Antimycin A-treated Cells Research Articles

Acute stimulation of glucose influx upon mitoenergetic dysfunction requires LKB1, AMPK, Sirt2 and mTOR-RAPTOR.

Mitochondria play a central role in cellular energy production, and their dysfunction can trigger a compensatory increase in glycolytic flux to sustain cellular ATP levels. Here, we studied the mechanism of this homeostatic phenomenon in C2C12 myoblasts. Acute (30 min) mitoenergetic dysfunction induced by the mitochondrial inhibitors piericidin A and antimycin A stimulated Glut1-mediated glucose uptake without altering Glut1 (also known as SLC2A1) mRNA or plasma membrane levels. The serine/threonine liver kinase B1 (LKB1; also known as STK11) and AMP-activated protein kinase (AMPK) played a central role in this stimulation. In contrast, ataxia-telangiectasia mutated (ATM; a potential AMPK kinase) and hydroethidium (HEt)-oxidizing reactive oxygen species (ROS; increased in piericidin-A- and antimycin-A-treated cells) appeared not to be involved in the stimulation of glucose uptake. Treatment with mitochondrial inhibitors increased NAD+ and NADH levels (associated with a lower NAD+:NADH ratio) but did not affect the level of Glut1 acetylation. Stimulation of glucose uptake was greatly reduced by chemical inhibition of Sirt2 or mTOR-RAPTOR. We propose that mitochondrial dysfunction triggers LKB1-mediated AMPK activation, which stimulates Sirt2 phosphorylation, leading to activation of mTOR-RAPTOR and Glut1-mediated glucose uptake.

Selective impairment on the proliferation of neural progenitor cells by oxidative phosphorylation disruption

Mitochondria produce ATP, regulate apoptosis, and maintain calcium homeostasis, and thus, mitochondrial dysfunction critically impairs nervous system development. Furthermore, the disruption of oxidative phosphorylation (OXPHOS) in mitochondria could lead to energy depletion and elevate oxidative stress. In the present study, the authors investigated how perturbation of the respiratory chain and bioenergetics affects neural progenitor cells (NPCs). Mitochondrial OXPHOS was impaired by inhibiting electron transfer using the antimycin A and ATP synthase inhibitor oligomycin. It was found that oligomycin impaired NPCs proliferation and was toxic at high concentrations, whereas antimycin A-treated cells showed no changes in NPCs proliferation. Although ROS production was elevated concentration-dependently by both inhibitors, oligomycin-treated C17.2 NPCs, but not antimycin A-treated NPCs, showed a significantly higher cell death rate and lower levels of intracellular ATP. These findings suggest that bioenergetic considerations are critically important for cell viability regulation in NPCs. Taken together, the present study shows that OXPHOS disruption can have a neurotoxic effect on NPCs, and thus, adversely influence the developing brain and the neurogenic capacity of the adult brain.

Enhancement of human embryonic stem cell pluripotency through inhibition of the mitochondrial respiratory chain

Human embryonic stem cell (hESC) pluripotency has been reported by several groups to be best maintained by culture under physiological oxygen conditions. Building on that finding, we inhibited complex III of the mitochondrial respiratory chain using antimycin A or myxothiazol to examine if specifically targeting the mitochondria would have a similar beneficial result for the maintenance of pluripotency. hESCs grown in the presence of 20 nM antimycin A maintained a compact morphology with high nuclear/cytoplasmic ratios. Furthermore, real-time PCR analysis demonstrated that the levels of Nanog mRNA were elevated 2-fold in antimycin A-treated cells. Strikingly, antimycin A was also able to replace bFGF in the media without compromising pluripotency, as long as autocrine bFGF signaling was maintained. Further analysis using low-density quantitative PCR arrays showed that antimycin A treatment reduced the expression of genes associated with differentiation, possibly acting through a ROS-mediated pathway. These results demonstrate that modulation of mitochondrial function results in increased pluripotency of the cell population, and sheds new light on the mechanisms and signaling pathways modulating hESC pluripotency.

Antimycin A induces apoptosis in As4.1 juxtaglomerular cells

Antimycin A, an inhibitor of electron transport in mitochondria, has been used as reactive oxygen species (ROS) generator in the biological system. Here, we investigated the in vitro effect of antimycin A on apoptosis in As4.1 juxtaglomerular cells. Antimycin A efficiently induced apoptosis in As4.1 cells as evidenced by flow cytometric detection of sub-G 1 DNA content, annexin V binding assay and DAPI staining. This apoptotic process was accompanied by loss of mitochondrial transmembrane potential (Δ Ψ m), Bcl-2 decrease, caspase-3 activation and PARP cleavage. All of caspase inhibitors tested in this experiment failed to rescue As4.1 cells from antimycin A-induced cell death at the time of 48 h in view of sub-G 1 cells and annexin V positive staining cells. However, with regard to the mitochondrial membrane potential (Δ Ψ m), pan caspase inhibitor (Z-VAD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK) at the concentration of 25 μM noticeably decreased the loss of mitochondrial membrane potential (Δ Ψ m) in antimycin A-treated cells. Taken together, we have demonstrated that antimycin A as an inhibitor of electron transport in mitochondria potently induces apoptosis in As4.1 juxtaglomerular cells.

The changes of intracellular H 2O 2 are an important factor maintaining mitochondria membrane potential of antimycin A-treated As4.1 juxtaglomerular cells

We investigated an involvement of ROS, such as H 2O 2 and O 2 − and GSH in the As4.1 cell death by antimycin A and examined whether ROS scavengers rescue antimycin A-induced As4.1 cell death and its mechanism. Levels of intracellular H 2O 2 and O 2 − were markedly increased in antimycin A-treated cells. Antimycin A reduced the intracellular GSH content. A ROS scavenger, Tiron down-regulated the production of intracellular H 2O 2. However, the reduction of intracellular H 2O 2 level did not change the apoptosis parameters, such as sub-G1 DNA content and annexin V binding. Interestingly, treatment of Tiron could partially prevent the loss of mitochondrial transmembrane potential (Δ Ψ m). Treatment of SOD and catalase also reduced the intracellular H 2O 2 and loss of mitochondrial transmembrane potential (Δ Ψ m) without reducing O 2 − level and apoptosis in antimycin A-treated As4.1 cells. All the ROS scavengers, SOD and catalase did not inhibit GSH depletion induced by antimycin A, resulting in failure of preventing the apoptosis. In addition, all the reagents including antimycin A did not induce any specific phase arrest of cell cycle in As4.1 cells. In summary, these results demonstrate that antimycin A generates potently ROS, H 2O 2 and O 2 − and induces the depletion of GSH content in As4.1 JG cells, and that Tiron, SOD and catalase inhibited partially the loss of mitochondrial transmembrane potential (Δ Ψ m) via the reduction of intracellular H 2O 2 level.

Mitochondria-mediated nuclear mutator phenotype in Saccharomyces cerevisiae.

Using Saccharomyces cerevisiae as a model organism, we analyzed the consequences of disrupting mitochondrial function on mutagenesis of the nuclear genome. We measured the frequency of canavanine-resistant colonies as a measure of nuclear mutator phenotype. Our data suggest that mitochondrial dysfunction leads to a nuclear mutator phenotype (i) when oxidative phosphorylation is blocked in wild-type yeast at mitochondrial complex III by antimycin A and (ii) in mutant strains lacking the entire mitochondrial genome (rho(0)) or those with deleted mitochondrial DNA (rho(-)). The nuclear mutation frequencies obtained for antimycin A-treated cells as well as for rho(-) and rho(0) cells were approximately 2- to 3-fold higher compared to untreated control and wild-type cells, respectively. Blockage of oxidative phosphorylation by antimycin A treatment led to increased intracellular levels of reactive oxygen species (ROS). In contrast, inactivation of mitochondrial activity (rho(-) and rho(0)) led to decreased intracellular levels of ROS. We also demonstrate that in rho(0) cells the REV1, REV3 and REV7 gene products, all implicated in error-prone translesion DNA synthesis (TLS), mediate mutagenesis in the nuclear genome. However, TLS was not involved in nuclear DNA mutagenesis caused by inhibition of mitochondrial function by antimycin A. Together, our data suggest that mitochondrial dysfunction is mutagenic and multiple pathways are involved in this nuclear mutator phenotype.

Perturbations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia.

The effects of ischemia on the maturation of secretory proteins are not well understood. Among several events that occur during ischemia-reperfusion are a rapid and extensive decrease in ATP levels and an alteration of cellular oxidative state. Since the normal folding and assembly of secretory proteins are mediated by endoplasmic reticulum (ER) molecular chaperones, the function of which depends on ATP and maintenance of an appropriate redox environment, ischemia might be expected to perturb folding of secretory proteins. In this study, whole animal and cultured cell models for the epithelial ischemic state were used to examine this possibility. After acute kidney ischemia, marked increases in the mRNA levels of the ER chaperones glucose-regulated protein (grp)78/immunoglobulin-binding protein (BiP), grp94, and ER protein (ERp)72 were noted. Likewise, when cellular ATP was depleted to less than 10% of control with antimycin A, mRNA levels of BiP, ERp72, and grp94 were increased in kidney and thyroid epithelial cell culture models. Since the signal for the up-regulation of these stress proteins is believed to be the accumulation of misfolded/misassembled secretory proteins in the ER, their induction after ischemia in vivo and antimycin treatment of cultured cells suggests that maturation of secretory proteins in the ER lumen might indeed be perturbed. To analyze the effects of antimycin A on the maturation of secretory proteins, we studied the fate of thyroglobulin (Tg), a large oligomeric secretory glycoprotein, the folding and assembly of which seems to require a variety of ER chaperones. Treatment of cultured thyroid epithelial cells with antimycin A greatly inhibited ( > 90%) the secretion of Tg. Sucrose density gradient analysis revealed that in antimycin A-treated cells Tg associates into large macromolecular complexes which, by immunofluorescence, appeared to localize to the ER. Furthermore, coimmunoprecipitation studies after antimycin A treatment demonstrated that Tg stably associates with BiP, grp94, and ERp72. Together, our results suggest that a key cellular lesion in ischemia is the misfolding of secretory proteins as they transit the ER, and this leads not only to increased expression of ER chaperones but also to their stable association with and the subsequent retention of at least some misfolded secretory proteins.