EFFECT OF INSULIN ON LEVELS AND TURNOVER OF INTERMEDIATES OF BRAIN CARBOHYDRATE METABOLISM IN VIVO

Abstract— –The rates of incorporation of ¹⁴C from [U-^l4C]glucose into intermediary metabolites have been measured in rat brain in vivo. The time course of labelling of glycogen was similar to that of glutamate and of glucose, which were all maximally labelled between 20 and 40min, but different from lactate, which lost radioactivity rapidly after 20min. The extent of labelling of glycogen (d.p.m./ μ mol of glucose) was of the same order as that of glutamate at 20 and 40 min after injection of [¹⁴C]glucose. However, calculations of turnover rates showed that glutamate turns over some 8-10 times faster than glycogen. Insulin, intracisternally applied, produced after 4-5 h a 60 per cent increase in glucose-6-P and a 50 per cent increase in glycogen. There was no change in the levels of glucose, glutamate or lactate, nor in the activity or properties of the particulate and soluble hexokinase of the brain. The injection of insulin affected neither the glycogen nor glucose contents of skeletal muscle from the same animals. The effects of insulin on the incorporation of ^l4C into the metabolites contrasted with its effects on their levels. The specific activities of glycogen and glucose were unchanged and there was a slight but non-significant increase in the specific activity of glutamate. The time course of incorporation into lactate was unaffected up to 20 min, but a significant delay in the loss of ¹⁴C after 20 min occurred as a result of the insulin injection. At 40 min, the specific activity of cerebral lactate was 60 per cent higher in insulin-treated animals than in control animals. The results are interpreted in terms of an effect of insulin on glucose uptake to the brain, with possibly an additional effect on a subsequent stage in metabolism, which involves lactate.     

METABOLISM IN VIVO OF BRAIN GALACTOLIPIDS: THE JIMPY MUTANT

Abstract— The incorporation in vivo of [U-¹⁴C]glucose into the galactolipids of the brain of control and Jimpy mutant mice was examined. Over a 24-h period of incorporation there was no indication of an increased rate of turnover of brain galactolipids in the mutant. The Jimpy mutant was identified at ages prior to and at the inception of myelination (7–10 days post partum) with a coat marker (Tabby). There was similar total radioactivity in galactolipids of the Jimpy at these ages but a reduction to 13 per Cent of control at 13 days and to 6 per cent at 16 days of postnatal age. This devetopmental pattern of galactolipid synthesis in Jimpy brain is not in accord with a primary defect in the biosynthesis of cerebrosides and sulphatides.     

UPTAKE AND METABOLISM OF GLUTAMATE IN ASTROCYTES CULTURED FROM DISSOCIATED MOUSE BRAIN HEMISPHERES

Abstract— Uptake kinetics of l -glutamate in cultured, normal glia cells obtained from the brain hemispheres of newborn mice were measured together with the activities of the glutamate metabolizing enzymes, glutamic-oxaloacetate-transaminase, glutamate dehydrogenase and glutamine synthetase. During 3 weeks of culturing, the activities of the enzymes rose from low neonatal values toward the levels in the adult brain (206, 12.3 and 25.9 nmol. min⁻¹. mg⁻¹ cell protein for the three enzymes, respectively). The uptake kinetics indicated an unsaturable component together with an uptake following Michaelis-Menten kinetics with a K_m of 220 μ m and a V _max of 7.9 nmol. min⁻¹. mg⁻¹ cell protein. The saturable glutamate uptake was inhibited by d -glutamate, l -aspartate and α-aminoadipate whereas l -glutamine, GABA and glutarate had no effect. The uptake which was Ca²⁺-independent had a K_m for sodium of 18m m and it was stimulated by an increase in the external potassium concentration from 5 to 10 and 25 m m. The results suggest that glia cells are important for the uptake of glutamate from synaptic clefts and for the subsequent metabolism of glutamate.     

THE EFFECTS OF HYPERKETONEMIA ON GLUTAMATE AND GLUTAMINE METABOLISM IN DEVELOPING RAT BRAIN

Abstract— The effect of increased exposure to ketone bodies in the developing rat brain suggest that intrauterine and postnatal hyperketonemia lead to an altered metabolism of glutamine and glutamate. It is postulated that this effect is related to the delayed development of glutaminase ( l -glutamine amido-hydrolase EC 3.5.1.2) and glutamate dehydrogenase ( l -glutamate: NAD oxidoreductase EC 1.4.1.2).
The specific activities of glutamate dehydrogenase (GDH), glutaminase and glutamine synthetase ( l -glutamate: ammonia ligase EC 6.3.1.2) in the brains of newborn rats increased during early development. A positive correlation was observed between the specific activity of glutaminase and the concentration of glutamate in the brain as well as between the concentrations of blood and brain glutamine and glutamate in both control and hyperketonemic pups. This indicates a different degree of permeability and metabolism for glutamine and glutamate in the brain during the neonatal period, as compared to adulthood.
In hyperketonemic pups, glutamine and glutamate metabolism were found to differ from that in control animals. The concentrations of glutamate were higher, and glutamine lower, in both the blood and brain as compared to that in controls. The concentrations of α-ketoglutarate were also lower in their brain. In the brains of hyperketonemic and control pups, the concentration of malate was the same. During the first 3 weeks of life the increase of spec. act. of GDH and glutaminase was found to be suppressed in the brains of hyperketonemic pups. However, the spec. act. of glutamine synthetase was similar to that of the control pups.     

EFFECTS OF LITHIUM TREATMENT IN VITRO AND IN VIVO ON ACETYLCHOLINE METABOLISM IN RAT BRAIN

Abstract— The effects of LiCl on cholinergic function in rat brain in vitro and in vivo have been investigated. The high affinity transport of choline and the synthesis of acetylcholine in synaptosomes were reduced when part (25-75%) of the NaCl in the buffer was replaced with LiCl or sucrose. This appeared to be due to lack of Na⁺ rather than to Li⁺, as addition of LiCl to normal buffer had little effect. Following an injection of LiCl (10mmol/kg, i.p.) into rats the concentration of a pulsed dose of [²H₄]choline (20 μmol/kg, i.v., 1 min) and its conversion to [²H₄]acetylcholine, and the concentrations of [²H₂]acetylcholine and [²H₀]choline were measured in the striatum, cortex, hippocampus and cerebellum. The [²H₄]choline and [²H₄]acetylcholine were initially (15 min after LiCl) reduced (to ?30% in the cortex) and later (24 h after LiCl) increased (to + 50% in the striatum). There was a corresponding initial increase (to +50% in the cerebellum) and later decrease (to ?30% in the hippocampus) of the endogenous acetylcholine and choline. These results indicate an initial decrease and later increase in the utilization of acetylcholine after acute treatment with LiCl. Following 10 days of treatment with LiCl there was an increased rate of synthesis of [²H₄]acetylcholine from pulsed [²H₄]choline in the striatum, hippocampus and cortex (P < 0.05). The high affinity transport of [²H₄]choline and its conversion to [²H₄]acetylcholine was activated (131% of control; P < 0.01) in synaptosomes isolated from brains of 10-day treated rats. Investigation of synaptosomes isolated from striatum, hippocampus and cortex revealed that only striatal [²H₄]acetylcholine synthesis was significantly stimulated. Kinetic analysis demonstrated that the apparent K_T for choline was decreased by 30% in striatal synaptosomes isolated from rats treated for 10 days with LiCl. Striatal synaptosomes from 10-day treated rats compared to striatal synaptosomes from untreated rats also released acetylcholine at a stimulated rate in a medium containing 35 mM-KCl. These results indicate that LiCl treatment stimulates cholinergic activity in certain brain regions and this may play a significant role in the therapeutic effect of LiCl in neuropsychiatric disorders.     

PRECONVULSIVE CHANGES IN BRAIN GLUCOSE METABOLISM FOLLOWING DRUGS INHIBITING GLUTAMATE DECARBOXYLASE

—Brain glucose and glycogen concentrations have been studied in mice treated with allylglycine, 4-deoxypyridoxine and isoniazid, and the effects compared with the preconvulsive increase in brain glucose and glycogen concentration that follows d , l -methionine sulphoximine treatment. Allylglycine (180 mg/kg), 4-deoxypyridoxine (250 mg/kg), isoniazid (150 mg/kg) and d ,l -methionine sulphoximine (300 mg/kg) when given to mice at room temperature, cause a fall in rectal temperature which can be prevented by maintaining the mice in an incubator at 33-34°C. An increase in brain glucose concentration is seen after allylglycine (+ 133%), d ,l -methionine sulphoximine (+ 113%) and 4-deoxypyridoxine (+ 70%) treatment when mice are kept at room temperature and killed before convulsions occur. This is associated with a rise in blood glucose concentration after allylglycine, but not after the other drugs. Preventing the fall in rectal temperature reduces, but does not abolish, the rise in brain glucose concentration seen after allylglycine, d ,l -methionine sulphoximine and 4-deoxypyridoxine. Brain glycogen concentration increases at room temperature after D,L-methionine sulphoximine and 4-deoxypyridoxine, but in mice with maintained body temperature only 4-deoxypyridoxine produces an increase in brain glycogen. Isoniazid does not increase brain glucose or glycogen at room temperature, but reduces their concentration in mice kept in the incubator. All four drugs are known to act on amino acid metabolism; d ,l -methionine sulphoximine potently inhibits glutamine synthetase whereas 4-deoxypyridoxine, allylglycine and isoniazid inhibit glutamate decarboxylase. The connection, if any, between a block in the further metabolism of glutamate and an increase in brain glucose and glycogen is unknown.     

THE LABELLING OF NERVE ENDING PHOSPHOLIPIDS IN GUINEA-PIG BRAIN IN VIVO AND THE EFFECT OF ELECTRICAL STIMULATION ON PHOSPHATIDYLINOSITOL METABOLISM IN PRELABELLED SYNAPTOSOMES

The intracerebral injection of ³²P_i into guinea-pig cortex resulted in a steady rate of incorporation into all phospholipids over a 20 h period. The specific radioactivities of phosphatidate and phos-phatidylinositol in synaptosomes prepared from cortex prelabelled, in vivo, were at a maximum after 2 h and the respective activities were 3–8 times higher than in whole cortex. This peak in labelling corresponded with the maximum specific activity of the brain ATP. No similar differential labelling pattern was observed for phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine. Electrical stimulation of the prelabelled synaptosomes produced a rapid drop in the specific activity of phosphatidylinositol and phosphatidate and an increase in the specific activity of CDP-diacylglycerol. The specific activity of synaptosomal ATP was not affected. Study of the subsynaptosomal fractions obtained after osmotic rupture of the synaptosomes revealed that the most highly labelled phosphatidylinositol was in the synaptic vesicle fraction (D) and the most active phosphatidate was in a ‘microsomal’ fraction (E). Electrical stimulation caused a loss of phosphatidylinositol radioactivity from fraction D and a loss of phosphatidate radioactivity from fraction E. The specific activity of these lipids in other fractions was not affected. A possible role for presynaptic phosphatidylinositol is suggested.     

EFFECT OF COPPER STATUS ON BRAIN NEUROTRANSMITTER METABOLISM IN THE LAMB

Abstract— Ataxic and non-ataxic lambs reared under field conditions which gave rise to low copper status were treated with copper intravenously. Untreated ataxic animals served as controls. The neurotransmitter amines, dopamine, norepinephrine and serotonin, were determined in the anterior and posterior regions of the brain stem. Dopamine levels in the anterior region, including the corpus striatum, were significantly lower in the untreated animals than in those treated with copper. Norepinephrine levels were also lower but serotonin concentrations were not different. Plasma amine oxidase activity was markedly higher in the copper treated animals but monoamine oxidase activity in brain stem homogenates was not significantly affected. The monoamine oxidase activity in cortical and cerebellar homogenates was significantly lower in the treated animals than in the untreated animals.     

IN VIVO METABOLISM OF [G-3H]LIGNOCERIC ACID IN DEVELOPING RAT BRAIN1

Abstract— [G-³H]Lignoceric acid (tetracosanoic acid) was injected into the brains of 20-day-old rats, and the animals were killed after 8, 24, or 72 h. Various lipids were isolated from these brains, and the distribution of radioactivity was determined. The injected free acid rapidly disappeared, and the radioactivity was incorporated into varying chain-length nonhydroxy- and hydroxy saturated fatty acids of sphingolipids and phospholipids. Little radioactivity was found in unsaturated acids, sphingo-sine, and cholesterol. A time-dependent shift of the label among various fatty acids was relatively small 8 h after injection, probably because of the metabolic stability of the brain sphingolipids. In cerebrosides, the radioactivity was equally distributed between nonhydroxy and x-hydroxy fatty acids of all chain lengths. C₂₃ and C₂₂ fatty acids contained equal total radioactivities; C₂₃ and C₂₄ fatty acids contained similar specific activities. These results confirm the significant role of a-hydroxylation and 2-oxidation in the synthesis of very long-chain fatty acids in brain. In total lipid fatty acids, docosanoic acid (22:0) contained more radioactivity than its α-oxidation precursor, α-hydroxytricosanoic acid (23h:0) at all times. In sphingolipid fatty acids, the specific activity of 21:0 was always higher than that of its ct-oxidation precursor 22:0. These observations indicate that part of the 22:0 and 21:0 was derived by β-oxidation from the injected lignoceric acid or its α-oxidation product, respectively.     

FLOW IN VIVO OF GLUCOSE CARBON TO BRAIN PROTEIN IN RATS: EFFECT OF STARVATION

—The conversion of plasma glucose into brain proteins in vivo was measured in rats after various periods of food deprivation. Rates of flow of glucose carbon into both soluble and insoluble brain proteins were calculated from the curve representing the decrease of plasma [¹⁴C]-glucose specific activity with time, and from the specific activity of brain protein 180 min after intravenous injection of a tracer dose of d -[¹⁴C]-glucose. Compared to the post-absorptive rats, food deprivation for 72 h caused a 30 per cent reduction in the rate of flow of glucose carbon into soluble brain proteins but did not affect the flow into insoluble proteins. Results of experiments in which the soluble brain proteins were separated by isoelectric focusing suggest that prolonged fasting in adult rats causes substantial differences in the conversion of glucose to different proteins.     

CHOLINERGIC NERVE ENDINGS IN OCTOPUS BRAIN

—Optic lobes of the brains of Octopus dofleini werehomogenized hasaline–sucrose medium and subjected to differential and density gradient centrifugation. The fractions and subfractions thus obtained were analysed for acetylcholine (ACh) (bioassay) and protein content and were subjected to electronmicroscopy. Bound ACh was associated with particulate fractions, and a large portion of it could be recovered in subfractions that contained predominantly nerve endings. Expressed in terms of amount of ACh per mg protein, the ACh content of the nerve ending fractions was nearly 100 times greater than that of corresponding fractions previously obtained by others from mammalian brain. Calculations show that this was the minimal amount of ACh to be expected if the isolated nerve endings were predominantly cholinergic. Octopus brain tissue is in therefore very promising for future studies on ACh metabolism and compartmentation cholinergic nerve endings.     

IDENTIFICATION OF D-ASPARTIC ACID IN THE BRAIN OF OCTOPUS VULGARIS LAM

Abstract— An assay method based on the use of specific oxidases has been used to search for D-amino acids in the organs of Octopus vulgaris . A high concentration of D-aspartate has been detected in the central brain and in the optic lobes but not in non-nervous organs.
Other D-amino acids appear to be absent from brain and from other tissues.     

褪黑素对谷氨酸钠致痫大鼠脑内一氧化氮含量的影响

目的观察褪黑素(Melatonin,MT)对谷氨酸钠致痫大鼠脑内一氧化氮(nitric oxide,NO)含量的影响,研究其抑制癫痫的作用机制。方法40只健康雄性SD大鼠随机分为4组(每组10只):生理盐水对照组(NS组);谷氨酸钠致痫组(Glu组);褪黑素+谷氨酸钠组(MT+Glu组);Luzidole+褪黑素+谷氨酸组(Luz+MT+Glu组)。观察大鼠行为变化,记录脑电图,用NADPH组织化学反应检测大鼠海马内NO含量变化。结果行为学观察和EEG显示,NS组无痫样发作和痫样放电,Glu组和Luz+MT+Glu组痫样发作重(Ⅲ-Ⅴ级),脑电图显示频发高幅的痫样波,MT+Glu组有轻微发作(0-Ⅱ级),脑电图上偶见散在单个微小痫样波;NADPH组织化学反应结果显示,Glu组和Luz+MT+Glu组大鼠大脑皮质及海马内NOS阳性细胞与对照组比较增多,差异性明显(P<0.05),MT+Glu组较Glu组和Luz+MT+Glu组内NOS阳性细胞减少,差异性明显(P<0.05)。结论MT对谷氨酸钠致痫大鼠痫样发作程度、痫样放电有抑制作用,其机制之一是经其特异性受体,减弱NO作用,进而发挥抑痫效应。     

THE EFFECT OF dl-ALLYLGLYCINE ON POLYAMINE AND GABA METABOLISM IN MOUSE BRAIN

Abstract— dl -Allylglycine, a potent inhibitor of glutamate decarboxylase in vivo when given intraperitoneally, causes a marked decrease in brain GABA concentration and at the same time a dramatic increase in l -ornithine decarboxylase activity and a simultaneous decrease in S -adenosyl- l -methionine decarboxylase activity followed by putrescine accumulation. It does not, however, alter the degree of GABA formation from putrescine. The timing of the recovery of glutamate decarboxylase activity after the injection of dl -allylglycine is concomitant with that of the GABA concentration, indicating that it is probably glutamate decarboxylase that is solely responsible for making up the GABA deficit caused by dl -allylglycine, and that the changes in polyamine metabolism are associated in some indirect way with the recovery process.