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Microsystems & Nanoengineering 2022Measurement of cell metabolism in moderate-throughput to high-throughput organ-on-chip (OOC) systems would expand the range of data collected for studying drug effects...
Measurement of cell metabolism in moderate-throughput to high-throughput organ-on-chip (OOC) systems would expand the range of data collected for studying drug effects or disease in physiologically relevant tissue models. However, current measurement approaches rely on fluorescent imaging or colorimetric assays that are focused on endpoints, require labels or added substrates, and lack real-time data. Here, we integrated optical-based oxygen sensors in a high-throughput OOC platform and developed an approach for monitoring cell metabolic activity in an array of membrane bilayer devices. Each membrane bilayer device supported a culture of human renal proximal tubule epithelial cells on a porous membrane suspended between two microchannels and exposed to controlled, unidirectional perfusion and physiologically relevant shear stress for several days. For the first time, we measured changes in oxygen in a membrane bilayer format and used a finite element analysis model to estimate cell oxygen consumption rates (OCRs), allowing comparison with OCRs from other cell culture systems. Finally, we demonstrated label-free detection of metabolic shifts in human renal proximal tubule cells following exposure to FCCP, a drug known for increasing cell oxygen consumption, as well as oligomycin and antimycin A, drugs known for decreasing cell oxygen consumption. The capability to measure cell OCRs and detect metabolic shifts in an array of membrane bilayer devices contained within an industry standard microtiter plate format will be valuable for analyzing flow-responsive and physiologically complex tissues during drug development and disease research.
PubMed: 36187891
DOI: 10.1038/s41378-022-00442-7 -
ELife Sep 2022Macrophages are a highly adaptive population of innate immune cells. Polarization with IFNγ and LPS into the 'classically activated' M1 macrophage enhances...
Macrophages are a highly adaptive population of innate immune cells. Polarization with IFNγ and LPS into the 'classically activated' M1 macrophage enhances pro-inflammatory and microbicidal responses, important for eradicating bacteria such as . By contrast, 'alternatively activated' M2 macrophages, polarized with IL-4, oppose bactericidal mechanisms and allow mycobacterial growth. These activation states are accompanied by distinct metabolic profiles, where M1 macrophages favor near exclusive use of glycolysis, whereas M2 macrophages up-regulate oxidative phosphorylation (OXPHOS). Here, we demonstrate that activation with IL-4 and IL-13 counterintuitively induces protective innate memory against mycobacterial challenge. In human and murine models, prior activation with IL-4/13 enhances pro-inflammatory cytokine secretion in response to a secondary stimulation with mycobacterial ligands. In our murine model, enhanced killing capacity is also demonstrated. Despite this switch in phenotype, IL-4/13 trained murine macrophages do not demonstrate M1-typical metabolism, instead retaining heightened use of OXPHOS. Moreover, inhibition of OXPHOS with oligomycin, 2-deoxy glucose or BPTES all impeded heightened pro-inflammatory cytokine responses from IL-4/13 trained macrophages. Lastly, this work identifies that IL-10 attenuates protective IL-4/13 training, impeding pro-inflammatory and bactericidal mechanisms. In summary, this work provides new and unexpected insight into alternative macrophage activation states in the context of mycobacterial infection.
Topics: Animals; Cytokines; Glucose; Humans; Interleukin-10; Interleukin-13; Interleukin-4; Lipopolysaccharides; Macrophage Activation; Macrophages; Mice; Oligomycins; Oxidative Phosphorylation
PubMed: 36173104
DOI: 10.7554/eLife.74690 -
Neuroreport Oct 2022Strokes represent as one of the leading causes of death and disability in the USA, however, there is no optimal treatment to reduce the occurrence or improve prognosis....
OBJECTIVE
Strokes represent as one of the leading causes of death and disability in the USA, however, there is no optimal treatment to reduce the occurrence or improve prognosis. Preconditioning of tissues triggers ischemic tolerance, a physiological state that may involve a metabolic switch (i.e. from glycolysis to oxidative phosphorylation or OxPhos) to preserve tissue viability under an ischemic insult. Here, we hypothesized that metabolic switching of energy source from glucose to galactose in cultured mesenchymal stem cells (MSCs) stands as an effective OxPhos-enhancing strategy.
METHODS
MSCs were grown under ambient condition (normal MSCs) or metabolic switching paradigm (switched MSCs) and then assayed for oxygen consumption rates (OCR) and extracellular acidification rate (ECAR) using the Seahorse technology to assess mitochondrial respiration.
RESULTS
Normal MSCs showed a lower OCR/ECAR ratio than switched MSCs at baseline (P < 0.0001), signifying that there were greater levels of OxPhos compared to glycolysis in switched MSCs. By modulating the mitochondrial metabolism with oligomycin (time points 4-6), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (7-9), and rotenone and antimycin (time points 10-12), switched MSCs greater reliance on OxPhos was further elucidated (time points 5-12; P < 0.0001; time point 4; P < 0.001).
CONCLUSION
The metabolic switch from glycolytic to oxidative metabolism amplifies the OxPhos potential of MSCs, which may allow these cells to afford more robust therapeutic effects against neurological disorders that benefit from ischemic tolerance.
Topics: Galactose; Glucose; Glycolysis; Mesenchymal Stem Cells; Oligomycins; Oxidative Phosphorylation; Rotenone
PubMed: 36126260
DOI: 10.1097/WNR.0000000000001828 -
Reproduction in Domestic Animals =... Jan 2023This study evaluated the bioenergetic map of mitochondria metabolism in cryopreserved bovine sperm. The detected oligomycin-sensitive basal respiration supported ATP...
This study evaluated the bioenergetic map of mitochondria metabolism in cryopreserved bovine sperm. The detected oligomycin-sensitive basal respiration supported ATP production; frozen-thawed spermatozoa were found to have a coupling efficiency higher than 0.80. Cell respiration, however, was not stimulated by the protonophoric action of FCCP, as its titration with 1, 2, 4 and 6 μM did not stimulate the uncoupling activity on oxidative phosphorylation as highlighted by unresponsive oxygen consumption. The unusual effect on the stimulation of maximal respiration was not related to fibronectin- or PDL-coated plates used for cellular metabolism analysis. Conversely, irradiation of frozen-thawed bovine sperm with the red light improved mitochondrial parameters. In effect, the maximal respiration of red-light-stimulated sperm in PDL-coated plates was higher than the non-irradiated. In spite of this, red-light irradiation had no impact on membrane integrity and mitochondrial activity evaluated by epifluorescence microscopy.
Topics: Male; Animals; Cattle; Semen; Spermatozoa; Energy Metabolism; Mitochondria; Cryopreservation; Sperm Motility; Semen Preservation
PubMed: 36107136
DOI: 10.1111/rda.14261 -
The Journal of Cell Biology Nov 2022Mitochondrial damage represents a dramatic change in cellular homeostasis. One rapid response is perimitochondrial actin polymerization, termed acute damage-induced...
Mitochondrial damage represents a dramatic change in cellular homeostasis. One rapid response is perimitochondrial actin polymerization, termed acute damage-induced actin (ADA). The consequences of ADA are not understood. In this study, we show evidence suggesting that ADA is linked to rapid glycolytic activation upon mitochondrial damage in multiple cells, including mouse embryonic fibroblasts and effector CD8+ T lymphocytes. ADA-inducing treatments include CCCP, antimycin, rotenone, oligomycin, and hypoxia. The Arp2/3 complex inhibitor CK666 or the mitochondrial sodium-calcium exchanger (NCLX) inhibitor CGP37157 inhibits both ADA and the glycolytic increase within 5 min, supporting ADA's role in glycolytic stimulation. Two situations causing chronic reductions in mitochondrial ATP production, mitochondrial DNA depletion and mutation to the NDUFS4 subunit of complex 1 of the electron transport chain, cause persistent perimitochondrial actin filaments similar to ADA. CK666 treatment causes rapid mitochondrial actin loss and a drop in ATP in NDUFS4 knock-out cells. We propose that ADA is necessary for rapid glycolytic activation upon mitochondrial impairment, to re-establish ATP production.
Topics: Actins; Adenosine Triphosphate; Animals; CD8-Positive T-Lymphocytes; Cells, Cultured; Electron Transport Complex I; Fibroblasts; Glycolysis; Mice; Mitochondria; Polymerization
PubMed: 36102863
DOI: 10.1083/jcb.202201160 -
Frontiers in Physiology 2022The temperature of a living cell is a crucial parameter for cellular events, such as cell division, gene expressions, enzyme activities and metabolism. We previously...
The temperature of a living cell is a crucial parameter for cellular events, such as cell division, gene expressions, enzyme activities and metabolism. We previously developed a quantifiable mitochondrial thermometry 1.0 based on rhodamine B methyl ester (RhB-ME) and rhodamine 800 (Rh800), and the theory for mitochondrial thermogenesis. Given that the synthesized RhB-ME is not readily available, thus, a convenient mitochondrial thermometry 2.0 based on tetra-methyl rhodamine methyl ester (TMRM) and Rh800 for the thermogenic study of brown adipocyte was further evolved. The fluorescence of TMRM is more sensitive (∼1.4 times) to temperature than that of RhB-ME, then the TMRM-based mito-thermometry 2.0 was validated and used for the qualitatively dynamic profiles for mitochondrial thermogenic responses and mitochondrial membrane potential in living cells simultaneously. Furthermore, our results demonstrated that the heterogenous thermogenesis evoked by β3 adrenoceptor agonist only used overall up to ∼46% of the thermogenic capacity evoked by CCCP stimulation. On the other hand, the results demonstrated that the maximum thermogenesis evoked by NE and oligomycin A used up to ∼79% of the thermogenic capacity, which suggested the maximum thermogenic capacity under physiological conditions by inhibiting the proton-ATPase function of the mitochondrial complex V, such as under the cold activation of sympathetic nerve and the co-release of sympathetic transmitters.
PubMed: 36091398
DOI: 10.3389/fphys.2022.977431 -
Frontiers in Cell and Developmental... 2022Beige adipocytes play key roles in organismal energy and metabolic balance. In this study, we assessed whether the supplementation of human white adipocytes,...
Nicotinamide-riboside shifts the differentiation of human primary white adipocytes to beige adipocytes impacting substrate preference and uncoupling respiration through SIRT1 activation and mitochondria-derived reactive species production.
Beige adipocytes play key roles in organismal energy and metabolic balance. In this study, we assessed whether the supplementation of human white adipocytes, differentiated from human adipose tissue-derived stem cells, with nicotinamide riboside (NR), a potent NAD + precursor, can shift differentiation to beige adipocytes (beiging). NR induced mitochondrial biogenesis and the expression of beige markers (TBX1 and UCP1) in white adipocytes demonstrating that NR can declutch beiging. NR did not induce PARP activity but supported SIRT1 induction, which plays a key role in beiging. NR induced etomoxir-resistant respiration, suggesting increases in the oxidation of carbohydrates, carbohydrate breakdown products, or amino acids. Furthermore, NR boosted oligomycin-resistant respiration corresponding to uncoupled respiration. Enhanced etomoxir and oligomycin-resistant respiration were dependent on mitochondrial reactive-species production. Taken together, NR supplementation can induce beiging and uncoupled respiration, which are beneficial for combatting metabolic diseases.
PubMed: 36072335
DOI: 10.3389/fcell.2022.979330 -
Frontiers in Immunology 2022The protozoan parasite is the causative agent of bovine coccidiosis, an enteric disease of global importance that significantly affects cattle productivity. Previous...
The protozoan parasite is the causative agent of bovine coccidiosis, an enteric disease of global importance that significantly affects cattle productivity. Previous studies showed that bovine NETosis-an important early host innate effector mechanism of polymorphonuclear neutrophil (PMN)-is elicited by stages. So far, the metabolic requirements of -triggered NET formation are unknown. We here studied early glycolytic and mitochondrial responses of PMN as well as the role of pH, distinct metabolic pathways, P2 receptor-mediated purinergic signaling, and monocarboxylate transporters 1 and 2 (MCT1, MCT2) in sporozoite-induced NET formation. Seahorse-based experiments revealed a rapid induction of both neutrophil oxygen consumption rate (OCR) and early glycolytic responses, thereby reflecting immediate PMN activation and metabolic changes upon confrontation with sporozoites. The impact of these metabolic changes on NET formation was studied chemical inhibition experiments targeting glycolysis and energy generation by the use of 2-fluor-2-deoxy-D-glucose (FDG), 6-diazo-5-oxo-L-norleucin (DON), sodium dichloroacetate (DCA), oxythiamine (OT), sodium oxamate (OXA), and oligomycin A (OmA) to block glycolysis, glutaminolysis, pyruvate dehydrogenase kinase, pyruvate dehydrogenase, lactate dehydrogenase, and mitochondrial ATP-synthase, respectively. Overall, sporozoite-induced NET formation was significantly diminished PMN pretreatments with OmA and OXA, thereby indicating a key role of ATP- and lactate-mediated metabolic pathways. Consequently, we additionally studied the effects of extracellular pH, MCT1, MCT2, and purinergic receptor inhibitors (AR-C141900, AR-C155858, theobromine, and NF449, respectively). Pretreatment with the latter inhibitors led to blockage of sporozoite-triggered DNA release from exposed bovine PMN. This report provides first evidence on the pivotal role of carbohydrate-related metabolic pathways and purinergic receptors being involved in sporozoite-induced NETosis.
Topics: Adenosine Triphosphate; Animals; Cattle; Cattle Diseases; Coccidiosis; Eimeria; Glycolysis; Sporozoites
PubMed: 36032127
DOI: 10.3389/fimmu.2022.842482 -
Computers in Biology and Medicine Sep 2022Gliomas are malignant tumors in the central nervous system. Cuproptosis is a newly discovered cell death mechanism targeting lipoylated tricarboxylic acid cycle...
Gliomas are malignant tumors in the central nervous system. Cuproptosis is a newly discovered cell death mechanism targeting lipoylated tricarboxylic acid cycle proteins. Previous studies have found that cuproptosis participates in tumor progression, but its role in gliomas is still elusive. Here, we systematically explored the bulk-tumor and single-cell transcriptome data to reveal its role in gliomas. The cuproptosis activity score (CuAS) was constructed based on cuproptosis-related genes, and machine learning techniques validated the score stability. High CuAS gliomas were more likely to have a poor prognosis and an aggressive mesenchymal (MES) subtype. Subsequently, the SCENIC algorithm predicted 20 CuAS-related transcription factors (TFs) in gliomas. Function enrichment and microenvironment analyses found that CuAS was associated with tumor immune infiltration. Accordingly, intercellular communications between neoplasm and immunity were explored by the R package "Cellchat". Five signaling pathways and 8 ligand-receptor pairs including ICAM1, ITGAX, ITGB2, ANXA1-FRR1, and the like, were identified to suggest how cuproptosis activity connected neoplastic and immune cells. Critically, 13 potential drugs targeting high CuAs gliomas were predicted according to the CTRP and PRISM databases, including oligomycin A, dihydroartemisinin, and others. Taken together, cuproptosis is involved in glioma aggressiveness, neoplasm-immune interactions, and may be used to assist in drug selection.
Topics: Humans; Brain Neoplasms; Gene Expression Regulation, Neoplastic; Glioma; Transcriptome; Tumor Microenvironment; Copper; Apoptosis
PubMed: 35964468
DOI: 10.1016/j.compbiomed.2022.105924 -
Scientific Reports Aug 2022Mitochondrial dysfunction promotes cancer aggressiveness, metastasis, and resistance to therapy. Similar traits are associated with epithelial mesenchymal transition...
Mitochondrial dysfunction promotes cancer aggressiveness, metastasis, and resistance to therapy. Similar traits are associated with epithelial mesenchymal transition (EMT). We questioned whether mitochondrial dysfunction induces EMT in head and neck cancer (HNC) cell lines. We induced mitochondrial dysfunction in four HNC cell lines with carbonyl cyanide-4(trifluoromethoxy)phenylhydrazone (FCCP), a mitochondrial electron transport chain uncoupling agent, and oligomycin, a mitochondrial ATP synthase inhibitor. Extracellular flux analyses and expression of the cystine/glutamate antiporter system xc (xCT) served to confirm mitochondrial dysfunction. Expression of the EMT-related transcription factor SNAI2, the mesenchymal marker vimentin and vimentin/cytokeratin double positivity served to detect EMT. In addition, holotomographic microscopy was used to search for morphological features of EMT. Extracellular flux analysis and xCT expression confirmed that FCCP/oligomycin induced mitochondrial dysfunction in all cell lines. Across the four cell lines, mitochondrial dysfunction resulted in an increase in relative SNAI2 expression from 8.5 ± 0.8 to 12.0 ± 1.1 (mean ± SEM; p = 0.007). This effect was predominantly caused by the CAL 27 cell line (increase from 2.2 ± 0.4 to 5.5 ± 1.0; p < 0.001). Similarly, only in CAL 27 cells vimentin expression increased from 2.2 ± 0.5 × 10 to 33.2 ± 10.2 × 10 (p = 0.002) and vimentin/cytokeratin double positive cells increased from 34.7 ± 5.1 to 67.5 ± 9.8% (p = 0.003), while the other 3 cell lines did not respond with EMT (all p > 0.1). Across all cell lines, FCCP/oligomycin had no effect on EMT characteristics in holotomographic microscopy. Mitochondrial dysfunction induced EMT in 1 of 4 HNC cell lines. Given the heterogeneity of HNC, mitochondrial dysfunction may be sporadically induced by EMT, but EMT does not explain the tumor promoting effects of mitochondrial dysfunction in general.
Topics: Cadherins; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Line, Tumor; Epithelial-Mesenchymal Transition; Head and Neck Neoplasms; Humans; Keratins; Mitochondria; Oligomycins; Vimentin
PubMed: 35918485
DOI: 10.1038/s41598-022-16829-5