Branched-chain amino acid catabolism: unique segregation of pathway enzymes in organ systems and peripheral nerves

Abstract

. Branched-chain amino acid catabolism: unique segregation of pathway enzymes in organ systems and peripheral nerves. Am J Physiol Endocrinol Metab 286: E64-E76, 2004. First published September 9, 2003 10.1152/ ajpendo.00276.2003We have examined the localization of the first two enzymes in the branched-chain amino acid (BCAA) catabolic pathway: the branched-chain aminotransferase (BCAT) isozymes (mitochondrial BCATm and cytosolic BCATc) and the branched-chain ␣-keto acid dehydrogenase (BCKD) enzyme complex. Antibodies specific for BCATm or BCATc were used to immunolocalize the respective isozymes in cryosections of rat tissues. BCATm was expressed in secretory epithelia throughout the digestive tract, with the most intense expression in the stomach. BCATm was also strongly expressed in secretory cells of the exocrine pancreas, uterus, and testis, as well as in the transporting epithelium of convoluted tubules in kidney. In muscle, BCATm was located in myofibrils. Liver, as predicted, was not immunoreactive for BCATm. Unexpectedly, BCATc was localized in elements of the autonomic innervation of the digestive tract, as well as in axons in the sciatic nerve. The distributions of BCATc and BCATm did not overlap. BCATm-expressing cells also expressed the second enzyme of the BCAA catabolic pathway, BCKD. In selected monkey and human tissues examined by immunoblot and/or immunohistochemistry, BCATm and BCATc were distributed in patterns very similar to those found in the rat. The results show that BCATm is in a position to regulate BCAA availability as protein precursors and anabolic signals in secretory portions of the digestive and other organ systems. The unique expression of BCATc in neurons of the peripheral nervous system, without coexpression of BCKD, raises new questions about the physiological function of this BCAT isozyme. digestive system; human; leucine; monkey; rat IN THE BODY, the nutritionally indispensable branched-chain amino acids (BCAAs) serve a number of important metabolic functions. BCAAs are key nitrogen donors for the synthesis of the metabolically significant dispensable amino acids glutamine and alanine. Glutamine is an important energy substrate for the gastrointestinal tract (38). Glutamine and alanine are also the major carriers of nitrogen from amino acid oxidation in skeletal muscle to the liver (7,20,33,48,56). In the central nervous system, BCAAs are thought to participate in an intercellular shuttle between neurons and astroglia that provides nitrogen for synthesis of the excitatory amino acid glutamate (3,4,31,39,40,64). In addition to the role of BCAAs in nitrogen metabolism, the BCAA leucine serves as an anabolic nutritional signal. Leucine stimulates protein synthesis in selected tissues via activation of the ribosomal protein S6 kinase 1 (12,19,42,61). Furthermore, high physiological concentrations of leucine stimulate secretion of insulin, and it has been postulated that this effect occurs in part via activation of glutamate dehydrogenase (43, 52).The initial reaction in the de...

Cytosolic Branched Chain Aminotransferase (BCATc) Regulates mTORC1 Signaling and Glycolytic Metabolism in CD4+ T Cells

“…For detection of phospho-E1␣, E1␣, and E2, an anti-E1␣ pS293, an anti-E2, and an E1␣ peptide antibody (27) were used. BCAT isozyme-specific antisera were raised in rabbits using purified recombinant human BCATm and BCATc and affinity purified as described (8,24). CsA was purchased from Enzo Life Sciences (Farmingdale, NY) and dissolved in dimethyl sulfoxide, rapamycin was purchased from LC Laboratories (Woburn, MA), NALA was purchased from BACHEM (Bubendorf, Switzerland).…”

“…BCAT Enzyme Activity-Total branched chain aminotransferase activity was measured in solubilized cells at 37°C in 25 mM potassium phosphate buffer, pH 7.8, using 1 mM ␣-keto [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]isovalerate and 12 mM Ile as described (7). A unit of enzyme activity was defined as 1 nmol of Val formed per min/mg of protein at 37°C.…”

“…For the Leu oxidation assay, the washed T cells were incubated in Krebs buffer in the presence of 1 mM [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]Leu (specific activity 200 dpm/nmol). The assay was carried out in a 20-ml scintillation vial, and cells were gassed with a 95% O 2 , 5% CO 2 mixture for 20 s, sealed with a rubber stopper, and incubated in Dubnoff metabolic shaker at 37°C for 120 min.…”

“…The BCATs catalyze the reversible transfer of the ␣-amino group of a BCAA to ␣-ketoglutarate, producing the branched chain ␣-keto acids (BCKAs) and Glu (6,7). BCKDC catalyzes irreversible oxidative decarboxylation of the BCKAs to produce the branched chain acyl-CoAs and NADH and contains multiple copies of three enzymes: branched chain ␣-keto acid decarboxylase/dehydrogenase (E1), dihydrolipoyl transcylase (E2), and dihydrolipoamide dehydrogenase (E3) (8). BCKDC is inactivated by phosphorylation of the E1␣ subunit of the E1 enzyme, catalyzed by a specific kinase (BDK), which is inhibited by the ␣-keto acid of Leu (KIC, ␣-ketoisocaproate) (9,10).…”

Branched-chain Amino Acid Metabolon

“…Interestingly, in freshly isolated rat islets, we found limited amounts of BCATm protein on Western blots. 3 Immunohistochemical results suggest the highest concentration of BCATm is actually in the exocrine pancreas (acinar cells), not in the islets (52), and KIC is also an insulin secretagogue. One might speculate that in vivo both leucine and KIC produced in the acinus play a role in the physiological response to leucine.…”