Control and Regulation of Substrate Selection in Cytoplasmic and Mitochondrial Catabolic Networks. A Systems Biology Analysis

Appropriate substrate selection between fats and glucose is associated with the success of interventions that maintain health such as exercise or caloric restriction, or with the severity of diseases such as diabetes or other metabolic disorders. Although the interaction and mutual inhibition between glucose and fatty-acids (FAs) catabolism has been studied for decades, a quantitative and integrated understanding of the control and regulation of substrate selection through central catabolic pathways is lacking. We addressed this gap here using a computational model representing cardiomyocyte catabolism encompassing glucose (Glc) utilization, pyruvate transport into mitochondria and oxidation in the tricarboxylic acid (TCA) cycle, β-oxidation of palmitate (Palm), oxidative phosphorylation, ion transport, pH regulation, and ROS generation and scavenging in cytoplasmic and mitochondrial compartments. The model is described by 82 differential equations and 119 enzymatic, electron transport and substrate transport reactions accounting for regulatory mechanisms and key players, namely pyruvate dehydrogenase (PDH) and its modulation by multiple effectors. We applied metabolic control analysis to the network operating with various Glc to Palm ratios. The flux and metabolites’ concentration control were visualized through heat maps providing major insights into main control and regulatory nodes throughout the catabolic network. Metabolic pathways located in different compartments were found to reciprocally control each other. For example, glucose uptake and the ATP demand exert control on most processes in catabolism while TCA cycle activities and membrane-associated energy transduction reactions exerted control on mitochondrial processes namely β-oxidation. PFK and PDH, two highly regulated enzymes, exhibit opposite behavior from a control perspective. While PFK activity was a main rate-controlling step affecting the whole network, PDH played the role of a major regulator showing high sensitivity (elasticity) to substrate availability and key activators/inhibitors, a trait expected from a flexible substrate selector strategically located in the metabolic network. PDH regulated the rate of Glc and Palm consumption, consistent with its high sensitivity toward AcCoA, CoA, and NADH. Overall, these results indicate that the control of catabolism is highly distributed across the metabolic network suggesting that fuel selection between FAs and Glc goes well beyond the mechanisms traditionally postulated to explain the glucose-fatty-acid cycle

A small erythropoietin derived non-hematopoietic peptide reduces cardiac inflammation, attenuates age associated declines in heart function and prolongs healthspan

BackgroundAging is associated with increased levels of reactive oxygen species and inflammation that disrupt proteostasis and mitochondrial function and leads to organism-wide frailty later in life. ARA290 (cibinetide), an 11-aa non-hematopoietic peptide sequence within the cardioprotective domain of erythropoietin, mediates tissue protection by reducing inflammation and fibrosis. Age-associated cardiac inflammation is linked to structural and functional changes in the heart, including mitochondrial dysfunction, impaired proteostasis, hypertrophic cardiac remodeling, and contractile dysfunction. Can ARA290 ameliorate these age-associated cardiac changes and the severity of frailty in advanced age?MethodsWe conducted an integrated longitudinal (n = 48) and cross-sectional (n = 144) 15 months randomized controlled trial in which 18-month-old Fischer 344 x Brown Norway rats were randomly assigned to either receive chronic ARA290 treatment or saline. Serial echocardiography, tail blood pressure and body weight were evaluated repeatedly at 4-month intervals. A frailty index was calculated at the final timepoint (33 months of age). Tissues were harvested at 4-month intervals to define inflammatory markers and left ventricular tissue remodeling. Mitochondrial and myocardial cell health was assessed in isolated left ventricular myocytes. Kaplan–Meier survival curves were established. Mixed ANOVA tests and linear mixed regression analysis were employed to determine the effects of age, treatment, and age-treatment interactions.ResultsChronic ARA290 treatment mitigated age-related increases in the cardiac non-myocyte to myocyte ratio, infiltrating leukocytes and monocytes, pro-inflammatory cytokines, total NF-κB, and p-NF-κB. Additionally, ARA290 treatment enhanced cardiomyocyte autophagy flux and reduced cellular accumulation of lipofuscin. The cardiomyocyte mitochondrial permeability transition pore response to oxidant stress was desensitized following chronic ARA290 treatment. Concurrently, ARA290 significantly blunted the age-associated elevation in blood pressure and preserved the LV ejection fraction. Finally, ARA290 preserved body weight and significantly reduced other markers of organism-wide frailty at the end of life.ConclusionAdministration of ARA290 reduces cell and tissue inflammation, mitigates structural and functional changes within the cardiovascular system leading to amelioration of frailty and preserved healthspan

Hexokinase II Detachment from Mitochondria Triggers Apoptosis through the Permeability Transition Pore Independent of Voltage-Dependent Anion Channels

Type II hexokinase is overexpressed in most neoplastic cells, and it mainly localizes on the outer mitochondrial membrane. Hexokinase II dissociation from mitochondria triggers apoptosis. The prevailing model postulates that hexokinase II release from its mitochondrial interactor, the voltage-dependent anion channel, prompts outer mitochondrial membrane permeabilization and the ensuing release of apoptogenic proteins, and that these events are inhibited by growth factor signalling. Here we show that a hexokinase II N-terminal peptide selectively detaches hexokinase II from mitochondria and activates apoptosis. These events are abrogated by inhibiting two established permeability transition pore modulators, the adenine nucleotide translocator or cyclophilin D, or in cyclophilin D knock-out cells. Conversely, insulin stimulation or genetic ablation of the voltage-dependent anion channel do not affect cell death induction by the hexokinase II peptide. Therefore, hexokinase II detachment from mitochondria transduces a permeability transition pore opening signal that results in cell death and does not require the voltage-dependent anion channel. These findings have profound implications for our understanding of the pathways of outer mitochondrial membrane permeabilization and their inactivation in tumors

Mitochondrial respiration and ROS emission during β-oxidation in the heart: An experimental-computational study

<div><p>Lipids are main fuels for cellular energy and mitochondria their major oxidation site. Yet unknown is to what extent the fuel role of lipids is influenced by their uncoupling effects, and how this affects mitochondrial energetics, redox balance and the emission of reactive oxygen species (ROS). Employing a combined experimental-computational approach, we comparatively analyze β-oxidation of palmitoyl CoA (PCoA) in isolated heart mitochondria from Sham and streptozotocin (STZ)-induced type 1 diabetic (T1DM) guinea pigs (GPs). Parallel high throughput measurements of the rates of oxygen consumption (VO₂) and hydrogen peroxide (H₂O₂) emission as a function of PCoA concentration, in the presence of L-carnitine and malate, were performed. We found that PCoA concentration < 200 nmol/mg mito protein resulted in low H₂O₂ emission flux, increasing thereafter in Sham and T1DM GPs under both states 4 and 3 respiration with diabetic mitochondria releasing higher amounts of ROS. Respiratory uncoupling and ROS excess occurred at PCoA > 600 nmol/mg mito prot, in both control and diabetic animals. Also, for the first time, we show that an integrated two compartment mitochondrial model of β-oxidation of long-chain fatty acids and main energy-redox processes is able to simulate the relationship between VO₂ and H₂O₂ emission as a function of lipid concentration. Model and experimental results indicate that PCoA oxidation and its concentration-dependent uncoupling effect, together with a partial lipid-dependent decrease in the rate of superoxide generation, modulate H₂O₂ emission as a function of VO₂. Results indicate that keeping low levels of intracellular lipid is crucial for mitochondria and cells to maintain ROS within physiological levels compatible with signaling and reliable energy supply.</p></div

Respiratory and ROS emission fluxes from Sham and diabetic mitochondria as a function of PCoA concentration.

<p>Freshly isolated heart mitochondria from Sham and diabetic GPs were assayed for β-oxidation driven respiration and H₂O₂ emission as described in detail under Materials and Methods. Depicted are the specific rates of O₂ consumption, VO₂, (A, B) and H₂O₂ emission fluxes (C, D) determined under states 4 (no ADP; A, C) and 3 (+1mM ADP; B, D) respiration in Sham and STZ-treated (diabetic) mitochondria, respectively. The specific rates of O₂ consumption and H₂O₂ emission, measured in parallel in the same mitochondrial preparations at different PCoA concentrations (panels A-D), were plotted against each other for Sham and diabetic under states 4 (E) and 3 (F) respiration. N = 12 technical repeats from 3 biological replicates (experiments/hearts) in each Sham or diabetic group. Raw traces of O₂ consumption and H₂O₂ emission from representative experiments with Sham and diabetic mitochondria are shown in Fig C in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005588#pcbi.1005588.s001" target="_blank">S1 Text</a>.</p

Mitochondrial antioxidant defense protein levels and ROS emission in the absence or the presence of inhibitors of the GSH and Trx antioxidant systems in Sham and STZ hearts.

<p>(A, B) Heart tissue from Sham or diabetic GPs was processed and the antioxidant proteins indicated were analyzed by Western Blot as described in Materials and Methods. Left panel shows representative Western Blot analysis of protein abundance. The bar plot on the right panel displays the statistical comparison between the results obtained with heart tissue from the two groups [n = 4, four experiments]. Protein was normalized to total protein abundance in a given lane based on Direct Blue 71 (DB71) staining of the membrane as described [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005588#pcbi.1005588.ref016" target="_blank">16</a>,<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005588#pcbi.1005588.ref021" target="_blank">21</a>]. Key to symbols: GR, glutathione reductase; Gpx4, glutathione peroxidase 4; Trx2, thioredoxin 2; TrxR2, thioredoxin reductase 2; Nnt, nicotinamide nucleotide transhydrogenase; SOD2, superoxide dismutase 2; Prx3, peroxiredoxin 3. (C, D) Freshly isolated heart mitochondria (100μg mitochondrial protein) from Sham or STZ-treated GP were preincubated without or with 50nM auranofin (AF) plus 10μM 1-chloro-2,4 dinitrobenzene (DNCB) [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005588#pcbi.1005588.ref013" target="_blank">13</a>,<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005588#pcbi.1005588.ref016" target="_blank">16</a>]. Monitoring of H₂O₂ was performed with an Amplex red assay during state 4 (with [A] 10μM PCoA/0.5mM malate/0.5mM L-carnitine or [B] 5mM each of G/M) and state 3 (+1 mM ADP) of mitochondrial respiration, using a wavelength scanning fluorometer (QuantaMaster; Photon Technology International, Inc.) [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005588#pcbi.1005588.ref013" target="_blank">13</a>,<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005588#pcbi.1005588.ref016" target="_blank">16</a>] in the same assay medium utilized for high throughput measurements. The specific fluxes of H₂O₂ emission are shown for PCoA (C) and G/M (D) in the absence or the presence of AF + DNCB. (E, F) Depicted are the model simulated rates of ROS emission and O₂ consumption upon inhibition of antioxidant defense activities, glutathione reductase (GR) and thioredoxin reductase (TrxR) to mimic the actions of the experimentally utilized inhibitors DNCB and auranofin, respectively (C, D). The plots display the steady state values of H₂O₂ emission (E) and VO₂ (F) obtained at high (control: 100% GR and TrxR activities, empty bars) and low (15% and 22% of GR and TrxR activities, respectively, grey bars) scavenging capacity, under states 4 and 3 respiration as indicated. For comparative purpose, in panel E are shown the experimental values of Sham depicted in panel C, corresponding to the absence (filled squares) or presence (filled triangles) of inhibitors, under states 4 and 3 respiration as indicated. The simulations correspond to 40μM PCoA with 5x10^-4mM and 0.1mM ADP in states 4 and 3, respectively, while the remaining parameters are described in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005588#pcbi.1005588.s001" target="_blank">S1 Text</a>.</p

Scheme of the two-compartment mitochondrial model including β-oxidation and lipid transport.

<p>The present model encompasses mitochondrial energetic and redox processes, their interactions, and transport between compartments, in addition to energy metabolism, ion transport (H⁺, Ca²⁺, Na⁺, Pi) and β-oxidation (top). O₂^.− is dismutated to H₂O₂ by matrix-localized superoxide dismutase (MnSOD) or transported to the extra-mitochondrial compartment, where it will be scavenged by Cu,ZnSOD. H₂O₂ can either diffuse from the matrix or be scavenged by the large capacity glutathione (GSH) and thioredoxin (Trx) systems, or by catalase (CAT) in the extra-mitochondrial compartment. The palmitate oxidation is linked to the TCA cycle via AcCoA and NADH. Also, NADH and FADH₂ (bound to the electron transfer flavoprotein, ETF) are involved in the interaction between β-oxidation and the respiratory chain. The multiple regulatory interactions involved are not represented for simplicity. <i>Key to symbols</i>: Concentric circles with an arrow across (upper left of the scheme) represent the ΔΨ_m. The scheme is modified from [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005588#pcbi.1005588.ref015" target="_blank">15</a>,<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005588#pcbi.1005588.ref063" target="_blank">63</a>].</p

Modeling studies of mitochondrial respiration, H₂O₂ emission and ROS scavenging intermediates as a function of PCoA concentration and proton conductance.

<p>The steady state behavior of VO₂ in the model was analyzed for the same range of PCoA as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005588#pcbi.1005588.g003" target="_blank">Fig 3</a> mimicking those used in the experiments under states 4 (A) and 3 (C) respiration (blue traces) shown in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005588#pcbi.1005588.g002" target="_blank">Fig 2A and 2B</a>. Also depicted in black traces are the rates of H₂O₂ emission corresponding to the same simulations. Panels B and D display the steady state concentration of main components of matrix antioxidant systems (GSH, Trx(SH)₂, NADPH) under both states 4 (B) and 3 (D) respiration. The parameterization and initial conditions employed in the simulations are described in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005588#pcbi.1005588.s001" target="_blank">S1 Text</a> together with the Matlab code for the full computational model.</p