Reduction Of Intracellular ATP Research Articles

Energy stress promotes P-bodies formation via lysine-63-linked polyubiquitination of HAX1.

Energy stress, characterized by the reduction of intracellular ATP, has been implicated in various diseases, including cancer. Here, we show that energy stress promotes the formation of P-bodies in a ubiquitin-dependent manner. Upon ATP depletion, the E3 ubiquitin ligase TRIM23 catalyzes lysine-63 (K63)-linked polyubiquitination of HCLS1-associated protein X-1 (HAX1). HAX1 ubiquitination triggers its liquid‒liquid phase separation (LLPS) and contributes to P-bodies assembly induced by energy stress. Ubiquitinated HAX1 also interacts with the essential P-body proteins, DDX6 and LSM14A, promoting their condensation. Moreover, we find that this TRIM23/HAX1 pathway is critical for the inhibition of global protein synthesis under energy stress conditions. Furthermore, high HAX1 ubiquitination, and increased cytoplasmic localization of TRIM23 along with elevated HAX1 levels, promotes colorectal cancer (CRC)-cell proliferation and correlates with poor prognosis in CRC patients. Our data not only elucidate a ubiquitination-dependent LLPS mechanism in RNP granules induced by energy stress but also propose a promising target for CRC therapy.

3-Epi-betulinic acid 3-O-β-D-glucopyranoside (eBAG) induces autophagy by activation of AMP-activated protein kinase in hepatocellular carcinoma

3-Epi-betulinic acid 3-O-β-D-glucopyranoside (eBAG) is a pentacyclic triterpene mainly distributed in food and medicinal plants, which exhibits various pharmacological properties. However, whether these functions are attributed to eBAG or additional components in these plants remain unknown. Herein, we report that eBAG exerted an inhibitory activity against hepatocellular carcinoma and esophageal cancer cells. EBAG induced non-apoptotic cell death in hepatocellular carcinoma cells. The eBAG-induced cell death was inhibited by knock-down of autophagy related gene (ATG) 5 and ATG7, by administration of 3-methyladenine, a selective autophagy inhibitor that suppresses phosphoinositide 3-kinase (PI3K), and by chloroquine, a classic autophagy flux inhibitor. We demonstrated that eBAG induced an autophagy-mediated cell death. Application of eBAG mimicked cellular bioenergetics depletion leading to the reduction of intracellular ATP, activation of AMP-activated protein kinase (AMPK), and inhibition of mTOR. Co-treatment with compound C, an AMPK inhibitor, abrogated cell death induced by eBAG. We further validated the anti-tumor effect of eBAG in the murine xenograft model of hepatocellular carcinoma and found that eBAG treatment promoted the induction of autophagy and reduction of tumor growth in mice. As a functional food ingredient, eBAG is a potential therapeutic agent for the treatment of hepatocellular carcinoma and esophageal cancer.

Mechanistic insights into severe pulmonary inflammation caused by silica stimulation: The role of macrophage pyroptosis

Respirable silica dust is a common hazard faced by occupational workers and prolonged exposure to this dust can lead to pulmonary inflammation, fibrosis and, in severe cases, silicosis. However, the underlying mechanism by which silica exposure causes these physical disorders is not yet understood. In this study, we aimed to shed light on this mechanism by establishing in vitro and in vivo silica exposure models from the perspective of macrophages. Our results showed that compared to the control group, silica exposure resulted in an upregulation of the pulmonary expression of P2X7 and Pannexin-1, but this effect was suppressed by treatment with MCC950, a specific inhibitor of NLRP3. Our in vitro studies showed that silica exposure induced mitochondrial depolarization in macrophages, which led to a reduction of intracellular ATP and an influx of Ca2+. Furthermore, we found that creating an extracellular high potassium environment by adding KCl to the macrophage medium inhibited the expression of pyroptotic biomarkers and pro-inflammatory cytokines such as NLRP3 and IL-1β. Treatment with BBG, a P2X7 antagonist, also effectively inhibited the expression of P2X7, NLRP3, and IL-1β. On the other hand, treatment with FCF, a Pannexin-1 inhibitor, suppressed the expression of Pannexin-1 but had no effect on the expression of pyroptotic biomarkers such as P2X7, NLRP3, and IL-1β. In conclusion, our findings suggest that silica exposure triggers the opening of P2X7 ion channels, resulting in intracellular K+ efflux, extracellular Ca2+ influx, and the assembly of the NLRP3 inflammasome, ultimately leading to macrophage pyroptosis and pulmonary inflammation.

Antibacterial activity and mechanism of slightly acidic electrolyzed water (SAEW) combined with ultraviolet light against Staphylococcus aureus

This study aimed to investigate the antibacterial effect and mechanism of Staphylococcus aureus (S. aureus) by combined treatment with slightly acidic electrolyzed water (SAEW) and ultraviolet light (UV, 253.7 nm) (SAEW + UV). The result shows that about 6.70 logs CFU/mL of S. aureus was inactivated after the SAEW + UV treatment (available chlorine concentration, ACC, 70 mg/L) for 90 s. The SAEW + UV treatment has a higher antimicrobial effect than UV or SAEW alone treatment, and the effect increases with time and ACC (P < 0.05). After treatment with SAEW + UV at ACC of 70 mg/L for 90 s, S. aureus showed a decrease in ATP and an accumulation of ROS, and an increase in the conductivity of its cell membrane, which indicates that the membrane was also damaged, but no evidence of nucleic acid and protein leakage was seen. The results showed that SAEW + UV might damage bacterial cell membranes, leading to the accumulation of intracellular ROS and the reduction of intracellular ATP, which leads to bacterial death. Moreover, the specific mechanisms of ROS production and ATP reduction of SAEW + UV on S. aureus need to be further investigated.

A FAM46C-Dependent Molecular Circuit Modulates Antibody Synthesis and Proliferation in Plasma Cell Dyscrasias

Plasma cells (PCs) are the terminal effectors of B lymphocyte differentiation in charge of antibody production. Such intensive task requires rapid endoplasmic reticulum (ER) expansion and the concerted induction of the machinery affording ER import, folding, and trafficking along the secretory route. Moreover, the by-products of massive immunoglobulin (Ig) production cause a peculiar dependence of PCs on protein degradative pathways with a crucial clinical consequence: theextraordinary success of proteasome inhibitors against multiple myeloma (MM) and systemic light chain amyloidosis (AL). Notably, an essential player in the proteomic reshaping occurring during PC differentiation is the non-canonical poly(A)polymerase FAM46C/TENT5C. Upon B cell activation, FAM46C is induced to polyadenylate and stabilize mRNAs encoding Igs and ER-targeted proteins, thus promoting humoral immune responses (Mroczek et al., 2017; Bilska et al., 2020). Interestingly, FAM46C locus on chromosome 1p12 is mutated or deleted in up to 20% MM patients (Chapman et al., 2011; Boyd et al., 2011, Barbieri et al., 2016), suggesting that MM cells are subject to a selective pressure to restrict FAM46C expression and activity, to ultimately curb Ig synthesis and secretion in favor of cell survival and tumor growth. Accordingly, exogenous re-expression of FAM46C in mutated MM cells raised the secretory capacity beyond sustainability, resulting in intracellular ATP reduction, ROS accumulation and increased apoptotic susceptibility (Fucci et al., 2020). Our research aims to discover the precise molecular circuits, centered on FAM46C, regulating PC secretory capacity in efficient coordination with degradative pathways, to discover unprecedented vulnerabilities of PCs and new therapeutic opportunities against PC dyscrasias. To clarify the molecular mechanisms underlying the effects of FAM46C on Ig secretion we combined cutting-edge protein biochemistry, imaging and unbiased proteomic assays and adopted in vitro and in vivo models of MM and AL. In these models, we disclosed that FAM46C concertedly promotes the expression of ER and Golgi proteins, potently expanding the secretory apparatus and light chain (LC) secretion. To assess the impact of FAM46C on PC proliferation and disease progression in vivo, we silenced or overexpressed FAM46C in AMLC-2 cells that bear a hemizygous loss in 1p12 region (Arendt et al., 2008). Following intravenous injection into Rag2-/- IL2rg-/- mice, we found remarkably reduced tumor growth and longer survival in recipients of FAM46C-overexpressing cells, despite higher initial circulating LC levels, as compared to other groups. Moreover, we observed intermediate disease progression in mice receiving mock-transduced ALMC-2 cells, attesting to dose-dependent impact of FAM46C. The in vivo association of higher secretory activity with drastically decreased PC proliferation reveals that FAM46C uncouples monoclonal component levels from disease burden. Finally, among FAM46C-modulated proteins, we found SRP components and RNA methyltransferases involved in ribosome assembly and tRNA processing. The data unveil a new role played by FAM46C in PCs, in addition to its mRNA stabilizing activity, indicating the existence of a previously unrecognized integrated network coordinating ribosome biogenesis, protein translation, and ER expansion, potentially crucial to harmonize intensive protein secretion with cellular homeostasis. Altogether, our studies disclose an unexpected molecular circuit orchestrating Ig synthesis and secretion, and regulating PC proliferation and survival that may be exploited to design new therapeutic strategies aimed to reduce pathogenic LC production, to eliminate MM and AL cells, and to improve the efficacy of current therapeutic regimens.

HIF-Dependent CKB Expression Promotes Breast Cancer Metastasis, Whereas Cyclocreatine Therapy Impairs Cellular Invasion and Improves Chemotherapy Efficacy.

Simple SummaryTargeting dysregulated cellular metabolism is a promising avenue to treat metastatic disease. The aim of our study was to identify genes downstream of the hypoxia-inducible factor (HIF)-1 transcription factor that are amenable to therapeutic intervention to treat metastatic breast cancer (MBC). We identified creatine kinase, brain isoform (CKB) as an HIF-dependent gene that strongly promotes invasion and metastasis in estrogen-receptor (ER)-negative breast cancer models. Deletion of Ckb also repressed glycolysis and mitochondrial respiration, leading to a reduction in intracellular ATP. Either the deletion of Ckb or inhibition of creatine kinase (CK) activity using the creatine analog cyclocreatine (cCr) repressed cell invasion, the formation of invadopodia and lung metastasis. In addition, when paired with paclitaxel or doxorubicin, cCr enhanced growth inhibition in an additive or synergistic manner. cCr may be an effective anti-metastatic agent in ER-negative, HIF-1α-positive breast cancers, targeting both cellular metabolism and motility, particularly when paired with conventional cytotoxic agents.The oxygen-responsive hypoxia inducible factor (HIF)-1 promotes several steps of the metastatic cascade. A hypoxic gene signature is enriched in triple-negative breast cancers (TNBCs) and is correlated with poor patient survival. Inhibiting the HIF transcription factors with small molecules is challenging; therefore, we sought to identify genes downstream of HIF-1 that could be targeted to block invasion and metastasis. Creatine kinase brain isoform (CKB) was identified as a highly differentially expressed gene in a screen of HIF-1 wild type and knockout mammary tumor cells derived from a transgenic model of metastatic breast cancer. CKB is a cytosolic enzyme that reversibly catalyzes the phosphorylation of creatine, generating phosphocreatine (PCr) in the forward reaction, and regenerating ATP in the reverse reaction. Creatine kinase activity is inhibited by the creatine analog cyclocreatine (cCr). Loss- and gain-of-function genetic approaches were used in combination with cCr therapy to define the contribution of CKB expression or creatine kinase activity to cell proliferation, migration, invasion, and metastasis in ER-negative breast cancers. CKB was necessary for cell invasion in vitro and strongly promoted tumor growth and lung metastasis in vivo. Similarly, cyclocreatine therapy repressed cell migration, cell invasion, the formation of invadopodia and lung metastasis. Moreover, in common TNBC cell line models, the addition of cCr to conventional cytotoxic chemotherapy agents was either additive or synergistic to repress tumor cell growth.

Reversing Multidrug Resistance by Inducing Mitochondrial Dysfunction for Enhanced Chemo-Photodynamic Therapy in Tumor.

Efficiency of standard chemotherapy is dramatically hindered by intrinsic multidrug resistance (MDR). Recently, to amplify therapeutic efficacy, photodynamic therapy (PDT)-induced mitochondrial dysfunction by decorating targeting moieties on nanocarriers has obtained considerable attention. Nevertheless, low targeting efficiency, complex synthesis routes, and difficulty in releasing contents become the major obstacles in further clinical application. Herein, an ingenious liposomal-based nanomedicine (L@BP) was fabricated by encapsulating a mitochondria-anchored photosensitizer (Cy-Br) and paclitaxel (PTX) for realizing enhanced cooperation therapy. At the cellular level, L@BP could hurdle endosomal traps to localize and implement PDT in mitochondria. Intriguingly, the PDT-induced in situ mitochondrial dysfunction led to intracellular ATP reduction, which triggered the downregulated P-glycoprotein transportation capacity and thus resulted in diminishing the efflux of chemotherapeutic agents and increasing drug uptake by drug-resistant cells. The prepared nanomedicine eminently accumulated in the tumor site and acquired enhanced therapeutic efficiency on PTX-resistant lung cancer cells, which possessed great potential in circumventing MDR tumors.

Caspase-11 promotes NLRP3 inflammasome activation via the cleavage of pannexin1 in acute kidney disease.

Ischemia/reperfusion (I/R) injury is a major cause of acute kidney injury (AKI) in clinic. The activation of NLRP3 inflammasome is associated with inflammation and renal injury in I/R-induced AKI. In the current study we explored the molecular and cellular mechanisms for NLRP3 inflammasome activation following renal I/R. Mice were subjected to I/R renal injury by clamping bilateral renal pedicles. We showed that I/R injury markedly increased caspase-11 expression and the cleavage of pannexin 1 (panx1) in the kidneys accompanied by NLRP3 inflammasome activation evidenced by the activation of caspase-1 and interlukin-1β (IL-1β) maturation. In Casp-11-/- mice, I/R-induced panx1 cleavage, NLRP3 inflammasome activation as well as renal functional deterioration and tubular morphological changes were significantly attenuated. In cultured primary tubular cells (PTCs) and NRK-52E cells, hypoxia/reoxygenation (H/R) markedly increased caspase-11 expression, NLRP3 inflammasome activation, IL-1β maturation and panx1 cleavage. Knockdown of caspase-11 attenuated all those changes; similar effects were observed in PTCs isolated from Casp-11-/- mice. In NRK-52E cells, overexpression of caspase-11 promoted panx1 cleavage; pretreatment with panx1 inhibitor carbenoxolone or knockdown of panx1 significantly attenuated H/R-induced intracellular ATP reduction, extracellular ATP elevation and NLRP3 inflammasome activation without apparent influence on H/R-induced caspase-11 increase; pretreatment with P2X7 receptor inhibitor AZD9056 also attenuated NLRP3 inflammasome activation. The above results demonstrate that the cleavage of panx1 by upregulated caspase-11 is involved in facilitating ATP release and then NLRP3 inflammasome activation in I/R-induced AKI. This study provides new insight into the molecular mechanism of NLRP3 inflammasome activation in AKI.

Modulation of Hippocampal GABAergic Neurotransmission and Gephyrin Levels by Dihydromyricetin Improves Anxiety.

Anxiety disorders are the most common mental illness in the U.S. and are estimated to consume one-third of the country’s mental health spending. Although anxiolytic therapies are available, many patients exhibit treatment-resistance, relapse, or substantial side effects. An urgent need exists to explore the underlying mechanisms of chronic anxiety and to develop alternative therapies. Presently, we identified dihydromyricetin (DHM), a flavonoid that has anxiolytic properties in a mouse model of isolation-induced anxiety. Socially isolated mice demonstrated increased anxiety levels and reduced exploratory behavior measured by elevated plus-maze and open-field tests. Socially isolated mice showed impaired GABAergic neurotransmission, including reduction in GABAA receptor-mediated extrasynaptic tonic currents, as well as amplitude and frequency of miniature inhibitory postsynaptic currents measured by whole-cell patch-clamp recordings from hippocampal slices. Furthermore, intracellular ATP levels and gephyrin expression decreased in anxious animals. DHM treatment restored ATP and gephyrin expression, GABAergic transmission and synaptic function, as well as decreased anxiety-like behavior. Our findings indicate broader roles for DHM in anxiolysis, GABAergic neurotransmission, and synaptic function. Collectively, our data suggest that reduction in intracellular ATP and gephyrin contribute to the development of anxiety, and represent novel treatment targets. DHM is a potential candidate for pharmacotherapy for anxiety disorders.

Abstract 5266: Kinetic measurements of intracellular ATP levels in co-culture models using live-cell analysis

Abstract Differential metabolic requirements of tumor cells have gained recognition as attractive therapeutic targets. For example, clinical trials are currently underway to test the efficacy of the glutaminase-1 (GLS1) inhibitor CB-839 in a variety of indications, including triple-negative breast cancer (TNBC). Standard approaches to monitoring drug induced metabolic perturbations are limited to endpoint assays that lack cell-specific data in complex co-culture models and provide limited kinetic information. Here we describe a live-cell approach to image and analyze intracellular ATP levels of tumor cells in mono- or co-culture over time using the IncuCyte® CytoATP Sensor. A panel of breast cancer cell lines stably expressing a genetically encoded, fluorescent ATP sensor or a control (non-ATP binding) sensor were generated and cultured alone or with a monolayer of stromal cells. The effect of CB-839 treatment on tumor cell ATP levels was monitored over days and analyzed using the IncuCyte® S3 for Metabolism, which is equipped with a specialized optical module and image acquisition mode. In concordance with previous results, TNBC cell lines were more sensitive to GLS1 inhibition than their receptor-positive counterparts. Across all TNBC lines tested, CB-839 treatment resulted in a reduction in intracellular ATP that was sustained for the duration of the 3-day time course. In contrast, most receptor-positive cell lines displayed little to no effect of GLS1 inhibition. In some cases, a transient reduction followed by recovery within 48 hours was observed, highlighting the value of kinetic analysis. Co-culture with CCD-1086SK fibroblasts attenuated the effect of CB-839 in TNBC cell lines. This was not due to drug buffering, as the reduction in ATP induced by CB-839 treatment of MCF7 cells was unaffected by co-culture with stromal cells. Visualization tools included in the IncuCyte® ATP Analysis Software Module provide a quick, qualitative assessment of data across the assay plate and a view into the heterogeneity of responses to treatment. Measurements of proliferation, cell death, and mitochondrial membrane potential provided additional data supporting the observations generated via ATP readout. Overall, these data highlight the value of live-cell, kinetic analysis of metabolic and cell health measurements. Citation Format: Cicely L. Schramm, Michael L. Bowe, Laura A. Skerlos, Grigory S. Filonov, Yong X. Chen, Daniel M. Appledorn. Kinetic measurements of intracellular ATP levels in co-culture models using live-cell analysis [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 5266.

Abstract 1616: Adjuvant supplementation of docosahexaenoic acid with 2-deoxyglucose, metformin, and aspirin on cellular metabolism in breast, lung, and prostate tumor cells

Abstract A standard treatment for cancer often involves the use of adjuvant supplementation with targeted therapy to maximize tumor suppression. Recent studies have shown that docosahexaenoic acid (DHA; C22:6 n-3), a long chain omega-3 polyunsaturated fatty acid, acts on an array of molecular oncogenes that are often targeted by anti-tumorigenic drugs. After a careful review of DHA’s mechanistic properties, 2-deoxyglucose (2DG), metformin, and aspirin were identified as drugs that share similar molecular targets as DHA. 2DG and metformin both target cellular metabolism by inhibiting intracellular ATP, albeit different mechanisms. 2DG inhibits glycolysis, while metformin targets the mitochondria. Aspirin inhibits inflammation via irreversible acetylation of platelet COX. The current study investigates the effects of DHA in combination with each drug on numerous cancer subtypes to determine if concurrent treatments can synergistically inhibit tumor proliferation. In BT-474 human breast ductal carcinoma, MDA-MB-231 human breast adenocarcinoma, and A549 human lung adenocarcinoma, DHA was shown to enhance the tumor-inhibiting effects of 2DG via intracellular ATP reduction. In contrast, DHA showed no effect with metformin on the same tumor cell lines. With limited studies localizing DHA’s metabolic effects, these results suggest that DHA may have potential selectivity for ATP inhibition via glycolysis. In response to DHA + aspirin, the PC-3 prostate adenocarcinoma and MDA-MB-231 displayed synergistic inhibition of cell viability, while LNCaP prostate carcinoma showed an additive effect. With studies showing DHA’s ability to inhibit endothelial COX, further studies will be done to determine whether the synergistic effects of DHA + aspirin expand to COX expression as well. Moreover, downstream targets of 2DG and metformin will be studied to determine the extent of DHA enhancement on oncogenic targets, such as mTOR, 4EB-P1, and GRB10. The results of this study provide preliminary evidence for the efficacy of adjuvant supplementation with DHA in combination with anti-tumorigenic drugs. With ongoing analysis, the potential for adjuvant nutritional supplementation will allow for additional treatment strategies for future patients. Citation Format: Irvin V. Ma, Michael Mouradian, Andrew R. Cooper, Erika D. Vicente, Ronald S. Pardini. Adjuvant supplementation of docosahexaenoic acid with 2-deoxyglucose, metformin, and aspirin on cellular metabolism in breast, lung, and prostate tumor cells [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 1616.

Eugenol Nanoencapsulated by Sodium Caseinate: Physical, Antimicrobial, and Biophysical Properties

To improve the application of essential oils as natural antimicrobial preservatives, the objective of the present study was to determine physical, antimicrobial, and biophysical properties of eugenol after nanoencapsulation by sodium caseinate (NaCas). Emulsions were prepared by mixing eugenol in 20.0 mg/mL NaCas solution at an overall eugenol content of 5.0–137.9 mg/mL using shear homogenization. Stable emulsions were observed up to 38.5 mg/mL eugenol, which had droplet diameters of smaller than 125 nm at pH 5–9 after ambient storage for up to 30 days. The encapsulated eugenol had similar minimal inhibitory and minimal bactericidal concentrations as free eugenol against Escherichia coli O157:H7 ATCC 43895, Listeria monocytogenes Scott A, and Salmonella Enteritidis but showed better inhibition of E. coli O157:H7 than free eugenol during incubation at 37 °C for 48 h. After 20 min interaction at 21 °C, bacteria treated with encapsulated eugenol had a greater reduction of intracellular ATP and a greater increase of extracellular ATP than free eugenol, suggesting the enhanced permeation of eugenol after nanoencapsulation. However, such overall trend was not observed when examining bacterial morphology and uptake of crystal violet, suggesting the possible membrane adaptation. Findings from this study showed the feasibility of preparing nanoemulsions with high loading of eugenol using NaCas.

Lipophilic triphenylphosphonium derivatives enhance radiation-induced cell killing via inhibition of mitochondrial energy metabolism in tumor cells

It has recently been reported that radiation enhances mitochondrial energy metabolism in various tumor cell lines. To examine how this radiation-induced alteration in mitochondrial function influences tumor cell viability, various lipophilic triphenylphosphonium (TPP+) cation derivatives and related compounds such as 4-hydroxy-2,2,6,6-tetramethyl-1-oxy-piperidin (Tempol) with TPP+ (named “Mito-”) were designed to inhibit the mitochondrial electron transport chain. Mito-(CH2)10-Tempol (M10T) and its derivatives, Mito-(CH2)5-Tempol (M5T), Mito-(CH2)10-Tempol-Methyl (M10T-Me), Mito-C10H21 (M10), and C10H21-Tempol (10T), were prepared. In HeLa human cervical adenocarcinoma cells and A549 human lung carcinoma cells, the fractional uptake of the compound into mitochondria was highest among the TTP+ analogs conjugated with Tempol (M10T, M5T, and 10T). M10T, M10T-Me, and M10 exhibited strong cytotoxicity and enhanced X-irradiation-induced reproductive cell death, while 10T and M5T did not. Furthermore, M10T, M10T-Me, and M10 decreased basal mitochondrial membrane potential and intracellular ATP. M10T treatment inhibited X-ray-induced increases in ATP production. These results indicate that the TPP cation and a long hydrocarbon linker are essential for radiosensitization of tumor cells. The reduction in intracellular ATP by lipophilic TPP+ is partly responsible for the observed radiosensitization.

Rapid sodium signaling couples glutamate uptake to breakdown of ATP in perivascular astrocyte endfeet.

Perivascular endfeet of astrocytes are highly polarized compartments that ensheath blood vessels and contribute to the blood-brain barrier. They experience calcium transients with neuronal activity, a phenomenon involved in neurovascular coupling. Endfeet also mediate the uptake of glucose from the blood, a process stimulated in active brain regions. Here, we demonstrate in mouse hippocampal tissue slices that endfeet undergo sodium signaling upon stimulation of glutamatergic synaptic activity. Glutamate-induced endfeet sodium transients were diminished by TFB-TBOA, suggesting that they were generated by sodium-dependent glutamate uptake. With local agonist application, they could be restricted to endfeet and immunohistochemical analysis revealed prominent expression of glutamate transporters GLAST and GLT-1 localized towards the neuropil vs. the vascular side of endfeet. Endfeet sodium signals spread at an apparent maximum velocity of ∼120 µm/s and directly propagated from stimulated into neighboring endfeet; this spread was omitted in Cx30/Cx43 double-deficient mice. Sodium transients resulted in elevation of intracellular magnesium, indicating a decrease in intracellular ATP. In summary, our results establish that excitatory synaptic activity and stimulation of glutamate uptake in astrocytes trigger transient sodium increases in perivascular endfeet which rapidly spread through gap junctions into neighboring endfeet and cause a reduction of intracellular ATP. The newly discovered endfeet sodium signaling thereby represents a fast, long-lived and inter-cellularly acting indicator of synaptic activity at the blood-brain barrier, which likely constitutes an important component of neuro-metabolic coupling in the brain. GLIA 2017;65:293-308.

Synergistic modulation of KCNQ1/KCNE1 K+ channels (IKs) by phosphatidylinositol 4,5-bisphosphate (PIP2) and [ATP

Cardiac KCNQ1/KCNE1 channels (IKs) are dependent on the concentration of membrane phosphatidylinositol-4,5-bisphosphate (PIP2) and on cytosolic ATP by two distinct mechanisms. In this study we measured IKs and FRET between PH-PLCδ-based fluorescent PIP2 sensors in a stable KCNQ1/KCNE1 CHO cell line. Effects of activating either a muscarinic M3 receptor or the switchable phosphatase Ci-VSP on IKs were analyzed. Recovery of IKs from inhibition induced by muscarinic stimulation was incomplete despite full PIP2 resynthesis. Recovery of IKs was completely suppressed under ATP-free conditions, but partially restored by the ATP analog AMP-PCP, providing evidence that depletion of intracellular ATP inhibits IKs independent of PIP2-depletion.Simultaneous patch-clamp and FRET measurements in cells co-expressing Ci-VSP and the PIP2-FRET sensor revealed a component of IKs inhibition directly related to dynamic PIP2-depletion. A second component of inhibition was independent of acute changes in PIP2 and could be mimicked by ATP-free pipette solution, suggesting that it results from intracellular ATP-depletion. The reduction of intracellular ATP upon Ci-VSP activation appears to be independent of its activity as a phosphoinositide phosphatase. Our data demonstrate that ATP-depletion slowed IKs activation but had no short-term effect on PIP2 regeneration, suggesting that impaired PIP2-resynthesis cannot account for the rapid IKs inhibition by ATP-depletion. Furthermore, the second component of IKs inhibition by Ci-VSP was reduced by AMP-PCP in the pipette filling solution, indicating that direct binding of ATP to the KCNQ1/KCNE1 complex is required for voltage activation of IKs. We suggest that fluctuations of the cellular metabolic state regulate IKs in parallel with Gq-coupled PLC activation and PIP2-depletion.

Fatty Acid Overload to Mitochondria Promotes Myocardial Insulin Resistance in Differentiated H9C2 Myocytes

OBJECTIVE: In chronic heart failure, it is well known that an increase in serum fatty acid (FA) due to adrenergic stimulation induces myocardial insulin-resistance, which further deteriorates myocardial function. Since precise mechanisms of pathogenesis in FA-induced myocardial insulin-resistance is still elusive, we in this study investigated the relationship between FA-induced myocardial insulin-resistance and mitochondrial dysfunction, using an ex vivo insulin-resistant myocytes model.METHODS and RESULTS: The differentiated H9c2 myocytes were treated with saturated FA (palmitate; 0.2 mM) for 24 hours to produce the ex vivo insulin-resistant myocytes. The palmitate-treated myocytes exhibited an impaired insulin (100 nM)-mediated 2-deoxy-D-glucose (2DG) uptake (1.0 ± 0.1 fold increase from without insulin vs. 1.7 ± 0.1 of control, P< 0.01) and attenuated phosphorylation of insulin signaling molecules (IRS-1 and AKT), indicating insulin-resistance. When myocytes were pretreated with perhexiline (2 μM; an inhibitor of mitochondrial FA uptake) the reduced 2-DG uptakes (1.2 ± 0.1 fold increase from without insulin vs. palmitate-treated myocytes, P< 0.01) and attenuated phosphorylation of insulin-signaling were restored. The palmitate-treated myocytes revealed the intracellular ATP reduction (74 ± 6 % decrease from control, P< 0.01; luciferase assay), mitochondrial membrane potential depolarization (JC-1 ratio of 590/528 nm: 2.9 ± 0.1 vs. 5.3 ± 0.2 of control, P< 0.01), indicating mitochondrial dysfunction. When the myocytes were pretreated with perhexiline, the reduced intracellular ATP levels (32±14 % increase from palmitate-treated myocytes, P< 0.01) and depolarized mitochondrial membrane potential (JC-1 ratio: 3.5 ± 0.1 vs. palmitate-treated myocytes, P< 0.01) by palmitate were restored, indicating the improvement in mitochondrial function.CONCLUSION: Our findings indicated that mitochondrial dysfunction via mitochondrial FA overload may underlie the pathogenesis of myocardial insulin-resistance under HF.

A Small-Molecule Inhibitor of Glucose Transporter 1 Downregulates Glycolysis, Induces Cell-Cycle Arrest, and Inhibits Cancer Cell Growth In Vitro and In Vivo

The functional and therapeutic importance of the Warburg effect is increasingly recognized, and glycolysis has become a target of anticancer strategies. We recently reported the identification of a group of novel small compounds that inhibit basal glucose transport and reduce cancer cell growth by a glucose deprivation-like mechanism. We hypothesized that the compounds target Glut1 and are efficacious in vivo as anticancer agents. Here, we report that a novel representative compound WZB117 not only inhibited cell growth in cancer cell lines but also inhibited cancer growth in a nude mouse model. Daily intraperitoneal injection of WZB117 at 10 mg/kg resulted in a more than 70% reduction in the size of human lung cancer of A549 cell origin. Mechanism studies showed that WZB117 inhibited glucose transport in human red blood cells (RBC), which express Glut1 as their sole glucose transporter. Cancer cell treatment with WZB117 led to decreases in levels of Glut1 protein, intracellular ATP, and glycolytic enzymes. All these changes were followed by increase in ATP-sensing enzyme AMP-activated protein kinase (AMPK) and declines in cyclin E2 as well as phosphorylated retinoblastoma, resulting in cell-cycle arrest, senescence, and necrosis. Addition of extracellular ATP rescued compound-treated cancer cells, suggesting that the reduction of intracellular ATP plays an important role in the anticancer mechanism of the molecule. Senescence induction and the essential role of ATP were reported for the first time in Glut1 inhibitor-treated cancer cells. Thus, WZB117 is a prototype for further development of anticancer therapeutics targeting Glut1-mediated glucose transport and glucose metabolism.

Mitochondrial complex I deficiency and ATP/ADP ratio in lymphocytes of amyotrophic lateral sclerosis patients

Objectives: Several lines of evidence suggest that mitochondrial dysfunction is involved in amyotrophic lateral sclerosis (ALS), but despite the fact that mitochondria play a central role in excitotoxicity, oxidative stress, and apoptosis, the intimate underlying mechanism linking mitochondrial defects to motor neuron degeneration in ALS still remains elusive. This study was performed to assess the mitochondrial respiratory chain dysfunction and cellular energy index (ATP/ADP ratio) in lymphocytes of ALS patients.Methods: In this study, activity of mitochondrial respiratory chain complex I (measured as NADH-ferricyanide reductase) and both intracellular ATP and ADP measurements were performed on lymphocytes of ALS patients (n = 14) and control subjects (n = 26). Then, ATP/ADP ratio was calculated.Results: Our finding showed that in patients compared with controls, complex I activity and intracellular ATP were significantly reduced (P = 0·001) and intracellular ADP content was increased (P<0·005) and ATP/ADP ratio subsequently was decreased and also we found strong correlation between complex I activity and intracellular ATP content and strong reverse correlation between complex I activity and intracellular ADP content in the patients with ALS (r2 = 0·90).Discussion: This study suggests that complex I deficiency and both reduction in intracellular ATP and increase in intracellular ADP content may be involved in the progression and pathogenesis of ALS.

Abstract 964: Nicotinamide phosphoribosyltransferase (Nampt) regulates growth of non-small cell lung cancer cells with c-Met overexpression

Abstract Background: Non-small cell lung cancer (NSCLC) often has an oncogene addiction, including epidermal growth factor receptor (EGFR) signaling or hepatocyte growth factor (HGF)/c-Met signaling. NSCLC cells with these addictions require a large amount of intracellular ATP to activate their receptor tyrosine kinases (RTKs). Nicotinamide phosphoribosyltransferase (Nampt) is an enzyme to synthesize Nicotinamide adenine dinucleotide (NAD), which regulates cellular redox reactions as well as intracellular ATP biosynthesis. We have reported that the inhibition of intracellular Nampt function suppressed tumor growth via intracellular ATP reduction in EGFR-gene mutated NSCLC. We have developed a novel Nampt inhibitor, which is encapsulated within poly (lactic-co-glycolic acid) (PLGA) microspheres, to enhance the tumoricidal activity in vivo. We hypothesized that the Nampt inhibitor has a high anti-tumor activity in NSCLC with c-MET overexpression, and we evaluated the role of Nampt in growth of NSCLC with c-Met overexpression. Methods: In vitro study, expression of Nampt protein and mRNA in NSCLC cell lines (H1975, H1648, H820, H1993, from ATCC) were studied using western blotting and real-time RT-PCR (Lightcycler 2.0, Roche). NSCLC cells were treated with Nampt-siRNA (Invitrogen) or a Nampt inhibitor, FK866 (Axon medchem), and cell proliferation was examined using BrdU assay (Roche). Cell apoptosis in the NSCLC was detected by Flow cytometry (BD FACSAria II, BD Bioscience). Intracellular ATP levels in the treated NSCLC cells were also evaluated using bioluminescence assay (Roche). In western blotting, expression of c-MET signal proteins in the treated NSCLC was also studied. In vivo study, NSCLC cells were transplanted into 7-10 weeks-old female nude mice (CD-1Foxn1, Charles River), and the mice were treated with or without FK866. We used PLGA-FK866 in the tumor-bearing mice model. In immunohistochemistry, c-Met and MAPK signal proteins in xenograft tumors was evaluated using immunofluorescence microscopy (FV1000-D, OLYMPUS). Results: Nampt mRNA was overexpressed in all NSCLC cell lines. BrdU assay showed that treatment of Nampt-siRNA and FK866 suppressed cell proliferation and induced apoptosis in NSCLC cells. The expression of c-MET and MAPK signals were suppressed in FK866-treated NSCLC. ATP bioluminescence assay exhibited that ATP levels were decreased in FK866-treated NSCLC (p&amp;lt;0.05). In the tumor-bearing mice model, PLGA-FK866 suppressed growth of xenograft tumors (p&amp;lt;0.05), and achieved downregulation of MAPK signal proteins in xenografts. Conclusion: In the present study, we founded that the Nampt inhibitor suppressed NSCLC growth via suppression of intracellular ATP levels, and the expression of c-Met and MAPK signal proteins. These results suggest that Nampt has a potential to become a novel therapeutic target in NSCLC with c-MET overexpression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 964. doi:10.1158/1538-7445.AM2011-964

Metabolic Stress Boosts Humoral Responses In Vivo Independently of Inflammasome and Inflammatory Reaction

Adjuvant formulations boost humoral responses by acting through several, yet incompletely elucidated pathways. In this study, we show that oligomycin or 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR) enhances Ab production when coinjected with T cell-dependent Ags. Oligomycin and AICAR lead to intracellular ATP reduction, suggesting that metabolic stress could be sensed by immune cells and leads to increased humoral responses. AICAR promotes IL-4 and IL-21 by naive Th cells but does not affect dendritic cell activation/maturation in vitro or in vivo. Accordingly, the adjuvant effect of AICAR or oligomycin does not require MyD88 or caspase-1 expression in vivo. Because AICAR is well tolerated in humans, this compound could represent a novel and safe adjuvant promoting humoral responses in vivo with a minimal reactogenicity.