Increase In ATP Concentration Research Articles

Embryonic Mice with Lung-Specific RAGE Upregulation Have Enhanced Mitochondrial Respiration

RAGE (receptor for advanced glycation end-products) represents a class of multi-ligand pattern recognition receptors highly expressed in the vertebrate lung. Our previous work demonstrated unique patterns of RAGE expression in the developing murine lung and regulation by key transcription factors including NKX2.1 and FoxA2. The current investigation employed conditional lung-specific upregulation via a TetOn transgenic mouse model (RAGE TG) and nontransgenic controls. RAGE expression was induced in RAGE TG mice throughout gestation (embryonic day, E0-E18.5) or from E15.5-E18.5 and compared to age-matched controls. High-resolution respirometry was used to assess mitochondrial respiration and context was provided by quantifying ATP and reactive oxygen species (ROS) generation. Lung lysates were also screened by immunoblotting for MAPK/PI3K signaling intermediates. RAGE upregulation increased mitochondrial oxygen consumption in the E0-E18.5 and E15.5-E18.5 groups compared to controls. RAGE TG mice also had increased ATP concentrations, which persisted even after controlling for oxygen consumption. In contrast, ROS generation was diminished in RAGE TG animals compared to controls. Lastly, in both RAGE TG groups, pERK and pp38 were significantly decreased, whereas pAKT was significantly elevated, suggesting that RAGE signaling is likely perpetuated via pAKT pathways. Together, these data demonstrate that despite lung hypoplasia in RAGE TG mice, the remaining tissue experiences a favorable shift in mitochondrial bioenergetics without excessive redox assault and a preference for AKT signaling over ERK or p38.

Immunomodulatory effects of laser-synthesized nanodiamonds on peripheral blood mononuclear cells: evaluation of unconjugated, PEGylated, and antibody-conjugated formulations

The application of laser-synthesized nanodiamonds (LNDs) is of great interest to biomedical researchers and drug developers because this emerging method of synthesis yields nanodiamonds of consistent size (<5 nm diameter) and surface chemistry that can be functionalized to perform a staggering range of highly specialized tasks. The present study assessed the threshold at which LNDs in various conjugations and concentrations triggered immune responses and cytotoxicity in peripheral mononuclear blood cells from healthy donors, as assessed by changes in ATP concentrations and induced secretion of the cytokines IFN-γ, IL-6 and TNF-α. Conjugations assessed were raw (unconjugated) NDs, PEGylated (PEG5k-NDs), and antibody conjugated to goat anti-mouse antibodies (IgG-NDs). Concentrations of each conjugation were prepared and tested at 50.0, 10.0, 2.0, 0.4, and 0.08 μg/mL. Results showed that pegylated and raw NDs were well tolerated, with the indicators of inflammation or minimal cytotoxicity emerging only at the highest concentrations tested (50.0 μg/mL). IgG-NDs showed signs of inflammatory responses at the two highest concentrations tested (10.0 and 50.0 μg/mL). There was some evidence that the dilutant vehicle used for ND suspension may have contributed to the immune response. All three ND configurations increased ATP concentration in a dose-dependent manner, up to a concentration of 10.0 μg/mL. At the highest concentration (50.0 μg/mL), the ND solutions showed minimal signs of cytotoxicity. Conclusion from this testing suggest that LNDs are likely to offer substantial utility in biomedical applications because of their capacity to evade the immune response at concentrations at least as high as 2.0 μg/mL and potentially up to 50.0 μg/mL.

Polyphosphate affects cytoplasmic and chromosomal dynamics in nitrogen-starved Pseudomonas aeruginosa

Polyphosphate (polyP) synthesis is a ubiquitous stress and starvation response in bacteria. In diverse species, mutants unable to make polyP have a wide variety of physiological defects, but the mechanisms by which this simple polyanion exerts its effects remain unclear. One possibility is that polyP's many functions stem from global effects on the biophysical properties of the cell. We characterize the effect of polyphosphate on cytoplasmic mobility under nitrogen-starvation conditions in the opportunistic pathogen Pseudomonas aeruginosa. Using fluorescence microscopy and particle tracking, we quantify the motion of chromosomal loci and cytoplasmic tracer particles. In the absence of polyP and upon starvation, we observe a 2- to 10-fold increase in mean cytoplasmic diffusivity. Tracer particles reveal that polyP also modulates the partitioning between a "more mobile" and a "less mobile" population: Small particles in cells unable to make polyP are more likely to be "mobile" and explore more of the cytoplasm, particularly during starvation. Concomitant with this larger freedom of motion in polyP-deficient cells, we observe decompaction of the nucleoid and an increase in the steady-state concentration of ATP. The dramatic polyP-dependent effects we observe on cytoplasmic transport properties occur under nitrogen starvation, but not carbon starvation, suggesting that polyP may have distinct functions under different types of starvation.

Inhibition of pannexin-1 does not restore electrolyte balance in precystic Pkd1 knockout mice.

Mutations in PKD1 and PKD2 cause autosomal dominant polycystic kidney disease (ADPKD), which is characterized by the formation of fluid-filled cysts in the kidney. In a subset of ADPKD patients, reduced blood calcium (Ca2+) and magnesium (Mg2+) concentrations are observed. As cystic fluid contains increased ATP concentrations and purinergic signaling reduces electrolyte reabsorption, we hypothesized that inhibiting ATP release could normalize blood Ca2+ and Mg2+ levels in ADPKD. Inducible kidney-specific Pkd1 knockout mice (iKsp-Pkd1-/-) exhibit hypocalcemia and hypomagnesemia in a precystic stage and show increased expression of the ATP-release channel pannexin-1. Therefore, we administered the pannexin-1 inhibitor brilliant blue-FCF (BB-FCF) every other day from Day 3 to 28 post-induction of Pkd1 gene inactivation. On Day 29, both serum Ca2+ and Mg2+ concentrations were reduced in iKsp-Pkd1-/- mice, while urinary Ca2+ and Mg2+ excretion was similar between the genotypes. However, serum and urinary levels of Ca2+ and Mg2+ were unaltered by BB-FCF treatment, regardless of genotype. BB-FCF did significantly decrease gene expression of the ion channels Trpm6 and Trpv5 in both control and iKsp-Pkd1-/- mice. Finally, no renoprotective effects of BB-FCF treatment were observed in iKsp-Pkd1-/- mice. Thus, administration of BB-FCF failed to normalize serum Ca2+ and Mg2+ levels.

Purinergic enzymes on extracellular vesicles: immune modulation on the go.

An increase in the extracellular concentration of ATP as a consequence of cellular stress or cell death results in the activation of immune cells. To prevent inflammation, extracellular ATP is rapidly metabolized to adenosine, which deploys an anti-inflammatory signaling cascade upon binding to P1 receptors on immune cells. The ectonucleotidases necessary for the degradation of ATP and generation of adenosine are present on the cell membrane of many immune cells, and their expression is tightly regulated under conditions of inflammation. The discovery that extracellular vesicles (EVs) carry purinergic enzyme activity has brought forward the concept of EVs as a new player in immune regulation. Adenosine-generating EVs derived from cancer cells suppress the anti-tumor response, while EVs derived from immune or mesenchymal stem cells contribute to the restoration of homeostasis after infection. Here we will review the existing knowledge on EVs containing purinergic enzymes and molecules, and discuss the relevance of these EVs in immune modulation and their potential for therapy.

General efficiency relation for dissipative molecular machines

Living systems use chemical fuel to process information, assemble structures and maintain fluxes. Many of these processes are dissipative: energy is consumed purely to maintain non-equilibrium steady-state outputs. How efficiently the input energy is transduced toward the output dissipation as opposed to being lost during intermediate steps, and whether the efficiency is constrained by general principles or specific fine-tuning, are open questions. Here, applying a recent mapping from non-equilibrium systems to battery-resistor circuits, an analytic expression for the efficiency of any dissipative molecular machine driven by a chemical potential difference is derived. This expression disentangles the chemical potential from the machine’s details, whose effect on the efficiency is fully specified by a constant called the load resistance. The efficiency passes through an inflection point separating totally inefficient machines from efficient machines if the balance between chemical potential difference and load resistance exceeds thermal noise. This explains all-or-none dynein stepping with increasing ATP concentration observed in single-molecule experiments. These results indicate that energy transduction in living systems is efficient not because of idiosyncratic optimization of biomolecules, but rather because the concentration of chemical fuel is kept above a threshold level.

Sperm induce a secondary increase in ATP levels in mouse eggs that is independent of Ca2+ oscillations.

Egg activation at fertilization in mouse eggs is caused by a series of cytosolic Ca2+ oscillations that are associated with an increase in ATP concentrations driven by increased mitochondrial activity. We have investigated the role of Ca2+ oscillations in these changes in ATP at fertilization by measuring the dynamics of ATP and Ca2+ in mouse eggs. An initial ATP increase started with the first Ca2+ transient at fertilization and then a secondary increase in ATP occurred ∼1 h later and this preceded a small and temporary increase in the frequency of Ca2+ oscillations. Other stimuli that caused Ca2+ oscillations such as PLCz1 or thimerosal, caused smaller or slower changes in ATP that failed to show the distinct secondary rise. Sperm-induced Ca2+ oscillations in the egg also triggered changes in the fluorescence of NADH which followed the pattern of Ca2+ spikes in a similar pattern to oscillations triggered by PLCz1 or thimerosal. When eggs were loaded with low concentrations of the Ca2+ chelator BAPTA, sperm triggered one small Ca2+ increase, but there were still extra phases of ATP increase that were similar to control fertilized eggs. Singular Ca2+ increases caused by thapsigargin were much less effective in elevating ATP levels. Together these data suggest that the secondary ATP increase at fertilization in mouse eggs is not caused by increases in cytosolic Ca2+. The fertilizing sperm may stimulate ATP production in eggs via both Ca2+ and by another mechanism that is independent of PLCz1 or Ca2+ oscillations.

Extracellular ATP-induced calcium oscillations regulating the differentiation of osteoblasts through aerobic oxidation metabolism pathways.

The increase of ATP concentration in the extracellular space represents one of the effective signals that stimulate the physiological activities of cells when the bone is exposed to external mechanical stimulation such as stretching and shear stress force throughout life. However, the effects of ATP on osteoblast differentiation and related mechanisms are not well understood. In this study, the roles of extracellular ATP on osteoblast differentiation, intracellular calcium ([Ca2+]i) levels, metabolomics, and the expression of proteins related to energy metabolism were investigated. Our results showed that 100μM extracellular ATP initiated intracellular calcium ([Ca2+]i) oscillations via the calcium-sensing receptor (P2R) and promoted the differentiation of MC3T3-E1 cells. Metabolomics analysis showed that the differentiation of MC3T3-E1 cells depended on aerobic oxidation, but little glycolysis. Moreover, the differentiation of MC3T3-E1 cells and aerobic oxidation were suppressed with the inhibition of AMP-activated protein kinase (AMPK). These results indicate that calcium oscillations triggered by extracellular ATP can activate aerobic oxidation through AMPK-related signaling pathways and thus promote osteoblast differentiation.

Oxidative Stress Response and E. coli Biofilm Formation under the Effect of Pristine and Modified Carbon Nanotubes.

The article investigates the expression of oxyR and soxS oxidative stress genes in E. coli under the effect of pristine multi-walled carbon nanotubes (MWCNTs) and pristine single-walled carbon nanotubes (SWCNTs), MWCNTs and SWCNTs functionalized with carboxyl groups (MWCNTs-COOH and SWCNTs-COOH, respectively), SWCNTs functionalized with amino groups (SWCNTs-NH2) and SWCNTs functionalized with octadecylamine (SWCNTs-ODA). Significant differences were found in the expression of the soxS gene, while no changes were observed in the expression level of the oxyR gene. The pro-oxidant effect of SWCNTs, SWCNTs-COOH, SWCNTs-NH2, and SWCNTs-ODA is presented, and the contrary antioxidant effect of pristine MWCNTs and MWCNTs-COOH in the presence of methyl viologen hydrate (paraquat) is shown. The article shows that SWCNTs-COOH, SWCNTs-NH2, and SWCNTs-ODA added to the medium generate the production of reactive oxygen species (ROS) in bacterial cells. SWCNTs-COOH intensified the E. coli biofilm formation, and the biofilm biomass exceeded the control by 25 times. Additionally, it is shown that the rpoS expression increased in response to MWCNTs-COOH and SWCNTs-COOH, and the effect of SWCNTs-COOH was more significant. SWCNTs-COOH and SWCNTs-NH2 initiated an increase in ATP concentration in the planktonic cells and a decrease in the biofilm cells. The atomic force microscopy (AFM) method showed that the volume of E. coli planktonic cells after the exposure to carbon nanotubes (CNTs) decreased compared to that without exposure, mainly due to a decrease in cell height. The absence of a strong damaging effect of functionalized SWCNTs on E. coli K12 cells, both in suspension and in biofilms, is shown. Contact with functionalized SWCNTs initiated the aggregation of the polymeric substances of the biofilms; however, the cells did not lyse. Among the studied CNTs, SWCNTs-COOH caused an increased expression of the soxS and rpoS, the formation of ROS, and stimulation of the biofilm formation.

Global translational induction during NLR-mediated immunity in plants is dynamically regulated by CDC123, an ATP-sensitive protein

The recognition of pathogen effectors by their cognate nucleotide-binding leucine-rich repeat (NLR) receptors activates effector-triggered immunity (ETI) in plants. ETI is associated with correlated transcriptional and translational reprogramming and subsequent death of infected cells. Whether ETI-associated translation is actively regulated or passively driven by transcriptional dynamics remains unknown. In a genetic screen using a translational reporter, we identified CDC123, an ATP-grasp protein, as a key activator of ETI-associated translation and defense. During ETI, an increase in ATP concentration facilitates CDC123-mediated assembly of the eukaryotic translation initiation factor 2 (eIF2) complex. Because ATP is required for the activation of NLRs as well as the CDC123 function, we uncovered a possible mechanism by which the defense translatome is coordinately induced during NLR-mediated immunity. The conservation of the CDC123-mediated eIF2 assembly suggests its possible role in NLR-mediated immunity beyond plants.

ОТ АКТИВНОСТИ К БЕЗДЕЙСТВИЮ И СНОВА К АКТИВНОСТИ. СИГНАЛЬНЫЕ ПРОЦЕССЫ В ПОСТУРАЛЬНОЙ МЫШЦЕ В ПЕРЕХОДНЫЙ ПЕРИОД

Experiments with tail-suspended rats exposed to Kepler's parabolic flight and human subjects in dry immersion showed repeatedly decreases, sometimes to the point of full inhibition, of the soleus m. electrical activity. Stopping the activity, closely resembling the temporal physiological rest that in rat's m. soleus is normally 8-10 hours a day, is much longer in experiments and can be characterized as a state of inactivity. Already after 24 hours of inactivity, expression of key muscle ubiquitin Е3 ligases increases with parallel reductions in protein synthesis and translational capacity. Expression of the slow isoform of myosin heavy chains reduces also. Purpose of the review is to summarize the data on dynamics of molecular events in postural m. soleus of the mammal after cessation of its contractile activity. Changes in the complex of signaling pathways in the transitional period are triggered by a group of molecular messengers which are directly dependent on the contractile activity. Established was the function of increasing ATP concentration and ensuing reduction in phosphorylation of АМP-activated protein kinase (АМPК), as well as expression of slow myosin, regulators of mitochondria and ribosomes biogenesis and resting membrane potential at the initial stage of muscle inactivity. ATP-dependent processes contribute to NO reduction and Ca2+ions accumulation in muscle fibers. Changes in these messengers precisely underlie the subsequent atrophic developments, transformation of myosin phenotype, muscle fiber atony and mitochondrial dysfunction. At the same time, due to still not clearly understood changes in excitability of spinal motoneurons, within three day of inactivity the postural muscle demonstrates spontaneous activity with a gradually growing amplitude of electromyographic signals. Results of the investigations of signal processes in the postural muscle during the transition from activity to inactivity and back to autonomic activity suggest difference and nonuniformity and nonlinearity of these processes and change of mechanisms driving the destructive events.

Ratiometric ECL sensor based on Apt-AuNS@Lu nanoprobe for analyzing cell swelling-induced ATP release.

A novel ratiometric electrochemiluminescence (ECL) system based on gold nanostars (AuNSs) support was constructed for the determination of hypotonicity-induced ATP release from HepG2 cells. AuNS@Lu nanoprobe was used as anodic luminophore and K2S2O8 as cathodic luminophore as well as anodic co-reactant. AuNS with the large specific surface was adopted to adsorb plentiful luminol to form solid-state probe and as affinity support to immobilize ATP aptamer (Apt). The obtained nanocomposite (Apt-AuNS@Lu) generated astrong ECL signal at + 0.4V (vs. Ag/AgCl) with co-reactant K2S2O8, because of excellent conductivity and catalytic activity of AuNS. Furthermore, graphene oxide was reduced onto indium tin oxide (ITO) electrodes to facilitate the electron transfer. Following, polydopamine (PDA) film was formed via self-polymerization, improving stability and adhesion of the electrode surface. To immobilize ATP capture aptamer (AptC), abounding AuNSs were attached to RGO/PDA surface. When the sensor was incubated in the mixture solution of Apt-AuNS@Lu and target ATP, the ECL signal of Apt-AuNS@Lu increased with the increase of ATP concentration, meanwhile, the signal of K2S2O8 declined. The ratio of the two luminophores was used for the quantitative determination of ATP. The linear range was 5 to 250nM, and the limit of detection was 1.4nM at (3σ)/S. The method was successfully applied to analyze ATP release from HepG2 cells stimulated by 0.45% NaCl hypotonic solution. The results showed that the release kinetics profile of ATP had a sigmoidal shape with rapid release within 10min and then slowed. Compared to the isotonic groups, the intracellular ATP concentration was 3.7 ± 0.3µM (n = 3) decreasing by 40.3% and the extracellular was 23.4 ± 1.2nM (n = 3) increasing by 9.2 times in the hypotonicity for 10min, which showed ATP release from cells and good agreement with commercial ELISA test. The proposed strategy would be beneficial to broadening application of ECL technology in studying cell biological functions.

Divergent kinase WNG1 is regulated by phosphorylation of an atypical activation sub-domain.

Apicomplexan parasites like Toxoplasma gondii grow and replicate within a specialized organelle called the parasitophorous vacuole. The vacuole is decorated with parasite proteins that integrate into the membrane after trafficking through the parasite secretory system as soluble, chaperoned complexes. A regulator of this process is an atypical protein kinase called WNG1. Phosphorylation by WNG1 appears to serve as a switch for membrane integration. However, like its substrates, WNG1 is secreted from the parasite dense granules, and its activity must, therefore, be tightly regulated until the correct membrane is encountered. Here, we demonstrate that, while another member of the WNG family can adopt multiple multimeric states, WNG1 is monomeric and therefore not regulated by multimerization. Instead, we identify two phosphosites on WNG1 that are required for its kinase activity. Using a combination of in vitro biochemistry and structural modeling, we identify basic residues that are also required for WNG1 activity and appear to recognize the activating phosphosites. Among these coordinating residues are the ‘HRD’ Arg, which recognizes activation loop phosphorylation in canonical kinases. WNG1, however, is not phosphorylated on its activation loop, but rather on atypical phosphosites on its C-lobe. We propose a simple model in which WNG1 is activated by increasing ATP concentration above a critical threshold once the kinase traffics to the parasitophorous vacuole.

Dynamics of Metabolic and Oxidative Parameters of Erythrocytes during Treatment of Chronic Heart Failure with Molecular Hydrogen.

We studied the effect of molecular hydrogen (H2) on the content of 2,3-diphosphoglyceric acid (2,3-DPG), ATP, malondialdehyde, and catalase activity in erythrocytes in chronic heart failure. Inhalation of 2% molecular hydrogen H2 was carried out for 40 min repeatedly (5 days) or once. Inhalation of H2 caused an increase in ATP concentration in both research groups, but was more pronounced after repeated inhalation. The content of 2,3-DPG increased after repeated exposure to H2. The increase in metabolic activity under the effect of H2 was accompanied by a decrease in malondialdehyde concentration and an increase in catalase activity. Thus, the application of H2 in chronic heart failure reduced oxidative stress and improved metabolism of erythrocytes, which contributes to improvement of microcirculation. This allows us to recommend H2 for protection against ischemic and reperfusion damage to the myocardium.

Protein kinase inhibitor ceritinib blocks ectonucleotidase CD39 – a promising target for cancer immunotherapy

BackgroundAn important mechanism, by which cancer cells achieve immune escape, is the release of extracellular adenosine into their microenvironment. Adenosine activates adenosine A2A and A2B receptors on immune cells constituting...

Osteocalcin reduces fat accumulation and inflammatory reaction by inhibiting ROS-JNK signal pathway in chicken embryonic hepatocytes

Osteocalcin (OCN) has a function in preventing fatty liver hemorrhagic syndrome (FLHS) in poultry. The aim of this study was to investigate the effects of OCN on fat emulsion stimulated chicken embryonic hepatocytes and related signaling pathways. The primary chicken embryonic hepatocytes were isolated from the incubated 15-day (E15) pathogen free eggs and cultured with dulbecco's modified eagle medium (DMEM). After the hepatocyte density reached 80%, the cells were divided into 5 groups: control group (CONT), fat emulsion group (FE, 10% FE, v/v), FE with ucOCN at 1 ng/mL (FE-LOCN), 3 ng/mL (FE-MOCN), and 9 ng/mL (FE-HOCN). In addition, 2 mM N-Acetyl-L-cysteine (NAC) a reactive oxygen species (ROS) scavenger, and 5 μM SP600125, a Jun N-terminal kinase (JNK) inhibitor, were added separately in to the DMEM with 10% FE to test effects of FE on the function of ROS-JNK signal pathway. The number of hepatocytes, cell ultra-microstructure, viability, and apoptosis were detected after 48 h treatment, and the protein expressions and enzyme concentrations were detected after 72 h treatment. The results showed that, compared to the control group, FE increased the triglyceride (TG) concentration and lipid droplets (LDs) in chicken embryonic hepatocytes (P < 0.05), and induced hepatocytic edema with obviously mitochondrial swelling, membrane damage, and cristae rupture. FE also decreased ATP concentration, increased ROS concentrations and mitochondrial DNA (mtDNA) copy number, promoted inflammatory interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-α) concentrations and hepatocytic apoptosis rate, and raised phospho-c-Jun N-terminal kinase (p-JNK) protein expressions. Compared to the FE group, ucOCN significantly increased hepatocyte viability, reduced hepatocytic TG concentrations and LDs numbers, and alleviated hepatocytic edema and mitochondrial swelling. Furthermore, ucOCN significantly decreased ROS concentrations, increased ATP concentrations, reduced IL-1, IL-6, TNF-α concentrations and hepatocytic apoptosis rate, and inhibited p-JNK protein expressions (P < 0.05). NAC had the similar functions of ucOCN reduced the ROS concentration and inhibited the TNF-α protein expression and p-JNK/JNK ration. Similarly, SP600125 reduced p-JNK/JNK protein expression, IL-1, IL-6, TNF-α, and TG concentrations without effects on ROS concentration and hepatocytic apoptosis. These results suggest that ucOCN alleviates FE-induced mitochondrial damage, cellular edema, and apoptosis of hepatocytes. These results reveal that the functions of ucOCN in reducing fat accumulation and inflammatory reaction in chicken embryonic hepatocytes are mostly via inhibiting the ROS-JNK signal pathway.

Deletion of glyceraldehyde-3-phosphate dehydrogenase (gapN) in Clostridium saccharoperbutylacetonicum N1-4(HMT) using CLEAVE™ increases the ATP pool and accelerates solvent production.

SummaryThe development and advent of mutagenesis tools for solventogenic clostridial species in recent years has allowed for the increased refinement of industrially relevant strains. In this study we have utilised CLEAVE™, a CRISPR/Cas genome editing system developed by Green Biologics Ltd., to engineer a strain of Clostridium saccharoperbutylacetonicum N1‐4(HMT) with potentially useful solvents titres and energy metabolism. As one of two enzymes responsible for the conversion of glyceraldehyde‐3‐phosphate (GAP) to 3‐phosphoglyceric acid in glycolysis, it was hypothesised that deletion of gapN would increase ATP and NADH production that could in turn improve solvent production. Herein, whole genome sequencing has been used to evaluate CLEAVE™ and the successful knockout of gapN, demonstrating a clean knockout with no other detectable variations from the wild type sequence. Elevated solvent levels were detected during the first 24 h of batch fermentation, indicating an earlier shift to solventogenesis. A 2.4‐fold increase in ATP concentration was observed, and quantitation of NAD(P)H derivatives revealed a more reducing cytoplasm for the gapN strain. These findings expand our understanding of clostridium carbon metabolism and report a new approach to optimising biofuel production.

Crocetin ameliorates non-alcoholic fatty liver disease by modulating mitochondrial dysfunction in L02 cells and zebrafish model

Ethnopharmacological relevanceTraditional Chinese medicine considers that the etiology and pathogenesis of non-alcoholic fatty liver disease (NAFLD) are related to liver depression and qi stagnation. Saffron and its active ingredient, crocetin (CCT), are used for the treatment of metabolic diseases owing to their “Liver deobstruent” and “Liver tonic” effects. However, the effect of CCT on NAFLD has not been fully elucidated. In the present study, the effect and potential molecular mechanism of CCT were explored in both in vivo and in vitro models of NAFLD. Materials and methodsCCT was isolated from saffron and purity and structure characterization were performed using HPLC, MS, 1H-NMR, and 13C-NMR. The effect of CCT on the viability of L02 cells and its maximum tolerable concentration (MTC) in zebrafish were investigated. Free fatty acids (FFA) and thioacetamide (TAA) were used to induce lipid accumulation in L02 cells and steatosis in zebrafish, respectively. The effects of CCT on indexes related to lipid metabolism, oxidative stress, and mitochondrial function in NAFLD models were explored using biochemical assay kits, Western blot analysis, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), histopathology analysis, and determination of mitochondrial membrane potential (ΔΨm). Morphological analysis of mitochondria was performed using transmission electron microscopy (TEM). ResultsThe levels of triglyceride (TG), total cholesterol (TC), malondialdehyde (MDA), and alanine/aspartate aminotransferases (ALT/AST) activities in FFA treated L02 cells were significantly reduced after CCT treatment. CCT treatment significantly increased ATP concentration, ΔΨm, and activities of superoxide dismutase (SOD), catalase (CAT), and cytochrome c oxidase (COX IV) in FFA treated L02 cells. TEM images showed restoration of mitochondrial morphology. CCT decreased ATP concentration and upregulated expression of B-cell lymphoma-2 (Bcl-2) and COX IV, whereas, CCT downregulated expression of BCL2-Associated X (Bax) and cleaved caspase-3 in TAA treated zebrafish. These findings indicated that mitochondrial dysfunction was alleviated after CCT treatment. Oil Red O staining of L02 cells and zebrafish showed that CCT treatment reversed the accumulation of lipid droplets. ConclusionIn summary, CCT treatment effectively alleviated the symptoms of NAFLD and restored mitochondrial function in L02 cells and zebrafish NAFLD model.

Phase 1 Single and Multiple Ascending Dose Study to Assess the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of AG-946 in Healthy Volunteers

Background: Glycolysis is the primary source of ATP in red blood cells (RBCs), which is critical for maintaining RBC health. Pyruvate kinase red cell isoform (PKR) catalyzes the final step of glycolysis to generate ATP. Enhancing ATP production via PKR activation is under investigation as a potential therapeutic approach in hemolytic anemias. Increased levels of the glycolytic metabolite 2,3-diphosphoglycerate (2,3-DPG) in sickle cell disease (SCD) decreases hemoglobin oxygen affinity and results in increased RBC sickling. PKR activation has been shown to reduce 2,3-DPG. In previous clinical studies in pyruvate kinase deficiency, thalassemia and SCD, treatment with mitapivat, a PKR activator, led to improvements in hemoglobin and markers of hemolysis.AG-946 is an investigational, next-generation, oral small molecule activator of wild-type and mutant PKR isoforms, with high potency. Modelling from preclinical studies suggest that AG-946 is likely to have a pharmacokinetic (PK) profile that allows for once daily (QD) dosing and long duration of pharmacodynamic (PD) effects in humans. Here we report preliminary blinded results from an ongoing study assessing the safety, tolerability, PK and PD of AG-946 in healthy volunteers (NCT04536792).Methods: In this phase 1, randomized, double-blind, placebo (P)-controlled study, single ascending oral doses (SAD) or multiple ascending oral doses (MAD) of AG-946 were administered under fasting conditions to healthy men and women (18-55 years of age) in sequential cohorts. In SAD (4 cohorts of 8 subjects each) and in MAD (2 cohorts of 8 subjects each) subjects were planned to be randomized to receive either AG-946 (n=6) or P (n=2). The dose levels studied so far are 1, 3, 10, and 30 mg in SAD and 1 mg QD and 2 mg QD for 14 days in MAD. Safety assessments included vital signs, physical exams, electrocardiograms, clinical laboratory parameters and adverse events (AEs). Serial blood samples were drawn for PK and PD (2,3-DPG; ATP) assessments at regular intervals throughout the study period.Results: As of June 02, 2021, 39 (median age 33 years; n = 33 male) subjects in SAD and 17 (median age 36 years; all male) subjects in MAD received AG-946 or P. There were 6 (SAD, n = 5; MAD, n = 1) early discontinuations; all were unrelated to study treatment. In SAD, 4/39 (10.3%) subjects experienced ≥ 1 treatment-emergent AE (TEAE); all TEAEs were assessed as mild (Grade [Gr] 1). In MAD 4/17 (23.5%) subjects experienced ≥ 1 TEAE; the majority of the TEAEs were mild (Gr 1), with 1 subject experiencing a serious AE (Gr 2) of exercise-induced rhabdomyolysis 14 days after last dose, considered unrelated to study treatment. All other AEs in SAD and MAD were also considered unrelated to study treatment.In both SAD and MAD, AG-946 exhibited rapid absorption with median T max (time to maximum concentration) ranging from 0.5 to 1 hour. Following SAD, dose-normalized AG-946 exposures (AUC and C max [area under the curve and maximum concentration observed]) increased with increasing AG-946 doses, suggesting a greater than dose proportional increase in exposure over the tested dose range. Following MAD, AG-946 exposures were higher on Day 14 compared with Day 1, with mean accumulation ratios based on AUC of 3.6 at 1 mg QD and 3.3 at 2 mg QD, and mean accumulation ratios based on C max of 1.95 at 1 mg QD and 1.6 at 2 mg QD.In both SAD and MAD, an increase in AG-946 dose was associated with a decrease in 2,3-DPG concentrations (Figure 1), and an increase in ATP concentrations (Figure 2). The PD changes were sustained up to 168 hours after a single dose and > 7 days after the last day of multiple QD dosing, consistent with the slow off-rate from PKR.As expected, no clinically significant changes in Hb have been observed in the SAD or MAD cohorts, to date.Conclusions: AG-946, a highly potent PKR activator, was well tolerated in healthy volunteers following single dose administrations up to 30 mg and multiple 14-day dosing with 1 mg QD and 2 mg QD. The PK profile of AG-946 supports QD dosing, and is accompanied by sustained dose-dependent increases in ATP and decreases in 2,3-DPG, consistent with activation of the glycolytic pathway. Enrollment into additional SAD and MAD cohorts is ongoing and will be followed by an open-label phase in subjects with SCD. [Display omitted] DisclosuresIyer: Novartis: Current equity holder in publicly-traded company; Agios Pharmaceuticals: Current Employment, Current holder of stock options in a privately-held company. Ronseaux: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Gamache: Agios Pharmaceuticals: Current Employment. Callaghan: Agios Pharmaceuticals: Current Employment.

Antioxidative Protective Effect of Ozone Therapy on Isolated Mitochondria from Human Sperm

Oxidative stress is implicated in male infertility and significantly higher reactive oxygen Species (ROS) are detected in 25% of infertile males. We showed that Ozone oxygen therapy (O2/O3) induces protective effects for oxidative stress factors and its consequences on isolated mitochondria obtained sperm which may provide insight into the role of Ozone oxygen in human infertility. The present study was carried out to further characterize and compare protective effect of Ozone oxygen on isolated mitochondria obtained from sperm. Semen was collected from human normal donors. We gained human sperm mitochondria by differential centrifugation and isolated mitochondria incubated with different concentrations (5µg/ml, 30 µg/ml, 80 µg/ml) of O2/O3. O2/O3 prevent significant decrease in reactive oxygen species formation and mitochondrial membrane potential collapse on isolated Human sperm mitochondria. Ozone oxygen therapy induced increase in ATP concentration on isolated mitochondria. Our findings showed that O2/O3 prevent toxicity in sperm, effect on mitochondrial respiratory chain and avoid to cytochrome c release and apoptosis signaling.