Α-ketoacid Dehydrogenase Activity Research Articles

Branched-chain α-ketoacid dehydrogenase deficiency (maple syrup urine disease): Treatment, biomarkers, and outcomes

Over the past three decades, we studied 184 individuals with 174 different molecular variants of branched-chain α-ketoacid dehydrogenase activity, and here delineate essential clinical and biochemical aspects of the maple syrup urine disease (MSUD) phenotype. We collected data about treatment, survival, hospitalization, metabolic control, and liver transplantation from patients with classic (i.e., severe; n = 176), intermediate (n = 6) and intermittent (n = 2) forms of MSUD. A total of 13,589 amino acid profiles were used to analyze leucine tolerance, amino acid homeostasis, estimated cerebral amino acid uptake, quantitative responses to anabolic therapy, and metabolic control after liver transplantation. Standard instruments were used to measure neuropsychiatric outcomes. Despite advances in clinical care, classic MSUD remains a morbid and potentially fatal disorder. Stringent dietary therapy maintains metabolic variables within acceptable limits but is challenging to implement, fails to restore appropriate concentration relationships among circulating amino acids, and does not fully prevent cognitive and psychiatric disabilities. Liver transplantation eliminates the need for a prescription diet and safeguards patients from life-threatening metabolic crises, but is associated with predictable morbidities and does not reverse pre-existing neurological sequelae. There is a critical unmet need for safe and effective disease-modifying therapies for MSUD which can be implemented early in life. The biochemistry and physiology of MSUD and its response to liver transplantation afford key insights into the design of new therapies based on gene replacement or editing.

Catabolic Defect of Branched-Chain Amino Acids Promotes Heart Failure.

Although metabolic reprogramming is critical in the pathogenesis of heart failure, studies to date have focused principally on fatty acid and glucose metabolism. Contribution of amino acid metabolic regulation in the disease remains understudied. Transcriptomic and metabolomic analyses were performed in mouse failing heart induced by pressure overload. Suppression of branched-chain amino acid (BCAA) catabolic gene expression along with concomitant tissue accumulation of branched-chain α-keto acids was identified as a significant signature of metabolic reprogramming in mouse failing hearts and validated to be shared in human cardiomyopathy hearts. Molecular and genetic evidence identified the transcription factor Krüppel-like factor 15 as a key upstream regulator of the BCAA catabolic regulation in the heart. Studies using a genetic mouse model revealed that BCAA catabolic defect promoted heart failure associated with induced oxidative stress and metabolic disturbance in response to mechanical overload. Mechanistically, elevated branched-chain α-keto acids directly suppressed respiration and induced superoxide production in isolated mitochondria. Finally, pharmacological enhancement of branched-chain α-keto acid dehydrogenase activity significantly blunted cardiac dysfunction after pressure overload. BCAA catabolic defect is a metabolic hallmark of failing heart resulting from Krüppel-like factor 15-mediated transcriptional reprogramming. BCAA catabolic defect imposes a previously unappreciated significant contribution to heart failure.

Dataset reporting BCKDK interference in a BCAA-catabolism restricted environment.

This data article contains complementary figures to the research article “Mitochondrial response to the BCKDK-deficiency: some clues to understand the positive dietary response in this form of autism” [1]. Herein we present data relative to the effect of knocking down BCKDK gene on the real time oxygen consumption rate of fibroblasts obtained from a Maple Syrup Urine Disease (MSUD) patient. Interference of BCKDK expression on such cells showing a reduced branched-chain α-ketoacid dehydrogenase (BCKDHc) activity; let us generate a scenario to study the direct effect of BCKDK absence in an environment of high branched-chain amino acids (BCAAs) concentrations. Data relative to the effectiveness of the knockdown together with the potentiality of the BCKDK-knockdown to increase the deficient branched-chain α-ketoacid dehydrogenase activity detected in MSUD patients are also shown.

Simvastatin increases liver branched-chain α-ketoacid dehydrogenase activity in rats fed with low protein diet.

The rate-limiting step in branched-chain amino acids (BCAAs) disposal is catalyzed by the mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDH). BCKDH activity is regulated mainly by a reversible dephosphorylation (activation)/phosphorylation (inactivation) cycle catalyzed by a specific phosphatase (BDP) and kinase (BDK). Current catalytic activity of BCKDH, described as BCKDH activity state, and thus also BCAAs catabolic rate depend directly on the portion of BCKDH occurring in its active dephosphorylated form. Liver BCKDH activity state alters in response to different nutritional factors. Feeding rats a low-protein diet decreases BCKDH activity. It has been previously shown that lipid lowering drugs, fibrates upregulate liver BCKDH activity and stimulate BCAAs catabolism, especially under the condition of dietary protein deprivation. Effect of statins on liver BCKDH activity has not been studied yet. The present study was aimed at investigating the in vivo effect of simvastatin on liver BCKDH activity, as well as E1, E2 and BDP and BDK mRNA levels in rats fed with either a standard (23% protein) or a low protein (8% protein) diet. For 14 days, simvastatin (80 mg/kg b wt/day) or the vehicle (0.3% methylcellulose) were administrated orally by gavage to the treated and control groups, respectively. The actual BCKDH and total BCKDH activities were assayed spectrophotometrically prior to and following incubation with lambda phosphatase, respectively. The mRNA levels of the selected genes were quantified by means of a semi-quantitative RT-PCR. In rats fed with the low protein diet simvastatin administration reversed physiological adaptation of liver BCKDH to protein restriction and increased liver BCKDH activity state by 39% (p<0.05). Changes in BCKDH activity did not correspond to any changes in mRNA levels for BCKDH catalytic and regulatory enzymes. On the contrary, in rats fed with standard diet liver BCKDH activity state did not alter substantially in response to simvastatin administration. In conclusion, the obtained results indicate that simvastatin stimulates liver BCKDH activity and BCAAs degradation in rats fed with the low protein diet, whereas it exerts no effect on BCKDH in rats fed with the standard diet. Stimulation of liver BCKDH activity can be attributed to altered phosphorylation status of the complex (increased dephosphorylation), and it is not associated with changes in mRNA levels for complex enzymes. It is conceivable that in protein malnourished rats simvastatin effect on liver BCKDH activity and BCAAs metabolism can contribute to the myotoxicity observed during treatment with this agent.

Structural and Biochemical Characterization of Human Mitochondrial Branched-chain α-Ketoacid Dehydrogenase Phosphatase

The branched-chain α-ketoacid dehydrogenase phosphatase (BDP) component of the human branched-chain α-ketoacid dehydrogenase complex (BCKDC) has been expressed in Escherichia coli and purified in the soluble form. The monomeric BDP shows a strict dependence on Mn(2+) ions for phosphatase activity, whereas Mg(2+) and Ca(2+) ions do not support catalysis. Metal binding constants for BDP, determined by competition isothermal titration calorimetry, are 2.4 nm and 10 μm for Mn(2+) and Mg(2+) ions, respectively. Using the phosphorylated decarboxylase component (p-E1b) of BCKDC as a substrate, BDP shows a specific activity of 68 nmol/min/mg. The Ca(2+)-independent binding of BDP to the 24-meric transacylase (dihydrolipoyl transacylase; E2b) core of BCKDC results in a 3-fold increase in the dephosphorylation rate of p-E1b. However, the lipoyl prosthetic group on E2b is not essential for BDP binding or E2b-stimulated phosphatase activity. Acidic residues in the C-terminal linker of the E2b lipoyl domain are essential for the interaction between BDP and E2b. The BDP structure was determined by x-ray crystallography to 2.4 Å resolution. The BDP structure is dominated by a central β-sandwich. There are two protrusions forming a narrow cleft ∼10 Å wide, which constitutes the active site. The carboxylate moieties of acidic residues Asp-109, Asp-207, Asp-298, and Asp-337 in the active-site cleft participate in binding two metal ions. Substitutions of these residues with alanine nullify BDP phosphatase activity. Alteration of the nearby Arg-104 increases the K(m) for p-E1b peptide by 60-fold, suggesting that this residue is critical for the recognition of the native p-E1b protein.

1,3-Dinitrobenzene–Induced Metabolic Impairment through Selective Inactivation of the Pyruvate Dehydrogenase Complex

Prolonged exposure to the chemical intermediate, 1,3-dinitrobenzene (1,3-DNB), produces neuropathology in the central nervous system of rodents analogous to that observed in various conditions of acute energy deprivation including thiamine deficiency and Leigh's necrotizing encephalopathy. Increased production of reactive intermediates in addition to induction of oxidative stress has been implicated in the neurotoxic mechanism of 1,3-DNB, but a clear metabolic target has not been determined. Here we propose that similar to thiamine deficiency, the effects of 1,3-DNB on metabolic status may be due to inhibition of the thiamine-dependent α-ketoacid dehydrogenase complexes. The effects of 1,3-DNB on astroglial metabolic status and α-ketoacid dehydrogenase activity were evaluated using rat C6 glioma cells. Exposure to 1,3-DNB resulted in altered morphology and biochemical dysfunction consistent with disruption of oxidative energy metabolism. Cotreatment with acetyl-carnitine or acetoacetate attenuated morphological and metabolic effects of 1,3-DNB exposure as well as increased cell viability. 1,3-DNB exposure inhibited pyruvate dehydrogenase complex (PDHc) and the inhibition correlated with the loss of lipoic acid (LA) immunoreactivity, suggesting that modification of LA is a potential mechanism of inhibition. Treatment with antioxidants and thiol-containing compounds failed to protect against loss of LA. Alternatively, inhibition of dihydrolipoamide dehydrogenase, the E3 component of the complex attenuated loss of LA. Collectively, these data suggest that 1,3-DNB impairs oxidative energy metabolism through direct inhibition of the PDHc and that this impairment is due to perturbations in the function of protein-bound LA.

Overexpression of Lactobacillus casei D-hydroxyisocaproic acid dehydrogenase in cheddar cheese.

Metabolism of aromatic amino acids by lactic acid bacteria is an important source of off-flavor compounds in Cheddar cheese. Previous work has shown that alpha-keto acids produced from Trp, Tyr, and Phe by aminotransferase enzymes are chemically labile and may degrade spontaneously into a variety of off-flavor compounds. However, dairy lactobacilli can convert unstable alpha-keto acids to more-stable alpha-hydroxy acids via the action of alpha-keto acid dehydrogenases such as d-hydroxyisocaproic acid dehydrogenase. To further characterize the role of this enzyme in cheese flavor, the Lactobacillus casei d-hydroxyisocaproic acid dehydrogenase gene was cloned into the high-copy-number vector pTRKH2 and transformed into L. casei ATCC 334. Enzyme assays confirmed that alpha-keto acid dehydrogenase activity was significantly higher in pTRKH2:dhic transformants than in wild-type cells. Reduced-fat Cheddar cheeses were made with Lactococcus lactis starter only, starter plus L. casei ATCC 334, and starter plus L. casei ATCC 334 transformed with pTRKH2:dhic. After 3 months of aging, the cheese chemistry and flavor attributes were evaluated instrumentally by gas chromatography-mass spectrometry and by descriptive sensory analysis. The culture system used significantly affected the concentrations of various ketones, aldehydes, alcohols, and esters and one sulfur compound in cheese. Results further indicated that enhanced expression of d-hydroxyisocaproic acid dehydrogenase suppressed spontaneous degradation of alpha-keto acids, but sensory work indicated that this effect retarded cheese flavor development.

Determination of branched-chain α-keto acid dehydrogenase activity state and branched-chain α-keto acid dehydrogenase kinase activity and protein in mammalian tissues

Determination of branched-chain α-keto acid dehydrogenase activity state and branched-chain α-keto acid dehydrogenase kinase activity and protein in mammalian tissues

Changes in hepatic branched-chain α-keto acid dehydrogenase activity in response to isoleucine imbalance in growing chickens

Three experiments were carried out to determine the influence of isoleucine imbalance on hepatic branched-chain α-keto acid dehydrogenase (BCKAD) activity in growing chickens. An isoleucine imbalance was induced by adding a 5% imbalancing amino acid mixture to a basal diet that contained adequate concentrations of all indispensable amino acids except isoleucine, which was marginally adequate (0.64–0.76% of the diet). The imbalancing mixture caused depressions ( P < 0.05) in feed intake and growth rate. The depression in feed intake appeared to occur prior to the depression in growth rate. The isoleucine concentration in plasma decreased ( P < 0.05), but not consistently, among experiments in response to the imbalancing mixture of amino acids. Basal and total activities of hepatic BCKAD were increased ( P < 0.05) 21% and 28%, respectively, within 24 hours in one experiment and were elevated ( P < 0.05) 19% and 14%, respectively, at the end of the 13 days of a second experiment. The moisture, protein, and fat contents of whole body and liver were not affected by the imbalancing mixture of amino acids. It appears likely that broiler chicks did not adapt to the imbalanced diets because the depressed feed intake and growth rate and alterations in plasma isoleucine and hepatic BCKAD activity persisted through 13 days of experiment. The isoleucine requirement, expressed as percent of diet, was increased by the imbalancing mixture of amino acids, and the efficiency of isoleucine utilization for growth (grams of weight gain per milligram of isoleucine intake) was decreased in two of three experiments. These results suggest that BCKAD may have a play in the increased isoleucine requirement of broiler chicks under conditions of isoleucine imbalance.

Stimulation of rat-liver branched-chain α-keto acid dehydrogenase activity by chronic metabolic acidosis

During chronic metabolic acidosis, the degradation of protein and amino acids reportedly increases. Branched-chain α-keto acid dehydrogenase complex (BCKDH) relates amino-acid catabolism and mitochondrial-energy metabolism. This study was designed to evaluate the effect of acidosis on the activity of liver BCKDH, the key regulatory enzyme in the catabolism of branched-chain amino acids. Experimental acidosis was induced in rats by ingestion of 0.28 M ammonium chloride solution for 10 days. We made two different liver-mitochondrial extracts to assay independently the active form of BCKDH and the total BCKDH activity. Acidosis significantly increased both active and total BCKDH specific activities ( P<0.05). The mean value for the active form of the BCKDH complex was 9.27±1.10 (S.E.M., n=7) mU/mg of mitochondrial protein in acidotic rats and 5.18±0.84 ( n=7) for the control rats. The value of the total complex was 16.10±1.22 ( n=7) for the acidosis and 11.51±0.58 ( n=7) for the control. No significant changes were found in the activity state of the complex. Citrate synthase activity did not show significant variations between treatments. The stimulation of liver BCKDH activities by the acidosis may contribute to maintaining the level of intermediates of the tricarboxylic-acid cycle in this metabolic situation in which the net release of glutamine are produced.

Regulation of Rat Liver Branched-Chain α-Keto Acid Dehydrogenase Activity by Meal Frequency and Dietary Protein

Effects of feeding frequency on liver branched-chain alpha-keto acid dehydrogenase (BCKAD) activity are unknown. In the present study, rats were trained to consume their daily allotment of food in 6 h (meal-feeding). Rats were fed diets containing 0, 9, 25 or 50% casein and after 10 d were killed before or 3 h after the meal. The enzyme in rats fed diets containing 0, 9 and 25% casein was activated three- to sixfold after meal consumption. Previous studies showed that the liver enzyme is essentially fully activated in post-absorptive rats fed an adequate protein diet ad libitum. Meal-feeding an adequate protein (25% casein) diet resulted in a marked decrease in the postabsorptive percentage of active complex compared to ad libitum feeding of the same diet (29 +/- 6% vs. 93 +/- 6% active). Administration of alpha-ketoisocaproate (200 mumol/100 g body weight, an inhibitor of BCKAD kinase) reversed the meal-feeding-induced inactivation of the complex within 10 min. We conclude that the frequency of food intake, in addition to the level of dietary protein, influences the proportion of liver BCKAD in the active state. Inactivation of hepatic BCKAD in rats trained to feed once a day may be an adaptive mechanism that results in increased efficiency of branched-chain amino acid utilization between meals.

Physiological covalent regulation of rat liver branched-chain α-ketoacid dehydrogenase

A radiochemical assay was developed for measuring branched-chain α-ketoacid dehydrogenase activity of Triton X-100 extracts of freeze-clamped rat liver. The proportion of active (dephosphorylated) enzyme was determined by measuring enzyme activities before and after activation of the complex with a broad-specificity phosphoprotein phosphatase. Hepatic branched-chain α-ketoacid dehydrogenase activity in normal male Wistar rats was 97% active but decreased to 33% active after 2 days on low-protein (8%) diet and to 13% active after 4 days on the same diet. Restricting protein intake of lean and obese female Zucker rats also caused inactivation of hepatic branched-chain α-ketoacid dehydrogenase complex. Essentially all of the enzyme was in the active state in rats maintained for 14 days on either 30 or 50% protein diets. This was also the case for rats maintained on a commercial chow diet (minimum 23% protein). However, maintaining rats on 20, 8, and 0% protein diets decreased the percentage of the active form of the enzyme to 58, 10, and 7% of the total, respectively. Fasting of chow-fed rats for 48 h had no effect on the activity state of hepatic branched-chain α-ketoacid dehydrogenase, i.e., 93% of the enzyme remained in the active state compared to 97% for chow-fed rats. However, hepatic enzyme of rats maintained on 8% protein diet was 10% active before starvation and 83% active after 2 days of starvation. Thus, dietary protein deficiency results in inactivation of hepatic branched-chain α-ketoacid dehydrogenase complex, presumably as a consequence of low hepatic levels of branched-chain α-ketoacids, established inhibitors of branched-chain α-ketoacid dehydrogenase kinase. With rats fed a low-protein diet and subsequently starved, inhibition of branched-chain α-ketoacid dehydrogenase kinase by branched-chain α-ketoacids generated as a consequence of endogenous proteolysis most likely promotes the greater branched-chain α-ketoacid dehydrogenase activity state.

Influence of Dietary Nitrogen Intake on Mammalian Branched Chain α-Keto Acid Dehydrogenase Activity

Male albino rats (100–130 g) were fed diets of varying casein contents for a period of six days. Increasing the casein content from 0.38% to 22% led to an elevation of hepatic branched chain α-keto acid dehydrogenase (BCKAD) activity. Km values measured with α-ketoisocaproate and α-ketoisovalerate as substrates were not influenced by the dietary treatment. Replacing the casein content (22%) of the diet by an equivalent amount of amino acids resulted in a significant decrease in hepatic BCKAD activity. The decrease in Vmax values was not accompanied by changes in Km-values. No enhancement of hepatic enzyme activity was observed when increasing the amino acid content of the diet from 17.5% to 25%. Changes in enzyme activity due to alterations of the dietary nitrogen source were not observed in kidney and brain.α-keto acid dehydrogenase casein amino acids Km-values

Studies on the regulation of leucine catabolism: Mechanism responsible for oxidizable substrate inhibition and dichloroacetate stimulation of leucine oxidation by the heart

In the absence of any other oxidizable substrate, the perfused rat heart oxidizes [1- 14C]leucine to 14CO 2 at a rapid rate and releases only small amounts of α-[1- 14C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such perfused hearts, is very active. Under such perfusion conditions, dichloroacetate has almost no effect on [1- 14C]leucine oxidation, α-[1- 14C]ketoisocaproate release, or branched-chain α-keto acid dehydrogenase activity. Perfusion of the heart with some other oxidizable substrate, e.g., glucose, pyruvate, ketone bodies, or palmitate, results in an inhibition of [1- 14C]leucine oxidation to 14CO 2 and the release of large amounts of α-[1- 14C]ketoisocaproate into the perfusion medium. The branched-chain α-keto acid dehydrogenase complex, assayed in extracts of mitochondria prepared from such hearts, is almost completely inactivated. The enzyme can be reactivated, however, by incubating the mitochondria at 30 °C without an oxidizable substrate. With hearts perfused with glucose or ketone bodies, dichloroacetate greatly increases [1- 14C]leucine oxidation, decreases α-[1- 14C]ketoisocaproate release into the perfusion medium, and activates the branched-chain α-keto acid dehydrogenase complex. Pyruvate may block dichloroacetate uptake because dichloroacetate neither stimulates [1- 14C]leucine oxidation nor activates the branched-chain α-keto acid dehydrogenase complex of pyruvate-perfused hearts. It is suggested that leucine oxidation by heart is regulated by the activity of the branched-chain α-keto acid dehydrogenase complex which is subject to interconversion between active and inactive forms. Oxidizable substrates establish conditions which inactivate the enzyme. Dichloroacetate, known to activate the pyruvate dehydrogenase complex by inhibition of pyruvate dehydrogenase kinase, causes activation of the branched-chain α-keto acid dehydrogenase complex, suggesting the existence of a kinase for this complex.

Branched-chain amino acid oxidation by isolated rat tissue preparations

Branched-chain amino acid transaminase activity, branched-chain α-keto acid dehydrogenase activity, and leucine oxidation were measured in homogenates and slices of several rat tissues. Transaminase activity was highest in heart, while dehydrogenase activity was highest in liver. Leucine oxidation in isolated tissues may be limited by either transaminase or dehydrogenase activity depending upon the relative activities of these two enzymes in the tissue. The results suggest that, as the load of branched-chain amino acids increases, the liver may become an increasingly important site for the degradation of branched-chain α-keto acids.

Subcellular distribution and cofactor function of human branched chain α-ketoacid dehydrogenase in normal and mutant cultured skin fibroblasts

Branched chain α-ketoacid dehydrogenase activity was examined in cultured fibroblasts from normal patients and compared with that from a family with genetically defective enzyme function. A freeze-thaw disrupted preparation of broken cells required CoA, NAD +, thiamine pyrophosphate (TPP), and Mg 2+ for reconstitution of the dehydrogenase activities toward both pyruvate and the branched chain α-ketoacids; α-ketoisovalerate, α-keto-β-methylvalerate, and α-ketoisocaproate. Using this system, different absolute activities and effects of cofactor additions were found for the three branched chain α-ketoacids and pyruvate dehydrogenases in normal cells. Activity toward all three branched chain α-ketoacids was impaired in homozygous affected cells, but pyruvate dehydrogenase activity was normal. Genotypes of presumed normal, heterozygous, and homozygous affected cells were discriminated when reconstituted with CoA and NAD + alone, but not with TPP and Mg 2+ alone. A low affinity, high capacity α-keto-β-methylvaleric acid dehydrogenase was reconstituted by TPP and Mg 2+ to normal levels in mutant cells. Subcellular distribution studies localized the dehydrogenase activity for all substrates to the mitochondrial inner membranematrix of these preparations. TPP and Mg 2+ stabilized the mitochondrial inner membrane dehydrogeanse activity toward α-keto-β-methylvalerate in both normal and mutant fibroblasts, but failed to stabilize activity toward α-ketoisocaproic acid. We conclude that at least one gene product of the branched chain α-ketoacid dehydrogenase complex is common to all three substrates, but that decarboxylation of α-ketoisovaleric, α-ketoisocaproic, and α-keto-β-methylvaleric acids is quantitatively and qualitatively different.

Thiamine increases the specific activity of human liver branched chain alpha-ketoacid dehydrogenase.

THIAMINE pyrophosphate (TPP), the active derivative of vitamin B1, functions as a coenzyme in dehydrogenase reactions such as the oxidative decarboxylation of α-ketoisovaleric (KIV), α-keto-β-methylvaleric (KMV) and α-ketoisocaproic (KIC) acids in humans1. Impairment of these reactions produces the group of disorders known as maple syrup urine disease2,3. In the classic form of the disease, the ketoacids and their amino acid precursors accumulate in homozygous affected individuals and depress the function of the central nervous system early in life. Therapy involves restriction of branched chain amino acids in the diet to decrease their concentrations in the body fluids4. Further reduction in plasma leucine, isoleucine and valine has been achieved by supplementing this diet with thiamine for patients with partial (60%) and severe (95%) reduction in enzyme activity5,6. Other investigators found no effect in patients lacking all enzyme activity7,8. We are seeking an explanation for the clinical response at the enzyme level and have already reported that branched chain α-ketoacid dehydrogenase activity was increased in peripheral white blood cells. This occurred after 3 weeks of oral thiamine treatment in patients with 5% activity and in normal controls5. In contrast, activity in mitochondrial inner membranes from cultured normal and mutant skin fibroblasts was not stimulated directly by TPP/Mg2+, but was prolonged by the presence of TPP/Mg2+, suggesting that the cofactor increases the half life of the dehydrogenase complex. We have now found that thiamine supplementation of normal diets increases normal human liver branched chain α-ketoacid dehydrogenase activity.

Change of α-Keto Acid Dehydrogenase Activity of Escherichia coli Related with its Thiamine Content

Change of α-Keto Acid Dehydrogenase Activity of Escherichia coli Related with its Thiamine Content