Glucose Deprivation-induced Cell Death Research Articles

Uridine prevents the glucose deprivation-induced death of immunostimulated astrocytes via the action of uridine phosphorylase

We previously reported that in immunostimulated astrocytes, glucose deprivation induced cell death via the loss of ATP, reduced glutathione, and mitochondrial transmembrane potential. The cytotoxicity was due to reactive nitrogen and oxygen species and blocked by adenosine, a purine nucleoside, via the preservation of cellular ATP. Here, we investigated whether uridine, a pyrimidine nucleoside, could prevent the glucose deprivation-induced cytotoxicity in LPS + IFN-γ-treated (immunostimulated) astrocytes. Glucose deprivation induced the death of immunostimulated cells, which was significantly reduced by uridine. Glucose deprivation rapidly decreased cellular ATP levels in immunostimulated astrocytes, which was also reversed by uridine. The inhibition of cellular uptake of uridine by S-(4-nitrobenzyl)-6-thioinosine attenuated the protective effect of uridine. mRNA and protein expression for uridine phosphorylase, an enzyme catalyzing reversible phosphorolysis of uridine, were observed in rat brain as well as primary astrocytes. 5-(Phenylthio)acyclouridine (PTAU), a specific inhibitor of uridine phosphorylase, inhibited the protective effect of uridine. Additionally, the loss of mitochondrial transmembrane potential and reduced glutathione by glucose deprivation in immunostimulated cells was attenuated by uridine, which was also reversed by PTAU. These results provide the first evidence that uridine protects immunostimulated astrocytes against the glucose deprivation-induced death by preserving intracellular ATP through the action of uridine phosphorylase.

Distinct roles of CysLT 1 and CysLT 2 receptors in oxygen glucose deprivation-induced PC12 cell death

Cysteinyl leukotrienes are involved in ischemic brain injury, and their receptors (CysLT 1 and CysLT 2) have been cloned. To clarify which subtype mediates the ischemic neuronal injury, we performed permanent transfection to increase CysLT 1 and CysLT 2 receptor expressions in PC12 cells. Oxygen glucose deprivation (OGD)-induced cell death was detected by Hoechst 33258 and propidium iodide fluorescent staining as well as by flow cytometry. OGD induced late phase apoptosis mainly and necrosis minimally. Over-expression of CysLT 1 receptor decreased and over-expression of CysLT 2 receptor increased OGD-induced cell death. An agonist LTD 4 (10 −7 M) also induced apoptosis, especially in CysLT 2 receptor over-expressing cells. A selective CysLT 1 receptor antagonist montelukast did not affect OGD-induced apoptosis; while non-selective CysLT receptor antagonist Bay u9773 inhibited OGD-induced apoptosis, especially in CysLT 2 receptor over-expressing cells. Thus, CysLT 1 and CysLT 2 receptors play distinct roles in OGD-induced PC12 cell death; CysLT 1 attenuates while CysLT 2 facilitates the cell death.

Mitogen-activated protein kinases (MAPKs) mediate SIN-1/glucose deprivation-induced death in rat primary astrocytes

Peroxynitrite is a potent neurotoxic molecule produced from a reaction between NO and superoxide and induces NO-mediated inflammation under neuropathological conditions. Previously, we reported that glucose deprivation induced ATP depletion and cell death in immunostimulated astrocytes, which was mainly due to peroxynitrite. In this study, the role of MAPKs (ERK1/2, p38MAPK, and JNK1SAPK) signal pathway in the SIN-1/glucose deprivation-induced death of astrocytes was examined. A combined treatment with glucose deprivation and 50 microM SIN-1, an endogenous peroxynitrite generator, rapidly and markedly increased the death in rat primary astrocytes. Also, SIN-1/glucose deprivation resulted in the activation of MAPKs, which was significantly blocked by the treatment with 20 microM MAPKs inhibitors (ERK1/2, PD98059; p38MAPK, SB203580; JNK/SAPK, SP600125). Interestingly, SIN-1/glucose deprivation caused the loss of intracellular ATP level, which was significantly reversed by MAPKs inhibitors. These results suggest that the activation of MAPKs plays an important role in SIN-1/glucose deprivation-induced cell death by regulating the intracellular ATP level.

Ionotropic glutamate receptors and glutamate transporters are involved in necrotic neuronal cell death induced by oxygen–glucose deprivation of hippocampal slice cultures

Organotypic hippocampal slice cultures represent a feasible model for studies of cerebral ischemia and the role of ionotropic glutamate receptors in oxygen–glucose deprivation-induced neurodegeneration. New results and a review of existing data are presented in the first part of this paper. The role of glutamate transporters, with special reference to recent results on inhibition of glutamate transporters under normal and energy-failure (ischemia-like) conditions is reviewed in the last part of the paper. The experimental work is based on hippocampal slice cultures derived from 7 day old rats and grown for about 3 weeks. In such cultures we investigated the subfield neuronal susceptibility to oxygen–glucose deprivation, the type of induced cell death and the involvement of ionotropic glutamate receptors. Hippocampal slice cultures were also used in our studies on glutamate transporters reviewed in the last part of this paper. Neurodegeneration was monitored and/or shown by cellular uptake of propidium iodide, loss of immunocytochemical staining for microtubule-associated protein 2 and staining with Fluoro-Jade B. To distinguish between necrotic vs. apoptotic neuronal cell death we used immunocytochemical staining for active caspase-3 (apoptosis indicator) and Hoechst 33342 staining of nuclear chromatin. Our experimental studies on oxygen–glucose deprivation confirmed that CA1 pyramidal cells were the most susceptible to this ischemia-like condition. Judged by propidium iodide uptake, a selective CA1 lesion, with only minor affection on CA3, occurred in cultures exposed to oxygen–glucose deprivation for 30 min. Nuclear chromatin staining by Hoechst 33342 and staining for active caspase-3 showed that oxygen–glucose deprivation induced necrotic cell death only. Addition of 10μM of the N-methyl- d-aspartate glutamate receptor antagonist MK-801, and 20μM of the non- N-methyl- d-aspartate glutamate receptor antagonist 2,3-dihyroxy-6-nitro-7-sulfamoyl-benzo( F)quinoxaline to the culture medium confirmed that both N-methyl- d-aspartate and non- N-methyl- d-aspartate ionotropic glutamate receptors were involved in the oxygen–glucose deprivation-induced cell death. Glutamate is normally quickly removed, from the extracellular space by sodium-dependent glutamate transporters. Effects of blocking the transporters by addition of the dl-threo-beta-benzyloxyaspartate are reviewed in the last part of the paper. Under normal conditions addition of dl-threo-beta-benzyloxyaspartate in concentrations of 25μM or more to otherwise untreated hippocampal slice cultures induced neuronal cell death, which was prevented by addition of 2,3-dihyroxy-6-nitro-7-sulfamoyl-benzo( F)quinoxaline and MK-801. In energy failure situations, like cerebral ischemia and oxygen–glucose deprivation, the transporters are believed to reverse and release glutamate to the extracellular space. Blockade of the transporters by a subtoxic (10μM) dose of dl-threo-beta-benzyloxyaspartate during oxygen–glucose deprivation (but not during the next 48 h after oxygen–glucose deprivation) significantly reduced the oxygen–glucose deprivation-induced propidium iodide uptake, suggesting a neuroprotective inhibition of reverse transporter activity by dl-threo-beta-benzyloxyaspartate during oxygen–glucose deprivation under these conditions. Adding to this, other results from our laboratory have demonstrated that pre-treatment of the slice cultures with glial cell-line derived neurotrophic factor upregulates glutamate transporters. As a logical, but in some glial cell-line derived neurotrophic factor therapy-related conditions clearly unwanted consequence the susceptibility for oxygen–glucose deprivation-induced glutamate receptor-mediated cell death is increased after glial cell-line derived neurotrophic factor treatment. In summary, we conclude that both ionotropic glutamate receptors and glutamate transporters are involved in oxygen–glucose deprivation-induced necrotic cell death in hippocampal slice cultures, which have proven to be a feasible tool in experimental studies on this topic.

Decreased glycolytic metabolism contributes to but is not the inducer of apoptosis following IL-3-starvation.

IL-3 regulates the glycolytic pathway. In Baf-3 cells IL-3 starvation leads to a decrease in glucose uptake and in lactate production. To determine if there is a link between the decreased metabolism induced by growth factor-starvation and the induction of cell death, we have compared the cell death characteristics and the metabolic modifications induced by IL-3-deprivation or glucose-deprivation in Baf-3 cells. We show that in both conditions cells die by an apoptotic process which involves the activation of similar Caspases. Different metabolic parameters (i.e. intracellular ATP levels and lactate accumulation in the culture medium) were measured. We show that IL-3 deprivation leads to a partial decrease in lactate production in contrast to glucose deprivation that completely inhibits lactate production. Similarly following IL-3-starvation a significant drop in the intracellular ATP levels in live cells is observed only after 16 h when a large fraction, more than 50 per cent of cells, is already apoptotic. On the contrary, glucose deprivation is followed by an abrupt decrease in ATP levels in the first 2 h of treatment. However, in the presence of IL-3, cells are able to survive for an extended time in these conditions since 70% of cells survived with low ATP levels for up to 16 h. This was not due to partial inhibition of the apoptotic process by the low level of ATP as glucose-deprivation in the absence of IL-3 led to faster death kinetics of Baf-3 cells compared with IL-3 starvation only. These results indicate that the drop in ATP levels and the triggering of apoptosis can be dissociated in time and that when the glycolytic pathway is strongly inhibited, cells are able to survive with relatively low ATP levels if IL-3 is present. Finally we show that induction of bcl-x by IL-3 protects cells from glucose-deprivation induced cell death.

Adenosine and Purine Nucleosides Protect Rat Primary Astrocytes from Peroxynitrite-Potentiated, Glucose Deprivation-Induced Death: Preservation of Intracellular ATP Level

Previously we have reported that immunostimulated astrocytes became highly vulnerable to glucose deprivation. In the present study we examined the effect of various kinds of nucleosides on the augmented death of glucose-deprived immunostimulated astrocytes. Preincubation with interferon-γ (100 U/ml) and lipopolysaccharide (1 μg/ml) for 48 h and continuous exposure to glucose deprivation (4 h) significantly induced the lactate dehydrogenase (LDH) release, as a marker of cell injury or death, from astrocytes. The glucose deprivation-induced augmented cell death in immunostimulated astrocytes was mimicked by exogenous peroxynitrite generator 3-morpholinosydnonimine (SIN-1). The increased death in immunostimulated or SIN-1-treated astrocytes deprived of glucose was blocked by adenosine and ATP. Other purine nucleos(t)ides, not pyrimidine nucleotides, also showed similar protective effects. Adenosine receptor agonist R(-)-N-(2-phenylisopropyl)-adenosine or N-cyclohexyladenosine did not alter the augmented cell death. Adenosine receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine, xanthine amine congener or 3,7-dimethyl-1-propargylxanthine also did not reverse the protective effect of adenosine. Intracellular ATP levels rapidly decreased prior to the LDH release in glucose-deprived immunostimulated astrocytes. The loss of intracellular ATP was prevented by adenosine and other purine nucleotides. The present results suggest that adenosine and their metabolites may protect astrocytes from peroxynitrite-potentiated, glucose deprivation-induced death by serving as substrates for intracellular ATP generation.

Neuroprotective and neurotoxic effects of monoamine oxidase-B inhibitors and derived metabolites under ischemia in PC12 cells

Selegiline and rasagiline are selective and irreversible monoamine oxidase-B inhibitors that exert neuroprotective effects in various preclinical models. The aim of the present study was to examine the effect of selegiline and its major metabolite, l-methamphetamine in comparison to rasagiline and its major metabolite, 1- R-aminoindan on oxygen–glucose deprivation induced cell death in nerve growth factor (NGF)-differentiated pheochromocytoma (PC12) cells. Our results show that selegiline reduces oxygen–glucose deprivation induced cell death by 30%. When the cultures were treated with rasagiline at similar concentrations, cell death induced by oxygen–glucose deprivation was reduced by 45–55%. l-methamphetamine, a major selegiline metabolite, but not 1- R-aminoindan, the major rasagiline metabolite, enhanced oxygen–glucose deprivation-induced cell death by 70%. Under normoxic conditions, both metabolites lack neurotoxicity. Concomitant exposure of the cultures under oxygen–glucose deprivation, to a combination of either selegiline and l-methamphetamine or rasagiline and 1- R-aminoindan, indicated that l-methamphetamine, but not 1- R-aminoindan, blocked the neuroprotective effect of the parental drug. These results suggest there may be a neuroprotective advantage of rasagiline over selegiline.

Glucocorticoids exacerbate peroxynitrite mediated potentiation of glucose deprivation-induced death of rat primary astrocytes

Glucocorticoids have been implicated in the exacerbation of several types of neurotoxicity in various neuropathological situations. In this study, we investigated the effect of a glucocorticoid dexamethasone on glucose deprivation induced cell death of immunostimulated rat primary astrocytes, which is dependent on the production of peroxynitrite from the immunostimulated cells [Choi et al. Glia, 31(2001) 155–164; J. Neuroimmunol. 112 (2001) 55–62]. Glucose deprivation in immunostimulated rat primary astrocytes results in the release of lactate dehydrogenase (LDH) after 5 h and co-treatment with dexamethasone (1–1000 nM) dose-dependently increased LDH release. Treatment of the exogenous peroxynitrite generator SIN-1 (20 μM), plus glucose deprivation, also increased LDH release after 6 h and co-treatment with dexamethasone dose-dependently increased LDH release. A glucocorticoid receptor antagonist, RU-486, reversed the potentiation of cell death by dexamethasone. Glucose deprivation in immunostimulated cells decreased the intracellular ATP levels, which preceded LDH release from the cell, and co-treatment with dexamethasone dose-dependently potentiated the depletion of intracellular ATP levels. In addition, dexamethasone further deteriorated SIN-1 plus glucose deprivation-induced decrease in mitochondrial transmembrane potential in rat primary astrocytes, which was reversed by RU-486. The results from the present study suggest that glucocorticoids may be detrimental to astrocytes in situations where activation of glial cells are observed, including ischemia and Alzheimer’s disease, by mechanisms involving depletion of intracellular ATP levels and deterioration of mitochondrial transmembrane potentials.

Protective role of Bcl2 in metabolic oxidative stress-induced cell death

Previous studies have shown that overexpression of Bcl2 protects cells from glucose deprivation-induced cell death in multidrug-resistant human breast carcinoma, MCF-7/ADR cells. In this study, we further investigated the protective role of Bcl2 in glucose deprivation-induced cytotoxicity. Although Bcl2 did not prevent a 3.2-fold increase in the level of hydroperoxide during glucose deprivation, it led to a compartmentalization of hydroperoxide molecules in the mitochondria. It also inhibited glucose deprivation-induced cytochrome c release from the mitochondria. It is possible that overexpression of Bcl2 prevents glucose deprivation-induced ceramide generation, probably by preventing the leakage of hydroperoxide from the mitochondria. We also observed that glucose deprivation induced a sixfold increase in oxidized glutathione content, as well as in thiol precursor content. Overexpression of Bcl2 suppressed an increase in oxidized glutathione content and thiol precursor content. Our results indicate that Bcl2 protects cells from metabolic oxidative stress-induced damage by inhibiting the leakage of hydroperoxide from the mitochondria and subsequently preventing ceramide generation. Preventing ceramide generation inhibits the signal transduction pathway and results in the suppression of cytochrome c release from the mitochondria.

Calcitonin Potentiates Oxygen–Glucose Deprivation-Induced Neuronal Death

Calcitonin is a hormone that decreases plasma calcium through inhibition of osteolysis. It is used in the treatment of osteoporosis and other bone disorders. Salmon calcitonin is typically utilized in individuals for whom use of estrogen is contraindicated, for example, women at high risk for breast cancer. In addition to actions on bone, calcitonin may have effects on the central nervous system. Receptors for calcitonin are present on central neurons, and salmon calcitonin has been shown to alter neuronal activity. Since salmon calcitonin is used clinically, and it can have actions on neurons, the present studies were designed to determine whether salmon calcitonin could alter death of cortical neurons. The effects of salmon calcitonin on neuronal death induced by exposure of murine cortical cultures to serum deprivation, staurosporine, oxygen–glucose deprivation, kainate, and NMDA were tested. Salmon calcitonin had no effect on apoptotic cell death in cortical cultures. However, acute treatment with salmon calcitonin (1–1000 nM) caused significant potentiation of neuronal death induced by oxygen–glucose deprivation. Similarly, salmon calcitonin potentiated cell death induced by exposure to kainate. In contrast, it did not potentiate cell death induced by exposure to NMDA. In fact, addition of a high concentration (1000 nM) of salmon calcitonin attenuated NMDA toxicity. These results indicate that calcitonin is not a survival factor for cortical neurons; however, it can alter excitotoxic cell death. The most interesting, and disturbing, effect is the ability of low concentrations of salmon calcitonin to potentiate oxygen–glucose deprivation-induced cell death.

Role of paxillin in metabolic oxidative stress-induced cytoskeletal reorganization: involvement of SAPK signal transduction pathway and PTP-PEST gene expression

Previous studies have shown that glucose deprivation-induced cell death is associated with apoptosis, which is characterized by cellular membrane blebbing in multi-drug-resistant human breast carcinoma MCF-7/ADR cells. In this study, we investigated the mechanism of glucose deprivation-induced cytoskeletal reorganization, which is known to be responsible for the morphological alterations. An increase in the formation of focal adhesion and stress fibers was observed during the early period of glucose deprivation (1–2 h). However, a disappearance of focal adhesion complexes and a loss of stress fiber formation along with membrane blebbing were observed when glucose deprivation continued. These alterations were delayed in MCF-7/ADR cells transfected with bcl-2 and completely suppressed by treatment with an antioxidant, N-acetyl- L-cysteine. These results indicated that glucose deprivation-induced oxidative stress caused the cytoskeletal reorganization. The glucose deprivation-induced alteration of cytoskeletal organization was further investigated by studying a modification of paxillin, one of the focal adhesion proteins. Immunoblotting with anti-paxillin antibody showed that the paxillin band shifted from 68 kDa to about 80 kDa during 1–4 h of glucose deprivation. The mobility shift indicated the modification of paxillin. This possibility was further studied by an immunoprecipitation assay with anti-paxillin/anti-phosphotyrosine antibody and phosphoamino acid analysis (PAA). The immunoprecipitation study revealed that the level of tyrosine phosphorylation of paxillin was maintained for 2 h and then markedly decreased without a change in the total level of paxillin. The PAA study showed that paxillin is dephosphorylated on tyrosine concurrent with phosphorylation on serine/threonine. Expression of a dominant-negative mutant of c-Jun NH 2-terminal kinase (JNK1) suppressed glucose deprivation-induced JNK1 activation, PTP-PEST gene expression, and alteration of paxillin. Taken together, these results suggest that the alteration of the phosphorylation/dephosphorylation of paxillin may be related to the cytoskeletal reorganization and these events are mediated by glucose deprivation-induced oxidative stress and the stress-activated protein kinase signal transduction pathway.

Dominant-negative Jun N-terminal protein kinase (JNK-1) inhibits metabolic oxidative stress during glucose deprivation in a human breast carcinoma cell line

Signal transduction pathways involved in glucose deprivation-induced oxidative stress were investigated in human breast carcinoma cells (MCF-7/ADR). In MCF-7/ADR, glucose deprivation-induced prolonged activation of c-Jun N-terminal kinase (JNK1) as well as cytotoxicity and the accumulation of oxidized glutathione. Glucose deprivation also caused significant increases in total glutathione, cysteine, γ-glutamylcysteine, and immunoreactive proteins corresponding to the catalytic as well as regulatory subunits of γ-glutamylcysteine synthetase, suggesting that the synthesis of glutathione increased as an adaptive response. Expression of a catalytically inactive dominant negative JNK1 in MCF-7/ADR inhibited glucose deprivation-induced cell death and the accumulation of oxidized glutathione as well as altered the duration of JNK activation from persistent (>2 h) to transient (30 min). In addition, stimulation of glutathione synthesis during glucose deprivation was not observed in cells expressing the highest levels of dominant negative protein. Finally, a linear dose response suppression of oxidized glutathione accumulation was noted for clones expressing increasing levels of dominant negative JNK1 during glucose deprivation. These results show that expression of a dominant negative JNK1 protein was capable of suppressing persistent JNK activation as well as oxidative stress and cytotoxicity caused by glucose deprivation in MCF-7/ADR. These findings support the hypothesis that JNK signaling pathways may control the expression of proteins contributing to cell death mediated by metabolic oxidative stress during glucose deprivation. Finally, these results support the concept that JNK signaling-induced shifts in oxidative metabolism may provide a general mechanism for understanding the diverse biological effects seen during the activation of JNK signaling cascades.

Brain-derived neurotrophic factor (BDNF) protects cultured rat cerebellar granule neurons against glucose deprivation-induced apoptosis.

In the present study, cell death induced by glucose deprivation in primary cultures of cerebellar granule neurons was examined. Glucose deprivation-induced apoptotic cell death was demonstrated using the terminal transferase-mediated (TdT) deoxyuridine triphosphate (d-UTP)-biotin nick end labeling (TUNEL) method and DNA fragmentation assays. When the effects of different neurotrophins on the survival of cerebellar granule neurons after glucose deprivation were assessed, BDNF, but not NT-3 or NGF, was found to protect cerebellar granule neurons against glucose deprivation-induced cell death. In addition, BDNF treatment increased c-Fos immunoreactivity in the cerebellar granule neurons. These results are consistent with the hypothesis that neuronal death due to glucose deprivation has a significant apoptotic component and that neurotrophins can protect against hypoglycemic damage.

Glucose deprivation-induced cytotoxicity in drug resistant human breast carcinoma MCF-7/ADR cells: role of c-myc and bcl-2 in apoptotic cell death.

We investigated the effect of glucose deprivation treatment on clonogenicity in multidrug-resistant human breast carcinoma MCF-7/ADR cells. Survival of MCF-7/ADR cells decreased exponentially up to 8 hours of incubation in the glucose-free medium. The surviving fraction of these cells for 8 hours of glucose-deprivation treatment was 1.5 x 10(-3). Photomicrographs and gel electrophoresis data suggest that glucose deprivation-induced cell death is associated with apoptosis. Data from western and northern blots showed an induction of c-myc gene expression during treatment with glucose-free medium in MCF-7/ADR cells. MCF-7/ADR cells transfected with c-myc antisense oligodeoxynucleotides became resistant to glucose deprivation-induced apoptosis. Overexpression of bcl-2 gene protected MCF-7/ADR cells from this apoptotic cell death. Taken together, these results indicate that c-myc expression is a necessary component of glucose-free medium induced apoptosis and bcl-2 prevents apoptotic death induced by c-myc.