Keto Acid Dehydrogenase Research Articles

Defective branched chain amino acid catabolism contributes to cardiac dysfunction and remodeling following myocardial infarction

Cardiac metabolic remodeling is a central event during heart failure (HF) development following myocardial infarction (MI). It is well known that myocardial glucose and fatty acid dysmetabolism contribute to post-MI cardiac dysfunction and remodeling. However, the role of amino acid metabolism in post-MI HF remains elusive. Branched chain amino acids (BCAAs) are an important group of essential amino acids and function as crucial nutrient signaling in mammalian animals. The present study aimed to determine the role of cardiac BCAA metabolism in post-MI HF progression. Utilizing coronary artery ligation-induced murine MI models, we found that myocardial BCAA catabolism was significantly impaired in response to permanent MI, therefore leading to an obvious elevation of myocardial BCAA abundance. In MI-operated mice, oral BCAA administration further increased cardiac BCAA levels, activated the mammalian target of rapamycin (mTOR) signaling, and exacerbated cardiac dysfunction and remodeling. These data demonstrate that BCAAs act as a direct contributor to post-MI cardiac pathologies. Furthermore, these BCAA-mediated deleterious effects were improved by rapamycin cotreatment, revealing an indispensable role of mTOR in BCAA-mediated adverse effects on cardiac function/structure post-MI. Of note, pharmacological inhibition of branched chain ketoacid dehydrogenase kinase (BDK), a negative regulator of myocardial BCAA catabolism, significantly improved cardiac BCAA catabolic disorders, reduced myocardial BCAA levels, and ameliorated post-MI cardiac dysfunction and remodeling. In conclusion, our data provide the evidence that impaired cardiac BCAA catabolism directly contributes to post-MI cardiac dysfunction and remodeling. Moreover, improving cardiac BCAA catabolic defects may be a promising therapeutic strategy against post-MI HF.

A Spontaneous Missense Mutation in Branched Chain Keto Acid Dehydrogenase Kinase in the Rat Affects Both the Central and Peripheral Nervous Systems.

A novel mutation, causing a phenotype we named frogleg because its most obvious characteristic is a severe splaying of the hind limbs, arose spontaneously in a colony of Sprague-Dawley rats. Frogleg is a complex phenotype that includes abnormalities in hind limb function, reduced brain weight with dilated ventricles and infertility. Using micro-satellite markers spanning the entire rat genome, the mutation was mapped to a region of rat chromosome 1 between D1Rat131 and D1Rat287. Analysis of whole genome sequencing data within the linkage interval, identified a missense mutation in the branched-chain alpha-keto dehydrogenase kinase (Bckdk) gene. The protein encoded by Bckdk is an integral part of an enzyme complex located in the mitochondrial matrix of many tissues which regulates the levels of the branched-chain amino acids (BCAAs), leucine, isoleucine and valine. BCAAs are essential amino acids (not synthesized by the body), and circulating levels must be tightly regulated; levels that are too high or too low are both deleterious. BCKDK phosphorylates Ser293 of the E1α subunit of the BCKDH protein, which catalyzes the rate-limiting step in the catabolism of the BCAAs, inhibiting BCKDH and thereby, limiting breakdown of the BCAAs. In contrast, when Ser293 is not phosphorylated, BCKDH activity is unchecked and the levels of the BCAAs will decrease dramatically. The mutation is located within the kinase domain of Bckdk and is predicted to be damaging. Consistent with this, we show that in rats homozygous for the mutation, phosphorylation of BCKDH in the brain is markedly decreased relative to wild type or heterozygous littermates. Further, circulating levels of the BCAAs are reduced by 70–80% in animals homozygous for the mutation. The frogleg phenotype shares important characteristics with a previously described Bckdk knockout mouse and with human subjects with Bckdk mutations. In addition, we report novel data regarding peripheral neuropathy of the hind limbs.

Abstract 104: Defective Branched-chain Amino Acid Catabolism Impairs Glucose Metabolism and Sensitizes the Heart to Ischemia/Reperfusion Injury

The branched-chain amino acids (BCAA) are essential amino acids required for protein homeostasis, energy balance, and nutrient signaling. It has been shown recently that elevations in the circulating BCAAs and related metabolites are associated with insulin resistance and ischemic heart disease, suggesting an important link between BCAA metabolism and cardiac pathology. BCAA catabolism in mitochondria is regulated by the branched-chain keto acid dehydrogenase complex (BCKDC). A mitochondria localized phosphatase 2C (PP2Cm) has been identified to activate BCKDC, serving as a key regulator of BCAA catabolism. Deletion of PP2Cm (KO) increased blood BCAA levels but did not alter cardiac function in unstressed mice. Using 13 C- and 31 P-NMR spectroscopy, we found a significant reduction in the contribution of glucose to oxidative metabolism in KO hearts (16±3 vs. 26±2% for KO and wild-type (WT), respectively, P <0.05), which was accompanied by an ~25% reduction in glucose uptake rate ( P <0.05). Cardiac glycogen content was also reduced (4.4±0.5 vs. 10.9±1.8 μmol glucose/g, P <0.05). In isolated mitochondria from KO or WT hearts treated with BCAAs, significant reductions in pyruvate supported respiration were observed. In enzyme kinetics experiment, acute exposure to BCAAs inhibited pyruvate dehydrogenase complex (PDH) activity in a dose-dependent manner. In isolated perfused hearts subjected to ischemia/reperfusion (I/R), KO hearts showed a greater increase of diastolic pressure during ischemia ( P <0.05), which persisted throughout the reperfusion ( P <0.05). The contractile function in KO barely recovered while it recovered to ~40% of baseline in WT at the end of reperfusion. Increasing glucose supply via overexpression of GLUT1, an insulin-independent glucose transporter, fully restored the impaired recovery to I/R in KO mice. These results show that BCAA catabolism deficiency, as a result of PP2Cm deletion, disrupts normal glucose metabolism and renders the heart highly sensitive to acute ischemia/reperfusion (I/R) injury. These findings reveal an important role of BCAA catabolism in regulating glucose metabolism and cardiac stress response.

Metformin inhibits Branched Chain Amino Acid (BCAA) derived ketoacidosis and promotes metabolic homeostasis in MSUD.

Maple Syrup Urine Disease (MSUD) is an inherited disorder caused by the dysfunction in the branched chain keto-acid dehydrogenase (BCKDH) enzyme. This leads to buildup of branched-chain keto-acids (BCKA) and branched-chain amino acids (BCAA) in body fluids (e.g. keto-isocaproic acid from the BCAA leucine), leading to numerous clinical features including a less understood skeletal muscle dysfunction in patients. KIC is an inhibitor of mitochondrial function at disease relevant concentrations. A murine model of intermediate MSUD (iMSUD) shows significant skeletal muscle dysfunction as by judged decreased muscle fiber diameter. MSUD is an orphan disease with a need for novel drug interventions. Here using a 96-well plate (liquid chromatography- mass spectrometry (LC-MS) based drug-screening platform we show that Metformin, a widely used anti-diabetic drug, reduces levels of KIC in patient-derived fibroblasts by 20–50%. This Metformin-mediated effect was conserved in vivo; Metformin-treatment significantly reduced levels of KIC in the muscle (by 69%) and serum (by 56%) isolated from iMSUD mice, and restored levels of mitochondrial metabolites (e.g. AMP and other TCA). The drug also decreased the expression of mitochondrial branched chain amino transferase (BCAT) which produces KIC in skeletal muscle. This suggests that Metformin can restore skeletal muscle homeostasis in MSUD by decreasing mitochondrial KIC production.

Branched-chain amino acid restriction in Zucker-fatty rats improves muscle insulin sensitivity by enhancing efficiency of fatty acid oxidation and acyl-glycine export.

ObjectiveA branched-chain amino acid (BCAA)-related metabolic signature is strongly associated with insulin resistance and predictive of incident diabetes and intervention outcomes. To better understand the role that this metabolite cluster plays in obesity-related metabolic dysfunction, we studied the impact of BCAA restriction in a rodent model of obesity in which BCAA metabolism is perturbed in ways that mirror the human condition.MethodsZucker-lean rats (ZLR) and Zucker-fatty rats (ZFR) were fed either a custom control, low fat (LF) diet, or an isonitrogenous, isocaloric LF diet in which all three BCAA (Leu, Ile, Val) were reduced by 45% (LF-RES). We performed comprehensive metabolic and physiologic profiling to characterize the effects of BCAA restriction on energy balance, insulin sensitivity, and glucose, lipid and amino acid metabolism.ResultsLF-fed ZFR had higher levels of circulating BCAA and lower levels of glycine compared to LF-fed ZLR. Feeding ZFR with the LF-RES diet lowered circulating BCAA to levels found in LF-fed ZLR. Activity of the rate limiting enzyme in the BCAA catabolic pathway, branched chain keto acid dehydrogenase (BCKDH), was lower in liver but higher in skeletal muscle of ZFR compared to ZLR and was not responsive to diet in either tissue. BCAA restriction had very little impact on metabolites studied in liver of ZFR where BCAA content was low, and BCKDH activity was suppressed. However, in skeletal muscle of LF-fed ZFR compared to LF-fed ZLR, where BCAA content and BCKDH activity were increased, accumulation of fatty acyl CoAs was completely normalized by dietary BCAA restriction. BCAA restriction also normalized skeletal muscle glycine content and increased urinary acetyl glycine excretion in ZFR. These effects were accompanied by lower RER and improved skeletal muscle insulin sensitivity in LF-RES fed ZFR as measured by hyperinsulinemic-isoglycemic clamp.ConclusionsOur data are consistent with a model wherein elevated circulating BCAA contribute to development of obesity-related insulin resistance by interfering with lipid oxidation in skeletal muscle. BCAA-dependent lowering of the skeletal muscle glycine pool appears to contribute to this effect by slowing acyl-glycine export to the urine.

Mitochondrial targeting increases specific activity of a heterologous valine assimilation pathway in Saccharomyces cerevisiae

Bio-based isobutantol is a sustainable ‘drop in’ substitute for petroleum-based fuels. However, well-studied production routes, such as the Ehrlich pathway, have yet to be commercialized despite more than a century of research. The more versatile bacterial valine catabolism may be a competitive alternate route producing not only an isobutanol precursor but several carboxylic acids with applications as biomonomers, and building blocks for other advanced biofuels. Here, we transfer the first two committed steps of the pathway from pathogenic Pseudomonas aeruginosa PAO1 to yeast to evaluate their activity in a safer model organism. Genes encoding the heteroligomeric branched chain keto-acid dehydrogenase (BCKAD; bkdA1, bkdA2, bkdB, lpdV), and the homooligomeric acyl-CoA dehydrogenase (ACD; acd1) were tagged with fluorescence epitopes and targeted for expression in either the mitochondria or cytoplasm of S. cerevisiae. We verified the localization of our constructs with confocal fluorescence microscopy before measuring the activity of tag-free constructs. Despite reduced heterologous expression of mitochondria-targeted enzymes, their specific activities were significantly improved with total enzyme activities up to 138% greater than those of enzymes expressed in the cytoplasm. In total, our results demonstrate that the choice of protein localization in yeast has significant impact on heterologous activity, and suggests a new path forward for isobutanol production.

High Leucine Diets Stimulate Cerebral Branched-Chain Amino Acid Degradation and Modify Serotonin and Ketone Body Concentrations in a Pig Model.

In addition to its role as an essential protein component, leucine (Leu) displays several other metabolic functions such as activation of protein synthesis. This property makes it an interesting amino acid for the therapy of human muscle atrophy and for livestock production. However, Leu can stimulate its own degradation via the branched-chain keto acid dehydrogenase complex (BCKDH). To examine the response of several tissues to excessive Leu, pigs were fed diets containing two- (L2) and four-fold (L4) higher Leu contents than the recommended amount (control). We found that the L4 diet led to a pronounced increase in BCKDH activity in the brain (2.5-fold, P < 0.05), liver (1.8-fold, P < 0.05) and cardiac muscle (1.7-fold, P < 0.05), whereas we found no changes in enzyme activity in the pancreas, skeletal muscle, adipose tissue and intestinal mucosa. The L2 diet had only weak effects on BCKDH activity. Both high Leu diets reduced the concentrations of free valine and isoleucine in nearly all tissues. In the brain, high Leu diets modified the amount of tryptophan available: for serotonin synthesis. Compared to the controls, pigs treated with the high Leu diets consumed less food, showed increased plasma concentrations of 3-hydroxybutyrate and reduced levels of circulating serotonin. In conclusion, excessive Leu can stimulate BCKDH activity in several tissues, including the brain. Changes in cerebral tryptophan, along with the changes in amino acid-derived metabolites in the plasma may limit the use of high Leu diets to treat muscle atrophy or to increase muscle growth.

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

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

GENETIC AND METABOLIC URGENCIES IN THE NEONATAL INTENSIVE CARE UNIT: MAPLE SYRUP URINE DISEASE

Maple syrup urine disease (MSUD) is a hereditary disorder of branched chain amino/keto acid metabolism, caused by a decreased activity of the branched-chain alpha- ketoacid dehydrogenase complex (BCKAD), which leads to abnormal elevated plasma concentrations of branched-chain amino acids (BCAAs) clinically manifested as a heavy burden for Central Nervous system. The toxic accumulation of substrates promotes the development of a severe and rapidly progressive neonatal encephalopathy if treatment is not immediately given. This disorder has a specific medical management in acute phase in order to minimize mortality and morbidity. For all those reasons, it is important to include the MSUD as a possible diagnosis in a encephalopathic newborn. We present a colombian newborn with classical MSUD with fatal outcome as an example of metabolic emergency and a differential diagnosis in the encephalopathic newborn.

Maternal Diabetes Leads to Adaptation in Embryonic Amino Acid Metabolism during Early Pregnancy.

During pregnancy an adequate amino acid supply is essential for embryo development and fetal growth. We have studied amino acid composition and branched chain amino acid (BCAA) metabolism at day 6 p.c. in diabetic rabbits and blastocysts. In the plasma of diabetic rabbits the concentrations of 12 amino acids were altered in comparison to the controls. Notably, the concentrations of the BCAA leucine, isoleucine and valine were approximately three-fold higher in diabetic rabbits than in the control. In the cavity fluid of blastocysts from diabetic rabbits BCAA concentrations were twice as high as those from controls, indicating a close link between maternal diabetes and embryonic BCAA metabolism. The expression of BCAA oxidizing enzymes and BCAA transporter was analysed in maternal tissues and in blastocysts. The RNA amounts of three oxidizing enzymes, i.e. branched chain aminotransferase 2 (Bcat2), branched chain ketoacid dehydrogenase (Bckdha) and dehydrolipoyl dehydrogenase (Dld), were markedly increased in maternal adipose tissue and decreased in liver and skeletal muscle of diabetic rabbits than in those of controls. Blastocysts of diabetic rabbits revealed a higher Bcat2 mRNA and protein abundance in comparison to control blastocysts. The expression of BCAA transporter LAT1 and LAT2 were unaltered in endometrium of diabetic and healthy rabbits, whereas LAT2 transcripts were increased in blastocysts of diabetic rabbits. In correlation to high embryonic BCAA levels the phosphorylation amount of the nutrient sensor mammalian target of rapamycin (mTOR) was enhanced in blastocysts caused by maternal diabetes. These results demonstrate a direct impact of maternal diabetes on BCAA concentrations and degradation in mammalian blastocysts with influence on embryonic mTOR signalling.

The Impact of the Branched-Chain Ketoacid Dehydrogenase Complex on Amino Acid Homeostasis in Arabidopsis.

The branched-chain amino acids (BCAAs) Leu, Ile, and Val are among nine essential amino acids that must be obtained from the diet of humans and other animals, and can be nutritionally limiting in plant foods. Despite genetic evidence of its importance in regulating seed amino acid levels, the full BCAA catabolic network is not completely understood in plants, and limited information is available regarding its regulation. In this study, transcript coexpression analyses revealed positive correlations among BCAA catabolism genes in stress, development, diurnal/circadian, and light data sets. A core subset of BCAA catabolism genes, including those encoding putative branched-chain ketoacid dehydrogenase subunits, is highly expressed during the night in plants on a diel cycle and in prolonged darkness. Mutants defective in these subunits accumulate higher levels of BCAAs in mature seeds, providing genetic evidence for their function in BCAA catabolism. In addition, prolonged dark treatment caused the mutants to undergo senescence early and overaccumulate leaf BCAAs. These results extend the previous evidence that BCAAs can be catabolized and serve as respiratory substrates at multiple steps. Moreover, comparison of amino acid profiles between mature seeds and dark-treated leaves revealed differences in amino acid accumulation when BCAA catabolism is perturbed. Together, these results demonstrate the consequences of blocking BCAA catabolism during both normal growth conditions and under energy-limited conditions.

Energy Expenditure in Chilean Children with Maple Syrup Urine Disease (MSUD).

Maple syrup urine disease (MSUD) is an autosomal recessive disorder caused by a blockage of branched-chain keto acid of BCAA (branched-chain keto acid dehydrogenase, BCKDH) leading to neurological damage induced by accumulation of leucine and metabolites. MSUD expenditure and energy requirement information is limited. To determine if basal/total energy expenditure (BEE/TEE) is comparable between different determination methods and if values agree with recommendations of energy in MSUD children, and whether they relate to nutritional status. Case-control study between MSUD (n = 16) and healthy children (n = 11) aged 6-18 years. Current nutritional status, physical activity level, body composition by DEXA and BEE/TEE by indirect calorimetry (BEEr) and predictive equations (FAO/WHO/ONU - WHO - and Schofield) were assessed; STATA 2013 (p < 0.05). When comparing the energy expenditure variables, there was no significant difference between groups. Moreover, compared to BEEr, equations underestimate according to BEE WHO and Schofield, respectively (P = 0.00; 0.02). The WHO equation had lower average calorie difference, greater concordance correlation and association with indirect calorimetry compared to the Schofield equation for both groups, being the best predictor of the BEE for MSUD group. Energy recommendations for MSUD children are according to energy expenditure; thus the use of WHO equation is a clinically and statistically feasible tool for its determination.

2014 AHA Late-Breaking Basic Science Abstracts

### 24178 ### MAVS Mediates Protection against Myocardial Ischemic Injury with Hydrogen Sulfide David Durrant, Adolfo G Mauro, Juan Valle Raleigh, Anindita Das, Jun He, Khoa Nguyen, Stefano Toldo, Antonio Abbate, Fadi N Salloum; Virginia Commonwealth Univ, Richmond, VA Background : Hydrogen sulfide (H2S) has been shown to protect against myocardial ischemic and inflammatory injury in part by preserving mitochondrial integrity. Since mitochondrial antiviral signaling (MAVS) protein has been implicated in attenuating Bax-mediated cytochrome c release from mitochondria caused by oxidative stress or ischemia, we sought to determine whether MAVS mediates the cardioprotective effects of H2S. Methods and Results : After baseline echocardiography, adult male wild type (WT) or MAVS KO mice underwent myocardial infarction (MI) by coronary artery ligation for 30 min. followed by 24 h reperfusion. Mice were pretreated with Na2S (100 μg/kg; ip ) or saline 1 h before MI. Infarct size, measured with TTC staining, was reduced and LV fractional shortening (FS) was preserved with Na2S at 24h post MI in WT mice as compared to saline-treated mice, but not in MAVS KO mice (Figs. A and B). The risk area was not different between the groups. Western blot analysis revealed a significant decline in myocardial MAVS expression at 24h post MI, which was preserved with Na2S (Fig. C). Another subset of mice was subjected to permanent coronary artery occlusion and treated with Na2S or saline daily for 28 days. LVFS decreased significantly at 28 days post-MI in the saline group, but was significantly preserved with Na2S (Fig. D). Moreover, LV infarct scar size, assessed by trichrome staining, was smaller in Na2S group (22.4 ± 2.7%) as compared to control (33.5 ± 2.1%, P<0.05). Survival rate was 2 fold higher with Na2S compared to saline (P<0.05). Western blot analysis confirmed a significant …

Branched-chain amino acids in metabolic signalling and insulin resistance

Branched-chain amino acids (BCAAs) are important nutrient signals that have direct and indirect effects. Frequently, BCAAs have been reported to mediate antiobesity effects, especially in rodent models. However, circulating levels of BCAAs tend to be increased in individuals with obesity and are associated with worse metabolic health and future insulin resistance or type 2 diabetes mellitus (T2DM). A hypothesized mechanism linking increased levels of BCAAs and T2DM involves leucine-mediated activation of the mammalian target of rapamycin complex 1 (mTORC1), which results in uncoupling of insulin signalling at an early stage. A BCAA dysmetabolism model proposes that the accumulation of mitotoxic metabolites (and not BCAAs per se) promotes β-cell mitochondrial dysfunction, stress signalling and apoptosis associated with T2DM. Alternatively, insulin resistance might promote aminoacidaemia by increasing the protein degradation that insulin normally suppresses, and/or by eliciting an impairment of efficient BCAA oxidative metabolism in some tissues. Whether and how impaired BCAA metabolism might occur in obesity is discussed in this Review. Research on the role of individual and model-dependent differences in BCAA metabolism is needed, as several genes (BCKDHA, PPM1K, IVD and KLF15) have been designated as candidate genes for obesity and/or T2DM in humans, and distinct phenotypes of tissue-specific branched chain ketoacid dehydrogenase complex activity have been detected in animal models of obesity and T2DM.

Effects of leucine supplementation and serum withdrawal on branched-chain amino acid pathway gene and protein expression in mouse adipocytes.

The essential branched-chain amino acids (BCAA), leucine, valine and isoleucine, are traditionally associated with skeletal muscle growth and maintenance, energy production, and generation of neurotransmitter and gluconeogenic precursors. Recent evidence from human and animal model studies has established an additional link between BCAA levels and obesity. However, details of the mechanism of regulation of BCAA metabolism during adipogenesis are largely unknown. We interrogated whether the expression of genes and proteins involved in BCAA metabolism are sensitive to the adipocyte differentiation process, and responsive to nutrient stress from starvation or BCAA excess. Murine 3T3-L1 preadipocytes were differentiated to adipocytes under control conditions and under conditions of L-leucine supplementation or serum withdrawal. RNA and proteins were isolated at days 0, 4 and 10 of differentiation to represent pre-differentiation, early differentiation and late differentiation stages. Expression of 16 BCAA metabolism genes was quantified by quantitative real-time PCR. Expression of the protein levels of branched-chain amino acid transaminase 2 (Bcat2) and branched-chain alpha keto acid dehydrogenase (Bckdha) was quantified by immunoblotting. Under control conditions, all genes displayed induction of gene expression during early adipogenesis (Day 4) compared to Day 0. Leucine supplementation resulted in an induction of Bcat2 and Bckdha genes during early and late differentiation. Western blot analysis demonstrated condition-specific concordance between gene and protein expression. Serum withdrawal resulted in undetectable Bcat2 and Bckdha protein levels at all timepoints. These results demonstrate that the expression of genes related to BCAA metabolism are regulated during adipocyte differentiation and influenced by nutrient levels. These results provide additional insights on how BCAA metabolism is associated with adipose tissue function and extends our understanding of the transcriptomic response of this pathway to variations in nutrient availability.

BCKDH: the missing link in apicomplexan mitochondrial metabolism is required for full virulence of Toxoplasma gondii and Plasmodium berghei.

While the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii are thought to primarily depend on glycolysis for ATP synthesis, recent studies have shown that they can fully catabolize glucose in a canonical TCA cycle. However, these parasites lack a mitochondrial isoform of pyruvate dehydrogenase and the identity of the enzyme that catalyses the conversion of pyruvate to acetyl-CoA remains enigmatic. Here we demonstrate that the mitochondrial branched chain ketoacid dehydrogenase (BCKDH) complex is the missing link, functionally replacing mitochondrial PDH in both T. gondii and P. berghei. Deletion of the E1a subunit of T. gondii and P. berghei BCKDH significantly impacted on intracellular growth and virulence of both parasites. Interestingly, disruption of the P. berghei E1a restricted parasite development to reticulocytes only and completely prevented maturation of oocysts during mosquito transmission. Overall this study highlights the importance of the molecular adaptation of BCKDH in this important class of pathogens.

Identification of Two Novel BCKDHB Mutations in Korean Siblings with Maple Syrup Urine Disease Showing Mild Clinical Presentation

defect in branched-chain α -keto acid dehydrogenase complex. Mutations have been identified in the BCKDHA, BCKDHB, or DBT genes, which encode different subunits of the BCKDH complex. Although encephalopathy and progressive neurodegeneration are its major manifestations, the severity of the disease may range from the severe classic type to milder intermediate variants. We report two Korean siblings with the milder intermediate MSUD who were diagnosed with MSUD by a combination of newborn screening tests using tandem mass spectrometry and family genetic screening for MSUD. At diagnosis, the patients’ plasma levels were elevated for leucine, isoleucine, valine, and alloisoleucine, and branched-chain α -keto acids and branched-chain α -hydroxy acids were detected in their urine. BCKDHA, BCKDHB, and DBT analysis was performed, and two novel mutations were identified in BCKDHB. Our patients were thought to have the milder intermediate variant of MSUD, rather than the classic form. Although MSUD is a typical metabolic disease with poor prognosis, better outcomes can be expected if early diagnosis and prompt management are provided, particularly for milder forms of the disease.

Genome instability in Maple Syrup Urine Disease correlates with impaired mitochondrial biogenesis

ObjectiveThe mitochondrial branched-chain ketoacid dehydrogenase (BCKD) catalyzes the degradation of branched-chain amino acids (BCAA), which have been shown to induce oxidative stress. Maple Syrup Urine Disease (MSUD) is caused by impaired activity of BCKD, suggesting that oxidative stress and resulting DNA damage could contribute to pathology. We evaluated the potential effect of BCKD deficiency on genome integrity and mitochondrial function as a downstream target. MethodsPrimary fibroblasts from MSUD patients and controls were either cultivated under normal conditions or exposed to metabolic or oxidative stress. DNA was analyzed for damage and mitochondrial function was evaluated by gene expression analyses, functional assays and immunofluorescent methods. ResultsPatient fibroblasts accumulated damage in mitochondrial DNA (mtDNA) and nuclear DNA, with a corresponding reduction in mitochondrial transcription, mtDNA copy number and pyruvate dehydrogenase. We found no evidence of increased level of reactive oxygen species (ROS) in patient fibroblasts under normal conditions, suggesting that the genotoxic effect is ascribed to accumulating metabolites. ConclusionsImpaired BCKD activity as in MSUD, results in accumulation of DNA damage and corresponding mitochondrial dysfunction.

Lipoic acid biosynthesis defects.

Lipoate is a covalently bound cofactor essential for five redox reactions in humans: in four 2-oxoacid dehydrogenases and the glycine cleavage system (GCS). Two enzymes are from the energy metabolism, α-ketoglutarate dehydrogenase and pyruvate dehydrogenase; and three are from the amino acid metabolism, branched-chain ketoacid dehydrogenase, 2-oxoadipate dehydrogenase, and the GCS. All these enzymes consist of multiple subunits and share a similar architecture. Lipoate synthesis in mitochondria involves mitochondrial fatty acid synthesis up to octanoyl-acyl-carrier protein; and three lipoate-specific steps, including octanoic acid transfer to glycine cleavage H protein by lipoyl(octanoyl) transferase 2 (putative) (LIPT2), lipoate synthesis by lipoic acid synthetase (LIAS), and lipoate transfer by lipoyltransferase 1 (LIPT1), which is necessary to lipoylate the E2 subunits of the 2-oxoacid dehydrogenases. The reduced form dihydrolipoate is reactivated by dihydrolipoyl dehydrogenase (DLD). Mutations in LIAS have been identified that result in a variant form of nonketotic hyperglycinemia with early-onset convulsions combined with a defect in mitochondrial energy metabolism with encephalopathy and cardiomyopathy. LIPT1 deficiency spares the GCS, and resulted in a combined 2-oxoacid dehydrogenase deficiency and early death in one patient and in a less severely affected individual with a Leigh-like phenotype. As LIAS is an iron-sulphur-cluster-dependent enzyme, a number of recently identified defects in mitochondrial iron-sulphur cluster synthesis, including NFU1, BOLA3, IBA57, GLRX5 presented with deficiency of LIAS and a LIAS-like phenotype. As in DLD deficiency, a broader clinical spectrum can be anticipated for lipoate synthesis defects depending on which of the affected enzymes is most rate limiting.

Branched-chain amino acid metabolism: from rare Mendelian diseases to more common disorders

Branched-chain amino acid (BCAA) metabolism plays a central role in the pathophysiology of both rare inborn errors of metabolism and the more common multifactorial diseases. Although deficiency of the branched-chain ketoacid dehydrogenase (BCKDC) and associated elevations in the BCAAs and their ketoacids have been recognized as the cause of maple syrup urine disease (MSUD) for decades, treatment options for this disorder have been limited to dietary interventions. In recent years, the discovery of improved leucine tolerance after liver transplantation has resulted in a new therapeutic strategy for this disorder. Likewise, targeting the regulation of the BCKDC activity may be an alternative potential treatment strategy for MSUD. The regulation of the BCKDC by the branched-chain ketoacid dehydrogenase kinase has also been implicated in a new inborn error of metabolism characterized by autism, intellectual disability and seizures. Finally, there is a growing body of literature implicating BCAA metabolism in more common disorders such as the metabolic syndrome, cancer and hepatic disease. This review surveys the knowledge acquired on the topic over the past 50 years and focuses on recent developments in the field of BCAA metabolism.