High Levels Of Leucine Research Articles

Control of Insulin Secretion by Production of Reactive Oxygen Species: Study Performed in Pancreatic Islets from Fed and 48-Hour Fasted Wistar Rats.

Mitochondria and NADPH oxidase are important sources of reactive oxygen species in particular the superoxide radical (ROS) in pancreatic islets. These molecules derived from molecular oxygen are involved in pancreatic β-cells signaling and control of insulin secretion. We examined the involvement of ROS produced through NADPH oxidase in the leucine- and/or glucose-induced insulin secretion by pancreatic islets from fed or 48-hour fasted rats. Glucose-stimulated insulin secretion (GSIS) in isolated islets was evaluated at low (2.8 mM) or high (16.7 mM) glucose concentrations in the presence or absence of leucine (20 mM) and/or NADPH oxidase inhibitors (VAS2870–20 μM or diphenylene iodonium—DPI—5 μM). ROS production was determined in islets treated with dihydroethidium (DHE) or MitoSOX Red reagent for 20 min and dispersed for fluorescence measurement by flow cytometry. NADPH content variation was examined in INS-1E cells (an insulin secreting cell line) after incubation in the presence of glucose (2.8 or 16.7 mM) and leucine (20 mM). At 2.8 mM glucose, VAS2870 and DPI reduced net ROS production (by 30%) and increased GSIS (by 70%) in a negative correlation manner (r = -0.93). At 16.7 mM glucose or 20 mM leucine, both NADPH oxidase inhibitors did not alter insulin secretion neither net ROS production. Pentose phosphate pathway inhibition by treatment with DHEA (75 μM) at low glucose led to an increase in net ROS production in pancreatic islets from fed rats (by 40%) and induced a marked increase (by 144%) in islets from 48-hour fasted rats. The NADPH/NADP+ ratio was increased when INS-1E cells were exposed to high glucose (by 4.3-fold) or leucine (by 3-fold). In conclusion, increased ROS production through NADPH oxidase prevents the occurrence of hypoglycemia in fasting conditions, however, in the presence of high glucose or high leucine levels, the increased production of NADPH and the consequent enhancement of the activity of the antioxidant defenses mitigate the excess of ROS production and allow the secretory process of insulin to take place.

FLCN Maintains the Leucine Level in Lysosome to Stimulate mTORC1

The intracellular amino acid pool within lysosome is a signal that stimulates the nutrient-sensing mTORC1 signalling pathway. The signal transduction cascade has garnered much attention, but little is known about the sequestration of the signalling molecules within the lysosome. Using human HEK293 cells as a model, we found that suppression of the BHD syndrome gene FLCN reduced the leucine level in lysosome, which correlated with decreased mTORC1 activity. Both consequences could be reversed by supplementation with high levels of leucine, but not other tested amino acids. Conversely, overexpressed FLCN could sequester lysosomal leucine and stimulate mTORC1 in an amino acid limitation environment. These results identify a novel function of FLCN: it controls mTORC1 by modulating the leucine signal in lysosome. Furthermore, we provided evidence that FLCN exerted this role by inhibiting the accumulation of the amino acid transporter PAT1 on the lysosome surface, thereby maintaining the signal level within the organelle.

High anabolic potential of essential amino acid mixtures in advanced nonsmall cell lung cancer

Conventional nutritional supplements are not or only partly successful in inducing protein accretion in advanced cancer, suggesting an attenuated anabolic response. To prevent muscle wasting and its deleterious consequences, generating an anabolic response is crucial. Dietary essential amino acids (EAA) have anabolic properties in other wasting diseases; however, data in advanced cancer are lacking. In 13 patients with advanced nonsmall-cell lung cancer (NSCLC) (stage III and IV) and 11 healthy age-matched subjects, we measured protein synthesis and breakdown of the whole body, and net protein anabolism (difference between protein synthesis and breakdown) after intake of 14 g of free EAA with high leucine levels (EAA/leucine) versus a balanced amino acid mixture containing both EAA and non-EAA as present in whey protein, according to a randomized, double-blind, crossover design. Protein synthesis and net protein anabolism were higher after intake of the EAA/leucine than the balanced amino acid mixture (P < 0.001), independent of presence of cancer. A highly significant linear relationship between net protein anabolism and the amount of EAA available in the systemic circulation (R(2): 0.85, P < 0.001) was found in both groups. The presence of muscle or recent weight loss, systemic inflammatory response, or length of survival did not influence this relationship. High leucine levels in the EAA/leucine mixture was of no anabolic benefit. There is no anabolic resistance or attenuated anabolic potential to intake of 14 g of EAA/leucine or balanced amino acid mixture in advanced (mainly stage III) NSCLC. The high anabolic potential of dietary EAA in cancer patients is independent of their nutritional status, systemic inflammatory response or disease trajectory, suggesting a key role of EAA in new nutritional approaches to prevent muscle loss, thereby improving outcome of patients with advanced cancer. NCT01172314.

Metabolic heterogeneity of follicular amino acids in polycystic ovary syndrome is affected by obesity and related to pregnancy outcome

BackgroundPolycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder frequently accompanied by obesity and by insulin resistance, and patients with this syndrome suffer from infertility and poor pregnancy outcome. Disturbances in plasma amino acid (AA) metabolism have been implicated in women with PCOS. However, direct evidence on follicular AA metabolic profiles in PCOS patients and their relationship with pregnancy outcome is sparse.MethodsWe conducted a prospective study in 63 PCOS patients and 48 controls in the Division of Reproductive Center, Peking University Third Hospital. Follicular AA levels were measured by the liquid chromatography-tandem mass spectrometric method, and the results were analyzed based on different grouping criteria.ResultsThe levels of aromatic amino acid (AAA) increased in PCOS patients independent of obesity (P < 0.05), whereas the levels of branched-chain amino acid (BCAA), glutamic acid, phenylalanine, alanine, and arginine increased with body mass index irrespective of the PCOS status (all P < 0.05). In addition, compared with non insulin resistant-PCOS patients and controls, insulin resistant-PCOS group had higher levels of leucine, valine and glutamic acid (all P < 0.05). In PCOS group, aspartic acid and serine levels were elevated in pregnant patients compared with the non-pregnant subjects (both P < 0.05). Moreover, the levels of BCAA and valine were higher in the non-pregnant group than in the pregnant group (both P < 0.05). The pregnancy rate (45.00%) of subjects with elevated BCAA level was significantly lower than that (66.67%) in control subjects (P = 0.036) at a BCAA cutoff value of 239.10 μM, while the abortion rate was much higher (33.33% versus 2.78%, P = 0.004).ConclusionsBoth PCOS and obesity were accompanied by follicular AA metabolic disturbances, with obesity exerting a more pronounced effect on AA metabolic profiles. The disruptions in specific AAs in the follicular fluid might account for the inferior pregnancy outcome in obese patients and increased risk of abortion in PCOS patients.

Dietary essential amino acids are highly anabolic in pediatric patients with cystic fibrosis

Current nutritional approaches have been partially successful in Cystic Fibrosis (CF). Essential amino acids mixtures with high Leucine levels (EAA) have anabolic properties in catabolic conditions, however data in CF are lacking. On two days according a randomized crossover design, 15 pediatric CF patients ingested 6.7 g EAA versus mixture of total amino acids as present in whey. Whole body protein and Arginine metabolism (as EAA lack Arginine) were assessed by stable isotope methodology. Protein synthesis (P<0.05) but not protein breakdown was higher after EAA and 70% higher values for net anabolism (P<0.001)were found both in patients with and without nutritional failure. Arginine turnover was lower (P<0.001) and de novo Arginine synthesis tended lower (P=0.09) after EAA. Nitric oxide synthesis was not different. CF patients are highly responsive to EAA intake independent of their nutritional status. Addition of Arginine to the EAA mixture may be warranted in CF.

The possible role of leucine in modulating glucose homeostasis under distinct catabolic conditions

Branched-chain amino acids (BCAA) (especially leucine) have been shown to activate protein synthesis pathways, decrease proteolysis and increase insulin sensitivity. Furthermore, it appears that leucine can be used as a nutritional therapy to avoid sarcopenia and skeletal muscle atrophy due to immobilization or glucocorticoid treatment. However, it is of note that all of these conditions are related to insulin resistance to varying degrees and affect different tissues, particularly skeletal muscle. Additionally, evidence from recent studies demonstrate that a combination of protein containing high levels of leucine with nutrients containing saturated fatty acids or an excess of leucine are capable of inducing insulin resistance. From this discussion, a few major questions arise. First, what is the role of a combination of macronutrients in inducing insulin resistance? Second, in insulin resistance, does leucine supplementation follow the same path observed under healthy conditions? Finally, what are the dose-dependent outcome and the latency of leucine effect under such conditions? The present article discusses these questions based on data from the literature and experiments performed by our group.

Constitutive activation of mTOR pathway by leucine causes heart hypertrophy which can be blocked by rapamycin

Mitochondrial branched chain aminotransferase (BCATm) KO mice fed an amino acid diet containing intermediate levels of branched chain amino acids (BCAA) to prevent toxicity and wild type mice fed an amino acid diet with normal levels of the BCAA were studied. To determine the mTOR specific effects of constitutively high concentration of BCAA in the blood, BCATm KO and wild type mice were fed diets with and without encapsulated rapamycin. Following thirteen days of feeding, the mice were sacrificed without fasting. The plasma levels of BCAAs were found to be higher in the KO mice without any effect by rapamycin. Despite higher concentration of leucine, food intake was higher for the KO mice suggesting that chronically high leucine levels cannot suppress food intake. BCATm KO mice exhibited heart but not skeletal muscle hypertrophy which was prevented by dietary rapamycin. In addition, feeding the KO mice with a diet containing rapamycin resulted in increased activity. Rapamycin feeding had no effect on heart size of the wild type mice although mouse activity was reduced. Western blotting showed the activation of mTOR signaling pathway in KO mice. Our results suggest that leucine induced protein synthesis through the activation of mTOR that leads to growth is tissue dependent and leucine alone is insufficient to induce skeletal muscle hypertrophy. (Support Ajinomoto and Virginia Tech)

High levels of leucine are required for the upregulation of amino acid transporters in human skeletal muscle following essential amino acid ingestion

The goal of this study was to determine whether the upregulation of select amino acid transporters in human skeletal muscle is dependent upon the amount of leucine in an essential amino acid (EAA) solution. Young, healthy individuals ingested 10g of EAA with either a leucine concentration similar to high quality protein (CTRL; 1.8g leucine [n=6]) or an increased leucine concentration (LEU; 3.5g leucine [n=7]). Muscle biopsies were obtained prior to and at 1 and 2h following EAA ingestion to examine select amino acid transporter mRNA expression. Following EAA ingestion, CTRL had little change in amino acid transporter mRNA expression, whereas at 1h post ingestion LEU experienced an increase in L-type (LAT1), sodium-coupled neutral (SNAT2), and proton-coupled (PAT1) amino acid transporter mRNA expression. These data highlight an important role for leucine availability in the upregulation of select amino acid transporters. Furthermore, additional leucine may sensitize the muscle cell to subsequent feedings by increasing the availability of amino acid transporters. Funding: NIH/NIAMS R01 AR049877, NIA P30AG024832, NIH 1UL1RR029876-01

Effect of dietary leucine and lysine levels on intramuscular fat content in finishing pigs

Feeding high leucine levels (2.0 and 3.0% total dietary leucine) to finishing pigs (73 to 127 kg liveweight) increased the intra muscular fat content of the longissimus muscle in pigs fed diets with low lysine levels (0.5% total dietary lysine) but not in animals fed high lysine levels (0.7 %). Key words: Lysine, leucine, growth performance, carcass and meat quality

Chronic exposure to high leucine impairs glucose-induced insulin release by lowering the ATP-to-ADP ratio.

Exposure of rat pancreatic islets to 20 mM leucine for 24 h reduced insulin release in response to glucose (16.7 and 22.2 mM). Insulin release was normal when the same islets were stimulated with leucine (40 mM) or glyburide (1 microM). To investigate the mechanisms responsible for the different effect of these secretagogues, we studied several steps of glucose-induced insulin secretion. Glucose utilization and oxidation rates in leucine-precultured islets were similar to those of control islets. Also, the ATP-sensitive K(+) channel-independent pathway of glucose-stimulated insulin release, studied in the presence of 30 mM K(+) and 250 microM diazoxide, was normal. In contrast, the ATP-to-ADP ratio after stimulation with 22.2 mM glucose was reduced in leucine-exposed islets with respect to control islets. The decrease of the ATP-to-ADP ratio was due to an increase of ADP levels. In conclusion, prolonged exposure of pancreatic islets to high leucine levels selectively impairs glucose-induced insulin release. This secretory abnormality is associated with (and might be due to) a reduced ATP-to-ADP ratio. The abnormal plasma amino acid levels often present in obesity and diabetes may, therefore, affect pancreatic islet insulin secretion in these patients.

The Isolation and Detection of Non-Collagenous Proteins from the Compact Bone of the Dinosaur Iguanodon

This report describes the isolation of guanidinium chloride extractable protein from deminer-alised bone extracts obtained from the 125–130 mya dinosaur Iguanodon. Protein products were isolated in the Mr. range 5,000–66,000 using SDS-PAGE and represent the first electro-phoretically defined proteins isolated from dinosaur tissues. The levels of glycine, aspartate and serine tentatively suggest the presence of phosphoproteins. Hydroxylysine and hydroxy-proline were not detected, confirming the presence of non-collagenous material. In addition the absence of ornithine confirmed lack of bacterial contamination. The relatively high level of leucine in the 2MNaCl NaCl fractions together with the abolition of alcian blue reactivity following protease-free chondroitinase digestion suggests the presence of proteoglycans. The study is of interest in describing the early proteins laid down in mineralised tissues for epitac-tic crystal growth and may provide evidence on evolutionary aspects of bone proteins.

Electrophoretic and amino acid compositions of the proteins of maize lines differing in the anthocyan coloration of the seeds

The electrophoretic and amino acid compositions of the proteins of lines of maize differing in the anthocyan coloration of their seeds have been investigated. The molecular masses of the quantitatively main components ranged from 11 to 42 kDa for the albumins and from 15 to 81 kDa for the globulins. Differences between the lines were observed in the levels of components with molecular masses of from 11 to 31 kDa. The amino acid compositions of the zeins of the lines studied were characterized by high levels of leucine, glutamic acid, aspartic acid, alanine and serine, and those of the other groups of proteins by high levels of arginine, glutamic acid, and glycine.

LEUCINE AS AN ANTAGONIST OF GLUCOCORTICOID-INDUCED MUSCLE ATROPHY 1300

This study was undertaken to examine whether leucine infusion is capable of preventing skeletal muscle atrophy associated with glucocorticoid treatment. Rats were administered hydrocortisone 21-acetate (CA) or the dosing vehicle(carboxymethyl cellulose (CMC) and were infused with saline (SAL) or leucine(LEU, 168 mM, 2.15 ml/hr) for 7 consecutive days. Quadriceps and gastrocnemius muscle masses following treatments showed the following pattern: LEU-CMC>SAL-CMC>LEU-CA>SAL-CA. When normalized to body mass, all groups were significantly different from each other with atrophy prevention≈50%. Leucine infusion resulted in higher serum leucine levels in CMC (52%) and in CA-treated (128%) rats as compared to their saline-infused counterparts. Serum glutamine or alanine levels were not altered by any treatment, but serum glutamate concentrations were reduced in CA-treated animals. Glutamine levels were similar (≈3.8 μmol/g) in fast-twitch red(deep quadriceps FTR) muscles among all groups. In fast-twitch white(superficial quadriceps FTW) muscles of saline-infused animals, glutamine concentrations were reduced by hormone treatment from 1.8±0.2 to 1.2±0.6 μmol/g. Leucine supplementation did not completely reverse this effect. Leucine infusion resulted in higher leucine levels in FTR(223±17 vs 97±9 nmol/g) and FTW (312±52 vs 104±17 nmol/g) muscles of glucocorticoid-treated rats. These data show the potential effects of leucine in counteracting muscle atrophy from glucocorticoids. The elevated skeletal muscle levels of leucine without concomitant changes in muscle glutamine may provide a sufficient stimulus to maintain a cellular environment, which is sufficient to attenuate the glucocorticoid-related catabolic effects on muscle mass.

The influence of short-term feeding of amino acid-deficient diets and high dietary leucine levels on the intramuscular fat content of pig muscle

AbstractThe objective of the study was to investigate the influence of amino acid-deficient diets and high dietary leucine levels offered for intervals of either 21 or 35 days pre-slaughter on the intramuscular fat content of pig muscle. Twenty-four hybrid gilts were offered individually ad libitum one of four diets that comprised combinations of amino acid levels (supplemented or deficient: 5·6 v. 4·0 g lysine per kg) and low or high leucine levels (10·3 v. 30·3 g leucine per kg). Live weights at the start of the study were 74·5 and 87·0 kg for pigs on the 35- and 21-day feeding regimens, respectively. There were no significant effects of dietary amino acid level, leucine level, or feeding interval on food intake, daily live-weight gain or food efficiency. Pigs given the amino acid-deficient diets had lower killing-out proportions (736 v. 747 (s.e. 3·4) g/kg; P &lt; 0·05) and longissimus dorsi muscle areas (32·6 v. 35·7 (s.e. 0·71) cm2, P &lt; 0·01) than those given amino acid-supplemented diets. Subjective score for longissimus dorsi colour indicated that pigs on the high leucine diet had darker muscle than those on the low leucine diet. Intramuscular fat contents of the longissimus dors i muscle at the Wth/llth rib and the 3rd/4th lumbar vertebra and of the semimembranosu s muscle were increased by 19, 18 and 18 g/kg, respectively (P &lt; 0·05), for pigs given amino acid-deficient compared with those given amino acid-supplemented diets. Dietary leucine level had no significant effect on intramuscular fat level. Pigs given the diets for 35, compared with 21, days had higher fat levels in the semimembranosu s muscle (52 v. 30 (s.e. 5·2) g/kg; P &lt; 0·01) but not in the other two muscle locations studied. This study suggests that relatively short-term feeding of amino acid-deficient diets can produce substantial increases in intramuscular fat levels.

Sequence and structural similarities between the leucine-specific binding protein and leucyl-tRNA synthetase of Escherichia coli.

A role for the leucyl-tRNA synthetase (EC 6.1.1.4) has been established for regulating the transport of leucine across the inner membrane of Escherichia coli by the leucine, isoleucine, valine (LIV-I) transport system. This transport system is mediated by interactions of periplasmic binding proteins with a complex of membrane-associated proteins, and transcription of the high-affinity branched-chain amino acid transport system genes is repressed by growth of E. coli on high levels of leucine. We now report results from sequence comparisons and structural modeling studies, which indicate that the leucine-specific binding protein, one of the periplasmic components of the LIV-I transport system, contains a 121-residue stretch, representing 36% of the mature protein, which displays both sequence and structural similarities to a region within the putative nucleotide-binding domain of leucyl-tRNA synthetase. Early fusion events between ancestral genes for the leucine-specific binding protein and leucyl-tRNA synthetase could account for the similarity and suggest that processes of aminoacylation and transport for leucine in E. coli may be performed by evolutionarily interrelated proteins.

Plasma level of branched chain alpha-amino acids following its alpha-keto acid administration to rats.

To detect the effective substance accelerating the conversion of branched chain alpha-keto acids (BCKA) to corresponding amino acids, some sugars, sugar alcohols, ethanol, octanoate and ATP were tested with intravenous injection of three BCKA to rats. Plasma leucine levels 30 min after BCKA injection in rats, which were infused with 2 M solution of fructose or sorbitol, reached the significantly high peak corresponding to 860% and 790% of the basal levels. Administration of ethanol (2 or 3.5 M) or octanoate (4.5 mM) also resulted in significantly high levels of leucine in plasma following BCKA injection as compared with control. Molar ratio of plasma branched chain amino acid (BCAA)/aromatic amino acid 30 min after BCKA injection increased to 12.9 in control rats. The ratios 60 and 120 min after BCKA administration were significantly higher in rats treated with ethanol (3.5 M) or octanoate (4.5 mM) than those in control. These results suggest that infusion of the metabolites inducing high NADH/NAD ratio in their metabolic pathways results in inhibition of BCKA decarboxylation and, as a result, enhancement of transamination of BCKA into BCAA.

Effects of Sulfaguanidine and Amino Acid Imbalances on the Induction of Niacin Deficiency in Mature and Immature Japanese Quail (Coturnix coturnix japonica)

Effects of sulfaguanidine, leucine, or leucine plus isoleucine in a niacin free diet on body weight changes, liver weight, pyridine nucleotide concentrations, and activities of enzymes in mature female or immature Japanese quail were measured. The inclusion of .5% of sulfaguanidine or a high level of leucine in a niacin free diet had no influence during an 8-week period on liver pyridine nucleotide levels and the development of niacin deficiency symptoms in mature female quail. This would suggest that the mature female quail has a low requirement for dietary niacin. Immature quail were also tested to study the effects of amino acid imbalance on the induction of niacin deficiency. Although there was a marked reduction in body weight gains by the administration of leucine or leucine plus isoleucine, these treatments did not appear to accentuate niacin deficiency symptoms in these animals. Also pyridine nucleotide levels and malic enzyme, glyceral-dehyde-3-phosphate dehydrogenase, and aldolase activities in liver tissues were not affected by these treatments.

Reconstitution of neutral amino acid transport from partially purified membrane components from Ehrlich ascites tumor cells.

Solubilized protein fractions have been obtained from plasma membranes of Ehrlich ascites cells either by extraction with 0.5% Triton X-100 or by extraction with 2% cholate. Partial purification of the solubilized protein fraction has been obtained by utilizing a combination of ammonium sulfate precipitation and column chromatography. Leucine-binding activity has been detected in the Triton X-100 solubilized membrane fraction. The leucine-binding activity was measured by equilibrium dialysis and was saturable with high levels of leucine or phenylalanine and is not strongly effected by alanine. These properties are similar to those previously identified as System L. In addition, the cholate extracted protein fraction was partially purified and reconstituted into liposomes. Sodium dependent uptake of alanine and leucine could be demonstrated in the reconstituted vesicles. Concentrative uptake was dependent upon a sodium gradient. A membrane potential produced by valinomycin mediated potassium diffusion in the presence of sodium also stimulated amino acid transport in reconstituted liposomes.