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

Doering, Christopher B.; Williams, Ifor R.; Danner, Dean J.

doi:10.1006/mben.2000.0164

Cited by 13 publications

(8 citation statements)

References 44 publications

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“…TREMK-4 cells, which contain a recombinant BCKD kinase gene under the control of a drug-inducible promoter, were a generous gift from Dr. Dean Danner (Emory School of Medicine, Atlanta, GA). The cells were maintained in cell culture, and BCKD kinase was induced as described previously (12). To examine phosphorylation end points, the cells were washed once and then incubated for 1 h with Krebs-Ringer-HEPES buffer (KRH) at 37°C.…”

Section: Methodsmentioning

confidence: 99%

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

Lynch

Halle²,

Fujii³

et al. 2003

American Journal of Physiology-Endocrinology and Metabolism

103

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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 α-keto acid dehydrogenase (BCKD) complex. The activity of this complex is regulated acutely by phosphorylation of the E1α-subunit at Ser293 (S293), which inactivates the complex. Because the α-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.

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Section: Methodsmentioning

confidence: 99%

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

Lynch

Halle²,

Fujii³

et al. 2003

American Journal of Physiology-Endocrinology and Metabolism

103

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“…The amount of BCKD in the unphosphorylated state relative to the total amount of BCKD (unphosphorylated ϩ phosphorylated) represents the BCKD activity state and is expressed as a percentage. Although a putative BCKD phosphatase is reported, the activity state of BCKD is more closely related to the expression of BCKD kinase than to a balance between steady-state activities of the kinase and phosphatase (13,14,18). For example, in liver BCKD kinase, protein levels are low, leaving most of the BCKD complex catalytically active.…”

mentioning

confidence: 99%

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

Nellis

Doering²,

Kasinski³

et al. 2002

American Journal of Physiology-Endocrinology and Metabolism

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Nellis, Mary M., Christopher B. Doering, Andrea Kasinski, and Dean J. Danner. Insulin increases branchedchain ␣-ketoacid dehydrogenase kinase expression in Clone 9 rat cells. Am J Physiol Endocrinol Metab 283: E853-E860, 2002. First published June 3, 2002 10.1152/ajpendo.00133.2002The branched-chain amino acids (BCAA) are committed to catabolism by the activity of the branched-chain ␣-ketoacid dehydrogenase (BCKD) complex. BCKD activity is regulated through the action of the complex-specific BCKD kinase that phosphorylates two serine residues in the E1␣ 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.hormone-controlled gene expression; branched-chain amino acids THE BRANCHED-CHAIN amino acids (BCAA), leucine, isoleucine, and valine, are classified as essential components of the mammalian diet because they cannot be synthesized de novo. BCAA account for up to 20% of the residues in the average protein. Catabolism of the BCAA provides energy, and their products serve as precursors for fatty acid synthesis. BCAA can modify other metabolic processes through a poorly understood mechanism whereby cells sense the concentration of these amino acids. Protein turnover, especially in the liver and muscles, is slowed when the BCAA concentration is maintained (6,12,32,37,38).Another player in the cellular regulation of protein turnover is insulin, which stimulates protein synthesis, especially in liver (11,15). A combination of insulin and leucine may be needed for the promotion of protein synthesis, and this action may occur through separate pathways to enhance assembly of the translation initiation complex (2, 5, 22). As expected, because tissues respond differently to insulin exposure, tissue-specific variation in these effects is reported (9,19,35,40).Maintenance of cellular BCAA concentration in mammals results from a balance between supply and catabolic loss. Supply results from dietary intake or breakdown of endogenous protein. Irreversible loss results from catabolism of the BCAA that begins with their reversible transamination to yield the branchedchain ␣-ketoacids (BCKA). Oxidative decarboxylation of the BCKA commits these compounds to their catabolic fate. The react...

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“…BCKD activity is regulated through the action of the complex-specific BCKD kinase that phosphorylates two serine residues in the E1α subunit and thereby inhibits BCKD. Notably, insulin stimulates BCKD kinase expression inhibiting BCKD increasing cellular BCAA levels [226][227][228][229][230][231]. Mechanistically, MEX miR-29b functions as an enhancer of insulin-mediated suppression of BCAA catabolism promoting mTORC1 activation at both the PI3K/AKT/TSC2/RHEB and the BCAA/RAG-Ragulator/RHEB pathway.…”

Section: Mir-29bmentioning

confidence: 99%

Lifetime Impact of Cow’s Milk on Overactivation of mTORC1: From Fetal to Childhood Overgrowth, Acne, Diabetes, Cancers, and Neurodegeneration

Melnik

2021

Biomolecules

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The consumption of cow’s milk is a part of the basic nutritional habits of Western industrialized countries. Recent epidemiological studies associate the intake of cow’s milk with an increased risk of diseases, which are associated with overactivated mechanistic target of rapamycin complex 1 (mTORC1) signaling. This review presents current epidemiological and translational evidence linking milk consumption to the regulation of mTORC1, the master-switch for eukaryotic cell growth. Epidemiological studies confirm a correlation between cow’s milk consumption and birthweight, body mass index, onset of menarche, linear growth during childhood, acne vulgaris, type 2 diabetes mellitus, prostate cancer, breast cancer, hepatocellular carcinoma, diffuse large B-cell lymphoma, neurodegenerative diseases, and all-cause mortality. Thus, long-term persistent consumption of cow’s milk increases the risk of mTORC1-driven diseases of civilization. Milk is a highly conserved, lactation genome-controlled signaling system that functions as a maternal-neonatal relay for optimized species-specific activation of mTORC1, the nexus for regulation of eukaryotic cell growth, and control of autophagy. A deeper understanding of milk´s impact on mTORC1 signaling is of critical importance for the prevention of common diseases of civilization.

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

Cited by 13 publications

References 44 publications

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

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

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

Lifetime Impact of Cow’s Milk on Overactivation of mTORC1: From Fetal to Childhood Overgrowth, Acne, Diabetes, Cancers, and Neurodegeneration

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