Activity Of Branched-chain Α-keto Acid Dehydrogenase Research Articles

Activation of Hepatic Branched-Chain α-Ketoacid Dehydrogenase Complex by Vitamin D Deficiency in Rats.

Branched-chain α-ketoacid dehydrogenase (BCKDH) complex is a rate-limiting enzyme in branched-chain amino acid catabolism and is subject to inactivation via phosphorylation by BCKDH kinase (BDK). In the present study, we examined the effects of vitamin D-deficiency on hepatic BCKDH and BDK activities in rats. Rats fed a vitamin D-deficient diet long-term showed a slight but significant decrease in plasma Ca concentration, which was associated with an elevation of BCKDH activity and a decrease in BDK activity. These results suggest that vitamin D deficiency promotes BCAA catabolism via BCKDH activation, which resulted from BDK suppression. It is proposed that Ca2+-dependent BDK inhibition by thiamine pyrophosphate may be involved in the BDK suppression.

Cell-autonomous effect of cardiomyocyte branched-chain amino acid catabolism in heart failure in mice

Parallel to major changes in fatty acid and glucose metabolism, defect in branched-chain amino acid (BCAA) catabolism has also been recognized as a metabolic hallmark and potential therapeutic target for heart failure. However, BCAA catabolic enzymes are ubiquitously expressed in all cell types and a systemic BCAA catabolic defect is also manifested in metabolic disorder associated with obesity and diabetes. Therefore, it remains to be determined the cell-autonomous impact of BCAA catabolic defect in cardiomyocytes in intact hearts independent from its potential global effects. In this study, we developed two mouse models. One is cardiomyocyte and temporal-specific inactivation of the E1α subunit (BCKDHA-cKO) of the branched-chain α-ketoacid dehydrogenase (BCKDH) complex, which blocks BCAA catabolism. Another model is cardiomyocyte specific inactivation of the BCKDH kinase (BCKDK-cKO), which promotes BCAA catabolism by constitutively activating BCKDH activity in adult cardiomyocytes. Functional and molecular characterizations showed E1α inactivation in cardiomyocytes was sufficient to induce loss of cardiac function, systolic chamber dilation and pathological transcriptome reprogramming. On the other hand, inactivation of BCKDK in intact heart does not have an impact on baseline cardiac function or cardiac dysfunction under pressure overload. Our results for the first time established the cardiomyocyte cell autonomous role of BCAA catabolism in cardiac physiology. These mouse lines will serve as valuable model systems to investigate the underlying mechanisms of BCAA catabolic defect induced heart failure and to provide potential insights for BCAA targeted therapy.

Downregulation of extramitochondrial BCKDH and its uncoupling from AMP deaminase in type 2 diabetic OLETF rat hearts.

Systemic branched-chain amino acid (BCAA) metabolism is dysregulated in cardiometabolic diseases. We previously demonstrated that upregulated AMP deaminase 3 (AMPD3) impairs cardiac energetics in a rat model of obese type 2 diabetes, Otsuka Long-Evans-Tokushima fatty (OLETF). Here, we hypothesized that the cardiac BCAA levels and the activity of branched-chain α-keto acid dehydrogenase (BCKDH), a rate-limiting enzyme in BCAA metabolism, are altered by type 2 diabetes (T2DM), and that upregulated AMPD3 expression is involved in the alteration. Performing proteomic analysis combined with immunoblotting, we discovered that BCKDH localizes not only to mitochondria but also to the endoplasmic reticulum (ER), where it interacts with AMPD3. Knocking down AMPD3 in neonatal rat cardiomyocytes (NRCMs) increased BCKDH activity, suggesting that AMPD3 negatively regulates BCKDH. Compared with control rats (Long-Evans Tokushima Otsuka [LETO] rats), OLETF rats exhibited 49% higher cardiac BCAA levels and 49% lower BCKDH activity. In the cardiac ER of the OLETF rats, BCKDH-E1α subunit expression was downregulated, while AMPD3 expression was upregulated, resulting in an 80% lower AMPD3-E1α interaction compared to LETO rats. Knocking down E1α expression in NRCMs upregulated AMPD3 expression and recapitulated the imbalanced AMPD3-BCKDH expressions observed in OLETF rat hearts. E1α knockdown in NRCMs inhibited glucose oxidation in response to insulin, palmitate oxidation, and lipid droplet biogenesis under oleate loading. Collectively, these data revealed previously unrecognized extramitochondrial localization of BCKDH in the heart and its reciprocal regulation with AMPD3 and imbalanced AMPD3-BCKDH interactions in OLETF. Downregulation of BCKDH in cardiomyocytes induced profound metabolic changes that are observed in OLETF hearts, providing insight into mechanisms contributing to the development of diabetic cardiomyopathy.

Evaluation of therapeutic strategies targeting BCAA catabolism using a systems pharmacology model.

Background: Abnormal branched-chained amino acids (BCAA) accumulation in cardiomyocytes is associated with cardiac remodeling in heart failure. Administration of branched-chain α-keto acid dehydrogenase (BCKD) kinase inhibitor BT2 has been shown to reduce cardiac BCAA levels and demonstrated positive effects on cardiac function in a preclinical setting. The current study is focused on evaluating the impact of BT2 on the systemic and cardiac levels of BCAA and their metabolites as well as activities of BCAA catabolic enzymes using a quantitative systems pharmacology model. Methods: The model is composed of an ordinary differential equation system characterizing BCAA consumption with food, disposal in the proteins, reversible branched-chain-amino-acid aminotransferase (BCAT)-mediated transamination to branched-chain keto-acids (BCKA), followed by BCKD-mediated oxidation. Activity of BCKD is regulated by the balance of BCKDK and protein phosphatase 2Cm (PP2Cm) activities, affected by BT2 treatment. Cardiac BCAA levels are assumed to directly affect left ventricular ejection fraction (LVEF). Biochemical characteristics of the enzymes are taken from the public domains, while plasma and cardiac BCAA and BCKA levels in BT2 treated mice are used to inform the model parameters. Results: The model provides adequate reproduction of the experimental data and predicts synchronous BCAA responses in the systemic and cardiac space, dictated by rapid BCAA equilibration between the tissues. The model-based simulations indicate maximum possible effect of BT2 treatment on BCAA reduction to be 40% corresponding to 12% increase in LVEF. Model sensitivity analysis demonstrates strong impact of BCKDK and PP2Cm activities as well as total BCKD and co-substrate levels (glutamate, ketoglutarate and ATP) on BCAA and BCKA levels. Conclusion: Model based simulations confirms using of plasma measurements as a marker of cardiac BCAA changes under BCKDK inhibition. The proposed model can be used for optimization of preclinical study design for novel compounds targeting BCAA catabolism.

Antihypertensive drug valsartan as a novel BDK inhibitor

Catabolism of branched-chain amino acids (BCAAs) is affected by various physiological conditions and its abnormality is associated with glucose metabolism, heart disease, and neurological dysfunction. The first two steps of the BCAA metabolic pathway are common to the three BCAAs (leucine, isoleucine, and valine). The second step is an irreversible rate-limited reaction catalyzed by branched-chain α-keto acid dehydrogenase (BCKDH), which is bound to a specific kinase, BCKDH kinase (BDK), and inactivated by phosphorylation. Here, we investigated potential new BDK inhibitors and discovered valsartan, an angiotensin II type 1 receptor (AT1R) blocker, as a new BDK inhibitor. BCKDH phosphorylation and the BCKDH-BDK interaction were inhibited by valsartan in vitro. Valsartan administration in rats resulted in increased BCKDH activity by decreasing the dephosphorylated level of BCKDH complex, bound forms of BDK from BCKDH complex as well as decreased plasma BCAA concentrations. Valsartan is a novel BDK inhibitor that competes with ATP, via a different mechanism from allosteric inhibitors. The BDK inhibitor has been shown to preserve cardiac function in pressure overload-induced heart failure mice and to attenuate insulin resistance in obese mice. Our findings suggest that valsartan is a potent seed compound for developing a powerful BDK inhibitor and useful medication for treating heart failure and metabolic diseases with suppressed BCAA catabolism.

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.

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

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

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.

Impaired adiponectin signaling contributes to disturbed catabolism of branched-chain amino acids in diabetic mice.

The branched-chain amino acids (BCAA) accumulated in type 2 diabetes are independent contributors to insulin resistance. The activity of branched-chain α-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(-/-)) 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 α-keto acids (BCKA) were significantly elevated. APN treatment markedly reverted PP2Cm, BDK, BCKD activity, and BCAA and BCKA levels in HD-fed APN(-/-) 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.

A Novel Regulatory Defect in the Branched-Chain α-Keto Acid Dehydrogenase Complex Due to a Mutation in thePPM1KGene Causes a Mild Variant Phenotype of Maple Syrup Urine Disease

This article describes a hitherto unreported involvement of the phosphatase PP2Cm, a recently described member of the branched-chain α-keto acid dehydrogenase (BCKDH) complex, in maple syrup urine disease (MSUD). The disease-causing mutation was identified in a patient with a mild variant phenotype, involving a gene not previously associated with MSUD. SNP array-based genotyping showed a copy-neutral homozygous pattern for chromosome 4 compatible with uniparental isodisomy. Mutation analysis of the candidate gene, PPM1K, revealed a homozygous c.417_418delTA change predicted to result in a truncated, unstable protein. No PP2Cm mutant protein was detected in immunocytochemical or Western blot expression analyses. The transient expression of wild-type PPM1K in PP2Cm-deficient fibroblasts recovered 35% of normal BCKDH activity. As PP2Cm has been described essential for cell survival, apoptosis and metabolism, the impact of its deficiency on specific metabolic stress variables was evaluated in PP2Cm-deficient fibroblasts. Increases were seen in ROS levels along with the activation of specific stress-signaling MAP kinases. Similar to that described for the pyruvate dehydrogenase complex, a defect in the regulation of BCKDH caused the aberrant metabolism of its substrate, contributing to the patient's MSUD phenotype--and perhaps others.

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.

MicroRNAs: A new class of regulatory genes affecting metabolism

MicroRNAs (miRNAs) are short noncoding RNAs that regulate gene expression by binding to target mRNAs, which leads to reduced protein synthesis and sometimes decreased steady-state mRNA levels. Although hundreds of miRNAs have been identified, much less is known about their biological function. Several studies have provided evidence that miRNAs affect pathways that are fundamental for metabolic control in higher organisms such as adipocyte and skeletal muscle differentiation. Furthermore, some miRNAs have been implicated in lipid, amino acid, and glucose homeostasis. These studies open the possibility that miRNAs may contribute to common metabolic diseases and point to novel therapeutic opportunities based on targeting of miRNAs.

Effects of liver failure on branched-chain α-keto acid dehydrogenase complex in rat liver and muscle: comparison between acute and chronic liver failure

Background/Aims: Branched-chain α-keto acid dehydrogenase (BCKDH) complex catalyses the committed step in the branched-chain amino acid (BCAA) catabolic pathway. In many cases of liver failure, the serum BCAAs/aromatic amino acids ratio (Fisher's ratio) decreases, and BCAAs have been administered to patients with liver failure to correct this ratio. We conducted an animal study to examine whether the effects on hepatic BCKDH complex differ between acute liver failure (ALF) and chronic liver failure (CLF). Methods: ALF and CLF was induced in rats by a single high-dose injection and 21 weeks of repeated low-dose injections of carbon tetrachloride, respectively. Plasma BCAA and branched-chain α-keto acid (BCKA) levels, and activities and protein amounts of hepatic BCKDH complex and kinase were measured. Results: ALF was characterized by elevated plasma BCAA and BCKA levels and decreased hepatic BCKDH activity. CLF was characterized by decreased plasma BCAA and BCKA levels and increased hepatic BCKDH activity. This increase in BCKDH activity in CLF was associated with the decreased BCKDH kinase, which is responsible for the BCKDH inactivation. Conclusions: The results obtained in the present study suggest that BCAA catabolism is suppressed in ALF and increased in CLF.

Leucine toxicity in a neuronal cell model with inhibited branched chain amino acid catabolism

Individuals with the inborn error of metabolism, maple syrup urine disease (MSUD), are identified by newborn screening programs and treated with protein-modified diets that allow near normal growth and development. However, regardless of cause, a protein insult leads to metabolic decompensation, resulting in brain cell damage. The mechanism responsible for the damage is not well characterized due, in part, to the lack of an appropriate experimental model system with impaired branched chain α-ketoacid dehydrogenase (BCKD) activity. Here, we describe the construction of a rat pheochromocytoma cell (PC12) model harboring a doxycycline-controlled BCKD-kinase transgene. When BCKD-kinase is over-expressed in these cells, the endogenous BCKD activity is decreased, blocking branched chain amino acid (BCAA) catabolism. In cells over-expressing BCKD-kinase, addition of 25 mM leucine to the medium results in cell death. This experimental cell model accurately mimics the neuronal dysfunction in maple syrup urine disease and should facilitate further understanding of the pathophysiology of this disease and neuronal cell branched chain amino acid metabolism in general.

Potential role of leucine metabolism in the leucine-signaling pathway involving mTOR.

Leucine has been shown to stimulate adipose tissue protein synthesis in vivo as well as leptin secretion, protein synthesis, hyper-plastic growth, and tissue morphogenesis in in vitro experiments using freshly isolated adipocytes. Recently, others have proposed that leucine oxidation in the mitochondria may be required to activate the mammalian target of rapamycin (mTOR), the cytosolic Ser/Thr protein kinase that appears to mediate some of these effects. The first irreversible and rate-limiting step in leucine oxidation is catalyzed by the branched-chain alpha-keto acid dehydrogenase (BCKD) complex. The activity of this complex is regulated acutely by phosphorylation of the E1alpha-subunit at Ser293 (S293), which inactivates the complex. Because the alpha-keto acid of leucine regulates the activity of BCKD kinase, it has been suggested as a potential target for leucine regulation of mTOR. To study the regulation of BCKD phosphorylation and its potential link to mTOR activation, a phosphopeptide-specific antibody recognizing this site was developed and characterized. Phospho-S293 (pS293) immunoreactivity in liver corresponded closely to diet-induced changes in BCKD activity state. Immunoreactivity was also increased in TREMK-4 cells after the induction of BCKD kinase by a drug-inducible promoter. BCKD S293 phosphorylations in adipose tissue and gastrocnemius (which is mostly inactive in vivo) were similar. This suggests that BCKD complex in epididymal adipose tissue from food-deprived rats is mostly inactive (unable to oxidize leucine), as is the case in muscle. To begin to test the leucine oxidation hypothesis of mTOR activation, the dose-dependent effects of orally administered leucine on acute activation of S6K1 (an mTOR substrate) and BCKD were compared using the pS293 antibodies. Increasing doses of leucine directly correlated with increases in plasma leucine concentration. Phosphorylation of S6K1 (Thr389, the phosphorylation site leading to activation) in adipose tissue was maximal at a dose of leucine that increased plasma leucine approximately threefold. Changes in BCKD phosphorylation state required higher plasma leucine concentrations. The results seem more consistent with a role for BCKD and BCKD kinase in the activation of leucine metabolism/oxidation than in the activation of the leucine signal to mTOR.

Stimulatory effect of cold acclimation on skeletal muscle branched-chain α-keto acid dehydrogenase in rats

The effects of cold-acclimation (3 weeks at 4°C) on the actual activities of branched-chain α-keto acid dehydrogenase (BCKDH) and pyruvate dehydrogenase (PDH) complexes in the skeletal muscle and liver of normal 6-week-old rats were studied. The activities of BCKDH and PDH complexes were assayed using [1–14C] α-ketoisocaproate and [1-14C]pyruvate, respectively. Cold-acclimation markedly stimulated the activity of BCKDH, and slightly but significantly increased the activities of PDH and citrate synthase (CS) in the skeletal muscle, but did not alter PDH, BCKDH or CS activities in the liver. The increase in muscle BCKDH activity (control rats, 1.45 ± 0.54 mU/g wet wt; cold-acclimated rats, 2.54 ± 0.50 mU/g wet wt; 75% increase: P = 0.001) was much greater than the increases in PDH activity (84 ± 16 mU/g wet wt and 111 ± 25 mU/g wet wt; 32% increase: P = 0.020) or CS activities (27 ± 3 μmol/min per g wet wt and 31 ± 2 μmol/min per g wet wt; 14% increase: P = 0.046). These results suggest that, unlike PDH, BCKDH is not directly associated with the mitochondrial oxidative activity of the skeletal muscle of cold-acclimated rats. This study provides the first evidence that BCKDH in skeletal muscle responds selectively to cold-acclimation.

Insulin increases branched-chain alpha-ketoacid dehydrogenase kinase expression in Clone 9 rat cells.

The branched-chain amino acids (BCAA) are committed to catabolism by the activity of the branched-chain alpha-ketoacid dehydrogenase (BCKD) complex. BCKD activity is regulated through the action of the complex-specific BCKD kinase that phosphorylates two serine residues in the E1alpha subunit. Greater BCKD kinase expression levels result in a lower activity state of BCKD and thus a decreased rate of BCAA catabolism. Activity state varies among tissues and can be altered by diet, exercise, hormones, and disease state. Within individual tissues, the concentration of BCKD kinase reflects the activity state of the BCKD complex. Here we investigated the effects of insulin, an important regulator of hepatic metabolic enzymes, on BCKD kinase expression in Clone 9 rat cells. Insulin effected a twofold increase in message levels and a twofold increase in BCKD kinase protein levels. The response was completely blocked by treatment with LY-294002 and partially blocked by rapamycin, thus demonstrating a dependence on phosphatidylinositol 3-kinase and mTOR function, respectively. These studies suggest that insulin acts to regulate BCAA catabolism through stimulation of BCKD kinase expression.

Controlled Overexpression of BCKD Kinase Expression: Metabolic Engineering Applied to BCAA Metabolism in a Mammalian System

A common metabolic complication of human disease is uncontrolled muscle protein breakdown or cachexia, which occurs in patients with chronic diseases such as cancer, AIDS, renal failure, and diabetes. Increased branched-chain amino acid catabolism is implicated as causal and has stimulated the investigation of methods to regulate the metabolism of these amino acids. Here we demonstrate doxycycline-controlled overexpression of a branched-chain α-ketoacid dehydrogenase (BCKD) kinase transgene in mammalian cell culture. This kinase functions to inactivate the BCKD complex by phosphorylation, thus preventing the catabolism of these essential, regulatory metabolites. In this study, doxycycline treatment leads to a 10-fold increase in BCKD kinase protein. The transgene-generated kinase is rapidly incorporated within mitochondria and functions correctly to inactivate the BCKD complex. The maximum reduction in basal BCKD activity achieved was 94%. Unexpectedly, total BCKD activity was also decreased by kinase overexpression despite no observable change in expression of the BCKD catalytic proteins. These results demonstrate that artificial regulation of branched-chain amino acid metabolism is possible through the controlled overexpression of a single endogenous enzyme and suggest the feasibility of clinical applications.