Competition for transport of amino acids into rat heart: effect of competitors on protein synthesis and degradation.

Transport of the neutral amino acids, 2-(methylamino)isobutyrate (MeAIB) and Phe, was examined in isolated rat hearts perfused by the Langendorff method. Hearts were perfused by recirculating for various time periods buffer containing [14C]-MeAIB or [14C]-Phe plus desired additions. Uptake of MeAIB was linear for approximately 30 minutes; Phe uptake was linear for a maximum of 5 minutes, and reached a steady state after 15 minutes. Km and Vmax for MeAIB were 1.1 +/- 0.03 mmol/L and 37.7 +/- 0.4 pmol/microL intracellular fluid (ICF)/min; values for Phe were 1.8 +/- 0.02 mmol/L and 364 +/- 5 pmol/microL ICF/minute. Uptake of MeAIB (0.2 mmol/L) was reduced 95% in the presence of Ser (10 mmol/L), and less severely by large neutral amino acids ([LNAA], 10 mmol/L) such as Phe and Leu (by 46% and 54%, respectively). Uptake of Phe (0.2 mmol/L) was reduced by LNAA such as Val, Leu, and Ile (by 51%, 78%, and 81%, respectively), or by commercial preparations used in parenteral nutrition, eg, Travasol or Travasol plus extra branched-chain amino acids (BCAA) (Branchamin); Ser had little effect (8% reduction). Insulin in the perfusion medium increased the fractional rate of protein synthesis. Individual BCAA at physiological concentrations (0.2 mmol/L) did not alter the rate of protein synthesis. Branchamin or Travasol plus Branchamin also had no effect on the rate of protein synthesis in heart, but did depress the rate of degradation. These studies suggest that amino acid transport into heart may be affected by normal levels of plasma amino acids, whereas protein synthesis is not.

Protein selection by rats adapted to high or moderately low levels of dietary protein.

After preliminary studies on flavor acceptability, patterns and indices of subsequent feeding behavior were monitored by computer in young rats which were adapted to 15% or 70% casein diets before being offered, sequentially, choices between flavored diet pairs in which the proportions of percentage casein were 5/65, 5/55, 5/45, 5/35 and 5/25. Similarly adapted rats received these choices in the reverse sequence. Rats adapted to 15% casein usually ate randomly from the diet pairs and selected approximately 15-30% casein; individual behaviors were prominent. The 70% casein groups avoided the higher casein diet, often within minutes (except for the first-offered 5/25 choice), and seldom selected more than 10% casein; individual differences were infrequent. Such rats also distinguished between flavored 70% and 65% casein diets. Sizes and numbers of meals and rates of eating differed for the paired diets, especially for rats adapted to 70% casein. A flavor added to the 70% casein adaptation diet was not avoided when present only in the 5% casein diet of a 5/65 choice. Rats adapted to 70% soy protein before receiving flavored 5/65 to 5/25 choices selected 20-28% soy protein, a level far above those of casein selections by rats adapted to 70% casein. Dietary adaptation and type of protein thus affect subsequent diet selection and feeding patterns and indices.

Neutral amino acid transport into rat skeletal muscle: competition, adaptive regulation, and effects of insulin.

Amino acid (AA) transport systems A and L, which transfer preferentially small neutral AA (SNAA) and large neutral AA (LNAA), respectively, were studied in the isolated soleus muscle with the specific models, 2-(methylamino)isobutyrate (MeAIB) and 2-aminobicyclo[2,2,1]heptane-2-carboxylate (BCH). Affinity for MeAIB was greater than for BCH (Km = 3.2 +/- 0.2 and 8.7 +/- 0.2 mm, respectively). Rate of transport of MeAIB (Vmax = 104 +/- 3 pmol/microL/min) was slower than for BCH (970 +/- 12 pmol/microL/min), but accumulation was far more concentrative; transport of BCH, but not MeAIB, rapidly reached a steady-state level. MeAIB transport was reduced in the presence of SNAA; BCH transport was reduced to a lesser extent only by LNAA. Mixtures of AA at concentrations resembling those in plasmas of rats fed either a 6% or 50% casein diet reduced transport of MeAIB, whereas BCH transport was low only with the latter mixture. Only MeAIB transport was stimulated by insulin. Preincubation of muscles for 5 hours in a AA-free medium stimulated subsequent MeAIB uptake by about twofold to fourfold; this effect was suppressed by inhibitors of protein synthesis. Selective differences were thus observed in transport by skeletal muscle of model AA for the A and L systems: increased transport resulting from various stimuli was limited to the model for the A system, and transport of either model was depressed with mixtures containing physiological levels of AA. Changes in dietary protein or AA intake may thus alter transport of certain neutral AA into skeletal muscle via changes in plasma AA pools.

Branched-chain and other amino acids in tissues of rats fed leucine-limiting amino acid diets containing norleucine.

Amino acid concentrations were measured in plasma, brain, muscle and liver from rats fed leucine-limiting diets containing varying proportions of other indispensable amino acids (IAA), the branched-chain amino acids (BCAA) and norleucine, a BCAA analog known to compete with large neutral amino acids (LNAA) for transport into tissues. Leucine was low and other IAA were high when dietary IAA were 125% and leucine was 65% of requirements; higher leucine and lower IAA concentrations occurred when dietary IAA were 75% of requirements. Tissue leucine was high and isoleucine and valine were low in rats fed excess leucine. Norleucine induced dose-dependent reductions in BCAA, especially in brain and muscle in which isoleucine or valine were sometimes undetectable. Leucine was not depressed further when control values were low as in the rats fed 125% IAA. Norleucine frequently prevented the high BCAA found after feeding additional BCAA. Other LNAA tended to be low in the brain and muscle of rats fed norleucine. Lysine was high only in the tissues of rats fed 75% IAA and norleucine; this effect was prevented when added leucine was given. Brain tryptophan, but not always serotonin, was low in rats fed norleucine. The results show transport-related, selective and usually marked depletions of tissue BCAA in rats fed norleucine; this suggests norleucine may be an aid in the treatment of clinical conditions involving excesses of BCAA.

Dietary disproportions of amino acids in the rat: effects on food intake, plasma and brain amino acids and brain serotonin.

Food intake, growth, plasma and brain amino acid, and brain serotonin and 5-hydroxyindole-3-acetic acid (5-HIAA) concentrations were measured in rats fed low protein diets containing disproportionate amounts of large neutral amino acids (LNAA) devoid of tryptophan or histidine (tryptophan or histidine imbalance). Five-day food intakes and weight gains of rats fed the imbalanced diets were depressed. The concentration of the limiting amino acid was low in brains of rats fed diets containing LNAA that compete with either tryptophan or histidine for entry into brain. Correlations were observed between the brain concentrations of most individual LNAA and either the ratios of the plasma concentration of that LNAA to the sum of the other LNAA, or the predicted rates of influx of that LNAA. Cumulative food intakes were correlated with brain concentrations of the limiting amino acid, tryptophan or histidine. Food intakes were not consistently correlated with concentrations of serotonin and 5-HIAA because these compounds were altered only in brains of rats in the tryptophan study. Competition among amino acids for uptake into brain appears to be involved in the feeding response of the rat to dietary disproportions of amino acids, but this response is not directly related to changes in brain concentrations of serotonin and 5-HIAA.

Norleucine: a branched-chain amino acid analog affecting feeding behavior of rats.

Norleucine, an isomer of leucine and isoleucine and a potent competitor of large neutral amino acid transport into brain, thereby depleting certain amino acid pools, was tested for its effects on growth and feeding behavior of rats fed an amino acid diet limiting in leucine. Growth and food intake were depressed in proportion to the dietary level of norleucine (0.2 to 1.1% of the diet). With suboptimal amounts of indispensable amino acids, leucine at 150% of the requirement reversed the effects of 0.2 and 0.5% norleucine; slight excesses of the other indispensable amino acids were required with extra leucine for maximum growth with 1.1% norleucine. Rats almost exclusively preferred the control to the norleucine diet, but not if the latter diet also contained leucine. Rats also strongly selected a nonprotein rather than norleucine diet when this was the first available choice. If the first choice was between the nonprotein and control diets, rats later almost exclusively selected the norleucine-containing rather than the nonprotein diet for varying periods (2 to 6 days). These studies suggest that amino acid analogs may be useful agents in the study of animal behavior associated with changes in brain amino acid pools.

Dietary branched-chain amino acids and protein selection by rats.

Consumption by rats of high protein diets is associated with elevated plasma and brain concentrations of branched-chain amino acids (BCAA). We examined the possibility that changes in BCAA concentrations in blood and brain might serve as modulators of protein consumption. After young rats had adjusted to selecting between a 10% or 25% casein diet and a 50% casein diet, a mixture of BCAA was included in the diet containing the lower amount of protein (10% + BCAA, 25% + BCAA). Supplementation of the 10% or 25% casein diets with BCAA and subsequent elevation of BCAA concentrations in plasma and brain were associated with increased selection of protein in rats given the 10% + BCAA/50% casein diet choice, but not in rats offered the 25% + BCAA/50% casein diets. When no alternative diet was available, addition of BCAA to a 15% casein diet depressed food intake, and rats given a choice between a 15% casein diet with or without added BCAA selected almost exclusively the diet without added BCAA. Although BCAA concentrations were high in plasma and brain in all experiments, concentrations of methionine, tyrosine, phenylalanine, tryptophan and histidine were low in brain in experiments in which rats altered their diet or protein selection after BCAA addition. High concentrations of BCAA in plasma and brain were not consistently associated with changes in protein selection.

Distribution of alpha-aminoisobutyric acid in tissues of lean and genetically obese mice.

Distribution of tracer amounts of the nonmetabolizable neutral amino acid alpha-[1-14C]aminoisobutyric acid (AIB) between blood and several tissues was measured in lean and ob/ob mice over an 8-hr period. As AIB was injected on the basis of body weight and as ob/ob mice have a relatively low blood volume, absolute concentrations of AIB in blood and tissues were almost always higher in the obese than the lean mice. However, the ratio of AIB concentration in the tissues to that in the blood was clearly higher in skeletal muscle, diaphragm, and brain, and possibly higher in liver of the lean than of the obese animals. Ratios in heart were similar. The results suggest that lean and genetically obese mice differ in their capacity to transport amino acids between blood and various tissues.

Some characteristics of threonine transport across the blood-brain barrier of the rat.

Threonine entry into brain is altered by diet-induced changes in concentrations of plasma amino acids, especially the small neutrals. To study this finding further, we compared effects of various amino acids (large and small neutrals, analogues, and transport models) on transport of threonine and phenylalanine across the blood-brain barrier. Threonine transport was saturable and was usually depressed more by natural large than small neutrals. Norvaline and 2-amino-n-butyrate (AABA) were stronger competitors than norleucine. 2-Aminobicyclo[2.2.1]heptane-2-carboxylate (BCH), a model in other preparations for the large neutral (L) system, and cysteine, a proposed model for the ASC system only in certain preparations, reduced threonine transport; 2-(methylamino)isobutyrate (MeAIB; a model for the A system for small neutrals) did not. Phenylalanine transport was most depressed by cold phenylalanine and other large neutrals; threonine and other small neutrals had little effect. Norleucine, but not AABA, was a strong competitor; BCH was more competitive than cysteine or MeAIB. Absence of sodium did not affect phenylalanine transport, but decreased threonine uptake by 25% (p less than 0.001). Our results with natural, analogue, and model amino acids, and especially with sodium, suggest that threonine, but not phenylalanine, may enter the brain partly by the sodium-dependent ASC system.

Dietary amino acid analogues and transport of lysine or valine across the blood-brain barrier in rats.

Studies were undertaken to determine if dietary disproportions of amino acids would alter flux into brain of the amino acid present in the diet in a growth-limiting concentration. Rats were adapted to a lysine-limiting diet before receiving a meal of this control diet, alone or with added lysine or homoarginine (a competitor for lysine transport) or both, before intravenous infusion of [14C]lysine. The brain-to-plasma radioactivity ratio was lower in rats fed extra lysine or homoarginine than in rats fed the control diet, whereas lysine flux and brain lysine concentration were high in rats fed extra lysine alone. Flux and concentration were lower in rats fed homoarginine + lysine than in rats fed extra lysine alone. Other rats were fed a valine-limiting diet containing added valine, norleucine (a competitor for valine transport) or both, before [14C]valine was infused. Valine flux and brain valine concentrations were higher in rats fed extra valine than in control rats, whereas flux was lower in the group fed norleucine alone. Valine flux was higher in rats fed norleucine + valine than in the rats fed norleucine alone. Our studies show that dietary disproportions of amino acids can alter the flux of specific amino acids across the blood-brain barrier.

Induction of conditioned taste aversion in rats by GABA or other amino acids.

GABA included in the diet is known to reduce food intake and growth of rats fed a low protein diet. Experiments were designed to determine if GABA or other small neutral amino acids would affect food intake if they were administered separately from the diet, and if such amino acids could induce a conditioned taste aversion (CTA) to saccharin. Intubated or injected GABA or alpha-aminoisobutyric acid (AIB), a non-metabolizable isomer of GABA, reduced food intake. When rats were fed a low protein diet, IP injection of threonine (2 mmoles/200 g rat) induced CTA but did not depress food intake; serine (3 mmoles/200 g rat) induced CTA and caused only a small reduction in food intake. Another isomer, alpha-amino-n-butyric acid did not affect food intake or induce CTA at the tested concentrations. Adaptation to a high protein diet, which increases enzymatic degradation of many amino acids including GABA and serine, lessened severity of GABA-induced CTA and eliminated that caused by serine. CTA to saccharin can be induced by certain amino acids; the mechanism is unknown but may involve malaise or other adverse sensations.

Acceptability by rats of aqueous solutions of amino acid analogues.

Preferences differed widely when rats were offered choices between water and solutions of various natural amino acids and structurally related analogues. They avoided the branched-chain amino acid valine but preferred solutions of its isomer norvaline and of norleucine. The hydrochloride forms of ornithine and arginine were preferred to water at concentrations up to about 100 mM and avoided at 410 mM; homoarginine.HCl was never preferred and was avoided at 39 and 78 mM. Rats were indifferent to taurine and beta-alanine at most concentrations but refused these amino acids at high concentrations (205 and 410 mM, respectively). In conjunction with earlier observations on feeding behavior in response to dietary additions of amino acids, the results show that selections by rats between water and amino acid solutions cannot be used to predict choices among amino acid-containing diets.

Threonine entry into rat brain after diet-induced changes in plasma amino acids.

Passage of amino acids across the blood-brain barrier is modified by the amino acid composition of the blood. Because blood amino acid concentrations respond to changes in protein intake, we have examined associations among diet, plasma amino acid patterns, and the rate of entry of threonine into the brain. Rats were adapted for 8 h/day for 7-10 days to diets containing 6, 18, or 50% casein before receiving a single, independently varied, final meal of a diet containing 0, 6, 18, or 50% casein. After 4-7 h, they were anesthetized and infused intravenously with [14C]threonine for 5 min before plasma and brain samples were taken for determination of radioactivity and amino acid content. Plasma and brain threonine concentrations decreased as protein content increased in the diets to which the rats had been adapted. Plasma threonine concentrations increased twofold, from 1.6 to 3.0 mM, when rats adapted to 6% casein meals received a single 50% casein meal rather than a nonprotein meal; a fivefold increase, from 0.13 to 0.69 mM, occurred when rats had been previously adapted to 50% casein meals. Increasing the protein content of the final meal did not increase brain threonine concentrations. Highest and lowest rates of threonine entry into the brain occurred, respectively, in rats adapted to 6 and 50% casein meals. Changes in plasma threonine concentrations and threonine flux into brain reflected protein content of both pretreatment and final meals.

Valine entry into rat brain after diet-induced changes in plasma amino acids.

Passage of amino acids across the blood-brain barrier is assumed to be modified by amino acid composition of the blood. To gain a better understanding of the effects of protein intake on brain amino acid uptake, we examined associations among diet, plasma amino acid patterns, and the rate of entry of valine into the brain. Rats were fed (8 h/day for 7-10 days) diets containing 6, 18, or 50% casein before receiving one meal of a diet containing 0, 6, 18, or 50% casein. After 4-7 h, they were anesthetized and infused intravenously with [14C]valine for 5 min before plasma and brain samples were taken for determination of radioactivity and content of individual amino acids. As protein content of the meal was increased from 0 to 50% casein, plasma and brain concentrations of valine and most other large neutral amino acids (LNAA) increased severalfold; also the ratio of [14C]valine in brain to that in plasma decreased by greater than 50%, and the rate of valine entry into the brain increased 3.5-fold. The increase in valine flux slowed as plasma levels of LNAA, competitors for valine transport, increased. The results were far more dependent on protein content of the final meal than on that of the adaptation diet; thus changes in protein intake, as reflected in altered plasma amino acid patterns, markedly altered valine entry into the brain.

Induction in rats of lysine imbalance by dietary homoarginine.

The use of dietary homoarginine (HA) to produce a lysine imbalance was examined in young rats. HA, a basic amino acid analogue, was shown earlier to compete with lysine for transport across the blood-brain barrier. Feeding a diet limiting in lysine and containing HA reduced food intake and growth; increasing dietary lysine content lessened these effects. A 3-fold increase in dietary lysine caused 10, 4, 20 and 50-fold increases in lysine concentrations in plasma, brain, liver and muscle, respectively, and reductions in ornithine concentration of brain and liver and in arginine concentration of brain. HA increased lysine levels in plasma, liver and muscle of rats fed the lysine-limiting control diet, but lysine concentrations were reduced by HA when dietary lysine was high. HA invariably lowered concentrations of lysine, ornithine and arginine in brain and ornithine concentrations in plasma and muscle. Brain concentrations of small and large neutral amino acids were unchanged by HA; an exception was glycine, which was high in the brains of rats fed HA. The HA-associated, selective reductions in levels of each basic amino acid in brain in the ratios of these concentrations to those in plasma support the concept that effects of an amino acid imbalance involve competition for amino acid entry into the brain from the blood.

Tissue amino acids in rats fed norleucine, norvaline, homoarginine or other amino acid analogues.

Concentrations of several large neutral amino acids (LNAA) were earlier shown to be low, especially in brain, in rats fed a low protein diet containing a mixture of LNAA analogues. The purpose of this study was to learn if individual analogues would induce similar effects. Four hours after first feeding one meal containing norleucine, norvaline, alpha-aminophenylacetic acid, or alpha-aminooctanoic acid, concentrations of branched-chain amino acids were low in plasma, brain, liver and muscle; tyrosine and phenylalanine were more effectively reduced in brain than in other tissues. Lysine and arginine concentrations were low in brains of rats fed the basic amino acid analogue, homoarginine; concentrations of large and small neutral amino acids were unchanged. Dopamine was not low in brains having low tyrosine levels; serotonin was low in rats receiving alpha-aminooctanoate, the only analogue associated with a significant depression in brain tryptophan. The results suggest that the analogues have differing abilities to alter concentrations of tissue components. Decreases, especially in brain amino acid concentrations, may result from selective competition by analogues of a given transport class with natural amino acids transported from blood into brain by the same system.

Choices by rats between water and solutions of GABA or other amino acids.

Responses differed widely when rats were offered choices between water and solutions of GABA, its isomers alpha-aminobutyric acid (AABA) and alpha-aminoisobutyric acid (AIB), or of another 4-carbon amino acid, threonine. They preferred solutions of threonine and AABA starting at concentrations of about 30 mM; preference for threonine declined when its concentration was 330 mM or above. Rats never preferred GABA or AIB, but instead avoided these amino acids when concentrations were approximately 100 mM or above. Control rats showed strong preferences for drinking from a given location. Limited studies with humans showed variations in the concentrations at which they could detect GABA; the mean was about 0.06 mM, a concentration far below that at which rats began to avoid this amino acid. The ability of dietary GABA to depress food intake of rats (as shown in earlier studies) does not seem related to a uniquely high sensitivity to its gustatory qualities.

Dietary GABA and food selection by rats.

To obtain further information pertaining to amino acid-induced alterations in feeding behavior, studies were performed to examine the food choices made by rats fed low protein diets made more or less aversive by the addition of various amino acids. When rats were allowed to choose between two diets, they preferred a low protein control, threonine-imbalanced or nonprotein diet to one containing 2.5% gamma-aminobutyric acid (GABA). Acceptance increased when GABA content was lowered to 1.5%; rats preferred this diet when the alternative diet was made sufficiently aversive. There were large individual differences among rats selecting from pairs of unacceptable diets. Avoidance of, or preference for, a given diet is clearly affected by the relative aversive qualities of the offered pair of diets.

Food intake, growth and tissue amino acids in rats fed acid analogues.

The objective of this study was to examine the effects of dietary additions of analogues of large neutral amino acids (LNAA), previously shown to inhibit entry of natural LNAA into brain, on food intake, growth and tissue concentrations of specific amino acids in young rats. A mixture of norleucine, norvaline, alpha-aminophenylacetate and alpha-aminooctanote (atypical amino acids, AAA) markedly depressed food intake and growth of rats fed a 6% protein diet (LP) for 10 d but not of rats fed a 50% protein diet (HP). Except in rats fed HP, dietary AAA usually decreased concentrations of LNAA more than of small neutral amino acids (SNAA) or lysine, especially in brain. Concentrations of LNAA, especially in brain and muscle of rats adapted to LP or HP meals and fed one LP-AAA meal were lower than in similar rats fed one LP meal without AAA; feeding an HP-AAA meal to such rats generally prevented or lessened these changes. AAA-induced changes in SNAA and lysine were usually small in meal-fed rats. When AAA induced decreases in LNAA, the branched-chain amino acids were usually most affected; valine and isoleucine sometimes were undetected in brain and muscle. Serotonin and dopamine concentrations were not low in brain despite low levels of tryptophan and tyrosine. Changes in tissue LNAA concentrations would appear to reflect in part competition by large neutral AAA for transport of natural LNAA from the blood.