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

Harris, Robert A.; Powell, Steven M.; Paxton, Ralph; Gillim, Sarah E.; Nagae, Hidetoshi

doi:10.1016/0003-9861(85)90531-4

Cited by 75 publications

(65 citation statements)

References 35 publications

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“…3). Consistent with previous findings (27), the liver enzyme was largely in the active state (92%) in animals fed a standard Teklad rat chow diet (24% protein). Increasing the protein content of the diet to 50% led to an increase in total BCKD activity, and essentially all of the BCKD was in the active state (99%).…”

Section: Characterization Of Ps293 E1␣ Antibodysupporting

confidence: 91%

“…Male Sprague-Dawley rats (75-125 g) were purchased from Charles River Laboratories (Cambridge, MA) and maintained for Ն7 days before the start of the treatment protocol on Teklad rat chow (24% protein) and water ad libitum. In some experiments, animals were then maintained for 7 days on Teklad diets containing 8, 24, or 50% protein (27). Subsequently, the livers were freeze-clamped and powdered under liquid nitrogen.…”

Section: Methodsmentioning

confidence: 99%

“…Harris et al (27) have previously shown that amounts of hepatic BCKD protein and activity are influenced by the amount of protein in the diet. By use of their protocol, rats were fed diets containing 8, 24, or 50% protein for 7 days.…”

Section: Characterization Of Ps293 E1␣ Antibodymentioning

confidence: 99%

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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: Characterization Of Ps293 E1␣ Antibodysupporting

confidence: 91%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

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

View full text Add to dashboard Cite

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“…Approx. 5i10 6 stably transformed cells were plated in 60-mm dishes and cultured in the presence or absence of 1 µM dexamethasone. After a 48 h incubation, cells were harvested and luciferase activity was measured in cell extracts and expressed as relative light units/10 3 cells.…”

Section: Figure 4 Promoter Activity Of the Bcod Kinase Gene In H4iie mentioning

confidence: 99%

“…The activity of the BCOD complex varies between tissues, and is regulated by nutritional and hormonal stimuli [5][6][7][8][9]. This provides an effective mechanism by which to modulate the flux of branched-chain amino acids through the BCOD complex.…”

Section: Introductionmentioning

confidence: 99%

Down-regulation of rat mitochondrial branched-chain 2-oxoacid dehydrogenase kinase gene expression by glucocorticoids

Huang

Chuang²

1999

Biochemical Journal

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The mammalian mitochondrial branched-chain 2-oxoacid dehydrogenase (BCOD) complex is regulated by a reversible phosphorylation (inactivation)\dephosphorylation (activation) cycle. In the present study, the effects of glucocorticoids on the level of BCOD kinase mRNA were investigated in rat hepatoma cell lines (H4IIE and FTO-2B), as well as in the rat. In H4IIE cells, dexamethasone was found to significantly reduce steadystate concentrations of BCOD kinase mRNA after a 48 h culture, and this was correlated with a 2-fold increase in the dephosphorylated form of the BCOD complex. The half-life of the kinase mRNA in H4IIE cells was not affected by dexamethasone treatment. Therefore, the decrease in the steady-state kinase mRNA level resulting from dexamethasone treatment was not caused by changes in mRNA stability, which raised the possibility of regulation at the level of gene transcription. To identify the negative glucocorticoid-responsive element in the kinase pro-

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Decarboxylation of branched‐chain α‐ketoacids in hepatocytes from alloxan‐diabetic rats. The effect of insulin

Sterniczuk

Wałajtys-Rode

Wojtczak

1991

Cell Biochemistry & Function

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The flux through branched-chain alpha-ketoacid dehydrogenase and the activity of the branched-chain alpha-ketoacid dehydrogenase complex were measured in hepatocytes isolated from fed, starved and alloxan diabetic rats. The highest rate of branched-chain alpha-ketoacid oxidation was found in hepatocytes isolated from starved rats, slightly lower in those from fed rats, and significantly lower in diabetic hepatocytes. The amount of the active form of branched-chain alpha-ketoacid dehydrogenase was only slightly diminished in diabetic hepatocytes, whereas the flux through the dehydrogenase was inversely correlated with the rate of endogenous ketogenesis. The same was observed in hepatocytes isolated from starved rats when branched-chain alpha-ketoacid oxidation was measured in the presence of added oleate. In both cases the diminished flux through the dehydrogenase, restored by a short preincubation of hepatocytes with insulin, was paralleled by a decrease of fatty acid-derived ketogenesis. The significance of these findings is discussed in relation to the role of insulin in branched-chain alpha-ketoacid oxidation in liver of diabetic rats.

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

Cited by 75 publications

References 35 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

Down-regulation of rat mitochondrial branched-chain 2-oxoacid dehydrogenase kinase gene expression by glucocorticoids

Decarboxylation of branched‐chain α‐ketoacids in hepatocytes from alloxan‐diabetic rats. The effect of insulin

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