Protein hydrolysates and tissue repair

In order to isolate mutants with impaired uptake of branched-chain amino acids, mutants were induced which on complex medium were sensitive to an inhibitor of branched-chain amino acid biosynthesis. Eighteen mutants of independent origin were found. Ten of them were assayed for branched-chain amino acid uptake. Three of these were impaired in the uptake of L-valine, L-isoleucine and L-leucine, while the rest were unaffected in uptake of any of the three amino acids. Kinetics of the uptake by one selected mutant and the parental strain S288C were compared to models for one or two systems obeying Michaelis-Menten kinetics. This analysis suggested that a high-affinity system for all three amino acids is absent in the mutant, whereas low-affinity uptake of L-isoleucine and L-leucine by one or more systems remains unaffected. Moreover, medium-affinity uptake components for L-valine and L-leucine, not originally seen in the wild type, were identified in the mutant. In the wild type, 10 mM of any of the amino acids L-alanine, L-cysteine, L-isoleucine, L-leucine, L-tryptophan and L-valine reduce uptake of any of the three branched-chain amino acids. We propose that a permease responsible for high-affinity uptake of the branched-chain amino acids in strain S288C is partially or completely inactive in the mutant. Tetrad analysis shows that the phenotype can be ascribed to a single Mendelian gene. The wild-type allele is denoted BAP1 for branched-chain amino acid permease. The BAP1-dependent system is different from the general amino acid permease.

Exercise training enhances oxidative capacity whereas detraining reduces mitochondrial content in skeletal muscle. The strategy to suppress the detraining-induced reduction of mitochondrial content has not been fully elucidated. As previous studies reported that branched-chain amino acid (BCAA) ingestion increased mitochondrial content in skeletal muscle, we evaluated whether BCAA supplementation could suppress the detraining-induced reduction of mitochondrial content. Six-week-old male Institute of Cancer Research (ICR) mice were randomly divided into four groups as follows: control (Con), endurance training (Tr), detraining (DeTr), and detraining with BCAA supplementation (DeTr + BCAA). Mice in Tr, DeTr, and DeTr + BCAA performed treadmill running exercises [20-30 m/min, 60 min, 5 times/week, 4 weeks]. Then, mice in DeTr and DeTr + BCAA were administered with water or BCAA [0.6mg/g of body weight, twice daily] for 2 weeks of detraining. In whole skeletal muscle, mitochondrial enzyme activities and protein content were decreased after 2 weeks of detraining, but the reduction was suppressed by BCAA supplementation. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) protein content, a master regulator of mitochondrial biogenesis, was decreased by detraining irrespective of BCAA ingestion. Regarding mitochondrial degradation, BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), a mitophagy-related protein, was significantly higher in the Tr group than in the DeTr + BCAA group, but not different from in the DeTr group. With respect to mitochondrial quality, BCAA ingestion did not affect oxygen consumption rate (OCR) and reactive oxygen species (ROS) production in isolated mitochondria. Our findings suggest that BCAA ingestion suppresses the detraining-induced reduction of mitochondrial content partly through inhibiting mitophagy.

Insulin stimulates branched chain amino acid uptake and diminishes nitrogen flux from skeletal muscle of injured patients

The current world population together with the predictions of further growth suggest that it is necessary to increase crop yields worldwide. Legumes are the second most important food crop after cereals, and thanks to their ability to establish a symbiotic relationship with soil bacteria, the impact of the use of nitrogen fertilizers on the environment is reduced. This symbiosis gives rise to the process known as biological nitrogen fixation (BNF), which consists in the reduction of molecular nitrogen to ammonium, from which plants synthesize organic nitrogenous products essential for their nutrition. Unfortunately, BNF is a very sensitive process to biotic and abiotic stresses such as salinity, drought, or nutrient limitation, among others. The general aim of this work was to gain further insights in the regulation of BNF and the physiological and biochemical mechanisms that plant activate in response to abiotic stresses. In order to counteract the negative effects of osmotic stresses, plant and bacteria are able to synthesise osmoprotectant compounds to maintain cell viability, e.g. the amino acid proline. A real-time monitoring of proline utilisation in both plant and bacterial systems is a first key step towards understanding the multiple roles of this molecule under osmotic stress situations. Our results in chapter one showed that, in bacteroids, proline accumulation does not occur during the stress phase, but during recovery, once optimal plant growth conditions are re-established. In chapter two, a proteomic and metabolic study was performed to gain further insights about amino acid metabolism in pea nodules. In the classical model of nutrient exchange between symbionts, plant supplies energy in the form of dicarboxylates to the N2-fixing bacteroids in exchange for ammonium. However, this classic model was challenged upon the observation that mutations in the general ABC amino-acid transporters AapJQMP and BraDEFGC in Rhizobium leguminosarum resulted in N starvation symptoms in both pea and bean plants. The uptake of branched-chain amino acids (BCAAs) from the plant by the bacteroid was found to be essential for an effective BNF at least in R. leguminosarum species. Another experimental approach to further understand the role of amino acid metabolism in nodules is the application of compounds that inhibit the biosynthesis of BCAAs in plant cells such as group B herbicides. These approaches allowed us to verify how the blockage of BCAA transport between symbionts had a greater effect on nodule metabolism than the inhibition of BCAA biosynthesis. In fact, BCAA biosynthesis was also inhibited due to the aap/bra double mutation. In chapter two, we also evaluate the effect of water deficit on nodule proteome, since among the strategies that plants use in response to abiotic stresses there are several related to amino acid metabolism. This study highlights the relevance of low abundant amino acids, such as methionine, aromatic amino acids or γ-aminobutyric acid, in the response to water deficit. Finally, until now no attempt has been made to carry out an integral approach in which possible changes caused by drought in carbon (C) allocation, and in addition, the effect on the consumption or accumulation of metabolites in all plant organs be analysed. For this purpose, in chapter three, the effect of drought on both the [U-13C]-sucrose distribution and ureides, organic acids and carbohydrates content were analysed. We found that drought decreased 13C transport to sink tissues and changed the priority of C allocation between sink organs.

Uptake of branched-chain amino acids by Saccharomyces cerevisiae from media containing a preferred nitrogen source is mediated by the permeases encoded by BAP2, BAP3, and VAP1/TAT1. The transcriptional activity of the BAP2 promoter is affected by a number of genes, including SSY1, which encodes an amino acid permease homologue that is necessary for transcription of BAP2. Other genes that control BAP2 encode known (Leu3p, Tup1p) and putative (Stp1p, Stp2p) transcription factors. We present evidence that the zinc-finger proteins Stp1p and Stp2p bind directly to the BAP2 promoter. Binding of Stplp to the BAP2 promoter in vivo and in vitro indicates that the STP gene family indeed encodes transcription factors. The presence of a Leu3p binding site in the BAP2 promoter is required for full promoter activity on synthetic complete medium. The capacity of Leu3p to activate BAP2 transcription correlates with conditions that affect the level of alpha-isopropyl malate. The effect of a tup1 deletion on BAP2 transcription depends on SSY1. In an ssy1 strain, the phenotype of tup1 conforms to the well-established role of Tup1p as part of a repressor complex, but in the SSY1 strain deletion of TUP1 causes a decrease in transcription, indicating that Tup1p may also have an activating role at the BAP2 promoter. Our results thus suggest a complex interplay between several transcription factors in the expression of BAP2.

ABSTRACTBiostimulant use is a promising agricultural strategy to maintain yield while decreasing mineral fertilisers and pesticides. Whereas the positive effect of protein hydrolysates (PH) on growth and yield has already been described, the underlying processes are not well understood. To better identify and characterise the physiological and molecular targets of PH, we first carried out in vitro experiments using the model species Arabidopsis thaliana. PH stimulated Arabidopsis root growth in a dose‐dependent manner, with high concentrations inhibiting growth. After having determined a stimulating PH concentration, we performed metabolomic and transcriptomic analyses. We observed that PH application increased amino acid levels in the plants, including a high level of glutamine, although it was only present in trace amounts in PH. These results suggest that amino acids are taken up and metabolized in the plant. PH also had a profound impact on the Arabidopsis transcriptome, with genes involved in nitrate and amino acid transport and assimilation showing a clear up‐ and down‐regulation, respectively. For example, the expression of the high‐affinity nitrate transporter NRT2.1 (NITRATE TRANSPORTER 2) was decreased, and consistently high‐affinity nitrate uptake and the development of lateral roots were diminished. The PH contained high levels of branched amino acids, which are known to induce the TOR kinase activity, a major driver of growth. Finally, high‐throughput phenotyping also showed that PH supply increased shoot growth. In conclusion, our findings show that PH has a clear and robust effect on Arabidopsis growth, but also on the transcriptome, metabolite levels, and physiological processes like nitrogen metabolism.

Ingestion of dietary protein is known to induce both insulin and glucagon secretion. These responses may be affected by the dose and the form (intact or hydrolysed) in which protein is ingested. The aim of the study was to investigate the effect of different amounts of intact protein and protein hydrolysate of a vegetable (soya) and animal (whey) protein on insulin and glucagon responses and to study the effect of increasing protein loads for both intact protein and protein hydrolysate in man. The study employed a repeated-measures design with Latin-square randomisation and single-blind trials. Twelve healthy non-obese males ingested three doses (0.3, 0.4 and 0.6 g/kg body weight) of intact soya protein (SPI) and soya protein hydrolysate (SPH). Another group of twelve healthy male subjects ingested three doses (0.3, 0.4 and 0.6 g/kg body weight) of intact whey protein (WPI) and whey protein hydrolysate (WPH). Blood was sampled before (t = 0) and 15, 30, 60, 90 and 120 min after protein ingestion for insulin, glucagon and glucose determination. SPI induced a higher total area under the curve for insulin and glucagon than SPH while no difference between WPI and WPH was found. Insulin and glucagon responses increased with increasing protein load for SPI, SPH, WPI and WPH, but the effect was more pronounced for glucagon. A higher dose of protein or its hydrolysate will result in a lower insulin:glucagon ratio, an important parameter for the control of postprandial substrate metabolism. In conclusion, insulin and glucagon responses were protein and hydrolysate specific.

Uptake of amino acids and inorganic nitrogen by two dominant temperate grasses

Amino acids can be utilized as a source of carbon and energy by many of the fungi in the class Oomycetes (Faro, 1971; Gleason, 1968; Gleason et al., 1970a, b). The number of amino acids which support the growth of a particular fungus as the single carbon source is quite variable among the Oomycetes studied. Furthermore, the rate of growth varies considerably with the amino acid provided. However, preliminary experiments with Leptomitus have shown that certain amino acids which cannot support growth individually will support growth when supplied as a mixture (Gleason, 1968). In their natural habitat Oomycetes come in contact with substrates rich in protein. These substrates must be attacked by extra-cellular proteases, releasing a mixture of amino acids which can be subsequently transported into the cell (Gleason et al., 1970b). Thus, these fungi are adapted to environmental conditions in which they are confronted with a mixture rather than a single amino acid. The uptake of individual amino acids from a mixture has not been measured in previous investigations on Oomycetes. This investigation was designed to measure the sequence and rate of uptake of 14 amino acids from a synthetic mixture by Saprolegnia sp. (47-15a). Each of 14 amino acids was added to a synthetic medium at a concentration of 0.3 g per liter: lysine, arginine, aspartic acid, threonine, serine, glutamic acid, proline, glycine, alanine, valine, methionine, isoleucine, leucine and phenylalanine. The total amount of the combined amino acids was 4.2 g per liter. A pH indicator (brom cresol purple), thiamin, and salts were added at concentrations given by Gleason (1968). The procedure for preparation of the medium, inoculation, and growth conditions are also described by Gleason (1968). During growth at 20 C on a rotary shaker, the pH was maintained close to 6.7 by aseptic addition of molar sulfuric acid. At intervals during growth 2-ml aliquots of the medium were removed from the culture vessel and

Some properties of the LIV-III transport system, a common factor in the entry of branched-chain amino acids in Salmonella typhimurium, have been studied using a double mutant, KA266 (livA brnQ), which is defective in transport via either the LIV-I or LIV-II system. The LIV-III system operates with a low affinity (Km: >15μM) for L-isoleucine, L-leucine, and L-valine. Isoleucine transport via the LIV-III system is inhibited competitively by leucine or valine, but non-competitively by L-cysteine. Uptake of branched-chain amino acids into cells is partially repressed when the bacteria are grown in the presence of 2mM glycyl-L-leucine or more. In the wild type, transport activity of the LIV-III system for isoleucine and leucine is not detected. The LIV-II system appears to show a low affinity for valine similar to the LIV-III system, hence the combined activity of these systems is expressed as the activity of the LIV-III system. The defect in transport via the LIV-II system leads to a great reduction in the transport of valine in the LIV-III system.

Hydrolysates can be obtained by enzymatic treatments and present various bioactivities and functional properties such as antioxidant, metal-chelation etc ., enabling their industrial application. These activities depend on amino acid composition, peptides size profile, peptide structure, which are related to proteolysis conditions. Hence, pea protein hydrolysates were produced by either Alcalase® followed by Flavourzyme® (Alc + Flav≤1 kDa) or Protamex® followed by Flavourzyme® (Prot + Flav≤1 kDa). Physico-chemical characterization of these hydrolysates shows that Prot + Flav≤1 kDa hydrolysate has higher degree of hydrolysis and and thus, higher small-size peptides quantity compared to Alc + Flav≤1 kDa. Besides, switchSENSE® technology allowed to unveil the presence of Ni 2+ chelating peptides, demonstrating the high potential of such innovative technology to screen metal-complexing peptides in hydrolysates. Moreover, the Prot + Flav≤1 kDa hydrolysate shows higher radical scavenging and reducing power, related to its higher degree of hydrolysis and small-size peptides quantity. The peptides identified in both hydrolysates by LC-MS/MS analysis contain high amount of aspartic acid, glutamic acid, proline, leucine, valine and glycine, which might be responsible of their antioxidant properties. The sequential proteolysis may increase metal chelating and antioxidant activities of hydrolysates. • SwitchSENSE evidences the presence of Ni 2+ chelating peptides in pea hydrolysates. •Pea hydrolysate can be valorized as antioxidants. •Metal chelating peptides with antioxidants activities were identified by LC/MS/MS. •Hydrolysates physicochemical properties were correlated to their antioxidant ones.

Staphylococcus aureus is an important pathogen that is associated with nosocomial infections, as well as food poisoning. This bacterium is resistant to antimicrobial agents and can survive in a wide range of environmental conditions. The aim of this study was to measure the uptake and release of amino acids by S. aureus at mid-exponential and stationary phases of growth following exposure to a combination of conditions including variations in temperature, pH and NaCl. Bacterial cells were grown up to mid-exponential and stationary phases in tryptic soy broth (TSB), where the supernatants were collected for analyses of amino acids to determine the uptake and release characteristics. The uptake/release of amino acids was estimated by subtracting the initial levels of the free amino acids in the media from those measured at mid-exponential and stationary phases of growth. When cells were grown at ideal conditions, the analyses revealed that significant uptake of amino acids had occurred by stationary phase compared with the mid-exponential phase. A substantial release of valine and tyrosine into the external media was observed by cells at stationary phase. At both phases, the uptake and release patterns were significantly different between cells grown under ideal control conditions, when compared with those grown under various combinations of sub-optimal environmental conditions. The analyses of the supernatants harvested from controls and treatment groups at exponential phase indicated that the total uptake of amino acids was reduced approximately five times by cells grown with addition of 2.5% NaCl or with pH6 at 35°C, and 2-fold by cells grown at pH8 at 35°C. However, the final quantities of amino acids taken up by cells grown to stationary phase did not significantly alter between control and treated samples. Valine was found to be the most abundant amino acid that was significantly released into the media at stationary phase by both control and treated samples. It was evident that diverse environmental conditions resulted in differential patterns of amino acid uptake and release during adaptation to designated conditions.

How cells utilize nutrients to produce the ATP needed for bioenergetic homeostasis has been well-characterized. What is less well-studied is how resting cells metabolically shift from an ATP-producing catabolic metabolism to a metabolism that supports anabolic growth. In metazoan organisms, the discovery of growth factors and the ability of their receptors to induce new transcription and translation led to the hypothesis that the bioenergetic and synthetic demands of cell growth were primarily met through the replacement of nutrients consumed during net macromolecular synthesis, a demand-based system of nutrient uptake. Recent data have challenged this hypothesis. Instead, there is increasing evidence that cellular nutrient uptake is a push system. Growth factor signaling has been linked to direct stimulation of nutrient uptake. The ability of growth factor signaling to increase the uptake of glucose, lipids, and amino acids to levels that exceed a cell's bioenergetic and synthetic needs has been documented in a wide variety of settings. In some tissues, this leads to the storage of the excess nutrients in the form of glycogen or fat. In others, the excess is secreted as lactate and certain nonessential amino acids. When growth factor signaling stimulates nutrient uptake to levels that exceed a cell's bioenergetic needs, adaptive changes in intermediate metabolism lead to the production of anabolic precursors that fuel the net synthesis of protein, lipids, and nucleic acids. Through the increased production of these precursors, growth factor signaling provides a supply-side stimulation of cell growth and proliferation.

The uptake of injected radioactive amino acids by the isolated islet and other tissues of the toadfish has been examined. The islet and most other tissues appear to actively transport most amino acids and, of the tissues studied, the islet is one of the most active in this regard. Most tissues accumulated the dicarboxylic acids to the greatest extent, with the basic amino acids and the neutral amino acids with unbranched side chains also being taken up well. However, each tissue showed a unique pattern of uptake of the different amino acids, and islet was distinguished by its high uptake of glycine 15 min after injection. The pattern of uptake of amino acids by toadfish islet does not appear to be related to their relative ability to stimulate insulin release from islets of other species or to their relative rates of metabolism, but does correlate well with the content of free amino acids in mouse islets and with the sum of the amino acid contents of anglerfish proinsulin and proglucagon.

We examined the acute effects of a 1-h strength training session (STS) on plasma amino acid concentration following orally ingestion of leucine, branched-chain amino acids (BCAAs) or glutamine in nine physically active men who participated in double-blinded and randomised experiments. The subjects took placebo, leucine, BCAAs, or glutamine capsules (50 mg/kg) in either rest (REST) or STS condition. Blood samples were taken before and at 30, 60, 90, and 120 min after the beginning of the treatment and they were assayed for plasma amino acids with HPLC. Following both leucine and BCAA ingestion the peak concentration of leucine was similar at rest (524 +/- 46 and 530 +/- 29 nmol/ml, respectively) and similar after STS (398 +/- 43 and 387 +/- 46 nmol/ml, respectively) but the rest and STS concentrations differed from each other (P < 0.01-0.001). The modelled polynomial data for the leucine treatment showed that the peak concentration of leucine occurred at 67 min at rest and at 90 min in STS (difference between REST and STS: P = 0.012). For the BCAA treatment the polynomial data showed that the peak concentration of leucine occurred at 72 min at rest and at 78 min in STS (P = 0.067). The peak concentration of glutamine was similar in both rest and STS condition and occurred at 60 min at rest and at 57 min in STS. In conclusion, 1-h of STS slows the increase in the peak concentration of plasma leucine similarly after oral ingestion of leucine or BCAAs but after oral ingestion of glutamine it has no slowing effect on glutamine concentration.