Efficiency Of Phosphorylation Research Articles

Haemodynamic-directed cardiopulmonary resuscitation promotes mitochondrial fusion and preservation of mitochondrial mass after successful resuscitation in a pediatric porcine model

ObjectiveCerebral mitochondrial dysfunction is a key mediator of neurologic injury following cardiac arrest (CA) and is regulated by the balance of fusion and fission (mitochondrial dynamics). Under stress, fission can decrease mitochondrial mass and signal apoptosis, while fusion promotes oxidative phosphorylation efficiency. This study evaluates mitochondrial dynamics and content in brain tissue 24h after CA between two cardiopulmonary resuscitation (CPR) strategies. InterventionsPiglets (1 month), previously randomized to three groups: (1) Std-CPR (n=5); (2) HD-CPR (n=5; goal systolic blood pressure 90mmHg, goal coronary perfusion pressure 20mmHg); (3) Shams (n=7). Std-CPR and HD-CPR groups underwent 7min of asphyxia, 10min of CPR, and standardized post-resuscitation care. Primary outcomes: (1) cerebral cortical mitochondrial protein expression for fusion (OPA1, OPA1 long to short chain ratio, MFN2) and fission (DRP1, FIS1), and (2) mitochondrial mass by citrate synthase activity. Secondary outcomes: (1) intra-arrest haemodynamics and (2) cerebral performance category (CPC) at 24h. ResultsHD-CPR subjects had higher total OPA1 expression compared to Std-CPR (1.52; IQR 1.02–1.69 vs 0.67; IQR 0.54−0.88, p=0.001) and higher OPA1 long to short chain ratio than both Std-CPR (0.63; IQR 0.46−0.92 vs 0.26; IQR 0.26−0.31, p=0.016) and shams. Citrate synthase activity was lower in Std-CPR than sham (11.0; IQR 10.15–12.29 vs 13.4; IQR 12.28–15.66, p=0.047), but preserved in HD-CPR. HD-CPR subjects had improved intra-arrest haemodynamics and CPC scores at 24h compared to Std-CPR. ConclusionsFollowing asphyxia-associated CA, HD-CPR exhibits increased pro-mitochondrial fusion protein expression, preservation of mitochondrial mass, improved haemodynamics and superior neurologic scoring compared to Std-CPR. Institutional protocol numberIAC 16-001023.

Combined preparation based on chelating magnesium by phosphorylated casein. Characteristics of its syntesis

The article is devoted to the development and synthesis of a new drug for use in animal husbandry and veterinary medicine as an immunostimulatory and adaptogenic agent. The basis of the new drug is a combination of magnesium, phosphorus, and casein of cow's milk.
 Given the important scientific and practical importance for veterinary medicine of innovative drugs that would have pronounced immunostimulatory and adaptogenic properties, the aim of our research is to develop a method of obtaining a new drug with an original composition and further studies of its effectiveness and safety.
 Modern materials and methods were used to achieve this goal. In particular, mass spectrometry was used on a Waters H-class UPLC liquid high-pressure liquid chromatography spectrometer with a Waters TQ-S micro three-quadrupole detector; atomic emission spectrometer with inductively coupled plasma Analytik-Jena Plasma Quant PQ 9000 Elite; liquid chromatographer with a three-quadrupole mass detector and with analytical column – Waters ACQUITY UPLC BEH C18 1.7μm 2.1x50mm.
 The result of the work was the development of a method of modification of the casein molecule, which was carried out in several stages: the first stage was the direct phosphorylation of the casein molecule; the second stage of the synthesis was the chelation of magnesium with casein.
 Based on the results of this work, the following conclusions were formed: 1) the synthesis of a new drug is carried out in two stages: the first – modification of casein by direct phosphorylation and the second stage – chelation of magnesium with casein; 2) it was found that the efficiency of phosphorylation directly correlates with the number of treatment cycles of the reaction mixture and is optimal for three treatment cycles

Functional segmentation of CoQ and cyt c pools by respiratory complex superassembly

Electron transfer between respiratory complexes is an essential step for the efficiency of the mitochondrial oxidative phosphorylation. Until recently, it was stablished that ubiquinone and cytochrome c formed homogenous single pools in the inner mitochondrial membrane which were not influenced by the presence of respiratory supercomplexes. However, this idea was challenged by the fact that bottlenecks in electron transfer appeared after disruption of supercomplexes into their individual complexes. The postulation of the plasticity model embraced all these observations and concluded that complexes and supercomplexes co-exist and are dedicated to a spectrum of metabolic requirements. Here, we review the involvement of superassembly in complex I stability, the role of supercomplexes in ROS production and the segmentation of the CoQ and cyt c pools, together with their involvement in signaling and disease. Taking apparently conflicting literature we have built up a comprehensive model for the segmentation of CoQ and cyt c mediated by supercomplexes, discuss the current limitations and provide a prospect of the current knowledge in the field.

The Role of Mitonuclear Incompatibility in Bipolar Disorder Susceptibility and Resilience Against Environmental Stressors

It has been postulated that mitochondrial dysfunction has a significant role in the underlying pathophysiology of bipolar disorder (BD). Mitochondrial functioning plays an important role in regulating synaptic transmission, brain function, and cognition. Neuronal activity is energy dependent and neurons are particularly sensitive to changes in bioenergetic fluctuations, suggesting that mitochondria regulate fundamental aspects of brain function. Vigorous evidence supports the role of mitochondrial dysfunction in the etiology of BD, including dysregulated oxidative phosphorylation, general decrease of energy, altered brain bioenergetics, co-morbidity with mitochondrial disorders, and association with genetic variants in mitochondrial DNA (mtDNA) or nuclear-encoded mitochondrial genes. Despite these advances, the underlying etiology of mitochondrial dysfunction in BD is unclear. A plausible evolutionary explanation is that mitochondrial-nuclear (mitonuclear) incompatibility leads to a desynchronization of machinery required for efficient electron transport and cellular energy production. Approximately 1,200 genes, encoded from both nuclear and mitochondrial genomes, are essential for mitochondrial function. Studies suggest that mitochondrial and nuclear genomes co-evolve, and the coordinated expression of these interacting gene products are essential for optimal organism function. Incompatibilities between mtDNA and nuclear-encoded mitochondrial genes results in inefficiency in electron flow down the respiratory chain, differential oxidative phosphorylation efficiency, increased release of free radicals, altered intracellular Ca2+ signaling, and reduction of catalytic sites and ATP production. This review explores the role of mitonuclear incompatibility in BD susceptibility and resilience against environmental stressors.

Polydopamine nanoparticles as dual-task platform for osteoarthritis therapy: A scavenger for reactive oxygen species and regulator for cellular powerhouses

• Excellent antioxidative/anti-inflammatory dual-task nanoplatform based on PDA NPs. • PDA NPs not only scavenge reactive oxygen species but also reduce their production. • PDA NPs increase cellular respiratory efficiency, thereby reducing oxidative stress. • Injectable PDA NPs serve as minimal invasive therapeutic agents for osteoarthritis. Because of the obscure etiology of osteoarthritis (OA) and unsatisfactory treatment outcomes, new effective and minimally invasive therapies are urgently needed. Oxidative stress elicited by excessive reactive oxygen species (ROS) accumulation has historically been considered a potential trigger of OA. Polydopamine (PDA), an emerging versatile biopolymer produced by self-polymerization of dopamine, has attracted considerable attention in biomedical applications by virtue of its excellent biocompatibility and intriguing ROS-scavenging capacity. Herein, an antioxidative/anti-inflammatory dual-task nanoplatform based on PDA nanoparticles (NPs) is introduced for the treatment of temporomandibular joint osteoarthritis (TMJ-OA). In addition to directly reacting with ROS as a reducing agent, PDA NPs also regulate ROS generation in mitochondria which are considered cellular powerhouses. Specifically, the efficiency of mitochondrial oxidative phosphorylation (OXPHOS) is significantly increased in the presence of PDA NPs, indicating that PDA NPs may increase the efficiency of mitochondrial respiration, hence reducing ROS production. This intriguing dual-antioxidative mechanism may account for the remarkable antioxidative capacity of PDA NPs. Moreover, the anti-inflammatory capacity of PDA NPs is revealed both in vitro and in vivo . This work not only opens a new avenue for OA treatment but also provides valuable insights into the design of biomaterials with multiple biomedical applications via regulation of cellular energy metabolism.

Role of mitochondrial calcium in hypochlorite induced oxidative damage of cells

Hypochlorite (HOCl) is one of the most important mediators of inflammatory processes. Recent evidence demonstrates that changes in intracellular calcium pool play a significant role in the damaging effects of hypochlorite and other oxidants. Mitochondria are shown to be one of the intracellular targets of hypochlorite. But little is known about the mitochondrial calcium pool changes in HOCl-induced mitochondrial dysfunction. Using isolated rat liver mitochondria, we showed the oxidative damage of mitochondria (GSH oxidation and mixed protein-glutathione formation without membrane lipid peroxidation) and alterations in the mitochondrial functional parameters (decrease of respiratory activity and efficiency of oxidative phosphorylation, NADH and FADH coenzyme levels, and membrane potential) under hypochlorite action (50–300 μM). Simultaneously, the mitochondrial calcium release and swelling were demonstrated. In the presence of EGTA, the damaging effects of HOCl were less pronounced, reflecting direct involvement of mitochondrial Ca2+ in mechanisms of oxidant-induced injury. Furthermore, exposure of HeLa cells to hypochlorite resulted in a considerable increase in cytoplasmic calcium concentrations and a decrease in mitochondrial ones. Applying specific inhibitors of calcium transfer systems, we demonstrated that mitochondria play a key role in the redistribution of cytoplasmic Ca2+ ions under hypochlorite action and act as mediators of calcium release from the endoplasmic reticulum into the cytoplasm.

The Autumn Low Milk Yield Syndrome in High Genetic Merit Dairy Cattle: The Possible Role of a Dysregulated Innate Immune Response.

Simple SummaryMilk yield worldwide is dominated by few cosmopolitan dairy cattle breeds producing high production levels in the framework of hygiene standards that have dramatically improved over the years. Yet, there is evidence that such achievements have gone along with substantial animal health and welfare problems for many years, exemplified by reduced life expectancy and high herd replacement rates. Also, these animals are very susceptible to diverse environmental stressors, among which hot summer climate plays a central role in the occurrence of diverse disease cases underlying early cull from the herd. Milk production is also affected by heat stress, both directly and indirectly, as shown by low milk yield in the following autumn period. This article highlights the low milk yield syndrome and sets it into a conceptual framework, based on the crucial role of the innate immune system in the response to non-infectious stressors and in adaptation physiology at large.The analysis of milk yield data shows that high genetic merit dairy cows do not express their full production potential in autumn. Therefore, we focused on metabolic stress and inflammatory response in the dry and peripartum periods as possible causes thereof. It was our understanding that some cows could not cope with the stress imposed by their physiological and productive status by means of adequate adaptation strategies. Accordingly, this study highlights the noxious factors with a potential to affect cows in the above transition period: hot summer climate, adverse genetic traits, poor coping with unfavorable environmental conditions, outright production diseases and consequences thereof. In particular, the detrimental effects in the dry period of overcrowding, photoperiod change and heat stress on mammary gland development and milk production are highlighted in the context of the autumn low milk yield syndrome. The latter could be largely accounted for by a “memory” effect on the innate immune system induced in summer by diverse stressors after dry-off, according to strong circumstantial and indirect experimental evidence. The “memory” effect is based on distinct epigenetic changes of innate immunity genes, as already shown in cases of bovine mastitis. Following a primary stimulation, the innate immune system would be able to achieve a state known as “trained immunity”, a sort of “education” which modifies the response to the same or similar stressors upon a subsequent exposure. In our scenario, the “education” of the innate immune system would induce a major shift in the metabolism of inflammatory cells following their reprogramming. This would entail a higher basal consumption of glucose, in competition with the need for the synthesis of milk. Also, there is strong evidence that the inflammatory response generated in the dry period leads to a notable reduction of dry matter intake after calving, and to a reduced efficiency of oxidative phosphorylation in mitochondria. On the whole, an effective control of the stressors in the dry period is badly needed for better disease control and optimal production levels in dairy cattle.

Energy substrate metabolism and mitochondrial oxidative stress in cardiac ischemia/reperfusion injury

The heart is the most metabolically flexible organ with respect to the use of substrates available in different states of energy metabolism. Cardiac mitochondria sense substrate availability and ensure the efficiency of oxidative phosphorylation and heart function. Mitochondria also play a critical role in cardiac ischemia/reperfusion injury, during which they are directly involved in ROS-producing pathophysiological mechanisms. This review explores the mechanisms of ROS production within the energy metabolism pathways and focuses on the impact of different substrates. We describe the main metabolites accumulating during ischemia in the glucose, fatty acid, and Krebs cycle pathways. Hyperglycemia, often present in the acute stress condition of ischemia/reperfusion, increases cytosolic ROS concentrations through the activation of NADPH oxidase 2 and increases mitochondrial ROS through the metabolic overloading and decreased binding of hexokinase II to mitochondria. Fatty acid-linked ROS production is related to the increased fatty acid flux and corresponding accumulation of long-chain acylcarnitines. Succinate that accumulates during anoxia/ischemia is suggested to be the main source of ROS, and the role of itaconate as an inhibitor of succinate dehydrogenase is emerging. We discuss the strategies to modulate and counteract the accumulation of substrates that yield ROS and the therapeutic implications of this concept.

Effect of hyperthermia and acidosis on equine skeletal muscle mitochondrial oxygen consumption

The skeletal muscle of exercising horses develops pronounced hyperthermia and acidosis during strenuous or prolonged exercise, with very high tissue temperature and low pH associated with muscle fatigue or damage. The purpose of this study was to evaluate the individual effects of physiologically relevant hyperthermia and acidosis on equine skeletal muscle mitochondrial function, using ex vivo measurement of oxygen consumption to assess the function of different mitochondrial elements. Fresh triceps muscle biopsies from 6 healthy unfit Thoroughbred geldings were permeabilised to permit diffusion of small molecular weight substrates through the sarcolemma and analysed in a high resolution respirometer at 38, 40, 42, and 44 °C, and pH=7.1, 6.5, and 6.1. Oxygen consumption was measured under conditions of non-phosphorylating (leak) respiration and phosphorylating respiration through Complex I and Complex II. Data were analysed using a one-way repeated measures ANOVA and data are expressed as mean ± standard deviation. Leak respiration was ~3-fold higher at 44 °C compared to 38 °C regardless of electron source (Complex I: 22.88±3.05 vs 8.08±1.92 pmol O2/mg/s), P=0.002; Complex II: 79.14±23.72 vs 21.43±11.08 pmol O2/mg/s, P=0.022), resulting in a decrease in efficiency of oxidative phosphorylation. Acidosis had minimal effect on mitochondrial respiration at pH=6.5, but pH=6.1 resulted in a 50% decrease in mitochondrial oxygen consumption. These results suggest that skeletal muscle hyperthermia decreases the efficiency of oxidative phosphorylation through increased leak respiration, thus providing a specific biochemical basis for hyperthermia-induced muscle fatigue. The effect of myocellular acidosis on mitochondrial respiration was minimal under typical levels of acidosis, but atypically severe acidosis can lead to impairment of mitochondrial function.

P.533 The two-injection start of aripiprazole once-monthly provides rapid attainment of therapeutic concentrations without the need for 14-day oral tablet supplementation

Acitretin is a synthetic retinoid used for severe extensive psoriasis and it has been shown to be an effective and a safe therapeutic drug for other diseases including cancer when used in combination with other agents. However, cases of acitretin-associated liver injury have been documented, but the possible mechanisms of acitretin-associated hepatotoxicity and apoptosis are not entirely clarified. This study reports that mitochondrial dysfunctions may play an important role in liver injury and apoptosis induced by this retinoid. Acitretin (5–20 μM) impaired mitochondrial phosphorylation efficiency as demonstrated by the decrease in the state 3 respiration and ATP levels, and by the increase in the lag phase of ADP phosphorylation cycle, without affecting the membrane potential. Acitretin induced Ca2+-mediated mitochondrial permeability transition (MPT) and decreased the adenine nucleotide translocase (ANT) content. Acitretin-induced MPT was not prevented by thiol group protecting and antioxidant agents, excluding the involvement of oxidative stress mechanisms. However, MPT was prevented by ANT ligands ATP, ADP, tamoxifen and 4-hydroxytamoxifen, implying that the MPT induction by acitretin is mediated by the ANT. ANT plays a major role in promoting apoptosis and ATP synthesis, and it is still considered as a structural component of the pore with a regulatory role in MPT formation. Therefore, our results, including the decrease in the state 3 respiration and the increase in the lag phase of phosphorylation cycle, the ATP depletion and the induction of Ca2+-mediated MPT, indicate that acitretin-associated liver toxicity and apoptosis is possibly related with mitochondrial dysfunctions due to interactions with the ANT. Additionally, the combination of acitretin with other drugs, such as antiestrogens, which are able to inhibit the MPT, may contribute to decrease the toxicity induced by acitretin.

Effects of Phosphorylation Duration on the Jute Extracted Cellulose Nanofibrils Using Ultra‐sonication

AbstractThe pretreatment process for the extraction of cellulose nanofibrils (CNFs) has been attracting the awareness of researchers. However, toxicity, complicated multistep procedures, and the use of costly chemicals are still some problems for chemical modification of cellulose fibers during the CNFs production. In this work, the phosphorylation reaction is introduced to pretreat jute fibers to extract CNFs with tiny diameter, and then the physicochemical properties of the obtained CNFs are analyzed. Experimental results exhibit that the phosphorylation process can affect the overall defibrillation process and the structure as well as properties of the involved products. This study also reports that the phosphorylation efficiency can be influenced by the existence of water, absence of urea, high temperature, and reaction duration. Even at a low degree of substitution, the phosphorylated CNFs exhibit better thermal stability and higher residual char compared to raw jute at 700 °C.

Western Diet Causes Obesity-Induced Nonalcoholic Fatty Liver Disease Development by Differentially Compromising the Autophagic Response

Nonalcoholic fatty liver disease (NAFLD) is characterized by the development of steatosis, which can ultimately compromise liver function. Mitochondria are key players in obesity-induced metabolic disorders; however, the distinct role of hypercaloric diet constituents in hepatic cellular oxidative stress and metabolism is unknown. Male mice were fed either a high-fat (HF) diet, a high-sucrose (HS) diet or a combined HF plus HS (HFHS) diet for 16 weeks. This study shows that hypercaloric diets caused steatosis; however, the HFHS diet induced severe fibrotic phenotype. At the mitochondrial level, lipidomic analysis showed an increased cardiolipin content for all tested diets. Despite this, no alterations were found in the coupling efficiency of oxidative phosphorylation and neither in mitochondrial fatty acid oxidation (FAO). Consistent with unchanged mitochondrial function, no alterations in mitochondrial-induced reactive oxygen species (ROS) and antioxidant capacity were found. In contrast, the HF and HS diets caused lipid peroxidation and provoked altered antioxidant enzyme levels/activities in liver tissue. Our work provides evidence that hepatic oxidative damage may be caused by augmented levels of peroxisomes and consequently higher peroxisomal FAO-induced ROS in the early NAFLD stage. Hepatic damage is also associated with autophagic flux impairment, which was demonstrated to be diet-type dependent. The HS diet induced a reduction in autophagosomal formation, while the HF diet reduced levels of cathepsins. The accumulation of damaged organelles could instigate hepatocyte injuries and NAFLD progression.

Chronic cold exposure induces mitochondrial plasticity in deer mice native to high altitudes.

Small mammals native to high altitude must sustain high rates of thermogenesis to cope with cold. Skeletal muscle is a key site of shivering and non-shivering thermogenesis, but the importance of mitochondrial plasticity in cold hypoxic environments remains unresolved. We examined high-altitude deer mice, which have evolved a high capacity for aerobic thermogenesis, to determine the mechanisms of mitochondrial plasticity during chronic exposure to cold and hypoxia, alone and in combination. Cold exposure in normoxia or hypoxia increased mitochondrial leak respiration and decreased phosphorylation efficiency and OXPHOS coupling efficiency, which may serve to augment non-shivering thermogenesis. Cold also increased muscle oxidative capacity, but reduced the capacity for mitochondrial respiration via complex II relative to complexes I and II combined. High-altitude mice had a more oxidative muscle phenotype than low-altitude mice. Therefore, both plasticity and evolved changes in muscle mitochondria contribute to thermogenesis at high altitude. Small mammals native to high altitude must sustain high rates of thermogenesis to cope with cold and hypoxic environments. Skeletal muscle is a key site of shivering and non-shivering thermogenesis, but the importance of mitochondrial plasticity in small mammals at high altitude remains unresolved. High-altitude deer mice (Peromyscus maniculatus) and low-altitude white-footed mice (P. leucopus) were born and raised in captivity, and chronically exposed as adults to warm (25°C) normoxia, warm hypoxia (12kPa O2 ), cold (5°C) normoxia, or cold hypoxia. We then measured oxidative enzyme activities, oxidative fibre density and capillarity in the gastrocnemius, and used a comprehensive substrate titration protocol to examine the function of muscle mitochondria by high-resolution respirometry. Exposure to cold in both normoxia or hypoxia increased the activities of citrate synthase and cytochrome oxidase. In lowlanders, this was associated with increases in capillary density and the proportional abundance of oxidative muscle fibres, but in highlanders, these traits were unchanged at high levels across environments. Environment had some distinct effects on mitochondrial OXPHOS capacity between species, but the capacity of complex II relative to the combined capacity of complexes I and II was consistently reduced in both cold environments. Both cold environments also increased leak respiration and decreased phosphorylation efficiency and OXPHOS coupling efficiency in both species, which may serve to augment non-shivering thermogenesis. These cold-induced changes in mitochondrial function were overlaid upon the generally more oxidative phenotype of highlanders. Therefore, both plasticity and evolved changes in muscle mitochondria contribute to thermogenesis at high altitudes.

PER1 interaction with GPX1 regulates metabolic homeostasis under oxidative stress

Metabolism serves mammalian feeding and active behavior, and is controlled by circadian clock. The molecular mechanism by which clock factors regulate metabolic homeostasis under oxidative stress is unclear. Here, we have characterized that the daily oxygen consumption rhythm was deregulated in Per1 deficient mice. Per1 deficiency impaired daily mitochondrial dynamics and deregulated cellular GPx-related ROS fluctuations in the peripheral organs. We identified that PER1 enhanced GPx activity through PER1/GPX1 interaction in cytoplasm, consequently improving the oxidative phosphorylation efficiency of mitochondria. Per1 expression was specifically elevated in the fasting peripheral organs for protecting mitochondrial from oxidation stress. These observations reveal that Per1-driven mitochondrial dynamics is a critical effector mechanism for the regulation of mitochondrial function in response to oxidation stress.

Abstract 4795: Metabolic vulnerability by UQCRH methylation in clear cell renal cell carcinoma

Abstract Background: UQCRH, subunit 6 of complex III in the electron transport chain of the mitochondria, is a hinge protein that stabilizes cytochrome c-c1 complex formation. UQCRH is the top-ranked gene by inverse correlation between DNA methylation and gene expression in clear cell renal carcinoma (ccRCC) in the Cancer Genome Atlas and Broad Institute Cancer Cell Line Encyclopedia databases. In the clinic, ccRCC patients with low UQCRH expression exhibit higher tumor stage and grade. While its downregulation is correlated with worse tumor prognosis, little is known about the mechanism through which UQCRH is involved in cancer progression. We propose that hypermethylation-induced downregulation of UQCRH inhibits complex III activity, thus lowering the efficiency of oxidative phosphorylation and shifting the metabolic balance of the cell toward aerobic glycolysis, which contributes to tumor proliferation. Methods: Two ccRCC cell lines were used, KMRC-2 and 786-O. We measured UQCRH and mRNA expression levels and identified which cell lines exhibited UQCRH downregulation. We used the global demethylation agent Decitabine (Dacogen) to test whether methylation of the UQCRH gene was present in the low-UQCRH cell line. UQCRH overexpression and knockout sublines were created for high and low-UQCRH cell lines, respectively. We characterized the metabolic preferences of the sublines and original cell lines and measured tumor proliferation in vitro and in vivo. We also studied the effect of ketogenic diet on cell proliferation in vitro. Results: While 786-O expressed normal levels of UQCRH, KMRC-2 expressed low levels of UQCRH, but its UQCRH expression was rescued by increasing concentrations of Decitabine. Sublines with low UQCRH, whether by hypermethylation or knockout, had higher extracellular acidification rate compared to their high-UQCRH counterparts. Although no significant difference was detected in cell proliferation in vitro, low-UQCRH tumors grew faster in vivo. Ketogenic diet experiments indicated that high UQCRH expression was positively correlated with more efficient beta-hydroxybutyrate metabolism and cellular proliferation. Beta-hydroxybutyrate was toxic to low-UQCRH sublines. Conclusion: Hypermethylation induced UQCRH downregulation in KMRC-2. Cells with lower UQCRH levels exhibited weaker mitochondrial membrane potential and were more reliant on aerobic glycolysis. While this metabolic reprogramming did not affect proliferation in vitro, low-UQCRH tumors grew faster in vivo. The metabolic shift also represents a vulnerability that could be targeted in the development of therapies to hinder tumor progression. The ketogenic diet showed promise as such a strategy. Citation Format: Yanting Luo, Louise Medina Bengtsson, Xin Lu. Metabolic vulnerability by UQCRH methylation in clear cell renal cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4795.

P0982PTEN-INDUCED KINASE 1 (PINK1) DEPLETION ALTERS MITOCHONDRIAL EFFICIENCY AND GLYCOLYSIS IN HUMAN PODOCYTES

Abstract Background and Aims Podocytes are terminally differentiated cells, which constitute an inner layer of the renal filtration barrier. Podocytes are characterized by high metabolic activity, and their elevated energy requirements are met by maintaining the appropriate mitochondrial number and quality, which depend on mitochondrial biogenesis and mitophagy. Alteration of mitochondrial dynamics is linked to the development of insulin resistance and diabetes. The main goal of the research was to determine the role of mitophagy in podocytes bioenergetics and to elucidate the effects of hyperglycemia on mitochondrial dynamics. Method In order to inhibit mitophagy, we generated a human podocyte cell line stably expressing PINK1 shRNA through lentiviral transduction. Biochemical analyses were performed to assess the oxidative phosphorylation efficiency (by measurement of oxygen consumption rate; OCR) and glycolysis contribution to the total cell energy production (by measurement of extracellular acidification rate; ECAR) in the differentiated shPINK1 podocytes. The expression levels of mRNAs and proteins were evaluated in podocytes cultured in standard glucose (11 mM) and high glucose (30 mM) concentrations using real-time PCR and western blot. Intracellular mitochondrial network was visualized by MitoTrackertTM staining. The co-localization of proteins in podocytes was analysed by double immunofluorescence labelling and confocal microscopy. Results PINK1-deficiency resulted in the significant decrease of maximal respiration and spare respiratory capacity in podocytes (by 40% and 70%, respectively). Non-mitochondrial respiration was also decreased by 45% in shPINK1 cells. Interestingly, basal respiration and ATP production appeared similar in shPINK1 and control podocytes. PINK1 depletion increased glycolytic flux by 70%, in addition, the accompanying decline in glycolytic capacity and glycolytic reserve was observed (by 38% and 63%, respectively). Moreover, we observed accumulation of small and ring-shaped mitochondria in PINK1-deficient podocytes compared with the control cells. We showed a decreased expression of PINK1 and Parkin (mRNA and protein) in normal human podocytes cultured in hyperglycemic medium, which was associated with an elevated levels of mitochondrial fission markers (DRP1, FIS1) and with a decreased levels of PGC1α and TFAM, which play a role in mitochondrial biogenesis and mtDNA replication. Conclusion In this research, we demonstrated a novel role of mitophagy in podocyte bioenergetics. Moreover, we showed that high glucose inhibits mitophagy and promotes mitochondrial fission leading to the accumulation of damaged mitochondria and podocyte injury, which underlies the pathogenesis of diabetic nephropathy.

Respiratory Mitochondrial Efficiency and DNA Oxidation in Human Sperm after In Vitro Myo-Inositol Treatment.

Semen samples are known to contain abnormal amounts of reactive oxygen species (ROS) and oxygen free radicals; therefore, the identification of antioxidant molecules able to counteract the oxidative damage caused by ROS is foresight. Indeed, improving semen quality in terms of motility and reduction in DNA damage, can significantly improve the fertilization potential of sperm in vitro. To this regard, myo-inositol, based on its antioxidant properties, has been reported to be effective in improving sperm quality and motility in oligoasthenozoospermic patients undergoing assisted reproduction techniques when used as a dietary supplementation. Moreover, in vitro treatment demonstrated a direct relationship between myo-inositol, mitochondrial membrane potential and sperm motility. This experimental study aimed to evaluate the effects of myo-inositol (Andrositol-lab) in vitro treatment on sperm motility, capacitation, mitochondrial oxidative phosphorylation and DNA damage. Our results demonstrate that myo-inositol induces a significant increase in sperm motility and in oxygen consumption, the main index of oxidative phosphorylation efficiency and ATP production, both in basal and in in vitro capacitated samples. Moreover, we provide evidence for a significant protective role of myo-inositol against oxidative damage to DNA, thus supporting the in vitro use of myo-inositol in assisted reproductive techniques. Even if further studies are needed to clarify the mechanisms underlying the antioxidant properties of myo-inositol, the present findings significantly extend our knowledge on human male fertility and pave the way to the definition of evidence-based guidelines, aiming to improve the in vitro procedure currently used in ART laboratory for sperm selection.

Energy metabolism and oxidative status of rat liver mitochondria in conditions of experimentally induced hyperthyroidism

The aim of the present work was to investigate the energy metabolism and antioxidant status of rat liver mitochondria using a model of hyperthyroidism. In experimental animals, the level of triiodothyronine and thyroxine was increased 3- and 4-fold, respectively, in comparison with that in the control group, indicating the development of hyperthyroidism in these animals. Oxygen consumption was found to be higher in rats with experimentally induced hyperthyroidism (from 20 to 60% depending on the experimental scheme used), with a slight decrease in the efficiency of oxidative phosphorylation and respiratory state ratio. It was shown for the first time that the level the respiratory complexes of the electron transport chain in hyperthyroid rats increased; however, the quantity of complexes III and V changed unreliably. The assay of respiratory chain enzymes revealed that the activities of complexes I, II, and citrate synthase increased, whereas the activities complexes II + III, III, IV decreased in liver mitochondria of the experimental animals. Alterations in the oxidative state in liver mitochondria were found: a 60% increase in the hydrogen peroxide production rate and a 45% increase in lipid peroxidation. The activities of superoxide dismutase and catalase in the liver of experimental rats were higher than in the control. At the same time, the activity of glutathione peroxidase did not change. The data obtained indicate that the known activation of metabolism and changes in the oxidative status in thyrotoxicosis are associated with variations in the respiratory chain functioning and the antioxidant enzymes of mitochondria.

Mitochondrial impairment and cytotoxicity effects induced by the marine epibenthic dinoflagellate Coolia malayensis

Mitochondria was used to clarify the effects of Coolia malayensis strain UNR-02 crude extract by studying mitochondrial membrane potential (ΔΨm) generation and the fluctuations of ΔΨm associated with the induction of mitochondrial permeability transition (MPT). The cytoxicity of C. malayensis was also determined using both HepG2 and H9c2(2−1) cells. C. malayensis extract significantly depressed the oxidative phosphorylation efficiency, as was inferred from the perturbations in ΔΨm and in the phosphorylative cycle induced by ADP. Increased susceptibility to Ca2+-induced MPT was also observed. At the cellular level, the extract significantly decreased cell mass of both cell lines.

Mitochondrial Costs of Being Hot: Effects of Acute Thermal Change on Liver Bioenergetics in Toads (Bufo bufo).

Global climatic warming is predicted to drive extreme thermal events, especially in temperate terrestrial environments. Hence, describing how physiological parameters are affected by acute temperature changes would allow us to understand the energy management of organisms facing such non-predictable and constraining events. As mitochondria play a key role in the conversion of energy from food into ATP but also produce harmful reactive oxygen species, the understanding of its functioning is crucial to determine the proximal causes of potential decline in an animal’s performance. Here we studied the effects of acute temperature changes (between 20 and 30°C) on mitochondrial respiration, ATP synthesis rate, oxidative phosphorylation efficiency (ATP/O), and H2O2 generation in isolated liver mitochondria of a terrestrial ectotherm, the common toad (Bufo bufo). Using succinate as the respiratory substrate, we found that the mitochondrial rates of oxygen consumption, ATP synthesis, and H2O2 generation increased as the temperature increased, being 65, 52, and 66% higher at 30°C than at 20°C, respectively. We also found that the mitochondrial coupling efficiency (ATP/O) decreased, while the oxidative cost of ATP production (H2O2/ATP ratio) increased. The present results further indicate that between 40 and 60% of temperature effects on mitochondrial ATP production and H2O2 generation was at minima driven by an action on the oxidative capacity of the mitochondria. These results suggest that B. bufo may need to allocate extra energy to maintain ATP production and protect cells from oxidative stress, reducing the energy allocable performances.