Decrease In ATP Levels Research Articles

Dapagliflozin attenuates steatosis in livers of high-fat diet-induced mice and oleic acid-treated L02 cells via regulating AMPK/mTOR pathway

Dapagliflozin (DAPA), a kind of sodium-glucose cotransporter 2(SGLT2) inhibitor is used to treat diabetes mellitus by inhibiting urine glucose reuptake. Recent clinical outcomes indicate that SGLT2 inhibitors may exert pharmacological activities against non-alcoholic fatty liver diseases. Nonetheless, the underlying molecular mechanisms are still poorly elucidated. In this study, we investigated the potential anti-fatty liver effects of DAPA in vivo and in vitro and assayed their underlying mechanisms. Male NIH (National Institutes of Health) mice were fed with a high-fat diet (HFD) and then treated with DAPA by gavage for 4 weeks. In the following experiments, L02 cells were treated with oleic acid (OA) and different concentrations of DAPA to assess lipid metabolism. Our results revealed that DAPA administration could remarkably suppress excessive fat accumulation in the liver tissues of HFD-fed mice and OA-treated L02 cells. Importantly, DAPA could downregulate the expression levels of proteins related to lipid synthesis and upregulate the expression levels of genes associated with fatty acid oxidation in vitro and in vivo. We also found that DAPA intervention could activate adenosine monophosphate-activated protein kinase (AMPK) phosphorylation but inhibit mammalian target of rapamycin (mTOR) phosphorylation in vitro and in vivo. AMPK activation might be mediated by increasing liver kinase B1 activity and decreasing ATP level. Furthermore, these ameliorative effects were completely eliminated by an AMPK inhibitor, compound C. This study suggested that DAPA might remarkably ameliorate hepatic steatosis mediated through the AMPK/mTOR pathway and thus could be a potential drug candidate for the treatment of fatty liver diseases.

Nitric Oxide-Dependent Electron Transport Chain Inhibition by the Cytochrome bc1 Inhibitor and Pretomanid Combination Kills Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of human tuberculosis, harbors a branched electron transport chain, preventing the bactericidal action of cytochrome bc1 inhibitors (e.g., TB47). Here, we investigated, using luminescent mycobacterial strains, the in vitro combination activity of cytochrome bc1 inhibitors and nitric oxide (NO) donors including pretomanid (PMD) and explored the mechanisms of combination activity. The TB47 and PMD combination quickly abolished the light emission of luminescent bacilli, as was the case for the combination of TB47 and aurachin D, a putative cytochrome bd inhibitor. The TB47 and PMD combination inhibited M. tuberculosis oxygen consumption, decreased ATP levels, and had a delayed bactericidal effect. The NO scavenger carboxy-PTIO prevented the bactericidal activity of the drug combination, suggesting the requirement for NO. In addition, cytochrome bc1 inhibitors were largely bactericidal when administered with DETA NONOate, another NO donor. Proteomic analysis revealed that the cotreated bacilli had a compromised expression of the dormancy regulon proteins, PE/PPE proteins, and proteins required for the biosynthesis of several cofactors, including mycofactocin. Some of these proteomic changes, e.g., the impaired dormancy regulon induction, were attributed to PMD. In conclusion, combination of cytochrome bc1 inhibitors with PMD inhibited M. tuberculosis respiration and killed the bacilli. The activity of cytochrome bc1 inhibitors can be greatly enhanced by NO donors. Monitoring of luminescence may be further exploited to screen cytochrome bd inhibitors.

Fucoxanthin induces prostate cancer PC-3 cell apoptosis by causing mitochondria dysfunction and oxidative stress

To investigate the apoptosis- inducing effect of fucoxanthin in human prostate cancer PC-3 cells and the underlying mechanism. The viability and apoptosis of PC-3 cells treated with fucoxanthin were analyzed using commercial kits, and the mitochondrial membrane potential, mitochondrial morphology and mitochondrial superoxide were detected using fluorescence probe staining. The contents of ATP, H2O2, malondialdehyde (MDA), superoxide and the total antioxidant capacity of PC-3 cells were determined. The protein expressions of Bcl-2, Bax and cytochrome c were detected with Western blotting, and the activity of caspase-9 and caspase- 3/7 was detected using corresponding kits. Fucoxanthin significantly inhibited the viability of PC-3 cells in a time- and dose-dependent manner, and dose-dependently induced apoptosis of the cells (P < 0.05). Fucoxanthin-treated PC-3 cells showed significantly decreased mitochondrial membrane potential, mitochondrial fragmentation and increased superoxide level in the mitochondria (P < 0.05), and these effects of fucoxanthin were dose- dependent. Fucoxanthin dose-dependently decreased ATP level and the total antioxidant capacity of PC-3 cells, increased the contents of H2O2, MDA and superoxide (all P < 0.05), enhanced the protein expressions of Bax and cytochrome c in the cytoplasm, and lowered the protein expressions of Bcl-2 and cytochromes in the mitochondria (P < 0.05). Fucoxanthin induces apoptosis of PC-3 cells by triggering mitochondrial dysfunction to cause oxidative stress and by activating mitochondria-mediated apoptotic signaling pathways, suggesting its potential in prostate cancer treatment.

Mutant p53L194F Harboring Luminal-A Breast Cancer Cells Are Refractory to Apoptosis and Cell Cycle Arrest in Response to MortaparibPlus, a Multimodal Small Molecule Inhibitor.

Simple SummaryTumor suppressor protein p53 is a master regulator that inhibits the process of oncogenesis by induction of cell senescence/cell cycle arrest/apoptosis during normal and stressed states of cells. It is functionally inactivated in the majority of cancers. Mortalin, a member of the Hsp70 family of proteins, enriched in cancer cells, is known to cause cytoplasmic sequestration and inactivation of the p53’s transcriptional activation function. Inhibition of mortalin–p53 interaction and reactivation of p53 functions by natural and synthetic drugs has emerged as a possible cancer therapeutic strategy. We recently reported a novel multimodal small molecule, named MortaparibPlus, that inhibited mortalin–p53 interaction and caused reactivation of p53 function in colorectal cancer cells. Here, we report its effect on breast cancer cells with wildtype (MCF-7) or mutant (T47D) p53 status.We previously performed a drug screening to identify a potential inhibitor of mortalin–p53 interaction. In four rounds of screenings based on the shift in mortalin immunostaining pattern from perinuclear to pan-cytoplasmic and nuclear enrichment of p53, we had identified MortaparibPlus (4-[(1E)-2-(2-phenylindol-3-yl)-1-azavinyl]-1,2,4-triazole) as a novel synthetic small molecule. In order to validate its activity and mechanism of action, we recruited Luminal-A breast cancer cells, MCF-7 (p53wild type) and T47D (p53L194F) and performed extensive biochemical and immunocytochemical analyses. Molecular analyses revealed that MortaparibPlus is capable of abrogating mortalin–p53 interaction in both MCF-7 and T47D cells. Intriguingly, upregulation of transcriptional activation function of p53 (as marked by upregulation of the p53 effector gene—p21WAF1—responsible for cell cycle arrest and apoptosis) was recorded only in MortaparibPlus-treated MCF-7 cells. On the other hand, MortaparibPlus-treated T47D cells exhibited hyperactivation of PARP1 (accumulation of PAR polymer and decrease in ATP levels) as a possible non-p53 tumor suppression program. However, these cells did not show full signs of either apoptosis or PAR-Thanatos. Molecular analyses attributed such a response to the inability of MortaparibPlus to disrupt the AIF–mortalin complexes; hence, AIF did not translocate to the nucleus to induce chromatinolysis and DNA degradation. These data suggested that the cancer cells possessing enriched levels of such complexes may not respond to MortaparibPlus. Taken together, we report the multimodal anticancer potential of MortaparibPlus that warrants further attention in laboratory and clinical studies.

Mechanisms of Metal-Induced Mitochondrial Dysfunction in Neurological Disorders.

Metals are actively involved in multiple catalytic physiological activities. However, metal overload may result in neurotoxicity as it increases formation of reactive oxygen species (ROS) and elevates oxidative stress in the nervous system. Mitochondria are a key target of metal-induced toxicity, given their role in energy production. As the brain consumes a large amount of energy, mitochondrial dysfunction and the subsequent decrease in levels of ATP may significantly disrupt brain function, resulting in neuronal cell death and ensuing neurological disorders. Here, we address contemporary studies on metal-induced mitochondrial dysfunction and its impact on the nervous system.

Overexpression of Transcripts Coding for Renin-b but Not for Renin-a Reduce Oxidative Stress and Increase Cardiomyoblast Survival under Starvation Conditions.

A stimulated renin-angiotensin system is known to promote oxidative stress, apoptosis, necrosis and fibrosis. Renin transcripts (renin-b; renin-c) encoding a cytosolic renin isoform have been discovered that may in contrast to the commonly known secretory renin (renin-a) exert protective effects Here, we analyzed the effect of renin-a and renin-b overexpression in H9c2 cardiomyoblasts on apoptosis and necrosis as well as on potential mechanisms involved in cell death processes. To mimic ischemic conditions, cells were exposed to glucose starvation, anoxia or combined oxygen–glucose deprivation (OGD) for 24 h. Under OGD, control cells exhibited markedly increased necrotic and apoptotic cell death accompanied by enhanced ROS accumulation, loss of mitochondrial membrane potential and decreased ATP levels. The effects of OGD on necrosis were exaggerated in renin-a cells, but markedly diminished in renin-b cells. However, with respect to apoptosis, the effects of OGD were almost completely abolished in renin-b cells but interestingly also moderately diminished in renin-a cells. Under glucose depletion we found opposing responses between renin-a and renin-b cells; while the rate of necrosis and apoptosis was aggravated in renin-a cells, it was attenuated in renin-b cells. Based on our results, strategies targeting the regulation of cytosolic renin-b as well as the identification of pathways involved in the protective effects of renin-b may be helpful to improve the treatment of ischemia-relevant diseases.

Does hyperbaric oxygen pretreatment with 100% oxygen attenuate tourniquet‐induced acute ischemia‐reperfusion injury in mouse hindlimb?

Tourniquet-induced ischemia-reperfusion (IR) leads to an increase in reactive oxygen species (ROS) production and tissue necrosis and apoptosis during reperfusion, which disrupts the recovery of skeletal muscle contraction. Hyperbaric oxygen (HBO) therapy exposes patients to high oxygen levels at an increased pressure, which increases the amount of ROS in tissues. Using HBO therapy as a pretreatment can promote ischemic tolerance by increasing antioxidant effects in these tissues before the IR injury. In this study, we observed the effects of HBO pretreatment with 100% oxygen at 2.5 ATA on tourniquet-induced IR injury in mice. C57/BL6 mice were treated with 100% oxygen for 60 minutes at 2.5 ATA in a HBO chamber. A rubber band was placed at the hip joint of the unilateral hindlimb of the mice to induce ischemia for 3 hours, which was followed by rubber band release and subsequent reperfusion for 48 hours. All staining and measurements were performed after the 48 hours of reperfusion. TTC staining of the gastrocnemius muscle showed no significant change in IR-induced infarct size after HBO pretreatment. ROS level was decreased in the gastrocnemius muscle of HBO-pretreated IR mice compared to non-HBO-pretreated IR mice. IR mice pretreated with HBO showed a decrease in ATP level in the gastrocnemius muscle compared to mice without HBO treatment. The data suggest that one hour HBO pretreatment with 100% oxygen at 2.5 ATA increases oxidative metabolism but does not decrease tissue necrosis in mice with tourniquet-induced IR injury.

Drp1-dependent peptide reverse mitochondrial fragmentation, a homeostatic response in Friedreich ataxia.

Friedreich ataxia is an autosomal recessive, neurodegenerative disease characterized by the deficiency of the iron‐sulfur cluster assembly protein frataxin. Loss of this protein impairs mitochondrial function. Mitochondria alter their morphology in response to various stresses; however, such alterations to morphology may be homeostatic or maladaptive depending upon the tissue and disease state. Numerous neurodegenerative diseases exhibit excessive mitochondrial fragmentation, and reversing this phenotype improves bioenergetics for diseases in which mitochondrial dysfunction is a secondary feature of the disease. This paper demonstrates that frataxin deficiency causes excessive mitochondrial fragmentation that is dependent upon Drp1 activity in Friedreich ataxia cellular models. Drp1 inhibition by the small peptide TAT‐P110 reverses mitochondrial fragmentation but also decreases ATP levels in frataxin‐knockdown fibroblasts and FRDA patient fibroblasts, suggesting that fragmentation may provide a homeostatic pathway for maintaining cellular ATP levels. The cardiolipin‐stabilizing compound SS‐31 similarly reverses fragmentation through a Drp1‐dependent mechanism, but it does not affect ATP levels. The combination of TAT‐P110 and SS‐31 does not affect FRDA patient fibroblasts differently from SS‐31 alone, suggesting that the two drugs act through the same pathway but differ in their ability to alter mitochondrial homeostasis. In approaching potential therapeutic strategies for FRDA, an important criterion for compounds that improve bioenergetics should be to do so without impairing the homeostatic response of mitochondrial fragmentation.

Green Synthesized Silver Nanoparticles: Antibacterial and Anticancer Activities, Biocompatibility, and Analyses of Surface-Attached Proteins.

The increasing number of multi-drug-resistant bacteria and cancer cases, that are a real threat to humankind, forces research world to develop new weapons to deal with it. Biogenic silver nanoparticles (AgNPs) are considered as a solution to this problem. Biosynthesis of AgNPs is regarded as a green, eco-friendly, low-priced process that provides small and biocompatible nanostructures with antimicrobial and anticancer activities and potential application in medicine. The biocompatibility of these nanoparticles is related to the coating with biomolecules of natural origin. The synthesis of AgNPs from actinobacterial strain was confirmed using UV-Vis spectroscopy while their morphology, crystalline structure, stability, and coating were characterized using, transmission electron microscopy (TEM), X-ray diffraction (XRD), Zeta potential and Fourier transform infrared spectroscopy (FTIR). Antibacterial activity of biogenic AgNPs was evaluated by determination of minimum inhibitory and minimum biocidal concentrations (MIC and MBC) against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. The potential mechanism of antibacterial action of AgNPs was determined by measurement of ATP level. Since the use of AgNPs in biomedical applications depend on their safety, the in vitro cytotoxicity of biosynthesized AgNPs on MCF-7 human breast cancer cell line and murine macrophage cell line RAW 264.7 using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, cell lactate dehydrogenase (LDH) release and measurement of reactive oxygen species (ROS) level were assessed. The nanoparticle protein capping agent that can be involved in reduction of silver ions to AgNPs and their stabilization was identified using LC-MS/MS. Nanoparticles were spherical in shape, small in size (mean 13.2 nm), showed crystalline nature, good stability (−18.7 mV) and presence of capping agents. They exhibited antibacterial activity (MIC of 8–128 μg ml−1, MBC of 64–256 μg ml−1) and significantly decreased ATP levels in bacterial cells after treatment with different concentrations of AgNPs. The in vitro analysis showed that the AgNPs demonstrated dose-dependent cytotoxicity against RAW 264.7 macrophages and MCF-7 breast cancer cells but higher against the latter than the former. Cell viability decrease was found to be 42.2–14.2 and 38.0–15.5% while LDH leakage 14.6–42.7% and 19.0–45.0%, respectively. IC50 values calculated for MTT assay was found to be 16.3 and 12.0 μg ml−1 and for LDH assay 102.3 and 76.2 μg ml−1, respectively. Moreover, MCF-7 cells released a greater amount of ROS than RAW 264.7 macrophages during stimulation with all tested concentrations of AgNPs (1.47–3.13 and 1.02–2.58 fold increase, respectively). The SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis revealed the presence of five protein bands at a molecular weight between 31.7 and 280.9 kDa. These proteins showed the highest homology to hypothetical proteins and porins from E. coli, Delftia sp. and Pseudomonas rhodesiae. Based on obtained results it can be concluded that biogenic AgNPs were capped with proteins and demonstrated potential as antimicrobial and anticancer agent.

Estrogen-Related Receptor Influences the Hemolymph Glucose Content by Regulating Midgut Trehalase Gene Expression in the Last Instar Larvae of Bombyx mori.

The expression of trehalase in the midgut of insects plays an important role in glucose supply to the hemolymph. Energy metabolism is usually regulated by the estrogen-related receptor (ERR). A decrease in ATP levels is caused by the ERR hindering glycolysis. However, the relationship between trehalose accumulation and ERR expression is still unclear. Here, we found that silkworm ERR (BmERR) is concentrated and BmERR expression is strongly correlated with trehalase in the midgut during the last instar silkworm larval stage. We cloned the promoter of the trehalase from Bombyx mori (BmTreh) and found that the ERR bound directly to the core response elements of the promoter. Cell level interference and the overexpression of ERR can reduce or enhance BmTreh transcription and promoter activity. Overexpressed transgenic BmERR can significantly increase the expression of BmTreh in the midgut of the last instar silkworm larvae, thereby hydrolyzing trehalose into glucose and releasing it into the hemolymph. Additionally, increased hemolymph glucose content reduces silkworm pupa weight but does not affect silk protein production from the silk gland. Our results suggest a novel function for BmERR through its involvement in BmTreh regulation and expand the understanding of ERR functions in insect trehalose metabolism.

FABP4 deactivates NF-κB-IL1α pathway by ubiquitinating ATPB in tumor-associated macrophages and promotes neuroblastoma progression.

Neuroblastoma (NB) is the most common and deadliest pediatric solid tumor. Targeting and reactivating tumor‐associated macrophages (TAMs) is necessary for reversing immune suppressive state and stimulating immune defense to exert tumoricidal function. However, studies on the function and regulation of TAMs in NB progression are still limited. Fatty acid binding protein 4 (FABP4) in TAMs was correlated with advanced clinical stages and unfavorable histology of NB. FABP4‐mediated macrophages increased migration, invasion, and tumor growth of NB cells. Mechanically, FABP4 could directly bind to ATPB to accelerate ATPB ubiquitination in macrophages. The consequently decreased ATP levels could deactivate NF‐κB/RelA‐IL1α pathway, which subsequently results in macrophages reprogrammed to an anti‐inflammatory phenotype. We also demonstrated that FABP4‐enhanced migration and invasion were significantly suppressed by IL1α blocking antibody. Furthermore, circulating FABP4 was also associated with the clinical stages of NB. Our findings suggest that FABP4‐mediated macrophages may promote proliferation and migration phenotypes in NB cells through deactivating NF‐κB‐IL1α pathway by ubiquitinating ATPB. This study reveals the pathologic and biologic role of FABP4‐mediated macrophages in NB development and exhibits a novel application of targeting FABP4 in macrophages for NB treatment.

Developmental co-exposure of TBBPA and titanium dioxide nanoparticle induced behavioral deficits in larval zebrafish

Both tetrabromobisphenol A (TBBPA) and titanium dioxide nanoparticle (TiO2 NP) have widespread commercial applications, resulting in their ubiquitous co-presence in the environment and biota. Although environmental chemicals exist as mixtures, toxicity studies are nearly always conducted with single chemicals. Few studies explore potential interactions of different chemical mixtures. In this study, we employ the sensitive developing nerve system in zebrafish to assess the neurotoxicity of TBBPA/TiO2 NP mixtures. Specifically, zebrafish embryos were exposed to solvent control (0.1% DMSO), 2 μM TBBPA, 0.1 mg/L TiO2 NP, and their mixture from 8 to 120 h post fertilization (hpf), and motor/social behavioral assessments were conducted on embryos/larvae at different developmental stages. Our results showed that TBBPA/TiO2 NP single or co-exposures increased spontaneous movement, decreased touch response and swim speed, and affected social behaviors of light/dark preference, shoaling, mirror attack and social contact. In particular, many of these phenotypes were manifested with higher magnitude of changes from the mixture exposure. These behavioral deficits were also accompanied with increased cell death in olfactory region and neuromasts in the lateral line system, increased ROS in gallbladder, pancreas, liver, and intestine, as well as increased lipid peroxidation and decreased ATP levels in whole larval tissue homogenates. Further, genes coding for key cell apoptosis marker and antioxidant enzyme were significantly upregulated by these two chemicals, in particular to their mixture. Interestingly, the co-presence of TBBPA also increased the mean particle size of TiO2 NP in the exposure solutions and the TiO2 NP content in larval tissue. Together, our analysis suggests that TBBPA/TiO2 NP induced behavioral changes may be due to physical accumulation of these two chemicals in the target organs, and TiO2 NP may serve as carriers for increased accumulation of TBBPA. To conclude, we demonstrated that TBBPA/TiO2 NP together cause increased bioaccumulation of TiO2, and heightened responses in behavior, cell apoptosis and oxidative stress. Our findings also highlight the importance of toxicity assessment using chemical mixtures.

NAMPT promotes hepatitis B virus replication and liver cancer cell proliferation through the regulation of aerobic glycolysis.

Nicotinamide phosphoribosyltransferase (NAMPT) is a critical rate-limiting enzyme involved in NAD synthesis that has been shown to contribute to the progression of liver cancer. However, the potential role and mechanism of NAMPT in hepatitis B virus (HBV)-associated liver cancer remain unclear. The present study assessed the expression of NAMPT in HBV-positive and -negative liver cancer cells, and investigated whether HBV-induced NAMPT expression is dependent on HBV X protein (HBx). In addition, the role of NAMPT in HBV replication and transcription, and in HBV-mediated liver cancer cell growth was explored. The effects of NAMPT on the glycolytic pathway were also evaluated. Reverse transcription-quantitative PCR and western blotting results revealed that NAMPT expression levels were significantly higher in HBV-positive liver cancer cells than in HBV-negative liver cancer cells, and this effect was HBx-dependent. Moreover, the activation of NAMPT was demonstrated to be required for HBV replication and transcription. The NAMPT inhibitor FK866 repressed cell survival and promoted cell death in HBV-expressing liver cancer cells, and these effects were attenuated by nicotinamide mononucleotide. Furthermore, the inhibition of NAMPT was associated with decreased glucose uptake, decreased lactate production and decreased ATP levels in HBV-expressing liver cancer cells, indicating that NAMPT may promote the aerobic glycolysis. Collectively, these findings reveal a positive feedback loop in which HBV enhances NAMPT expression and the activation of NAMPT promotes HBV replication and HBV-mediated malignant cell growth in liver cancer. The present study highlights the important role of NAMPT in the regulation of aerobic glycolysis in HBV-mediated liver cancer, and suggests that NAMPT may be a promising treatment target for patients with HBV-associated liver cancer.

Hyperbaric Oxygen Preconditioning Protects Against Cerebral Ischemia/Reperfusion Injury by Inhibiting Mitochondrial Apoptosis and Energy Metabolism Disturbance.

Hyperbaric oxygen (HBO) therapy is considered a safe and feasible method that to provide neuroprotection against ischemic stroke. However, the therapy mechanisms of HBO have not been fully elucidated. We hypothesized that the mechanism underlying the protective effect of HBO preconditioning (HBO-PC) against cerebral ischemia/reperfusion injury was related to inhibition of mitochondrial apoptosis and energy metabolism disorder. To test this hypothesis, an ischemic stroke model was established by middle cerebral artery occlusion (MCAO) in rats. HBO-PC involved five consecutive days of pretreatment before MCAO. In additional experiments, X chromosome-linked inhibitor of apoptosis protein (XIAP) and second mitochondria-derived activator of caspases (SMAC) shRNA and NC plasmids were intraventricularly injected into rat brains after MCAO (2h). After 24h, all rats underwent motor function evaluation, which was assessed by modified Garcia scores. TTC staining for the cerebral infarct and cerebral edema, and TUNEL staining for cell apoptosis, were also analyzed. Reactive oxygen species and antioxidative enzymes in rat brains were detected, as well as mitochondrial complex enzyme activities, ATP levels, and Na+/K+ ATPase activity. Western blot was used to detect apoptotic proteins including Bcl-2, Bax, caspase-3, caspase-9, cyc-c, XIAP, and SMAC. HBO-PC remarkably reduced the infarct volume and improved neurological deficits. Furthermore, HBO-PC alleviated oxidative stress and regulated the expression of apoptosis-related proteins. Moreover, HBO-PC inhibited the decrease in ATP levels, mitochondrial complex enzyme activities, and Na+/K+ ATPase activity to maintain stable energy metabolism. XIAP knockdown weakened the protective effect of HBO, whereas SMAC knockdown strengthened its protective effect. The effects of HBO-PC can be attributed to inhibition of ischemia/hypoxia-induced mitochondrial apoptosis and energy metabolism disturbance. The action of HBO-PC is related to the XIAP and SMAC signaling pathways.

3 Modulation of glycolysis alters histone acetylation and gene expression in bovine blastocysts produced invitro

Changes in the dynamics of energy metabolism can affect the sophisticated molecular control of different cell types including embryonic stem cells (Zhang et al. 2018 Cell Metab. 27, 332-338; https://doi.org/10.1016/j.cmet.2018.01.008). In this study, we modulated pathways related to acetyl-CoA generation, the major donor for histone acetylation, and explored how this affects histone acetylation and the transcriptional profile of bovine blastocysts. Embryos were produced invitro by conventional protocols. On Day 4 of culture (Fertilization=Day 0), embryos were randomly allocated into 3 experimental groups according to culture medium [synthetic oviductal fluid with amino acids (SOFaa) + 4% bovine serum albumin] supplementation: Control (no additional supplementation), sodium iodoacetate (IA; 2 µM; glycolysis inhibitor), and sodium dichloroacetate (DCA; 2 mM; acetyl-CoA conversion stimulator). Expanded blastocysts were collected on Day 7 and assessed for ATP levels (luminescence), mitochondrial activity (MitoTracker Red CMXRos; ThermoFisher Scientific), histone 3 lysine 9 and 27 acetylation (H3K9ac and H3K27ac; immunostaining) and transcriptional profiling by RNA sequencing (RNA-Seq) of isolated inner cell mass. Data were submitted to normality test and treatment groups were compared to control using t-test or Mann–Whitney test for non-parametric data (mean±s.e.) considering P&amp;lt;0.05. RNA-Seq data were analysed by DESEqn 2 and transcripts with Padj &amp;lt;0.05 were submitted to gene ontology by DAVID (https://david.ncifcrf.gov/). Mitochondrial activity was higher in DCA compared with Control (control: 53078±2747 AU vs. DCA: 57520±902 AU; P=0.0034), which explains the higher ATP levels found in this group (control: 1.49±0.65 µM vs. DCA: 41.56±15.69 µM; P=0.03). However, although mitochondrial activity was expectedly lower in IA compared to control (control: 53078±2747 A.U. vs. DCA: 36249±3200 A.U.; P=0.0013), we did not observe a decrease in ATP levels (control: 1.49±0.65µM vs. IA: 3.23±1.13 µM; P=0.12). Confirming our hypothesis, modulation of acetyl-CoA generation affected histone acetylation, with levels of H3K9ac and H3K27ac being higher in DCA and lower in IA compared with Control (H3K9ac Control: 988.3±22.82 AU, DCA: 1301±32.28 AU, IA: 684±23.7 AU; P&amp;lt;0.0001 and H3K27ac Control: 502.5±13.64 AU, DCA: 667.2±12.19 AU, IA: 417.2±12.03 AU; P&amp;lt;0.0001). Finally, 905 genes were differentially expressed, 599 up- and 306 downregulated in DCA compared with Control. Another 675 genes were differentially expressed between control and IA, (385 up- and 290 downregulated in IA). Gene ontology indicated that, compared with control, the biological functions upregulated in DCA were related to developmental process and the downregulated functions were associated with metabolism regulation, indicating a role of metabolic pathways in the developmental competence of the embryo. IA, in contrast, had catabolic activity upregulated and nucleotide metabolism downregulated compared with control, supporting the depletion of metabolic activity and lower acetylation. Taken together, our results demonstrate that the modulation of energy metabolism affects the epigenetic status of bovine embryos with consequences for the transcriptional profile of pathways involved in embryo quality and viability.

The mitochondrial NADH pool is involved in hydrogen sulfide signaling and stimulation of aerobic glycolysis

Hydrogen sulfide is synthesized by enzymes involved in sulfur metabolism and oxidized via a dedicated mitochondrial pathway that intersects with the electron transport chain at the level of complex III. Studies with H2S are challenging since it is volatile and also reacts with oxidized thiols in the culture medium, forming sulfane sulfur species. The half-life of exogenously added H2S to cultured cells is unknown. In this study, we first examined the half-life of exogenously added H2S to human colonic epithelial cells. In plate cultures, H2S disappeared with a t1/2 of 3 to 4 min at 37 °C with a small fraction being trapped as sulfane sulfur species. In suspension cultures, the rate of abiotic loss of H2S was slower, and we demonstrated that sulfide stimulated aerobic glycolysis, which was sensitive to the mitochondrial but not the cytoplasmic NADH pool. Oxidation of mitochondrial NADH using the genetically encoded mito-LbNOX tool blunted the cellular sensitivity to sulfide-stimulated aerobic glycolysis and enhanced its oxidation to thiosulfate. In contrast, sulfide did not affect flux through the oxidative pentose phosphate pathway or the TCA cycle. Knockdown of sulfide quinone oxidoreductase, which commits H2S to oxidation, sensitized cells to sulfide-stimulated aerobic glycolysis. Finally, we observed that sulfide decreased ATP levels in cells. The dual potential of H2S to activate oxidative phosphorylation at low concentrations, but inhibit it at high concentrations, suggests that it might play a role in tuning electron flux and, therefore, cellular energy metabolism, particularly during cell proliferation.

Ozone Exposure Induces Metabolic Disorders and NAD+ Depletion Through PARP1 Activation in Spinal Cord Neurons.

Background and Objective: Ozone therapy has shown therapeutic efficacy in different disorders particularly low back pain (LBP). However, ozone therapy has been associated with toxic effects on the respiratory, endocrine, cardiovascular systems as well as nervous system because of its strong oxidizing capacity. Recent studies have reported possible associations between ozone exposure and metabolic disorders, but the findings are controversial and little is known on the mechanisms of action. This study aims to investigate the cytotoxic effects of ozone exposure and possible mechanism of action in the animal model.Methods: Wistar neonate rats with the age of 24 h after birth were sacrificed by cervical dislocation under general anesthesia, then immersed in 75% alcohol and iodophor for 5 min, respectively. The spinal cord was isolated and cut to samples of ~1 mm3 and prepared for further experiments. The spinal cord neurons (SCNs) were exposed to ozone at different concentrations and then cultured in 96-well plates with glass bottom for 7 days. The cell viability, ATP levels and the NAD+ concentration were determined and compared between the different experimental groups and the control group.Results: Analyses of the data by non-targeted liquid chromatography-mass spectrometry (LC-MS) analysis determined the metabolic disorder in SCNs following the ozone exposure. Moreover, our assessments showed that ozone exposure resulted in DNA damage, poly (ADP)-ribose polymerase-1 (PARP1) excessive activation, nicotinamide adenine dinucleotide (NAD+) depletion and decrease of ATP level in SCNs. The PARP1 inhibitor can inhibit the cytotoxic effect of ozone to SCNs without inhibiting the activation of AMP-activated protein kinase (AMPK). Our findings revealed that the cytotoxic effects of ozone to SCNs might be mediated by excessive PARP1 activation and subsequent NAD+ depletion. Moreover, using PARP1 inhibitor can protect SCNs from cytotoxic effects of ozone by preventing NAD+ depletion during ozone exposure.Conclusion: Ozone exposure seems to induce metabolic disorders and NAD+ depletion through excessive PARP1 activation in SCNs.

Synergistic Combination of Calcium and Citrate in Mesoporous Nanoparticles Targets Pleural Tumors

Summary Conventional chemotherapy leads to severe adverse effects since it involves systemic administration of toxic drugs at high dosage. Unlike traditional chemotherapeutics, calcium phosphate and citrate have both been discussed as very promising anticancer agents and are not inherently toxic. Yet, their breakthrough has been hampered by the lack of an administration approach that overcomes the strict regulatory mechanisms of the cell. Here, we present a combinatorial administration of calcium, phosphate, and citrate as colloidal, amorphous nanoparticles (CPCs) that selectively kill cancer cells without involvement of inherently toxic drugs. The particles are toxic neither before endosomal release nor after their degradation. This highly selective toxicity allowed us to successfully treat two different aggressive pleural tumors in mice, reducing their size by about 40% and 70% after only two local applications. Safety assessment studies over 2 months show no signs of adverse effects except for slightly enhanced pleural thickening after eight applications.

Inhibition of oxidative metabolism by nitric oxide restricts EMCV replication selectively in pancreatic beta-cells

Environmental factors, such as viral infection, are proposed to play a role in the initiation of autoimmune diabetes. In response to encephalomyocarditis virus (EMCV) infection, resident islet macrophages release the pro-inflammatory cytokine IL-1β, to levels that are sufficient to stimulate inducible nitric oxide synthase (iNOS) expression and production of micromolar levels of the free radical nitric oxide in neighboring β-cells. We have recently shown that nitric oxide inhibits EMCV replication and EMCV-mediated β-cell lysis and that this protection is associated with an inhibition of mitochondrial oxidative metabolism. Here we show that the protective actions of nitric oxide against EMCV infection are selective for β-cells and associated with the metabolic coupling of glycolysis and mitochondrial oxidation that is necessary for insulin secretion. Inhibitors of mitochondrial respiration attenuate EMCV replication in β-cells, and this inhibition is associated with a decrease in ATP levels. In mouse embryonic fibroblasts (MEFs), inhibition of mitochondrial metabolism does not modify EMCV replication or decrease ATP levels. Like most cell types, MEFs have the capacity to uncouple the glycolytic utilization of glucose from mitochondrial respiration, allowing for the maintenance of ATP levels under conditions of impaired mitochondrial respiration. It is only when MEFs are forced to use mitochondrial oxidative metabolism for ATP generation that mitochondrial inhibitors attenuate viral replication. In a β-cell selective manner, these findings indicate that nitric oxide targets the same metabolic pathways necessary for glucose stimulated insulin secretion for protection from viral lysis.

Bioenergetics impairment of Trypanosoma cruzi by the antihypertensive manidipine: A drug repurposing strategy

Considering the lack of effective and safe therapy for the treatment of Chagas disease, the antihypertensive drug manidipine (MDP) was in vitro evaluated against Trypanosoma cruzi. The bioenergetics of trypomastigotes was studied in the presence of the drug using fluorimetric and luminescent assays. Manidipine showed a potent antiparasitic activity, with IC50 values of 0.1 μM (intracellular amastigotes) and 3 μM (trypomastigotes), resulting in a promising selectivity index against the amastigotes (>1459). Using fluorimetric analysis, the drug showed depolarisation of the electric potential of the plasma membrane with no alteration of the permeability. A decrease in ATP levels suggested a bioenergetic alteration of the mitochondria, which was confirmed by the depolarisation of the mitochondrial membrane potential and a slight increase of the ROS levels. This is the first study to show the promising in vitro effectiveness of the antihypertensive MDP against T. cruzi, which may represent a candidate for future investigations in animal models.