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We have investigated NADH and succinate aerobic oxidation in frozen and thawed swine heart mitochondria. Simultaneous oxidation of NADH and succinate showed complete additivity under a variety of experimental conditions, suggesting that the electron fluxes originating from NADH and succinate are completely independent and do not mix at the level of the so-called mobile diffusible components. We ascribe the results to mixing of the fluxes at the level of cytochrome c in bovine mitochondria: the Complex IV flux control coefficient in NADH oxidation was high in swine mitochondria but very low in bovine mitochondria, suggesting a stronger interaction of cytochrome c with the supercomplex in the former. This was not the case in succinate oxidation, in which Complex IV exerted little control also in swine mitochondria. We interpret the data in swine mitochondria as restriction of the NADH flux by channelling within the I-III 2 -IV supercomplex, whereas the flux from succinate shows pool mixing for both Coenzyme Q and probably cytochrome c. The difference between the two types of mitochondria may be ascribed to different lipid composition affecting the cytochrome c binding properties, as suggested by breaks in Arrhenius plots of Complex IV activity occurring at higher temperatures in bovine mitochondria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

An efficient, eco-compatible, and very cheap method for the construction of fully substituted pyrazoles (Pzs) via eliminative nitrilimine-alkene 1,3-dipolar cycloaddition (ENAC) reaction was developed in excellent yield and high regioselectivity. Enaminones and nitrilimines generated in situ were selected as dipolarophiles and dipoles, respectively. A deep screening of the employed base, solvent, and temperature was carried out to optimize reaction conditions. Recycling tests of ionic liquid were performed, furnishing efficient performance until six cycles. Finally, a plausible mechanism of cycloaddition was proposed. Then, the effect of three different structures of Pzs was evaluated on the F 1 F O -ATPase activity and mitochondrial permeability transition pore (mPTP) opening. The Pz derivatives' titration curves of 6a , 6h , and 6o on the F 1 F O -ATPase showed a reduced activity of 86%, 35%, and 31%, respectively. Enzyme inhibition analysis depicted an uncompetitive mechanism with the typical formation of the tertiary complex enzyme-substrate-inhibitor ( ESI ). The dissociation constant of the ESI complex ( K i had a lower order of magnitude than other Pzs. The pyrazole core might set the specific mechanism of inhibition with the F 6a F 1 F O -ATPase, whereas specific functional groups of Pzs might modulate the binding affinity. The mPTP opening decreased in Pz-treated mitochondria and the Pzs' inhibitory effect on the mPTP was concentration-dependent with 6a and 6o . Indeed, the mPTP was more efficiently blocked with 0.1 mM 6a than with 1 mM 6a . On the contrary, 1 mM 6o . The F 6o F 1 F O -ATPase is a target of Pzs blocking mPTP formation.

The compromised viability and function of cardiovascular cells are rescued by small molecules of triazole derivatives (Tzs), identified as 3a and 3b, by preventing mitochondrial dysfunction. The oxidative phosphorylation improves the respiratory control rate in the presence of Tzs independently of the substrates that energize the mitochondria. The F 1 F O -ATPase, the main candidate in mitochondrial permeability transition pore (mPTP) formation, is the biological target of Tzs and hydrophilic F 1 domain of the enzyme is depicted as the binding region of Tzs. The protective effect of Tz molecules on isolated mitochondria was corroborated by immortalized cardiomyocytes results. Indeed, mPTP opening was attenuated in response to ionomycin. Consequently, increased mitochondrial roundness and reduction of both length and interconnections between mitochondria. In in-vitro and ex-vivo models of cardiovascular pathologies (i.e., hypoxia-reoxygenation and hypertension) were used to evaluate the Tzs cardioprotective action. Key parameters of porcine aortic endothelial cells (pAECs) oxidative metabolism and cell viability were not affected by Tzs. However, in the presence of either 1 μM 3a or 0.5 μM 3b the impaired cell metabolism of pAECs injured by hypoxia-reoxygenation was restored to control respiratory profile. Moreover, endothelial cells isolated from SHRSP exposed to high-salt treatment rescued the Complex I activity and the endothelial capability to form vessel-like tubes and vascular function in presence of Tzs. As a result, the specific biochemical mechanism of Tzs to block Ca 2+ -activated F 1 F O -ATPase protected cell viability and preserved the pAECs bioenergetic metabolism upon hypoxia-reoxygenation injury. Moreover, SHRSP improved vascular dysfunction in response to a high-salt treatment., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Salvatore Nesci reports financial support was provided by University of Bologna. Speranza Rubattu reports financial support was provided by Italian Ministry of Health. Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The beneficial effects of bergamot polyphenolic fraction (BPF) on the mitochondrial bioenergetics of porcine aortic endothelial cells (pAECs) were verified under the cardiotoxic action of doxorubicin (DOX). The cell viability of pAECs treated for 24 h with different concentrations of DOX was reduced by 50%, but the negative effect of DOX was reversed in the presence of increasing doses of BPF (100 µg/mL and 200 µg/mL BPF). An analysis of the protective effect of BPF on the toxic action of DOX was also carried out on cell respiration. We observed the inhibition of the mitochondrial activity at 10 µM DOX, which was not restored by 200 µg/mL BPF. Conversely, the decrease in basal respiration and ATP production caused by 0.5 or 1.0 µM DOX were improved in the presence of 100 or 200 µg/mL BPF, respectively. After 24 h of cell recovery with 100 µg/mL or 200 µg/mL BPF on pAECs treated with 0.5 µM or 1.0 µM DOX, respectively, the mitochondrial parameters of oxidative metabolism impaired by DOX were re-boosted.

LMNA mutation is associated with type-2 familial partial lipodystrophy ( FPLD2 ). The disease causes a disorder characterized by anomalous accumulation of body fat in humans. The dysfunction at the molecular level is triggered by a lamin A/C mutation, impairing the cell metabolism. In human fibroblasts and preadipocytes, a trend for ATP production, mainly supported by mitochondrial oxidative metabolism, is detected. Moreover, primary cell lines with FPLD2 mutation decrease the mitochondrial ATP production if compared with the control , even if no differences are observed in the oxygen consumption rate of bioenergetic parameters (i.e., basal and maximal respiration, spare respiratory capacity, and ATP turnover). Conversely, glycolysis is only inhibited in FPLD2 fibroblast cell lines. We notice that the amount of ATP produced in the fibroblasts is higher than in the preadipocytes, and likewise in the control , with respect to FPLD2 , due to a more active oxidative phosphorylation (OXPHOS) and glycolysis. Moreover, the proton leak parameter, which characterizes the transformation of white adipose tissue to brown/beige adipose tissue, is unaffected by FPLD2 mutation. The metabolic profile of fibroblasts and preadipocytes is confirmed by the ability of these cell lines to increase the metabolic potential of both OXPHOS and glycolysis under energy required independently by the FPLD2 mutation.

The effects and specificity of dietary supplementation of EconomasE TM (EcoE), mainly consisting of organic selenium (0.15 or 0.30 mg/kg feed; Se) or of vitamin E (100 or 200 mg/kg feed; VE), on lipid oxidation were evaluated in rabbit hamburgers during refrigerated storage. Oxidation data obtained by thiobarbituric acid-reactive substances (TBARS) spectrophotometric analysis and solid-phase microextraction (SPME) coupled with gas chromatography (GC) to determine hexanal content were compared. The relationships between oxidation levels, colour and pH and the discriminability of EcoE- or VE-treatment compared with control were also examined. TBARS content decreased in both VE and EcoE groups, while hexanal showed lower values only in the VE100 dietary group. The colour parameters were compatible with acceptable product quality and seemed to parallel the TBARS values up to the second day storage. Both VE and EcoE improved oxidative stability without affecting the sensory properties, but the VE effect appeared to more specifically hamper lipid oxidation, as evidenced by the determination and quantification of hexanal, a specific product of fatty acid peroxidation.

The bergamot polyphenolic fraction (BPF) was evaluated in the F 1 F O -ATPase activity of swine heart mitochondria. In the presence of a concentration higher than 50 µg/mL BPF, the ATPase activity of F 1 F O -ATPase, dependent on the natural cofactor Mg 2+ , increased by 15%, whereas the enzyme activity in the presence of Ca 2+ was inhibited by 10%. By considering this opposite BPF effect, the F 1 F O -ATPase activity involved in providing ATP synthesis in oxidative phosphorylation and triggering mitochondrial permeability transition pore (mPTP) formation has been evaluated. The BPF improved the catalytic coupling of oxidative phosphorylation in the presence of a substrate at the first phosphorylation site, boosting the respiratory control ratios (state 3/state 4) by 25% and 85% with 50 µg/mL and 100 µg/mL BPF, respectively. Conversely, the substrate at the second phosphorylation site led to the improvement of the state 3/state 4 ratios by 15% only with 100 µg/mL BPF. Moreover, the BPF carried out its beneficial effect on the mPTP phenomenon by desensitizing the pore opening. The acute effect of the BPF on the metabolism of porcine aortica endothelial cells (pAECs) showed an ATP rate index greater than one, which points out a prevailing mitochondrial oxidative metabolism with respect to the glycolytic pathway, and this ratio rose by about three times with 100 µg/mL BPF. Consistently, the mitochondrial ATP turnover, in addition to the basal and maximal respiration, were higher in the presence of the BPF than in the controls, and the MTT test revealed an increase in cell viability with a BPF concentration above 200 µg/mL. Therefore, the molecule mixture of the BPF aims to ensure good performance of the mitochondrial bioenergetic parameters.

The fat-soluble vitamin K (VK) has long been known as a requirement for blood coagulation, but like other vitamins, has been recently recognized to play further physiological roles, particularly in cell development and homeostasis. Vertebrates cannot de novo synthesize VK, which is essential, and it can only be obtained from the diet or by the activity of the gut microbiota. The IPEC-J2 cell line, obtained from porcine small intestine, which shows strong similarities to the human one, represents an excellent functional model to in vitro study the effect of compounds at the intestinal level. The acute VK treatments on the bioenergetic features of IPEC-J2 cells were evaluated by Seahorse XP Agilent technology. VK exists in different structurally related forms (vitamers), all featured by a naphtoquinone moiety, but with distinct effects on IPEC-J2 energy metabolism. The VK1, which has a long hydrocarbon chain, at both concentrations (5 and 10 μM), increases the cellular ATP production due to oxidative phosphorylation (OXPHOS) by 5% and by 30% through glycolysis. The VK2 at 5 μM only stimulates ATP production by OXPHOS. Conversely, 10 μM VK3, which lacks the long side chain, inhibits OXPHOS by 30% and glycolysis by 45%. However, even if IPEC-J2 cells mainly prefer OXPHOS to glycolysis to produce ATP, the OXPHOS/glycolysis ratio significantly decreases in VK1-treated cells, is unaffected by VK2, and only significantly increased by 10 μM VK3. VK1, at the two concentrations tested, does not affect the mitochondrial bioenergetic parameters, while 5 μM VK2 increases and 5 μM VK3 reduces the mitochondrial respiration (i.e., maximal respiration and spare respiratory capacity). Moreover, 10 μM VK3 impairs OXPHOS, as shown by the increase in the proton leak, namely the proton backward entry to the matrix space, thus pointing out mitochondrial toxicity. Furthermore, in the presence of both VK1 and VK2 concentrations, the glycolytic parameters, namely the glycolytic capacity and the glycolytic reserve, are unaltered. In contrast, the inhibition of glycoATP production by VK3 is linked to the 80% inhibition of glycolysis, resulting in a reduced glycolytic capacity and reserve. These data, which demonstrate the VK ability to differently modulate IPEC-J2 cell energy metabolism according to the different structural features of the vitamers, can mirror VK modulatory effects on the cell membrane features and, as a cascade, on the epithelial cell properties and gut functions: balance of salt and water, macromolecule cleavage, detoxification of harmful compounds, and nitrogen recycling., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bernardini, Algieri, La Mantia, Trombetti, Pagliarani, Forni and Nesci.)

Under aerobic conditions, mitochondrial oxidative phosphorylation (OXPHOS) converts the energy released by nutrient oxidation into ATP, the currency of living organisms. The whole biochemical machinery is hosted by the inner mitochondrial membrane (mtIM) where the protonmotive force built by respiratory complexes, dynamically assembled as super-complexes, allows the F 1 F O -ATP synthase to make ATP from ADP + Pi. Recently mitochondria emerged not only as cell powerhouses, but also as signaling hubs by way of reactive oxygen species (ROS) production. However, when ROS removal systems and/or OXPHOS constituents are defective, the physiological ROS generation can cause ROS imbalance and oxidative stress, which in turn damages cell components. Moreover, the morphology of mitochondria rules cell fate and the formation of the mitochondrial permeability transition pore in the mtIM, which, most likely with the F 1 F O -ATP synthase contribution, permeabilizes mitochondria and leads to cell death. As the multiple mitochondrial functions are mutually interconnected, changes in protein composition by mutations or in supercomplex assembly and/or in membrane structures often generate a dysfunctional cascade and lead to life-incompatible diseases or severe syndromes. The known structural/functional changes in mitochondrial proteins and structures, which impact mitochondrial bioenergetics because of an impaired or defective energy transduction system, here reviewed, constitute the main biochemical damage in a variety of genetic and age-related diseases.

Recently, the F 1 F O -ATP synthase, due to its dual role of life enzyme as main adenosine triphosphate (ATP) maker and of death enzyme, as ATP dissipator and putative structural component of the mitochondrial permeability transition pore (mPTP), which triggers cell death, has been increasingly considered as a drug target. Accordingly, the enzyme offers new strategies to counteract the increased antibiotic resistance. The challenge is to find or synthesize compounds able to discriminate between prokaryotic and mitochondrial F 1 F O -ATP synthase, exploiting subtle structural differences to kill pathogens without affecting the host. From this perspective, the eukaryotic enzyme could also be made refractory to macrolide antibiotics by chemically produced posttranslational modifications. Moreover, because the mitochondrial F 1 F O -ATPase activity stimulated by Ca 2+ instead of by the natural modulator Mg 2+ is most likely involved in mPTP formation, effectors preferentially targeting the Ca 2+ -activated enzyme may modulate the mPTP. If the enzyme involvement in the mPTP is confirmed, Ca 2+ -ATPase inhibitors may counteract conditions featured by an increased mPTP activity, such as neurodegenerative and cardiovascular diseases and physiological aging. Conversely, mPTP opening could be pharmacologically stimulated to selectively kill unwanted cells. On the basis of recent literature and promising lab findings, the action mechanism of F 1 and F O inhibitors is considered. These molecules may act as enzyme modifiers and constitute new drugs to kill pathogens, improve compromised enzyme functions, and limit the deathly enzyme role in pathologies. The enzyme offers a wide spectrum of therapeutic strategies to fight at the molecular level diseases whose treatment is still insufficient or merely symptomatic.

In spite of the known widespread toxicity of mercury, its impact on mitochondrial bioenergetics is a still poorly explored topic. Even if many studies have dealt with mercury poisoning of mitochondrial respiration, as far as we are aware Hg 2+ effects on individual complexes are not so clear. In the present study changes in swine heart mitochondrial respiration and F 1 F O -ATPase (F-ATPase) activity promoted by micromolar Hg 2+ concentrations were investigated. Hg 2+ was found to inhibit the respiration of NADH-energized mitochondria, whereas it was ineffective when the substrate was succinate. Interestingly, the same micromolar Hg 2+ doses which inhibited the NADH-O 2 activity stimulated the F-ATPase, most likely by interacting with adjacent thiol residues. Accordingly, Hg 2+ dose-dependently decreased protein thiols and all the elicited effects on mitochondrial complexes were reversed by the thiol reducing agent DTE. These findings clearly indicate that Hg 2+ interacts with Cys residues of these complexes and differently modulate their functionality by modifying the redox state of thiol groups. The results, which cast light on some implications of metal-thiol interactions up to now not fully explored, may contribute to clarify the molecular mechanisms of mercury toxicity to mitochondria., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The effect of the administration of commercial diets supplemented with 9 mg kg(-1) 3,5,3'-triiodo-l-thyronine (T(3)) or 10% (w/w) NaCl was evaluated on the ouabain-insensitive Na+-ATPase activity in rainbow trout gill microsomes. The trial, carried out following the seasonal trend from March to mid-May, included a treatment phase in freshwater and a subsequent transfer to brackish water (22 per thousand salinity) where trout were not treated. pH dependence, apparent Km values for Mg(2+) and Na+, and Hill coefficients evaluated throughout the trial for Na+-ATPase were generally not affected by the treatments and habitat change. In comparison with the control group, in both treated groups, Na+-ATPase activity was lower during the freshwater phase and higher after brackish-water transfer. As compared with untreated trout, gill (Na++K+)-ATPase activity during the freshwater phase was stimulated by NaCl treatment and also by T(3) treatment after transfer to brackish water. The results indicate that NaCl and T(3) administration act differently on the two ATPase activities involved in Na+ regulation and suggest a prevalent role of Na+-ATPase activity in hypoosmotic conditions.