The genetic architecture of branched-chain amino acid accumulation in tomato fruits

Previous studies of the genetic architecture of fruit metabolic composition have allowed us to identify four strongly conserved co-ordinate quantitative trait loci (QTL) for the branched-chain amino acids (BCAAs). This study has been extended here to encompass the other 23 enzymes described to be involved in the pathways of BCAA synthesis and degradation. On coarse mapping the chromosomal location of these enzymes, it was possible to define the map position of 24 genes. Of these genes eight co-localized, or mapped close to BCAA QTL including those encoding ketol-acid reductoisomerase (KARI), dihydroxy-acid dehydratase (DHAD), and isopropylmalate dehydratase (IPMD). Quantitative evaluation of the expression levels of these genes revealed that the S. pennellii allele of IPMD demonstrated changes in the expression level of this gene, whereas those of KARI and DHAD were invariant across the genotypes. Whilst the antisense inhibition of IPMD resulted in increased BCAA, the antisense inhibition of neither KARI nor DHAD produced a clear effect in fruit BCAA contents. The results are discussed both with respect to the roles of these specific enzymes within plant amino acid metabolism and within the context of current understanding of the regulation of plant branched-chain amino acid metabolism.     

Autophagy contributes to sulfonylurea herbicide tolerance via GCN2-independent regulation of amino acid homeostasis

Sulfonylurea (SU) herbicides inhibit branched-chain amino acid (BCAA) biosynthesis by targeting acetolactate synthase. Plants have evolved target-site resistance and metabolic tolerance to SU herbicides; the GCN2 (general control non-repressible 2) pathway is also involved in SU tolerance. Here, we report a novel SU tolerance mechanism, autophagy, which we call ‘homeostatic tolerance,’ is involved in amino acid signaling in Arabidopsis. The activation and reversion of autophagy and GCN2 by the SU herbicide tribenuron-methyl (TM) and exogenous BCAA, respectively, confirmed that TM-induced BCAA starvation is responsible for the activation of autophagy and GCN2. Genetic and biochemical analyses revealed a lower proportion of free BCAA and more sensitive phenotypes in atg5, atg7, and gcn2 single mutants than in wild-type seedlings after TM treatment; the lowest proportion of free BCAA and the most sensitive phenotypes were found in atg5 gcn2 and atg7 gcn2 double mutants. Immunoblotting and microscopy revealed that TM-induced activation of autophagy and GCN2 signaling do not depend on the presence of each other, and these 2 pathways may serve as mutually compensatory mechanisms against TM. TM inhibited the TOR (target of rapamycin), and activated autophagy in an estradiol-induced TOR RNAi line, suggesting that TM-induced BCAA starvation activates autophagy, probably via TOR inactivation. Autophagy and GCN2 were also activated, and independently contributed to TM tolerance in plants conferring metabolic tolerance. Together, these data suggest that autophagy is a proteolytic process for amino acid recycling and contributes to GCN2-independent SU tolerance, probably by its ability to replenish fresh BCAA.     

Metabolism of branched-chain amino acids in starved rats: the role of hepatic tissue

Parameters of branched-chain amino acids (BCAA; leucine, isoleucine and valine) and protein metabolism were evaluated using L-[1-(14)C]leucine and alpha-keto[1-(14)C]isocaproate (KIC) in the whole body and in isolated perfused liver (IPL) of rats fed ad libitum or starved for 3 days. Starvation caused a significant increase in plasma BCAA levels and a decrease in leucine appearance from proteolysis, leucine incorporation into body proteins, leucine oxidation, leucine-oxidized fraction, and leucine clearance. Protein synthesis decreased significantly in skeletal muscle and the liver. There were no significant differences in leucine and KIC oxidation by IPL. In starved animals, a significant increase in net release of BCAA and tyrosine by IPL was observed, while the effect on other amino acids was non-significant. We conclude that the protein-sparing phase of uncomplicated starvation is associated with decreased whole-body proteolysis, protein synthesis, branched-chain amino acid (BCAA) oxidation, and BCAA clearance. The increase in plasma BCAA levels in starved animals results in part from decreased BCAA catabolism, particularly in heart and skeletal muscles, and from a net release of BCAA by the hepatic tissue.     

Acute hyperammonemia activates branched-chain amino acid catabolism and decreases their extracellular concentrations: different sensitivity of red and white muscle

Hyperammonemia is considered to be the main cause of decreased levels of the branched-chain amino acids (BCAA), valine, leucine, and isoleucine, in liver cirrhosis. In this study we investigated whether the decrease in BCAA is caused by the direct effect of ammonia on BCAA metabolism and the effect of ammonia on BCAA and protein metabolism in different types of skeletal muscle. M. soleus (SOL, slow-twitch, red muscle) and m. extensor digitorum longus (EDL, fast-twitch, white muscle) of white rat were isolated and incubated in a medium with or without 500 μM ammonia. We measured the exchange of amino acids between the muscle and the medium, amino acid concentrations in the muscle, release of branched-chain keto acids (BCKA), leucine oxidation, total and myofibrillar proteolysis, and protein synthesis. Hyperammonemia inhibited the BCAA release (81% in SOL and 60% in EDL vs. controls), increased the release of BCKA (133% in SOL and 161% in EDL vs. controls) and glutamine (138% in SOL and 145% in EDL vs. controls), and increased the leucine oxidation in EDL (174% of controls). Ammonia also induced a significant increase in glutamine concentration in skeletal muscle. The effect of ammonia on intracellular BCAA concentration, protein synthesis and on total and myofibrillar proteolysis was insignificant. The data indicates that hyperammonemia directly affects the BCAA metabolism in skeletal muscle which results in decreased levels of BCAA in the extracellular fluid. The effect is associated with activated synthesis of glutamine, increased BCAA oxidation, decreased release of BCAA, and enhanced release of BCKA. These metabolic changes are not directly associated with marked changes in protein turnover. The effect of ammonia is more pronounced in muscles with high content of white fibres.     

Reprograming of proteasomal degradation by branched chain amino acid metabolism

Branched‐chain amino acid (BCAA) metabolism is a central hub for energy production and regulation of numerous physiological processes. Controversially, both increased and decreased levels of BCAAs are associated with longevity. Using genetics and multi‐omics analyses in Caenorhabditis elegans, we identified adaptive regulation of the ubiquitin‐proteasome system (UPS) in response to defective BCAA catabolic reactions after the initial transamination step. Worms with impaired BCAA metabolism show a slower turnover of a GFP‐based proteasome substrate, which is suppressed by loss‐of‐function of the first BCAA catabolic enzyme, the branched‐chain aminotransferase BCAT‐1. The exogenous supply of BCAA‐derived carboxylic acids, which are known to accumulate in the body fluid of patients with BCAA metabolic disorders, is sufficient to regulate the UPS. The link between BCAA intermediates and UPS function presented here sheds light on the unexplained role of BCAAs in the aging process and opens future possibilities for therapeutic interventions.     

Plasma amino acid imbalance in alcoholic liver cirrhosis

Plasma amino acid concentrations and plasma glucagon and serum insulin levels were studied in male patients with compensated alcoholic and nonalcoholic liver cirrhosis. Age, nutritional status, and liver function tests were similar in both groups; none of the patients presented hepatic encephalopathy. Plasma valine and leucine concentrations were lower, and tyrosine, higher in alcoholic than nonalcoholic liver cirrhosis. As a result, the molar ratios of branched-chain amino acids (BCAA) to aromatic amino acids (AAA) were reduced markedly in this group. Although correlation coefficients comparing BCAA/AAA ratios and KICG in alcoholic and nonalcoholic liver cirrhosis were similar, a steeper regression line was observed in alcoholics. Plasma glucagon and proline levels were significantly higher in alcoholic than nonalcoholic liver cirrhosis, the former correlated with AAA concentrations only in alcoholic liver cirrhosis, but not with BCAA levels. These results indicated that alcoholic liver cirrhosis presented a more deranged plasma amino acid pattern than nonalcoholic, and the amino acid imbalances, except for depressed BCAA and elevated proline, were derived, in part, from the hyperglucagonemia.     

Total branched-chain amino acids requirement in patients with maple syrup urine disease by use of indicator amino acid oxidation with L-[1-13C]phenylalanine

Maple syrup urine disease (MSUD) is an autosomal recessive disorder caused by defects in the mitochondrial multienzyme complex branched-chain alpha-keto acid dehydrogenase (BCKD; EC 1.2.4.4), responsible for the oxidative decarboxylation of the branched-chain ketoacids (BCKA) derived from the branched-chain amino acids (BCAA) leucine, valine, and isoleucine. Deficiency of the enzyme results in increased concentrations of the BCAA and BCKA in body cells and fluids. The treatment of the disease is aimed at keeping the concentration of BCAA below the toxic concentrations, primarily by dietary restriction of BCAA intake. The objective of this study was to determine the total BCAA requirements of patients with classical MSUD caused by marked deficiency of BCKD by use of the indicator amino acid oxidation (IAAO) technique. Five MSUD patients from the MSUD clinic of The Hospital for Sick Children participated in the study. Each was randomly assigned to different intakes of BCAA mixture (0, 20, 30, 50, 60, 70, 90, 110, and 130 mg.kg(-1).day(-1)), in which the relative proportion of BCAA was the same as that in egg protein. Total BCAA requirement was determined by measuring the oxidation of l-[1-(13)C]phenylalanine to (13)CO(2). The mean total BCAA requirement was estimated using a two-phase linear regression crossover analysis, which showed that the mean total BCAA requirement was 45 mg.kg(-1).day(-1), with the safe level of intake (upper 95% confidence interval) at 62 mg.kg(-1).day(-1). This is the first time BCAA requirements in patients with MSUD have been determined directly.     

Peptide inhibitors MAP the way towards fighting anthrax pathogenesis

BCAAs (branched-chain amino acids) are indispensable (essential) amino acids that are required for body protein synthesis. Indispensable amino acids cannot be synthesized by the body and must be acquired from the diet. The BCAA leucine provides hormone-like signals to tissues such as skeletal muscle, indicating overall nutrient sufficiency. BCAA metabolism provides an important transport system to move nitrogen throughout the body for the synthesis of dispensable (non-essential) amino acids, including the neurotransmitter glutamate in the central nervous system. BCAA metabolism is tightly regulated to maintain levels high enough to support these important functions, but at the same time excesses are prevented via stimulation of irreversible disposal pathways. It is well known from inborn errors of BCAA metabolism that dysregulation of the BCAA catabolic pathways that leads to excess BCAAs and their alpha-keto acid metabolites results in neural dysfunction. In this issue of Biochemical Journal, Joshi and colleagues have disrupted the murine BDK (branched-chain alpha-keto acid dehydrogenase kinase) gene. This enzyme serves as the brake on BCAA catabolism. The impaired growth and neurological abnormalities observed in this animal show conclusively the importance of tight regulation of indispensable amino acid metabolism.     

Catabolism of nutritionally essential amino acids in developing porcine enterocytes

This study was conducted using the piglet model to test the hypothesis that mucosal cells of the neonatal small intestine can degrade nutritionally essential amino acids (EAA). Enterocytes were isolated from the jejunum of 0-, 7-, 14-, and 21-day-old pigs, and incubated for 45 min in Krebs buffer containing plasma concentrations of amino acids and one of the following L-[1-¹⁴C]- or L-[U-¹⁴C]-amino acids plus unlabeled tracees at 0.5, 2, or 5 mM: histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine. In these cells, branched-chain amino acids (BCAA) were extensively transaminated and 15–50% of decarboxylated branched-chain α-ketoacids (BCKA) were oxidized to CO₂ depending on the age of piglets. BCAA transamination increased but their decarboxylation decreased between 0 and 14 days of age. Addition of 1 and 2 mM α-ketoglutarate to incubation medium dose-dependently stimulated BCAA transamination without affecting their decarboxylation. Western blot analysis revealed that the abundance of mitochondrial BCAA aminotransferase declined but cytosolic BCAA aminotransferase increased between 0 and 14 days of age, with the cytosolic protein being the major isoform in 7- to 21-day-old pigs. BCKA dehydrogenase protein existed primarily as the phosphorylated (inactive) form in enterocytes of newborn pigs and its levels were markedly reduced in older pigs. All measured parameters of BCAA metabolism did not differ between 14- and 21-day-old pigs. In contrast to BCAA, catabolism of methionine and phenylalanine was negligible and that of other EAA was absent in enterocytes from all ages of piglets due to the lack of key enzymes. These results indicate that enterocytes are an important site for substantial degradation of BCAA but not other EAA in the neonatal gut.     

General Control Nonderepressible 2 (GCN2) Kinase Protects Oligodendrocytes and White Matter during Branched-chain Amino Acid Deficiency in Mice

Branched-chain amino acid (BCAA) catabolism is regulated by branched-chain α-keto acid dehydrogenase, an enzyme complex that is inhibited when phosphorylated by its kinase (BDK). Loss of BDK function in mice and humans causes BCAA deficiency and epilepsy with autistic features. In response to amino acid deficiency, phosphorylation of eukaryotic initiation factor 2α (eIF2∼P) by general control nonderepressible 2 (GCN2) activates the amino acid stress response. We hypothesized that GCN2 functions to protect the brain during chronic BCAA deficiency. To test this idea, we generated mice lacking both Gcn2 and Bdk (GBDK) and examined the development of progeny. GBDK mice appeared normal at birth, but they soon stopped growing, developed severe ataxia, tremor, and anorexia, and died by postnatal day 15. BCAA levels in brain were diminished in both Bdk^−/− and GBDK pups. Brains from Bdk^−/− pups exhibited robust eIF2∼P and amino acid stress response induction, whereas these responses were absent in GBDK mouse brains. Instead, myelin deficiency and diminished expression of myelin basic protein were noted in GBDK brains. Genetic markers of oligodendrocytes and astrocytes were also reduced in GBDK brains in association with apoptotic cell death in white matter regions of the brain. GBDK brains further demonstrated reduced Sod2 and Cat mRNA and increased Tnfα mRNA expression. The data are consistent with the idea that loss of GCN2 during BCAA deficiency compromises glial cell defenses to oxidative and inflammatory stress. We conclude that GCN2 protects the brain from developing a lethal leukodystrophy in response to amino acid deficiencies.     

Effects of branched-chain amino acids on muscles under hyperammonemic conditions

The aim was to determine the effects of enhanced availability of branched-chain amino acids (BCAAs; leucine, isoleucine, and valine) on ammonia detoxification to glutamine (GLN) and protein metabolism in two types of skeletal muscle under hyperammonemic conditions. Isolated soleus (SOL, slow-twitch) and extensor digitorum longus (EDL, fast-twitch) muscles from the left leg of white rats were incubated in a medium with 1 mM ammonia (NH3 group), BCAAs at four times the concentration of the controls (BCAA group) or high levels of both ammonia and BCAA (NH3 + BCAA group). The muscles from the right leg were incubated in basal medium and served as paired controls. L-[1-¹⁴C]leucine was used to estimate protein synthesis and leucine oxidation, and 3-methylhistidine release was used to evaluate myofibrillar protein breakdown. We observed decreased protein synthesis and glutamate and α-ketoglutarate (α-KG) levels and increased leucine oxidation, GLN levels, and GLN release into medium in muscles in NH3 group. Increased leucine oxidation, release of branched-chain keto acids and GLN into incubation medium, and protein synthesis in EDL were observed in muscles in the BCAA group. The addition of BCAAs to medium eliminated the adverse effects of ammonia on protein synthesis and adjusted the decrease in α-KG found in the NH3 group. We conclude that (i) high levels of ammonia impair protein synthesis, activate BCAA catabolism, enhance GLN synthesis, and decrease glutamate and α-KG levels and (ii) increased BCAA availability enhances GLN release from muscles and attenuates the adverse effects of ammonia on protein synthesis and decrease in α-KG.     

The effect of ovariectomy of serum amino acids and cholesterol in the rat

Summary As steroid hormones are known to influence amino acid metabolism we tested the hypothesis that ovariectomy should lead to significant changes in this system.We found that after ovariectomy serum alanine was significantly decreased (p = 0.0006) in contrast to serum glycine and branched chain amino acids (BCAA). The ratio of glycine/BCAA, a parameter for anabolism or catabolism was not changed after ovariectomy. If, however, the amino acid alanine as the link to carbohydrate and lipid metabolism was introduced the alanine/BCAA ratio was significantly altered (p = 0.01).Although serum cholesterol was altered as well (increased,p = 0.03), no significant correlation with alanine was found. We can therefore assume that there are two independent mechanisms for lipid and amino acid changes after ovariectomy.The most prominent finding was that estradiol replacement after ovariectomy restored increased cholesterol levels but did not restore alanine levels. Other ovarial hormones must be incriminated for the regulation of alanine metabolism. The anabolic effects of estradiol as decreasing glycine and BCAA were noticed which rules out insufficient estradiol replacement.     

Acute and Chronic Administration of the Branched-Chain Amino Acids Decreases Nerve Growth Factor in Rat Hippocampus

Maple syrup urine disease (MSUD) is a neurometabolic disorder caused by deficiency of the activity of the mitochondrial enzyme complex branched-chain α-keto acid dehydrogenase leading to accumulation of the branched-chain amino acids (BCAA) and their corresponding branched-chain α-keto acids. In this study, we examined the effects of acute and chronic administration of BCAA on protein levels and mRNA expression of nerve growth factor (NGF) considering that patients with MSUD present neurological dysfunction and cognitive impairment. Considering previous observations, it is suggested that oxidative stress may be involved in the pathophysiology of the neurological dysfunction of MSUD. We also investigated the influence of antioxidant treatment (N-acetylcysteine and deferoxamine) in order to verify the influence of oxidative stress in the modulation of NGF levels. Our results demonstrated decreased protein levels of NGF in the hippocampus after acute and chronic administration of BCAA. In addition, we showed a significant decrease in the expression of ngf in the hippocampus only following acute administration in 10-day-old rats. Interestingly, antioxidant treatment was able to prevent the decrease in NGF levels by increasing ngf expression. In conclusion, the results suggest that BCAA is involved in the regulation of NGF in the developing rat. Thus, it is possible that alteration of neurotrophin levels during brain maturation could be of pivotal importance in the impairment of cognition provoked by BCAA. Moreover, the decrease in NGF levels was prevented by antioxidant treatment, reinforcing that the hypothesis of oxidative stress can be an important pathophysiological mechanism underlying the brain damage observed in MSUD.     

The esg locus of Myxococcus xanthus encodes the E1α and E1β subunits of a branched-chain keto acid dehydrogenase

The esg locus of Myxococcus xanthus appears to control the production of a signal that must be transmitted between cells for the completion of multicellular development DNA sequence analysis suggested that the esg locus encodes the E1 decarboxylase (composed of E1α and E1β subunits) of a branched-chain keto acid dehydrogenase (BCKAD) that is involved in branched-chain amino acid (BCAA) metabolism. The properties of an esg ::Tn5 insertion mutant supported this conclusion. These properties include: (i) the growth yield of the mutant was reduced with increasing concentrations of the BCAAs in the medium while the growth yield of wild-type cells increased, (ii) mutant extracts were deficient in BCKAD activity, and (iii) growth of the mutant in media with short branched-chain fatty acids related to the expected products of the BCKAD helped to correct the mutant defects in growth, pigmentation and development. The esg BCKAD appears to be involved in the synthesis of long branched-chain fatty acids since the mutant contained reduced levels of this class of compounds. Our results are consistent with a model in which the esg-encoded enzyme is involved in the synthesis of branched-chain fatty acids during vegetative growth, and these compounds are used later in cell-cell signalling during development.