Publications by authors named "Gloria A Benavides"

40 Publications

A role for GLUT3 in glioblastoma cell invasion that is not recapitulated by GLUT1.

Cell Adh Migr 2021 Dec;15(1):101-115

Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA.

The multifaceted roles of metabolism in invasion have been investigated across many cancers. The brain tumor glioblastoma (GBM) is a highly invasive and metabolically plastic tumor with an inevitable recurrence. The neuronal glucose transporter 3 (GLUT3) was previously reported to correlate with poor glioma patient survival and be upregulated in GBM cells to promote therapeutic resistance and survival under restricted glucose conditions. It has been suggested that the increased glucose uptake mediated by GLUT3 elevation promotes survival of circulating tumor cells to facilitate metastasis. Here we suggest a more direct role for GLUT3 in promoting invasion that is not dependent upon changes in cell survival or metabolism. Analysis of glioma datasets demonstrated that GLUT3, but not GLUT1, expression was elevated in invasive disease. In human xenograft derived GBM cells, GLUT3, but not GLUT1, elevation significantly increased invasion in transwell assays, but not growth or migration. Further, there were no changes in glycolytic metabolism that correlated with invasive phenotypes. We identified the GLUT3 C-terminus as mediating invasion: substituting the C-terminus of GLUT1 for that of GLUT3 reduced invasion. RNA-seq analysis indicated changes in extracellular matrix organization in GLUT3 overexpressing cells, including upregulation of osteopontin. Together, our data suggest a role for GLUT3 in increasing tumor cell invasion that is not recapitulated by GLUT1, is separate from its role in metabolism and survival as a glucose transporter, and is likely broadly applicable since GLUT3 expression correlates with metastasis in many solid tumors.
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http://dx.doi.org/10.1080/19336918.2021.1903684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8043167PMC
December 2021

Bioenergetic maladaptation and release of HMGB1 in calcineurin inhibitor-mediated nephrotoxicity.

Am J Transplant 2021 Mar 16. Epub 2021 Mar 16.

Division of Nephrology, University of Alabama at Birmingham, Birmingham, Alabama, USA.

Calcineurin inhibitors (CNIs) are potent immunosuppressive agents, universally used following solid organ transplantation to prevent rejection. Although effective, the long-term use of CNIs is associated with nephrotoxicity. The etiology of this adverse effect is complex, and effective therapeutic interventions remain to be determined. Using a combination of in vitro techniques and a mouse model of CNI-mediated nephrotoxicity, we found that the CNIs, cyclosporine A (CsA), and tacrolimus (TAC) share a similar mechanism of tubular epithelial kidney cell injury, including mitochondrial dysfunction and release of High-Mobility Group Box I (HMGB1). CNIs promote bioenergetic reprogramming due to mitochondrial dysfunction and a shift toward glycolytic metabolism. These events were accompanied by diminished cell-to-cell adhesion, loss of the epithelial cell phenotype, and release of HMGB1. Notably, Erk1/2 inhibitors effectively diminished HMGB1 release, and similar inhibitor was observed on inclusion of pan-caspase inhibitor zVAD-FMK. In vivo, while CNIs activate tissue proremodeling signaling pathways, MAPK/Erk1/2 inhibitor prevented nephrotoxicity, including diminished HMGB1 release from kidney epithelial cells and accumulation in urine. In summary, HMGB1 is an early indicator and marker of progressive nephrotoxicity induced by CNIs. We suggest that proremodeling signaling pathway and loss of mitochondrial redox/bioenergetics homeostasis are crucial therapeutic targets to ameliorate CNI-mediated nephrotoxicity.
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http://dx.doi.org/10.1111/ajt.16561DOI Listing
March 2021

Pyrazole-Based Lactate Dehydrogenase Inhibitors with Optimized Cell Activity and Pharmacokinetic Properties.

J Med Chem 2020 10 27;63(19):10984-11011. Epub 2020 Sep 27.

Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, 9000 Rockville Pike, Bethesda, Maryland 20892, United States.

Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate, with concomitant oxidation of reduced nicotinamide adenine dinucleotide as the final step in the glycolytic pathway. Glycolysis plays an important role in the metabolic plasticity of cancer cells and has long been recognized as a potential therapeutic target. Thus, potent, selective inhibitors of LDH represent an attractive therapeutic approach. However, to date, pharmacological agents have failed to achieve significant target engagement , possibly because the protein is present in cells at very high concentrations. We report herein a lead optimization campaign focused on a pyrazole-based series of compounds, using structure-based design concepts, coupled with optimization of cellular potency, drug-target residence times, and PK properties, to identify first-in-class inhibitors that demonstrate LDH inhibition . The lead compounds, named () and (), possess desirable attributes for further studying the effect of LDH inhibition.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830743PMC
October 2020

A novel approach to measure mitochondrial respiration in frozen biological samples.

EMBO J 2020 07 20;39(13):e104073. Epub 2020 May 20.

Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA.

Respirometry is the gold standard measurement of mitochondrial oxidative function, as it reflects the activity of the electron transport chain complexes working together. However, the requirement for freshly isolated mitochondria hinders the feasibility of respirometry in multi-site clinical studies and retrospective studies. Here, we describe a novel respirometry approach suited for frozen samples by restoring electron transfer components lost during freeze/thaw and correcting for variable permeabilization of mitochondrial membranes. This approach preserves 90-95% of the maximal respiratory capacity in frozen samples and can be applied to isolated mitochondria, permeabilized cells, and tissue homogenates with high sensitivity. We find that primary changes in mitochondrial function, detected in fresh tissue, are preserved in frozen samples years after collection. This approach will enable analysis of the integrated function of mitochondrial Complexes I to IV in one measurement, collected at remote sites or retrospectively in samples residing in tissue biobanks.
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http://dx.doi.org/10.15252/embj.2019104073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327496PMC
July 2020

Differential effects of REV-ERBα/β agonism on cardiac gene expression, metabolism, and contractile function in a mouse model of circadian disruption.

Am J Physiol Heart Circ Physiol 2020 06 1;318(6):H1487-H1508. Epub 2020 May 1.

Division of Cardiovascular Disease, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama.

Cell-autonomous circadian clocks have emerged as temporal orchestrators of numerous biological processes. For example, the cardiomyocyte circadian clock modulates transcription, translation, posttranslational modifications, ion homeostasis, signaling cascades, metabolism, and contractility of the heart over the course of the day. Circadian clocks are composed of more than 10 interconnected transcriptional modulators, all of which have the potential to influence the cardiac transcriptome (and ultimately cardiac processes). These transcriptional modulators include BMAL1 and REV-ERBα/β; BMAL1 induces REV-ERBα/β, which in turn feeds back to inhibit BMAL1. Previous studies indicate that cardiomyocyte-specific BMAL1-knockout (CBK) mice exhibit a dysfunctional circadian clock (including decreased REV-ERBα/β expression) in the heart associated with abnormalities in cardiac mitochondrial function, metabolism, signaling, and contractile function. Here, we hypothesized that decreased REV-ERBα/β activity is responsible for distinct phenotypical alterations observed in CBK hearts. To test this hypothesis, CBK (and littermate control) mice were administered with the selective REV-ERBα/β agonist SR-9009 (100 mg·kg·day for 8 days). SR-9009 administration was sufficient to normalize cardiac glycogen synthesis rates, cardiomyocyte size, interstitial fibrosis, and contractility in CBK hearts (without influencing mitochondrial complex activities, nor normalizing substrate oxidation and Akt/mTOR/GSK3β signaling). Collectively, these observations highlight a role for REV-ERBα/β as a mediator of a subset of circadian clock-controlled processes in the heart.
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http://dx.doi.org/10.1152/ajpheart.00709.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311693PMC
June 2020

Nuclear receptor binding factor 2 (NRBF2) is required for learning and memory.

Lab Invest 2020 09 29;100(9):1238-1251. Epub 2020 Apr 29.

Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA.

The mechanisms which underlie defects in learning and memory are a major area of focus with the increasing incidence of Alzheimer's disease in the aging population. The complex genetically-controlled, age-, and environmentally-dependent onset and progression of the cognitive deficits and neuronal pathology call for better understanding of the fundamental biology of the nervous system function. In this study, we focus on nuclear receptor binding factor-2 (NRBF2) which modulates the transcriptional activities of retinoic acid receptor α and retinoid X receptor α, and the autophagic activities of the BECN1-VPS34 complex. Since both transcriptional regulation and autophagic function are important in supporting neuronal function, we hypothesized that NRBF2 deficiency may lead to cognitive deficits. To test this, we developed a new mouse model with nervous system-specific knockout of Nrbf2. In a series of behavioral assessment, we demonstrate that NRBF2 knockout in the nervous system results in profound learning and memory deficits. Interestingly, we did not find deficits in autophagic flux in primary neurons and the autophagy deficits were minimal in the brain. In contrast, RNAseq analyses have identified altered expression of genes that have been shown to impact neuronal function. The observation that NRBF2 is involved in learning and memory suggests a new mechanism regulating cognition involving the role of this protein in regulating networks related to the function of retinoic acid receptors, protein folding, and quality control.
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http://dx.doi.org/10.1038/s41374-020-0433-4DOI Listing
September 2020

Mitochondrial Oxidative Phosphorylation Regulates the Fate Decision between Pathogenic Th17 and Regulatory T Cells.

Cell Rep 2020 02;30(6):1898-1909.e4

Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA. Electronic address:

Understanding metabolic pathways that regulate Th17 development is important to broaden therapeutic options for Th17-mediated autoimmunity. Here, we report a pivotal role of mitochondrial oxidative phosphorylation (OXPHOS) for lineage specification toward pathogenic Th17 differentiation. Th17 cells rapidly increase mitochondrial respiration during development, and this is necessary for metabolic reprogramming following T cell activation. Surprisingly, specific inhibition of mitochondrial ATP synthase ablates Th17 pathogenicity in a mouse model of autoimmunity by preventing Th17 pathogenic signature gene expression. Notably, cells activated under OXPHOS-inhibited Th17 conditions preferentially express Foxp3, rather than Th17 genes, and become suppressive Treg cells. Mechanistically, OXPHOS promotes the Th17 pioneer transcription factor, BATF, and facilitates T cell receptor (TCR) and mTOR signaling. Correspondingly, overexpression of BATF rescues Th17 development when ATP synthase activity is restricted. Together, our data reveal a regulatory role of mitochondrial OXPHOS in dictating the fate decision between Th17 and Treg cells by supporting early molecular events necessary for Th17 commitment.
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http://dx.doi.org/10.1016/j.celrep.2020.01.022DOI Listing
February 2020

A precision medicine approach to defining the impact of doxorubicin on the bioenergetic-metabolite interactome in human platelets.

Redox Biol 2020 01 7;28:101311. Epub 2019 Sep 7.

Mitochondrial Medicine Laboratory, Center for Free Radical Biology, Department of Pathology, University of Alabama at Birmingham, USA. Electronic address:

Non-invasive measures of the response of individual patients to cancer therapeutics is an emerging strategy in precision medicine. Platelets offer a potential dynamic marker for metabolism and bioenergetic responses in individual patients since they have active glycolysis and mitochondrial oxidative phosphorylation and can be easily isolated from a small blood sample. We have recently shown how the bioenergetic-metabolite interactome can be defined in platelets isolated from human subjects by measuring metabolites and bioenergetics in the same sample. In the present study, we used a model system to assess test the hypothesis that this interactome is modified by xenobiotics using exposure to the anti-cancer drug doxorubicin (Dox) in individual donors. We found that unsupervised analysis of the metabolome showed clear differentiation between the control and Dox treated group. Dox treatment resulted in a concentration-dependent decrease in bioenergetic parameters with maximal respiration being most sensitive and this was associated with significant changes in over 166 features. A metabolome-wide association study of Dox was also conducted, and Dox was found to have associations with metabolites in the glycolytic and TCA cycle pathways. Lastly, network analysis showed the impact of Dox on the bioenergetic-metabolite interactome and revealed profound changes in the regulation of reserve capacity. Taken together, these data support the conclusion that platelets are a suitable platform to predict and monitor therapeutic efficacy as well as anticipate susceptibility to toxicity in the context of precision medicine.
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http://dx.doi.org/10.1016/j.redox.2019.101311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6812033PMC
January 2020

Targeting Glycolysis through Inhibition of Lactate Dehydrogenase Impairs Tumor Growth in Preclinical Models of Ewing Sarcoma.

Cancer Res 2019 10 20;79(19):5060-5073. Epub 2019 Aug 20.

Pediatric Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.

Altered cellular metabolism, including an increased dependence on aerobic glycolysis, is a hallmark of cancer. Despite the fact that this observation was first made nearly a century ago, effective therapeutic targeting of glycolysis in cancer has remained elusive. One potentially promising approach involves targeting the glycolytic enzyme lactate dehydrogenase (LDH), which is overexpressed and plays a critical role in several cancers. Here, we used a novel class of LDH inhibitors to demonstrate, for the first time, that Ewing sarcoma cells are exquisitely sensitive to inhibition of LDH. EWS-FLI1, the oncogenic driver of Ewing sarcoma, regulated LDH A (LDHA) expression. Genetic depletion of LDHA inhibited proliferation of Ewing sarcoma cells and induced apoptosis, phenocopying pharmacologic inhibition of LDH. LDH inhibitors affected Ewing sarcoma cell viability both and by reducing glycolysis. Intravenous administration of LDH inhibitors resulted in the greatest intratumoral drug accumulation, inducing tumor cell death and reducing tumor growth. The major dose-limiting toxicity observed was hemolysis, indicating that a narrow therapeutic window exists for these compounds. Taken together, these data suggest that targeting glycolysis through inhibition of LDH should be further investigated as a potential therapeutic approach for cancers such as Ewing sarcoma that exhibit oncogene-dependent expression of LDH and increased glycolysis. SIGNIFICANCE: LDHA is a pharmacologically tractable EWS-FLI1 transcriptional target that regulates the glycolytic dependence of Ewing sarcoma.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-0217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774872PMC
October 2019

Acute increases in -GlcNAc indirectly impair mitochondrial bioenergetics through dysregulation of LonP1-mediated mitochondrial protein complex turnover.

Am J Physiol Cell Physiol 2019 06 13;316(6):C862-C875. Epub 2019 Mar 13.

Division of Molecular and Cellular Pathology, Department of Pathology, University of Alabama , Birmingham, Alabama.

The attachment of -linked β--acetylglucosamine (-GlcNAc) to the serine and threonine residues of proteins in distinct cellular compartments is increasingly recognized as an important mechanism regulating cellular function. Importantly, the -GlcNAc modification of mitochondrial proteins has been identified as a potential mechanism to modulate metabolism under stress with both potentially beneficial and detrimental effects. This suggests that temporal and dose-dependent changes in -GlcNAcylation may have different effects on mitochondrial function. In the current study, we found that acutely augmenting -GlcNAc levels by inhibiting -GlcNAcase with Thiamet-G for up to 6 h resulted in a time-dependent decrease in cellular bioenergetics and decreased mitochondrial complex I, II, and IV activities. Under these conditions, mitochondrial number was unchanged, whereas an increase in the protein levels of the subunits of several electron transport complex proteins was observed. However, the observed bioenergetic changes appeared not to be due to direct increased -GlcNAc modification of complex subunit proteins. Increases in -GlcNAc were also associated with an accumulation of mitochondrial ubiquitinated proteins; phosphatase and tensin homolog induced kinase 1 (PINK1) and p62 protein levels were also significantly increased. Interestingly, the increase in -GlcNAc levels was associated with a decrease in the protein levels of the mitochondrial Lon protease homolog 1 (LonP1), which is known to target complex IV subunits and PINK1, in addition to other mitochondrial proteins. These data suggest that impaired bioenergetics associated with short-term increases in -GlcNAc levels could be due to impaired, LonP1-dependent, mitochondrial complex protein turnover.
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http://dx.doi.org/10.1152/ajpcell.00491.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6620580PMC
June 2019

Identification of Compounds That Decrease Glioblastoma Growth and Glucose Uptake in Vitro.

ACS Chem Biol 2018 08 15;13(8):2048-2057. Epub 2018 Jun 15.

Department of Cell, Developmental and Integrative Biology , University of Alabama at Birmingham , Birmingham , Alabama , United States.

Tumor heterogeneity has hampered the development of novel effective therapeutic options for aggressive cancers, including the deadly primary adult brain tumor glioblastoma (GBM). Intratumoral heterogeneity is partially attributed to the tumor initiating cell (TIC) subset that contains highly tumorigenic, stem-like cells. TICs display metabolic plasticity but can have a reliance on aerobic glycolysis. Elevated expression of GLUT1 and GLUT3 is present in many cancer types, with GLUT3 being preferentially expressed in brain TICs (BTICs) to increase survival in low nutrient tumor microenvironments, leading to tumor maintenance. Through structure-based virtual screening (SBVS), we identified potential novel GLUT inhibitors. The screening of 13 compounds identified two that preferentially inhibit the growth of GBM cells with minimal toxicity to non-neoplastic astrocytes and neurons. These compounds, SRI-37683 and SRI-37684, also inhibit glucose uptake and decrease the glycolytic capacity and glycolytic reserve capacity of GBM patient-derived xenograft (PDX) cells in glycolytic stress test assays. Our results suggest a potential new therapeutic avenue to target metabolic reprogramming for the treatment of GBM, as well as other tumor types, and the identified novel inhibitors provide an excellent starting point for further lead development.
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http://dx.doi.org/10.1021/acschembio.8b00251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6425477PMC
August 2018

Methods for assessing mitochondrial quality control mechanisms and cellular consequences in cell culture.

Redox Biol 2018 07 10;17:59-69. Epub 2018 Apr 10.

Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States; VA Medical Center, University of Alabama at Birmingham, Birmingham, AL 35294, United States. Electronic address:

Mitochondrial quality is under surveillance by autophagy, the cell recycling process which degrades and removes damaged mitochondria. Inadequate autophagy results in deterioration in mitochondrial quality, bioenergetic dysfunction, and metabolic stress. Here we describe in an integrated work-flow to assess parameters of mitochondrial morphology, function, mtDNA and protein damage, metabolism and autophagy regulation to provide the framework for a practical assessment of mitochondrial quality. This protocol has been tested with cell cultures, is highly reproducible, and is adaptable to studies when cell numbers are limited, and thus will be of interest to researchers studying diverse physiological and pathological phenomena in which decreased mitochondrial quality is a contributory factor.
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http://dx.doi.org/10.1016/j.redox.2018.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6006680PMC
July 2018

Poldip2 is an oxygen-sensitive protein that controls PDH and αKGDH lipoylation and activation to support metabolic adaptation in hypoxia and cancer.

Proc Natl Acad Sci U S A 2018 02 6;115(8):1789-1794. Epub 2018 Feb 6.

Division of Cardiology, Department of Medicine, Emory University, Atlanta, GA 30322;

Although the addition of the prosthetic group lipoate is essential to the activity of critical mitochondrial catabolic enzymes, its regulation is unknown. Here, we show that lipoylation of the pyruvate dehydrogenase and α-ketoglutarate dehydrogenase (αKDH) complexes is a dynamically regulated process that is inhibited under hypoxia and in cancer cells to restrain mitochondrial respiration. Mechanistically, we found that the polymerase-δ interacting protein 2 (Poldip2), a nuclear-encoded mitochondrial protein of unknown function, controls the lipoylation of the pyruvate and α-KDH dihydrolipoamide acetyltransferase subunits by a mechanism that involves regulation of the caseinolytic peptidase (Clp)-protease complex and degradation of the lipoate-activating enzyme Ac-CoA synthetase medium-chain family member 1 (ACSM1). ACSM1 is required for the utilization of lipoic acid derived from a salvage pathway, an unacknowledged lipoylation mechanism. In Poldip2-deficient cells, reduced lipoylation represses mitochondrial function and induces the stabilization of hypoxia-inducible factor 1α (HIF-1α) by loss of substrate inhibition of prolyl-4-hydroxylases (PHDs). HIF-1α-mediated retrograde signaling results in a metabolic reprogramming that resembles hypoxic and cancer cell adaptation. Indeed, we observe that Poldip2 expression is down-regulated by hypoxia in a variety of cell types and basally repressed in triple-negative cancer cells, leading to inhibition of lipoylation of the pyruvate and α-KDH complexes and mitochondrial dysfunction. Increasing mitochondrial lipoylation by forced expression of Poldip2 increases respiration and reduces the growth rate of cancer cells. Our work unveils a regulatory mechanism of catabolic enzymes required for metabolic plasticity and highlights the role of Poldip2 as key during hypoxia and cancer cell metabolic adaptation.
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http://dx.doi.org/10.1073/pnas.1720693115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828627PMC
February 2018

Addition of carbonic anhydrase 9 inhibitor SLC-0111 to temozolomide treatment delays glioblastoma growth in vivo.

JCI Insight 2017 12 21;2(24). Epub 2017 Dec 21.

Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama, USA.

Tumor microenvironments can promote stem cell maintenance, tumor growth, and therapeutic resistance, findings linked by the tumor-initiating cell hypothesis. Standard of care for glioblastoma (GBM) includes temozolomide chemotherapy, which is not curative, due, in part, to residual therapy-resistant brain tumor-initiating cells (BTICs). Temozolomide efficacy may be increased by targeting carbonic anhydrase 9 (CA9), a hypoxia-responsive gene important for maintaining the altered pH gradient of tumor cells. Using patient-derived GBM xenograft cells, we explored whether CA9 and CA12 inhibitor SLC-0111 could decrease GBM growth in combination with temozolomide or influence percentages of BTICs after chemotherapy. In multiple GBMs, SLC-0111 used concurrently with temozolomide reduced cell growth and induced cell cycle arrest via DNA damage in vitro. In addition, this treatment shifted tumor metabolism to a suppressed bioenergetic state in vivo. SLC-0111 also inhibited the enrichment of BTICs after temozolomide treatment determined via CD133 expression and neurosphere formation capacity. GBM xenografts treated with SLC-0111 in combination with temozolomide regressed significantly, and this effect was greater than that of temozolomide or SLC-0111 alone. We determined that SLC-0111 improves the efficacy of temozolomide to extend survival of GBM-bearing mice and should be explored as a treatment strategy in combination with current standard of care.
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http://dx.doi.org/10.1172/jci.insight.92928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752277PMC
December 2017

Glutaminolysis is required for transforming growth factor-β1-induced myofibroblast differentiation and activation.

J Biol Chem 2018 01 8;293(4):1218-1228. Epub 2017 Dec 8.

From the Division of Pulmonary, Allergy and Critical Care Medicine.

Myofibroblasts participate in physiological wound healing and pathological fibrosis. Myofibroblast differentiation is characterized by the expression of α-smooth muscle actin and extracellular matrix proteins and is dependent on metabolic reprogramming. In this study, we explored the role of glutaminolysis and metabolites of TCA in supporting myofibroblast differentiation. Glutaminolysis converts Gln into α-ketoglutarate (α-KG), a critical intermediate in the TCA cycle. Increases in the steady-state concentrations of TCA cycle metabolites including α-KG, succinate, fumarate, malate, and citrate were observed in TGF-β1-differentiated myofibroblasts. The concentration of glutamate was also increased in TGF-β1-differentiated myofibroblasts compared with controls, whereas glutamine levels were decreased, suggesting enhanced glutaminolysis. This was associated with TGF-β1-induced expression of the glutaminase (GLS) isoform, GLS1, which converts Gln into glutamate, at both the mRNA and protein levels. The stimulation of GLS1 expression by TGF-β1 was dependent on both SMAD3 and p38 mitogen-activated protein kinase activation. Depletion of extracellular Gln prevented TGF-β1-induced myofibroblast differentiation. The removal of extracellular Gln postmyofibroblast differentiation decreased the expression of the profibrotic markers fibronectin and hypoxia-inducible factor-1α and reversed TGF-β1-induced metabolic reprogramming. Silencing of GLS1 expression, in the presence of Gln, abrogated TGF-β1-induced expression of profibrotic markers. Treatment of GLS1-deficient myofibroblasts with exogenous glutamate or α-KG restored TGF-β1-induced expression of profibrotic markers in GLS1-deficient myofibroblasts. Together, these data demonstrate that glutaminolysis is a critical component of myofibroblast metabolic reprogramming that regulates myofibroblast differentiation.
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http://dx.doi.org/10.1074/jbc.RA117.000444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5787800PMC
January 2018

Discovery and Optimization of Potent, Cell-Active Pyrazole-Based Inhibitors of Lactate Dehydrogenase (LDH).

J Med Chem 2017 11 9;60(22):9184-9204. Epub 2017 Nov 9.

National Center for Advancing Translational Sciences, National Institutes of Health , 9800 Medical Center Drive, Rockville, Maryland 20850, United States.

We report the discovery and medicinal chemistry optimization of a novel series of pyrazole-based inhibitors of human lactate dehydrogenase (LDH). Utilization of a quantitative high-throughput screening paradigm facilitated hit identification, while structure-based design and multiparameter optimization enabled the development of compounds with potent enzymatic and cell-based inhibition of LDH enzymatic activity. Lead compounds such as 63 exhibit low nM inhibition of both LDHA and LDHB, submicromolar inhibition of lactate production, and inhibition of glycolysis in MiaPaCa2 pancreatic cancer and A673 sarcoma cells. Moreover, robust target engagement of LDHA by lead compounds was demonstrated using the cellular thermal shift assay (CETSA), and drug-target residence time was determined via SPR. Analysis of these data suggests that drug-target residence time (off-rate) may be an important attribute to consider for obtaining potent cell-based inhibition of this cancer metabolism target.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5894102PMC
November 2017

Regulation of autophagy, mitochondrial dynamics, and cellular bioenergetics by 4-hydroxynonenal in primary neurons.

Autophagy 2017 29;13(11):1828-1840. Epub 2017 Sep 29.

a Center for Free Radical Biology , University of Alabama at Birmingham , Birmingham , AL , USA.

The production of reactive species contributes to the age-dependent accumulation of dysfunctional mitochondria and protein aggregates, all of which are associated with neurodegeneration. A putative mediator of these effects is the lipid peroxidation product 4-hydroxynonenal (4-HNE), which has been shown to inhibit mitochondrial function, and accumulate in the postmortem brains of patients with neurodegenerative diseases. This deterioration in mitochondrial quality could be due to direct effects on mitochondrial proteins, or through perturbation of the macroautophagy/autophagy pathway, which plays an essential role in removing damaged mitochondria. Here, we use a click chemistry-based approach to demonstrate that alkyne-4-HNE can adduct to specific mitochondrial and autophagy-related proteins. Furthermore, we found that at lower concentrations (5-10 μM), 4-HNE activates autophagy, whereas at higher concentrations (15 μM), autophagic flux is inhibited, correlating with the modification of key autophagy proteins at higher concentrations of alkyne-4-HNE. Increasing concentrations of 4-HNE also cause mitochondrial dysfunction by targeting complex V (the ATP synthase) in the electron transport chain, and induce significant changes in mitochondrial fission and fusion protein levels, which results in alterations to mitochondrial network length. Finally, inhibition of autophagy initiation using 3-methyladenine (3MA) also results in a significant decrease in mitochondrial function and network length. These data show that both the mitochondria and autophagy are critical targets of 4-HNE, and that the proteins targeted by 4-HNE may change based on its concentration, persistently driving cellular dysfunction.
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http://dx.doi.org/10.1080/15548627.2017.1356948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5788494PMC
June 2019

O-GlcNAc regulation of autophagy and α-synuclein homeostasis; implications for Parkinson's disease.

Mol Brain 2017 07 19;10(1):32. Epub 2017 Jul 19.

Department of Pathology, Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL, 35294-0017, USA.

Post-translational modification on protein Ser/Thr residues by O-linked attachment of ß-N-acetyl-glucosamine (O-GlcNAcylation) is a key mechanism integrating redox signaling, metabolism and stress responses. One of the most common neurodegenerative diseases that exhibit aberrant redox signaling, metabolism and stress response is Parkinson's disease, suggesting a potential role for O-GlcNAcylation in its pathology. To determine whether abnormal O-GlcNAcylation occurs in Parkinson's disease, we analyzed lysates from the postmortem temporal cortex of Parkinson's disease patients and compared them to age matched controls and found increased protein O-GlcNAcylation levels. To determine whether increased O-GlcNAcylation affects neuronal function and survival, we exposed rat primary cortical neurons to thiamet G, a highly selective inhibitor of the enzyme which removes the O-GlcNAc modification from target proteins, O-GlcNAcase (OGA). We found that inhibition of OGA by thiamet G at nanomolar concentrations significantly increased protein O-GlcNAcylation, activated MTOR, decreased autophagic flux, and increased α-synuclein accumulation, while sparing proteasomal activities. Inhibition of MTOR by rapamycin decreased basal levels of protein O-GlcNAcylation, decreased AKT activation and partially reversed the effect of thiamet G on α-synuclein monomer accumulation. Taken together we have provided evidence that excessive O-GlcNAcylation is detrimental to neurons by inhibition of autophagy and by increasing α-synuclein accumulation.
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http://dx.doi.org/10.1186/s13041-017-0311-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5517830PMC
July 2017

NADPH Oxidase 4 (Nox4) Suppresses Mitochondrial Biogenesis and Bioenergetics in Lung Fibroblasts via a Nuclear Factor Erythroid-derived 2-like 2 (Nrf2)-dependent Pathway.

J Biol Chem 2017 02 3;292(7):3029-3038. Epub 2017 Jan 3.

From the Division of Pulmonary, Allergy, and Critical Care Medicine.

Mitochondrial bioenergetics are critical for cellular homeostasis and stress responses. The reactive oxygen species-generating enzyme, NADPH oxidase 4 (Nox4), regulates a number of physiological and pathological processes, including cellular differentiation, host defense, and tissue fibrosis. In this study we explored the role of constitutive Nox4 activity in regulating mitochondrial function. An increase in mitochondrial oxygen consumption and reserve capacity was observed in murine and human lung fibroblasts with genetic deficiency (or silencing) of Nox4. Inhibition of Nox4 expression/activity by genetic or pharmacological approaches resulted in stimulation of mitochondrial biogenesis, as evidenced by elevated mitochondrial-to-nuclear DNA ratio and increased expression of the mitochondrial markers transcription factor A (TFAM), citrate synthase, voltage-dependent anion channel (VDAC), and cytochrome oxidase subunit 4 (COX IV). Induction of mitochondrial biogenesis was dependent on TFAM up-regulation but was independent of the activation of the peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α). The enhancement of mitochondrial bioenergetics as well as the increase in mitochondrial proteins in Nox4-deficient lung fibroblasts is inhibited by silencing of nuclear factor erythroid-derived 2-like 2 (Nrf2), supporting a key role for Nrf2 in control of mitochondrial biogenesis. Together, these results indicate a critical role for both Nox4 and Nrf2 in counter-regulation of mitochondrial biogenesis and metabolism.
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http://dx.doi.org/10.1074/jbc.M116.752261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5314196PMC
February 2017

Inhibition of autophagy with bafilomycin and chloroquine decreases mitochondrial quality and bioenergetic function in primary neurons.

Redox Biol 2017 04 18;11:73-81. Epub 2016 Nov 18.

Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294, United States; VA Medical Center, University of Alabama at Birmingham, Birmingham, AL 35294, United States. Electronic address:

Autophagy is an important cell recycling program responsible for the clearance of damaged or long-lived proteins and organelles. Pharmacological modulators of this pathway have been extensively utilized in a wide range of basic research and pre-clinical studies. Bafilomycin A1 and chloroquine are commonly used compounds that inhibit autophagy by targeting the lysosomes but through distinct mechanisms. Since it is now clear that mitochondrial quality control, particularly in neurons, is dependent on autophagy, it is important to determine whether these compounds modify cellular bioenergetics. To address this, we cultured primary rat cortical neurons from E18 embryos and used the Seahorse XF96 analyzer and a targeted metabolomics approach to measure the effects of bafilomycin A1 and chloroquine on bioenergetics and metabolism. We found that both bafilomycin and chloroquine could significantly increase the autophagosome marker LC3-II and inhibit key parameters of mitochondrial function, and increase mtDNA damage. Furthermore, we observed significant alterations in TCA cycle intermediates, particularly those downstream of citrate synthase and those linked to glutaminolysis. Taken together, these data demonstrate a significant impact of bafilomycin and chloroquine on cellular bioenergetics and metabolism consistent with decreased mitochondrial quality associated with inhibition of autophagy.
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http://dx.doi.org/10.1016/j.redox.2016.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5124357PMC
April 2017

Mitochondrial thiol modification by a targeted electrophile inhibits metabolism in breast adenocarcinoma cells by inhibiting enzyme activity and protein levels.

Redox Biol 2016 08 8;8:136-48. Epub 2016 Jan 8.

Department of Pathology, University of Alabama at Birmingham, AL, USA; Center for Free Radical Biology, University of Alabama at Birmingham, AL, USA. Electronic address:

Many cancer cells follow an aberrant metabolic program to maintain energy for rapid cell proliferation. Metabolic reprogramming often involves the upregulation of glutaminolysis to generate reducing equivalents for the electron transport chain and amino acids for protein synthesis. Critical enzymes involved in metabolism possess a reactive thiolate group, which can be modified by certain oxidants. In the current study, we show that modification of mitochondrial protein thiols by a model compound, iodobutyl triphenylphosphonium (IBTP), decreased mitochondrial metabolism and ATP in MDA-MB 231 (MB231) breast adenocarcinoma cells up to 6 days after an initial 24h treatment. Mitochondrial thiol modification also depressed oxygen consumption rates (OCR) in a dose-dependent manner to a greater extent than a non-thiol modifying analog, suggesting that thiol reactivity is an important factor in the inhibition of cancer cell metabolism. In non-tumorigenic MCF-10A cells, IBTP also decreased OCR; however the extracellular acidification rate was significantly increased at all but the highest concentration (10µM) of IBTP indicating that thiol modification can have significantly different effects on bioenergetics in tumorigenic versus non-tumorigenic cells. ATP and other adenonucleotide levels were also decreased by thiol modification up to 6 days post-treatment, indicating a decreased overall energetic state in MB231 cells. Cellular proliferation of MB231 cells was also inhibited up to 6 days post-treatment with little change to cell viability. Targeted metabolomic analyses revealed that thiol modification caused depletion of both Krebs cycle and glutaminolysis intermediates. Further experiments revealed that the activity of the Krebs cycle enzyme, aconitase, was attenuated in response to thiol modification. Additionally, the inhibition of glutaminolysis corresponded to decreased glutaminase C (GAC) protein levels, although other protein levels were unaffected. This study demonstrates for the first time that mitochondrial thiol modification inhibits metabolism via inhibition of both aconitase and GAC in a breast cancer cell model.
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http://dx.doi.org/10.1016/j.redox.2016.01.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4732023PMC
August 2016

Upregulation of autophagy decreases chlorine-induced mitochondrial injury and lung inflammation.

Free Radic Biol Med 2015 Aug 13;85:83-94. Epub 2015 Apr 13.

Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA; Pulmonary Injury and Repair Center, University of Alabama at Birmingham, Birmingham, AL 35294, USA; Center for Free Radical Biology, School of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA. Electronic address:

The mechanisms of toxicity during exposure of the airways to chlorinated biomolecules generated during the course of inflammation and to chlorine (Cl2) gas are poorly understood. We hypothesized that lung epithelial cell mitochondria are damaged by Cl2 exposure and activation of autophagy mitigates this injury. To address this, NCI-H441 (human lung adenocarcinoma epithelial) cells were exposed to Cl2 (100 ppm/15 min) and bioenergetics were assessed. One hour after Cl2, cellular bioenergetic function and mitochondrial membrane potential were decreased. These changes were associated with increased MitoSOX signal, and treatment with the mitochondrial redox modulator MitoQ attenuated these bioenergetic defects. At 6h postexposure, there was significant increase in autophagy, which was associated with an improvement of mitochondrial function. Pretreatment of H441 cells with trehalose (an autophagy activator) improved bioenergetic function, whereas 3-methyladenine (an autophagy inhibitor) resulted in increased bioenergetic dysfunction 1h after Cl2 exposure. These data indicate that Cl2 induces bioenergetic dysfunction, and autophagy plays a protective role in vitro. Addition of trehalose (2 vol%) to the drinking water of C57BL/6 mice for 6 weeks, but not 1 week, before Cl2 (400 ppm/30 min) decreased white blood cells in the bronchoalveolar lavage fluid at 6h after Cl2 by 70%. Acute administration of trehalose delivered through inhalation 24 and 1h before the exposure decreased alveolar permeability but not cell infiltration. These data indicate that Cl2 induces bioenergetic dysfunction associated with lung inflammation and suggests that autophagy plays a protective role.
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http://dx.doi.org/10.1016/j.freeradbiomed.2015.03.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4508227PMC
August 2015

Metabolic plasticity in resting and thrombin activated platelets.

PLoS One 2015 13;10(4):e0123597. Epub 2015 Apr 13.

Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; UAB Mitochondrial Medicine Laboratory, University of Alabama at Birmingham, Birmingham, Alabama, United States of America; Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama, United States of America.

Platelet thrombus formation includes several integrated processes involving aggregation, secretion of granules, release of arachidonic acid and clot retraction, but it is not clear which metabolic fuels are required to support these events. We hypothesized that there is flexibility in the fuels that can be utilized to serve the energetic and metabolic needs for resting and thrombin-dependent platelet aggregation. Using platelets from healthy human donors, we found that there was a rapid thrombin-dependent increase in oxidative phosphorylation which required both glutamine and fatty acids but not glucose. Inhibition of fatty acid oxidation or glutamine utilization could be compensated for by increased glycolytic flux. No evidence for significant mitochondrial dysfunction was found, and ATP/ADP ratios were maintained following the addition of thrombin, indicating the presence of functional and active mitochondrial oxidative phosphorylation during the early stages of aggregation. Interestingly, inhibition of fatty acid oxidation and glutaminolysis alone or in combination is not sufficient to prevent platelet aggregation, due to compensation from glycolysis, whereas inhibitors of glycolysis inhibited aggregation approximately 50%. The combined effects of inhibitors of glycolysis and oxidative phosphorylation were synergistic in the inhibition of platelet aggregation. In summary, both glycolysis and oxidative phosphorylation contribute to platelet metabolism in the resting and activated state, with fatty acid oxidation and to a smaller extent glutaminolysis contributing to the increased energy demand.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0123597PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4395425PMC
April 2016

Cardiomyocyte mitochondrial oxidative stress and cytoskeletal breakdown in the heart with a primary volume overload.

Am J Physiol Heart Circ Physiol 2015 Mar 16;308(6):H651-63. Epub 2015 Jan 16.

Department of Veterans Affairs Medical Center, Birmingham, Alabama; UAB Comprehensive Cardiovascular Center, University of Alabama at Birmingham, Birmingham, Alabama; Department of Medicine, Division of Cardiovascular Disease, University of Alabama at Birmingham, Birmingham, Alabama;

Left ventricular (LV) volume overload (VO) results in cardiomyocyte oxidative stress and mitochondrial dysfunction. Because mitochondria are both a source and target of ROS, we hypothesized that the mitochondrially targeted antioxidant mitoubiquinone (MitoQ) will improve cardiomyocyte damage and LV dysfunction in VO. Isolated cardiomyocytes from Sprague-Dawley rats were exposed to stretch in vitro and VO of aortocaval fistula (ACF) in vivo. ACF rats were treated with and without MitoQ. Isolated cardiomyocytes were analyzed after 3 h of cyclical stretch or 8 wk of ACF with MitoSox red or 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate to measure ROS and with tetramethylrhodamine to measure mitochondrial membrane potential. Transmission electron microscopy and immunohistochemistry were used for cardiomyocyte structural assessment. In vitro cyclical stretch and 8-wk ACF resulted in increased cardiomyocyte mitochondrial ROS production and decreased mitochondrial membrane potential, which were significantly improved by MitoQ. ACF had extensive loss of desmin and β₂-tubulin that was paralleled by mitochondrial disorganization, loss of cristae, swelling, and clustering identified by mitochondria complex IV staining and transmission electron microscopy. MitoQ improved mitochondrial structural damage and attenuated desmin loss/degradation evidenced by immunohistochemistry and protein expression. However, LV dilatation and fractional shortening were unaffected by MitoQ treatment in 8-wk ACF. In conclusion, although MitoQ did not affect LV dilatation or function in ACF, these experiments suggest a connection of cardiomyocyte mitochondria-derived ROS production with cytoskeletal disruption and mitochondrial damage in the VO of ACF.
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http://dx.doi.org/10.1152/ajpheart.00638.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4360051PMC
March 2015

Enhanced cardiac Akt/protein kinase B signaling contributes to pathological cardiac hypertrophy in part by impairing mitochondrial function via transcriptional repression of mitochondrion-targeted nuclear genes.

Mol Cell Biol 2015 Mar 22;35(5):831-46. Epub 2014 Dec 22.

Program in Molecular Medicine and Division of Endocrinology, Metabolism, and Diabetes, University of Utah, School of Medicine, Salt Lake City, Utah, USA Fraternal Order of Eagles Diabetes Research Center and Division of Endocrinology and Metabolism, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA

Sustained Akt activation induces cardiac hypertrophy (LVH), which may lead to heart failure. This study tested the hypothesis that Akt activation contributes to mitochondrial dysfunction in pathological LVH. Akt activation induced LVH and progressive repression of mitochondrial fatty acid oxidation (FAO) pathways. Preventing LVH by inhibiting mTOR failed to prevent the decline in mitochondrial function, but glucose utilization was maintained. Akt activation represses expression of mitochondrial regulatory, FAO, and oxidative phosphorylation genes in vivo that correlate with the duration of Akt activation in part by reducing FOXO-mediated transcriptional activation of mitochondrion-targeted nuclear genes in concert with reduced signaling via peroxisome proliferator-activated receptor α (PPARα)/PGC-1α and other transcriptional regulators. In cultured myocytes, Akt activation disrupted mitochondrial bioenergetics, which could be partially reversed by maintaining nuclear FOXO but not by increasing PGC-1α. Thus, although short-term Akt activation may be cardioprotective during ischemia by reducing mitochondrial metabolism and increasing glycolysis, long-term Akt activation in the adult heart contributes to pathological LVH in part by reducing mitochondrial oxidative capacity.
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http://dx.doi.org/10.1128/MCB.01109-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4323486PMC
March 2015

The Bioenergetic Health Index: a new concept in mitochondrial translational research.

Clin Sci (Lond) 2014 Sep;127(6):367-73

¶Department of Biostatistics, University of Alabama at Birmingham, Birmingham, AL 35294, U.S.A.

Bioenergetics has become central to our understanding of pathological mechanisms, the development of new therapeutic strategies and as a biomarker for disease progression in neurodegeneration, diabetes, cancer and cardiovascular disease. A key concept is that the mitochondrion can act as the 'canary in the coal mine' by serving as an early warning of bioenergetic crisis in patient populations. We propose that new clinical tests to monitor changes in bioenergetics in patient populations are needed to take advantage of the early and sensitive ability of bioenergetics to determine severity and progression in complex and multifactorial diseases. With the recent development of high-throughput assays to measure cellular energetic function in the small number of cells that can be isolated from human blood these clinical tests are now feasible. We have shown that the sequential addition of well-characterized inhibitors of oxidative phosphorylation allows a bioenergetic profile to be measured in cells isolated from normal or pathological samples. From these data we propose that a single value-the Bioenergetic Health Index (BHI)-can be calculated to represent the patient's composite mitochondrial profile for a selected cell type. In the present Hypothesis paper, we discuss how BHI could serve as a dynamic index of bioenergetic health and how it can be measured in platelets and leucocytes. We propose that, ultimately, BHI has the potential to be a new biomarker for assessing patient health with both prognostic and diagnostic value.
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http://dx.doi.org/10.1042/CS20140101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4202728PMC
September 2014

The role of GABARAPL1/GEC1 in autophagic flux and mitochondrial quality control in MDA-MB-436 breast cancer cells.

Autophagy 2014 Jun;10(6):986-1003

Department of Pathology; University of Alabama at Birmingham; Birmingham, AL USA; Center for Free Radical Biology; University of Alabama at Birmingham; Birmingham, AL USA; Department of Veterans Affairs; Birmingham VA Medical Center; Birmingham, AL USA.

GABARAPL1/GEC1 is an early estrogen-induced gene which encodes a protein highly conserved from C. elegans to humans. Overexpressed GABARAPL1 interacts with GABAA or kappa opioid receptors, associates with autophagic vesicles, and inhibits breast cancer cell proliferation. However, the function of endogenous GABARAPL1 has not been extensively studied. We hypothesized that GABARAPL1 is required for maintaining normal autophagic flux, and plays an important role in regulating cellular bioenergetics and metabolism. To test this hypothesis, we knocked down GABARAPL1 expression in the breast cancer MDA-MB-436 cell line by shRNA. Decreased expression of GABARAPL1 activated procancer responses of the MDA-MB-436 cells including increased proliferation, colony formation, and invasion. In addition, cells with decreased expression of GABARAPL1 exhibited attenuated autophagic flux and a decreased number of lysosomes. Moreover, decreased GABARAPL1 expression led to cellular bioenergetic changes including increased basal oxygen consumption rate, increased intracellular ATP, increased total glutathione, and an accumulation of damaged mitochondria. Taken together, our results demonstrate that GABARAPL1 plays an important role in cell proliferation, invasion, and autophagic flux, as well as in mitochondrial homeostasis and cellular metabolic programs.
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http://dx.doi.org/10.4161/auto.28390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4091181PMC
June 2014

Inhibition of autophagy and glycolysis by nitric oxide during hypoxia-reoxygenation impairs cellular bioenergetics and promotes cell death in primary neurons.

Free Radic Biol Med 2013 Dec 19;65:1215-1228. Epub 2013 Sep 19.

Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294-0017, USA; Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35294-0017, USA; Department of Veterans Affairs, Birmingham VA Medical Center, Birmingham, AL 35233, USA. Electronic address:

Excessive nitric oxide (NO) production is known to damage mitochondrial proteins and the autophagy repair pathway and so can potentially contribute to neurotoxicity. Accordingly, we hypothesized that protection against protein damage from reactive oxygen and nitrogen species under conditions of low oxygen by the autophagy pathway in neurons would be impaired by NO and enhance bioenergetic dysfunction. Rat primary cortical neurons had the same basal cellular respiration in hypoxia as in normoxia, whereas NO-exposed cells exhibited a gradual decrease in mitochondrial respiration in hypoxia. Upon reoxygenation, the respiration in NO-treated cells did not recover to prehypoxic levels. Hypoxia-reoxygenation in the presence of NO was associated with inhibition of autophagy, and the inability to recover during reoxygenation was exacerbated by an inhibitor of autophagy, 3-methyladenine. The effects of hypoxia could be recapitulated by inhibiting glycolytic flux under normoxic conditions. Under both normoxic and hypoxic conditions NO exposure induced immediate stimulation of glycolysis, but prolonged NO exposure, associated with irreversible inhibition of mitochondrial respiration in hypoxia, inhibited glycolysis. Importantly, we found that NO inhibited basal respiration under normoxic conditions only when glucose was absent from the medium or glycolysis was inhibited by 2-deoxy-d-glucose, revealing a novel NO-dependent mechanism for the inhibition of mitochondrial respiration that is modulated by glycolysis. Taken together these data suggest an oxygen-dependent interaction between mitochondrial respiration, glycolysis, and autophagy in protecting neuronal cells exposed to NO. Importantly, they indicate that mitochondrial dysfunction is intimately linked to a failure of glycolytic flux induced by exposure to NO. In addition, these studies provide new insights into the understanding of how autophagy and NO may play interactive roles in neuroinflammation-induced cellular damage, which is pertinent to our understanding of the pathology of neurodegenerative diseases in which excessive NO is generated.
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http://dx.doi.org/10.1016/j.freeradbiomed.2013.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859859PMC
December 2013

Bioenergetic and autophagic control by Sirt3 in response to nutrient deprivation in mouse embryonic fibroblasts.

Biochem J 2013 Sep;454(2):249-57

Center for Free Radical Biology, University of Alabama at Birmingham, 35294, USA.

Sirt3 (sirtuin 3) is an NAD-dependent deacetylase localized to mitochondria. Sirt3 expression is increased in mouse muscle and liver by starvation, which could protect against the starvation-dependent increase in oxidative stress and protein damage. Damaged proteins and organelles depend on autophagy for removal and this is critical for cell survival, but the role of Sirt3 is unclear. To examine this, we used Sirt3-KO (knockout) mouse embryonic fibroblast cells, and found that, under basal conditions, Sirt3-KO cells exhibited increased autophagy flux compared with WT (wild-type) cells. In response to nutrient deprivation, both WT and KO cells exhibited increased basal and ATP-linked mitochondrial respiration, indicating an increased energy demand. Both cells exhibited lower levels of phosphorylated mTOR (mammalian target of rapamycin) and higher autophagy flux, with KO cells exhibiting lower maximal mitochondrial respiration and reserve capacity, and higher levels of autophagy than WT cells. KO cells exhibit higher phospho-JNK (c-Jun N-terminal kinase) and phospho-c-Jun than WT cells under starvation conditions. However, inhibition of JNK activity in Sirt3-KO cells did not affect LC3-I (light chain 3-I) and LC3-II levels, indicating that Sirt3-regulated autophagy is independent of the JNK pathway. Caspase 3 activation and cell death are significantly higher in Sirt3-KO cells compared with WT cells in response to nutrient deprivation. Inhibition of autophagy by chloroquine exacerbated cell death in both WT and Sirt3-KO cells, and by 3-methyadenine exacerbated cell death in Sirt3-KO cells. These data suggest that nutrient deprivation-induced autophagy plays a protective role in cell survival, and Sirt3 decreases the requirement for enhanced autophagy and improves cellular bioenergetics.
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http://dx.doi.org/10.1042/BJ20130414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3927421PMC
September 2013

Mitochondria-targeted heme oxygenase-1 decreases oxidative stress in renal epithelial cells.

Am J Physiol Renal Physiol 2013 Aug 29;305(3):F255-64. Epub 2013 May 29.

Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

Mitochondria are both a source and target of the actions of reactive oxygen species and possess a complex system of inter-related antioxidants that control redox signaling and protect against oxidative stress. Interestingly, the antioxidant enzyme heme oxygenase-1 (HO-1) is not present in the mitochondria despite the fact that the organelle is the site of heme synthesis and contains multiple heme proteins. Detoxification of heme is an important protective mechanism since the reaction of heme with hydrogen peroxide generates pro-oxidant ferryl species capable of propagating oxidative stress and ultimately cell death. We therefore hypothesized that a mitochondrially localized HO-1 would be cytoprotective. To test this, we generated a mitochondria-targeted HO-1 cell line by transfecting HEK293 cells with a plasmid construct containing the manganese superoxide dismutase mitochondria leader sequence fused to HO-1 cDNA (Mito-HO-1). Nontargeted HO-1-overexpressing cells were generated by transfecting HO-1 cDNA (HO-1) or empty vector (Vector). Mitochondrial localization of HO-1 with increased HO activity in the mitochondrial fraction of Mito-HO-1 cells was observed, but a significant decrease in the expression of heme-containing proteins occurred in these cells. Both cytosolic HO-1- and Mito-HO-1-expressing cells were protected against hypoxia-dependent cell death and loss of mitochondrial membrane potential, but these effects were more pronounced with Mito-HO-1. Furthermore, decrement in production of tricarboxylic acid cycle intermediates following hypoxia was significantly mitigated in Mito-HO-1 cells. These data suggest that specific mitochondrially targeted HO-1 under acute pathological conditions may have beneficial effects, but the selective advantage of long-term expression is constrained by a negative impact on the synthesis of heme-containing mitochondrial proteins.
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http://dx.doi.org/10.1152/ajprenal.00160.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3742869PMC
August 2013