lipogenesis, expression of AMPKalpha2-CA led to fatty liver due to the accumulation of lipids released from adipose tissue. The relative scarcity of glucose due to AMPKalpha2-CA expression led to an increase in hepatic fatty acid oxidation and ketone bodies production as an alternative source of energy for peripheral tissues. Thus, short-term AMPK activation in the liver reduces blood glucose levels and results in a switch from glucose to fatty acid utilization to supply energy needs.

Skeletal muscle glucose uptake during exercise : How is it regulated?

Abstract: The increase in skeletal muscle glucose uptake during exercise results from a coordinated increase in rates of glucose delivery (higher capillary perfusion), surface membrane glucose transport, and intracellular substrate flux through glycolysis. The mechanism behind the movement of GLUT4 to surface membranes and the subsequent increase in transport by muscle contractions is largely unresolved, but it is likely to occur through intracellular signaling involving Ca2+-calmodulin-dependent protein kinase, 5′-AMP-activated protein kinase, and possibly protein kinase C.

Polyunsaturated fatty acids suppress glycolytic and lipogenic genes through the inhibition of ChREBP nuclear protein translocation

Abstract: Dietary polyunsaturated fatty acids (PUFAs) are potent inhibitors of hepatic glycolysis and lipogenesis. Recently, carbohydrate-responsive element-binding protein (ChREBP) was implicated in the regulation by glucose of glycolytic and lipogenic genes, including those encoding L-pyruvate kinase (L-PK) and fatty acid synthase (FAS). The aim of our study was to assess the role of ChREBP in the control of L-PK and FAS gene expression by PUFAs. We demonstrated in mice, both in vivo and in vitro, that PUFAs [linoleate (C18:2), eicosapentanoic acid (C20:5), and docosahexaenoic acid (C22:6)] suppressed ChREBP activity by increasing ChREBP mRNA decay and by altering ChREBP translocation from the cytosol to the nucleus, independently of an activation of the AMP-activated protein kinase, previously shown to regulate ChREBP activity. In contrast, saturated [stearate (C18)] and monounsaturated fatty acids [oleate (C18:1)] had no effect. Since glucose metabolism via the pentose phosphate pathway is determinant for ChREBP nuclear translocation, the decrease in xylulose 5-phosphate concentrations caused by a PUFA diet favors a PUFA-mediated inhibition of ChREBP translocation. In addition, overexpression of a constitutive nuclear ChREBP isoform in cultured hepatocytes significantly reduced the PUFA inhibition of both L-PK and FAS gene expression. Our results demonstrate that the suppressive effect of PUFAs on these genes is primarily caused by an alteration of ChREBP nuclear translocation. In conclusion, we describe a novel mechanism to explain the inhibitory effect of PUFAs on the genes encoding L-PK and FAS and demonstrate that ChREBP is a pivotal transcription factor responsible for coordinating the PUFA suppression of glycolytic and lipogenic genes.

Mitochondria and cancer: Warburg addressed.

Abstract: Otto Warburg recognized that cancer cells generate excessive lactate in the presence of oxygen (aerobic glycolysis). It now appears that this phenomenon is the product of two factors: a return to the more glycolytic metabolism of the embryo and alterations in oxidative phosphorylation (OXPHOS) to increase mitochondrial reactive oxygen species (ROS) production. Alterations in the Ras-PI3K-Akt signal transduction pathway can result in induction of hexokinase II and its attachment to mitochondrial porin redirecting mitochondrial ATP to phosphorylate glucose and drive glycolysis. Furthermore, partial inhibition of OXPHOS by mitochondrial gene mutations (germ-line or somatic) can reduce electron flux through the electron transport chain, increasing mitochondrial ROS production. The increased ROS mutagenizes nuclear proto-oncogenes (initiation) and drives nuclear replication (promotion), resulting in cancer. Therefore, hexokinase II and mitochondrial ROS may be useful alternate targets for cancer therapeutics.

Cells die with increased cytosolic ATP during apoptosis: a bioluminescence study with intracellular luciferase

Abstract: Apoptosis is a distinct form of cell death, which requires energy. Here, we made real-time continuous measurements of the cytosolic ATP level throughout the apoptotic process in intact HeLa, PC12 and U937 cells transfected with the firefly luciferase gene. Apoptotic stimuli (staurosporine (STS), tumor necrosis factor alpha (TNFalpha), etoposide) induced significant elevation of the cytosolic ATP level. The cytosolic ATP level remained at a higher level than in the control for up to 6 h during which activation of caspase-3 and internucleosomal DNA fragmentation took place. When the STS-induced ATP response was abolished by glucose deprivation-induced inhibition of glycolysis, both caspase activation and DNA laddering were completely inhibited. Annexin V-binding induced by STS or TNFalpha was largely suppressed by glycolysis inhibition. Thus, it is suggested that the cells die with increased cytosolic ATP, and elevation of cytosolic ATP level is a requisite to the apoptotic cell death process.

Perspective: emerging evidence for signaling roles of mitochondrial anaplerotic products in insulin secretion.

Abstract: The importance of mitochondrial biosynthesis in stimulus secretion coupling in the insulin-producing beta-cell probably equals that of ATP production. In glucose-induced insulin secretion, the rate of pyruvate carboxylation is very high and correlates more strongly with the glucose concentration the beta-cell is exposed to (and thus with insulin release) than does pyruvate decarboxylation, which produces acetyl-CoA for metabolism in the citric acid cycle to produce ATP. The carboxylation pathway can increase the levels of citric acid cycle intermediates, and this indicates that anaplerosis, the net synthesis of cycle intermediates, is important for insulin secretion. Increased cycle intermediates will alter mitochondrial processes, and, therefore, the synthesized intermediates must be exported from mitochondria to the cytosol (cataplerosis). This further suggests that these intermediates have roles in signaling insulin secretion. Although evidence is quite good that all physiological fuel secretagogues stimulate insulin secretion via anaplerosis, evidence is just emerging about the possible extramitochondrial roles of exported citric acid cycle intermediates. This article speculates on their potential roles as signaling molecules themselves and as exporters of equivalents of NADPH, acetyl-CoA and malonyl-CoA, as well as alpha-ketoglutarate as a substrate for hydroxylases. We also discuss the "succinate mechanism," which hypothesizes that insulin secretagogues produce both NADPH and mevalonate. Finally, we discuss the role of mitochondria in causing oscillations in beta-cell citrate levels. These parallel oscillations in ATP and NAD(P)H. Oscillations in beta-cell plasma membrane electrical potential, ATP/ADP and NAD(P)/NAD(P)H ratios, and glycolytic flux are known to correlate with pulsatile insulin release. Citrate oscillations might synchronize oscillations of individual mitochondria with one another and mitochondrial oscillations with oscillations in glycolysis and, therefore, with flux of pyruvate into mitochondria. Thus citrate oscillations may synchronize mitochondrial ATP production and anaplerosis with other cellular oscillations.

NAD+ as a metabolic link between DNA damage and cell death

Abstract: DNA damage occurs in ischemia, excitotoxicity, inflammation, and other disorders that affect the central nervous system (CNS). Extensive DNA damage triggers cell death and in the mature CNS, this occurs primarily through activation of the poly(ADP-ribose) polymerase-1 (PARP-1) cell death pathway. PARP-1 is an abundant nuclear enzyme that, when activated by DNA damage, consumes nicotinamide adenine dinucleotide (NAD)+ to form poly(ADP-ribose) on acceptor proteins. The mechanisms by which PARP-1 activation leads to cell death are not understood fully. We used mouse astrocyte cultures to explore the bioenergetic effects of NAD+ depletion by PARP-1 and the role of NAD+ depletion in this cell death program. PARP-1 activation was induced by the DNA alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), using medium in which glucose was the only exogenous energy substrate. PARP-1 activation led to a rapid but incomplete depletion of astrocyte NAD+, a near-complete block in glycolysis, and eventual cell death. Repletion of intracellular NAD+ restored glycolytic function and prevented cell death. The addition of non-glucose substrates to the medium, pyruvate, glutamate, or glutamine, also prevented astrocyte death after PARP-1 activation. These studies suggest PARP-1 activation leads to rapid depletion of the cytosolic but not the mitochondrial NAD+ pool. Depletion of the cytosolic NAD+ pool renders the cells unable to utilize glucose as a metabolic substrate. Under conditions where glucose is the only available metabolic substrate, this leads to cell death. This cell death pathway is particularly germane to brain because glucose is normally the only metabolic substrate that is transported rapidly across the blood-brain barrier.

Proteomic Analysis of Global Changes in Protein Expression during Bile Salt Exposure of Bifidobacterium longum NCIMB 8809

Abstract: Adaptation to and tolerance of bile stress are among the main limiting factors to ensure survival of bifidobacteria in the intestinal environment of humans. The effect of bile salts on protein expression patterns of Bifidobacterium longum was examined. Protein pattern comparison of strains grown with or without bile extract allowed us to identify 34 different proteins whose expression was regulated. The majority of these proteins were induced after both a minor (0.6 g liter−1) and a major (1.2 g liter−1) exposure to bile. These include general stress response chaperones, proteins involved in transcription and translation and in the metabolism of amino acids and nucleotides, and several enzymes of glycolysis and pyruvate catabolism. Remarkably, xylulose 5-phosphate/fructose 6-phosphate phosphoketolase, the key enzyme of the so-called bifidobacterial shunt, was found to be upregulated, and the activity on fructose 6-phosphate was significantly higher for protein extracts of cells grown in the presence of bile. Changes in the levels of metabolic end products (acetate and lactate) were also detected. These results suggest that bile salts, to which bifidobacteria are naturally exposed, induce a complex physiological response rather than a single event in which proteins from many different functional categories take part. This study has extended our understanding of the molecular mechanism underlying the capacity of intestinal bifidobacteria to tolerate bile.

Phosphorylation of the 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase/PFKFB3 family of glycolytic regulators in human cancer.

Abstract: Purpose: Fructose 2,6-bisphosphate (F2,6BP) is a potent activator of phosphofructokinase, which is a rate-limiting enzyme of glycolysis. The concentration of F2,6BP depends on the activity of the bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase). Four genes encoding PFK-2/FBPase have been identified and termed PFKFB1 to PFKFB4 . PFKFB3 protein is expressed in high levels in human tumors in situ . The purpose of this study was to determine the role of functional interactions between the phosphorylation of PFKFB3 and activated glycolysis in human cancer cells. Experimental Design: cDNA from several human tumor cell lines and human colon carcinoma were analyzed by reverse transcription-PCR to identify different splicing variants of PFKFB3. The effect of phosphorylation of Ser 461 was studied by recombinantly replacing this residue with glutamate (PFKFB3 S461E ). The phosphorylation of PFKFB3 protein in human cancer was determined by immunostaining using an anti-phospho-PFK-2(PFKFB3) antibody. Results: Two splicing variants of PFKFB3 are expressed in human cancer cell lines: PFKFB3-ACG and PFKFB3-AG. Quantitative, real-time PCR analysis confirmed the overexpression of PFKFB3 mRNA in colon carcinoma, with the dominant variant being the PFKFB3-ACG isoform that contains a phosphorylation site at Ser 461 . Forced expression of PFKFB3-ACG in COS-7 cells resulted in enhanced glycolysis. Introduction of PFKFB3-ACG S461E into COS-7 cells led to increased the lactate production and cell proliferation. Highly phosphorylated PFKFB3 protein was found in human tumor cells, vascular endothelial cells, and smooth muscle cells, as determined by immunostaining with an anti-phospho-PFK-2(PFKFB3) antibody. Conclusions: These findings support a potential role for the phosphorylation of PFKFB3 protein in the progression of cancer and angiogenesis.

Tricarboxylic Acid Cycle and Glyoxylate Bypass.

Abstract: The tricarboxylic acid (TCA) cycle plays two essential roles in metabolism. First, under aerobic conditions the cycle is responsible for the total oxidation of acetyl-CoA that is derived mainly from the pyruvate produced by glycolysis. Second, TCA cycle intermediates are required in the biosynthesis of several amino acids. Although the TCA cycle has long been considered a "housekeeping" pathway in Escherichia coli and Salmonella enterica, the pathway is highly regulated at the transcriptional level. Much of this control is exerted in response to respiratory conditions. The TCA cycle gene-protein relationship and mutant phenotypes have been well studied, although a few loose ends remain. The realization that a "shadow" TCA cycle exists that proceeds through methylcitrate has cleared up prior ambiguities. The glyoxylate bypass has long been known to be essential for growth on carbon sources such as acetate or fatty acids because this pathway allowsnet conversion of acetyl-CoA to metabolic intermediates. Strains lacking this pathway fail to grow on these carbon sources, since acetate carbon entering the TCA cycle is quantitatively lost as CO2 resulting in the lack of a means to replenish the dicarboxylic acids consumed in amino acid biosynthesis. The TCA cycle gene-protein relationship and mutant phenotypes have been well studied, although the identity of the small molecule ligand that modulates transcriptional control of the glyoxylate cycle genes by binding to the IclR repressor remains unknown. The activity of the cycle is also exerted at the enzyme level by the reversible phosphorylation of the TCA cycle enzyme isocitrate dehydrogenase catalyzed by a specific kinase/phosphatase to allow isocitratelyase to compete for isocitrate and cleave this intermediate to glyoxylate and succinate.

Glucose metabolism heterogeneity in human and mouse malignant glioma cell lines.

Abstract: The current study examined specific bioenergetic markers associated with the metabolic phenotype of several human and mouse glioma cell lines. Based on preliminary studies, we hypothesized that glioma cells would express one of at least two different metabolic phenotypes, possibly acquired through progression. The D-54MG and GL261 glioma cell lines displayed an oxidative phosphorylation (OXPHOS)-dependent phenotype, characterized by extremely long survival under glucose starvation, and low tolerance to poisoning of the electron transport chain (ETC). Alternatively, U-251MG and U-87MG glioma cells exhibited a glycolytic-dependent phenotype with functional OXPHOS. These cells displayed low tolerance to glucose starvation and were resistant to a ETC blocker. Moreover, these cells could be rescued in low glucose conditions by oxidative substrates (e.g., lactate, pyruvate). Finally, these two phenotypes could be distinguished by the differential expression of LDH isoforms. OXPHOS-dependent cells expressed both LDH-A and -B isoforms whereas glycolytic-dependent glioma cells expressed only LDH-B. In the latter case, LDH-B would be expected to be essential for the use of extracellular lactate to fuel cell activities. These observations raise the possibility that the heterogeneity in glucose metabolism and, in particular, the sole expression of LDH-B, might identify an important biological marker of glioma cells that is critical for their progression and that might afford a new target for anticancer drugs.

Age-related changes in ATP-producing pathways in human skeletal muscle in vivo.

Abstract: Energy for muscle contractions is supplied by ATP generated from 1) the net hydrolysis of phosphocreatine (PCr) through the creatine kinase reaction, 2) oxidative phosphorylation, and 3) anaerobic glycolysis. The effect of old age on these pathways is unclear. The purpose of this study was to examine whether age may affect ATP synthesis rates from these pathways during maximal voluntary isometric contractions (MVIC). Phosphorus magnetic resonance spectroscopy was used to assess high-energy phosphate metabolite concentrations in skeletal muscle of eight young (20-35 yr) and eight older (65-80 yr) men. Oxidative capacity was assessed from PCr recovery after a 16-s MVIC. We determined the contribution of each pathway to total ATP synthesis during a 60-s MVIC. Oxidative capacity was similar across age groups. Similar rates of ATP synthesis from PCr hydrolysis and oxidative phosphorylation were observed in young and older men during the 60-s MVIC. Glycolytic flux was higher in young than older men during the 60-s contraction (P < 0.001). When expressed relative to the overall ATP synthesis rate, older men relied on oxidative phosphorylation more than young men (P = 0.014) and derived a smaller proportion of ATP from anaerobic glycolysis (P < 0.001). These data demonstrate that although oxidative capacity was unaltered with age, peak glycolytic flux and overall ATP production from anaerobic glycolysis were lower in older men during a high-intensity contraction. Whether this represents an age-related limitation in glycolytic metabolism or a preferential reliance on oxidative ATP production remains to be determined.

The pyruvate requirement of some members of the Mycobacterium tuberculosis complex is due to an inactive pyruvate kinase: implications for in vivo growth

Abstract: Through examination of one of the fundamental in vitro characteristics of Mycobacterium bovis--its requirement for pyruvate in glycerol medium--we have revealed a lesion in central metabolism that has profound implications for in vivo growth and nutrition. Not only is M. bovis unable to use glycerol as a sole carbon source but the lack of a functioning pyruvate kinase (PK) means that carbohydrates cannot be used to generate energy. This disruption in sugar catabolism is caused by a single nucleotide polymorphism in pykA, the gene which encodes PK, that substitutes glutamic acid residue 220 with an aspartic acid residue. Substitution of this highly conserved amino acid residue renders PK inactive and thus blocks the ATP generating roles of glycolysis and the pentose phosphate pathway. This mutation was found to occur in other members of the M. tuberculosis complex, namely M. microti and M. africanum. With carbohydrates unable to act as carbon sources, the importance of lipids and gluconeogenesis for growth in vivo becomes apparent. Complementation of M. bovis with the pykA gene from M. tuberculosis H37Rv restored growth on glycerol. Additionally, the presence of a functioning PK caused the colony morphology of the complemented strain to change from the characteristic dysgonic growth of M. bovis to eugonic growth, an appearance normally associated with M. tuberculosis. We also suggest that the glycerol-soaked potato slices used for the derivation of the M. bovis bacillus Calmette and Guerin (BCG) vaccine strain selected for an M. bovis PK+ mutant, a finding that explains the alteration in colony morphology noted during the derivation of BCG. In summary, the disruption of a key step in glycolysis divides the M. tuberculosis complex into two groups with distinct carbon source utilization.

Pyruvate dehydrogenase complex: Metabolic link to ischemic brain injury and target of oxidative stress

Abstract: The mammalian pyruvate dehydrogenase complex (PDHC) is a mitochondrial matrix enzyme complex (greater than 7 million Daltons) that catalyzes the oxidative decarboxylation of pyruvate to form acetyl CoA, nicotinamide adenine dinucleotide (the reduced form, NADH), and CO(2). This reaction constitutes the bridge between anaerobic and aerobic cerebral energy metabolism. PDHC enzyme activity and immunoreactivity are lost in selectively vulnerable neurons after cerebral ischemia and reperfusion. Evidence from experiments carried out in vitro suggests that reperfusion-dependent loss of activity is caused by oxidative protein modifications. Impaired enzyme activity may explain the reduced cerebral glucose and oxygen consumption that occurs after cerebral ischemia. This hypothesis is supported by the hyperoxidation of mitochondrial electron transport chain components and NAD(H) that occurs during reperfusion, indicating that NADH production, rather than utilization, is rate limiting. Additional support comes from the findings that immediate postischemic administration of acetyl-L-carnitine both reduces brain lactate/pyruvate ratios and improves neurologic outcome after cardiac arrest in animals. As acetyl-L-carnitine is converted to acetyl CoA, the product of the PDHC reaction, it follows that impaired production of NADH is due to reduced activity of either PDHC or one or more steps in glycolysis. Impaired cerebral energy metabolism and PDHC activity are associated also with neurodegenerative disorders including Alzheimer's disease and Wernicke-Korsakoff syndrome, suggesting that this enzyme is an important link in the pathophysiology of both acute brain injury and chronic neurodegeneration.