Basal Oxygen Consumption Research Articles

The Effects of Elevated Sodium on Mitochondrial Function in Peripheral Blood Mononuclear Cells

INTRODUCTIONDiets high in sodium are associated with impaired vascular function independent of blood pressure. Recent data suggests that sodium accumulation from high salt diets reduces mitochondrial respiration in monocytes and macrophages, resembling what occurs during an inflammatory response. A similar response in circulating peripheral blood mononuclear cells (PBMCs) could implicate systemic inflammation as a mechanism contributing to high salt‐induced vascular dysfunction. Therefore, we aimed to test the hypothesis that exposure to a high sodium environment reduces PBMC mitochondrial respiration.METHODSPBMCs were separated from whole blood of ten healthy subjects (7 men/3 women, 18‐30 years, blood pressure ≤ 130/80mmHg; BMI < 30 kg/m2). PBMCs were then seeded at 2x105 cells/well in Poly‐D‐lysine treated cell culture plates. Plated cells were incubated for 24 hours in RPMI 1640 medium with 10% FBS at 137.7 mM sodium (normal sodium, NS) and 180 mM sodium (high sodium, HS). Basal, maximal, and ATP‐linked oxygen consumption rates were assessed post‐incubation by respirometry assay via Seahorse XFp analyzer (Agilent). Spare respiratory capacity was calculated as the difference between maximal and basal oxygen consumption rate.RESULTSBasal oxygen consumption rate was significantly reduced in HS compared to NS (65.0 ± 12.7 vs. 39.6 ± 6.5 pmol/min, p = 0.03), as was maximal oxygen consumption rate (198.3 ± 32.6 vs. 148.4 ± 27.2 pmol/min, p = 0.008). Basal ATP‐linked oxygen consumption rate was also significantly reduced in HS compared to NS (57.3 ± 9.8 vs. 34.4 ± 5.4 pmol/min, p = 0.01). Spare respiratory capacity in HS was significantly reduced compared to NS (133.2 ± 23.4 vs. 108.8 ± 21.2 pmol/min, p = 0.03).CONCLUSIONReduced basal and maximal oxygen consumption rates and spare respiratory capacity provide evidence that elevated sodium exposure reduces PBMC mitochondrial respiration. Additionally, high sodium exposure reduced ATP‐linked respiration potentially indicating alterations in PBMC energetics, resembling what occurs during inflammation. Collectively, our preliminary findings warrant further investigation into the role of sodium induced mitochondrial dysfunction in PBMCs as a potential mechanism contributing to high salt‐induced vascular dysfunction.

Plate‐Based Respirometry to Assess Zebrafish Embryo Bioenergetics in situ in Changing Environmental Conditions

All species require bioenergetic adaptation to maintain life functions, especially in changing environmental conditions. Global warming and deteriorating water quality may pose threats to aquatic species by interfering with bioenergetic mechanisms. Mitochondrial oxidative metabolism is a key component for bioenergetic responses as it provides the major proportion of cellular energy in the form of adenosine triphosphate (ATP). In our studies, we aim to establish the assessment of zebrafish mitochondrial bioenergetics and investigate the mechanisms, adaptation, and plasticity to temperature, pollutants, and the combination of both to gain insights into the upcoming threats for their energy homeostasis and survival. Using plate‐based respirometry of the Seahorse XF96 analyzer, we measured zebrafish (Danio rerio) embryo oxygen consumption rates in situ. To understand how temperature alters oxidative phosphorylation, we exposed 48 hours post‐fertilization zebrafish embryos to five different water temperatures before measuring oxygen consumption at various assay temperatures. The oxygen consumption rates were challenged using respiratory chain inhibitors and chemical uncouplers to reveal bioenergetic mechanisms and adjustments of mitochondrial efficiency. We show that at 18°C assay temperature, basal oxygen consumption of the embryo is low due to decreased ATP‐linked respiration and not limited by maximum substrate oxidation. Basal oxygen consumption rates increase with assay temperature and remain stable up to 37°C, before rapidly deteriorating at 41°C and above. The pre‐exposure of embryos to 37°C, however, expands the thermal window to higher limits, indicated by stable basal oxygen consumption rates at 41°C. Proton leak respiration, which wastes nutrient energy in parallel to ATP production, increases at assay temperatures above 28°C, thereby reducing the efficiency of ATP synthesis. This is seen as a decrease in coupling efficiency, which is defined as the fraction of oxygen consumption that drives ATP synthesis. Collectively, temperature affects the performance of zebrafish embryos by reducing cellular ATP turnover at lower temperatures and by increasing proton leak at higher temperatures, resulting in an optimum energy conversion temperature that can be shifted by thermal adaptation. In current experiments, we measure how pollutants impact zebrafish bioenergetics and its relation to thermal adaptation.

Characterizing a Canine Urothelial Carcinoma Model of Acquired MEK1/2 Inhibitor Resistance

Activating BRAF mutations are drivers of oncogenesis in several human cancers and in over 70% of canine urothelial carcinomas (cUC). Selective BRAF inhibitors are effective alone and in combination with MEK inhibitors in approximately half of BRAF mutant melanoma cases, but the majority of these patients ultimately develop resistance. Thus, intrinsic and acquired resistance to MAP kinase pathway targeted agents remains a challenge requiring the development of novel therapeutic strategies. Using canine urothelial carcinoma as a spontaneous cancer model to explore treatment of BRAF mutant cancers and acquired drug resistance, we sought to identify determinants of MEK1/2 inhibitor sensitivity and resistance. We applied the human‐derived MAPK Pathway Activity Score (MPAS), a predictor of sensitivity to MEK inhibition in human cancers based on expression levels of 10 downstream targets, to a panel of 32 canine cancer cell lines and found that MPAS correlates with trametinib sensitivity in this canine dataset. MPAS genes with significant (p<0.05) Pearson correlations to sensitivity were CCND1, DUSP6, ETV4, ETV5, and SPRY2. We also identified 30 non‐MPAS genes whose expression levels are significantly correlated with trametinib sensitivity, suggesting their role in de novo sensitivity to trametinib. Each of the 5 urothelial carcinoma lines had high MPAS scores and trametinib IC50 values <10nM. CRISPR‐Cas9 knockouts in two canine urothelial carcinoma lines also identified MEK1 as a critical dependency. To investigate mechanisms of acquired resistance, we generated trametinib‐resistant (TramR) clonal derivatives of the BRAF mutant Tyler1 canine urothelial carcinoma cell line (Tyler1‐TramR), with trametinib IC50 values greater than 500 nM versus 1 nM in parental Tyler1. Tyler1‐TramR cell lines exhibited sustained inhibition of ERK1/2 phosphorylation when treated with trametinib and were also resistant to pharmacologic inhibition of ERK1/2. Collectively, these data suggest an ERK‐independent mechanism of resistance. Gene expression analysis of two Tyler1‐TramR clones identified dramatic downregulation of epithelial markers accompanied by an increase in expression of mesenchymal genes and transcription factors that regulate the epithelial‐to‐mesenchymal transition (EMT). One Tyler1‐TramR clone exhibited metabolic alterations including decreased basal and maximal oxygen consumption rates, diminished spare respiratory capacity, and reduced glycolytic capacity. Overall, the findings in this study suggest that the mechanisms of acquired and intrinsic MEK inhibitor resistance are conserved between man and dog, further elucidating the translational value of cUC as a model to optimize therapies for Ras/Raf/MAP kinase driven cancers.

Characterizing Metabolic Changes in Cancer Cells after Treatment with an Extract Created from Walnuts

One of the main hallmarks of cancer cells is the reprogramming of energy metabolism to support the high energy and metabolite demands of constant proliferation. Although previously observed as mainly a shift from oxidative phosphorylation to glycolysis, also known as the Warburg Effect, recent research has shown that cancer cells can utilize both pathways to make energy, which has been referred to as metabolic flux. This suggests that cancer cells can adapt to their environment by reactivating or continuing to use the oxidative phosphorylation pathway to make energy. Thus, the energy metabolism of cancer cells is of therapeutic interest. The triple‐negative breast cancer cell line MDA‐MB‐231 can undergo metabolic flux, making it an ideal cell line to study the effects of metabolism‐altering drugs. Previous work from our lab showed that treatment with a Walnut Extract (WE) induced cell death in MDA‐MB‐231 cells, as well as HeLa cells. We also observed that the WE treatment targets the mitochondria by destabilizing the mitochondrial outer membrane potential, suggesting that the extract may be able to impact oxidative phosphorylation. To examine the effects on energy production, we have taken advantage of the Agilent Seahorse XFe96 Cell Metabolic Analyzer to monitor, in real time, metabolic changes in MDA‐MB‐231 and HeLa cells treated with WE. WE treatment had impacts on both glycolytic and oxidative phosphorylation processes in both cell lines. Both time‐ and dose‐dependent responses were observed, with WE treated MDA‐MB‐231 cells exhibiting a 70% reduction in basal oxygen consumption rate compared to vehicle treated cells. Additionally, basal glycolytic rate was reduced by up to 41%, and compensatory glycolysis was reduced 70%. In HeLa cells, basal oxygen consumption rate was reduced by 74%, with a 56% reduction in basal glycolytic rate and a 76% reduction in compensatory glycolysis. Quantification of ATP production rate showed an overall reduction of 73%, with a 91% reduction in mitochondrially contributed ATP production, as well as a 47% reduction in glycolytic ATP production in MDA‐MB‐231 cells. Quantitation of total ATP within the cell using a separate assay shows an 62% decrease in ATP in treated HeLa cells. We have also looked at the status of mTOR and AMPK in the cells after treatment with the WE, because of their role in energy sensing and energy production. Interestingly, our preliminary data suggests that phosphorylated mTOR levels do not change and we also do not detect any phosphorylation of AMPK, suggesting that the WE is not effecting its changes on these proteins despite the reduction in overall ATP in the cell. Taken together, our results suggest that the WE treatment can alter and reduce the rates of glycolysis and oxidative phosphorylation, and by extension, the metabolic flux of these cancer cells.

Improving the metabolic efficiency of human T cells through AMPK agonist treatment

Abstract BACKGROUND Adoptive immunotherapies are limited by the increased differentiation and metabolic exhaustion of the T cell product. The cellular energy sensor AMP-activated protein kinase (AMPK) controls many metabolic pathways. We hypothesized that increasing AMPK signaling during T cell expansion would create more metabolically efficient T cells. RESULTS Human T cells were stimulated in vitro, expanded with or without AMPK agonist A769662, and restimulated under varying glucose concentrations. Agonist treatment increased T cell expansion by manual cell counts (1.5× increase in cell number, p<0.01), and produced a higher percentage of CD62L+CD27+ cells by flow cytometry (10% increase, p<0.01). Twenty-four hours after re-stimulation, agonist-treated cells exhibited decreased basal oxygen consumption and extracellular acidification rates (45% and 20% decrease, p<0.001) but increased spare respiratory capacity (SRC) (97% increase, p<0.001). At 72 hours, control cells decreased proliferation in a glucose dose-dependent manner, as measured by BrDU uptake, whereas treated cells maintained a higher level of proliferation despite limited glucose availability. DISCUSSION Increasing AMPK signaling during T cell expansion improves doubling time while maintaining higher CD27 positivity, suggesting a greater yield of less differentiated cells. Upon re-stimulation, treated cells initiate less metabolic activity but maintain greater proliferation and SRC under glucose-limiting conditions. These results suggest that greater metabolic efficiency in agonist-treated cells allows for continued function despite nutrient restriction, a trait which could confer significant advantages to adoptive immunotherapies. Supported by grants from NIH (5K12 HD052892) and the St. Baldrick's Foundation (St. Baldrick's Fellowship Grant)

Gene therapy of yeast NDI1 on mitochondrial complex I dysfunction in rotenone-induced Parkinson\u2019s disease models in vitro and vivo

PurposeParkinson's disease (PD) is the second most common neurodegenerative disease without cure or effective treatment. This study explores whether the yeast internal NADH-quinone oxidoreductase (NDI1) can functionally replace the defective mammalian mitochondrial complex I, which may provide a gene therapy strategy for treating sporadic PD caused by mitochondrial complex I dysfunction.MethodRecombinant lentivirus expressing NDI1 was transduced into SH-SY5Y cells, or recombinant adeno-associated virus type 5 expressing NDI1 was transduced into the right substantia nigra pars compacta (SNpc) of mouse. PD cell and mouse models were established by rotenone treatment. The therapeutic effects of NDI1 on rotenone-induced PD models in vitro and vivo were assessed in neurobehavior, neuropathology, and mitochondrial functions, by using the apomorphine-induced rotation test, immunohistochemistry, immunofluorescence, western blot, complex I enzyme activity determination, oxygen consumption detection, ATP content determination and ROS measurement.ResultsNDI1 was expressed and localized in mitochondria in SH-SY5Y cells. NDI1 resisted rotenone-induced changes in cell morphology, loss of cell viability, accumulation of α-synuclein and pS129 α-synuclein, mitochondrial ROS production and mitochondria-mediated apoptosis. The basal and maximal oxygen consumption, mitochondrial coupling efficiency, basal and oligomycin-sensitive ATP and complex I activity in cell model were significantly increased in rotenone + NDI1 group compared to rotenone + vector group. NDI1 was efficiently expressed in dopaminergic neurons in the right SNpc without obvious adverse effects. The rotation number to the right side (NDI1-treated side) was significantly increased compared to that to the left side (untreated side) in mouse model. The number of viable dopaminergic neurons, the expression of tyrosine hydroxylase, total and maximal oxygen consumption, mitochondrial coupling efficiency and complex I enzyme activity in right substantia nigra, and the content of dopamine in right striatum were significantly increased in rotenone + NDI1 group compared to rotenone + vector group.ConclusionYeast NDI1 can rescue the defect of oxidative phosphorylation in rotenone-induced PD cell and mouse models, and ameliorate neurobehavioral and neuropathological damages. The results may provide a basis for the yeast NDI1 gene therapy of sporadic PD caused by mitochondrial complex I dysfunction.

Anti-obesity effects of heat-transformed green tea extract through the activation of adipose tissue thermogenesis

BackgroundAdipose tissue thermogenesis is a potential therapeutic target to increase energy expenditure and thereby combat obesity. The aim of the present study was to investigate the thermogenic and anti-obesity effects of heat-transformed green tea extract (HTGT) and enzymatically modified isoquercetin (EMIQ).MethodsImmortalized brown pre-adipocytes and C3H10T1/2 cells were used for in vitro analyses. A high-fat diet (HFD)-induced obesity mouse model and CIDEA-reporter mice were used for in vivo experiments. The effects of HTGT and EMIQ on mitochondrial metabolism were evaluated by immunoblot, mitochondrial staining, and oxygen consumption rate analyses. In vivo anti-obesity effects of HTGT and EMIQ were measured using indirect calorimetry, body composition analyses, glucose tolerance tests, and histochemical analyses.ResultsCo-treatment with HTGT and EMIQ (50 μg/mL each) for 48 h increased brown adipocyte marker and mitochondrial protein levels (UCP1 and COXIV) in brown adipocytes by 2.9-fold, while the maximal and basal oxygen consumption rates increased by 1.57- and 1.39-fold, respectively. Consistently, HTGT and EMIQ treatment increased the fluorescence intensity of mitochondrial staining in C3H10T1/2 adipocytes by 1.68-fold. The combination of HTGT and EMIQ (100 mg/kg each) increased the expression levels of brown adipocyte markers and mitochondrial proteins in adipose tissue. Two weeks of HTGT and EMIQ treatment (100 mg/kg each) led to a loss of 3% body weight and 7.09% of body fat. Furthermore, the treatment increased energy expenditure by 8.95% and improved glucose tolerance in HFD-fed mice.ConclusionsThe current study demonstrated that HTGT and EMIQ have in vivo anti-obesity effects partly by increasing mitochondrial metabolism in adipocytes. Our findings suggest that a combination of HTGT and EMIQ is a promising therapeutic agent for the treatment of obesity and related metabolic diseases.

The Cardiac Dysfunction Caused by Metabolic Alterations in Alzheimer's Disease.

A progressive defect in the energy generation pathway is implicated in multiple aging-related diseases, including cardiovascular conditions and Alzheimer's Disease (AD). However, evidence of the pathogenesis of cardiac dysfunction in AD and the associations between the two organ diseases need further elucidation. This study aims to characterize cellular defects resulting in decreased cardiac function in AD-model. 5XFAD mice, a strain expressing five mutations in human APP and PS1 that shows robust Aβ production with visible plaques at 2 months and were used in this study as a model of AD. 5XFAD mice and wild-type (WT) counterparts were subjected to echocardiography at 2-, 4-, and 6-month, and 5XFAD had a significant reduction in cardiac fractional shortening and ejection fraction compared to WT. Additionally, 5XFAD mice had decreased observed electrical signals demonstrated as decreased R, P, T wave amplitudes. In isolated cardiomyocytes, 5XFAD mice showed decreased fraction shortening, rate of shortening, as well as the degree of transient calcium influx. To reveal the mechanism by which AD leads to cardiac systolic dysfunction, the immunoblotting analysis showed increased activation of AMP-activated protein kinase (AMPK) in 5XFAD left ventricular and brain tissue, indicating altered energy metabolism. Mito Stress Assays examining mitochondrial function revealed decreased basal and maximal oxygen consumption rate, as well as defective pyruvate dehydrogenase activity in the 5XFAD heart and brain. Cellular inflammation was provoked in the 5XFAD heart and brain marked by the increase of reactive oxygen species accumulation and upregulation of inflammatory mediator activities. Finally, AD pathological phenotype with increased deposition of Aβ and defective cognitive function was observed in 6-month 5XFAD mice. In addition, elevated fibrosis was observed in the 6-month 5XFAD heart. The results implicated that AD led to defective mitochondrial function, and increased inflammation which caused the decrease in contractility of the heart.

Differential Effects of 2-Deoxyglucose and Glucose Deprivation on 4-Hydroxynonenal Dependent Mitochondrial Dysfunction in Primary Neurons

Mitochondrial dysfunction and metabolic decline are prevalent features of aging and age-related disorders, including neurodegeneration. Neurodegenerative diseases are associated with a progressive loss of metabolic homeostasis. This pathogenic decline in metabolism is the result of several factors, including decreased mitochondrial function, increased oxidative stress, inhibited autophagic flux, and altered metabolic substrate availability. One critical metabolite for maintaining neuronal function is glucose, which is utilized by the brain more than any other organ to meet its substantial metabolic demand. Enzymatic conversion of glucose into its downstream metabolites is critical for maintaining neuronal cell growth and overall metabolic homeostasis. Perturbation of glycolysis could significantly hinder neuronal metabolism by affecting key metabolic pathways. Here, we demonstrate that the glucose analogue 2-deoxyglucose (2DG) decreases cell viability, as well as both basal and maximal mitochondrial oxygen consumption in response to the neurotoxic lipid 4-hydroxynonenal (HNE), whereas glucose deprivation has a minimal effect. Furthermore, using a cell permeabilization assay we found that 2DG has a more pronounced effect on HNE-dependent inhibition of mitochondrial complex I and II than glucose deprivation. Importantly, these findings indicate that altered glucose utilization plays a critical role in dictating neuronal survival by regulating the mitochondrial response to electrophilic stress.

Oocyte metabolic function, lipid composition, and developmental potential are altered by diet in older mares.

Dietary supplementation is the most feasible method to improve oocyte function and developmental potential in vivo. During three experiments, oocytes were collected from maturing, dominant follicles of older mares to determine whether short-term dietary supplements can alter oocyte metabolic function, lipid composition, and developmental potential. Over approximately 8 weeks, control mares were fed hay (CON) or hay and grain products (COB). Treated mares received supplements designed for equine wellness and gastrointestinal health, flaxseed oil, and a proprietary blend of fatty acid and antioxidant support (reproductive support supplement (RSS)) intended to increase antioxidant activity and lipid oxidation. RSS was modified for individual experiments with additional antioxidants or altered concentrations of n-3 to n-6 fatty acids. Oocytes from mares supplemented with RSS when compared to COB had higher basal oxygen consumption, indicative of higher aerobic metabolism, and proportionately more aerobic to anaerobic metabolism. In the second experiment, oocytes collected from the same mares prior to (CON) and after approximately 8 weeks of RSS supplementation had significantly reduced oocyte lipid abundance. In the final experiment, COB was compared to RSS supplementation, including RSS modified to proportionately reduce n-3 fatty acids and increase n-6 fatty acids. The ability of sperm-injected oocytes to develop into blastocysts was higher for RSS, regardless of fatty acid content, than for COB. We demonstrated that short-term diet supplementation can directly affect oocyte function in older mares, resulting in oocytes with increased metabolic activity, reduced lipid content, and increased developmental potential.

Transdermal Delivery of a Hydrogen Sulphide Donor, ADT-OH Using Aqueous Gel Formulations for the Treatment of Impaired Vascular Function: an Ex Vivo Study

PurposeHydrogen sulphide (H2S) is an important signalling molecule involved in the regulation of several physiological and pathophysiological processes. The objective of this study was to investigate the feasibility of transdermal delivery of ADT-OH, a H2S donor, by investigating the transdermal flux of aqueous gels loaded with penetration enhancers or liposomes. Furthermore, we explored the ability of permeated ADT-OH to promote angiogenesis and mitochondrial bioenergetics in HUVEC cells.MethodsAqueous hypromellose gels (5% w/v) were prepared with up to 10% v/v propylene glycol (PG) or deformable liposomes with 0.025% w/w ADT-OH. ADT-OH permeation from formulations across excised murine skin into PBS was quantified over 24 h using HPLC-UV detection. Media was collected and applied to HUVEC cells to evidence ADT-OH functionality following permeation. Tube formation assays were performed as indicative of angiogenesis and mitochondrial oxygen consumption was evaluated using a Seahorse XF24.ResultsIncreasing the loading of PG caused an increase in ADT-OH permeation rate across skin and a decrease in dermal drug retention whereas liposomal gels produced a slow-release profile. Treatment of HUVEC’s using conditioned media collected from the ADT-OH loaded permeation studies enhanced tube formation and the basal oxygen consumption rates after 30 min of treatment.ConclusionsThese findings demonstrate that transdermal delivery of ADT-OH may provide a promising approach in the treatment of impaired vascular function. Gels prepared with 10% v/v PG have the potential for use in conditions requiring rapid H2S release whereas liposomal loaded gels for treatment requiring sustained H2S release.

Lipopolysaccharide From E. coli Increases Glutamate-Induced Disturbances of Calcium Homeostasis, the Functional State of Mitochondria, and the Death of Cultured Cortical Neurons

Lipopolysaccharide (LPS), a fragment of the bacterial cell wall, specifically interacting with protein complexes on the cell surface, can induce the production of pro-inflammatory and apoptotic signaling molecules, leading to the damage and death of brain cells. Similar effects have been noted in stroke and traumatic brain injury, when the leading factor of death is glutamate (Glu) excitotoxicity too. But being an amphiphilic molecule with a significant hydrophobic moiety and a large hydrophilic region, LPS can also non-specifically bind to the plasma membrane, altering its properties. In the present work, we studied the effect of LPS from Escherichia coli alone and in combination with the hyperstimulation of Glu-receptors on the functional state of mitochondria and Ca2+ homeostasis, oxygen consumption and the cell survival in primary cultures from the rats brain cerebellum and cortex. In both types of cultures, LPS (0.1–10 μg/ml) did not change the intracellular free Ca2+ concentration ([Ca2+]i) in resting neurons but slowed down the median of the decrease in [Ca2+]i on 14% and recovery of the mitochondrial potential (ΔΨm) after Glu removal. LPS did not affect the basal oxygen consumption rate (OCR) of cortical neurons; however, it did decrease the acute OCR during Glu and LPS coapplication. Evaluation of the cell culture survival using vital dyes and the MTT assay showed that LPS (10 μg/ml) and Glu (33 μM) reduced jointly and separately the proportion of live cortical neurons, but there was no synergism or additive action. LPS-effects was dependent on the type of culture, that may be related to both the properties of neurons and the different ratio between neurons and glial cells in cultures. The rapid manifestation of these effects may be the consequence of the direct effect of LPS on the rheological properties of the cell membrane.

Extracellular Flux Analysis of Mitochondrial Function in Pluripotent Stem Cells.

Mitochondrial function and energy metabolism are increasingly recognized not only as regulators of pluripotent stem cell function and fate, but also as critical targets in disease pathogenesis and aging. Therefore across the downstream applications of pluripotent stem cells, including development and diseasemodeling, drug screening, and cell-based therapies, it is crucial to be able to measure mitochondrial function and metabolism in a high-throughput, real-time and label-free manner. Here we describe the application of Seahorse extracellular flux analysis to measure mitochondrial function in pluripotent stem cells and their derivatives. Specifically, we highlight two assays, the Mitochondrial Stress Test, which quantifies overall mitochondrial function including basal, maximal and ATP-couple oxygen consumption rates, and the Electron Transport Chain Complex Specific assay, that quantifies function of individual complexes within the electron transport chain.

Adapted suspension tumor cells rewire metabolic pathways for anchorage-independent survival through AKT activation

Metastatic spread of cancer cells is the main cause of cancer-related death. As cancer cells adapt themselves in a suspended state in the blood stream before penetration and regrowth at distal tissues, understanding their survival strategy in an anchorage-independent condition is important to develop appropriate therapeutics. We have previously generated adapted suspension cells (ASCs) from parental adherent cancer cells to study the characteristics of circulating tumor cells. In this study, we explored metabolic rewiring in MDA-MB-468 ASCs to adapt to suspension growth conditions through extracellular flux analyses and various metabolic assays. We also determined the relationship between AKT activation and metabolic rewiring in ASCs using the AKT inhibitor, MK2206. ASCs reprogramed metabolism to enhance glycolysis and basal oxygen consumption rate. RNA-sequencing analysis revealed the upregulation in the genes related to glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation. The changes in the metabolic program led to a remarkable dependency of ASCs on carbohydrates as an energy source for proliferation as compared to parental adherent cells (ADs). AKT activation was observed in ASCs and those generated from pancreatic and other breast cancer cells, and AKT activation inhibition in ASCs decreased glycolysis and oxygen consumption. AKT activation is an important strategy for obtaining energy through the enhancement of glycolysis in ASCs. The regulation of AKT activity and/or glycolysis may provide a strong therapeutic strategy to prevent the metastatic spread of cancer cells.

Sodium butyrate potentiates insulin secretion from rat islets at the expense of compromised expression of \u03b2 cell identity genes

Short-chain fatty acids (SCFAs) produced by the gut microbiota have been well demonstrated to improve metabolic homeostasis. However, the role of SCFAs in islet function remains controversial. In the present study, none of the sodium acetate, sodium propionate, and sodium butyrate (SB) displayed acute impacts on insulin secretion from rat islets, whereas long-term incubation of the three SCFAs significantly potentiated pancreatic β cell function. RNA sequencing (RNA-seq) revealed an unusual transcriptome change in SB-treated rat islets, with the downregulation of insulin secretion pathway and β cell identity genes, including Pdx1, MafA, NeuroD1, Gck, and Slc2a2. But these β cell identity genes were not governed by the pan-HDAC inhibitor trichostatin A. Overlapping analysis of H3K27Ac ChIP-seq and RNA-seq showed that the inhibitory effect of SB on the expression of multiple β cell identity genes was independent of H3K27Ac. SB treatment increased basal oxygen consumption rate (OCR), but attenuated glucose-stimulated OCR in rat islets, without altering the expressions of genes involved in glycolysis and tricarboxylic acid cycle. SB reduced the expression of Kcnj11 (encoding KATP channel) and elevated basal intracellular calcium concentration. On the other hand, SB elicited insulin gene expression in rat islets through increasing H3K18bu occupation in its promoter, without stimulating CREB phosphorylation. These findings indicate that SB potentiates islet function as a lipid molecule at the expense of compromised expression of islet β cell identity genes.

The FoxQ1 transcription factor is a novel regulator of electron transport chain complex I subunits in human breast cancer cells.

The FoxQ1 is an oncogenic transcription factor that is overexpressed in basal-like and luminal-type human breast cancers when compared to the normal mammary tissue. The FoxQ1 is implicated in mammary tumor progression. However, the mechanism by which FoxQ1 promotes mammary tumorigenesis is not fully understood. In this study, we present experimental evidence for a novel function of FoxQ1 in the regulation of complex I activity of the electron transport chain. The RNA-seq data from FoxQ1 overexpressing basal-like SUM159 cells revealed a statistically significant increase in the expression of complex I subunits NDUFS1 and NDUFS2 when compared to the empty vector (EV) transfected control cells. Consistent with these results, the basal and ATP-linked oxygen consumption rates were significantly increased by FoxQ1 overexpression in SUM159 and luminal-type MCF-7 cells. The FoxQ1 overexpression in both cell lines resulted in increased intracellular levels of pyruvate, lactate, and ATP that was associated with overexpression of pyruvate dehydrogenase and pyruvate carboxylase proteins. Activity and assembly of complex I were significantly enhanced by FoxQ1 overexpression in SUM159 and MCF-7 cells that correlated with increased mRNA and/or protein levels of complex I subunits NDUFS1, NDUFS2, NDUFV1, and NDUFV2. The chromatin immunoprecipitation assay revealed the recruitment of FoxQ1 at the promoters of both NDUFS1 and NDUFV1. The cell proliferation of SUM159 and MCF-7 cells was increased significantly by overexpression of NDUFS1 as well as NDUFV1 proteins. In conclusion, we propose that increased complex I-linked oxidative phosphorylation is partly responsible for oncogenic role of FoxQ1 at least in human breast cancer cells.

TRAP1 suppresses oral squamous cell carcinoma progression by reducing oxidative phosphorylation metabolism of Cancer-associated fibroblasts

BackgroundGlucose metabolism in cancer associated fibroblasts (CAFs) within the tumor microenvironment is a material and energy source for tumorigenesis and tumor development. However, the characteristics and important regulatory mechanisms of glucose metabolism in fibroblasts associated with oral squamous cell carcinoma (OSCC) are still unknown.MethodsWe successfully isolated, cultured, purified and identified CAFs and normal fibroblasts (NFs). Cell culture, immunohistochemistry (IHC) and CCK8, flow cytometry, Seahorse XF Analyzer, MitoTracker assay, western blotting (WB), transmission electron microscope, Quantitative real-time PCR (qPCR), immunofluorescence (IF), and Label-free quantitative proteomics assay, animal xenograft model studies and statistical analysis were applied in this study.ResultsWe demonstrated that the proliferation activity of CAFs was significantly enhanced as compared to NFs, while the apoptosis rate was significantly decreased. CAFs in OSCC preferentially use oxidative phosphorylation (OXPHOS) rather than glycolysis. Moreover, CAFs showed stronger maximal respiration, a larger substantial mitochondrial spare respiratory capacity (SRC) and higher adenosine triphosphate (ATP) production capacity than NFs. The results of mitotracker green fluorescence staining showed that compared with NFs, CAFs exhibited stronger green fluorescence. The results of WB showed the expression level of Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) obviously increased in CAFs compared to NFs. These results confirmed that CAFs have greater mitochondrial activity and function than NFs. Furthermore, Label-free quantitative proteomics assays showed that both ATP synthase subunit O (ATP5O) and tumor necrosis factor receptor-associated protein 1 (TRAP1) are important differentially expressed proteins in the mitochondria of CAFs/NFs. Overexpression of TRAP1 in CAFs increased basal oxygen consumption rate (OCR), maximal respiration, ATP production and SRC. In vivo, overexpression TRAP1 expression in CAFs suppress tumor growth.ConclusionTaken together, the results indicated that TRAP1 is an important regulatory molecule of CAFs glucose metabolism and promotes OSCC progression by regulating the OXPHOS of CAFs.

Effects of fluoroquinolones and tetracyclines on mitochondria of human retinal MIO-M1 cells

Our goal was to explore the detrimental impacts of ciprofloxacin (CPFX) and tetracycline (TETRA) on human retinal Müller (MIO-M1) cells in vitro. Cells were exposed to 30, 60 and 120 μg/ml of CPFX and TETRA. The cellular metabolism was measured with the MTT assay. The JC-1 and CM-H2DCFDA assays were used to evaluate the levels of mitochondrial membrane potential (MMP) and ROS (reactive oxygen species), respectively. Mitochondrial DNA (mtDNA) copy number, along with gene expression levels associated with apoptotic (BAX, BCL2-L13, BCL2, CASP-3 and CASP-9), inflammatory (IL-6, IL-1β, TGF-α, TGF-β1 and TGF-β2) and antioxidant pathways (SOD2, SOD3, GPX3 and NOX4) were analyzed via Quantitative Real-Time PCR (qRT-PCR). Bioenergetic profiles were measured using the Seahorse® XF Flux Analyzer. Cells exposed 24 h to 120 μg/ml TETRA demonstrated higher cellular metabolism compared to vehicle-treated cells. At each time points, (i) all TETRA concentrations reduced MMP levels and (ii) ROS levels were reduced by TETRA 120 μg/ml treatment. TETRA caused (i) higher expression of CASP-3, CASP-9, TGF-α, IL-1B, GPX3 and SOD3 but (ii) decreased levels of TGF-B2 and SOD2. ATP production and spare respiratory capacity declined with TETRA treatment. Cellular metabolism was reduced with CPFX 120 μg/ml in all cultures and 60 μg/ml after 72 h. The CPFX 120 μg/ml reduced MMP in all cultures and ROS levels (72 h). CPFX treatment (i) increased expression of CASP-3, CASP-9, and BCL2-L13, (ii) elevated the basal oxygen consumption rate, and (iii) lowered the mtDNA copy numbers and expression levels of TGF-B2, IL-6 and IL-1B compared to vehicle-control cells. We conclude that clinically relevant dosages of bactericidal and bacteriostatic antibiotics can have negative effects on the cellular metabolism and mitochondrial membrane potential of the retinal MIO-M1 cells in vitro. It is noteworthy to mention that apoptotic and inflammatory pathways in exposed cells were affected significantly This is the first study showing the negative impact of fluoroquinolones and tetracyclines on mitochondrial behavior of human retinal MIO-M1 cells.

Sirtuin 3 Inhibition Targets AML Stem Cells through Perturbation of Fatty Acid Oxidation

Acute myeloid leukemia (AML) in adults has a 5-year survival of approximately 30% and a high rate of disease recurrence in part due to our inability to eliminate the disease-initiating leukemic stem cells (LSCs) (Shlush et al. Nature, 2017). Previous studies have shown that LSCs uniquely rely on oxidative phosphorylation (OXPHOS) for survival (Lagadinou et al. Cell Stem Cell, 2013). Thus, novel therapies that are designed to target LSC metabolism have the potential to improve patient outcomes.Work from our group and others has demonstrated that a critical metabolite for OXPHOS regulation in LSCs is the coenzyme NAD + (Jones et al. Cell Stem Cell, 2020; Mitchell et al. Blood Advances 2019). One family of NAD + dependent proteins important in cancer biology, and AML specifically (Yan et al. Blood Cancer Discovery, 2021), are sirtuins. To determine if any sirtuins are important in LSC function we knocked down each sirtuin family member (sirtuin 1-7) with siRNA in four primary AML specimens and measured viability and colony forming ability. Knockdown of sirtuin 3 (SIRT3) decreased viability and colony forming potential of all AML specimens tested. SIRT3 is a mitochondrial de-acetylase with a multi-faceted role in metabolic regulation and oncogenesis (Finley, et al. Trends in Molecular Medicine, 2016). SIRT3 interacts with pathways upstream of OXPHOS including the tricarboxylic acid (TCA) and fatty acid oxidation (FAO). Importantly, a SIRT3 inhibitor (YC8-02) has been developed and has been shown to be effective pre-clinically for the treatment of B-cell lymphoma (Li et al. Cancer Cell, 2019).To further understand the significance of SIRT3 in LSCs, we assessed viability and colony forming potential upon YC8-02 treatment. LSCs were enriched from primary specimens based upon relative reactive oxygen species (ROS) level as previously described (Lagadinou et al. Cell Stem Cell, 2013). LSCs and blasts enriched from ten primary AML, and four AML cell lines (MOLM13, TEX, OCI-AML2, OCI-AML3) were cultured for 48 hours with or without YC8-02 before assessing viability and colony forming ability. YC8-02 treatment resulted in a significant decrease in colony forming potential of AML cells compared to control (data not shown). Similarly, LSCs, blasts, and cell lines showed a significant decrease in viability upon YC8-02 treatment (Fig 1A and data not shown). Cord blood and mobilized peripheral blood samples conversely did not show a change in colony forming potential following SIRT3 knockdown or YC8-02 treatment, respectively (data not shown). To assess YC8-02's effect on LSC function, three AML samples were treated with 10µM of drug for 24 hours and transplanted into NSG-S mice. YC8-02 treatment resulted in a significant decrease in AML engraftment, indicating a decrease in LSC function (Fig 1B).To determine the mechanism by which SIRT3 inhibition causes cell death, LSCs enriched from three primary specimens were treated with YC8-02; metabolite and lipid levels were determined by mass spectrometry. This analysis revealed a significant accumulation of fatty acids post YC8-02 treatment. To further characterize these changes, MOLM13 cells were treated with 13C 16-palmitic acid following 4 hours of incubation with 10µM YC8-02. Cells were collected 4 and 16 hours after introduction of palmitic acid and metabolic tracing was assessed by mass spectrometry. We found an accumulation of long and very long chain fatty acids and a decrease in TCA cycle intermediates (Fig 1C). FAO normally supplies TCA with intermediate acetyl-CoA; thus, these data indicate a decrease in FAO upon YC8-02 treatment. Accordingly, we measured changes in OXPHOS in response to treatment with YC8-02, in primary LSCs (Fig 1D) and AML cell lines (data not shown) and found a significant decrease in basal oxygen consumption. Further, ATP levels were significantly decreased upon YC8-02 treatment in LSCs (Fig 1E).In conclusion, we show that SIRT3 plays a pivotal role in FAO and LSC function. When SIRT3 is inhibited, FAO activity decreases resulting in the accumulation of long and very long chain fatty acids. This change in FAO activity reduces the availability of products for the TCA cycle, limiting necessary intermediates for OXPHOS, decreasing ATP production, and ultimately causing cell death. Therefore, our data suggests that SIRT3 is a potential therapeutic target for LSCs and should be considered in future pre-clinical and clinical investigations. [Display omitted] DisclosuresMelnick: Constellation: Consultancy; Epizyme: Consultancy; Daiichi Sankyo: Research Funding; Sanofi: Research Funding; Janssen Pharmaceuticals: Research Funding; KDAC Pharma: Membership on an entity's Board of Directors or advisory committees. Minden: Astellas: Consultancy. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Inhibition of Pre-BCR Signaling Mediates a Metabolic Switch in B-Cell Progenitor Acute Lymphoblastic Leukemia

Despite improvements in overall survival for children with B-cell progenitor acute lymphoblastic leukemia (BCP-ALL), it remains the second-leading cause of cancer related death in children with approximately 200 deaths per year in the U.S. Thus, there remains a critical need for a definitive cure to prevent relapse for patients with BCP ALL. The accumulation of BCP ALL blasts results from the disruption of normal developmental checkpoints. One of these checkpoints, as pro-B cells transition to become pre-B cells, involves surface expression of the precursor-B-cell receptor (pre-BCR). Prior work has categorized BCP ALL into pre-BCR positive and pre-BCR negative subtypes based on the protein expression of Ig light chain and active signaling of SRC family kinases, SYK, BTK. Combining single cell analysis and machine learning, we previously identified pre-B cells with activation of pre-BCR signaling, namely CREB, 4EBP1, rpS6 and SYK, that are present at diagnosis and highly predictive of relapse. We call these relapse predictive cells. Relapse predictive cells were enriched in relapse samples, demonstrating their persistence from diagnosis to relapse and making them an actionable target to prevent relapse altogether.To better understand relapse predictive cells, we enriched pre-B cells from patients with known relapse status and performed whole transcriptome sequencing. Relapse predictive cells demonstrated significant upregulation of genes in the oxidative phosphorylation (OXPHOS), glycolysis, and reactive oxygen species (ROS) pathways compared to pre-B-like leukemia cells from patients who will not go on to relapse. Analysis of public genome-wide CRISPR screen datasets in 2 pre-BCR+ and 4 pre-BCR- cell lines found 69 essential genes uniquely present in pre-BCR+ cell lines, related to mitochondria translation, OXPHOS and TCA cycle pathway.We performed CRISPR knock down of proximal pre-BCR related tyrosine kinase SYK in pre-BCR+ (Nalm6, Kasumi-2) and pre-BCR- (697, REH, SUPB15) cell lines to understand how activated pre-BCR impacts cellular metabolism in pre-BCR+ and pre-BCR- cells. CyTOF analysis of pre-BCR signaling demonstrated effective inhibition of downstream pre-BCR pathway members in the KD cells (pSYK, pBLNK, pBTK). RNA sequencing demonstrated upregulation of mitochondrial translation and OXPHOS pathways with downregulation of hypoxia pathways in pre-BCR+ but not pre-BCR- SYK KD cells. Functional extracellular flux experiments by Seahorse confirmed pre-BCR+ SYK KD cells to have higher basal oxygen consumption rate (OCR) and lower extracellular acidification rate (ECAR) compared to wild-type pre-BCR+ cells, indicating a switch from highly glycolytic to aerobic metabolism.To determine the interplay between pre-BCR signaling and cellular metabolism at the single cell level, we performed CYTOF with a panel examining pre-BCR pathway members, developmental phenotype and metabolism in these cell lines as well as matched diagnosis-relapse patient-derived xenografts. These results indicate, in line with the RNA sequencing and Seahorse data, that inhibiting pre-BCR signaling is accompanied by inhibition of glycolysis with lower protein expression of glycolytic related enzymes HIF1A, GLUT1, PFKFB4, GAPDH, ENO1 and LDHA. Further, we observed in cells completely deficient in the ability to initiate pre-BCR signal (SYK knock out), activated p4EBP1 indicating signaling feedback from the PI3K-AKT pathway and a metabolic adaption indicating utilization of energy sources other than glucose in cells surviving SYK loss. Finally, to determine the impact of loss of pre-BCR signaling on proliferation, in vitro competition assays demonstrated SYK KD cells to be less proliferative in all the cell lines except pre-BCR- cell line 697. In vivo, SYK KO demonstrated significantly slower engraftment (median %hCD45: 84% vs 54%, P=0.009) in NSG mice and significantly longer survival time than the mice xenografted with wild-type cells (median survival 28 vs 39 days, P=0.0004).Together, our data indicate that individual BCP ALL cells with active pre-BCR signaling are associated with relapse and that these cells have a unique metabolic state that relies on active glycolysis and metabolic flexibility supporting proliferation in vitro as well as engraftment and aggressivity in vivo. Further metabolomics experiments and characterization of primary patient samples are underway. DisclosuresMullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company. Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding.