Branched-chain Α-keto Acid Research Articles

Metabolite Regulation of Nuclear Localization of Carbohydrate-response Element-binding Protein (ChREBP): ROLE OF AMP AS AN ALLOSTERIC INHIBITOR

The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis.

Catabolic Defect of Branched-Chain Amino Acids Promotes Heart Failure.

Although metabolic reprogramming is critical in the pathogenesis of heart failure, studies to date have focused principally on fatty acid and glucose metabolism. Contribution of amino acid metabolic regulation in the disease remains understudied. Transcriptomic and metabolomic analyses were performed in mouse failing heart induced by pressure overload. Suppression of branched-chain amino acid (BCAA) catabolic gene expression along with concomitant tissue accumulation of branched-chain α-keto acids was identified as a significant signature of metabolic reprogramming in mouse failing hearts and validated to be shared in human cardiomyopathy hearts. Molecular and genetic evidence identified the transcription factor Krüppel-like factor 15 as a key upstream regulator of the BCAA catabolic regulation in the heart. Studies using a genetic mouse model revealed that BCAA catabolic defect promoted heart failure associated with induced oxidative stress and metabolic disturbance in response to mechanical overload. Mechanistically, elevated branched-chain α-keto acids directly suppressed respiration and induced superoxide production in isolated mitochondria. Finally, pharmacological enhancement of branched-chain α-keto acid dehydrogenase activity significantly blunted cardiac dysfunction after pressure overload. BCAA catabolic defect is a metabolic hallmark of failing heart resulting from Krüppel-like factor 15-mediated transcriptional reprogramming. BCAA catabolic defect imposes a previously unappreciated significant contribution to heart failure.

A novel liquid chromatography tandem mass spectrometry method for simultaneous determination of branched-chain amino acids and branched-chain α-keto acids in human plasma.

Branched-chain amino acids (BCAAs) and branched-chain α-keto acids (BCKAs) play significant biological roles as they are involved in protein and neurotransmitter synthesis as well as energy metabolism pathways. To routinely and accurately study the dynamics of BCAAs and BCKAs in human diseases, e.g. cerebral infarction, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated. The plasma samples were deproteinized with acetonitrile, and then separated on a reversed phase C18 column with a mobile phase of 0.1% formic acid (solvent A)-methanol (solvent B) using gradient elution. The detection of BCAAs and BCKAs was conducted in multiple reaction monitoring with positive/negative electrospray ionization switching mode. Biologically relevant isomers such as leucine and isoleucine were individually quantified by combining chromatographic separation and fragmentation. Good linearity (R (2)>0.99) was obtained for all six analytes with the limits of detection from 0.1 to 0.2µg/mL. The intra-day and inter-day accuracy ranged from 93.7 to 108.4% and the relative standard deviation (RSD) did not exceed 15.0%. The recovery was more than 80% with RSD less than 14.0%. The main improvements compared to related, state-of-the-art methods included enhanced sensitivity, enhanced separation of isomers, and reduced complexity of sample processing. Finally, the validated method was applied to analyze the plasma samples of healthy volunteers and patients suffering cerebral infarction, and significant differences in the concentration levels of BCAAs and BCKAs were observed.

Dataset reporting BCKDK interference in a BCAA-catabolism restricted environment.

This data article contains complementary figures to the research article “Mitochondrial response to the BCKDK-deficiency: some clues to understand the positive dietary response in this form of autism” [1]. Herein we present data relative to the effect of knocking down BCKDK gene on the real time oxygen consumption rate of fibroblasts obtained from a Maple Syrup Urine Disease (MSUD) patient. Interference of BCKDK expression on such cells showing a reduced branched-chain α-ketoacid dehydrogenase (BCKDHc) activity; let us generate a scenario to study the direct effect of BCKDK absence in an environment of high branched-chain amino acids (BCAAs) concentrations. Data relative to the effectiveness of the knockdown together with the potentiality of the BCKDK-knockdown to increase the deficient branched-chain α-ketoacid dehydrogenase activity detected in MSUD patients are also shown.

Developmental Defects of Caenorhabditis elegans Lacking Branched-chain α-Ketoacid Dehydrogenase Are Mainly Caused by Monomethyl Branched-chain Fatty Acid Deficiency

Branched-chain α-ketoacid dehydrogenase (BCKDH) catalyzes the critical step in the branched-chain amino acid (BCAA) catabolic pathway and has been the focus of extensive studies. Mutations in the complex disrupt many fundamental metabolic pathways and cause multiple human diseases including maple syrup urine disease (MSUD), autism, and other related neurological disorders. BCKDH may also be required for the synthesis of monomethyl branched-chain fatty acids (mmBCFAs) from BCAAs. The pathology of MSUD has been attributed mainly to BCAA accumulation, but the role of mmBCFA has not been evaluated. Here we show that disrupting BCKDH in Caenorhabditis elegans causes mmBCFA deficiency, in addition to BCAA accumulation. Worms with deficiency in BCKDH function manifest larval arrest and embryonic lethal phenotypes, and mmBCFA supplementation suppressed both without correcting BCAA levels. The majority of developmental defects caused by BCKDH deficiency may thus be attributed to lacking mmBCFAs in worms. Tissue-specific analysis shows that restoration of BCKDH function in multiple tissues can rescue the defects, but is especially effective in neurons. Taken together, we conclude that mmBCFA deficiency is largely responsible for the developmental defects in the worm and conceivably might also be a critical contributor to the pathology of human MSUD.

Identification of six novel mutations in Iranian patients with maple syrup urine disease and their in silico analysis​

Maple syrup urine disease (MSUD) is a rare inborn error of branched-chain amino acid metabolism. The disease prevalence is higher in populations with elevated rate of consanguineous marriages such as Iran. Different types of disease causing mutations have been previously reported in BCKDHA, BCKDHB, DBT and DLD genes known to be responsible for MSUD phenotype.In this study, two sets of multiplex polymorphic STR (Short Tandem Repeat) markers linked to the above genes were used to aid in homozygosity mapping in order to find probable pathogenic change(s) in the studied families. The families who showed homozygote haplotype for the BCKDHA gene were subsequently sequenced. Our findings showed that exons 2, 4 and 6 contain most of the mutations which are novel. The changes include two single nucleotide deletion (i.e. c. 143delT and c.702delT), one gross deletion covering the whole exon four c.(375+1_376-1)_(8849+1_885-1), two splice site changes (c.1167+1G>T, c. 288+1G>A), and one point mutation (c.731G>A). Computational approaches were used to analyze these two novel mutations in terms of their impact on protein structure. Computational structural modeling indicated that these mutations might affect structural stability and multimeric assembly of branched-chain α-keto acid dehydrogenase complex (BCKDC).

Mitochondrial response to the BCKDK-deficiency: Some clues to understand the positive dietary response in this form of autism

Mutations on the mitochondrial-expressed Branched Chain α-Keto acid Dehydrogenase Kinase (BCKDK) gene have been recently associated with a novel dietary-treatable form of autism. But, being a mitochondrial metabolism disease, little is known about the impact on mitochondrial performance. Here, we analyze the mitochondrial response to the BCKDK-deficiency in patient's primary fibroblasts by measuring bioenergetics, ultra-structural and dynamic parameters. A two-fold increase in superoxide anion production, together with a reduction in ATP-linked respiration and intracellular ATP levels (down to 60%) detected in mutants fibroblasts point to a general bioenergetics depletion that could affect the mitochondrial dynamics and cell fate. Ultrastructure analysis of BCKDK-deficient fibroblasts shows an increased number of elongated mitochondria, apparently associated with changes in the mediator of inner mitochondria membrane fusion, GTPase OPA1 forms, and in the outer mitochondrial membrane, mitofusin 2/MFN2. Our data support a possible hyperfusion response of BCKDK-deficient mitochondria to stress. Cellular fate also seems to be affected as these fibroblasts show an altered proportion of the cells on G0/G1 and G2/M phases. Knockdown of BCKDK gene in control fibroblasts recapitulates most of these features. Same BCKDK-knockdown in a MSUD patient fibroblasts unmasks the direct involvement of the accelerated BCAAs catabolism in the mitochondrial dysfunction. All these data give us a clue to understand the positive dietary response to an overload of branched-chain amino acids. We hypothesize that a combination of the current therapeutic option with a protocol that considers the oxidative damage and energy expenditure, addressing the patients' individuality, might be useful for the physicians.

BCAA Metabolism and NH3 Homeostasis.

The branched chain amino acids (BCAA) are essential amino acids required not only for growth and development, but also as nutrient signals and as nitrogen donors to neurotransmitter synthesis and glutamate/glutamine cycling. Transamination and oxidative decarboxylation of the BCAAs are catalysed by the branched-chain aminotransferase proteins (BCATm, mitochondrial and BCATc, cytosolic) and the branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC), respectively. These proteins show tissue, cell compartmentation, and protein-protein interactions, which call for substrate shuttling or channelling and nitrogen transfer for oxidation to occur. Efficient regulation of these pathways is mediated through the redox environment and phosphorylation in response to dietary and hormonal stimuli. The wide distribution of these proteins allows for effective BCAA utilisation. We discuss how BCAT, BCKDC, and glutamate dehydrogenase operate in supramolecular complexes, allowing for efficient channelling of substrates. The role of BCAAs in brain metabolism is highlighted in rodent and human brain, where differential expression of BCATm indicates differences in nitrogen metabolism between species. Finally, we introduce a new role for BCAT, where a change in function is triggered by oxidation of its redox-active switch. Our understanding of how BCAA metabolism and nitrogen transfer is regulated is important as many studies now point to BCAA metabolic dysregulation in metabolic and neurodegenerative conditions.

Branched-Chain Aminotransferases Control TORC1 Signaling in Saccharomyces cerevisiae.

The conserved target of rapamycin complex 1 (TORC1) integrates nutrient signals to orchestrate cell growth and proliferation. Leucine availability is conveyed to control TORC1 activity via the leu-tRNA synthetase/EGOC-GTPase module in yeast and mammals, but the mechanisms sensing leucine remain only partially understood. We show here that both leucine and its α-ketoacid metabolite, α-ketoisocaproate, effectively activate the yeast TORC1 kinase via both EGOC GTPase-dependent and -independent mechanisms. Leucine and α-ketoisocaproate are interconverted by ubiquitous branched-chain aminotransferases (BCAT), which in yeast are represented by the mitochondrial and cytosolic enzymes Bat1 and Bat2, respectively. BCAT yeast mutants exhibit severely compromised TORC1 activity, which is partially restored by expression of Bat1 active site mutants, implicating both catalytic and structural roles of BCATs in TORC1 control. We find that Bat1 interacts with branched-chain amino acid metabolic enzymes and, in a leucine-dependent fashion, with the tricarboxylic acid (TCA)-cycle enzyme aconitase. BCAT mutation perturbed TCA-cycle intermediate levels, consistent with a TCA-cycle block, and resulted in low ATP levels, activation of AMPK, and TORC1 inhibition. We propose the biosynthetic capacity of BCAT and its role in forming multicomplex metabolons connecting branched-chain amino acids and TCA-cycle metabolism governs TCA-cycle flux to activate TORC1 signaling. Because mammalian mitochondrial BCAT is known to form a supramolecular branched-chain α-keto acid dehydrogenase enzyme complex that links leucine metabolism to the TCA-cycle, these findings establish a precedent for understanding TORC1 signaling in mammals.

Glucose supplementation stimulates peripheral branched-chain amino acid catabolism in lactating dairy cows during essential amino acid infusions

To determine how glucose modulates protein synthesis when essential AA are in abundant supply, 5 early-lactation, rumen-fistulated Holstein dairy cows were fed a diet containing 6.95 MJ/kg of net energy for lactation and 12.4% crude protein and abomasally infused for 5d with saline, 844 or 1,126g/d of a complete essential AA mix, with and without the inclusion of 1,000g/d of glucose, in a 5×5 Latin square design. Infusion of essential AA increased milk yield by 4.1kg/d, milk protein by 256g/d, milk fat by 95g/d, and milk urea nitrogen by 70% compared with saline, with no differences between the level of essential AA infusion. The addition of glucose to essential AA infusate did not stimulate milk protein yield or concentration, but reduced milk urea nitrogen by 17% and decreased milk fat yield. Arterial concentrations of total essential AA increased 3- to 4-fold, mammary clearance decreased 61%, and mammary uptake of essential AA increased 65% in response to essential AA infusion. Arterial branched-chain AA concentrations declined 29% in response to glucose and mammary clearance increased 48%, but mammary AA uptake was unchanged. Essential AA infusion increased plasma 3-methylhistidine by 50% and reduced muscle branched-chain α-keto acid dehydrogenase kinase abundance by 14%, indicating stimulation of muscle protein turnover and branched-chain AA catabolism, respectively. Glucose had no further effect on muscle branched-chain α-keto acid dehydrogenase kinase abundance but decreased mRNA expression of branched chain aminotransferase 1. Lack of further increases in plasma 3-methylhistidine or greater stimulation of muscle branched-chain AA catabolism indicates that muscle protein degradation was unchanged with glucose but that accretion may have been stimulated. The decrease in circulating branched-chain AA concentrations and nitrogen excretion in response to glucose suggests that surplus essential AA were redirected to peripheral, extra-mammary tissues.

Intracerebroventricular administration of α-ketoisocaproic acid decreases brain-derived neurotrophic factor and nerve growth factor levels in brain of young rats.

Maple syrup urine disease (MSUD) is an inherited aminoacidopathy resulting from dysfunction of the branched-chain keto acid dehydrogenase complex, leading to accumulation of the branched-chain amino acids (BCAA) leucine, isoleucine and valine as well as their corresponding transaminated branched-chain α-ketoacids. This disorder is clinically characterized by ketoacidosis, seizures, coma, psychomotor delay and mental retardation whose pathophysiology is not completely understood. Recent studies have shown that oxidative stress may be involved in neuropathology of MSUD. However, the effect of accumulating α-ketoacids in MSUD on neurotrophic factors has not been investigated. Thus, the objective of the present study was to evaluate the effects of acute intracerebroventricular administration of α-ketoisocaproic acid (KIC) on brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) levels in the brains of young male rats. Ours results showed that intracerebroventricular administration of KIC decreased BDNF levels in hippocampus, striatum and cerebral cortex, without induce a detectable change in pro-BDNF levels. Moreover, NGF levels in the hippocampus were reduced after intracerebroventricular administration of KIC. In conclusion, these data suggest that the effects of KIC on demyelination and memory processes may be mediated by reduced trophic support of BDNF and NGF. Moreover, lower levels of BDNF and NGF are consistent with the hypothesis that a deficit in this neurotrophic factor may contribute to the structural and functional alterations of brain underlying the psychopathology of MSUD, supporting the hypothesis of a neurodegenerative process in MSUD.

Abstract 16240: Tristetraprolin (TTP) is a Novel Regulator of Branched-chain Amino Acid (BCAA) Catabolism in the Heart

Introduction: Heart failure (HF) is associated with the accumulation of branched-chain amino acids (BCAAs) in heart and plasma. Besides, BCAAs are essential for normal cell growth and function. Therefore, tight regulation of BCAA metabolism is crucial to maintain normal cellular homeostasis. Tristetraprolin (TTP), an mRNA binding protein that promotes the degradation of its targets and regulates a number of biological processes including cellular metabolism. In in silico analyses, we identified the E2 subunit of branched-chain α-ketoacid dehydrogenase complex (BCKDC), the rate-limiting enzyme complex in BCAA catabolism, as a potential target of TTP. Here we assess the hypothesis that TTP plays a regulatory role in BCAA catabolism through regulating BCKDC-E2 in the heart. We also investigate TTP’s contribution in the pathogenesis of HF by modulating BCAA catabolism. Results: We observed a significant increase in TTP levels in cardiac tissue of human HF and mouse myocardial infarct induced HF samples. We also showed that deletion of TTP significantly decreases BCAA levels in vitro. As BCAA accumulation in the heart is associated with the pathogenesis of HF, we investigated whether TTP can regulate cardiac BCAA catabolism. Computational analysis of mRNAs of enzymes, which are involved in BCAA catabolism, revealed highly conserved TTP binding sites in BCKDC-E2 mRNA, suggesting that it is a potential target of TTP. BCKDC-E2 mRNA levels were significantly higher in the heart of TTP knock-out mice and TTP siRNA treated cardiomyocytes. Additionally, the mRNA stability of BCKDC-E2 was higher in cardiomyocytes with TTP downregulation, further confirming that TTP regulates the stability of this mRNA. Moreover, RNA co-immunoprecipitation studies demonstrated that TTP physically interacts with the BCKDC-E2 mRNA. Finally, we showed that BCKDC-E2 mRNA levels were significantly decreased in human HF samples. Conclusion: Our data indicate that TTP levels are increased in HF samples and TTP regulates BCAA catabolism in the heart by binding to and degrading BCKDC-E2 mRNA. These findings suggest that alterations in TTP expression may play a role in the development of HF based on its effect upon BCKDC-E2 and BCAA catabolism.

L-carnitine Prevents Oxidative Stress in the Brains of Rats Subjected to a Chemically Induced Chronic Model of MSUD.

Maple syrup urine disease (MSUD), or branched-chain α-keto aciduria, is an inherited disorder that is caused by a deficiency in branched-chain α-keto acid dehydrogenase complex (BCKAD) activity. Blockade of this pathway leads to the accumulation of the branched-chain amino acids (BCAAs), leucine, isoleucine, and valine, and their respective ketoacids in tissues. The main clinical symptoms presented by MSUD patients include ketoacidosis, hypoglycemia, opisthotonos, poor feeding, apnea, ataxia, convulsions, coma, psychomotor delay, and mental retardation. Although increasing evidence indicates that oxidative stress is involved in the pathophysiology of this disease, the mechanisms of the brain damage caused by this disorder remain poorly understood. In the present study, we investigated the effect of BCAAs on some oxidative stress parameters and evaluated the efficacy of L-carnitine (L-car), an efficient antioxidant that may be involved in the reduction of oxidative damage observed in some inherited neurometabolic diseases, against these possible pro-oxidant effects of a chronic MSUD model in the cerebral cortex and cerebellum of rats. Our results showed that chronic BCAA administration was able to promote both lipid and protein oxidation, impair brain antioxidant defenses, and increase reactive species production, particularly in the cerebral cortex, and that L-car was able to prevent these effects. Taken together, the present data indicate that chronic BCAA administration significantly increased oxidative damage in the brains of rats subjected to a chronic model of MSUD and that L-car may be an efficient antioxidant in this disorder.

Increased valinomycin production in mutants of Streptomyces sp. M10 defective in bafilomycin biosynthesis and branched-chain α-keto acid dehydrogenase complex expression.

Streptomyces sp. M10 is a valinomycin-producing bacterial strain that shows potent bioactivity against Botrytis blight of cucumber plants. During studies to increase the yield of valinomycin (a cyclododecadepsipeptide) in strain M10, additional antifungal metabolites, including bafilomycin derivatives (macrolide antibiotics), were identified. To examine the effect of bafilomycin biosynthesis on valinomycin production, the bafilomycin biosynthetic gene cluster was cloned from the genome of strain M10, as were two branched-chain α-keto acid dehydrogenase (BCDH) gene clusters related to precursor supply for bafilomycin biosynthesis. A null mutant (M10bafm) of one bafilomycin biosynthetic gene (bafV) failed to produce bafilomycin, but resulted in a 1.2- to 1.5-fold increase in the amount of valinomycin produced. In another null mutant (M10bkdFm) of a gene encoding a subunit of the BCDH complex (bkdF), bafilomycin production was completely abolished and valinomycin production increased fourfold relative to that in the wild-type M10 strain. The higher valinomycin yield was likely the result of redistribution of the metabolic flux from bafilomycin to valinomycin biosynthesis, because the two antibiotics share a common precursor, 2-ketoisovaleric acid, a deamination product of valine. The results show that directing precursor flux toward active ingredient biosynthesis could be used as a prospective tool to increase the competence of biofungicides.

Production of C4 and C5 branched-chain alcohols by engineered Escherichia. coli.

Higher alcohols, longer chain alcohols, contain more than 3 carbon atoms, showed close energy advantages as gasoline, and were considered as the next generation substitution for chemical fuels. Higher alcohol biosynthesis by native microorganisms mainly needs gene expression of heterologous keto acid decarboxylase and alcohol dehydrogenases. In the present study, branched-chain α-keto acid decarboxylase gene from Lactococcus lactis subsp. lactis CICC 6246 (Kivd) and alcohol dehydrogenases gene from Zymomonas mobilis CICC 41465 (AdhB) were transformed into Escherichia coli for higher alcohol production. SDS-PAGE results showed these two proteins were expressed in the recombinant strains. The resulting strain was incubated in LB medium at 37 °C in Erlenmeyer flasks and much more 3-methyl-1-butanol (104 mg/L) than isobutanol (24 mg/L) was produced. However, in 5 g/L glucose-containing medium, the production of two alcohols was similar, 156 and 161 mg/L for C4 (isobutanol) and C5 (3-methyl-1-butanol) alcohol, respectively. Effects of fermentation factors including temperature, glucose content, and α-keto acid on alcohol production were also investigated. The increase of glucose content and the adding of α-keto acids facilitated the production of C4 and C5 alcohols. The enzyme activities of pure Kivd on α-ketoisovalerate and α-ketoisocaproate were 26.77 and 21.24 μmol min(-1) mg(-1), respectively. Due to its ability on decarboxylation of α-ketoisovalerate and α-ketoisocaproate, the recombinant E. coli strain showed potential application on isoamyl alcohol and isobutanol production.

Molecular analysis of maple syrup urine disease in Jordanian families

Maple syrup urine disease (MSUD) is an autosomal-recessive inborn error of amino acid metabolism characterized by the accumulation of three branched-chain amino acids (BCAAs) in patients' cells due to reduced activity of the branched-chain α-ketoacid dehydrogenase complex (BCKD). In this study, we aimed to sequence the coding exons of the BCKDHA, BCKDHB, and DBT genes in five Jordanian families with MSUD and one family from Iraq with MSUD. BCAA levels were measured in probands initially presenting with developmental delay and encephalopathy. All of the coding exons and flanking intronic sequences of the BCKDHA, BCKDHB, and DBT genes were amplified and subjected to direct DNA sequencing. Four different mutations in the BCKDHA gene were identified, including a novel frame-shift mutation, c.908_909delTG, in family 4. Two novel missense mutations at residues Met263 and Gly353 in the DBT gene were also found to be cosegregated with the MSUD phenotype in families 5 and 6, respectively. Structural analyses suggested that these two mutations may affect the assembly of the intermediate E2 trimer. No BCKDHB gene mutations were found in Jordanian patients. The mutation profiles described in this study provide a basis for the early detection of MSUD disease. To our knowledge, this is the first molecular study of MSUD in Jordan and Middle Eastern Arabic countries.

Hepatic cumulative net appearance of amino acids and related gene expression response to different protein diets in pigs

The liver is an important organ responsible for amino acids (AA) conversion, and catabolism. To investigate the metabolic characteristic of AA in liver, eighteen castrated male pigs (20±1kg), implanted with arterial–portal–hepatic–mesenteric catheters, were fed three crude protein (CP) diets (14%, 17%, 20%) with six pigs per group. Blood was sampled for 7.5h post-feeding. Hepatic tissue was sampled from additional fifteen pigs (three CP diets groups, 5 per group) on 3h post-feeding. Portal vein plasma flow (PVPF), hepatic vein plasma flow (HVPF), hepatic cumulative net appearance and related gene expression were analyzed. There were no difference (P>0.05) for PVPF and HVPF among the three CP diets. Compared with the 20% CP diet, the 14% CP diet increased (P<0.05) the hepatic metabolism of lysine (59.50% vs. 43.00%) and threonine (44.30% vs. 30.22%). Likewise, the mRNA levels of α-aminoadipate δ-semialdehyde synthase (AASS) and l-threonine 3-dehydrogenase (TDH) were highest (P<0.05) in 14% CP group. The branched-chain AA (BCAA) had a low metabolic rate (3.4–13.4%) in liver. Morever, both the branched-chain amino-transferase 2 (BCAT2) and branched-chain α-keto acid dehydrogenase (BCKDHA) genes had a low expression. In contrast, aromatic AA (AAA) were almost entirely metabolized (63–93%) and the genes of phenylalanine hydroxylase (PAH) and tyrosine aminotransferase (TAT) had a high expression in liver. Notably, compared with the 20% CP diet, the 14% CP diet increased (P<0.05) hepatic metabolism of BCAA, AAA and gene expressions of BCAT, BCKDHA, PAH and TAT. These results revealed pigs fed on a low CP diets, only supplemented with lysine, methionine, threonine and tryptophan, had a numerical increasing EAA metabolism and EAA related metabolic gene expression in liver.

Abstract A07: MNT-dependent suppression of branched chain amino acid catabolism: A window into the role of MNT:MAX and MNT:MLX complexes in controlling MYC activity

Abstract The MYC family of transcription factors participate in a variety of physiological process and can be oncogenic when dysregulated. A key function of MYC is to reprogram metabolic pathways to meet the biomass needs for both normal the cancer cell proliferation. We previously showed that either too much or too little of the MAX-interacting bHLHZip transcriptional repressor MNT can suppress MYC induced oncogenesis. To begin to investigate the complicated relationship between MYC and MNT, we employed microarray and RNAseq analyses to investigate the gene expression profiles between WT and MNT deficient cells during log phase cell proliferation and during the process of serum stimulated cell cycle entry. Key genes found to be significantly upregulated in MNT deficient cells included ones that promote glycolysis, glutaminolysis, polyamine synthesis, and branched chain amino acid (BCAA) catabolism. Data from metabolomic analysis of MNT-deficient cells was also supportive of elevated glycolysis, glutaminolysis and BCAA catabolism. The high glycolysis and glutaminolysis profiles were similar to MYC-induced metabolic reprograming in tumor cells and T cell activation, but BCAA catabolic activity appeared to be unique to MNT deficient cells. Branched chain α-keto acid dehydrogenase E1α (BCKDH E1α), a subunit of the rate-limit enzyme complex in BCAA catabolism, was found to be one of the most upregulated genes in MNT deficient cells. BCKDH E1α protein was also strongly upregulated in MNT deficient cells, and MNT was found to bind a highly conserved Ebox element in the BCKDH E1αgene and repress its transcription. In contrast to MNT, MYC was found to not regulate BCKDH E1α. Replacing the MNT bHLHZip with the MYC bHLHZip region negated repression and replacing the MYC bHLHZip region with the MNT bHLHZip allowed MYC to activate BCKDH E1α transcription. In contrast to BCKDH E1α, either an engineered Ebox reporter or proximal promoters for specific E-box-containing MYC target genes involved in glycolysis and glutaminolysis that were activated by MYC were repressed by MNT. Protein interaction studies suggest that the differential target gene specificity of MNT compared to MYC is related to the ability of MNT to interact with both MAX and with MLX. Finally, since BCKDH E1α complex was identified as one of the major sources of Reactive Oxygen Species (ROS) in cells and MNT deficient cells have high levels of ROS that sensitizes them to apoptosis (Link et al. PNAS, 2012), we investigated the role of BCKDH E1α in the high ROS in MNT deficient cells and in inhibition of MYC-induced cell transformation. Our findings suggest that elevated BCKDH E1α and BCKDH E1α-induced BCAA catabolism and ROS production significantly contribute to the anti-oncogenic effect of MNT deficiency. Together, our findings suggest that MNT and MYC function as antagonists in some key metabolic pathways, but not in the BCAA catabolism pathway and that the BCAA catabolism pathway may be a potential target for anti-cancer drug development. Citation Format: Guang Yang, Jason M. Link, Peter J. Hurlin, Peter J. Hurlin. MNT-dependent suppression of branched chain amino acid catabolism: A window into the role of MNT:MAX and MNT:MLX complexes in controlling MYC activity. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr A07.

Impact of combined resistance and aerobic exercise training on branched-chain amino acid turnover, glycine metabolism and insulin sensitivity in overweight humans.

Obesity is associated with decreased insulin sensitivity (IS) and elevated plasma branched-chain amino acids (BCAAs). The purpose of this study was to investigate the relationship between BCAA metabolism and IS in overweight (OW) individuals during exercise intervention. Whole-body leucine turnover, IS by hyperinsulinaemic-euglycaemic clamp, and circulating and skeletal muscle amino acids, branched-chain α-keto acids and acylcarnitines were measured in ten healthy controls (Control) and nine OW, untrained, insulin-resistant individuals (OW-Untrained). OW-Untrained then underwent a 6 month aerobic and resistance exercise programme and repeated testing (OW-Trained). IS was higher in Control vs OW-Untrained and increased significantly following exercise. IS was lower in OW-Trained vs Control expressed relative to body mass, but was not different from Control when normalised to fat-free mass (FFM). Plasma BCAAs and leucine turnover (relative to FFM) were higher in OW-Untrained vs Control, but did not change on average with exercise. Despite this, within individuals, the decrease in molar sum of circulating BCAAs was the best metabolic predictor of improvement in IS. Circulating glycine levels were higher in Control and OW-Trained vs OW-Untrained, and urinary metabolic profiling suggests that exercise induces more efficient elimination of excess acyl groups derived from BCAA and aromatic amino acid (AA) metabolism via formation of urinary glycine adducts. A mechanism involving more efficient elimination of excess acyl groups derived from BCAA and aromatic AA metabolism via glycine conjugation in the liver, rather than increased BCAA disposal through oxidation and turnover, may mediate interactions between exercise, BCAA metabolism and IS. Clinicaltrials.gov NCT01786941.

Effect of low doses of bezafibrate and fenofibrate on liver 2-oxo-glutarate dehydrogenase complex in low-protein diet fed rats

&lt;p&gt;Multienzyme 2-oxoglutarate complex (2-OGDH) together with branched chain α-ketoacid dehydrogenase (BCKDH) and pyruvate dehydrogenase belong to the family of mitochondrial 2-oxoacid dehydrogenases. Hypolipidemic drugs, bezafibrate and fenofibrate, up-regulate liver BCKDH. The present study has been undertaken to determine the effect of low doses of bezafibrate and fenofibrate on liver 2-OGDH. Fibrates were administrated to rats fed low-protein diet at 5, 10 or 20 mg/kg. In rats treated with increasing doses of bezafibrate 2-OGDH activity increased by 7, 35 and 42%, while in rats administered with fenofibrate by 8, 18, and 56% (p&amp;lt;0.05 for bezafibrate 10 and 20, and fenofibrate 20 mg/kg). Changes in 2-OGDH activity did not correspond with changes in mRNA levels of the complex enzymes. Moreover, mRNA levels of PPARα remained unaltered. It is conceivable that stimulation of 2-OGDH activity by low doses of fibrates is the result of post-transcriptional events and may have a significant effect on liver metabolism.&lt;/p&gt;&lt;p&gt; &lt;/p&gt;