Oxygen Consumption Rate Research Articles

Deuterium Metabolic Imaging Differentiates Glioblastoma Metabolic Subtypes and Detects Early Response to Chemoradiotherapy.

Deuterium magnetic resonance spectroscopic imaging of glucose metabolism has the potential to differentiate between glycolytic and mitochondrial metabolic subtypes in glioblastoma and to evaluate early treatment responses, which could guide patient treatment.

SCAR32: Functional characterization and expansion of the clinical-genetic spectrum.

Biallelic mutations in PRDX3 have been linked to autosomal recessive spinocerebellar ataxia type 32. In this study, which aims to contribute to the growing body of knowledge on this rare disease, we identified two unrelated patients with mutations in PRDX3. We explored the impact of PRDX3 mutation in patient skin fibroblasts and the role of the gene in neurodevelopment. We performed trio exome sequencing that identified mutations in PRDX3 in two unrelated patients. We also performed functional studies in patient skin fibroblasts and generated a "crispant" zebrafish (Danio rerio) model to investigate the role of the gene during nervous system development. Our study reports two additional patients. Patient 1 is a 19-year-old male who showed a novel homozygous c.525_535delGTTAGAAGGTT (p. Leu176TrpfsTer11) mutation as the genetic cause of cerebellar ataxia. Patient 2 is a 20-year-old male who was found to present the known c.425C>G/p. Ala142Gly variant in compound heterozygosity with the p. Leu176TrpfsTer11 one. While the fibroblast model failed to recapitulate the pathological features associated with PRDX3 loss of function, our functional characterization of the prdx3 zebrafish model revealed motor defects, increased susceptibility to reactive oxygen species-triggered apoptosis, and an impaired oxygen consumption rate. We identified a new variant, thereby expanding the genetic spectrum of PRDX3-related disease. We developed a novel zebrafish model to investigate the consequences of prdx3 depletion on neurodevelopment and thus offered a potential new tool for identifying new treatment opportunities.

Convolutional neural networks to classify human stress that occurs during in‐field sugarcane harvesting: A case study

AbstractAssessing human stress in agriculture proves to be a complex and time‐intensive endeavor within the field of ergonomics, particularly for the development of agricultural systems. This methodology involves the utilization of instrumentation and the establishment of a dedicated laboratory setup. The complexity arises from the need to capture and analyze various physiological and psychological indicators, such as heart rate (HR), muscle activity, and subjective feedback to comprehensively assess the impact of farm operations on subjects. The instrumentation typically includes wearable devices, sensors, and monitoring equipment to gather real‐time data of subject during the performance of farm operations. Deep learning (DL) models currently achieve human performance levels on real‐world face recognition tasks. In this study, we went beyond face recognition and experimented with the recognition of human stress based on facial features during the drudgery‐prone agricultural operation of sugarcane harvesting. This is the first research study for deploying artificial intelligence‐driven DL techniques to identify human stress in agriculture instead of monitoring several ergonomic characteristics. A total of 20 (10 each for male and female) subjects comprising 4300 augmented RGB images (215 per subject) were acquired during sugarcane harvesting seasons and then these images were deployed for training (80%) and validation (20%). Human stress and nonstress states were determined based on four ergonomic physiological parameters: heart rate (ΔHR), oxygen consumption rate (OCR), energy expenditure rate (EER), and acceptable workload (AWL). Stress was defined when ΔHR, OCR, EER, and AWL reached or exceeded certain standard threshold values. Four convolutional neural network‐based DL models (1) DarkNet53, (2) InceptionV3, (3) MobileNetV2 and (4) ResNet50 were selected due to their remarkable feature extraction abilities, simple and effective implementation to edge computation devices. In all four DL models, training performance results delivered training accuracy ranging from 73.8% to 99.1% at combinations of two mini‐batch sizes and four levels of epochs. The maximum training accuracies were 99.1%, 99.0%, 97.7%, and 95.4% at the combination of mini‐batch size 16 and 25 epochs for DarkNet53, InceptionV3, ResNet50, and MobileNetV2, respectively. Due to the best performance, DarkNet53 was tested further on an independent data set of 100 images and found 89.8%–93.3% confident to classify stressed images for female subjects while 92.2%–94.5% for male subjects, though it was trained on the integrated data set. The comparative classification of the developed model and ergonomic measurements for stress classification was carried out with a net accuracy of 88% where there were few instances of wrong classifications.

Extracellular vesicles activated cancer-associated fibroblasts promote lung cancer metastasis through mitophagy and mtDNA transfer.

Studies have shown that oxidative stress and its resistance plays important roles in the process of tumor metastasis, and mitochondrial dysfunction caused by mitochondrial DNA (mtDNA) damage is an important molecular event in oxidative stress. In lung cancer, the normal fibroblasts (NFs) are activated as cancer-associated fibroblasts (CAFs), and act in the realms of the tumor microenvironment (TME) with consequences for tumor growth and metastasis. However, its activation mechanism and whether it participates in tumor metastasis through antioxidative stress remain unclear. The role and signaling pathways of tumor cell derived extracellular vesicles (EVs) activating NFs and the characteristic of induced CAFs (iCAFs) were measured by the transmission electron microscopy, nanoparticle tracking analysis, immunofluorescence, collagen contraction assay, quantitative PCR, immunoblotting, luciferase reporter assay and mitochondrial membrane potential detection. Mitochondrial genome and single nucleotide polymorphism sequencing were used to investigate the transport of mtDNA from iCAFs to ρ0 cells, which were tumor cells with mitochondrial dysfunction caused by depletion of mtDNA. Further, the effects of iCAFs on mitochondrial function, growth and metastasis of tumor cells were analysed in co-culture models both in vitro and in vivo, using succinate dehydrogenase, glutathione and oxygen consumption rate measurements, CCK-8 assay, transwell assay, xenotransplantation and metastasis experiments as well as in situ hybridization and immunohistochemistry. Our findings revealed that EVs derived from high-metastatic lung cancer cells packaged miR-1290 that directly targets MT1G, leading to activation of AKT signaling in NFs and inducing NFs conversion to CAFs. The iCAFs exhibit higher levels of autophagy and mitophagy and more mtDNA release, and reactive oxygen species (ROS) could further promote this process. After cocultured with the conditioned medium (CM) of iCAFs, the ρ0 cells may restore its mitochondrial function by acquisition of mtDNA from CAFs, and further promotes tumor metastasis. These results elucidate a novel mechanism that CAFs activated by tumor-derived EVs can promote metastasis by transferring mtDNA and restoring mitochondrial function of tumor cells which result in resistance of oxidative stress, and provide a new therapeutic target for lung cancer metastasis.

Revealing and targeting metabolic drivers contributing to treatment escape in diffuse midline glioma.

10038 Background: Diffuse midline glioma (DMG) is an aggressive pediatric brain tumor that predominately affects children, with a median survival of 9-11 months. DMGs are driven by self-renewing, stem-like glioma cells that have been stalled in an oligodendrocyte precursor cell (OPC)-like state and that highly express PDGFRA. This overexpression of PDGFRA has been shown to be pivotal in DMG development. Thus, targeting PDGFRA may serve as a viable treatment approach for DMG. In our previous studies, we revealed that avapritinib, a next generation tyrosine kinase inhibitor of PDGFRA, is highly effective in DMG cells. While a subset of patients receiving treatment with avapritinib experienced a promising clinical response, all patients eventually experienced tumor progression due to treatment escape. In this study, we investigated which mechanisms DMG cells use to escape avapritinib treatment, and how we can therapeutically exploit these resistance mechanisms. Methods: Transcriptomic profiling and functional assays were performed on patient-derived DMG cell lines. A combinatorial drug screening identified drug candidates evaluated in this study. Results: Bulk RNA sequencing analysis of two patient derived DMG cell lines revealed an upregulation of genes associated with both fatty acid metabolism and oxidative phosphorylation (OXPHOS) following avapritinib treatment. Functional assays confirmed elevated OXPHOS in avapritinib-treated cells with significant increases in mitochondrial energy transduction, palmitate- (a product of fatty acid metabolism) driven oxygen consumption rates, and incorporation of palmitate-derived carbons into the tricarboxylic acid (TCA) cycle. CRISPR-Cas9-mediated knockout of PDGFRAin one DMG cell line confirmed the metabolic changes observed following avapritinib treatment. Analysis of bulk RNA sequencing data revealed an upregulation of key fatty acid- related transcription factors in avapritinib-treated DMG cells. Specifically, Peroxisome Proliferator-Activated Receptor alpha (PPAR-alpha), Sterol Regulatory Binding Element Binding Transcription Factors 1 and 2, and Fatty Acid Synthase (FASN) were most prominently upregulated. To determine which therapies could target the dependency of avapritinib-treated cells on fatty acid metabolism, we performed a combinatorial drug screening and found three lipid pathway inhibitors that have synergistic cytotoxic effects with avapritinib. Specifically, a FASN inhibitor, cholesterol pathway inhibitor, and PPAR-alpha inhibitor. Conclusions: In this study, we revealed metabolic drivers that may contribute to avapritinib resistance in DMG cells, and identified compounds capable of inhibiting these drivers that demonstrate synergy with avapritinib. We now intend on testing these combination therapies in vivo as we aim to provide a long-term clinical benefit to DMG patients receiving avapritinib.

Mitochondrial function of dairy calf lymphocytes from birth to immunologic maturity

The inability of dairy calves to fully respond to immune stimuli until they reach maturity at 6 mo of age severely limits the use of parenteral vaccines to protect calves against disease. Immune responses are metabolically demanding, and immune cells rely on mitochondrial metabolites for their functionality. Due to the essential role of mitochondria in driving T-cell responses necessary for vaccine efficacy, we hypothesized that the mitochondrial function of dairy calf lymphocytes changes with age, from birth to immunologic maturity. In this cross-sectional study, groups of dairy calves (n = 4/group) were blood sampled at birth before colostrum intake and at 1, 2, 3, 4, 6, 8, 16, and 24 wk of age. Mid-lactation adult cows (n = 4) were also sampled to reference fully mature immune cell populations. B, CD4+, CD8+, and γδ T lymphocytes were enriched using magnetic-activated cell sorting, and their mitochondrial function was assessed with an extracellular flux analyzer. Non-mitochondrial oxygen consumption, basal respiration, maximal respiration, spare respiratory capacity, proton leak, and the oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) ratio were reported. Results were compared among groups using a Kruskal-Wallis test. The OCR to ECAR ratio is an indicator of the relative proportions of oxidative phosphorylation and aerobic glycolysis which is associated with effector functions in lymphocytes. The ratio was lower in 0 wk than adults in CD4+ T-cells. For CD8+ T-cells, the OCR to ECAR ratio for the 2 wk group was lower than the 3 wk group. A lower OCR to ECAR ratio indicates more reliance on glycolytic metabolism than oxidative phosphorylation. Maximal respiration is an indication of mitochondrial efficiency and is often associated with mitochondrial mass. For γδ T-cells, the 3 wk group had higher maximal respiration than the 16 wk group, whereas for B cells maximal respiration was higher in the 1 wk compared with the 16 wk group. Basal respiration indicates all cell functions that require oxygen and was lower in the 0 wk group than the 1 wk and 3 wk groups for CD4+ T-cells. γδ T-cells exhibited lower basal respiration in the 2 wk group than the 24 wk one. Although we found minimal differences in the mitochondrial outcomes reported from non-stimulated lymphocytes from birth through 6 mo of age and mid-lactation adults who served as mature immune cell populations, these results align with previous reports from weaning aged calf and adult CD4+ T-cells. In conclusion, there was insufficient evidence to suggest that the mitochondria in the lymphocytes of dairy calves from birth through immunologic maturity had functional changes associated with age. In conclusion, the capacity of unstimulated calf mitochondria to perform oxidative phosphorylation is not associated with age.

Assessing the impact of imidacloprid, glyphosate, and their mixtures on multiple biomarkers in Corbicula largillierti

Pesticide mixtures are frequently utilized in agriculture, yet their cumulative effects on aquatic organisms remain poorly understood. Aquatic animals can be effective bioindicators and invasive bivalves, owing to their widespread distribution, provide an opportunity to assess these impacts. Glyphosate and imidacloprid, among the most prevalent pesticides globally, are frequently detected in freshwater systems in South America. This study aims to understand the cumulative effects of pesticide mixtures on aquatic organisms, using invasive Corbicula largillierti clams from a natural stream in northwestern Argentina. We conducted 48-hour exposure experiments using two concentrations of imidacloprid (20 and 200 μg L−1 a.i), two concentrations of glyphosate (0.3 and 3 mg L−1 a.i), and two combinations of these pesticides (both at low and high concentrations, respectively), simulating the direct contamination of both pesticides based on their agronomic recipe and observed values in Argentine aquatic environments. Clam metabolism was assessed through the examination of multiple oxidative stress parameters and measuring oxygen consumption rate as a proxy for standard metabolic rate (SMR). Our findings revealed that imidacloprid has a more pronounced effect compared to glyphosate. Imidacloprid significantly decreased clam SMR and cellular levels of reduced glutathione (GSH). However, when both pesticides were present, also cellular glycogen and thiobarbituric acid-reactive substances (TBARS) were affected. Proteins and glutathione S-Transferase (GST) activity were unaffected by either pesticide or their mixture at the assayed concentrations, highlighting the need to test several stress parameters to detect toxicological impacts. Our results indicated additive effects of imidacloprid and glyphosate across all measured parameters. The combination of multiple physiological and cytological biomarkers in invasive bivalves offers significant potential to enhance biomonitoring sensitivity and obtain insights into the origins and cellular mechanisms of chemical impacts. These studies can improve pollution regulatory policies and pesticide management.

CircRNA6448-14/miR-455-3p/OTUB2 axis stimulates glycolysis and stemness of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a gastrointestinal malignancy with high incidence. This study aimed to reveal the complete circRNA-miRNA-mRNA regulatory network in ESCC and validate its function mechanism. Expression of OTU Domain-Containing Ubiquitin Aldehyde-Binding Protein 2 (OTUB2) in ESCC was analyzed by bioinformatics to find the binding sites between circRNA6448-14 and miR-455-3p, as well as miR-455-3p and OTUB2. The binding relationships were verified by RNA Immunoprecipitation (RIP) and dual-luciferase assay. The expressions of circRNA6448-14, miR-455-3p, and OTUB2 were detected by quantitative real-time polymerase chain reaction (qRT-PCR). MTT assay measured cell viability, and the spheroid formation assay assessed the ability of stem cell sphere formation. Western blot (WB) determined the expression of marker proteins of stem cell surface and rate-limiting enzyme of glycolysis. The Seahorse XFe96 extracellular flux analyzer measured the rate of extracellular acidification rate and cellular oxygen consumption. Corresponding assay kits assessed cellular glucose consumption, lactate production, and adenosine triphosphate (ATP) generation. In ESCC, circRNA6448-14 and OTUB2 were highly expressed in contrast to miR-455-3p. Knocking down circRNA6448-14 could prevent the glycolysis and stemness of ESCC cells. Additionally, circRNA6448-14 enhanced the expression of OTUB2 by sponging miR-455-3p. Overexpression of OTUB2 or silencing miR-455-3p reversed the inhibitory effect of knockdown of circRNA6448-14 on ESCC glycolysis and stemness. This research demonstrated that the circRNA6448-14/miR-455-3p/OTUB2 axis induced the glycolysis and stemness of ESCC cells. Our study revealed a novel function of circRNA6448-14, which may serve as a potential therapeutic target for ESCC.

Human induced pluripotent cardiomyocytes as a model for cardiomyopathy with a Myozap gene mutation

Abstract Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): 1- Central Department of Missions, Egyptian Ministry of Higher education and Scientific Research. 2- small fund from Tampere University. The contractility of the heart is based on cardiomyocytes (CM). These cardiomyocytes are connected through a complex network of proteins forming the intercalated disc (ID) which is crucial to ensure mechanical and electrochemical coupling. Dilated cardiomyopathy (DCM) is a disease of the myocardium that can lead to heart failure and life-threatening arrhythmias. Up to 30% of DCM cases are caused by mutations in genes encoding proteins of the sarcomere (the basic contractile unit) or desmosome (a cell-to-cell adhesion structure) and the structural cytoskeleton. Myozap (Myocardium-Enriched Zonula Occludens-1-Associated Protein) is a distinctive cardiac-enriched ID protein. It is crucial for biomechanical stress adaption in CMs due to its outstanding role in the Rho/SRF (Serum Response Factor) signalling pathway. In this study, we have identified two Finnish families with DCM and carrying a novel variant in myozap. Our aim is to characterize myozap gene mutation-both heterozygous and homozygous truncating variants-as well as its implication in DCM. We have established patient-specific human induced pluripotent stem-cell lines and derived cardiomyocyte (hiPSC-CM) to study the disease at the cellular level. Myozap expression, compared to the control cell line, was evaluated by immunohistochemistry. Electrophysiological methods, including microelectrode array (MEA) and patch clamping, were implemented to measure various parameters with emphasis on those ones related to cell-cell connection e.g., the conduction velocity. Moreover, metabolic stress indicators, including oxygen consumption rate (OCR), were estimated by Seahorse. Additionally, qPCR and ELISA were performed to measure the colocalised and closely related genes to ID and protein level of troponin, respectively. Immunohistochemistry revealed downregulation and absence of myozap expression in heterozygous and homozygous cell lines, respectively. Compared to the control one, differences in BPM, FPD were observed. Metabolic analysis of the myozap mutant elucidated mitochondrial impairment compared to the control. Some SRF and sarcomeric target genes, including MYH7, MYH6 and Myl-2 were downregulated. On the contrary, apoptotic genes (e.g., caspase 3) were upregulated. Tight junction protein (TJP) was upregulated, and this is very importance since it is closely related to the myozap in the ID proteins network. Moreover, troponin protein level, measured by ELISA, showed upregulation in mutant myozap. In conclusion, our study provides a new insight into understanding myozap’s significance in cardiac biology and in cardiomyopathy pathophysiology.

Targeting glutaminolysis pathway in trained immunity induced by oxidized low-density lipoprotein

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft DFG Atherosclerosis is a chronic inflammatory disease of the arterial wall that causes cardiovascular disease. Monocyte-derived macrophages importantly contribute to atherogenesis. Monocytes can be primed to exhibit increased responsiveness upon a secondary non-related stimulus, a phenomenon called trained immunity. Intracellular metabolic and epigenetic reprogramming lays the basis of trained immunity. One of the stimuli that can induce trained immunity is oxidized low-density lipoprotein (oxLDL). oxLDL-trained monocytes exhibit an increase in metabolic pathways such as glycolysis, OXPHOS, and cholesterol biosynthesis. The glutaminolysis pathway that fuels the tricarboxylic acid (TCA) cycle is essential for trained immunity induction by b-glucan, the cell wall component of Candida albicans. The goal of this project is to investigate the involvement of the glutaminolysis pathway in oxLDL-induced trained immunity in isolated human primary monocytes. We exposed monocytes for 24 hours to oxLDL, allowed subsequent differentiation into macrophages for six days in culture medium, and assessed cytokine production capacity upon restimulation. Co-incubation of oxLDL with the inhibitor that blocks glutaminase, the enzyme that generates glutamate from glutamine, significantly reduced cytokine production capacity in oxLDL-trained cells. We further showed that glutaminolysis is necessary for oxLDL-induced increase in oxygen consumption (OCR) and glycolysis rate (ECAR). Single nucleotide polymorphisms in glutaminolysis genes were shown to correlate with ex vivo oxLDL-induced trained immunity in 243 healthy volunteers. These data suggest an important role of glutaminolysis in the establishment of oxLDL-induced trained immunity. To unravel the molecular mechanisms behind this observation we plan to perform epigenetic as well as metabolic analyses of the monocytes trained in the presence of the inhibitor.

Mitochondrial players as novel therapeutic options in arrhythmogenic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Joint Project Südtirol- FWF (grant number 23623; Italy-Austria) and the Department of Innovation, Research and University of the Autonomous Province of Bolzano-South Tyrol (Italy) Background Mitochondria are dynamic organelles that sense and respond to environmental, nutritional, and pathological changes. These organelles can smartly adapt to different stressful conditions, by changing the way they network or the energy sources they use. Over the past years, numerous disorders have been associated with mitochondrial dysfunction, including cardiomyopathies. Arrhythmogenic Cardiomyopathy (ACM) is a cardiac disease characterized by the progressive replacement of the myocardium with fibro-fatty tissue. The current treatment primarily focuses on managing the symptoms rather than addressing the underlying issue of tissue substitution. Purpose: Therefore, in this study, we explored the mitochondrial and metabolic phenotype in ACM and tested the efficacy of a mitochondria-targeting library to rescue the fibro-fatty phenotype in ACM Methods: Human primary cardiac stromal cells (CStCs) were cultured in adipogenic medium (AM), a condition used to model the ACM fibro-fatty phenotype. Mitochondria-related analyses of ultrastructure, membrane potential, network connection, respiratory capacity, ROS production and redox system were performed at day (d) d0, d3, d7, d15 of AM exposure. A mitochondria-targeting library (MedChem Express) was tested on ACM-CStCs exposed to AM for 7 days. The extent of lipid accumulation was determined using the neutral lipid staining dye BODIPY 493/503, cell numbers were measured using Hoechst counterstaining, and images acquired using a high-content imaging system Results: At d0, the mitochondrial network was more fragmented in ACM compared to healthy CStCs (CTR). This result was in line with the observation of a decrease in mitochondrial respiratory capacity and an increase in ROS production. In addition, AM exposure led to faster and higher lipid accumulation in ACM CStCs, which was associated with higher mitochondrial oxygen consumption rates observed in ACM CStCs after 7 days of AM exposure. When ACM CStCs were simultaneously exposed to AM and 407 mitochondrial-directed compounds, about 25% of the compounds demonstrated the ability to reduce lipid accumulation Conclusion: ACM CStCs demonstrate an increased risk for mitochondrial network impairment. Moreover, prolonged exposure of ACM CStCs cells to AM negatively affects lipid deposition as well as mitochondrial and metabolic parameters. This highlights the potential for using specific mitochondria-targeting compounds to reduce lipid accumulation.

Smooth muscle cell-specific deletion of S100A4 modifies smooth muscle cell fate and inflammation in murine atherosclerotic lesions

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation (SNSF) S100A4, a calcium-binding protein, is crucial in smooth muscle cell (SMC) phenotypic switch, yet its role in atherosclerotic plaque development and SMC plasticity remains unclear. Our previous experiments using an S100A4 neutralizing antibody approach in ApoE-/- atherosclerotic-prone mice showed reduced atherosclerotic burden but did not establish a direct link between the various S100A4-expressing cells and atherosclerosis. Herein, we show that aortic Oil RedO staining in ApoE-/-; S100A4-/- mice subjected to a 12-week high-cholesterol-diet (HCD) revealed reduced lesion areas compared to ApoE-/- mice (Figure 1, from 21.29% of the total aorta in ApoE-/-; S100A4+/+ mice to 13.27% in ApoE-/-; S100A4-/- mice), highlighting a pivotal role for S100A4 in atherosclerotic plaque development. We then used a lineage tracing ApoE-/- mouse model, in which we induced an SMC-specific deletion of S100A4 (SMC-S100A4Δ/Δ). After 12 weeks of HCD, SMC-S100A4Δ/Δ and control mice (SMC-S100A4wt/wt) were sacrificed, and aortas were processed for en face Oil Red O staining, immunohistochemistry, and single-cell RNA sequencing (scRNA-seq). SMC-specific deletion of S100A4 reduced the necrotic core area (from 29.75% of total plaque area in SMC-S100A4wt/wt to 18.23% in SMC-S100A4Δ/Δ mice) without affecting total atherosclerotic plaque size suggesting a more potent impact on plaque composition. ScRNA-seq analysis from SMC-S100A4Δ/Δ and SMC-S100A4wt/wt mice showed that the inflammatory macrophage-like SMC phenotype was highly suppressed in the plaque and associated with increased SMC repair phenotypes as well as a global reduction of myeloid inflammatory cells. SMCs also retained specific contractile SMC markers, namely Acta2 and Myh11 (fold change of 2.285 and 2.492, respectively) hinting towards a more stable fibrous cap. Immunofluorescence staining on aortic roots plaque (Figure 2) confirmed those results with an increase of SMCs (from 11.02% to 16.18%) as well as a-smooth muscle actin (a-SMA, Acta2)-positive SMCs (from 3.25% to 5.1%) in SMC-S100A4 Δ/Δ mice. Furthermore, the Gene Set Enrichment Analysis (GSEA) of scRNAseq data revealed that, in SMC-S100A4Δ/Δ mice, genes related to mitochondrial metabolism and oxidative respiration were highly downregulated in all SMC clusters. We investigated SMC mitochondrial fitness in vitro and showed that S100A4-/- SMCs have an increased basal oxygen consumption rate and an increased proton leak (fold change of 1.37 and 1.96 respectively) suggestive of differences in cellular metabolic adaptability. In conclusion, our research emphasizes the significant role of S100A4 in plaque development, demonstrating how SMC-derived S100A4 knockdown affects plaque evolution, reducing inflammation, and promoting stability likely through influencing SMC phenotypic switch through metabolic pathways.

Lactate-induced IGF1R protein lactylation promotes proliferation and metabolic reprogramming of lung cancer cells.

Lung cancer (LC) is regarded as a fatal cancer, and insulin-like growth factor 1 (IGF1) and its receptor (IGF1R) have been found to play a key role in regulating tumor glycolytic metabolism. The aim of this study is to investigate LC proliferation regulated by metabolite-mediated IGF1R lactylation. IGF1R was highly expressed in LC tissues and cells, and the effects of IGF1R on protein stability were inhibited by Lactate dehydrogenase A (LDHA) inhibition. Moreover, the tightness of IGF1R binding to IGF1 was also enhanced by exogenous lactic acid but suppressed by LDHA silencing, while cell viability and proliferation were promoted by over-expression of IGF1R. Exogenous lactic acid further exacerbated the effects of the IGF1R gene, while LDHA knocking down reduced the IGF1R-induced malignant behaviors. The IGF1R and exogenous lactic acid were also found to increase extracellular acidification rate (ECAR) and decrease oxygen consumption rate to regulate glycolysis, which was inhibited by LDHA deficiency in LC cells. The study concluded that IGF1R-mediated aggressive behaviors of LC cells were associated with higher levels of IGF1R lactylation. Moreover, lactic acid can improve the protein stability of the IGF1R oncogene, thus promoting glycolysis and generating lactic acid, forming a closed loop. Therefore, targeting IGF1R is envisaged to provide a novel strategy for developing therapeutic agents against LC.

Transport activity regulates mitochondrial bioenergetics and biogenesis in renal tubules.

Renal tubules are featured with copious mitochondria and robust transport activity. Mutations in mitochondrial genes cause congenital renal tubulopathies, and changes in transport activity affect mitochondrial morphology, suggesting mitochondrial function and transport activity are tightly coupled. Current methods of using bulk kidney tissues or cultured cells to study mitochondrial bioenergetics are limited. Here, we optimized an extracellular flux analysis (EFA) to study mitochondrial respiration and energy metabolism using microdissected mouse renal tubule segments. EFA detects mitochondrial respiration and glycolysis by measuring oxygen consumption and extracellular acidification rates, respectively. We show that both measurements positively correlate with sample sizes of a few centimeter-length renal tubules. The thick ascending limbs (TALs) and distal convoluted tubules (DCTs) critically utilize glucose/pyruvate as energy substrates, whereas proximal tubules (PTs) are significantly much less so. Acute inhibition of TALs' transport activity by ouabain treatment reduces basal and ATP-linked mitochondrial respiration. Chronic inhibition of transport activity by 2-week furosemide treatment or deletion of with-no-lysine kinase 4 (Wnk4) decreases maximal mitochondrial capacity. In addition, chronic inhibition downregulates mitochondrial DNA mass and mitochondrial length/density in TALs and DCTs. Conversely, gain-of-function Wnk4 mutation increases maximal mitochondrial capacity and mitochondrial length/density without increasing mitochondrial DNA mass. In conclusion, EFA is a sensitive and reliable method to investigate mitochondrial functions in isolated renal tubules. Transport activity tightly regulates mitochondrial bioenergetics and biogenesis to meet the energy demand in renal tubules. The system allows future investigation into whether and how mitochondria contribute to tubular remodeling adapted to changes in transport activity.

Oxygen consumption rate of flatworms under the influence of wake- and sleep-promoting neurotransmitters.

Flatworms are among the best studied animal models for regeneration; however, they also represent an emerging opportunity to investigate other biological processes as well. For instance, flatworms are nocturnal and sleep during the day, a state that is regulated by sleep/wake history and the action of the sleep-promoting neurotransmitter gamma-aminobutyric acid (or GABA). Sleep is widespread across the animal kingdom, where it serves many nonexclusive functions. Notably, sleep saves energy by reducing metabolic rate and by not doing something more energetically taxing. Whether the conservation of energy is apparent in sleeping flatworms is unclear. We measured the oxygen consumption rate (OCR) of flatworms dosed with either (1) GABA (n = 29) which makes flatworms inactive or (2) dopamine (n = 20) which stimulates flatworms to move, or (3) day and night neurotransmitter-free controls (n = 28 and 27, respectively). While OCRdid not differ between the day and night, flatworms treated with GABA used less oxygen than those treated with dopamine, and less than the day-time control. Thus, GABA affected flatworm physiology, ostensibly by enforcing energy-conserving sleep. Evidence that dopamine increased metabolism was less strong. This work broadens our understanding of flatworm physiology and expands the phylogenetic applicability of energy conservation as a function of sleep.

Heat stress upregulates arachidonic acid to trigger autophagy in sertoli cells via dysfunctional mitochondrial respiratory chain function

As a key factor in determining testis size and sperm number, sertoli cells (SCs) play a crucial role in male infertility. Heat stress (HS) reduces SCs counts, negatively impacting nutrient transport and supply to germ cells, and leading to spermatogenesis failure in humans and animals. However, how HS affects the number of SCs remains unclear. We hypothesized that changes in SC metabolism contribute to the adverse effects of HS. In this study, we first observed an upregulation of arachidonic acid (AA), an unsaturated fatty acid after HS exposure by LC-MS/MS metabolome detection. By increasing ROS levels, expression of KEAP1 and NRF2 proteins as well as LC3 and LAMP2, 100 µM AA induced autophagy in SCs by activating oxidative stress (OS). We observed adverse effects of AA on mitochondria under HS with a decrease of mitochondrial number and an increase of mitochondrial membrane potential (MMP). We also found that AA alternated the oxygen transport and absorption function of mitochondria by increasing glycolysis flux and decreasing oxygen consumption rate as well as the expression of mitochondrial electron transport chain (ETC) proteins Complex I, II, V. However, pretreatment with 5 mM NAC (ROS inhibitor) and 2 µM Rotenone (mitochondrial ETC inhibitor) reversed the autophagy induced by AA. In summary, AA modulates autophagy in SCs during HS by disrupting mitochondrial ETC function, inferring that the release of AA is a switch-like response, and providing insight into the underlying mechanism of high temperatures causing male infertility.

Prdx1 regulates macrophage polarization by maintaining mitochondrial homeostasis

In order to investigate the role of Prdx1 in macrophage polarization, mouse leukemia cells of monocyte macrophage (RAW264.7) were treated with lipopolysaccharides (LPS)+ interferon gamma (IFNγ) or IL-4 to induce type 1 macrophage (M1) and type 1 macrophage (M2) macrophages, respectively. The Prdx1 gene knockout cells (Prdx1-/-) were used for the study. Flow cytometry was conducted to detect M1/M2 macrophage markers, and ELISA kits were used to measure M1/M2 cytokine levels. Inducible nitric-oxide synthase (iNOS) activity, arginase-1 (Arg-1) activity, and oxidative damage were also assessed. The Seahorse XFe24 Extracellular Flux Analyzer was employed to measure extracellular acidification rate and oxygen consumption rate. The mitochondrial membrane potential was analyzed using the mitochondrial membrane potential dye (JC-1) fluorescent probe, and mitochondrial superoxide was detected through fluorescence staining. Additionally, the impact of adding a mitochondrial reactive oxygen species (ROS) scavenger on RAW264.7 macrophage polarization was examined. The results demonstrated an increase in ROS, hydrogen peroxide, and 8-hydroxy-2 deoxyguanosine (8-OHDG). Cytotoxicity and mitochondrial toxic effects, including mitochondrial superoxide accumulation, decreased adenosine-triphosphate (ATP) production, reduced mitochondrial membrane potential, and decreased mitochondrial DNA copy number, were observed. Furthermore, down-regulation of translocase of inner mitochondrial membrane 23 (TIM23) mitochondrial protein and mitochondrial stress protein heat shock protein 60 (HSP60) was noted. The extra cellular acidification rate (ECAR) in M1 macrophage polarization in RAW264.7 cells was increased, while oxygen consumption rate (OCR) in M2 macrophages was reduced. These findings indicate that Prdx1 knockout in RAW264.7 cells can inhibit M2 macrophage polarization but promote M1 macrophage polarization by impairing mitochondrial function and reducing oxidative phosphorylation.

Understanding tightened muscle in knee osteoarthritis and the impacts of Fu's subcutaneous needling: A pilot trial with shear-wave elastography and near-infrared spectroscopy.

Given the scarce reports on the interplay between Fu's subcutaneous needling (FSN), tightened muscle, and therapeutic effects, we developed a clinical research protocol to synchronously collect data on clinical efficacy and muscle characteristics in patients with knee osteoarthritis, exploring the mechanism of FSN action. The primary aim was to assess the feasibility and safety of this protocol, guiding future trials and their sample size calculations. In this prospective, single-blind, self-controlled study, 19 patients with early to mid-stage unilateral knee osteoarthritis underwent FSN therapy on both knees over 1 week (4 sessions, every other day). We measured local elastic modulus, muscle thickness, blood flow volume, and oxygen consumption rate of bilateral vastus lateralis muscles using shear-wave elastography and near-infrared spectroscopy (NIRS) before and after the first and fourth treatments. Additionally, real-time NIRS indicators (oxygenated hemoglobin [O2Hb], deoxyhemoglobin [HHb], total hemoglobin [THb], and tissue saturation index [TSI]) were recorded during these treatments. Pain intensity (visual analogue scale [VAS]), functional status (Western Ontario and McMaster Universities Osteoarthritis Index [WOMAC]), and active range of motion were evaluated before these treatments. All 19 participants completed the trial without serious adverse events. After 3 FSN treatments, significant changes were observed in VAS and WOMAC scores (VAS: P < .001; WOMAC: P < .001), and knee flexion (P < .001) and external rotation (P = .02), except for internal rotation. No meaningful significant differences were observed in muscle characteristics at baseline or between pre- and post-treatment periods. NIRS results during treatments indicated significant increases in local O2Hb and THb post-FSN therapy (First treatment: O2Hb: P = .005; THb: P = .006. Fourth treatment: O2Hb: P = .002; THb: P = .004); however, no significant increases were observed for HHb (First treatment: P = .06; Fourth treatment: P = .28). No linear correlation was found between therapeutic effects and changes in tightened muscle indices. FSN reduces pain and improves joint function in knee osteoarthritis, while also enhancing blood flow and oxygenation in the vastus lateralis muscle of the affected side. Further revisions of this protocol are warranted based on our insights.

#1492 Metabolic switch in CD8+ T cells may influence immune response in chronic antibody-mediated rejection (CAMR)

Abstract Background and Aims CAMR is the main cause of chronic graft injury and subsequent graft loss, but its pathogenesis is still largely unclear. Our group demonstrated an increase in CD8+ T cells infiltrating biopsies from CAMR patients (PMID: 20595199), but their role remains to be defined. Recently, immunometabolism has emerged as a field connecting cell energy metabolism with the immune cell differentiation and functions. Particularly, the permanent aerobic glycolysis of CD8+ lymphocytes induces their transition from naïve to activated state and effector T cells (PMID: 37315709). Thus, the aim of the study was to investigate the molecular mechanisms underlying the development of CAMR by the analysis of gene expression profiles of CD8+ T lymphocytes in conjunction with their metabolic state. Method We enrolled 14 patients with biopsy-proven CAMR and 12 stable transplant recipients with normal graft histology and function (control group). CD8+ T cells were purified by CD8 MicroBeads (Miltenyi Biotec) then counted (TC20™ Automated Cell Counter). Gene expression profiles of CD8+ T lymphocytes isolated from both groups were assessed using Agilent microarrays. Data obtained were statistically and functionally evaluated (IPA software) and validated by qPCR. Oxygen consumption rate (OCR) in CD8+ and CD8- cells was measured by a Resipher oxygen sensing lid (Lucid Scientific Inc., USA). The contribution of mitochondrial respiration to OCR was investigated in isolated CD8+ T cells from CAMR and controls treated with rotenone/antimycin A (Rot/AA), oligomycin (Olygo) or carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to evaluate spare respiratory capacity (SRC), ATP-linked respiration, proton leak and maximal respiration. Results Gene expression profiles of CD8+ T cells showed a characteristic alteration of lipid metabolism in CAMR patients with the triglyceride biosynthesis pathway (p = 2.69E-3) as the most altered pathway and several genes de-regulated all potentially involved in the altered metabolic state of CD8+ T cells. Among others, we validated the increased expression of Nicotinamide Phosphoribosyltransferase (NAMPT) (FC = +2.39), the rate-limiting enzyme of nicotinamide adenine dinucleotide (NAD+) salvage pathway involved in the glycolysis progression, mostly activated in effector T cells. We then decided to evaluate the OCR and the metabolic state of CD8+T cells. Basal OCR was higher in CD8+ rather than CD8- cells, but with different trends among cells isolated from control and CAMR patients. Indeed, CD8+ cells from CAMR patients showed higher OCR compared to control, while OCR was lower in CD8- from CAMR rather than control patients. CD8+ OCR from both CAMR and control patients was mostly dependent on mitochondrial respiration, since it was deeply reduced by Rot/AA treatment. Interestingly, inhibitory response to Oligo treatment by CD8+ cells from CAMR patients was modest, while OCR was noticeably stimulated by FCCP in these cells, suggesting that mitochondrial oxygen uptake was not ATP linked but rather associated with increased uncoupling. These data suggest that CD8+ T cells from CAMR patients are activated effector T cells primarily utilizing aerobic glycolysis. To confirm this hypothesis, we evaluated the gene expression levels of: i) HIF1a, which involves permanent dependence on glycolysis even in the presence of oxygen (Warburg effect); ii) fatty acid transporter CD36, whose levels mimic an energy dependence on the beta oxidation of fatty acids, a process activated in memory T cells. We confirmed an increased expression of HIF1a and a reduced expression of CD36 in CD8+T cells from CAMR patients compared to controls (p&amp;lt;0.05), that confirm the T effector phenotype. Conclusion Our data suggest that CD8+ T cell from CAMR patients showed permanent dependency on glycolysis leading to altered immune phenotype and response, thus indicating novel opportunities to modulate the immune system for therapeutic purposes in kidney transplanted patients and reduce graft rejection.

Fructose Reduces Mitochondrial Metabolism and Increases Extracellular BCAA during Insulin Resistance in C2C12 Myotubes.

C2C12 mouse myoblasts were treated with fructose at a concentration of 100 µM (which approximates physiologically attainable concentrations in peripheral circulation) both with and without hyperinsulinemic-mediated insulin resistance. Gene expression was assessed by qRT-PCR, and protein expression was assessed by Western blot. Oxygen consumption rate and extracellular acidification rate were used to assess mitochondrial oxidative and glycolytic metabolism, respectively. Liquid chromatography-mass spectrometry was utilized to analyze leucine, isoleucine and valine concentration values. Fructose significantly reduced peak glycolytic and peak mitochondrial metabolism without altering related gene or protein expression. Similarly, no effect of fructose on BCAA catabolic enzymes was observed; however, fructose treatment resulted in elevated total extracellular BCAA in insulin-resistant cells. Collectively, these observations demonstrate that fructose at physiologically attainable levels does not appear to alter insulin sensitivity or BCAA catabolic potential in cultured myotubes. However, fructose may depress peak cell metabolism and BCAA utilization during insulin resistance.