Differential Effects of Itaconate and its Esters on the Glutathione and Glucose Metabolism of Cultured Primary Rat Astrocytes.

Itaconate is produced as endogenous metabolite by decarboxylation of the citric acid cycle intermediate cis-aconitate. As itaconate has anti-microbial and anti-inflammatory properties, this substance is considered as potential therapeutic drug for the treatment of inflammation in various diseases including traumatic brain injury and stroke. To test for potential adverse effects of itaconate on the viability and metabolism of brain cells, we investigated whether itaconate or its membrane permeable derivatives dimethyl itaconate (DI) and 4-octyl itaconate (OI) may affect the basal glucose and glutathione (GSH) metabolism of cultured primary astrocytes.

ATP Restoration by ATP-Deprived Cultured Primary Astrocytes.

A high cellular concentration of adenosine triphosphate (ATP) is essential to fuel many important functions of brain astrocytes. Although cellular ATP depletion has frequently been reported for astrocytes, little is known on the metabolic pathways that contribute to ATP restoration by ATP-depleted astrocytes. Incubation of cultured primary rat astrocytes in glucose-free buffer for 60 min with the mitochondrial uncoupler BAM15 lowered the cellular ATP content by around 70%, the total amount of adenosine phosphates by around 50% and the adenylate energy charge (AEC) from 0.

Consequences of a 2-Deoxyglucose Exposure on the ATP Content and the Cytosolic Glucose Metabolism of Cultured Primary Rat Astrocytes.

The glucose analogue 2-deoxyglucose (2DG) has frequently been used as a tool to study cellular glucose uptake and to inhibit glycolysis. Exposure of primary cultured astrocytes to 2DG caused a time- and concentration-dependent cellular accumulation of 2-deoxyglucose-6-phosphate (2DG6P) that was accompanied by a rapid initial decline in cellular ATP content. Inhibitors of mitochondrial respiration as well as inhibitors of mitochondrial uptake of pyruvate and activated fatty acids accelerated the ATP loss, demonstrating that mitochondrial ATP regeneration contributes to the partial maintenance of the ATP content in 2DG-treated astrocytes.

Modulation of Pyruvate Export and Extracellular Pyruvate Concentration in Primary Astrocyte Cultures.

Astrocyte-derived pyruvate is considered to have neuroprotective functions. In order to investigate the processes that are involved in astrocytic pyruvate release, we used primary rat astrocyte cultures as model system. Depending on the incubation conditions and medium composition, astrocyte cultures established extracellular steady state pyruvate concentrations in the range between 150 µM and 300 µM.

Exogenous Substrates Prevent the Decline in the Cellular ATP Content of Primary Rat Astrocytes During Glucose Deprivation.

Brain astrocytes are well known for their broad metabolic potential. After glucose deprivation, cultured primary astrocytes maintain a high cellular ATP content for many hours by mobilizing endogenous substrates, but within 24 h the specific cellular ATP content was lowered to around 30% of the initial ATP content. This experimental setting was used to test for the potential of various exogenous substrates to prevent a loss in cellular ATP in glucose deprived astrocytes.

Local Administrations of Iron Oxide Nanoparticles in the Prefrontal Cortex and Caudate Putamen of Rats Do Not Compromise Working Memory and Motor Activity.

Iron oxide nanoparticles (IONPs) have come into focus for their use in medical applications although possible health risks for humans, especially in terms of brain functions, have not yet been fully clarified. The present study investigates the effects of IONPs on neurobehavioural functions in rats. For this purpose, we infused dimercaptosuccinic acid-coated IONPs into the medial prefrontal cortex (mPFC) and caudate putamen (CPu).

Experimental insights into electrocatalytic [Cp*Rh(bpy)Cl] mediated NADH regeneration.

NADH plays a crucial role in many enzymatically catalysed reactions. Due to the high costs of NADH a regeneration mechanism of this cofactor can enlarge the applications of enzymatic reactions dramatically. This paper gives a thorough system analysis of the mediated electrochemical regeneration of active NADH using cyclic voltammograms and potentiostatic measurements with varying pH, electrode potential, and electrolyte solution, highlighting the system's limiting conditions, elucidating optimal working parameters for the electrochemical reduction of NAD, and bringing new insight on the oxidation of inactive reduction products.

Modulation of Cellular Levels of Adenosine Phosphates and Creatine Phosphate in Cultured Primary Astrocytes.

Adenosine triphosphate (ATP) is the main energy currency of all cells, while creatine phosphate (CrP) is considered as a buffer of high energy-bond phosphate that facilitates rapid regeneration of ATP from adenosine diphosphate (ADP). Astrocyte-rich primary cultures contain ATP, ADP and adenosine monophosphate (AMP) in average specific contents of 36.0 ± 6.

Modulation of Multidrug Resistance Protein 1-mediated Transport Processes by the Antiviral Drug Ritonavir in Cultured Primary Astrocytes.

The Multidrug Resistance Protein 1 (Mrp1) is an ATP-dependent efflux transporter and a major facilitator of drug resistance in mammalian cells during cancer and HIV therapy. In brain, Mrp1-mediated GSH export from astrocytes is the first step in the supply of GSH precursors to neurons. To reveal potential mechanisms underlying the drug-induced modulation of Mrp1-mediated transport processes, we investigated the effects of the antiviral drug ritonavir on cultured rat primary astrocytes.

G6PDi-1 is a Potent Inhibitor of G6PDH and of Pentose Phosphate pathway-dependent Metabolic Processes in Cultured Primary Astrocytes.

Glucose-6-phosphate dehydrogenase (G6PDH) catalyses the rate limiting first step of the oxidative part of the pentose phosphate pathway (PPP), which has a crucial function in providing NADPH for antioxidative defence and reductive biosyntheses. To explore the potential of the new G6PDH inhibitor G6PDi-1 to affect astrocytic metabolism, we investigated the consequences of an application of G6PDi-1 to cultured primary rat astrocytes. G6PDi-1 efficiently inhibited G6PDH activity in lysates of astrocyte cultures.

Endogenous Energy Stores Maintain a High ATP Concentration for Hours in Glucose-Depleted Cultured Primary Rat Astrocytes.

Adenosine triphosphate (ATP) is the central energy currency of all cells. Cultured primary rat astrocytes contain a specific cellular ATP content of 27.9 ± 4.

β-lapachone-mediated WST1 Reduction as Indicator for the Cytosolic Redox Metabolism of Cultured Primary Astrocytes.

Electron cycler-mediated extracellular reduction of the water-soluble tetrazolium salt 1 (WST1) is frequently used as tool for the determination of cell viability. We have adapted this method to monitor by determining the extracellular WST1 formazan accumulation the cellular redox metabolism of cultured primary astrocytes via the NAD(P)H-dependent reduction of the electron cycler β-lapachone by cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1). Cultured astrocytes that had been exposed to β-lapachone in concentrations of up to 3 µM remained viable and showed an almost linear extracellular accumulation of WST1 formazan for the first 60 min, while higher concentrations of β-lapachone caused oxidative stress and impaired cell metabolism.

Consumption and Metabolism of Extracellular Pyruvate by Cultured Rat Brain Astrocytes.

Brain astrocytes are considered as glycolytic cell type, but these cells also produce ATP via mitochondrial oxidative phosphorylation. Exposure of cultured primary astrocytes in a glucose-free medium to extracellular substrates that are known to be metabolised by mitochondrial pathways, including pyruvate, lactate, beta-hydroxybutyrate, alanine and acetate, revealed that among the substrates investigated extracellular pyruvate was most efficiently consumed by astrocytes. Extracellular pyruvate was consumed by the cells almost proportional to time over hours in a concentration-dependent manner with apparent Michaelis-Menten kinetics [K = 0.

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.

Effects of Local Administration of Iron Oxide Nanoparticles in the Prefrontal Cortex, Striatum, and Hippocampus of Rats.

Iron oxide nanoparticles (IONPs) are used for diverse medical approaches, although the potential health risks, for example adverse effects on brain functions, are not fully clarified. Several in vitro studies demonstrated that the different types of brain cells are able to accumulate IONPs and reported a toxic potential for IONPs, at least for microglia. However, little information is available for the in vivo effects of direct application of IONPs into the brain over time.

LDHA is enriched in human islet alpha cells and upregulated in type 2 diabetes.

The lactate dehydrogenase isoform A (LDHA) is a key metabolic enzyme that preferentially catalyzes the conversion of pyruvate to lactate. Whereas LDHA is highly expressed in many tissues, its expression is turned off in the differentiated adult β-cell within the pancreatic islets. The repression of LDHA under normal physiological condition and its inappropriate upregulation under a diabetogenic environment is well-documented in rodent islets/β-cells but little is known about LDHA expression in human islet cells and whether its abundance is altered under diabetic conditions.

Sila-Ibuprofen.

The synthesis, characterization, biological activity, and toxicology of sila-ibuprofen, a silicon derivative of the most common nonsteroidal anti-inflammatory drug, is reported. The key improvements compared with ibuprofen are a four times higher solubility in physiological media and a lower melting enthalpy, which are attributed to the carbon-silicon switch. The improved solubility is of interest for postsurgical intravenous administration.

β-Lapachone Induces Acute Oxidative Stress in Rat Primary Astrocyte Cultures that is Terminated by the NQO1-Inhibitor Dicoumarol.

β-lapachone (β-lap) is reduced in tumor cells by the enzyme NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1) to a labile hydroquinone which spontaneously reoxidises to β-lap, thereby generating reactive oxygen species (ROS) and oxidative stress. To test for the consequences of an acute exposure of brain cells to β-lap, cultured primary rat astrocytes were incubated with β-lap for up to 4 h. The presence of β-lap in concentrations of up to 10 µM had no detectable adverse consequences, while higher concentrations of β-lap compromised the cell viability and the metabolism of astrocytes in a concentration- and time-dependent manner with half-maximal effects observed for around 15 µM β-lap after a 4 h incubation.

Iron-Doping of Copper Oxide Nanoparticles Lowers Their Toxic Potential on C6 Glioma Cells.

Copper oxide nanoparticles (CuO-NPs) are well known for their cytotoxicity which in part has been attributed to the release of copper ions from CuO-NPs. As iron-doping has been reported to reduce the susceptibility of CuO-NPs to dissolution, we have compared pure CuO-NPs and CuO-NPs that had been doped with 10% iron (CuO-Fe-NPs) for copper release and for their toxic potential on C6 glioma cells. Physicochemical characterization revealed that dimercaptosuccinate (DMSA)-coated CuO-NPs and CuO-Fe-NPs did not differ in their size or zeta potential.

The Menadione-Mediated WST1 Reduction by Cultured Astrocytes Depends on NQO1 Activity and Cytosolic Glucose Metabolism.

The reduction of water-soluble tetrazolium salts (WSTs) is frequently used to determine the metabolic integrity and the viability of cultured cells. Recently, we have reported that the electron cycler menadione can efficiently connect intracellular oxidation reactions in cultured astrocytes with the extracellular reduction of WST1 and that this menadione cycling reaction involves an enzyme. The enzymatic reaction involved in the menadione-dependent WST1 reduction was found strongly enriched in the cytosolic fraction of cultured astrocytes and is able to efficiently use both NADH and NADPH as electron donors.

Uptake of Intact Copper Oxide Nanoparticles Causes Acute Toxicity in Cultured Glial Cells.

Copper oxide nanoparticles (CuO-NPs) dispersions are known for their high cell toxic potential but contaminating copper ions in such dispersions are a major hurdle in the investigation of specific nanoparticle-mediated toxicity. In order to distinguish between the adverse effects exhibited by CuO-NPs and/or by contaminating ionic copper, the membrane-impermeable copper chelator bathocuproine disulfonate (BCS) was added in a low molar ratio (20% of the total copper applied) in order to chelate the copper ions that had been released extracellularly from the CuO-NPs before or during the incubation. Physicochemical characterization of synthesized CuO-NPs revealed that the presence of this low concentration of BCS did not alter the size or zeta potential of the CuO-NPs.

Quantification and biodegradability assessment of meso-2,3-dimercaptosuccinic acid adsorbed on iron oxide nanoparticles.

Many interesting applications of magnetic iron oxide nanoparticles (IONPs) have recently been developed based on their magnetic properties and promising catalytic activity. Depending on their intended use, such nanoparticles (NPs) are frequently functionalized with proteins, polymers, or other organic molecules such as meso-2,3-dimercaptosuccinic acid (DMSA) to improve their colloidal stability or biocompatibility. Although the coating strongly affects the colloidal properties and environmental behaviour of NPs, quantitative analysis of the coating is often neglected.

Exposure of Cultured Astrocytes to Menadione Triggers Rapid Radical Formation, Glutathione Oxidation and Mrp1-Mediated Export of Glutathione Disulfide.

Menadione (2-methyl-1,4-naphthoquinone) is a synthetic derivative of vitamin K that allows rapid redox cycling in cells and thereby generates reactive oxygen species (ROS). To test for the consequences of a treatment of brain astrocytes with menadione, we incubated primary astrocyte cultures with this compound. Incubation with menadione in concentrations of up to 30 µM did not affect cell viability.

Consequences of a Metabolic Glucose-Depletion on the Survival and the Metabolism of Cultured Rat Astrocytes.

Brain astrocytes are considered to be highly glycolytic, but these cells also produce ATP via mitochondrial oxidative phosphorylation. To investigate how a metabolic depletion of glucose will affect the metabolism of astrocytes, we applied glucose at an initial concentration of 2 mM to cultured primary astrocytes and monitored the cell viability and various metabolic parameters during an incubation for up to 2 weeks. Already within 2 days of incubation the cells had completely consumed the applied glucose and lactate had accumulated in the medium to a concentration of around 3 mM.

Dicoumarol Inhibits Multidrug Resistance Protein 1-Mediated Export Processes in Cultured Primary Rat Astrocytes.

Dicoumarol is frequently used as inhibitor of the detoxifying enzyme NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1). In order to test whether dicoumarol may also affect the cellular glutathione (GSH) metabolism, we have exposed cultured primary astrocytes to dicoumarol and investigated potential effects of this compound on the cell viability as well as on the cellular and extracellular contents of GSH and its metabolites. Incubation of astrocytes with dicoumarol in concentrations of up to 100 µM did not acutely compromise cell viability nor was any GSH consumption or GSH oxidation to glutathione disulfide (GSSG) observed.