Metabolism and transport of gamma-carboxyglutamic acid.

gamma-Carboxyglutamic acid residues have beeh shown to be present in prothrombin, the other vitamin K-dependent clotting factors, and more recently in bone and kidney proteins. This amino acid is formed by a posttranslational vitamin K-dependent carboxylation of glutamyl residues in polypeptide precursors of these protens. It has now been demonstrated that this amino acid, either in the free or peptide-bound form, is not metabolically degraded by the rat, but is quantitatively excreted in the urine. In nephrectomized rats, the tissue concentration of intravenously administered gamma-carboxyglutamic acid is increased, but there is still no evidence of any oxidative metabolism of this amino acid. These amino acid is transported by kidney slices against a concentration gradient, but does not accumulate in liver, intestinal or brain tissues. Preliminary data suggest that gamma-carboxyglutamic acid may be concentrated by a carrier system different from that utilized by other amino acids.

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.

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.

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.

Alleviation in the rat of a GABA-induced reduction in food intake and growth.

Cold exposure and diet dilution which stimulate food intake of normal rats lessened depressions of food intake and growth induced by dietary GABA. During a 3-day adaptation to the cold, rats fed a diet containing 4.5% GABA lost weight; thereafter, food intake and growth rate differed little from those of cold control rats and were usually greater than those of normal rats fed GABA. Hepatic GABA-aminotransferase activity of cold-exposed rats fed the GABA diet increased to about twice that of normal control rats. Rats fed a control diet diluted by half with cellulose ate 50% more of this diet than of the undiluted diet but gained only 20% less weight. Rats ate twice as much of a diluted, 9% GABA diet as of an undiluted, 4.5% GABA diet (thus doubling their GABA intake) and gained three times as much weight. A novel food (condensed milk) barely lessened the adverse responses to GABA. These results show that conditions requiring rats to increase their food intake in order to maintain body weight can also increase their acceptance of a diet high in GABA.

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.

Avoidance of GABA-containing diets by olfactory bulbectomized rats.

In order to test the importance of olfaction in the avoidance of GABA-containing diets by intact rats, olfactory bulbectomized and sham-operated control rats were allowed to choose between a low protein control diet and this diet supplemented with 2.5% GABA; the specificity of the response was examined by also testing for responses to other amino acids. Both groups of rats markedly avoided the GABA diet initially; the bulbectomized rats later tended to increase their intake of this diet. The bulbectomized rats chose similar amounts from the control diet and one supplemented with alanine whereas the control rats ultimately strongly preferred the latter diet. Both groups only moderately avoided a threonine-supplemented diet. The results suggest that (1) the odor of GABA is not critical in the avoidance of diets containing this amino acid; and (2) patterns of food selection from diets containing GABA differ from choices from diets containing other small neutral amino acids such as alanine or threonine.

Effect of dietary protein and GABA on food intake, growth and tissue amino acids in cats.

GABA at 5%, but not 3%, of a low protein diet depressed food intake and growth of kittens. Adaptation to high protein prevented these effects. When cats adapted to low or high protein were fed a meal containing GABA, plasma GABA concentration after 2 hr was 8-fold higher in the low than in the high protein group; clearance was almost complete within 6 hr. Concentrations of proline, branched-chain, other large neutral and basic (especially ornithine) amino acids increased more when cats were fed a high rather than a low protein meal; glycine decreased. At 6 hr, concentrations had consistently returned to initial levels only in the low protein group. Feeding the high protein diet ad lib increased tissue concentrations of threonine, proline and the branched-chain amino acids. Hepatic or renal GABA-aminotransferase activity was not altered in kittens fed the high protein diet. Kidney activity was 10-fold that of liver, which may contribute to the better tolerance of GABA by cats than by rats.

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.

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.

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.

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.

Food and protein intake, glucagon, and distribution of alpha-aminoisobutyrate in the rat.

Distribution of alpha-aminoisobutyric acid (AIB) in the rat was modified by food, dietary protein, and glucagon. In rats last fed 24 h before AIB injection, AIB clearance from plasma and uptake into liver were greater in rats fed a high-protein diet (60% casein) than in rats fed the control diet (18% casein); AIB clearance from plasma and uptake into muscle were lowered by a low-protein diet (6% casein). Feeding rats lowered clearance of AIB from plasma in low- and high-protein groups. Distribution ratios (AIB concentration in tissue water/AIB in plasma) were low in all tissues but liver during the first 7 h after feeding high protein when compared to the control values; ratios were low in muscle, heart, and kidney after feeding low protein. Maximum ratios occurred at different times for different tissues; the time was delayed by the high-protein diet in all tissues but liver. Glucagon increased all ratios in rats fed the control or low-protein diets, with the smallest changes occurring in liver and muscle from low-protein rats.

alpha-Aminoisobutyric acid transport in liver slices from rats fed low protein meals.

The effect of low protein intake on alpha-aminioisobutyric acid (AIB) transport has been examined in liver slices from meal-fed rats. Treatments (force-feeding casein or gelatin hydrolysates, glycine, or potassium chloride; injecting glucagon, or preincubating liver slices with cyclic AMP) which stimulated transport in control rat fed 3-hour meals containing 18% casein were less effective in rats fed 6% casein meals for 8 days. Responses of protein-depleted rats to glucagon or cyclic AMP had become essentially normal after they had consumed 18% casein meals for 2 days, whereas between 6 to 10 days were required for complete recovery of the response to casein hydrolysate. Stimulation of AIB transport by casein hydrolysate was also normal in depleted rats after they had consumed 40% casein meals for 2 days. Basal, non-stimulated transport of AIB was not significantly depressed in rats fed the low protein diet. Casein hydrolysate-induced increases in hepatic cyclic AMP concentrations were smaller in rats fed low protein meals. The results show that, although various stimuli of hepatic AIB transport become less effective in the protein-deprived rat, relatively rapid recovery of the response can occur upon refeeding adequate or high levels of protein.