Mitochondrial ATP Production Research Articles

Chimeric SFT2D2-TBX19 Promotes Prostate Cancer Progression by Encoding TBX19-202 Protein and Stabilizing Mitochondrial ATP Synthase through ATP5F1A Phosphorylation.

Specific chimeric RNAs and their products are consistently regarded as ideal tumor diagnostic markers and therapeutic targets. Chimeric RNAs can mediate tumor cell plasticity, neuroendocrine processes, polarization of tumor-associated macrophages, and resistance to chemotherapy and immunotherapy. However, the discovery of chimeric RNAs in prostate cancer is still in its early stages. This study identifies the chimeric SFT2D2-TBX19 as a novel transcript encoding the TBX19-202 protein. Both TBX19-202 and its parental TBX19, which share homologous amino acid sequences, enhance prostate cancer cell proliferation, migration, and invasion. Additionally, SFT2D2-TBX19 also functions as a lncRNA, interacting with the ATP synthase F1 subunit ATP5F1A, thereby increasing ATP5F1A phosphorylation mediated by TNK2/ACK1, which stabilizes the interaction between ATP5F1A and ATP5F1B. The region spanning 1801-2400bp of SFT2D2-TBX19 and the intermediate structural domain of ATP5F1A are crucial functional areas. This stabilization of ATP5F1A and ATP5F1B enhances mitochondrial ATP synthase activity and ATP production. Even under conditions of mitochondrial vulnerability, SFT2D2-TBX19 protects mitochondrial structural stability to maintain prostate cancer cell proliferation. This research provides comprehensive evidence that chimeric SFT2D2-TBX19 promotes prostate cancer progression by encoding the TBX19-202 protein and stabilizing mitochondrial ATP synthase via ATP5F1A phosphorylation. These findings highlight SFT2D2-TBX19 as a potential therapeutic target for prostate cancer.

Abstract 4138716: A novel Urocortin-2 analog COR-1167 corrects cardiac and renal dysfunction on top of Empagliflozin in a rat model of acute decompensated heart failure

Introduction: Urocortin-2 (UCN-2) is a peptide belonging to the corticotropin-releasing factor family that has cardiovascular, renal, metabolic and anti-inflammatory actions and improves cardiac function in human heart failure (HF) and cardiorenal dysfunction in chronic HF models. However, UCN-2 has a short half-life and intravenous delivery limits its therapeutic use. COR-1167 was discovered as a stable UCN-2 analog with qd pharmacological effects when injected subcutaneously. Hypothesis: Whether COR-1167 exerts chronic cardiorenal protection in Acute Decompensated HF (ADHF) is unknown, and in particular on background of SGLT2 inhibition, therefore both treatments were tested in a rat model of ADHF. Methods: Rats had coronary artery ligation to induce MI and a reduced ejection fraction phenotype. Three months later, rats were treated with vehicle or Empagliflozin (Empa) at 10 mg/kg/day PO. One month after starting Empa, two acute decompensation events were induced by administrating NaCl solution (1.8 g/kg, PO) on Day 0 and Day 14. Empa treated ADHF rats received vehicle or COR-1167 (3 µg/kg/day) by SC injections starting at 12 hours after the first decompensation and all treatments continued for 14 days. The groups were ADHF, ADHF + Empa and ADHF + Empa + COR-1167 (n=7-10/group). Left ventricular (LV) hemodynamics (cardiac catheters), cardiac output (echocardiography) and LV tissue perfusion (magnetic resonance imaging), cardiac mitochondrial coupling and ATP production (Seahorse) were measured 1 day after the second decompensation, while urine volume (uVol) and urine Na + excretion (uNa + ) collected from metabolic cages were analyzed 7 days after the first decompensation. Results: Compared to ADHF control rats, Empa treatment had no effect on systolic blood pressure (SBP), cardiac output (CO), LV end systolic pressure volume relationship (LVESPVR), LV end diastolic pressure volume relationship (LVEDPR), mitochondrial coupling and ATP production, but it increased myocardial perfusion, uVol and uNa + excretion. Compared to ADHF + Empa rats, COR-1167 treatment increased SBP, CO, LVESPVR, myocardial perfusion, mitochondrial coupling and ATP production, uVol, uNa + , while decreasing LVEDPVR. Conclusion: In the setting of ADHF, significant cardiorenal benefits and decongestion occurred when COR-1167 was administered on top of an SGLT2 inhibitor.

EXTH-22. TARGETING MITOCHONDRIAL PROTEASE IN DIFFUSE GLIOMAS

Abstract BACKGROUND Targeting mitochondrial function has emerged as a promising therapeutic strategy in cancer treatment. TR107 is characterized as a specific activator of caseinolytic protease proteolytic subunit (ClpP) in the mitochondria, potentially affecting oncogenic pathways. Here, we investigated the effects of TR107 on adult malignant gliomas, IDH-wildtype and IDH-mutant METHODS We utilized patient-derived glioma cells to determine the efficacy of TR107 with cell viability, proliferation, cell cycle, and apoptosis assays. Immunofluorescent staining and electron microscopy (EM) were employed to uncover organelle morphological changes upon treatment. Mitochondrial function and ATP production were assessed in live cells utilizing Seahorse analysis. Global proteomics and RNA sequencing were performed to identify treatment-induced dysregulated pathways in glioma cells RESULTS Both IDH-wildtype and IDH-mutant glioma cell lines were highly sensitive to TR107, showing at least 100 times lower IC50 compared to the related compound ONC201. Genetic knock out of ClpP revealed that the growth inhibitory effects of TR107 are ClpP-dependent. Interestingly, IDH-mutant cell lines showed a more profound disintegration of mitochondrial morphology and dysregulation of mitochondrial function evidenced by an enhanced suppression of oxidative phosphorylation, mitochondrial complexes expression, mtDNA copy number, complex I activity, and ATP production, compared to IDH-wildtype cells. Six days of treatment with TR107 led to a complete inhibition of cell growth and up to 97% of cell death in IDH-mutant cells, while 50% of IDH-wildtype cells remained viable. Western blot, EM, and RNAsequencing analyses uncovered autophagy as a potential pro-survival mechanism in IDH-wildtype cells. Importantly, multiple metabolic and DNA repair-related pathways were affected by the treatment, suggested by global proteomics and RNA sequencing. Finally, TR107 was not affected by ABC transporters, supporting its potential use in brain tumor patients CONCLUSION TR107 demonstrated potent cytotoxic effects in patient-derived glioma cells by disrupting mitochondrial structural and functional integrity. TR107 efficacy in vivo is under investigation.

Synthesis and Biological Evaluation of N-(1H-Indol-6-ylmethyl)benzenesulfonamide Analogs as Metabolic Inhibitors of Mitochondrial ATP Production in Pancreatic Cancer Cells.

A library of 26 indolyl sulfonamides and 12 amide and ester analogs based upon the 6-indolyl framework has been synthesized in an effort to target pancreatic cancer. The cytotoxicity of the indolyl sulfonamide compounds has been determined using a traditional (48-h compound exposure) assay against 7 pancreatic cancer cell lines and 1 non-cancerous cell line. The potential role of the compounds as metabolic inhibitors of ATP production was evaluated using a rapid screening (2-h compound exposure) assay developed within our laboratories. The IC50 values of the active compounds were determined using the rapid assay and six compounds displayed an IC50 value <5 μM against one or more pancreatic cancer cell lines. The ester analogs also display activity as potential metabolic inhibitors of ATP production with four of the six compounds displaying an IC50 value <5 μM against one or more pancreatic cancer cell lines.

Breast cancer cells utilize T3 to trigger proliferation through cellular Ca2+ modulation

High levels of thyroid hormones are linked to increased risk and advanced stages of breast cancer. Our previous work demonstrated that the biologically active triiodothyronine (T3) facilitates mitochondrial ATP production by upregulating Ca2+ handling proteins, thereby boosting mitochondrial Ca2+ uptake and Krebs cycle activity. In this study, different cell types were utilized to investigate whether T3 activates a Ca2+-induced signaling pathway to boost cancer cell proliferation. Using live-cell imaging, biochemical assays, and molecular profiling, differences in intracellular signaling among MCF7 and MDA-MB-468 breast cancer cells, non-cancerous breast cells hTERT-HME1, and PC3 prostate carcinoma cells, previously found to be insensitive to thyroid hormones in terms of proliferation, were investigated. Our findings revealed that T3 upregulates 1,4,5-trisphosphate receptor 3 via thyroid hormone receptor α. This boosts mitochondrial Ca2+ uptake, reduction equivalent yield, and mitochondrial ATP production, supporting the viability and proliferation of breast cancer cells without affecting non-cancerous hTERT-HME1 or PC3 prostate carcinoma cells. Understanding the interplay between T3 signaling, organellar interaction, and breast cancer metabolism could lead to targeted therapies that exploit cancer cell vulnerabilities. Our findings highlight T3 as a crucial regulator of cancer metabolism, reinforcing its potential as a therapeutic target in breast cancer.Graphical

Role of the PGAM5-CypD mitochondrial pathway in methylglyoxal-induced bone loss in diabetic osteoporosis

Diabetic osteoporosis (DOP) is a skeletal complication with a high rate of disability. It results in a great burden to the patient's family and society. Methylglyoxal (MG) is a toxic by-product of the glycolytic process that occurs during diabetic conditions. It causes osteoblastic injury and con-tributes to the initiation and development of DOP. Disruption of mitochondrial homeostasis has been implicated as a cause of dysregulated osteo-blastogenesis, an essential step in bone formation. It is unclear whether mitochondrial dysfunction is involved in MG-induced osteoblast dysfunction. In this study, we showed that mitochondrial dysfunction contributes to MG-induced MC3T3-E1 cell apoptosis and impaired differentiation. A significant reduction of mitochondrial membrane potential (MMP) and ATP production occurred in MG-induced osteoblasts as well as increasing mitochondrial reactive oxygen species (mtROS) and intracellular Ca2+. Classical antioxidant N-Acetylcysteine (NAC) significantly attenuated mitochondrial dysfunction as well as osteoblast apoptosis and osteogenic differentiation damage induced by MG. More importantly, we found that activating phosphoglycerate mutase family member 5 (PGAM5) and cyclophilin D (CypD), which contributes to mitochondrial homeostasis, is involved in MG-induced osteoblast injury. Both PGAM5 and CypD knockdown effectively reversed osteoblast viability and function, whereas PGAM5 or CypD overexpression aggravated osteoblast injury caused by MG. Moreover, the result of co-transfection revealed that PGAM5 is an upstream signaling molecule of CypD. By constructing type I diabetes mouse models, we further found that the expression of PGAM5 and CypD were both increased in the femur along with a reduction of ATP and increased TUNEL-positive cells. These results, for the first time, suggest that MG-induced mitochondrial dysfunction induces osteoblast injury through the PGAM5-CypD pathway. This study provides insight into the prevention and treatment of DOP. Lay summaryThis study highlights the role of mitochondria in regulating osteoblast viability and function under conditions of diabetic osteoporosis (DOP). We found that the PGAM5-CypD mitochondrial pathway is activated following glycolytic by-product methylglyoxal (MG) treatment, which contributes to mitochondrial dysfunction and osteogenic dysfunction. This mechanism implicates mitochondria as a potential therapeutic target for osteoporosis.

The cardioprotective potential of soluble urokinase Plasminogen Activator Receptor (suPAR) in myocardial ischemia

Abstract Background and aims: Increased circulating soluble urokinase plasminogen activator receptor (suPAR) levels are associated with adverse cardiovascular outcomes, however, any cardiovascular-related functions remain unknown. This study aimed to (i) document haemodynamic profiles in isolated rat hearts under the influence of suPAR, and to (ii) explore any mechanisms triggered by suPAR following myocardial ischaemia-reperfusion. Methods We undertook a 4-pronged approach (Figure 1). A Langendorff isolated rat heart model was used to perfuse all hearts with Krebs-Henseleit solution flowing retrogradely through the aorta at 37°C. A balloon inserted into the left ventricle allowed measurements of ventricular pressure, and a pressure transducer placed above the aorta recorded perfusion pressure for coronary flow rates. The experimental protocol consisted an initial 30-minute stabilisation period, followed by 40 minutes of ischemia via buffer flow cessation. Hearts were subsequently recovered during 90 minutes of reperfusion, receiving either suPAR treatment or perfusion buffer as controls. An inhibitor arm comprising co-infusion of suPAR and a monoclonal antibody capable of binding to suPAR was also assessed. Troponin T was measured in outflow perfusates collected at specific intervals. Proteins from hearts in treatment groups were separated using gel electrophoresis, with Western blotting to visualise the phosphorylation status of kinases (Akt, STAT). High-flow respirometry and fluorometry were investigated in cardiac mitochondria to determine respiration rates and ATP production in ischaemia reperfusion under the influence of suPAR. Statistical analysis was conducted with ANOVA using SPSS. Results Hearts receiving suPAR infusion at reperfusion (n = 16) showed a significant increase in developed pressure (p = .017) and lower perfusion pressure (p = .02) compared to controls (n = 16), revealing that suPAR-treated hearts recovered with better contractility and vasodilatory properties post-ischaemia. suPAR’s haemodynamic profiles in the antibody group were similar to controls, attenuating suPAR-induced effects (n = 8). Outflow effluents in suPAR-treated hearts obtained &amp;gt;3 fold lower serial Troponin T values than controls (p &amp;lt; .001). Heart tissues receiving suPAR treatment revealed a 1.7-fold increase in Akt (p = .001) and a 3.8-fold increase in STAT3 phosphorylation compared to controls (p &amp;lt; .001) and the inhibitor group (p = .04). Finally, suPAR increased mitochondrial ATP production by 28% (p = .013) compared to controls after ischaemia. Conclusion This is a world-first demonstration of suPAR’s cardioprotective influences in isolated rat hearts recovering from ischaemia. suPAR’s ability to promote cardiac contractility and vasodilation was associated with lesser injury as shown by Troponin T reduction. Mechanistically, suPAR infusion upregulated two key cardioprotective pathways, which may also function to protect cardiac mitochondria.Figure 1 - Schematic of Methods

Advances in myocardial energy metabolism: metabolic remodeling in heart failure and beyond.

The very high energy demand of the heart is primarily met by ATP production from mitochondrial oxidative phosphorylation, with glycolysis providing a smaller amount of ATP production. This ATP production is markedly altered in heart failure, primarily due to a decrease in mitochondrial oxidative metabolism. Although an increase in glycolytic ATP production partly compensates for the decrease in mitochondrial ATP production, the failing heart faces an energy deficit, that contributes to the severity of contractile dysfunction. The relative contribution of the different fuels for mitochondrial ATP production dramatically changes in the failing heart, which depends to a large extent on the type of heart failure. A common metabolic defect in all forms of heart failure (including HFrEF, HFpEF, and diabetic cardiomyopathies) is a decrease in mitochondrial oxidation of pyruvate originating from glucose (i.e. glucose oxidation). This decrease in glucose oxidation occurs regardless of whether glycolysis is increased, resulting in an uncoupling of glycolysis from glucose oxidation that can decrease cardiac efficiency. The mitochondrial oxidation of fatty acids by the heart increases or decreases, depending on the type of heart failure. For instance, in HFpEF and diabetic cardiomyopathies myocardial fatty acid oxidation increases, while in HFrEF myocardial fatty acid oxidation either decreases or remains unchanged. The oxidation of ketones (which provides the failing heart with an important energy source) also differs depending on the type of heart failure, being increased in HFrEF, and decreased in HFpEF and diabetic cardiomyopathies. The alterations in mitochondrial oxidative metabolism and glycolysis in the failing heart are due to transcriptional changes in key enzymes involved in the metabolic pathways, as well as alterations in redox state, metabolic signaling, and posttranslational epigenetic changes in energy metabolic enzymes. Of importance, targeting the mitochondrial energy metabolic pathways has emerged as a novel therapeutic approach to improving cardiac function and cardiac efficiency in the failing heart.

RNA-seq analysis of mitochondria-related genes regulated by AMPK in the human trophoblast cell line BeWo.

How AMP activated protein kinase (AMPK) signaling regulates mitochondrial functions and mitophagy in human trophoblast cells remains unclear. This study was designed to investigate potential players mediating the regulation of AMPK on mitochondrial functions and mitophagy by next generation RNA-seq. We compared ATP production in protein kinase AMP-activated catalytic subunit alpha 1/2 (PRKAA1/2) knockdown (AKD) and control BeWo cells using the Seahorse real-time ATP rate test, then analyzed gene expression profiling by RNA-seq. Differentially expressed genes (DEG) were examined by Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. Then protein-protein interactions (PPI) among mitochondria related genes were further analyzed using Metascape and Ingenuity Pathway Analysis (IPA) software. Both mitochondrial and glycolytic ATP production in AKD cells were lower than in the control BeWo cells (CT), with a greater reduction of mitochondrial ATP production. A total of 1092 DEGs were identified, with 405 upregulated and 687 downregulated. GO analysis identified 60 genes associated with the term 'mitochondrion' in the cellular component domain. PPI analysis identified three clusters of mitochondria related genes, including aldo-keto reductase family 1 member B10 and B15 (AKR1B10, AKR1B15), alanyl-tRNA synthetase 1 (AARS1), mitochondrial ribosomal protein S6 (MRPS6), mitochondrial calcium uniporter dominant negative subunit beta (MCUB) and dihydrolipoamide branched chain transacylase E2 (DBT). In summary, this study identified multiple mitochondria related genes regulated by AMPK in BeWo cells, and among them, three clusters of genes may potentially contribute to altered mitochondrial functions in response to reduced AMPK signaling.

Systematic Review and Meta-Analysis on the Association Between Daily Niacin Intake and Glaucoma.

Glaucoma is a progressive optic neuropathy, characterised by a complex pathophysiology, with mitochondrial dysfunction playing a significant role in the cellular damage and apoptosis of ganglion cells. Niacin is a precursor to several molecules acting as coenzymes in the mitochondrial production of ATP, in DNA repair and in the reduction of reactive oxygen species. The objective of this systematic review is to assess the impact of daily niacin intake on glaucoma. Case-control and cohort studies regarding niacin and glaucoma, indexed in PubMed, Web of Science, Cochrane and Scopus, were included. Other study methodologies, studies regarding niacin in other ocular disease or other nutrients in glaucoma were excluded. Bias was assessed using the Newcastle-Ottawa Scale. The study protocol was registered in the PROSPERO database (no. CRD42024578889). Five case-control studies were included. In the pooled analysis, a significantly higher proportion of patients with high niacin consumption was found in the group without glaucoma compared to those with glaucoma as defined by ISGEO criteria (p-value < 0.00001; OR = 0.66, 95% CI 0.55-0.79) or as defined by retinal imaging (p-value = 0.02; OR = 0.63, 95% CI 0.43-0.94). Daily dietary intake of niacin is significantly lower in patients with glaucoma compared to the general population. Given different average daily intakes of niacin in these populations, different glaucoma definitions and several confounding variables which weaken the associations, large sample, standardised randomised controlled trials are needed to confirm the potential benefits of niacin in glaucoma.

Abstract A051: Bitter agonists increase macrophage phagocytosis of head and neck squamous cell carcinoma cells

Abstract Head and neck squamous cell carcinoma (HNSCC) is the 7th most commonly diagnosed cancer worldwide and is expected to increase in incidence by 30% by 2030. The HNSCC microenvironment is characterized by substantial macrophage infiltration. However, tumor-associated macrophages often cultivate an immunosuppressive environment that aids tumor progression. Previous work has shown that macrophages express bitter taste receptors (T2Rs), a class of GPCRs that can be activated with bitter compounds. Previous work has also demonstrated that stimulation of T2Rs in macrophages with bitter agonists, such as denatonium benzoate, increases phagocytosis of bacteria, but the impact of bitterants on macrophage phagocytosis of tumor cells is unknown. The goal of this study was to assess the impact of bitter compounds on macrophage phagocytosis of HNSCC cells in vitro. Human peripheral blood mononuclear cells obtained from healthy donors were used to generate monocyte-derived macrophages (MDMs). To investigate the effects of bitter agonist stimulation on macrophage phagocytosis, CFSE-labeled MDMs and DAPI-labeled HNSCC cell line SCC47, were co-stimulated with denatonium benzoate, and macrophage phagocytosis was quantified. This revealed that treatment with denatonium benzoate led to a 70% increase in macrophage phagocytosis compared to controls. Additional analysis revealed a 1.8-fold increase in F-actin in MDMs in response to denatonium benzoate measured via CellMask™ Actin staining, and that macrophages had generated F-actin positive tunneling nanotubes (TNTs). Further characterization with live cell imaging showed that these TNTs were being utilized as a means of mitochondrial transfer as observed by MitoTracker™ and CellMask™ Actin staining. Furthermore, there was a 2.5-fold decrease in mitochondrial ATP production in MDMs stimulated with denatonium benzoate, possibly indicating metabolic reprogramming. Timelapse imaging of MDMs demonstrated a reduction in mitochondrial ATP production, with a concomitant increase in F-actin production. These observations were confirmed in Phorbol 12-myristate 13-acetate differentiated THP-1 cells. Taken together, these results indicate that bitter taste receptor stimulation may contribute to a more activated macrophage phenotype denoted by a decrease in mitochondrial ATP production, increased F-actin staining, and increased phagocytosis of tumor cells. Citation Format: Brianna L Hill, Sarah M Sywanycz, Zoey A Miller, Robert J Lee, Ryan M Carey. Bitter agonists increase macrophage phagocytosis of head and neck squamous cell carcinoma cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr A051.

Kaempferol Reduces Cardiopulmonary Load and Muscular Damage in Repeated 400‐m Sprints: A Double‐Blind, Randomized, Placebo‐Controlled Trial

ABSTRACTPlants exposed to hypoxic conditions have been suggested to produce more biologically active phytochemicals than those exposed to normal oxygen levels. Previously, we investigated 314 highland crop species and showed that the flavonoid kaempferol extracted from a highland quinoa grain markedly increased mitochondrial metabolism and ATP production in a hypoxic environment in vitro. Thus, we hypothesized that kaempferol would be effective during exercise under harsh conditions, in which anaerobic metabolism occurs. This study adopted a double‐blind, placebo‐controlled, crossover design to investigate the effect of a single oral dose of kaempferol (10 mg) on the athletic performance‐related indicators of 13 male university athletes (20.8 ± 0.7 years) who performed two consecutive 400‐m runs with the shortest 90‐min interval assuming qualifying and main races. Although no significant differences were observed in the 400‐m race times between the placebo and kaempferol groups, kaempferol intake markedly reduced the respiratory and heart rates during the second run (p &lt; 0.05). In addition, kaempferol intake reduced the levels of muscle damage markers, myoglobin, and aspartate transaminase (p &lt; 0.05). A single oral dose of kaempferol reduced the cardiopulmonary burden and muscle damage in individuals participating in 400‐m runs. Kaempferol may be a useful supplement for relieving the physical load, particularly in individuals performing strenuous exercises with high oxygen demand.Trial Registration: UMIN Clinical Trials Registry in Japan: UMIN000049588

Novel ultrathin ferrous sulfide nanosheets: Towards replacing black phosphorus in anticancer nanotheranostics

Biodegradable two-dimensional nanomaterials could be a significant breakthrough in the field of oncology nanotheranostic agents, which are rapidly emerging as promising candidates for tumor theranostic applications. Herein, a novel biodegradable ferrous sulfide nanosheet (FeS NS) is developed. Compared to the traditional photothermal material, black phosphorus nanosheet (BP NS), FeS demonstrates superior degradability and enhanced photothermal performance, and making it ideal for efficient photothermal therapy (PTT) of tumors. In the acidic tumor microenvironment, FeS degrades and releases H2S, which inhibits mitochondrial respiration and ATP production. This process leads to a reduction in heat shock protein expression, lowering the resistance of tumor cells to photothermal stimulation, and improving the efficacy of PTT. The released Fe2+ exhibits efficient peroxidase activity, triggering ferroptosis in tumor cells. Furthermore, due to its superparamagnetic nature, FeS NSs could accumulate at the tumor site and provide a strong magnetic resonance imaging (MRI) signal for imaging-guided tumor therapy. Overall, as a promising alternative to BP, the FeS NSs are a potentially innovative nanotheranostic agent of tumors, offering a synergistic approach to ferroptosis−PTT with MRI guidance.

High mitochondrial DNA content is a key determinant of stemness, proliferation, cell migration, and cancer metastasis in vivo

Here, we examined the potential role of mitochondrial DNA (mtDNA) levels in conveying aggressive phenotypes in cancer cells, using two widely-used breast cell lines as model systems (MCF7[ER+] and MDA-MB-231[ER-]). These human breast cancer cell lines were fractionated into mtDNA-high and mtDNA-low cell sub-populations by flow cytometry, using SYBR Gold as a vital probe to stain mitochondrial nucleoids in living cells. Enrichment of mtDNA-high and mtDNA-low cell sub-populations was independently validated, using a specific DNA-binding mAb probe (AC-30-10), and mitochondrial-based functional assays. As predicted, mtDNA-high MCF7 cells showed significant increases in mitochondrial mass, membrane potential, and superoxide production, as well as increased mitochondrial respiration and ATP production. Moreover, mtDNA-high MCF7 cells demonstrated increases in stemness features, such as anchorage-independent growth and CD44 levels, as well as drug-resistance to Gemcitabine and Tamoxifen. Proliferation rates were also significantly increased, with a dramatic shift towards the S- and G2/M-phases of the cell cycle; this was indeed confirmed by RNA-Seq analysis. Complementary results were obtained with MDA-MB-231 cells. More specifically, mtDNA-high MDA-MB-231 cells showed increases in stemness features and ATP production, as well as rapid cell cycle progression. Moreover, mtDNA-high MDA-MB-231 cells also exhibited increases in both cell migration and invasion, suggesting a role for mtDNA in distant metastasis. To test this hypothesis more directly, a preclinical in vivo model was utilized. For this purpose, MDA-MB-231 tumour cell grafts were treated with an established mtDNA synthesis inhibitor, namely Alovudine (3’-deoxy-3’-fluorothymidine). As expected, drug-induced depletion of mtDNA led to a shift from mitochondrial to glycolytic metabolism. Interestingly, Alovudine very effectively reduced the formation of spontaneous metastases by nearly 70%, but minimally inhibited tumour growth by approximately 20%. Taken together, these data suggest that high mtDNA content is a key driver of stemness, proliferation, and migration, as well as cancer cell metastasis.

7086 Disrupted Mitochondrial Function in Alström Syndrome- A Monogenic Model of Insulin Resistance and Obesity

Abstract Disclosure: S. Ali: None. S. Heising: None. V. Veeranna: None. A. Vincent: None. G.S. Xavier: None. T. Geberhiwot: None. Alström syndrome (ALMS) is a rare monogenic disease typified by severe insulin resistance (IR) and obesity. IR and obesity are cardinal features of metabolic syndrome. The metabolic effects of insulin at key target tissues leads to metabolic haemostasis by promoting glucose and lipid uptake and metabolism to generate energy in cells. Although insulin is a major regulator of fuel metabolism, its effects on mitochondrial ATP production in severe insulin resistance are unclear. RNASeq data from adipose tissue (AT) from patients with ALMS show downregulation of the expression of genes associated with mitochondrial respiratory chain function, pointing towards possible altered mitochondrial function in ALMS. We therefore undertook a detailed assessment of mitochondrial oxidative phosphorylation capacity (OXPHOS) in skeletal muscle (SM) in ALMS. We performed SM biopsy in 10 ALMS and 10 healthy control volunteers that were age, gender and Body Mass Index matched. The SM was subjected to high resolution respirometry, the gold standard method to quantify mitochondrial function ex vivo, using OROBOROS Oxygraph -2k (O2k) system. Overall OXPHOS was higher in control SM compared to ALMS, with maximal respiration of 62.96±19.2 and 44.24 ± 12.53 pmol/(s*mg), respectively (p&amp;lt;0.05).Reduced oxygen influx was observed in ALMS SM in comparison to controls for fatty acid ß oxidation (6.7±5.96 vs 7.87±2.7 pmol/(s*mg);p&amp;lt;0.05, at complex I (19.4 ± 8.98 vs 29.7 ±5.3 pmol/(s*mg);p&amp;lt;0.05, and at complex II (34.98 ±11.75 vs 53.52± 13.2 pmol/(s*mg) ;p&amp;lt;0.05. Mitochondrial membrane integrity was unaffected during these experiments. Therefore, our analysis unveils a consistent reduction in mitochondrial respiration across all the electron transport pathway. This noteworthy finding suggests impairment in respiratory function and the electron transport chain in mitochondria, a crucial aspect of cellular energy metabolism. Several studies have previously linked IR with mitochondrial dysfunction, where insulin-resistant rodents and humans have shown blunted mitochondrial response to ATP generation. Our results align with this body of evidence emphasizing the significance of investigating the underlying mechanism contributing to this phenomenon. These findings could pave the way to exploring potential mitochondrial-targeted therapies to restore mitochondrial function and therefore, improve insulin sensitivity to tackle IR in ALMS. Presentation: 6/2/2024

The dose-dependent dual effects of alpha-ketoglutarate (AKG) on cumulus oocyte complexes during in vitro maturation

In this study, we reported for the first time the dose-dependent dual effects of Alpha-Ketoglutarate (AKG) on cumulus oocyte complexes (COCs) during in vitro maturation (IVM). AKG at appropriate concentration (30 µM) has beneficial effects on IVM. This includes improved cumulus expansion, oocyte quality, and embryo development. These effects are mediated through multiple underlying mechanisms. AKG reduced the excessive accumulation of reactive oxygen species (ROS) in cumulus cells, reduced the consumption of GSH and NADPH. Cumulus GSH and NADPH were transported to oocytes via gap junctions, thereby reducing the oxidative stress, apoptosis and maintaining the redox balance in oocytes. In addition, AKG improved the mitochondrial function by regulating the mitochondrial complex 1 related gene expression in oocytes to maintain mitochondrial membrane potential and ATP production. On the other hand, oocyte generated GDF9 could also be transported to cumulus cells to promote cumulus expansion. Conversely, a high concentration of AKG (750 µM) exerted adverse effects on IVM and suppressed the cumulus expansion as well as reduced the oocyte quality. The suppression of the cumulus expansion caused by high concentration of AKG could be rescued with GDF9 supplementation in COCs, indicating the critical role of GDF9 in IVM. The results provide valuable information on the variable effects of AKG at different concentrations on reproductive physiology.Graphical abstract

Disruption of mitochondrial electron transport impairs urinary concentration via AMPK-dependent suppression of aquaporin-2.

Urinary concentration is an energy-dependent process that minimizes body water loss by increasing aquaporin-2 (AQP2) expression in collecting duct (CD) principal cells. To investigate the role of mitochondrial (mt) ATP production in renal water clearance, we disrupted mt electron transport in CD cells by targeting ubiquinone (Q) binding protein QPC (UQCRQ), a subunit of mt complex III essential for oxidative phosphorylation. QPC-deficient mice produced less concentrated urine than controls, both at baseline and after type 2 vasopressin receptor stimulation with desmopressin. Impaired urinary concentration in QPC-deficient mice was associated with reduced total AQP2 protein levels in CD tubules, while AQP2 phosphorylation and membrane trafficking remained unaffected. In cultured inner medullary CD cells treated with mt complex III inhibitor antimycin A, the reduction in AQP2 abundance was associated with activation of 5' adenosine monophosphate-activated protein kinase (AMPK) and was reversed by treatment with AMPK inhibitor SBI-0206965. In summary, our studies demonstrated that the physiological regulation of AQP2 abundance in principal CD cells was dependent on mt electron transport. Furthermore, our data suggested that oxidative phosphorylation in CD cells was dispensable for maintaining water homeostasis under baseline conditions, but necessary for maximal stimulation of AQP2 expression and urinary concentration.

Quercetin Promotes the Repair of Mitochondrial Function in H9c2 Cells Through the miR-92a-3p/Mfn1 Axis.

Cardiocerebrovascular disease is a severe threat to human health. Quercetin has a wide range of pharmacological effects such as antitumor and antioxidant. In this study, we aimed to determine how quercetin regulates mitochondrial function in H9c2 cells. An H9c2 cell oxygen glucose deprivation/reoxygenation (OGD/R) model was constructed. The expression of miR-92a-3p and mitofusin 1 (Mfn1) mRNA in the cells was detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Changes in the mitochondrial membrane potential of cells were examined by JC-1 staining. ATP production in the cells was detected using a biochemical assay. Mitochondrial morphological changes were observed using transmission electron microscopy. Detection of miR-92a-3p binding to Mfn1 was done using dual luciferase. Western blotting was used to detect the protein expression of Mfn1 in the cells. miR-92a-3p is essential in regulating cell viability, apoptosis, and tumor cell metastasis. OGD/R induced miR-92a-3p expression, decreased mitochondrial membrane potential and mitochondrial ATP production, and increased mitochondrial damage. Mitochondria are the most critical site for ATP production. Continued opening of the mitochondrial permeability transition pore results in an abnormal mitochondrial transmembrane potential. Both quercetin and inhibition of miR-29a-3p were able to downregulate miR-29a-3p levels, increase cell viability, mitochondrial membrane potential, and ATP levels, and improve mitochondrial damage morphology. Furthermore, we found that downregulation of miR-29a-3p upregulated the protein expression of Mfn1 in cells. Additionally, miR-92a-3p was found to bind to Mfn1 in a luciferase assay. miR- 29a-3p overexpression significantly inhibited the protein expression level of Mfn1. Quercetin treatment partially reversed the effects of miR-29a-3p overexpression in H9c2 cells. Quercetin promoted the recovery of mitochondrial damage in H9c2 cells through the miR-92a-3p/Mfn1 axis.

Aldosterone, mitochondria and regulation of cardiovascular metabolic disease.

Aldosterone is a mineralocorticoid hormone involved in controlling electrolyte balance, blood pressure, and cellular signaling. It plays a pivotal role in cardiovascular and metabolic physiology. Excess aldosterone activates mineralocorticoid receptors, leading to subsequent inflammatory responses, increased oxidative stress, and tissue remodeling. Various mechanisms have been reported to link aldosterone with cardiovascular and metabolic diseases. However, mitochondria, responsible for energy generation through oxidative phosphorylation, have received less attention regarding their potential role in aldosterone-related pathogenesis. Excess aldosterone leads to mitochondrial dysfunction, and this may play a role in the development of cardiovascular and metabolic diseases. Aldosterone has the potential to affect mitochondrial structure, function, and dynamic processes, such as mitochondrial fusion and fission. In addition, aldosterone has been associated with the suppression of mitochondrial DNA, mitochondria-specific proteins, and ATP production in the myocardium through mineralocorticoid receptor, nicotinamide adenine dinucleotide phosphate oxidase, and reactive oxygen species pathways. In this review, we explore the mechanisms underlying aldosterone-induced cardiovascular and metabolic mitochondrial dysfunction, including mineralocorticoid receptor activation and subsequent inflammatory responses, as well as increased oxidative stress. Furthermore, we review potential therapeutic targets aimed at restoring mitochondrial function in the context of aldosterone-associated pathologies. Understanding these mechanisms is vital, as it offers insights into novel therapeutic strategies to mitigate the impact of aldosterone-induced mitochondrial dysfunction, thereby potentially improving the outcomes of individuals affected by cardiovascular and metabolic disorders.

A Newly Identified Protective Role of C5a Receptor 1 in Kidney Tubules against Toxin-Induced Acute Kidney Injury

Acute kidney injury (AKI) remains a major reason for hospitalization with limited therapeutic options. While complement activation is implicated in AKI, the role of C5a receptor 1 (C5aR1) in kidney tubular cells is unclear. We used aristolochic acid nephropathy (AAN) and folic acid nephropathy (FAN) models to establish the role of C5aR1 in kidney tubules during AKI in germline C5ar1-/- mice, myeloid cell-specific, and kidney tubule-specific C5ar1 knockout mice. After aristolochic acid and folic acid injection, C5ar1-/- mice had increased AKI severity and a higher degree of tubular injury. Macrophage depletion in C5ar1-/- mice or myeloid cell-specific C5ar1 deletion did not affect the outcomes of AA-induced AKI. RNA-sequencing data from RTECs showed that C5ar1 deletion was associated with the downregulation of mitochondrial metabolism and ATP production transcriptional pathways. Metabolic studies confirmedreduced mitochondrial membrane potential at baseline and increased mitochondrial oxidative stress after injury in C5ar1-/- RTECs. Moreover, C5ar1-/- RTECs had enhanced glycolysis, glucose uptake, and lactate production upon injury, corroborated by metabolomics analysis of kidneys from AAN mice. Kidney tubule-specific C5ar1 knockout mice recapitulated exacerbated AKI observed in C5ar1-/- mice in AAN and FAN. Our data indicate that C5aR1 signaling in kidney tubules exerts renoprotective effects against toxin-induced AKI by limiting overt glycolysis and maintaining mitochondrial function, revealing a novel link between the complement system and tubular cell metabolism.