Mitochondrial control of hypoxia-induced pathological retinal angiogenesis.

OBJECTIVE: Pathological retinal neovascularization is vision-threatening. In mouse oxygen-induced retinopathy (OIR) we sought to define mitochondrial respiration changes longitudinally during hyperoxia-induced vessel loss and hypoxia-induced neovascularization, and to test interventions addressing those changes to prevent neovascularization. METHODS: OIR was induced in C57BL/6J mice and retinal vasculature was examined at maximum neovessel formation. We assessed total proteome changes and the ratio of mitochondrial to nuclear DNA copy numbers (mtDNA/nDNA) of OIR vs. control retinas, and mitochondrial oxygen consumption rates (OCR) in ex vivo OIR vs. control retinas (BaroFuse). Pyruvate vs. vehicle control was supplemented to OIR mice either prior to or during neovessel formation. RESULTS: In OIR vs. control retinas, global proteomics showed decreased retinal mitochondrial respiration at peak neovascularization. OCR and mtDNA/nDNA were also decreased at peak neovascularization suggesting impaired mitochondrial respiration. In vivo pyruvate administration during but not prior to neovessel formation (in line with mitochondrial activity time course) suppressed NV. CONCLUSIONS: Mitochondrial energetics were suppressed during retinal NV in OIR. Appropriately timed supplementation of pyruvate may be a novel approach in neovascular retinal diseases.

Harmonization of experimental procedures to assess mitochondrial respiration in human permeabilized skeletal muscle fibers.

AIM: High-resolution respirometry in human permeabilized muscle fibers is extensively used for analysis of mitochondrial adaptions to nutrition and exercise interventions, and is linked to athletic performance. However, the lack of standardization of experimental conditions limits quantitative inter- and intra-laboratory comparisons. METHODS: In our study, an international team of investigators measured mitochondrial respiration of permeabilized muscle fibers obtained from three biopsies (vastus lateralis) from the same healthy volunteer to avoid inter-individual variability. High-resolution respirometry assays were performed together at the same laboratory to assess whether the heterogenity in published results are due to the effects of respiration media (MiR05 versus Z) with or without the myosin inhibitor blebbistatin at low- and high-oxygen regimes. RESULTS: Our findings reveal significant differences between respiration media for OXPHOS and ET capacities supported by NADH&succinate-linked substrates at different oxygen concentrations. Respiratory capacities were approximately 1.5-fold higher in MiR05 at high-oxygen regimes compared to medium Z near air saturation. The presence or absence of blebbistatin in human permeabilized muscle fiber preparations was without effect on oxygen flux. CONCLUSION: Our study constitutes a basis to harmonize and establish optimum experimental conditions for respirometric studies of permeabilized human skeletal muscle fibers to improve reproducibility.

Elucidation of the Role of L-Arginine and Nω-Nitro-L-Arginine in the Treatment of Rats with Different Levels of Hypoxic Tolerance and Exposure to Lead Nitrate.

BACKGROUND/AIMS: Individual resistance to hypoxia is an important feature of the physiological profile of an organism, particularly in relation to lead-induced toxicity. METHODS: Our study focused on evaluating parameters of mitochondrial oxygen consumption, microsomal oxidation, intensity of lipoperoxidation processes and antioxidant defences in the liver of rats with low (LR) and high (HR) resistance to hypoxia to elucidate the mechanisms of action of L-arginine and the NO synthase inhibitor L-NNA before or after exposure to lead nitrate. RESULTS: Our study suggests that the redistribution of oxygen-dependent processes towards mitochondrial processes under the influence of the nitric oxide precursor amino acid L-arginine is an important mechanism for maintaining mitochondrial respiratory chain function during per os lead nitrate exposure (3.6 mg lead nitrate/kg bw per day for 30 days). Animals were given L-arginine at a dose of 600 mg/kg bw (i.p., 30 min) before and after exposure to lead nitrate or the NO synthase inhibitor Nω-nitro-L-arginine (L-NNA) at a dose of 35 mg/kg bw (i.p., 30 min) before and after exposure to lead nitrate. Our experiments demonstrated the efficacy of using lead nitrate to simulate lead-related toxic processes via Pb levels in liver tissue; we demonstrated significantly reduced levels of nitrites and nitrates, i.e. stable metabolites of the nitric oxide system, in both LR and HR animals. The effect of the amino acid L-arginine stabilised the negative effects of lead nitrate exposure in both groups of LR and HR rats. We observed the efficiency of mitochondrial energy supply processes and showed a greater vulnerability of NADH-dependent oxidation during lead nitrate exposure in the liver of HR rats. CONCLUSION: L-arginine initiated the processes of oxidation of NADH-dependent substrates in the LR group, whereas in the HR group this directionality of processes was more effective when the role of the nitric oxide system was reduced (use of L-NNA). Our study of key antioxidant enzyme activities in rat liver tissue during lead nitrate exposure revealed changes in the catalase-peroxidase activity ratio. We found different activities of antioxidant enzymes in the liver tissue of rats treated with lead nitrate and L-arginine or L-NNA, with a significant increase in GPx activity in the LR group when L-arginine was administered both before and after exposure to lead nitrate.

CCL2 and Lactate from Chemotherapeutics-Treated Fibroblasts Drive Malignant Traits by Metabolic Rewiring in Low-Migrating Breast Cancer Cell Lines.

While cytostatic chemotherapy targeting DNA is known to induce genotoxicity, leading to cell cycle arrest and cytokine secretion, the impact of these drugs on fibroblast-epithelial cancer cell communication and metabolism remains understudied. Our research focused on human breast fibroblast RMF-621 exposed to nonlethal concentrations of cisplatin and doxorubicin, revealing reduced proliferation, diminished basal and maximal mitochondrial respirations, heightened mitochondrial ROS and lactate production, and elevated MCT4 protein levels. Interestingly, RMF-621 cells enhanced glucose uptake, promoting lactate export. Breast cancer cells MCF-7 exposed to conditioned media (CM) from drug-treated stromal RMF-621 cells increased MCT1 protein levels, lactate-driven mitochondrial respiration, and a significantly high mitochondrial spare capacity for lactate. These changes occurred alongside altered mitochondrial respiration, mitochondrial membrane potential, and superoxide levels. Furthermore, CM with doxorubicin and cisplatin increased migratory capacity in MCF-7 cells, which was inhibited by MCT1 (BAY-8002), glutamate dehydrogenase (EGCG), mitochondrial pyruvate carrier (UK5099), and complex I (rotenone) inhibitors. A similar behavior was observed in T47-D and ZR-75-1 breast cancer cells. This suggests that CM induces metabolic rewiring involving elevated lactate uptake to sustain mitochondrial bioenergetics during migration. Treatment with the mitochondrial-targeting antioxidant mitoTEMPO in RMF-621 and the addition of an anti-CCL2 antibody in the CM prevented the promigratory MCF-7 phenotype. Similar effects were observed in THP1 monocyte cells, where CM increased monocyte recruitment. We propose that nonlethal concentrations of DNA-damaging drugs induce changes in the cellular environment favoring a promalignant state dependent on mitochondrial bioenergetics.

The Impact of Weight Loss on Inflammation, Oxidative Stress, and Mitochondrial Function in Subjects with Obesity.

Inflammation, oxidative stress, and mitochondrial function are implicated in the development of obesity and its comorbidities. The purpose of this study was to assess the impact of weight loss through calorie restriction on the metabolic profile, inflammatory and oxidative stress parameters, and mitochondrial respiration in an obese population. A total of 109 subjects underwent two cycles of a very low-calorie diet alternated with a low-calorie diet (24 weeks). We analyzed biochemical and inflammatory parameters in serum, as well as oxidative stress markers, mRNA antioxidant gene expression, and mitochondrial respiration in peripheral blood mononuclear cells (PBMCs). After the intervention, there was an improvement in both insulin resistance and lipid profiles, including cholesterol subfractions. Weight loss produced a significant reduction in mitochondrial ROSs content and an increase in glutathione levels, coupled with an enhancement in the mRNA expression of antioxidant systems (SOD1, GSR, and CAT). In addition, a significant improvement in basal oxygen consumption, maximal respiration, and ATP production was observed. These findings demonstrate that moderate weight loss can improve insulin resistance, lipid profiles and subfractions, inflammatory and oxidative stress parameters, and mitochondrial respiration. Therefore, we can affirm that dietary intervention can simultaneously achieve significant weight loss and improve metabolic profile and mitochondrial function in obesity.

Autonomous Oscillatory Mitochondrial Respiratory Activity: Results of a Systematic Analysis Show Heterogeneity in Different In Vitro-Synchronized Cancer Cells.

Circadian oscillations of several physiological and behavioral processes are an established process in all the organisms anticipating the geophysical changes recurring during the day. The time-keeping mechanism is controlled by a transcription translation feedback loop involving a set of well-characterized transcription factors. The synchronization of cells, controlled at the organismal level by a brain central clock, can be mimicked in vitro, pointing to the notion that all the cells are endowed with an autonomous time-keeping system. Metabolism undergoes circadian control, including the mitochondrial terminal catabolic pathways, culminating under aerobic conditions in the electron transfer to oxygen through the respiratory chain coupled to the ATP synthesis according to the oxidative phosphorylation chemiosmotic mechanism. In this study, we expanded upon previous isolated observations by utilizing multiple cell types, employing various synchronization protocols and different methodologies to measure mitochondrial oxygen consumption rates under conditions simulating various metabolic stressors. The results obtained clearly demonstrate that mitochondrial respiratory activity undergoes rhythmic oscillations in all tested cell types, regardless of their individual respiratory proficiency, indicating a phenomenon that can be generalized. However, notably, while primary cell types exhibited similar rhythmic respiratory profiles, cancer-derived cell lines displayed highly heterogeneous rhythmic changes. This observation confirms on the one hand the dysregulation of the circadian control of the oxidative metabolism observed in cancer, likely contributing to its development, and on the other hand underscores the necessity of personalized chronotherapy, which necessitates a detailed characterization of the cancer chronotype.

Gallic Acid Enhances the Efficacy of BCR::ABL1 Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia through Inhibition of Mitochondrial Respiration and Modulation of Oncogenic Signaling Pathways.

While BCR::ABL1 tyrosine kinase inhibitors have transformed the treatment paradigm for chronic myeloid leukemia (CML), disease progression and treatment resistance due to BCR::ABL1-dependent and BCR::ABL1-independent mechanisms remain a therapeutic challenge. Natural compounds derived from plants have significantly contributed to cancer pharmacotherapy. This study investigated the efficacy of an active component of Leea indica, a local medicinal plant, in CML. Using high-performance liquid chromatography-electrospray ionization-mass spectrometry, a chemical constituent from L. indica extract was isolated and identified as gallic acid. Commercially obtained gallic acid was used as a chemical standard. Gallic acid from L. indica inhibited proliferation and induced apoptosis in CML cell lines, as did the chemical standard. Furthermore, gallic acid induced apoptosis and decreased the colony formation of primary CML CD34+ cells. The combination of isolated gallic acid or its chemical standard with BCR::ABL1 tyrosine kinase inhibitors resulted in a significantly greater inhibition of colony formation and cell growth compared to a single drug alone. Mechanistically, CML cells treated with gallic acid exhibited the disruption of multiple oncogenic pathways including ERK/MAPK, FLT3 and JAK/STAT, as well as impaired mitochondrial respiration. Rescue studies showed that gallic acid is significantly less effective in inducing apoptosis in mitochondrial respiration-deficient ρ0 cells compared to wildtype cells, suggesting that the action of gallic acid is largely through the inhibition of mitochondrial respiration. Our findings highlight the therapeutic potential of L. indica in CML and suggest that gallic acid may be a promising lead chemical constituent for further development for CML treatment.

The exposure to extremely low frequency electromagnetic-fields inhibits the growth and potentiates the sensitivity to chemotherapy of bidimensional and tridimensional human osteosarcoma models.

We previously established a thermodynamical model to calculate the specific frequencies of extremely low frequency-electromagnetic field (ELF-EMF) able to arrest the growth of cancer cells. In the present study, for the first time, we investigated the efficacy of this technology on osteosarcoma, and we applied a precise frequency of the electromagnetic field on three human osteosarcoma cell lines, grown as adherent cells and spheroids. We evaluated the antitumour efficacy of irradiation in terms of response to chemotherapeutic treatments, which is usually poor in this type of cancer. Importantly, the results of this novel combinatorial approach revealed that the specific exposure can potentiate the efficacy of several chemotherapeutic drugs, both on bidimensional and tridimensional cancer models. The effectiveness of cisplatinum, methotrexate, ifosfamide and doxorubicin was greatly increased by the concomitant application of the specific ELF-EMF. Moreover, our experiments confirmed that ELF-EMF inhibited the proliferation and modulated the mitochondrial metabolism of all cancer models tested, whereas mesenchymal cells were not affected. The latter finding is extremely valuable, given the importance of preserving the cell reservoir necessary for tissue regeneration after chemotherapy. Altogether, this novel evidence opens new avenues to the clinical applications of ELF-EMF in oncology.

Lactate promotes fatty acid oxidation by the TCA cycle and mitochondrial respiration in muscles of obese mice.

Lower oxidative capacity in skeletal muscles (SKMs) is a prevailing cause of metabolic diseases. Exercise not only enhances the fatty acid oxidation (FAO) capacity of SKMs but also increases lactate levels. Given that lactate may contribute to tricarboxylic acid cycle (TCA) flux and impact monocarboxylate transporter 1 in the SKMs, we hypothesize that lactate can influence glucose and fatty acid (FA) metabolism. To test this hypothesis, we investigated the mechanism underlying lactate-driven FAO regulation in the SKM of mice with diet-induced obesity (DIO). Lactate was administered to DIO mice immediately after exercise over three weeks. We found that increased lactate levels enhanced energy expenditure mediated by fat metabolism during exercise recovery and decreased triglyceride levels in DIO mice SKMs. To determine the lactate-specific effects without exercise, we administered lactate to mice on a high-fat diet (HFD) for eight weeks. Similar to our exercise conditions, lactate increased FAO, TCA cycle activity, and mitochondrial respiration in the SKMs of HFD-fed mice. Additionally, under sufficient FA conditions, lactate increased uncoupling protein-3 abundance via the NADH/NAD+ shuttle. Conversely ATP synthase abundance decreased in the SKMs of HFD mice. Taken together, our results suggest that lactate amplifies the adaptive increase in FAO capacity mediated by the TCA cycle and mitochondrial respiration in SKMs under sufficient FA abundance.

Perturbations in mitochondrial metabolism associated with defective cardiolipin biosynthesis: An in-organello real-time NMR study.

Mitochondria are central to cellular metabolism; hence, their dysfunction contributes to a wide array of human diseases including cancer, cardiopathy, neurodegeneration, and heritable pathologies such as Barth syndrome. Cardiolipin, the signature phospholipid of the mitochondrion promotes proper cristae morphology, bioenergetic functions, and directly affects metabolic reactions carried out in mitochondrial membranes. To match tissue-specific metabolic demands, cardiolipin typically undergoes an acyl tail remodeling process with the final step carried out by the phospholipid-lysophospholipid transacylase tafazzin. Mutations in the tafazzin gene are the primary cause of Barth syndrome. Here, we investigated how defects in cardiolipin biosynthesis and remodeling impact metabolic flux through the tricarboxylic acid cycle and associated pathways in yeast. Nuclear magnetic resonance was used to monitor in real-time the metabolic fate of 13C3-pyruvate in isolated mitochondria from three isogenic yeast strains. We compared mitochondria from a wild-type strain to mitochondria from a Δtaz1 strain that lacks tafazzin and contains lower amounts of unremodeled cardiolipin, and mitochondria from a Δcrd1 strain that lacks cardiolipin synthase and cannot synthesize cardiolipin. We found that the 13C-label from the pyruvate substrate was distributed through about twelve metabolites. Several of the identified metabolites were specific to yeast pathways, including branched chain amino acids and fusel alcohol synthesis. Most metabolites showed similar kinetics amongst the different strains but mevalonate and α-ketoglutarate, as well as the NAD+/NADH couple measured in separate nuclear magnetic resonance experiments, showed pronounced differences. Taken together, the results show that cardiolipin remodeling influences pyruvate metabolism, tricarboxylic acid cycle flux, and the levels of mitochondrial nucleotides.

HOXC11-mediated regulation of mitochondrial function modulates chemoresistance in colorectal cancer.

BACKGROUND: Chemoresistance remains a significant challenge in colorectal cancer (CRC) treatment, necessitating a deeper understanding of its underlying mechanisms. HOXC11 has emerged as a potential regulator in various cancers, but its role in CRC chemoresistance remains unclear. METHODS: Sulforhodamine B assay was employed to assess the cell viability of CRC cells following treatment with chemotherapeutic drugs. Immunofluorescence staining was performed to examine the subcellular localization of HOXC11 in normal and chemoresistant CRC cells. The Seahorse mito stress test was conducted to evaluate the mitochondrial respiratory function of CRC cells. Real-time PCR was utilized to measure the expression level and copy number of mitochondrial DNA (mtDNA). RESULTS: Our findings revealed that HOXC11 was overexpressed in CRC cells compared to normal colorectal cells and correlated with poorer prognosis in CRC patients. Knockout of HOXC11 reversed acquired chemoresistance in CRC cells. Furthermore, we observed a functional subset of HOXC11 localized to the mitochondria in chemoresistant CRC cells, which regulated mitochondrial function by modulating mtDNA transcription, thereby affecting chemoresistance. CONCLUSIONS: In summary, our study reveals that HOXC11 regulates mitochondrial function through the modulation of mtDNA transcription, impacting chemoresistance in colorectal cancer cells. These findings underscore the significance of understanding the molecular mechanisms underlying chemoresistance and highlight the potential therapeutic implications of targeting mitochondrial function in CRC treatment.

METTL3-mediated NDUFB5 m6A modification promotes cell migration and mitochondrial respiration to promote the wound healing of diabetic foot ulcer.

BACKGROUND: Diabetic foot ulcer (DFU) is the most devastating complication of diabetes mellitus (DM) and plays a major role in disability and death in DM patients. NADH: ubiquinone oxidoreductase subunit B5 (NDUFB5) plays an important role in maintaining mitochondrial respiration, but whether it is involved in regulating the progression of advanced glycation end products (AGEs)-mediated DFU is still unclear. METHODS: Firstly, the role of AGEs on cell viability, migration, and mitochondrial respiration in human umbilical vein endothelial cells (HUVECs) was explored in vitro. Next, NDUFB5 expression was detected in human samples and AGEs-treated HUVECs, and NDUFB5's effect on AGEs-induced HUVECs injury and skin wound in diabetic mice was further clarified. In addition, the role of m6A modification mediated by methyltransferase-like 3 (METTL3) in regulating NDUFB5 expression and AGEs-induced HUVECs injury was investigated. RESULTS: NDUFB5 promoted cell viability, migration, and mitochondrial respiration in AGEs-treated HUVECs, whereas mitochondrial fusion promoter M1 facilitated cell viability, migration, and mitochondrial oxiadative respiration in NDUFB5 knockdown HUVECs. Meanwhile, NDUFB5 promotes skin wound healing in diabetic mice. Besides, METTL3-mediated m6A modification and insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2) enhanced NDUFB5 expression in HUVECs. Furthermore, METTL3 promoted cell viability, migration, and mitochondrial respiration in AGEs-treated HUVECs by increasing NDUFB5. CONCLUSION: METTL3-mediated NDUFB5 m6A modification inhibits AGEs-induced cell injury in HUVECs. METTL3 and NDUFB5 might serve as potential targets for DFU therapy in the future.

Impact of the SIK3 Pathway Inhibition on Osteoclast Differentiation via Oxidative Phosphorylation.

Maintenance of bone homeostasis and the balance between bone resorption and formation are crucial for maintaining skeletal integrity. This study sought to investigate the role of salt-inducible kinase 3 (SIK3), a key regulator in cellular energy metabolism, during the differentiation of osteoclasts. Despite osteoclasts being high energy-consuming cells essential for breaking down mineralized bone tissue, the specific function of SIK3 in this process remains unclear. To address this issue, we generated osteoclast-specific SIK3 conditional knockout mice and assessed the impact of SIK3 deletion on bone homeostasis. Our findings revealed that SIK3 conditional knockout mice exhibited increased bone mass and an osteopetrosis phenotype, suggesting a pivotal role for SIK3 in bone resorption. Moreover, we assessed the impact of pterosin B, a SIK3 inhibitor, on osteoclast differentiation. The treatment with pterosin B inhibited osteoclast differentiation, reduced the numbers of multinucleated osteoclasts, and suppressed resorption activity in vitro. Gene expression analysis demonstrated that SIK3 deletion and pterosin B treatment influence a common set of genes involved in osteoclast differentiation and bone resorption. Furthermore, pterosin B treatment altered intracellular metabolism, particularly affecting key metabolic pathways, such as the tricarboxylic acid cycle and oxidative phosphorylation. These results provide valuable insights into the involvement of SIK3 in osteoclast differentiation and the molecular mechanisms underlying osteoclast function and bone diseases.

Osteoporosis is a disease that causes bones to become weak and fragile, increasing the risk of fractures especially in elderly. It is caused by an imbalance between the formation of new bone and the destruction of old bone. Cells called osteoclasts are responsible for breaking down old bone. Excessive osteoclast activity results in bone loss and osteoporosis. Our research has identified a LKB1-SIK3 pathway, which acts as an energy sensor in osteoclasts. We found that this pathway is activated when osteoclast activity is increased, and we were able to reduce osteoclast activity by genetically removing or inhibiting SIK3. These findings suggest that targeting the LKB1-SIK3 pathway may be a promising new approach for the treatment of osteoporosis. Developing drugs that inhibit SIK3 may slow bone loss and reduce the risk of fractures in osteoporotic patients.

FERMT1 suppression induces anti-tumor effects and reduces stemness in glioma cancer cells.

OBJECTIVE: Glioma is a leading cause of mortality worldwide, its recurrence poses a major challenge in achieving effective treatment outcomes. Cancer stem cells (CSCs) have emerged as key contributors to tumor relapse and chemotherapy resistance, making them attractive targets for glioma cancer therapy. This study investigated the potential of FERMT1 as a prognostic biomarker and its role in regulating stemness through cell cycle in glioma. METHODS: Using data from TCGA-GBM, GSE4290, GSE50161 and GSE147352 for analysis of FERMT1 expression in glioma tissues. Then, the effects of FERMT1 knockdown on cell cycle, proliferation, sphere formation ability, invasion and migration were investigated. The influences of FERMT1 on expression of glycolysis-related proteins and levels of ATP, glucose, lactate and G6PDH were also explored. Furthermore, the effects of FERMT1 knockdown on cellular metabolism were evidenced. RESULTS: Significant upregulation of FERMT1 in glioma tissues was observed. Silencing FERMT1 not only affected the cell cycle but also led to a notable reduction in proliferation, invasion and migration. The expression of glycolysis-associated proteins including GLUT1, GLUT3, GLUT4, and SCO2 were reduced by FERMT1 knockdown, resulted in increased ATP and glucose as well as decreased lactic acid and G6PDH levels. FERMT1 knockdown also inhibited cellular metabolism. Moreover, FERMT1 knockdown significantly reduced sphere diameter, along with inhibiting the expression of transcription factors associated with stemness in glioma cells. CONCLUSION: These findings demonstrated that FERMT1 could be an ideal target for the advancement of innovative strategies against glioma treatment via modulating cellular process involved in stemness regulation and metabolism.

Effects of substrate availability and mitochondrial disruption on oxidative metabolism and sperm motility in fertile dogs.

In several mammalian species, the measurement of mitochondrial oxygen consumption (MITOX) under different metabolic conditions has demonstrated a positive correlation with sperm motility and may be a sensitive indicator of mitochondrial health. In general, the maintenance of sperm motility and many key sperm functions and fertilizing events are heavily energy-dependent processes, and some species-specific substrate preferences exist. Although canine sperm have been known to undergo capacitation and maintain motility with supplementation of a wide range of energy substrates, the relationship between mitochondrial function, and the maintenance of oxidative metabolism and sperm motility remain unclear. The objective of this study was to explore the metabolic flexibility of canine sperm, and to investigate the relationship between mitochondrial function, and maintenance of motility under differing nutrient conditions. We explored substrate preferences and the bioenergetics underlying maintenance of canine sperm motility by monitoring mitochondrial oxidative function and sperm kinematics in the presence of mitochondrial effector drug treatments: FCCP, antimycin (ANTI), and oligomycin (OLIGO). We hypothesized that canine sperm possess the ability to use compensatory pathways and utilize diverse nutrient sources in the maintenance of motility. Oxygen consumption (change in pO2, oxygen partial pressure) and sperm kinematics (CASA) were measured concurrently (t0-t30) to assess the relationship between oxidative metabolism and maintenance of sperm motility in dogs. Four media were tested: containing glucose, lactate, and pyruvate (GLP), containing glucose (G), fructose (F), or lactate and pyruvate (LP). In the absence of pharmacological inhibition of the electron transport chain, energetic substrate had no effect on sperm kinematics in fertile dogs. Following mitochondrial disruption by ANTI and OLIGO, mitochondrial oxygen consumption was negatively correlated with several sperm motility parameters in GLP, G, F, and LP media. In every media, FCCP treatment quickly induced significantly higher oxygen consumption than in untreated sperm, and spare respiratory capacity, the maximal inducible oxidative metabolism, was high. With respiratory control ratios RCR >1 there was no indication of bioenergetic dysfunction in any media type, indicating that sperm mitochondria of fertile dogs have a high capacity for substrate oxidation and ATP turnover regardless of substrate. Our results suggest MITOX assessment is a valuable tool for assessing mitochondrial functionality, and that canine sperm employ flexible energy management systems which may be exploited to improve sperm handling and storage.

Mitochondrial functions and fatty acid profiles in fish heart: an insight into physiological limitations linked with thermal tolerance and age.

Heart failure is among the first major consequences of heat stress in aquatic ectotherms. Mitochondria produce most of the ATP used by the heart and represent almost half of the volume in cardiac cells. It has therefore been hypothesized that mitochondrial dysfunctions may be highly involved in heart failure associated with heat stress. The present study aims to investigate if CTmax is linked to the thermal sensitivity of three-spined sticklebacks' (G. aculeatus) cardiac mitochondria, and if it is influenced by heart fatty acid composition and age. To do so, we measured the CTmax of 30 fish. The cardiac mitochondrial oxygen consumption was measured by high resolution respirometry at three temperatures and heart lipid profiles were obtained by Gas chromatography (GC) coupled with a Flame Ionization Detector (FID). Fish age was estimated via otolith readings. Fatty acid profiles showed no correlation with CTmax, but EPA levels were higher in older individuals. Mitochondrial respiration was measured in 35 fish using high resolution respirometry. It was strongly affected by temperature and showed a drastic drop in OXPHOS respiration fed by Complex I and Complex I+II, while uncoupled respiration plateaued at CTmax temperature. Our results suggest that Complex I is an important modulator of the impact of temperature on mitochondrial respiration at high temperatures but is not the main limiting factor in physiological conditions (maximal OXPHOS). Mitochondrial respiration was also affected by fish age, showing a general decrease in older individuals.

MALSU1-mediated regulation of mitochondrial function governs proliferation and doxorubicin resistance in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) poses a formidable challenge in oncology due to its aggressive nature and limited treatment options. Although doxorubicin, a widely used chemotherapeutic agent, shows efficacy in TNBC treatment, acquired resistance remains a significant obstacle. Our study explores the role of MALSU1, a regulator of mitochondrial translation, in TNBC and its impact on cell proliferation and doxorubicin resistance. We observed increased MALSU1 expression in TNBC, correlating with poor patient prognosis. MALSU1 knockdown in TNBC cells significantly reduced proliferation, indicating its pivotal role in sustaining cell growth. Mechanistically, MALSU1 depletion resulted in decreased activities of mitochondrial respiratory chain complexes, cellular ATP levels, and mitochondrial respiration. Notably, exogenous addition of normal mitochondria restored proliferation and mitochondrial respiration in MALSU1-depleted TNBC cells. Importantly, MALSU1 knockdown enhanced the sensitivity of doxorubicin-resistant TNBC cells to doxorubicin treatment. Furthermore, pharmacological inhibition of mitochondrial translation using tigecycline and chloramphenicol mimicked the effects of MALSU1 knockdown, suggesting mitochondrial translation as a potential therapeutic target. Taken together, our findings not only elucidate the intricate role of MALSU1 in TNBC biology and doxorubicin resistance but also lay the groundwork for future investigations targeting MALSU1 and/or mitochondrial translation as a promising avenue for developing innovative therapeutic strategies against TNBC.

Adaptive Effects of Intermittent Hypoxia Training on Oxygen-Dependent Processes as a Potential Therapeutic Strategy Tool.

BACKGROUND/AIMS: Important benefits of intermittent hypoxic training (IHT) have emerged as an effective tool for enhancing adaptive potential in different pathological states, among which acute hypoxia dominates. Therefore, the aim of our study was to evaluate the mechanisms related to the effects of the nitric oxide system (nitrites, nitrates, carbamide, and total polyamine content) on ADP-stimulated oxygen consumption and oxidative phosphorylation in heart and liver mitochondria and biomarkers of oxidative stress in the blood, heart, and liver of rats exposed to the IHT method and acute hypoxia and treated with the amino acid L-arginine (600 mg/kg, 30 min) or the NO synthase inhibitor L-NNA (35 mg/kg, 30 min) prior to each IHT session. METHODS: We analysed the modulation of the system of oxygen-dependent processes (mitochondrial respiration with the oxygraphic method, microsomal oxidation, and lipoperoxidation processes using biochemical methods) in tissues during IHT in the formation of short-term and long-term effects (30, 60, and 180 days after the last IHT session) with simultaneous administration of L-arginine. In particular, we investigated how mitochondrial functions are modulated during intermittent hypoxia with the use of oxidation substrates (succinate or α-ketoglutarate) in bioenergetic mechanisms of cellular stability and adaptation. RESULTS: The IHT method is associated with a significant increase in the production of endogenous nitric oxide measured by the levels of its stable metabolite, nitrite anion, in both plasma (almost 7-fold) and erythrocytes (more than 7-fold) of rats. The intensification of nitric oxide-dependent pathways of metabolic transformations in the energy supply processes in the heart and liver, accompanied by oscillatory mechanisms of adaptation in the interval mode, causes a probable decrease in the production of urea and polyamines in plasma and liver, but not in erythrocytes. The administration of L-arginine prior to the IHT sessions increased the level of the nitrite-reducing component of the nitric oxide cycle, which persisted for up to 180 days of the experiment. CONCLUSION: Thus, the efficacy of IHT and its nitrite-dependent component shown in this study is associated with the formation of long-term adaptive responses by preventing the intensification of lipoperoxidation processes in tissues due to pronounced changes in the main enzymes of antioxidant defence and stabilisation of erythrocyte membranes, which has a pronounced protective effect on the system of regulation of oxygen-dependent processes as a whole.

FABP4 Is an Indispensable Factor for Regulating Cellular Metabolic Functions of the Human Retinal Choroid.

The purpose of the current study was to elucidate the physiological roles of intraocularly present fatty acid-binding protein 4 (FABP4). Using four representative intraocular tissue-derived cell types, including human non-pigmented ciliary epithelium (HNPCE) cells, retinoblastoma (RB) cells, adult retinal pigment epithelial19 (ARPE19) cells and human ocular choroidal fibroblast (HOCF) cells, the intraocular origins of FABP4 were determined by qPCR analysis, and the intracellular functions of FABP4 were investigated by seahorse cellular metabolic measurements and RNA sequencing analysis using a specific inhibitor for FABP4, BMS309403. Among these four different cell types, FABP4 was exclusively expressed in HOCF cells. In HOCF cells, both mitochondrial and glycolytic functions were significantly decreased to trace levels by BMS309403 in a dose-dependent manner. In the RNA sequencing analysis, 67 substantially up-regulated and 94 significantly down-regulated differentially expressed genes (DEGs) were identified in HOCF cells treated with BMS309403 and those not treated with BMS309403. The results of Gene Ontology enrichment analysis and ingenuity pathway analysis (IPA) revealed that the DEGs were most likely involved in G-alpha (i) signaling, cAMP-response element-binding protein (CREB) signaling in neurons, the S100 family signaling pathway, visual phototransduction and adrenergic receptor signaling. Furthermore, upstream analysis using IPA suggested that NKX2-1 (thyroid transcription factor1), HOXA10 (homeobox A10), GATA2 (gata2 protein), and CCAAT enhancer-binding protein A (CEBPA) were upstream regulators and that NKX homeobox-1 (NKX2-1), SFRP1 (Secreted frizzled-related protein 1) and TREM2 (triggering receptor expressed on myeloid cells 2) were causal network master regulators. The findings in this study suggest that intraocularly present FABP4 originates from the ocular choroid and may be a critical regulator for the cellular homeostasis of non-adipocyte HOCF cells.

STL Inhibited Angiogenesis of DPSCs Through Depressing Mitochondrial Respiration by Enhancing RNF217.

Angiogenesis is the determining factor during dental pulp regeneration. Six-twelve leukemia (STL) is identified as a key regulatory factor on the biological function of dental pulp stem cells (DPSCs) under hypoxic conditions, but its effect on angiogenesis is unclear. Co-culture of DPSCs and human umbilical vein endothelial cells (HUVECs) is used to detect tubule formation ability in vitro and the angiogenesis ability in vivo. RNA-seq and bioinformatic analyses are performed to screen differentially expressed genes. Seahorse Cell Mito Stress Test is proceeded to exam mitochondrial respiration. STL decreased tubule formation and mitochondrial respiration of DPSCs in vitro and restrained the number of blood vessels and the expression of VEGF in new formed tissue in vivo. Furthermore, pretreating STL-depleted DPSCs with rotenone, a mitochondrial respiration inhibitor, counteracted the promoting effect of STL knockdown on tubule formation. Then, RNA-seq and bioinformatic analyses identified some angiogenesis relevant genes and pathways in STL-depleted DPSCs. And STL enhanced expression of mRNA-ring finger protein 217 (RNF217), which inhibited the tubule formation and mitochondrial respiration of DPSCs. STL inhibited the angiogenesis of DPSCs through depressing mitochondrial respiration by enhancing RNF217, indicating that STL is a potential target for angiogenesis of DPSCs.