Mitochondrial F1F0-ATPase Research Articles

Abnormalities of neural stem cells in Lesch–Nyhan disease

Lesch–Nyhan disease (LND) is a neurodevelopmental disorder caused by variants in the HPRT1 gene, which encodes the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGprt). HGprt deficiency provokes numerous metabolic changes which vary among different cell types, making it unclear which changes are most relevant for abnormal neural development. To begin to elucidate the consequences of HGprt deficiency for developing human neurons, neural stem cells (NSCs) were prepared from 6 induced pluripotent stem cell (iPSC) lines from individuals with LND and compared to 6 normal healthy controls. For all 12 lines, gene expression profiles were determined by RNA-seq and protein expression profiles were determined by shotgun proteomics. The LND lines revealed significant changes in expression of multiple genes and proteins. There was little overlap in findings between iPSCs and NSCs, confirming the impact of HGprt deficiency depends on cell type. For NSCs, gene expression studies pointed towards abnormalities in WNT signaling, which is known to play a role in neural development. Protein expression studies pointed to abnormalities in the mitochondrial F0F1 ATPase, which plays a role in maintaining cellular energy. These studies point to some mechanisms that may be responsible for abnormal neural development in LND.

Hexane fraction of Globimetula braunii induces mitochondria-mediated apoptosis in Plasmodium berghei-infected mice

BackgroundApoptosis is a common pathology in malaria and most antimalarial drugs induce apoptosis during chemotherapy. Globimetula braunii is an African mistletoe used for the treatment of malaria but its effect on mitochondria-mediated apoptosis is not known. MethodsMalarial infection was induced by the intraperitoneal injection of NK 65 strain Plasmodium berghei-infected erythrocytes into mice which were treated with graded doses (100–400 mg/kg) of methanol extract (ME), and fractions of n-hexane, dichloromethane, ethylacetate and methanol (HF, DF, EF and MF) for 9 days after the confirmation of parasitemia. Artequine (10 mg/kg) was used as control drug. The fraction with the highest antiplasmodial activity was used (same dose) to treat mice infected with chloroquine-resistant (ANKA) strain for 5 consecutive days after the confirmation of parasitemia. P-alaxin (10 mg/kg) was used as control drug. On the last day of the treatment, liver mitochondria were isolated and mitochondrial Permeability Transition (mPT) pore opening, mitochondrial F0F1 ATPase (mATPase) activity, lipid peroxidation (mLPO) and liver deoxyribonucleic acid (DNA) fragmentation were assessed spectrophotometrically. Caspases 3 and 9 were determined by Enzyme-Linked Immunosorbent Assay (ELISA) technique. Cytochrome c, P53, Bcl-2-associated X protein (Bax), and B-cell lymphoma-2 (Bcl2) were determined via immunohistochemistry. Phytochemical constituents of the crude methanol extract of Globimetula braunii were determined via the Gas Chromatography-Mass Spectrometry (GC-MS) analysis ResultsThere was large amplitude mPT induction by malaria parasites, extract and fractions of Globimetula braunii. At 400 mg/kg, HF significantly (p < 0.01) downregulated mATPase activity, and mLPO in both (susceptible and resistant) models, caused DNA fragmentation (P < 0.0001), induced caspases activation, P53, bax and cytochrome c release but downregulated Bcl2 in both models. The GC-MS analysis of methanol extract of Globimetula braunii showed that α-amyrin is the most abundant phytochemical. ConclusionThe n-hexane fraction of Globimetula braunii induced mitochondrial-mediated apoptosis through the opening of the mitochondrial pore, fragmentation of genomic DNA, increase in the levels of P53, bax, caspase 3 and 9 activation and cytochrome c release with concomitant decrease in the level of Bcl2. α-Amyrin is a triterpene with apoptotic effects.

Curcumin modulates multiple cell death, matrix metalloproteinase activation and cardiac protein release in susceptible and resistant Plasmodium berghei-infected mice

Pro-inflammatory signaling, cell death, and metalloproteinases activation are events in Plasmodium infection. However, it is not known if treatment with mefloquine (MF), and curcumin (CM) supplementation, will modulate these conditions. Malaria was induced in two different studies using susceptible (NK 65, study 1) and resistant (ANKA, study 2) strains of mouse malaria parasites (Plasmodium berghei) in thirty male Swiss mice (n = 5) in each study. Following confirmation of parasitemia, mice received 10 mL/kg distilled water (infected control), MF (10 mg/kg), MF and CM (25 mg/kg), MF and CM (50 mg/kg), CM (25 mg/kg) and CM (50 mg/kg). Five mice (not infected) were used as control. After treatment, the animals were sacrificed, serum obtained and liver mitochondria were isolated. Serum Tumour Necrosis Factor alpha (TNF-α), C-reactive protein (CRP), Interleukins-1 beta (IL-1β) and Interleukins-6 (IL-6) as well as caspases-3, 9 (C3 and C9), p53, serum troponin I (TI) and creatine kinase (CK), were assayed using ELISA techniques. Mitochondrial membrane permeability transition (mPT) pore opening, mitochondrial F0F1 ATPase activity, and lipid peroxidation (mLPO) were determined spectrophotometrically. Matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) expressions were determined using electrophoresis. CM supplementation (25 mg/kg) significantly decreased serum p53, TNF-α, CRP and IL-6 compared with MF. In the resistant model, CM prevented mPT pore opening, significantly decreased F0F1 ATPase activity and mLPO. MF activated caspase-3 while supplementation with CM significantly decreased this effect. Furthermore, MMP-2 and MMP-9 were selectively expressed in the susceptible model. Malarial treatment with mefloquine elicits different cell death responses while supplementation with curcumin decreased TI level and CK activities.

Mitoquinol mesylate (MITOQ) attenuates diethyl nitrosamine-induced hepatocellular carcinoma through modulation of mitochondrial antioxidant defense systems.

Diethyl nitrosamine (DEN) induced cirrhosis-hepatocellular carcinoma (HCC) model associates cancer progression with oxidative stress and mitochondrial dysfunction. This study investigated the effects of mitoquinol mesylate (MitoQ), a mitochondrial-targeted antioxidant on DEN-induced oxidative damage in HCC Wistar rats. Fifty male Wistar rats were randomly divided into five groups. Healthy control, DEN, and MitoQ groups were orally administered exactly 10mg/kgof distilled water, DEN, and MitoQ, respectively for 16weeks. Animals in the MitoQ + DEN group were pre-treated with MitoQ for a week followed by co-administration of 10mg/kg each of MitoQ and DEN. DEN + MitoQ group received DEN for 8weeks, then co-administration of 10mg/kg each of DEN and MitoQ till the end of 16th week. Survival index, tumour incidence, hematological profile, liver function indices, lipid profile, mitochondrial membrane composition, mitochondrial respiratory enzymes, and antioxidant defense status in both mitochondrial and post-mitochondrial fractions plus expression of antioxidant genes were assessed. In MitoQ + DEN and DEN + MitoQ groups, 80% survival occurred while tumour incidence decreased by 60% and 40% respectively, compared to the DEN-only treated group. Similarly, MitoQ-administered groups showed a significant (p<0.05) decrease in the activities of liver function enzymes while hemoglobin concentration, red blood cell count, and packed cell volume were significantly elevated compared to the DEN-only treated group. Administration of MitoQ to the DEN-intoxicated groups successfully enhanced the activities of mitochondrial F1F0-ATPase and succinate dehydrogenase; and up-regulated the expression and activities of SOD2, CAT, and GPx1. Macroscopic and microscopic features indicated a reversal of DEN-induced hepatocellular degeneration in the MitoQ + DEN and DEN + MitoQ groups. These data revealed that MitoQ intervention attenuated DEN-induced oxidative stress through modulation of mitochondrial antioxidant defense systems and alleviated the burden of HCC as a chemotherapeutic agent.

Identification of New Proteins and Potential Mitochondrial F1F0-ATPase Inhibitor Factor 1-Associated Mechanisms in Arabidopsis thaliana Using iTRAQ-Based Quantitative Proteomic Analysis

The mitochondrial synthesis of ATP makes a vital contribution to the growth and development of biological organisms, in which the enzyme mitochondrial F1F0-ATP synthase plays a pivotal role, in that it can either synthesize or hydrolyze cellular ATP. The finding of our previous study revealed that mitochondrial F1F0-ATPase inhibitor factor 1 (IF1) in Arabidopsis thaliana has a conserved function as an endogenous inhibitor affecting cellular energy status and plays an important role in plant growth and reproduction, particularly in fertility. In this study, to gain an insight into IF1-related traits, we performed isobaric tags for relative and absolute quantitation labeling analysis. In total, 67 of 4778 identified proteins were identified as differentially expressed proteins (DEPs; 59 up-regulated and 8 down-regulated) between wild-type and if1 mutant Arabidopsis thaliana seedlings. Gene ontology enrichment analysis revealed that these DEPs were the most significantly enriched in pathways such as “long-day photoperiodism, flowering,” “positive regulation of protein import into chloroplast stroma,” and “pollen sperm cell differentiation,” which are closely associated with reproductive development. Moreover, Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that photosynthesis was the pathway most significantly enriched with DEPs. Collectively, our results revealed a global shift in protein abundance patterns corresponding to AtIF1 mutation, entailing changes in the abundance of multiple key proteins and metabolic processes, which will provide a valuable proteomic foundation for future studies.

Anti-Inflammatory Potentials of the n-Hexane Fraction of Alstonia boonei Stem Bark in Lipopolysaccharide-Induced Inflammation in Wistar Rats.

BackgroundInflammation is a protective response of the host to infections and tissue damage and medicinal plants have been used to regulate inflammatory response. The phytochemical contents of the n-hexane fraction of Alstonia boonei and their anti-inflammatory potentials in lipopolysaccharide-induced inflammation were investigated in rat liver.Materials and MethodsA quantity of 5 mg/kg lipopolysaccharide (LPS) was used to induce inflammation in twenty-five male Wistar rats, grouped (n = 5) and treated as follows: negative control (10 mL/kg saline), positive control (1 mg/kg ibuprofen); 50, 100 and 20 mg/kg of the n-hexane fraction of Alstonia boonei were administered to test groups. In another experiment, twenty rats (n = 5, without LPS) were administered the same doses of the n-hexane fraction of A. boonei and ibuprofen for seven days. At the end of the experiment, animals were sacrificed, serum was obtained from blood and liver mitochondria isolated in a refrigerated centrifuge. Mitochondrial permeability transition (mPT) pore opening and mitochondrial F0F1 ATPase (mATPase) were determined spectrophotometrically. Serum interleukins 1β, 6 (IL-1β, IL-6), tumour necrosis factor alpha (TNF-α), C-reactive protein (CRP) and creatine kinase (CK), gamma glutamyl transferase (GGT), aspartate and alanine aminotransferases (AST and ALT,) of the animals in which inflammation was induced using LPS but treated with graded doses of n-hexane fraction of A. boonei were determined using the ELISA technique. The phytochemical contents of the n-hexane fraction of A. boonei were determined using ultra performance liquid chromatography-tandem mass spectrometer (UHPLC-MS).ResultsCalcium induced mPT in 8 fold and LPS induced mPT 14 fold in the negative control while the n-hexane fraction reversed mPT in the treated groups (50, 100 and 200 mg/kg) to 2, 4, 4 folds, respectively. LPS treatment of the negative group enhanced F0F1 mATPase activity, increased CRP, TNF-α, IL-1β, IL-6 levels as well as CK, AST, ALT and GGT activities. These values were significantly reduced by 100 and 200 mg/kg of the n-hexane fraction. UHPLC-MS analysis of the fraction revealed the presence of terpenoids, phenolics and sphingolipids.ConclusionThese results showed that bioactive phytochemicals present in the n-hexane fraction of A. boonei were not toxic, have an anti-inflammatory effect and could be used for the treatment of inflammatory diseases.

Betulinic acid purified from Alstonia boonei inhibits folate biosynthesis in malarial Plasmodium, enhances mitochondrial pore opening and F1F0 ATPase in mice

Activity-guided assay of antimalarial properties of Alstonia boonei led to the purification of betulinic acid from the chloroform fraction of the methanol stem bark extract. The structure and stereochemistry of betulinic acid from A. boonei was established by 1H, 13C, COSYHMBC, HSQC, LC-MS and DEPT techniques. This compound demonstrated antiplasmodial activities using chloroquine susceptible (NK 65) and resistant (ANKA) strains of Plasmodium berghei. Betulinic acid (BA) opens mitochondrial permeability transition pore (mPT), increased mitochondrial F1F0 ATPase activity, decreased lipid peroxidation and prevented DNA fragmentation. Molecular dynamics simulation studies against falcipain-2, dihydrofolate reductase, dihydropteroate synthase and mitochondrial F1F0 ATPase (F-2-BA, DHFR-BA, DHPS-BA and mATPase-BA, respectively) revealed that betulinic acid (BA) mechanistically disrupted the secondary structure of mATPase (mouse) with RMSD > 2 (RMSD=2.34 Å). Binding effects of BA was more prominent on DHFR as evidenced by high structural deviations (RMSD = 2.24 Å). However, Falcipain-2 had the least deviation (RMSD = 1.77 Å) correlative with the reduced activity of the compound. DHPS, on the other hand, had a mean RMSD value of 1.93 Å. The disruption of mATPase could explain the effect of the compound on the opening of mPT as experimentally reported.

Studies on the mitochondrial, immunological and inflammatory effects of solvent fractions of Diospyros mespiliformis Hochst in Plasmodium berghei-infected mice

The use of medicinal plants in the treatment of malaria is gaining global attention due to their efficacy and cost effectiveness. This study evaluated the bioactivity-guided antiplasmodial efficacy and immunomodulatory effects of solvent fractions of Diospyros mespiliformis in mice infected with a susceptible strain of Plasmodium berghei (NK 65). The crude methanol extract of the stem of D. mespiliformis (DM) was partitioned between n-hexane, dichloromethane, ethyl acetate and methanol. Male Swiss mice (20 ± 2 g) infected with P. berghei were grouped and treated with vehicle (10 mL/kg, control), Artemether lumefantrine (10 mg/kg), 100, 200 and 400 mg/kg of n-hexane, dichloromethane, ethyl acetate and methanol fractions of D. mespiliformis for seven days. Blood was obtained for heme and hemozoin contents while serum was obtained for inflammatory cytokines and immunoglobulins G and M assessments. Liver mitochondria were isolated for mitochondrial permeability transition (mPT), mitochondrial F1F0 ATPase (mATPase) and lipid peroxidation (mLPO) assays. The GC–MS was used to identify the compounds present in the most potent fraction. The dichloromethane fraction had the highest parasite clearance and improved hematological indices relative to the drug control. The heme values increased, while the hemozoin content significantly (P < 0.05) decreased compared with the drug control. The highest dose of HF and MF opened the mPT pore while the reversal effects of DF on mPT, mATPase and mLPO were dose-dependent. The levels of IgG, IgM and TNFα in the DF group were significantly higher than the drug control, while the IL-1β and IL-6 values did not vary linearly with the dose. Lupeol and Stigmastan-3,5-diene were the most abundant phytochemicals in the DF. The outcome of this study showed that the DF has immunomodulatory effects in infected mice, reduced proliferation of the malaria parasite and thus protect liver cells.

New Mechanistic Insights on Carbon Nanotubes' Nanotoxicity Using Isolated Submitochondrial Particles, Molecular Docking, and Nano-QSTR Approaches.

Simple SummaryCarbon nanotubes are revolutionary materials with applications in a lot of different areas. However, there is a rising concern regarding unlikely toxicity effects these materials may trigger. Due to this, the main aim of this paper is to develop a comprehensive approach to study toxicity effect of carbon nanotubes on the mitochondria F0F1-ATPase. We have employed a combination of experimental and computational study. In so doing, we have combined in vitro inhibition responses in submitochondrial particles with docking elastic network models, fractal surface analysis, and Nano-quantitative structure toxicity relationship models (Nano-QSTR models). Results show that this method may be used for the fast prediction of the nanotoxicity induced by single walled carbon nanotubes (SWCNT), avoiding time- and money-consuming techniques, and may open new avenues toward to the better understanding and prediction of new nanotoxicity mechanisms.Single-walled carbon nanotubes can induce mitochondrial F0F1-ATPase nanotoxicity through inhibition. To completely characterize the mechanistic effect triggering the toxicity, we have developed a new approach based on the combination of experimental and computational study, since the use of only one or few techniques may not fully describe the phenomena. To this end, the in vitro inhibition responses in submitochondrial particles (SMP) was combined with docking, elastic network models, fractal surface analysis, and Nano-QSTR models. In vitro studies suggest that inhibition responses in SMP of F0F1-ATPase enzyme were strongly dependent on the concentration assay (from 3 to 5 µg/mL) for both pristine and COOH single-walled carbon nanotubes types (SWCNT). Besides, both SWCNTs show an interaction inhibition pattern mimicking the oligomycin A (the specific mitochondria F0F1-ATPase inhibitor blocking the c-ring F0 subunit). Performed docking studies denote the best crystallography binding pose obtained for the docking complexes based on the free energy of binding (FEB) fit well with the in vitro evidence from the thermodynamics point of view, following an affinity order such as: FEB (oligomycin A/F0-ATPase complex) = −9.8 kcal/mol > FEB (SWCNT-COOH/F0-ATPase complex) = −6.8 kcal/mol ~ FEB (SWCNT-pristine complex) = −5.9 kcal/mol, with predominance of van der Waals hydrophobic nano-interactions with key F0-ATPase binding site residues (Phe 55 and Phe 64). Elastic network models and fractal surface analysis were performed to study conformational perturbations induced by SWCNT. Our results suggest that interaction may be triggering abnormal allosteric responses and signals propagation in the inter-residue network, which could affect the substrate recognition ligand geometrical specificity of the F0F1-ATPase enzyme in order (SWCNT-pristine > SWCNT-COOH). In addition, Nano-QSTR models have been developed to predict toxicity induced by both SWCNTs, using results of in vitro and docking studies. Results show that this method may be used for the fast prediction of the nanotoxicity induced by SWCNT, avoiding time- and money-consuming techniques. Overall, the obtained results may open new avenues toward to the better understanding and prediction of new nanotoxicity mechanisms, rational drug design-based nanotechnology, and potential biomedical application in precision nanomedicine.

Nanozymes as Enzyme Inhibitors.

Nanozyme is a type of nanomaterial with intrinsic enzyme-like activity. Following the discovery of nanozymes in 2007, nanozyme technology has become an emerging field bridging nanotechnology and biology, attracting research from multi-disciplinary areas focused on the design and synthesis of catalytically active nanozymes. However, various types of enzymes can be mimicked by nanomaterials, and our current understanding of nanozymes as enzyme inhibitors is limited. Here, we provide a brief overview of the utility of nanozymes as inhibitors of enzymes, such as R-chymotrypsin (ChT), β-galactosidase (β-Gal), β-lactamase, and mitochondrial F0F1-ATPase, and the mechanisms underlying inhibitory activity. The advantages, challenges and future research directions of nanozymes as enzyme inhibitors for biomedical research are further discussed.

Identification and characterization of Arabidopsis thaliana mitochondrial F1F0-ATPase inhibitor factor 1

Mitochondrial F1F0-ATP synthase (F1F0-ATPase) inhibitor factor 1 (IF1) has been extensively characterized as an endogenous inhibitor that prevents the hydrolysis of adenosine-5'-triphosphate (ATP) by mitochondrial ATPases in mammals and yeasts; however, IF1’s functions in plants remain unclear. Here, a comprehensive bioinformatic analysis was performed to identify plant mitochondrial F1F0-ATPase IF1 orthologs. Plant IF1s contain a conserved F1F0-ATPase inhibitory domain, but lack the antiparallel α-helical coiled-coil structure compared with mammalian IF1s. A subcellular localization analysis in Arabidopsis thaliana revealed that AtIF1-green fluorescent protein was present only in mitochondria. Additionally, AtIF1 was widely expressed in diverse organs and intense β-glucuronidase staining was observed in reproductive tissues and germinating seeds. Compared with the wild-type and p35S:AtIF1-if1 etiolated seedlings, the ATP/ADP ratio was significantly lower in the AtIF1 T-DNA knockout seedlings (if1 mutant) growing under dark conditions, suggesting that AtIF1 can influence the energy state of cells. A significant reduction in seed yield and strong growth retardation under dark conditions were observed in the if1 mutant line. Furthermore, if1 plants exhibited a substantially decreased sensitivity to abscisic acid. Thus, the A. thaliana mitochondrial IF1, which is a conserved F1F0-ATPase inhibitor, is crucial for plant growth and responses to abscisic acid.

Mitoubiquinol Mesylate Attenuated Diethyl Nitrosamine–Induced Hepatocellular Carcinoma Through Modulation of Mitochondrial Antioxidant Defense Systems

PURPOSE Hepatocellular carcinoma (HCC) is a highly malignant cancer, with a high recurrence rate and a poor prognosis. Diethyl nitrosamine (DEN) cirrhosis HCC–induced model has revealed an association of cancer progression with oxidative stress and mitochondrial dysfunction. This study investigated the effects of mitoubiquinol mesylate (MitoQ), a mitochondrial targeted antioxidant derivative from ubiquinone on DEN-induced oxidative damage in HCC Wistar rats. METHODS Fifty male Wistar rats were randomly divided into 5 groups, 10 rats per group. Groups A, B, and C received distilled water 10 mL/kg DEN, and MitoQ orally for 16 weeks, respectively. Animals in group D were pretreated with MitoQ for 1 week followed by coadministration of MitoQ and DEN (protective effect), whereas group E received DEN for 8 weeks, then coadministration of DEN and MitoQ (therapeutic effect) until the end of the study. Survival index, tumor incidence, liver function indices, hematologic profile, mitochondrial respiratory enzymes, and antioxidant defense status were assessed. RESULTS Data obtained show that rats in groups D and E had 80% survival and decreased tumor incidence (40% and 60%, respectively) compared with group B. Similarly, MitoQ significantly ( P &lt; .05) decreased the activities of liver function enzymes, while hemoglobin concentration, red blood cell count, and lymphocytes levels were significantly elevated compared with the DEN-only group. Furthermore, MitoQ significantly ( P &lt; .05) protected the liver from DEN-induced oxidative damage; however, there was no significant difference ( P &gt; .05) between activities of mitochondrial F1F0-ATPase and succinate dehydrogenase of groups A, B, D, and E, respectively, although these enzyme activities were significantly ( P &lt; .05) elevated in group C. Macroscopic and microscopic features indicated a reversal of DEN-induced cellular degeneration in hepatocytes. CONCLUSION These data suggest that MitoQ treatment for 16 weeks attenuated DEN-induced oxidative stress indices via modulation of mitochondrial antioxidant defense systems and could alleviate the burden of HCC as a chemotherapeutic agent.

P15 Effects of coptisine on glucose metabolism and the mechanisms

Aim: Coptis chinensis (Chinese goldthread) has been widely used in Traditional Chinese Medicine for centuries and has a variety of pharmacological effects. In this study, we investigated the effect of coptisine on glucose related metabolic regulation in vitro and in vivo . Methods: Normal ICR mice, alloxan-induced type 1 diabetic mice and type 2 diabetic mice (KKay mice) were employed to explore the effect of coptisine on glucose metabolism in vivo . Human hepatoma cells (HepG2) and rat skeletal muscle myotube cells (L6) were used to investigate the effect of coptisine on the glucose metabolism in vitro . Results: In normal ICR mice, coptisine (10 mg/kg) decreased the fasting and fed blood glucose significantly without any significant effect on body weight. In alloxan-induced type 1 diabetic mice, coptisine decreased fasting and fed blood glucose. In type 2 diabetic Kkay mice, coptisine improved insulin tolerance and glucose tolerance but did not change LDL, TG, HDL or CHO level in serum. Our in vitro results showed that coptisine increased the glucose consumption and glucose uptake in HepG2 cells and L6 myotube cells. Coptisine significantly increased ATP level in HepG2 cells and decreased mitochondrial membrane potential in HepG2 cells. Furthermore, coptisine promoted hepatic mitochondrial F0F1-ATPase activity. Conclusion: In summary, coptisine has been shown to be effective in improving glucose metabolism in animal models. The effects of coptisine may be closely related with enhanced function of the mitochondria.

The antioxidant effect of ubiquinone and combined therapy on mitochondrial function in blood cells in non-proliferative diabetic retinopathy: A randomized, double-blind, phase IIa, placebo-controlled study

Objectives: To evaluate the effect of ubiquinone and combined antioxidant therapy on mitochondrial function in non-proliferative diabetic retinopathy (NPDR) in a randomized, double-blind, phase IIa, placebo-controlled, clinical trial. Three groups of 20 patients were formed: Group 1, ubiquinone; Group 2, combined therapy; and Group 3, placebo (one daily dose for 6 months).Methods: Fluidity of the submitochondrial membrane in platelets was determined by examining intensity of fluorescence between the monomer (Im) and excimer (Ie). Hydrolytic activity of the mitochondrial F0F1-ATPase was evaluated with the spectrophotometric method.Results: Normal, baseline submitochondrial membrane fluidity, 0.24 ± 0.01 Ie/Im, was significantly diminished in the three study groups vs. normal values (P < 0.0001); placebo, 0.14 ± 0.01 Ie/Im; ubiquinone, 0.14 ± 0.01 Ie/Im; and combined therapy, 0.13 ± 0.00 Ie/Im. Afterward, it increased significantly (P < 0.0001), the ubiquinone group 0.22 ± 0.01 Ie/Im, combined therapy group, 0.19 ± 0.01 Ie/Im; with no changes the placebo group. Baseline hydrolytic activity of the F0F1-ATPase enzyme increased in the three study groups vs. normal values (184.50 ± 7.84 nmol PO4), placebo, 304.12 ± 22.83 nmol PO4 (P < 0.002); ubiquinone, 312.41 ± 25.63 nmol PO4 (P < 0.009); and combined therapy, 371.28 ± 33.50 nmol PO4 (P < 0.002). Afterward, a significant decrease the enzymatic activity: ubiquinone, 213.25 ± 14.19 nmol PO4 (P < 0.001); and combined therapy, 225.55 ± 14.48 nmol PO4 (P < 0.0001).Discussion: Mitochondrial dysfunction significantly improved in groups of NPDR patients treated with antioxidants.

Calorie restriction does not restore brain mitochondrial function in P301L tau mice, but it does decrease mitochondrial F0F1-ATPase activity.

Calorie restriction (CR) has been shown to increase lifespan and delay aging phenotypes in many diverse eukaryotic species. In mouse models of Alzheimer's disease (AD), CR has been shown to decrease amyloid-beta and hyperphosphorylated tau levels and preserve cognitive function. Overexpression of human mutant tau protein has been shown to induce deficits in mitochondrial electron transport chain complex I activity. Therefore, experiments were performed to determine the effects of 4-month CR on brain mitochondrial function in Tg4510 mice, which express human P301L tau. Expression of mutant tau led to decreased ADP-stimulated respiratory rates, but not uncoupler-stimulated respiratory rates. The membrane potential was also slightly higher in mitochondria from the P301L tau mice. As shown previously, tau expression decreased mitochondrial complex I activity. The decreased complex I activity, decreased ADP-stimulated respiratory rate, and increased mitochondrial membrane potential occurring in mitochondria from Tg4510 mice were not restored by CR. However, the CR diet did result in a genotype independent decrease in mitochondrial F0F1-ATPase activity. This decrease in F0F1-ATPase activity was not due to lowered levels of the alpha or beta subunits of F0F1-ATPase. The possible mechanisms through which CR reduces the F0F1-ATPase activity in brain mitochondria are discussed.

Inhibition of glycogen synthase kinase 3 ameliorates liver ischemia/reperfusion injury via an energy-dependent mitochondrial mechanism

The mechanisms of glycogen synthase kinase-3 (GSK-3)-mediated cytoprotection during liver ischemia/reperfusion (I/R) remain controversial, particularly in older organs. This study explores the role and potential mechanisms of GSK-3 in young and aging livers. A rodent partial warm I/R model was used to evaluate the therapeutic potential of GSK-3 modulation during hepatic I/R in young and aging Sprague-Dawley rats. GSK-3 inhibition through IPC or SB216763 (SB21) preconditioning protected young rats from I/R-induced liver injury. This protection was absent in old animals but could be restored by glucose infusion prior to the I/R insult. The protection conferred by GSK-3 inhibition depended on mitochondrial metabolism regulation. Indeed, the inhibition of GSK-3 suppressed mitochondrial permeability transition pore (MPTP) opening, triggering mitohormesis in young animals, whereas insufficient fuel suppressed mitochondrial metabolism and inactivated the GSK-3-related protection in old animals. SB21 and glucose reactivated the mitochondrial F0F1-ATPase and subsequent protective cascades in the senescent liver. These effects were antagonized by an ATPase inhibitor and by an MPTP opener. The protection conferred by GSK-3 inhibition during hepatic I/R insult is energy dependent, particularly in senescent livers. These findings demonstrate a key role for GSK-3-related mitochondrial energy homeostasis, which may shed new light on the clinical use of GSK-3 inhibitors to protect liver function in surgical settings, particularly for older patients.

Su1387 F1F0-ATPase Modulation Is Efficacious in Models in IBD and Induces Apoptosis of Human CCR9+ Gut-Tropic T-cells

Background: Targeting immune metabolism may represent a novel approach for therapeutic intervention in autoimmunity. The mitochondrial F1F0-ATPase is the final complex in the electron transport chain. Small molecule, allosteric modulators of this complex cause increase in superoxide formation and apoptosis of susceptible cells. Because of their distinctive metabolic phenotype, chronically activated lymphocytes are preferentially sensitive to ATPase Modulator-induced apoptosis vs resting or acutely activated lymphocytes. In inflammatory bowel disease (IBD), mucosal lymphocytes are chronically activated, overexpress anti-apoptotic proteins and show resistance to activation-induced cell death (AICD). Thus, restoring lymphocyte AICD may be a potential therapeutic strategy. Here a set of F1F0-ATPase Modulators was evalulated in murine IBD models and in gut-tropic T cells from IBD patients. Methods: Acute IBD was induced by sensitizing mice with topical TNBS followed 7 days later by intracolonic TNBS challenge. ATPase Modulators were dosed orally on Day1 and Day2 after TNBS challenge. On Day4 mice were sacrificed and the colon was collected for histological analysis or isolation of lamina propria mononuclear cells which were analyzed for viability and inflammatory molecules by flow cytometry or RT-PCR. Peripheral blood mononuclear cells (PBMCs) from healthy donors or IBD patients were treated with ATPase Modulators for 20 hours. Cell death of activated, gut-tropic T cells (CD3+CCR9+) vs other T cells (CD3+ CCR9-) was detected by 7ADD uptake and Annexin V staining. Statistical analyses used unpaired Student's T test. Results: In acute murine TNBS-induced colitis, oral dosing at of ATPase Modulators attenuated weight loss, improved histologic assessments, decreased IFNγ and TNF production and significantly increased LP lymphocyte cell death (%viability: vehicle 15.8±3.3 vs LYC-51194 27.3±6.6, p<0.05). In PBMC from healthy subjects (n=24), ATPase Modulators were able to induce cell death but only in CCR9+ gut-tropic T cells (%cell death: CD3+CCR9+ vehicle 22.9±11.7 vs LYC-53661 56.6±11.7, p<0.0001; CD3+CCR9vehicle 0.47±0.51 vs LYC-53661 3.2±2.97). The induction of cell death in gut-tropic T cells was preceded by an increase in reactive oxygen species and was prevented by the ROS scavenger vitamin E. Similar experiments were performed with PBMC from IBD patients (CD n=15, UC n=15). LYC-51661, and similarly LYC-51194, induced cell death in CCR9+ T cells (%cell death: CD vehicle 17.9±13 vs LYC-51661 61.3±16.6 p< 0.0001; UC vehicle 18.8±9.5 vs LYC-51661 55.2±16.1 p< 0.0001) while sparing CD3+CCR9negative cells. Conclusions: The apparently selective effect on chronically activated lymphocytes may provide an advantage over current immunosuppressive treatments and suggest that ATPase Modulators may be a promising approach for treating IBD.

594 Anti-Inflammatory Effects of ApoA-I in Experimental Colitis Through Inhibition of Intestinal Monocyte Infiltration and Activiation

Background: There is an increasing recognition that targeting immune metabolism may represent a new approach for therapeutic intervention in autoimmune diseases such as inflammatory bowel disease (IBD). Small molecules that allosterically modulate the activity of the mitochondrial F1F0-ATPase induce apoptosis in susceptible cells and have shown robust efficacy in several models of immune-mediated disease. Chronically activated lymphocytes have a distinctive metabolic phenotype that sensitizes these cells to ATPase Modulatorinduced apoptosis compared to acutely activated cells. This may provide an advantage over current less specific immunosuppressive treatments. In IBD, lamina propria (LP) lymphocytes are chronically activated, overexpress anti-apoptotic proteins and are resistant to activationinduced cell death (AICD). Restoring AICD of lymphocytes by inhibiting ATPase activity is a potential therapeutic strategy. Here, three ATPase Modulators were evaluated in murine IBD models and in LP cells from IBD patient biopsies.Methods: A relapsing-remitting model of chronic murine IBD was induced by intracolonic administration of 4-5 escalating doses of TNBS. ATPase modulators were dosed orally after 2-3 TNBS challenges. Mucosal LP mononuclear cells were isolated from mice treated with vehicle or LYC-51194 and analyzed for viability and inflammatory molecules by flow cytometry and RT-PCR. LP cells isolated from IBD patient biopsies were treated with LYC-51194 or anti-Fas mAb and viability assessed by flow cytometry. Results: Bz-423, LYC-51661 and LYC-51194 inhibited the F1F0-ATPase and induced lymphocyte apoptosis in vitro. In a relapsing-remitting model of chronic murine IBD, ATPase modulators did not show any overt toxicity and produced benefits including attenuated weight loss, normalized histology, decreased inflammatory cytokine production (IFNγ, IL-6, TNF) and increased the percentage of cell death in LP T cells (vehicle 22±6 vs LYC-51194 53±10 p<0.0009). To translate these findings to human biology, LP cells isolated from IBD patient biopsies were treated with LYC-51194 or antiFas mAb. In agreement with previously published results, LP T cells from inflamed areas from CD subjects showed resistance to Fas-induced cell death (% cell death: vehicle 22.5±7.7 n=10 vs anti-Fas 22±12 n=7). However, similar to what was observed in rodent disease models, LYC-51194 treatment was able to induce apoptosis in LP T cells from inflamed IBD patient samples (% cell death: vehicle 22.5±7.7 n=10 vs LYC-51194 49.3±8.1 n=9; p= 0.0003). Conclusions: Taken together, these results suggest that induction of apoptosis by application of ATPase Modulator compounds may be a promising approach for treating IBD.

593 Modulation of the F1F0-ATPase Induces Apoptosis of Mouse and Human Lamina Propria T-cells and Is Efficacious in Models of IBD

Background: There is an increasing recognition that targeting immune metabolism may represent a new approach for therapeutic intervention in autoimmune diseases such as inflammatory bowel disease (IBD). Small molecules that allosterically modulate the activity of the mitochondrial F1F0-ATPase induce apoptosis in susceptible cells and have shown robust efficacy in several models of immune-mediated disease. Chronically activated lymphocytes have a distinctive metabolic phenotype that sensitizes these cells to ATPase Modulatorinduced apoptosis compared to acutely activated cells. This may provide an advantage over current less specific immunosuppressive treatments. In IBD, lamina propria (LP) lymphocytes are chronically activated, overexpress anti-apoptotic proteins and are resistant to activationinduced cell death (AICD). Restoring AICD of lymphocytes by inhibiting ATPase activity is a potential therapeutic strategy. Here, three ATPase Modulators were evaluated in murine IBD models and in LP cells from IBD patient biopsies.Methods: A relapsing-remitting model of chronic murine IBD was induced by intracolonic administration of 4-5 escalating doses of TNBS. ATPase modulators were dosed orally after 2-3 TNBS challenges. Mucosal LP mononuclear cells were isolated from mice treated with vehicle or LYC-51194 and analyzed for viability and inflammatory molecules by flow cytometry and RT-PCR. LP cells isolated from IBD patient biopsies were treated with LYC-51194 or anti-Fas mAb and viability assessed by flow cytometry. Results: Bz-423, LYC-51661 and LYC-51194 inhibited the F1F0-ATPase and induced lymphocyte apoptosis in vitro. In a relapsing-remitting model of chronic murine IBD, ATPase modulators did not show any overt toxicity and produced benefits including attenuated weight loss, normalized histology, decreased inflammatory cytokine production (IFNγ, IL-6, TNF) and increased the percentage of cell death in LP T cells (vehicle 22±6 vs LYC-51194 53±10 p<0.0009). To translate these findings to human biology, LP cells isolated from IBD patient biopsies were treated with LYC-51194 or antiFas mAb. In agreement with previously published results, LP T cells from inflamed areas from CD subjects showed resistance to Fas-induced cell death (% cell death: vehicle 22.5±7.7 n=10 vs anti-Fas 22±12 n=7). However, similar to what was observed in rodent disease models, LYC-51194 treatment was able to induce apoptosis in LP T cells from inflamed IBD patient samples (% cell death: vehicle 22.5±7.7 n=10 vs LYC-51194 49.3±8.1 n=9; p= 0.0003). Conclusions: Taken together, these results suggest that induction of apoptosis by application of ATPase Modulator compounds may be a promising approach for treating IBD.

Mitochondrial metabolism contributes to oxidative stress and reveals therapeutic targets in chronic lymphocytic leukemia

AbstractAlterations of cellular metabolism represent a hallmark of cancer. Numerous metabolic changes are required for malignant transformation, and they render malignant cells more prone to disturbances in the metabolic framework. Despite the high incidence of chronic lymphocytic leukemia (CLL), metabolism of CLL cells remains a relatively unexplored area. The examined untreated CLL patients displayed a metabolic condition known as oxidative stress, which was linked to alterations in their lymphoid compartment. Our studies identified mitochondrial metabolism as the key source for abundant reactive oxygen species (ROS). Unlike in other malignant cells, we found increased oxidative phosphorylation in CLL cells but not increased aerobic glycolysis. Furthermore, CLL cells adapted to intrinsic oxidative stress by upregulating the stress-responsive heme-oxygenase-1 (HO-1). Our data implicate that HO-1 was, beyond its function as an antioxidant, involved in promoting mitochondrial biogenesis. Thus ROS, adaptation to ROS, and mitochondrial biogenesis appear to form a self-amplifying feedback loop in CLL cells. Taking advantage of the altered metabolic profile, we were able to selectively target CLL cells by PK11195. This benzodiazepine derivate blocks the mitochondrial F1F0-ATPase, leads to a surplus production of mitochondrial superoxide, and thereby induces cell death in CLL cells. Taken together, our findings depict how bioenergetics and redox characteristics could be therapeutically exploited in CLL.