Branched-chain Alpha-keto Acid Research Articles

Branched-chain amino acid catabolic defect promotes α-cell proliferation via activating mTOR signaling

Elevated circulating level of branched-chain amino acids (BCAAs) is closely related to the development of type 2 diabetes. However, the role of BCAA catabolism in various tissues in maintaining glucose homeostasis remains largely unknown. Pancreatic α-cells have been regarded as amino acid sensors in recent years. Therefore, we generated α-cell specific branched-chain alpha-ketoacid dehydrogenase E1α subunit (BCKDHA) knockout (BCKDHA-αKO) mice to decipher the effects of BCAA catabolism in α-cells on whole-body energy metabolism. BCKDHA-αKO mice showed normal body weight, body fat, and energy expenditure. Plasma glucagon level and glucose metabolism also remained unchanged in BCKDHA-αKO mice. Whereas, the deletion of BCKDHA led to increased α-cell number due to elevated cell proliferation in neonatal mice. In vitro, only leucine among BCAAs promoted aTC1-6 cell proliferation, which was blocked by the agonist of BCAA catabolism BT2 and the inhibitor of mTOR Rapamycin. Like Rapamycin, BT2 attenuated leucine-stimulated phosphorylation of S6 in αTC1-6 cells. Elevated phosphorylation level of S6 protein in pancreatic α-cells was also observed in BCKDHA-αKO mice. These results suggest that local accumulated leucine due to defective BCAA catabolism promotes α-cell proliferation through mTOR signaling, which is insufficient to affect glucagon secretion and whole-body glucose homeostasis.

Reactive nitrogen species inhibit branched chain alpha-ketoacid dehydrogenase complex and impact muscle cell metabolism

Branched chain α-ketoacid dehydrogenase complex (BCKDC) is the rate-limiting enzyme in branched chain amino acid (BCAA) catabolism, a metabolic pathway with great importance for human health. BCKDC belongs to the mitochondrial α-ketoacid dehydrogenase complex family, which also includes pyruvate dehydrogenase complex and oxoglutarate dehydrogenase complex. Here, we revealed that BCKDC can be substantially inhibited by reactive nitrogen species (RNS) via a mechanism similar to what we recently discovered with pyruvate dehydrogenase complex and oxoglutarate dehydrogenase complex-RNS can cause inactivating covalent modifications of the lipoic arm on its E2 subunit. In addition, we showed that such reaction between RNS and the lipoic arm of the E2 subunit can further promote inhibition of the E3 subunits of α-ketoacid dehydrogenase complexes. We examined the impacts of this RNS-mediated BCKDC inhibition in muscle cells, an important site of BCAA metabolism, and demonstrated that the nitric oxide production induced by cytokine stimulation leads to a strong inhibition of BCKDC activity and BCAA oxidation in myotubes and myoblasts. More broadly, nitric oxide production reduced the level of functional lipoic arms across the multiple α-ketoacid dehydrogenases and led to intracellular accumulation of their substrates (α-ketoacids), decrease of their products (acyl-CoAs), and a lower cellular energy charge. In sum, this work revealed a new mechanism for BCKDC regulation, demonstrated that RNS can generally inhibit all α-ketoacid dehydrogenases, which has broad physiological implications across multiple cell types, and elucidated the mechanistic connection between RNS-driven inhibitory modifications on the E2 and E3 subunits of α-ketoacid dehydrogenases.

Sex differences in cachexia outcomes, anabolic signalling and BCAA metabolism following chemotherapy

Sex is a risk factor for cancer. Affecting nearly 80% of cancer patients is cachexia, a body and skeletal muscle-wasting syndrome. Poor nutritional status, tumour related factors and chemotherapy contribute to cachexia. Although negative effects of chemotherapy on skeletal muscle are documented, the majority of studies are completed in male animals. Although the branched-chain amino acids (BCAA: leucine, isoleucine and valine) activate anabolic signalling in skeletal muscle and have been shown to reduce some of the effects of cachexia, BCAA nutritional support does not reverse cachexia and few studies have investigated the effects of chemotherapy on BCAA metabolism in skeletal muscle. Therefore, the objective of this study is to compare the effects of chemotherapy on cachexia outcomes and BCAA metabolism between male and female mice. Three-month-old CD2F1 male and female mice were treated with either the chemotherapy drug cocktail folfiri (50mg/kg 5-fluorouracil (5FU), 90mg/kg Leucovorin and 24mg/kg CPT11) (drug) or vehicle (10% DMSO in saline) for 6-weeks. Within sex, drug treatment led to reductions in body and skeletal muscle weights. Between sex, drug-treated female mice lost more body and gastrocnemius muscle weight compared to drug-treated males. Within sex, drug treated animals exhibited reductions in anabolic signalling, specifically AKT, S6K1, S6 and 4E-BP1. Between sex, drug-treated females mice showed greater loss of anabolic signalling compared to drug-treated males. Within sex, the drug cocktail reduced amino acid transporters SNAT1 and LAT1, while reducing BCAA concentrations in the skeletal muscle. Drug-treated female mice exhibited greater loss of LAT1 compared to male-drug treated mice. However, male-drug treated mice had greater decreases in BCAA concentrations compared to female drug-treated mice. In addition, minimal differences were found within sex for key enzymes involved in BCAA metabolism. However, the inhibitory phosphorylation of branched-chain alpha-ketoacid dehydrogenase complex (BCKD) was elevated only in drug-treated male animals. Lastly, within sex, drug treatment showed elevated levels of BCAA concentrations in the liver, but only male drug-treated mice showed elevated plasma BCAAs compared to male vehicle-treated mice. Our data suggests a link between muscle atrophy and decreased BCAA metabolism during chemotherapy. We found that drug-treated female mice had worsened outcomes for cachexia measures such as body, skeletal muscle weight and anabolic signalling compared to drug-treated male mice. However, drug-treated males exhibited greater decreases in BCAA concentrations and their metabolism compared to drug-treated females. Findings from this study suggest that altered availability and metabolism of the BCAAs may contribute to muscle wasting differently in males and females during chemotherapy and interventions that correct such alterations may need to consider sex in order to better manage cachexia. Nserc, York University This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Melatonin improves behavioral parameters and oxidative stress in zebrafish submitted to a leucine-induced MSUD protocol.

Maple syrup urine disease (MSUD) is an inherited metabolic disorder caused by a deficiency in branched-chain alpha-ketoacid dehydrogenase complex (BCKAC). The treatment is a standard therapy based on a protein-restricted diet with low branched-chain amino acids (BCAA) content to reduce plasma levels and, consequently, the effects of accumulating their metabolites, mainly in the central nervous system. Although the benefits of dietary therapy for MSUD are undeniable, natural protein restriction may increase the risk of nutritional deficiencies, resulting in a low total antioxidant status that can predispose and contribute to oxidative stress. As MSUD is related to redox and energy imbalance, melatonin can be an important adjuvant treatment. Melatonin directly scavenges the hydroxy radical, peroxyl radical, nitrite anion, and singlet oxygen and indirectly induces antioxidant enzyme production. Therefore, this study assesses the role of melatonin treatment on oxidative stress in brain tissue and behavior parameters of zebrafish (Danio rerio) exposed to two concentrations of leucine-induced MSUD: leucine 2 mM and 5mM; and treated with 100 nM of melatonin. Oxidative stress was assessed through oxidative damage (TBARS, DCF, and sulfhydryl content) and antioxidant enzyme activity (SOD and CAT). Melatonin treatment improved redox imbalance with reduced TBARS levels, increased SOD activity, and normalized CAT activity to baseline. Behavior was analyzed with novel object recognition test. Animals exposed to leucine improved object recognition due to melatonin treatment. With the above, we can suggest that melatonin supplementation can protect neurologic oxidative stress, protecting leucine-induced behavior alterations such as memory impairment.

Identification of a novel homozygous mutation of the BCKDHB gene in an Iranian patient with maple syrup disease using next-generation sequencing

BackgroundMaple syrup urine disease (MSUD) is an autosomal recessive disorder caused by decreased branched-chain alpha-keto acid dehydrogenase (BCKD) activity in human tissues. The BCKD enzyme is mostly active in the human body in the muscles and the brain; thus, genetic deficiency of the BCKD complex can result in neurological damage in patients. Deleterious mutations in BCKDHB, BCKDHA, DBT, and DLD genes have a significant impact on BCKD activity. MethodsIn this study, we described a case of MSUD in a consanguineous Iranian family without a history of metabolic disorders. A 7-year-old boy was diagnosed 21 days after birth with lethargy, feeding problems, and vomiting. Plasma amino acid concentration and level of ammonia were measured. And then, whole-exome sequencing (WES) 100× was used to identify causative mutation along with some bioinformatics analysis. ResultsA novel pathogenic homozygous mutation, c.552_553insA; P185Tfs*17, was detected in the BCKDHB gene's exon5. This variant was predicted as a pathogenic mutation using bioinformatics tools including Mutationtaster, Fathmm-mkl, Provean, DEOGEN2, SNPs&GO, PhyloP, and PhastCons. This mutation leads to a truncated protein in which conserved regions are lost. Besides, the variant is completely co-segregated with the disease in this family. ConclusionA novel pathogenic mutation c.552_553insA in the BCKDHB gene related to MSUD was diagnosed in an Iranian family. This finding from the present study can be efficient in upgrading mutation databases and recognizing MSUD causes.

The mitochondrial BCKD complex interacts with hepatic apolipoprotein E in cultured cells in vitro and mouse livers in vivo

Background and aimsApolipoprotein E (APOE) is known for its role in lipid metabolism and its association with age-related disease pathology. The aim of the present work was to identify previously unknown functions of APOE based on the detection of novel APOE protein–protein interaction candidates.Approach and resultsAPOE targeted replacement mice and transfected cultured hepatocytes expressing the human isoforms APOE3 and APOE4 were used. For 7 months, APOE3 and APOE4 mice were fed a high-fat and high-sugar diet to induce obesity, while a subgroup was subjected to 30% dietary restriction. Proteomic analysis of coimmunoprecipitation products from APOE mouse liver extracts revealed 28 APOE-interacting candidate proteins, including branched-chain alpha-keto acid dehydrogenase (BCKD) complex subunit alpha (BCKDHA) and voltage-dependent anion-selective channel 1 (VDAC1). The binding of APOE and BCKDHA was verified in situ by proximity ligation assay in cultured cells. The activity of the BCKD enzyme complex was significantly higher in obese APOE4 mice than in APOE3 mice, while the plasma levels of branched-chain amino acids and mTOR signalling proteins were not different. However, the protein–protein interaction with VDAC1 was strongly induced in APOE3 and APOE4 mice upon dietary restriction, suggesting a prominent role of APOE in mitochondrial function.ConclusionsThe protein–protein interactions of APOE with BCKDHA and VDAC1 appear to be of physiological relevance and are modulated upon dietary restriction. Because these are mitochondrial proteins, it may be suggested that APOE is involved in mitochondria-related processes and adaptation to hepatic energy demands.

Inhibition of branched-chain alpha-keto acid dehydrogenase kinase augments the sensitivity of ovarian and breast cancer cells to paclitaxel

ContextMany cancer patients who initially respond to chemotherapy eventually develop chemoresistance, and to address this, we previously conducted a RNAi screen to identify genes contributing to resistance. One of the hits from the screen was branched-chain α-keto acid dehydrogenase kinase (BCKDK). BCKDK controls the metabolism of branched-chain amino acids (BCAAs) through phosphorylation and inactivation of the branched-chain α-keto acid dehydrogenase complex (BCKDH), thereby inhibiting catabolism of BCAAs.MethodsWe measured the impact on paclitaxel sensitivity of inhibiting BCKDK in ovarian and breast cancer cell lines.ResultsInhibition of BCKDK using siRNA or two chemical inhibitors (BCKDKi) was synergistic with paclitaxel in both breast and ovarian cancer cells. BCKDKi reduced levels of BCAA and the addition of exogenous BCAA suppressed this synergy. BCKDKi inactivated the mTORC1-Aurora pathway, allowing cells to overcame M-phase arrest induced by paclitaxel. In some cases, cells almost completed cytokinesis, then reverted to a single cell, resulting in multinucleate cells.ConclusionBCKDK is an attractive target to augment the sensitivity of cancer cells to paclitaxel.

Chemotherapy Drugs Induce Cachexia and Alter Muscle Protein Metabolism

Chemotherapy is the main regimen to treat neoplastic tumours. Maintenance of muscle mass during treatment is associated with greater patient prognosis and reduces toxicity and severe side effects. However, chemotherapy induces cachexia, a wasting disorder characterized by muscle weakness and a loss of muscle mass. Although cancer, a disease treated with chemotherapy, is often diagnosed in older individuals, studies investigating chemotherapy‐induced cachexia are done exclusively in young animals at an age equivalent to a 20‐year‐old human. Therefore, studies investigating the effects of chemotherapy‐induced cachexia at an age range relevant to humans is needed. The objective of this study is to compare the severity and mechanisms of chemotherapy‐induced cachexia in 3‐month‐old adult (equivalent to a 20‐year‐old human) and 18‐month‐old (equivalent to a 56‐year‐old human) mice. Three‐month‐old adult CD2F1 male mice were treated with either folfiri (50mg/kg 5‐fluorouracil (5FU), 90mg/kg Leucovorin and 24mg/kg CPT11) (drug) or vehicle (10% DMSO in saline) for 6‐weeks. Male mice aged to 18‐months will undergo similar treatments. Adult mice treated with the drug cocktail exhibited significant body weight loss (~10%, n=4, p=0.0117) with no differences in food intake. Decreases in body weight were associated with a loss of muscle mass in the gastrocnemius (~25%, n=4, p=0.006), quadriceps (~30%, n=4, p=0.019) and tibialis anterior (~40%, n=4, p<0.0001). Insulin tolerance tests at weeks 3 and 6 of treatment were also performed and glucose levels were found to be non‐significantly higher in drug‐treated mice. Signalling factors related to the mammalian/mechanistic target of rapamycin complex 1 (mTORC1), protein synthesis, autophagy and the ubiquitin proteasome system will be measured. Key enzymes responsible for the breakdown of the branched chain amino acids (BCAAs), mainly branched‐chain alpha‐ketoacid dehydrogenase complex (BCKD) and branched‐chain amino acid transferase 2 (BCAT2) will also be measured in the skeletal muscle. Along this line, in cell culture, we have previously shown decreased (~20%) activity of the BCKD enzyme, corresponding with decreased amino acid concentrations of leucine (~40%), isoleucine (~60%), valine (~50%), alanine (~30%), arginine (~60%), phenylalanine (50%), serine (50%) and glutamate (50%) in chemotherapy‐treated myotubes. We also observed decreased (~65%) expression of amino acid transporters sodium‐coupled neutral amino acid transporter 1 (SNAT1) and large neutral amino acid transporter 1 (LAT1). Timepoint analysis showed SNAT1 expression to be decreased prior to decreases in BCKD activity, signifying that alterations in BCAA metabolism may be a result of decreased levels of BCAAs and amino acids in chemotherapy‐treated myotubes. Therefore, these findings stemmed our interest in how the BCAAs, and their metabolites are affected in‐vivofollowing chemotherapy treatment. Findings from this study will help to elucidate whether unique signatures of cachexia are experienced in older mice. Better understanding of the mechanisms underlying muscle mass regulation following chemotherapy treatment may help combat cachexia.

Exposure to leucine induces oxidative stress in the brain of zebrafish.

Maple Syrup Urine Disease (MSUD) is an autosomal recessive inherited disorder caused by a deficiency in the activity of the branched-chain alpha-ketoacid dehydrogenase complex leading to the accumulation of branched-chain amino acids (BCAA) leucine, isoleucine, and valine and their respective branched-chain α-ketoacids and corresponding hydroxy acids. Considering that Danio rerio, known as zebrafish, has been widely used as an experimental model in several research areas because it has favorable characteristics that complement other experimental models, this study aimed to evaluate oxidative stress parameters in zebrafish exposed to high levels of leucine (2mM and 5mM), in a model similar of MSUD. Twenty-four hours after exposure, the animals were euthanized, and the brain content dissected for analysis of oxidative stress parameters: thiobarbituric acid reactive substances (TBARS), 2',7'-dichlorofluorescein oxidation assay (DCF); content of sulfhydryl, and superoxide dismutase (SOD) and catalase (CAT) activities. Animals exposed to 2mM and 5mM leucine showed an increase in the measurement of TBARS and decreased sulfhydryl content. There were no significant changes in DCF oxidation. In addition, animals exposed to 2mM and 5mM leucine were found to have decreased SOD activity and increased CAT activity. Based on these results, exposure of zebrafish to high doses of leucine can act as a promising animal model for MSUD, providing a better understanding of the toxicity profile of leucine exposure and its use in future investigations and strategies related to the pathophysiology of MSUD.

EP010: Conservative management with serial biochemical monitoring for newborn screen detected Maple Syrup Urine Disease (MSUD) patients without metabolic decompensation

Maple syrup urine disease (MSUD) is a rare inborn error of metabolism characterized by deficiency of branched-chain alpha-keto acid dehydrogenase that metabolizes the three branched-chain amino acids (BCAAs) leucine, isoleucine, and valine. The result of this enzyme deficiency is a toxic buildup of metabolites, typically within the first 24-48 hours of life. The main neurotoxic effects are due to accumulation of large amounts of leucine which can lead to severe cerebral edema. Treatment requires immediate medical intervention to lower leucine levels.

Presence of a Mitovirus Is Associated with Alteration of the Mitochondrial Proteome, as Revealed by Protein-Protein Interaction (PPI) and Co-Expression Network Models in Chenopodium quinoa Plants.

Simple SummaryPlants often harbor persistent plant virus infection transmitted only vertically through seeds, resulting in no obvious symptoms (cryptic infections). Several studies have shown that such cryptic infections provide resilience against abiotic (and biotic) stress. We have recently discovered a new group of cryptic plant viruses infecting mitochondria (plant mitovirus). Mitochondria are cellular organelles displaying a pivotal role in protecting cells from the stress of nature . Here, we look at the proteomic alterations caused by the mitovirus cryptic infection of Chenopodium quinoa by Systems Biology approaches allowing one to evaluate data at holistic level. Quinoa is a domesticated plant species with many exciting features of abiotic stress resistance, and it is distinguished by its exceptional nutritional characteristics, such as the content and quality of proteins, minerals, lipids, and tocopherols. These features determined the growing interest for the quinoa crop by the scientific community and international organizations since they provide opportunities to produce high-value grains in arid, high-salt and high-UV agroecological environments. We discovered that quinoa lines hosting mitovirus activate some metabolic processes that might help them face drought. These findings present a new perspective for breeding crop plants through the augmented genome provided by accessory cryptic viruses to be investigated in the future.Plant mitoviruses belong to Mitoviridae family and consist of positive single-stranded RNA genomes replicating exclusively in host mitochondria. We previously reported the biological characterization of a replicating plant mitovirus, designated Chenopodium quinoa mitovirus 1 (CqMV1), in some Chenopodium quinoa accessions. In this study, we analyzed the mitochondrial proteome from leaves of quinoa, infected and not infected by CqMV1. Furthermore, by protein–protein interaction and co-expression network models, we provided a system perspective of how CqMV1 affects mitochondrial functionality. We found that CqMV1 is associated with changes in mitochondrial protein expression in a mild but well-defined way. In quinoa-infected plants, we observed up-regulation of functional modules involved in amino acid catabolism, mitochondrial respiratory chain, proteolysis, folding/stress response and redox homeostasis. In this context, some proteins, including BCE2 (lipoamide acyltransferase component of branched-chain alpha-keto acid dehydrogenase complex), DELTA-OAT (ornithine aminotransferase) and GR-RBP2 (glycine-rich RNA-binding protein 2) were interesting because all up-regulated and network hubs in infected plants; together with other hubs, including CAT (catalase) and APX3 (L-ascorbate peroxidase 3), they play a role in stress response and redox homeostasis. These proteins could be related to the higher tolerance degree to drought we observed in CqMV1-infected plants. Although a specific causative link could not be established by our experimental approach at this stage, the results suggest a new mechanistic hypothesis that demands further in-depth functional studies.

AICAR stimulates mitochondrial biogenesis and BCAA catabolic enzyme expression in C2C12 myotubes

Type 2 diabetes is characterized by reduced insulin sensitivity, elevated blood metabolites, and reduced mitochondrial metabolism. Insulin resistant populations often exhibit reduced expression of genes governing mitochondrial metabolism such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α). Interestingly, PGC-1α regulates the expression of branched-chain amino acid (BCAA) metabolism, and thus, the consistently observed increased circulating levels of BCAA in diabetics may be partially explained by reduced PGC-1α expression. Conversely, PGC-1α upregulation appears to increase BCAA catabolism. PGC-1α activity is regulated by 5′-AMP-activated protein kinase (AMPK), however, only limited experimental data exists on the effect of AMPK activation in the regulation of BCAA catabolism. The present report examined the effects of the commonly used AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) on the metabolism and expression of several related targets (including BCAA catabolic enzymes) of cultured myotubes. C2C12 myotubes were treated with AICAR at 1 mM for up to 24 h. Mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Metabolic gene and protein expression were assessed via qRT-PCR and western blot, respectively. AICAR treatment significantly increased mitochondrial content and peak mitochondrial capacity. AICAR treatment also increased AMPK activation and mRNA expression of several regulators of mitochondrial biogenesis but reduced glycolytic metabolism and mRNA expression of several glycolytic enzymes. Interestingly, branched-chain alpha-keto acid dehydrogenase a (BCKDHa) protein was significantly increased following AICAR-treatment suggesting increased overall BCAA catabolic capacity in AICAR-treated cells. Together, these experiments demonstrate AICAR/AMPK activation can upregulate BCAA catabolic machinery in a model of skeletal muscle.

Muscle-directed AAV gene therapy rescues the maple syrup urine disease phenotype in a mouse model

Maple syrup urine disease (MSUD) is a rare, inherited metabolic disorder characterized by a dysfunctional mitochondrial enzyme complex, branched-chain alpha-keto acid dehydrogenase (BCKDH), which catabolizes branched-chain amino acids (BCAAs). Without functional BCKDH, BCAAs and their neurotoxic alpha-keto intermediates can accumulate in the blood and tissues. MSUD is currently incurable and treatment is limited to dietary restriction or liver transplantation, meaning there is a great need to develop new treatments for MSUD. We evaluated potential gene therapy applications for MSUD in the intermediate MSUD (iMSUD) mouse model, which harbors a mutation in the dihydrolipoamide branched-chain transacylase E2 (DBT) subunit of BCKDH. Systemic delivery of an adeno-associated virus (AAV) vector expressing DBT under control of the liver-specific TBG promoter to the liver did not sufficiently ameliorate all aspects of the disease phenotype. These findings necessitated an alternative therapeutic strategy. Muscle makes a larger contribution to BCAA metabolism than liver in humans, but a muscle-specific approach involving a muscle-specific promoter for DBT expression delivered via intramuscular (IM) administration only partially rescued the MSUD phenotype in mice. Combining the muscle-tropic AAV9 capsid with the ubiquitous CB7 promoter via IM or IV injection, however, substantially increased survival across all assessed doses. Additionally, near-normal serum BCAA levels were achieved and maintained in the mid- and high-dose cohorts throughout the study; this approach also protected these mice from a lethal high-protein diet challenge. Therefore, administration of a gene therapy vector that expresses in both muscle and liver may represent a viable approach to treating patients with MSUD.

Maple syrup urine disease: Characteristics of diagnosis and treatment in 45 patients in Chile.

Maple urine syrup disease (MSUD) is an autosomal recessive disorder characterized by deficient activity of the branched-chain alpha ketoacid dehydrogenase (BCKAD) enzymatic complex due to biallelic variants in the alpha (BCKDHA) or beta (BCKDHB) subunits or the acyltransferase component (DBT). Treatment consists in leucine (LEU), isoleucine (ILE), and valine (VAL) (branched-chain amino acids) dietary restriction and strict metabolic control. to determine the characteristics of the Chilean cohort with MSUD currently in follow-up at Instituto de Nutrición y Tecnología de los Alimentos, during the 1990-2017 period Retrospective analytical study in 45 MSUD cases. Measured: biochemical parameters (LEU, ILE, and VAL), anthropometric evaluation, and neurocognitive development. In 18 cases undergoing genetic study were analyzed according to the gene and protein location, number of affected alleles, and type of posttranslational modification affected. Then, 45 patients with MSUD diagnosis were identified during the period: 37 were alive at the time of the study. Average diagnosis age was 71 ± 231 days. Average serum diagnosis LEU concentrations: 1.463 ± 854.1 μmol/L, VAL 550 ± 598 μmol/L and ILE 454 ± 458 μmol/L. BCKDHB variants explain 89% cases, while BCKDHA and DBT variants explain 5.5% of cases each. Variants p.Thr338Ile in BCKDHA, p.Pro240Thr and p.Ser342Asn in BCKDHB have not been previously reported in literature. Average serum follow-up LEU concentrations were 252.7± 16.9μmol/L in the <5 years group and 299 ± 123.2μmol/L in ≥5 years. Most cases presented some degree of developmental delay. Early diagnosis and treatment is essential to improve the long-term prognosis. Frequent blood LEU measurements are required to optimize metabolic control and to establish relationships between different aspects analyzed.

Methionine and Arginine Supply Alters Abundance of Amino Acid, Insulin Signaling, and Glutathione Metabolism-Related Proteins in Bovine Subcutaneous Adipose Explants Challenged with N-Acetyl-d-sphingosine.

Simple SummaryIncreased circulating concentrations of ceramides (Ce) in dairy cows contribute to subcutaneous adipose tissue (SAT) lipolysis and could enhance the risk of developing metabolic disorders. Dietary supply of methionine (Met) or arginine (Arg) alters cellular metabolism in key tissues including SAT. Whether Met or Arg directly affect SAT metabolism when Ce concentrations are elevated is unknown. We propose that Met or Arg could have a beneficial effect within adipose tissue in terms of alleviating potential inflammatory and pro-oxidant effects associated with the transition into lactation.The objective was to perform a proof-of-principle study to evaluate the effects of methionine (Met) and arginine (Arg) supply on protein abundance of amino acid, insulin signaling, and glutathione metabolism-related proteins in subcutaneous adipose tissue (SAT) explants under ceramide (Ce) challenge. SAT from four lactating Holstein cows was incubated with one of the following media: ideal profile of amino acid as the control (IPAA; Lys:Met 2.9:1, Lys:Arg 2:1), increased Met (incMet; Lys:Met 2.5:1), increased Arg (incArg; Lys:Arg 1:1), or incMet plus incArg (Lys:Met 2.5:1 Lys:Arg 1:1) with or without 100 μM exogenous cell-permeable Ce (N-Acetyl-d-sphingosine). Ceramide stimulation downregulated the overall abundance of phosphorylated (p) protein kinase B (AKT), p-mechanistic target of rapamycin (mTOR), and p-eukaryotic elongation factor 2 (eEF2). Without Ce stimulation, increased Met, Arg, or Met + Arg resulted in lower p-mTOR. Compared with control SAT stimulated with Ce, increased Met, Arg, or Met + Arg resulted in greater activation of mTOR (p-mTOR/total mTOR) and AKT (p-AKT/total AKT), with a more pronounced response due to Arg. The greatest protein abundance of glutathione S-transferase Mu 1 (GSTM1) was detected in response to increased Met supply during Ce stimulation. Ceramide stimulation decreased the overall protein abundance of the Na-coupled neutral amino acid transporter SLC38A1 and branched-chain alpha-ketoacid dehydrogenase kinase (BCKDK). However, compared with controls, increased Met or Arg supply attenuated the downregulation of BCKDK induced by Ce. Circulating ceramides might affect amino acid, insulin signaling, and glutathione metabolism in dairy cow adipose tissue. Further in vivo studies are needed to confirm the role of rumen-protected amino acids in regulating bovine adipose function.

Functional assay for analysis of variants of uncertain significance in maple syrup urine disease

Introduction: Enhanced molecular characterisation using high-throughput sequencing has significantly improved patient diagnosis and management. However, the identification of variants of uncertain significance (VUS) limits its use for clinical decision making, such as prenatal diagnosis in subsequent pregnancies. Case report: We report a female infant who presented with mild gross motor delay at 4 months and developed seizures with hypoglycaemia at 5 months. Plasma amino acid and urine organic acid findings were consistent with Maple syrup urine disease (MSUD), despite a normal newborn screening test for this condition. MSUD results in deficiency of branched-chain alpha-ketoacid dehydrogenase, responsible for leucine, isoleucine, and valine degradation, caused by biallelic pathogenic variants in BCKDHA, BCKDHB, or DBT genes. Results: Molecular analysis in this patient identified two VUS, c.434-15_434-4del and c.365A>G (p. Tyr122Cys) in the DBT gene. Functional testing, in the form of leucine decarboxylation studies in fibroblasts revealed deficient function and mRNA splicing studies provided additional data that permitted variant reclassification and subsequent prenatal testing. Conclusion: This case reinforces the need for specialist biochemical genetic laboratories to retain their ability to perform functional assays to aid the resolution of VUS.

Nitric Oxide Mediates Direct Restriction of Pyruvate Dehydrogenase Complex via Generation of Nitroxyl During Macrophage Polarization

M1 macrophages “commit” to a metabolic state where increased aerobic glycolysis sustains fast ATP production, independently of Oxidative Phosphorylation (OXPHOS). This is associated with a “broken” mitochondrial TCA cycle and accumulation of metabolites like citrate, succinate and itaconate, important for cellular functions. We have shown that induced Nitric Oxide (NO) levels in Bone Marrow Derived Macrophages (BMDMs) from Wild Type (WT) mice activated with lipopolysaccharide and interferon gamma (LPS + IFNγ) are necessary and sufficient for the repression of OXPHOS and for metabolic reprogramming at mitochondrial Aconitase (ACO2) and Pyruvate Dehydrogenase (PDH). We hypothesize that NOdirectly targets the PDH complex via its critical cofactor lipoate, interacting with its thiol motifs. High-sensitivity proteomics on PDH complex shows that stimulated BMDM and cells exposed to NO have stalled enzymatic machinery, and this correlates with NO-associated lipoate on E2 subunit of PDH. Moreover, through native in gel activity assays we found decreased enzymatic activity of PDH-E3 and shifts in molecular weight of both PDH-E3 and E3 binding protein (E3Bp); in addition we found these positive for nitrogen specie-mediated modifications, which were not easily reversible with common reducing agents. Using chemical approaches we show that interaction of lipoate with NO can generate a reduced NO radical, nitroxyl (HNO), which can interact with thiols to form unstable intermediates that spontaneously rearrange to a sulfinamide; this represents a nearly irreversible thiol modification. Consistently, mass spectrometry analysis of recombinant protein reveals HNO-dependent post translational modification (PTM) of PDHE3. Structurally, HNO-specific modifications on E3 strongly impair homodimer formation, essential for activity. Our work is the first to demonstrate that HNO is produced physiologically in proinflammatory macrophages, and it is locally released within the interaction with lipoate moiety at the level of PDH-E2 and E3Bp, facilitating irreversible modification of distal E3. In turn this leads to inability for deacetylation of lipoate and global impairment of enzyme function. Finally, since the same E3 subunit is shared with two other key cellular metabolic enzymes, Oxoglutarate and Branched-chain alpha-keto acid Dehydrogenases (OGDH and BCKDH) we predict that NO could be responsible for orchestrating macrophage energy metabolism during inflammation also by limiting the activity of these dehydrogenases through irreversible modifications, opening the possibility for new therapeutic manipulations.

Metabolism of the Branched‐Chain Amino Acids are Disrupted by Chemotherapy Drugs

Cachexia is a devastating muscle-wasting condition found in many diseases, including cancer, chronic kidney disease and heart failure. Aside from both tumour burden and disease-related malnutrition, the development of cachexia is associated with chemotherapy treatment. Branched- chain amino acids (BCAA: leucine, isoleucine and valine) are critical regulators of skeletal muscle protein anabolism due to their activation of the mammalian/mechanistic target of rapamycin complex 1 (mTORC1). However, BCAA supplementation/nutritional support does not fully reverse chemotherapy-induced cachexia. Therefore, we investigated whether breakdown of BCAAs is affected by chemotherapy drugs. On day 4 of differentiation, L6 myotubes were treated with vehicle (1.4μL/mL DMSO) or a common chemotherapy drug cocktail, folfiri (a mixture of CPT-11 (20μg/mL), leucovorin (10μg/mL), and 5-fluorouracil (50μg/mL)) for 24-48h. Myotubes treated with folfiri exhibited ~30% reductions in myotube diameter (p < 0.05, n=3) and ~50% reductions in abundance of myofibrillar proteins myosin heavy chain-1 (MHC) and troponin (p < 0.05). Protein content of branched-chain alpha-ketoacid dehydrogenase complex (BCKD), the enzyme responsible for the irreversible decarboxylation of the BCAA ketoacids, was unchanged following folfiri treatment. However, the activity of this enzyme complex was significantly decreased (~20%) 24 and 48h following treatment with folfiri (p < 0.05). Branched-chain alpha-ketoacid dehydrogenase complex kinase (BDK), a negative regulator of BCKD, was increased 24h (~20%), but unchanged at 48h following folfiri treatment. Compared to vehicle, folfiri-treated myotubes showed a non-significant reduction (~30%) in phenylalanine incorporation into proteins. In line with studies showing a link between impaired BCAA catabolism and insulin resistance, our data suggest a link between chemotherapy-induced muscle atrophy and altered BCAA catabolism.

Comb-like EEG pattern in maple syrup urine disease: A case report

Maple syrup urine disease (MSUD) is a rare autosomal recessive metabolic disorder with neonatal-onset feeding difficulties, movement disorder, encephalopathy and seizures. If not treated in a timely manner, fatal progressive neurological deterioration and respiratory failure ensue [1,2]. This serious condition is caused by various degrees of deficiency in the branched-chain alpha-ketoacid dehydrogenase leading to the accumulation of the branched-chain amino acids, leucine, isoleucine, and valine and their toxic by-products in the brain.

Maple syrup urine disease: magnetic resonance imaging findings in three patients.

Maple syrup urine disease (MSUD) is an autosomal recessive inherited metabolic disorder, caused by branched-chain alpha-ketoacid dehydrogenase (BCKD) deficiency, leading to toxic accumulation of branched-chain amino acids (BCAAs) including leucine, isoleucine and valine and their corresponding a-ketoacids. The diagnosis of MSUD is based on elevated BCAAs and allo-isoleucine in plasma, and branched-chain hydroxyacids and ketoacids in urine. The identification of alloisoleucine >5 µmol/L is considered pathognomonic. Moreover, brain magnetic resonance imaging (MRI) showing atypical signal intensity and oedema is characteristic of MSUD. Recognition of the classical neuro-radiological findings of MSUD is particularly useful in local settings as many healthcare facilities lack the resources to measure Plasma Amino Acids (PAA). We report three cases of MSUD, in whom the disorder was strongly suspected at presentation, based on classical brain MRI findings, which was urgently confirmed by PAA analysis.