The Interaction of Transport and Metabolism on Brain Glucose Utilization: A Reevaluation of the Lumped Constant

Abstract: The relative cerebral cortical metabolism of glucose (GLU) and 2-deoxy-D-glucose (DG) was measured in vivo in control and insulin-treated hypoglycemic rats. The ratio of the utilization rate constants for the two hexoses, i.e., K _DG/ K _CLU is defined as the Hexose Utilization Index (HUI). The HUI was found to be invariant in rats whose cerebral glucose content exceeded 1 μmo1.g⁻¹ wet weight (HUI = 0.48 ± 0.07). Severe hypoglycemia (plasma glucose <2 mM) effected a shift in the HUI to 1.04 ± 0.21. The results are consistent with a model in which the interpretation of the HUI is determined by the rate of transport into brain, or subsequent phosphorylation, as the rate-limiting step for hexose utilization.     

No Effect of Hyperketonemia on Local Cerebral Glucose Utilization in Conscious Rats

Abstract: Local cerebral glucose utilization was measured by the [¹⁴C]2-deoxy- d -glucose method in conscious control and hyperketonemic rats. Hyperketonemia was induced by 3 days of starvation or by infusion of 3- hydroxybutyrate in fed rats. These treatments produced combined blood ketone body concentrations (acetoacetate + 3-hydroxybutyrate) of from 1.2 to 2.4 mM. Neither treatment significantly affected glucose utilization in any of the 15 brain regions studied. These observations indicate that hyperketonemia in resting, conscious rats does not interfere with brain uptake and phosphorylation of glucose.     

Simultaneous Measurement of Nitrite and Nitrate Levels as Indices of Nitric Oxide Release in the Cerebellum of Conscious Rats

Abstract: We examined the modulation of nitric oxide production in vivo by measuring levels of nitrite (NO₂⁻) and nitrate (NO₃⁻) in the dialysate of the cerebellum in conscious rats, by using an in vivo brain microdialysis technique. The levels of both NO₂⁻ and NO₃⁻ were decreased by the intraperitoneal injection of N ^G-nitro- l -arginine methyl ester, an inhibitor of nitric oxide synthase, whereas N ^G-nitro- d -arginine methyl ester had no effect. l -Arginine by itself increased NO₂⁻ and NO₃⁻ levels and diminished the reduction of their levels caused by N ^G-nitro- l -arginine methyl ester. Direct infusion of l -glutamate, N -methyl- d -aspartate, or KCl into the cerebellum through a dialysis probe resulted in an increase in NO₂⁻ and/or NO₃⁻ levels. The effects of N -methyl- d -aspartate and KCl were dependent on extracellular calcium. Furthermore, the stimulatory effects of l -glutamate and N -methyl- d -aspartate were inhibited by N ^G-nitro- l -arginine methyl ester and (±)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), an N -methyl- d -aspartate receptor antagonist. These results suggest that NO₂⁻ and NO₃⁻ levels may be related to nitric oxide production in vivo.     

Increased Nitric Oxide Synthase Activities and l-[3H]Arginine Uptake in Brain Following Portacaval Anastomosis

Abstract: Glutamatergic synaptic dysfunction has been proposed as a causal factor in portal-systemic encephalopathy. Increased in vitro and in vivo glutamate release and decreased glutamate binding to NMDA receptors were previously reported in the brains of portacaval-shunted rats. Such changes could lead to alterations in the second messenger systems coupled to glutamate receptors. As NMDA receptors have been shown to act via the nitric oxide/cyclic GMP second messenger system, we studied the activities of constitutive nitric oxide synthase (NOS), in the brains of rats following portacaval shunting. Results demonstrate that NOS activities are significantly increased in cerebellum (by 54%, p < 0.01), cerebral cortex (by 65%, p < 0.01), hippocampus (by 88%, p < 0.01), and striatum (by 64%, p < 0.01) of shunted rats compared with sham-operated controls. As l -arginine transport is a prerequisite for nitric oxide production, we also studied l -[³H]arginine transport into cerebellar and cerebral cortical synaptosomes prepared from the brains of portacaval-shunted and sham-operated rats. l -[³H]Arginine uptake was significantly increased (by ∼50%, p < 0.01) in both cerebellum and cortex. Increased NOS activities of neuronal and/or astrocytic origin and the resultant increased production of nitric oxide in brain could be the consequence of increased NMDA receptor activation following portacaval shunting. Furthermore, increased nitric oxide production could contribute to the increased cerebral blood flow consistently observed following portacaval shunting.     

MEASUREMENT OF THE RATE OF GLUCOSE UTILIZATION BY RAT BRAIN IN VIVO

Abstract— A method is described by which the rate of glucose utilization by whole brain of conscious rats may be measured. The basis is the uptake of ¹⁴C derived front [2-¹⁴C] glucose into the acid-soluble metabolite pool of brain. Catheters are placed in the femoral artery and vein under light ether anesthesia. After full recovery of consciousness a single intravenous injection of [2-¹⁴C] glucose is given and arterial blood samples taken at intervals. Simultaneous with the last sample the brain is removed and frozen within 1 s. The accumulation of ¹⁴C into the acid-soluble metabilite pool is measured and the rate of glucose utilization is calculated according to the equation:

The integral is calculated from the plasma glucose specific activity curve and evidence is presented to justify this procedure. The rate of glucose utilization measured by this method was 0·62 μmol/min per g in conscious rats and 0·28 μmol/min per g in sodium pentobarbital anesthetized rats.     

Free Fatty Acids in the Rat Brain in Moderate and Severe Hypoxia

Abstract: The effects of mild, moderate, and severe hypoxia on cerebral cortical concentrations of free fatty acids (FFAs) were investigated in artificially ventilated rats under nitrous oxide anaesthesia. No change occurred during either mild (arterial Po₂ 35–40 mm Hg) or moderate (Po₂ 25–30 mm Hg) hypoxia. The effects of severe hypoxia (Po₂ about 20 mm Hg) combined with hypotension (mean arterial blood pressure 80–85 mm Hg) varied with the EEG pattern and the tissue energy state. Thus, a major increase in total as well as in individual FFAs occurred first when EEG was severely depressed (almost isoelectric) and energy homeostasis disrupted. On a relative basis the greatest change occurred in free arachidonic acid. It is concluded that hypoxia is associated with an increase in the concentrations of FFAs in brain tissue, provided that tissue oxygen deficiency is severe enough to cause tissue energy failure. However, an increase in FFAs does not invariably accompany minor reductions in the adenylate energy charge (EC) of the tissue.     

Effect of aluminum on the blood-brain barrier permeability during nitric oxide-blockade-induced chronic hypertension in rats

We examined the effect of aluminum on the permeability of the blood-brain barrier (BBB) during nitric oxide-blockade-induced chronic hypertension in rats. Animals were given the inhibitor of nitric oxide synthase, l-NAME (N ^ω-nitro-l-arginine methyl ester), for 4 wk to induce chronic hypertension. Two groups of rats were given an intraperitoneal injection of aluminum chloride. The integrity of the BBB was assessed by a quantitative measurement for Evans blue (EB) dye. The arterial blood pressure in l-NAME- and l-NAME plus aluminum-treated animals was significantly elevated from 115±2.8 and 110±1.7 mm Hg to 174±5.2 and 175±4.8 mm Hg, respectively (p<0.01). The EB dye content in the brain regions of the rats in the l-NAME group was increased, but there was no statistical significance compared to the saline group. The extravasation of EB dye was significantly increased in the brain regions of the animals treated with aluminum compared to the rats treated with saline (p<0.05). A significantly higher EB dye content in the brain regions was observed in the l-NAME plus aluminium group compared to l-NAME, aluminum, and saline groups (p<0.01). These findings indicate that exposure to a high level of aluminum leads to an additional increase in BBB permeability where nitric oxide-blockade-induced chronic hypertension potentiates the effect of aluminum to enhance BBB permeability to EB dye.     

Cerebral Cortical Glucose Utilization in the Conscious Rat: Evidence for a Circadian Rhythm

Abstract: The presence of a circadian rhythm of glucose utilization was demonstrated in vivo in rat cerebral cortex. The activity pattern of the rats, living in a controlled lighting regimen with lights on from 7 a.m. to 7 p. m., appeared to coincide with the rate of glucose consumption in the brain. The rate of utilization was measured at 3-h intervals throughout the day and was found to fall from a maximum at 3 a.m. of 0.98 ± 0.13 μmol min⁻¹ g⁻¹ to a minimum of 0.70 ± 0.08 μmol min⁻¹ g⁻¹ at 3 p. m. Brain glucose also varied with time and its fluctuating level weakly correlated with its rate of utilization. Animals entrained on a 5-h (4: 30-9: 30 p. m.) feeding schedule had a similar circadian rhythm, with only a slight increase in amplitude. Reversal of the light cycle caused a disruption in the normal rhythm, but utilization still varied significantly with time of day. The results both indicate the potential error that can be encountered in experiments done at different times of the day and stress the need for awareness of time of day as a factor in measurements of alterations of metabolic rate in the brain.     

The Effects of Apomorphine upon Local Cerebral Glucose Utilization in Conscious Rats and in Rats Anesthetized with Chloral Hydrate

The effects of the dopaminergic agonist apomorphine (1 mg . kg-1 i.v.) upon local cerebral glucose utilization in 43 anatomically discrete regions of the CNS were examined in conscious, lightly restrained rats and in rats anesthetized with chloral hydrate by means of the quantitative autoradiographic [14C]2-deoxyglucose technique. In animals anesthetized with chloral hydrate, glucose utilization was reduced throughout all regions of the CNS from the levels observed in conscious animals, although the magnitude of the reductions in glucose use displayed considerable regional heterogeneity. With chloral hydrate anesthesia, the proportionately most marked reductions in glucose use (by 40-60% from conscious levels) were noted in primary auditory nuclei, thalmaic relay nuclei, and neocortex, and the least pronounced reductions in glucose use (by 15-25% from conscious levels) were observed in limbic areas, some motor relay nuclei, and white matter. In conscious, lightly restrained rats, the administration of apomorphine (1 mg . kg-1) effected significant increased in glucose utilization in 15 regions of the CNS (e.g., subthalamic nucleus, ventral thalamic nucleus, rostral neocortex, substantia nigra, pars reticulata), and significant reductions in glucose utilization in two regions of the CNS (lateral habenular nucleus and anterior cingulate cortex). In rats anesthetized with chloral hydrate, the effects of apomorphine upon local glucose utilization were less widespread and less marked than in conscious animals. In only two of the regions (the globus pallidus and septal nucleus), which displayed increased glucose use following apomorphine in conscious rats, were significant increases in local glucose utilization observed with this agent in chloral hydrate-anesthetized rats. In the pars compacta of the substantia nigra, in which apomorphine increased glucose utilization in conscious animals, significant reductions in glucose utilization were observed following apomorphine in rats anesthetized with chloral hydrate. The profound effects of chloral hydrate anesthesia upon local cerebral glucose use, and the modification by this anesthetic regime of the local metabolic responses to apomorphine, emphasize the difficulties which exists in the extrapolation of data from anesthetized animals to the conditions which prevail in the conscious animal.     

Mechanisms Involved in the Neuroprotective Activity of a Nitric Oxide Synthase Inhibitor During Focal Cerebral Ischemia

Abstract: We have reported previously that posttreatment with N ^G-nitro-L-arginine methyl ester (L-NAME), an inhibitor of the nitric oxide synthase, reduced the volume of cortical and striatal infarct induced by middle cerebral artery occlusion in rats. In the present study, we investigated the mechanisms by which L-NAME (3 mg/kg i.p.) is neuroprotective in this model of cerebral ischemia. First, we have shown the reversal of the neuroprotective effect of L-NAME by a coinjection of L-arginine. Second, in order to determine by which mechanism nitric oxide exacerbates neuronal damage produced by focal cerebral ischemia, we studied the effect of the inhibition of nitric oxide synthase by L-NAME on the histological consequences of a focal injection of N -methyl-D-aspartate (NMDA) in the striatum, and on the striatal overflow of glutamate and aspartate induced either by K⁺ depolarization or by focal cerebral ischemia. We have found that L-NAME treatment reduced the excitotoxic damage produced by NMDA injection. By using microdialysis, we have shown that the K⁺- and the ischemia-induced glutamate efflux was reduced by 52 and 30%, respectively, after the L-NAME treatment. These results indicate that nitric oxide synthesis induced by the NMDA receptor overstimulation is one of the major events leading to neuronal damage. One possible mechanism by which nitric oxide may contribute to the excitotoxic process is by facilitating the ischemia-induced glutamate overflow.     

Nitroarginine,an inhibitor of nitric oxide synthetase,attenuates ammonia toxicity and ammonia-induced alterations in brain metabolism

We have proposed that acute ammonia toxicity is mediated by activation of the N-methyl-D-aspartate type of glutamate receptors. MK-801, a selective antagonist of these receptors, prevents death of animals induced by acute ammonia intoxication as well as ammonia-induced depletion of ATP. It seems therefore that, following activation of the N-methyl-D-aspartate receptors, the subsequent events in ammonia toxicity should be similar to those involved in glutamate neurotoxicity. As it has been shown that inhibitors of nitric oxide synthetase such as nitroargnine prevent glutamate toxicity, we have tested whether nitroarginine prevents ammonia toxicity and ammonia-induced alterations in brain energy and ammonia metabolites. It is shown that nitroarginine prevents partially (50%), but significantly death of mice induced by acute ammonia intoxication. Nitroarginine also prevents partially ammonia-induced depletion of brain ATP. It also prevents completely the rise in glucose and pyruvate and partially that in lactate. Injection of nitroarginine alone, in the absence of ammonia, induces a remarkable accumulation of glutamine and a decrease in glutamate. The results reported indicate that nitroarginine attenuates acute ammonia toxicity and ammonia-induced alterations in brain energy metabolites. The effects of MK-801 and of nitroarginine are different, suggesting that ammonia can induce nitric oxide synthetase by mechanisms other than activation of N-methyl-D-aspartate receptors.     

The effect of the calcium antagonist nimodipine upon local cerebral glucose utilization in the rat brain

The new calcium antagonist Nimodipine has been shown to have more powerful dilator action on cerebral than peripheral vessels. The effect of the drug on cerebral metabolism was studied in conscious rats using the /14C/-2-deoxyglucose quantitative autoradiographic technique. Intravenous injection of Nimodipine, 2 mcg/Kg, determined significant increases in local cerebral glucose utilization that appeared to be homogeneous in magnitude and anatomic distribution throughout the brain. This study raises the question whether Nimodipine affects brain functions by other mechanisms than an increase in cerebral blood flow.     

Glucose Metabolism in the Developing Cerebral Cortex as Detected by 1H{13C} Nuclear Magnetic Resonance Spectroscopy Ex Vivo

Abstract: Metabolism of [1-¹³C]glucose was monitored in superfused cerebral cortex slice preparations from 1-, 2-, and 5-week-old rats using ¹H-observed/¹³C-edited (¹H{¹³C}) NMR spectroscopy. The rate of label incorporation into glutamate C-4 did not differ among the three age groups: 0.52–0.67% of total ¹H NMR-detected glutamate/min. This was rather unexpected, as oxygen uptake proceeded at 1.1 ± 0.1, 1.9 ± 0.1, and 2.0 ± 0.1 µmol/min/g wet weight in brain slices prepared from 1-, 2-, and 5-week-old animals, respectively. Steady-state glutamate C-4 fractional enrichments in the slice preparations were ∼23% in all age groups. In the acid extracts of slices glutamate C-4 enrichments were smaller, however, in 1- and 2-week-old (17.8 ± 1.7 and 16.8 ± 0.8%, respectively) than in 5-week-old rats (22.7 ± 0.7%) after 75 min of incubation with 5 m M [1-¹³C]glucose. We add a new assignment to the ¹H{¹³C} NMR spectroscopy, as acetate C-2 was detected in slice preparations from 5-week-old animals. In the acid extracts of slice preparations acetate C-2 was labeled by ∼30% in 5-week-old rats but by 15% in both 1- and 2-week-old animals, showing that the turnover rate was increased in 5-week-old animals. In the extracts 3–4% of the C-6 of N -acetyl-aspartate (NAA; CH₃ of the acetyl group) contained label as determined by both NMR and mass spectrometry, which indicated that there was no significant labeling to other carbons in NAA. NAA accumulated label from [1-¹³C]glucose but not from [2-¹³C]acetate, and the rate of label incorporation increased by threefold on cerebral maturation.     

α-Guanidinoglutaric Acid, an Endogenous Convulsant, as a Novel Nitric Oxide Synthase Inhibitor

Abstract: The effects of α-guanidinoglutaric acid (GGA), the levels of which were increased in the cobalt-induced epileptic focus tissue in the cerebral cortex of cats, on brain nitric oxide synthase (NOS) activity were observed. GGA inhibited NOS activity in a linear mixed manner ( K _i = 2.69 µ M ) and was as effective as N ^G-monomethyl- l -arginine (MeArg; K _i = 3.51 µ M ), a well-known NOS inhibitor. Although MeArg was synthesized by substituting the guanidino nitrogen of l -arginine (Arg), GGA was a non-guanidino nitrogen-substituted guanidino compound. On the other hand, Arg, which is an endogenous NOS substrate, elevates the threshold of seizures induced by GGA. There is evidence that GGA is an endogenous, potent, and non-guanidino nitrogen-substituted NOS inhibitor and that suppression of nitric oxide biosynthesis may be involved in GGA-induced convulsions. Therefore, GGA may be a useful tool in elucidating the chemical nature of NOS and the physiological function of nitric oxide.     

Influence of an extract of Ginkgo biloba on cerebral blood flow and metabolism

The purpose of the present investigation was to examine the effects of an extract of Ginkgo biloba (EGB) on blood glucose levels, on local cerebral blood flow as well as on cerebral glucose concentration and consumption. The local cerebral blood flow (LCBF) was measured in conscious rats by means of the 14C-iodoantipyrine technique and local cerebral glucose utilization (LCGU) by 14C-2-deoxy-glucose autoradiography. EGB increased the LCBF in 39 analyzed, anatomically defined brain structures by 50 to 100 per cent. No influence of EGB on LCGU was demonstrable. However, EGB enhanced the blood glucose level dose-dependently. Substrates and metabolites of energy metabolism were measured in the cortex of the isolated rat brain perfused at constant rate and with 7 mmol/l glucose added to the perfusion medium. In these experiments EGB decreased the cortical glucose concentration without other substrate levels being changed. These results suggest that glucose uptake may be inhibited by EGB. It is argued that the effects of EGB on brain glucose concentration and blood flow may contribute to its protection of brain tissue against ischemic or hypoxic damage.     

Possible Role of Nitric Oxide in Catecholamine Secretion by Chromaffin Cells in the Presence and Absence of Cultured Endothelial Cells

Abstract: We studied the effect of cultured endothelial cells on the secretion of catecholamines by cultured bovine chromaffin cells. Chromaffin cell catecholamine secretion was stimulated by either boluses of potassium (K⁺) or the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP). Endothelial cells inhibited the catecholamine release and stimulatory effects of K⁺ and DMPP. This inhibition increased with time, and in 25 min the initial stimulated secretory response (100%) to 30 m M K⁺ or 25 μ M DMPP dropped to 45 ± 3% and 53.5 ± 2.3%, respectively. This endothelial cell-induced inhibition was blocked by the nitric oxide synthase inhibitors N -nitro- l -arginine methyl ester ( l -NAME) and N -monoethyl- l -arginine ( l -NMMA), and by the guanylate cyclase inhibitor methylene blue, indicating that the l -arginine/nitric oxide/ cyclic GMP pathway is involved in this endothelial cell-chromaffin cell interaction. In the absence of endothelial cells, incubation of chromaffin cells with l -NAME, l -NMMA, or methylene blue also augmented the secretagogue-induced catecholamine secretion, indicating that nitric oxide from chromaffin cells could be implicated in an autoinhibitory process of catecholamine release. These results provide indirect evidence for the presence of nitric oxide synthase in bovine adrenomedullary chromaffin cells. Our results show that there is an autoinhibitory mechanism of catecholamine release in chromaffin cells and that an additional level of inhibition is observed when cultured vascular endothelial cells are present. These two inhibitory processes may have different origins, but they appear to converge into a common pathway, the l -arginine/nitric oxide synthase/guanylate cyclase pathway.     

Modulation of Glutamine Synthesis in Cultured Astrocytes by Nitric Oxide

1. Previous results suggest that glutamine synthesis in brain could be modulated by nitrix oxide. The aim of this work was to assess this possibility.2. As glutamine synthetase in brain is located mainly in astrocytes, we used primary cultures of astrocytes to assess the effects of increasing or decreasing nitrix oxide levels on glutamine synthesis in intact astrocytes.3. Nitric oxide levels were decreased by adding nitroarginine, an inhibitor of nitric oxide synthase. To increase nitric oxide we used S-nitroso-N-acetylpenicillamine, a nitric oxide generating agent.4. It is shown that S-nitroso-N-acetylpenicillamine decreases glutamine synthesis in intact astrocytes by 40–50%. Nitroarginine increases glutamine synthesis slightly in intact astrocytes.5. These results indicate that brain glutamine synthesis may be modulated in vivo by nitric oxide.     

Endothelial nitric oxide synthase activation contributes to post-exercise hypotension in spontaneously hypertensive rats

We investigated the role that endothelial nitric oxide synthase plays in post-exercise hypotension in spontaneously hypertensive rats. To accomplish this, rats were subjected to a single bout of dynamic exercise on a treadmill at 15 m/min for 20 min. l-Nitroarginine methyl ester (l-NAME, 40 mg/kg, i.p.) significantly inhibited post-exercise hypotension (25 ± 11 and 5 ± 3 mm Hg, respectively; P < 0.05). In addition, the superoxide anion generation was decreased, while the plasma nitrite production and serine phosphorylation of endothelial nitric oxide synthase were significantly elevated in spontaneously hypertensive rats at 30 min after the termination of exercise. Taken together, these data demonstrate that the increased phosphorylation of endothelial nitric oxide synthase plays a crucial role in the reduction of arterial pressure following a single bout of dynamic exercise in spontaneously hypertensive rats.     

Endothelin-Stimulated Capacitative Calcium Entry in Enteric Glial Cells: Synergistic Effects of Protein Kinase C Activity and Nitric Oxide

Abstract: Depletion of intracellular calcium stores by agonist stimulation is coupled to calcium influx across the plasma membrane, a process termed capacitative calcium entry. Capacitative calcium entry was examined in cultured guinea pig enteric glial cells exposed to endothelin 3. Endothelin 3 (10 n M ) caused mobilization of intracellular calcium stores followed by influx of extracellular calcium. This capacitative calcium influx was inhibited by Ni²⁺ (89 ± 2%) and by La³⁺ (78 ± 2%) but was not affected by L-, N-, or P-type calcium channel blockers. Chelerythrine, a specific antagonist of protein kinase C, dose-dependently inhibited capacitative calcium entry. The nitric oxide synthase inhibitor N ^G-nitro- l -arginine decreased calcium influx in a dose-dependent manner. The combination of chelerythrine and N ^G-nitro- l -arginine produced synergistic inhibitory effects. Capacitative calcium entry occurs in enteric glial cells via lanthanum-inhibitable channels through a process regulated by protein kinase C and nitric oxide.