Decrease In Oxidative Phosphorylation Research Articles

PCramoll and rCramoll lectins induce cell death in human prostate adenocarcinoma (PC-3) cells by impairment of mitochondrial homeostasis

Lectins from Cratylia mollis seed have shown potential in vivo antitumor actions, however the mechanism have not yet been addressed. Here we evaluated the antitumor effects of native (pCramoll) and recombinant (rCramoll) lectins from C. mollis against human prostate adenocarcinoma (PC-3) cells. The viability of PC-3 cells was analyzed with the MTT assay and ANNEXIN V/propidium iodide staining. The actions of pCramoll or rCramoll on mitochondrial superoxide production, free cytosolic calcium concentration and mitochondrial membrane potential were evaluated using fluorescent probes (MitoSox Red, Fura 2-AM and safranin O, respectively). pCramoll and rCramoll reduced the viability of PC-3 cells in a dose-dependent manner. Both lectins increased the generation of mitochondrial superoxide as well as the concentration of cytosolic calcium. These changes led to a decrease in oxidative phosphorylation, which impaired the formation of ATP. The resulting cell death was not blocked by MPT (mitochondrial permeability transition) inhibitors (Debio 025 or bongkrekic acid). Thus pCramoll and rCramoll promote PC-3 cell death through calcium signaling, leading to mitochondrial collapse. This work provides more insights into the action of pCramoll and rCramoll against cancer cells. These lectins represent valuable tools for biomedical research.

Microbial stimulation of different Toll-like receptor signalling pathways induces diverse metabolic programmes in human monocytes.

Microbial stimuli such as lipopolysaccharide (LPS) induce robust metabolic rewiring in immune cells known as the Warburg effect. It is unknown whether this increase in glycolysis and decrease in oxidative phosphorylation (OXPHOS) is a general characteristic of monocytes that have encountered a pathogen. Using CD14+ monocytes from healthy donors, we demonstrated that most microbial stimuli increased glycolysis, but that only stimulation of Toll-like receptor (TLR) 4 with LPS led to a decrease in OXPHOS. Instead, activation of other TLRs, such as TLR2 activation by Pam3CysSK4 (P3C), increased oxygen consumption and mitochondrial enzyme activity. Transcriptome and metabolome analysis of monocytes stimulated with P3C versus LPS confirmed the divergent metabolic responses between both stimuli, and revealed significant differences in the tricarboxylic acid cycle, OXPHOS and lipid metabolism pathways following stimulation of monocytes with P3C versus LPS. At a functional level, pharmacological inhibition of complex I of the mitochondrial electron transport chain diminished cytokine production and phagocytosis in P3C- but not LPS-stimulated monocytes. Thus, unlike LPS, complex microbial stimuli and the TLR2 ligand P3C induce a specific pattern of metabolic rewiring that involves upregulation of both glycolysis and OXPHOS, which enables activation of host defence mechanisms such as cytokine production and phagocytosis.

Influence of mitochondrial dynamics by selenium in trophoblasts

Influence of mitochondrial dynamics by selenium in trophoblasts

Defining the effects of storage on platelet bioenergetics: The role of increased proton leak

The quality of platelets decreases over storage time, shortening their shelf life and potentially worsening transfusion outcomes. The changes in mitochondrial function associated with platelet storage are poorly defined and to address this we measured platelet bioenergetics in freshly isolated and stored platelets. We demonstrate that the hypotonic stress test stimulates both glycolysis and oxidative phosphorylation and the stored platelets showed a decreased recovery to this stress. We found no change in aggregability between the freshly isolated and stored platelets. Bioenergetic parameters were changed including increased proton leak and decreased basal respiration and this was reflected in a lower bioenergetic health index (BHI). Mitochondrial electron transport, measured in permeabilized platelets, showed only minor changes which are unlikely to have a significant impact on platelet function. There were no changes in basal glycolysis between the fresh and stored platelets, however, glycolytic rate was increased in stored platelets when mitochondrial ATP production was inhibited. The increase in proton leak was attenuated by the addition of albumin, suggesting that free fatty acids could play a role in increasing proton leak and decreasing mitochondrial function. In summary, platelet storage causes a modest decrease in oxidative phosphorylation driven by an increase in mitochondrial proton leak, which contributes to the decreased recovery to hypotonic stress.

Analyses of the transcriptome and metabolome demonstrate that HIF1α mediates altered tumor metabolism in clear cell renal cell carcinoma.

Hypoxia inducible factor 1 alpha (HIF1α) is a transcription factor that is frequently stabilized and active in human clear cell renal cell carcinoma (ccRCC). We have found that constitutively active HIF1α is sufficient to cause neoplastic transformation in a murine model of ccRCC termed the TRACK model. RNA sequencing (RNAseq) and untargeted metabolomics analyses of samples from TRACK kidneys demonstrate that HIF1α activates the transcription of genes that cause increased glucose uptake, glycolysis, and lactate production, as well as a decrease in the flux of pyruvate entering the tricarboxylic acid (TCA) cycle and a decrease in oxidative phosphorylation; these changes are identical to those observed in human ccRCC samples. These studies show that a constitutively active HIF1α promotes tumorigenesis in TRACK mice by mediating a metabolic switch to aerobic glycolysis, i.e., the Warburg effect, and suggest that TRACK mice are a valid model to test novel therapies targeting metabolic changes to inhibit human ccRCC.

Serine deprivation enhances antineoplastic activity of biguanides.

Metformin, a biguanide widely used in the treatment of type II diabetes, clearly exhibits antineoplastic activity in experimental models and has been reported to reduce cancer incidence in diabetics. There are ongoing clinical trials to evaluate its antitumor properties, which may relate to its fundamental activity as an inhibitor of oxidative phosphorylation. Here, we show that serine withdrawal increases the antineoplastic effects of phenformin (a potent biguanide structurally related to metformin). Serine synthesis was not inhibited by biguanides. Instead, metabolic studies indicated a requirement for serine to allow cells to compensate for biguanide-induced decrease in oxidative phosphorylation by upregulating glycolysis. Furthermore, serine deprivation modified the impact of metformin on the relative abundance of metabolites within the citric acid cycle. In mice, a serine-deficient diet reduced serine levels in tumors and significantly enhanced the tumor growth-inhibitory actions of biguanide treatment. Our results define a dietary manipulation that can enhance the efficacy of biguanides as antineoplastic agents that target cancer cell energy metabolism.

Stabilization of LKB1 and Akt by neddylation regulates energy metabolism in liver cancer.

The current view of cancer progression highlights that cancer cells must undergo through a post-translational regulation and metabolic reprogramming to progress in an unfriendly environment. In here, the importance of neddylation modification in liver cancer was investigated. We found that hepatic neddylation was specifically enriched in liver cancer patients with bad prognosis. In addition, the treatment with the neddylation inhibitor MLN4924 in Phb1-KO mice, an animal model of hepatocellular carcinoma showing elevated neddylation, reverted the malignant phenotype. Tumor cell death in vivo translating into liver tumor regression was associated with augmented phosphatidylcholine synthesis by the PEMT pathway, known as a liver-specific tumor suppressor, and restored mitochondrial function and TCA cycle flux. Otherwise, in protumoral hepatocytes, neddylation inhibition resulted in metabolic reprogramming rendering a decrease in oxidative phosphorylation and concomitant tumor cell apoptosis. Moreover, Akt and LKB1, hallmarks of proliferative metabolism, were altered in liver cancer being new targets of neddylation. Importantly, we show that neddylation-induced metabolic reprogramming and apoptosis were dependent on LKB1 and Akt stabilization. Overall, our results implicate neddylation/signaling/metabolism, partly mediated by LKB1 and Akt, in the development of liver cancer, paving the way for novel therapeutic approaches targeting neddylation in hepatocellular carcinoma.

Metabolic Shift In Hypoxia-Adapted CML Cells and Mechanisms Of Acquired Dasatinib Resistance Uncovered By Proteomic iTRAQ Profiling

Hypoxia is one of the essential components of the leukemia bone marrow (BM) microenvironment that promotes leukemia cell homing, survival and chemoresistance (Benito, PlosOne 2011). Tyrosine kinase inhibitors (TKIs) do not eradicate the total mass of chronic myeloid leukemia (CML) cells including primitive and quiescent cells. Persistence of CML cells in the hypoxic BM niche after cessation of TKI therapy may result in disease relapse. We have reported that hypoxia adapted CML cells acquire TKI resistance associated with higher glyoxalase-1 (Glo-1) enzyme activity which detoxifies methylglyoxal, a cytotoxic by-product of glycolysis (Takeuchi, Cell Death Differ 2010).In this study, we employed a proteomic approach based on isobaric tags for relative and absolute quantification (iTRAQ, Applied Biosystems) to investigate additional molecular mechanisms of CML adaptation to hypoxia and acquired resistance to dasatinib under hypoxic conditions. The specific pathway alterations were identified by KEGG(Kyoto University) and MetaCore (GeneGo). We utilized two hypoxia-adapted (HA) subclones of CML cell lines, KCL22-HA and KBM5-HA cells, which were selected over a month under 1.0 % oxygen culture conditions. The growth rate of both HA-CML cell lines was slower than that of the corresponding parental cells (ratio of incremental increase in cell numbers, 0.39 for KCL22-HA/KCL22, 0.54 for KBM5-HA/KBM5 at 48hrs). Although parental KCL22 cells were sensitive to dasatinib, KCL22-HA cultured under hypoxia acquired resistance to dasatinib (IC50: KCL22 0.1 nM, KCL22-HA >20nM, at 48hrs by MTT). In contrast, dasatinib induced more prominent cell growth inhibition in KBM5-HA cells cultured under hypoxia compared to KBM5 parental cells (IC50: KBM5 1.3 nM, KBM-5/HA 0.3 nM). Dasatinib effectively downregulated the phosphorylation levels of Stat5 and ERK in parental KCL22 and KBM5 cells. Notably, the baseline levels of p-Stat5 and p-ERK were markedly diminished in both KCL22-HA and KBM5-HA cells. We next performed iTRAQ proteomic analysis and detected more than 1,300 proteins in each cell type. Comparison of the basal proteome of KCL22 vs KCL22-HA and KBM5 vs KBM5-HA cells showed differential expression of 54 proteins in KCL22 isogenic cells (37 upregulated, 17 downregulated) and of 159 proteins in KBM5 cells (56 upregulated, 103 downregulated). These alterations included consistent activation of glycolysis and gluconeogenesis pathway (p<0.0001) along with increased ATP synthase (p=0.02) and hypoxia induced HIF1 activation (p<0.0001) in both cells adapted to grow under hypoxia. In dasatinib-resistant KCL-HA cells, these changes were partially reversed by dasatinib which affected expression of 19 proteins (5 up-regulated / 14 down-regulated) with significant decrease in oxidative phosphorylation (p=0.01), ATP synthase (p=0.02), spliceosome (p=0.02) and Cytochrome C (p=0.04). On the contrary, in dasatinib-sensitive KBM5-HA cells dasatinib upregulated 296 proteins including 39 apoptogenic proteins such as Cytochrome C (p<0.001) and apoptosis related mitochondrial chaperones HSP10 and HSP60 (p<0.001), along with further upregulation of the enzymes involved in the glycolytic energy production (p<0.001) and stimulation of nuclear mRNA splicing via spliceosome (p<0.001), known to play a crucial role in the control of gene expression of the apoptosis-regulating genes. We observed the consistent upregulation of glycolysis and increases of ribosome and spliceosome after dasatinib treatment in dasatinib-sensitive parental KCL22 and KBM5 cells. Interestingly, dasatinib-resistant KBM5-STI cells harboring T315 Bcr-Abl mutation exhibited significantly lower levels of baseline gluconeogenesis (p<0.0001) and translation regulation of translation initiation pathways (p<0.0001) compared to the parental KBM5 cells.In summary, these findings demonstrate the dramatic metabolic changes in hypoxia adjusted CML cells and indicate that hypoxia adapted / dasatinib resistant CML cells repress their stimulated glycolic metabolic state and oxidative phosphorylation along with spliceosomal mRNA splicing after dasatinib exposure, which allow them to enter quiescent stage and escape from apoptosis. Altogether these data indicate novel mechanisms of acquired bone marrow microenvironment-mediated resistance to TKI in CML. Disclosures:Tabe:Bristol-Myers Squibb: Research Funding.

Polymicrobial sepsis is associated with decreased hepatic oxidative phosphorylation and an altered metabolic profile

BackgroundOrgan failure in sepsis accounts for significant mortality worldwide. Mitochondrial and metabolic responses are central to the overall response of the cell, and thus of the organ and organism. Adaptive responses in metabolism are critical to the recovery at the cellular level. The purpose of these investigations was to test the hypothesis that sepsis is associated with decreased aerobic respiration and significant metabolic changes in the liver. MethodsC57BL/6 mice underwent cecal ligation and puncture (CLP) with a 21 gauge needle or an operation without CLP. Mice were euthanized from 0–24 h after the procedure and liver tissue was harvested. Tissue oxygen consumption and mitochondrial complex activity were measured. Global biochemical profiles of 311 metabolites were performed at the 8-h time point (n = 8/group) and analyzed by gas chromatography–mass spectrometry and liquid chromatography tandem mass spectrometry platforms by Metabolon (Durham, North Carolina). The influence of lipopolysaccharide (LPS) on aerobic and anaerobic respiration in primary mouse hepatocytes was also investigated. ResultsCLP in vivo or LPS in vitro resulted in a significant decrease in hepatic oxygen consumption. There was a significant decrease in oxidative phosphorylation measured at 12 h. LPS also resulted in a significant increase in anaerobic respiration in hepatocytes. Interestingly, the metabolomic analysis resulted in a metabolic shift in the liver from carbohydrate-based energy to utilization of fatty acids and amino acids. This included an increase in every tricarboxylic acid cycle intermediate and derivative, suggesting an increased flux into the cycle from fatty acid beta-oxidation and anaplerotic contributions from amino acids. ConclusionsSepsis results in a metabolic response and profile consistent with increased anaerobic respiration, which occurs prior to significant changes in hemodynamics. The metabolic responses of cells and organs may be important adaptive responses to prevent organ failure and death.

Abstract 1695: Quantification of mitochondria in MCF-10A, MDA-MB-231, and SUM149 cells to understand potential defects in oxidative phosphorylation in cancer.

Abstract Cancer cells are known to exhibit altered metabolism. The Warburg effect, whereby cancer cells preferentially use glycolysis instead of oxidative phosphorylation to produce ATP, plays an important role in the tumorigenic capacity of breast cancer. Previously, we found that SUM149 (inflammatory breast cancer) and MDA-MB-231 (invasive breast cancer) cells have decreased oxidative phosphorylation. Since mitochondria are the powerhouses of oxidative phosphorylation, examining their concentrations, phenotypes, and the distribution of their key enzymes can provide crucial information. These data can be used to further clarify whether the observed metabolic discrepancies stem from abundance differences or from other sources, such as defective enzymes and/or intermediate participation in alternate metabolic pathways. The purpose of our research was to elucidate if differential mitochondria concentrations accounted for the observed marked decrease in oxidative phosphorylation in cancer cells. Specifically, we focused on quantifying the number of mitochondria in two aggressive cancer cell lines, MDA-MB-231 and SUM149, and a normal-like breast cell line, MCF-10A. In order to measure the number of mitochondria in the cancerous and noncancerous cells, a commercially available antibody conjugated to green fluorescent protein (GFP) and directed against the E1-alpha subunit of pyruvate dehydrogenase was used in each cell line. The resulting cells were imaged under high-resolution confocal microscopy and their relative fluorescence assessed. The average intensity of GFP in each cell was calculated and used as an indicator for the amount of mitochondria in each cell line. Additionally, these results were compared to transmission electron microscopy (TEM) images of each of these cell lines to validate the immunofluorescent quantifications. Our results showed that there is no significant difference among the number of mitochondria in MDA-MB-231, SUM149, and MCF-10A cells. Additionally, the distribution of mitochondria among all three cell lines was relatively uniform, with a slightly greater abundance of mitochondria around the nuclei. These findings are surprising given that the published research on the Warburg effect, as well as our own prior observations on these three specific cell lines, suggested that cancer cells may have different numbers of mitochondria, although the previous work did not offer definitive proof of this interpretation. Therefore, these results contradict that hypothesis, and direct further studies to identify qualitative functional defects within mitochondria such as mutated or silenced mitochondrial genes, and to explore alternate pathways that breast cancer cells use to derive energy. Our investigation highlights the importance of taking a multi-facetted approach to studying cancer cell metabolism. Citation Format: Sepideh Ashrafzadeh, Lauren D. Van Wassenhove, Sofia D. Merajver. Quantification of mitochondria in MCF-10A, MDA-MB-231, and SUM149 cells to understand potential defects in oxidative phosphorylation in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1695. doi:10.1158/1538-7445.AM2013-1695

Cardioprotection by the TSPO ligand 4′-chlorodiazepam is associated with inhibition of mitochondrial accumulation of cholesterol at reperfusion

The translocator protein (TSPO) is located on the outer mitochondrial membrane where it is responsible for the uptake of cholesterol into mitochondria of steroidogenic organs. TSPO is also present in the heart where its role remains uncertain. We recently showed that TSPO ligands reduced infarct size and improved mitochondrial functions after ischaemia-reperfusion. This study, thus, sought to determine whether cholesterol could play a role in the cardioprotective effect of TSPO ligands. In a model of 30 min coronary occlusion/15 min reperfusion in Wistar rat, we showed that reperfusion induced lipid peroxidation as demonstrated by the increase in conjugated diene and thiobarbituric acid reactive substance formation and altered mitochondrial function (decrease in oxidative phosphorylation and increase in the sensitivity of mitochondrial permeability transition pore opening) in ex-vivo isolated mitochondria. This was associated with an increase in mitochondrial cholesterol uptake (89.5 ± 12.2 vs. 39.9 ± 3.51 nmol/mg protein in controls, P < 0.01) and a subsequent strong generation of auto-oxidized oxysterols, i.e. 7α- and 7β-hydroxycholesterol, 7-ketocholesterol, cholesterol-5α,6α-epoxide, and 5β,6β-epoxide (+173, +149, +165, +165, and +193%vs. controls, respectively; P < 0.01). Administration of the selective TSPO ligand 4'-chlorodiazepam inhibited oxidative stress, improved mitochondrial function, and abolished both mitochondrial cholesterol accumulation and oxysterol production. This was also observed with the new TSPO ligand TRO40303. These data suggest that 4'-chlorodiazepam inhibits oxidative stress and oxysterol formation by reducing the accumulation of cholesterol in the mitochondrial matrix at reperfusion and prevents mitochondrial injury. This new and original mechanism may contribute to the cardioprotective properties of TSPO ligands.

Potential anti-aging agents suppress the level of constitutive mTOR- and DNA damage- signaling

Two different mechanisms are considered to be the primary cause of aging. Cumulative DNA damage caused by reactive oxygen species (ROS), the by-products of oxidative phosphorylation, is one of these mechanisms (ROS concept). Constitutive stimulation of mitogen- and nutrient-sensing mTOR/S6 signaling is the second mechanism (TOR concept). The flow- and laser scanning- cytometric methods were developed to measure the level of the constitutive DNA damage/ROS- as well as of mTOR/S6- signaling in individual cells. Specifically, persistent activation of ATM and expression of γH2AX in untreated cells appears to report constitutive DNA damage induced by endogenous ROS. The level of phosphorylation of Ser235/236-ribosomal protein (RP), of Ser2448-mTOR and of Ser65-4EBP1, informs on constitutive signaling along the mTOR/S6 pathway. Potential gero-suppressive agents rapamycin, metformin, 2-deoxyglucose, berberine, resveratrol, vitamin D3 and aspirin, all decreased the level of constitutive DNA damage signaling as seen by the reduced expression of γH2AX in proliferating A549, TK6, WI-38 cells and in mitogenically stimulated human lymphocytes. They all also decreased the level of intracellular ROS and mitochondrial trans-membrane potential ΔΨm, the marker of mitochondrial energizing as well as reduced phosphorylation of mTOR, RP-S6 and 4EBP1. The most effective was rapamycin. Although the primary target of each on these agents may be different the data are consistent with the downstream mechanism in which the decline in mTOR/S6K signaling and translation rate is coupled with a decrease in oxidative phosphorylation, (revealed by ΔΨm) that leads to reduction of ROS and oxidative DNA damage. The decreased rate of translation induced by these agents may slow down cells hypertrophy and alleviate other features of cell aging/senescence. Reduction of oxidative DNA damage may lower predisposition to neoplastic transformation which otherwise may result from errors in repair of DNA sites coding for oncogenes or tumor suppressor genes. The data suggest that combined assessment of constitutive γH2AX expression, mitochondrial activity (ROS, ΔΨm) and mTOR signaling provides an adequate gamut of cell responses to evaluate effectiveness of gero-suppressive agents.

Metabolism of HeLa cells revealed through autofluorescence lifetime upon infection with enterohemorrhagic &lt;italic&gt;Escherichia coli&lt;/italic&gt;

Fluorescence lifetime imaging microscopy (FLIM) is a sensitive technique in monitoring functional and conformational states of nicotinamide adenine dinucleotide reduced (NADH) and flavin adenine dinucleotide (FAD),main compounds participating in oxidative phosphorylation in cells. In this study, we have applied FLIM to characterize the metabolic changes in HeLa cells upon bacterial infection and made comparison with the results from the cells treated with staurosporine (STS), a well-known apoptosis inducer. The evolving of NADH's average autofluorescence lifetime during the 3 h after infection with enterohemorragic Escherichia coli (EHEC) or STS treatment has been observed. The ratio of the short and the long lifetime components' relative contributions of NADH increases with time, a fact indicating cellular metabolic activity, such as a decrease of oxidative phosphorylation over the course of infection, while opposite dynamics is observed in FAD. Being associated with mitochondria, FAD lifetimes and redox ratio could indicate heterogeneous mitochondrial function, microenvironment with bacterial infection, and further pathway to cell death. The redox ratios for both EHEC-infected and STS-treated HeLa cells have been observed and these observations also indicate possible apoptosis induced by bacterial infection.

Silencing of nicotinamide nucleotide transhydrogenase impairs cellular redox homeostasis and energy metabolism in PC12 cells

Mitochondrial NADPH generation is largely dependent on the inner-membrane nicotinamide nucleotide transhydrogenase (NNT), which catalyzes the reduction of NADP+ to NADPH utilizing the proton gradient as the driving force and NADH as the electron donor. Small interfering RNA (siRNA) silencing of NNT in PC12 cells results in decreased cellular NADPH levels, altered redox status of the cell in terms of decreased GSH/GSSG ratios and increased H2O2 levels, thus leading to an increased redox potential (a more oxidized redox state). NNT knockdown results in a decrease of oxidative phosphorylation while anaerobic glycolysis levels remain unchanged. Decreased oxidative phosphorylation was associated with a) inhibition of mitochondrial pyruvate dehydrogenase (PDH) and succinyl-CoA:3-oxoacid CoA transferase (SCOT) activity; b) reduction of NADH availability, c) decline of mitochondrial membrane potential, and d) decrease of ATP levels. Moreover, the alteration of redox status actually precedes the impairment of mitochondrial bioenergetics. A possible mechanism could be that the activation of the redox-sensitive c-Jun N-terminal kinase (JNK) and its translocation to the mitochondrion leads to the inhibition of PDH (upon phosphorylation) and induction of intrinsic apoptosis, resulting in decreased cell viability. This study supports the notion that oxidized cellular redox state and decline in cellular bioenergetics – as a consequence of NNT knockdown – cannot be viewed as independent events, but rather as an interdependent relationship coordinated by the mitochondrial energy-redox axis. Disruption of electron flux from fuel substrates to redox components due to NNT suppression induces not only mitochondrial dysfunction but also cellular disorders through redox-sensitive signaling.

Alterations in mitochondrial respiratory function in response to endurance training and endurance racing

Limited information exists about the muscle mitochondrial respiratory function changes that occur in horses during an endurance season. To determine effects of training and racing on muscle oxidative phosphorylation (OXPHOS) and electron transport system (ETS) capacities in horses with high resolution respirometry (HRR). Mitochondrial respiration was measured in microbiopsies taken from the triceps brachii (tb) and gluteus medius (gm) muscles in 8 endurance horses (7 purebred Arabians and 1 crossbred Arabian) before training (T0), after two 10 week training periods (T1, T2) and after 2 CEI** endurance races (R1, R2). Muscle OXPHOS capacity was determined using 2 titration protocols without (SUIT 1) or with pyruvate (SUIT 2) as substrate. Electrons enter at the level of Complex I, Complex II or both complexes simultaneously (Complexes I+II). Muscle ETS capacity was obtained by uncoupling Complexes I+II sustained respiration. T1 improved OXPHOS and ETS capacities in the tb as demonstrated by the significant increase of oxygen fluxes vs. T0 (Complex I: +67%; ETS: +37%). Training improved only OXPHOS in the gm (Complex I: +34%). Among horses that completed the race, a significant decrease in OXPHOS (Complex I: ∼ -35%) and ETS (-22%) capacities was found in the tb with SUIT 2 indicating a reduced aerobic glycolysis. Significant correlations between CK activities and changes in OXPHOS were found suggesting a relationship between exercise-induced muscle damage and depression of mitochondrial respiration. For the first time, OXPHOS and ETS capacities in equine muscle at different steps of an endurance season have been determined by HRR. Significant alterations in mitochondrial respiratory function in response to endurance training and endurance racing have been observed although these changes appeared to be muscle group specific.

Mitochondrial DNA Mutations Induce Mitochondrial Dysfunction, Apoptosis and Sarcopenia in Skeletal Muscle of Mitochondrial DNA Mutator Mice

BackgroundAging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established.Methodology/Principal FindingsWe investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase γ, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Δψm). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage.Conclusions/SignificanceThese findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.

009 Normobaric hypoxia impairs cardiac energetics and diastolic function in normal human volunteers

<h3>Background</h3> Left ventricular dysfunction is consistently observed in human heart in the first few days of hypoxic exposure, yet the cellular mechanisms underlying the dysfunction are poorly understood. We postulated that normobaric hypoxia impairs cardiac energetics leading to cardiac dysfunction. Healthy males (n=12, age 24±2) underwent 20 h of normobaric hypoxia in purpose-built hypoxia chambers. The partial pressure of oxygen during end tidal expiration (PETO2) was kept between 50 and 60 mmHg, whilst keeping peripheral oxygen saturation (SaO2) above 80%. Cardiac function was measured using magnetic resonance imaging (MRI) and echocardiography before and after hypoxic exposure. High energy phosphate metabolism was measured as the ratio of phosphocreatine to ATP (PCr/ATP) using 31Phosphorus magnetic resonance spectroscopy (MRS) before and after 20 h of hypoxia. During hypoxia, PETO₂ and SaO₂ averaged 55±1 mmHg and 83.6±0.4%, respectively. There was a 15% reduction in cardiac PCr/ATP, from 2.0±0.1 before to 1.7±0.1 after hypoxia (p&lt;0.01), and reduced diastolic function, measured as E/E9, from 6.1±0.4 to 7.5±0.7, (p&lt;0.01). Short term normobaric hypoxia led to rapid changes in cardiac energy metabolism and alterations in diastolic function in normal human hearts. Increased cardiac energy demand and reduced oxygen-limited decrease in oxidative phosphorylation may explain the low PCr/ATP and cardiac dysfunction observed after hypoxic exposure, whether in health or disease.

Induction of the Warburg effect by Kaposi's sarcoma herpesvirus is required for the maintenance of latently infected endothelial cells

Kaposi's sarcoma (KS) is the most commonly reported tumor in parts of Africa and is the most common tumor of AIDS patients world-wide. KS-associated herpesvirus (KSHV) is the etiologic agent of KS. Although KS tumors contain many cell types, the predominant cell is the spindle cell, a cell of endothelial origin that maintains KSHV latency. KSHV activates many cell-signaling pathways but little is known about how KSHV alters cellular metabolism during latency. The Warburg effect, a common metabolic alteration of most tumor cells, is defined by an increase in aerobic glycolysis and a decrease in oxidative phosphorylation as an energy source. The Warburg effect adapts cells to tumor environments and is necessary for the survival of tumor cells. During latent infection of endothelial cells, KSHV induces aerobic glycolysis and lactic acid production while decreasing oxygen consumption, thereby inducing the Warburg effect. Inhibitors of glycolysis selectively induce apoptosis in KSHV-infected endothelial cells but not their uninfected counterparts. Therefore, similar to cancer cells, the Warburg effect is necessary for maintaining KSHV latently infected cells. We propose that KSHV induction of the Warburg effect adapts infected cells to tumor microenvironments, aiding the seeding of KS tumors. Additionally, inhibitors of glycolysis may provide a unique treatment strategy for latent KSHV infection and ultimately KS tumors.

Abstract 3934: Quantitative assessment of mtDNA damage differentiates long-term and short-term survivors with glioblastoma

Abstract The role of mitochondrial dysfunction in cancer has long been under scientific scrutiny. Emerging evidence suggests that a critical mechanism of anticancer therapy induced apoptosis may be the generation of mitochondrial reactive oxygen species (ROS) that can damage vital biomolecules, including mitochondrial DNA (mtDNA). We have developed methods to quantify mtDNA damage in human cells and undertook this investigation in glioblastoma to determine if there is a correlation between mtDNA damage and patient survival. Fresh-frozen tumor tissue was collected from 17 patients with glioblastoma before initiation of anti-tumor therapy. The patients were classified as short-term survivors (STS, survival 9 months or less) or long-term survivors (LTS, survival 24 months or more). Following initial resection, all of the patients underwent adjuvant therapy, usually consisting of radiation therapy plus temozolomide (1). We modified a protocol to assess mtDNA damage in which quantitative long-range PCR is performed on an 8.9kb segment of the mtDNA circle (2). The amount of amplified DNA generated is inversely proportional to the extent of mtDNA damage that blocks polymerase synthesis. Total DNA was extracted from the samples, quantified and evaluated qualitatively by gel electrophoresis. The amplified mtDNA was normalized to mtDNA copy number, which was determined by Real-Time PCR of the mitochondrial ND1 gene using DNA standards. A statistically significant correlation was found between the level of mtDNA damage and survival. Higher levels of mtDNA damage - less amplifiable mtDNA - were found in the tumors of LTS than in the tumors of STS. The mean amplifiable mtDNA in nanograms per million copies of ND1 was 22.2 for STS tumors and 15.8 for LTS tumors (two-tail t-test P value = 0.038). These results are consistent with the hypothesis that reactive oxygen species must be generated in order to trigger apoptosis in response to chemotherapeutic drugs, or other treatment modalities. LTS glioblastomas have less amplifiable mtDNA, therefore more damage per mtDNA molecule, indicating that these tumors have greater basal ROS-generating metabolism, less effective anti-oxidant buffering, or poor mtDNA repair or elimination mechanisms. In comparison, STS glioblastomas may “shut down” basal mitochondrial production of ROS, possibly by a more pronounced Warburg effect (decrease in oxidative phosphorylation), buffer the ROS via anti-oxidant mechanisms, or more effectively repair or eliminate their damaged mtDNA. Increased amounts of mtDNA damage therefore may indicate susceptibility to anti-cancer regimens in glioblastoma.

Decrease in oxidative phosphorylation with aging correlates with the aging phenotype

The goal of the present study was to evaluate the implication of mitochondrial function in the age associated decrease in cardiac contractile function. Mitochondrial respiratory capacity was measured in permeabilized fibers from left ventricles of young and old rats from 3 different stains, presenting or not the aging phenotype. The Fisher 344 rats show severe age related pathology compare to the brown Norway rats. In contrast, hybrids Fisher 344 X Brown Norway showed a longer life expectancy and significantly fewer pathological lesions related to aging compared to the two other strains. A dramatic decrease of around 45% in coupled oxidative phosphorylation (OXPHOS) was observed in the old (24‐26 months) compared to the young (6 months) Fisher 344. The effect of aging on respiration was similar for complexes I, II, and IV, supporting either a decrease in mitochondrial content, an oxidative defect, or both with aging. The same trend, but without significant differences, was observed in cardiac fibers from the brown Norway, with a 15‐20% decrease in OXHPOS in the aged rats. The hybrids rats, which show better resistance to aging, do not have any loss of mitochondrial function. The severity of the age‐related pathology in the different strains correlates strongly with the presence of mitochondrial dysfunction. Our results suggest the importance mitochondrial defect in the deterioration of cardiac function during aging.