Alpha-keto Acid Dehydrogenase Research Articles

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.

Hyperleucinosis during infections in maple syrup urine disease post liver transplantation

Maple syrup urine disease (MSUD) is due to biallelic variants in one of the three genes: BCKDHA, BCKDHB, and DBT. Branched-chain alpha-ketoacid dehydrogenase complex deficiency and elevated leucine, valine, isoleucine and alloisoleucine in body fluids are the results. We report hyperleucinosis during intercurrent illnesses in six patients with MSUD post liver transplantation. Patient charts were retrospectively reviewed. Data was entered into an Excel Database. Literature was reviewed. Six patients with MSUD were included who had post liver transplantation hyperleucinosis during an intercurrent illness. Five had encephalopathy. One received hemodialysis for the management of hyperleucinosis. All patients had unrestricted diet. Additionally, there were five patients (one patient included into the current study) reported in the literature. We suggested management considerations for the follow-up of patients with MSUD post liver transplantation after the first episode of unexplained encephalopathy or signs of acute hyperleucinosis during intercurrent illness due to our clinical experience: 1) Healthy: Unrestricted diet and monitoring of leucine levels; 2) Illness: a) home illness management: increased carbohydrate intake b) illness management at hospital: intravenous dextrose, intravenous lipid and daily plasma amino acid monitoring. We report hyperleucinosis and/or encephalopathy as a rare event post liver transplantation in MSUD as a multicenter case series. Hyperleucinosis and/or encephalopathy may occur in both related and unrelated donor liver transplantation. Based on the long-term follow-up of those patients, these suggested management considerations may be revised as per the patients' needs.

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.

Identification of secreted and cellular antigens of Staphylococcus aureus causing dairy sheep mastitis and their potential for vaccine development

Staphylococcus aureus is the leading cause of clinical mastitis and is associated with persistent subclinical infections in ewes, significantly compromising the quality and quantity of milk productions. To date, vaccines intended for use in sheep have been mainly focused on biofilm production traits, but many S. aureus pathogenic isolates do not produce biofilm, including those circulating in Sardinia, one of the leading sheep milk producers in Europe. The aim of this work was to identify suitable immunodominant, alternative candidates to biofilm components for vaccine and diagnostic development. An immunoproteomics study was carried out by testing sera from naturally infected sheep with a prevalent S. aureus lineage against cellular and secreted antigens, followed by tandem mass spectrometry identification of the most prominent immunogens. Four cellular and three secreted S. aureus antigens elicited a strong humoral host immune response. The four cellular antigens were the housekeeping proteins pyruvate kinase, elongation Factor Tu, dihydrolipoyl dehydrogenase, and alpha-keto acid dehydrogenase. The three secreted antigens were the bifunctional autolysin (Atl) and the two components of the Panton-Valentine leukocidin, lukF-PV/lukM, demonstrating the carriage of prophage phiPV83 in a sheep isolate and the strong response of the sheep host against them. In consideration of the key role played by these secreted proteins in S. aureus replication and immune evasion, these antigens may represent suitable candidates for developing vaccines eliciting a more successful immunological protection in areas where non-biofilm forming Staphylococcus spp. are the most widespread intramammary pathogens.

Leucinosis, or maple syrup urine disease (lecture and a clinical case)

Maple syrup urine disease (leucinosis, short-chain ketoaciduria, branched-chain disease, branched-chain ketonuria) is an autosomal recessive disorder which is a consequence of the deficient branched-chain alpha ketoacid dehydrogenase complex. There are five subtypes of the disease: classical, intermediate, intermittent, thiamine-dependent and E3-deficient. Leucinosis is characterized by high plasma levels of branched-chain amino acids (leucine, isoleucine and valine) and high urine levels of branched-chain ketoacids, as well as of lactate and pyruvate. Tandem mass spectrometry can be used as a screening method in newborns. Mild disease cannot be identified at screening. The diagnosis should be based on tandem mass spectrometry of a blood sample and aminoacid analysis by gas chromatography of a urine sample. Prenatal diagnosis requires molecular genetic tests. Treatment of maple syrup urine disease is aimed at normalization of plasma branched-chain amino acids levels and includes two main components, namely, life-long diet therapy and active treatment of acute metabolic deterioration episodes. A favorable course of the disease is possible only with early (pre-symptomatic) initiation of treatment. The development of cognitive functions depends on plasma leucine levels. We present a clinical case of delayed diagnosis of leucinosis, despite its early clinical manifestation, leading to irreversible consequences for the patient.

1919-P: Dysfunction of BCAA Catabolism in Pancreatic Alpha Cells Is Associated with Higher Glucagon Secretion in Diabetic Mice

Background: Circulating plasma glucagon level is known to increase in diabetic state. We found that postprandial plasma glucagon level significantly increased in diabetic mice more than that in the control group. However, it’s still unknown which nutrient stimulates glucagon secretion as well as the underlying mechanism of glucagon secretion enhancement in diabetic state. Therefore, we aimed to answer these questions by using diabetic mouse models and isolated islets. Methods: Plasma glucagon levels were measured by sandwich ELISA in several diabetic model mice after oral administration of various nutrients. The effects of nutrients on glucagon secretion were assessed using isolated islets from diabetic mice. The expression levels of nutrient catabolism-related factors were analyzed in diabetic mice compared with control mice. Results and Discussions: Rather than carbohydrate or lipid, protein increased plasma glucagon levels in all diabetic models including high-fat diet mice, STZ mice, and db/db mice. Among amino acids, branched-chain amino acids (BCAA), but not the other essential or nonessential amino acids, increased plasma glucagon. BCAA directly stimulated glucagon secretion in isolated islets from diabetic mice, which was suppressed by amino acid transporter inhibitor (BCH) or branched-chain alpha-ketoacid dehydrogenase (BCKDK) inhibitor (BT2). This suggests that BCAA’s stimulation of glucagon secretion is mediated by BCAA catabolism in alpha cells. We also found that the expression level of BCKDK was higher, while branched-chain amino acid transaminase 2 (BCAT2) was lower, in diabetic islets than control islets. Considering that dysfunction of BCAA catabolism has been reported in both adipocytes and hepatocytes of diabetic mice, the dysfunction of BCAA catabolism might be a common pathogenesis for metabolic disorders observed in diabetes. Disclosure E. Wada: None. M. Kobayashi: None. O. Kikuchi: None. D. Kohno: None. T. Kitamura: None.

Data on the proliferation and differentiation of C2C12 myoblast treated with branched-chain ketoacid dehydrogenase kinase inhibitor.

The catabolism of branched chain amino acids (BCAAs) is mainly carried out in skeletal muscle myofibers. It is mediated by branched chain aminotransferase 2 and branched chain alpha ketoacid dehydrogenase (BCKDH) in mitochondria for energy supply, especially during exercise. BCKDH kinase (BCKDK) is a negative regulator of BCAAs catabolism by its inhibitory phosphorylation of the BCKDH E1a subunit. The data presented in this article are related to the research article that we previously have reported entitled “Energy metabolism profile of the effects of amino acid treatment on skeletal muscle cells: Leucine inhibits glycolysis of myotubes” (Suzuki et al., 2020)[1]. In this report, we have demonstrated that 1hour treatment of BT2, an inhibitor of BCKDK, decreased the glycolysis of C2C12 differentiated myotubes compared to the control. Although BCAAs metabolism is basically assumed to be carried out in differentiated myofibers, BCKDK is expressed in both undifferentiated myoblasts and differentiated myotubes, and the biological and physiological significance of BCAAs metabolism in myoblasts is still unclear. Present data demonstrate an in vitro assessment of BT2 on C2C12 myoblasts proliferation and differentiation. The data suggest that activation of BCAAs catabolism by the BCKDK inhibitor BT2 impairs C2C12 myoblasts proliferation and differentiation.

Bio-production of high-purity propionate by engineering L-threonine degradation pathway in Pseudomonas putida.

Propionic acid (PA) is widely used in the food, agricultural, and pharmaceutical industries. Since the petrochemical PA is unsustainable, biological production of PA from renewable substrates is gaining attention. In this study, we engineered the strain Pseudomonas putida KT2440 to transform L-threonine to PA with only CO2 released as by-product. The cell factory was created by chromosomal incorporation of heterologous L-threonine deaminase, permease, and acyl-CoA thioesterase, deletion of branch pathways as well as overproduction of the endogenous branched-chain alpha-keto acid dehydrogenase complex. The final engineered strain could produce 399mM PA from 400mM L-threonine in a batch biotransformation process, with a molar yield of 99.8% under the optimized conditions in 48h. The PA titer further reached to 50.3g/L (679mM) with a productivity of 0.6g/L/h in a fed-batch conversion process. No obvious by-products, such as acetate and succinate, were detected in the broth, which would significantly facilitate downstream purification steps. Thus, this study offers an alternative route for biological production of PA.

Effect of valine on myotube insulin sensitivity and metabolism with and without insulin resistance.

Population data have consistently demonstrated a correlation between circulating branched-chain amino acids (BCAA) and insulin resistance. Most recently valine catabolite, 3-hydroxyisobutyrate, has emerged as a potential cause of BCAA-mediated insulin resistance; however, it is unclear if valine independently promotes insulin resistance. It is also unclear if excess valine influences the ability of cells to degrade BCAA. Therefore, this study investigated the effect of valine on muscle insulin signaling and related metabolism in vitro. C2C12 myotubes were treated with varying concentrations (0.5mM-2mM) of valine for up to 48h. qRT-PCR and western blot were used to measure metabolic gene and protein expression, respectively. Insulin sensitivity (indicated by pAkt:Akt), metabolic gene and protein expression, and cell metabolism were also measured following valine treatment both with and without varying levels of insulin resistance. Mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Valine did not alter regulators of mitochondrial biogenesis or glycolysis; however, valine reduced branched-chain alpha-keto acid dehydrogenase a (Bckdha) mRNA (but not protein) expression which was exacerbated by insulin resistance. Valine treatment had no effect on pAkt:Akt following either acute or 48-h treatment, regardless of insulin stimulation or varying levels of insulin resistance. In conclusion, despite consistent population data demonstrating a relationship between circulating BCAA (and related metabolites) and insulin resistance, valine does not appear to independently alter insulin sensitivity or worsen insulin resistance in the myotube model of skeletal muscle.

Exclusive neuronal detection of KGDHC-specific subunits in the adult human brain cortex despite pancellular protein lysine succinylation

The ketoglutarate dehydrogenase complex (KGDHC) consists of three different subunits encoded by OGDH (or OGDHL), DLST, and DLD, combined in different stoichiometries. DLD subunit is shared between KGDHC and pyruvate dehydrogenase complex, branched-chain alpha-keto acid dehydrogenase complex, and the glycine cleavage system. Despite KGDHC’s implication in neurodegenerative diseases, cell-specific localization of its subunits in the adult human brain has never been investigated. Here, we show that immunoreactivity of all known isoforms of OGDHL, OGDH, and DLST was detected exclusively in neurons of surgical human cortical tissue samples identified by their morphology and visualized by double labeling with fluorescent Nissl, while being absent from glia expressing GFAP, Aldhl1, myelin basic protein, Olig2, or IBA1. In contrast, DLD immunoreactivity was evident in both neurons and glia. Specificity of anti-KGDHC subunits antisera was verified by a decrease in staining of siRNA-treated human cancer cell lines directed against the respective coding gene products; furthermore, immunoreactivity of KGDHC subunits in human fibroblasts co-localized > 99% with mitotracker orange, while western blotting of 63 post-mortem brain samples and purified recombinant proteins afforded further assurance regarding antisera monospecificity. KGDHC subunit immunoreactivity correlated with data from the Human Protein Atlas as well as RNA-Seq data from the Allen Brain Atlas corresponding to genes coding for KGDHC components. Protein lysine succinylation, however, was immunohistochemically evident in all cortical cells; this was unexpected, because this posttranslational modification requires succinyl-CoA, the product of KGDHC. In view of the fact that glia of the human brain cortex lack succinate-CoA ligase, an enzyme producing succinyl-CoA when operating in reverse, protein lysine succinylation in these cells must exclusively rely on propionate and/or ketone body metabolism or some other yet to be discovered pathway encompassing succinyl-CoA.

An Updated View on the Molecular Pathomechanisms of Human Dihydrolipoamide Dehydrogenase Deficiency in Light of Novel Crystallographic Evidence

Dihydrolipoamide dehydrogenase (LADH, E3) deficiency is a rare (autosomal, recessive) genetic disorder generally presenting with an onset in the neonatal age and early death; the highest carrier rate has been found among Ashkenazi Jews. Acute clinical episodes usually involve severe metabolic decompensation and lactate acidosis that result in neurological, cardiological, and/or hepatological manifestations. Clinical severity is due to the fact that LADH is a common E3 subunit to the alpha-ketoglutarate, pyruvate, alpha-ketoadipate, and branched-chain alpha-keto acid dehydrogenase complexes, and is also a constituent in the glycine cleavage system, thus a loss in LADH function adversely affects multiple key metabolic routes. However, the severe clinical pictures frequently still do not parallel the LADH activity loss, which implies the involvement of auxiliary biochemical mechanisms; enhanced reactive oxygen species generation as well as affinity loss for multienzyme complexes proved to be key auxiliary exacerbating pathomechanisms. This review provides an overview and an up-to-date molecular insight into the pathomechanisms of this disease in light of the structural conclusions drawn from the first crystal structure of a disease-causing hE3 variant determined recently in our laboratory.

Quantitative Analysis of the Whole-Body Metabolic Fate of Branched-Chain Amino Acids

Elevations in branched-chain amino acids (BCAAs) associate with numerous systemic diseases, including cancer, diabetes, and heart failure. However, an integrated understanding of whole-body BCAA metabolism remains lacking. Here, we employ invivo isotopic tracing to systemically quantify BCAA oxidation in healthy and insulin-resistant mice. We find that most tissues rapidly oxidize BCAAs into the tricarboxylic acid (TCA) cycle, with the greatest quantity occurring in muscle, brown fat, liver, kidneys, and heart. Notably, pancreas supplies 20% of its TCA carbons from BCAAs. Genetic and pharmacologic suppression of branched-chain alpha-ketoacid dehydrogenase kinase, a clinically targeted regulatory kinase, induces BCAA oxidation primarily in skeletal muscle of healthy mice. While insulin acutely increases BCAA oxidation in cardiac and skeletal muscle, chronically insulin-resistant mice show blunted BCAA oxidation in adipose tissues and liver, shifting BCAA oxidation toward muscle. Together, this work provides a quantitative framework for understanding systemic BCAA oxidation in health and insulin resistance.

Liver transplantation from a live donor to a patient with maple syrup urine disease: Two case reports

Liver transplantation were reported in patients with classic maple syrup urine disease in the literature. Branched chain alpha keto acid dehydrogenase activity can be improved in patients after transplant, and a protein-restricted diet is usually not needed. The first patient was a boy aged 2,5 years who presented with frequent ketosis attacks and epileptic seizures, and the second patient was an 11-month-old boy who also presented with frequent ketosis episodes, both despite adherence to diet therapy. Both patients received liver transplantations from live donors. A low protein diet was no longer required and no decline in cognitive functions was observed in either patient in the follow-up. We wanted to present these cases to show that despite a normal diet, plasma levels of branched- chain amino acids remained normal without any decline in cognitive function after liver transplantation in patients with classic maple syrup urine disease patients.