Mitochondrial Respiration Rate Research Articles

Orthogonal Persulfide Generation through Precision Tools Provides Insights into Mitochondrial Sulfane Sulfur.

The sulfane sulfur pool, comprised of persulfide (RS-SH) and polysulfide (RS-SnH) derived from hydrogen sulfide (H2S), has emerged as a major player in redox biochemistry. Mitochondria, besides energy generation, serve as significant cellular redox hubs, mediate stress response and cellular health. However, the effects of endogenous mitochondrial sulfane sulfur (MSS) remain largely uncharacterized as compared with their cytosolic counterparts, cytosolic sulfane sulfur (CSS). To investigate this, we designed a novel artificial substrate for mitochondrial 3-mercaptopyruvate sulfurtransferase (3-MST), a key enzyme involved in MSS biosynthesis. Using cells expressing a mitochondrion-localized persulfide biosensor, we demonstrate this tool's ability to selectively enhance MSS. While H2S was previously known to suppress human immunodeficiency virus (HIV-1), we found that MSS profoundly affected the HIV-1 life cycle, mediating viral reactivation from latency. Additionally, we provide evidence for the role of the host's mitochondrial redox state, membrane potential, apoptosis, and respiration rates in managing HIV-1 latency and reactivation. Together, dynamic fluctuations in the MSS pool have a significant and possibly conflicting effect on HIV-1 viral latency. The precision tools developed herein allow for orthogonal generation of persulfide within both mitochondria and the cytosol and will be useful in interrogating disease biology.

Chronic changes in developmental oxygen have little effect on mitochondria and tracheal density in the endothermic moth Manduca sexta.

Oxygen availability during development is known to impact the development of insect respiratory and metabolic systems. Drosophila adult tracheal density exhibits developmental plasticity in response to hypoxic or hyperoxic oxygen levels during larval development. Respiratory systems of insects with higher aerobic demands, such as those that are facultative endotherms, may be even more responsive to oxygen levels above or below normoxia during development. The moth Manduca sexta is a large endothermic flying insect that serves as a good study system to start answering questions about developmental plasticity. In this study, we examined the effect of developmental oxygen levels (hypoxia: 10% oxygen, and hyperoxia: 30% oxygen) on the respiratory and metabolic phenotype of adult moths, focusing on morphological and physiological cellular and intercellular changes in phenotype. Mitochondrial respiration rate in permeabilized and isolated flight muscle was measured in adults. We found that permeabilized flight muscle fibers from the hypoxic group had increased mitochondrial oxygen consumption, but this was not replicated in isolated flight muscle mitochondria. Morphological changes in the trachea were examined using confocal imaging. We used transmission electron microscopy to quantify muscle and mitochondrial density in the flight muscle. The respiratory morphology was not significantly different between developmental oxygen groups. These results suggest that the developing M. sexta trachea and mitochondrial respiration have limited developmental plasticity when faced with rearing at 10% or 30% oxygen.

Disruption in glutathione metabolism and altered energy production in the liver and kidney after ischemic acute kidney injury in mice

Acute kidney injury (AKI) is a systemic disease that affects energy metabolism in various remote organs in murine models of ischemic AKI. However, AKI-mediated effects in the liver have not been comprehensively assessed. After inducing ischemic AKI in 8–10-week-old, male C57BL/6 mice, mass spectrometry metabolomics revealed that the liver had the most distinct phenotype 24 h after AKI versus 4 h and 7 days. Follow up studies with in vivo [13C6]-glucose tracing on liver and kidney 24 h after AKI revealed 4 major findings: (1) increased flux through glycolysis and the tricarboxylic (TCA) cycle in both kidney and liver; (2) depleted hepatic glutathione levels and its intermediates despite unchanged level of reactive oxygen species, suggesting glutathione consumption exceeds production due to systemic oxidative stress after AKI; (3) hepatic ATP depletion despite unchanged rate of mitochondrial respiration, suggesting increased ATP consumption relative to production; (4) increased hepatic and renal urea cycle intermediates suggesting hypercatabolism and upregulation of the urea cycle independent of impaired renal clearance of nitrogenous waste. Taken together, this is the first study to describe the hepatic metabolome after ischemic AKI in a murine model and demonstrates that there is significant liver-kidney crosstalk after AKI.

In Vitro Hypoxia/Reoxygenation Induces Mitochondrial Cardiolipin Remodeling in Human Kidney Cells.

Renal ischemia/reperfusion is a serious condition that not only causes acute kidney injury, a severe clinical syndrome with high mortality, but is also an inevitable part of kidney transplantation or other kidney surgeries. Alterations of oxygen levels during ischemia/reperfusion, namely hypoxia/reoxygenation, disrupt mitochondrial metabolism and induce structural changes that lead to cell death. A signature mitochondrial phospholipid, cardiolipin, with many vital roles in mitochondrial homeostasis, is one of the key players in hypoxia/reoxygenation-induced mitochondrial damage. In this study, we analyze the effect of hypoxia/reoxygenation on human renal proximal tubule epithelial cell (RPTEC) cardiolipins, as well as their metabolism and mitochondrial functions. RPTEC cells were placed in a hypoxic chamber with a 2% oxygen atmosphere for 24 h to induce hypoxia; then, they were replaced back into regular growth conditions for 24 h of reoxygenation. Surprisingly, after 24 h, hypoxia cardiolipin levels substantially increased and remained higher than control levels after 24 h of reoxygenation. This was explained by significantly elevated levels of cardiolipin synthase and lysocardiolipin acyltransferase 1 (LCLAT1) gene expression and protein levels. Meanwhile, hypoxia/reoxygenation decreased ADP-dependent mitochondrial respiration rates and oxidative phosphorylation capacity and increased reactive oxygen species generation. Our findings suggest that hypoxia/reoxygenation induces cardiolipin remodeling in response to reduced mitochondrial oxidative phosphorylation in a way that protects mitochondrial function.

Activation of the hypoxia-inducible factor pathway by roxadustat improves glucose metabolism in human primary myotubes from men.

Hypoxia-inducible factor prolyl 4-hydroxylase (HIF-P4H) enzymes regulate adaptive cellular responses to low oxygen concentrations. Inhibition of HIF-P4Hs leads to stabilisation of hypoxia-inducible factors (HIFs) and activation of the HIF pathway affecting multiple biological processes to rescue cells from hypoxia. As evidence from animal models suggests that HIF-P4H inhibitors could be used to treat metabolic disorders associated with insulin resistance, we examined whether roxadustat, an HIF-P4H inhibitor approved for the treatment of renal anaemia, would have an effect on glucose metabolism in primary human myotubes. Primary skeletal muscle cell cultures, established from biopsies of vastus lateralis muscle from men with normal glucose tolerance (NGT) (n=5) or type 2 diabetes (n=8), were treated with roxadustat. Induction of HIF target gene expression was detected with quantitative real-time PCR. Glucose uptake and glycogen synthesis were investigated with radioactive tracers. Glycolysis and mitochondrial respiration rates were measured with a Seahorse analyser. Exposure to roxadustat stabilised nuclear HIF1α protein expression in human myotubes. Treatment with roxadustat led to induction of HIF target gene mRNAs for GLUT1 (also known as SLC2A1), HK2, MCT4 (also known as SLC16A4) and HIF-P4H-2 (also known as PHD2 or EGLN1) in myotubes from donors with NGT, with a blunted response in myotubes from donors with type 2 diabetes. mRNAs for LDHA, PDK1 and GBE1 were induced to a similar degree in myotubes from donors with NGT or type 2 diabetes. Exposure of myotubes to roxadustat led to a 1.4-fold increase in glycolytic rate in myotubes from men with NGT (p=0.0370) and a 1.7-fold increase in myotubes from donors with type 2 diabetes (p=0.0044), with no difference between the groups (p=0.1391). Exposure to roxadustat led to a reduction in basal mitochondrial respiration in both groups (p<0.01). Basal glucose uptake rates were similar in myotubes from donors with NGT (20.2 ± 2.7 pmol mg-1 min-1) and type 2 diabetes (25.3 ± 4.4 pmol mg-1 min-1, p=0.4205). Treatment with roxadustat enhanced insulin-stimulated glucose uptake in myotubes from donors with NGT (1.4-fold vs insulin-only condition, p=0.0023). The basal rate of glucose incorporation into glycogen was lower in myotubes from donors with NGT (233 ± 12.4 nmol g-1 h-1) than in myotubes from donors with type 2 diabetes (360 ± 40.3 nmol g-1 h-1, p=0.0344). Insulin increased glycogen synthesis by 1.9-fold (p=0.0025) in myotubes from donors with NGT, whereas roxadustat did not affect their basal or insulin-stimulated glycogen synthesis. Insulin increased glycogen synthesis by 1.7-fold (p=0.0031) in myotubes from donors with type 2 diabetes. While basal glycogen synthesis was unaffected by roxadustat, pretreatment with roxadustat enhanced insulin-stimulated glycogen synthesis in myotubes from donors with type 2 diabetes (p=0.0345). Roxadustat increases glycolysis and inhibits mitochondrial respiration in primary human myotubes regardless of diabetes status. Roxadustat may also improve insulin action on glycogen synthesis in myotubes from donors with type 2 diabetes.

Analysis of arsenic-modulated expression of hypothalamic estrogen receptor, thyroid receptor, and peroxisome proliferator-activated receptor gamma mRNA and simultaneous mitochondrial morphology and respiration rates in the mouse.

Arsenic has been identified as an environmental toxicant acting through various mechanisms, including the disruption of endocrine pathways. The present study assessed the ability of a single intraperitoneal injection of arsenic, to modify the mRNA expression levels of estrogen- and thyroid hormone receptors (ERα,β; TRα,β) and peroxisome proliferator-activated receptor gamma (PPARγ) in hypothalamic tissue homogenates of prepubertal mice in vivo. Mitochondrial respiration (MRR) was also measured, and the corresponding mitochondrial ultrastructure was analyzed. Results show that ERα,β, and TRα expression was significantly increased by arsenic, in all concentrations examined. In contrast, TRβ and PPARγ remained unaffected after arsenic injection. Arsenic-induced dose-dependent changes in state 4 mitochondrial respiration (St4). Mitochondrial morphology was affected by arsenic in that the 5 mg dose increased the size but decreased the number of mitochondria in agouti-related protein- (AgRP), while increasing the size without affecting the number of mitochondria in pro-opiomelanocortin (POMC) neurons. Arsenic also increased the size of the mitochondrial matrix per host mitochondrion. Complex analysis of dose-dependent response patterns between receptor mRNA, mitochondrial morphology, and mitochondrial respiration in the neuroendocrine hypothalamus suggests that instant arsenic effects on receptor mRNAs may not be directly reflected in St3-4 values, however, mitochondrial dynamics is affected, which predicts more pronounced effects in hypothalamus-regulated homeostatic processes after long-term arsenic exposure.

Quantifying variability associated with high-resolution respirometry in human permeabilized skeletal muscle fibers

Rates of skeletal muscle mitochondrial respiration are often measured using an Oroboros Oxygraph-2K (O2K) high-resolution respirometer. The purpose of this investigation was to determine the coefficients of variation (CV) and technical error of measurement (TEM) for rates of mitochondrial respiration obtained from two permeabilized skeletal muscle fiber bundles from a single biopsy (intra-biopsy) and on two experimental visits (inter-biopsy) using an O2K high-resolution respirometer. We hypothesized that our CV and TEM values would be similar to those reported by other research groups. We also hypothesized that there would be no statistically significant differences between rates of mitochondrial respiration obtained on two experimental visits separated by one week. Biopsies of the vastus lateralis were obtained from 14 healthy young individuals (female: 10; male: 4; age: 23 ± 3 years; height: 1.73 ± 0.1 m; body mass: 73.3 ± 20.4 kg) from the same leg on two experimental visits separated by an average of 6 ± 2 days. We examined rates of pyruvate and malate-supported, adenosine diphosphate (ADP)-stimulated mitochondrial respiration in the presence and absence of 20 mM creatine. Mean average intra- and inter-biopsy CVs for maximal complex I+II-supported, ADP-stimulated mitochondrial respiration were 11.5 % and 9.7 %, respectively. Mean average intra- and inter-biopsy TEM values for maximal complex I+II-supported, ADP-stimulated mitochondrial respiration were 10.6 pmol/s/mg wet weight and 8.6 pmol/s/mg wet weight, respectively. Statistically significant differences for rates of mitochondrial respiration were not observed between experimental visits regardless of creatine condition (non-creatine or 20 mM creatine). Our data are consistent with previously reported CV and TEM values and demonstrate that changes in maximal mitochondrial respiration of approximately 15 % can be reliably detected with a sample size of ~10 participants.

Na/K-ATPase α1/Src Signaling Regulates Mitochondrial Metabolic Function and ROS Production in Human iPSC-derived Cardiomyocytes

Through an isoform specific mechanism, the Na/K-ATPase (NKA) α1 polypeptide-mediated regulation of Src kinase has emerged as a novel regulator of mitochondrial function in various cell types. Mitochondrial metabolism ensures adequate myocardial performance and adaptation to physiological demand. It is also a critical cellular determinant of cardiac repair and remodeling. To investigate the importance of the proposed NKA/Src regulatory signaling on cardiac mitochondrial metabolic function independently from the classic Na/K-ATPase enzymatic function, we used a gene targeting approach in cardiomyocytes. Human induced pluripotent stem cells (hiPSC) expressing a Src-signaling null mutant (A420P) form of NKA α1 were generated using CRISPR/Cas9-mediated genome editing. NKA isoform protein expression and enzymatic activity remained unchanged. However, baseline levels of Src and ERK1/2 phosphorylation were decreased by 55% and 80%, respectively in the A420P hiPSC (n=3, p&lt;0.05). Both the WT and A420P hiPSC differentiated into cardiomyocytes (iCM) as assessed by morphology, spontaneous cell contraction, marker gene expression, and subcellular striations. Cell metabolism assessed by Seahorse extracellular flux analysis revealed significant reductions in basal and maximal rates of mitochondrial respiration (50%), spare respiratory capacity (50%), ATP production (60%), and coupling effciency (20%) (n=4, p&lt;0.05). ROS production was monitored in live cells by measuring the oxidation of the CM-H2DCFDA dye using a fluorescence microscope. The slope of the ROS production rate curve was dramatically reduced in A420P iCM (80%, n=3, p&lt;0.05). Protein carbonylation levels, analyzed by ELISA as markers of oxidative damage, were also decreased in A420P iCM (n=4, p&lt;0.05). Taken together, these data provide genetic evidence for a role of NKA α1/Src in the tonic stimulation of basal mitochondrial metabolism and ROS production in human cardiac myocytes. This has potential significance in cardiometabolic diseases and suggests that targeting the NKA α1/Src signaling axis may offer a novel approach to address mitochondrial dysfunction. NIH Grant R15 HL145666, NIH Grant P20GM103434 to the West Virginia IDeA Network of Biomedical Research Excellence, and American Heart Association Postdoctoral Fellowship — 22POST917776 — to Marco T. Pessoa. 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.

Praseodymium and warming interactions in mussels: Comparison between observed and predicted results

Being a crucial element for technological development, praseodymium (Pr) has been increasingly used, leading to a rise in its concentration in aquatic systems. However, its potential threats to organisms remain poorly understood. Besides contamination, organisms are also threatened by climate change-related factors, including warming. It is important to evaluate how climate change-related factors may influence the effects of contaminants. To address this, histopathological and biochemical analyses were performed in adult mussels of Mytilus galloprovincialis, following a 28-day exposure to Pr (10 μg/L) and warming (4 °C increase) separately, and in combination. Additionally, biochemical and physiological alterations were analysed in the sperm of mussels after 30-min exposure to the same treatments. Furthermore, it was used the Independent Action model to predict the interaction between Pr and warming. The results showed, in the case of adults exposed to Pr, an increase in superoxide dismutase (SOD) and glutathione S-transferases (GSTs) activities. However, it was insufficient, leading to histopathological injuries, redox imbalance, and cellular damage. In the case of sperm, Pr induced an increase of mitochondrial activity and respiration rate, in response to the increase in systemic metabolic rate and oxygen demand. Warming increased the metabolism, and induced redox imbalance and cellular damage in adults. In sperm, a rise in temperature induced lipid peroxidation and a decrease in velocity. Warming induced some alterations in how adult mussels responded to Pr, activating catalase instead of SOD, and in addition to GSTs, also activated carboxylesterases. However, it was not enough to avoid redox imbalance and cellular damage. In the case of sperm, the combination induced a decrease in H2O2 production, and higher oxygen demand, which prevented the decrease in motility and velocity. This study highlights the limitations of using models and emphasizes the importance of studying the impacts of emerging contaminants, such as rare earth elements, and their combination with climate change-related factors. Under environmental conditions, chronic exposure to the combined effect of different stressors might generate impacts at higher biological levels. This may affect organisms' respiratory and filtration capacity, nutrient absorption, defence capacity against infections or diseases, and sperm viability, ultimately resulting in reduced growth and reproduction, with consequences at the population level.

Captivity affects mitochondrial aerobic respiration and carotenoid metabolism in the house finch (Haemorhous mexicanus).

In many species of animals, red carotenoid-based coloration is produced by metabolizing yellow dietary pigments, and this red ornamentation can be an honest signal of individual quality. However, the physiological basis for associations between organism function and the metabolism of red ornamental carotenoids from yellow dietary carotenoids remains uncertain. A recent hypothesis posits that carotenoid metabolism depends on mitochondrial performance, with diminished red coloration resulting from altered mitochondrial aerobic respiration. To test for an association between mitochondrial respiration and red carotenoids, we held wild-caught, molting male house finches in either small bird cages or large flight cages to create environmental challenges during the period when red ornamental coloration is produced. We predicted that small cages would present a less favorable environment than large flight cages and that captivity itself would decrease both mitochondrial performance and the abundance of red carotenoids compared with free-living birds. We found that captive-held birds circulated fewer red carotenoids, showed increased mitochondrial respiratory rates, and had lower complex II respiratory control ratios - a metric associated with mitochondrial efficiency - compared with free-living birds, though we did not detect a difference in the effects of small cages versus large cages. Among captive individuals, the birds that circulated the highest concentrations of red carotenoids had the highest mitochondrial respiratory control ratio for complex II substrate. These data support the hypothesis that the metabolism of red carotenoid pigments is linked to mitochondrial aerobic respiration in the house finch, but the mechanisms for this association remain to be established.

Aerobic capacity and mitochondrial function in bipolar disorder: a longitudinal study during acute phases and after clinical remission.

Aerobic capacity has shown to predict physical and mental health-related quality of life in bipolar disorder (BD). However, the correlation between exercise respiratory capacity and mitochondrial function remains understudied. We aimed to assess longitudinally intra-individual differences in these factors during mood episodes and remission in BD. This study included eight BD patients admitted to an acute psychiatric unit. Incremental cardiopulmonary exercise test (CPET) was conducted during acute episodes (T0), followed by constant work rate cycle ergometry (CWRCE) to evaluate endurance time, oxygen uptake at peak exercise (VO2peak) and at the anaerobic threshold. The second test was repeated during remission (T1). Mitochondrial respiration rates were assessed at T0 and T1 in peripheral blood mononuclear cells. Endurance time, VO2peak, and anaerobic threshold oxygen consumption showed no significant variations between T0 and T1. Basal oxygen consumption at T1 tended to inversely correlate with maximal mitochondrial respiratory capacity (r=-0.690, p=0.058), and VO2peak during exercise at T1 inversely correlated with basal and minimum mitochondrial respiration (r=-0.810, p=0.015; r=-0.786, p=0.021, respectively). Our preliminary data showed that lower basal oxygen consumption may be linked to greater mitochondrial respiratory capacity, and maximum oxygen uptake during the exercise task was associated with lower basal mitochondrial respiration, suggesting that lower oxygen requirements could be associated with greater mitochondrial capacity. These findings should be replicated in larger samples stratified for manic and depressive states.

Abstract 435: A key regulatory role of the translation initiation factor DAP5 in the metabolic reprogramming of metastatic breast cancer

Abstract Triple negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer, accounting for 10-15% of all breast cancers in women. The frequency of metastasis in TNBC is disproportionally high compared to other subtypes of breast cancer and is the primary cause of patient mortality. At the cellular level, TNBC's aggressive nature demands rapid adaptation of the tumor cells to navigate cellular stressors for survival and metastatic transformation. These survival adaptations include a cancer cell’s ability to reprogram its metabolism and regulate mRNA translation control, coordinating alternative pathways to retain a selective proteome and metabolites that will aid in survival, proliferation, and migration. Advanced cancers, including TNBC, are generally highly glycolytic, and have lower rates of mitochondrial respiration (OXPHOS), a phenomenon known as the Warburg effect. This metabolic shift prioritizes the generation of essential metabolic intermediates, to facilitate the production of cellular biomass and fuel proliferation. Another notable alternative pathway is mediated by the ubiquitously expressed translation initiation factor, Death Associated Protein 5 (DAP5). DAP5 is a homolog of eIF4G and mediates translation independently of the canonical mTORC1/eIF4E pathway. Our findings suggest that this alternative translation initiation plays a crucial role in regulating the metabolic reprogramming of metastatic breast cancer. This non-canonical pathway via DAP5 facilitates the selective translation of 20-30% cap-dependent mRNAs, many of which are essential in cancer cell survival, migration, and metabolism. Importantly, it has recently been shown that DAP5 is critical for breast cancer metastasis but not in primary tumor development. Furthermore, TNBC patient data shows that elevated levels of DAP5 mRNA are correlated with poor survival and metastasis. Our data, using the highly metastatic TNBC cell line, MDA-MB-231, demonstrated that silencing DAP5 significantly reduced glycolytic rate and glycolytic capacity. Consistent with these findings, translatome profiling, showed that DAP5 silencing reduced numerous metabolic targets including glycolytic enzymes and metabolite transporters. Calcium (Ca2+) signaling, which has an important role in cancer progression, migration, and cellular bioenergetics is also affected by DAP5 in TNBC cells. Collectively, our findings indicate that breast cancer cells utilize the translation initiation factor DAP5 to selectively translate mRNAs that support metabolic reprogramming and cell migration, suggesting that DAP5 has a key regulatory role in the cancer’s transformation. Citation Format: Erna Mitaishvili, Guilherme Henrique Souza Bomfim, Anna Zou, Moira Sauane, Rodrigo Lacruz, Columba de la Parra. A key regulatory role of the translation initiation factor DAP5 in the metabolic reprogramming of metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 435.

BDE-99 stimulates generation of aberrant brown/beige adipocytes

Adipose tissue compromises one of the principal depots where brominated flame retardants (BFR) accumulate in vivo, yet whether BFR disturb thermogenic brown/beige adipocytes is still not referred to date. Herein, effects of BDE-99, a major congener of polybrominated diphenyl ethers (PBDEs) detected in humans, on brown/beige adipocytes were explored for the first time, aiming to provide new knowledge evaluating the obesogenic and metabolic disrupting effects of BFR. Our results firstly demonstrated that exposure to BDE-99 during the lineage commitment period significantly promoted C3H10T1/2 MSCs differentiating into brown/beige adipocytes, evidenced by the increase of brown/beige adipocyte marker UCP1, Cidea as well as mitochondrial membrane potential and basal respiration rate, which was similar to pharmacological PPARγ agonist rosiglitazone. Unexpectedly, the mitochondrial maximal respiration rate of BDE-99 stimulated brown/beige adipocytes was not synchronously enhanced and resulted in a significant reduction of mitochondrial spare respiration capacity (SRC) compared to control or rosiglitazone stimulated adipocytes, indicating a deficient energy-dissipating capacity of BDE-99 stimulated thermogenic adipocytes. Consistently with compromised mitochondrial SRC, lipidomic analysis further revealed that the lipids profile of mitochondria derived from BDE-99 stimulated brown/beige adipocytes were quite different from control or rosiglitazone stimulated cells. In detail, BDE-99 group contains more free fatty acid (FFA) and lyso-PE in mitochondria. In addition to energy metabolism, our results also demonstrated that BDE-99 stimulated brown/beige adipocytes were deficient in endocrine, which secreted more adverse adipokine named resistin, coinciding with comparable beneficial adipokine adiponectin compared with that of rosiglitazone. Taken together, our results showed for the first time that BDE-99 stimulated brown/beige adipocytes were aberrant in energy metabolism and endocrine, which strongly suggests that BDE-99 accumulated in human adipose tissue could interfere with brown/beige adipocytes to contribute to the occurrence of obesity and relevant metabolic disorders.

Fatty Acids Play a Critical Role in Mitochondrial Oxidative Phosphorylation in Effector T Cells in Graft-versus-Host Disease.

Although the role of aerobic glycolysis in activated T cells has been well characterized, whether and how fatty acids (FAs) contribute to donor T cell function in allogeneic hematopoietic stem cell transplantation is unclear. Using xenogeneic graft-versus-host disease (GVHD) models, this study demonstrated that exogenous FAs serve as a crucial source of mitochondrial respiration in donor T cells in humans. By comparing human T cells isolated from wild-type NOD/Shi-scid-IL2rγnull (NOG) mice with those from MHC class I/II-deficient NOG mice, we found that donor T cells increased extracellular FA uptake, the extent of which correlates with their proliferation, and continued to increase FA uptake during effector differentiation. Gene expression analysis showed the upregulation of a wide range of lipid metabolism-related genes, including lipid hydrolysis, mitochondrial FA transport, and FA oxidation. Extracellular flux analysis demonstrated that mitochondrial FA transport was required to fully achieve the mitochondrial maximal respiration rate and spare respiratory capacity, whereas the substantial disruption of glucose supply by either glucose deprivation or mitochondrial pyruvate transport blockade did not impair oxidative phosphorylation. Taken together, FA-driven mitochondrial respiration is a hallmark that differentiates TCR-dependent T cell activation from TCR-independent immune response after hematopoietic stem cell transplant.

The glutaminase inhibitor CB-839 targets metabolic dependencies of JAK2-mutant hematopoiesis in MPN

AbstractHyperproliferation of myeloid and erythroid cells in myeloproliferative neoplasms (MPN) driven by the JAK2-V617F mutation is associated with altered metabolism. Given the central role of glutamine in anabolic and catabolic pathways, we examined the effects of pharmacologically inhibiting glutaminolysis, that is, the conversion of glutamine (Gln) to glutamate (Glu), using CB-839, a small molecular inhibitor of the enzyme glutaminase (GLS). We show that CB-839 strongly reduced the mitochondrial respiration rate of bone marrow cells from JAK2-V617F mutant (VF) mice, demonstrating a marked dependence of these cells on Gln-derived ATP production. Consistently, in vivo treatment with CB-839 normalized blood glucose levels, reduced splenomegaly and decreased erythrocytosis in VF mice. These effects were more pronounced when CB-839 was combined with the JAK1/2 inhibitor ruxolitinib or the glycolysis inhibitor 3PO, indicating possible synergies when cotargeting different metabolic and oncogenic pathways. Furthermore, we show that the inhibition of glutaminolysis with CB-839 preferentially lowered the proportion of JAK2-mutant hematopoietic stem cells (HSCs). The total number of HSCs was decreased by CB-839, primarily by reducing HSCs in the G1 phase of the cell cycle. CB-839 in combination with ruxolitinib also strongly reduced myelofibrosis at later stages of MPN. In line with the effects shown in mice, proliferation of CD34+ hematopoietic stem and progenitor cells from polycythemia vera patients was inhibited by CB-839 at nanomolar concentrations. These data suggest that inhibiting GLS alone or in combination with inhibitors of glycolysis or JAK2 inhibitors represents an attractive new therapeutic approach to MPN.

Abstract A027: Targeting MYC-induced reprogramming of mitochondrial metabolism in breast cancer

Abstract Oncogenic MYC is frequently overexpressed in breast cancer, and its overexpression is associated with aggressiveness and unfavorable prognosis of breast cancer. MYC overexpression promotes tumor growth by stimulating cell cycle progression and rewiring metabolism – creating cancer vulnerabilities that can be therapeutically targeted via synthetic lethal strategies. A large part of our current understanding of the MYC overexpression biology and related synthetic lethal pathways is based on findings in various inducible MYC overexpression systems, which demonstrate the acute effects of MYC activation but may poorly represent long-term MYC overexpression status in real cancers. Here we classified 15 triple-negative breast cancer (TNBC) cell lines according to both MYC target gene set expression and MYC protein expression into MYC-high and MYC-low categories and explored the MYC-high status-coupled therapeutic responses and cancer-relevant metabolic changes. We show that mitochondrial respiration inhibiting biguanide drug metformin inhibits cell proliferation and generates senescence-like features in MYC-high TNBC cells. Furthermore, high-resolution respirometry experiments show that MYC-high TNBC cells have higher mitochondrial respiration rates than MYC-low TNBC cells, a phenotype not explained by a higher number of mitochondria. Also, targeting mitochondrial respiration with small molecule inhibitors specifically reduces proliferation in MYC-high TNBC cells and sensitizes them to apoptosis – indicating that MYC-high TNBC cells have higher dependency on mitochondrial respiration than the MYC-low cells. Targeted metabolomic flux experiments demonstrate that small molecule inhibitors of mitochondrial respiration rewire TCA cycle, resulting in suppression of MYC-driven glutamine anaplerosis, which offers a possible explanation for the apoptotic sensitization. In conclusion, our findings provide new insight into the long-term MYC overexpression-associated metabolic changes – highlighting mitochondrial respiration as a potential target for the development of MYC-dependent synthetic lethal strategies against breast cancer. Citation Format: Johanna M. Anttila, Mariel Savelius, Daniel Nicorici, Ryan Awadhpersad, Pauliina M. Munne, Linda Id, Topi A. Tervonen, Christopher Jackson, Anni I. Nieminen, Juha Klefström. Targeting MYC-induced reprogramming of mitochondrial metabolism in breast cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A027.

Anti-inflammatory effects of pulsed magnetic field on the immune system

When an inflammatory response occurs in the body, primary immune cells, such as macrophages, phagocytize and present antigens, and secondary immune cells, such as T cells, produce antibodies against the presented antigens to prevent infection in the body. Abnormalities in the immune system releases pro-inflammatory cytokines such as TNF-α and IL-6, leading to rheumatoid arthritis and sepsis. This study aimed to confirm the change in inflammatory activity across innate immunity and acquired immunity while investigating the effects of pulsed magnetic field (PMF) on the immune system to regulate excessive inflammation. Raw 264.7 and EL4 cells were co-cultured in a DMEM medium using permeable cell culture inserts and inflammation was induced using LPS. Our PMF stimulator has the maximum intensity of 4700 G at a transition time of 136 μs with pulse intervals of 1 Hz. In order to confirm anti-inflammatory effects of PMF, the level of IL-6, pH and mitochondrial respiration rate (MRR) were measured using ELISA, pH meter and MTT assay, respectively. It was observed in vitro that inflammation-induced macrophages induce activation of T cells, as evidenced by the faster acidification in pH, increased IL-6 expression, and enhanced MRR, compared to T cells. But after PMF stimulation, pH levels improved, the concentration of IL-6 reduced, and MRR decreased in both macrophages and T-cells, which indicates inflammation relief. Our results show that macrophages, the primary immune cells, are first involved in the inflammatory response and recovery and influence T cells, the secondary immune cells. Therefore PMF stimulus is thought to affect the immune system of the human body by balancing the activation and suppression of immune cells and improve anti-inflammation. For clinical use, non-invasive PMF can be developed as a medical devices modulating immune system with optimizing the PMF conditions such as pulse shape, duration, or repetition rate.

Overexpression of manganese superoxide dismutase mitigates ACL injury-induced muscle atrophy, weakness and oxidative damage

Oxidative stress has been implicated in the etiology of skeletal muscle weakness following joint injury. We investigated longitudinal patient muscle samples following knee injury (anterior cruciate ligament tear). Following injury, transcriptomic analysis revealed downregulation of mitochondrial metabolism-related gene networks, which were supported by reduced mitochondrial respiratory flux rates. Additionally, enrichment of reactive oxygen species (ROS)-related pathways were upregulated in muscle following knee injury, and further investigation unveiled marked oxidative damage in a progressive manner following injury and surgical reconstruction. We then investigated whether antioxidant protection is effective in preventing muscle atrophy and weakness after knee injury in mice that overexpress Mn-superoxide dismutase (MnSOD+/−). MnSOD+/− mice showed attenuated oxidative damage, atrophy, and muscle weakness compared to wild type littermate controls following ACL transection surgery. Taken together, our results indicate that ROS-related damage is a causative mechanism of muscle dysfunction after knee injury, and that mitochondrial antioxidant protection may hold promise as a therapeutic target to prevent weakness and development of disability.

Need for speed: Human fast-twitch mitochondria favor power over efficiency

ObjectiveHuman skeletal muscle consists of a mixture of slow- and fast-twitch fibers with distinct capacities for contraction mechanics, fermentation, and oxidative phosphorylation. While the divergence in mitochondrial volume favoring slow-twitch fibers is well established, data on the fiber type-specific intrinsic mitochondrial function and morphology are highly limited with existing data mainly being generated in animal models. This highlights the need for more human data on the topic. MethodsHere, we utilized THRIFTY, a rapid fiber type identification protocol to detect, sort, and pool fast- and slow-twitch fibers within 6 h of muscle biopsy sampling. Respiration of permeabilized fast- and slow-twitch fiber pools was then analyzed with high-resolution respirometry. Using standardized western blot procedures, muscle fiber pools were subsequently analyzed for control proteins and key proteins related to respiratory capacity. ResultsMaximal complex I+II respiration was 25% higher in human slow-twitch fibers compared to fast-twitch fibers. However, per mitochondrial volume, the respiratory rate of mitochondria in fast-twitch fibers was approximately 50% higher for complex I+II, which was primarily mediated through elevated complex II respiration. Furthermore, the abundance of complex II protein and proteins regulating cristae structure were disproportionally elevated in mitochondria of the fast-twitch fibers. The difference in intrinsic respiratory rate was not reflected in fatty acid–or complex I respiration. ConclusionMitochondria of human fast-twitch muscle fibers compensate for their lack of volume by substantially elevating intrinsic respiratory rate through increased reliance on complex II.

The Na/K-ATPase α1/Src Signaling Axis Regulates Mitochondrial Metabolic Function and Redox Signaling in Human iPSC-Derived Cardiomyocytes.

Na/K-ATPase (NKA)-mediated regulation of Src kinase, which involves defined amino acid sequences of the NKA α1 polypeptide, has emerged as a novel regulatory mechanism of mitochondrial function in metazoans. Mitochondrial metabolism ensures adequate myocardial performance and adaptation to physiological demand. It is also a critical cellular determinant of cardiac repair and remodeling. To assess the impact of the proposed NKA/Src regulatory axis on cardiac mitochondrial metabolic function, we used a gene targeting approach in human cardiac myocytes. Human induced pluripotent stem cells (hiPSC) expressing an Src-signaling null mutant (A420P) form of the NKA α1 polypeptide were generated using CRISPR/Cas9-mediated genome editing. Total cellular Na/K-ATPase activity remained unchanged in A420P compared to the wild type (WT) hiPSC, but baseline phosphorylation levels of Src and ERK1/2 were drastically reduced. Both WT and A420P mutant hiPSC readily differentiated into cardiac myocytes (iCM), as evidenced by marker gene expression, spontaneous cell contraction, and subcellular striations. Total NKA α1-3 protein expression was comparable in WT and A420P iCM. However, live cell metabolism assessed functionally by Seahorse extracellular flux analysis revealed significant reductions in both basal and maximal rates of mitochondrial respiration, spare respiratory capacity, ATP production, and coupling efficiency. A significant reduction in ROS production was detected by fluorescence imaging in live cells, and confirmed by decreased cellular protein carbonylation levels in A420P iCM. Taken together, these data provide genetic evidence for a role of NKA α1/Src in the tonic stimulation of basal mitochondrial metabolism and ROS production in human cardiac myocytes. This signaling axis in cardiac myocytes may provide a new approach to counteract mitochondrial dysfunction in cardiometabolic diseases.