Oral branched-chain amino acid supplements that reduce brain serotonin during exercise in rats also lower brain catecholamines

Exercise raises brain serotonin release and is postulated to cause fatigue in athletes; ingestion of branched-chain amino acids (BCAA), by competitively inhibiting tryptophan transport into brain, lowers brain tryptophan uptake and serotonin synthesis and release in rats, and reputedly in humans prevents exercise-induced increases in serotonin and fatigue. This latter effect in humans is disputed. But BCAA also competitively inhibit tyrosine uptake into brain, and thus catecholamine synthesis and release. Since increasing brain catecholamines enhances physical performance, BCAA ingestion could lower catecholamines, reduce performance and thus negate any serotonin-linked benefit. We therefore examined in rats whether BCAA would reduce both brain tryptophan and tyrosine concentrations and serotonin and catecholamine synthesis. Sedentary and exercising rats received BCAA or vehicle orally; tryptophan and tyrosine concentrations and serotonin and catecholamine synthesis rates were measured 1 h later in brain. BCAA reduced brain tryptophan and tyrosine concentrations, and serotonin and catecholamine synthesis. These reductions in tyrosine concentrations and catecholamine synthesis, but not tryptophan or serotonin synthesis, could be prevented by co-administering tyrosine with BCAA. Complete essential amino acid mixtures, used to maintain or build muscle mass, were also studied, and produced different effects on brain tryptophan and tyrosine concentrations and serotonin and catecholamine synthesis. Since pharmacologically increasing brain catecholamine function improves physical performance, the finding that BCAA reduce catecholamine synthesis may explain why this treatment does not enhance physical performance in humans, despite reducing serotonin synthesis. If so, adding tyrosine to BCAA supplements might allow a positive action on performance to emerge.     

Acute effects of caffeine injection on neutral amino acids and brain monoamine levels in rats

The injection of caffeine (100 mg/kg, i.p.) into male rats acutely increased brain levels of trytophan, serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA). Blood levels of glucose, nonesterified fatty acids (NEFA) and insulin also increased, while those of the aromatic and branched-chain amino acids fell. Serum tryptophan levels either did not fall, or increased. Consequently, the serum ratio of trypthopahn to the sum of other large neutral amino acids (LNAA) increased. Less consistently noted were increases in serum free tryptophan levels. Brain tyrosine levels were not appreciably altered by caffeine, nor was the serum tyrosine ratio. In dose-response studies, 25 mg/kg of caffeine was the minimal effective dose needed to raise brain tryptophan, but only the 100 mg/kg dose elevated all three indoles in brain. In no experiments did caffeine, at any time or dose, alter brain levels of dopamine or norepinephrine. Caffeine thus probably raises brain tryptophan levels by causing insulin secretion, and thereby changing plasma amino acid levels to favor increased tryptophan uptake into brain. The rises in brain 5-HT and 5-HIAA may follow from the increase in brain tryptophan, although further data are required clearly to establish such a mechanism.     

BRAIN TYROSINE HYDROXYLATION: ALTERATION OF OXYGEN AFFINITY IN VIVO BY IMMOBILIZATION OR ELECTROSHOCK IN THE RAT

Abstract— The rates of brain tyrosine and tryptophan hydroxylation, estimated in vivo from the accumulation of DOPA and 5-hydroxytryptophan after the administration of a decarboxylase inhibitor, appear dependent on the availability of oxygen as a substrate. During two types of physical stress, electroshock and curare-immobilization, the rate of brain tyrosine hydroxylation was greater than in unstressed controls and was not significantly decreased when the stresssed animals were made hypoxic. The loss of oxygen dependence by brain tyrosine hydroxylation during stress was observed in several brain regions and was not associated with alterations in the concentrations of brain tyrosine. tryptophan, serotonin, dopamine or norepinephrine. The rate of brain tryptophan hydroxylation was not affected by stress and remained oxygen dependent. The increase in catecholamine synthesis during stress appears to be the result of increased catecholaminergic nerve impulse flow. These experiments are consistent with the hypothesis that during neuronal stimulation an allosteric change in tyrosine hydroxylase increases the affinity of the enzyme for oxygen allowing greater catecholamine synthesis despite limiting concentrations of this substrate.     

Amino acids and central fatigue

Summary. There is an increasing interest in the mechanisms behind central fatigue, particularly in relation to changes in brain monoamine metabolism and the influence of specific amino acids on fatigue. Several studies in experimental animals have shown that physical exercise increases the synthesis and metabolism of brain 5-hydroxytryptamine (5-HT). Support for the involvement of 5-HT in fatigue can be found in studies where the brain concentration of 5-HT has been altered by means of pharmacological agents. When the 5-HT level was elevated in this way the performance was impaired in both rats and human subjects, and in accordance with this a decrease in the 5-HT level caused an improvement in running performance in rats. The precursor of 5-HT is the amino acid tryptophan and the synthesis of 5-HT in the brain is thought to be regulated by the blood supply of free tryptophan in relation to other large neutral amino acids (including the branched-chain amino acids, BCAA) since these compete with tryptophan for transport into the brain. Studies in human subjects have shown that the plasma ratio of free tryptophan/BCAA increases during and, particularly, after sustained exercise. This would favour the transport of tryptophan into the brain and also the synthesis and release of 5-HT which may lead to central fatigue. Attempts have been made to influence the 5-HT level by giving BCAA to human subjects during different types of sustained heavy exercise. The results indicate that ingestion of BCAA reduces the perceived exertion and mental fatigue during exercise and improves cognitive performance after the exercise. In addition, in some situations ingestion of BCAA might also improve physical performance; during exercise in the heat or in a competitive race when the central component of fatigue is assumed to be more pronounced than in a laboratory experiment. However, more experiments are needed to further clarify the effect of BCAA and also of tryptophan ingestion on physical performance and mental fatigue. Received January 3, 2000 / Accepted February 1, 2000     

Effects of hemorrhagic hypotension on tyrosine concentrations in rat spinal cord and plasma

Tyrosine is the precursor for catecholamine neurotransmitters. When catecholamine-containing neurons are physiologically active (as sympathoadrenal cells are in hypotension), tyrosine administration increases catecholamine synthesis and release. Since hypotension can alter plasma amino acid composition, we examined the effects of an acute hypotensive insult on tyrosine concentrations in plasma and spinal cord. Rats were cannulated and bled until the systolic blood pressure was 50 mmHg, or were kept normotensive for 1 h. Tyrosine and other large neutral amino acids (LNAA) known to compete with tyrosine for brain uptake were assayed in plasma and spinal cord. The rate at which intra-arterial [3H]tyrosine disappeared from the plasma was also estimated in hemorrhaged and control rats. In plasma of hemorrhaged animals, both the tyrosine concentration and the tyrosine/LNAA ratio was elevated; moreover, the disappearance of [3H]tyrosine was slowed. Tyrosine concentrations also increased in spinal cords of hemorrhaged-hypotensive rats when compared to normotensive controls. Changes in plasma amino acid patterns may thus influence spinal cord concentrations of amino acid precursors for neurotransmitters during the stress of hemorrhagic shock.     

Adaptations of neurotransmitter synthesis to chronic hypoxia in cell culture

Tyrosine hydroxylase and tryptophan hydroxylase are widely held to be rate-limiting for the synthesis of the catecholamines and serotonin, respectively. Both enzymes are oxygen-requiring and kinetic properties suggest that oxygen availability may limit synthesis of these neurotransmitters in the brain. Using pheochromocytoma cells as a cell culture model for catecholamine synthesis, and neuroblastoma cells as a model for serotonin synthesis, enzyme activity was measured under control and hypoxic conditions. Both tyrosine hydroxylase and tryptophan hydroxylase activity increased substantially with chronic exposure but not with acute exposure. In the case of tyrosine hydroxylase, increased enzyme content with hypoxia accounts for increased activity. This suggests a mechanism for the maintenance of neurotransmitter synthesis with chronic hypoxia. Measurement of intracellular metabolites revealed no change in dopamine or norepinephrine in hypoxic pheochromocytoma cells, consistent with a simple adaptive mechanism. However, in neuroblastoma cells, hypoxia was associated with an increase in serotonin concentration. The reasons for this are still unclear.     

Brain Serotonin Content: Physiological Regulation by Plasma Neutral Amino Acids

When plasma tryptophan is elevated by the injection of tryptophan or insulin, or by the consumption of carbohydrates, brain tryptophan and serotonin also rise; however, when even larger elevations of plasma tryptophan are produced by the ingestion of protein-containing diets, brain tryptophan and serotonin do not change. The main determinant of brain tryptophan and serotonin concentrations does not appear to be plasma tryptophan alone, but the ratio of this amino acid to other plasma neutral amino acids (that is, tyrosine, phenylalanine, leucine, isoleucine, and valine) that compete with it for uptake into the brain.     

Correlation between brain tryptophan and plasma neutral amino acid levels following food consumption in rats

Brain tryptophan increases significantly within two hr of the time that rats begin to consume a diet containing carbohydrate and fat, but fails to rise if the diet also contains 18–24% protein. The effects of particular diets on brain tryptophan are not well correlated with plasma tryptophan concentrations alone, but do correlate well with the ratio of plasma tryptophan to individual neutral amino acids (leucine, isoleucine, valine, tyrosine, phenylalanine) or to their sums. (These amino acids compete with tryptophan for uptake into the brain.) Carbohydrate ingestion raises brain tryptophan by elevating plasma tryptophan and depressing the plasma levels of the competing neutral amino acids; protein consumption prevents an increase in brain tryptophan by raising the plasma concentrations of the competing amino acids more than of tryptophan.     

Maize based diets and possible neurobehavioural after-effects among some populations in the world

Maize is a cereal particularly lacking in tryptophan, which is the precursor of serotonin, an important neurotransmitter. Altough complementary foods may eliminate tryptophan deficiency, serotonin deficiency may often continue to exist because of competition made by other Large Neutral Amino Acids (LNAA) against tryptophan for neuron access, since they use the same carrier to cross the blood-brain barrier. Thus serotonin synthesis depends on two variables: the amount of tryptophan and the trp/LNAA ratio (R). “R” is lowest for common maize, low for beans and, as a rule, for most vegetable foods, higher for meat. So, when maize is the preponderant food in the meal, the “R” value lowers and so in parallel serotonin synthesis does. Serotonin deficiency involves several behavioural consequences, such as the tendency towards aggressive behaviour or the religious fanaticism. Among native american populations, these consequences appear, as a rule, positively correlated with maize alimentary dependence (Aztecs appear as those who greatly suffered from serotonin deficiency). In the world these are thinkable for some african populations (i.e. Zulu) or european (i.e. Balkan peoples).     

Alterations of tryptophan metabolism in a rat strain (Osborne-Mendel) predisposed to obesity

1. Osborne-Mendel (O-M) rats displayed differences in brain and systemic tryptophan metabolism. O-M rats had decreased brainstem tryptophan-5-hydroxylase activity and decreased serotonin (5-HT) levels as compared to Sprague-Dawley rats. However, brain tryptophan levels were actually increased in O-M rats. Norepinephrine, dopamine and 5-hydroxyindole-3-acetic acid levels were not different between strains. 2. Pineal serotonin levels were increased in O-M rats. 3. Liver tryptophan 2,3-dioxygenase activity was increased in O-M rats while tyrosine aminotransferase activity was not different between strains. 4. Total blood cholesterol was decreased in O-M rats while triglycerides, free fatty acids and albumin was not different between strains. Total serum tryptophan was not different between strains while O-M rats had an increased level of free (unbound) tryptophan.     

Selective effect of a maize diet in reducing serum and brain tryptophan contents and blood and brain serotonin levels.

The Maize diet used selectively lowers the tryptophan, serotonin and 5-hydroxyindoleacetic acid levels in the central nervous system without affecting catecholamine content. These changes are maximal as early as 48 hours after exposure to the Maize diet. The brain serotonin decrease is reversed to normal when the Maize diet is supplemented with Tryptophan, while nicotinic acid is ineffective. The maize diet seems to be a rapid and selective means of reducing brain serotonin content. The hypothesis that the psychic depression observed in patients with Pellagra may be related to an altered serotonin metabolism is discussed.     

Effect of experimental hyperphenylalaninemia on biogenic amine synthesis at later stages of brain development

The effects of experimental hyperphenylalaninemia on catecholamine and serotonin synthesis in brain at a later stage of brain development were investigated. A group of 35-day-old rats treated with normal chow supplemented with 5% Phe + 0.4% alpha-methylphenylalanine, alpha MP, for the previous 10 days showed decreases in dopa, norepinephrine, and epinephrine versus controls. A group treated with a normal diet supplemented with 0.4% alpha MP showed similar decreases and these differences could be attributed to the presence of the phenylalanine hydroxylase and tyrosine hydroxylase inhibitor, alpha MP, rather than the hyperphenylalaninemia condition. No differences in dopamine were observed. Serotonin and 5-hydroxyindoleacetic acid (5HIAA) were decreased 50% in the HyPhe condition and were unaffected in the presence of alpha MP alone, indicating that the decreases in serotonin and 5HIAA were due to the increases in phenylalanine rather than the presence of the inhibitor. These abnormalities in serotonin metabolism at later stages of brain development may be relevant to early discontinuation of dietary therapy in the PKU patient and implies a role in tryptophan supplementation to increase intracerebral serotonin values.     

Hepatocyte transplantation (HTx) corrects selected neurometabolic abnormalities in murine intermediate maple syrup urine disease (iMSUD)

Skvorak et al. [1] demonstrated the therapeutic efficacy of HTx in a murine model of iMSUD, confirming significant metabolic improvement and survival. To determine the effect of HTx on extrahepatic organs, we examined the metabolic effects of HTx in brain from iMSUD animals. Amino acid analysis revealed that HTx corrected increased ornithine, partially corrected depleted glutamine, and revealed a trend toward alloisoleucine correction. For amino acid and monoamine neurotransmitters, decreased GABA was partially corrected with HTx, while the l-histidine dipeptide of GABA, homocarnosine, was decreased in iMSUD mice and hypercorrected following HTx. Elevated branched-chain amino acids (BCAA; leucine, isoleucine, and valine) in MSUD can deplete brain tyrosine and tryptophan (the precursors of monoamine neurotransmitters, dopamine (DA) and serotonin (5-hydroxytryptamine; 5-HT)) through competition via the large neutral amino acid transporter. HTx corrected decreased DA levels and the DA metabolite, 3-methoxytyramine, and partially corrected the DA intermediate 3,4-dihydroxyphenylacetate (DOPAC) and 5-HT levels, despite normal tyrosine and tryptophan levels in iMSUD mouse brain. We further observed enhanced intracellular turnover of both DA and 5-HT in iMSUD mouse brain, both of which partially corrected with HTx. Our results suggest new pathomechanisms of neurotransmitter metabolism in this disorder and support the therapeutic relevance of HTx in iMSUD mice, while providing proof-of-principle that HTx has corrective potential in extrahepatic organs.     

Tryptophan Uptake and Hydroxylation in Rat Forebrain Synaptosomes

Tryptophan uptake, hydroxylation, and decarboxylation in isolated synaptosomes were studied to assess how their properties may determine the rate of serotonin synthesis in the presynaptic nerve terminals of the brain. Simultaneous measurements of the rates of uptake, hydroxylation, and decarboxylation in the presence and absence of various inhibitors showed that tryptophan hydroxylase is rate-limiting for serotonin synthesis in this model system. There was significant direct decarboxylation of tryptophan to tryptamine. Measurement of tryptophan hydroxylase flux with varying internal concentrations of tryptophan allowed the determination of the Km of tryptophan hydroxylase in synaptosomes for tryptophan of 120 +/- 15 microM. Depolarisation of synaptosomes with veratridine caused both a reduction in the internal tryptophan concentration and an apparent activation of tryptophan hydroxylase. This activation did not occur in the absence of Ca2+ or in the presence of trifluoperazine. Synaptosomal serotonin synthesis and brain stem-soluble tryptophan hydroxylase were inhibited by low concentrations of noradrenaline or dopamine. Dibutyryl cyclic AMP, glucagon, insulin, and vasopressin were observed to have no effect on tryptophan uptake or hydroxylation in synaptosomes.     

Effects of Dietary Amino Acids on Brain Amino Acids and Transmitter Amines in Rats with a Portacaval Shunt

The etiologic relationship between disturbances in metabolism of amino acids and amines and hepatic coma was investigated by examining the effects of diets containing various mixtures of amino acids on brain amine metabolism in rats with a portacaval shunt, using a method for simultaneous analysis of amino acids and amines. Rats with a portacaval shunt were fed on four different amino acid compositions with increased amounts of various amino acids suspected to be etiologically related to hepatic coma, such as methionine, phenylalanine, tyrosine, and tryptophan. The animals were killed 4 weeks after operation. During the experimental period, these animals did not become comatose, but exhibited various behavioral abnormalities. Marked increase in the plasma and brain levels of the augmented amino acids, especially methionine and tyrosine, were observed in rats with a portacaval shunt. Brain noradrenaline, dopamine, and serotonin levels were significantly decreased when the brain tyrosine level was increased. These results indicate that in rats with a portacaval shunt the dietary levels of amino acids greatly influence the brain levels of both amino acids and transmitter amines.     

Acute effects of aspartame on large neutral amino acids and monoamines in rat brain

The dipeptide aspartame (APM; aspartylphenylalanine methylester), an artificial sweetener, was studied $\text{in vivo}$ for its ability to influence brain levels of the large neutral amino acids and the rates of hydroxylation of the aromatic amino acids. The administration by gavage of APM (200 mg/kg) caused large increments in blood and brain levels of phenylalanine and tyrosine by 60 minutes. Brain tryptophan level was occasionally reduced significantly, but the brain levels of the branched-chain amino acids were always unaffected. Smaller doses (50, 100 mg/kg) also raised blood and brain tyrosine and phenylalanine, but did not reduce brain tryptophan levels. At the highest dose (200 mg/kg), APM gavage caused an insignificant increase in dopa accumulation (after NSD-1015), and a modest reduction in 5-hydroxytryptophan accumulation. No changes in the brain levels of serotonin, 5-hydroxyindoleacetic acid, dopamine, dihydroxyphenylacetic acid, homovanillic acid, or norepinephrine were produced by APM administration (200 mg/kg). These results thus indicate that APM, even when administered in amounts that cause large increments in brain tyrosine and phenylalanine, produce minimal effects on the rates of formation of monoamine transmitters.     

Regulation of catecholamine synthesis in rat brain synaptosomes

Abstract— Catecholamine synthesis in synaptosomal preparations of rat striatum, cortex and brain stem was investigated. The striatum had much higher activity than either the cortex or brain stem. Equilibration of labelled tyrosine between tissue and incubation medium was completed within 2 min. The apparent K_m of tyrosine hydroxylase (EC 1.14.3a) and of the overall catecholamine synthetic pathway were both approximately 5 ± 10^?6m for tyrosine. The following amines were found to inhibit striatal dopamine synthesis: dopamine, 25% inhibition at 5 ± 10^?7m ; noradrenaline, 25% inhibition at 5 ± 10^?6m ;and serotonin, 30% inhibition at 10^?5m . The catecholamine-induced inhibition of synthesis was antagonized by pre-incubation with cocaine. Increasing the potassium concentration from 5 to 55 mm caused a release of amines into the medium which was accompanied by a 40% increase in dopamine synthesis, when synthesis was measured during the first 5 min of exposure to elevated potassium. These results indicate that synaptosomal catecholamine synthesis is inhibited by increases in intra-synaptosomal amine levels, and that short-term exposure to depolarizing concentrations of potassium can increase synthesis.     

Effect of Chronic Corn Consumption on Serotonin Content of Rat Brain

SEROTONIN, a putative neurotransmitter in the mammalian central nervous system, is synthesized in the brain by the 5-hydroxylation and decarboxylation of the essential amino-acid L-tryptophan^1,2. The control of serotonin biosynthesis seems to involve a different mechanism from that responsible for catecholamine biosynthesis^3,4 in its dependence on the availability of the amino-acid precursor^5,6. Thus, small doses of tryptophan that do not increase brain or plasma tryptophan concentrations beyond their normal daily ranges cause significant increases in the serotonin concentration of rat brain⁷. Conversely, the chronic ingestion of diets lacking in tryptophan (with casein hydrolysates or amino-acid mixtures substituted for natural proteins) depresses brain serotonin levels^8–10. The dependence of serotonin biosynthesis on tryptophan availability probably arises from the unusually high substrate K _M that characterizes tryptophan hydroxylase¹. It seems likely that this enzyme normally functions in an unsaturated state; hence physiological increases in intraneuronal tryptophan could drive the hydroxylation of the amino-acid and, ultimately, its conversion to serotonin.     

Synthesis and High Affinity Uptake of Serotonin and Dopamine by Human Y79 Retinoblastoma Cells

Human Y79 retinoblastoma cells are capable of synthesizing the putative retinal neurotransmitters dopamine and serotonin. Separation of the catecholamines and indolamines by high performance liquid chromatography combined with electrochemical detection showed that the cells readily convert tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and, to a lesser extent, dopamine. When DOPA was added, a large quantity of dopamine was produced, as well as norepinephrine, epinephrine, and 3,4-dihydroxyphenylacetic acid. Exogenous tryptophan added to the cells was partially converted to 5-hydroxytryptophan and serotonin. A larger quantity of serotonin was produced when 5-hydroxytryptophan was added. Y79 cells have a high- and low-affinity uptake system for dopamine and serotonin. The K'm and V'max for the high-affinity uptake of dopamine and serotonin are 2.34 +/- 0.64 and 3.63 +/- 1.15 microM and 4.77 +/- 1.12 and 3.20 +/- 1.20 pmol min-1 mg protein-1, respectively. These kinetic parameters are similar to those reported for other retinal preparations where dopamine and serotonin have been suggested to function as neurotransmitters. Tyrosine and tryptophan, the physiologic precursors of dopamine and serotonin, respectively, and phenylalanine are also taken up by high- and low-affinity transport systems. The kinetic parameters for their high-affinity uptake systems are all very similar, suggesting that they may be taken up by the same transporter. These studies show that a tumor cell line derived from the human retina synthesizes dopamine and serotonin and has high-affinity uptake systems for these compounds and their precursors.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of insulin deficiency, dietary protein intake, and plasma amino acid concentrations on brain amino acid levels in rats

The effect of diabetes (streptozotocin, 65 mg/kg ip), dietary protein intake (15-60%), and plasma amino acid concentrations on brain large neutral amino acid levels in rats was examined. After 20 days, the plasma concentrations of methionine and the branched chain amino acids (BCAA), valine, isoleucine, and leucine were increased in diabetic rats. In brain tissue, methionine and valine levels were increased but threonine, tyrosine, and tryptophan concentrations were depressed. Increased protein consumption promoted a diabetic-like plasma amino acid pattern in normal rats while enhancing that of diabetic animals. However, with the exception of threonine, glycine, valine, and tyrosine, there was little effect on brain amino acid levels. A good association was found between the calculated brain influx rate and the actual brain concentration of threonine, methionine, tyrosine, and tryptophan in diabetic animals. There was no correlation, however, between brain influx rate and brain BCAA levels. Thus, the brain amino acid pattern in diabetes represents the combined effects of insulin insufficiency and composition of the diet ingested on plasma amino acid levels as well as metabolic adaptation within the brain itself.