Measurement Of Oxygen Consumption Rate Research Articles

Rapid metabolic compensation in response to temperature change in the intertidal copepod, Tigriopus californicus

Animals living in the intertidal zone must adapt to thermal variability, including adjustments in metabolism. We examined metabolic responses to temperature in the copepod, Tigriopus californicus, which inhabits supratidal splash pools along the Pacific coast of North America. We maintained three populations of T. californicus at 20 °C, one from southern California (San Diego, “SD”) and two from Oregon (Fogarty Creek, “FCN”, Boiler Bay, “BOB”) and examined possible geographic patterns in metabolism. We measured oxygen consumption rate (V̇o2) at 20 °C and following 48 h (chronic) acclimation to 25, 27.5 and 30 °C. V̇o2 was temperature-independent, with temperature quotients (Q10) values ≤1 in all populations, indicative of metabolic compensation. We detected no variation in V̇o2 or survival between populations. To explore the time course of metabolic compensation, we performed an acute acclimation experiment in which V̇o2 was measured at 20 °C, following immediate exposure to 25 °C, and following 2 h, 4 h and 6 h exposure to 25 °C. This acute acclimation experiment revealed that V̇o2 increased immediately in SD and FCN, but was no longer different than 20 °C levels by 2 h and 4 h at 25 °C, respectively. BOB showed no significant change in V̇o2, which may indicate complete temperature-independent metabolism or different mechanisms of compensation between populations. This study demonstrates a time course of rapid metabolic compensation in response to temperature that occurs in a small intertidal animal, and suggests intertidal invertebrates can thermally acclimate within a few hours of a significant temperature change.

Field metabolic rates of teleost fishes are recorded in otolith carbonate

Field metabolic rate (FMR) is key to understanding individual and population-level responses to environmental changes, but is challenging to measure in field conditions, particularly in aquatic environments. Here we show that FMR can be estimated directly from the isotopic composition of carbon in fish otoliths (δ13Coto). We describe the relationship between δ13Coto values and oxygen consumption rate, and report results from laboratory experiments relating individual-level measurements of oxygen consumption rates to δ13Coto values in Atlantic cod (Gadus morhua). We apply our new δ13Coto metabolic proxy to existing δ13Coto data from wild cod and four deepwater fish species to test the validity of inferred FMR estimates. The δ13Coto metabolic proxy offers a new approach to study physiological ecology in free-ranging wild fishes. Otolith-based proxies for FMR are particularly promising as they allow retrospective assessment of time-integrated, individual-level FMR throughout an individual fish’s life history.

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments.

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell's metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measurement of Respiration Rate in Live Caenorhabditis elegans

Mitochondrial function and dysfunction are at the core of aging and involved in many age-dependent diseases. Rate of oxygen consumption is a measure of mitochondrial function and energy production rate. The nematode Caenorhabditis elegans (C. elegans) offers an opportunity to study "living" mitochondria without the need for mitochondrial extraction, purification and associated artifacts. Oxygen consumption rate (OCR) is traditionally measured using single-chamber Clark electrodes with or without the addition of metabolic modulators. More recently, multi-well oxygen electrodes with automated injection system have been developed to enable rapid measurement of OCR under different conditions. Here, we describe a detailed protocol that we have adapted from existing protocols to measure coupled and uncoupled mitochondrial respiration (with and without metabolic modulators) in live respiring nematodes using a Seahorse XFe96 extracellular flux analyzer. We present details on our protocol, including preparation of nematode culture, use of metabolic modulators, execution of Seahorse XF assay as well as post-experimental data analysis. As a reference, we provide results of a series of experiments in which the metabolic activity of N2 wild-type nematodes was compared to N2 nematode treated with paraquat, a compound that generates reactive oxygen species (ROS), thus causing oxidative damage and mitochondrial dysfunction. These data illustrate the kind of insights that can be obtained even using a low number of nematodes (10 animals only per well).

A New Efficient Method for Measuring Oxygen Consumption Rate Directly ex vivo in Human Epidermal Biopsies.

Skin cells are constantly exposed to environmental influences such as air pollution, chemicals, pathogens and UV radiation. UV radiation can damage different biological structures, but most importantly cellular DNA. Mitochondria contain their own genome and accumulate UV-induced DNA mutations to a large extent. This can result, e.g., in accelerated skin aging. Understanding the impact of harmful external influences on mitochondrial function is therefore essential for a better view on the development of age-related diseases. Previous studies have been carried out on cell cultures derived from primary cells, which does not fully represent the real situation in the skin, while the mitochondrial parameters were considered barely or not at all. Here we describe a method to measure mitochondrial respiratory parameters in epithelial tissue derived from human skin biopsies using an Agilent Seahorse XF24 Flux Analyzer. Before the assay, epidermis and dermis are separated enzymatically, we then used the XF24 Islet capture microplates to position the epidermis samples to measure oxygen consumption rates (OCR) and extracellular acidification rates (ECAR). In these plates, small nets can be fixed to the plate bottom. The epidermis was placed with the vital-basal-side on the net. Active ingredients in the three ports were injected consecutively to determine the effect of each compound. This allows determining the efficiency of the individual complexes within the respiratory chain. This protocol enables the testing of toxic substances and their influence on the mitochondrial respiration parameters in human epithelial tissue.

Chronic Lymphocytic Leukemia B Cells Display IgM and IgD Isotype-Restricted Features That Affect Association with Co-Receptors, BCR Signaling, and Leukemic B-Cell Growth In Vivo

The leukemic cells in patients with chronic lymphocytic leukemia (CLL) are highly dependent on B-cell receptor (BCR) mediated signaling. Despite this and the fact that >90% of CLL clones co-express IgM and IgD, the composition and molecular mechanisms regulating BCR signaling regarding the two isotypes and the co-receptors with which they associate is lacking. Here we have addressed these issues.First, using Imaging Flow Cytometry, we evaluated BCR organization on the surface membrane of CLL cells from 11 patients who had participated in a 2H2O-labeling study that determined in vivoCLL B-cell birth rates (BR). We found that in all cases mIgM resided in more and larger surface clusters than mIgD. Also, a statistically significant, direct correlation was observed for IgM density and in vivoCLL-cell BR, with patients exhibiting more recently-divided cells having the highest expression of IgM. This was not the case for IgD.BCR signaling requires co-receptors that can co-localize differently with the two isotypes. Thus, we tested co-localization of stimulatory (CD20) and inhibitory (CD22) co-receptors with mIgM and mIgD, using the proximity ligation assay technique that discriminates 10 to 40 nm distances. Higher IgM:CD20 and lower IgD:CD20 co-localization ratios directly associated with in vivo BR. Conversely, patients whose CLL B cells showed greater IgM to CD22 co-localization ratios had lower BRs. Thus, association of IgM with stimulatory versus inhibitory co-receptors correlated with positive or negative regulation of CLL growth in vivo.Next, we questioned the extent that the observed differences in BCR organization affected the entire clone by measuring a marker of single cell metabolic activity - cell size. IgM and BR associated with entire clonal populations that were skewed toward larger, more active cells. Similarly, high BR CLLs displayed an increased mitochondrial maximal respiration and glycolytic activity and capacity, based on measurements of oxygen consumption rate and extracellular acidification rate, respectively.Since our findings supported a link between IgM- but not IgD-BCRs, growth rate in vivoand clonal metabolic activity, we questioned whether intrinsic, constitutive CLL BCR autonomous signaling differed for these two isotypes. To address this, we examined the signaling capacities of CLL-derived BCRs expressed as IgM or IgD isotypes, while maintaining the original IGHV-D-J and IGLV-J rearrangements. We used B cells that do not express endogenous BCR-related molecules but do express an inducible ERT2- SLP-65 fusion protein which enables examining Ca++influx. All BCRs expressed as IgM effectively mobilized Ca++ without need for an external ligand, indicating autonomous signaling. In contrast, BCRs expressed as IgD did not signal autonomously but required crosslinking with anti-BCR. Thus, only mIgM BCRs naturally transduce a signal in the absence of antigen.To determine the extent that BCR signaling influences clonal activity and in vivoBR, we compared cell size of CLL B cells taken from patients before and after 4 weeks of treatment with the Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib (iBTK). Ibrutinib had a strong treatment effect on cell activity, reducing overall cell size in 10/11 patients. A comparison of single cell areas for patients with lower (BR = 0.54%) and higher (BR = 1.42%) BRs showed an overall reduction of the median cell size for both cases. Thus, iBTK treatment leads to an equilibration of the cell size profile among the cases differing in BR, indicating that ibrutinib acts proportionally more potently on more metabolically active CLL B cells. Likewise, these findings are consistent with BCR signaling, transduced through BTK, being responsible for the increased cellular activity of aggressive CLL clones.In conclusion, increased mIgM density and proximity of mIgM to stimulatory receptors is linked to greater metabolic activity clones and increased rate of proliferationin vivo. Conversely, proximity of mIgM to inhibitory receptors has the opposite correlations.Moreover, only mIgM carries out autonomous signaling, providing another biologic trait linking all these features. Thus, our data support a tight, isotype-dependent regulation of BCR signaling and its consequences for CLL B cells. Further understanding these mechanisms should help generate novel therapies to modify the quality of BCR-transduced signaling and thus cell fate. DisclosuresBarrientos:Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics/AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Rai:Cellectis: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees. Chiorazzi:AR Pharma: Equity Ownership; Janssen, Inc: Consultancy.

EVI1 in Acute Myeloid Leukemia Triggers Metabolic Reprograming Associated with Leukemogenesis and Increases Sensitivity to L-Aspalaginase

Leukemia cells survive and proliferate under conditions of metabolic stress by acquiring mutations that increase energy metabolism. Here, we aimed to identify a specific metabolic inhibitor and examine transcription factor-enhanced changes in energy metabolism by refractory leukemia cells. Overexpression of Ecotropic Virus Integration site 1 protein homolog (EVI1) in adults and children with mixed lineage leukemia-rearrangement acute myeloid leukemia (MLL-r AML) has a very poor prognosis. We focused on metabolic reprograming of MLL leukemia cells expressing EVI1, since the metabolic relationship between MLL and EVI1 is unclear. We used an extracellular flux analyze to examine metabolic changes during leukemia development in a mouse model of MLL-r AML expressing high levels of EVI1 (EVI1+).To examine whether EVI1 regulates energy metabolism in MLL-rearranged leukemia cells, we used transgenic mice expressing EVI1 (TG) in LSK and GMP cells model in which AML is driven by the MLL-AF9 oncogene. We measured oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using a flux analyzer. TG MLL-AF9 mice showed a significantly higher basal and capacity of OCR than WT MLL-AF9 mice ex vivo. EVI1+ cells showed accelerated oxidative phosphorylation (OXPHOS) prior to activation of glycolysis, and higher dependency on glutamine as an energy source. To identify the metabolic pathways regulated by EVI1, we performed capillary electrophoresis time-of-fight mass spectrometry-based metabolome profiling of WT and TG MLL-AF9 leukemia cells. We found significant differences between the cells in terms of the amounts of metabolites derived from the glycolytic and TCA cycles. Fructose 1,6-bisphosphate and lactate were up-regulated in TG MLL-AF9 cells, implying activation of glycolysis. Moreover, the amounts of fumarate and malate (metabolites of the TCA cycle) were significantly higher in TG MLL-AF9 cells. EVI1 played a role in glycolysis as well as driving expression of genes engaged in the tricarboxylic acid cycle. Next, we tested whether pharmacological inhibition of glycolysis and glutaminolysis suppresses MLL-AF9. L-asparaginase (ASP) [which catalyzes hydrolysis of asparagine (Asn) and glutamine (Gln) to asparatic acid or glutamic acid, respectively] markedly suppressed proliferation of TG MLL-AF9 cells, EVI1highAML cell lines. To examine the therapeutic potential of ASP in vivo, we treated secondary recipients of TG MLL-AF9 AML cells with ASP or control (vehicle), beginning 5 days post-transplantation. Mice then received intraperitoneal injections (five times per week) of distilled water or ASP (1000 U/kg). ASP led to a significant reduction in the number of GFP+ AML cells in the peripheral blood and increased the survival of recipient mice. Next, we examined an AML xenograft model. Two groups of NOG mice were injected subcutaneously with UCSD/AML1 cells and then treated with ASP or control. ASP -treated mice showed a significant reduction in the growth of AML tumors. Overall, these findings indicate that ASP -mediated inhibition of OXPHOS is a potential treatment for AML.We clarified that increased glutamine dependency by MLL-r AML cells showing high EVI1 expression makes them sensitive to ASP. We found that the energy advantage of AML cells is acquired via transcription factor-mediated activation of mitochondrial metabolism, leading to a poor prognosis. Furthermore, we show that new therapeutic options can be identified by examining the energy-based metabolic characteristics of leukemia cells. DisclosuresNo relevant conflicts of interest to declare.

Carbenoxolone Decreases the Microenvironment-Induced Chemoresistance of Acute Myeloid Leukemia Cells

IntroductionBone marrow niche of acute myeloid leukemia (AML) is a highly specialized microenvironment that regulates leukemic progression by favoring leukemic cell quiescence and chemoresistance. Niche components, especially mesenchymal stromal/stem cells (MSCs), have pro-survival effects on leukemia cells by protecting them from chemotherapy-induced apoptosis. The protection mechanisms of AML cells by the microenvironment are extensively studied to identify pharmacological targets to prevent AML relapse after chemotherapy. Direct intercellular communications between hematopoietic stem cells and MSCs involve connexins, such as connexin 43 (Taniguchi Ishikawa E et al. Connexin-43 prevents hematopoietic stem cell senescence through transfer of reactive oxygen species to bone marrow stromal cells. Proc Natl Acad Sci U S A. 2012,109:9071-6). The connexins are key components of gap junctions and we postulated in this study that blocking their assembly could modify cell-cell interactions in the leukemic niche and consequently the chemoresistance. To this end we evaluated the effects of carbenoxolone (CBX), a glycyrrhetinic acid derivative known to block gap junctions (Rozental R et al. How to close a gap junction channel. Efficacies and potencies of uncoupling agents. Methods Mol Biol 2001;154:447-76), already evaluated in the treatment of human diseases.MethodsPrimary AML blasts isolated from bone marrow patients and 8 AML cell lines (KG1-a, KG1, HL-60, THP-1, MOLM-13, MV4-11, ML2, NB4) were exposed to CBX to assess cell viability and proliferation (Trypan blue exclusion and MTT assays). To study the combined drug effects of CBX and cytarabine (Ara-C), isobolograms were determined by co-treatment of AML cell lines with various doses of these two molecules. The metabolic effects of CBX on AML live cells were investigated with Seahorse® analyzer to measure oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) and high-throughput metabolic profiling was performed by OmniLog® technology. Direct cell-cell interactions were studied in a co-culture system of AML cells and bone marrow primary MSCs combining or not CBX (150 µM) and Ara-C (1 µM). After 48h (37°C, 5% CO2), AML cells adherent to MSCs were collected and the two populations were discriminated by flow cytometry studies (AML cells: CD45+CD90- / MSCs: CD45-CD90+) to quantify cell number apoptosis/necrosis (AnnexinV/7AAD assay). Gap junction inhibition by CBX was checked by calcein transfer (dye transferred mainly by gap junction channels) from labeled MSCs to AML cells in the co-culture system. Statistical analyses were performed with Mann-Whitney-Wilcoxon test.ResultsTreatment of AML cell lines with CBX reduced cell growth and viability in a time- and dose-dependent manner and the CBX IC50 was around 150 µM. Moreover, the exposure for 48h of primary AML blast to this dose of CBX induced a two time decrease in the number of viable cells. Interestingly, the isobolograms of the 8 AML cells lines identified three different profiles of resistance to chemotherapy and a synergistic effect between CBX and Ara-C. Regarding the energy metabolism, the analysis of all AML cell lines showed a major reduction of OCR and ECAR after treatment with CBX (150 µM, 48h) regardless their chemoresistance to Ara-C. Moreover, treated AML cells lacked their metabolic capacities for utilization of numerous substrates. In the co-culture experiments, contact with MSCs induced resistance to Ara-C; the apoptosis/necrosis rate observed after Ara-C exposure was 4.3- and 6.4-times decreased by MSC-contact for KG1a cells (n=5, p=0.0115) and primary blasts (n=5, p=0.0001), respectively. Co-treatment of leukemic cells with CBX and Ara-C reversed these deleterious effects of MSC-contact; the apoptosis/necrosis rate observed in MSC-contact leukemic cells was 5- and 2-times increased by CBX co-treatment for KG1a cells (n=5, p=0.007) and primary blasts (n=5, p=0.042), respectively. Finally, CBX induced a 48%-decrease in calcein transfer between leukemic cells and MSCs, highlighting its role as a gap junction inhibitor.ConclusionNiche-induced chemoresistance is associated with the mechanism of AML relapse after initial well-conducted chemotherapy. Combined to chemotherapy, CBX could be of interest to reduce the deleterious effects of leukemic niche by targeting gap junctions. DisclosuresNo relevant conflicts of interest to declare.

Preadipocytes of obese humans display gender-specific bioenergetic responses to glucose and insulin

Background/ObjectivesAlthough the prevalence of obesity and its associated metabolic disorders is increasing in both sexes, the clinical phenotype differs between men and women, highlighting the need for individual treatment options. Mitochondrial dysfunction in various tissues, including white adipose tissue (WAT), has been accepted as a key factor for obesity-associated comorbidities such as diabetes. Given higher expression of mitochondria-related genes in the WAT of women, we hypothesized that gender differences in the bioenergetic profile of white (pre-) adipocytes from obese (age- and BMI-matched) donors must exist. Subjects/MethodsUsing Seahorse technology, we measured oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) of (pre-)adipocytes from male (n = 10) and female (n = 10) deeply-phenotyped obese donors under hypo-, normo- and hyperglycemic (0, 5 and 25 mM glucose) and insulin-stimulated conditions. Additionally, expression levels (mRNA/protein) of mitochondria-related genes (e.g. UQCRC2) and glycolytic enzymes (e.g. PKM2) were determined. ResultsDissecting cellular OCR and ECAR into different functional modules revealed that preadipocytes from female donors show significantly higher mitochondrial to glycolytic activity (higher OCR/ECAR ratio, p = 0.036), which is supported by a higher ratio of UQCRC2 to PKM2 mRNA levels (p = 0.021). However, no major gender differences are detectable in in vitro differentiated adipocytes (e.g. OCR/ECAR, p = 0.248). Importantly, glucose and insulin suppress mitochondrial activity (i.e. ATP-linked respiration) significantly only in preadipocytes of female donors, reflecting their trends towards higher insulin sensitivity. ConclusionsCollectively, we show that preadipocytes, but not in vitro differentiated adipocytes, represent a model system to reveal gender differences with clinical importance for metabolic disease status. In particular preadipocytes of females maintain enhanced mitochondrial flexibility, as demonstrated by pronounced responses of ATP-linked respiration to glucose.

Beta-Hydroxybutyrate Promotes Proliferation, Migration and Stemness in a Subpopulation of 5FU Treated SW480 Cells: Evidence for Metabolic Plasticity in Colon Cancer

Background:Beta-hydroxybutyrate (BHB) as a ketone body is the metabolic fuel in oxidative phosphorylation pathway. So far the effects of BHB on the biology of tumor cells is contradictory. Therefore, we investigated the effect of BHB on viability, metabolism, proliferation and migration of 5FU treated SW480 colon cancer cell line.Methods:we treated the SW480 cells with IC50 dose of 5-fluorouracil (5FU) for 72 h to isolate a subpopulation of 5FU treated cells that were resistant to it. Effects of BHB on cell viability was investigated by MTT assay. Measurement of oxygen consumption rate (OCR) in parallel with extracellular acidification rate (ECAR) upon BHB treatment was used for determination of metabolic profile of these cells. Investigating the relationship between metabolic phenotype and the status of differentiation and stemness was done by analyzing the expression of PGC-1α, c-MYC, NANOG, ALPi and KRT20 genes by qRT-PCR. Clonogenic and scratch assay were performed to determine the proliferation and migration abilities of incubated with BHB compared to untreated cells.Results:BHB increased cell viability in SW480 and 5FU treated SW480 cells. The results showed a significantly decreased ECAR and increased OCR in both cell types following BHB treatment reflecting the superiority of oxidative phosphorylation profile compared to glycolysis in both cell types. Also, treatment with BHB increases the expression of genes normally associated with stemness and mitochondrial biogenesis and decreases the expression of genes related to glycolytic program and differentiation in 5FU treated cells. Self-renewal and migration potential of BHB treated cells increased significantly.Conclusion:These findings suggest that BHB utilization via oxidative mitochondrial metabolism can fuel proliferation, migration and stemness in 5FU treated SW480 colon cancer cells.

The online monitoring and assessment of thallium stress using oxygen consumption rate and carbon dioxide excretion rate of zebrafish (Danio rerio)

An online monitoring and assessment system of metabolism to measure oxygen consumption rate (OC) and carbon dioxide (CO2) excretion rate (CR) of zebrafish (Danio rerio) was used to illustrate changes in stressful states in 15 days’ (360 h) 0.1 μg/L Tl exposure. Tl had a significant inhibition on zebrafish OC and CR (p < 0.01). OC was more suitable for Tl stress assessment than CR, considering that the OC response was more stable and discernible from the control comparing with CR. However, CR is a suitable alternative to characterize toxic effects on different metabolic substrates. Both OC and CR were integrated to present the respiratory quotient (RQ) analysis. RQ was efficient in differentiating between CO2 produced by respiration in the control group (RQ less than 0.7) and CO2 used for urination or stored in tissues after Tl exposure (some RQs larger than 1.0). Circadian rhythm was observed in RC and CR in the controls and persisted in 0.1 μg/L Tl treatments. The rhythm was relatively more disordered in CR. OC and CR would be suitable for indicating physiological stress in the online system as sensitive physiological indices.

Impact of in vitro heavy metal exposure on pancreatic β-cell function

Susceptibility to type-2 diabetes mellitus (DM) is determined, in part, by a variety of environmental factors, including exposure to metals. Heavy metals including inorganic arsenic (iAs), zinc (Zn), manganese (Mn), and cadmium (Cd) have been reported to affect glucose homeostasis or DM risk in population-based and/or laboratory studies. Previous evidence from our lab has shown that iAs can increase DM risk by impairing mitochondrial metabolism, one of the key steps in the regulation of glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells. The goal of the current study was to compare the effects of iAs on GSIS and mitochondrial function in INS-1 832/13 β-cells with those of Cd, Mn, and Zn, and to evaluate effects of binary mixtures of these metals. As expected, 24-hour exposure to iAs (arsenite, ≥1 μM) significantly inhibited GSIS as did Cd (5 μM) and Mn (12.5, 25, or 50 μM). Zn had no effects on GSIS at concentrations up to 50 μM. Mitochondrial function was assessed by measuring oxygen consumption rate (OCR) after glucose stimulation and during simulated mitochondrial stress. While both iAs and Mn impaired mitochondrial function (inhibiting OCR, maximal respiration, and/or spare respiratory capacity of mitochondria), no significant effects were found in cells exposed to Cd. Interestingly, no additive or synergistic effects on GSIS or OCR were observed in binary mixtures of iAs with either Mn or Cd. These data suggest that Mn, like iAs, may inhibit GSIS by impairing mitochondrial function, whereas Cd may target other mechanisms that regulate GSIS in β-cells.

PO-246 Nandrolone affects cell growth and differentiation in hepatoma cells

IntroductionHepatocellular carcinoma (HCC) represents the sixth leading cancer and the third most common cause of death from cancer. Many different aetiological factors are involved in the development of HCC, which may be modulated by both estrogens and androgens hormones during its initiation, progression and metastasis. The misuse of anabolic androgenic steroids (AAS) is associated with serious adverse effects to the liver, including cellular adenomas and adenocarcinomas, and is considered a factor risk of developing hepatic sex hormone related tumours. The purpose of this study was to investigate the role of Nandrolone, one of the most commonly used AAS, in regulating proliferation and differentiation of HCC.Material and methodsHuman HCC cell line HepG2 was treated with Nandrolone, a synthetic androgen ligand, for 48 hs and its viability and proliferation was assessed by MTS and cell cycle analysis, respectively. The expression of protein involved in cell cycle regulation and differentiation markers were analysed by western blot and real time PCR. Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were performed using Seahorse XF96 extracellular flux analyzer. Respiratory chain complex activities were assayed spectrophotometrically. Stemness surface markers expression was detected by FACSCalibur flow cytometer.Results and discussionsNandrolone treatment caused cell growth inhibition associated to a downregulation of cyclin D1 and an upregulation of the cyclin-dependent kinase inhibitors p21Waf1/Cip1 leading to cell cycle arrest in the G2 phase. Moreover, a significant overall impairment of mitochondrial functions, resulting in a reduced OCR and impairment of OXPHOS complexes activities were also observed, thus suggesting a role in the control of the metabolic reprogramming. Finally, a significant increase of the stemness markers was detected following Nandrolone treatment, also confirmed in additional human stem cell types and in an in vivo mouse model.ConclusionNandrolone shows a strong anti-proliferative effect in differentiated tumour cells, promoting cancer cells stemness through cellular metabolic reprogramming. These results could have important public health implications in order to improve the primary prevention such as revising altered lifestyles, like AAS abuse.

PO-405 Blockade of endothelial cell glycolysis to improve current immunotherapy

IntroductionTumour blood vessels are highly abnormal, they are leaky and poor perfused. This creates a hypoxic microenvironment and limits the delivery of nutrients, chemotherapy and immune cells. Tumour vessel abnormalities are due to pathological angiogenesis by hyperglycolytic tumour endothelial cells (TECs). Cantelmo et al recently showed that blocking the glycolytic activator PFKFB3 in TECs by the small compound 3PO normalised the tumour vasculature, improved oxygenation and the delivery and efficacy of cytoxic agents.Here, we investigate if tumour vessel normalisation (TVN) induced by PFKFB3 blockade enhances intratumoral T cell infiltration and activation, and increases the efficacy of immune-checkpoint inhibition. We also characterised the effect of TVN on cancer cell metabolism, to make them more vulnerable to immune-mediated killing.Material and methodsWe injected B16 mouse melanoma cells subcutaneously in wild-type mice and treated the tumor-bearing mice with either 3PO or vehicle. We analysed the intratumoral T cell infiltration and activation by immunofluorescence and FACS analysis. We also treated tumor-bearing mice with the immune-checkpoint inhibitor anti-PD1 alone or in combination with 3PO. Cancer cell metabolism upon TVN was assessed by radioactive flux analysis and measurement of oxygen consumption rate in freshly isolated cancer cells exposed to 3PO in vivo.Results and discussionsOngoing work suggests that 3PO treatment significantly increases intratumoral T cell infiltration compared to controls. Tumor-infiltrating T cells appeared to be more activated and proliferative. Furthermore, upon 3PO treatment, cytotoxic short-lived effector (CD44+ KLRG1+ CD127-) and IFNg+ T cells increased compared to controls. The combination of 3PO with anti-PD1 significantly inhibited tumour growth in comparison to 3PO or anti-PD1 monotherapy. Metabolic analysis of isolated cancer cells showed increased oxygen consumption, glucose and fatty acid oxidation in tumours treated with 3PO.ConclusionOur data suggest that TVN induced by PFKFB3 blockade in TECs: (i) increases the immunogenecity of intrinsically resistant tumours, both in terms of T cell infiltration and activation; (ii) reprograms cancer cells towards a more oxidative metabolism. This results in increased cancer cell susceptibility to immune responses and in overall increased efficacy of immune-checkpoint inhibition.Further research will elucidate how TVN regulates T cell activation and reprogramms cancer cell metabolism.

Effect of Insulin Deprivation on Brain Mitochondrial ATP Production and Mitochondrial Proteome in Diabetic Mice

Accumulating evidence indicates a strong association between diabetic status and degenerative brain disease, including cognitive impairment. Here we hypothesized that although insulin is not necessary for glucose metabolism in brain, like in skeletal muscle insulin is critical for mitochondrial function in brain. We measured oxygen consumption rate (OCR), maximal ATP production rate (MAPR), and reactive oxygen species (ROS) production in isolated brain mitochondria of streptozotocin (STZ) diabetic mice. STZ mice deprived of insulin treatment for 96 hours (D-I), in comparison to nondiabetic mice (ND) showed a significant reduction in State 3 MAPR [1.77±0.14 vs. 2.35±0.27 (pmol/s/ug)], phosphorylation efficiency [0.42±0.04 vs. 0.54±0.07 (mol ATP produced/mol of oxygen consumed)] and respiratory control ratio [2.95±0.16 vs. 3.12±0.18] indicating reduced mitochondrial coupling efficiency during insulin deprivation. Citrate synthase and cytochrome c oxidase activities were significantly decreased in the hypothalamus of D-I compared ND. Surprisingly, no significant differences in ROS emission were detected between ND and D-I suggesting additional factors, potentially elevated ketone bodies, may protect against increased oxidative stress in the D-I brain. Proteomics analysis of the cerebrum found post-translational modification involving oxidative damage of several mitochondrial proteins that potentially cause tau phosphorylation and neurofibrillary degeneration in D-I vs. ND. In STZ mice with continued insulin treatment (D+I), state 3 OCR, MAPR, and phosphorylation efficiency were comparable to ND, demonstrating the restorative effects of insulin and euglycemia on brain mitochondrial function. We conclude that insulin deprivation and hyperglycemia significantly alter brain mitochondrial function. Insulin treatment and control of glycemia could have salutary effects on these parameters. Disclosure G. Ruegsegger: None. M. Shankarappa: None. P. Summer: None. S. Gopala: None. P. Zabielski: None. K. Klaus: None. S. Dasari: None. I.R. Lanza: None. K. Nair: None.

Measurement of respiratory function in isolated cardiac mitochondria using Seahorse XFe24 Analyzer: applications for aging research.

Mitochondria play a critical role in the cardiomyocyte physiology by generating majority of the ATP required for the contraction/relaxation through oxidative phosphorylation (OXPHOS). Aging is a major risk factor for cardiovascular diseases (CVD) and mitochondrial dysfunction has been proposed as potential cause of aging. Recent technological innovations in Seahorse XFe24 Analyzer enhanced the detection sensitivity of oxygen consumption rate and proton flux to advance our ability study mitochondrial function. Studies of the respiratory function tests in the isolated mitochondria have the advantages to detect specific defects in the mitochondrial protein function and evaluate the direct mitochondrial effects of therapeutic/pharmacological agents. Here, we provide the protocols for studying the respiratory function of isolated murine cardiac mitochondria by measuring oxygen consumption rate using Seahorse XFe24 Analyzer. In addition, we provide details about experimental design, measurement of various respiratory parameters along with interpretation and analysis of data.

PO-106 Unique and overlapping role of HIF proteins in radiation response and tumour cell metabolism

Introduction Hypoxia is a common feature of human solid tumours and caused by limited diffusion and structural aberrations of tumour vasculature. Tumour hypoxia in cancer is linked to worse outcome and treatment resistance. Hypoxic cells are also characterised by changes in their energy requirements and radiation response. Many of these adaptations are driven by the hypoxia-inducible transcription factors or HIFs. Mammalian cells encode for three HIF1–3 proteins which are O2 regulated. The similarities and differences between HIF proteins in regulating tumour cell metabolism and radiation response are still understudied. Material and methods Using gene-editing with the CRISPR/CAS9 system we generated isogenic series of cells lacking HIF1, HIF2 or both HIF1,2 in the H1299 non-small cell lung cancer cell line. We analysed the differences in hypoxia response on downstream target gene activation. In parallel, we analysed the upregulation of HIF protein expression under hypoxic conditions for each cellular model. Proliferative capacity and hypoxic tolerance were assessed for each cell line. We determined the metabolic consequences of silencing HIF by measuring oxygen consumption rate, extracellular acidification rate, extracellular pH and lipid droplet accumulation. We determined the radiation response of each cellular model under both normoxic and hypoxic conditions using clonogenic survival assays, gamma-H2AX staining and cell cycle analysis. Results and discussions We found that HIF2 compensates for the depletion of HIF1, but HIF1 is not upregulated upon HIF2 depletion. Both HIF1 and HIF2 proteins appear to be crucial for the upregulation of downstream HIF target genes such as CAIX, GLUT1 or TWIST. HIF depleted cells appear to be less glycolytic and at the same time trigger mitochondrial activity. The radiation response of the different cellular models shows a specific profile in terms of clonogenic survival and double-strand break formation in both normoxic and hypoxic conditions. Conclusion Taken together these genetically-modified cell models may help us to further define the radiation response of hypoxic tumour cells for therapeutic interventions.

Prediction of the in vitro developmental competence of early-cleavage-stage human embryos with time-lapse imaging and oxygen consumption rate measurement.

PurposeTo assess an embryo's ability to develop into a good‐quality blastocyst during the early‐cleavage stage using time‐lapse imaging and the oxygen consumption rate.MethodsIn total, 942 zygotes had their oxygen consumption rates measured. In total, 282 zygotes were assessed by using time‐lapse imaging. In total, 121 zygotes were examined by using both their oxygen consumption rate and time‐lapse imaging.ResultsThe embryos with moderate respiration rates of between 0.41 and 0.61 (×1014/mol s−1) on day 3 had a 22.1% chance of becoming good‐quality blastocysts; those outside that range had a 14.3% chance. With the time‐lapse system, when the first division was within 24 hours, 22.3% of the embryos grew to good blastocysts. After 24 hours, the rate dropped to 8.6%. The intervals between two consecutive cleavages were calculated and the duration of the second cell cycle was defined. When the time was between nine hours and 13 hours, there was a higher rate of good blastocysts. Regarding both criteria, when the embryos had progressed in the optimal range, a high percentage of them had become good blastocysts; it was 8.0% outside of that range.ConclusionIndividual embryos with the potential to develop into good‐quality blastocysts could be selected at day 3 of culture using these systems.

Ca2+/Calmodulin‐Dependent Protein Kinase Kinase β Negatively Regulates Progesterone Mediated PGRMC1 Signaling and the Warburg Effect

ObjectivesCa2+/calmodulin‐dependent protein kinase kinase β (CaMKKβ) signaling cascades directly regulate a variety of physiological processes and have been implicated in several human diseases, including cancer and neurodegenerative disorders. We discovered progesterone‐receptor membrane component 1 (PGRMC1) is differentially phosphorylated in CaMKKβ knockout (KO) HEK293 cells. PGRMC1 is a member of the membrane‐associated progesterone receptor (MAPR) family with a cytochrome b5‐like heme‐binding region. The gene is known to be involved in diverse functions, including regulation of cytochrome P450, steroidogenesis, vesicle trafficking, progesterone signaling and mitotic spindle and cell cycle regulation. PGRMC1 is associated with multiple progesterone‐dependent effects in diverse cell types. However, direct progesterone binding to PGRMC1 is yet to be demonstrated. The lack of credible ligand binding to PGRMC1 may be due to the fact that bacterially expressed PGRMC1 preparations may not possess the necessary post‐translational modifications (PTMs) required for progesterone binding. Therefore, we hypothesized that CaMKKβ is an upstream kinase that mediates phosphorylation of PGRMC1 and, thereby, regulates progesterone signaling.MethodologyWe used TiO2 column enrichment followed by mass spectrometry to identify differentially expressed phosphopeptides derived from CaMKKβ KO versus wild type HEK293 cells. We interrogated cellular metabolism by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as an indicator of mitochondrial respiration and glycolysis, respectively. Further, we used isolectric focusing (IEF) followed by SDS‐PAGE and immunoblotting to identify different charged fractions of PGRMC1.ResultsLoss of CaMKKβ significantly decreased PGRMC1 protein expression in multiple CaMKKβ KO cell lines. TiO2 column enriched phosphopeptide analysis revealed the presence of phosphorylated S57, T178, Y180 and S181 peptides in CaMKKβ KO cells whereas only S181 phosphorylation was found in wild type cells. In addition, IEF analysis revealed multiple charged fractions of PGRMC1. The ~pI/pH‐3 fraction was significantly higher in CaMKKβ KO cells which may correspond to increased phosphorylation. Loss of CaMKKβ significantly increased the rate of glycolysis but reduced mitochondrial OCR. Treatment with 10μM progesterone significantly increased the rate of glycolysis within 30 mins in wild type cells; however it failed to show a similar effect in CaMKKβ KO cells. In contrast, progesterone treatment significantly lowered OCR in both wild type and CaMKKβ KO cells within 30 mins.ConclusionCaMKKβ negatively regulates PRGMC1 phosphorylation which may, in turn, control the relative turnover of the protein. CaMKKβ negatively regulates the progesterone‐mediated Warburg effect which may explain the role of CaMKKβ in tumorigenesis and neurodegenerative disease. Our study identifies a link between CaMKKβ and progesterone signaling which may be used for therapeutic targeting.Support or Funding InformationSupported by CIHR grant # MOP‐130282 (PF)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel Marine Compounds Modulate Mitochondrial Function in H9c2 Cells: Potential New Pharmaceutical Targets to Control Cardiac Metabolism

IntroductionThe global burden of cardiovascular disease is increasing and is the leading cause of morbidity and mortality worldwide. As the heart is a highly metabolic organ, mitochondrial health is critical to normal cardiac function and is compromised in a number of cardiac diseases. Therefore, new pharmaceutical agents and screening tools to modulate and evaluate cardiac metabolism may offer novel therapeutics for cardiovascular disease.MethodsA number of marine compounds have been curated over the years by faculty at the Scripps Institution of Oceanography. Chemical structures have been largely elucidated through nuclear magnetic resonance spectroscopy and mass spectrometry, and these have been broadly available for biological screening through the UCSD Center for Compound Resources. Seventy‐three promising marine compounds were evaluated by application on H9c2 rat cardiomyoblasts while measuring oxygen consumption rate and extracellular acidification rate with a Seahorse XF96 extracellular flux analyzer. Mitochondrial metabolism was tested at a range of compound concentrations (0.1–1.6 mM). Subsequently, dose response curves of compounds that showed biological activity in the initial screen were performed. Dose response assay concentrations ranged from 0.01 nM to 3 μM with vehicle only as the control.ResultsEight compounds showed significant dose dependent alterations in mitochondrial metabolism in the initial screen. Dose response assays of glycospongosine revealed an increase in ATP linked‐maximal respiration ratio. In contrast, higher glycospongosine concentrations led to a gradual dose dependent decrease in reserve capacity. Injection of leptochelin A first increased basal respiration of the cardiomyoblasts, after the first increase, basal respiration gradually decreased. Increasing concentrations of honaucin A lowered the maximal mitochondrial respiration dose dependently. In addition, kabiramide C decreased maximal respiration normalized to basal respiration.ConclusionWe present here the results of screening multiple marine natural products in a cell based metabolic assay. Marine compounds have a demonstrated potential to modulate cardiac metabolism in a variety of settings; however, further research in disease models is necessary to determine if such modulation via these compounds is beneficial or harmful to cardiovascular disease.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.