Increased ATP Synthesis Research Articles

14,15-EET promotes mitochondrial biogenesis and protects cortical neurons against oxygen/glucose deprivation-induced apoptosis

14,15-Epoxyeicosatrienoic acid (14,15-EET), a metabolite of arachidonic acid, is enriched in the brain cortex and exerts protective effect against neuronal apoptosis induced by ischemia/reperfusion. Although apoptosis has been well recognized to be closely associated with mitochondrial biogenesis and function, it is still unclear whether the neuroprotective effect of 14,15-EET is mediated by promotion of mitochondrial biogenesis and function in cortical neurons under the condition of oxygen–glucose deprivation (OGD). In this study, we found that 14,15-EET improved cell viability and inhibited apoptosis of cortical neurons. 14,15-EET significantly increased the mitochondrial mass and the ratio of mitochondrial DNA to nuclear DNA. Key makers of mitochondrial biogenesis, peroxisome proliferator activator receptor gamma-coactivator 1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), were elevated at both mRNA and protein levels in the cortical neurons treated with 14,15-EET. Moreover, 14,15-EET markedly attenuated the decline of mitochondrial membrane potential, reduced ROS, while increased ATP synthesis. Knockdown of cAMP-response element binding protein (CREB) by siRNA blunted the up-regulation of PGC-1α and NRF-1 stimulated by 14,15-EET, and consequently abolished the neuroprotective effect of 14,15-EET. Our results indicate that 14,15-EET protects neurons from OGD-induced apoptosis by promoting mitochondrial biogenesis and function through CREB mediated activation of PGC-1α and NRF-1.

AMP kinase (PRKAA1)

The PRKAA1 gene encodes the catalytic α-subunit of 5′ AMP-activated protein kinase (AMPK). AMPK is a cellular energy sensor that maintains energy homeostasis within the cell and is activated when...

SIRT5 is under the control of PGC‐1α and AMPK and is involved in regulation of mitochondrial energy metabolism

The sirtuins (SIRTs; SIRT1-7) are a family of NAD(+)-dependent enzymes that dynamically regulate cellular physiology. Apart from SIRT1, the functions and regulatory mechanisms of the SIRTs are poorly defined. We explored regulation of the SIRT family by 2 energy metabolism-controlling factors: peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and AMP-activated protein kinase (AMPK). Overexpression of PGC-1α in mouse primary hepatocytes increased SIRT5 mRNA expression 4-fold and also the protein in a peroxisome proliferator-activated receptor α (PPARα)- and estrogen-related receptor α (ERRα)-dependent manner. Furthermore, food withdrawal increased SIRT5 mRNA 1.3-fold in rat liver. Overexpression of AMPK in mouse hepatocytes increased expression of SIRT1, SIRT2, SIRT3, and SIRT6 <2-fold. In contrast, SIRT5 mRNA was down-regulated by 58%. The antidiabetes drug metformin (1 mM), an established AMPK activator, reduced the mouse SIRT5 protein level by 44% in cultured hepatocytes and by 31% in liver in vivo (300 mg/kg, 7 d). Metformin also induced hypersuccinylation of mitochondrial proteins. Moreover, SIRT5 overexpression increased ATP synthesis and oxygen consumption in HepG2 cells, but did not affect mitochondrial biogenesis. In summary, our results identified SIRT5 as a novel factor that controls mitochondrial function. Moreover, SIRT5 levels are regulated by PGC-1α and AMPK, which have opposite effects on its expression.-Buler, M., Aatsinki, S.-M., Izzi, V., Uusimaa, J., Hakkola, J. SIRT5 is under the control of PGC-1α and AMPK and is involved in regulation of mitochondrial energy metabolism.

Proton Gradient Regulation 5-Mediated Cyclic Electron Flow under ATP- or Redox-Limited Conditions: A Study of ƊATPase pgr5 and ƊrbcL pgr5 Mutants in the Green Alga Chlamydomonas reinhardtii

The Chlamydomonas reinhardtii proton gradient regulation5 (Crpgr5) mutant shows phenotypic and functional traits similar to mutants in the Arabidopsis (Arabidopsis thaliana) ortholog, Atpgr5, providing strong evidence for conservation of PGR5-mediated cyclic electron flow (CEF). Comparing the Crpgr5 mutant with the wild type, we discriminate two pathways for CEF and determine their maximum electron flow rates. The PGR5/proton gradient regulation-like1 (PGRL1) ferredoxin (Fd) pathway, involved in recycling excess reductant to increase ATP synthesis, may be controlled by extreme photosystem I acceptor side limitation or ATP depletion. Here, we show that PGR5/PGRL1-Fd CEF functions in accordance with an ATP/redox control model. In the absence of Rubisco and PGR5, a sustained electron flow is maintained with molecular oxygen instead of carbon dioxide serving as the terminal electron acceptor. When photosynthetic control is decreased, compensatory alternative pathways can take the full load of linear electron flow. In the case of the ATP synthase pgr5 double mutant, a decrease in photosensitivity is observed compared with the single ATPase-less mutant that we assign to a decreased proton motive force. Altogether, our results suggest that PGR5/PGRL1-Fd CEF is most required under conditions when Fd becomes overreduced and photosystem I is subjected to photoinhibition. CEF is not a valve; it only recycles electrons, but in doing so, it generates a proton motive force that controls the rate of photosynthesis. The conditions where the PGR5 pathway is most required may vary in photosynthetic organisms like C. reinhardtii from anoxia to high light to limitations imposed at the level of carbon dioxide fixation.

Metabolomic Heterogeneity of Pulmonary Arterial Hypertension

Although multiple gene and protein expression have been extensively profiled in human pulmonary arterial hypertension (PAH), the mechanism for the development and progression of pulmonary hypertension remains elusive. Analysis of the global metabolomic heterogeneity within the pulmonary vascular system leads to a better understanding of disease progression. Using a combination of high-throughput liquid-and-gas-chromatography-based mass spectrometry, we showed unbiased metabolomic profiles of disrupted glycolysis, increased TCA cycle, and fatty acid metabolites with altered oxidation pathways in the human PAH lung. The results suggest that PAH has specific metabolic pathways contributing to increased ATP synthesis for the vascular remodeling process in severe pulmonary hypertension. These identified metabolites may serve as potential biomarkers for the diagnosis of PAH. By profiling metabolomic alterations of the PAH lung, we reveal new pathogenic mechanisms of PAH, opening an avenue of exploration for therapeutics that target metabolic pathway alterations in the progression of PAH.

Enhanced synaptic transmission at the squid giant synapse by artificial seawater based on physically modified saline

Superfusion of the squid giant synapse with artificial seawater (ASW) based on isotonic saline containing oxygen nanobubbles (RNS60 ASW) generates an enhancement of synaptic transmission. This was determined by examining the postsynaptic response to single and repetitive presynaptic spike activation, spontaneous transmitter release, and presynaptic voltage clamp studies. In the presence of RNS60 ASW single presynaptic stimulation elicited larger postsynaptic potentials (PSP) and more robust recovery from high frequency stimulation than in control ASW. Analysis of postsynaptic noise revealed an increase in spontaneous transmitter release with modified noise kinetics in RNS60 ASW. Presynaptic voltage clamp demonstrated an increased EPSP, without an increase in presynaptic ICa++ amplitude during RNS60 ASW superfusion. Synaptic release enhancement reached stable maxima within 5–10 min of RNS60 ASW superfusion and was maintained for the entire recording time, up to 1 h. Electronmicroscopic morphometry indicated a decrease in synaptic vesicle density and the number at active zones with an increase in the number of clathrin-coated vesicles (CCV) and large endosome-like vesicles near junctional sites. Block of mitochondrial ATP synthesis by presynaptic injection of oligomycin reduced spontaneous release and prevented the synaptic noise increase seen in RNS60 ASW. After ATP block the number of vesicles at the active zone and CCV was reduced, with an increase in large vesicles. The possibility that RNS60 ASW acts by increasing mitochondrial ATP synthesis was tested by direct determination of ATP levels in both presynaptic and postsynaptic structures. This was implemented using luciferin/luciferase photon emission, which demonstrated a marked increase in ATP synthesis following RNS60 administration. It is concluded that RNS60 positively modulates synaptic transmission by up-regulating ATP synthesis, thus leading to synaptic transmission enhancement.

Acetylcholine Promotes ROS Detoxification Against Hypoxia/reoxygenation-Induced Oxidative Stress Through FoxO3a/PGC-1α Dependent Superoxide Dismutase

Background/Aims: Acetylcholine (ACh) is known to modulate the cardiac redox environment and thereby suppress reactive oxygen species (ROS) generation during oxidative stress. However, there is little information about its regulation on ROS clearance. Here we investigate the beneficial effects of ACh on superoxide dismutase (SOD) as key ROS-detoxifying enzyme system in cultured rat cardiomyoblasts. Methods: H9c2 cells were subjected to hypoxia/reoxygenation (H/R) to mimic oxidative stress. Western blot was used to detect the expression of SOD and related signaling molecules. Specific protein knockdown was performed with siRNA transfection. Results: ACh treatment on the beginning of reoxygenation decreased ROS and apoptosis. ACh increased ATP synthesis and mitochondrial DNA. Furthermore, ACh significantly reversed H/R-induced reduction in protein expressions and activities of SOD. ACh stimulated peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α) and decreased forkhead box subfamily O3a (FoxO3a) phosphorylation. Atropine (muscarinic receptor antagonist) abolished the cytoprotection afforded by ACh. PGC-1α siRNA blocked ACh-induced invigorating effects on SOD2, whereas it did not alter SOD1 and FoxO3a phosphorylation. FoxOSa siRNA drastically decreased the expressions of SOD2 and PGC-1α, while it did not affect SOD1. Conclusion: ACh activates SOD2 within mitochondria through FoxO3a/PGC-1α pathway and up-regulates SOD1 in the cytoplasm, thus protecting against oxidative injury induced by H/R. Our findings provide new insights into mechanisms underlying the cardioprotection of ACh on ROS detoxifying. © 2014 S. Karger AG, Basel

Critical Role of the mTOR Pathway in Development and Function of Myeloid-Derived Suppressor Cells in lal−/− Mice

Lysosomal acid lipase (LAL) is essential for the hydrolysis of cholesteryl esters and triglycerides to generate cholesterol and free fatty acids in cellular lysosomes. Ablation of the lal gene (lal(-/-)) systemically increased expansion of cluster of differentiation molecule 11b (CD11b), lymphocyte antigen 6G (Ly6G) myeloid-derived suppressor cells (MDSCs) that caused myeloproliferative neoplasms in mice. Study of lal(-/-) bone marrow Ly6G(+) MDSCs via transcriptional profiling showed increases in mammalian target of rapamycin (mTOR) signaling pathway transcripts. Injection of mTOR pharmacologic inhibitors into lal(-/-) mice significantly reduced bone marrow myelopoiesis and systemic CD11b(+)Ly6G(+) cell expansion. Rapamycin treatment of lal(-/-) mice stimulated a shift from immature CD11b(+)Ly6G(+) cells to CD11b(+) single-positive cells in marrow and tissues and partially reversed the increased cell proliferation, decreased apoptosis, increased ATP synthesis, and increased cell cycling of bone marrow CD11b(+)Ly6G(+) cells obtained from lal(-/-) mice. Pharmacologic and siRNA suppression of mTOR, regulatory-associated protein of mTOR, rapamycin-insensitive companion of mTOR, and Akt1 function corrected CD11b(+)Ly6G(+) cell in lal(-/-) mice development from Lin(-) progenitor cells and reversed the immune suppression on T-cell proliferation and function in association with decreased reactive oxygen species production, and recovery from impairment of mitochondrial membrane potential compared with control mutant cells. These results indicate a crucial role of LAL-regulated mTOR signaling in the production and function of CD11b(+)Ly6G(+) cells. The mTOR pathway may serve as a novel target to modulate the emergence of MDSCs in those pathophysiologic states in which these cells play an immunosuppressive role.

Long-term therapy with Bendavia (MTP-131), a novel mitochondria-targeting peptide, improves left ventricular systolic function in dogs with chronic heart failure

Background: Mitochondria (MITO) of the failing heart manifest structural and functional abnormalities evidenced by hyperplasia, reduced organelle size and diminished respiration that lead to a defect in ATP synthesis. Bendavia (MTP-131), a novel, first in class, MITO-targeting peptide, improves the efficiency of mitochondria electron transfer chain and enhances ATP synthesis in multiple organs including heart, kidney and skeletal muscle. The present study examined the effects of long-term therapy with Bendavia on LV systolic function and ATP synthesis in dogs with chronic heart failure (HF) (LV ejection fraction, EF∼30%). Methods: Fourteen dogs with coronary microembolization-induced HF were randomized to 3 months therapy with subcutaneous injections of Bendavia (0.5 mg/kg once daily, n=7) or saline (Control, n=7). LV end-diastolic (EDV) and end-systolic (ESV) volumes, EF, heart rate (HR), mean aortic pressure (AP), LV end-diastolic pressure (EDP) and peak LV +dP/dt were measured before (PRE) and after 3 months of therapy (POST). Treatment effect Δ, within each group, was calculated as the difference between PRE and POST. MITO ATP synthesis and ATP/ADP ratio were measured in isolated LV cardiomyocytes obtained at the end of 3 months of therapy using the bioluminescent ApoSENSORTM ATP/ADP assay kit. Results: Bendavia had no effect on HR or mAP. Compared to Control, Bendavia decreased EDV, ESV and EDP, increased EF and peak LV +dP/dt and increased ATP synthesis and ratio of ATP/ADP (Table). View this table: Indexes of LV function and ATP Synthesis Conclusions: In HF dogs, long-term monotherapy with Bendavia improves LV systolic function without increasing HR or decreasing mAP. Bendavia increased ATP synthesis; a likely mediator of improved LV function.

Long-term therapy with Bendavia (MTP-131), a novel mitochondria-targeting peptide, reverses mitochondrial abnormalities in left ventricular myocardium of dogs with advanced heart failure

Background: Mitochondria (MITO) of failed human hearts and hearts of dogs with experimental heart failure manifest functional abnormalities characterized by reduced state-3 respiration, reduced membrane potential (Δψm) and increased opening of membrane permeability transition pores (mPTP). These abnormalities lead to reduced ATP synthesis that adversely impacts LV function. Bendavia (MTP-131), a novel, first in class, MITO-targeting peptide, has been shown to improve LV systolic function in dogs with chronic heart failure and to improve ATP synthesis in multiple organs including heart, kidney and skeletal muscle in other animal models of disease. The present study tested the hypothesis that long-term therapy with Bendavia reverses functional MITO abnormalities in dogs with chronic heart failure. Methods: Studies were performed in cardiomyocytes isolated from LV myocardium of 14 HF dogs produced by intracoronary microembolizations (LV ejection fraction ∼30%). Dogs were randomized to 3 months monotherapy with subcutaneous injections of Bendavia (0.5 mg/kg once daily, n=7) or saline (Control, n=7). MITO state-3 respiration was measured using a Clark electrode. Δψm was measured using the dye JC-1 and mPTP was measured based on the rate of calcein exit from MITO. MITO ATP synthesis was measured using the bioluminescent ApoSENSORTM assay kit. Results: Compared to Control, Bendavia significantly improved MITO state-3 respiration, increased Δψm, decreased the rate of calcein exit from MITO thus decreasing mPTP opening and increased in ATP synthesis (Table). View this table: Indexes of mitochondria function Conclusions: Therapy with Bendavia reverses abnormalities of MITO function in LV myocardium of dogs with chronic heart failure and increases ATP synthesis. The latter can explain the observed improvement of LV systolic function following long-term therapy with Bendavia in dogs with HF.

Abstract 090: Long-term Therapy with a Bendavia (MTP-131), a Novel Mitochondria-Targeting Peptide, Normalizes Cardiolipin Biosynthesis and Remodeling in Left Ventricular Myocardium of Dogs with Advanced Heart Failure

Background: Cardiolipin (CL) is a signature phospholipid of the mitochondrial inner membrane and responsible for modulation of activities of various enzymes essential for oxidative phosphorylation. Cardiac CL is biosynthesized in a series of steps from phosphatidic acid and remodeled into a form which contains four 18:2 fatty acid chains, tetralinoleol CL [(18:2) 4 CL]. Long-term therapy with Bendavia (MTP-131), a novel mitochondria-targeting peptide, was previously shown to improve left ventricular (LV) function, increase ATP synthesis, and increase the activity and protein levels of mitochondria cytochrome c oxidase (complex IV) in LV myocardium of dogs with advanced heart failure (HF). This study examined the effects of Bendavia on total CL and (18:2) 4 CL in LV myocardium of dogs with HF. Methods: LV tissue was obtained from 12 dogs with microembolization-induced HF randomized to 3 months therapy with subcutaneous injections of Bendavia (0.5 mg/kg once daily, n=7) or saline (Control, n=7). LV tissue from 7 normal dogs was used for comparison. Total CL and (18:2) 4 CL were measured using electrospray ionization mass spectroscopy and quantified in nmol/mg of non-collagen protein (NCP). Results: Total CL was significantly decreased in LV myocardium of Control dogs compared to normal dogs (19.1±1.1 vs. 26.7±1.4 nmol/mg, p&lt;0.05) as was (18:2) 4 CL (14.2±0.9 vs. 20.5±1.2 nmol/mg, p&lt;0.05). Long-term therapy with Bendavia significantly increased both total CL (23.6±1.1 nmol/mg) and (18:2) 4 CL (17.8±1.0 nmol/mg) to near normal levels in LV myocardium of treated HF dogs compared to untreated HF Controls (p&lt;0.05). Conclusions: Total CL and (18:2) 4 CL are decreased in LV myocardium of dogs with HF. Treatment with Bendavia normalizes total CL and (18:2) 4 CL. These findings are consistent with observed normalization of mitochondrial complex IV activity, normalization of rate of ATP synthesis and improvement of global LV performance in dogs with HF after long-term therapy with Bendavia.

Nicotinamide N-methyltransferase expression in SH-SY5Y neuroblastoma and N27 mesencephalic neurones induces changes in cell morphology via ephrin-B2 and Akt signalling.

Nicotinamide N-methyltransferase (NNMT, E.C. 2.1.1.1) N-methylates nicotinamide to produce 1-methylnicotinamide (MeN). We have previously shown that NNMT expression protected against neurotoxin-mediated cell death by increasing Complex I (CxI) activity, resulting in increased ATP synthesis. This was mediated via protection of the NDUFS3 subunit of CxI from degradation by increased MeN production. In the present study, we have investigated the effects of NNMT expression on neurone morphology and differentiation. Expression of NNMT in SH-SY5Y human neuroblastoma and N27 rat mesencephalic dopaminergic neurones increased neurite branching, synaptophysin expression and dopamine accumulation and release. siRNA gene silencing of ephrin B2 (EFNB2), and inhibition of Akt phosphorylation using LY294002, demonstrated that their sequential activation was responsible for the increases observed. Incubation of SH-SY5Y with increasing concentrations of MeN also increased neurite branching, suggesting that the effects of NNMT may be mediated by MeN. NNMT had no significant effect on the expression of phenotypic and post-mitotic markers, suggesting that NNMT is not involved in determining phenotypic fate or differentiation status. These results demonstrate that NNMT expression regulates neurone morphology in vitro via the sequential activation of the EFNB2 and Akt cellular signalling pathways.

Crosstalk from Non-Cancerous Mitochondria Can Inhibit Tumor Properties of Metastatic Cells by Suppressing Oncogenic Pathways

Mitochondrial-nucleus cross talks and mitochondrial retrograde regulation can play a significant role in cellular properties. Transmitochondrial cybrid systems (cybrids) are an excellent tool to study specific effects of altered mitochondria under a defined nuclear background. The majority of the studies using the cybrid model focused on the significance of specific mitochondrial DNA variations in mitochondrial function or tumor properties. However, most of these variants are benign polymorphisms without known functional significance. From an objective of rectifying mitochondrial defects in cancer cells and to establish mitochondria as a potential anticancer drug target, understanding the role of functional mitochondria in reversing oncogenic properties under a cancer nuclear background is very important. Here we analyzed the potential reversal of oncogenic properties of a highly metastatic cell line with the introduction of non-cancerous mitochondria. Cybrids were established by fusing the mitochondria DNA depleted 143B TK- ρ0 cells from an aggressive osteosarcoma cell line with mitochondria from benign breast epithelial cell line MCF10A, moderately metastatic breast cancer cell line MDA-MB-468 and 143B cells. In spite of the uniform cancerous nuclear background, as observed with the mitochondria donor cells, cybrids with benign mitochondria showed high mitochondrial functional properties including increased ATP synthesis, oxygen consumption and respiratory chain activities compared to cybrids with cancerous mitochondria. Interestingly, benign mitochondria could reverse different oncogenic characteristics of 143B TK- cell including cell proliferation, viability under hypoxic condition, anti-apoptotic properties, resistance to anti-cancer drug, invasion, and colony formation in soft agar, and in vivo tumor growth in nude mice. Microarray analysis suggested that several oncogenic pathways observed in cybrids with cancer mitochondria are inhibited in cybrids with non-cancerous mitochondria. These results suggest the critical oncogenic regulation by mitochondrial-nuclear cross talk and highlights rectifying mitochondrial functional properties as a promising target in cancer therapy.

Effect of supernatants from Lactobacillus acidophilus culture on ATP levels in human gingival fibroblasts

Bacteria of Lactobacillus genus comprise around 1% of physiological flora in oral cavity. Despite numerous studies on Lactobacillus bacteria, their interaction with cells of host’s oral cavity has not been fully recognized. Studies were performed on effects of super natants obtained from bacterial cultures of Lactobacillus acidophilus strains on ATP levels in human gingival fibroblasts (HGF-1) and on their viability. ATP levels were evaluated using luminescence test and cell viability was estimated using a fluorescence test. Mean levels of ATP in cultures of control fibroblasts, HGF-1, supplemented with 10% PBS amounted to 4.90 ± 0.32 mln of RLU (rela tive light units). In turn, mean level of ATP in cul tures of HGF-1 fibroblasts supplemented with supernatants of H2O2-producing L. acidophilus cultures amounted to 5.94 ± 0.31 mln of RLU, and in the cul tures sup ple mented with su per natants of L. acidophilus producing no H2O2 it amounted to 5.88 ± 0.28 mln of RLU. The lev els of ATP ob tained in HGF-1 cultures with supernatants of L. acidophilus were significantly higher than those in control cultures. On the other hand, ATP levels in HGF-1 cultures with supernatants of H2O2-producing L. acidophilus cultures and with supernatants of H2O2-not producing L. acidophilus cultures showed no significant differences. The presented for the first time in this study increase in ATP synthesis in gingival fibroblasts under effect of extracellular products of L. acidophilus cultures may represent an important protective mechanism in which oral lactobacilli influence human gingival fibroblasts.

Abstract 1888: Metabolomic analysis provides insights into uncoupling-induced cancer resistance in the K5-UCP3 mouse model.

Abstract Metabolic reprogramming is a hallmark of tumorigenesis and malignancy across a variety of cancer types. Reduced oxidative phosphorylation and increased ATP synthesis by glycolytic upregulation is thought to provide both the energy and metabolic precursors necessary to meet the increased biosynthetic and energetic demands associated with malignancy. We hypothesized that since uncoupling protein 3 (UCP3) dissipates the mitochondrial electrochemical proton gradient, it may lead to the consumption or depletion of biosynthetic substrates necessary for carcinogenesis. In support, previous studies have shown that mice expressing a keratin 5-UCP3 transgene are completely resistant to chemically- and Ras-induced skin carcinogenesis, however the metabolic consequences of UCP3 overexpression in relation to cancer resistance are unclear. To examine the metabolic changes associated with UCP3 expression and this profound skin cancer resistance, we performed metabolomic analysis of more than 350 metabolites in epidermis from K5-UCP3 and wild type FVB/N littermate mice. Results of this analysis show significant UCP3-dependent changes in metabolites indicative of increased energy harvesting via glycolysis, beta-oxidation and branched chain amino acid metabolism. Consistent with decreased energy reserve, K5-UCP3 epidermis exhibits increased uncoupled respiration, decreased ATP and increased AMP levels, which correspond to increased AMP Kinase (AMPK) activation (Thr172 phosphorylation). Taken together, these data suggest that uncoupling proteins might function to oppose the metabolic demands of tumorigenesis and suggest that targeting mitochondrial uncoupling may be a novel and effective strategy in cancer prevention and treatment. Citation Format: Ashley Solmonson, Mignon Keaton, Sara M. Nowinski, Elizabeth Morin-Kensicki, Edward M. Mills. Metabolomic analysis provides insights into uncoupling-induced cancer resistance in the K5-UCP3 mouse model. [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 1888. doi:10.1158/1538-7445.AM2013-1888

Abstract LB-131: Mieap, a p53-inducible protein, controls mitochondrial quality by repairing or eliminating unhealthy mitochondria.

Abstract The accumulation of unhealthy mitochondria results in mitochondrial dysfunction, which has been implicated in aging, cancer, and a variety of degenerative diseases. However, the mechanism by which mitochondrial quality is regulated remains unclear. Here, we show that Mieap, a novel p53-inducible protein, plays a critical role in mitochondrial quality control. Mieap expression is directly regulated by p53 and is frequently lost in human cancer as result of DNA methylation. Mieap dramatically induces the accumulation of lysosomal proteins within mitochondria and mitochondrial acidic condition without destroying the mitochondrial structure (designated MALM, for Mieap-induced accumulation of lysosome-like organelles within mitochondria) in response to mitochondrial damage. MALM is involved in the degradation of oxidized mitochondrial proteins, leading to increased ATP synthesis and decreased reactive oxygen species generation. However, when MALM is inhibited, Mieap induces vacuole-like structures (designated as MIV for Mieap-induced vacuole), which engulf and degrade the unhealthy mitochondria by accumulating lysosomes. The inactivation of p53 severely impairs both MALM and MIV generation. These results suggest that Mieap plays a pivotal role in mitochondrial quality control by repairing or eliminating unhealthy mitochondria via MALM or MIV generation, respectively. Cancer cells might accumulate unhealthy mitochondria due to p53 mutations and/or Mieap methylation, representing a potential cause of the Warburg effect. Citation Format: Yasuyuki Nakamura, Hiroki Kamino, Hitoya Sano, Hirofumi Arakawa. Mieap, a p53-inducible protein, controls mitochondrial quality by repairing or eliminating unhealthy mitochondria. [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 LB-131. doi:10.1158/1538-7445.AM2013-LB-131

Mitochondria‐targeted antioxidant promotes recovery of skeletal muscle mitochondrial function after burn trauma assessed byin vivo31P nuclear magnetic resonance and electron paramagnetic resonance spectroscopy

Burn injury causes a major systemic catabolic response that is associated with mitochondrial dysfunction in skeletal muscle. We investigated the effects of the mitochondria-targeted peptide antioxidant Szeto-Schiller 31 (SS-31) on skeletal muscle in a mouse burn model using in vivo phosphorus-31 nuclear magnetic resonance ((31)P NMR) spectroscopy to noninvasively measure high-energy phosphate levels; mitochondrial aconitase activity measurements that directly correlate with TCA cycle flux, as measured by gas chromatography mass spectrometry (GC-MS); and electron paramagnetic resonance (EPR) to assess oxidative stress. At 6 h postburn, the oxidative ATP synthesis rate was increased 5-fold in burned mice given a single dose of SS-31 relative to untreated burned mice (P=0.002). Furthermore, SS-31 administration in burned animals decreased mitochondrial aconitase activity back to control levels. EPR revealed a recovery in redox status of the SS-31-treated burn group compared to the untreated burn group (P<0.05). Our multidisciplinary convergent results suggest that SS-31 promotes recovery of mitochondrial function after burn injury by increasing ATP synthesis rate, improving mitochondrial redox status, and restoring mitochondrial coupling. These findings suggest use of noninvasive in vivo NMR and complementary EPR offers an approach to monitor the effectiveness of mitochondrial protective agents in alleviating burn injury symptoms.

Abstract TP126: The Ketones Acetoacetate And Beta-hydroxybutyrate Alleviate Ischemic Injury Following Occlusion Of The Middle Cerebral Artery

Acute ischemic stroke is treated with thrombolysis but therapeutic benefits are modest. One potential reason for the limited clinical improvement is the apoptotic, delayed cell death that occurs prominently in the periphery of the ischemic core, the penumbra, despite successful reperfusion. Apoptotic cell death has been attributed to glutamate excitotoxicity, a consequence of ischemic energy depletion as well as a cause of persistent mitochondrial dysfunction that perpetuates energetic failure. We have previously shown that glutamate excitotoxicity and mitochondrial dysfunction are alleviated by the ketones acetoacetate and beta-hydroxybutyrate, a combination of physiological compounds produced by liver during calorie restriction to sustain cellular energy requirements. Ketones enhance mitochondrial respiration driven by complex I of the electron transport chain, decrease oxidative stress and increase ATP synthesis. Here, we investigated the effects of ketones in 3 month-old mice subjected to middle cerebral artery (MCA) occlusion with a filament for 90 min. Ketones (50 mg/kg) were administered intraperitoneally 30 min after the onset of ischemia and significantly improved outcomes. MCA occlusion caused significant neuronal loss 24 hours later in the subcortical and cortical regions of the ipsilateral hemisphere. Damage was most severe in the subcortical region and was quite evident on a 7 Tesla MRI. The results were confirmed by non-invasive imaging of apoptosis with the fluorescent indicator Annexin Vivo 750 using a Xenogen IVIS Spectrum (Caliper). Treating mice with ketones significantly decreased neuronal damage based on histology, MRI and fluorescence imaging of apoptosis with Annexin Vivo 750. Furthermore, ketones improved motor activity on the Rotarod and Open Field tests. Untreated mice exhibited evident decreases in the times they remained on the rods whereas ketone-treated mice were almost back to baseline. Consistently, untreated animals were less active in the open field. In sum, our data show that a combination of physiological ketones previously shown to enhance mitochondrial activity and alleviate glutamate excitotoxicity decreases cellular loss and improves motor function in a mouse model of ischemic stroke.

G13 Abnormal responses to visual cortex activation in early stage Huntington disease patients using 31P-NMR spectroscopy

Objective To test the hypothesis that brain energy metabolism is abnormal in patients at an early stage of Huntington disease (HD). Background Energy metabolism has been a major focus of HD research for many years due to several observations in both patients and models of the disease. However, there are currently no in vivo biomarkers of brain energy metabolism in HD. Methods We coupled noninvasive 31P-NMR spectroscopy with activation of the occipital cortex in order to measure the levels of ATP, phosphocreatine (PCr) and inorganic phosphate (Pi) before, during and after a visual stimulus. We studied 15 HD patients at an early stage of the disease (mean motor UHDRS =18±9) and 15 age- and sex-matched controls. Results In controls, we observed an 11% increase in Pi/PCr ratio (p=0.024) and a 13% increase in Pi/ATP ratio (p=0.016) during brain activation, reflecting increased ATP synthesis and ADP levels. Subsequently, controls had a return to baseline levels during recovery (p=0.012 et 0.022 respectively). In HD patients, both Pi/PCr and Pi/ATP ratios were unchanged during and after visual stimulation, reflecting altered mitochondrial bioenergetics. In addition, in HD patients the ratio of Pi/ATP correlated with the UHDRS score during the activation (p=0.014) and recovery periods (p=0.009), while Pi/PCr ratio correlated with the UHDRS score during recovery (p=0.016), reflecting a correlation between brain energy metabolism and disease severity in HD. Conclusions 31Phosphorus nuclear magnetic resonance spectroscopy could provide functional biomarkers of brain energy deficit to monitor therapeutic efficacy in Huntington disease.

Membrane Sodium Potassium ATPase Inhibition Mediated ATP Synthesis Induced by Digoxin, Photoinduction and Electromagnetic Fields

Aims and Objectives: Endogenous digoxin, exposure to sunlight and electric fields can produce membrane sodium potassium ATPase inhibition. Membrane sodium potassium ATPase in the setting of digoxin induced inhibition can synthesize ATP. The membrane sodium potassium ATPase mediated ATP synthesis can serve as a source for cellular energetics. This was studied and reported in this paper. Methodology: The following groups were included in the study: -endomyocardial fibrosis, alzheimer’s disease, multiple sclerosis, non-hodgkin’s lymphoma, metabolic syndrome x with cerebrovascular thrombosis and coronary artery disease, schizophrenia, autism, seizure disorder, creutzfeldt jakob disease and acquired immunodeficiency syndrome. There were 10 patients in each group and each patient had an age and sex matched healthy control selected randomly from the general population. The blood samples were drawn in the fasting state before treatment was initiated. Membrane sodium potassium ATPase was inhibited by the addition of digoxin at concentration 1 mg/ml and exposure to sunlight and low intensity electromagnetic fields for 1 hour. The ATP synthesis by membrane sodium potassium ATPase in the inhibited state was studied. Results: The results of the study showed increased ATP synthesis by RBC membrane sodium potassium ATPase in the presence of added digoxin, exposure to sunlight and low intensity electromagnetic fields. The added digoxin, exposure to sunlight and low intensity electromagnetic fields produce sodium potassium ATPase inhibition. Membrane sodium potassium ATPase in its inhibited state can synthesize ATP. The results are expressed as percentage change in the parameters after 1 hour incubation as compared to the values at zero time. There was RBC membrane sodium potassium ATPase mediated ATP synthesis in the patients with schizophrenia, malignancy, metabolic syndrome x, autoimmune disease and neuronal degeneration in the presence of added digoxin, exposure to sunlight and low intensity electromagnetic fields. Conclusion: The membrane sodium potassium ATPase inhibition mediated ATP synthesis may serve as a major source of cellular energetics. The sunlight induced photic induction of membrane sodium potassium ATPase inhibition may be a primitive source of cellular energy before the evolution of mitochondrial oxidative phosphorylation. The electromagnetic induction of membrane sodium potassium ATPase inhibition mediated ATP synthesis may also be a similar primitive source of cellular energy. The photic and electromagnetic inhibition mediated membrane sodium potassium ATPase inhibition related ATP synthesis still serves as a major source of cellular energetics despite the presence of mitochondrial oxidative phosphorylation and anaerobic glycolysis. Key words: Membrane sodium potassium ATPase; Digoxin; Photic induction; Electromagnetic fields; ATP synthesis