Altered Cellular Energy Metabolism Research Articles

The purinergic receptor P2X7 as a modulator of viral vector-mediated antigen cross-presentation.

Modified Vaccinia Virus Ankara (MVA) is a safe vaccine vector inducing long- lasting and potent immune responses. MVA-mediated CD8+T cell responses are optimally induced, if both, direct- and cross-presentation of viral or recombinant antigens by dendritic cells are contributing. To improve the adaptive immune responses, we investigated the role of the purinergic receptor P2X7 (P2RX7) in MVA-infected feeder cells as a modulator of cross-presentation by non-infected dendritic cells. The infected feeder cells serve as source of antigen and provide signals that help to attract dendritic cells for antigen take up and to license these cells for cross-presentation. We demonstrate that presence of an active P2RX7 in major histocompatibility complex (MHC) class I (MHCI) mismatched feeder cells significantly enhanced MVA-mediated antigen cross-presentation. This was partly regulated by P2RX7-specific processes, such as the increased availability of extracellular particles as well as the altered cellular energy metabolism by mitochondria in the feeder cells. Furthermore, functional P2RX7 in feeder cells resulted in a delayed but also prolonged antigen expression after infection. We conclude that a combination of the above mentioned P2RX7-depending processes leads to significantly increased T cell activation via cross- presentation of MVA-derived antigens. To this day, P2RX7 has been mostly investigated in regards to neuroinflammatory diseases and cancer progression. However, we report for the first time the crucial role of P2RX7 for antigen- specific T cell immunity in a viral infection model.

Immunotherapy Approaches in Isocitrate-Dehydrogenase-Mutant Low-Grade Glioma.

Low-grade gliomas (LGGs) are slow-growing tumors in the central nervous system (CNS). Patients characteristically show the onset of seizures or neurological deficits due to the predominant LGG location in high-functional brain areas. As a molecular hallmark, LGGs display mutations in the isocitrate dehydrogenase (IDH) enzymes, resulting in an altered cellular energy metabolism and the production of the oncometabolite D-2-hydroxyglutarate. Despite the remarkable progress in improving the extent of resection and adjuvant radiotherapy and chemotherapy, LGG remains incurable, and secondary malignant transformation is often observed. Therefore, novel therapeutic approaches are urgently needed. In recent years, immunotherapeutic strategies have led to tremendous success in various cancer types, but the effect of immunotherapy against glioma has been limited due to several challenges, such as tumor heterogeneity and the immunologically "cold" tumor microenvironment. Nevertheless, recent preclinical and clinical findings from immunotherapy trials are encouraging and offer a glimmer of hope for treating IDH-mutant LGG patients. Here, we aim to review the lessons learned from trials involving vaccines, T-cell therapies, and IDH-mutant inhibitors and discuss future approaches to enhance the efficacy of immunotherapies in IDH-mutant LGG.

Abstract 787: Aberrantly increased expression of ZIC2 is correlated with altered cellular metabolism in prostate cancer

Abstract Prostate cancer incidence and mortality rates are consistently reported to be higher in American men of recent African ancestry when compared to American men of recent European or Asian ancestry. However, actionable factors contributing to these different rates remain undefined. In order to identify common molecular events to assist in the design of interventions aimed at prevention and treatment of lethal prostate cancer, we analyzed existing public RNA-Seq data and report that Zic family member 2 (ZIC2) RNA levels are significantly higher in prostate and other cancers. ZIC2 encodes a C2H2 zinc finger domain containing protein that is expressed during embryonic development in metazoans. Altered ZIC2 expression results in holoprosencephaly in humans. The ZIC2 locus also encodes several RNA species that have been co-purified with members of the polycomb repressive complex 2 (PRC2). In prostate cancer, we found that ZIC2 levels correlated significantly with primary cancer grade, i.e., the highest ZIC2 expression was found in lethal, metastatic prostate cancer. Therefore, we hypothesize that increased ZIC2 contributes to cancer initiation and progression. To establish models for identifying functional roles for ZIC2 in cancer, we targeted the first exon in ZIC2 by CRISPR/Cas9, with the intent of abrogating ZIC2 expression. We established clonal cell lines from PC-3, E006AA-Par and 293T cells with mutant ZIC2 loci. ZIC2 mutant cells displayed significant decreases in proliferation and altered phenotypes. RNA-Seq revealed expression patterns consistent with reduced glycolysis and increased oxidative phosphorylation, as well as many altered signaling pathways, in cells with ZIC2 mutations. Metabolism was monitored by extracellular flux analysis and revealed increased oxygen consumption and significantly higher respiration rates in ZIC2 mutant cells. ZIC2 localization to mitochondria was predicted using computational methods and confirmed by sub cellular fractionation and immunoblot analysis as well as by GFP-tagged microscopy. JC-1 fluorescence indicated increased mitochondrial membrane potential in ZIC2 mutant cells. We present these results and suggest that ZIC2 promotes anabolic metabolism consistent with embryonic development and cancer cell proliferation. We found that altering ZIC2 expression in PC-3 cells increased oxidative phosphorylation and produced a 5X increase in the number of apoptotic cells. Our results suggest that ZIC2 expression contributes to the cancer phenotype, given that altered cellular energy metabolism and evasion of apoptosis are defining characteristics of nearly all cancers. We further propose that the targeted inhibition of ZIC2 in cancer cells will decrease mortality rates in men diagnosed with lethal prostate cancer. Citation Format: Nathan J. Bowen, Alira Danaher. Aberrantly increased expression of ZIC2 is correlated with altered cellular metabolism in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 787.

Aberrant Cerebral Blood Flow in Response to Hunger and Satiety in Women Remitted from Anorexia Nervosa.

The etiology of pathological eating in anorexia nervosa (AN) remains poorly understood. Cerebral blood flow (CBF) is an indirect marker of neuronal function. In healthy adults, fasting increases CBF, reflecting increased delivery of oxygen and glucose to support brain metabolism. This study investigated whether women remitted from restricting-type AN (RAN) have altered CBF in response to hunger that may indicate homeostatic dysregulation contributing to their ability to restrict food. We compared resting CBF measured with pulsed arterial spin labeling in 21 RAN and 16 healthy comparison women (CW) when hungry (after a 16-h fast) and after a meal. Only remitted subjects were examined to avoid the confounding effects of malnutrition on brain function. Compared to CW, RAN demonstrated a reduced difference in the Hungry − Fed CBF contrast in the right ventral striatum, right subgenual anterior cingulate cortex (pcorr < 0.05) and left posterior insula (punc < 0.05); RAN had decreased CBF when hungry versus fed, whereas CW had increased CBF when hungry versus fed. Moreover, decreased CBF when hungry in the left insula was associated with greater hunger ratings on the fasted day for RAN. This represents the first study to show that women remitted from AN have aberrant resting neurovascular function in homeostatic neural circuitry in response to hunger. Regions involved in homeostatic regulation showed group differences in the Hungry − Fed contrast, suggesting altered cellular energy metabolism in this circuitry that may reduce motivation to eat.

Requirement of ABC transporter inhibition and Hoechst 33342 dye deprivation for the assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

BackgroundElucidating the precise properties of cancer stem cells (CSCs) is indispensable for the development of effective therapies against tumors, because CSCs are key drivers of tumor development, metastasis and relapse. We previously reported that the Hoechst 33342 dye-low staining side population (SP) method can enrich for CSCs in the C6 glioma cell line, and that the positively stained main population (MP) cells are non-CSCs. Presence of cancer stem-like SP cells is reported in various types of cancer. Although altered cellular energy metabolism is a hallmark of cancer, very little has been studied on the applicability of fluorescent probes for the understanding of CSC energy metabolism.MethodsThe metabolic status of C6 SP and MP cells are evaluated by CellROX, MitoTracker Green (MTG) and JC-1 for cellular oxidative stress, mitochondrial amount, and mitochondrial membrane potential, respectively.ResultsSP cells were found to exhibit significantly lower fluorescent intensities of CellROX and MTG than MP cells. However, inhibition of ATP binding cassette (ABC) transporters by verapamil enhanced the intensities of these probes in SP cells to the levels similar to those in MP cells, indicating that SP cells expel the probes outside of the cells through ABC transporters. Next, SP cells were stained with JC-1 dye which exhibits membrane potential dependent accumulation in mitochondrial matrix, followed by formation of aggregates. The mitochondrial membrane potential indicated by the aggregates of JC-1 was 5.0-fold lower in SP cells than MP cells. Inhibition of ABC transporters enhanced the fluorescent intensities of the JC-1 aggregates in both SP and MP cells, the former of which was still 2.2-fold lower than the latter. This higher JC-1 signal in MP cells was further found to be due to the Hoechst 33342 dye existing in MP cells. When SP and MP cells were recultured to deprive the intracellular Hoechst 33342 dye and then stained with JC-1 in the presence of verapamil, the intensities of JC-1 aggregates in such SP and MP cells became comparable.ConclusionInhibiting ABC transporters and depriving Hoechst 33342 dye are required for the accurate assessment of side population-defined C6 glioma stem cell metabolism using fluorescent probes.

Abstract 214: Intracellular ATP depletion in cancer cells by a synthetic ATP-binding protein

Abstract Introduction: Altered cellular energy metabolism is a crucial characteristic of most cancers regardless of tissue or cellular origin. In contrast to normal cells, which generate most of their adenosine triphosphate (ATP) through oxidative phosphorylation, cancer cells are dependent on aerobic glycolysis to meet energy demands (Warburg effect). However, recent studies demonstrate an upregulation in mitochondrial activity (O2 consumption and ATP production) when cancer cells are subjected to stress, such as radiation/chemotherapy treatment. To understand the significance of ATP produced by oxidative phosphorylation in cancer cells, a synthetic ATP-binding protein, DX, was targeted to the mitochondria in HeLa cells to reduce bioavailable ATP. Hypothesis: Reduced available ATP, due to DX in the mitochondria of HeLa cells, will negatively affect cellular metabolism, viability and chemoresistance. Methods: For controlled expression, DX was cloned into the pcDNA™5/TO expression vector under the control of a tetracycline (Tet)-regulated promoter. The DX construct was designed to translocate into the matrix of the mitochondria using the mitochondria targeting sequence and the 3’ UTR of a nuclear SOD2 gene. Cultured T-REx™ HeLa cells (which stably express the Tet-repressor protein) were transfected with pcDNA™5/TO::DX-MITO. As a control, T-REx™ HeLa cells were transfected with the parent plasmid, pcDNA™5/TO, and cultured under identical conditions. RT-PCR was performed to confirm DX expression, while western blot analysis was performed to confirm DX localization to the mitochondria. The impact of DX on cell viability and morphology was assessed using a tetrazolium-based colorimetric assay, caspase 3/7 assay, and by light/fluorescent microscopy. To correlate phenotypic change with DX activity, the level of bioavailable ADP /ATP was measured at specific time points following DX induction. Results: RT-PCR and western blot analysis confirmed DX expression and translation in HeLa cells, respectively, following Tet induction. Cells expressing DX experienced a significant reduction in viability and alteration in morphology compared to control HeLa cells. Contemporaneous with DX expression, bioavailable ATP decreased and the levels of caspases 3/7 increased suggesting that a majority of cells undergo apoptosis in response to energy crisis. Conclusion: Reduced intracellular ATP levels, mediated by a synthetic ATP-binding protein, negatively affects the viability of cervical cancer cells. The consequence of this on biochemical pathways, mitochondrial function and cell viability requires further investigation. We suggest DX, could be used to control and regulate energy metabolism and represents a new approach for cancer chemotherapy. Citation Format: Eddie Khav, Bernardo Chavira, Matt Hamada, Nagaraj Vinay Janthakahalli, Shaleen Korch. Intracellular ATP depletion in cancer cells by a synthetic ATP-binding protein. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 214.

Fusarium mycotoxin enniatin B: Cytotoxic effects and changes in gene expression profile

The mycotoxin enniatin B, a cyclic hexadepsipeptide produced by the plant pathogen Fusarium, is prevalent in grains and grain-based products in different geographical areas. Although enniatins have not been associated with toxic outbreaks, they have caused toxicity in vitro in several cell lines. In this study, the cytotoxic effects of enniatin B were assessed in relation to cellular energy metabolism, cell proliferation, and the induction of apoptosis in Balb 3T3 and HepG2 cells. The mechanism of toxicity was examined by means of whole genome expression profiling of exposed rat primary hepatocytes. Enniatin B altered cellular energy metabolism and reduced cell proliferation in Balb 3T3 and HepG2 cell lines. Furthermore, the proportion of apoptotic cell populations of Balb 3T3 cells slightly increased. On the other hand, enniatin B caused necrotic cell death in primary hepatocytes. Gene expression studies revealed the alteration of energy metabolism due to effects on mitochondrial organization and function and the assembly of complex I of the electron transport chain.

Genotype-Dependent Brown Adipose Tissue Activation in Patients With Pheochromocytoma and Paraganglioma.

Patients with pheochromocytomas and paragangliomas (PGLs) may have brown adipose tissue (BAT) activation induced by catecholamine excess. (18)F-fluorodeoxyglucose ((18)F-FDG) positron emission tomography (PET)/computed tomography (CT) can be used for the localization of both PGLs and BAT. It is unknown whether BAT is specifically affected by altered cellular energy metabolism in patients with SDHx- and VHL-related PGLs. The objective of the study was to determine endocrine and paracrine effects of catecholamine excess on BAT activation in patients with PGLs as detected by (18)F-FDG PET/CT, taking into account genetic variation. Patients with PGLs who were fully genetically characterized underwent presurgical (18)F-FDG PET/CT imaging for tumor localization and to quantify BAT activation. The study was conducted at a single Dutch tertiary referral center. Seventy-three patients, aged 52.4 ± 15.4 years, with a body mass index of 25.2 ± 4.1 kg/m(2), mean ± SD, were grouped into sporadic, cluster 1 (SDHx, VHL) and cluster 2 (RET, NF1, MAX) mutations. (18)F-FDG mean standard uptake values were assessed in predefined BAT locations, including perirenal fat. Twenty-one of 73 patients (28.8%) exhibited BAT activation. BAT activation was absent in all six patients with nonsecreting PGLs. No difference in (18)F-FDG uptake by perirenal fat on the side of the pheochromocytoma and the contralateral side was observed (mean standard uptake value of 0.80 vs 0.78, respectively, P = .42). The prevalence of BAT activation did not differ between sporadic (28.9%), cluster 1 (40.0%), and cluster 2 patients (15.4%, P= .36). Patients with PGLs exhibit a high prevalence of BAT activation on (18)F-FDG PET/CT. This is likely due to systemic catecholamine excess. BAT activation is not associated with specific germline mutations.

OP0295 Hypoxic Dysregulation of Energy Metabolism in the Inflamed Arthritic Joint

BackgroundHypoxia in the inflamed joint is a powerful trigger of synovial fibroblasts and endothelial cell (EC) activation, proliferation and survival. The metabolic turnover of the inflamed synovium outpaces the dysfunctional...

Serum metabolomics indicate altered cellular energy metabolism in children with cystic fibrosis

Cystic fibrosis (CF) is a multi-system disease affecting multiple organs and cells besides the respiratory system. Metabolomic profiling allows simultaneous detection of biochemicals originating from cells, organs, or exogenous origin that may be valuable for monitoring of disease severity or in diagnosis. We hypothesized that metabolomics using serum from children would differentiate CF from non-CF lung disease subjects and would provide insight into metabolism in CF. Serum collected from children with CF (n = 31) and 31 age and gender matched children with other lung diseases was used for metabolomic profiling by gas- and liquid-chromatography. Relative concentration of metabolites was compared between the groups using partial least square discriminant analyses (PLS-DA) and linear modeling. A clear separation of the two groups was seen in PLS-DA. Linear model found that among the 459 detected metabolites 92 differed between CF and non-CF. These included known biochemicals in lipid metabolism, oxidants, and markers consistent with abnormalities in bile acid processing. Bacterial metabolites were identified and differed between the groups indicating intestinal dysbiosis in CF. As a novel finding several pathways were markedly different in CF, which jointly point towards decreased activity in the β-oxidation of fatty acids. These pathways include low ketone bodies, low medium chain carnitines, elevated di-carboxylic acids and decreased 2-hydroxybutyrate from amino acid metabolism in CF compared to non-CF. Serum metabolomics discriminated CF from non-CF and show altered cellular energy metabolism in CF potentially reflecting mitochondrial dysfunction. Future studies are indicated to examine their relation to the underlying CF defect and their use as biomarkers for disease severity or for cystic fibrosis transmembrane regulator (CFTR) function in an era of CFTR modifying drugs.

Metabolic Oversupply and Mitochondrial Dysfunction as a Cause of Diaphragmatic Failure after Mechanical Ventilation

Mechanical ventilation (MV) unloads the diaphragm and leads to ventilator‐induced diaphragmatic dysfunction (VIDD). The cellular basis of VIDD is poorly understood, but oxidative stress is involved. A known cause of oxidative stress and skeletal muscle impairment is mitochondrial dysfunction, which can result from altered cellular energy metabolism. Therefore, we investigated indices of mitochondrial function and energy metabolism in human diaphragm after MV. Compared to controls, MV diaphragms had abnormal intramyocellular lipid accumulation (+219%, p&lt;0.01), de‐phosphorylation of the cellular metabolic sensor AMPK (−64%, p&lt;0.01), and downregulation of SIRT1 (−50%, p&lt;0.01). Mitochondrial biogenesis and antioxidant gene systems were globally downregulated, as indicated by gene expression profiling. Consequently, whole muscle mitochondrial enzyme activities were decreased by 35–70%. MV diaphragms contained abnormally high levels of mitochondrial cytochrome c oxidase (COX) deficient myofibers that co‐localized with intracellular lipid accumulation, and which contained high levels of clonally‐expanded mtDNA deletions. MV‐induced contractile inactivity results in an overabundance of energy substrate supply relative to demand, a potent metabolic imbalance that triggers mitochondrial ROS production, and may lead to compromised diaphragmatic function.

Increased thalamic N-acetylaspartate in male patients with familial bipolar I disorder

N-Acetylaspartate (NAA) in the anterior and mediodorsal thalamic regions was measured using proton magnetic resonance spectroscopic imaging ( 1H-MRSI) in 15 euthymic male patients with familial bipolar I disorder and compared to values in 15 male control subjects to determine if there was evidence for altered neuronal/axonal integrity. MRI tissue segmentation methods were also utilized to obtain tissue-contribution estimates for each MRSI voxel. Relative to the comparison group, the patients with bipolar I disorder demonstrated significantly higher NAA and creatine in both the right and left thalamus. NAA was also significantly higher in the left thalamus compared to the right in both bipolar I patients and controls. There were no group or lateralized differences in the percentages of different tissue types within the MRSI voxels, suggesting that the thalamic NAA and creatine alterations were not an artifact of variations in tissue type percentages in the MRSI voxels. There was also no significant association between NAA or creatine and illness duration. The findings of increased thalamic NAA bilaterally may represent neuronal hypertrophy or hyperplasia, reduced glial cell density, or abnormal synaptic and dendritic pruning. Increased thalamic creatine bilaterally may represent altered cellular energy metabolism and is consistent with prior studies demonstrating changes in thalamic metabolism in mood disorders.

Mitochondrial Enzyme Deficiency Causing Exercise Limitation in Normal-Appearing Adults

Deficiencies of mitochondrial enzymes cause a number of severe neurologic syndromes in pediatric patients. Isolated myopathy secondary to enzymatic deficiency only rarely has been recognized and reported in adults. Three normal-appearing patients with unexplained dyspnea on exertion, tachycardia, and fatigue were studied with progressive incremental and constant load exercise testing with hemodynamic and gas exchange measurements. Subsequently, muscle biopsies were performed and enzymatic profiles determined. Exercise testing suggested altered cellular energy metabolism. Oxygen transport and gas exchange at rest and with exercise were normal. During exercise, ventilation increased excessively for the work performed, but it was appropriate for carbon dioxide production. Cardiac outputs increased significantly (196 to 305%), heart rates reached maximum levels, and lactic acid levels increased (385 to 833%) during exercise at 25 W. Muscle morphology was normal, but mitochondrial enzyme assays demonstrated one or more deficiencies in all three patients. Mitochondrial enzymatic deficiency can be a cause of unexplained exercise symptomatology and limitation in adults. Our experience would suggest the incidence of the disorder may be greater than that previously recognized or expected. Symptoms of exercise limitation associated with dyspnea, tachycardia, or muscle fatigue should alert the clinician to a possible abnormality in cellular energy metabolism.

Effect of prostaglandins I2 (prostacyclin) and F2 alpha on function, energy metabolism, and calcium uptake in ischaemic/reperfused hearts.

The aim was to examine the effect on cardiac function, energy metabolism, and calcium uptake of either prostaglandin I2 (PGI2, prostacyclin) or prostaglandin F2 alpha (both 28.6 nM) on the response of isolated rat hearts to 25 min of total global ischaemia with or without 30 min reperfusion. Rat hearts were perfused by the Langendorff method and function assessed by left ventricular pressure. Energy metabolites were measured using enzymatic techniques and 45Ca2+ uptake determined by radioisotopic analysis. Although there was no effect of either prostaglandin on contractile depression during ischaemia, both compounds accelerated the onset of and increased the magnitude of ischaemic contracture. High energy phosphate content at the end of the ischaemic period was not affected by prostaglandin treatment; however, tissue lactate levels were increased by PGI2 as was tissue calcium content. Under control conditions mean recovery of left ventricular developed pressure ranged from 66% to 83%. In the presence of PGI2 and PGF2 alpha, recovery of developed pressure was reduced to 20% and 38% of preischaemic values, respectively. The reduced recovery in developed pressure was accompanied by an approximately threefold increase in diastolic pressure (p < 0.05). The depression of functional recovery in reperfused hearts treated with prostaglandins was associated with various disturbances of cellular metabolism including depressed ATP and creatine phosphate content and increased tissue lactate and calcium following 30 min of reperfusion. A significant correlation was found between the changes in developed pressure and diastolic pressure during reperfusion and the reduction in ATP and creatine phosphate repletion. The deficit in recovery of ventricular function also correlated significantly with increased lactate and calcium accumulation in the reperfused heart. Low concentrations of PGI2 and PGF2 alpha can depress contractile recovery of the globally ischaemic heart through a mechanism associated with altered cellular energy metabolism and increased calcium accumulation.