Uncoupling Of Phosphorylation Research Articles

Biochemical basis and therapeutic potential of mitochondrial uncoupling in cardiometabolic syndrome.

Mild uncoupling of oxidative phosphorylation is an intrinsic property of all mitochondria, allowing for adjustments in cellular energy metabolism to maintain metabolic homeostasis. Small molecule uncouplers have been extensively studied for their potential to increase metabolic rate, and recent research has focused on developing safe and effective mitochondrial uncoupling agents for the treatment of obesity and cardiometabolic syndrome (CMS). Here, we provide a brief overview of CMS and cover the recent mechanisms by which chemical uncouplers regulate CMS-associated risk-factors and comorbidities, including dyslipidemia, insulin resistance, steatotic liver disease, type 2 diabetes, and atherosclerosis. Additionally, we review the current landscape of uncoupling agents, focusing on repurposed FDA-approved drugs and compounds in advanced preclinical or early-stage clinical development. Lastly, we discuss recent molecular insights by which chemical uncouplers enhance cellular energy expenditure, highlighting their potential as a new addition to the current CMS drug landscape, and outline several limitations that need to be addressed before these agents can successfully be introduced into clinical practice.

Uncoupling as initiating event in mitochondrial dysfunction after diuron exposure

Diuron, a herbicide derived from urea, has been shown to induce urinary bladder urothelial tumors in rodents, leading the U.S. Environmental Protection Agency (USEPA) to designate it as a ‘known/likely’ human carcinogen. In our laboratory, a series of studies investigating the carcinogenic mode of action (MoA) of Diuron have consistently revealed its cytotoxic effects on the urinary bladder urothelium. Prolonged exposure to relatively high doses of Diuron results in urothelial necrosis, regenerative hyperplasia, and eventually, the development of tumors. The hypothesis posited is that Diuron and its metabolites exert toxicity by causing damage to mitochondria, a phenomenon referred to as mitotoxicity. Our research focuses on evaluating how Diuron and its metabolites affect mitochondria isolated from both the urothelium and the liver, the primary organ for Diuron biotransformation. In this context, we present and discuss data pertaining to mitochondria isolated from the liver of Wistar rats exposed to Diuron or its metabolites 3-(3,4-diclorofenil)-1-metilureia (DCPMU) or 3,4-dichloroaniline (DCA) at concentrations ranging from 0.5 to 500 µM in vitro. The findings indicate that, at concentrations of 100 and 500 µM, the tested chemicals induce uncoupling of oxidative phosphorylation, as evidenced by the dissipation of mitochondrial membrane potential and basal oxygen consumption. Notably, at 500 µM, DCA causes mitochondrial swelling, a morphofunctional indicator of severe organelle damage. These outcomes underscore the classification of Diuron and its metabolites, DCA and DCPMU, as mitotoxic to liver cells, given the pronounced mitochondrial dysfunction they induce.

Mitochondria dysfunction is one of the causes of diclofenac toxicity in the green alga Chlamydomonas reinhardtii.

Non-steroidal anti-inflammatory drugs (NSAIDs), such as diclofenac (DCF), form a significant group of environmental contaminants. When the toxic effects of DCF on plants are analyzed, authors often focus on photosynthesis, while mitochondrial respiration is usually overlooked. Therefore, an in vivo investigation of plant mitochondria functioning under DCF treatment is needed. In the present work, we decided to use the green alga Chlamydomonas reinhardtii as a model organism. Synchronous cultures of Chlamydomonas reinhardtii strain CC-1690 were treated with DCF at a concentration of 135.5 mg × L-1, corresponding to the toxicological value EC50/24. To assess the effects of short-term exposure to DCF on mitochondrial activity, oxygen consumption rate, mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (mtROS) production were analyzed. To inhibit cytochrome c oxidase or alternative oxidase activity, potassium cyanide (KCN) or salicylhydroxamic acid (SHAM) were used, respectively. Moreover, the cell's structure organization was analyzed using confocal microscopy and transmission electron microscopy. The results indicate that short-term exposure to DCF leads to an increase in oxygen consumption rate, accompanied by low MMP and reduced mtROS production by the cells in the treated populations as compared to control ones. These observations suggest an uncoupling of oxidative phosphorylation due to the disruption of mitochondrial membranes, which is consistent with the malformations in mitochondrial structures observed in electron micrographs, such as elongation, irregular forms, and degraded cristae, potentially indicating mitochondrial swelling or hyper-fission. The assumption about non-specific DCF action is further supported by comparing mitochondrial parameters in DCF-treated cells to the same parameters in cells treated with selective respiratory inhibitors: no similarities were found between the experimental variants. The results obtained in this work suggest that DCF strongly affects cells that experience mild metabolic or developmental disorders, not revealed under control conditions, while more vital cells are affected only slightly, as it was already indicated in literature. In the cells suffering from DCF treatment, the drug influence on mitochondria functioning in a non-specific way, destroying the structure of mitochondrial membranes. This primary effect probably led to the mitochondrial inner membrane permeability transition and the uncoupling of oxidative phosphorylation. It can be assumed that mitochondrial dysfunction is an important factor in DCF phytotoxicity. Because studies of the effects of NSAIDs on the functioning of plant mitochondria are relatively scarce, the present work is an important contribution to the elucidation of the mechanism of NSAID toxicity toward non-target plant organisms.

P-171 Mitochondrial complex I, a key component of the mitochondrial respiratory chain, is necessary for maintaining chromosomal stability during the final stages of oocyte maturation

Abstract Study question What is the role of mitochondrial complex I during the final stages of human oocyte maturation? Summary answer Mitochondrial complex I is necessary for the cytoplasmic maturation of germinal vesicle (GV) oocytes and the subsequent organization of the meiotic spindle. What is known already Mitochondria play a pivotal role in oogenesis. Primordial human oocytes are characterized by an abundance of immature mitochondria, which exhibit low activity and a diminished capacity to produce adenosine triphosphate (ATP). During oocyte recruitment, mitochondrial fusion and fission play an essential role, contributing to oocyte maturation and oocyte developmental competence. Furthermore, an increase in ATP levels during polar body extrusion has been associated with higher fertilization rates, suggesting a reliance on mitochondria as ATP suppliers during oocyte maturation. However, the precise role of mitochondria during the final stages of human oocyte maturation is yet to be understood. Study design, size, duration At total of 99 immature oocytes, including 56 GVs and 40 metaphase I (MI) oocytes, were included in the study. These were obtained from 40 oocyte donation cycles, performed at a single IVF centre between August 2023 and September 2023. Participants/materials, setting, methods Immature denuded oocytes were cultured in G2TM PLUS medium to reach nuclear maturation (rescue in vitro maturation; rIVM). rIVM was performed in the absence (0.1% DMSO, control) or presence of 0.5 µM Rotenone (an inhibitor of mitochondrial complex I) or 10 µM FCCP (an uncoupler of mitochondrial oxidative phosphorylation). To assess mitochondrial and nuclear status, we performed immunofluorescence analyses for TOMM20 and the meiotic spindle (DNA and TUBA). Main results and the role of chance After 40 hours of culture, 64.3% of control GV oocytes progressed to the MII stage, 7.1% reached MI, while 21.4% arrested, and 7.1% degenerated. Control MI oocytes showed a 90% rIVM rate after 24 hours, with none arresting and 10% degenerating. Different mitochondrial inhibitors had varied effects on GV and MI oocytes. FCCP resulted in the degeneration of all treated oocytes. When treated with rotenone, 87.5% of GVs advanced to the MI stage, after which they arrested, but no signs of degeneration were observed. Notably, 100% of MIs and MIIs derived from GVs subjected to rotenone treatment presented with misaligned chromosomes, compared to 33.3% in the control group. When starting from MIs, the rIVM rate decreased to 41.7% following rotenone treatment, with no signs of degeneration. Here, misalignments were observed in 40% of the rIVM-MII oocytes and 43% of those that arrested, compared to 33.3% in controls. These results suggest that both GV and MI oocytes have adequate complex I activity, essential for proper nuclear maturation at the MII stage. We further highlight the importance of complex I activity, especially in GVs, for processes such as polar body extrusion during oocyte maturation. Limitations, reasons for caution Rescue in vitro maturation poses a challenge due to its limited effectiveness in achieving sufficient MII developmental competence. Consequently, mitochondrial inhibitors may have unforeseen effects on mitochondria and spindle formation. The specific role of complex I should be confirmed by employing other complex I inhibitors not related to rotenone. Wider implications of the findings Our results suggest that complex I activity, a key component of the mitochondrial respiratory chain, plays a crucial role in spindle organization during oocyte maturation. This relationship between mitochondrial function and chromosomal stability in oocytes suggests that interventions targeting the mitochondrial respiratory chain may be promising for enhancing oocyte quality. Trial registration number Not applicable

Chlorfenapyr poisoning: a systematic review

Introduction Chlorfenapyr, a N-substituted halogenated pyrrole, is a broad-spectrum insecticide. The insecticidal activity of chlorfenapyr depends on its biotransformation by hepatic cytochrome P450 monooxygenases to tralopyril, which uncouples mitochondrial oxidative phosphorylation and disrupts adenosine triphosphate production. Neither the metabolism of chlorfenapyr nor the mechanism of tralopyril is completely elucidated. Acute human chlorfenapyr poisoning is not well characterized, and best practice in management following acute exposure is unclear. The purpose of this review is to characterize acute human chlorfenapyr poisoning by its clinical course, laboratory investigations, and imaging findings and propose a management plan for acute human chlorfenapyr exposure. Methods We systematically searched PubMed, Web of Science, Google Scholar, and EMBASE from inception to April 2024 across all languages for human chlorfenapyr and tralopyril cases, with descriptions of exposure, clinical manifestations, and clinical course included. Only manuscripts and abstracts from scientific conferences with sufficient clinical data following acute human exposures were included. In vitro studies, animal studies, agricultural studies, environmental impact studies, and non-clinical human studies were excluded. We then reviewed citations of included studies for additional eligible publications. Non-English publications were translated using Google Translate or primarily translated by our authors. The study adhered to Preferred Reporting for Systematic Reviews and Meta-analyses (PRISMA) guidelines for systematic reviews. Results We identified 3,376 publications of which 48 met study inclusion criteria, describing 75 unique cases of human poisoning from ingestion, inhalation, dermal exposure, and intra-abdominal injection of chlorfenapyr. No cases of tralopyril exposure were identified. The median time from exposure to symptom onset was six hours (interquartile range 1–48 hours). The most frequent initial or presenting signs/symptoms included diaphoresis, nausea and/or vomiting, and altered mental status. While hyperthermia (≥38 degrees centigrade) was less common at presentation, hyperthermia developed in 61 percent of all patients and was temporally associated with clinical deterioration and death. Most common laboratory abnormalities included elevated blood creatine kinase activity, hepatic aminotransferase activities, and lactate concentration. Imaging studies of the central nervous system often showed extensive symmetrical white matter abnormalities with swelling. Case fatality was 76 percent, and survivors commonly experienced sustained neurological sequelae. Management strategies were highly varied, and the effectiveness of specific medical interventions was unclear. Discussion Acute human chlorfenapyr poisoning is characterized by a latent period as long as 14 days, deterioration over hours to days, and can result in serious morbidity and mortality. Development of hyperthermia, likely driven by oxidative phosphorylation uncoupling by tralopyril, is an ominous clinical sign and is temporally associated with clinical decompensation and death. Laboratory abnormalities, particularly elevated creatine kinase activity, hepatic aminotransferase activities, and lactate concentration, were common, but only creatine kinase activity differed amongst survivors and fatalities. Best clinical practice in the management of patients exposed to chlorfenapyr is unclear, and we opine that a conservative approach with close clinical monitoring and supportive care is prudent. Limitations The limitations of all reviews include their inherent retrospective and observational nature as well as publication bias that emphasizes severe outcomes, thus impacting the spectrum of illness and skewing mortality percentage. In addition, we interrogated a finite number of databases for publications on human chlorfenapyr exposure and there were limited cases with laboratory testing to confirm chlorfenapyr poisoning. Analysis of our systematic review was not powered to detect differences between groups, comparative statistics were not performed, and significance is not reported. Conclusions Acute human chlorfenapyr toxicity is characterized by a latent period following exposure, development of new or progression of established signs/symptoms, potential for critical illness, rapid deterioration, serious morbidity, and mortality. A conservative approach to patient management is prudent.

Theoretical and Experimental Study of the Interaction of Protonophore Uncouplers and Decoupling Agents with Functionally Active Mitochondria.

The purpose of this work was to quantitatively characterize the effectiveness of oxidative phosphorylation uncouplers and decoupling agents in functionally active mitochondria, taking into account their content in the hydrophobic region of the inner membrane of these organelles. When conducting theoretical studies, it is accepted that uncouplers and decouplers occupy part of the volume of mitochondria to exhibit their activity, which is defined as the effective volume. The following quantities characterizing the action of these reagents are considered: (1) concentrations of reagents that cause double stimulation of mitochondrial respiration in state 4 ( ); (2) effective distribution coefficient ( ) - the ratio of the amount of reagents in the effective volume of mitochondria and the water volume; (3) the relative amount of reagents associated with the effective volume of mitochondria ( ); (4) specific activity of reagents localized in the effective volume of mitochondria ( ). We have developed methods for determining these values, based on an analysis of the dependence of the rate of mitochondrial respiration on the concentration of uncouplers and decoupling agents at two different concentrations of mitochondrial protein in the incubation medium. During experimental studies, we compared the effects of the classical protonophore uncouplers 2,4-dinitrophenol (DNP) and сarbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), the natural uncouplers lauric and palmitic acids, and the natural decouplers α,ω-tetradecanedioic (TDA) and α,ω-hexadecanedioic (HDA) acids that differ both in the structure of the molecule and in the degree of solubility in lipids. Using the developed methods, we have clarified the dependence of the degree of activity of these uncouplers and decoupling agents on the distribution of their molecules between the effective volume of mitochondria and the water volume.

Influence of the oxidative phosphorylation uncoupler m-chlorophenol on activated sludge production, nutrients removal efficiency and bacterial community structure of a membrane reactor system

In this work, the effects of the metabolic uncoupler meta-chlorophenol (m-CP) on excess sludge production and performance of an MBR system, as well as on activated sludge communities, were investigated. MLVSS were significantly reduced from 8410 mg/L to 5175 mg/L during the first week of the addition of 125 mg/L m-CP, while, thereafter, biosolids were restored to the level prior to uncoupler’s addition. Adding m-CP resulted in more severe effects on nitrifiers rather than organoheterotrophs. Both alkalinity, dissolved oxygen (DO), electrical conductivity (EC) and pH data evidenced the adverse effect of m-CP on the nitrification process. PO43--P concentration drastically raised immediately after m-CP addition from 2.48 ± 0.35 mg/L to 11.84 mg/L, possibly due to the uncoupling of P-release and the hydrolysis of phosphorus reserves. Diversity was highly affected by the addition of m-CP, resulting in the significant decrease of Chao1, Shannon and Fisher indices. By establishing stable operating conditions after m-CP addition, Pseudomonas, Arcanobacterium, Trichococcus, Kocuria and Actinobaculum raised from almost undetectable levels (0.12 % of the total relative abundance) to 35.9 %, 17.8 %, 15.1 %, 11.5 % and 5.7 %, respectively, indicating a massive shift in bacterial community structure, while no nitrifying taxa were detected after m-CP addition.

Insulin reduces endoplasmic reticulum stress-induced apoptosis by decreasing mitochondrial hyperpolarization and caspase-12 in INS-1 pancreatic β-cells.

Pancreatic β-cell mass is a critical determinant of insulin secretion. Severe endoplasmic reticulum (ER) stress causes β-cell apoptosis; however, the mechanisms of progression and suppression are not yet fully understood. Here, we report that the autocrine/paracrine function of insulin reduces ER stress-induced β-cell apoptosis. Insulin reduced the ER-stress inducer tunicamycin- and thapsigargin-induced cell viability loss due to apoptosis in INS-1 β-cells. Moreover, the effect of insulin was greater than that of insulin-like growth factor-1 at physiologically relevant concentrations. Insulin did not attenuate the ER stress-induced increase in unfolded protein response genes. ER stress did not induce cytochrome c release from mitochondria. Mitochondrial hyperpolarization was induced by ER stress and prevented by insulin. The protonophore/mitochondrial oxidative phosphorylation uncoupler, but not the antioxidants N-acetylcysteine and α-tocopherol, exhibited potential cytoprotection during ER stress. Both procaspase-12 and cleaved caspase-12 levels increased under ER stress. The caspase-12 inhibitor Z-ATAD-FMK decreased ER stress-induced apoptosis. Caspase-12 overexpression reduced cell viability, which was diminished in the presence of insulin. Insulin decreased caspase-12 levels at the post-translational stages. These results demonstrate that insulin protects against ER stress-induced β-cell apoptosis in this cell line. Furthermore, mitochondrial hyperpolarization and increased caspase-12 levels are involved in ER stress-induced and insulin-suppressed β-cell apoptosis.

Dynamic Shift Towards Increased O2 Consumption and Reduced Lactate Uptake During Acute Volume Expansion in Sprague Dawley (SD) Rats

Rationale: In a recent study (PMID: 37575482), we established a method to assess alterations in O2 and substance metabolism in the kidneys in conscious free moving rat. This involved the repeated collection of renal arteriovenous blood and urine, alongside continuous measurements (24/7) of renal blood flow (RBF) and blood pressure (BP). Our previous studies demonstrated that in SD rats, there is an augmentation in RBF and GFR following chronic salt loading. Additionally, total renal O2 consumption (VO2) increases while there is a decrease in the net consumption of lactate with a tendency for its increased production. We hypothesize that these changes are not exclusive to the chronic phase of salt-loading but can also manifest during rapid increases of RBF and GFR. The primary aim of this study was to establish an experimental model reflecting these dynamics. Methods: SD rat (male, 10 wk age fed 0.4 NaCl (LS) diet, n=8) were studied. The jugular vein and femoral vein and artery were catheterized together with the renal vein. FITC-inulin (2 mg/mL) dissolved in 2%BSA saline was infused at 1.0 mL/100 g body wt/h from jugular vein. RBF was measured with an ultrasonic flow probe (Transonic, Inc) on the left renal artery and BP was measured through an arterial catheter. Whole blood, collected from donor rats, was diluted by 20% with saline, and a 6 mL volume expansion was performed with this solution over 1 hour. Blood was collected from both the renal venous and arterial catheters and urine from a ureteral catheter before and after volume expansion. O2 content was measured by radiometer and plasma lactate measured with a high sensitivity L-lactate fluorescent assay kit (ab169557, abcam). The same experiment was performed without volume expansion (i.e., sham study). O2 and lactate delivery and consumption were calculated as follows: Delivery = arterial content (A) x RBF; Consumption = (A − renal venous content (RV)) x RBF; Extraction = (A-RV) / A. Results: BP increased from 101 ± 3 to 111 ± 4 mmHg (p<0.05), RBF increased from 5.4 ± 0.5 to 7.9 to 0.6 mL/min/g kidney weight (gkw) (p<0.05) by volume expansion. GFR increased from 0.94 ± 0.06 to 1.19 ± 0.08 mL/min/gkw (p<0.05). O2 delivery increased from 1.06 ± 0.09 to 1.61 ± 0.12 mL/min/gkw (p<0.05). O2 consumption (VO2) increased from 0.06 ± 0.01 to 0.08 ± 0.01 mL/min/gkw (p<0.05). The O2 extraction ratio remained unchanged. Both lactate consumption (0.94 ± 0.11 to 0.47 ± 0.11 mM/gkw, p<0.05) and extraction ratio (0.18 ± 0.01 to 0.11 ± 0.02, p<0.05) were reduced by volume expansion. Conclusion: The increase of O2 consumption and reduction of lactate consumption with acute volume expansion model appear to be similar to those that we observed when SD rats were subjected to a chronic step increase of salt intake (from 0.4 to 4.0% NaCl). In contrast, however, an increase in O2 extraction was not observed with acute volume expansion as was the case with a high salt diet in which our studies have indicated that a prolonged tubular reabsorptive workload leads to enhanced oxidative stress with an uncoupling of oxidative phosphorylation. R01 HL151587, AHA 23POST1008714. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

DGK5 phosphorylation finetunes PA homeostasis in plant immunity

Phosphatidic acid (PA) as a universal second messenger is transiently and rapidly produced upon immune activation in plants. A recent study by Kong et al. elucidated a mechanism for maintaining PA homeostasis via two uncoupled phosphorylation events of DIACYLGLYCEROL KINASE 5 (DGK5) at different phosphorylation sites by two distinct kinases.

Changes in energy metabolism of rat myocardium during exposure to ionizing radiation

Objective. To study the effect of incorporated 137Cs on the energy metabolism of the myocardium of white rats. Materials and methods. The work was carried out on 8 male white rats divided into 2 experimental groups, which were fed products contaminated with 137Cs for 7 days. Polarography was used to study the rate of oxygen consumption by myocardial tissue preparations on endogenous substrates, with the addition of exogenous succinic and glutamic acids, and with the addition of an uncoupler of oxidative phosphorylation, 2,4-dinitrophenol.Results. An increase in the rate of respiration on endogenous substrates caused by the uncoupling of oxidative phosphorylation was noted. At the same time, no statistically significant change in the amount of endogenous substrates was found. Conclusion. The revealed violation of energy production in the myocardium of experimental animals is caused by the uncoupling of oxidative phosphorylation, and can be explained by a change in the operation of potassium channels, which can explain the development of cardiovascular pathology under the action of ionizing radiation.

POSTTRANSLATIONAL MODIFICATIONS OF THE SULFHYDRYL GROUP OF THE CYSTEINE RESIDUE OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

This review considers the main types of oxidative posttranslational modi cations of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) targeting the sulfhydryl group of the catalytic cysteine residue Cys152. The highly reactive sulfhydryl group of Cys152 in the active centre of GAPDH undergoes oxidation and S-nitrosylation, leading to enzyme inactivation and destabilization. Upon reversible oxidation of the sulfhydryl group to form cysteine-sulfenic acid, the enzyme loses dehydrogenase activity, but gains the ability to catalyze the acyl-phosphatase reaction. Hydrolysis of the product of the dehydrogenase reaction, 1,3-diphosphoglycerate, under the action of the oxidized GAPDH leads to uncoupling of oxidation and phosphorylation at this stage of glycolysis. The action of nitric oxide results in S-nitrosylation of Cys152 GAPDH and the subsequent formation of cysteine-sulfenic acid due to hydrolysis of the S-NO-group. Data are presented on the relationship between S-nitrosylation of the catalytic Cys152 of GAPDH and its oxidation followed by S-glutathionylation of the enzyme at Cys152. The role of posttranslational modi cations of the sulfhydryl group of the catalytic cysteine residue in the regulation of enzyme activity, as well as the mechanisms ensuring the reversibility of such modi cations are discussed.

Divergent Roles of Escherichia Coli Encoded Lon Protease in Imparting Resistance to Uncouplers of Oxidative Phosphorylation: Roles of marA, rob, soxS and acrB.

Uncouplers of oxidative phosphorylation dissipate the proton gradient, causing lower ATP production. Bacteria encounter several non-classical uncouplers in the environment, leading to stress-induced adaptations. Here, we addressed the molecular mechanisms responsible for the effects of uncouplers in Escherichia coli. The expression and functions of genes involved in phenotypic antibiotic resistance were studied using three compounds: two strong uncouplers, i.e., Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 2,4-Dinitrophenol (DNP), and one moderate uncoupler, i.e., Sodium salicylate (NaSal). Quantitative expression studies demonstrated induction of transcripts encoding marA, soxS and acrB with NaSal and DNP, but not CCCP. Since MarA and SoxS are degraded by the Lon protease, we investigated the roles of Lon using a lon-deficient strain (Δlon). Compared to the wild-type strain, Δlon shows compromised growth upon exposure to NaSal or 2, 4-DNP. This sensitivity is dependent on marA but not rob and soxS. On the other hand, the Δlon strain shows enhanced growth in the presence of CCCP, which is dependent on acrB. Interestingly, NaSal and 2,4-DNP, but not CCCP, induce resistance to antibiotics, such as ciprofloxacin and tetracycline. This study addresses the effects of uncouplers and the roles of genes involved during bacterial growth and phenotypic antibiotic resistance. Strong uncouplers are often used to treat wastewater, and these results shed light on the possible mechanisms by which bacteria respond to uncouplers. Also, the rampant usage of some uncouplers to treat wastewater may lead to the development of antibiotic resistance.

Association of a high rate of glycolysis and the activity of the uncoupling of oxidation and phosphorylation in the blood cells of patients with late-stage knee osteoarthritis and the development of postoperative pain

Objective: to investigate the relationship between the expression of genes mediating cellular energy production and the development of chronic postoperative pain (CPP) after total knee arthroplasty (TKA) in patients with osteoarthritis (OA).Material and methods. Prior to TKA, the blood of 50 patients with stage III–IV knee OA and complaints of constant pain and joint dysfunction was analyzed. The control group consisted of 26 healthy individuals. Pain intensity was assessed using a visual analogue scale (VAS), a short BPI questionnaire and the WOMAC index, and the presence of neuropathic pain was assessed using the DN4 and PainDETECT questionnaires. The development of CPP was determined 3 and 6 months after TKA. Total RNA isolated from blood was used to determine the expression of PKM2, LDH, SDH, AMPKα, PDH, IDH, MDH and ATP synthase genes by real-time quantitative reverse transcriptase-polymerase chain reaction.Results and discussion. CPP ≥30 mm according to VAS was detected in 17 patients. Before TKA, the expression of all analyzed genes was significantly increased compared to that of the control group. However, there were no differences in clinical, pain-related and functional indicators in the analyzed group of patients with OA. Before surgery, patients who subsequently developed CPP had significantly higher expression of genes related to glycolysis (PKM2, LDH), Krebs cycle – KC (SDH) and master regulator of energy metabolism (AMPKα) than patients who were satisfied with the results of TKA. At the same time, no differences were found in the expression of PDH and other KC enzyme genes (IDH, MDH) and ATP synthase in patients with and without CPP.Conclusion. The development of CPP is associated with a higher rate of glycolysis and energy deficiency, presumably due to the higher uncoupling activity of oxidation and phosphorylation that can be observed before TKA.

The acute and chronic low-temperature stress responses in Porites lutea from a relatively high-latitude coral reef of the South China Sea

Relatively high-latitude coral reefs could be potential “refuges” for corals under climate change. One of the most important aspects limiting their availability as refuges is low-temperature stress. However, the mechanisms underlying the response of coral holobionts to low-temperature stress is unclear. In this study, we aimed to explore the underlying mechanisms by recording the maximum quantum yields of photosystem II (Fv/Fm) and transcriptome responses of Porites lutea under acute (1–2 weeks) and chronic (6–12 weeks) low-temperature stress at 20°C and 14°C. The P. lutea samples were collected from a relatively high-latitude coral reef in the South China Sea (109°00′–109°15′E and 21°00′–21°10′ N). The study suggested that: (1) Under acute low-temperature stress, the Fv/Fm of Symbiodiniaceae dropped by 64%, which was significantly higher than the 49% observed under chronic stress. Low-temperature stress inhibited photosystem II(PSII) functioning, with greater inhibition under acute stress. (2) Downregulation of sugar metabolism-related genes under low-temperature stress implied that the decrease in energy was due to obstruction of PSII. (3) Under low-temperature stress, calcification-related genes were downregulated in coral hosts, possibly because of energy deprivation caused by inhibited photosynthesis, Symbiodiniaceae expulsion, and oxidative phosphorylation uncoupling in mitochondria. (4) Acute low-temperature stress induced the upregulation of genes related to the TNF signaling pathway and endoplasmic reticulum stress, promoting apoptosis and coral bleaching. However, these phenomena were not observed during chronic stress, suggesting acclimation to chronic low-temperature stress and a greater survival pressure of acute low-temperature stress on coral holobionts. In conclusion, low-temperature stress inhibits Symbiodiniaceae PSII functioning, reducing energy production and affecting calcification in coral holobionts. Acute low-temperature stress is more threatening to coral holobionts than chronic stress.

Pyrazinoic acid, the active form of the anti-tuberculosis drug pyrazinamide, and aromatic carboxylic acid analogs are protonophores.

Pyrazinoic acid is the active form of pyrazinamide, a first-line antibiotic used to treat Mycobacterium tuberculosis infections. However, the mechanism of action of pyrazinoic acid remains a subject of debate, and alternatives to pyrazinamide in cases of resistance are not available. The work presented here demonstrates that pyrazinoic acid and known protonophores including salicylic acid, benzoic acid, and carbonyl cyanide m-chlorophenyl hydrazone all exhibit pH-dependent inhibition of mycobacterial growth activity over a physiologically relevant range of pH values. Other anti-tubercular drugs, including rifampin, isoniazid, bedaquiline, and p-aminosalicylic acid, do not exhibit similar pH-dependent growth-inhibitory activities. The growth inhibition curves of pyrazinoic, salicylic, benzoic, and picolinic acids, as well as carbonyl cyanide m-chlorophenyl hydrazone, all fit a quantitative structure-activity relationship (QSAR) derived from acid-base equilibria with R2 values > 0.95. The QSAR model indicates that growth inhibition relies solely on the concentration of the protonated forms of these weak acids (rather than the deprotonated forms). Moreover, pyrazinoic acid, salicylic acid, and carbonyl cyanide m-chlorophenyl hydrazone all caused acidification of the mycobacterial cytoplasm at concentrations that inhibit bacterial growth. Thus, it is concluded that pyrazinoic acid acts as an uncoupler of oxidative phosphorylation and that disruption of proton motive force is the primary mechanism of action of pyrazinoic acid rather than the inhibition of a classic enzyme activity.

The study of the protective effect of mitochondrial uncouplers during acute toxicity of the fungicide difenoconazole in different organs of mice.

Pesticides represent a serious problem for agricultural workers due to their neurotoxic effects. The aim of this study was to evaluate the ability of pharmacological oxidative phosphorylation uncouplers to reduce the effect of the difenoconazole fungicide on mitochondrial DNA (mtDNA) of various organs in mice. Injections of difenoconazole caused cognitive deficits in mice, and the protonophore 2,4-dinitrophenol (2,4-DNP) and Azur I (AzI), a demethylated metabolite of methylene blue (MB), prevented the deterioration of cognitive abilities in mice induced by difenoconazole. Difenoconazole increased the rate of reactive oxygen species (ROS) production, likely through inhibition of complex I of the mitochondrial respiratory chain. After intraperitoneal administration of difenoconazole lungs, testes and midbrain were most sensitive to the accumulation of mtDNA damage. In contrast, the cerebral cortex and hippocampus were not tolerant to the effects of difenoconazole. The protonophore 2,4-DNP reduced the rate of ROS formation and significantly reduced the amount of mtDNA damage caused by difenoconazole in the midbrain, and partially, in the lungs and testes. MB, an alternative electron carrier capable of bypassing inhibited complex I, had no effect on the effect of difenoconazole on mtDNA, while its metabolite AzI, a demethylated metabolite of MB, was able to protect the mtDNA of the midbrain and testes. Thus, mitochondria-targeted therapy is a promising approach to reduce pesticide toxicity for agricultural workers.

Mitochondrial permeability transition regulator, cyclophilin D, is transcriptionally activated by C/EBP during adipogenesis

Age-related bone loss is associated with decreased bone formation, increased bone resorption and accumulation of bone marrow fat. During aging, differentiation potential of bone marrow stromal (a.k.a. mesenchymal stem) cells (BMSCs) is shifted towards an adipogenic lineage and away from an osteogenic lineage. In aged bone tissue, we previously observed pathological opening of the mitochondrial permeability transition pore (MPTP) which leads to mitochondrial dysfunction, oxidative phosphorylation uncoupling, and cell death. Cyclophilin D (CypD) is a mitochondrial protein that facilitates opening of the MPTP. We found earlier that CypD is downregulated during osteogenesis of BMSCs leading to lower MPTP activity and, thus, protecting mitochondria from dysfunction. However, during adipogenesis, a fate alternative to osteogenesis, the regulation of mitochondrial function and CypD expression is still unclear. In this study, we observed that BMSCs have increased CypD expression and MPTP activity, activated glycolysis, and fragmented mitochondrial network during adipogenesis. Adipogenic C/EBPα acts as a transcriptional activator of expression of the CypD gene, Ppif, during this process. Inflammation-associated transcription factor NF-κB shows a synergistic effect with C/EBPα inducing Ppif expression. Overall, we demonstrated changes in mitochondrial morphology and function during adipogenesis. We also identified C/EBPα as a transcriptional activator of CypD. The synergistic activation of CypD by C/EBPα and the NF-κB p65 subunit during this process suggests a potential link between adipogenic signaling, inflammation, and MPTP gain-of-function, thus altering BMSC fate during aging.

Low-Dose Exposure of WS2 Nanosheets Induces Differential Apoptosis in Lung Epithelial Cells.

Escalating the production and application of tungsten disulfide (WS2) nanosheets inevitably increases environmental human exposure and warrants the necessity of studies to elucidate their biological impacts. Herein, we assessed the toxicity of WS2 nanosheets and focused on the impacts of low doses (≤10 μg/mL) on normal (BEAS-2B) and tumorigenic (A549) lung epithelial cells. The low doses, which approximate real-world exposures, were found to induce cell apoptosis, while doses ≥ 50 μg/mL cause necrosis. Focused studies on low-dose exposure to WS2 nanosheets revealed more details of the impacts on both cell lines, including reduction of cell metabolic activity, induction of lipid peroxidation in cell membranes, and uncoupling of mitochondrial oxidative phosphorylation that led to the loss of ATP production. These phenomena, along with the expression situations of a few key proteins involved in apoptosis, point toward the occurrence of mitochondria-dependent apoptotic signaling in exposed cells. Substantial differences in responses to WS2 exposure between normal and tumorigenic lung epithelial cells were noticed as well. Specifically, BEAS-2B cells experienced more adverse effects and took up more nanosheets than A549 cells. Our results highlight the importance of dose and cell model selection in the assessment of nanotoxicity. By using doses consistent with real-world exposures and comparing normal and diseased cells, we can gain knowledge to guide the development of safety precautions for mitigating the adverse impacts of nanomaterial exposure on human health.

Mitochondria-targeted Uncouplers Decrease Inflammatory Reactions in Endothelial Cells by Enhancing Methylation of the ICAM1 Gene Promoter.

The study aimed to investigate the effects of low concentrations of mitochondrial uncouplers in endothelial cells on the CpG dinucleotide methylation of the ICAM1 gene promoter. The excessive inflammatory response in the endothelium is responsible for the development of many cardiovascular diseases. Mitochondria are important regulators of endothelial cell functions. Mild uncoupling of oxidative phosphorylation and respiration in endothelial mitochondria exerts a long lasting anti-inflammatory effect. However, the detailed mechanism of the anti-inflammatory activity of mitochondrial uncouplers remains unclear.We hypothesized that mild mitochondrial uncoupling leads to epigenetic changes in genomic DNA contributing to the anti-inflammatory response. We studied the long-term effects of mitochondria-targeted compounds with the uncoupler's activities: the antioxidant plastoquinonyl-decyltriphenylphosphonium (SkQ1), dodecyl-triphenylphosphonium (C12TPP), and 2,4-dinitrophenol (DNP). The mRNA expression of the intercellular adhesion molecule 1 (ICAM1), a marker of inflammatory activation of endothelial cells, was measured by RT-qPCR. Cytosine methylation in the CpG sites of the ICAM1 gene promoter was estimated by bisulfite sequencing of individual clones. It was found that downregulation of ICAM1 expression caused by DNP and C12TPP was accompanied by an increase in the methylation of CpG sites in the ICAM1 gene promoter. None of the compounds affected intracellular or intramitochondrial ATP levels. Low concentrations of mitochondrial oxidative phosphorylation uncouplers are able to increase methylation of ICAM1 gene promoter, which corresponds to the observed decrease in the levels of mRNA of this gene. Thus, the change in methylation of the ICAM1 gene promoter may underlie the mechanism of decreased ICAM1 expression caused by mild mitochondrial depolarization. Mitochondrial uncouplers may be exploited as possible therapeutic candidates to treat excessive inflammation in endothelium, by changing the methylation status of genomic DNA.