Presence Of Pyruvate Research Articles

Вплив етанолу та циклоспорину А на дихання ізольованих гепатоцитів щурів

It is known that ethanol affects the oxidative processes in liver mitochondria, which can be one of the mechanisms of the development of the alcoholic liver disease. A well-known immunosuppressant cyclosporine A is also an inhibitor of mitochondrial permeabi­lity transition pore. However, the pharmacological interaction of ethanol and cyclosporin A is not sufficiently studied. The aim of the study was to investigate the effect of ethanol and cyclosporine A on the respiration rate of isolated rat hepatocytes. Five male Wistar rats were used in the study. Hepatocytes were isolated by perfusion with collagenase. Cell plasma membrane integrity was assessed with trypan blue staining (0.1 %). Intact cell number was 85.7±0.92 %. The rate of oxygen consumption was measured using a polarographic device based on a Clark electrode. Protonophore FCCP was used to study the uncoupled respiration. Cell respiration was studied in the presence of glucose, pyruvate or monomethylsuccinate in solution. Statistical analysis was performed with Origin Pro 2018 software. Significance of difference between groups was evaluated with analysis of variance test. After one hour incubation with ethanol (50 mM), the rate of basal respiration of hepatocytes oxidizing glucose increased by a meager 8 %. Similar result was obtained upon the presence of pyruvate in solution. Monomethylsuccinate, however, abolished the effect of ethanol on basal respiration. he but not uncoupled respiration of hepatocytes upon glucose or pyruvate oxidation. Ethanol did not affect the uncoupled respiration of hepatocytes in presence of glucose, pyruvate of monomethylsuccinate. Incubation with cyclocporin A did not cause any changes in cell oxygen consumption in all experiments. Cyclosporin A also did not modify the effects of ethanol on basal respiration of hepatocytes. Therefore, no pharmacological no interaction between ethanol and cyclosporin A was detected, which could be evidenced by the change of mitochondrial respiration of isolated hepatocytes.

Antiseizure effect of 2-deoxyglucose is not dependent on the presynaptic vacuole ATP pump or the somatic ATP-sensitive K+ channel.

Inhibition of glycolysis with 2-deoxyglucose (2-DG) produces antiseizure effects in brain slices and animal models, yet the mechanisms remain elusive. Here, we examined two glycolysis-derived ATP-associated mechanisms: vacuole ATP pump (V-ATPase) and ATP-sensitive K+ channel (KATP). Epileptiform bursts were generated in the CA3 area of hippocampal slices by 0 Mg2+ and 4-aminopyridine. 2-DG consistently abolished epileptiform bursts in the presence of pyruvate (to sustain tricarboxylic acid cycle for oxidative ATP production) at 30-33°C but not at room temperature (22°C). Under physiological conditions, 2-DG did not reduce the amplitude of evoked excitatory postsynaptic currents (EPSCs) or the paired-pulse ratio in CA3 neurons. During repetitive high-frequency (20 Hz, 20-50 pulses) stimulation, 2-DG did not accelerate the decline of EPSCs (i.e., depletion of transmitter release), even when preincubated with 8 mM K+ to enhance activity-dependent uptake of 2-DG. In addition, in 2-DG tetanic stimulation (200 Hz, 1 s) dramatically increased rather than diminished the occurrence of spontaneous EPSCs immediately after stimulation (i.e., no transmitter depletion). Moreover, a V-ATPase blocker (concanamycin) failed to block epileptiform bursts that were subsequently abolished by 2-DG. Furthermore, 2-DG did not induce detectable KATP current in hippocampal neurons. Finally, epileptiform bursts were not affected by either a KATP opener (diazoxide) or a KATP blocker (glibenclamide) but were blocked by 2-DG in the same slices. Altogether, these data suggest that 2-DG's antiseizure action is temperature dependent and achieved exclusively by inhibition of glycolysis and is not likely to be mediated by the two membrane-bound ATP-associated machinery mechanisms, V-ATPase and KATP.NEW & NOTEWORTHY Inhibition of glycolysis with 2-deoxyglucose (2-DG) represents a novel metabolic antiseizure approach, yet the mechanisms remain elusive. Here, we show that 2-DG's antiseizure action is both glycolysis and temperature dependent but not mediated by the vacuole ATP pump (V-ATPase) or ATP-sensitive K+ channel (KATP). Our data provide new insights to understand 2-DG's cellular mechanisms of action and, more broadly, neuronal metabolism and excitability.

Endocannabinoids effect on oxidative status of human platelets.

Reactive oxygen species (ROS) are known to regulate platelet activation. Since endocannabinoids behave as platelet agonists, we investigated the effect of two endocannabinoids, 2-arachidonoylglycerol (2AG) and anandamide (AEA) on the oxidative status of human platelets. We have demonstrated that 2AG and AEA stimulate ROS production, superoxide anion formation and lipid peroxidation. The effect is dose and time dependent and mainly occurs through the involvement of cannabinoid receptor 1 (CB1) since all tested parameters are greatly reduced by SR141716, the CB1 specific inhibitor. The specific inhibitor of cannabinoid receptor 2 (CB2) SR144528 produces a very small inhibition. The involvement of syk/PI3K/AKT/mTor pathway in oxidative stress induced by endocannabinoids is shown. Nicotinamide adenine dinucleotide phosphate oxidase seems to be poorly involved in the endocannabinoids effect. Concerning the aerobic metabolism, it has been demonstrated that endocannabinoids reduce the oxygen consumption and adenosine triphosphate synthesis, both in the presence of pyruvate + malate or succinate. In addition, endocannabinoids inhibit the activity of respiratory complexes II, III and IV and increase the activity of respiratory complex I. The endocannabinoids effect on aerobic metabolism seems to be also a CB1 mediated mechanism. Thus, in human platelets oxidative stress induced by endocannabinoids, mainly generated in the respiratory chain through the activation of complex I and the inhibition of complex II, III and IV, may lead to thrombotic events, contributing to cardiovascular diseases.

Influence of electrode potential, pH and NAD+ concentration on the electrochemical NADH regeneration

Electrochemical NAD+ reduction is a promising method to regenerate NADH for enzymatic reactions. Many different electrocatalysts have been tested in the search for high yields of the 1,4-isomer of NADH, the active NADH, but aside from electrode material, other system parameters such as pH, electrode potential and educt concentration also play a role in NADH regeneration. The effect of these last three parameters and the mechanisms behind their influence on NADH regeneration was systematically studied and presented in this paper. With percentages of active NADH ranging from 10 to 70% and faradaic efficiencies between 1 and 30%, it is clear that all three system parameters drastically affect the reaction outcome. As a proof of principle, the NAD+ reduction in the presence of pyruvate and lactate dehydrogenase was performed. It could be shown that the electrochemical NADH regeneration can also be done successfully in parallel to enzymatically usage of the regenerated cofactor.

DJ-1 attenuates the glycation of mitochondrial complex I and complex III in the post-ischemic heart

DJ-1 is a ubiquitously expressed protein that protects cells from stress through its conversion into an active protease. Recent work found that the active form of DJ-1 was induced in the ischemic heart as an endogenous mechanism to attenuate glycative stress—the non-enzymatic glycosylation of proteins. However, specific proteins protected from glycative stress by DJ-1 are not known. Given that mitochondrial electron transport proteins have a propensity for being targets of glycative stress, we investigated if DJ-1 regulates the glycation of Complex I and Complex III after myocardial ischemia–reperfusion (I/R) injury. Initial studies found that DJ-1 localized to the mitochondria and increased its interaction with Complex I and Complex III 3 days after the onset of myocardial I/R injury. Next, we investigated the role DJ-1 plays in modulating glycative stress in the mitochondria. Analysis revealed that compared to wild-type control mice, mitochondria from DJ-1 deficient (DJ-1 KO) hearts showed increased levels of glycative stress following I/R. Additionally, Complex I and Complex III glycation were found to be at higher levels in DJ-1 KO hearts. This corresponded with reduced complex activities, as well as reduced mitochondrial oxygen consumption ant ATP synthesis in the presence of pyruvate and malate. To further determine if DJ-1 influenced the glycation of the complexes, an adenoviral approach was used to over-express the active form of DJ-1(AAV9-DJ1ΔC). Under I/R conditions, the glycation of Complex I and Complex III were attenuated in hearts treated with AAV9-DJ1ΔC. This was accompanied by improvements in complex activities, oxygen consumption, and ATP production. Together, this data suggests that cardiac DJ-1 maintains Complex I and Complex III efficiency and mitochondrial function during the recovery from I/R injury. In elucidating a specific mechanism for DJ-1’s role in the post-ischemic heart, these data break new ground for potential therapeutic strategies using DJ-1 as a target.

Radical-free hyperpolarized MRI using endogenously occurring pyruvate analogues and UV-induced nonpersistent radicals.

It was recently demonstrated that nonpersistent radicals can be generated in frozen solutions of metabolites such as pyruvate by irradiation with UV light, enabling radical‐free dissolution dynamic nuclear polarization. Although pyruvate is endogenous, the presence of pyruvate may interfere with metabolic processes or the detection of pyruvate as a metabolic product, making it potentially unsuitable as a polarizing agent. Therefore, the aim of the current study was to characterize solutions containing endogenously occurring alternatives to pyruvate as UV‐induced nonpersistent radical precursors for in vivo hyperpolarized MRI. The metabolites alpha‐ketovalerate (αkV) and alpha‐ketobutyrate (αkB) are analogues of pyruvate and were chosen as potential radical precursors. Sample formulations containing αkV and αkB were studied with UV–visible spectroscopy, irradiated with UV light, and their nonpersistent radical yields were quantified with electron spin resonance and compared with pyruvate. The addition of 13C‐labeled substrates to the sample matrix altered the radical yield of the precursors. Using αkB increased the 13C‐labeled glucose liquid‐state polarization to 16.3% ± 1.3% compared with 13.3% ± 1.5% obtained with pyruvate, and 8.9% ± 2.1% with αkV. For [1–13C]butyric acid, polarization levels of 12.1% ± 1.1% for αkV, 12.9% ± 1.7% for αkB, 1.5% ± 0.2% for OX063 and 18.7% ± 0.7% for Finland trityl, were achieved. Hyperpolarized [1–13C]butyrate metabolism in the heart revealed label incorporation into [1–13C]acetylcarnitine, [1–13C]acetoacetate, [1–13C]butyrylcarnitine, [5‐13C]glutamate and [5‐13C]citrate. This study demonstrates the potential of αkV and αkB as endogenous polarizing agents for in vivo radical‐free hyperpolarized MRI. UV‐induced, nonpersistent radicals generated in endogenous metabolites enable high polarization without requiring radical filtration, thus simplifying the quality‐control tests in clinical applications.

Kinetic, spectral, and structural studies of the slow-binding inhibition of the Escherichia coli dihydrodipicolinate synthase by 2, 4-oxo-pentanoic acid

Dihydrodipicolinate synthase (DHDPS) catalyzes the first step in the biosynthetic pathway for production of l-lysine in bacteria and plants. The enzyme has received interest as a potential drug target owing to the absence of the enzyme in mammals. The DHDPS reaction is the rate limiting step in lysine biosynthesis and involves the condensation of l-aspartate-β-semialdehyde and pyruvate to form 2, 3-dihydrodipicolinate. 2, 4-oxo-pentanoic acid (acetopyruvate) is a slow-binding inhibitor of DHDPS that is competitive versus pyruvate with an initial Ki of about 20 μM and a final inhibition constant of about 1.4 μM. The enzyme:acetopyruvate complex displays an absorbance spectrum with a λmax at 304 nm and a longer wavelength shoulder. The rate constant for formation of the complex is 86 M−1 s−1. The enzyme forms a covalent enamine complex with the first substrate pyruvate and can be observed spectrally with a λmax at 271 nm. The spectra of the enzyme in the presence of pyruvate and acetopyruvate shows the initial formation of the pyruvate enamine intermediate followed by the slower appearance of the E:acetopyruvate spectra with a rate constant of about 0.013 s−1. The spectral studies suggest the formation of a Schiff base between acetopyruvate and K161 on enzyme that subsequently deprotonates to form a resonance stabilized anion similar to the enamine intermediate formed with pyruvate. The crystal structure of the E:acetopyruvate complex confirms the formation of the Schiff base between acetopyruvate and K161.

Transient Changes of Metabolism at the Pronuclear Stage in Mice Influences Skeletal Muscle Phenotype in Adulthood.

Skeletal muscle has a remarkable plasticity, and its phenotype is strongly influenced by hormones, transcription factors, and physical activity. However, whether skeletal phenotype can be oriented or not during early embryonic stages has never been investigated. Here, we report that pyruvate as the only source of carbohydrate in the culture medium of mouse one cell stage embryo influenced the establishment of the muscular phenotype in adulthood. We found that pyruvate alone induced changes in the contractile phenotype of the skeletal muscle in a sexually dependent manner. For male mice, a switch to a more glycolytic phenotype was recorded, whereas, in females, the pyruvate induced a switch to a more oxidative phenotype. In addition, the influence of pyruvate on the contractile phenotypes was confirmed in two mouse models of muscle hypertrophy: the well-known myostatin deficient mouse (Mstn−/−) and a mouse carrying a specific deletion of p43, a mitochondrial triiodothyronine receptor. Finally, to understand the link between these adult phenotypes and the early embryonic period, we assessed the levels of two histone H3 post-translational modifications in presence of pyruvate alone just after the wave of chromatin reprogramming specific of the first cell cycle. We showed that H3K4 acetylation level was decreased in Mstn−/− 2-cell embryos, whereas no difference was found for H3K27 trimethylation level, whatever the genotype. These findings demonstrate for the first time that changes in the access of energy substrate during the very first embryonic stage can induce a precocious orientation of skeletal muscle phenotype in adulthood.

Skeletal muscle mitochondrial fragmentation and impaired bioenergetics from nutrient overload are prevented by carbon monoxide.

Nutrient excess increases skeletal muscle oxidant production and mitochondrial fragmentation that may result in impaired mitochondrial function, a hallmark of skeletal muscle insulin resistance. This led us to explore whether an endogenous gas molecule, carbon monoxide (CO), which is thought to prevent weight gain and metabolic dysfunction in mice consuming high-fat diets, alters mitochondrial morphology and respiration in C2C12 myoblasts exposed to high glucose (15.6 mM) and high fat (250 µM BSA-palmitate) (HGHF). Also, skeletal muscle mitochondrial morphology, distribution, respiration, and energy expenditure were examined in obese resistant (OR) and obese prone (OP) rats that consumed a high-fat and high-sucrose diet for 10 wk with or without intermittent low-dose inhaled CO and/or exercise training. In cells exposed to HGHF, superoxide production, mitochondrial membrane potential (ΔΨm), mitochondrial fission regulatory protein dynamin-related protein 1 (Drp1) and mitochondrial fragmentation increased, while mitochondrial respiratory capacity was reduced. CO decreased HGHF-induced superoxide production, Drp1 protein levels and mitochondrial fragmentation, maintained ΔΨm, and increased mitochondrial respiratory capacity. In comparison with lean OR rats, OP rats had smaller skeletal muscle mitochondria that contained disorganized cristae, a normal mitochondrial distribution, but reduced citrate synthase protein expression, normal respiratory responses, and a lower energy expenditure. The combination of inhaled CO and exercise produced the greatest effect on mitochondrial morphology, increasing ADP-stimulated respiration in the presence of pyruvate, and preventing a decline in resting energy expenditure. These data support a therapeutic role for CO and exercise in preserving mitochondrial morphology and respiration during metabolic overload.

Mitochondrial threshold for H2O2 release in skeletal muscle of mammals

The aim of the study was to evaluate the interplay between mitochondrial respiration and H2O2 release during the transition from basal non-phosphorylating to maximal phosphorylating states. We conducted a large scale comparative study of mitochondrial oxygen consumption, H2O2 release and electron leak (% H2O2/O) in skeletal muscle mitochondria isolated from mammal species ranging from 7 g to 500 kg. Mitochondrial fluxes were measured at different steady state rates in presence of pyruvate, malate, and succinate as respiratory substrates. Every species exhibited a burst of H2O2 release from skeletal muscle mitochondria at a low rate of oxidative phosphorylation, essentially once the activity of mitochondrial oxidative phosphorylation reached 26% of the maximal respiration. This threshold for ROS generation thus appears as a general characteristic of skeletal muscle mitochondria in mammals. These findings may have implications in situations promoting succinate accumulation within mitochondria, such as ischemia or hypoxia.

β-Hydroxybutyrate Increases Exercise Capacity Associated with Changes in Mitochondrial Function in Skeletal Muscle.

β-hydroxybutyrate is the main ketone body generated by the liver under starvation. Under these conditions, it can sustain ATP levels by its oxidation in mitochondria. As mitochondria can modify its shape and function under different nutritional challenges, we study the chronic effects of β-hydroxybutyrate supplementation on mitochondrial morphology and function, and its relation to exercise capacity. Male C57BL/6 mice were supplemented with β-hydroxybutyrate mineral salt (3.2%) or control (CT, NaCl/KCl) for six weeks and submitted to a weekly exercise performance test. We found an increase in distance, maximal speed, and time to exhaustion at two weeks of supplementation. Fatty acid metabolism and OXPHOS subunit proteins declined at two weeks in soleus but not in tibialis anterior muscles. Oxygen consumption rate on permeabilized fibers indicated a decrease in the presence of pyruvate in the short-term treatment. Both the tibialis anterior and soleus showed decreased levels of Mitofusin 2, while electron microscopy assessment revealed a significant reduction in mitochondrial cristae shape in the tibialis anterior, while a reduction in the mitochondrial number was observed only in soleus. These results suggest that short, but not long-term, β-hydroxybutyrate supplementation increases exercise capacity, associated with modifications in mitochondrial morphology and function in mouse skeletal muscle.

Mitochondrial Utilization of Competing Fuels Is Altered in Insulin Resistant Skeletal Muscle of Non-obese Rats (Goto-Kakizaki).

AimInsulin-resistant skeletal muscle is characterized by metabolic inflexibility with associated alterations in substrate selection, mediated by peroxisome-proliferator activated receptor δ (PPARδ). Although it is established that PPARδ contributes to the alteration of energy metabolism, it is not clear whether it plays a role in mitochondrial fuel competition. While nutrient overload may impair metabolic flexibility by fuel congestion within mitochondria, in absence of obesity defects at a mitochondrial level have not yet been excluded. We sought to determine whether reduced PPARδ content in insulin-resistant rat skeletal muscle of a non-obese rat model of T2DM (Goto-Kakizaki, GK) ameliorate the inhibitory effect of fatty acid (i.e., palmitoylcarnitine) on mitochondrial carbohydrate oxidization (i.e., pyruvate) in muscle fibers.MethodsBioenergetic function was characterized in oxidative soleus (S) and glycolytic white gastrocnemius (WG) muscles with measurement of respiration rates in permeabilized fibers in the presence of complex I, II, IV, and fatty acid substrates. Mitochondrial content was measured by citrate synthase (CS) and succinate dehydrogenase activity (SDH). Western blot was used to determine protein expression of PPARδ, PDK isoform 2 and 4.ResultsCS and SDH activity, key markers of mitochondrial content, were reduced by ∼10–30% in diabetic vs. control, and the effect was evident in both oxidative and glycolytic muscles. PPARδ (p < 0.01), PDK2 (p < 0.01), and PDK4 (p = 0.06) protein content was reduced in GK animals compared to Wistar rats (N = 6 per group). Ex vivo respiration rates in permeabilized muscle fibers determined in the presence of complex I, II, IV, and fatty acid substrates, suggested unaltered mitochondrial bioenergetic function in T2DM muscle. Respiration in the presence of pyruvate was higher compared to palmitoylcarnitine in both animal groups and fiber types. Moreover, respiration rates in the presence of both palmitoylcarnitine and pyruvate were reduced by 25 ± 6% (S), 37 ± 6% (WG) and 63 ± 6% (S), 57 ± 8% (WG) compared to pyruvate for both controls and GK, respectively. The inhibitory effect of palmitoylcarnitine on respiration was significantly greater in GK than controls (p < 10–3).ConclusionWith competing fuels, the presence of fatty acids diminishes mitochondria ability to utilize carbohydrate derived substrates in insulin-resistant muscle despite reduced PPARδ content.

Aerobic degradation of cis-dichloroethene by the marine bacterium Marinobacter salsuginis strain 5N-3.

An aerobic bacterium, designated strain 5N-3 (NBRC 113055), that degrades cis-dichloroethene (cDCE) was isolated from a sea sediment in Japan. Strain 5N-3 was able to degrade a certain amount of cDCE in the presence of pyruvate without the action of inducers. In the presence of inducers, such as phenol and benzene, the strain completely removed cDCE. By the application of 16S ribosomal RNA (16S rRNA) gene sequencing and average nucleotide identity analyses, the strain 5N-3 was identified as Marinobacter salsuginis. On the other hand, identified species of Marinobacter are not known to degrade cDCE at all. A draft genome sequence analysis of the strain 5N-3 suggested that the dmp-homologous operon (operon for phenol degradation) may be contributing to the aerobic degradation of cDCE. This is the first report on an aerobic marine bacterium that has been found to degrade cDCE.

Mitochondria Are a Subset of Extracellular Vesicles Released by Activated Monocytes and Induce Type I IFN and TNF Responses in Endothelial Cells.

Extracellular vesicles, including microvesicles, are increasingly recognized as important mediators in cardiovascular disease. The cargo and surface proteins they carry are considered to define their biological activity, including their inflammatory properties. Monocyte to endothelial cell signaling is a prerequisite for the propagation of inflammatory responses. However, the contribution of microvesicles in this process is poorly understood. To elucidate the mechanisms by which microvesicles derived from activated monocytic cells exert inflammatory effects on endothelial cells. LPS (lipopolysaccharide)-stimulated monocytic cells release free mitochondria and microvesicles with mitochondrial content as demonstrated by flow cytometry, quantitative polymerase chain reaction, Western Blot, and transmission electron microscopy. Using RNAseq analysis and quantitative reverse transcription-polymerase chain reaction, we demonstrated that both mitochondria directly isolated from and microvesicles released by LPS-activated monocytic cells, as well as circulating microvesicles isolated from volunteers receiving low-dose LPS-injections, induce type I IFN (interferon), and TNF (tumor necrosis factor) responses in endothelial cells. Depletion of free mitochondria significantly reduced the ability of these microvesicles to induce type I IFN and TNF-dependent genes. We identified mitochondria-associated TNFα and RNA from stressed mitochondria as major inducers of these responses. Finally, we demonstrated that the proinflammatory potential of microvesicles and directly isolated mitochondria were drastically reduced when they were derived from monocytic cells with nonrespiring mitochondria or monocytic cells cultured in the presence of pyruvate or the mitochondrial reactive oxygen species scavenger MitoTEMPO. Mitochondria and mitochondria embedded in microvesicles constitute a major subset of extracellular vesicles released by activated monocytes, and their proinflammatory activity on endothelial cells is determined by the activation status of their parental cells. Thus, mitochondria may represent critical intercellular mediators in cardiovascular disease and other inflammatory settings associated with type I IFN and TNF signaling.

Distinct differences in rates of oxygen consumption and ATP synthesis of regionally isolated non-synaptic mouse brain mitochondria.

Brain mitochondrial dysfunction has been implicated in several neurodegenerative diseases. The distribution and efficiency of mitochondria display large heterogeneity throughout the regions of the brain. This may imply that the selective regional susceptibility of neurodegenerative diseases could be mediated through inherent differences in regional mitochondrial function. To investigate regional cerebral mitochondrial energetics, the rates of oxygen consumption and adenosine-5'-triphosphate (ATP) synthesis were assessed in isolated non-synaptic mitochondria of the cerebral cortex, hippocampus, and striatum of the male mouse brain. Oxygen consumption rates were assessed using a Seahorse XFe96 analyzer and ATP synthesis rates were determined by an online luciferin-luciferase coupled luminescence assay. Complex I- and complex II-driven respiration and ATP synthesis, were investigated by applying pyruvate in combination with malate, or succinate, as respiratory substrates, respectively. Hippocampal mitochondria exhibited the lowest basal and adenosine-5'-diphosphate (ADP)-stimulated rate of oxygen consumption when provided pyruvate and malate. However, hippocampal mitochondria also exhibited an increased proton leak and an elevated relative rate of oxygen consumption in response to the uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), showing a large capacity for uncoupled respiration in the presence of pyruvate. When the complex II-linked substrate succinate was provided, striatal mitochondria exhibited the highest respiration and ATP synthesis rate, whereas hippocampal mitochondria had the lowest. However, the mitochondrial efficiency, determined as ATP produced/O2 consumed, was similar between the three regions. This study reveals inherent differences in regional mitochondrial energetics and may serve as a tool for further investigations of regional mitochondrial function in relation to neurodegenerative diseases.

Pyruvate secreted from patient-derived cancer-associated fibroblasts supports survival of primary lymphoma cells.

Cancer‐associated fibroblasts (CAF) are a key component in the tumor microenvironment and play functional roles in tumor metastasis and resistance to chemotherapies. We have previously reported that CAF isolated from lymphoma samples increase anaerobic glycolysis and decrease intracellular production of reactive oxygen species, promoting the survival of tumor cells. Herein, we analyzed the mechanisms underlying this support of tumor‐cell survival by CAF. As direct contact between lymphoma cells and CAF was not indispensable to survival support, we identified that the humoral factor pyruvate was significantly secreted by CAF. Moreover, survival of lymphoma cells was promoted by the presence of pyruvate, and this promotion was canceled by inhibition of monocarboxylate transporters. Metabolome analysis of lymphoma cells in coculture with CAF demonstrated that intermediates in the citric acid cycle were significantly increased, indicating that tumor cells produced energy by aerobic metabolism. These findings indicate that energy production in lymphoma cells is regulated in coordination not only with anaerobic glycolysis, but also with aerobic metabolism termed the reverse‐Warburg effect, involving the secretion of pyruvate from CAF resulting in increased use of the citric acid cycle in lymphoma cells.

Effect of carbohydrates on lipid metabolism during porcine oocyte IVM.

Porcine oocytes contain a large amount of endogenous lipid, which is thought to function as an intracellular source of energy. The aim of this study was to determine the effects of stimulating or inhibiting lipid metabolism using l-carnitine or etomoxir respectively on the IVM of porcine oocytes cultured in media of varying carbohydrate composition. In the presence of pyruvate and lactate, exclusion of glucose inhibited oocyte nuclear and cytoplasmic maturation compared with oocytes matured in media containing low (1.5mM) and high (4.0mM) concentrations of glucose. In the absence of pyruvate and lactate in low-glucose medium only, a greater proportion of l-carnitine-treated oocytes progressed to the MII stage compared with untreated oocytes. The inclusion of pyruvate and lactate significantly altered the distribution of cytoplasmic lipid droplets and elevated the ATP content of oocytes, whereas the l-carnitine treatment did not. Further, the inhibitory effect of etomoxir on nuclear maturation was decreased in high- compared with low-glucose medium. The results indicate that carbohydrate substrates are absolutely necessary for effective porcine oocyte maturation, and that l-carnitine supplementation can only partially compensate for deficiencies in carbohydrate provision.

Myocardial GLP-1 Receptor Activation in the Presence of Glucose: Strong Partners

Glucagon-like peptide 1 (GLP-1) receptor agonists increase the intracellular calcium levels of atrial cardiomyocytes in a protein kinase A dependent manner. This elicits a positive inotropic effect. Furthermore, the translocation of GLUT1 is promoted. The relevance of the latter process is unclear, therefore we assessed the influence of energy substrates. Muscle strips (trabeculae; n = 86) were isolated from human right atrial appendages obtained from patients undergoing heart surgery (n = 34), mounted on hooks, electrically stimulated (1 Hz) and treated with a single dose of exenatide (15 nM), GLP-1(7–36) amide (180 nM), GLP-1(9–36) amide (200 nM), isoproterenol (100 nM), or increasing concentrations of calcium (4.0 and 7.2 mM). Either 11.2 mM glucose or 22.4 mM pyruvate served as the energy substrate. Developed force, diastolic tension and relaxation parameters were recorded and analyzed. Administration of exenatide and GLP-1(7–36) amide, but not GLP-1(9–36) amide, led to a transient positive inotropic effect in the presence of pyruvate and glucose. This effect tended to be more pronounced in glucose-treated muscle strips at maximal developed force and steady state conditions. Both isoproterenol and calcium exerted a strong positive inotropic effect with no difference regarding the energy substrate. In conclusion, the positive inotropic effect of GLP-1 receptor agonists is more pronounced in glucose enriched Tyrode’s solution, which might be linked to the previously reported translocation of GLUT1.

Protective activity of pyruvate against vanadium-dependent cytotoxicity in Chinese hamster ovary (CHO-K1) cells.

With increasing human exposure to vanadium-containing compounds and growing concern over their impact on human health, identification of safe methods for efficient treatment of vanadium poisoning may be of value. In this study, using Chinese hamster ovary (CHO-K1) cells, we show that the toxicity of vanadyl sulphate (VOSO4) is mitigated in the presence of sodium pyruvate. The exposure of CHO-K1 cells to 100 μM VOSO4 for 48 h induced significant cytotoxicity (measured with a resazurin assay) and elevation of the contents of malondialdehyde (MDA), a lipid peroxidation product, in the examined cells. When added simultaneously with VOSO4 to the culture medium, pyruvate (4.5 mM) reduced VOSO4-mediated cytotoxicity by twofold and inhibited MDA formation. Phase-contrast microscopy confirmed that the general morphology of cell cultures treated with 100 μM VOSO4 and 4.5 mM pyruvate was improved compared to VOSO4-only treated cells. The two-way analysis of variance revealed that the reduction of the adverse effects of VOSO4 in the presence of pyruvate was due to the independent action of pyruvate as well as antagonistic interaction between VOSO4 and pyruvate. From these data, it can be concluded that the pyruvate treatment may play a beneficial role in reducing vanadium-triggered health hazards.

A Breathing Atom-Transfer Radical Polymerization: Fully Oxygen-Tolerant Polymerization Inspired by Aerobic Respiration of Cells.

The first well-controlled aqueous atom-transfer radical polymerization (ATRP) conducted in the open air is reported. This air-tolerant ATRP was enabled by the continuous conversion of oxygen to carbon dioxide catalyzed by glucose oxidase (GOx), in the presence of glucose and sodium pyruvate as sequential sacrificial substrates. Controlled polymerization using initiators for continuous activator regeneration (ICAR) ATRP of oligo(ethylene oxide) methyl ether methacrylate (OEOMA, Mn =500) yielded polymers with low dispersity (1.09≤Đ≤1.29) and molecular weights (MWs) close to theoretical values in the presence of pyruvate. Without added pyruvates, lower MWs were observed due to generation of new chains by H2 O2 formed by reaction of O2 with GOx. Successful chain extension of POEOMA500 macroinitiator with OEOMA300 (Đ≤1.3) and Bovine Serum Albumin bioconjugates (Đ≤1.22) confirmed a well-controlled polymerization. The reactions in the open air in larger scale (25 mL) were also successful.