Determination of Branched-Chain α-Keto Acid Dehydrogenase Activity State and Branched-Chain α-Keto Acid Dehydrogenase Kinase Activity and Protein in Mammalian Tissues

Nakai, Norihiro; Kobayashi, Rumi; Popov, Kirill M.; Harris, Robert A.; Shimomura, Yoshiharu

doi:10.1016/s0076-6879(00)24218-3

Cited by 48 publications

(37 citation statements)

References 28 publications

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“…**P Ͻ 0.01, before vs. after surgery. was significantly decreased by low-protein diets, 48-h starvation, alloxan-diabetes, and once-a-day meal feeding in rats (3,38,45 (41), and this also could be true for the regulation of BCKD kinase expression. Fat has about twice as much BCATm per milligram of protein than muscle and about five times more dehydrogenase (36).…”

Section: Discussionmentioning

confidence: 80%

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Obesity-related elevations in plasma leucine are associated with alterations in enzymes involved in branched-chain amino acid metabolism

She

Horn²,

Reid³

et al. 2007

American Journal of Physiology-Endocrinology and Metabolism

441

459

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Section: Discussionmentioning

confidence: 80%

“…A unit of activity was defined as 1 mol of valine formed per minute at 37°C. Tissue extraction and determination of BCKD activity were carried out as described previously (41). BCKD complex was precipitated from whole tissue extracts using 9% polyethylene glycol.…”

Section: Methodsmentioning

confidence: 99%

Obesity-related elevations in plasma leucine are associated with alterations in enzymes involved in branched-chain amino acid metabolism

She

Horn²,

Reid³

et al. 2007

American Journal of Physiology-Endocrinology and Metabolism

441

459

View full text Add to dashboard Cite

show abstract

“…Branched-chain α-keto acid dehydrogenase (BCKD) activity was assayed spectrophotometrically using α-ketoisocaproate (α-KIC) as the substrate as described (13). The assay reaction mixture consisted of 1 mM α-KIC, 30 mM potassium phosphate, 2 mM MgCl 2 , 0.4 mM thiamine pyrophosphate, 0.4 mM CoA, 1 mM NAD + , 0.1% (w/v) Triton X-100, 2 mM DTT, pig heart dihydrolipoamide dehydrogenase (5 units/ml), and 2 µl of tissue extract.…”

Section: Methodsmentioning

confidence: 99%

ENU mutagenesis identifies mice with mitochondrial branched-chain aminotransferase deficiency resembling human maple syrup urine disease

Kao

et al. 2004

J. Clin. Invest.

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Tandem mass spectrometry was applied to detect derangements in the pathways of amino acid and fatty acid metabolism in N-ethyl-N-nitrosourea-treated (ENU-treated) mice. We identified mice with marked elevation of blood branched-chain amino acids (BCAAs), ketoaciduria, and clinical features resembling human maple syrup urine disease (MSUD), a severe genetic metabolic disorder caused by the deficiency of branched-chain α-keto acid dehydrogenase (BCKD) complex. However, the BCKD genes and enzyme activity were normal. Sequencing of branched-chain aminotransferase genes (Bcat) showed no mutation in the cytoplasmic isoform (Bcat-1) but revealed a homozygous splice site mutation in the mitochondrial isoform (Bcat-2). The mutation caused a deletion of exon 2, a marked decrease in Bcat-2 mRNA, and a deficiency in both BCAT-2 protein and its enzyme activity. Affected mice responded to a BCAA-restricted diet with amelioration of the clinical symptoms and normalization of the amino acid pattern. We conclude that BCAT-2 deficiency in the mouse can cause a disease that mimics human MSUD. These mice provide an important animal model for study of BCAA metabolism and its toxicity. Metabolomics-guided screening, coupled with ENU mutagenesis, is a powerful approach in uncovering novel enzyme deficiencies and recognizing important pathways of genetic metabolic disorders.

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“…Tissue extraction and assessment of BCKD activity were performed as previously described (30). BCKD complex was concentrated from whole tissue extracts using 9% polyethylene glycol.…”

Section: Bckd Enzyme Activity Assaysmentioning

confidence: 99%

Impaired Adiponectin Signaling Contributes to Disturbed Catabolism of Branched-Chain Amino Acids in Diabetic Mice

et al. 2014

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The branched-chain amino acids (BCAA) accumulated in type 2 diabetes are independent contributors to insulin resistance. The activity of branched-chain a-keto acid dehydrogenase (BCKD) complex, rate-limiting enzyme in BCAA catabolism, is reduced in diabetic states, which contributes to elevated BCAA concentrations. However, the mechanisms underlying decreased BCKD activity remain poorly understood. Here, we demonstrate that mitochondrial phosphatase 2C (PP2Cm), a newly identified BCKD phosphatase that increases BCKD activity, was significantly downregulated in ob/ob and type 2 diabetic mice. Interestingly, in adiponectin (APN) knockout (APN 2/2 ) mice fed with a high-fat diet (HD), PP2Cm expression and BCKD activity were significantly decreased, whereas BCKD kinase (BDK), which inhibits BCKD activity, was markedly increased. Concurrently, plasma BCAA and branched-chain a-keto acids (BCKA) were significantly elevated. APN treatment markedly reverted PP2Cm, BDK, BCKD activity, and BCAA and BCKA levels in HD-fed APN 2/2 and diabetic animals.Additionally, increased BCKD activity caused by APN administration was partially but significantly inhibited in PP2Cm knockout mice. Finally, APN-mediated upregulation of PP2Cm expression and BCKD activity were abolished when AMPK was inhibited. Collectively, we have provided the first direct evidence that APN is a novel regulator of PP2Cm and systematic BCAA levels, suggesting that targeting APN may be a pharmacological approach to ameliorating BCAA catabolism in the diabetic state.The branched-chain amino acids (BCAA) are essential amino acids such as leucine, isoleucine, and valine; their homeostasis is determined largely by catabolic activities in a number of organs including liver, muscle and adipose tissue (1-3). The first step of BCAA catabolism generates a set of corresponding branched-chain a-keto acids (BCKA), which are irreversibly decarboxylated by the branched-chain a-keto acid dehydrogenase (BCKD) complex (4). As with most nutrients, maintaining of the physiological level of BCAA is critical for cell metabolism and survival. However, many researchers have described increased BCAA and BCKA levels in diabetes and obesity (3,(5)(6)(7)(8). Furthermore, BCAA and their catabolites are strongly associated with insulin resistance (9-11), and elevated BCAA contributes to the development of insulin resistance (10,12). Mechanistically, elevated BCAA levels activate mTOR/p70S6 kinase, resulting in an increased I insulin receptor substrate-1 phosphorylation, thereby inhibiting phosphatidylinositol 3-kinase. This inhibition of phosphatidylinositol 3-kinase in turn leads to impaired insulin signaling (13,14). It is also reported that BCAA are independent predictors of insulin resistance, diabetes, and cardiovascular events (15-17). Therefore, it is necessary to determine the mechanisms of abnormal BCAA catabolism in order to better understand their association with metabolic-related pathogenesis. The BCKD complex is the rate-limiting enzyme in BCAA catabolism (4,12); regulation o...

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Determination of Branched-Chain α-Keto Acid Dehydrogenase Activity State and Branched-Chain α-Keto Acid Dehydrogenase Kinase Activity and Protein in Mammalian Tissues

Cited by 48 publications

References 28 publications

Obesity-related elevations in plasma leucine are associated with alterations in enzymes involved in branched-chain amino acid metabolism

Obesity-related elevations in plasma leucine are associated with alterations in enzymes involved in branched-chain amino acid metabolism

ENU mutagenesis identifies mice with mitochondrial branched-chain aminotransferase deficiency resembling human maple syrup urine disease

Impaired Adiponectin Signaling Contributes to Disturbed Catabolism of Branched-Chain Amino Acids in Diabetic Mice

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