Measurement Of Oxygen Consumption Rate Research Articles

Abstract TMP109: Labeling And Isolating Cell Specific Neuronal Mitochondria And Their Functional Analysis In Mice Post Stroke

Introduction: In vivo neuronal mitochondria dysfunction post stroke has not been well investigated. Methods: Using Mito-tag mice in combine with local infection of AAV9 produced from plasmids carrying Cre-recombinase controlled by neuronal promoters, we generated reporter mice expressing a hemagglutinin (HA)-tagged EGFP that localizes to mitochondrial outer membranes in synaptophysin (SYN) and calmodulin-kinase II (CaMKII) glutamatergic neurons localized to cerebral cortex. These mice were subjected to permanent middle cerebral artery occlusion and sacrificed 14 days post stroke. Fresh brain tissues were excised from the ischemic core (IC), ischemic boundary zone (IBZ) as well as from the homologous contralateral hemisphere, and the crude mitochondrial fractions (CMF) were extracted. Neuronal mitochondria were then isolated from the CMF by anti-HA magnetic beads (Thermo Scientific) and their specificity and oxidative phosphorylation (OXPHOS) complexes were confirmed by means of Western-blot. Seahorse assay was employed to measure oxygen consumption rate (OCR) of the isolated mitochondria as an indicator of mitochondrial function. Results: The bead pulled components were co-precipitated with GFP and enriched with mitochondrial markers, e.g. voltage-dependent anion channel, cytochrome C, and COX IV, but lacked the Golgi protein RCAS1 as well as endoplasmic reticulum markers: Heme-oxygenase 1 and Calnexin, indicating that specific neuronal mitochondria have been selectively isolated. Western-blot data showed that OXPHOS components in SYN + and CAMKII + neuronal mitochondria were significantly downregulated in the IBZ and further decreased in the IC compared to the contralateral tissue, which was associated with the significant reductions of mitochondrial function measured by OCR (p<0.05, respectively, for both neuron types). These data suggesting dysfunction of neuronal mitochondria post stroke. Conclusions: We, for the first time, measure mitochondrial function in 2 types of neurons post stroke. By introducing specific neuronal promoter controlled Cre recombinase to Mito-tag mouse, mitochondria from individual neuronal linage cells can be efficiently labeled and isolated to investigate functional change post stroke.

Immuno-metabolic effect of pancreastatin inhibitor PSTi8 in diet induced obese mice: In vitro and in vivo findings

AimsPancreastatin (PST), an anti-insulin peptide derived from chromogranin A. Its levels increase in cases of obesity, which contributes to adipose tissue inflammation and insulin resistance. This study aims to investigate the immunometabolic effect of PST inhibitor (PSTi8) against PST by using in vitro and in vivo finding. Main methods3T3-L1 cells were differentiated with or without PSTi8, and Oil Red O staining was performed. J774A.1 cells were used for macrophage polarization study. The diet-induced obesity and T2DM model was developed in C57BL/6 mice through high-fat diet for 8 weeks. Alzet osmotic pumps were filled with PSTi8 (release rate: 2 mg/kg/day) and implanted in mice for eight weeks. Further, insulin and glucose tolerance tests were performed. Liver and eWAT sections were stained with hematoxylin and eosin. FACS was used to measure mitochondrial ROS and membrane potential, while Oroboros O2k was used to measure oxygen consumption rate. Immunocytochemistry and qRT-PCR were done for protein and gene expression, respectively. Key findingsPSTi8 inhibited the expression of lipolytic genes and proteins in 3T3-L1 adipocytes. PSTi8 improved the inulin sensitivity, lipid profile, MMP, and OCR levels in the 3T3-L1 adipocyte and eWAT. It also increased the M1 to M2 macrophage polarization in J77A.1 cells and eWAT. Further, PSTi8 attenuated inflammatory CD4+ T, CD8+ T cells and increased the anti-inflammatory T-reg and eosinophil populations in the eWAT. It also reduced the expression of pro-inflammatory genes like Mcp1, Tnfα, and Il-6. SignificanceCollectively, PSTi8 exerted its beneficial effect on adipose tissue inflammation and restored energy expenditure against diet-induced obesity.

Dietary essential amino acids for the treatment of heart failure with reduced ejection fraction.

Heart failure with reduced ejection fraction (HFrEF) is a leading cause of mortality worldwide, requiring novel therapeutic and lifestyle interventions. Metabolic alterations and energy production deficit are hallmarks and thereby promising therapeutic targets for this complex clinical syndrome. We aim to study the molecular mechanisms and effects on cardiac function in rodents with HFrEF of a designer diet in which free essential amino acids-in specifically designed percentages-substituted for protein. Wild-type mice were subjected to transverse aortic constriction (TAC) to induce left ventricle (LV) pressure overload or sham surgery. Whole-body glucose homeostasis was studied with glucose tolerance test, while myocardial dysfunction and fibrosis were measured with echocardiogram and histological analysis. Mitochondrial bioenergetics and morphology were investigated with oxygen consumption rate measurement and electron microscopy evaluation. Circulating and cardiac non-targeted metabolite profiles were analyzed by ultrahigh performance liquid chromatography-tandem mass spectroscopy, while RNA-sequencing was used to identify signalling pathways mainly affected. The amino acid-substituted diet shows remarkable preventive and therapeutic effects. This dietary approach corrects the whole-body glucose metabolism and restores the unbalanced metabolic substrate usage-by improving mitochondrial fuel oxidation-in the failing heart. In particular, biochemical, molecular, and genetic approaches suggest that renormalization of branched-chain amino acid oxidation in cardiac tissue, which is suppressed in HFrEF, plays a relevant role. Beyond the changes of systemic metabolism, cell-autonomous processes may explain at least in part the diet's cardioprotective impact. Collectively, these results suggest that manipulation of dietary amino acids, and especially essential amino acids, is a potential adjuvant therapeutic strategy to treat systolic dysfunction and HFrEF in humans.

Measuring Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) in Muscle Stem Cells Using a Seahorse Analyzer: Applicability for Aging Studies.

In recent years, evidence showing metabolism as a fundamental regulator of stem cell functions has emerged. In skeletal muscle, its stem cells (satellite cells) sustain muscle regeneration, although they lose their regenerative potential with aging, and this has been attributed, at least in part, to changes in their metabolism. In this chapter, we describe a protocol to analyze the metabolism of satellite cells using the Seahorse technology, which can be applied to aging mice.

Benzo(a)pyrene-induced mitochondrial respiration and glycolysis disturbance in human neuroblastoma cells.

Mammalian cells generate ATP through mitochondrial respiration and glycolysis. Mitochondria not only play a key role in cell energy metabolism but also in cell cycle regulation. As a neurotoxic pollutant, benzo(a)pyrene (BaP) can trigger neuronal oxidative damage and apoptosis. However, the features of BaP-induced energy metabolism disturbance in SH-SY5Y cells has rarely been addressed. This study aimed to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as indications of respiratory activities and glycolytic. SH-SY5Y cells were treated with BaP to establish a cytotoxicity model, and butylated hydroxy anisole (BHA) was used to alleviate the damages induced by BaP. Using the Seahorse Extracellular Flux analyzer (XFp), we found that BaP significantly reduced basal respiration, ATP-linked OCR in SH-SY5Y cells with dose- and time-dependent. BHA supplementation recovered the mitochondrial respiration, synchronously attenuated intracellular ROS generation and lipid peroxidation, and simultaneously reversed the abnormal changes in antioxidant biomarkers, then rescued BaP-induced cell apoptosis. But long-term exposure to BaP or exposure to a high dosage of BaP could decrease OCR associated with maximal respiratory, spare capacity, and glycolysis metabolism. At the same time, the damage to cells is also more severe with the rate of apoptosis and mitochondrial membrane potential (ΔΨm) loss rising sharply, which were not entirely reversed by BHA. This study provides energy metabolism-related, indicative biomarkers of cytotoxicity induced by BaP, which might provide information for early prevention and intervention.

Fluorinated triphenylphosphonium analogs improve cell selectivity and invivo detection of mito-metformin.

Triphenylphosphonium (TPP+) conjugated compounds selectively target cancercells by exploiting their hyperpolarized mitochondrial membrane potential. Todate, studies have focused on modifying either the linker or the cargo of TPP+-conjugated compounds. Here, we investigated the biological effects ofdirect modification to TPP+ to improve the efficacy and detection of mito-metformin (MMe), a TPP+-conjugated probe we have shown to have promising preclinical efficacy against solid cancer cells. We designed, synthesized, and tested trifluoromethyl and methoxy MMe analogs (pCF3-MMe, mCF3-MMe, and pMeO-MMe) against multiple distinct human cancer cells. pCF3-MMe showed enhanced selectivity toward cancer cells compared to MMe, while retaining thesame signaling mechanism. Importantly, pCF3-MMe allowed quantitative monitoring of cellular accumulation via 19F-NMR invitro and invivo. Furthermore, adding trifluoromethyl groups to TPP+ reduced toxicity invivo while retaining anti-tumor efficacy, opening an avenue to de-risk these next-generation TPP+-conjugated compounds.

Dysregulated energy metabolism impairs chondrocyte function in osteoarthritis

Metabolic pathways are a series of chemical reactions by which cells take in nutrient substrates for energy and building blocks needed to maintain critical cellular processes. Details of chondrocyte metabolism and how it rewires during the progression of osteoarthritis (OA) are unknown. This research aims to identify what changes in the energy metabolic state occur in OA cartilage. Patient matched OA and non-OA cartilage specimens were harvested from total knee replacement patients. Cartilage was first collected for metabolomics, proteomics, and transcriptomics analyses to study global alterations in OA metabolism. We then determined the metabolic routes by tracking [U-13C] isotope with liquid chromatography-mass spectrometry (LC-MS). We further evaluated cellular bioenergetic profiles by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) and investigated the effects of low-dose and short-term effects of 2-deoxyglucose (2DG) on chondrocytes. OA chondrocytes showed increased basal ECAR and more lactate production compared to non-OA chondrocytes. [U-13C] glucose labelling revealed that less glucose-derived carbon entered the tricarboxylic acid (TCA) cycle. On the other hand, mitochondrial respiratory rates were markedly decreased in the OA chondrocytes compared to non-OA chondrocytes. These changes were accompanied by decreased cellular ATP production, mitochondrial membrane potential and disrupted mitochondrial morphology. We further demonstrated in vitro that short-term inhibition of glycolysis suppressed matrix degeneration gene expression in chondrocytes and bovine cartilage explants cultured under inflammatory conditions. This study represents the first comprehensive comparative analysis of metabolism in OA chondrocytes and lays the groundwork for therapeutic targeting of metabolism in OA.

Regulation of Oxidative Energy Metabolism By G0S2 in FLT3+ AML

Introduction: Acute myeloid leukemia (AML) patients with mutations in the FMS-like tyrosine kinase 3 (FLT3) gene represent 20-30% of all AML cases. These patients can be treated with tyrosine kinase inhibitors (TKIs) targeting FLT3; however, up to 60% of patients develop resistance or experience adverse side effects, which makes identifying mechanisms of drug resistance a priority. Our previous work demonstrated a tumor suppressor role for G0/G1 switch gene 2 (G0S2) in chronic myeloid leukemia (CML), which is profoundly downregulated in TKI resistance (Gonzalez MA, et al. under review). However, the role of G0S2 in FLT3+ AML is unknown. G0S2 regulates multiple cellular functions, including lipolysis, de novo lipogenesis, and oxidative phosphorylation; therefore, we hypothesized that reduced G0S2 expression in FLT3+ AML results in altered oxidative energy metabolism, promoting therapy resistance. Methods: To address this hypothesis, we first used shRNA lentiviral vectors to transduce the FLT3+ AML cell lines, MOLM-13 and MOLM-14, with a non-targeting control vector (shNT), a G0S2 knockdown vector (shG0S2), and an ectopic G0S2 expression vector (pLVX G0S2). In primary FLT3+ AML MNCs, we used an empty vector (EV) and pLVX G0S2. We assessed colony formation ability by plating cells in 0.9% Methocult plus cytokines for primary cells, and measured apoptosis using AnnexinV and 7-aminoactinomycin D (7AAD). Cell lines were treated with Doxycycline (100 ng/ml) ± FLT3 inhibitors, either Quizartinib (20nM) or Midostaurin (50nM). We performed immunoblotting using SDS-page, PVDF membranes, and a human antibody cocktail for the 5-electron transport chain (ETC) complexes. For the in vivo experiments, NOD-scid IL2Rgammanull (NSG) mice received intravenous injections of 3 million cells/mouse of the MOLM-14 cell lines with the shNT, shG0S2, and pLVX G0S2 vectors, and all mice were immediately placed in doxycycline hyclate chow to induce vector expression. To assess the role of G0S2 in FTL3+ AML cells, we used the correlation feature available at UALCAN to calculate the genes co-expressed with G0S2 in The Cancer Genome Atlas (TCGA) AML data. Finally, we measured oxygen consumption rates (OCR) using the Cell Mito Stress Test kit for the Agilent Seahorse XFp Bioanalyzer. Results: Using publically available data, we found that G0S2 mRNA expression does not correlate with overall survival (OS) in AML, but is significantly downregulated in AML versus normal mononuclear cells (MNCs, p<0.001), and further reduced in AML patients with mutated FLT3 (p=0.006). In colony formation assays, the FLT3 TKI Midostaurin reduced colony number in the presence of G0S2 ectopic expression when compared to its control. In contrast, FLT3 TKIs had less of an effect on apoptosis in the presence of G0S2 ectopic expression. In primary FLT3+ AML MNCs, ectopic G0S2 expression resulted in a significant reduction of colony formation, which correlated with increased induction of apoptosis when compared to the empty vector control. Importantly, pathway enrichment analysis using data from The Cancer Genome Atlas (TCGA) revealed that the genes co-expressed with G0S2 in AML implicated a role in energy metabolism. After confirming G0S2 ectopic expression in the MOLM-13 cell line, immunoblotting showed reduced expression of complexes III and V of the ETC, which correlated with reductions in oxygen consumption rates using the Agilent Seahorse Bioanalyzer. In vivo, G0S2 knockdown in FLT3+ AML cell lines resulted in a significant reduction in overall survival of NSG mice compared with controls (Figure 1). Conclusion: Altogether, our data demonstrate that loss of G0S2 expression in FLT3+ AML dysregulates oxygen metabolism by altering expression of ETC components. Ultimately, we showed that G0S2 is a tumor suppressor that is downregulated in FLT3+ AML, which might promote therapy resistance by altering oxidative energy metabolism. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Disruption of Intracellular Metal Homeostasis As a Vulnerability in SF3B1 Mutated AML

Acute Myeloid Leukemia (AML) is a heterogeneous disease in which outcome is highly dependent on cytogenetics and mutational profile. Adverse risk AML, recently defined and updated in the 2022 European LeukemiaNet (ELN) genetic risk classification, is associated with a poor overall survival. To identify novel therapeutic approaches for AML, we screened a library of 10,000 compounds across 56 genetically diverse primary AML specimens. We then looked for active compounds with increased activity towards adverse risk AML samples and with low cytotoxicity to normal hematopoietic cells. Among the hits identified, we were able to validate that S767 molecule is preferentially active on adverse risk AML samples. Additionally, we found a strong association between SF3B1 mutations and sensitivity to S767. To gain insights into S767 mechanism of action, we performed a CRISPR/Cas9 genome-wide functional screen, and identified multiples genes involved in mitochondrial processes both in synthetic rescue and synthetic lethal genes. In particular, knock-down of enzymes involved in heme biogenesis and iron-sulfur cluster biosynthesis appear to interfere with S767 cytotoxic activity. Given the central role of iron in these two synthetic pathways, and the structure of S767, we hypothesized that our molecule binds and disrupts intracellular iron homeostasis. Using UV-spectrometry analysis, we determined that S767 has metal affinity and binds to Fe2+, but also Cu2+ and Zn2+. We then quantified intracellular metal concentrations by ICP-MS (inductively coupled plasma mass spectrometry) and found that S767 exposure significantly decreases intracellular iron concentrations while increasing that of copper. Since metals are critical cellular cofactors involved in multiple enzymatic reactions, disruption of intracellular metal homeostasis can affect many processes. Mitochondrial respiratory chain is particularly sensitive to these perturbations as copper and iron-sulfur clusters are present in complexes I-IV of the electron transport chain and are essential for mitochondrial complexes function. By measuring oxygen consumption rate in leukemic cells exposed to S767, we found that the molecule indeed disrupts mitochondrial respiration. SF3B1 is a splicing factor recurrently mutated in myeloid malignancies including myelodysplastic syndromes (MDS) and AML. Heterozygous mutations typically affect SF3B1 splicing functions and induces consequently mis-splicing of target mRNAs, including the ABCB7 iron-sulfur cluster transporter. Analysis of the Leucegene transcriptomic data of primary AML specimens revealed mis-splicing and downregulation of ABCB7 in SF3B1 mutated patients. Strikingly, ABCB7 was one of the top synthetic lethal gene identified by chemogenomic CRISPR/Cas9 screen following exposure to S767 molecule. Given that ABCB7 exports iron-sulfur clusters out of the mitochondria to be assembled by cytosolic proteins and plays a role in iron homeostasis, we hypothesized that its aberrant expression in SF3B1 mutated AML participates to the high sensitivity of these leukemias to S767. We thus identified a novel molecule with selective activity in AML. Our data suggest that S767 is not a protein-targeting molecule but rather acts through the disruption of intracellular metal homeostasis, and ultimately affects mitochondrial respiration. Its mechanism of action highlighted a vulnerability in SF3B1 mutated AML which present a defect in ABCB7 expression. Importantly, SF3B1 mutations are now recognized in the 2022 ELN risk classification as an adverse prognostic marker if they are associated with intermediate or adverse risk AML, giving a unique opportunity to develop novel therapeutic approaches for this subgroup of patients with poor clinical outcome. In addition, SF3B1 mutations are found in 25% of MDS cases, which are associated with ring sideroblasts and accumulation of mitochondrial iron, and that could also benefit from such therapeutic approach.

Inhibition of mitochondrial function: An alternative explanation for the antipyretic and hypothermic actions of acetaminophen

AimsAcetaminophen is the medication of choice when treating fever because of its limited anti-inflammatory effects. However at overdose it can cause mitochondrial dysfunction and damage, often associated with metabolism to N-acetyl-p-benzoquinone imine (NAPQI). What has never been investigated is whether the inhibition of mitochondrial function, particularly fatty acid uptake and oxidation could be the key to its antipyretic and hypothermic properties. MethodsMitochondrial function and fatty acid oxidation (FAO) was determined by measuring oxygen consumption rate (OCR) in isolated mitochondria and in 3T3-L1 adipocytes using the XFp Analyser. Basal fatty acids and adrenergic stimulated OCR of mitochondria and 3T3-L1 adipocytes were assessed with acetaminophen and compared to NAPQI, etomoxir, and various mitochondrial stress compounds. Key findingsUsing the XFp Analyser, acetaminophen (10 mM) decreased FAO by 31 % and 29 % in basal and palmitate stimulated adipocytes. NAPQI (50 μM) caused a 63 % decrease in both basal and palmitate stimulated FAO. Acetaminophen (10 mM) caused a 34 % reduction in basal and adrenergic stimulated OCR. In addition acetaminophen also inhibited complex I and II activity at 5 mM. NAPQI was far more potent at reducing mitochondrial respiratory capacity, maximum respiratory rates and ATP production than acetaminophen. SignificanceThese studies demonstrate the direct inhibition of mitochondrial function by acetaminophen at concentrations which have been shown to reduce fever and hypothermia in mammals. Understanding how antipyretics directly affect mitochondrial function and heat generation could lead to the development of new antipyretics which are not compromised by the anti-inflammatory and toxicity of the current medications.

Thermal acclimation and metabolic scaling of a groundwater asellid in the climate change scenario

Metabolic rate has long been used in animal adaptation and performance studies, and individual oxygen consumption is used as proxy of metabolic rate. Stygofauna are organisms adapted to groundwater with presumably lower metabolic rates than their surface relatives. How stygofauna will cope with global temperature increase remains unpredictable. We studied the thermal acclimation and metabolic scaling with body mass of a stygobitic crustacean, Proasellus lusitanicus, in the climate change scenario. We measured oxygen consumption rates in a thermal ramp-up experiment over four assay temperatures and tested two hypotheses: (i) P. lusitanicus exhibits narrow thermal plasticity, inadequate for coping with a fast-increasing thermal regime; and (ii) oxygen consumption rates scale with the body mass by a factor close to 0.75, as commonly observed in other animals. Our results show that P. lusitanicus has low thermal plasticity in a fast-increasing thermal regime. Our data also suggest that oxygen consumption rates of this species do not follow mass-dependent scaling, potentially representing a new trait of metabolic optimization in groundwater habitats, which are often limited in food and oxygen. Species with limited dispersal capacities and rigid metabolic guilds face extinction risk due to climate change and omitting groundwater ecosystems from climate change agendas emphasizes the unprotected status of stygofauna.

Developing Bi-Gold Compound BGC2a to Target Mitochondria for the Elimination of Cancer Cells.

Reactive oxygen species (ROS) homeostasis and mitochondrial metabolism are critical for the survival of cancer cells, including cancer stem cells (CSCs), which often cause drug resistance and cancer relapse. Auranofin is a mono-gold anti-rheumatic drug, and it has been repurposed as an anticancer agent working by the induction of both ROS increase and mitochondrial dysfunction. Hypothetically, increasing auranofin’s positive charges via incorporating more gold atoms to enhance its mitochondria-targeting capacity could enhance its anti-cancer efficacy. Hence, in this work, both mono-gold and bi-gold compounds were designed and evaluated to test our hypothesis. The results showed that bi-gold compounds generally suppressed cancer cells proliferation better than their mono-gold counterparts. The most potent compound, BGC2a, substantially inhibited the antioxidant enzyme TrxR and increased the cellular ROS. BGC2a induced cell apoptosis, which could not be reversed by the antioxidant agent vitamin C, implying that the ROS induced by TrxR inhibition might not be the decisive cause of cell death. As expected, a significant proportion of BGC2a accumulated within mitochondria, likely contributing to mitochondrial dysfunction, which was further confirmed by measuring oxygen consumption rate, mitochondrial membrane potential, and ATP production. Moreover, BGC2a inhibited colony formation and reduced stem-like side population (SP) cells of A549. Finally, the compound effectively suppressed the tumor growth of both A549 and PANC-1 xenografts. Our study showed that mitochondrial disturbance may be gold-based compounds’ major lethal factor in eradicating cancer cells, providing a new approach to developing potent gold-based anti-cancer drugs by increasing mitochondria-targeting capacity.

Measurement of oxygen consumption rates of human renal proximal tubule cells in an array of organ-on-chip devices to monitor drug-induced metabolic shifts

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.

Anticancer Effect of Cathelicidin LL-37, Protegrin PG-1, Nerve Growth Factor NGF, and Temozolomide: Impact on the Mitochondrial Metabolism, Clonogenic Potential, and Migration of Human U251 Glioma Cells.

Glioblastoma (GBM) is one of the most aggressive and lethal malignancy of the central nervous system. Temozolomide is the standard of care for gliomas, frequently results in resistance to drug and tumor recurrence. Therefore, further research is required for the development of effective drugs in order to guarantee specific treatments to succeed. The aim of current study was to investigate the effects of nerve growth factor (NGF), human cathelicidin (LL-37), protegrin-1 (PG-1), and temozolomide on bioenergetic function of mitochondria, clonogenicity, and migration of human U251 glioma cells. Colony formation assay was used to test the ability of the glioma cells to form colonies in vitro. The U251 glioma cells migration was evaluated using wound-healing assay. To study the mitochondrial metabolism in glioma cells we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) using a Seahorse XF cell Mito stress test kit and Seahorse XF cell Glycolysis stress kit, respectively. We revealed that LL-37, NGF, and TMZ show strong anti-tumorigenic activity on GMB. LL-37 (4 μM), TMZ (155 μM), and NGF (7.55 × 10−3 μM) inhibited 43.9%–60.3%, 73.5%–81.3%, 66.2% the clonogenicity of glioma U251 cells for 1–2 days, respectively. LL-37 (4 μM), and NGF (7.55 × 10−3 μM) inhibited the migration of U251 glioma cells on the third and fourth days. TMZ also inhibited the migration of human glioma U251 cells over 1–3 days. In contrast, PG-1 (16 μM) stimulated the migration of U251 glioma cells on the second, fourth, and sixth days. Anti-mitogenic and anti-migration activities of NGF, LL-37, and TMZ maybe are relation to their capacity to reduce the basal OCR, ATP-synthetase, and maximal respiration of mitochondria in human glioma U251 cells. Glycolysis, glycolytic capacity and glycolytic spare in glioma U251 cells haven`t been changed under the effect of NGF, LL-37, PG-1, and TMZ in regard to control level. Thus, LL-37 and NGF inhibit migration and clonogenicity of U251 glioma cells, which may indicate that these compounds have anti-mitogenic and anti-migration effects on human glioma cells. The study of the mechanisms of these effects may contribute in the future to the use of NGF and LL-37 as therapeutic agents for gliomas.

Short Communication: Beta-adrenergic agonists alter oxidative phosphorylation in primary myoblasts.

Beta-adrenergic agonists (β-AAs) are widely used supplements in beef and pork production to improve feed efficiency and increase lean muscle mass, yet little is known about the molecular mechanism by which β-AAs achieve this outcome. Our objective was to identify the influence of ractopamine HCl and zilpaterol HCl on mitochondrial respiratory activity in muscle satellite cells isolated from crossbred beef steers (N = 5), crossbred barrows (N = 2), Yorkshire-cross gilts (N = 3), and commercial weather lambs (N = 5). Real-time measurements of oxygen consumption rates (OCRs) were recorded using extracellular flux analyses with a Seahorse XFe24 analyzer. After basal OCR measurements were recorded, zilpaterol HCl, ractopamine HCl, or no β-AA was injected into the assay plate in three technical replicates for each cell isolate. Then, oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, and rotenone were injected into the assay plate sequentially, each inducing a different cellular state. This allowed for the measurement of OCR at these states and for the calculation of the following measures of mitochondrial function: basal respiration, non-mitochondrial respiration, maximal respiration, proton leak, adenosine triphosphate (ATP)-linked respiration, and spare respiratory capacity. Incubation of bovine cells with either zilpaterol HCl or ractopamine HCl increased maximal respiration (P = 0.046) and spare respiratory capacity (P = 0.035) compared with non-supplemented counterparts. No difference (P > 0.05) was observed between zilpaterol HCl and ractopamine HCl for maximal respiration and spare respiratory capacity in bovine cell isolates. No measures of mitochondrial function (basal respiration, non-mitochondrial respiration, maximal respiration, proton leak, ATP-linked respiration, and spare respiratory capacity) were altered by β-AA treatment in ovine or porcine cells. These findings indicate that β-AAs in cattle may improve the efficiency of oxidative metabolism in muscle satellite cells by modifying mitochondrial respiratory activity. The lack of response by ovine and porcine cells to β-AA incubation also demonstrates differing physiological responses to β-AA across species, which helps to explain the variation in its effectiveness as a growth supplement.

Mitochondrial Respiration Inhibition Suppresses Papillary Thyroid Carcinoma Via PI3K/Akt/FoxO1/Cyclin D1 Pathway.

BackgroundPapillary thyroid carcinoma (PTC) is the most common thyroid malignancy, but little is known regarding PTC metabolic phenotypes and the effects of mitochondrial activity on PTC progression. The great potential of mitochondria-targeting therapy in cancer treatment promoted us to use tool compounds from a family of Mito-Fu derivatives to investigate how the regulation of mitochondrial respiration affected tumor progression characteristics and molecular changes in PTC.MethodsMito-Fu L20, a representative of 12 synthetic derivatives, was chosen for mitochondrial inhibition experiments. Sample sections from PTC patients were collected and processed to explore potential molecular alterations in tumor lymph node metastasis (LNM). In vitro analyses were performed using human PTC cell lines (K1 and TPC-1), with the human normal thyroid follicular cell line (Nthy) as a control. K1 cells were injected into nude mice to generate an animal model. The mice were injected with normal saline or Mito-Fu L20 at 20 or 50 mg/kg every other day; their body weights and tumor volumes were also measured over time. To elucidate the resulting metabolic phenotype, we measured oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), cellular adenosine triphosphate (ATP) levels and reactive oxygen species (ROS) production, and mitochondrial membrane potential. Wound healing and Transwell assays, cell cycle assays, real-time fluorescence quantitative PCR, Western blotting, and immunohistochemical staining were performed to explore glycolysis-dominant metabolism in PTC.ResultsCyclin D1 and mitochondrial complex IV were detected in tumor samples from PTC patients with LNM. Mito-Fu L20 showed dose-independent and reversible modulation of mitochondrial respiration in PTC. In addition to mitochondrial dysfunction and early apoptosis, G1/S phase arrest. Notably, reversible mitochondrial inhibition yielded durable suppression of tumor proliferation, migration, and invasion via the PI3K/Akt/FoxO1/Cyclin D1 pathway. In vivo experiments demonstrated that Mito-Fu L20 has a good safety profile and specific restorative effect on mitochondrial activity in the liver. In addition, Mito-Fu L20 showed antitumor effects, alleviated tumor angiogenesis, and improved thyroid function.ConclusionReversible inhibition of ATP production and durable suppression of PTC growth indicates that the downregulation of mitochondrial function has a negative impact on tumor progression and LNM via the PI3K/Akt/FoxO1/Cyclin D1 pathway. The results provide new insights into the antitumor potential and clinical translation of mitochondrial inhibitors.

Abstract 5807: MDM2 mitochondrial translocation mediates metabolic reprogramming towards OXPHOS in TKI-resistant oncogene-addicted cancer

Abstract There is mounting evidence of oxidative phosphorylation (OXPHOS) dependency in cancers resistant to tyrosine kinase inhibitors (TKI), but the cause of this metabolic switch remains elusive. Mitochondria-bound MDM2 (mtMDM2), the fraction of MDM2 oncoprotein which is actively imported into the mitochondria, has been found to dysregulate electron transport chain (ETC) complex I function and OXPHOS. AKT is known to be a regulator of MDM2 protein stability. Given that AKT phosphorylation is commonly detected in oncogene-addicted tumours, we investigated the role of mtMDM2 and AKT in promoting TKI resistance through reprogrammed cellular metabolism. EGFR+ NSCLC [HCC827 and HCC827-GR (gefitinib-resistant)] and BRAF+ melanoma [A375 and A375-VR (vemurafenib-resistant)] cells were established via repetitive pulsed strategy. Translocation of MDM2 was examined through subcellular fractionation and Western Blotting. To investigate the role of MDM2 in mediating OXPHOS, genetic knockdown of MDM2 was performed via siRNA followed by measurement of oxygen consumption rate (Seahorse Analyzer); mRNA expression of mtDNA-encoded ETC subunits by real-time PCR (qPCR). ChIP-qPCR analysis was performed to examine the binding affinity of MDM2 and TFAM (mitochondrial transcription factor A) to mitochondrial DNA (mtDNA). Mitochondrial translocation of MDM2 was predominant in TKI-sensitive cells; MDM2 silencing upregulated OXPHOS and induced mRNA expression of mtDNA-encoded ETC complex I subunits, suggesting that mtMDM2 inhibits OXPHOS. ChIP analysis revealed competitive binding of MDM2 and TFAM at the LSP region of mtDNA. Conversely, MDM2 was primarily localized the cytoplasm in TKI-resistant cells and was associated with increased OXPHOS in TKI-resistant cells. Mechanistically, both pAKT and pMDM2 (Ser166/Ser186) were higher in TKI-resistant cells. Collectively, we describe a novel observation that OXPHOS upregulation underpinning TKI resistance in cancer cells could be mediated by AKT upregulation and MDM2 localisation. Citation Format: Jie Qing Eu, Li Ren Kong, Jayshree Hirpara, Boon Cher Goh, Andrea LA Wong, Shazib Pervaiz. MDM2 mitochondrial translocation mediates metabolic reprogramming towards OXPHOS in TKI-resistant oncogene-addicted cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5807.

Abstract 1990: Exploring the immune-metabolic mechanisms of adipose tissue in oesophageal adenocarcinoma

Abstract Background: Oesophageal Adenocarcinoma (OAC) is the most strongly associated cancer with obesity. Approximately 75% of OAC patients are obese which results in chronic systemic low-grade inflammation, which is believed to drive carcinogenesis as well as influencing radiation treatment response. Adipose tissue is a regulatory organ with many downstream effects which are still not understood. This study aims to elucidate what influence adipose tissue metabolism and secretome have in treatment resistance and whether obesity alters this response. Methods: Following patient consent, ex-vivo Visceral Adipose Tissue (VAT) explants were exposed to increasing doses of radiation with/without Palmitate and Oleate. Agilent Seahorse Xfe24 was used to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in VAT explants and mitochondrial metabolic parameters in dendritic cells (DCs) following treatment with Adipose Conditioned Media (ACM). Levels of DC maturation following irradiated ACM exposure and Macrophage (Mɸ) polarisation following ACM or Tumour Conditioned Media (TCM) exposure were analyzed by flow cytometry. Irradiated ACM and TCM were analyzed via MSD 54plex ELISA to assess secreted factors on angiogenic, chemokine, cytokine, inflammatory, TH17 or vascular injury panels. Results: VAT explant energy metabolism showed a significant increase for OCR and ECAR with obesity and increasing radiation exposure however OCR was significantly decreased following Palmitate treatment compared with untreated and Oleate treated adipose explants (p&amp;lt;0.05). DCs treated with iACM showed decreased expression of CD11c, CD80, CD86, HLA-DR, CD40 and PD-L1 with increasing radiation in a dose-dependent manner and decreased HLA-DR and CD54 following ACM exposure from obese versus non-obese patients (p&amp;lt;0.05). Mɸ showed increased expression of pro-inflammatory and anti-inflammatory markers following ACM exposure but following TCM exposure only anti-inflammatory markers were increased (p&amp;lt;0.05). DCs showed altered Proton Leak, Basal and ATP linked respiration following exposure to ACM (p&amp;lt;0.05). Altered secretion of proinflammatory mediators was observed from the obese adipose secretome compared with non-obese patients and with increasing radiation doses (p&amp;lt;0.05). Conclusion: We have demonstrated that obesity and increasing radiation doses can significantly alter the immune-metabolic influences of adipose tissue and further studies are profiling the lipidomic and metabolomic landscape of the adipose secretome. Alterations in the secretome of adipose tissue could potentiate the tumor microenvironment and deleteriously affect immune cell function therefore further interrogation is required to fully elucidate the influence adipose tissue may have in treatment response. Citation Format: Fiona O'Connell, Eimear Mylod, Ailsing B. Heeran, Noel E. Donlon, Maria Davern, Christine Butler, Anshul Bhardwaj, Claire Donohoe, Narayanasamy Ravi, John V. Renyolds, Margaret R. Dunne, Helen M. Roche, Jacintha O'Sullivan. Exploring the immune-metabolic mechanisms of adipose tissue in oesophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1990.

Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice

Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice

Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice.

Mitochondria play an important role in cellular ATP production, reactive oxygen species regulation, and Ca2+ concentration control. Mitochondrial dysfunction has been implicated in the pathogenesis of multiple neurodegenerative diseases, including Parkinson's disease (PD), Huntington's disease, and Alzheimer's disease. To study the role of mitochondria in models of these diseases, we can measure mitochondrial respiration via oxygen consumption rate (OCR) as a proxy for mitochondrial function. OCR has already been successfully measured in cell cultures, as well as isolated mitochondria. However, these techniques are less physiologically relevant than measuring OCR in acute brain slices. To overcome this limitation, the authors developed a new method using a Seahorse XF analyzer to directly measure the OCR in acute striatal slices from adult mice. The technique is optimized with a focus on the striatum, a brain area involved in PD and Huntington's disease. The analyzer performs a live cell assay using a 24-well plate, which allows the simultaneous kinetic measurement of 24 samples. The method uses circular-punched pieces of striatal brain slices as samples. We demonstrate the effectiveness of this technique by identifying a lower basal OCR in striatal slices of a mouse model of PD. This method will be of broad interest to researchers working in the field of PD and Huntington's disease.