MITODYN: An Open Source Software for Quantitative Modeling of Mitochondrial and Cellular Energy Metabolic Flux Dynamics in Health and Disease.

Mitochondrial respiratory chain (RC) transforms the reductive power of NADH or FADH2 oxidation into a proton gradient between the matrix and cytosolic sides of the inner mitochondrial membrane, that ATP synthase uses to generate ATP. This process constitutes a bridge between carbohydrates' central metabolism and ATP-consuming cellular functions. Moreover, the RC is responsible for a large part of reactive oxygen species (ROS) generation that play signaling and oxidizing roles in cells. Mathematical methods and computational analysis are required to understand and predict the possible behavior of this metabolic system. Here we propose a software tool that helps to analyze individual steps of respiratory electron transport in their dynamics, thus deepening understanding of the mechanism of energy transformation and ROS generation in the RC. This software's core is a kinetic model of the RC represented by a system of ordinary differential equations (ODEs). This model enables the analysis of complex dynamic behavior of the RC, including multistationarity and oscillations. The proposed RC modeling method can be applied to study respiration and ROS generation in various organisms and naturally extended to explore carbohydrates' metabolism and linked metabolic processes.

Unveiling a key role of oxaloacetate-glutamate interaction in regulation of respiration and ROS generation in nonsynaptic brain mitochondria using a kinetic model.

Glutamate plays diverse roles in neuronal cells, affecting cell energetics and reactive oxygen species (ROS) generation. These roles are especially vital for neuronal cells, which deal with high amounts of glutamate as a neurotransmitter. Our analysis explored neuronal glutamate implication in cellular energy metabolism and ROS generation, using a kinetic model that simulates electron transport details in respiratory complexes, linked ROS generation and metabolic reactions. The analysis focused on the fact that glutamate attenuates complex II inhibition by oxaloacetate, stimulating the latter's transformation into aspartate. Such a mechanism of complex II activation by glutamate could cause almost complete reduction of ubiquinone and deficiency of oxidized form (Q), which closes the main stream of electron transport and opens a way to massive ROS generating transfer in complex III from semiquinone radicals to molecular oxygen. In this way, under low workload, glutamate triggers the respiratory chain (RC) into a different steady state characterized by high ROS generation rate. The observed stepwise dependence of ROS generation on glutamate concentration experimentally validated this prediction. However, glutamate's attenuation of oxaloacetate's inhibition accelerates electron transport under high workload. Glutamate-oxaloacetate interaction in complex II regulation underlies the observed effects of uncouplers and inhibitors and acceleration of Ca2+ uptake. Thus, this theoretical analysis uncovered the previously unknown roles of oxaloacetate as a regulator of ROS generation and glutamate as a modifier of this regulation. The model predicted that this mechanism of complex II activation by glutamate might be operative in situ and responsible for excitotoxicity. Spatial-time gradients of synthesized hydrogen peroxide concentration, calculated in the reaction-diffusion model with convection under a non-uniform local approximation of nervous tissue, have shown that overproduction of H2O2 in a cell causes excess of its level in neighbor cells.

The landscape of tiered regulation of breast cancer cell metabolism.

Altered metabolism is a hallmark of cancer, but little is still known about its regulation. In this study, we measure transcriptomic, proteomic, phospho-proteomic and fluxomics data in a breast cancer cell-line (MCF7) across three different growth conditions. Integrating these multiomics data within a genome scale human metabolic model in combination with machine learning, we systematically chart the different layers of metabolic regulation in breast cancer cells, predicting which enzymes and pathways are regulated at which level. We distinguish between two types of reactions, directly and indirectly regulated. Directly-regulated reactions include those whose flux is regulated by transcriptomic alterations (~890) or via proteomic or phospho-proteomics alterations (~140) in the enzymes catalyzing them. We term the reactions that currently lack evidence for direct regulation as (putative) indirectly regulated (~930). Many metabolic pathways are predicted to be regulated at different levels, and those may change at different media conditions. Remarkably, we find that the flux of predicted indirectly regulated reactions is strongly coupled to the flux of the predicted directly regulated ones, uncovering a tiered hierarchical organization of breast cancer cell metabolism. Furthermore, the predicted indirectly regulated reactions are predominantly reversible. Taken together, this architecture may facilitate rapid and efficient metabolic reprogramming in response to the varying environmental conditions incurred by the tumor cells. The approach presented lays a conceptual and computational basis for mapping metabolic regulation in additional cancers.

Restrictions in ATP diffusion within sarcomeres can provoke ATP-depleted zones impairing exercise capacity in chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by the inability of patients to sustain a high level of ventilation resulting in perceived exertional discomfort and limited exercise capacity of leg muscles at average intracellular ATP levels sufficient to support contractility. METHODS: Myosin ATPase activity in biopsy samples from healthy and COPD individuals was implemented as a local nucleotide sensor to determine ATP diffusion coefficients within myofibrils. Ergometric parameters clinically measured during maximal exercise tests in both groups were used to define the rates of myosin ATPase reaction and aerobic ATP re-synthesis. The obtained parameters in combination with AK- and CK-catalyzed reactions were implemented to compute the kinetic and steady-state spatial ATP distributions within control and COPD sarcomeres. RESULTS: The developed reaction-diffusion model of two-dimensional sarcomeric space identified similar, yet extremely low nucleotide diffusion in normal and COPD myofibrils. The corresponding spatio-temporal ATP distributions, constructed during imposed exercise, predicted in COPD sarcomeres a depletion of ATP in the zones of overlap between actin and myosin filaments along the center axis at average cytosolic ATP levels similar to healthy muscles. CONCLUSIONS: ATP-depleted zones can induce rigor tension foci impairing muscle contraction and increase a risk for sarcomere damages. Thus, intra-sarcomeric diffusion restrictions at limited aerobic ATP re-synthesis can be an additional risk factor contributing to the muscle contractile deficiency experienced by COPD patients. GENERAL SIGNIFICANCE: This study demonstrates how restricted substrate mobility within a cellular organelle can provoke an energy imbalance state paradoxically occurring at abounding average metabolic resources.

(13)C metabolic flux analysis shows that resistin impairs the metabolic response to insulin in L6E9 myotubes.

BACKGROUND: It has been suggested that the adipokine resistin links obesity and insulin resistance, although how resistin acts on muscle metabolism is controversial. We aimed to quantitatively analyse the effects of resistin on the glucose metabolic flux profile and on insulin response in L6E9 myotubes at the metabolic level using a tracer-based metabolomic approach and our in-house developed software, Isodyn. RESULTS: Resistin significantly increased glucose uptake and glycolysis, altering pyruvate utilisation by the cell. In the presence of resistin, insulin only slightly increased glucose uptake and glycolysis, and did not alter the flux profile around pyruvate induced by resistin. Resistin prevented the increase in gene expression in pyruvate dehydrogenase-E1 and the sharp decrease in gene expression in cytosolic phosphoenolpyruvate carboxykinase-1 induced by insulin. CONCLUSIONS: These data suggest that resistin impairs the metabolic activation of insulin. This impairment cannot be explained by the activity of a single enzyme, but instead due to reorganisation of the whole metabolic flux distribution.

A key role for mitochondrial gatekeeper pyruvate dehydrogenase in oncogene-induced senescence.

In response to tenacious stress signals, such as the unscheduled activation of oncogenes, cells can mobilize tumour suppressor networks to avert the hazard of malignant transformation. A large body of evidence indicates that oncogene-induced senescence (OIS) acts as such a break, withdrawing cells from the proliferative pool almost irreversibly, thus crafting a vital pathophysiological mechanism that protects against cancer. Despite the widespread contribution of OIS to the cessation of tumorigenic expansion in animal models and humans, we have only just begun to define the underlying mechanism and identify key players. Although deregulation of metabolism is intimately linked to the proliferative capacity of cells, and senescent cells are thought to remain metabolically active, little has been investigated in detail about the role of cellular metabolism in OIS. Here we show, by metabolic profiling and functional perturbations, that the mitochondrial gatekeeper pyruvate dehydrogenase (PDH) is a crucial mediator of senescence induced by BRAF(V600E), an oncogene commonly mutated in melanoma and other cancers. BRAF(V600E)-induced senescence was accompanied by simultaneous suppression of the PDH-inhibitory enzyme pyruvate dehydrogenase kinase 1 (PDK1) and induction of the PDH-activating enzyme pyruvate dehydrogenase phosphatase 2 (PDP2). The resulting combined activation of PDH enhanced the use of pyruvate in the tricarboxylic acid cycle, causing increased respiration and redox stress. Abrogation of OIS, a rate-limiting step towards oncogenic transformation, coincided with reversion of these processes. Further supporting a crucial role of PDH in OIS, enforced normalization of either PDK1 or PDP2 expression levels inhibited PDH and abrogated OIS, thereby licensing BRAF(V600E)-driven melanoma development. Finally, depletion of PDK1 eradicated melanoma subpopulations resistant to targeted BRAF inhibition, and caused regression of established melanomas. These results reveal a mechanistic relationship between OIS and a key metabolic signalling axis, which may be exploited therapeutically.

Relevance of the MEK/ERK signaling pathway in the metabolism of activated macrophages: a metabolomic approach.

The activation of immune cells in response to a pathogen involves a succession of signaling events leading to gene and protein expression, which requires metabolic changes to match the energy demands. The metabolic profile associated with the MAPK cascade (ERK1/2, p38, and JNK) in macrophages was studied, and the effect of its inhibition on the specific metabolic pattern of LPS stimulation was characterized. A [1,2-[(13)C](2)]glucose tracer-based metabolomic approach was used to examine the metabolic flux distribution in these cells after MEK/ERK inhibition. Bioinformatic tools were used to analyze changes in mass isotopomer distribution and changes in glucose and glutamine consumption and lactate production in basal and LPS-stimulated conditions in the presence and absence of the selective inhibitor of the MEK/ERK cascade, PD325901. Results showed that PD325901-mediated ERK1/2 inhibition significantly decreased glucose consumption and lactate production but did not affect glutamine consumption. These changes were accompanied by a decrease in the glycolytic flux, consistent with the observed decrease in fructose-2,6-bisphosphate concentration. The oxidative and nonoxidative pentose phosphate pathways and the ratio between them also decreased. However, tricarboxylic acid cycle flux did not change significantly. LPS activation led to the opposite responses, although all of these were suppressed by PD325901. However, LPS also induced a small decrease in pentose phosphate pathway fluxes and an increase in glutamine consumption that were not affected by PD325901. We concluded that inhibition of the MEK/ERK cascade interferes with central metabolism, and this cross-talk between signal transduction and metabolism also occurs in the presence of LPS.

Edelfosine-induced metabolic changes in cancer cells that precede the overproduction of reactive oxygen species and apoptosis.

BACKGROUND: Metabolic flux profiling based on the analysis of distribution of stable isotope tracer in metabolites is an important method widely used in cancer research to understand the regulation of cell metabolism and elaborate new therapeutic strategies. Recently, we developed software Isodyn, which extends the methodology of kinetic modeling to the analysis of isotopic isomer distribution for the evaluation of cellular metabolic flux profile under relevant conditions. This tool can be applied to reveal the metabolic effect of proapoptotic drug edelfosine in leukemia Jurkat cell line, uncovering the mechanisms of induction of apoptosis in cancer cells. RESULTS: The study of 13C distribution of Jukat cells exposed to low edelfosine concentration, which induces apoptosis in ≤5% of cells, revealed metabolic changes previous to the development of apoptotic program. Specifically, it was found that low dose of edelfosine stimulates the TCA cycle. These metabolic perturbations were coupled with an increase of nucleic acid synthesis de novo, which indicates acceleration of biosynthetic and reparative processes. The further increase of the TCA cycle fluxes, when higher doses of drug applied, eventually enhance reactive oxygen species (ROS) production and trigger apoptotic program. CONCLUSION: The application of Isodyn to the analysis of mechanism of edelfosine-induced apoptosis revealed primary drug-induced metabolic changes, which are important for the subsequent initiation of apoptotic program. Initiation of such metabolic changes could be exploited in anticancer therapy.

Modeling of spatial metabolite distributions in the cardiac sarcomere.

Although a high ATP diffusion rate implies homogeneous distribution of the principal energetic currency in the cytosol, local diffusion barriers represented by macromolecular structures can render ATP concentrations to be inhomogeneous. A method is presented here that provides apparent diffusion coefficient values in local intracellular regions and allows the estimation of spatial metabolite distribution. The apparent local diffusion coefficient for ATP in cardiac myofibrils was determined from the analysis of diffusion-dependent rightward shift of the substrate dependence for actomyosin ATPase activity using the reaction-diffusion model, which accounted for the properties of phosphotransfer reactions. This functional analysis, which took into account the local diffusional ATP delivery to the active sites, provided an apparent value that was three orders of magnitude lower than that defined by direct methods for the cytosol. The low value of the diffusion coefficient was shown to define unusual properties of the intracellular space in working heart, where small reductions in ATP levels in the surrounding cytosol result in a large drop in [ATP] inside myofibrils. This drop is critical for vital cellular functions, and the analysis presented here defines its physical basis. The diffusion barriers thus defined explain the coexistence of pathological energy deficit with almost normal average ATP levels.

Rapid simulation and analysis of isotopomer distributions using constraints based on enzyme mechanisms: an example from HT29 cancer cells.

MOTIVATION: Addition of labeled substrates and the measurement of the subsequent distribution of the labels in isotopomers in reaction networks provide a unique method for assessing metabolic fluxes in whole cells. However, owing to insufficiency of information, attempts to quantify the fluxes often yield multiple possible sets of solutions that are consistent with a given experimental pattern of isotopomers. In the study of the pentose phosphate pathways, the need to consider isotope exchange reactions of transketolase (TK) and transaldolase (TA) (which in past analyses have often been ignored) magnifies this problem; but accounting for the interrelation between the fluxes known from biochemical studies and kinetic modeling solves it. The mathematical relationships between kinetic and equilibrium constants restrict the domain of estimated fluxes to the ones compatible not only with a given set of experimental data, but also with other biochemical information. METHOD: We present software that integrates kinetic modeling with isotopomer distribution analysis. It solves the ordinary differential equations for total concentrations (accounting for the kinetic mechanisms) as well as for all isotopomers in glycolysis and the pentose phosphate pathway (PPP). In the PPP the fluxes created in the TK and TA reactions are expressed through unitary rate constants. The algorithms that account for all the kinetic and equilbrium constant constraints are integrated with the previously developed algorithms, which have been further optimized. The most time-consuming calculations were programmed directly in assembly language; this gave an order of magnitude decrease in the computation time, thus allowing analysis of more complex systems. The software was developed as C-code linked to a program written in Mathematica (Wolfram Research, Champaign, IL), and also as a C++ program independent from Mathematica. RESULTS: Implementing constraints imposed by kinetic and equilibrium constants in the isotopomer distribution analysis in the data from the cancer cells eliminated estimates of fluxes that were inconsistent with the kinetic mechanisms of TK and TA. Fluxes measured experimentally in cells can be used to estimate better the kinetics of TK and TA as they operate in situ. Thus, our approach of integrating various methods for in situ flux analysis opens up the possibility of designing new types of experiments to probe metabolic interrelationships, including the incorporation of additional biochemical information. AVAILABILITY: Software is available freely at: http://www.bq.ub.es/bioqint/selivanov.htm CONTACT: martacascante@ub.edu