Elevated Oxygen Consumption Rate Research Articles

Toxic effect of fluorene on Perinereis aibuhitensis body wall and its corresponding defense mechanisms: A metabolomics perspective

Fluorene is a coastal sediment pollutant with high ecological risk. Perinereis aibuhitensis is an ecotoxicological model used for polycyclic aromatic hydrocarbon bioremediation; however, the effects of fluorene on the physiological metabolism of P. aibuhitensis and its corresponding responses remain unclear. This study explored the tolerance and defense responses of P. aibuhitensis in sediments with different fluorene concentrations using histology, ecological biomarkers, and metabolic responses. Metabolomics analyses revealed that P. aibuhitensis has high tolerance to fluorene in sediments. Fluorene stress disrupted the normal metabolism of the P. aibuhitensis body wall, resulting in excessive glycosphospholipid and stearamide accumulation and elevated oxygen consumption rates. To mitigate this, P. aibuhitensis has adopted tail cutting, yellowing, and modulation of metabolite contents in the body wall. This study provides novel insights into the potential ecological risk of fluorene pollution in marine sediments and proposes the use of P. aibuhitensis in the bioremediation of fluorene-contaminated sediments.

Dimorphic effect of TFE3 in determining mitochondrial and lysosomal content in muscle following denervation

BackgroundMuscle atrophy is a common consequence of the loss of innervation and is accompanied by mitochondrial dysfunction. Mitophagy is the adaptive process through which damaged mitochondria are removed via the lysosomes, which are regulated in part by the transcription factor TFE3. The role of lysosomes and TFE3 are poorly understood in muscle atrophy, and the effect of biological sex is widely underreported.MethodsWild-type (WT) mice, along with mice lacking TFE3 (KO), a transcriptional regulator of lysosomal and autophagy-related genes, were subjected to unilateral sciatic nerve denervation for up to 7 days, while the contralateral limb was sham-operated and served as an internal control. A subset of animals was treated with colchicine to capture mitophagy flux.ResultsWT females exhibited elevated oxygen consumption rates during active respiratory states compared to males, however this was blunted in the absence of TFE3. Females exhibited higher mitophagy flux rates and greater lysosomal content basally compared to males that was independent of TFE3 expression. Following denervation, female mice exhibited less muscle atrophy compared to male counterparts. Intriguingly, this sex-dependent muscle sparing was lost in the absence of TFE3. Denervation resulted in 45% and 27% losses of mitochondrial content in WT and KO males respectively, however females were completely protected against this decline. Decreases in mitochondrial function were more severe in WT females compared to males following denervation, as ROS emission was 2.4-fold higher. In response to denervation, LC3-II mitophagy flux was reduced by 44% in females, likely contributing to the maintenance of mitochondrial content and elevated ROS emission, however this response was dysregulated in the absence of TFE3. While both males and females exhibited increased lysosomal content following denervation, this response was augmented in females in a TFE3-dependent manner.ConclusionsFemales have higher lysosomal content and mitophagy flux basally compared to males, likely contributing to the improved mitochondrial phenotype. Denervation-induced mitochondrial adaptations were sexually dimorphic, as females preferentially preserve content at the expense of function, while males display a tendency to maintain mitochondrial function. Our data illustrate that TFE3 is vital for the sex-dependent differences in mitochondrial function, and in determining the denervation-induced atrophy phenotype.

Abstract 1603: Intra-pancreatic fat promotes the progression of PDAC by activating thermogenesis

Abstract Background: The presence of minimal intra-pancreatic fat deposition (IPFD) in the healthy human pancreas has been demonstrated in numerous studies. But excess IPFD, or fatty pancreas disease, leads to a wide array of diseases, including but not limited to diabetes, pancreatitis, and pancreatic ductal adenocarcinoma (PDAC). Remarkably, PDAC patients with excessive intra-pancreatic fat accumulation are commonly associated with a poor prognosis compared to those with minimal deposition. However, the interaction between tumor and intra-pancreatic adipose tissue remains elusive. Here, we evaluated the pancreatic fat infiltration in normal people and patients with PDAC respectively, and explored the mechanism of how intra-pancreatic fat affects the progression of PDAC. Methods: Magnetic resonance imaging (MRI) and clinical biopsy specimens were used to assess the degree of pancreatic fat infiltration in PDAC patients and healthy control individuals. The C57BL/6J mice injected orthotopically with KPC cells and adipocytes, high-fat diet (HFD)-induced obesity KC (Pdx1-Cre; KrasLSL-G12D/WT) mice, and KC; ob/ob mice were established to dynamically observe how adipocytes affect tumor progression in vivo. The snRNA-seq of tumor samples and proteomics of conditional medium of PDAC cell lines were performed to identify the mechanism of interaction between cancer cells and adipocytes. Results: We found that patients with PDAC presented excess intra-pancreatic fat deposition more frequently than healthy individuals, and the degree of fat infiltration was positively correlated with the tumor burden of PDAC patients. HFD-induced obesity KC mice and KC; ob/ob mice showed a higher degree of IPFD, which contributed to tumor growth and low survival rates of mice. Additionally, the expression of uncoupling protein 1 was upregulated in PDAC patients and KPC (Pdx1-Cre; KrasLSL-G12D/WT; p53LSL-R172H/WT) mice compared to normal controls. Constantly, the snRNA-seq of PDAC samples demonstrated the increase in thermogenic signature. In vitro, C3H10T1/2 cells treated with conditional medium from pancreatic cancer cell lines or co-cultured with pancreatic cancer cell lines showed increased expression of browning markers and elevated oxygen consumption rate. Specifically, combined analysis of the proteomic of T3M4 and CFPAC-1 conditional medium and the snRNA-seq revealed that pancreatic cancer cells secreted integrin β4 (ITGB4) to improve the thermogenesis of intra-pancreatic adipocytes (IPA), which in turn promoted cancer cell progression. Conclusion: Parts of PDAC patients present excess intra-pancreatic fat accumulation, a process associated with tumor growth and poor prognosis. Mechanistically, pancreatic cancer cells activate the thermogenesis of IPA by secreting ITGB4, and then the thermogenetic adipocytes fuel the progression of the tumor in response. Citation Format: Xinpeng Yin, Yuan Chen, Ruiyuan Xu, Chenglin Hu, Chengcheng Wang, Yupei Zhao. Intra-pancreatic fat promotes the progression of PDAC by activating thermogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1603.

Maternal pre-eclampsia serum increases neurite growth and mitochondrial function through a potential IL-6-dependent mechanism in differentiated SH-SY5Y cells.

Introduction: Pre-eclampsia (PE) is a common and serious hypertensive disorder of pregnancy, which affects 3%-5% of first-time pregnancies and is a leading cause of maternal and neonatal morbidity and mortality. Prenatal exposure to PE is associated with an increased risk of neurodevelopmental disorders in affected offspring, although the cellular and molecular basis of this increased risk is largely unknown. Methods: Here, we examined the effects of exposure to maternal serum from women with PE or a healthy uncomplicated pregnancy on the survival, neurite growth and mitochondrial function of neuronally differentiated human SH-SY5Y neuroblastoma cells, which are commonly used to study neurite growth. Neurite growth and mitochondrial function are two strongly linked neurodevelopmental parameters in which alterations have been implicated in neurodevelopmental disorders. Following this, we investigated the pleiotropic cytokine interleukin-6 (IL-6) levels as a potential mechanism. Results: Cells exposed to 3% (v/v) PE serum for 72h exhibited increased neurite growth (p < 0.05), which was validated in the human neural progenitor cell line, ReNcell® VM (p < 0.01), and mitochondrial respiration (elevated oxygen consumption rate (p < 0.05), basal mitochondrial respiration, proton leak, ATP synthesis, and non-mitochondrial respiration) compared to control serum-treated cells. ELISA analysis showed elevations in maternal IL-6 in PE sera (p < 0.05) and placental explants (p < 0.05). In support of this, SH-SY5Y cells exposed to 3% (v/v) PE serum for 24h had increased phospho-STAT3 levels, which is a key intracellular mediator of IL-6 signalling (p < 0.05). Furthermore, treatment with anti-IL-6 neutralizing antibody blocked the effects of PE serum on neurite growth (p < 0.05), and exposure to IL-6 promoted neurite growth in SH-SY5Y cells (p < 0.01). Discussion: Collectively these data show elevated serum levels of maternal IL-6 in PE, which increases neurite growth and mitochondrial function in SH-SY5Y cells. This rationalizes the further study of IL-6 as a potential mediator between PE exposure and neurodevelopmental outcome in the offspring.

Cellular and metabolic characteristics of pre-leukemic hematopoietic progenitors with GATA2 haploinsufficiency.

Mono-allelic germline disruptions of the transcription factor GATA2 result in a propensity for developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), affecting more than 85% of carriers. How a partial loss of GATA2 functionality enables leukemic transformation years later is unclear. This question has remained unsolved mainly due to the lack of informative models, as Gata2 heterozygote mice do not develop hematologic malignancies. Here we show that two different germline Gata2 mutations (TgErg/Gata2het and TgErg/Gata2L359V) accelerate AML in mice expressing the human hematopoietic stem cell regulator ERG. Analysis of Erg/Gata2het fetal liver and bone marrow-derived hematopoietic cells revealed a distinct pre-leukemic phenotype. This was characterized by enhanced transition from stem to progenitor state, increased proliferation, and a striking mitochondrial phenotype, consisting of highly expressed oxidative-phosphorylation-related gene sets, elevated oxygen consumption rates, and notably, markedly distorted mitochondrial morphology. Importantly, the same mitochondrial gene-expression signature was observed in human AML harboring GATA2 aberrations. Similar to the observations in mice, non-leukemic bone marrows from children with germline GATA2 mutation demonstrated marked mitochondrial abnormalities. Thus, we observed the tumor suppressive effects of GATA2 in two germline Gata2 genetic mouse models. As oncogenic mutations often accumulate with age, GATA2 deficiency-mediated priming of hematopoietic cells for oncogenic transformation may explain the earlier occurrence of MDS/AML in patients with GATA2 germline mutation. The mitochondrial phenotype is a potential therapeutic opportunity for the prevention of leukemic transformation in these patients.

The Hidden Notes of Redox Balance in Neurodegenerative Diseases.

Reactive oxygen species (ROS) are versatile molecules that, even if produced in the background of many biological processes and responses, possess pleiotropic roles categorized in two interactive yet opposite domains. In particular, ROS can either function as signaling molecules that shape physiological cell functions, or act as deleterious end products of unbalanced redox reactions. Indeed, cellular redox status needs to be tightly regulated to ensure proper cellular functioning, and either excessive ROS accumulation or the dysfunction of antioxidant systems can perturb the redox homeostasis, leading to supraphysiological concentrations of ROS and potentially harmful outcomes. Therefore, whether ROS would act as signaling molecules or as detrimental factors strictly relies on a dynamic equilibrium between free radical production and scavenging resources. Of notice, the mammalian brain is particularly vulnerable to ROS-mediated toxicity, because it possesses relatively poor antioxidant defenses to cope with the redox burden imposed by the elevated oxygen consumption rate and metabolic activity. Many features of neurodegenerative diseases can in fact be traced back to causes of oxidative stress, which may influence both the onset and progression of brain demise. This review focuses on the description of the dual roles of ROS as double-edge sword in both physiological and pathological settings, with reference to Alzheimer’s and Parkinson’s diseases.

MiR-652-5p elevated glycolysis level by targeting TIGAR in T-cell acute lymphoblastic leukemia

The effect of glycolysis remains largely elusive in acute T lymphoblastic leukemia (T-ALL). Increasing evidence has indicated that the dysregulation of miRNAs is involved in glycolysis, by targeting the genes coding glycolysis rate-limiting enzymes. In our previous studies, we found that overexpression of the ARRB1-derived miR-223 sponge repressed T-ALL progress and reduced the expression of miR-652-5p. However, little is known about miR-652-5p on T-ALL. Here, we showed that impaired miR-652-5p expression inhibited growth, promoted apoptosis of T-ALL cells in vitro and prolonged overall survival (OS) in vivo. Based on the GO enrichment of miR-652-5p target genes, we uncovered that impaired miR-652-5p decreased glycolysis, including reduced the lactate, pyruvate, ATP level and the total extracellular acidification rate (ECAR), elevated oxygen consumption rate (OCR) in T-ALL cell lines. Mechanically, miR-652-5p targeted the 3ʹUTR of Tigar mRNA and inhibited its expression. Furthermore, the alteration of glycosis level was attributed to Tigar overexpression, consistent with the effect of impaired miR-652-5p. Additionally, Tigar suppressed the expression of PFKFB3, a glycolysis rate-limiting enzyme, in vivo and in vitro. Taken together, our results demonstrate that impaired miR-652-5p/Tigar axis could repress glycolysis, thus to slow growth of T-ALL cells, which support miR-652-5p as a novel potential drug target for T-ALL therapeutics.

The glycolysis inhibitor 2-deoxyglucose ameliorates adjuvant-induced arthritis by regulating macrophage polarization in an AMPK-dependent manner

Macrophages are highly plastic cells critical for the development of rheumatoid arthritis (RA). Macrophages exhibit a high degree of pro-inflammatory plasticity in RA, accompanied by a metabolic reprogramming from oxidative phosphorylation (OXPHOS) to glycolysis. 2-deoxyglucose (2-DG), a glycolysis inhibitor, has previously been shown to exhibit anti-inflammatory and anti-arthritic properties. However, the specific mechanisms of inflammatory modulation by 2-DG remain unclear. This study used 2-DG to treat rats with adjuvant arthritis (AA) and investigated its specific anti-arthritic mechanisms in the murine-derived macrophage cell line RAW264.7 in vitro. 2-DG reduced the arthritis index as well as alleviated cellular infiltration, synovial hyperplasia, and bone erosion in AA rats. Moreover, 2-DG treatment modulated peritoneal macrophage polarization, increasing levels of the arginase1 (Arg1) and decreasing expression of the inducible nitric oxide synthase (iNOS). 2-DG activated AMP-activated protein kinase (AMPK) via phosphorylation and reduced activation of the nuclear factor κB (NF-κB) in peritoneal macrophages of AA rats. In vitro, we verified that 2-DG promoted macrophage transition from M1 to M2-type by upregulating the expression of p-AMPKα and suppressing NF-κB activation in LPS-stimulated RAW264.7 cells. LPS-induced macrophages exhibited a metabolic shift from glycolysis to OXPHOS following 2-DG treatment, as observed by reduced extracellular acidification rate (ECAR), lactate export, glucose consumption, as well as an elevated oxygen consumption rate (OCR) and intracellular ATP concentration. Importantly, changes in polarization and metabolism in response to 2-DG were dampened after AMPKα knockdown. These findings indicate that the anti-arthritic 2-DG effect is mediated by a modulation of macrophage polarization in an AMPK-dependent manner.

Bottom Boundary Layer Oxygen Fluxes During Winter on the Oregon Shelf

AbstractIn this study, lander‐based eddy covariance measurements made 30 cm above a sandy seafloor are combined with ocean‐observing and ship‐based measurements to evaluate magnitudes of oxygen fluxes within the bottom boundary layer (BBL) during winter on the Oregon shelf. The total oxygen fluxes observed at a mid‐shelf station were −12.3 ± 7.6 and −18.6 ± 16.9 mmol m−2d−1 during February 2018 and January 2019 deployments, respectively, and −51.8 ± 34.9 mmol m−2d−1 during a deployment on the inner shelf in January 2019. These mean (±SD) flux derivations are greater than fluxes determined previously at nearly the same locations during the summer upwelling season, which points to the activation of benthic respiration by winter wave‐dominated BBL dynamics. To determine the direct flux contributions (or potential bias) at wave frequencies, a phase‐based method of spectral decomposition was applied to separate wave‐induced and turbulence‐induced components of velocity and dissolved oxygen time‐series. This approach identified 25.4, 18.6, and 23.3% of the average total EC oxygen fluxes from the three deployments as carried by waves or possibly turbulence stretched by waves. Additionally, we observed evidence of large waves regularly resuspending sediments and transporting entrained particles past the EC sensors. We apply this evidence to infer that wave frequencies must also contribute significantly to the real spectral domain of benthic oxygen fluxes and that the total fluxes of this study reflect elevated oxygen consumption rates without artifacts. This assessment may not be true for all data sets and may be dependent on wave properties and the quality and treatment of the measurements.

A developed serum-free medium and an optimized chemical cocktail for direct conversion of human dermal fibroblasts into brown adipocytes

Brown adipocytes coordinate systemic energy metabolism associated with the pathogenesis of obesity and related metabolic diseases including type 2 diabetes. We have previously reported chemical compound-induced brown adipocytes (ciBAs) converted from human dermal fibroblasts without using transgenes. In this study, to reveal a precise molecular mechanism underlying the direct conversion and human adipocyte browning, we developed serum-free brown adipogenic medium (SFBAM) with an optimized chemical cocktail consisting of Rosiglitazone, Forskolin, and BMP7. During the direct conversion, treatment with BMP7 enhanced Ucp1 expression rather than the conversion efficiency in the absence of BMP signalling inhibitors. Moreover, treatment with a TGF-β signalling pathway inhibitor was no longer required in the serum-free medium, likely because the TGF-β pathway was already suppressed. SFBAM and the chemical cocktail efficiently converted human dermal fibroblasts into ciBAs within four weeks. The ciBAs exhibited increased mitochondrial levels, elevated oxygen consumption rate, and a response to β-adrenergic receptor agonists. Thus the ciBAs converted by the serum-free medium and the chemical cocktail provide a novel model of human brown (beige) adipocytes applicable for basic research, drug screening, and clinical applications.

OR12-2 Obesity Induces Changes in T Cell Metabolism and Function

Low-grade inflammation is responsible for many obesity-associated pathologies such as insulin resistance, type 2 diabetes and reduction in protective immunity. T cells play an important role in mounting an effective immune response and the modulation of inflammation, and have been identified as a key player in obesity-associated inflammation. Altered T cell metabolism can influence T cell differentiation and function; however, it is not yet known if or how obesity influences the T cell metabolic state. We hypothesized that diet induced obesity promotes changes in T cell metabolism leading to increased pro-inflammatory T cells function. To test our hypothesis, we assessed the effect of diet-induced obesity (DIO) on T cell subpopulation number and metabolism in the visceral adipose tissue (VAT) and peripheral lymphoid tissue (spleen). To achieve that, C57BL/6 mice were placed on high fat (DIO mice) or regular chow diet (control mice) at weaning and remained on diet for 18 weeks. VAT and spleen were collected and T cells were isolated and analyzed by flow cytometry for: T cells subpopulation markers, glucose uptake, and cytokine production. In addition, the expression of almost 240 enzymes involved in several metabolic pathways were measured in CD4+ T cells by RT-qPCR. We found that proportions of Treg cells were decreased in the VAT of DIO mice while CD8+ T cells increased, indicating an inflammatory environment in the adipose tissue of the DIO mice. CD4+ T cells isolated from the spleen of DIO mice were also found to be metabolically altered with increased glucose uptake and elevated oxygen consumption rate (OCR; a marker of mitochondrial metabolism). The spare respiratory capacity, an indicator of mitochondrial reserve, of CD4+ T cells from DIO mice was found to be reduced by the fatty acids oxidation inhibitor Etomoxir, while the pyruvate transport inhibitor UK5099 was found to have no significant effect, indicating a preference toward the use of fatty acids as a mitochondrial fuel source. The metabolic qPCR panel showed slight differences in expression between control vs. DIO mice with a tendency toward a decrease in the expression of enzymes involved in oxidative phosphorylation and fatty acid transport along with an increase in enzymes that positively influence glycolysis in DIO mice. Lastly, the antidiabetic drug metformin was found to inhibit OCR and alter the expression of several activation markers in CD4+ cells in vitro and in vivo, indicating that metformin may be an effective candidate to reverse T cell metabolic dysfunction and thereby restore T cell dysfunction in obesity. Research Support: National Institutes of Health grant R01-DK106090 and Derfner Foundation Research Award 2018.

Pseudomonas aeruginosa accentuates inflammatory responses under HIV infection through TREM‐1 activation

ObjectiveHuman immunodeficiency virus (HIV) threatens human health worldwide. Anti‐retroviral therapy (ART) extends survival but does not eliminate HIV from its cellular reservoirs. Patients accepting ART show altered immunity and immune activation, and continue to present increased risk of bacteria associated pneumonia, which is a major cause of morbidity and mortality. As an important opportunistic human pathogen, Pseudomonas aeruginosa causes a wide variety of severe nosocomial infections. Triggering receptor expressed on myeloid cells‐1 (TREM‐1) is an amplifier of inflammation, and has been recognized as a critical immunomodulator in inflammatory diseases of both infectious and non‐infectious etiologies. However, molecular mechanisms underlying the functional role of TREM‐1 in lung infections of HIV patients remains to be defined.MethodsWild type C57 BL/6J and HIV transgenic mice were treated with P. aeruginosa strain PAO1 through intranasal administration, mouse lung injury status was observed through Hematoxylin and Eosin (H&amp;E) staining. Total RNA of lung tissues was isolated, real‐time RT‐PCR and western immunoblots were performed to quantify gene and protein expression. THP‐1 derived macrophages and human monocyte‐derived macrophages were treated with recombinant HIV viral proteins Tat and gp120, expression of miR‐155 and SOCS1 were quantified, and oxidative phosphorylation (OXPHOS) was measured through mitostress assay. Co‐culture model was used to analyze intracellular communications between macrophage and lung epithelial cells, and exosomes secreted by macrophages were isolated and cocultured with recipient cells, then expression of key tight junction proteins involved in epithelial barrier function were analyzed.ResultsCompared to wild type control, PAO1 accentuates acute neutrophilic inflammation in HIV transgenic mice with increase in neutrophil influx, expression levels of TREM‐1 and proinflammatory cytokines, particularly IL‐1β, TNF‐α and IL‐6. TREM‐1 silencing in macrophages exposed to HIV‐related proteins Tat or gp120, resulted in significant attenuation of miR‐155 expression with increased SOCS1 expression, and reduced mitochondrial reactive oxygen species (mtROS) and elevated oxygen consumption rate (OCR). Furthermore, TREM‐1 silencing in Tat‐treated macrophages reversed the disruption of tight junction barrier integrity in co‐cultured lung epithelial cells through modulation of macrophage‐derived exosome shuttling.ConclusionTREM‐1 escalates neutrophilic lung inflammation in HIV transgenic mice treated with PAO1, results in mitochondrial dysfunction of macrophages and disrupts tight junction barrier integrity of epithelia cells in lung microenvironment. These data suggest that TREM‐1 inhibition might be an important immunomodulatory therapeutic strategy for HIV‐infected patients with lung infections.Support or Funding InformationThis work is supported by VA Merit Review 2I01BX001786‐06A1 (Ruxana T Sadikot) and R01 HL125042(David M Guidot).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Deletion of Neuronal GLT-1 in Mice Reveals Its Role in Synaptic Glutamate Homeostasis and Mitochondrial Function.

The glutamate transporter GLT-1 is highly expressed in astrocytes but also in neurons, primarily in axon terminals. We generated a conditional neuronal GLT-1 KO using synapsin 1-Cre (synGLT-1 KO) to elucidate the metabolic functions of GLT-1 expressed in neurons, here focusing on the cerebral cortex. Both synaptosomal uptake studies and electron microscopic immunocytochemistry demonstrated knockdown of GLT-1 in the cerebral cortex in the synGLT-1 KO mice. Aspartate content was significantly reduced in cerebral cortical extracts as well as synaptosomes from cerebral cortex of synGLT-1 KO compared with control littermates. 13C-Labeling of tricarboxylic acid cycle intermediates originating from metabolism of [U-13C]-glutamate was significantly reduced in synGLT-1 KO synaptosomes. The decreased aspartate content was due to diminished entry of glutamate into the tricarboxylic acid cycle. Pyruvate recycling, a pathway necessary for full glutamate oxidation, was also decreased. ATP production was significantly increased, despite unaltered oxygen consumption, in isolated mitochondria from the synGLT-1 KO. The density of mitochondria in axon terminals and perisynaptic astrocytes was increased in the synGLT-1 KO. Intramitochondrial cristae density of synGLT-1 KO mice was increased, suggesting increased mitochondrial efficiency, perhaps in compensation for reduced access to glutamate. SynGLT-1 KO synaptosomes exhibited an elevated oxygen consumption rate when stimulated with veratridine, despite a lower baseline oxygen consumption rate in the presence of glucose. GLT-1 expressed in neurons appears to be required to provide glutamate to synaptic mitochondria and is linked to neuronal energy metabolism and mitochondrial function.SIGNIFICANCE STATEMENT All synaptic transmitters need to be cleared from the extracellular space after release, and transporters are used to clear glutamate released from excitatory synapses. GLT-1 is the major glutamate transporter, and most GLT-1 is expressed in astrocytes. Only 5%-10% is expressed in neurons, primarily in axon terminals. The function of GLT-1 in axon terminals remains unknown. Here, we used a conditional KO approach to investigate the significance of the expression of GLT-1 in neurons. We found multiple abnormalities of mitochondrial function, suggesting impairment of glutamate utilization by synaptic mitochondria in the neuronal GLT-1 KO. These data suggest that GLT-1 expressed in axon terminals may be important in maintaining energy metabolism and biosynthetic activities mediated by presynaptic mitochondria.

PS02.174: THE ACTION OF A NOVEL RADIOSENSITISER WITHIN THE OESOPHAGEAL ADENOCARCINOMA TUMOUR MICROENVIROMENT

Abstract Background Oesophageal Cancer (OAC) is an aggressive disease with survival rates of ∼15-20%. Current therapeutic regimes focus on neo-adjuvant therapy (chemo-radiation) prior to surgery. Unfortunately, only 20–30% of patients show a beneficial response, with 70–80% of patients as non-responders. This major clinical challenge of treatment resistance reinforces the need for the identification of novel treatments which can act as radio-sensitisers in the neo-adjuvant setting. Methods Through a drug screening approach in-vivo, we have identified a novel anti-angiogenic and anti-metabolic compound, 11B_CC8 with radiosensitising activity. The ability of 11B_CC8 to act as an anti-metabolic agent under hypoxia was evaluated using the XFe24 Seahorse analyser and the Don Whitley i2 workstation. The ability of 11B_CC8 to radiosensitise our isogenic OAC cells under hypoxic conditions was assessed by clonogenic assay. The effect of 11B_CC8 on inflammatory, metabolic and angiogenic protein secretions from OAC treatment naïve tumour conditioned media (TCM) was evaluated by multiplex ELISA. Fresh treatment naïve patient biopsies were screened for their metabolic activity using the XFe24 seahorse analyser at baseline and post- treatment with 11B_CC8. The elucidation of the possible mechanism of action of 11B_CC8 was evaluated by Mass-Spectrometry. Results Our novel anti-angiogenic and anti-metabolic agent can enhance radiosensitivity in our isogenic model of OAC under both normoxic and hypoxic (0.5% O 2) conditions. 11B_CC8 significantly reduces oxygen consumption rate (OCR) under normoxic but not hypoxic conditions. Ex-vivo, 11B_CC8 significantly reduced the secretion of IL1β (P = 0.0117). Real-time ex-vivo metabolic rate analysis of our treatment naïve OAC biopsies showed significantly elevated OCR, when compared to Extracellular Acidification rate, a measure of glycolysis (P = 0.0059). Treatment with 11B_CC8 produced a reduction in OCR (P = 0.0039). Conclusion Our novel anti-angiogenic and anti-metabolic agent can enhance radiosensitivity in-vitro under both normoxic and hypoxic conditions. Ex-vivo, treatment naïve OAC human patient samples, 11B_CC8 can significantly reduce the secretion of IL1β and altered metabolic programming, specifically oxidative phosphorylation in human explants. Disclosure All authors have declared no conflicts of interest.

Regulation of neural stem cell fate decisions by mitochondrial dynamics.

Regulation of neural stem cell fate decisions by mitochondrial dynamics.

Perillyl alcohol, a pleiotropic natural compound suitable for brain tumor therapy, targets free radicals.

Monoterpenes such as limonene and perillyl alcohol (POH) are promising natural compounds with pro-oxidant properties partly due to endoplasmic reticulum (ER) stress-induced cytotoxicity, and antioxidant activity owing to their activity as free radical scavengers, inhibition of coenzyme Q synthesis, activation of antioxidant-responsive elements (inducing detoxification enzymes) and induction of apoptosis. Activation of ER-stress responses generates reactive oxygen species (ROS), which are highly reactive free radicals mainly produced during mitochondrial electron transfer for adenosine triphosphate (ATP) synthesis. When cells are subjected to oxidative stress conditions, there is an accumulation of ROS that can lead to irreversible cell injury caused primarily by lipid peroxidation, protein aggregation and/or DNA damage. Malignant tumors, such as glioblastoma multiforme, display elevated rates of oxygen consumption, necrosis and abnormal structural microvasculature. Alterations in the tumor microenvironment are tightly linked to tumor progression and occur as a result of activation of complex signaling networks involving inter-clonal cooperation, cell-matrix interactions and an ongoing inflammatory response leading to genetic and epigenetic alterations. This review will focus on the pro- and anti-oxidant activities of POH, which are greatly dependent on the respective ROS levels within the tumor microenvironment and involve the ER stress response system. As well, some critical aspects of tumor-associated metabolic changes and the consequences of endogenous ROS production for tumor progression will be discussed.

Prostaglandin E2 Alters the Differentiation and Function of Antigen-Specific T Cells By Targeting the Metabolic Gene Regulatory Network Downstream of mTORC1

Umbilical cord blood transplantation (UCBT) has extended the availability of hematopoietic stem cell transplantation to patients without compatible adult donors. Studies in zebrafish and mouse models have shown that the prostaglandin compound, 16,16 dimethyl prostaglandin E2 (PGE2), increases HSC number and homing. In a human clinical trial of double UCBT, PGE2 decreased time to engraftment, promoted early T cell chimerism, favored generation of long-lived memory CD8+ cells and reduced the incidence of CMV viremia. To obtain mechanistic insight of how PGE2 affects the differentiation program of antigen-specific T cells, we used a well-established model of T effector (TEFF) vs. T memory (TM) differentiation of TCR-transgenic OTI T cells. OTI cells were stimulated with Ova257-264 plus APC with or without PGE2, followed by incubation with either IL-2 or IL-7, which are critical for the development of CD8+ TEFF and TM, respectively. Antigen-specific stimulation with or without PGE2 followed by IL-2 resulted in differentiation to CD44+CD62L- TEFF cells. In contrast, antigen-specific stimulation followed by IL-7 resulted in generation of CD44+CD62L+ central memory T cells. Under these conditions, PGE2 treatment gave rise to a phenotype of CD44-CD62L+Bcl2+Sca1+ cells, consistent with T stem cell memory. Two key pathways regulated by AMPK and mTOR have a decisive role on TM differentiation. AMPK can promote the generation of TM cells. However, we found that PGE2 inhibited AMPK activation indicating that differentiation of TM by PGE2 is not mediated by AMPK. Because the mTORC1-specific inhibitor rapamycin can promote the generation of TM, we focused our studies on the effects of PGE2 on mTORC1. Two classes of direct downstream targets of mTORC1 have been well characterized. mTOR phosphorylates the ribosomal protein S6 kinases (S6K1/2) and the eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BP1/2), both of which control specific steps in the initiation of cap-dependent translation. mTORC1 activation can stimulate glycolysis as well as lipid biosynthesis. This is achieved through the activation of a transcriptional program affecting metabolic gene targets of hypoxia inducible factor 1a (HIF1a) and sterol regulatory element-binding protein (SREBP1/2). HIF1a is regulated downstream of 4E-BP whereas SREBP is regulated downstream of S6K1. Although the mTORC1 inhibitor rapamycin can promote differentiation of TM and Treg cells, recent studies revealed that mTORC1, via its effects on lipid metabolism has a mandatory role on Treg differentiation. We found that PGE2 treatment during antigen-specific stimulation of OTI cells with Ova257-264 resulted in activation of mTOR as determined by phosphorylation of mTOR, 4E-BP and S6K and phosphorylation of Akt on the mTOR-specific site. Surprisingly, when antigen-specific stimulation was followed by IL-7, PGE2 treatment inhibited expression of the 4E-BP downstream targets Myc, HIF1a, the glycolysis genes Glut1, HK2, LDH-A, and the uptake of glucose. Phosphorylation of S6K was also impaired and lipid biosynthesis was suppressed as determined by decreased expression of fatty acid synthase FASN. In contrast, a metabolic program of lipid utilization was activated, characterized by increase of CPT1a, which promotes fatty acid transport in the mitochondria for b-oxidation, and the lipid oxidase Acox1. Mitochondria biogenesis analyzed by Mitotracker staining and expression of mitochondrial genes MTOC-1, TIMM50 and COX5a were also enhanced. In bioenergetics studies control-treated antigen-specific T cells had a glycolytic phenotype with elevated extracellular acidification rate (ECAR) whereas PGE2-treated cells had elevated oxygen consumption rate (OCR) and increased OCR/ECAR ratio, indicating preferential use of oxidative phosphorylation to generate energy. Thus, mTORC1 might regulate differentiation of antigen-specific T cells to TM by promoting lipid biosynthesis upon engaging distinct downstream targets in response to extracellular cues, thereby providing the required fuel for the bioenergetic demands of TM cells. Our studies reveal an unexpected mechanism by which PGE2 regulates the functional fate of T cells by modifying mTORC1 downstream signals and altering T cell metabolic imprints. These findings have implications for harnessing immune memory in the context of tumor-specific and pathogen-specific immunity. DisclosuresNo relevant conflicts of interest to declare.

TAPP adaptors control Akt-dependent metabolic activation of germinal center B cells and suppress autoimmunity via binding to the SHIP product PI(3,4)P2

Abstract The phosphoinositide phosphatase SHIP regulates B cell activation by converting the PI 3-kinase product PI(3,4,5)P3 to PI(3,4)P2. While this activity of SHIP appears critical for generation of effective adaptive immunity while avoiding autoimmunity, the function of PI(3,4)P2 in B cells remains relatively unexplored. Tandem PH domain containing proteins (TAPPs) are adaptor proteins that specifically bind to PI(3,4)P2 via their C-terminal PH domains, targeting them to the plasma membrane. Mice bearing inactivating mutations in the PH domains of both TAPP1 and TAPP2, uncoupling them from PI(3,4)P2, exhibit hypergammaglobulinemia and develop lupus-like characteristics including anti-DNA antibodies and deposition of immune complexes in kidneys. Here we find that TAPP KI mice develop chronic germinal centres (GCs) and age-associated increases in B cell expression of activation, exhaustion and memory markers, all of which are B cell intrinsic and dependent on the co-stimulatory molecule ICOS. TAPP KI B cells exhibit elevated phosphorylation of Akt and its target GSK3, and increased survival. Strikingly, TAPP KI B cells exhibit increased metabolic activity associated with elevated oxygen consumption rate, increased extracellular acidification rate, increased expression of glucose transporter Glut1 and increased glucose transport rate. Together our findings suggest that TAPP-PI(3,4)P2 interaction is important for regulating signalling via Akt, B cell metabolism and development of autoimmunity.

Elevated oxygen consumption rate in response to acute low-glucose stress: Metformin restores rate to normal level

Cardiovascular disease (CVD) continues to be the leading cause of mortality among all age demographics in the United States, with the highest occurrence in populations aged 65 and older.Glucose levels, particularly hyperglycemia, are associated with the premature onset of age-related diseases including CVD. A major challenge in the treatment of elderly patients with chronically elevated blood glucose is the frequency of hypoglycemic episodes. Molecular mechanisms of hypoglycemia remain unclear, but are associated with premature onset of age-related-diseases. Here we report a mitochondrial metabolic profile assessing short-term (up to six hours) and longer-term (12–24h) durations of low-glucose stress. We observed that the anti-diabetic biguanide and mitochondrial complex I inhibitor, metformin, can lower and restore the elevated oxygen consumption rate during shorter-term glucose stress to levels similar to that of cells cultured in normal glucose. This effect appears, in part, to involve activation of the 5′ AMP-activated protein kinase (AMPK).

Biological oxygen demand optode analysis of coral reef-associated microbial communities exposed to algal exudates

Algae-derived dissolved organic matter has been hypothesized to induce mortality of reef building corals. One proposed killing mechanism is a zone of hypoxia created by rapidly growing microbes. To investigate this hypothesis, biological oxygen demand (BOD) optodes were used to quantify the change in oxygen concentrations of microbial communities following exposure to exudates generated by turf algae and crustose coralline algae (CCA). BOD optodes were embedded with microbial communities cultured from Montastraea annularis and Mussismilia hispida, and respiration was measured during exposure to turf and CCA exudates. The oxygen concentrations along the optodes were visualized with a low-cost Submersible Oxygen Optode Recorder (SOOpR) system. With this system we observed that exposure to exudates derived from turf algae stimulated higher oxygen drawdown by the coral-associated bacteria than CCA exudates or seawater controls. Furthermore, in both turf and CCA exudate treatments, all microbial communities (coral-, algae-associated and pelagic) contributed significantly to the observed oxygen drawdown. This suggests that the driving factor for elevated oxygen consumption rates is the source of exudates rather than the initially introduced microbial community. Our results demonstrate that exudates from turf algae may contribute to hypoxia-induced coral stress in two different coral genera as a result of increased biological oxygen demand of the local microbial community. Additionally, the SOOpR system developed here can be applied to measure the BOD of any culturable microbe or microbial community.