Analysis of proteins showing differential changes during ATP oscillations in chondrogenesis

Prechondrogenic condensation is a critical step for skeletal pattern formation. Our previous study showed that ATP oscillations play an essential role in prechondrogenic condensation because they induce oscillatory secretion. However, the molecular mechanisms that underlie ATP oscillations remain poorly understood. We examined how differential changes in proteins are implicated in ATP oscillations during chondrogenesis by using liquid chromatography/mass spectrometry. Our analysis showed that a number of proteins involved in ATP synthesis/consumption, catabolic/anabolic processes, actin dynamics, cell migration and adhesion were detected at either the peak or the trough of ATP oscillations, which implies that these proteins have oscillatory expression patterns that are coupled to ATP oscillations. On the basis of the results, we suggest that (1) the oscillatory expression of proteins involved in ATP synthesis/consumption and catabolic/anabolic processes can contribute to the generation or maintenance of ATP oscillations and that (2) the oscillatory expression of proteins involved in actin dynamics, cell migration and adhesion plays key roles in prechondrogenic condensation by inducing collective adhesion and migration in cooperation with ATP oscillations. Copyright © 2014 John Wiley & Sons, Ltd.     

Exposure to high glutamate concentration activates aerobic glycolysis but inhibits ATP‐linked respiration in cultured cortical astrocytes

Astrocytes play a key role in removing the synaptically released glutamate from the extracellular space and maintaining the glutamate below neurotoxic level in the brain. However, high concentration of glutamate leads to toxicity in astrocytes, and the underlying mechanisms are unclear. The purpose of this study was to investigate whether energy metabolism disorder, especially impairment of mitochondrial respiration, is involved in the glutamate‐induced gliotoxicity. Exposure to 10‐mM glutamate for 48 h stimulated glycolysis and respiration in astrocytes. However, the increased oxygen consumption was used for proton leak and non‐mitochondrial respiration, but not for oxidative phosphorylation and ATP generation. When the exposure time extended to 72 h, glycolysis was still activated for ATP generation, but the mitochondrial ATP‐linked respiration of astrocytes was reduced. The glutamate‐induced astrocyte damage can be mimicked by the non‐metabolized substrate d ‐aspartate but reversed by the non‐selective glutamate transporter inhibitor TBOA. In addition, the glutamate toxicity can be partially reversed by vitamin E. These findings demonstrate that changes of bioenergetic profile occur in cultured cortical astrocytes exposed to high concentration of glutamate and highlight the role of mitochondria respiration in glutamate‐induced gliotoxicity in cortical astrocytes. Copyright © 2014 John Wiley & Sons, Ltd.     

Cell energy metabolism: An update

In living cells, growth is the result of coupling between substrate catabolism and multiple metabolic processes that take place during net biomass formation and maintenance processes. During growth, both ATP/ADP and NADH/NAD⁺ molecules play a key role. Cell energy metabolism hence refers to metabolic pathways involved in ATP synthesis linked to NADH turnover. Two main pathways are thus involved in cell energy metabolism: glycolysis/fermentation and oxidative phosphorylation. Glycolysis and mitochondrial oxidative phosphorylation are intertwined through thermodynamic and kinetic constraints that are reviewed herein. Further, our current knowledge of short-term and long term regulation of cell energy metabolism will be reviewed using examples such as the Crabtree and the Warburg effect.     

Mitochondrial superoxide plays a crucial role in the development of mitochondrial dysfunction during high glucose exposure in rat renal proximal tubular cells

Diabetic nephropathy is the leading cause of end-stage renal disease in the United States. Despite several studies indicating a role for mitochondrial oxidative stress and mitochondrial dysfunction in the development of diabetic complications, the precise mechanisms underlying renal mitochondrial dysfunction and renal cell injury remain unclear. The hypothesis of the current study was that high-glucose-mediated generation of mitochondrial superoxide is a key early event that leads to mitochondrial injury in renal proximal tubular cells. To ascertain the role of mitochondrial superoxide we have tested whether overexpression of the primary mitochondrial antioxidant, manganese superoxide dismutase (MnSOD), protects against hyperglycemia-induced renal injury using normal rat renal proximal tubular cells (NRK). NRK cells were exposed to high glucose (25 mM) and the changes in the mitochondrial membrane potential, ATP levels, and superoxide generation and the loss of cell viability were measured at 24 and 48 h after high glucose exposure. Our results indicate that high glucose first induced superoxide generation and hyperpolarization in the mitochondria, followed by a secondary event, which involved a decline in ATP levels, partial Complex III inactivation, and loss of cell viability. These high-glucose-induced changes were completely prevented by overexpression of MnSOD in NRK cells. However, MnSOD activity was not changed after high glucose exposure in vitro or during the early stages of diabetes using the streptozotocin rat model. These findings show for the first time that hyperglycemic induction of superoxide production within the mitochondria initiates specific mitochondrial injury (i.e., Complex III) via a mechanism independent of MnSOD inactivation.     

Glucose modulates [Ca2+]i oscillations in pancreatic islets via ionic and glycolytic mechanisms

Pancreatic islets of Langerhans display complex intracellular calcium changes in response to glucose that include fast (seconds), slow ( approximately 5 min), and mixed fast/slow oscillations; the slow and mixed oscillations are likely responsible for the pulses of plasma insulin observed in vivo. To better understand the mechanisms underlying these diverse patterns, we systematically analyzed the effects of glucose on period, amplitude, and plateau fraction (the fraction of time spent in the active phase) of the various regimes of calcium oscillations. We found that in both fast and slow islets, increasing glucose had limited effects on amplitude and period, but increased plateau fraction. In some islets, however, glucose caused a major shift in the amplitude and period of oscillations, which we attribute to a conversion between ionic and glycolytic modes (i.e., regime change). Raising glucose increased the plateau fraction equally in fast, slow, and regime-changing islets. A mathematical model of the pancreatic islet consisting of an ionic subsystem interacting with a slower metabolic oscillatory subsystem can account for these complex islet calcium oscillations by modifying the relative contributions of oscillatory metabolism and oscillatory ionic mechanisms to electrical activity, with coupling occurring via K(ATP) channels.     

Selective actions of mitochondrial fission/fusion genes on metabolism-secretion coupling in insulin-releasing cells

Mitochondria form filamentous networks that undergo continuous fission/fusion. In the pancreatic beta-cells, mitochondria are essential for the transduction of signals linking nutrient metabolism to insulin granule exocytosis. Here we have studied mitochondrial networks in the insulinoma cell line INS-1E, primary rat and human beta-cells. We have further investigated the impact of mitochondrial fission/fusion on metabolism-secretion coupling in INS-1E cells. Overexpression of hFis1 caused dramatic mitochondrial fragmentation, whereas Mfn1 evoked hyperfusion and the aggregation of mitochondria. Cells overexpressing hFis1 or Mfn1 showed reduced mitochondrial volume, lowered cellular ATP levels, and as a consequence, impaired glucose-stimulated insulin secretion. Decreased mitochondrial ATP generation was partially compensated for by enhanced glycolysis as indicated by increased lactate production in these cells. Dominant-negative Mfn1 elicited mitochondrial shortening and fragmentation of INS-1E cell mitochondria, similar to hFis1. However, the mitochondrial volume, cytosolic ATP levels, and glucose-stimulated insulin secretion were little affected. We conclude that mitochondrial fragmentation per se does not impair metabolism-secretion coupling. Through their impact on mitochondrial bioenergetics and distribution, hFis1 and Mfn1 activities influence mitochondrial signal generation thereby insulin exocytosis.     

The end effector of circadian heart rate variation: the sinoatrial node pacemaker cell

Cardiovascular function is regulated by the rhythmicity of circadian, infradian and ultradian clocks. Specific time scales of different cell types drive their functions: circadian gene regulation at hours scale, activation-inactivation cycles of ion channels at millisecond scales, the heart''s beating rate at hundreds of millisecond scales, and low frequency autonomic signaling at cycles of tens of seconds. Heart rate and rhythm are modulated by a hierarchical clock system: autonomic signaling from the brain releases neurotransmitters from the vagus and sympathetic nerves to the heart’s pacemaker cells and activate receptors on the cell. These receptors activating ultradian clock functions embedded within pacemaker cells include sarcoplasmic reticulum rhythmic spontaneous Ca²⁺ cycling, rhythmic ion channel current activation and inactivation, and rhythmic oscillatory mitochondria ATP production. Here we summarize the evidence that intrinsic pacemaker cell mechanisms are the end effector of the hierarchical brain-heart circadian clock system. [BMB Reports 2015; 48(12): 677-684]     

Glucose,glycolysis, and neurodegenerative diseases

Prolonged survival of a typical postmitotic neuron hinges on a balance between multiple processes, among these are a sustenance of ATP production and protection against reactive oxygen species. In neuropathological conditions, mitochondrial defects often lead to both a drop in ATP levels, as well as increase reactive oxygen species production from inefficient electron transport processes and NADPH-oxidases activities. The former often resulted in the phenomenon of compensatory aerobic glycolysis. The latter stretches the capacity of the cell's redox buffering capacity, and may lead to damages of key enzymes involved in energy metabolism. Several recent reports have indicated that enhancing glucose availability and uptake, as well as increasing glycolytic flux via pharmacological or genetic manipulation of glycolytic enzymes, could be protective in animal models of several major neurodegenerative diseases, including Parkinson's disease, Huntington's disease, and Amyotrophic lateral sclerosis. Activation of canonical Wnt signaling, which improves disease symptoms in mouse models of Alzheimer's disease also appears to work via an elevation of glycolytic enzymes and enhance glucose metabolism. Here, I discuss these findings and the possible underlying mechanisms of how an increase in glucose uptake and glycolysis could be neuroprotective. Increased glycolytic production of ATP would help alleviate energy deficiency, and ATP's hydrotropic effect may enhance solubility and clearance of toxic aggregates prevalent in many neurodegenerative diseases. Furthermore, channeling of glucose into the Pentose Phosphate Pathway would increase the redox buffering capacity of the cell.     

Sequence of insulin effects on cytoskeletal and cytosolic phosphofructokinase, mitochondrial hexokinase, glucose 1,6-bisphosphate and fructose 2,6-bisphosphate levels, and the antagonistic action of calmodulin inhibitors, in diaphragm muscle.

1. Time-curves of insulin effects on energy-producing systems in different cellular compartments of rat diaphragm muscle have revealed: (a) a rapid (within minutes) and transient stimulatory effect of insulin on cytoskeletal phosphofructokinase and aldolase and mitochondrial hexokinase. (b) A slower and consistent stimulatory effect on glucose 1,6-bisphosphate level, with concomitant gradual activation of cytosolic phosphofructokinase. Fructose 2,6-bisphosphate levels were not changed by insulin. (c) Lactate concentration correlated with the stimulation of cytoskeletal and cytosolic glycolysis. 2. Calmodulin antagonists, trifluoperazine or CGS 9343B, prevented all these effects of insulin. 3. These results suggest that cytoskeletal glycolysis and mitochondrial oxidation are the source of ATP for the rapid actions of insulin, whereas cytosolic glycolysis is the source of ATP for the slow actions of insulin. Calmodulin is involved in all these effects of insulin.     

A computational model integrating electrophysiology, contraction, and mitochondrial bioenergetics in the ventricular myocyte

An intricate network of reactions is involved in matching energy supply with demand in the heart. This complexity arises because energy production both modulates and is modulated by the electrophysiological and contractile activity of the cardiac myocyte. Here, we present an integrated mathematical model of the cardiac cell that links excitation-contraction coupling with mitochondrial energy generation. The dynamics of the model are described by a system of 50 ordinary differential equations. The formulation explicitly incorporates cytoplasmic ATP-consuming processes associated with force generation and ion transport, as well as the creatine kinase reaction. Changes in the electrical and contractile activity of the myocyte are coupled to mitochondrial energetics through the ATP, Ca2+, and Na+ concentrations in the myoplasmic and mitochondrial matrix compartments. The pseudo steady-state relationship between force and oxygen consumption at various stimulus frequencies and external Ca2+ concentrations is reproduced in both model simulations and direct experiments in cardiac trabeculae under normoxic conditions, recapitulating the linearity between cardiac work and respiration in the heart. Importantly, the model can also reproduce the rapid time-dependent changes in mitochondrial NADH and Ca2+ in response to abrupt changes in workload. The steady-state and dynamic responses of the model were conferred by ADP-dependent stimulation of mitochondrial oxidative phosphorylation and Ca2+ -dependent regulation of Krebs cycle dehydrogenases, illustrating how the model can be used as a tool for investigating mechanisms underlying metabolic control in the heart.     

Oscillatory signaling processes: the how, the why and the where

Oscillatory processes in biological signal transduction have come under progressively increasing scrutiny in terms of their functional significance and mechanisms of emergence and regulation. Since oscillatory processes can be a by-product of rapid adaptation and can also easily emerge if the feedback underlying adaptive processes is inadvertently artificially enhanced, one needs to exercise caution in both claiming the existence of in vivo oscillations and seeking to assign to them a specific functional significance. Nevertheless, oscillations can be a powerful means of encoding and transferring information both in time and in space, thus possessing important potential advantages for evolutionary selection and stabilization. Thus periodicity in the cell responses to diverse persistent external stimuli might become a more recognized and even expected feature of signaling processes.     

Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells

Increased conversion of glucose to lactic acid associated with decreased mitochondrial respiration is a unique feature of tumors first described by Otto Warburg in the 1920s. Recent evidence suggests that the Warburg effect is caused by oncogenes and is an underlying mechanism of malignant transformation. Using a novel approach to measure cellular metabolic rates in vitro, the bioenergetic basis of this increased glycolysis and reduced mitochondrial respiration was investigated in two human cancer cell lines, H460 and A549. The bioenergetic phenotype was analyzed by measuring cellular respiration, glycolysis rate, and ATP turnover of the cells in response to various pharmacological modulators. H460 and A549 cells displayed a dependency on glycolysis and an ability to significantly upregulate this pathway when their respiration was inhibited. The converse, however, was not true. The cell lines were attenuated in oxidative phosphorylation (OXPHOS) capacity and were unable to sufficiently upregulate mitochondrial OXPHOS when glycolysis was disabled. This observed mitochondrial impairment was intimately linked to the increased dependency on glycolysis. Furthermore, it was demonstrated that H460 cells were more glycolytic, having a greater impairment of mitochondrial respiration, compared with A549 cells. Finally, the upregulation of glycolysis in response to mitochondrial ATP synthesis inhibition was dependent on AMP-activated protein kinase activity. In summary, our results demonstrate a bioenergetic phenotype of these two cancer cell lines characterized by increased rate of glycolysis and a linked attenuation in their OXPHOS capacity. These metabolic alterations provide a mechanistic explanation for the growth advantage and apoptotic resistance of tumor cells. oxygen consumption; oxidative phosphorylation; Warburg effect; real time     

The mechanism underlying the effects of the cell surface ATP synthase on the regulation of intracellular acidification during acidosis

The F1F0 ATP synthase has recently become the focus of anti‐cancer research. It was once thought that ATP synthases were located strictly on the inner mitochondrial membrane; however, in 1994, it was found that some ATP synthases localized to the cell surface. The cell surface ATP synthases are involved in angiogenesis, lipoprotein metabolism, innate immunity, hypertension, the regulation of food intake, and other processes. Inhibitors of this synthase have been reported to be cytotoxic and to induce intracellular acidification. However, the mechanisms by which these effects are mediated and the molecular pathways that are involved remain unclear. In this study, we aimed to determine whether the inhibition of cell proliferation and the induction of cell apoptosis that are induced by inhibitors of the cell surface ATP synthase are associated with intracellular acidification and to investigate the mechanism that underlines the effects of this inhibition, particularly in an acidic tumor environment. We demonstrated that intracellular acidification contributes to the cell proliferation inhibition that is mediated by cell surface ATP synthase inhibitors, but not to the induction of apoptosis. Intracellular acidification is only one of the mechanisms of ecto‐ATP synthase‐targeted antitumor drugs. We propose that intracellular acidification in combination with the inhibition of cell surface ATP generation induce cell apoptosis after cell surface ATP synthase blocked by its inhibitors. A better understanding of the mechanisms activated by ecto‐ATP synthase‐targeted cancer therapies may facilitate the development of potent anti‐tumor therapies, which target this enzyme and do not exhibit clinical limitations. J. Cell. Biochem. 114: 1695–1703, 2013. © 2013 Wiley Periodicals, Inc.     

Reciprocal periodicity in cyclic AMP binding and phosphorylation of differentiating Dictyostelium discoideum cells.

The binding of [³H]cyclic AMP to cell surface receptors of differentiated D. discoideum cells at 25° is an oscillatory process with a periodicity of 2 min. This alternating change in the cells' binding capacity for cyclic AMP may be the basis for the refractory period to cyclic AMP stimulation, an essential feature of the chemotactic system. The incorporation of [³²P] by whole cells from [γ³²P]ATP is also oscillatory with a periodicity identical to that of [³H]cyclic AMP binding. However, the two processes are inversely related in time such that periods of maximal cyclic AMP binding correspond to periods of minimal cellular phosphorylation. These results suggest a receptor kinase/phosphatase mediated desensitization of the cyclic AMP receptor.     

Regulation of Glycolytic Oscillations by Mitochondrial and Plasma Membrane H-ATPases

We investigated the coupling between glycolytic and mitochondrial membrane potential oscillations in Saccharomyces cerevisiae under semianaerobic conditions. Glycolysis was measured as NADH autofluorescence, and mitochondrial membrane potential was measured using the fluorescent dye 3,3′-diethyloxacarbocyanine iodide. The responses of glycolytic and membrane potential oscillations to a number of inhibitors of glycolysis, mitochondrial electron flow, and mitochondrial and plasma membrane H⁺-ATPase were investigated. Furthermore, the glycolytic flux was determined as the rate of production of ethanol in a number of different situations (changing pH or the presence and absence of inhibitors). Finally, the intracellular pH was determined and shown to oscillate. The results support earlier work suggesting that the coupling between glycolysis and mitochondrial membrane potential is mediated by the ADP/ATP antiporter and the mitochondrial F₀F₁-ATPase. The results further suggest that ATP hydrolysis, through the action of the mitochondrial F₀F₁-ATPase and plasma membrane H⁺-ATPase, are important in regulating these oscillations. We conclude that it is glycolysis that drives the oscillations in mitochondrial membrane potential.     

Inhibition of oxidative metabolism by nitric oxide restricts EMCV replication selectively in pancreatic beta-cells

Environmental factors, such as viral infection, are proposed to play a role in the initiation of autoimmune diabetes. In response to encephalomyocarditis virus (EMCV) infection, resident islet macrophages release the pro-inflammatory cytokine IL-1β, to levels that are sufficient to stimulate inducible nitric oxide synthase (iNOS) expression and production of micromolar levels of the free radical nitric oxide in neighboring β-cells. We have recently shown that nitric oxide inhibits EMCV replication and EMCV-mediated β-cell lysis and that this protection is associated with an inhibition of mitochondrial oxidative metabolism. Here we show that the protective actions of nitric oxide against EMCV infection are selective for β-cells and associated with the metabolic coupling of glycolysis and mitochondrial oxidation that is necessary for insulin secretion. Inhibitors of mitochondrial respiration attenuate EMCV replication in β-cells, and this inhibition is associated with a decrease in ATP levels. In mouse embryonic fibroblasts (MEFs), inhibition of mitochondrial metabolism does not modify EMCV replication or decrease ATP levels. Like most cell types, MEFs have the capacity to uncouple the glycolytic utilization of glucose from mitochondrial respiration, allowing for the maintenance of ATP levels under conditions of impaired mitochondrial respiration. It is only when MEFs are forced to use mitochondrial oxidative metabolism for ATP generation that mitochondrial inhibitors attenuate viral replication. In a β-cell selective manner, these findings indicate that nitric oxide targets the same metabolic pathways necessary for glucose stimulated insulin secretion for protection from viral lysis.     

Tight Coupling of Metabolic Oscillations and Intracellular Water Dynamics in Saccharomyces cerevisiae

We detected very strong coupling between the oscillating concentration of ATP and the dynamics of intracellular water during glycolysis in Saccharomyces cerevisiae. Our results indicate that: i) dipolar relaxation of intracellular water is heterogeneous within the cell and different from dilute conditions, ii) water dipolar relaxation oscillates with glycolysis and in phase with ATP concentration, iii) this phenomenon is scale-invariant from the subcellular to the ensemble of synchronized cells and, iv) the periodicity of both glycolytic oscillations and dipolar relaxation are equally affected by D₂O in a dose-dependent manner. These results offer a new insight into the coupling of an emergent intensive physicochemical property of the cell, i.e. cell-wide water dipolar relaxation, and a central metabolite (ATP) produced by a robustly oscillating metabolic process.     

Metabolome and metaboproteome remodeling in nuclear reprogramming

Nuclear reprogramming resets differentiated tissue to generate induced pluripotent stem (iPS) cells. While genomic attributes underlying reacquisition of the embryonic-like state have been delineated, less is known regarding the metabolic dynamics underscoring induction of pluripotency. Metabolomic profiling of fibroblasts vs. iPS cells demonstrated nuclear reprogramming-associated induction of glycolysis, realized through augmented utilization of glucose and accumulation of lactate. Real-time assessment unmasked downregulated mitochondrial reserve capacity and ATP turnover correlating with pluripotent induction. Reduction in oxygen consumption and acceleration of extracellular acidification rates represent high-throughput markers of the transition from oxidative to glycolytic metabolism, characterizing stemness acquisition. The bioenergetic transition was supported by proteome remodeling, whereby 441 proteins were altered between fibroblasts and derived iPS cells. Systems analysis revealed overrepresented canonical pathways and interactome-associated biological processes predicting differential metabolic behavior in response to reprogramming stimuli, including upregulation of glycolysis, purine, arginine, proline, ribonucleoside and ribonucleotide metabolism, and biopolymer and macromolecular catabolism, with concomitant downregulation of oxidative phosphorylation, phosphate metabolism regulation, and precursor biosynthesis processes, prioritizing the impact of energy metabolism within the hierarchy of nuclear reprogramming. Thus, metabolome and metaboproteome remodeling is integral for induction of pluripotency, expanding on the genetic and epigenetic requirements for cell fate manipulation.     

What couples glycolysis to mitochondrial signal generation in glucose-stimulated insulin secretion?

Pancreatic islet beta-cells are poised to generate metabolic messengers in the mitochondria that link glucose metabolism to insulin exocytosis. This is accomplished through the tight coupling of glycolysis to mitochondrial activation. The messenger molecules ATP and glutamate are produced after the metabolism of glycolysis-derived pyruvate in the mitochondria. The entry of monocarboxylates such as pyruvate into the beta cell is limited, explaining why overexpression of monocarboxylate transporters unravels pyruvate-stimulated insulin secretion. NADH generated by glycolysis is efficiently reoxidized by highly active mitochondrial shuttles rather than by lactate dehydrogenase. Overexpression of this enzyme does not alter glucose-stimulated insulin secretion, suggesting that NADH availability restricts the conversion of pyruvate to lactate in the beta cell. These metabolic features permit the fuel function of glucose to be extended to the generation of signaling molecules, which increases cytosolic Ca2+ and promotes insulin exocytosis.