Monocarboxylates and glucose utilization as energy substrates in rat brain slices under selective glial poisoning – a 31P NMR study

We have investigated effects of various energy substrates including glucose, lactate and pyruvate on the recovery of the high energy phosphate levels after high-K⁺ stimulation in rat brain slices by using ³¹P NMR. It was found that lactate, pyruvate and glucose almost equally supported the recovery of phosphocreatine (PCr) levels after high-K⁺ stimulation (60 mM, 8 min) in artificial cerebrospinal fluid (ACSF). In iodoacetic acid (IAA) and fluorocitrate (FC)-pretreated slices, whereas glucose was unable to be utilized, the recovery of the PCr level after high-K⁺ stimulation in ACSF containing lactate was completely abolished, the recovery of the PCr in ACSF containing pyruvate was unaffected. These results indicate that neurons themselves can utilize pyruvate as an exogenous energy substrate, but not lactate, without glial support. In intact brain, glucose may be metabolized to pyruvate in glial cells and then transported to neurons as an energy substrate. These suggest an astrocyte-neuron pyruvate shuttle mechanism of the brain energy metabolism in vivo.We also investigated the effect of ischemic-preconditioning in FC-pretreated slices, which showed that the PCr levels recovered substantially in ACSF containing lactate after high-K⁺ stimulation. This indicates that after the preconditioning, such as ischemia, neurons themselves acquired the ability to utilize lactate as an energy substrate.     

Monocarboxylates and glucose utilization as energy substrates in rat brain slices under selective glial poisoning – a 31P NMR study

Molecular and Cellular Biochemistry -     

Rate of utilization of glucose and `compartmentation'' of α-oxoglutarate and glutamate in rat brain

1. The rate of incorporation of ¹⁴C into pyruvate, α-oxoglutarate, lactate and glucose of rat tissues was measured after the subcutaneous injection of uniformly labelled glucose. 2. In rat brain the specific radioactivities of lactate and glucose were similar to that of alanine. In liver the specific radioactivity of glucose was considerably higher than that of lactate or alanine. 3. The specific radioactivities of α-oxo acids of rat brain were lower than those of corresponding amino acids, alanine and glutamate. These findings have been explained in relation to metabolic compartments in vivo. 4. The approximate estimated rate of glucose utilization in rat brain in vivo is 0·96μmole/g. of brain/min.     

Incorporation of label from [1-14C]ethanol into the glutamate–glutamine pools of rat brain in vivo

Glutamate and glutamine were isolated from the brains of rats given [1-(14)C]ethanol by three different routes of injection with and without carrier ethanol or acetate. Without exception, the specific radioactivity of brain glutamine was 3- to 5-fold that of brain glutamate. The route of injection had no consistent effect on the results. These results indicate that small amounts of ethanol are oxidized by rat brain in the compartment responsible for the so-called ;small metabolic pool' of brain glutamate.     

Decreased glutamate, glutamine and citrulline concentrations in plasma and muscle in endotoxemia cannot be reversed by glutamate or glutamine supplementation: a primary intestinal defect?

Endotoxemia affects intestinal physiology. A decrease of circulating citrulline concentration is considered as a reflection of the intestinal function. Citrulline can be produced in enterocytes notably from glutamate and glutamine. The aim of this work was to determine if glutamate, glutamine and citrulline concentrations in blood, intestine and muscle are decreased by endotoxemia, and if supplementation with glutamate or glutamine can restore normal concentrations. We induced endotoxemia in rats by an intraperitoneal injection of 0.3?mg?kg^?1 lipopolysaccharide (LPS). This led to a rapid anorexia, negative nitrogen balance and a transient increase of the circulating level of IL-6 and TNF-α. When compared with the values measured in pair fed (PF) animals, almost all circulating amino acids (AA) including citrulline decreased, suggesting a decrease of intestinal function. However, at D2 after LPS injection, most circulating AA concentrations were closed to the values recorded in the PF group. At that time, among AA, only glutamate, glutamine and citrulline were decreased in gastrocnemius muscle without change in intestinal mucosa. A supplementation with 4% monosodium glutamate (MSG) or an isomolar amount of glutamine failed to restore glutamate, glutamine and citrulline concentrations in plasma and muscle. However, MSG supplementation led to an accumulation of glutamate in the intestinal mucosa. In conclusion, endotoxemia rapidly but transiently decreased the circulating concentrations of almost all AA and more durably of glutamate, glutamine and citrulline in muscle. Supplementation with glutamate or glutamine failed to restore glutamate, glutamine and citrulline concentrations in plasma and muscles. The implication of a loss of the intestinal capacity for AA absorption and/or metabolism in endotoxemia (as judged from decreased citrulline plasma concentration) for explaining such results are discussed.     

Localization of lipocaline-type prostaglandin D synthase in rat brain: immunoelectron microscopic study

The distribution of lipocaline-type prostaglandin D synthase (L-PGDS) in rat brain was investigated by immunoelectron microscopy using a protein A-gold technique. In perivascular cells adjacent to the basement membrane of arterioles in the pia-arachnoid and of blood vessels in the subpial cortex, gold labeling was confined to the lumen of the dilated rough endoplasmic reticulum, and not found in the few lysosomes present in the cytoplasm. The results suggest that the perivascular cells secrete L-PGDS and seem not to degrade lipophilic molecules carried by L-PGDS. Moreover, gold particles representing the antigenic sites of L-PGDS were found in the Golgi apparatus, rough endoplasmic reticulum, vesicles, and nuclear envelope of arachnoid trabecular cells, arachnoid barrier cells, and arachnoid pia mater cells. The labeling was less detectable in the same organelles of choroid plexus epithelial cells, compared with leptomeningeal cells. In meningeal macrophages and parenchymal microglia, L-PGDS was detected in lysosomes, multivesicular bodies, and endocytic vesicles. The production of L-PGDS in perivascular cells is important to the various functions of this enzyme in brain parenchyma.     

Regional distribution of α-neo-endorphin in rat brain and pituitary

α-Neo-endorphin was isolated as the first form of “big” Leu-enkephalin and its complete amino acid sequence has recently been established. Using an antiserum raised against synthetic α-neo-endorphin, a highly sentitive and specific radioimmunoassay was developed. The antiserum practically possesses no cross-reactivity to Leu-enkephalin, dynorphin[1–13] and PH-8P, and very little to β-neo-endorphin. Distribution of α-neo-endorphin has been determined in rat brain and pituitary by the use of the highly specific antiserum. The highest concentration was observed at posterior lobe of pituitary. Furthermore, immunoreactive α-neo-endorphin was characterized by gel-filtration and high performance liquid chromatography, and shown to be identical with authentic α-neo-endorphin.     

The concentration of soluble extracellular amyloid-β protein in acute brain slices from CRND8 mice

Background

Many recent studies of the effects of amyloid-β protein (Aβ) on brain tissue from amyloid precursor protein (APP) overexpressing mice have concluded that Aβ oligomers in the extracellular space can profoundly affect synaptic structure and function. As soluble proteins, oliomers of Aβ can diffuse through brain tissue and can presumably exit acute slices, but the rate of loss of Aβ species by diffusion from brain slices and the resulting reduced concentrations of Aβ species in brain slices are unknown.

Methodology/Principal Findings

Here I combine measurements of Aβ_1–42 diffusion and release from acute slices and simple numerical models to measure the concentration of Aβ_1–42 in intact mice (in vivo) and in acute slices from CRND8 mice. The in vivo concentration of diffusible Aβ_1–42 in CRND8 mice was 250 pM at 6 months of age and 425 pM at 12 months of age. The concentration of Aβ_1–42 declined rapidly after slice preparation, reaching a steady-state concentration within one hour. 50 µm from the surface of an acute slice the steady-state concentration of Aβ was 15–30% of the concentration in intact mice. In more superficial regions of the slice, where synaptic physiology is generally studied, the remaining Aβ is less than 15%. Hence the concentration of Aβ_1–42 in acute slices from CRND8 mice is less than 150 pM.

Conclusions/Significance

Aβ affects synaptic plasticity in the picomolar concentration range. Some of the effects of Aβ may therefore be lost or altered after slice preparation, as the extracellular Aβ concentration declines from the high picomolar to the low picomolar range. Hence loss of Aβ by diffusion may complicate interpretation of the effects of Aβ in experiments on acute slices from APP overexpressing mice.     

Chronic olanzapine treatment decreases arachidonic acid turnover and prostaglandin E₂ concentration in rat brain

The atypical antipsychotic, olanzapine (OLZ), is used to treat bipolar disorder, but its therapeutic mechanism of action is not clear. Arachidonic acid (AA, 20:4n-6) plays a critical role in brain signaling and an up-regulated AA metabolic cascade was reported in postmortem brains from bipolar disorder patients. In this study, we tested whether, similar to the action of the mood stabilizers lithium, carbamazepine and valproate, chronic OLZ treatment would reduce AA turnover in rat brain. We administered OLZ (6 mg/kg/day) or vehicle i.p. to male rats once daily for 21 days. A washout group received 21 days of OLZ followed by vehicle on day 22. Two hours after the last injection, [1-1?C]AA was infused intravenously for 5 min, and timed arterial blood samples were taken. After the rat was killed at 5 min, its brain was microwaved, removed and analyzed. Chronic OLZ decreased plasma unesterified AA concentration, AA incorporation rates and AA turnover in brain phospholipids. These effects were absent after washout. Consistent with reduced AA turnover, OLZ decreased brain cyclooxygenase activity and the brain concentration of the proinflammatory AA-derived metabolite, prostaglandin E?, In view of up-regulated brain AA metabolic markers in bipolar disorder, the abilities of OLZ and the mood stabilizers to commonly decrease prostaglandin E?, and AA turnover in rat brain phospholipids, albeit by different mechanisms, may be related to their efficacy against the disease.     

Changes in dialysate concentrations of glutamate and GABA in the brain: an index of volume transmission mediated actions?

Brain microdialysis has become a frequently used method to study the extracellular concentrations of neurotransmitters in specific areas of the brain. For years, and this is still the case today, dialysate concentrations and hence extracellular concentrations of neurotransmitters have been interpreted as a direct index of the neuronal release of these specific neurotransmitter systems. Although this seems to be the case for neurotransmitters such as dopamine, serotonin and acetylcholine, the extracellular concentrations of glutamate and GABA do not provide a reliable index of their synaptic exocytotic release. However, many microdialysis studies show changes in extracellular concentrations of glutamate and GABA under specific pharmacological and behavioural stimuli that could be interpreted as a consequence of the activation of specific neurochemical circuits. Despite this, we still do not know the origin and physiological significance of these changes of glutamate and GABA in the extracellular space. Here we propose that the changes in dialysate concentrations of these two neurotransmitters found under specific treatments could be an expression of the activity of the neurone-astrocyte unit in specific circuits of the brain. It is further proposed that dialysate changes of glutamate and GABA could be used as an index of volume transmission mediated actions of these two neurotransmitters in the brain. This hypothesis is based firstly on the assumption that the activity of neurones is functionally linked to the activity of astrocytes, which can release glutamate and GABA to the extracellular space; secondly, on the existence of extrasynaptic glutamate and GABA receptors with functional properties different from those of GABA receptors located at the synapse; and thirdly, on the experimental evidence reporting specific electrophysiological and neurochemical effects of glutamate and GABA when their levels are increased in the extracellular space. According to this concept, glutamate and GABA, once released into the extracellular compartment, could diffuse and have long-lasting effects modulating glutamatergic and/or GABAergic neurone-astrocytic networks and their interactions with other neurotransmitter neurone networks in the same areas of the brain.     

Purification and characterization of a β-galactoside-binding soluble lectin from rat and bovine brain

A β-galactoside-binding activity has been detected in mammalian brain extracts using a hemagglutination test and a nerve cell aggragation assay. Inhibition studies suggested the involvement of lectin-carbohydrate interactions in these processes. In an attempt to explore further the biological role of brain lectins, the β-galactoside-binding activity has been purified to apparent homogeneity from bovine and rat brain by salt extraction of the brain tissue and affinity chromatography on asialofetuin-agarose. The molecular weights determined by gel filtration, under native conditions on Ultrogel AcA-34, were 30 000 for the bovine brain lectin and 32 000 for the rat brain lectin; polyacrylamide gel electrophoresis in SDS gave molecular weights of 15 000 and 16 000, respectively, suggesting that the two brain lectins are dimers. Both lectins have an isoelectric point of 3.9. Amino acid composition data indicate that both lectins contain high proportions of glycine and acidic amino acids. The lectins are specific for β-D-galactosides and related sugars and the configuration of carbon atoms 1, 2 and 4 seems of primary importance. Moreover, the nerve cell aggregation-promoting activity of the purified lectin is 300-fold that of the crude extracts.     

A comparative study of hematological parameters of α and β thalassemias in a high prevalence zone: Saudi Arabia

BACKGROUND AND AIMS:

Saudi Arabia falls in the high prevalent zone of αα and β thalassemias. Early screening for the type of thalassemia is essential for further investigations and management. The study was carried out to differentiate the type of thalassemia based on red cell indices and other hematological parameters.

MATERIALS AND METHODS:

The study was carried out on 991 clinically suspected cases of thalassemias in Riyadh, Saudi Arabia. The hematological parameters were studied on Coulter STKS. Cellulose acetate hemoglobin electrophoresis and high-performance liquid chromatography (HPLC) were performed on all the blood samples. Gene deletion studies were carried out by restriction fragment length polymorphism (RFLP) technique using the restriction endonucleases Bam HI.

STATISTICAL ANALYSIS:

Statistical analysis was performed on SPSS 11.5 version.

RESULTS:

The hemoglobin electrophoresis and gene studies revealed that there were 406 (40.96%) and 59 (5.95 %) cases of β thalassemia trait and β thalassemia major respectively including adults and children. 426 cases of various deletion forms of α thalassemias were seen. Microcytosis was a common feature in β thalassemias trait and (-α/-α) and (--/αα) types of α thalassemias. MCH was a more significant distinguishing feature among thalassemias. β thalassemia major and α thalassemia (-α/αα) had almost normal hematological parameters.

CONCLUSION:

MCV and RBC counts are not statistically significant features for discriminating between α and β thalassemias. There is need for development of a discrimination index to differentiate between α and β thalassemias traits on the lines of discriminatory Indices available for distinguishing β thalassemias trait from iron deficiency anemia.     

Metabolic profiling of vitamin C deficiency in Gulo?/? mice using proton NMR spectroscopy

Nutrient deficiencies are an ongoing problem in many populations and ascorbic acid is a key vitamin whose mild or acute absence leads to a number of conditions including the famously debilitating scurvy. As such, the biochemical effects of ascorbate deficiency merit ongoing scrutiny, and the Gulo knockout mouse provides a useful model for the metabolomic examination of vitamin C deficiency. Like humans, these animals are incapable of synthesizing ascorbic acid but with dietary supplements are otherwise healthy and grow normally. In this study, all vitamin C sources were removed after weaning from the diet of Gulo-/- mice (n?=?7) and wild type controls (n?=?7) for 12?weeks before collection of serum. A replicate study was performed with similar parameters but animals were harvested pre-symptomatically after 2-3?weeks. The serum concentration of 50 metabolites was determined by quantitative profiling of 1D proton NMR spectra. Multivariate statistical models were used to describe metabolic changes as compared to control animals; replicate study animals were used for external validation of the resulting models. The results of the study highlight the metabolites and pathways known to require ascorbate for proper flux.     

Inhibition of GABAA ligand-gated Cl− channels by zinc in adult rat brain: A regional study

Zinc (Zn²⁺) was shown to invariably inhibit muscimol-stimulated³⁶Cl⁻ uptake by synaptoneurosomes in the cerebral cortex, hippocampus and cerebellum. The Zn²⁺ sensitivity of the GABA_A receptor-gated³⁶Cl⁻ uptake in the cerebral cortex was comparable to that in the hippocampus, whereas the uptake in the cerebellum was less sensitive to Zn²⁺. Although diazepam-potentiation of muscimol-stimulated³⁶Cl⁻ uptake was unaltered by 100 μM Zn²⁺ in the cerebellum. Zn²⁺ inhibited [³H]diazepam binding significantly at 1 mM in the cerebral cortex and cerebellum, whereas Ni²⁺ increased the binding in a concentration-dependent manner in both regions. Although lower concentrations of Zn²⁺ did not affect [³H]Ro 15-4513 binding to diazepam-sensitive sites, higher concentrations of Zn²⁺ increased the binding in both regions. Unlike the diazepam-sensitive sites the diazepam-insensitive [³H]Ro 15-4513 binding was not affected by Zn²⁺ or Ni²⁺ at any of the tested concentrations. These results suggest that the GABA_A ligand-gated Cl⁻ flux and its diazepam-potentiation are heterogeneously modulated in various brain regions. It is also suggested that cerebellar diazepam-insensitive [³H]Ro 15-4513 binding sites are insensitive to Zn²⁺ and Ni²⁺.     

Decreased glutamine synthetase,increased citrulline–nitric oxide cycle activities,and oxidative stress in different regions of brain in epilepsy rat model

To understand their role in epilepsy, the nitric oxide synthetase (NOS), argininosuccinate synthetase (AS), argininosuccinate lyase (AL), glutamine synthetase (GS), and arginase activities, along with the concentration of nitrate/nitrite (NOx), thiobarbituric acid reactive substances (TBARS), and total antioxidant status (TAS), were estimated in different regions of brain in rats subjected to experimental epilepsy induced by subcutaneous administration of kainic acid (KA). The short-term (acute) group animals were killed after 2 h and the long term (chronic) group animals were killed after 5 days of single injection of KA (15 mg/kg body weight). After decapitation of rats, the brain regions were separated and in their homogenates, the concentration of NOx, TBARS and TAS and the activities of NOS, AS, AL, arginase and glutamine synthetase were assayed by colorimetric methods. The results of the study demonstrated the increased activity of NOS and formation of NO in acute and chronic groups epilepsy. The activities of AS and AL were increased and indicate the effective recycling of citrulline to arginine. The activity of glutamine synthetase was decreased in acute and chronic groups of epilepsy compared to control group and indicate the modulation of its activity by NO in epilepsy. The activity of arginase was not changed in acute group; however it was decreased in chronic group and may favor increased production of NO in this condition. The concentration TBARS were increased and TAS decreased in acute and chronic groups of epilepsy and supports the oxidative stress in epilepsy.     

Differential effects of veratridine and calcium on the release of [3H]noradrenaline and [14C]α-aminoisobutyrate from rat brain cortex slices

The efflux of [³H]noradrenaline (NA) and of the non transmitter, non metabolizable, amino acid [¹⁴C]α-aminoisobutyrate (AIB), was followed simultaneously from superfused rat brain cortex thin slices, that had been preloaded with those substances. Short (2 min) “pulses” of increasing veratridine concentrations were applied at 10 min intervals. When calcium in the superfusion fluid was 1 mM, [³H]NA efflux increased progressively with pulses of 1, 3, 10 and 30 μM veratridine, but further increase to 100 μM resulted in a decrease of the induced ³H-efflux. Veratridine-enhanced [³H]NA efflux decreased considerably in 0.1 mM calcium and was virtually suppressed when no calcium was added to the superfusion fluid. In 1 mM calcium, the efflux of [¹⁴C] AIB was increased progressively by pulses of 10, 30 and 100 μM veratridine, but no increase in efflux was seen with 1 or 3 μM drug. In 0.1 mM, or without added calcium, the induced efflux of [¹⁴C]AIB was markedly increased. Similar findings were seen when a long (10 min) pulse of 10 μM veratridine was given. After such long pulses there was a rapid return of AIB efflux to pre-veratridine levels if calcium was 1 mM, but in the absence of added calcium, the return to baseline levels of both [³H]NA and, especially, that of [¹⁴C]AIB efflux, was greatly impaired. The veratridine enhanced efflux of both NA and AIB was entirely blocked by 1 μM tetrodotoxin.     

Metabolic pathways in the periphery and brain: Contribution to mental disorders?

The association between mental disorders and diabetes has a long history. Recent large-scale, well-controlled epidemiological studies confirmed a link between diabetes and psychiatric illnesses. The scope of this review is to summarize our current understanding of this relationship from a molecular perspective. We first discuss the potential contribution of diabetes-associated metabolic impairments to the etiology of mental conditions. Then, we focus on possible shared molecular risk factors and mechanisms. Simple comorbidity, shared susceptibility loci, and common pathophysiological processes in diabetes and mental illnesses have changed our traditional way of thinking about mental illness. We conclude that schizophrenia and affective disorders are not limited to an imbalance in dopaminergic and serotoninergic neurotransmission in the brain. They are also systemic disorders that can be considered, to some extent, as metabolic disorders.     

The agmatine-degrading enzyme agmatinase: a key to agmatine signaling in rat and human brain?

Agmatinase, an ureohydrolase belonging to the arginase family, is widely expressed in mammalian tissues including the brain. Here, it may serve two different functions, the inactivation of the arginine derivative agmatine, a putative neurotransmitter, and the formation of the diamine putrescine. In order to identify the cellular sources of agmatinase expression in the brain, we generated a polyclonal monospecific antibody against recombinant rat agmatinase. With immunocytochemistry, selected areas of rat and human brain were screened. Clearly, in both species agmatinase-like immunoreactivity was predominantly detected in distinct populations of neurons, especially cortical interneurons. Also, principal neurons in limbic regions like the habenula and in the cerebellum robustly expressed agmatinase protein. When comparing the overall agmatinase expression with immunocytochemical data available for agmatine and polyamine biosynthetic enzymes, the observed pattern may argue in favor of an agmatine inactivating function rather than fueling the alternative pathway of polyamine synthesis. The putative neurotransmitter agmatine is seemingly involved with mental disorders. Therefore, agmatinase may be similarly important for pathogenesis. The normal expression profile of the protein as described here may therefore be altered under pathological conditions.     

Vesicular glutamate transporter 2 immunoreactivity in putative vagal mechanosensor terminals of mouse and rat esophagus: indication of a local effector function?

Intraganglionic laminar endings (IGLEs) represent the major vagal afferent structures throughout the gastrointestinal tract. Previous ultrastructural investigations have revealed synaptic contacts of IGLEs on myenteric neurons. Thus, in addtion to functioning probably as mechanosensors, IGLEs may also synaptically influence myenteric neurons. In search of clues for potential transmitters in IGLEs, we investigated, by combined neuronal tracing and immunocytochemistry in the esophagus, the correlation between IGLEs and vesicular glutamate transporter 2 (VGLUT2), which is considered a reliable marker for glutamatergic neurons. In rat esophagus, IGLEs were immunostained with calretinin. In the mouse, anterograde wheat germ agglutinin/horseradish peroxidase (WGA-HRP) tracing from nodose ganglion was used in order to label esophageal IGLEs. Confocal laser scanning microscopy demonstrated that VGLUT2 immunoreactivity was highly colocalized with synaptophysin and that both calretinin and tyramide amplified WGA-HRP in rat and mouse esophagus, respectively. No colocalization was found with calcitonin gene-related peptide, a marker for spinal primary afferents. Thus, VGLUT2 is found in vagal afferent endings in the esophagus, suggesting that glutamate is contained in, and probably released from, synaptic vesicles previously described in IGLEs. Functional evidence pending, this finding is in favor of a local effector function of IGLEs onto myenteric neurons.