Increase In Oxidative Metabolism Research Articles

Dynamics of Redox Metabolism during Complete Metamorphosis of Insects: Insights from the Sunflower Caterpillar Chlosyne lacinia (Lepidoptera).

Complete insect metamorphosis requires substantial metabolic and physiological adjustments. Although oxidative stress has been implicated in metamorphosis, details on redox metabolism during larva-to-pupa and pupa-to-adult remain scarce. This study explores redox metabolism during metamorphosis of a lepidopteran (Chlosyne lacinia), focusing on core metabolism, antioxidant systems and oxidative stress. The larva-to-pupa transition was characterized by increased lactate dehydrogenase and glutathione peroxidase (GPX) activities, coupled with depletion of reduced glutathione (GSH), high disulfide-to-total-glutathione ratio (GSSG/tGSH), and increased lipid peroxidation. As metamorphosis progressed, metabolic enzyme activities, citrate synthase and glucose 6-phosphate dehydrogenase increased, indicating heightened oxidative metabolism associated with adult development. Concurrently, GSH and GPX levels returned to larval levels and GSSG/tGSH reached its most reduced state right before adult emergence. Adult emergence was marked by a further increase in oxidative metabolism, accompanied by redox imbalance and enhanced antioxidant mechanisms. These findings highlight a fluctuation in redox balance throughout metamorphosis, with periods of oxidative eustress followed by compensatory antioxidant responses. This study is the first to identify concurrent changes in metabolism, antioxidants, redox balance and oxidative stress throughout metamorphosis. Our findings extend knowledge on redox metabolism adjustments and highlight redox adaptations and oxidative stress as natural components of complete insect metamorphosis.

A9 LOSS OF INTESTINAL EPITHELIAL NCOR1 INCREASES OXIDATIVE METABOLISM

Abstract Background The nuclear co-repressor NCOR1 acts as a central platform of a complex that represses gene expression associated with inflammatory response. Literature also recently uncovered its possible role in oxidative metabolism in some tissues and organs. Although intestinal epithelial NCOR1 is crucial to restrict IBD symptoms during experimental colitis, its precise role in controlling intestinal epithelial cell biology has not been established. Aims We aimed to investigate the role of intestinal epithelial NCOR1 in oxidative metabolism. Methods Crossing Villin-Cre and Ncor1loxP/loxP mice was performed to delete Ncor1 in the whole intestinal epithelium conditionally. Crypts from control and NCOR1ΔIEC littermates were isolated to generate colonoids 3D cultures. Phenotypic, genotypic, and transcriptomic analyses were performed on those colonoids. Results Colonoids from NCOR1ΔIEC mice were phenotypically smaller but were found to display drastic higher intestinal stem cell propagation features during organoid clonogenic assays. A Gene Set Enrichment Analysis from RNA sequencing predicted an increase in oxidative metabolism in NCOR1ΔIEC colonoids. Several genes associated with oxidative metabolism were confirmed to be modulated in both NCOR1ΔIEC colonoids and the colon of NCOR1ΔIEC mice. Mitochondrial mass was also significantly increased in colonoids when NCOR1 was absent. Loss of the co-repressor did not affect the expression of PGC1-α, a known antagonist of NCOR1. However, transcripts of cytochrome c somatic (Cycs), a target gene of PGC1-α, were found higher in NCOR1ΔIEC colonoids, suggesting that the coactivator activity is increased in the absence of NCOR1. Conclusions Our results highlight a new role for NCOR1 in the oxidative metabolism of intestinal epithelial cells. Its action via PGC1-α activity could potentially be targeted as a therapeutic measure during inflammatory bowel diseases. Funding Agencies CIHR

Emodin alleviates CRS4-induced mitochondrial damage via activation of the PGC1α signaling.

Cardiorenal syndrome type 4 (CRS4), a progressive deterioration of cardiac function secondary to chronic kidney disease (CKD), is a leading cause of death in patients with CKD. In this study, we aimed to investigate the cardioprotective effect of emodin on CRS4. C57BL/6 mice with 5/6 nephrectomy and HL-1 cells stimulated with 5% CKD mouse serum were used for in vivo and in vitro experiments. To assess the cardioprotective potential of emodin, we employed a comprehensive array of methodologies, including echocardiography, tissue staining, immunofluorescence staining, biochemical detection, flow cytometry, real-time quantitative PCR, and western blot analysis. Our results showed that emodin exerted protective effects on the function and structure of the residual kidney. Emodin also reduced pathologic changes in the cardiac morphology and function of these mice. These effects may have been related to emodin-mediated suppression of reactive oxygen species production, reduction of mitochondrial oxidative damage, and increase of oxidative metabolism via restoration of PGC1α expression and that of its target genes. In contrast, inhibition of PGC1α expression significantly reversed emodin-mediated cardioprotection in vivo. In conclusion, emodin protects the heart from 5/6 nephrectomy-induced mitochondrial damage via activation of the PGC1α signaling. The findings obtained in our study can be used to develop effective therapeutic strategies for patients with CRS4.

Evaluation of clinical features of bronchial asthma of varying severity and the level of modified proteins

The article presents the results of a study of 62 patients with bronchial asthma (ВА), which were divided into 2 groups: the first group included patients (n=34) with moderate severity of asthma, the second group included patients (n=28) with severe asthma. The worsening of the course of bronchial asthma in the examined patients is due to the duration of the disease, the presence of harmful risk factors, among which adverse living conditions with the development of sensitization to them are of the greatest importance. In severe bronchial asthma, an uncontrolled course of the disease prevails with a 1.8-fold decrease in asthma-control test values (p<0.05). With an increase in the severity of the inflammatory process in the airways, the level of AOРP in the blood plasma increases. In patients with bronchial asthma, the following features of oxidative metabolism were established: in the blood plasma and erythrocytes of patients with bronchial asthma of varying severity, a tendency to an increase in reactive carbonyl derivatives of proteins, malondialdehyde, AOPP compared with the control was observed, which indicates an increase in oxidative metabolism with the development of pathology.

Clinical features and evaluation of modified proteins in bronchial asthma of various severity

The article presents the results of a study of 62 patients with bronchial asthma (ВА), which were divided into 2 groups: the first group included patients (n = 34) with moderate severity of asthma, the second group included patients (n = 28) with severe asthma. The worsening of the course of bronchial asthma in the examined patients is due to the duration of the disease, the presence of harmful risk factors, among which adverse living conditions with the development of sensitization to them are of the greatest importance. In severe bronchial asthma, an uncontrolled course of the disease prevails with a 1.8-fold decrease in asthma-control test values (p<0.05). With an increase in the severity of the inflammatory process in the airways, the level of AOРP in the blood plasma increases. In patients with bronchial asthma, the following features of oxidative metabolism were established: in the blood plasma and erythrocytes of patients with bronchial asthma of varying severity, a tendency to an increase in reactive carbonyl derivatives of proteins, malondialdehyde, AOPP compared with the control was observed, which indicates an increase in oxidative metabolism with the development of pathology.

Different patterns of chronic hypoxia lead to hierarchical adaptive mechanisms in goldfish metabolism.

Some hypoxia-tolerant species, such as goldfish, experience intermittent and severe hypoxia in their natural habitat, causing them to develop multiple physiological adaptations. However, in fish, the metabolic impact of regular hypoxic exposure on swimming performance in normoxia is less well understood. Therefore, we experimentally tested whether chronic exposure to constant (30 days at 10% air saturation) or intermittent hypoxia (3 h in normoxia and 21 h in hypoxia, 5 days a week) would result in similar metabolic and swimming performance benefits after reoxygenation. Moreover, half of the normoxic and intermittent hypoxic fish were put on a 20-day normoxic training regime. After these treatments, metabolic rate (standard and maximum metabolic rates: SMR and MMR) and swimming performance [critical swimming speed (Ucrit) and cost of transport (COT)] were assessed. In addition, enzyme activities [citrate synthase (CS), cytochrome c oxidase (COX) and lactate dehydrogenase (LDH)] and mitochondrial respiration were examined in red muscle fibres. We found that acclimation to constant hypoxia resulted in (1) metabolic suppression (-45% SMR and -27% MMR), (2) increased anaerobic capacity (+117% LDH), (3) improved swimming performance (+80% Ucrit, -71% COT) and (4) no changes at the mitochondrial level. Conversely, the enhancement of swimming performance was reduced following acclimation to intermittent hypoxia (+45% Ucrit, -41% COT), with a 55% decrease in aerobic scope, despite a significant increase in oxidative metabolism (+201% COX, +49% CS). This study demonstrates that constant hypoxia leads to the greatest benefit in swimming performance and that mitochondrial metabolic adjustments only provide minor help in coping with hypoxia.

Dynamic changes in macrophage metabolism modulate induction and suppression of Type I inflammatory responses.

During microbial infection, macrophages link recognition of microbial stimuli to the induction of Type I inflammatory responses. Such inflammatory responses coordinate host defense and pathogen elimination but induce significant tissue damage if sustained, so macrophages are initially activated to induce inflammatory responses but then shift to a tolerant state to suppress inflammatory responses. Macrophage tolerance is regulated by induction of negative regulators of TLR signaling, but its metabolic basis was not known. Here, we review recent studies that indicate that macrophage metabolism changes dynamically over the course of microbial exposure to influence a shift in the inflammatory response. In particular, an initial increase in oxidative metabolism boosts the induction of inflammatory responses, but is followed by a shutdown of oxidative metabolism that contributes to suppression of inflammatory responses. We propose a unifying model for how dynamic changes to oxidative metabolism influences regulation of macrophage inflammatory responses during microbial exposure.

Human hyperpolarized [1-13C] pyruvate CMR and adenosine stress test

Abstract Background Hyperpolarized (HP) [1-13C]pyruvate cardiac magnetic resonance (CMR) imaging can visualize myocardial perfusion and metabolism beyound glucose uptake. Depending on the prevailing metabolic conditions, buid-up of either [1-13C]lactate or 13C-bicarbonate can be measured. The aim of the present study was to assess cardiac metabolism using HP [1-13C]pyruvate rest-stress CMR. Methods Six healthy volunteers underwent cine CMR and HP [1-13C]pyruvate CMR at rest and during an adenosine stress test. Signal from HP [1-13C]pyruvate and its downstream metabolites was measured at the mid-left-ventricle (LV) level. We did semi-quantitative assessment of first-pass myocardial [1-13C]pyruvate perfusion. Pressure-volume loops were assessed non-invasively. Results Myocardial [1-13C]pyruvate perfusion was significantly increased during stress with a reduction in time-to-peak from 6.2±2.8 sec to 2.7±1.3 sec, p=0.04. This higher perfusion was accompanied by an overall increased myocardial uptake and metabolism. The conversion rate constant (kPL) for lactate increased from 0.011±0.009 sec–1 to 0.020±0.010 sec–1, p=0.04. The pyruvate oxidation (kPB) increased from 0.004±0.004 sec–1 to 0.012±0.007 sec–1, p=0.008. This increase in oxidative metabolism was positively correlated with heart rate (R2=0.44, p=0.02). Conclusion We observed a significant increase in carbohydrate oxidation during cardiac stress in the healthy human heart. The present study forms the basis for comparisons in future research in patients with heart failure and coronary artery disease. HP [1-13C]pyruvate CMR could be a possible alternative to PET in the future. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Danish Heart FoundationIndependent Research Fund, Denmark Increased conversion rate of pyruvateIncreased metabolite signal in LV

Efficacy of Combined in-vivo Electroporation-Mediated Gene Transfer of VEGF, HGF, and IL-10 on Skin Flap Survival, Monitored by Label-Free Optical Imaging: A Feasibility Study.

Preventing surgical flaps necrosis remains challenging. Laser Doppler imaging and ultrasound can monitor blood flow in flap regions, but they do not directly measure the cellular response to ischemia. The study aimed to investigate the efficacy of synergistic in-vivo electroporation-mediated gene transfer of interleukin 10 (IL-10) with either hepatocyte growth factor (HGF) or vascular endothelial growth factor (VEGF) on the survival of a modified McFarlane flap, and to evaluate the effect of the treatment on cell metabolism, using label-free fluorescence lifetime imaging. Fifteen male Wistar rats (290–320 g) were randomly divided in three groups: group-A (control group) underwent surgery and received no gene transfer. Group-B received electroporation mediated hIL-10 gene delivery 24 h before and VEGF gene delivery 24 h after surgery. Group-C received electroporation mediated hIL-10 gene delivery 24 h before and hHGF gene delivery 24 h after surgery. The animals were assessed clinically and histologically. In addition, label-free fluorescence lifetime imaging was performed on the flap. Synergistic electroporation mediated gene delivery significantly decreased flap necrosis (P = 0.0079) and increased mean vessel density (P = 0.0079) in treatment groups B and C compared to control group-A. NADH fluorescence lifetime analysis indicated an increase in oxidative phosphorylation in the epidermis of the group-B (P = 0.039) relative to controls. These findings suggested synergistic in-vivo electroporation-mediated gene transfer as a promising therapeutic approach to enhance viability and vascularity of skin flap. Furthermore, the study showed that combinational gene therapy promoted an increase in tissue perfusion and a relative increase in oxidative metabolism within the epithelium.

Abstract P1-01-08: Optical metabolic imaging of the effects of acute and intermittent hypoxia in murine breast cancer cells

Abstract About 90% of the patients diagnosed with breast cancer succumb due to metastases and not from primary tumor. There are currently few methods that can distinguish indolent and aggressive breast tumors. Recent studies have shown differences in the metabolic profiles of primary, circulating, and metastatic breast cancer cells. One of the key regulators of metastases in breast cancer is the transcription factor Twist. The goal of our study was to determine the relationship between Twist expression and cellular metabolism, and the effect of this relationship on metastatic potential. Multiphoton microscopy can provide non-invasive, label-free and reproducible measurements of cellular metabolism. An optical redox ratio of endogenous fluorescence from nicotinamide adenine dicnucleotide (NADH) and flavin adenine dinucleotide (FAD) (calculated as FAD/NADH+FAD) can reflect the balance between mitochondrial oxidative phosphorylation and glucose catabolism in the cell. Because hypoxia is known to promote metastasis, we studied the effect of both intermittent and acute hypoxia on cellular metabolism in 4T1 murine breast cancer cells. We generated a 4T1-Twist− cell line using CRISPR-Cas9 technology. Western blots of Twist protein expression confirmed that Twist expression was significantly lower in the selected cell population compared with the parental 4T1 line. Both cell lines were exposed to 0.5% oxygen for 3 hours and one hour of reoxygenation (acute hypoxia), and 3 cycles of one-hour hypoxia followed by one-hour reoxygenation (intermittent hypoxia). Multiphoton imaging of endogenous fluorescence as well as metabolic flux analysis (Seahorse Biosciences) was performed for both hypoxic exposures in both cell lines. Our results indicate a significant increase in the optical redox ratio in 4T1 cells when exposed to acute hypoxia, indicating a possible increase in oxidative metabolism. There were no signfiicant differences in the 4T1-Twist− line. On the other hand, we observed a decrease in the optical redox ratio in response to intermittent hypoxia in the 4T1 cells and the opposite effect in the 4T1-Twist− line. In addition to these cell lines, we will also present data from sibling cell lines of the 4T1 with varying Twist levels and hence metastatic potential. These data indicate indicate potentially measurable metabolic differences between indolent and aggressive breast cancer cells when subject to a hypoxia-reoxygenation regimen. Citation Format: Lisa Rebello, Narasimhan Rajaram. Optical metabolic imaging of the effects of acute and intermittent hypoxia in murine breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-01-08.

67 Immunometabolism – emerging concepts and potential applications in livestock

Abstract Our understanding of the immune system emerged from the study of disease processes and the communication networks used by various cell types to respond to pathogens. As with many aspects of physiology, this initial view was colored by the techniques available at the time. With technical advances beginning in the 1990, research in sepsis and obesity began to identify critical interactions between the immune system and metabolism. Our current understanding of these interactions is informed by two active but largely distinct research communities. Many in the field of immunology are utilizing cellular metabolism tools to understand mitochondrial function and fuel use in response to activation of innate and adaptive immune cells, especially as these relate to cancer. From another vantage point, many metabolic physiologists are now seeking to understand the importance of tissue-resident immune cells and immune signaling molecules in metabolic homeostasis and pathologies. Beyond human health implications of recent findings, a number of immunometabolism insights have informed our understanding of livestock health. In inflammatory events, phagocytic cells are activated, and the dramatic increase in oxidative metabolism is driven primarily by glucose use. Metabolism of healthy animals is also influenced by secretions from immune cells. Studies in mice indicate that appropriate host/microbe interactions (balancing protection and tolerance) are mediated by a network of immune cell types in the gut, which is critical to both absorptive and barrier functions of the gut. Adipose tissue immune cells regulate lipolytic rate, insulin sensitivity, and perhaps whole-body inflammatory tone. Local immune cell impacts on metabolism of other organs, including the liver and pancreas, are also emerging. Immunity and metabolism are tightly interwoven, and the evolving understanding of these links may enable nutritional or pharmacological strategies to enhance resilience to disease and alter nutrient partitioning in livestock.

H1/H2 Histamine Receptor Blockade Alters the Glutathione Redox Status in Skeletal Muscle Following a Bout of Prolonged Exercise

The effects of an acute bout of prolonged exercise on muscle include increases in oxidative metabolism and altered cellular redox environment. Histamine mediates many acute exercise responses, including sustained vasodilation and associated delivery of substrates to muscle. Previously, we demonstrated that H1 and H2 histamine receptor blockade with oral antihistamines resulted in a diminished capacity for mitochondrial H2O2 emission in skeletal muscle following an acute bout of prolonged exercise. Here, we tested the hypothesis that along with diminishing mitochondrial H2O2 emission, H1 and H2 histamine receptor blockade would attenuate exercise‐induced changes in skeletal muscle glutathione redox status. Adult male Wistar rats (83 ± 5.5 days old; 440.04 ± 40.70 g) were assigned to one of four groups (n = 12/group): exercise, in the form of one hour of continuous treadmill running at 20 cm/sec (Group 1); mass‐specific oral gavage with H1 antagonist fexofenadine (7.11 mg/kg) and H2 antagonist ranitidine (3.95 mg/kg) one hour prior to the same exercise protocol (Group 2); a group receiving oral H1 and H2 antagonists, but not exercised (Group 3); and a control group, which received neither antihistamines nor exercise (Group 4). An hour post‐exercise, total, reduced (GSH) and oxidized glutathione (GSSG) were assessed in red gastrocnemius (mixed fiber types) and soleus (oxidative) skeletal muscles. Redox status was expressed as GSH/GSSG. While no changes were observed in total glutathione for any treatment in either muscle, in soleus, antihistamine treatment, but not exercise decreased GSH/GSSG (main effect; p < 0.05). In red gastrocnemius, exercise, but not antihistamine treatment decreased GSH/GSSH (main effect; p < 0.05). These observations trend with those of a blunted capacity for mitochondrial H2O2 emission in skeletal muscle with antihistamine treatment and suggest an altered redox response to exercise following H1/H2 histamine receptor blockade in soleus. Given the widespread and routine use of antihistamines, further research into the effects of these antagonists on redox status in muscle is warranted.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada, Nova Scotia Health Research Foundation, Canada Foundation for Innovation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PO-258 Telmisartan induces melanoma cell apoptosis via generation of ROS and has synergistic effects with targeted therapy in vitro

IntroductionMelanoma is one of the most aggressive malignancies. A half of melanoma tumours carry BRAF V600E mutation, but despite dramatic initial effects of BRAF inhibitors in the clinic, patients eventually relapse, suggesting that combination therapies are needed to overcome resistance. BRAF inhibitors suppress glycolysis, yet the subsequent increase in oxidative metabolism limits their efficacy. Therefore, agents that target different aspects of cell metabolism may improve melanoma therapy efficacy. Telmisartan is the AT1R inhibitor and PPARγ agonist. PPARγ is a nuclear receptor that is an important regulator of lipid and glucose metabolism. Activation of PPARγ in melanoma cells has growth inhibitory effect, by inducing cell cycle arrest. We hypothesised that telmisartan could inhibit melanoma cell viability and potentially improve BRAF inhibitor therapy by modulating cell metabolism.Material and methodsPublicly available databases were analysed for expression of AT1R and PPARγ in melanoma patient tumour samples and various cell lines. A panel of melanoma cell lines was tested in MTT, apoptosis and metabolic assays in presence of telmisartan by flow cytometry.Results and discussionsBoth telmisartan targets were expressed in melanoma patient samples, but PPARγ levels significantly decreased in melanoma compared to uninvolved skin. Telmisartan decreased viability of melanoma cells in vitro to a similar extent as the direct PPARγ agonist pioglitazone, while pure AT1R inhibitor losartan had no influence. While pioglitazone induced cell cycle arrest in A375 cells, telmisartan induced apoptosis at same concentrations through induction of ROS and dissipation of mitochondrial potential. As expected for PPARγ activation, telmisartan increased glucose uptake in A375 cells. Melanoma cells have intrinsically high levels of oxidative stress due to the accelerated metabolism, which renders them more susceptible to oxidative stress-induced cell death than the normal cells. In line with this notion, telmisartan-induced cell death could be ameliorated with N-acetyl cysteine. In addition, telmisartan synergized with both dacarbazine and vemurafenib in vitro and was still effective in A375R cell line, vemurafenib resistant cell line.ConclusionTelmisartan has anti-melanoma potential in vitro and could increase the effectiveness of melanoma therapeutics, both conventional and BRAF inhibitor therapy. Our future efforts include elucidation of the exact mechanism by which telmisartan exerts these effects.

Progressive Upregulation of Oxidative Metabolism Facilitates Plasmablast Differentiation to a T-Independent Antigen

SUMMARYTransitioning from a metabolically quiescent naive B cell to an antibody-secreting plasmablast requires division-dependent cellular differentiation. Though cell division demands significant ATP and metabolites, the metabolic processes used for ATP synthesis during plasmablast formation are not well described. Here, the metabolic requirements for plasmablast formation were determined. Following T-independent stimulation with lipopolysaccharide, B cells increased expression of the oxidative phosphorylation machinery in a stepwise manner. Such activated B cells have increased capacity to perform oxidative phosphorylation but showed dependency on glycolysis. Plasmablasts displayed higher oxidative metabolism to support antibody secretion, as inhibiting oxidative ATP production resulted in decreased antibody titers. Differentiation by Blimp1 was required for this increase in oxidative metabolism, as Blimp1-deficient cells proliferate but do not upregulate oxidative phosphorylation. Together, these findings identify a shift in metabolic pathways as B cells differentiate, as well as the requirement for increased metabolic potential to support antibody production.

Consumption of Alcopops During Brain Maturation Period: Higher Impact of Fructose Than Ethanol on Brain Metabolism.

Alcopops are flavored alcoholic beverages sweetened by sodas, known to contain fructose. These drinks have the goal of democratizing alcohol among young consumers (12–17 years old) and in the past few years have been considered as fashionable amongst teenagers. Adolescence, however, is a key period for brain maturation, occurring in the prefrontal cortex and limbic system until 21 years old. Therefore, this drinking behavior has become a public health concern. Despite the extensive literature concerning the respective impacts of either fructose or ethanol on brain, the effects following joint consumption of these substrates remains unknown. Our objective was to study the early brain modifications induced by a combined diet of high fructose (20%) and moderate amount of alcohol in young rats by 13C Nuclear Magnetic Resonance (NMR) spectroscopy. Wistar rats had isocaloric pair-fed diets containing fructose (HF, 20%), ethanol (Et, 0.5 g/day/kg) or both substrates at the same time (HFEt). After 6 weeks of diet, the rats were infused with 13C-glucose and brain perchloric acid extracts were analyzed by NMR spectroscopy (1H and 13C). Surprisingly, the most important modifications of brain metabolism were observed under fructose diet. Alterations, observed after only 6 weeks of diet, show that the brain is vulnerable at the metabolic level to fructose consumption during late-adolescence throughout adulthood in rats. The main result was an increase in oxidative metabolism compared to glycolysis, which may impact lactate levels in the brain and may, at least partially, explain memory impairment in teenagers consuming alcopops.

Progressive upregulation of oxidative metabolism facilitates plasmablast differentiation to a T-independent antigen

Abstract Transitioning from a metabolically quiescent naïve B cell to an antibody-secreting plasmablast requires division-dependent cellular differentiation. Though cellular processes such as cell division and antibody secretion demand significant ATP and metabolites, the metabolic processes used to generate ATP during plasmablast formation are not well described. Here, the metabolic machinery requirements for plasmablast formation were determined. Following T-independent stimulation with lipopolysaccharide, B cells increased expression of the oxidative phosphorylation machinery in a step-wise manner. Such activated B cells have increased capacity to perform oxidative phosphorylation, but showed additional dependency on glycolytic metabolism. Plasmablasts displayed higher oxidative metabolism to support antibody secretion, as inhibiting oxidative ATP production with oligomycin resulted in decreased antibody titres. Differentiation orchestrated by Blimp1 was required for this increase in oxidative metabolism as Blimp1-deficient cells proliferate normally, but neither differentiate to plasmablasts nor upregulate oxidative phosphorylation. Therefore, Blimp1 expression is required for full metabolic activity, and proliferation alone is insufficient to induce full oxidative phosphorylation. Together, these findings identify a shift in metabolic pathways as B cells differentiate, as well as the requirement for increased metabolic potential to support antibody production.

MTORC1-dependent increase in oxidative metabolism in POMC neurons regulates food intake and action of leptin

ObjectiveNutrient availability modulates reactive oxygen species (ROS) production in the hypothalamus. In turn, ROS regulate hypothalamic neuronal activity and feeding behavior. The mechanistic target of rapamycin complex 1 (mTORC1) pathway is an important cellular integrator of the action of nutrients and hormones. Here we tested the hypothesis that modulation of mTORC1 activity, particularly in Proopiomelanocortin (POMC)-expressing neurons, mediates the cellular and behavioral effects of ROS. MethodsC57BL/6J mice or controls and their knockout (KO) littermates deficient either for the mTORC1 downstream target 70-kDa ribosomal protein S6 kinase 1 (S6K1) or for the mTORC1 component Rptor specifically in POMC neurons (POMC-rptor-KO) were treated with an intracerebroventricular (icv) injection of the ROS hydrogen peroxide (H2O2) or the ROS scavenger honokiol, alone or, respectively, in combination with the mTORC1 inhibitor rapamycin or the mTORC1 activator leptin. Oxidant-related signal in POMC neurons was assessed using dihydroethidium (DHE) fluorescence. ResultsIcv administration of H2O2 decreased food intake, while co-administration of rapamycin, whole-body deletion of S6K1, or deletion of rptor in POMC neurons impeded the anorectic action of H2O2. H2O2 also increased oxidant levels in POMC neurons, an effect that hinged on functional mTORC1 in these neurons. Finally, scavenging ROS prevented the hypophagic action of leptin, which in turn required mTORC1 to increase oxidant levels in POMC neurons and to inhibit food intake. ConclusionsOur results demonstrate that ROS and leptin require mTORC1 pathway activity in POMC neurons to increase oxidant levels in POMC neurons and consequently decrease food intake.

Perturbations in carotenoid and porphyrin status result in differential photooxidative stress signaling and antioxidant responses

We examined differential photooxidative stress signaling and antioxidant responses in rice plants treated with norflurazon (NF) and oxyfluorfen (OF), which are inhibitors of carotenoid and porphyrin biosynthesis, respectively. Plants treated with OF markedly increased levels of cellular leakage and malondialdehyde, compared with NF-treated plants, showing that OF plants suffered greater oxidative damage with respect to membrane integrity. The enhanced production of H2O2 in response to OF, but not NF, indicates the important role of H2O2 in activation of photooxidative stress signaling in OF plants. In response to NF and OF, the increased levels of free salicylic acid as well as maintenance of the redox ratio of ascorbate and glutathione pools to a certain level are considered to be crucial factors in the protection against photooxidation. Plants treated with OF greatly up-regulated catalase (CAT) activity and Cat transcript levels, compared with NF-treated plants. Interestingly, NF plants showed no noticeable increase in oxidative metabolism, although they did show considerable increases in ascorbate peroxidase (APX) and peroxidase activities and transcript levels of APX, as in OF plants. Our results suggest that perturbations in carotenoid and porphyrin status by NF and OF can be sensed by differential photooxidative stress signaling, such as that involving H2O2, redox state of ascorbate and glutathione, and salicylic acid, which may be responsible for at least part of the induction of ROS-scavenging enzymes.

Abstract 1821: Sympathetic nerves regulate a metabolic switch promoting angiogenesis through adrenergic signaling in prostate cancer

Abstract Nerves pattern the vasculature during development and regeneration. Recent studies have shown that the sympathetic nervous system (SNS) is coopted to promote carcinogenesis, and that depletion of SNS β-adrenergic receptors (βARs) in the prostate stromal compartment inhibits tumor growth. As the vasculature is in direct contact with the SNS, and regulates tumor glucose and oxygen delivery, we aimed to identify the stromal populations that mediate SNS signaling and the mechanisms by which loss of βAR signaling inhibits prostate cancer progression. In vivo xenograft tumor growth was measured in Adrb2-/-; Adrb3-/- mice using an orthotopic PC3-luciferase model. Prostate cancer progression in the transgenic autochthonous HI-myc model was assessed after conditional Adrb2 deletion in stromal populations by intercrossing Adrb2fl/fl line to stromal Cre lines. Metabolism was measured ex vivo by flow quantification of the lipophilic cationic dye TMRE whose accumulation reflects mitochondrial membrane potential, and by NADH autofluorescence under UV excitation. In vitro oxidative phosphorylation and glycolytic flux were measured in primary prostate endothelial cells (VeraVec) using the Seahorse bioanalyzer and targeted gas chromatography-mass spectrometry (GC/MS). Deletion of the SNS βAR neural receptors in the microenvironment synergized to arrest exponential tumor growth. Imaging the tumor vasculature revealed a defect in vessel branching, a phenotype also observed after chemical sympathectomy, suggesting altered angiogenesis in the absence of βAR stimulation. Histology revealed an increase in vascular innervation and density during the transition from low to high grade pre-neoplastic PIN. Conditional Adrb2 deletion (Adrb2cKO) in endothelial cells (ECs), but not myeloid cells or pericytes, not only inhibited angiogenesis, but also reduced disease progression throughout all histopathological cancer stages. Transcriptome analysis of flow isolated Adrb2cKO ECs suggested an increase in oxidative metabolism and upregulation of mitochondrial Coa6. Ex vivo analysis of ECs revealed a metabolic switch during the low to high grade PIN transition, and this switch was inhibited by Adrb2cKO. Further in vitro characterization by Adrb2 knockdown revealed a metabolic shift in ECs to oxidative phosphorylation, which was mediated by Coa6 upregulation. As Coa6 cooperates with Cox10 in respiratory complex IV assembly, conditional co-deletion of Adrb2 and Cox10 in the HI-myc mouse rescued aerobic glycolysis, angiogenesis, and cancer progression. Our data thus demonstrates that the SNS mediates a pro-angiogenic switch during the early stages of prostate carcinogenesis. Moreover, we identified ECs as SNS stromal targets, and that abrogation of βAR signaling inhibits the angio-metabolic switch and delays prostate cancer progression, suggesting a novel therapeutic target for prostate cancer treatment. Citation Format: Ali Zahalka, Anna Arnal-Estape, Maria Maryanovich, Fumio Nakahara, Cristian Cruz, Paul S. Frenette. Sympathetic nerves regulate a metabolic switch promoting angiogenesis through adrenergic signaling in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1821. doi:10.1158/1538-7445.AM2017-1821

Complex I inhibition augments dichloroacetate cytotoxicity through enhancing oxidative stress in VM-M3 glioblastoma cells.

The robust glycolytic metabolism of glioblastoma multiforme (GBM) has proven them susceptible to increases in oxidative metabolism induced by the pyruvate mimetic dichloroacetate (DCA). Recent reports demonstrate that the anti-diabetic drug metformin enhances the damaging oxidative stress associated with DCA treatment in cancer cells. We sought to elucidate the role of metformin’s reported activity as a mitochondrial complex I inhibitor in the enhancement of DCA cytotoxicity in VM-M3 GBM cells. Metformin potentiated DCA-induced superoxide production, which was required for enhanced cytotoxicity towards VM-M3 cells observed with the combination. Similarly, rotenone enhanced oxidative stress resultant from DCA treatment and this too was required for the noted augmentation of cytotoxicity. Adenosine monophosphate kinase (AMPK) activation was not observed with the concentration of metformin required to enhance DCA activity. Moreover, addition of an activator of AMPK did not enhance DCA cytotoxicity, whereas an inhibitor of AMPK heightened the cytotoxicity of the combination. Our data indicate that metformin enhancement of DCA cytotoxicity is dependent on complex I inhibition. Particularly, that complex I inhibition cooperates with DCA-induction of glucose oxidation to enhance cytotoxic oxidative stress in VM-M3 GBM cells.