Lower Oxygen Consumption Rate Research Articles

Mitochondria from the systemic heart of Pacific hagfish (Eptatretus stoutii) are insensitive to one hour of anoxia followed by reoxygenation

Pacific hagfish (Eptatretus stoutii) are an ancient agnathan vertebrate known to be anoxia tolerant. To study their metabolic organization and the role of the mitochondria in anoxia tolerance we developed a novel protocol to measure mitochondrial function in permeabilized cardiomyocytes and how this is affected by one hour of anoxia followed by reoxygenation. When measured at 10 °C the mitochondria had a respiration rate of 2.1 ± 0.1pmol/s/mg WW during OXPHOS with saturating concentrations of glutamate, malate, and succinate. This is comparatively low compared to other ectothermic species. The functional characteristics of the mitochondria were quantified with mitochondrial control ratios. These demonstrated that proton leak contributed to just under 50% of the oxygen flux, with the remainder going towards ATP phosphorylation. Finally, when the preparations were exposed to an anoxia-reoxygenation protocol there was no difference in respiration compared to that of a heart sample from the same animal maintained under normoxia for the same time. When Complex I alone or Complex I and II were stimulated following one hour of anoxia there was no decline in oxygen flux observed. However, if Complex II was activated alone there was a significant decline in respiration. This decrease was however also observed in the mitochondria maintained in normoxia for one hour. In conclusion, Pacific hagfish cardiac mitochondria demonstrated a low rate of oxygen consumption, a loosely coupled electron transfer system, and a resistance to one hour of anoxia.

Multi-omic analysis of bat versus human fibroblasts reveals altered central metabolism.

Bats have unique characteristics compared to other mammals, including increased longevity and higher resistance to cancer and infectious disease. While previous studies have analyzed the metabolic requirements for flight, it is still unclear how bat metabolism supports these unique features, and no study has integrated metabolomics, transcriptomics, and proteomics to characterize bat metabolism. In this work, we performed a multi-omics data analysis using a computational model of metabolic fluxes to identify fundamental differences in central metabolism between primary lung fibroblast cell lines from the black flying fox fruit bat (Pteropus alecto) and human. Bat cells showed higher expression levels of Complex I components of electron transport chain (ETC), but, remarkably, a lower rate of oxygen consumption. Computational modeling interpreted these results as indicating that Complex II activity may be low or reversed, similar to an ischemic state. An ischemic-like state of bats was also supported by decreased levels of central metabolites and increased ratios of succinate to fumarate in bat cells. Ischemic states tend to produce reactive oxygen species (ROS), which would be incompatible with the longevity of bats. However, bat cells had higher antioxidant reservoirs (higher total glutathione and higher ratio of NADPH to NADP) despite higher mitochondrial ROS levels. In addition, bat cells were more resistant to glucose deprivation and had increased resistance to ferroptosis, one of the characteristics of which is oxidative stress. Thus, our studies revealed distinct differences in the ETC regulation and metabolic stress responses between human and bat cells.

Consuming a Linoleate-Rich Diet Increases Concentrations of Tetralinoleoyl Cardiolipin in Mouse Liver and Alters Hepatic Mitochondrial Respiration

BackgroundHepatic mitochondrial dysfunction is a major cause of fat accumulation in the liver. Individuals with fatty liver conditions have hepatic mitochondrial structural abnormalities and a switch in the side chain composition of the mitochondrial phospholipid, cardiolipin, from poly- to monounsaturated fatty acids. Linoleic acid (LA), an essential dietary fatty acid, is required to remodel nascent cardiolipin (CL) to its tetralinoleoyl cardiolipin (L4CL, CL with 4 LA side chains) form, which is integral for mitochondrial membrane structure and function to promote fatty acid oxidation. It is unknown, however, whether increasing LA in the diet can increase hepatic L4CL concentrations and improve mitochondrial respiration in the liver compared with a diet rich in monounsaturated and saturated fatty acids. ObjectivesThe main aim of this study was to test the ability of a diet fortified with LA-rich safflower oil (SO), compared with the one fortified with lard (LD), to increase concentrations of L4CL and improve mitochondrial respiration in the livers of mice. MethodsTwenty-four (9-wk-old) C57 BL/J6 male mice were fed either the SO or LD diets for ∼100 d, whereas food intake and body weight, fasting glucose, and glucose tolerance tests were performed to determine any changes in glycemic control. ResultsLivers from mice fed SO diet had higher relative concentrations of hepatic L4CL species compared with LD diet-fed mice (P value = 0.004). Uncoupled mitochondria of mice fed the SO diet, compared with LD diet, had an increased baseline oxygen consumption rate (OCR) and succinate-driven respiration (P values = 0.03 and 0.01). SO diet-fed mice had increased LA content in all phospholipid classes compared with LD-fed mice (P < 0.05). ConclusionsOur findings reveal that maintaining or increasing hepatic L4CL may result in increased OCR in uncoupled hepatic mitochondria in healthy mice whereas higher oleate content of CL reduced mitochondrial function shown by lower OCR in uncoupled mitochondria.

Mitochondrial H2O2 Is a Central Mediator of Diclofenac-Induced Hepatocellular Injury.

Nonsteroidal anti-inflammatory drug (NSAID) use is associated with adverse consequences, including hepatic injury. The detrimental hepatotoxicity of diclofenac, a widely used NSAID, is primarily connected to oxidative damage in mitochondria, which are the primary source of reactive oxygen species (ROS). The primary ROS responsible for inducing diclofenac-related hepatocellular toxicity and the principal antioxidant that mitigates these ROS remain unknown. Peroxiredoxin III (PrxIII) is the most abundant and potent H2O2-eliminating enzyme in the mitochondria of mammalian cells. Here, we investigated the role of mitochondrial H2O2 and the protective function of PrxIII in diclofenac-induced mitochondrial dysfunction and apoptosis in hepatocytes. Mitochondrial H2O2 levels were differentiated from other types of ROS using a fluorescent H2O2 indicator. Upon diclofenac treatment, PrxIII-knockdown HepG2 human hepatoma cells showed higher levels of mitochondrial H2O2 than PrxIII-expressing controls. PrxIII-depleted cells exhibited higher mitochondrial dysfunction as measured by a lower oxygen consumption rate, loss of mitochondrial membrane potential, cardiolipin oxidation, and caspase activation, and were more sensitive to apoptosis. Ectopic expression of mitochondrially targeted catalase in PrxIII-knockdown HepG2 cells or in primary hepatocytes derived from PrxIII-knockout mice suppressed the diclofenac-induced accumulation of mitochondrial H2O2 and decreased apoptosis. Thus, we demonstrated that mitochondrial H2O2 is a key mediator of diclofenac-induced hepatocellular damage driven by mitochondrial dysfunction and apoptosis. We showed that PrxIII loss results in the critical accumulation of mitochondrial H2O2 and increases the harmful effects of diclofenac. PrxIII or other antioxidants targeting mitochondrial H2O2 could be explored as potential therapeutic agents to protect against the hepatotoxicity associated with NSAID use.

#69 : Decline in Mitochondrial Function of Human Embryo with Maternal Aging

Background and Aims: Female fertility declines with age, due to increased chromosomal aneuploidy and possible reduced mitochondrial function in the embryo. Method: We outlined how mitochondrial function in human embryos, as predicted from oxygen consumption rate (OCR) measurements, changes in pre-implantation stage, and what factors, particularly maternal age, affect mitochondrial function in embryos. Results: The structure of the mitochondrial inner membrane and its respiratory function developed with embryo development, while the copy number of mitochondrial DNA per specimen was transiently reduced compared to that of the oocyte. The undifferentiated state of the inner cell mass cells appears to be associated with a low OCR. In contrast, the copy number of mitochondrial DNA increased in trophoblast cells and mitochondrial aerobic metabolism increased. The OCRs at morulae stage decreased with maternal age, but there was no relationship between maternal age and the copy number of mitochondrial DNA at any stages. The higher oxygen spent at the morula stage; the shorter time was needed for development to the mid-stage blastocyst. Conclusion: The mitochondrial respiratory function of human embryos developed along with embryonic growth. Mitochondrial function at morula stage declined with their maternal age and reduced mitochondrial function decreased the rate of development from morula to blastocyst.

Abstract 17976: Delivery of Allogeneic Mitochondria Accelerates Revascularization in a Murine Model of Peripheral Arterial Disease and Reverses Metabolic Signatures of Endothelial Senescence

Introduction: Peripheral arterial disease (PAD) is characterized by limb ischemia and biological therapeutics to treat PAD are limited. PAD associated with mitochondrial dysfunction in both endothelial cells and skeletal muscle. Hypothesis: Therefore, we hypothesized that mitochondrial transplantation (mito-transfer) can alleviate the cellular abnormalities linked to mitochondrial dysfunction in PAD. Methods: Mitochondria from allogeneic fibroblasts were transferred into human microvascular endothelial cells (ECs) of varying passages (p5-14) by centrifugation. After mito-transfer (24h), the metabolic activity and proliferative capacity of ECs were respectively quantified by extracellular flux analysis and EdU incorporation into DNA. Results: Compared to non-manipulated ECs, mito-transferred ECs had lower oxygen consumption rates (OCR) related to mitochondrial respiration (basal, maximal, and spare respiratory capacity, and mitochondrial ATP production). No significant differences were observed in the extracellular acidification rates related to the glycolytic activity of ECs. However, the ATP index (ratio of mitochondrial ATP/ glycolytic ATP) of late passage (p10) ECs significantly decreased after mito-transfer. Furthermore, compared to non-manipulated ECs, mito-transferred ECs of late passage (p10-14) showed a 25% increase in proliferation. Mito-transfer to the ischemic limb induced a 2-fold increase in limb perfusion after 14 days (Fig. 1). Conclusions: Collectively, these data show that EC aging is associated with increased oxidative phosphorylation metabolism and that mito-transfer decreased the senescence-associated oxidative glucose metabolism of ECs, enhanced EC proliferation, as well as accelerated revascularization in PAD. Mito-transfer is a promising strategy to treat PAD to improve revascularization and mitochondrial function.

Microbiota influences host exercise capacity via modulation of skeletal muscle glucose metabolism in mice

The microbiota enhances exercise performance and regulates host physiology and energy metabolism by producing beneficial metabolites via bacterial fermentation. In this study, we discovered that germ-free (GF) mice had a reduced capacity for aerobic exercise as well as low oxygen consumption rates and glucose availability. Surprisingly, GF mice showed lower body weight gain and lower fat mass than specific pathogen-free (SPF) mice. Therefore, we hypothesized that these paradoxical phenotypes could be mediated by a compensatory increase in lipolysis in adipose tissues owing to impaired glucose utilization in skeletal muscle. Our data revealed that gut microbiota depletion impairs host aerobic exercise capacity via the deterioration of glucose storage and utilization. The improved browning ability of GF mice may have contributed to the lean phenotype and negatively affected energy generation. These adaptations limit obesity in GF mice but impede their immediate fuel supply during exercise, resulting in decreased exercise performance.

Antibiotics reduce Pocillopora coral-associated bacteria diversity, decrease holobiont oxygen consumption and activate immune gene expression.

Corals are important models for understanding invertebrate host-microbe interactions; however, to fully discern mechanisms involved in these relationships, experimental approaches for manipulating coral-bacteria associations are needed. Coral-associated bacteria affect holobiont health via nutrient cycling, metabolic exchanges and pathogen exclusion, yet it is not fully understood how bacterial community shifts affect holobiont health and physiology. In this study, a combination of antibiotics (ampicillin, streptomycin and ciprofloxacin) was used to disrupt the bacterial communities of 14 colonies of the reef framework-building corals Pocillopora meandrina and P. verrucosa, originally collected from Panama and hosting diverse algal symbionts (family Symbiodiniaceae). Symbiodiniaceae photochemical efficiencies and holobiont oxygen consumption (as proxies for coral health) were measured throughout a 5-day exposure. Antibiotics altered bacterial community composition and reduced alpha and beta diversity, however, several bacteria persisted, leading to the hypothesis that these bacteria are either antibiotics resistant or occupy internal niches that are shielded from antibiotics. While antibiotics did not affect Symbiodiniaceae photochemical efficiency, antibiotics-treated corals had lower oxygen consumption rates. RNAseq revealed that antibiotics increased expression of Pocillopora immunity and stress response genes at the expense of cellular maintenance and metabolism functions. Together, these results reveal that antibiotic disruption of corals' native bacteria negatively impacts holobiont health by decreasing oxygen consumption and activating host immunity without directly impairing Symbiodiniaceae photosynthesis, underscoring the critical role of coral-associated bacteria in holobiont health. They also provide a baseline for future experiments that manipulate Pocillopora corals' symbioses by first reducing the diversity and complexity of coral-associated bacteria.

Abstract 138: Delivery of Allogeneic Mitochondria Accelerates Revascularization in a Murine Model of Peripheral Arterial Disease and Reverses Metabolic Signatures of Endothelial Senescence

Peripheral arterial disease (PAD) is associated with mitochondrial dysfunction in both endothelial cells and skeletal muscle. We examined whether mitochondrial transplantation (mito-transfer) can alleviate the cellular abnormalities linked to mitochondrial dysfunction in PAD. Mitochondria from allogeneic fibroblasts were transferred into human microvascular endothelial cells (ECs) of varying passages (p5-14) by centrifugation. After mito-transfer (24h), the metabolic activity and proliferative capacity of ECs were respectively quantified by extracellular flux analysis (Mito-Stress Test, Glycolysis Stress Test, and ATP rate assays) and EdU incorporation into DNA. To test the efficacy of mito-transfer for the treatment of PAD, mitochondria were injected into the murine ischemic limb and blood perfusion recovery was monitored. Compared to non-manipulated ECs, mito-transferred ECs had lower oxygen consumption rates (OCR) related to mitochondrial respiration (basal, maximal, and spare respiratory capacity, and mitochondrial ATP production). No significant differences were observed in the extracellular acidification rates related to the glycolytic activity of ECs. However, the ATP index (ratio of mitochondrial ATP/ glycolytic ATP) of late passage (p10) ECs significantly decreased after mito-transfer. Furthermore, compared to non-manipulated ECs, mito-transferred ECs of late passage (p10-14) showed a 25% increase in proliferation. Mito-transfer to the ischemic limb induced a 2-fold increase in limb perfusion after 14 days (Fig. 1). Collectively, these data show that EC aging is associated with increased oxidative phosphorylation metabolism and that mito-transfer decreased the senescence-associated oxidative glucose metabolism of ECs, enhanced EC proliferation, as well as accelerated revascularization in PAD. Mito-transfer is a promising strategy to treat PAD to improve revascularization and mitochondrial function.

Activation of PINK1-mediated mitophagy protects bovine mammary epithelial cells against lipopolysaccharide-induced mitochondrial and inflammatory damage in vitro.

Increased metabolic stress during early lactation results in damage of mitochondria and inflammatory responses in bovine mammary epithelial cells, both of which could be aggravated by inhibition of mitophagy. PTEN-induced putative kinase 1 (PINK1)-mediated mitophagy is essential in the removal of damaged mitochondria and the regulation of inflammatory responses. The aim of the present study was to elucidate the role of PINK1-mediated mitophagy on mitochondrial damage and inflammatory responses in bovine mammary epithelial cells challenged with lipopolysaccharide (LPS). Exogenous LPS activated mitophagy and led to lower protein abundance of oxidative phosphorylation (OXPHOS) complexes (COI-V) and lower oxygen consumption rate (OCR) along with increased mitochondrial reactive oxygen species (Mito-ROS) content. These effects were also associated with increased protein abundance of Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) in a time-dependent manner. Pretreatment with 3-Methyladenine (3-MA) or knockdown of PINK1 aggravated the downregulation of COI-V protein abundance, the increase in Mito-ROS content, and the protein abundance of NLRP3, Cleaved-Caspase-1 and IL-1β induced by LPS. Overexpression of PINK1 activated mitophagy and alleviated LPS-induced NLRP3 inflammasome activation by reducing Mito-ROS production. Overall, the data suggested that PINK1-mediated mitophagy is a crucial anti-inflammatory mechanism that removes damaged mitochondria in bovine mammary epithelial cells experiencing an increased inflammatory load.

Increased stromal PFKFB3-mediated glycolysis in inflammatory bowel disease contributes to intestinal inflammation.

Inflammatory bowel disease (IBD) is a chronic relapsing inflammation of the intestinal tract with currently not well-understood pathogenesis. In addition to the involvement of immune cells, increasing studies show an important role for fibroblasts in the pathogenesis of IBD. Previous work showed that glycolysis is the preferred energy source for fibroblasts in fibrotic diseases. 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) is a key kinase supporting glycolysis. Increased expression of PFKFB3 in several cancers and inflammatory diseases has been previously reported, but the metabolic status of fibroblasts and the role of PFKFB3 in patients with IBD are currently unknown. Therefore, in this study, we evaluated the role of glycolysis and PFKFB3 expression in IBD. Single-sample gene set enrichment analysis (ssGSEA) revealed that glycolysis was significantly higher in IBD intestinal samples, compared to healthy controls, which was confirmed in the validation cohorts of IBD patients. Single-cell sequencing data indicated that PFKFB3 expression was higher in IBD-derived stromal cells. In vitro, PFKFB3 expression in IBD-derived fibroblasts was increased after the stimulation with pro-inflammatory cytokines. Using seahorse real-time cell metabolic analysis, inflamed fibroblasts were shown to have a higher extracellular acidification rate and a lower oxygen consumption rate, which could be reversed by inhibition of JAK/STAT pathway. Furthermore, increased expression of pro-inflammatory cytokines and chemokines in fibroblasts could be reverted by PFK15, a specific inhibitor of PFKFB3. In vivo experiments showed that PFK15 reduced the severity of dextran sulfate sodium (DSS)- and Tcell transfer induced colitis, which was accompanied by a reduction in immune cell infiltration in the intestines. These findings suggest that increased stromal PFKFB3 expression contributes to inflammation and the pathological function of fibroblasts in IBD. Inhibition of PFKFB3 suppressed their inflammatory characteristics.

Divergent Cellular Energetics, Glutamate Metabolism, and Mitochondrial Function Between Human and Mouse Cerebral Cortex.

Disruptions of brain energy and neurotransmitter metabolism are associated with several pathological conditions including neurodegenerative diseases such as Alzheimer's disease. Transgenic rodent models, and in vitro preparations hereof, are often applied for studying pathological aspects of brain metabolism. However, despite the conserved cerebral development across mammalian species, distinct differences in cellular composition and structure may influence metabolism of the rodent and human brain. To address this, we investigated the metabolic function of acutely isolated brain slices and non-synaptic mitochondria obtained from the cerebral cortex of mice and neurosurgically resected neocortical tissue of humans. Utilizing dynamic isotope labeling with 13C-enriched metabolic substrates, we show that metabolism of glucose, acetate, β-hydroxybutyrate, and glutamine operates at lower rates in human cerebral cortical slices when compared to mouse slices. In contrast, human cerebral cortical slices display a higher capacity for converting exogenous glutamate into glutamine, which subsequently supports neuronal GABA synthesis, whereas mouse slices primarily convert glutamate into aspartate. In line with the reduced metabolic rate of the human brain slices, isolated non-synaptic mitochondria of the human cerebral cortex have a lower oxygen consumption rate when provided succinate as substrate. However, when provided pyruvate and malate, human mitochondria display a higher coupled respiration and lower proton leak, signifying a more efficient mitochondrial coupling compared to mouse mitochondria. This study reveals key differences between mouse and human brain metabolism concerning both neurons and astrocytes, which must be taken into account when applying in vitro rodent preparations as a model system of the human brain.

Larger Nile tilapia struggle in hot water

When your metabolism depends on the temperature of your surroundings, climate change represents a unique and alarming challenge. As temperatures increase, your metabolic rate rises too, requiring that you consume more oxygen to maintain your body at a faster pace. ‘At warmer temperatures, larger individuals may be prone to limitations in their capacity for oxygen uptake’, says David McKenzie, at the CNRS's Centre for Marine Biodiversity Exploitation and Conservation, Montpellier, France, with the consequence that larger fish may struggle to survive at higher temperatures. ‘If true, this would be important in projecting how global warming will affect fish populations’, says McKenzie; the size distribution of fish populations could become smaller as temperatures rise. ‘But evidence is lacking’, McKenzie admits. So, he and colleagues from the Federal University of São Carlos, Brazil, decided to find out how well Nile tilapia (Oreochromis niloticus), ranging in size from 21 to 313 g, managed to keep swimming at high speed as the temperature rose. First, the team tested how resilient the resting fish were as the water temperature rose. Initially, the fish began behaving erratically but eventually they toppled over when the temperature was too high. However, the largest fish were as resilient as the smallest; getting enough oxygen was not an issue when the fish were resting. ‘They may lose their balance for another reason when they are warmed up while resting in a tank’, McKenzie says.He and his colleagues then set the fish a challenge, encouraging them to swim at 85% of their top speed while the researchers gently increased the water temperature, and they found that the largest animals struggled most as the temperature rose. They gave up swimming at lower temperatures (around 37°C), while the smaller fish managed to keep going at temperatures up to 39.5°C. And when the team investigated the fish's oxygen consumption as the temperature increased, they found the largest fish got out of breath at far lower oxygen consumption rates (∼18 mmol O2 kg−1 h−1) than their smaller counterparts (41 mmol O2 kg−1 h−1).So, larger tilapia struggle to keep swimming as the temperature rises and they seem to find it difficult to get enough oxygen, which could have implications for the size of fish in future populations. McKenzie suspects that ‘Global warming may favour the small’, as smaller individuals may be able to take the heat while larger fish may have more trouble surviving in hot water.

Thermal tolerance of fish to heatwaves in agricultural streams: What does not kill you makes you stronger?

Abstract Heatwaves are increasing in frequency and intensity under climate change. Freshwater ecosystems are among the most thermally impacted systems, within which agricultural streams are experiencing the most extreme heatwaves and deserve prioritised focus. Heatwaves are approaching the upper thermal limits of many fishes but have received little attention to date. To study whether and how fish tolerate heatwaves from a physiological perspective, we simulated single, multiple, and extended heatwaves at 32 and 34°C in the laboratory, based on high‐resolution summer temperatures recorded in agricultural versus forested streams in Illinois, U.S.A. By investigating the effects of heatwaves on 25°C acclimated fathead minnow Pimephales promelas, an important prey species across North America, we witnessed its high thermal resilience, including a rapid return to metabolic homeostasis after single and multiple heatwaves, measured by oxygen consumption rate. During an extended heatwave, fathead minnow were still able to partially lower oxygen consumption rate after the initial exposure. We also found transient increases in their critical thermal maximum, especially after higher intensity and frequency of heatwaves. However, the thermal resilience of fathead minnow did come with costs, including reduced anaerobic capacity indicated by decreased lactate dehydrogenase activity and impaired antioxidant defence indicated by reduced superoxide dismutase in white muscle. By monitoring metabolic costs and physiological adjustments of fish during and after heatwaves, we showed that fathead minnow were resilient to simulated current and near‐future heatwaves, which may allow them to cope with thermal extremes expected in agricultural streams. Overall, the real‐time monitoring of fish responses to heatwaves incorporates natural dynamics of thermal patterns. It facilitates mechanistic understandings of how fish react to thermal challenges in the real world and offers opportunities to incorporate high‐resolution metabolic costs into future bioenergetic modelling.

Abstract 888: Elucidating the mechanisms of eATP-induced changes in cell growth, energy metabolism, and cancer stem cells in human non-small lung cancer A549 cells

Abstract The tumor microenvironment (TME) has concentrations of extracellular ATP (eATP) 103-104 times higher than normal healthy tissues, implying that eATP may have important roles in tumor development and progression. Our previous study, cited by a Nature Reviews Cancer paper, showed that the extracellular ATP (eATP) is internalized by tumors via macropinocytosis, a type of endocytosis, to increase intracellular ATP (iATP) levels and support tumor cells growth, drug resistance, and cancer cell movement by initiating epithelial to mesenchymal transition (EMT). Recently, our lab found that eATP functions in promoting cancer cell growth, metastasis are similar to those of TGF-β, a well-known EMT and cancer stem cells (CSCs) inducer. Increasing reports state that the EMT and CSCs share some molecular links, but the detailed mechanisms and inductor of them are still far from clear. In this report, eATP’s function in CSCs induction was investigated first. Result shows that eATP treatment significantly induces the protein expression level of CSC related makers, increases cell colony numbers, and cell populations with CSC markers expressed in human lung cancer A549 cells. In addition, RNA-sequencing analysis reveals the gene Stanniocalcin 1 (STC1) is significantly upregulated in both eATP and TGF-β treatments. STC1 gene has been reported to be a competitive biomarker in the tracking of cancers’ progressing. Besides, STC1 exhibits concentration-dependent stimulation effects on electron transportation chain (ETC) and respiration. However, the detail function of STC1 gene in eATP induced cell growth is unknown. To find out the mechanism, CRISPR-Cas9 technique was used to generate STC1 gene targeted knockout (STC1 KO) A549 cells. STC1 KO cells show slower cell growth rate, lower intracellular ATP level and lower oxygen consumption rate (OCR) when compared with normal A549 cells in vitro. These results strongly indicate STC1 gene involved in eATP induced cell growth by alteration iATP level and metabolic profile. Overall, this study not only unravels new role of eATP in CSC induction, but also provides new mechanism of eATP induced cell growth by altering the protein expression level of STC1. Citation Format: Jingwen Song, Xiaozhuo Chen. Elucidating the mechanisms of eATP-induced changes in cell growth, energy metabolism, and cancer stem cells in human non-small lung cancer A549 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 888.

Patient-derived microphysiological model identifies the therapeutic potential of metformin for thoracic aortic aneurysm.

SummaryBackgroundThoracic aortic aneurysm (TAA) is the permanent dilation of the thoracic aortic wall that predisposes patients to lethal events such as aortic dissection or rupture, for which effective medical therapy remains scarce. Human-relevant microphysiological models serve as a promising tool in drug screening and discovery.MethodsWe developed a dynamic, rhythmically stretching, three-dimensional microphysiological model. Using patient-derived human aortic smooth muscle cells (HAoSMCs), we tested the biological features of the model and compared them with native aortic tissues. Drug testing was performed on the individualized TAA models, and the potentially effective drug was further tested using β-aminopropionitrile-treated mice and retrospective clinical data.FindingsThe HAoSMCs on the model recapitulated the expressions of many TAA-related genes in tissue. Phenotypic switching and mitochondrial dysfunction, two disease hallmarks of TAA, were highlighted on the microphysiological model: the TAA-derived HAoSMCs exhibited lower alpha-smooth muscle actin expression, lower mitochondrial membrane potential, lower oxygen consumption rate and higher superoxide accumulation than control cells, while these differences were not evidently reflected in two-dimensional culture flasks. Model-based drug testing demonstrated that metformin partially recovered contractile phenotype and mitochondrial function in TAA patients’ cells. Mouse experiment and clinical investigations also demonstrated better preserved aortic microstructure, higher nicotinamide adenine dinucleotide level and lower aortic diameter with metformin treatment.InterpretationThese findings support the application of this human-relevant microphysiological model in studying personalized disease characteristics and facilitating drug discovery for TAA. Metformin may regulate contractile phenotypes and metabolic dysfunctions in diseased HAoSMCs and limit aortic dilation.FundingThis work was supported by grants from National Key R&D Program of China (2018YFC1005002), National Natural Science Foundation of China (82070482, 81771971, 81772007, 51927805, and 21734003), the Science and Technology Commission of Shanghai Municipality (20ZR1411700, 18ZR1407000, 17JC1400200, and 20YF1406900), Shanghai Municipal Science and Technology Major Project (2017SHZDZX01), and Shanghai Municipal Education Commission (Innovation Program 2017-01-07-00-07-E00027). Y.S.Z. was not supported by any of these funds; instead, the Brigham Research Institute is acknowledged.

Genetic Ablation of Na,K-ATPase α4 Results in Sperm Energetic Defects.

The Na,K-ATPase alpha 4 isoform (NKAα4) is expressed specifically in the male germ cells of the testes and is particularly abundant in mature spermatozoa. Genetic deletion of NKAα4 in mice (NKAα4 KO mice) results in complete infertility of male, but not female mice. The reduced fecundity of NKAα4 KO male mice is due to a series of defects, including a severe impairment in total and hyperactive sperm motility. In this work, we show that deletion of NKAα4 also leads to major defects in sperm metabolism and energetics. Thus, compared to wild-type sperm, sperm from NKAα4 KO mice display a significant reduction in the extracellular acidification rate (ECAR), indicative of impaired glycolytic flux. In addition, mitochondrial function is disrupted in sperm lacking NKAα4, as indicated by a reduction in the mitochondrial membrane potential and lower oxygen consumption rate (OCR). Moreover, the ratio between the oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD/NADH) is increased in NKAα4 KO sperm, indicating a shift in the cellular redox state. These metabolic changes are associated with augmented reactive oxygen species (ROS) production and increased lipid peroxidation in NKAα4 KO sperm. Altogether, these findings reveal a novel link between NKAα4 activity and sperm energetics, highlighting the essential role of this ion transporter in sperm physiology.

Metabolic Reprogramming in Response to Alterations of Mitochondrial DNA and Mitochondrial Dysfunction in Gastric Adenocarcinoma

We used gastric cancer cell line AGS and clinical samples to investigate the roles of mitochondrial DNA (mtDNA) alterations and mitochondrial respiratory dysfunction in gastric adenocarcinoma (GAC). A total of 131 clinical samples, including 17 normal gastric mucosa (N-GM) from overweight patients who had received sleeve gastrectomy and 57 paired non-cancerous gastric mucosae (NC-GM) and GAC from GAC patients who had undergone partial/subtotal/total gastrectomy, were recruited to examine the copy number and D310 sequences of mtDNA. The gastric cancer cell line AGS was used with knockdown (KD) mitochondrial transcription factor A (TFAM) to achieve mitochondrial dysfunction through a decrease of mtDNA copy number. Parental (PT), null-target (NT), and TFAM-KD-(A/B/C) represented the parental, control, and TFAM knocked-down AGS cells, respectively. These cells were used to compare the parameters reflecting mitochondrial biogenesis, glycolysis, and cell migration activity. The median mtDNA copy numbers of 17 N-GM, 57 NC-GM, and 57 GAC were 0.058, 0.055, and 0.045, respectively. The trend of decrease was significant (p = 0.030). In addition, GAC had a lower mean mtDNA copy number of 0.055 as compared with the paired NC-GM of 0.078 (p < 0.001). The mean mtDNA copy number ratio (mtDNA copy number of GAC/mtDNA copy number of paired NC-GM) was 0.891. A total of 35 (61.4%) GAC samples had an mtDNA copy number ratio ≤0.804 (p = 0.017) and 27 (47.4%) harbored a D310 mutation (p = 0.047), and these patients had shorter survival time and poorer prognosis. After effective knockdown of TFAM, TFAM-KD-B/C cells expressed higher levels of hexokinase II (HK-II) and v-akt murine thymoma viral oncogene homolog 1 gene (AKT)-encoded AKT, but lower levels of phosphorylated pyruvate dehydrogenase (p-PDH) than did the NT/PT AGS cells. Except for a higher level of p-PDH, the expression levels of these proteins remained unchanged in TFAM-KD-A, which had a mild knockdown of TFAM. Compared to those of NT, TFAM-KD-C had not only a lower mtDNA copy number (p = 0.050), but also lower oxygen consumption rates (OCR), including basal respiration (OCRBR), ATP-coupled respiration (OCRATP), reserve capacity (OCRRC), and proton leak (OCRPL)(all with p = 0.050). In contrast, TFAM-KD-C expressed a higher extracellular acidification rate (ECAR)/OCRBR ratio (p = 0.050) and a faster wound healing migration at 6, 12, and 18 h, respectively (all with p = 0.050). Beyond a threshold, the decrease in mtDNA copy number, the mtDNA D310 mutation, and mitochondrial dysfunction were involved in the carcinogenesis and progression of GACs. Activation of PDH might be considered as compensation for the mitochondrial dysfunction in response to glucose metabolic reprogramming or to adjust mitochondrial plasticity in GAC.

Nischarin Deletion Reduces Oxidative Metabolism and Overall ATP: A Study Using a Novel NISCHΔ5-6 Knockout Mouse Model

Nischarin (Nisch) is a cytosolic scaffolding protein that harbors tumor-suppressor-like characteristics. Previous studies have shown that Nisch functions as a scaffolding protein and regulates multiple biological activities. In the current study, we prepared a complete Nisch knockout model, for the first time, by deletion of exons 5 and 6. This knockout model was confirmed by Qrt–PCR and Western blotting with products from mouse embryonic fibroblast (MEF) cells. Embryos and adult mice of knockouts are significantly smaller than their wild-type counterparts. Deletion of Nisch enhanced cell migration, as demonstrated by wound type and transwell migration assays. Since the animals were small in size, we investigated Nisch’s effect on metabolism by conducting several assays using the Seahorse analyzer system. These data indicate that Nisch null cells have lower oxygen consumption rates, lower ATP production, and lower levels of proton leak. We examined the expression of 15 genes involved in lipid and fat metabolism, as well as cell growth, and noted a significant increase in expression for many genes in Nischarin null animals. In summary, our results show that Nischarin plays an important physiological role in metabolic homeostasis.

P028 Inhibition of stromal glycolysis by targeting PFKFB3 decreases experimental colitis

Abstract Background Studies in fibrotic diseases revealed that glycolysis is the preferred energy source for fibroblasts. 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) has the highest kinase activity to shunt glucose toward glycolysis. Therefore inhibition of PFKFB3 has been proposed as a potential target for several cancers and inflammatory diseases. However, the metabolic status of fibroblasts in patients with inflammatory bowel disease (IBD) and the role of PFKFB3 are currently unknown. Methods Single-sample gene set enrichment analysis (ssGSEA) of GSE16879 was performed to evaluate metabolic changes in IBD. Seahorse real-time cell metabolic analysis was performed to explore the metabolic activity of fibroblasts. Next the expression of PFKFB3 in primary patient derived intestinal fibroblast was determined by quantitative PCR (qPCR) and western blot under normal and inflammatory conditions. Proliferation and migration of fibroblasts were measured using colony formation and wound healing assays. In order to evaluate the effect of the inhibition of PFKFB3 in vivo, PFK15, a specific inhibitor of PFKFB3, was intraperitoneal injected in mice with dextran sodium sulfate (DSS)-induced colitis and in the T-cell transfer model for colitis. Next to clinical parameters, the abundance of α-smooth muscle actin (α-SMA) expressing fibroblasts, immune cells (CD45) and endothelial cells (CD105) was determined by immunohistochemistry. Results The ssGSEA analysis revealed glycolysis was significantly higher in IBD patients, compared to healthy controls. Consistently, the expression of PFKFB3 was also elevated in a cohort of inflamed intestinal tissues from IBD patients compared to non-inflamed sites from the same patient or healthy controls. On the cellular levels, this analysis showed that PFKB3 expression was higher in IBD-derived stromal cells compared to healthy or non-inflamed stromal cells. In vitro PFKFB3 expression in fibroblasts was increased after the stimulation with pro-inflammatory cytokines like TNF-α and a mix of cytokines often upregulated in IBD patients: interleukin (IL)-17A, oncostatin M (OSM) and IL-1β. As for the metabolic changes, inflamed fibroblasts had a higher extracellular acidification rate and a lower oxygen consumption rate, which could be reverted by inhibition of PFKFB3 using PFK15. Furthermore, PFK15 suppressed the proliferation and migration of fibroblasts. The in vivo experiments showed that PFK15 reduced the severity of the colitis, accompanied by a reduction of the total amount of immune cells (CD45), activated fibroblasts (α-SMA) and angiogenesis (CD105). Conclusion Increased PFKFB3 expression seems to contribute the inflammation and the pathological function of fibroblasts in IBD.