Assessing bioenergetic function in response to oxidative stress by metabolic profiling

It is now clear that mitochondria are an important target for oxidative stress in a broad range of pathologies, including cardiovascular disease, diabetes, neurodegeneration, and cancer. Methods for assessing the impact of reactive species on isolated mitochondria are well established but constrained by the need for large amounts of material to prepare intact mitochondria for polarographic measurements. With the availability of high-resolution polarography and fluorescence techniques for the measurement of oxygen concentration in solution, measurements of mitochondrial function in intact cells can be made. Recently, the development of extracellular flux methods to monitor changes in oxygen concentration and pH in cultures of adherent cells in multiple-sample wells simultaneously has greatly enhanced the ability to measure bioenergetic function in response to oxidative stress. Here we describe these methods in detail using representative cell types from renal, cardiovascular, nervous, and tumorigenic model systems while illustrating the application of three protocols to analyze the bioenergetic response of cells to oxidative stress.     

Regulation of vascular smooth muscle cell proliferation, migration and death by heparan sulfate 6-O-endosulfatase1

Migration, proliferation and death of vascular smooth muscle cells (VSMC) are important events in vascular pathology regulated by heparan sulfate proteoglycans and hence potentially by cell surface HS 6-O-endosulfatase1 (sulf1). Sulf1 mRNA expression was increased in cultured VSMC compared to rat aorta. Furthermore, adenovirus mediated overexpression of quail sulf1 decreased adhesion, and increased proliferation and apoptosis of VSMC. Overexpression of a dominant negative variant also decreased adhesion of VSMC and increased proliferation, apoptosis, migration and chemotaxis of VSMC. Our results imply that only normal levels of 6-O-sulfation maintained by sulf1 are optimal for several functions of VSMC.     

Regulation of interleukin-4 signaling by extracellular reduction of intramolecular disulfides

Interleukin-4 (IL-4) contains three structurally important intramolecular disulfides that are required for the bioactivity of the cytokine. We show that the cell surface of HeLa cells and endotoxin-activated monocytes can reduce IL-4 intramolecular disulfides in the extracellular space and inhibit binding of IL-4 to the IL-4Rα receptor. IL-4 disulfides were in vitro reduced by thioredoxin 1 (Trx1) and protein disulfide isomerase (PDI). Reduction of IL-4 disulfides by the cell surface of HeLa cells was inhibited by auranofin, an inhibitor of thioredoxin reductase that is an electron donor to both Trx1 and PDI. Both Trx1 and PDI have been shown to be located at the cell surface and our data suggests that these enzymes are involved in catalyzing reduction of IL-4 disulfides. The pro-drug N-acetylcysteine (NAC) that promotes T-helper type 1 responses was also shown to mediate the reduction of IL-4 disulfides. Our data provides evidence for a novel redox dependent pathway for regulation of cytokine activity by extracellular reduction of intramolecular disulfides at the cell surface by members of the thioredoxin enzyme family.     

Upregulation of S100 calcium-binding protein A9 is required for induction of smooth muscle cell proliferation by a periodontal pathogen

We investigated the effect of a periodontal pathogen, Porphyromonas gingivalis, on human aortic smooth muscle cell (hAOSMC) proliferation as mechanisms of atherosclerosis. Cultured hAOSMCs exposed to the supernatant of plasma incubated with P. gingivalis showed a marked transformation from a contractile to proliferative phenotype, resulting in enhancement of cell growth. DNA microarray analysis revealed a P. gingivalis-dependent upregulation of S100A9 in hAOSMCs. Small interference-RNA for S100A9 dramatically attenuated the effect of P. gingivalis on transformation and proliferation of hAOSMCs. Our data suggested that upregulation of S100A9 mediated by P. gingivalis is an important event in the development of aortic intimal hyperplasia.     

Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor

Obstructive sleep apnea is characterized by intermittent hypoxia (IH), and associated with cardiovascular diseases, such as stroke and heart failure. These cardiovascular diseases have a relation to atherosclerosis marked by the proliferation of vascular smooth muscle cells (VSMCs). In this study, we investigated the influence of IH on cultured rat aortic smooth muscle cell (RASMC). The proliferation of RASMC was significantly increased by IH without changing the level of apoptosis. In order to see what induces RASMC proliferation, we investigated the influence of normoxia (N)-, IH- and sustained hypoxia (SH)-treated cell conditioned media on RASMC proliferation. IH-treated cell conditioned medium significantly increased RASMC proliferation compared with N-treated cell conditioned medium, but SH-treated cell conditioned medium did not. We next investigated the epidermal growth factor (EGF) family as autocrine growth factors. Among the EGF family, we found significant increases in mRNAs for epiregulin (ER), amphiregulin (AR) and neuregulin-1 (NRG1) in IH-treated cells and mature ER in IH-treated cell conditioned medium. We next investigated the changes in erbB family receptors that are receptors for ER, AR and NRG1, and found that erbB2 receptor mRNA and protein expressions were increased by IH, but not by SH. Phosphorylation of erbB2 receptor at Tyr-1248 that mediates intracellular signaling for several physiological effects including cell proliferation was increased by IH, but not by SH. In addition, inhibitor for erbB2 receptor suppressed IH-induced cell proliferation. These results provide the first demonstration that IH induces VSMC proliferation, and suggest that EGF family, such as ER, AR and NRG1, and erbB2 receptor could be involved in the IH-induced VSMC proliferation.     

Redox regulation of the tumor suppressor PTEN by glutathione

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expressed in Saccharomyces cerevisiae was reversibly oxidized by hydrogen peroxide and reduced by cellular reductants. Reduction of hPTEN was delayed in each of S. cerevisiae gsh1Δ and gsh2Δ mutants. Expression of γ-glutamylcysteine synthetase Gsh1 in the gsh1Δ mutant rescued regeneration rate of hPTEN. Oxidized hPTEN was reduced by glutathione in a concentration- and time-dependent manner. Glutathionylated PTEN was detected. Incubation of 293T cells with BSO and knockdown expression of GCLc in HeLa cells by siRNA resulted in the delay of reduction of oxidized PTEN. Also, in HeLa cells transfected with GCLc siRNA, stimulation with epidermal growth factor resulted in the increase of oxidized PTEN and phosphorylation of Akt. These results suggest that the reduction of oxidized hPTEN is mediated by glutathione.     

Solubilization,purification and reconstitution of Ca-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: Preservation of native structure and function of the enzyme by DHPC

The properties of Ca²⁺-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C₁₂E₈) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca²⁺-ATPase with a greater specific activity than solubilization with C₁₂E₈ or Triton X-100. DHPC was determined to be superior to C₁₂E₈; while that the C₁₂E₈ was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca²⁺-ATPase retained the E1Ca−E1*Ca conformational transition as that observed for native microsomes; whereas the C₁₂E₈ and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca²⁺ transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C₁₂E₈ and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca²⁺-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C₁₂E₈ and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca²⁺ uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca²⁺-ATPase retained more organized and native secondary conformation compared to C₁₂E₈ and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C₁₂E₈ and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca²⁺-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C₁₂E₈ and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein–lipid interactions in the function of the membrane-bound enzyme.     

Differentiation of SH-SY5Y cells to a neuronal phenotype changes cellular bioenergetics and the response to oxidative stress

Cell differentiation is associated with changes in metabolism and function. Understanding these changes during differentiation is important in the context of stem cell research, cancer, and neurodegenerative diseases. An early event in neurodegenerative diseases is the alteration of mitochondrial function and increased oxidative stress. Studies using both undifferentiated and differentiated SH-SY5Y neuroblastoma cells have shown distinct responses to cellular stressors; however, the mechanisms remain unclear. We hypothesized that because the regulation of glycolysis and oxidative phosphorylation is modulated during cellular differentiation, this would change bioenergetic function and the response to oxidative stress. To test this, we used retinoic acid (RA) to induce differentiation of SH-SY5Y cells and assessed changes in cellular bioenergetics using extracellular flux analysis. After exposure to RA, the SH-SY5Y cells had an increased mitochondrial membrane potential, without changing mitochondrial number. Differentiated cells exhibited greater stimulation of mitochondrial respiration with uncoupling and an increased bioenergetic reserve capacity. The increased reserve capacity in the differentiated cells was suppressed by the inhibitor of glycolysis 2-deoxy-d-glucose. Furthermore, we found that differentiated cells were substantially more resistant to cytotoxicity and mitochondrial dysfunction induced by the reactive lipid species 4-hydroxynonenal or the reactive oxygen species generator 2,3-dimethoxy-1,4-naphthoquinone. We then analyzed the levels of selected mitochondrial proteins and found an increase in complex IV subunits, which we propose contributes to the increase in reserve capacity in the differentiated cells. Furthermore, we found an increase in MnSOD that could, at least in part, account for the increased resistance to oxidative stress. Our findings suggest that profound changes in mitochondrial metabolism and antioxidant defenses occur upon differentiation of neuroblastoma cells to a neuron-like phenotype.     

Promising effects of the 4HPR-BSO combination in neuroblastoma monolayers and spheroids

To enhance the efficacy of fenretinide (4HPR)-induced reactive oxygen species (ROS) in neuroblastoma, 4HPR was combined with buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, in neuroblastoma cell lines and spheroids, the latter being a three-dimensional tumor model. 4HPR exposure (2.5-10 μM, 24 h) resulted in ROS induction (114-633%) and increased GSH levels (68-120%). A GSH depletion of 80% of basal levels was observed in the presence of BSO (25-100 μM, 24 h). The 4HPR-BSO combination resulted in slightly increased ROS levels (1.1- to 1.3-fold) accompanied by an increase in cytotoxicity (110-150%) compared to 4HPR treatment alone. A correlation was observed between the ROS-inducing capacity of each cell line and the increase in cytotoxicity induced by 4HPR-BSO compared to 4HPR. No significant correlation between baseline antioxidant levels and sensitivity to 4HPR or BSO was observed. In spheroids, 4HPR-BSO induced a strong synergistic growth retardation and induction of apoptosis. Our data show that BSO increased the cytotoxic effects of 4HPR in neuroblastoma monolayers and spheroids in ROS-producing cell lines. This indicates that the 4HPR-BSO combination might be a promising new strategy in the treatment of neuroblastoma.     

Induction of necrotic cell death and mitochondrial permeabilization by heme binding protein 2/SOUL

We found that heme-binding protein 2/SOUL sensitised NIH3T3 cells to cell death induced by A23187 and etoposide, but it did not affect reactive oxygen species formation. In the presence of sub-threshold calcium, recombinant SOUL provoked mitochondrial permeability transition (mPT) in vitro that was inhibited by cyclosporine A (CsA). This effect was verified in vivo by monitoring the dissipation of mitochondrial membrane potential. Flow cytometry analysis showed that SOUL promoted necrotic death in A23187 and etoposide treated cells, which effect was prevented by CsA. These data suggest that besides its heme-binding properties SOUL promotes necrotic cell death by inducing mPT.     

Binding preference of eIF4E for 4E-binding protein isoform and function of eIF4E N-terminal flexible region for interaction, studied by SPR analysis

To investigate the binding preference of eIF4E for the three eIF4E-binding isoforms (4E-BP1-3) and the function of N-terminal flexible region of eIF4E for their interactions, the binding parameters of recombinant full-length and N-terminal residues-deleted eIF4Es with 4E-BP1-3 were investigated by the surface plasmon resonance (SPR) analysis. Consequently, it was clarified that 4E-BP2 exhibits the highest binding affinity for both m7GTP-bound and -unbound full-length eIF4Es when compared with 4E-BP1 and 4E-BP3. This is primarily due to the difference among their dissociation rates, because their association rates are almost the same. Interestingly, the deletion of the 33 N-terminal residues of eIF4E increased its binding affinities for 4E-BP1 and 4E-BP2 markedly, whereas such a change was not observed by at least the N-terminal deletion up to 26 residues. In contrast, the binding parameters of 4E-BP3 were hardly influenced by N-terminal deletion up to 33 residues. From the comparison of the amino acid sequences of 4E-BP1-3, the present result indicates the importance of N-terminal flexible region of eIF4E for the suppressive binding with 4E-BP1 and 2, together with the possible contribution of N-terminal sequence of 4E-BP isoform to the regulative binding to eIF4E.     

Kinetic model of the inhibition of respiration by endogenous nitric oxide in intact cells

Nitric oxide (NO) inhibits mitochondrial respiration by decreasing the apparent affinity of cytochrome c oxidase (CcO) for oxygen. Using iNOS-transfected HEK 293 cells to achieve regulated intracellular NO production, we determined NO and O₂ concentrations and mitochondrial O₂ consumption by high-resolution respirometry over a range of O₂ concentrations down to nanomolar. Inhibition of respiration by NO was reversible, and complete NO removal recovered cell respiration above its routine reference values. Respiration was observed even at high NO concentrations, and the dependence of IC₅₀ on [O₂] exhibits a characteristic but puzzling parabolic shape; both these features imply that CcO is protected from complete inactivation by NO and are likely to be physiologically relevant. We present a kinetic model of CcO inhibition by NO that efficiently predicts experimentally determined respiration at physiological O₂ and NO concentrations and under hypoxia, and accurately predicts the respiratory responses under hyperoxia. The model invokes competitive and uncompetitive inhibition by binding of NO to the reduced and oxidized forms of CcO, respectively, and suggests that dissociation of NO from reduced CcO may involve its O₂-dependent oxidation. It also explains the non-linear dependence of IC₅₀ on O₂ concentration, and the hyperbolic increase of c₅₀ as a function of NO concentration.     

Proteasomal degradation of the multifunctional regulator YB-1 is mediated by an F-Box protein induced during programmed cell death

F-Box proteins (FBPs) are variable adaptor proteins that earmark protein substrates for ubiquination and destruction by the proteasome. Through their N-terminal F-box motif, they couple specific protein substrates to a catalytic machinery known as SCF (Skp-1/Cul1/F-Box) E3-ubiquitin ligase. Typical FBPs bind the specific substrates in a phosphorylation dependent manner via their C-termini using either leucine rich repeats (LRR) or tryptophan-aspartic acid (WD40) domains for substrate recognition. By using a gene trap strategy that selects for genes induced during programmed cell death, we have isolated the mouse homolog of the hypothetical human F-Box protein 33 (FBX33). Here we identify FBX33 as a component of an SCF E3-ubiquitin ligase that targets the multifunctional regulator Y-box binding protein 1 (YB-1)/dbpB/p50 for polyubiquitination and destruction by the proteasome. By targeting YB-1 for proteasomal degradation, FBX33 negatively interferes with YB-1 mediated functions. In contrast to typical FBPs, FBX33 has no C-terminal LRR or WD40 domains and associates with YB-1 via its N-terminus. The present study confirms the existence of a formerly hypothetical F-Box protein in living cells and describes one of its substrates.     

The direct effect of leptin on skeletal muscle thermogenesis is mediated by substrate cycling between de novo lipogenesis and lipid oxidation

We report here studies that integrate data of respiration rate from mouse skeletal muscle in response to leptin and pharmacological interference with intermediary metabolism, together with assays for phosphatidylinositol 3-kinase (PI3K) and AMP-activated protein kinase (AMPK). Our results suggest that the direct effect of leptin in stimulating thermogenesis in skeletal muscle is mediated by substrate cycling between de novo lipogenesis and lipid oxidation, and that this cycle requires both PI3K and AMPK signaling. This substrate cycling linking glucose and lipid metabolism to thermogenesis provides a novel thermogenic mechanism by which leptin protects skeletal muscle from excessive fat storage and lipotoxicity.     

Amelioration of cadmium-induced cardiac impairment by taurine

The present study has been designed to investigate the protective role of taurine (2-aminoethanesulfonic acid), a sulfur containing conditionally essential amino acid, against cadmium-induced cardiac dysfunction in mice. Cadmium chloride (CdCl(2)) was used as the source of cadmium and it was administered orally at a dose of 4mg/kg body weight for 6 days. Cadmium exposure caused significant accumulation of the cadmium and iron in mice hearts tissue. Levels of serum specific markers related to cardiac impairments, e.g. total cholesterol, HDL cholesterol and triglyceride were altered due to cadmium toxicity. Reduction in the activities of antioxidant enzymes, namely, superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx) and glucose-6-phosphate dehydrogenase (G6PD) have been observed in cadmium exposed mice. Cadmium intoxication also decreased the cardiac glutathione (GSH) and total thiols contents and increased the levels of oxidized glutathione (GSSG), lipid peroxidation end products, protein carbonyl content and the extent of DNA fragmentation. Oral administration of taurine at a dose of 100mg/kg body weight for 5 days, however, prevented all the toxin-induced oxidative impairments mentioned above. "Ferric Reducing/Antioxidant Power (FRAP) assay" showed that taurine could protect the cardiac tissue by preventing cadmium-induced reduction of the intracellular antioxidant power. Histological examination of cardiac segments also supported the beneficial role of taurine against cadmium-induced damages in the murine hearts. Effect of a well established antioxidant, vitamin C has been included in the study as a positive control. Combining all, results suggest that taurine attenuates cadmium-induced impairment in mice hearts.     

Availability of the key metabolic substrates dictates the respiratory response of cancer cells to the mitochondrial uncoupling

Active glycolysis and glutaminolysis provide bioenergetic stability of cancer cells in physiological conditions. Under hypoxia, metabolic and mitochondrial disorders, or pharmacological treatment, a deficit of key metabolic substrates may become life-threatening to cancer cells. We analysed the effects of mitochondrial uncoupling by FCCP on the respiration of cells fed by different combinations of Glc, Gal, Gln and Pyr. In cancer PC12 and HCT116 cells, a large increase in O₂ consumption rate (OCR) upon uncoupling was only seen when Gln was combined with either Glc or Pyr. Inhibition of glutaminolysis with BPTES abolished this effect. Despite the key role of Gln, addition of FCCP inhibited respiration and induced apoptosis in cells supplied with Gln alone or Gal/Gln. For all substrate combinations, amplitude of respiratory responses to FCCP did not correlate with Akt, Erk and AMPK phosphorylation, cellular ATP, and resting OCR, mitochondrial Ca^2 + or membrane potential. However, we propose that proton motive force could modulate respiratory response to FCCP by regulating mitochondrial transport of Gln and Pyr, which decreases upon mitochondrial depolarisation. As a result, an increase in respiration upon uncoupling is abolished in cells, deprived of Gln or Pyr (Glc). Unlike PC12 or HCT116 cells, mouse embryonic fibroblasts were capable of generating pronounced response to FCCP when deprived of Gln, thus exhibiting lower dependence on glutaminolysis. Overall, the differential regulation of the respiratory response to FCCP by metabolic environment suggests that mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function, and can be explored for selective cancer treatment.