Successful treatment of severe MSUD in Bckdhb-/- mice with neonatal AAV gene therapy.

Maple syrup urine disease (MSUD) is rare autosomal recessive metabolic disorder caused by the dysfunction of the mitochondrial branched-chain 2-ketoacid dehydrogenase (BCKD) enzyme complex leading to massive accumulation of branched-chain amino acids and 2-keto acids. MSUD management, based on a life-long strict protein restriction with nontoxic amino acids oral supplementation represents an unmet need as it is associated with a poor quality of life, and does not fully protect from acute life-threatening decompensations or long-term neuropsychiatric complications. Orthotopic liver transplantation is a beneficial therapeutic option, which shows that restoration of only a fraction of whole-body BCKD enzyme activity is therapeutic. MSUD is thus an ideal target for gene therapy. We and others have tested AAV gene therapy in mice for two of the three genes involved in MSUD, BCKDHA and DBT. In this study, we developed a similar approach for the third MSUD gene, BCKDHB. We performed the first characterization of a Bckdhb-/- mouse model, which recapitulates the severe human phenotype of MSUD with early-neonatal symptoms leading to death during the first week of life with massive accumulation of MSUD biomarkers. Based on our previous experience in Bckdha-/- mice, we designed a transgene carrying the human BCKDHB gene under the control of a ubiquitous EF1α promoter, encapsidated in an AAV8 capsid. Injection in neonatal Bckdhb-/- mice at 1014 vg/kg achieved long-term rescue of the severe MSUD phenotype of Bckdhb-/- mice. These data further validate the efficacy of gene therapy for MSUD opening perspectives towards clinical translation.

Outcomes from a Single Transplant Center of 5 Pediatric Cases of Domino Liver Transplantation from Live Donors with Maple Syrup Urine Disease.

BACKGROUND Maple syrup urine disease (MSUD) is a rare genetic deficiency of the branched-chain alpha-keto acid dehydrogenase (BCKAD) complex that breaks down amino acids, resulting in multi-organ failure. This report is of 5 pediatric cases of domino liver transplantation (DLT) from live donors with MSUD from a single transplant center in Beijing. CASE REPORT All MSUD donors were confirmed to have disease-causing mutations in BCKDHA (branched-chain keto acid dehydrogenase E1, alpha polypeptide) or BCKDHB (branched-chain keto acid dehydrogenase E1, ß polypeptide) genes by peripheral blood whole-exon sequencing. Serum leucine and valine concentrations were significantly higher than normal values. Recipients ranged in age from 0.75 to 9 years old. Three patients underwent auxiliary liver transplantation, and the other children all underwent liver or partial liver transplantation. This case report was followed up for 25 to 79 months. The prognosis, growth, and development of patients were followed up. By the end of the last follow-up, all children had survived. All patients had normal serum leucine and valine concentrations after surgery. In case 1, portal vein stenosis post-operatively. In case 2, stenosis of hepatic artery and bile duct occurred. In case 5, hepatic artery and portal vein stenosis occurred, resulting in graft loss.   CONCLUSIONS The findings from our center support the findings from other pediatric liver transplant centers that liver transplantation using MSUD donors can have successful outcomes without the development of MSUD in the recipient.

Neonatal gene therapy achieves sustained disease rescue of maple syrup urine disease in mice.

Maple syrup urine disease (MSUD) is a rare recessively inherited metabolic disorder causing accumulation of branched chain amino acids leading to neonatal death, if untreated. Treatment for MSUD represents an unmet need because the current treatment with life-long low-protein diet is challenging to maintain, and despite treatment the risk of acute decompensations and neuropsychiatric symptoms remains. Here, based on significant liver contribution to the catabolism of the branched chain amino acid leucine, we develop a liver-directed adeno-associated virus (AAV8) gene therapy for MSUD. We establish and characterize the Bckdha (branched chain keto acid dehydrogenase a)-/- mouse that exhibits a lethal neonatal phenotype mimicking human MSUD. Animals were treated at P0 with intravenous human BCKDHA AAV8 vectors under the control of either a ubiquitous or a liver-specific promoter. BCKDHA gene transfer rescued the lethal phenotype. While the use of a ubiquitous promoter fully and sustainably rescued the disease (long-term survival, normal phenotype and correction of biochemical abnormalities), liver-specific expression of BCKDHA led to partial, though sustained rescue. Here we show efficacy of gene therapy for MSUD demonstrating its potential for clinical translation.

Branched-chain ketoacid overload inhibits insulin action in the muscle.

Branched-chain α-keto acids (BCKAs) are catabolites of branched-chain amino acids (BCAAs). Intracellular BCKAs are cleared by branched-chain ketoacid dehydrogenase (BCKDH), which is sensitive to inhibitory phosphorylation by BCKD kinase (BCKDK). Accumulation of BCKAs is an indicator of defective BCAA catabolism and has been correlated with glucose intolerance and cardiac dysfunction. However, it is unclear whether BCKAs directly alter insulin signaling and function in the skeletal and cardiac muscle cell. Furthermore, the role of excess fatty acids (FAs) in perturbing BCAA catabolism and BCKA availability merits investigation. By using immunoblotting and ultra-performance liquid chromatography MS/MS to analyze the hearts of fasted mice, we observed decreased BCAA-catabolizing enzyme expression and increased circulating BCKAs, but not BCAAs. In mice subjected to diet-induced obesity (DIO), we observed similar increases in circulating BCKAs with concomitant changes in BCAA-catabolizing enzyme expression only in the skeletal muscle. Effects of DIO were recapitulated by simulating lipotoxicity in skeletal muscle cells treated with saturated FA, palmitate. Exposure of muscle cells to high concentrations of BCKAs resulted in inhibition of insulin-induced AKT phosphorylation, decreased glucose uptake, and mitochondrial oxygen consumption. Altering intracellular clearance of BCKAs by genetic modulation of BCKDK and BCKDHA expression showed similar effects on AKT phosphorylation. BCKAs increased protein translation and mTORC1 activation. Pretreating cells with mTORC1 inhibitor rapamycin restored BCKA's effect on insulin-induced AKT phosphorylation. This study provides evidence for FA-mediated regulation of BCAA-catabolizing enzymes and BCKA content and highlights the biological role of BCKAs in regulating muscle insulin signaling and function.

Phosphorylation of BCKDK of BCAA catabolism at Y246 by Src promotes metastasis of colorectal cancer.

Branched-chain α-keto acid dehydrogenase kinase (BCKDK), the key enzyme of branched-chain amino acids (BCAAs) metabolism, has been reported to promote colorectal cancer (CRC) tumorigenesis by upregulating the MEK-ERK signaling pathway. However, the profile of BCKDK in metastatic colorectal cancer (mCRC) remains unknown. Here, we report a novel role of BCKDK in mCRC. BCKDK is upregulated in CRC tissues. Increased BCKDK expression was associated with metastasis and poor clinical prognosis in CRC patients. Knockdown of BCKDK decreased CRC cell migration and invasion ex vivo, and lung metastasis in vivo. BCKDK promoted the epithelial mesenchymal transition (EMT) program, by decreasing the expression of E-cadherin, epithelial marker, and increasing the expression of N-cadherin and Vimentin, which are mesenchymal markers. Moreover, BCKDK-knockdown experiments in combination with phosphoproteomics analysis revealed the potent role of BCKDK in modulating multiple signal transduction pathways, including EMT and metastasis. Src phosphorylated BCKDK at the tyrosine 246 (Y246) site in vitro and ex vivo. Knockdown and knockout of Src downregulated the phosphorylation of BCKDK. Importantly, phosphorylation of BCKDK by Src enhanced the activity and stability of BCKDK, thereby promoting the migration, invasion, and EMT of CRC cells. In summary, the identification of BCKDK as a novel prometastatic factor in human CRC will be beneficial for further diagnostic biomarker studies and suggests novel targeting opportunities.

Genome-wide screening identifies novel genes implicated in cellular sensitivity to BRAFV600E expression.

The V600E mutation of BRAF (BRAFV600E), which constitutively activates the ERK/MAPK signaling pathway, is frequently found in melanoma and other cancers. Like most other oncogenes, BRAFV600E causes oncogenic stress to normal cells, leading to growth arrest (senescence) or apoptosis. Through genome-wide screening, we identified genes implicated in sensitivity of human skin melanocytes and fibroblasts to BRAFV600E overexpression. Among the identified genes shared by the two cell types are proto-oncogenes ERK2, a component of the ERK/MAPK pathway, and VAV1, a guanine nucleotide exchange factor for Rho family GTPases that also activates the ERK/MAPK pathway. CDKN1A, which has been known to promote senescence of fibroblasts but not melanocytes, is implicated in sensitivity of the fibroblasts but not the melanocytes to BRAFV600E overexpression. Disruptions of GPR4, a pH-sensing G-protein coupled receptor, and DBT, a subunit of the branched chain α-keto acid dehydrogenase that is required for the second and rate-limiting step of branched amino acid catabolism and implicated in maple syrup urine disease, are the most highly selected in the melanocytes upon BRAFV600E overexpression. Disruption of DBT severely attenuates ERK/MAPK signaling, p53 activation, and apoptosis in melanocytes, at least in part due to accumulation of branched chain α-keto acids. The expression level of BRAF positively correlates with that of DBT in all cancer types and with that of GPR4 in most cancer types. Overexpression of DBT kills all four melanoma cell lines tested regardless of the presence of BRAFV600E mutation. Our findings shed new lights on regulations of oncogenic stress signaling and may be informative for development of novel cancer treatment strategies.

Paroxysmal spasticity of lower extremities as the initial symptom in two siblings with maple syrup urine disease.

Maple syrup urine disease (MSUD) is a rare autosomal recessive metabolic disorder caused by mutations in genes that encode subunits of the branched­chain α­ketoacid dehydrogenase (BCKD) complex. Impairment of the BCKD complex results in an abnormal accumulation of branched­chain amino acids and their corresponding branched­chain keto acids in the blood and cerebrospinal fluid, which are neurovirulent and may become life­threatening. An 11­day­old boy was admitted to the hospital with paroxysmal spasticity of lower extremities. Of note, his 10­year­old sister presented similar symptoms during the neonatal period, and her condition was diagnosed as MSUD when she was 1.5 years old. Genetic screening was performed, and the boy and his sister exhibited two novel compound heterozygous mutations in the branched chain keto acid dehydrogenase E1 subunit ß (BCKDHB) gene: A substitution from guanine to adenine in the coding region at position 1,076 (c.1,076G>A) in exon 10 and a deletion of a thymine at position 705 (c.705delT) in exon 6. The missense mutation c.1076G>A results in an amino acid substitution from arginine to lysine at position 359 (p.Arg359Lys), whereas the mutation c.705delT results in the replacement of a cysteine at position 235 with a stop codon (p.Cys235Ter). Neither of the BCKDHB alleles in the compound heterozygote patients is able to generate normal E1ß subunits, resulting in a possible impairment of the activity of the BCKD complex. In the present study, it was hypothesized that the two novel heterozygous mutations in the BCKDHB gene found in the Chinese family may be responsible for the phenotype of the two siblings with MSUD.

Changes in tissue abundance and activity of enzymes related to branched-chain amino acid catabolism in dairy cows during early lactation.

Branched-chain α-keto acid dehydrogenase (BCKDH) complex catalyzes the irreversible oxidative decarboxylation of branched-chain α-keto acids. This reaction is considered as the rate-limiting step in the overall branched-chain amino acid (BCAA) catabolic pathway in mammals. For characterizing the potential enzymatic involvement of liver, skeletal muscle, adipose tissue (AT), and mammary gland (MG) in BCAA metabolism during early lactation, tissue and blood samples were examined on d 1, 42, and 105 after parturition from 25 primiparous Holstein cows. Serum BCAA profiles were analyzed and the mRNA and protein abundance as well as the activity in the different tissues were assessed for the BCAA catabolic enzymes, partly for the branched-chain aminotransferase and completely for BCKDH. Total BCAA concentration in serum was lowest on d 1 after parturition and increased thereafter to a steady level for the duration of the experiment. Pronounced differences between the tissues were observed at all molecular levels. The mRNA abundance of the mitochondrial isoform of branched-chain aminotransferase (BCATm) was greatest in AT as compared with the other tissues studied, indicating that AT might be an important contributor in the initiation of BCAA catabolism in dairy cows. From the different subunits of the BCKDH E1 component, only the mRNA for the ß polypeptide (BCKDHB), not for the α polypeptide (BCKDHA), was elevated in liver. The BCKDHA mRNA abundance was similar across all tissues except muscle, which tended to lower values. Highest BCKDHA protein abundance was observed in both liver and MG, whereas BCKDHB protein was detectable in these tissues but could not be quantified. Adipose tissue and muscle only displayed abundance of the α subunit, with muscle having the lowest BCKDHA protein of all tissues. We found similarities in protein abundance for both BCKDH E1 subunits in liver and MG; however, the corresponding overall BCKDH enzyme activity was 7-fold greater in liver compared with MG, allowing for hepatic oxidation of BCAA transamination products. Reduced BCKDH activity in MG associated with no measurable activity in AT and muscle may favor sparing of BCAA for the synthesis of the different milk components, including nonessential AA. Deviating from previously published data on BCAA net fluxes and isotopic tracer studies in ruminants, our observed results might in part be due to complex counter-regulatory mechanisms during early lactation.

Regulatory and biosynthetic effects of the bkd gene clusters on the production of daptomycin and its analogs A21978C1-3.

Daptomycin is a cyclic lipopeptide antibiotic produced by Streptomyces roseosporus in an acidic peptide complex A21978C. In this complex, A21978C1-3 is most abundant and contains branched-chain fatty acyl groups, while daptomycin has a straight decanoic acyl group. The branched-chain α-keto acid dehydrogenase complex (BCDH complex), encoded by bkd gene clusters in Streptomyces, is responsible for the early step of converting branched-chain amino acids into branched-chain fatty acids. In a daptomycin industrial producer S. roseosporus L30, two alleles of bkd gene clusters, bkdA1B1C1/bkdA2B2C2, and a regulatory gene bkdR located upstream of bkdA2B2C2 are identified. We show that BkdR positively regulated bkdA2B2C2 expression and was negatively auto-regulated, but is not directly involved in regulation of daptomycin gene cluster expression. However, BkdR is required for both daptomycin and A21978C1-3 production. Furthermore, deletion of bkdA2B2C2 only led to partial reduction of A21978C1-3 production, while the ΔbkdA1B1C1 mutant shows very weak production of A21978C1-3, and the double bkd mutant has a similar production profile as the single ΔbkdA1B1C1 mutant, suggesting that bkdA1B1C1 gene cluster plays a dominant role in branched-chain fatty acid biosynthesis. So we reveal a unique regulatory function of BkdR and genetic engineered a bkd null strain for daptomycin production with reduced impurities.

Two novel mutations in the BCKDHB gene that cause maple syrup urine disease.

BACKGROUND: Maple syrup urine disease (MSUD) is a rare metabolic disorder of autosomal recessive inheritance caused by decreased activity of branched-chain α-ketoacid dehydrogenase complex (BCKD). Mutations in the three genes (BCKDHA, BCKDHB and DBT) are associated with MSUD. Here, we describe the presenting symptoms, clinical course and gene mutation analysis of a Chinese boy with MSUD. METHODS: Plasma amino acid analysis was performed by tandem mass spectrometry and the levels of organic acids in urine were measured with gas chromatography-mass spectrometry. The BCKDHB gene was sequenced by Sanger method. Furthermore, the significance of the novel mutations was predicted by Polyphen and Mutationtaster. After diagnosis, the patient was fed with protein-restricted diet to reduce intake of BCAA and was treated with l-carnitine. Metabolic parameters, clinical presentation and mental development were followed up. RESULTS: The patient was diagnosed as MSUD. Two novel BCKDHB mutations (c.523 T > C and c.478-25_552del100) were identified. In silico analysis predicted that the two mutations were "disease causing". The boy tolerated the treatment well and had symptomatic improvement. He presented with mild hypotonia and had nearly normal DQ scores at the age of 10 months. The two novel mutations resulted in the clinical manifestations of MSUD. Our results may reflect the heterogeneity of the pathogenic variants found in patients with MSUD.

miR-124-3p affects the formation of intramuscular fat through alterations in branched chain amino acid consumption in sheep.

Intramuscular fat is used to determine meat quality in animals; however, factors affecting branched chain amino acid (BCAA) catabolism, which fuels adipogenesis and lipogenesis, remain unclear. To better understand the post-transcriptional influence on BCAA catabolism during adipogenesis, we investigated the role of miR-124-3p. Stromal vascular fraction (SVF) cells were isolated from skeletal muscle of sheep, and induced to differentiate. We determined the roles of miR-124-3p and its predicted target, branched chain keto acid dehydrogenase E1, alpha polypeptide (BCKDHA), in adipogenic differentiation and lipogenesis of SVFs after overexpressing or inhibiting miR-124-3p or BCKDHA, respectively. miR-124-3p altered the luciferase activity of constructs containing 3'-UTR of BCKDHA and the formation of lipid droplets, along with the adipogenic markers and BCAA consumption. Besides, the adipogenic performance and BCAA consumption in BCKDHA-overexpressing or knocked-down SVFs and the expression of adipogenic marker genes were altered. We demonstrate that miR-124-3p is an important factor for adipogenesis and provide insights into the formation of intramuscular fat in animals.

O-Acyl Sugars Protect a Wild Tobacco from Both Native Fungal Pathogens and a Specialist Herbivore.

O-Acyl sugars (O-AS) are abundant trichome-specific metabolites that function as indirect defenses against herbivores of the wild tobacco Nicotiana attenuata; whether they also function as generalized direct defenses against herbivores and pathogens remains unknown. We characterized natural variation in O-AS among 26 accessions and examined their influence on two native fungal pathogens, Fusarium brachygibbosum U4 and Alternaria sp. U10, and the specialist herbivore Manduca sexta At least 15 different O-AS structures belonging to three classes were found in N. attenuata leaves. A 3-fold quantitative variation in total leaf O-AS was found among the natural accessions. Experiments with natural accessions and crosses between high- and low-O-AS accessions revealed that total O-AS levels were associated with resistance against herbivores and pathogens. Removing O-AS from the leaf surface increased M. sexta growth rate and plant fungal susceptibility. O-AS supplementation in artificial diets and germination medium reduced M. sexta growth and fungal spore germination, respectively. Finally, silencing the expression of a putative branched-chain α-ketoacid dehydrogenase E1 ß-subunit-encoding gene (NaBCKDE1B) in the trichomes reduced total leaf O-AS by 20% to 30% and increased susceptibility to Fusarium pathogens. We conclude that O-AS function as direct defenses to protect plants from attack by both native pathogenic fungi and a specialist herbivore and infer that their diversification is likely shaped by the functional interactions among these biotic stresses.

Alterations of plasma metabolite profiles related to adipose tissue distribution and cardiometabolic risk.

Metabolomic profiling of obese individuals revealed altered concentrations of many metabolites, especially branched-chain amino acids (BCAA), possibly linked to altered adipose tissue BCAA catabolism. We tested the hypothesis that some features of this metabolite signature relate closely to visceral obesity and concomitant alterations in cardiometabolic risk factors. We also postulated that alterations in BCAA-catabolizing enzymes are predominant in visceral adipose tissue. Fifty-nine women (BMI 20-41 kg/m(2)) undergoing gynecologic surgery were recruited and characterized for overall and regional adiposity, blood metabolite levels using targeted metabolomics, and cardiometabolic risk factors. Adipose samples (visceral and subcutaneous) were obtained and used for gene expression and Western blot analyses. Obese women had significantly higher circulating BCAA and kynurenine/tryptophan (Kyn/Trp) ratio than lean or overweight women (P < 0.01). Principal component analysis confirmed that factors related to AA and the Kyn/Trp ratio were positively associated with BMI, fat mass, visceral or subcutaneous adipose tissue area, and subcutaneous adipocyte size (P ≤ 0.05). AA-related factor was positively associated with HOMA-IR (P ≤ 0.01). Factors reflecting glycerophospholipids and sphingolipids levels were mostly associated with altered blood lipid concentrations (P ≤ 0.05). Glutamate level was the strongest independent predictor of visceral adipose tissue area (r = 0.46, P < 0.001). Obese women had lower expression and protein levels of BCAA-catabolizing enzymes in visceral adipose tissue than overweight or lean women (P ≤ 0.05). We conclude that among metabolites altered in obesity plasma concentrations of BCAA and the Kyn/Trp ratio are closely related to increased adiposity. Alterations in expression and protein levels of BCAA-catabolizing enzymes are predominant in visceral adipose tissue.

[Maple syrup urine disease caused by two novel BCKDHB gene mutations in a Chinese neonate].

OBJECTIVE: Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disorder that is caused by mutations in the subunits of the branched chain α-ketoacid dehydrogenase (BCKD) complex. This report presents a Han ethnic Chinese newborn infant with the severe classic form of MSUD caused by two novel missense mutations in the BCKDHB gene. METHOD: The clinical and biochemical data of a Chinese neonate with classic form of MSUD were analyzed, and the DNA sequences of BCKDHA, BCKDHB, DBT and DLD genes were investigated for mutations. Then the DNA samples of the proband and the patient's parents were tested with Sanger sequencing. RESULT: The manifestations of this patient were poor feeding, low reaction, and compensatory metabolic acidosis. Tandem mass spectrometry (MS/MS) showed that leucine and valine were significantly higher than normal. Urine gas chromatography-mass spectrometry (GC/MS) showed significant abnormality. Brain CT scan showed white matter changes. We identified two previously unreported mutations in the BCKDHB gene, p.Leu194Phe (c.580 C>T) and p.Ser199Arg (c.597 T>G) in exon 5. Segregation analysis showed that the novel mutation p.Ser199Arg was maternally inherited and the novel mutation p.Leu194Phe was paternally inherited. Neither mutation was found in the 186 alleles of 93 normal Han ethnic Chinese individuals. In human BCKDHB protein crystal structure, the 194th and 199th amino acids changes are likely to affect the spatial structure of the protein. The 194th and 199th amino acid of human BCKDHB protein was conserved among species. PolyPhen protein function prediction indicated that the 194th and 199th amino acid changes were likely to affect protein function. CONCLUSION: Two novel missense mutations were identified in the BCKDHB gene in the Chinese patient with MSUD.

Adipose transplant for inborn errors of branched chain amino acid metabolism in mice.

Liver transplantation appears to be quite beneficial for treatment of maple syrup urine disease (MSUD, an inherited disorder of branched chain amino acid metabolism); however, there is a limited availability of donor livers worldwide and the first year costs of liver transplants are quite high. Recent studies have suggested that intact adipose tissue, already widely used in reconstructive surgery, may have an underappreciated high capacity for branched chain amino acid (BCAA) metabolism. Here we examined the potential for adipose tissue transplant to lower circulating BCAAs in two models of defective BCAA metabolism, BCATm and PP2Cm [branched chain keto acid dehydrogenase complex (BCKDC) phosphatase] knockout (KO) mice. After 1-2g fat transplant, BCATm and PP2Cm KO mice gained or maintained body weight 3weeks after surgery and consumed similar or more food/BCAAs the week before phlebotomy. Transplant of fat into the abdominal cavity led to a sterile inflammatory response and nonviable transplanted tissue. However when 1-2g of fat was transplanted subcutaneously into the back, either as small (0.1-0.3g) or finely minced pieces introduced with an 18-ga. needle, plasma BCAAs decreased compared to Sham operated mice. In two studies on BCATm KO mice and one study on PP2Cm KO mice, fat transplant led to 52-81% reductions in plasma BCAAs compared to baseline plasma BCAA concentrations of untreated WT type siblings. In PP2Cm KO mice, individual BCAAs in plasma were also significantly reduced by fat transplant, as were the alloisoleucine/Phe ratios. Therefore, subcutaneous fat transplantation may have merit as an adjunct to dietary treatment of MSUD. Additional studies are needed to further refine this approach.

Placental stem cell correction of murine intermediate maple syrup urine disease.

UNLABELLED: There is improved survival and partial metabolic correction of a mouse intermediate maple syrup urine disease (iMSUD) model after allogenic hepatocyte transplantation, confirming that a small number of enzyme-proficient liver-engrafted cells can improve phenotype. However, clinical shortages of suitable livers for hepatocyte isolation indicate a need for alternative cell sources. Human amnion epithelial cells (hAECs) share stem cell characteristics without the latter's safety and ethical concerns and differentiate to hepatocyte-like cells. Eight direct hepatic hAEC transplantations were performed in iMSUD mice over the first 35 days beginning at birth; animals were provided a normal protein diet and sacrificed at 35 and 100 days. Treatment at the neonatal stage is clinically relevant for MSUD and may offer a donor cell engraftment advantage. Survival was significantly extended and body weight was normalized in iMSUD mice receiving hAEC transplantations compared with untreated iMSUD mice, which were severely cachectic and died ≤28 days after birth. Branched chain α-keto acid dehydrogenase enzyme activity was significantly increased in transplanted livers. The branched chain amino acids leucine, isoleucine, valine, and alloisoleucine were significantly improved in serum and brain, as were other large neutral amino acids. CONCLUSION: Placental-derived stem cell transplantation lengthened survival and corrected many amino acid imbalances in a mouse model of iMSUD. This highlights the potential for their use as a viable alternative clinical therapy for MSUD and other liver-based metabolic diseases.

Regulation of leucine catabolism by metabolic fuels in mammary epithelial cells.

Lactation is associated with elevated catabolism of branched-chain amino acids (BCAA) in mammary glands to produce glutamate, glutamine, alanine, aspartate, and asparagine. This study determined effects of metabolic fuels on the catabolism of leucine (a representative BCAA) in bovine mammary epithelial cells. Cells were incubated at 37 °C for 2 h in Krebs buffer containing 0.5 mM L-leucine and either L-[1-(14)C]leucine or L-[U-(14)C]leucine. The medium also contained 0-5 mM D-glucose, 0-2 mM L-glutamine, 0-4 mM DL-ß-hydroxybutyrate, or 0-2 mM oleic acid. Rates of leucine decarboxylation were 60 % lower, but rates of α-ketoisocaproate production were 34 % higher, in the presence of 2 mM glucose than in its absence. All variables of leucine catabolism did not differ between 2 and 5 mM glucose or between 0 and 4 mM DL-ß-hydroxybutyrate. Compared with 0-0.25 mM glutamine, 0.5 and 2 mM L-glutamine reduced leucine transport, transamination, and decarboxylation. In contrast, increasing the concentration of oleic acid from 0 to 2 mM dose-dependently stimulated leucine transamination, decarboxylation, and oxidation of carbons 2-6. Oleic acid also enhanced the abundance of cytosolic BCAA transaminase, while reducing the phosphorylated level (inactive state) of the E1α subunit of the mitochondrial branched-chain α-ketoacid dehydrogenase complex. Thus, hypoglycemia or ketosis in early lactation does not likely affect BCAA metabolism in mammary epithelial cells. Increasing circulating levels of BCAA and oleic acid may have great potential to increase the syntheses of glutamate, glutamine, aspartate, alanine, and asparagine by lactating mammary glands, thereby leading to enhanced production of milk for suckling neonates.

Branched-chain fatty acids promote Listeria monocytogenes intracellular infection and virulence.

Anteiso-branched-chain fatty acids (BCFA) represent the dominant group of membrane fatty acids and have been established as crucial determinants in resistance against environmental stresses in Listeria monocytogenes, a facultative intracellular pathogen. Here, we investigate the role of anteiso-BCFA in L. monocytogenes virulence by using mutants deficient in branched-chain alpha-keto acid dehydrogenase (BKD), an enzyme complex involved in the synthesis of BCFA. In tissue culture models of infection, anteiso-BCFA contributed to intracellular growth and survival in macrophages and significantly enhanced plaque formation upon prolonged infection in L2 fibroblasts. The intracellular defects observed could be attributed partially to insufficient listeriolysin O (LLO) production, indicating a requirement for anteiso-BCFA in regulating virulence factor production. In a murine model of infection, the BKD-deficient mutant was highly attenuated, further emphasizing the importance of BKD-mediated metabolism in L. monocytogenes virulence. This study demonstrates an underappreciated role for BCFA in bacterial pathogenesis, which may provide insight into the development and application of antimicrobial agents.

Functional characterization of the novel intronic nucleotide change c.288+9C>T within the BCKDHA gene: understanding a variant presentation of maple syrup urine disease.

Mutations in any of the three different genes--BCKDHA, BCKDHB, and DBT--encoding for the E1α, E1ß, and E2 catalytic components of the branched-chain α-ketoacid dehydrogenase complex can cause maple syrup urine disease (MSUD). Disease severity ranges from the classic to the mildest variant types and precise genotypes, mostly based on missense mutations, have been associated to the less severe presentations of the disease. Herein, we examine the consequences at the messenger RNA (mRNA) level of the novel intronic alteration c.288+9C>T found in heterozygous fashion in a BCKDHA variant MSUD patient who also carries the nucleotide change c.745G>A (p.Gly249Ser), previously described as a severe change. Direct analysis of the processed transcripts from the patient showed--in addition to a low but measurable level of normal mRNA product--an aberrantly spliced mRNA containing a 7-bp fragment of intron 2, which could be rescued when the patient's cells were treated with emetine. This aberrant transcript with a premature stop codon would be unstable, supporting the possible activation of nonsense-mediated mRNA decay pathway. Consistent with this finding, minigene splicing assays demonstrated that the point mutation c.288+9C>T is sufficient to create a cryptic splice site and cause the observed 7-bp insertion. Furthermore, our results strongly suggest that the c.288+9C>T allele in the patient generates both normal and aberrant transcripts that could sustain the variant presentation of the disease, highlighting the importance of correct genotyping to establish genotype-phenotype correlations and as basis for the development of therapeutic interventions.

Molecular cloning, sequence identification and tissue expression profile of three novel sheep (Ovis aries) genes - BCKDHA, NAGA and HEXA.

The complete coding sequences of three sheep genes- BCKDHA, NAGA and HEXA were amplified using the reverse transcriptase polymerase chain reaction (RT-PCR), based on the conserved sequence information of the mouse or other mammals. The nucleotide sequences of these three genes revealed that the sheep BCKDHA gene encodes a protein of 313 amino acids which has high homology with the BCKDHA gene that encodes a protein of 447 amino acids that has high homology with the Branched chain keto acid dehydrogenase El, alpha polypeptide (BCKDHA) of five species chimpanzee (93%), human (96%), crab-eating macaque (93%), bovine (98%) and mouse (91%). The sheep NAGA gene encodes a protein of 411 amino acids that has high homology with the alpha-N-acetylgalactosaminidase (NAGA) of five species human (85%), bovine (94%), mouse (91%), rat (83%) and chicken (74%). The sheep HEXA gene encodes a protein of 529 amino acids that has high homology with the hexosaminidase A(HEXA) of five species bovine (98%), human (84%), Bornean orangután (84%), rat (80%) and mouse (81%). Finally these three novel sheep genes were assigned to GenelDs: 100145857, 100145858 and 100145856. The phylogenetic tree analysis revealed that the sheep BCKDHA, NAGA, and HEXA all have closer genetic relationships to the BCKDHA, NAGA, and HEXA of bovine. Tissue expression profile analysis was also carried out and results revealed that sheep BCKDHA, NAGA and HEXA genes were differentially expressed in tissues including muscle, heart, liver, fat, kidney, lung, small and large intestine. Our experiment is the first to establish the primary foundation for further research on these three sheep genes.