Switching Between Cooperation and Competition in the Use of Extracellular Glucose

This paper addresses some questions related to the evolution of cooperative behaviors, in the context of energetic metabolism. Glycolysis can perform either under a dissipative working regime suitable for rapid proliferation or under an efficient regime that entails a good modus operandi under conditions of glucose shortage. A cellular mechanism allowing switching between these two regimes may represent an evolutionary achievement. Thus, we have explored the conditions that might have favored the emergence of such an accommodative mechanism. Because of an inevitable conflict for resources between individual interests and the common good, rapid and inefficient use of glucose is always favored by natural selection in spatially homogeneous environment, regardless of the external conditions. In contrast, when the space is structured, the behavior of the system is determined by its free energy content. If the fuel is abundant, the dissipative strategy dominates the space. However, under famine conditions the efficient regime represents an evolutionary stable strategy in a Harmony game. Between these two extreme situations, both metabolic regimes are engaged in a Prisoner’s Dilemma game, where the output depends on the extracellular free energy. The energy transition values that lead from one domain to another have been calculated. We conclude that an accommodative mechanism permitting alternation between dissipative and efficient regimes might have evolved in heterogeneous and highly fluctuating environments. Overall, the current work shows how evolutionary optimization and game-theoretical approaches can be complementary in providing useful insights into biochemical systems. Reviewing Editor: Dr. Antony Dean     

What Process Is Glycolytic Stoichiometry Optimal For?

It has been proposed that the glycolytic stoichiometry of 2 ATP per glucose is the result of an optimization that maximizes the rate of ATP production. However, using a nonequilibrium thermodynamic approach, we show here that glycolysis operates under optimal output power and not at optimal flow of ATP production. Furthermore, it can be proved that the same maximal output power can be achieved with different stoichiometries. However, changes in the glycolytic stoichiometry would dramatically affect the efficiency of all those cellular processes powered by ATP. Our results suggest that the stoichiometric coefficient, as found in most contemporary cells, may be the outcome of an evolutionary process leading to yield an operative quantum energy for the hydrolysis of ATP. [Reviewing Editor: Dr. Antony Dean]     

An Early and Anaerobic Scenario for the Transition to Undifferentiated Multicellularity

Glycolysis, an ancient energy-processing pathway, can operate either under an efficient but slow regime or, alternatively, under a dissipative but fast-working regime. Trading an increase in efficiency for a decrease in rate represents a cooperative behavior, while a dissipative metabolism can be regarded as a cheating strategy. Herein, using irreversible thermodynamic principles and methods derived from game theory, we investigate whether, and under what conditions, the interplay between these two metabolic strategies may have promoted the clustering of undifferentiated cells. In the current model, multicellularity implies the loss of motility, which represents a hindrance rather than a improvement when competing with mobile single-celled organisms. Despite that, when considering glycolysis as the only energy-processing pathway, we conclude that cells endowed with a low basal anabolic metabolism may have benefited from clustering when faced to compete with cells exhibiting a high anabolic activity. The current results suggest that the transition to multicellularity may have taken place much earlier than hitherto thought, providing support for an extended period of Precambrian metazoan diversification.     

Synaptosomal bioenergetics. The role of glycolysis, pyruvate oxidation and responses to hypoglycaemia

The bioenergetic interaction between glycolysis and oxidative phosphorylation in isolated nerve terminals (synaptosomes) from guinea-pig cerebral cortex is characterized. Essentially all synaptosomes contain functioning mitochondria. There is a tight coupling between glycolytic rate and respiration: uncoupler causes a tenfold increase in glycolysis and a sixfold increase in respiration. Synaptosomes contain little endogenous glycolytic substrate and glycolysis is dependent on external glucose. In glucose-free media, or following addition of iodoacetate, synaptosomes continue to respire and to maintain high ATP/ADP ratios. In contrast to glucose, the endogenous substrate can neither maintain high respiration in the presence of uncoupler nor generate ATP in the presence of cyanide. Pyruvate, but not succinate, is an excellent substrate for intact synaptosomes. The in-situ mitochondrial membrane potential (delta psi m) is highly dependent upon the availability of glycolytic or exogenous pyruvate; glucose deprivation causes a 20-mV depolarization, while added pyruvate causes a 6-mV hyperpolarization even in the presence of glucose. Inhibition of pyruvate dehydrogenase by arsenite or pyruvate transport by alpha-cyano-4-hydroxycinnamate has little effect on ATP/ADP ratios; however respiratory capacity is severely restricted. It is concluded that synaptosomes are valuable models for studying the control of mitochondrial substrate supply in situ.     

Glucose metabolism in perfused mouse hearts overexpressing human GLUT-4 glucose transporter

Glucose and fatty acid metabolism was assessed in isolated working hearts from control C57BL/KsJ-m+/+db mice and transgenic mice overexpressing the human GLUT-4 glucose transporter (db/+-hGLUT-4). Heart rate, coronary flow, cardiac output, and cardiac power did not differ between control hearts and hearts overexpressing GLUT-4. Hearts overexpressing GLUT-4 had significantly higher rates of glucose uptake and glycolysis and higher levels of glycogen after perfusion than control hearts, but rates of glucose and palmitate oxidation were not different. Insulin (1 mU/ml) significantly increased glycogen levels in both groups. Insulin increased glycolysis in control hearts but not in GLUT-4 hearts, whereas glucose oxidation was increased by insulin in both groups. Therefore, GLUT-4 overexpression increases glycolysis, but not glucose oxidation, in the heart. Although control hearts responded to insulin with increased rates of glycolysis, the enhanced entry of glucose in the GLUT-4 hearts was already sufficient to maximally activate glycolysis under basal conditions such that insulin could not further stimulate the glycolytic rate.     

The role of glucose in the survival and `recovery'' of the anoxic isolated perfused rat heart

Studies with the isolated perfused working rat heart were carried out to investigate factors that may enable the heart to recover after periods of anoxia. It was found that the presence of glucose in the perfusion fluid during anoxia was essential for complete post-anoxic recovery and the presence of a high concentration of K(+) increased not only the rate of recovery but also the final extent of recovery. In an attempt to clarify the roles played by glucose and K(+) in aiding the survival and recovery of the anoxic myocardium the concentrations of parameters associated with energy liberation and anaerobic glycolysis (ATP, ADP, AMP, P(i), creatine phosphate, glycogen and lactate) were measured in the presence and absence of glucose during the anoxic phase. Determinations of these parameters were carried out during the working aerobic control period, the anoxic period (K(+) arrest) and the recovery period. The results demonstrated that glucose acted as an energy source during anoxia and thus maintained myocardial concentrations of high-energy phosphates, particularly ATP. These studies have also shown a direct relationship between the ability of the heart to recover and the concentration of myocardial ATP at the time of reoxygenation.     

HK2/hexokinase-II integrates glycolysis and autophagy to confer cellular protection

Hexokinases (HKs) catalyze the first step of glucose metabolism, phosphorylating glucose to glucose 6-phosphate (G6P). HK2/hexokinase-II is a predominant isoform in insulin-sensitive tissues such as heart, skeletal muscle, and adipose tissues and is also upregulated in many types of tumors associated with enhanced aerobic glycolysis (the Warburg effect). Accumulating evidence indicates that HK2 plays an important role not only in glycolysis but also in cell survival. Although there is increasing recognition that cellular metabolism and cell survival are closely related, the molecular link between metabolism and autophagic pathways has not been fully elucidated. We recently discovered that HK2 facilitates autophagy in response to glucose deprivation (HK substrate deprivation) to protect cardiomyocytes, and suggest that HK2 functions as a molecular switch from glycolysis to autophagy to ensure cellular energy homeostasis under starvation conditions.     

The Warburg effect: Evolving interpretations of an established concept

Metabolic reprogramming and altered bioenergetics have emerged as hallmarks of cancer and an area of active basic and translational cancer research. Drastically upregulated glucose transport and metabolism in most cancers regardless of the oxygen supply, a phenomenon called the Warburg effect, is a major focuses of the research. Warburg speculated that cancer cells, due to defective mitochondrial oxidative phosphorylation (OXPHOS), switch to glycolysis for ATP synthesis, even in the presence of oxygen. Studies in the recent decade indicated that while glycolysis is indeed drastically upregulated in almost all cancer cells, mitochondrial respiration continues to operate normally at rates proportional to oxygen supply. There is no OXPHOS-to-glycolysis switch but rather upregulation of glycolysis. Furthermore, upregulated glycolysis appears to be for synthesis of biomass and reducing equivalents in addition to ATP production. The new finding that a significant amount of glycolytic intermediates is diverted to the pentose phosphate pathway (PPP) for production of NADPH has profound implications in how cancer cells use the Warburg effect to cope with reactive oxygen species (ROS) generation and oxidative stress, opening the door for anticancer interventions taking advantage of this. Recent findings in the Warburg effect and its relationship with ROS and oxidative stress controls will be reviewed. Cancer treatment strategies based on these new findings will be presented and discussed.     

Evolutionary optimization of metabolic pathways. Theoretical reconstruction of the stoichiometry of ATP and NADH producing systems

The structural design of ATP and NADH producing systems, such as glycolysis and the citric acid cycle (TCA), is analysed using optimization principles. It is assumed that these pathways combined with oxidative phosphorylation have reached, during their evolution, a high efficiency with respect to ATP production rates. On the basis of kinetic and thermodynamic principles, conclusions are derived concerning the optimal stoichiometry of such pathways. Extending previous investigations, both the concentrations of adenine nucleotides as well as nicotinamide adenine dinucleotides are considered variable quantities. This implies the consideration of the interaction of an ATP and NADH producing system, an ATP consuming system, a system coupling NADH consumption with ATP production and a system consuming NADH decoupled from ATP production. It is examined in what respect real metabolic pathways can be considered optimal by studying a large number of alternative pathways. The kinetics of the individual reactions are described by linear or bilinear functions of reactant concentrations. In this manner, the steady-state ATP production rate can be calculated for any possible ATP and NADH producing pathway. It is shown that most of the possible pathways result in a very low ATP production rate and that the very efficient pathways share common structural properties. Optimization with respect to the ATP production rate is performed by an evolutionary algorithm. The following results of our analysis are in close correspondence to the real design of glycolysis and the TCA cycle. (1) In all efficient pathways the ATP consuming reactions are located near the beginning. (2) In all efficient pathways NADH producing reactions as well as ATP producing reactions are located near the end. (3) The number of NADH molecules produced by the consumption of one energy-rich molecule (glucose) amounts to four in all efficient pathways. A distance measure and a measure for the internal ordering of reactions are introduced to study differences and similarities in the stoichiometries of metabolic pathways.     

Glycolysis plays a major role for adenosine triphosphate supplementation in mouse sperm flagellar movement

The mammalian sperm must be highly motile for a long time to fertilize a egg. It has been supposed that ATP required for sperm flagellar movement depends predominantly on mitochondrial respiration. We assessed the contribution of mitochondrial respiration to mouse sperm motility. Mouse sperm maintained vigorous motility with high beat frequency in an appropriate solution including a substrate such as glucose. The active sperm contained a large amount of ATP. When carbonyl cyanide m-chlorophenylhydrazone (CCCP) was applied to suppress the oxidative phosphorylation in mitochondria, the vigorous motility was maintained and the amount of ATP was kept at the equivalent level to that without CCCP. When pyruvate or lactate was provided instead of glucose, both sperm motility and the amount of ATP were high. However, they were drastically decreased when oxidative phosphorylation was suppressed by addition of CCCP. We also found that sperm motility could not be maintained in the presence of respiratory substrates when glycolysis was suppressed. 2-Deoxy-d-glucose (DOG) had no effect on mitochondrial respiration assessed by a fluorescent probe, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), but, it inhibited motility and decreased ATP content when pyruvate or lactate were provided as substrates. The present results suggest that glycolysis has an unexpectedly important role in providing the ATP required for sperm motility throughout the length of the sperm flagellum.     

Energetics of Anaerobic Sodium Transport by the Fresh Water Turtle Bladder

Certain of the metabolic events associated with anaerobic sodium transport by the isolated bladder of the fresh water turtle have been investigated. The data suggest that energy for this transport arises from glycolysis and that endogenous glycogen was the major and perhaps the sole source of substrate. The rate of anaerobic glycolysis, as determined by lactate formation, correlates well with the rate as determined by glycogen utilization. Using lactate formation as the index of anaerobic glycolysis, a linear relationship was observed between glycolysis and net anaerobic sodium transport. In the absence of sodium transport, glycolysis decreased by approximately 45 per cent. Tissue ATP concentrations were maintained at about the same level under anaerobic as under aerobic conditions. Finally if it is assumed that in the conversion of glycogen to lactate anaerobically, 3 moles of ATP are generated per mole of glucose residue, an average of over 15 equivalents of sodium were transported for every mole of ATP generated.     

Glycolysis Is Governed by Growth Regime and Simple Enzyme Regulation in Adherent MDCK Cells

Due to its vital importance in the supply of cellular pathways with energy and precursors, glycolysis has been studied for several decades regarding its capacity and regulation. For a systems-level understanding of the Madin-Darby canine kidney (MDCK) cell metabolism, we couple a segregated cell growth model published earlier with a structured model of glycolysis, which is based on relatively simple kinetics for enzymatic reactions of glycolysis, to explain the pathway dynamics under various cultivation conditions. The structured model takes into account in vitro enzyme activities, and links glycolysis with pentose phosphate pathway and glycogenesis. Using a single parameterization, metabolite pool dynamics during cell cultivation, glucose limitation and glucose pulse experiments can be consistently reproduced by considering the cultivation history of the cells. Growth phase-dependent glucose uptake together with cell-specific volume changes generate high intracellular metabolite pools and flux rates to satisfy the cellular demand during growth. Under glucose limitation, the coordinated control of glycolytic enzymes re-adjusts the glycolytic flux to prevent the depletion of glycolytic intermediates. Finally, the model''s predictive power supports the design of more efficient bioprocesses.     

Role of phosphate in the central metabolism of two lactic acid bacteria--a comparative systems biology approach

Lactic acid-producing bacteria survive in distinct environments, but show common metabolic characteristics. Here we studied the dynamic interactions of the central metabolism in Lactococcus lactis, extensively used as a starter culture in the dairy industry, and Streptococcus pyogenes, a human pathogen. Glucose-pulse experiments and enzymatic measurements were performed to parameterize kinetic models of glycolysis. Significant improvements were made to existing kinetic models for L. lactis, which subsequently accelerated the development of the first kinetic model of S. pyogenes glycolysis. The models revealed an important role for extracellular phosphate in the regulation of central metabolism and the efficient use of glucose. Thus, phosphate, which is rarely taken into account as an independent species in models of central metabolism, should be considered more thoroughly in the analysis of metabolic systems in the future. Insufficient phosphate supply can lead to a strong inhibition of glycolysis at high glucose concentrations in both species, but this was more severe in S. pyogenes. S. pyogenes is more efficient at converting glucose to ATP, showing a higher tendency towards heterofermentative energy metabolism than L. lactis. Our comparative systems biology approach revealed that the glycolysis of L. lactis and S. pyogenes have similar characteristics, but are adapted to their individual natural habitats with respect to phosphate regulation.     

Futile cycles revisited: a markov chain model of simultaneous glycolysis and gluconeogenesis

We have used a random walk model of glycolysis and gluconeogenesis to investigate the bioenergetic implications of considering the cell cytoplasm to be a uniform well-mixed compartment. Radiotracer studies conducted on hepatocytes harvested from fasted rats and incubated with 40 mM glucose and 10 mM lactate demonstrated simultaneous glycolysis and gluconeogenesis, with net glycolysis. Tracer introduced as glycerol was incorporated both into glucose (via gluconeogenesis) and into pyruvate (via glycolysis). The data allow us to place a lower bound on the energetic cost of futile cycles involving adenosine triphosphate (ATP) hydrolysis in the early phosphorylation steps of glycolysis. Applying the Markov Chain model for glucose undergoing metabolism to pyruvate, the expected number of ATP molecules hydrolysed is not less than 15 ATP molecules per glucose molecule. The data suggest that, in hepatocytes under the circumstances of this experiment, either glycolysis is a net consumer of ATP, or glycolysis and gluconeogenesis are compartmentalized to a greater extent than is generally supposed.     

Selection for rapid uptake of scarce or fluctuating resource explains vulnerability of glycolysis to imbalance

Glycolysis is a conserved central pathway in energy metabolism that converts glucose to pyruvate with net production of two ATP molecules. Because ATP is produced only in the lower part of glycolysis (LG), preceded by an initial investment of ATP in the upper glycolysis (UG), achieving robust start-up of the pathway upon activation presents a challenge: a sudden increase in glucose concentration can throw a cell into a self-sustaining imbalanced state in which UG outpaces LG, glycolytic intermediates accumulate and the cell is unable to maintain high ATP concentration needed to support cellular functions. Such metabolic imbalance can result in “substrate-accelerated death”, a phenomenon observed in prokaryotes and eukaryotes when cells are exposed to an excess of substrate that previously limited growth. Here, we address why evolution has apparently not eliminated such a costly vulnerability and propose that it is a manifestation of an evolutionary trade-off, whereby the glycolysis pathway is adapted to quickly secure scarce or fluctuating resource at the expense of vulnerability in an environment with ample resource. To corroborate this idea, we perform individual-based eco-evolutionary simulations of a simplified yeast glycolysis pathway consisting of UG, LG, phosphate transport between a vacuole and a cytosol, and a general ATP demand reaction. The pathway is evolved in constant or fluctuating resource environments by allowing mutations that affect the (maximum) reaction rate constants, reflecting changing expression levels of different glycolytic enzymes. We demonstrate that under limited constant resource, populations evolve to a genotype that exhibits balanced dynamics in the environment it evolved in, but strongly imbalanced dynamics under ample resource conditions. Furthermore, when resource availability is fluctuating, imbalanced dynamics confers a fitness advantage over balanced dynamics: when glucose is abundant, imbalanced pathways can quickly accumulate the glycolytic intermediate FBP as intracellular storage that is used during periods of starvation to maintain high ATP concentration needed for growth. Our model further predicts that in fluctuating environments, competition for glucose can result in stable coexistence of balanced and imbalanced cells, as well as repeated cycles of population crashes and recoveries that depend on such polymorphism. Overall, we demonstrate the importance of ecological and evolutionary arguments for understanding seemingly maladaptive aspects of cellular metabolism.     

Oscillations and efficiency in glycolysis

We suggest that temporal oscillations of concentrations of intermediates in biochemical reaction systems may enhance the efficiency of free energy conversion (reduce dissipation) in those reactions. Experiments on glycolysis are used to estimate the Gibbs free energy changes along the glycolysis mechanism, and to postulate a construct for the glycolysis "machine" which involves: the PFK reaction as the primary oscillophor; the GAPDH reaction as a phase-shifting device; and the PK reaction with the property of intrinsic oscillatory response at resonance with the driving frequency. Analysis of a simple reaction mechanism with these postulated properties shows that the conversion of free energy from reactants to products is more efficient in an oscillatory than a steady state operation. The efficiency of free energy conversion in glycolysis from glucose + ADP to products + ATP is estimated to be increased by 5--10% due to oscillations. This may have been relevant for the evolutionary development of oscillations such as in glycolysis, especially in anaerobic cells.     

Glucose withdrawal induces oxidative stress followed by apoptosis in glioblastoma cells but not in normal human astrocytes

Tumor cells rely preferentially on anaerobic glycolysis rather than on respiration for ATP generation, a phenomenon known as the Warburg effect. We explored the effects of glucose withdrawal on glioblastoma multiforme-derived cell lines and their nontransformed counterparts, normal human astrocytes. We found that glucose withdrawal induces extensive apoptosis in glioblastoma multiforme cells but not in normal astrocytes. In all cells examined, ATP levels are sustained on glucose withdrawal due to elevation of fatty acid oxidation and ensuing respiration; however, we show that oxidative stress generated in the mitochondrial respiratory chain is the direct cause of cell death in glioblastoma multiforme cells. Oxidative stress that only occurs in glioblastoma multiforme cells underlies the selective susceptibility to glucose withdrawal-induced apoptosis documented in the malignant cells. This study implicates glycolysis as a potentially efficient and selective target for glioblastoma multiforme treatment.     

Effects of dinitrophenol and oligomycin on the coupling between anaerobic metabolism and anaerobic sodium transport by the isolated turtle bladder

Dinitrophenol (1 x 10^-5 M) has been found to inhibit anaerobic sodium transport by the isolated urinary bladder of the fresh water turtle. Concurrently, anaerobic glycolysis was stimulated markedly. However, tissue ATP levels diminished only modestly, remaining at approximately 75% of values observed under anaerobic conditions without DNP. The utilization of glucose (from endogenous glycogen) corresponded closely to that predicted from the molar quantities of lactate formed. Thus the glycolytic pathway was completed in the presence of DNP and if ATP were synthesized normally during glycolysis, synthesis should have been increased. On the other hand, the decrease in Na transport should have decreased ATP utilization. Oligomycin did not block sodium transport either aerobically or anaerobically, but ATP concentrations did decrease. When anaerobic glycolysis was blocked by iodoacetate, pyruvate did not sustain sodium transport thus suggesting that no electron acceptors were available in the system. Two explanations are entertained for the anaerobic effect of DNP: (a) Stimulation by DNP of plasma membrane as well as mitochondrial ATPase activity; (b) inhibition of a high energy intermediate derived from glycolytic ATP or from glycolysis per se. The arguments relevant to each possibility are presented in the text. Although definitive resolution is not possible, we believe that the data favor the hypothesis that there was a high energy intermediate in the anaerobic system and that this intermediate, rather than ATP, served as the immediate source of energy for the sodium pump.     

Regulation of sugar transport and metabolism in lactic acid bacteria

Abstract The phosphoenolpyruvate (PEP)-dependent lactose: phosphotransferase system (PTS), P-β-galactosidase, and enzymes of the d -tagatose-6P pathway, are prerequisite for rapid homolactic fermentation of lactose by Group N ('starter') streptococci. Moreover, the reactions of transport and catabolism constitute an open cycle in which ATP and lactic acid are metabolic products. The efficient and controlled operation of this cycle requires 'fine-control' mechanisms to ensure: (i) tight coupling between transport and energy-yielding reactions, (ii) co-metabolism of both glucose and galactose moieties of the disaccharide, and (iii) coordination of the rate of sugar transport to the rate of sugar catabolism. The elucidation of these fine-control mechanisms in intact cells of Streptococcus lactis has required the isolation of glucokinase (GK) and mannose-PTS defective mutants, the synthesis of novel lactose analogs, and the use of high resolution [³¹P]NMR spectroscopy. It has been established that PEP provides the crucial link between transport and energy-yielding reactions of the PTS: glycolysis cycle, and that both ATP-dependent glucokinase and PEP-dependent mannose-PTS can participate in the phosphorylation of intracellular glucose. Finally, evidence has been obtained in vivo, for modulation of pyruvate kinase activity in response to fluctuation in, concentrations of positive (FDP), and negative (P_i) effectors of the allosteric enzyme. Fine-control of pyruvate kinase activity may in turn regulate: (i) the distribution of PEP to either the PTS or ATP synthesis, (ii) overall activity of the PTS: glycolysis cycle, and (iii) the formation of the endogenous PEP-potential in starved organisms. The accumulation of non-metabolizable PTS sugars (e.g., 2-deoxy- d -glucose) by growing cells can perturb these fine-control mechanisms and, by establishment of a PEP-dissipating futile cycle, may result in bacteriostasis.