Leucine In Rats Research Articles

Analysis of the Stimulative Effect of Tryptophan on Hepatic Protein Synthesis in Rats.

Tryptophan is an essential amino acid important as a protein building block, but it also serves as substrate for the generation of several bioactive compounds with important physiological roles. Furthermore, tryptophan has been reported to have a unique role as a nutritional signaling molecule that regulates protein synthesis in mouse and rat liver. In the present study, the acute effects of tryptophan on protein synthesis were confirmed and compared with those of leucine in rats. Eighteen hours fasted rats were orally administered of tryptophan or leucine at a dose of 135 mg/100 g body weight by gavage and then sacrificed 1 h after administration. The effects of tryptophan and leucine on the rate of protein synthesis were evaluated by the surface sensing of translation (SUnSET) method. We also examined the ability of tryptophan to induce activation of the mTOR pathway by measuring phosphorylation of 4E-BP1 and S6K1. Oral administration of tryptophan led to a stimulation of the rate of protein synthesis concomitant with activation of mTOR pathway in the liver, but not in skeletal muscle. We also investigated the sensitivity of liver protein synthesis to tryptophan administration. The half-maximal effective doses (ED50) of tryptophan in stimulating 4E-BP1 and S6K1 phosphorylation were both about 60% of daily intake. The effect of tryptophan on hepatic protein synthesis was similar to that of leucine on muscle protein synthesis, and the sensitivity of liver protein synthesis to tryptophan administration appeared to be almost the same or slightly lower than that of muscle protein synthesis to leucine administration.

Protein distribution affects muscle mass based on differences in postprandial muscle protein synthesis and plasma leucine in rats

Methods Male Sprague Dawley rats (275g) were fed isocaloric/isonitrogenous meals containing whey protein, with protein either distributed evenly at 16% of total energy over 3 meals (ED-Whey) or unevenly distributed over 3 meals (UD-Whey) with the first 2 meals containing only 8% of energy from whey protein and only the dinner meal containing sufficient protein to optimize muscle protein synthesis (MPS) (27.5% of total energy from whey protein) for 11 weeks. Measurements were taken to assess postprandial rates of MPS, plasma amino acids, mammalian target of rapamycin (mTOR) signaling, and the animals’ body composition was assessed by Dual energy X-ray absorptiometry (DXA). Hind limb muscle weights were taken to asses differences in muscle mass.

Is there In Vivo Evidence for Amino Acid Shuttles Carrying Ammonia from Neurons to Astrocytes?

The high in vivo flux of the glutamate/glutamine cycle puts a strong demand on the return of ammonia released by phosphate activated glutaminase from the neurons to the astrocytes in order to maintain nitrogen balance. In this paper we review several amino acid shuttles that have been proposed for balancing the nitrogen flows between neurons and astrocytes in the glutamate/glutamine cycle. All of these cycles depend on the directionality of glutamate dehydrogenase, catalyzing reductive glutamate synthesis (forward reaction) in the neuron in order to capture the ammonia released by phosphate activated glutaminase, while catalyzing oxidative deamination of glutamate (reverse reaction) in the astrocytes to release ammonia for glutamine synthesis. Reanalysis of results from in vivo experiments using (13)N and (15)N labeled ammonia and (15)N leucine in rats suggests that the maximum flux of the alanine/lactate or branched chain amino acid/branched chain amino acid transaminase shuttles between neurons and astrocytes are approximately 3-5 times lower than would be required to account for the ammonia transfer from neurons to astrocytes needed for glutamine synthesis (amide nitrogen) to sustain the glutamate/glutamine cycle. However, in the rat brain both the total ammonia fixation rate by glutamate dehydrogenase and the total branched chain amino acid transaminase activity are sufficient to support a branched chain amino acid/branched chain keto acid shuttle, as proposed by Hutson and coworkers, which would support the de novo synthesis of glutamine in the astrocyte to replace the ~20 % of neurotransmitter glutamate that is oxidized. A higher fraction of the nitrogen needs of total glutamate neurotransmitter cycling could be supported by hybrid cycles in which glutamate and tricarboxylic acid cycle intermediates act as a nitrogen shuttle. A limitation of all in vivo studies in animals conducted to date is that none have shown transfer of nitrogen for glutamine amide synthesis, either as free ammonia or via an amino acid from the neurons to the astrocytes. Future work will be needed, perhaps using methods for selectively labeling nitrogen in neurons, to conclusively establish the rate of amino acid nitrogen shuttles in vivo and their coupling to the glutamate/glutamine cycle.

An Approach to Defining the Upper Safe Limits of Amino Acid Intake

The existing data on the safe upper limits of amino acid intake in humans is essentially observational; how much do individuals ingest and what side effects do they have? There are numerous studies in humans comparing the effects of high doses of amino acids given as protein bound vs. as free amino acids. These studies have shown that protein-bound amino acids have much less effect on plasma levels of the test amino acid, because protein intake stimulates protein synthesis as another sink for the increased amino acid intake. In practice, the highest amino acid intakes occur with free amino acid supplements that may be ingested by athletes who believe that the amino acids will benefit them in training and/or performance. Previously, in a piglet study, we were able to define the point at which maximal phenylalanine oxidation occurred, above which plasma phenylalanine concentration and body balance rose exponentially. We regard this value of maximal disposal (oxidation) of an amino acid as one metabolic marker of the upper limit of intake. Recently, others have demonstrated a similar maximal oxidation rate for leucine in rats. Based on these experimental data and the paucity of published human data in controlled experiments, we think that a systematic approach needs to be undertaken to define the maximal oxidation rate for all dietary indispensable amino acids and other amino acids that may be ingested in excess by humans. We believe that this will provide a rational basis to begin to define the upper limits of tolerance for dietary amino acids. However, some amino acids, such as threonine and methionine, will be more difficult to study, because they have more than 1 route of disposal or very complex metabolic regulation, in which case defining their upper limits will be more multifaceted.

Regulation of muscle protein degradation, not synthesis, by dietary leucine in rats fed a protein-deficient diet

The aim of this study was to elucidate the effects of long-term intake of leucine in dietary protein malnutrition on muscle protein synthesis and degradation. A reduction in muscle mass was suppressed by leucine-supplementation (1.5% leucine) in rats fed protein-free diet for 7 days. Furthermore, the rate of muscle protein degradation was decreased without an increase in muscle protein synthesis. In addition, to elucidate the mechanism involved in the suppressive effect of leucine, we measured the activities of degradation systems in muscle. Proteinase activity (calpain and proteasome) and ubiquitin ligase mRNA (Atrogin-1 and MuRF1) expression were not suppressed in animals fed a leucine-supplemented diet, whereas the autophagy marker, protein light chain 3 active form (LC3-II), expression was significantly decreased. These results suggest that the protein-free diet supplemented with leucine suppresses muscle protein degradation through inhibition of autophagy rather than protein synthesis.

Rapid suppression of protein degradation in skeletal muscle after oral feeding of leucine in rats

A diet containing adequate amounts of protein rapidly suppresses myofibrillar protein degradation in rats and mice. This study determined whether dietary amino acids inhibit postprandial protein degradation in rat skeletal muscle. When rats fed on a 20% casein diet for 1 h after 18 h starvation, the rate of myofibrillar protein degradation measured by N τ-methylhistidine release from the isolated extensor digitorum longus muscle was significantly ( p < 0.05) decreased at 4 h after refeeding. A diet containing an amino acid mixture which is the same composition as casein also reduced myofibrillar protein degradation at 4 h after refeeding ( p < 0.05). An essential amino acid mixture (15.1%, corresponding to casein composition) and a leucine (2.9%) diets reduced the rate of myofibrillar protein degradation after refeeding ( p < 0.05), whereas a protein free diet did not. Administration of leucine alone (0.135 g/100 g body weight) by a feeding tube induced a decrease in the rate of myofibrillar protein degradation at 2 h after administration ( p < 0.05), whereas the serum insulin concentration was constant after leucine administration. These results suggested that leucine is one of regulating factors of myofibrillar protein degradation after refeeding of a protein diet.

Dietary protein regimens and chronic ethanol administration effects on sodium- and proton-dependent solute uptake in rat intestine

The effect of feeding ethanol daily for 40 days has been studied on intestinal uptake of glucose, glycine, and leucine in rats fed control, 8% protein (LP), and 30% protein (HP) diets. Na +-dependent uptake of glucose and glycine both at pH 7.2 and pH 5.5 was significantly depressed ( p < 0.001) in ethanol or LP diet-fed animals and remained unaffected in HP-fed rats compared to the controls. But ethanol administration to protein-malnourished rats enhanced the Na +-linked glucose and glycine uptakes. Leucine uptake remained unaffected under these conditions. Glucose uptake remained unaltered whereas glycine uptake was reduced when ethanol was administered to rats given HP diet. In the absence of Na +, uptake of glucose, glycine, and leucine was more at acidic pH compared to that at pH 7.2 under all the experimental conditions investigated. Proton-linked uptake of solutes was unaffected by feeding ethanol, LP, or HP diet in rats. Thus, chronic ethanol feeding specifically depresses the Na +-dependent uptake of glucose and glycine. Dietary protein content modifies ethanol effects on intestinal solute uptake in rats.

Time-dependent potentiation of insulin release induced by alpha-ketoisocaproate and leucine in rats: possible involvement of phosphoinositide hydrolysis.

The ability of the amino acid leucine and its keto acid, alpha-ketoisocaproate, to induce insulin release, to initiate phosphoinositide hydrolysis, and to amplify the subsequent insulin secretory response to glucose was assessed. In islets whose inositol-containing lipids were prelabelled with myo[2-3H]inositol, the addition of either compound resulted in an increase in insulin output, an increase in 3H efflux, rapid and significant increases in labelled inositol phosphate accumulation and a sustained increase in 3H efflux after removal of the stimulant. Direct measurements of labelled inositol phosphate accumulation in islets previously stimulated with alpha-ketoisocaproate demonstrate that this sustained increase in 3H efflux was the result of a persistent increase in phosphoinositide hydrolysis and was not simply a consequence of the hydrolysis of preformed inositol phosphates into more membrane permeable species. Prior exposure of islets to alpha-ketoisocaproate or leucine also resulted in an amplified secretory response to a subsequent glucose (10 mmol/l) stimulus. While peak first phase insulin release averaged 66 +/- 4 (mean +/- SEM, n = 18) pg.islet-1. min-1 from control islets, this value increased to 204 +/- 14 and 246 +/- 11 pg.islet-1.min-1 in the leucine or alpha-keto-isocaproate pretreated islets respectively. The duration of this amplified response paralleled the duration of the persistent increase in 3H efflux. Prior alpha-ketoisocaproate exposure also amplified the subsequent insulin secretory response to tolbutamide and glyceraldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of the level of dietary protein on the utilization of alpha-ketoisocaproate for protein synthesis

The efficiency of α-ketoisocaproate (KIC) as a dietary substitute for leucine in rats on varying protein intake was estimated by an isotopic method, previously shown to yield the same results as comparative growth experiments. 14C-KIC and 3H-leucine are injected orally. Six hours later the ratio, R, of 14C/3H in isolated proteins, divided by the same ratio in the injectate is measured. This ratio has been shown to be approximately equal to nutritional efficiency of KIC relative to leucine. As dietary protein increased from 6.3% to 48.3%, whole body protein R decreased from 0.515 ± 0.045 to 0.299 ± 0.016. Variations with protein intake were noted in R of protein isolated from individual organs. The magnitude of R in these organs varied two-fold, in the following sequence: brain > heart ≥ skeletal muscle ≥ salivary gland ≥ kidney > liver. Whole body protein R could be confidently predicted (r2 = 0.992) from R in the protein of kidney and muscle. Thus the nutritional efficiency of KIC as a dietary substitute for leucine in individual organs as well as in the whole animal is strongly dependent on the level of protein intake.

Effect of branched chain alpha-keto acids on kynurenine 3-hydroxylase from rat liver.

In the preceding paper, we found that branched chain alpha-keto acids, alpha-ketoglutarate and pyruvate were inhibitory to kynurenine 3-hydroxylase [EC 1.14.1.2] from Saccharomyces (Shin, M. et al. (1982): J. Nutr. Sci. vitaminol., 28, 191-201). As kynurenine 3-hydroxylase is reported to be a rate-limiting enzyme in the tryptophan-NAD pathway in rats, branched chain amino acids, branched chain alpha-keto acids and several other keto acids were tested for their effects on kynurenine 3-hydroxylase activity in the mitochondrial outer membrane fraction prepared from rat liver. In contrast with the yeast enzyme, the rat liver enzyme was resistant to alpha-keto acid-inhibition and more than 70% of the enzyme activity was maintained even in the presence of 20 mM of each alpha-keto acid. The present result implies that the mechanism of the hepatic NAD-lowering effect of leucine in rats might be completely different from that operative in Saccharomyces.

Effect of L-tryptophan and L-leucine on biosynthesis of niacin-related compounds in Saccharomyces carlsbergensis.

As a model system for investigating the mechanism of the hepatic NAD-lowering effect of leucine in rats, aerobically grown Saccharomyces carlsbergensis was used in this paper. Tryptophan supplementation of the medium doubled total niacin production by S. carlsbergensis. This elevation in total niacin was mainly due to increases in niacin (14 times) and niacinamide nucleotides (2 times). Among nucleotides, the NAD level doubled whereas NADH, NADP and NADPH levels dropped significantly. Simultaneous supplementation of the medium with leucine suppressed the elevation in total and free niacin levels. In the presence of tryptophan, approximately 50% of the total niacin was secreted in the medium in the form of free niacin, while in the presence of both tryptophan and leucine most of the total niacin remained in the cell. The specific activity of quinolinate phosphoribosyltransferase [EC 2.4.2.19] was not affected by supplementation of the medium with tryptophan and/or leucine. In contrast, the specific activity of nicotinamide deamidase [EC 3.5.1.19] increased fivefold in the presence of tryptophan. Simultaneous supplementation of the medium with leucine tryptophan. Simultaneous supplementation of the medium with leucine suppressed the increase in nicotinamide deamidase. Cellular incorporation of tryptophan was not affected by leucine simultaneously added as a supplement to the medium. Leucine did not have any inhibitory effect on total niacin synthesis from 3-hydroxyanthranilate. From the results, a possible mechanism for the inhibitory effect of leucine on the tryptophan-NAD pathway was discussed.

Studies on the Synthesis and Degradation of Light and Heavy Chains of Cardiac Myosin

Abstract The relative rate of degradation of the light and heavy chains of cardiac myosin was studied by the double isotope technique in which a single injection of [3H]leucine was administered 12 days prior to [14C]leucine in rats. As indicated by 14C:3H ratios of the purified myosin fractions, the light and heavy chains were degraded at heterogeneous rates, the heavy chains having a turnover rate twice that of the light chains.