Branched-chain Α-keto Acid Research Articles

Two novel mutations in the BCKDHB gene (R170H, Q346R) cause the classic form of maple syrup urine disease (MSUD)

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. BCKD is a mitochondrial complex encoded by four nuclear genes (BCKDHA, BCKDHB, DBT, and DLD) and is involved in the metabolism of branched-chain amino acids (BCAAs). In this study, we investigated the DNA sequences of BCKDHA, BCKDHB and DBT genes for mutations in a Chinese newborn with the classic form of MSUD and predicted the associated conformational changes using molecular modeling. We identified two previously unreported mutations in the BCKDHB gene, R170H (c.509G>A) in exon 5 and Q346R (c.1037 A>G) in exon 9. In silico analysis of the two novel missense mutations revealed that the mutation R170H-β alters the spatial orientation with both Y195-β′ and S206-α, which results in unstable β–β′ assembly and an unstable K+ ion binding loop of the α subunit, respectively; The Q346R mutation is predicted to disrupt the spatial conformation between Q346-β and I357-β′, which reduces the affinity of the β–β′ subunits. These results indicate that R170-β and Q346-β are crucial for the activity of the E1 component. These two novel mutations, R170H and Q346R result in the patient's clinical manifestation of the classic form of MSUD.

Expression of mitochondrial branched-chain aminotransferase and α-keto-acid dehydrogenase in rat brain: implications for neurotransmitter metabolism

In the brain, metabolism of the essential branched chain amino acids (BCAAs) leucine, isoleucine, and valine, is regulated in part by protein synthesis requirements. Excess BCAAs are catabolized or excreted. The first step in BCAA catabolism is catalyzed by the branched chain aminotransferase (BCAT) isozymes, mitochondrial BCATm and cytosolic BCATc. A product of this reaction, glutamate, is the major excitatory neurotransmitter and precursor of the major inhibitory neurotransmitter γ-aminobutyric acid (GABA). The BCATs are thought to participate in a α-keto-acid nitrogen shuttle that provides nitrogen for synthesis of glutamate from α-ketoglutarate. The branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC) catalyzes the second, irreversible step in BCAA metabolism, which is oxidative decarboxylation of the branched-chain α-keto acid (BCKA) products of the BCAT reaction. Maple Syrup Urine Disease (MSUD) results from genetic defects in BCKDC, which leads to accumulation of toxic levels of BCAAs and BCKAs that result in brain swelling. Immunolocalization of BCATm and BCKDC in rats revealed that BCATm is present in astrocytes in white matter and in neuropil, while BCKDC is expressed only in neurons. BCATm appears uniformly distributed in astrocyte cell bodies throughout the brain. The segregation of BCATm to astrocytes and BCKDC to neurons provides further support for the existence of a BCAA-dependent glial-neuronal nitrogen shuttle since the data show that BCKAs produced by glial BCATm must be exported to neurons. Additionally, the neuronal localization of BCKDC suggests that MSUD is a neuronal defect involving insufficient oxidation of BCKAs, with secondary effects extending beyond the neuron.

Lipoic acid metabolism of Plasmodium--a suitable drug target.

α-Lipoic acid (6,8-thioctic acid; LA) is a vital co-factor of α-ketoacid dehydrogenase complexes and the glycine cleavage system. In recent years it was shown that biosynthesis and salvage of LA in Plasmodium are necessary for the parasites to complete their complex life cycle. LA salvage requires two lipoic acid protein ligases (LplA1 and LplA2). LplA1 is confined to the mitochondrion while LplA2 is located in both the mitochondrion and the apicoplast. LplA1 exclusively uses salvaged LA and lipoylates α-ketoglutarate dehydrogenase, branched chain α-ketoacid dehydrogenase and the H-protein of the glycine cleavage system. LplA2 cannot compensate for the loss of LplA1 function during blood stage development suggesting a specific function for LplA2 that has yet to be elucidated. LA salvage is essential for the intra-erythrocytic and liver stage development of Plasmodium and thus offers great potential for future drug or vaccine development.LA biosynthesis, comprising octanoyl-acyl carrier protein (ACP) : protein N-octanoyltransferase (LipB) and lipoate synthase (LipA), is exclusively found in the apicoplast of Plasmodium where it generates LA de novo from octanoyl-ACP, provided by the type II fatty acid biosynthesis (FAS II) pathway also present in the organelle. LA is the co-factor of the acetyltransferase subunit of the apicoplast located pyruvate dehydrogenase (PDH), which generates acetyl-CoA, feeding into FAS II. LA biosynthesis is not vital for intra-erythrocytic development of Plasmodium, but the deletion of several genes encoding components of FAS II or PDH was detrimental for liver stage development of the parasites indirectly suggesting that the same applies to LA biosynthesis.These data provide strong evidence that LA salvage and biosynthesis are vital for different stages of Plasmodium development and offer potential for drug and vaccine design against malaria.

Clofibrate‐Induced Reduction of Plasma Branched‐Chain Amino Acid Concentrations Impairs Glucose Tolerance in Rats

It has been reported that branched-chain amino acid (BCAA) administration stimulates glucose uptake into muscles and whole body glucose oxidation in rats. The authors examined the effect of decreased plasma BCAA concentrations induced by clofibrate treatment on glucose tolerance in rats. Since clofibrate, a drug for hyperlipidemia (high serum triglyceride concentration), is a potent inhibitor of the branched-chain α-keto acid dehydrogenase kinase, clofibrate treatment (0.2 g/kg body weight) activated the hepatic branched-chain α-keto acid dehydrogenase complex, resulting in decreased plasma BCAA concentrations by 30% to 50% from the normal level. An intraperitoneal glucose tolerance test was conducted after clofibrate administration, and the results showed that peak plasma glucose concentration and the area under the curve of glucose concentration during the intraperitoneal glucose tolerance test were significantly higher in clofibrate-treated rats than in control rats. This impaired glucose tolerance in the clofibrate-treated rats was ameliorated by administration of BCAAs (0.45 g/kg body weight, leucine:isoleucine:valine = 2:1:1), which kept plasma BCAA concentrations at normal levels during the intraperitoneal glucose tolerance test. These results suggest that plasma BCAAs play an important role in maintaining normal glucose tolerance in rats.

Branched-chain amino acids (BCAAs) as nutraceuticals for exercise

Branched-chain amino acids (BCAAs: leucine, isoleucine and valine) are essential amino acids for humans and animals. It has been shown that BCAA oxidation is promoted by exercise through activation of branched-chain α-keto acid dehydrogenase complex (BCKDC), which is a rate-limiting enzyme in the catabolic pathway of the BCAAs, and the elevated enzyme activity in skeletal muscle is quickly downregulated after exercise. This tight regulation of the BCKDC is consistent with stimulation of protein synthesis after exercise in skeletal muscle. With this background, it is interesting to consider the BCAAs as a supplement for sports. We conducted a human study to examine the effects of BCAA supplementation on the delayed-onset muscle soreness (DOMS) induced by squatting exercises. The results clearly showed that BCAA supplementation significantly reduced DOMS evaluated by a visual analog scale method and maintained leg muscle force during the period of DOMS. Other studies using different types of exercise support our findings, suggesting that BCAA supplements are useful for conditioning of skeletal muscle after exercise.

Dissecting Complex Metabolic Integration Provides Direct Genetic Evidence for CodY Activation by Guanine Nucleotides

The global regulator CodY controls the expression of dozens of metabolic genes and genes mediating adaptation to nutrient availability in many low-G+C Gram-positive bacteria. Branched-chain amino acids L-isoleucine, L-leucine, and L-valine (ILV) activate CodY both in vivo and in vitro, and genes that direct their synthesis (ilv, ybgE, and ywaA) are highly repressed by CodY, creating a potential negative feedback loop. The nucleoside triphosphate GTP also activates CodY in vitro, but the evidence for activation by GTP in vivo is limited and indirect. We constructed a Bacillus subtilis strain (ybgE bcd ywaA) that is unable to convert branched-chain α-keto acids to ILV or to use ILV as a precursor for branched-chain fatty acid synthesis. Unexpectedly, the strain was not viable on rich medium. Supplementing rich medium with short, branched-chain fatty acids or derepressing expression of genes for de novo ILV synthesis bypassed the original lethality, restoring growth and showing that the lack of viability was due to insufficient intracellular production of the precursors of branched-chain fatty acids. Spontaneous extragenic suppressor mutants that arose in the triple mutant population proved to have additional mutations in guaA or guaB or codY. Expression of ILV biosynthetic genes in codY mutants was increased. The gua mutations caused guanine/guanosine auxotrophy and led to partial derepression of direct CodY-repressed targets, including ILV biosynthetic genes, under conditions similar to those that caused the original lethality. We conclude that a guanine derivative, most likely GTP, controls CodY activity in vivo.

Structural and Thermodynamic Basis for Weak Interactions between Dihydrolipoamide Dehydrogenase and Subunit-binding Domain of the Branched-chain α-Ketoacid Dehydrogenase Complex

The purified mammalian branched-chain α-ketoacid dehydrogenase complex (BCKDC), which catalyzes the oxidative decarboxylation of branched-chain α-keto acids, is essentially devoid of the constituent dihydrolipoamide dehydrogenase component (E3). The absence of E3 is associated with the low affinity of the subunit-binding domain of human BCKDC (hSBDb) for hE3. In this work, sequence alignments of hSBDb with the E3-binding domain (E3BD) of the mammalian pyruvate dehydrogenase complex show that hSBDb has an arginine at position 118, where E3BD features an asparagine. Substitution of Arg-118 with an asparagine increases the binding affinity of the R118N hSBDb variant (designated hSBDb*) for hE3 by nearly 2 orders of magnitude. The enthalpy of the binding reaction changes from endothermic with the wild-type hSBDb to exothermic with the hSBDb* variant. This higher affinity interaction allowed the determination of the crystal structure of the hE3/hSBDb* complex to 2.4-Å resolution. The structure showed that the presence of Arg-118 poses a unique, possibly steric and/or electrostatic incompatibility that could impede E3 interactions with the wild-type hSBDb. Compared with the E3/E3BD structure, the hE3/hSBDb* structure has a smaller interfacial area. Solution NMR data corroborated the interactions of hE3 with Arg-118 and Asn-118 in wild-type hSBDb and mutant hSBDb*, respectively. The NMR results also showed that the interface between hSBDb and hE3 does not change significantly from hSBDb to hSBDb*. Taken together, our results represent a starting point for explaining the long standing enigma that the E2b core of the BCKDC binds E3 far more weakly relative to other α-ketoacid dehydrogenase complexes.

Discovery of a Metagenome-Derived Enzyme that Produces Branched-Chain Acyl-(Acyl-Carrier-Protein)s from Branched-Chain α-Keto Acids

Clones that confer the production of N-acyl amino acids to model cultured bacterial hosts are frequently identified in activity-based screens of soil DNA libraries. The N-acyl amino acids encoded by these clones are synthesized from acyl-(acyl-carrier-protein)s (acyl-ACPs, ACP) and amino acids using a diverse group of bacterial enzymes referred to as N-acyl amino acid synthases (NASs).[1, 2] The acyl-ACP substrates of NAS enzymes are common intermediates in the de novo synthesis of fatty acids by the type II fatty acid synthase system (FAS II).[3] In addition to N-acyl amino acids, FAS II-derived acyl-ACPs also serve as acyl-donors in the production of N-acyl homoserine lactone autoinducers, the cofactor lipoic acid and several lipopeptide antibiotics (Figure 1A, compounds 1–4).[4–9] During a recent screen of soil metagenomic libraries for clones exhibiting antimicrobial activity, we uncovered an NAS-containing clone (clone EC5) that confers the production of long-chain N-acyl-phenylalanines and N-acyl-tryptophans to multiple Gram-negative host species.[10] The NAS-encoding gene found on clone EC5, nasA, is the first gene in a predicted two gene operon, nasAB (GenBank Accession No. {type:entrez-nucleotide,attrs:{text:GQ869383,term_id:260177223,term_text:GQ869383}}GQ869383). The second gene in this operon, nasB, encodes for a putative 1118 residue protein that contains six well-characterized Pfam homology domains (Figure 1B).[11] Based on generic functional predictions for these domains, we hypothesized that this enzyme was likely to be involved in the formation of ACP-linked fatty acids similar to those produced by bacterial FAS II systems. Here we report results from a series of heterologous expression experiments in Burkholderia graminis C4D1M, which show that nasB functions to provide branched-chain acyl-ACPs for nasA. Many bacteria, particularly Gram-negative species, do not natively produce branched-chain fatty acids.[3] NasB and its homologs are likely used by bacteria with straight-chain specific FAS II systems for generating branched-chain acyl-ACP substrates and should be useful as tools for synthesizing these substrates in model heterologous expression systems in which they are not natively produced (e.g. E. coli).

Production of volatile compounds by Lactobacillus sakei from branched chain α-keto acids

Lactobacillus sakei belongs to the main flora of raw fermented sausages and is used as starter culture. Bacterial starter cultures can convert amino acids to α-keto acids by aminotransferases. These α-keto acids are the precursors of aroma active aldehydes, alcohols and carboxylic acids. In this study the formation of aldehydes, alcohols and carboxylic acids from leucine, isoleucine, valine and the corresponding α-keto acids are analysed in model fermentations with two different strains of L sakei. In the absence and upon addition of leucine, isoleucine and valine they produced 1 μg/ml 3-methylbutanoic, 0.2 μg/ml 2-methylbutanoic acid and 3 μg/ml 2-methylpropanoic acid, respectively. Upon addition of α-ketoisocaproic acid, α-keto-3-methyl-pentanoic acid or α-ketoisovaleric acid the amount of the corresponding carboxylic acid was increased to 40 μg/ml 3-methylbutanoic acid, 20 μg/ml 2-methylbutanoic acid and 35 μg/ml 2-methylpropanoic acid. The response patterns of the strains and amounts of carboxylic acids produced were similar. This behaviour was typical when compared with other strains of L. sakei and suggests general lack of transaminase activity and a limit in the transport of branched chain amino acids and their conversion to volatiles, some of which can contribute to the aroma of fermented sausages.

Interaction between glutamate dehydrogenase (GDH) and l-leucine catabolic enzymes: Intersecting metabolic pathways

Branched-chain amino acids (BCAAs) catabolism follows sequential reactions and their metabolites intersect with other metabolic pathways. The initial enzymes in BCAA metabolism, the mitochondrial branched-chain aminotransferase (BCATm), which deaminates the BCAAs to branched-chain α-keto acids (BCKAs); and the branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC), which oxidatively decarboxylates the BCKAs, are organized in a supramolecular complex termed metabolon. Glutamate dehydrogenase (GDH1) is found in the metabolon in rat tissues. Bovine GDH1 binds to the pyridoxamine 5′-phosphate (PMP)-form of human BCATm (PMP-BCATm) but not to pyridoxal 5′-phosphate (PLP)-BCATm in vitro. This protein interaction facilitates reamination of the α-ketoglutarate (αKG) product of the GDH1 oxidative deamination reaction. Human GDH1 appears to act like bovine GDH1 but human GDH2 does not show the same enhancement of BCKDC enzyme activities. Another metabolic enzyme is also found in the metabolon is pyruvate carboxylase (PC). Kinetic results suggest that PC binds to the E1 decarboxylase of BCKDC but does not effect BCAA catabolism. The protein interaction of BCATm and GDH1 promotes regeneration of PLP-BCATm which then binds to BCKDC resulting in channeling of the BCKA products from BCATm first half reaction to E1 and promoting BCAA oxidation and net nitrogen transfer from BCAAs. The cycling of nitrogen through glutamate via the actions of BCATm and GDH1 releases free ammonia. Formation of ammonia may be important for astrocyte glutamine synthesis in the central nervous system. In peripheral tissue association of BCATm and GDH1 would promote BCAA oxidation at physiologically relevant BCAA concentrations.

Abstract 984: The role of branched-chain amino acids (BCAAs) oxidation in the survival of brain metastatic breast cancer cells

Abstract Background: Glucose is the main source of energy for most cells. When the glucose is limited some cells can use amino acids to support their survival. This ability of oxidizing branched chain amino acids (BCAAs) of the brain metastatic breast cancer cells was investigated. Methods: Breast cancer cell lines, MDA-MB-231 cell and its brain metastatic derivative MDA-MB-Br3 were used in this study. The survivability, ability of oxidizing BCAAs and expression of BCAA oxidation related enzymes of these cells were determined. Results: MDA-MB-Br3 cells survived well and oxidized more BCAAs than MDA-MB-231 cells when cultured in glucose free medium. Accordingly, the activity of branched-chain a-ketoacid dehydrogenase complex of MDA-MB-Br3 cells is significantly higher than that of the MDA-MB-231 cells. Conclusions: The breast cancer cell line MDA-MB-Br3 can use BCAAs as a resource of energy for survival in the environment with very low level of glucose. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 984. doi:10.1158/1538-7445.AM2011-984

Movement disorders in adult surviving patients with maple syrup urine disease

Maple syrup urine disease is a rare metabolic disorder caused by mutations in the branched-chain α-keto acid dehydrogenase complex gene. Patients generally present early in life with a toxic encephalopathy because of the accumulation of the branched-chain amino acids leucine, isoleucine, and valine and the corresponding ketoacids. Movement disorders in maple syrup urine disease have typically been described during decompensation episodes or at presentation in the context of a toxic encephalopathy, with complete resolution after appropriate dietary treatment. Movement disorders in patients surviving childhood are not well documented. We assessed 17 adult patients with maple syrup urine disease (mean age, 27.5 years) with a special focus on movement disorders. Twelve (70.6%) had a movement disorder on clinical examination, mainly tremor and dystonia or a combination of both. Parkinsonism and simple motor tics were also observed. Pyramidal signs were present in 11 patients (64.7%), and a spastic-dystonic gait was observed in 6 patients (35.2%). In summary, movement disorders are common in treated adult patients with maple syrup urine disease, and careful neurological examination is advisable to identify those who may benefit from specific therapy. © 2011 Movement Disorder Society.

In vivo neuroprotective effect of L-carnitine against oxidative stress in maple syrup urine disease

Maple syrup urine disease (MSUD) is an autosomal recessive inborn error of metabolism caused by deficiency of the activity of the mitochondrial enzyme complex branched-chain α-keto acid dehydrogenase (BCKAD) leading to accumulation of the branched-chain amino acids (BCAA) leucine, isoleucine and valine and their corresponding branched-chain α-keto acids. Affected patients present severe brain dysfunction manifested such as ataxia, seizures, coma, psychomotor delay and mental retardation. The mechanisms of brain damage in this disease remain poorly understood. Recent studies have shown that oxidative stress may be involved in neuropathology of MSUD. L-Carnitine (L-Car) is considered a potential antioxidant through its action against peroxidation as a scavenger of reactive oxygen species and by its stabilizing effect of damage to cell membranes. In this study we evaluate the possible neuroprotective in vivo effects of L-Car against pro-oxidative effects of BCAA in cerebral cortex of rats. L-Car prevented lipoperoxidation, measured by thiobarbituric acid-reactive substances, protein damage, measured by sulfhydryl and protein carbonyl content and alteration on catalase and glutathione peroxidase activity in rat cortex from a chemically-induced model of MSUD. Our data clearly show that L-Car may be an efficient antioxidant, protecting against the oxidative stress promoted by BCAA. If the present results are confirmed in MSUD patients, this could represent an additional therapeutic approach to the patients affected by this disease.

Dietary carbohydrate-to-protein ratio affects TOR signaling and metabolism-related gene expression in the liver and muscle of rainbow trout after a single meal

Most teleost fish are known to require high levels of dietary proteins. Such high-protein intake could have significant effects, particularly on insulin-regulated gene expression. We therefore analyzed the effects of an increase in the ratio of dietary carbohydrates/proteins on the refeeding activation of the Akt-target of rapamycin (TOR) signaling pathways in rainbow trout and the effects on the expression of several genes related to hepatic and muscle metabolism and known to be regulated by insulin, amino acids, and/or glucose. Fish were fed once one of three experimental diets containing high (H), medium (M), or low (L) protein (P) or carbohydrate (C) levels after 48 h of feed deprivation. Activation of the Akt/TOR signaling pathway by refeeding was severely impaired by decreasing the proteins-to-carbohydrates ratio. Similarly, postprandial regulation of several genes related to glucose (Glut4, glucose-6-phosphatase isoform 1), lipid (fatty acid synthase, ATP-citrate lyase, sterol responsive element binding protein, carnitine palmitoyltransferase 1, and 3-hydroxyacyl-CoA dehydrogenase), and amino acid metabolism (serine dehydratase and branched-chain α-keto acid dehydrogenase E2 subunit) only occurred when fish were fed the high-protein diet. On the other hand, diet composition had a low impact on the expression of genes related to muscle protein degradation. Interestingly, glucokinase was the only gene of those monitored whose expression was significantly upregulated by increased carbohydrate intake. In conclusion, this study demonstrated that macro-nutrient composition of the diet strongly affected the insulin/amino acids signaling pathway and expression pattern of genes related to metabolism.

Activity assay of Lipoamidase, an expected modulator of metabolic fate of externally administered lipoic acid

α-Lipoic acid (LA) is a cofactor functioning in four multienzymes: pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched-chain α-keto acid dehydrogenase, and the glycine cleavage enzyme systems. In each enzyme, LA is covalently bound to the e-amino group of lysine residues. LA has been used as a medicine for treating diabetic condition in Europe and is also currently a popular supplement resource, and thus the metabolic fate after orally intake of LA attracts much attention whether externally administered LA is incorporated into protein bound form because large fraction of administered dose is not fully explained yet. Lipoamidase is the enzyme catalyzing formation and breakage of amide bond between LA and lysine e-amino groups in the protein and thus is expected to play critical role in LA metabolism and function. In order to know how the enzyme lipoamidase is involved in the fate of LA in the body, a simple assay method is requested for determining lipoamidase activity in tissues. The method using simple HPLC detection of newly synthesized fluorescent substrate, dansyl-α-lipoyllysine, is discussed together with other previously reported methods.

Phenylbutyrate therapy for maple syrup urine disease

Therapy with sodium phenylacetate/benzoate or sodium phenylbutyrate in urea cycle disorder patients has been associated with a selective reduction in branched-chain amino acids (BCAA) in spite of adequate dietary protein intake. Based on this clinical observation, we investigated the potential of phenylbutyrate treatment to lower BCAA and their corresponding α-keto acids (BCKA) in patients with classic and variant late-onset forms of maple syrup urine disease (MSUD). We also performed in vitro and in vivo experiments to elucidate the mechanism for this effect. We found that BCAA and BCKA are both significantly reduced following phenylbutyrate therapy in control subjects and in patients with late-onset, intermediate MSUD. In vitro treatment with phenylbutyrate of control fibroblasts and lymphoblasts resulted in an increase in the residual enzyme activity, while treatment of MSUD cells resulted in the variable response which did not simply predict the biochemical response in the patients. In vivo phenylbutyrate increases the proportion of active hepatic enzyme and unphosphorylated form over the inactive phosphorylated form of the E1α subunit of the branched-chain α-keto acid dehydrogenase complex (BCKDC). Using recombinant enzymes, we show that phenylbutyrate prevents phosphorylation of E1α by inhibition of the BCKDC kinase to activate BCKDC overall activity, providing a molecular explanation for the effect of phenylbutyrate in a subset of MSUD patients. Phenylbutyrate treatment may be a valuable treatment for reducing the plasma levels of neurotoxic BCAA and their corresponding BCKA in a subset of MSUD patients and studies of its long-term efficacy are indicated.

Ornish and Pritikin Programs Approved by CMS

Maple syrup urine disease (MSUD) is an inherited aminoacidopathy caused by a deficiency in branched-chain α-keto acid dehydrogenase complex activity that leads to the accumulation of the branched-chain amino acids (BCAAs) leucine (Leu), isoleucine, and valine and their respective α-keto-acids, α-ketoisocaproic acid (KIC), α keto-β-methylvaleric acid, and α-ketoisovaleric acid. The major clinical features presented by MSUD patients include ketoacidosis, failure to thrive, poor feeding, apnea, ataxia, seizures, coma, psychomotor delay, and mental retardation; however, the pathophysiology of this disease is poorly understood. MSUD treatment consists of a low protein diet supplemented with a mixture containing micronutrients and essential amino acids but excluding BCAAs. Studies have shown that oxidative stress may be involved in the neuropathology of MSUD, with the existence of lipid and protein oxidative damage in affected patients. In recent years, studies have demonstrated the antioxidant role of l-carnitine (l-Car), which plays a central function in cellular energy metabolism and for which MSUD patients have a deficiency. In this work, we investigated the in vitro effect of Leu and KIC in the presence or absence of l-Car on DNA damage in peripheral whole blood leukocytes using the alkaline comet assay with silver staining and visual scoring. Leu and KIC resulted in a DNA damage index that was significantly higher than that of the control group, and l-Car was able to significantly prevent this damage, mainly that due to KIC.

Glucose promotion of GABA metabolism contributes to the stimulation of insulin secretion in β-cells

We have demonstrated recently that branched-chain α-keto acid stimulation of insulin secretion is dependent on islet GABA (γ-aminobutyric acid) metabolism: GABA transamination to succinic semialdehyde is increased by 2-oxoglutarate, generated in α-keto acid transamination to its corresponding α-amino acid. The present work was aimed at investigating whether glucose also promotes islet GABA metabolism and whether the latter contributes to the stimulation of insulin secretion. Glucose (20 mM) decreased both the content and release of islet GABA. Gabaculine (1 mM), a GABA transaminase inhibitor, partially suppressed the secretory response of rat perifused islets to 20 mM glucose at different L-glutamine concentrations (0, 1 and 10 mM), as well as the glucose-induced decrease in islet GABA. The drug also reduced islet ATP content and the ATP/ADP ratio at 20 mM glucose. Exogenous succinic semialdehyde induced a dose-dependent increase in islet GABA content by reversal of GABA transamination and a biphasic insulin secretion in the absence of glucose. It depolarized isolated β-cells and triggered action potential firing, accompanied by a reduction of membrane currents through ATP-sensitive K(+) channels. The gene expression and enzyme activity of GABA transaminase were severalfold higher than that of 2-oxoglutarate dehydrogenase in islet homogenates. We conclude that, at high glucose concentrations, there is an increased diversion of glucose metabolism from the citric acid cycle into the 'GABA shunt'. Semialdehyde succinic acid is a cell-permeant 'GABA-shunt' metabolite that increases ATP and the ATP/ADP ratio, depolarizes β-cells and stimulates insulin secretion. In summary, an increased islet GABA metabolism may trigger insulin secretion.

VGLUTs—Potential Targets for the Treatment of Seizures?

Vesicular glutamate transporters (VGLUTs) load glutamate into synaptic vesicles. In this issue of Neuron, Juge etal. report that ketone bodies compete with chloride-dependent activation of VGLUTs, leading to suppression of glutamate release and seizures. These findings provide a surprising explanation for the efficacy of the ketogenic diet in controlling epilepsy.

Four novel mutations identified in Norwegian patients result in intermittent maple syrup urine disease when combined with the R301C mutation

Maple syrup urine disease (MSUD) is caused by a defect in branched chain α-ketoacid dehydrogenase complex (BCKD), an essential metabolon for the catabolism of the branched chain amino acids. Here, we report four novel mutations in the DBT gene, encoding the transacylase subunit (E2) of BCKD, resulting in intermittent MSUD in seven Norwegian patients. The patients had episodes with neurological symptoms including lethargy and/or ataxia during childhood infections. All seven patients were heterozygous for the annotated R301C mutation. The second allelic mutations were identified in five patients; one nonsense mutation (G62X), two missense mutations (W84C and R376C) and a mutation in the 3′ untranslated region (UTR; c.∗358A>C) in two patients. These four novel mutations result in near depletion of E2 protein, and the common R301C protein contributes predominantly to the residual (14%) cellular BCKD activity. Structural analyses of the mutations implied that the W84C and R376C mutations affect stability of intramolecular domains in E2, while the R301C mutation likely disturbs E2 trimer assembly as previously reported. The UTR mutated allele coincided with a strong reduction in mRNA levels, as did the non-R301C specific allele in two patients where the second mutation could not be identified. In summary, the pathogenic effect of the novel mutations is depletion of cellular protein, and the intermittent form of MSUD appears to be attributed to the residual R301C mutant protein in these patients.