Mitochondrial ATP Production Rate Research Articles

Photodynamic priming modulates cellular ATP levels to overcome P-glycoprotein-mediated drug efflux in chemoresistant triple-negative breast cancer.

P-glycoprotein (P-gp, ABCB1) is a well-researched ATP-binding cassette (ABC) drug efflux transporter linked to the development of cancer multidrug resistance (MDR). Despite extensive studies, approved therapies to safely inhibit P-gp in clinical settings are lacking, necessitating innovative strategies beyond conventional inhibitors or antibodies to reverse MDR. Photodynamic therapy is a globally approved cancer treatment that uses targeted, harmless red light to activate non-toxic photosensitizers, confining its cytotoxic photochemical effects to disease sites while sparing healthy tissues. This study demonstrates that photodynamic priming (PDP), a sub-cytotoxic photodynamic therapy process, can inhibit P-gp function by modulating cellular respiration and ATP levels in light accessible regions. Using chemoresistant (VBL-MDA-MB-231) and chemosensitive (MDA-MB-231) triple-negative breast cancer cell lines, we showed that PDP decreases mitochondrial membrane potential by 54.4% ± 30.4 and reduces mitochondrial ATP production rates by 94.9% ± 3.46. Flow cytometry studies showed PDP can effectively improve the retention of P-gp substrates (calcein) by up to 228.4% ± 156.3 in chemoresistant VBL-MDA-MB-231 cells, but not in chemosensitive MDA-MB-231 cells. Further analysis revealed that PDP did not alter the cell surface expression level of P-gp in VBL-MDA-MB-231 cells. These findings indicate that PDP can reduce cellular ATP below the levels that is required for the function of P-gp and improve intracellular substrate retention. We propose that PDP in combination with chemotherapy drugs, might improve the efficacy of chemotherapy and overcome cancer MDR.

The Specific ROCK2 Inhibitor KD025 Alleviates Glycolysis through Modulating STAT3-, CSTA- and S1PR3-Linked Signaling in Human Trabecular Meshwork Cells.

To investigate the biological significance of Rho-associated coiled-coil-containing protein kinase (ROCK) 2 in the human trabecular meshwork (HTM), changes in both metabolic phenotype and gene expression patterns against a specific ROCK2 inhibitor KD025 were assessed in planar-cultured HTM cells. A seahorse real-time ATP rate assay revealed that administration of KD025 significantly suppressed glycolytic ATP production rate and increased mitochondrial ATP production rate in HTM cells. RNA sequencing analysis revealed that 380 down-regulated and 602 up-regulated differentially expressed genes (DEGs) were identified in HTM cells treated with KD025 compared with those that were untreated. Gene ontology analysis revealed that DEGs were more frequently related to the plasma membrane, extracellular components and integral cellular components among cellular components, and related to signaling receptor binding and activity and protein heterodimerization activity among molecular functions. Ingenuity Pathway Analysis (IPA) revealed that the detected DEGs were associated with basic cellular biological and physiological properties, including cellular movement, development, growth, proliferation, signaling and interaction, all of which are associated with cellular metabolism. Furthermore, the upstream regulator analysis and causal network analysis estimated IL-6, STAT3, CSTA and S1PR3 as possible regulators. Current findings herein indicate that ROCK2 mediates the IL-6/STAT3-, CSTA- and S1PR3-linked signaling related to basic biological activities such as glycolysis in HTM cells.

Effect of lactate supplementation to skeletal muscle cells on fiber type transition and cellular bioenergetics

It is well-known that lactate (LAC) is the primary cellular end-product of glycolysis. However, during intense exercise and transient hypoxic conditions, the intracellular LAC levels are significantly elevated. Recently, our in vitro biochemical and biophysical studies have revealed that LAC avidly binds to oxy-myoglobin (oxy-Mb) in acidic conditions and rapidly releases oxygen (O2). Based on these results, we hypothesize that LAC plays a significant role in tissue O2 management combating hypoxic conditions in high demanding exercising tissues, such as Mb rich skeletal muscles type I oxidative fibers. Here, we have investigated three types of mouse C2C12 cell line models, i.e. Wild-type (WT) cells, Mb knock-out (MbKO) cells, and Empty vector (EV) cells. 5-day old differentiated C2C12 cells were supplemented with varying concentrations (3 mM, 5 mM and 8 mM) of sodium lactate added to the DMEM growth medium. Thereafter, cells were incubated in temperature regulated CO2 incubator for varying time periods (3 h, 6 h, and 24 h) to evaluate the early and prolonged effect on tissue fiber type transition and cellular bioenergetics. Metabolic assays such as hexokinase activity, LAC dehydrogenase (LDH) activity, and glucose uptake were performed using the intracellular cell lysate whereas extracellular LAC levels were measured using LAC assay kit. Seahorse experiments were conducted to measure the cellular glycolytic and mitochondrial ATP production rates. Our findings revealed that, there was no significant difference in myotube width in C2C12 cells either with or without LAC supplementation across all the test conditions. Intriguingly, at lower LAC supplementation (1.8 mM) conditions, the extracellular LAC levels were found to be significantly increased in WT cells compared to MbKO cells. However, at mimicking hyperlactatemia (5 mM LAC) and lactic acidosis (8 mM LAC) conditions, distinct extracellular LAC levels were recorded with WT and MbKO cells. Our results clearly suggests that Mb containing cells effciently utilize LAC for cellular bioenergetics and other biochemical processes. Similarly, in response to increasing LAC supplementation levels, LDH activity was also found to higher in WT cells compared to MbKO cells. Furthermore, glucose uptake in MbKO cells was significantly decreased. Finally, significantly lower rates of glycolytic ATP production was observed when compared to mitochondrial ATP production in WT cells with similar overall ATP production rates in all treatment conditions and incubation periods. However, marginal decrease in both glycolytic and mitochondrial ATP production rates were observed in MbKO cells. Overall, this study clearly demonstrated that external supplementation of LAC to C2C12 cells had no significant effect on fiber type transition but reveals that Mb plays a significant role in LAC utilization. USDA-ARS project 6026-51000-012-06S, Sturgis Foundation Pilot Grant G1-54750-01 and ACRI-ABI Investigator Initiated Grant Award GR037175-4450S ABI. 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.

Ceramides as Mediators of Mitochondrial Dysfunction Driving Kidney Injury

Objective. Perturbations in proximal tubule mitochondrial function and lipid metabolism potentiate acute kidney injury and failed repair. Sphingolipids such as ceramides are recognized as lipotoxic drivers of metabolic dysfunction and cell death. We hypothesized that ceramides drive mitochondrial dysfunction and tubular insult and that lowering ceramides would prevent acute kidney injury in a mouse model of bilateral ischemia reperfusion injury (IRI). Methods. We examined acute effects of ceramide treatment on primary mouse proximal tubule epithelial cell (PTEC) mitochondrial oxygen consumption and mitochondrial supercomplex assembly by Seahorse respirometry and blue native PAGE, respectively. In vivo, we profiled accumulation of ceramides via HPLC-MS/MS in kidney cortex tissue of mice challenged with warm bilateral IRI. We lowered whole-body ceramides via Degs1 inducible global knockout ( Degs1iGKO) in male C57Bl/6J mice prior to challenge with IRI. Clinical, transcriptional, and histopathological renal outcomes were compared to sham-operated and Degs1fl/fl controls 24 hours after kidney reperfusion. Results. Treatment of PTECs with 5-50uM C2-ceramide resulted in acute and dose-dependent decreases in mitochondrial ATP production rates (15-65% decrease from baseline, respectively, p<0.05), as well as disruption of electron transport chain supercomplex structures (i.e., III2+IV and I+III2+IV). In mice, IRI led to a 25% increase in total cortex ceramides (sham: 995±110 vs IRI: 1239±185 pmol lipid/mg protein, p<0.05). Lipid species within the de novo ceramide synthesis pathway were elevated as early as 5 minutes after IRI (e.g., 34% increase in Cer(d18:0/16:0), sham: 2.38±0.27 vs 5 minutes: 3.20±0.81 pmol lipid/mg protein, p<0.05) and continued to accumulate throughout the progression of the AKI up to 24 hours after reperfusion (e.g., 122% increase in Cer(d18:0/16:0), sham: 2.38±0.27 vs 24 hours: 5.29±1.71 pmol lipid/mg protein, p<0.001). Cortex ceramides, particularly deleterious long-chain (e.g., Cer(d18:1/16:0)) species, correlated positively with kidney injury biomarkers (plasma creatinine, Pearson r=0.57; BUN, r=0.74; and cortex expression of Lcn2, r=0.60 , and Havcr1, r=0.62 , p<0.05). Degs1 global knockout decreased cortex ceramide levels by 56% (fl/fl: 995±110 vs KO: 435±174 pmol lipid/mg protein, p<0.001). Degs1iGKO animals had improved cortex histopathology, blood urea nitrogen (fl/fl: 81.02±2.13 vs KO: 49.82±6.34 mg/dL, p <0.001), plasma creatinine (fl/fl: 1.28±0.4 vs KO: 0.32±0.11 mg/dL, p<0.001), and cortex Lcn2 expression (fl/fl: 36.36±21.50 vs KO: 5.31±4.61, p<0.01) following IRI. Conclusions. Renal ceramides are implicated as drivers of kidney injury and mitochondrial dysfunction. Global Degs1 knockout prevented AKI induced by ischemia reperfusion injury. Thus, ceramide-lowering interventions may be an effective strategy to prevent AKI. This work was supported by funding from the National Institutes of Health [R01KD130296 (SAS, WLH), R01DK131609 (SAS), U01CA272529 (SAS), R44DK116450 (SAS), R01DK112826 (WLH), T32DK091317 (RJN), F31DK134088 (RJN)]. 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.

Pioglitazone reverses alcohol-induced alterations in alveolar macrophage mitochondrial phenotype.

People with alcohol use disorder (AUD) have an increased risk of developing pneumonia and pulmonary diseases. Alveolar macrophages (AMs) are immune cells of the lower respiratory tract that are necessary for clearance of pathogens. However, alcohol causes AM oxidative stress, mitochondrial damage and dysfunction, and diminished phagocytic capacity, leading to lung injury and immune suppression. AMs were isolated by bronchoalveolar lavage from people with AUD and male and female C57BL/6J mice given chronic ethanol (20% w/v, 12 weeks) in drinking water. The peroxisome proliferator-activated receptor γ ligand, pioglitazone, was used to treat human AMs exvivo (10 μM, 24 h) and mice invivo by oral gavage (10 mg/kg/day). Levels of AM mitochondrial superoxide and hypoxia-inducible factor-1 alpha (HIF-1α) mRNA, a marker of oxidative stress, were measured by fluorescence microscopy and RT-qPCR, respectively. Mouse AM phagocytic ability was determined by internalized Staphylococcus aureus, and mitochondrial capacity, dependency, and flexibility for glucose, long-chain fatty acid, and glutamine oxidation were measured using an extracellular flux analyzer. Invitro studies used a murine AM cell line, MH-S (±0.08% ethanol, 72 h) to investigate mitochondrial fuel oxidation and ATP-linked respiration. Pioglitazone treatment decreased mitochondrial superoxide in AMs from people with AUD and ethanol-fed mice and HIF-1α mRNA in ethanol-fed mouse lungs. Pioglitazone also reversed mouse AM glutamine oxidation and glucose or long-chain fatty acid flexibility to meet basal oxidation needs. Invitro, ethanol decreased the rate of AM mitochondrial and total ATP production, and pioglitazone improved changes in glucose and glutamine oxidation. Pioglitazone reversed chronic alcohol-induced oxidative stress in human AM and mitochondrial substrate oxidation flexibility and superoxide levels in mouse AM. Decreased ethanol-induced AM HIF-1α mRNA with pioglitazone suggests that this pathway may be a focus for metabolic-targeted therapeutics to improve morbidity and mortality in people with AUD.

An adenosine derivative promotes mitochondrial supercomplexes reorganization and restoration of mitochondria structure and bioenergetics in a diethylnitrosamine-induced hepatocellular carcinoma model

Hepatocellular carcinoma (HCC) progression is associated with dysfunctional mitochondria and bioenergetics impairment. However, no data about the relationship between mitochondrial supercomplexes (hmwSC) formation and ATP production rates in HCC are available. Our group has developed an adenosine derivative, IFC-305, which improves mitochondrial function, and it has been proposed as a therapeutic candidate for HCC. We aimed to determine the role of IFC-305 on both mitochondrial structure and bioenergetics in a sequential cirrhosis-HCC model in rats. Our results showed that IFC-305 administration decreased the number and size of liver tumors, reduced the expression of tumoral markers, and reestablished the typical architecture of the hepatic parenchyma. The livers of treated rats showed a reduction of mitochondria number, recovery of the mtDNA/nDNA ratio, and mitochondrial length. Also, IFC-305 increased cardiolipin and phosphatidylcholine levels and promoted hmwSC reorganization with changes in the expression levels of hmwSC assembly-related genes. IFC-305 in HCC modified the expression of several genes encoding elements of electron transport chain complexes and increased the ATP levels by recovering the complex I, III, and V activity. We propose that IFC-305 restores the mitochondrial bioenergetics in HCC by normalizing the quantity, morphology, and function of mitochondria, possibly as part of its hepatic restorative effect.

Environmentally Relevant Concentrations of Tetrabromobisphenol A Exposure Impends Neurovascular Formation through Perturbing Mitochondrial Metabolism in Zebrafish Embryos and Human Primary Endothelial Cells.

Tetrabromobisphenol A (TBBPA), the most extensively utilized brominated flame retardant, has raised growing concerns regarding its environmental and health risks. Neurovascular formation is essential for metabolically supporting neuronal networks. However, previous studies primarily concerned the neuronal injuries of TBBPA, its impact on the neurovascularture, and molecular mechanism, which are yet to be elucidated. In this study, 5, 30, 100, 300 μg/L of TBBPA were administered to Tg (fli1a: eGFP) zebrafish larvae at 2-72 h postfertilization (hpf). The findings revealed that TBBPA impaired cerebral and ocular angiogenesis in zebrafish. Metabolomics analysis showed that TBBPA-treated neuroendothelial cells exhibited disruption of the TCA cycle and the Warburg effect pathway. TBBPA induced a significant reduction in glycolysis and mitochondrial ATP production rates, accompanied by mitochondrial fragmentation and an increase in mitochondrial reactive oxygen species (mitoROS) production in neuroendothelial cells. The supplementation of alpha-ketoglutaric acid, a key metabolite of the TCA cycle, mitigated TBBPA-induced mitochondrial damage, reduced mitoROS production, and restored angiogenesis in zebrafish larvae. Our results suggested that TBBPA exposure impeded neurovascular injury via mitochondrial metabolic perturbation mediated by mitoROS signaling, providing novel insight into the neurovascular toxicity and mode of action of TBBPA.

Alcohol and its metabolites dysregulate cellular bioenergetics and induce oxidative and endoplasmic reticulum stress in primary human bronchial epithelial cells.

Chronic alcohol consumption/misuse is a significant risk factor for pneumonia and lung infection leading to the development of chronic pulmonary disorders such as chronic obstructive pulmonary disease (COPD) and lung fibrosis. In this study, we sought to delineate the mechanism of alcohol-associated lung disease. We did so by measuring invitro mitochondrial, endoplasmic reticulum (ER) oxidative stress in human bronchial epithelial cells (hBECs) treated with ethanol and its oxidative (acetaldehyde) and nonoxidative (fatty acid ethyl esters or FAEEs) metabolites. Primary hBECs from a normal subject were treated with relevant concentrations of ethanol and its metabolites and incubated at 37°C for 24 h. Viability and cytotoxicity were determined using cell viability and lactate dehydrogenase (LDH) assay kits, respectively. Oxidized glutathione (GSSG) and reduced glutathione (GSH) were measured by colorimetric reaction, and 4-hydroxynenonal (4HNE) by immunohistochemistry. Endoplasmic reticulum stress and dysregulated cellular bioenergetics were determined by western blot analysis. Mitochondrial stress and real-time ATP production rates were determined using a Seahorse Extracellular Flux analyzer. Amelioration of ethanol-induced oxidative/ER stress and mitochondrial energetics was determined using an AMPKα agonist. Human bronchial epithelial cells treated with ethanol, acetaldehyde, and FAEEs showed a concentration-dependent increase in the secretion of LDH, oxidative/ER stress, deactivation of AMPKα phosphorylation and mitochondrial stress (decreased spare respiratory capacity) with concomitant decreases in mitochondrial and glycolytic ATP production rates. FAEEs caused greater cytotoxicity, ER stress, and dysregulated cellular bioenergetics than those ethanol and its oxidative metabolite. AMPKα agonist-pretreated cells significantly ameliorated ethanol-induced oxidative/ER stress, deactivation of AMPKα, and dysregulated cellular bioenergetics. Findings of this study suggest that ethanol and its metabolites contribute to cytotoxicity, oxidative/ER stress, and dysregulation of cellular bioenergetics in hBECs. The attenuation of ethanol-induced ER/oxidative stress and mitochondrial respiration by an AMPKα agonist may reflect a potential for it to be developed as a therapeutic agent for chronic alcohol-associated lung disease.

Novel Relationship between Mitofusin 2-Mediated Mitochondrial Hyperfusion, Metabolic Remodeling, and Glycolysis in Pulmonary Arterial Endothelial Cells.

The disruption of mitochondrial dynamics has been identified in cardiovascular diseases, including pulmonary hypertension (PH), ischemia-reperfusion injury, heart failure, and cardiomyopathy. Mitofusin 2 (Mfn2) is abundantly expressed in heart and pulmonary vasculature cells at the outer mitochondrial membrane to modulate fusion. Previously, we have reported reduced levels of Mfn2 and fragmented mitochondria in pulmonary arterial endothelial cells (PAECs) isolated from a sheep model of PH induced by pulmonary over-circulation and restoring Mfn2 normalized mitochondrial function. In this study, we assessed the effect of increased expression of Mfn2 on mitochondrial metabolism, bioenergetics, reactive oxygen species production, and mitochondrial membrane potential in control PAECs. Using an adenoviral expression system to overexpress Mfn2 in PAECs and utilizing 13C labeled substrates, we assessed the levels of TCA cycle metabolites. We identified increased pyruvate and lactate production in cells, revealing a glycolytic phenotype (Warburg phenotype). Mfn2 overexpression decreased the mitochondrial ATP production rate, increased the rate of glycolytic ATP production, and disrupted mitochondrial bioenergetics. The increase in glycolysis was linked to increased hypoxia-inducible factor 1α (HIF-1α) protein levels, elevated mitochondrial reactive oxygen species (mt-ROS), and decreased mitochondrial membrane potential. Our data suggest that disrupting the mitochondrial fusion/fission balance to favor hyperfusion leads to a metabolic shift that promotes aerobic glycolysis. Thus, therapies designed to increase mitochondrial fusion should be approached with caution.

In vitro reduction of bovine oocyte ATP production with oligomycin affects embryo epigenome.

Epigenetic programming is a crucial process during early embryo development that can have a significant impact on the results of assisted reproductive technology (ART) and offspring health. Here we show evidence using a bovine in vitro experiment that embryo epigenetic programing is dependent on oocyte mitochondrial bioenergetic activity during maturation. This study investigated if oocyte and early embryo epigenetic programming are dependent on oocyte mitochondrial ATP production. A bovine in vitro experiment was performed in which oocyte mitochondrial ATP production was reduced using 5 nmol/L oligomycin A (OM; ATP synthase inhibitor) during in vitro maturation (IVM) compared to control (CONT). OM exposure significantly reduced mitochondrial ATP production rate in MII oocytes (34.6% reduction, P = 0.018) and significantly decreased embryo cleavage rate at 48 h post insemination (7.6% reduction, P = 0.031). Compared to CONT, global DNA methylation (5mC) levels were decreased in OM-exposed MII oocytes (9.8% reduction, P = 0.019) while global histone methylation (H3K9me2) was increased (9.4% increase, P = 0.024). In zygotes, OM exposure during IVM increased 5mC (22.3% increase, P < 0.001) and histone acetylation (H3K9ac, 17.3% increase, P = 0.023) levels, while H3K9me2 levels were not affected. In morulae, 5mC levels were increased (10.3% increase, P = 0.041) after OM exposure compared to CONT, while there was no significant difference in H3K9ac and H3K9me2 levels. These epigenetic alterations were not associated with any persistent effects on embryo mitochondrial ATP production rate or mitochondrial membrane potential (assessed at the four-cell stage). Also, epigenetic regulatory genes were not differentially expressed in OM-exposed zygotes or morulae. Finally, apoptotic cell index in blastocysts was increased after OM exposure during oocyte maturation (41.1% increase, P < 0.001). We conclude that oocyte and early embryo epigenetic programming are dependent on mitochondrial ATP production during IVM.

Abstract A108: Metabolic oriented treatment: efficacy of sr59230a 𝜷3-adrenergic receptor antagonist, and sr plus buformin® in Ewing sarcoma

Abstract Purpose of study: Metabolic targeted therapies may represent an innovative strategy in Ewing sarcoma (ES). Introduction: Ewing sarcoma (ES) is highly aggressive round cell mesenchymal neoplasm, most often occurring in children and young adults. Metabolic peculiarities of ES cells are indeed involved in tumor growth, survival and resistance to therapy. We have further investigated apoptotic role of 𝜷3-adrenergic receptor (𝜷3-AR) SR59230A (SR) antagonist for its ability to reduce cells viability due increased oxidative stress, now we underline the metabolic role of SR. Materials &amp; Methods: The metabolic profile after SR administration in A673 ES cell model was studied using the Seahorse XF Analyzer. Viability was evaluated with MTS. Changes in gene expression was evaluated with RNA sequencing. Western blot analysis and Immunofluorescence was performed for redox signature and metabolic alteration. Ros production was assessed by flow cytometry. Results: Quantification of transcript abundance using Salmon in A673 after SR treatment showed decrease in response to reactive oxygen species, increase of Thioredoxin Interacting Protein (TXNIP) inhibits the antioxidative function of thioredoxin, moreover reduced glucose uptake, consequently decrease NADPH/NADP resulting in the accumulation of ROS coincident with a decrease of GLUT1, SOD2, TrDX an increase of LC3IIA protein expression. SR enhances A673 pyruvate dependency, and decrease glyco and mito ATP production rate, more over observed under glucose deprivation. To inibite mitochondrial ATP production rate we proposed the metabolic oriented treatment with Buformin® (BUF) an oral antidiabetic drug of the biguanide class. BUF completely shut down mitochondrial respiration acted on complex I of electron transport chain (ETC). Buf combined with SR decrease ATP production and up-regulated ROS production in ES cells. Conclusion: SR antagonist of 𝜷3-AR and combination of SR with BUF could be an innovative therapeutic option to raise awareness ES. Citation Format: Cristina Banella, Laura Zocca, Alessia Boaretto, Gianluca Mattei, Marina Mola, Lara Ballerini, Lorito Nicla, Annalisa Tondo, Maura Calvani. Metabolic oriented treatment: efficacy of sr59230a 𝜷3-adrenergic receptor antagonist, and sr plus buformin® in Ewing sarcoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A108.

Bioenergetic effects of pristine and ultraviolet-weathered polydisperse polyethylene terephthalate and polystyrene nanoplastics on human intestinal Caco-2 cells

The ubiquitous human exposure to nanoplastics (NPs) increasingly raises concerns regarding impact on our health. However, little is known on the biological effects of complex mixtures of weathered NPs with heterogenous size and irregular shape present in the environment. In this study, the bioenergetic effects of four such NPs mixtures on human intestinal Caco-2 cells were investigated. To this aim, Caco-2 cells were exposed to polydisperse nanoPET (<800 nm) and nanoPS (mixture of 100 and 750 nm) samples with and without ultraviolet (UV) weathering at low concentration range (102–107 particles/mL) for 48 h. Mitochondrial respiration, glycolytic functions and ATP production rates of exposed cells were measured by Seahorse XFe96 Analyzer. Among four NPs samples, polydisperse nanoPET with irregular shapes induced significant stimulation of mitochondrial respiration, glycolysis and ATP production rates in Caco-2 cells. Spherical nanoPS caused significant stimulation on glycolytic functions of Caco-2 cells at the highest concentration used (106 particles/mL). ATR-FTIR spectra and carbonyl index indicated formation of carbonyl groups in nanoPET and nanoPS after UV weathering. UV weathering could alleviate bioenergetic stress caused by NPs in Caco-2 cells and even shifted the energy pathways from mitochondrial respiration to glycolysis due to electrostatic repulsion between negatively charged UV-aged NPs and cell membranes. This research is the first to study in-vitro bioenergetic responses of NPs samples with multidimensional features (polymer type, irregular shape, heterogenous size, UV-weathering) on human health. It highlights that effects between pristine and weathered NPs are different at a bioenergetic level, which has important implications for the risk assessment of NPs on human health.

Respiratory syncytial virus co-opts hypoxia-inducible factor-1α-mediated glycolysis to favor the production of infectious virus.

Respiratory syncytial virus (RSV) is the leading etiological agent of lower respiratory tract illness. However, efficacious vaccines or antiviral drugs for treating RSV infections are currently not available. Indeed, RSV depends on host cells to provide energy needed to produce progeny virions. Glycolysis is a series of oxidative reactions used to metabolize glucose and provide energy to host cells. Therefore, glycolysis may be helpful for RSV infection. In this study, we show that RSV increases glycolysis by inducing the stabilization, transcription, translation, and activation of hypoxia-inducible factor (HIF)-1α in infected cells, which is important for the production of progeny RSV virions. This study contributes to understanding the molecular mechanism by which HIF-1α-mediated glycolysis controls RSV infection and reveals an effective target for the development of highly efficient anti-RSV drugs.

Glycolytic reprogramming in macrophages and MSCs during inflammation.

Dysregulated inflammation is associated with many skeletal diseases and disorders, such as osteolysis, non-union of fractures, osteonecrosis, osteoarthritis and orthopaedic infections. We previously showed that continuous infusion of lipopolysaccharide (LPS) contaminated polyethylene particles (cPE) caused prolonged inflammation and impaired bone formation. However, the metabolic and bioenergetic processes associated with inflammation of bone are unknown. Mitochondria are highly dynamic organelles that modulate cell metabolism and orchestrate the inflammatory responses that involve both resident and recruited cells. Glycolytic reprogramming, the shift from oxidative phosphorylation (OXPHOS) to glycolysis causes inappropriate cell activation and function, resulting in dysfunctional cellular metabolism. We hypothesized that impaired immunoregulation and bone regeneration from inflammatory states are associated with glycolytic reprogramming and mitochondrial dysfunction in macrophages (Mφ) and mesenchymal stromal cells (MSCs). We used the Seahorse XF96 analyzer and real-time qPCR to study the bioenergetics of Mφ and MSCs exposed to cPE. To understand the oxygen consumption rate (OCR), we used Seahorse XF Cell Mito Stress Test Kit with Seahorse XF96 analyzer. Similarly, Seahorse XF Glycolytic Rate Assay Kit was used to detect the extracellular acidification rate (ECAR) and Seahorse XF Real-Time ATP Rate Assay kit was used to detect the real-time ATP production rates from OXPHOS and glycolysis. Real-time qPCR was performed to analyze the gene expression of key enzymes in glycolysis and mitochondrial biogenesis. We further detected the gene expression of proinflammatory cytokines in Mφ and genes related to cell differentiation in MSC during the challenge of cPE. Our results demonstrated that the oxidative phosphorylation of Mφ exposed to cPE was significantly decreased when compared with the control group. We found reduced basal, maximal and ATP-production coupled respiration rates, and decreased proton leak in Mφ during challenge with cPE. Meanwhile, Mφ showed increased basal glycolysis and proton efflux rates (PER) when exposed to cPE. The percentage (%) of PER from glycolysis was higher in Mφ exposed to cPE, indicating that the contribution of the glycolytic pathway to total extracellular acidification was elevated during the challenge of cPE. In line with the results of OCR and ECAR, we found Mφ during cPE challenge showed higher glycolytic ATP (glycoATP) production rates and lower mitochondrial ATP (mitoATP) production rates which is mainly from OXPHOS. Interestingly, MSCs showed enhanced glycolysis during challenge with cPE, but no significant changes in oxygen consumption rates (OCR). In accordance, seahorse assay of real-time ATP revealed glycoATP rates were elevated while mitoATP rates showed no significant differences in MSC during challenge with cPE. Furthermore, Mφ and MSCs exposed to cPE showed upregulated gene expression levels of glycolytic regulators and Mφ exposed to cPE expressed higher levels of pro-inflammatory cytokines. This study demonstrated the dysfunctional bioenergetic activity of bone marrow-derived Mφ and MSCs exposed to cPE, which could impair the immunoregulatory properties of cells in the bone niche. The underlying molecular defect related to disordered mitochondrial function could represent a potential therapeutic target during the resolution of inflammation.

Cell bioenergetics and ATP production of boar spermatozoa

Cellular metabolism is an important feature of spermatozoa that deserves more insights to be fully understood, in particular in porcine semen physiology. The present study aims to characterize the balance between glycolytic and oxidative metabolism in boar sperm cells. Agilent Seahorse technology was used to assess both oxygen consumption rate (OCR), as an oxidative metabolism index, and extracellular acidification rate (ECAR), as a glycolytic index. Different metabolic parameters were studied on freshly ejaculated sperm cells (identified as day zero sample, d0) and after one day of storage at 17 °C in Androhep extender (d1). Mitochondrial ATP production rate (MitoATP) was higher than the glycolytic ATP production rate (glycoATP) at both d0 and d1 while at d1 the amount of ATP production decreased, in particular, due to OXPHOS reduction. Conversely, glycoATP was not significantly different between d0 and d1. Interestingly, OCR profile showed no different bioenergetic parameters (i.e. ATP turnover, basal or maximal respiration, and spare respiration) between d0 and d1, thus indicating that sperm cell metabolism was reversibly decreased by preservation conditions. Other metabolic parameters showed the same trend, irrespective of the storage time: under stressed conditions (oligomycin plus FCCP), spermatozoa showed an increase in mitochondrial respiration while the metabolic potential of glycolysis did not undergo variations when compared to baseline metabolism. The rate of oxidation of fuel substrates – glucose, fatty acids, and glutamine – showed that sperm reliance on glucose oxidation to maintain baseline respiration was higher than fatty acids or glutamine. Interestingly spermatozoa demonstrated to have a low “capacity” parameter, which indicates that they cannot use only a single fuel substrate to produce energy. This feature of sperm metabolism to be unable to increase oxidation of a particular fuel to compensate for inhibition of alternative fuel pathway(s) was demonstrated by the negative value of “flexibility”. Our results showed that ATP production in boar sperm cells relied on mitochondrial oxidative metabolism in freshly ejaculated cells, while, under liquid storage conditions, their oxidative metabolism decreased while the glycolysis remained constant. These results open new fields of research in the preservation techniques of boar sperm cells.

FTY720-P, a Biased S1PR Ligand, Increases Mitochondrial Function through STAT3 Activation in Cardiac Cells.

FTY720 is an FDA-approved sphingosine derivative drug for the treatment of multiple sclerosis. This compound blocks lymphocyte egress from lymphoid organs and autoimmunity through sphingosine 1-phosphate (S1P) receptor blockage. Drug repurposing of FTY720 has revealed improvements in glucose metabolism and metabolic diseases. Studies also demonstrate that preconditioning with this compound preserves the ATP levels during cardiac ischemia in rats. The molecular mechanisms by which FTY720 promotes metabolism are not well understood. Here, we demonstrate that nanomolar concentrations of the phosphorylated form of FTY720 (FTY720-P), the active ligand of S1P receptor (S1PR), activates mitochondrial respiration and the mitochondrial ATP production rate in AC16 human cardiomyocyte cells. Additionally, FTY720-P increases the number of mitochondrial nucleoids, promotes mitochondrial morphology alterations, and induces activation of STAT3, a transcription factor that promotes mitochondrial function. Notably, the effect of FTY720-P on mitochondrial function was suppressed in the presence of a STAT3 inhibitor. In summary, our results suggest that FTY720 promotes the activation of mitochondrial function, in part, through a STAT3 action.

MiR-130a-3p regulates FUNDC1-mediated mitophagy by targeting GJA1 in myocardial ischemia/reperfusion injury

Understanding the complex pathogenesis in myocardial ischemia/reperfusion (I/R) injury (IRI) is an urgent problem in clinical trials. Increasing pieces of evidence have suggested that miRNAs are involved in the occurrence and development of heart diseases by regulating mitochondria-related gene expression. Mitochondria have been acknowledged as the key triggers of cardiac I/R injury. However, the potential impact of miR-130a on mitochondria remains unclear in myocardial IRI. Exploring the regulatory mechanism of miR-130a on mitochondria may provide a new target for IRI therapy. In the present study, we found that miR-130a significantly increased in acute myocardial infarction (AMI) patients and myocardial I/R rats. MiR-130a could downregulate the viability of cardiomyocytes and the knockdown of miR-130a could protect the viability of cardiomyocytes under hypoxia-reoxygenation (HR). Over-expression of miR-130a resulted in mitochondrial dysfunction. It was evidenced by decreases in mitochondrial ATP production, mitochondrial membrane potential (MMP), and an increase in reactive oxygen species (ROS) production. However, suppression of miR-130a could protect against mitochondrial damage, show elevation of mitochondrial ATP production rate and MMP, and reduce ROS production. We further explored the effect of miR-130a on the mitochondrial quality control (QMC) system by determining mitochondrial-protein-specific proteases and analyzed mitochondrial morphology by fluorescence imaging and electron microscopy, respectively. It was noted that miR-130a could suppress mitochondrial fusion and FUNDC1-mediated mitophagy to accelerate myocardial IRI. Moreover, we investigated the potential miR-130a targeted mitochondria-related genes to understand the regulatory mechanism of miR-130a in the setting of myocardial IRI. It was revealed that miR-130a targeted GJA1, and GJA1 rescued IRI by enhancing ATP production rate and oxidative phosphorylation, meanwhile protecting cell viability, MMP, and activating mitophagy. In addition, the knockdown of miR-130a significantly activated FUNDC1-mediated mitophagy, while the knockdown of GJA1 reversed the relevant response. Collectively, our findings suggest that miR-130a regulates FUNDC1-mediated mitophagy by targeting GJA1 in myocardial IRI.

Molecular mechanism of glycolytic assembly on mitochondria through O-GlcNAcylation.

Glucose is the primary fuel of cells, and its metabolism starts with the activity of the first rate-limiting enzyme Hexokinase (HK). HK1 is the dominant isoform in the brain and is mainly localized near the mitochondrial outer membrane. The positioning of HK1 on mitochondria is critical because it couples two energy generation pathways: Glycolysis and mitochondrial oxidative phosphorylation. Here, we report a new molecular mechanism that regulates HK1 activity and mitochondrial localization via the metabolic sensor enzyme O-GlcNAc transferase (OGT). OGT catalyzes a reversible post-translational modification by adding a GlcNAc sugar moiety to serine and threonine residues (O-GlcNAcylation). By combining experiments and molecular simulations, this study shows that the dynamical modification of HK1 with O-GlcNAcylation at its regulatory domain disrupts the product, G6P, from binding to this non-catalytic domain. We further demonstrate that G6P binding to the non-catalytic domain plays a role in controlling mitochondrial localization through electrostatic repulsion due to the additional negative charge from G6P. In addition, we demonstrated that O-GlcNAc modification induces a conformational change which increases mitochondrial HK1 and enhances glycolytic and mitochondrial ATP production rates. By computing binding affinities of the O-GlcNAc modified and unmodified HK1, we see that the O-GlcNAcylation decreases G6P and increases ATP binding to HK1. Furthermore, by mutating the O-GlcNAcylation site of HK1, we see a decrease in both glycolytic and mitochondrial ATP production rate and dysfunction of presynaptic vesicle releasing in neurons. Our findings may reveal key molecular pathways that couple neuronal metabolism to mitochondrial function via OGT and how their dysregulation leads to neurological disorders.

Tipping the balance toward stemness in trophoblast: Metabolic programming by Cox6B2.

Metabolic effector(s) driving cell fate is an emerging concept in stem cell biology. Here we showed that Cytochrome C Oxidase Subunit 6B2 (Cox6B2) is essential to maintain the stemness of trophoblast stem (TS) cells. RNA interference of Cox6b2 resulted in decreased mitochondrial Complex IV activity, ATP production, and oxygen consumption rate in TS cells. Furthermore, depletion of Cox6b2 in TS cells led to decreased self-renewal capacity indicated by compromised BrdU incorporation, Ki67 staining, and decreased expression of TS cell genetic markers. As expected, the consequence of Cox6b2 knockdown was the induction of differentiation. TS cell stemness factor CDX2 transactivates Cox6b2 promoter in TS cells. In differentiated cells, Cox6b2 is post-transcriptionally regulated by two microRNAs, miR-322-5p and miR-503-5p,leading to its downregulation as demonstrated by the gain-in or loss of function of these miRNAs. Cox6b2 transcripts gradually rise in placental trophoblast gestation progresses in both mice and rats with predominant expression in labyrinthine trophoblast. Cox6b2 expression is compromised in the growth-restricted placenta of rats with reciprocal up-regulation of miR-322-5p and miR-503-5p. These data highlight the importance of Cox6B2 in the regulation of TS cell state and uncompromised placental growth.

Bioenergetic and Metabolic Impairments in Induced Pluripotent Stem Cell-Derived Cardiomyocytes Generated from Duchenne Muscular Dystrophy Patients.

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene and dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality in DMD patients. We tested the hypothesis that DCM is caused by metabolic impairments by employing induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from four DMD patients; an adult male, an adult female, a 7-year-old (7y) male and a 13-year-old (13y) male, all compared to two healthy volunteers. To test the hypothesis, we measured the bioenergetics, metabolomics, electrophysiology, mitochondrial morphology and mitochondrial activity of CMs, using respirometry, LC–MS, patch clamp, electron microscopy (EM) and confocal microscopy methods. We found that: (1) adult DMD CMs exhibited impaired energy metabolism and abnormal mitochondrial structure and function. (2) The 7y CMs demonstrated arrhythmia-free spontaneous firing along with “healthy-like” metabolic status, normal mitochondrial morphology and activity. In contrast, the 13y CMs were mildly arrhythmogenic and showed adult DMD-like bioenergetics deficiencies. (3) In DMD adult CMs, mitochondrial activities were attenuated by 45–48%, whereas the 7y CM activity was similar to that of healthy CMs. (4) In DMD CMs, but not in 7y CMs, there was a 75% decrease in the mitochondrial ATP production rate compared to healthy iPSC-CMs. In summary, DMD iPSC-CMs exhibit bioenergetic and metabolic impairments that are associated with rhythm disturbances corresponding to the patient’s phenotype, thereby constituting novel targets for alleviating cardiomyopathy in DMD patients.