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

Andrew J. Sweatt,1 Mac Wood,1 Agus Suryawan,1 Reidar Wallin,2 Mark C. Willingham,3 and Susan M. Hutson1

1Department of Biochemistry, 2Section on Rheumatology, Department of Internal Medicine, and 3Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157

Submitted 19 June 2003 ; accepted in final form 3 September 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We 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 {alpha}-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 degradation of most indispensable amino acids is essentially irreversible, with excess amino acids being oxidized primarily in the liver. Thus the major fate of indispensable amino acids in peripheral tissues is synthesis into tissue proteins. Catabolism of the BCAAs differs markedly from that of the other indispensable amino acids in that the first step is reversible. In addition, BCAA catabolic enzymes appear to be distributed throughout the body, including in tissues of the digestive tract (18, 28, 36). The physiological significance of BCAA metabolism in tissues other than skeletal muscle and brain is not understood.

The first step in breakdown of the BCAAs is the reversible transfer of the {alpha}-amino group to {alpha}-ketoglutarate to form glutamate and the respective branched-chain {alpha}-keto acids in a reaction catalyzed by the branched-chain aminotransferase (BCAT) isozymes (mitochondrial BCATm and cytosolic BCATc); reviewed in Refs. 23, 35). The next and first irreversible step in BCAA catabolism is the oxidative decarboxylation of the branched-chain {alpha}-keto acid products of the transamination reaction. This step is catalyzed by the mitochondrial branched-chain {alpha}-keto acid dehydrogenase (BCKD) multienzyme complex, which contains multiple copies of three enzymes: a branched-chain {alpha}-keto acid decarboxylase (E1), a dihydrolipoyl transacylase (E2), and a dihydrolipoyl dehydrogenase (E3). Activity of BCKD is regulated by phosphorylation/dephosphorylation of the E1{alpha} subunit (21, 49). On the basis of observed tissue-specific differences in the activity of BCAT and the BCKD complex, it is thought that oxidation of BCAA involves extensive movement of metabolites between tissues (10, 24, 28, 30, 34, 53, 56).

The distribution of the BCAT isozymes in different tissues has been determined from measurements of enzyme activity and by Western blot analysis of BCAT proteins. In the rat, BCATm is found in most tissues, with very high BCAT activity found in the stomach, pancreas, and salivary glands (36, 56). BCATc appears to have more limited expression than BCATm. BCATc activity has been identified in rat brain, ovary, and placenta (18, 35). In the rat, BCKD activity is found in most organs, with highest activities occurring in the liver and kidney (56). In other organs, including the stomach, intestine, and brain, BCKD activities are up to two orders of magnitude lower than for liver and kidney (56).

The localization of the BCAA catabolic enzymes to particular cell types within a tissue has been investigated only for cells of the central nervous system. Immunostaining of cell cultures derived from rat brain revealed that BCAT isozymes are differentially expressed, with BCATm expressed in astroglia and BCATc expressed in neurons (4, 31, 40). In contrast, BCKD appeared to be expressed in both cell types (3). In this study, we report on the immunolocalization of BCAA catabolic enzymes to specific cell types in tissues known to have BCAA catabolic activities or to express mRNA coding for the catabolic enzymes. Particular attention was focused on tissues of the digestive tract, in which we demonstrate expression of BCATc in peripheral nerves, without concomitant expression of other BCAA catabolic enzymes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tissues. Rat tissues (salivary gland, esophagus, stomach, pancreas, duodenum, jejunum, ileum, colon, skeletal muscle, kidney, pancreas, ovary, uterus, testis, liver) were removed from male and female Sprague-Dawley or Long-Evans rats (225-250 g) and either flash-frozen in liquid nitrogen and stored at -80°C or cryoembedded in OCT Compound (Sakura Finetek, Torrance, CA) by freezing in liquid nitrogen and stored at -80°C. Monkey tissues (brain, skeletal muscle, adipose, liver, kidney, pancreas, jejunum, stomach) were obtained from monkeys involved in a study of the effect of dietary cholesterol on lipoprotein metabolism (50). Portions of the monkey tissues had been used previously to measure tissue BCAT and BCKD activities (56). Experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of the Wake Forest University School of Medicine. Human tissues (brain, muscle, adipose, liver, kidney, pancreas, jejunum, heart) were collected as described in a previously published study (56). Specimens were obtained from patients undergoing surgical procedures. All samples were obtained from tissues that would have been discarded following pathological examination during surgery. Stomach was obtained from a single organ donor who had given consent for use of the tissue for research purposes. All tissues were flash-frozen and stored in liquid nitrogen until analyzed. The protocol for human tissues was approved by the Institutional Review Board at Wake Forest University School of Medicine.

Antibodies. For identification of BCATm in rat tissues, a polyclonal antibody raised in rabbits against purified human recombinant BCATm was used. Characterization and affinity purification of this antibody have been described previously (40). With the exception of rat stomach, where three different BCATc antibodies were tested to verify the localization of BCATc (see Fig. 4), an antibody raised in rabbits against purified human recombinant BCATc was used for the immmunolocalization of BCATc in rat tissues. Affinity purification of this antibody is described below. The other two BCATc antibodies, used with rat stomach, were an immunoaffinity purified rabbit anti-rat BCATc peptide antibody, directed against the first 50 amino acids of the rat enzyme (40), and an IgG fraction of an antiserum that was raised in rabbits against purified rat brain BCATc protein (18). These two BCATc antibodies and the BCATm antibody have been used previously to identify BCATc and BCATm in rat tissues or rat brain primary cell cultures by immunoblotting and/or immunohistochemistry (3, 4, 18, 31, 40). Antiserum for localization of BCKD was generated against the purified E2 subunit of the rat liver BCKD complex and was the gift of Dr. Yoshiharu Shimomura (Nagoya Institute of Technology, Nagoya, Japan). An IgG fraction of the E2 antiserum has been used previously to identify BCKD in primary cell cultures derived from rat brain (3, 25).



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Fig. 4. BCATc antibodies identify BCATc in nervous elements in rat stomach. A: control (secondary antibody only). B: anti-rat BCATc NH2-terminal peptide antibody. C: anti-rat BCATc, IgG fraction. D: anti-recombinant human BCATc antibody. Deep zone of gastric mucosa is at top of each panel; smooth muscle layers are at the bottom. All 3 antibodies react with elements lying between the inner circular and outer longitudinal muscle layers. The immunoreactive structures are the ganglia and neural fibers of the intrinsic innervation of the gut (Auerbach's plexus, large arrows). D: additional labeling of fine innervating fibers is seen within the circular muscle layer. Small arrows, eosinophils showing nonimmunological background activity. Magnification for all panels, x140. Scale bar, 100 µm.

 

For preparation of the affinity-purified human BCATc and E2 antibodies, the antisera were made 50% saturated with ammonium sulfate and centrifuged for 10 min at 10,000 g. The supernatants were discarded, and the pellets were washed in PBS. Subsequently, the protein pellets were dissolved in PBS, followed by dialysis against PBS, and loaded onto an affinity resin having human BCATc or rat liver BCKD complex as a ligand. The columns were washed with PBS, and the fraction of antigen-specific antibodies was eluted in 0.1 M sodium acetate buffer (pH 4.0) containing 4 M urea and 0.5 M NaCl. The affinity-purified antibodies were dialyzed overnight against 50% glycerol-water at 4°C. The dialyzed antibodies were aliquoted and stored at -80°C. Human BCATc-Sepharose and rat liver BCKDSepharose were prepared by coupling the purified human recombinant BCATc or purified rat liver BCKD complex to Affigel 10 support (Bio-Rad, Richmond, CA) according to the manufacturer's directions.

Immunoblotting. Tissues for SDS-PAGE/immunoblot were pulverized with mortar and pestle while submerged in liquid nitrogen. Proteins were extracted from tissue powders by three rounds of freeze-thaw sonication in 25 mM HEPES (pH 7.4) containing 0.4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 10 µg/ml leupeptin, 5 mM benzamidine, and 1 mM diisopropyl fluorophosphate. Proteins in aliquots of tissue extracts (10-60 µg of protein) were separated by SDS-PAGE using 10% gels. Purified recombinant human BCATc and BCATm proteins (11) were included as standards. Proteins were transferred to Immobilon P membranes. Membranes were blocked with 5% nonfat milk-PBS or 1% BSA-PBS and incubated with immunoaffinity-purified rabbit anti-human BCATm (0.3-0.5 µg/ml), anti-rat peptide BCATc (0.4-0.6 µg/ml), or anti-human BCATc (0.4-0.6 µg/ml) antibodies. The immunoreactive protein bands were visualized using the enhanced chemiluminescence (ECL; for monkey and human tissue blots) or ECL Plus (for rat tissue blots) detection system according to the manufacturer's instructions (Amersham Biosciences, Piscataway NJ) and detected on X-ray film (Amersham Biosciences). Immunoreactive band intensities were analyzed for film exposures producing signals below saturation (bands were translucent). Band intensities were quantified in scanned images of the film (ImageQuant software, Amersham Biosciences) and are reported as arbitrary units per microgram of protein loaded. For quantification of BCATm in monkey tissues, band intensities for individual samples from each tissue were compared with intensities of a series of purified recombinant human BCATm or human BCATc standards (2-8 ng of purified BCAT). Extract protein concentrations were adjusted so that band intensities were within the linear range of the BCAT standards.

Immunohistochemistry. Frozen sections, 6-8 µm in thickness, were collected directly on slides or on adhesive tape and transferred to adhesive-coated slides by use of a UV-crosslinking system (Instrumedics CryoJane System, Hackensack, NJ). Sections were fixed by immersion in acetone (10 min), followed by lyophilization and storage at 25°C. Sections were rehydrated by immersion in PBS for 10 min. Nonspecific binding sites were blocked by treatment with 1% BSA-PBS for 15 min. Sections were incubated with the primary antibodies diluted to 5-10 µg/ml in BSA-PBS for 30-60 min and rinsed three times with PBS before incubation with horseradish peroxidase (HRP)- or FITC-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). After washing with PBS, color was developed for HRP conjugates by means of diaminobenzidine-H2O2. Controls consisted of incubations of sections with secondary antibodies only or with primary antibodies that had been preincubated overnight with a 10-fold excess of competing antigen, followed by secondary antibodies. In some experiments, immunolabeled sections were counterstained with hematoxylin, a nuclear stain, to aid in the identification of cell types. To visualize myelin in sciatic nerve, cryosections were fixed with 4% formaldehyde in PBS and stained with Oil Red O by standard methods (22). Tissues were viewed with a Zeiss AxioPlan 2 microscope, and images were obtained using an AxioCam digital camera and AxioVision imaging software (Carl Zeiss USA, Thornwood, NY). Images were adjusted and assembled using Adobe Photoshop 6.0 (Adobe Systems, San Jose, CA).

RNA extraction and RT-PCR. RT-PCR was used to determine whether BCATc mRNA was present in tissues outside the brain that exhibited BCATc-specific immunostaining in nervous elements. Flash-frozen tissue that had been powdered under liquid nitrogen (100-300 mg) and stored at -80°C was used for preparation of total RNA. Total RNA was extracted from the frozen tissue powder using TRIzol reagent according to manufacturer's instructions (Life Technologies, Rockville, MD). First-strand cDNA was synthesized using oligo(dT) primer and SuperScript II reverse transcriptase (GIBCO BRL, Gaithersburg MD) with 10 µg of RNA per sample. The integrity of the cDNA was confirmed by PCR using primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH): sense primer 5'-CCTTCATTGACCTCAACTACATGG-3'; antisense primer 5'-TCCACCACCCTGTTGCTGTAGC-3'. Rat BCATc was amplified with the following sequences: sense primer 5'-TCATGGCCTACTTGTCCCGG-3'; antisense primer 5'-CCATTAGGGCAACTCCAGTGT-3'. The predicted PCR product of 1241 bp includes all 1236 nucleotides of the BCATc coding sequence: ATG start through TAA stop (26), plus five nucleotides derived from the ends of the primers.

For PCR, cDNA (1.5 µl) was added to each reaction mixture (50 µl total volume) containing 10 mM Tris·HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates, and 0.2 µM of each primer. After an initial heat-denaturation step, 0.5 µl of Taq polymerase (5 unit/µl) was added to each reaction. An amplification program of denaturation (94°C, 1 min), annealing (60°C, 2 min), and extension (72°C, 1 min) was used for 34 cycles followed by a final elongation step at 72°C for 10 min. A second round of PCR was conducted by adding 1.5 µl of the first PCR reaction to a fresh reaction mixture. Aliquots of each reaction mixture (10 µl) were analyzed on a 2% agarose gel followed by staining with ethidium bromide.


    RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
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Expression of BCATm and BCATc in rat tissues. Immunoblotting with affinity-purified hBCATm antibodies showed ubiquitous but variable expression of BCATm protein in rat digestive tissues. To provide an estimate of the relative amounts of BCATm in digestive tissues, it was necessary to load different amounts of extract protein for each tissue on gels used for Western blotting (Fig. 1A). Immunoreactive band intensities were then converted to intensity per microgram of protein for each tissue, and the value for pancreas was taken as 100%. As shown in Fig. 1B, there was a 20-fold range of BCATm protein concentrations in the digestive system tissues. Consistent with previous reports (18, 24, 35), the highest relative concentrations of BCATm were found in pancreas and stomach. The other parts of the upper digestive tract (esophagus, duodenum) and the salivary glands had BCATm concentrations that were 24-40% of those of the pancreas. The lowest concentrations of BCATm were found in jejunum, ileum, and colon, which had 5-8% of the pancreatic levels. In tissues outside the digestive system and as shown previously (24, 56), heart and kidney had the highest BCATm concentrations, and all other tissues examined contained BCATm, including testis, spleen, uterus, lung, kidney, and thymus (data not shown).



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Fig. 1. Mitochondrial branched-chain aminotransferase (BCATm) is detectable by immunoblot throughout the rat gastrointestinal tract, whereas cytosolic BCAT (BCATc) is detectable only by RT-PCR. A: immunoblot of BCATm. Amounts of tissue extract loaded onto the gel ranged from 10 to 60 µg protein/lane. Recombinant human BCATm (8 ng) was used as a standard. B: relative tissue concentrations of BCATm. Immunoreactive band intensity per µg protein loaded was calculated for each tissue and is presented as a percentage of the value for pancreas, which had the highest concentration (taken as 100%). C: immunoblot for BCATc. Forty micrograms of tissue extract protein were loaded onto the gel, except for brain, where 20 µg were used. D: RT-PCR for BCATc mRNA. cDNA was reverse-transcribed from mRNA from rat gastrointestinal tissues, used as the template for PCR with BCATc-specific primers, and reamplified in a second round of PCR (see MATERIALS AND METHODS). Lanes are in the same order as for the immunoblot except for the positive control, for which a BCATc-encoding plasmid was used as the PCR template.

 

In the rat, BCATc protein has been detected by immunological methods in brain, ovary, and placenta (18). As shown in Fig. 1C, BCATc protein was not detected by immunoblotting in whole tissue extracts of the BCATm-expressing tissues of the digestive system. Similar results were obtained with other BCATm-expressing tissues (data not shown). Thus the immunoblot results are consistent with the current view that, in the rat, BCATc is not expressed outside the brain and female reproductive tissues. In other species, it has been reported that BCATc is expressed in a wider range of tissues (5, 17). Therefore, we used RT-PCR to determine whether BCATc mRNA was detectable in tissues of the digestive system. After a single round of PCR (34 cycles), a 1241-bp band corresponding to the BCATc mRNA was observed in several digestive tissues (ileum, jejunum, colon; data not shown). As shown in Fig. 1D, when an aliquot of the first-round PCR reaction was used in a second round of PCR amplification, a BCATc mRNA-derived band was observed in all tissues of the digestive system, although at almost undetectable levels in the esophagus. Low concentrations of BCATc mRNA were also found in testis, kidney, spleen, thymus, heart, lung, and liver (data not shown). The results raised the possibility that BCATc could be expressed at low levels in rat tissues or in a selected cell type within a tissue.

Localization of BCATm protein in rat tissues. BCATm immunoreactivity was found in epithelial cells in all portions of the digestive tract (Fig. 2). Controls for stomach and colon (no secondary antibody) are shown in Fig. 2, G and H. In the submandibular salivary gland, labeling was strongest in the serous-secreting components of the secretory epithelium; mucus-secreting epithelia were not stained (Fig. 2A). Throughout the stomach, the heaviest immunolabeling for BCATm was observed in the epithelial cells of the middle and deep zones of the gastric mucosa (Fig. 2B). Parietal cells, which have abundant, peripherally located mitochondria, were stained intensely for BCATm (Fig. 2B and inset). Chief cells were also labeled. The superficial zone of the mucosa, including much of the mucus-secreting epithelium, was not labeled (not shown). The longitudinal and circular smooth muscle layers, as well as the thin muscularis mucosae underlying the gastric mucosa, showed light immunolabeling. Light BCATm staining of the smooth muscle was seen throughout the digestive tract.



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Fig. 2. BCATm is present in epithelial cells in all portions of the rat gastrointestinal tract. A: salivary gland. B: stomach. C: duodenum. D: jejunum. E: ileum. F: colon. G: stomach control (secondary antibody only). H: colon control. Images in B-H are oriented such that the mucosa is at the top; the smooth muscle layers are at the bottom. In all tissues, eosinophils display nonimmunologically based reactivity (small arrows). Labeling for BCATm is most prominent over serous-secreting portions of the salivary gland and over the middle (neck) and deeper zones, including crypts, of the gastric mucosa (large arrows). B, inset: immunofluorescent localization of BCATm in the deep zone of the gastric epithelium. Glands are seen in longitudinal (*) and cross section (L, lumen of gland). Immunolabel is strongest in the peripheral cytoplasm of the epithelial cells. Magnification for all panels, x250. Scale bar, 100 µm.

 

In the small intestine, labeling of the duodenum was principally of cells in the deep zone of the mucosa, in particular those lining the crypts (Fig. 2C). Labeling in the jejunum and ileum was diffuse and much lighter than in the duodenum, with the highest concentrations of label again found in the crypts (Fig. 2, D and E). Cell types in the small intestinal crypts include lysozyme-secreting Paneth cells, although some endocrine and stem cells are also present and may be immunolabeled. Immunoreactivity for BCATm was not seen in the absorptive cells lying in the more superficial zone of the small intestine mucosa. The colon was labeled in much the same pattern as the duodenum, with the crypt epithelium being most heavily labeled (Fig. 2F). The epithelial cells of the crypt are precursor cells for the absorptive, mucus, and enteroendocrine cells of the more superficial mucosal epithelium.

BCATm also showed cell-specific localization in tissues outside the digestive system (Fig. 3). In Fig. 3, BCATmspecific immunostaining is seen as brown, and the cell nuclei are stained with hematoxylin (blue/purple). In skeletal muscle (Fig. 3A) and heart (data not shown), BCATm was found in myofibrils. In kidney, BCATm was located in the cortex. Intense labeling with the BCATm antibody was seen in epithelial cells lining short tubule segments in the cortex (Fig. 3B). The smaller lumina of these tubules are indicative of distal convoluted tubules, as are the more apical positions of the nuclei of the immunoreactive epithelial cells. Less intense staining was observed in cells of the glomerulus. Immunoreactive cells were seen in interstitial capillaries (Fig. 3B). These cells, which may be endothelial cells, were not labeled in the controls (data not shown). BCATm-specific immunostaining was not evident in proximal tubules and collecting ducts. In the pancreas, there was intense immunolabeling for BCATm in acinar cells of the exocrine pancreas, whereas immunolabeling was weak in the islets of Langerhans and in the cells lining the intercalated ducts (Fig. 3C). Light staining was observed in cells at the periphery and in cells in the interior of the islets. BCATm-specific immunostaining was not observed in the immunoadsorbed control shown in Fig. 3I. BCATm was also found in the lung, where it was localized to the bronchiolar and alveolar epithelium (data not shown). Consistent with earlier reports (32), BCATm was not seen in sections of liver (compare Fig. 3, G with H).



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Fig. 3. BCATm is present in specific cell types in rat tissues. In this figure and other color figures, cell nuclei were counterstained with hematoxylin (blue/purple) to allow identification of cell types in the tissues. Specific immunolabel appears brown. A: skeletal muscle, x600. B: kidney, x440. Arrows, immunoreactive distal convoluted tubules. Occasional immunoreactive cells are seen in the interstitial capillaries (glom, glomerulus). C: pancreas, x280 (islet, islet of Langerhans). D: ovary, x170. Arrows, immunoreactive theca interna of an antral follicle. E: uterus, x1,040. F: testis, x200. Arrows, immunoreactive interstitial Leydig cells (tubule, seminiferous tubule). G: liver, x560. H: liver control, x520. Primary antibody (5-10 µg/ml) was preadsorbed with recombinant BCATm. I: pancreas, x440. Immunoadsorbed control. Scale bar: 50 µm for C, 100 µm for F, and 25 µm for all other panels.

 

All tissues of the reproductive tract of male and female rats that were examined expressed BCATm. In ovarian follicles, labeling for BCATm was seen in the secretory cells that make up the theca interna (arrows in Fig. 3D). The follicular epithelial cells (granulosum) and stromal fibroblasts (theca externa) were unlabeled. BCATm was localized in the uterus to secretory epithelial cells in the deep portions of endometrial glands (Fig. 3E). Surrounding smooth muscle was not labeled. In the testis, BCATm was restricted to the interstitial tissues, with the most intense labeling in Leydig cells (Fig. 3F). Seminiferous tubules did not show appreciable BCATm-specific immunoreactivity.

Localization of BCATc in rat tissues. The observation that low levels of BCATc mRNA were present in rat tissues raised the possibility that BCATc protein might also be present in these tissues, albeit at levels too low to be detected easily by immunoblotting of whole tissue extracts. Therefore, several different BCATc-specific antibodies were used to look for BCATc-specific staining in rat stomach (see MATERIALS AND METHODS). Rat stomach was chosen because it expresses the BCATc mRNA, and the high concentration of BCATm in this tissue would provide a good test for the specificity of the BCATc antibodies. All three anti-BCATc antibodies exhibited a similar staining pattern. Cells and processes of the neural elements of the stomach exhibited intense staining for BCATc (Fig. 4, B-D). Intense BCATc-specific labeling was found in the myenteric (Auerbach's) plexus lying between the circular and longitudinal smooth muscle layers. Immunolabeling was also observed in fine processes of the submucosal (Meissner's) plexus (not shown). Because the affinity-purified anti-human BCATc antibody showed high specificity and low background (Fig. 4D), it was used to localize BCATc in other rat tissues.

BCATc-specific immunostaining was found in neural elements of all gastrointestinal tract tissues that were examined (Fig. 5). In the salivary gland (Fig. 5A), bundles and finer processes of immunoreactive neurons were seen in the vascular/connective tissue beds. BCATc-specific staining was also seen in nerves in the pancreas (Fig. 5B). From the stomach to the colon, BCATc was immunolocalized in the nerves and ganglia of the myenteric nerve plexus (Fig. 5, C-G). It appeared that cell bodies as well as neuronal processes were immunolabeled. In these and all other tissues, BCATc immunostaining was not evident in nonneuronal cells. Salivary gland and jejunum controls are shown in Fig. 5, H and I, respectively.



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Fig. 5. BCATc is localized in nerves of the rat gastrointestinal tract tissues. Cryosections were probed with affinity-purified anti-recombinant human BCATc, followed by secondary antibody and peroxidase substrate, as described in MATERIALS AND METHODS. A: salivary gland. B: pancreas. C: stomach. D: duodenum. E: jejunum. F: ileum. G: colon. H: salivary gland control (secondary antibody only). I: jejunum control. Labeling for BCATc is restricted to nervous elements lying between lobes of the salivary gland and to nerves and ganglia in Auerbach's plexus in the gastrointestinal tract (large arrows). Small arrows, eosinophil background activity. Magnification, x500 for A and B and x250 for all other panels. Scale bar, 50 µm for A and B and 100 µm for all other panels.

 

To determine whether BCATc was also present in nerves outside the digestive tract, rat sciatic nerve was examined for BCATc-specific immunoreactivity. Nerve-specific localization of BCATc was confirmed in cross sections of the sciatic nerve (Fig. 6). Labeling within the cross-sectioned nerve appeared as well-defined small circular and polygonal profiles (Fig. 6A). Companion sections of the sciatic nerve were stained with the lipid stain Oil Red O to visualize the myelin sheath surrounding the nerve axons (Fig. 6B). Comparison of Fig. 6, A and B, suggests that BCATc immunoreactivity is restricted to the nerve axons and not found in the myelin sheath. As the nerve enters the muscle compartment, immunoreactivity for BCATc could also be seen in tangentially and cross-sectioned axons (Fig. 6C).



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Fig. 6. BCATc is expressed in axons in the sciatic nerve. A: rat sciatic nerve was immunolabeled with affinity-purified anti-recombinant human BCATc. Arrows, immunoreactive axons. Arrowheads, structures stained in B. B: sciatic nerve was stained with Oil Red O to reveal lipid-rich elements (myelin, indicated by arrowheads). C: rat gastrocnemius was labeled with anti-recombinant human BCATc. N, nerve; m, muscle. Magnification for all panels, x920. Scale bar, 25 µm.

 

Localization of BCKD in rat tissues. In general, the distribution of the E2 subunit of BCKD in rat tissues paralleled that of BCATm (Fig. 7). BCKD E2-specific staining was found in the epithelial cells of the middle and deep zones of the gastric mucosa (compare Fig. 7A with Fig. 2B). As seen with BCATm, cells with parietal cell morphology exhibited intense E2 immunostaining. There was no specific labeling for BCKD E2 in the myenteric nerve plexus. E2 was present in the exocrine and endocrine pancreas, although there was less difference in intensity of immunolabeling between acinar cells and islets than was observed with BCATm (compare Fig. 7B with Fig. 3C). BCKD immunoreactivity was the same as that observed with BCATm in skeletal muscle and heart, whereas in kidney BCKD E2-specific staining was found in the proximal as well as in the distal convoluted tubules (data not shown). Consistent with reports showing that rat liver has the highest concentration of BCKD activity and protein (21, 56, 59), intense BCKD E2-specific staining was found in liver hepatocytes (Fig. 7C). Immunoadsorbed liver and pancreas controls are shown in Fig. 7, G and H, respectively.



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Fig. 7. Branched-chain {alpha}-keto acid dehydrogenase (BCKD) is expressed ubiquitously in rat tissues. E2 subunit of BCKD was immunolocalized in selected rat tissues. In all tissues except stomach, cell nuclei were counterstained with hematoxylin. A: stomach. Large arrows, immunoreactive epithelial cells in the neck region of the gastric mucosa. Eosinophils display background activity (small arrows). B: pancreas. Circle shows approximate boundary of an islet of Langerhans. C: liver. D: ovary. Arrow, immunoreactive cells of theca interna. E: uterus. Arrow, immunoreactive epithelial cells lining endometrial cavity; *lumen of an endometrial gland with immunoreactive epithelium. F: testis. Arrow, immunoreactive interstitial Leydig cells. G and H: respective liver and pancreas controls (primary antibody immunoadsorbed with purified BCKD complex). Magnification in A and E, x250; D, x125; all other panels, x500. Scale bar, 100 µm for A and E, 200 µm for D, and 50 µm for all other panels.

 

In the female reproductive tract, immunoreactivity for BCKD E2 was seen in cells comprising the granulosa and theca interna in the ovary (Fig. 7D) and in the epithelial cells of the uterine endometrium (Fig. 7E). As observed with BCATm, BCKD E2 was found in the Leydig cells of the testis and not in the seminiferous tubule (Fig. 7F). Thus the distribution of BCKD is very similar to that of BCATm.

Expression of BCAT isozymes in selected human and nonhuman primate tissues. Immunoblotting of African Green monkey and human tissues with isozyme-specific, affinitypurified anti-BCAT antibodies confirmed the ubiquitous expression of BCATm and expression of BCATc in monkey and human brain tissue (Fig. 8). BCATm protein levels varied considerably in both monkey and human tissues. The human recombinant proteins were used as standards to estimate the level of the BCAT proteins in the monkey tissues. The results indicated that the highest concentration of BCATm protein is found in pancreas and kidney, followed by muscle, stomach, and jejunum. Some degradation of BCATm can be observed in the pancreas extracts. Significant concentrations of BCATc protein were observed only in brain tissue. In monkey brain, BCATc represented ~80% of total BCAT protein (BCATm ~20%), which is similar to what has been reported for rat brain (24, 35, 36). BCATm was near the limits of detection in liver and subcutaneous fat.



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Fig. 8. In the monkey and the human, BCAT isozymes show tissue distributions similar to those in the rat. Extracts of monkey and human tissues were analyzed by immunoblot with antibodies directed against recombinant human BCATm or BCATc. Recombinant human BCATm and BCATc (20 ng each) were used as standards. Human tissues are in the same order as monkey tissues, except for heart, which was used instead of stomach. Tissue extracts were loaded at 30 µg protein/lane. Inset table: concentrations of BCATc and BCATm in monkey tissues, as determined by densitometry in separate blots (see MATERIALS AND METHODS).

 

The human BCATm and rat BCKD antibodies were used to examine the cellular localization of these proteins in available monkey tissues. In most tissues, the localization of these enzymes was similar to what was observed in rat tissues. An exception was monkey liver, which, like human liver, has measurable BCAT activity (56). As shown in Fig. 9A, BCATm immunoreactivity was not found in hepatocytes. Immunoreactivity appeared to be localized in cells that have the location and morphology of Kupffer cells. As in the rat, BCKD E2 immunoreactivity was found in hepatocytes. For other tissues (stomach, pancreas, kidney, skeletal muscle, and heart), the localization of BCATm immunoreactivity paralleled the localization of this protein in the rat (data not shown). As in the rat, distal convoluted tubules of monkey kidney were labeled, although the glomerulus was not immunoreactive for BCATm. For BCKD, the labeling pattern in liver, pancreas, skeletal, and heart muscle was the same as in the corresponding rat tissues (data not shown).



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Fig. 9. In the monkey, BCATm is localized to Kupffer cells in liver and to distal convoluted tubules in kidney. In these sections, nuclei were counterstained with hematoxylin. A: liver. Arrows, immunoreactive Kupffer cells in the liver sinusoidal lining. Inset: control section exposed to immunoadsorbed antibody. B: kidney. Arrows, immunoreactive epithelium of convoluted tubules. Inset: control section. Magnification for both panels, x230. Scale bar, 100 µm.

 


    DISCUSSION
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A novel finding of the present work is the consistent localization of BCAA catabolic enzymes in secretory epithelial cells. This is particularly marked for BCATm, which appears along the course of the digestive tract in cells that secrete either salivary components, acid, enzymes, or enzyme precursors. BCATm was not found in absorptive cells or in goblet cells, which secrete mucus components. In other organ systems, BCATm is found in protein- and steroid-secreting cells (e.g., ovarian endometrium and testicular Leydig cells, respectively) as well as in transporting epithelia (kidney distal convoluted tubules). The BCATm-containing cells are variously derived from ecto-, endo-, and mesoderm. For the most part, these cells have in common only their position at an interface with the lumen of an organ (i.e., epithelial). Second, our results show that, with a few exceptions, BCATm is expressed with BCKD, the second enzyme of the BCAA catabolic pathway.

The preferential localization of BCATm in secretory epithelial cells raises interesting questions about the function(s) of the BCAA in these cells. For example, high concentrations of BCATm and BCKD are found in the pancreatic acini that synthesize and secrete proteins, rather than in the islet, where leucine and {alpha}-ketoisocaproate (KIC, the transamination product of leucine) are known to stimulate insulin secretion (43, 60, 61). The high concentrations of BCATm in the pancreatic acini could function to provide KIC as a signaling molecule to the islet. Alternatively, BCATm activity in the acini may serve to dampen leucine signaling in the islet. KIC release from pancreas has not been measured directly. However, even though differences did not reach statistical significance, portal KIC concentrations were 6.4 µM higher than arterial KIC concentrations in fed dogs (40.6 vs. 34.2 µM; Ref. 63). The portal vein receives blood from the stomach, pancreas, and intestine. Leucine stimulates protein synthesis in several tissues (15, 42, 46, 60). If leucine or KIC is a nutrient signal in the exocrine pancreas, then the high concentrations of BCATm may regulate the availability of the active metabolite.

In the gastrointestinal tract, BCAA catabolic enzymes are found in the secretory epithelial cells rather than in the absorptive epithelial cells of the intestinal mucosa. Therefore, only a small fraction of enterally fed BCAAs would be expected to be oxidized by the intestinal epithelium during the absorptive process. This interpretation is consistent with results from studies using leucine and KIC tracers, in which it has been shown that rates of nitrogen transfer (BCAA transamination) are higher than rates of oxidation (BCKD step) and that oxidation by the gut is limited (2, 44, 45, 62, 63). For example, Matthews and coworkers (44, 45) calculated that <2% of nasogastrically delivered leucine and ~5% of KIC were oxidized on the first pass in humans. Yu et al. (63) measured metabolism of arterially delivered leucine across the midgut (duodenum to transverse colon) and liver in dogs. The gut accounted for only 4% of whole body leucine oxidation in fasted dogs and for 13% in fed dogs.

Across species, stomach has one of the highest specific activities of BCAT (35, 56). Although a number of studies have examined leucine metabolism across the splanchnic bed, where amino acids derived from hydrolysis in the small intestine are plentiful (2, 13, 16, 44, 45, 47, 62, 63), information on the stomach is limited. Amino acid transporters that transport BCAA have been identified in the stomach. Sobrevia et al. (55) have reported that an L-type Na+-independent transport system is present in the basolateral surface of the oxyntic glands, which contain both parietal and chief cells. This transporter is likely to mediate BCAA uptake by the gastric epithelium from the arterial circulation. Another amino acid transporter, the ATB0+ protein, is located on the luminal surface of lung epithelial cells and is also expressed in mouse stomach (54). This indicates that free BCAA, if available in the stomach, can also be taken up at the luminal surface of the gastric epithelium. On the other hand, mRNA encoding a recently cloned peptide transport protein was expressed in the small intestine but not in stomach or colon in several animal species (8). Thus it is likely that most stomach BCAA metabolism is derived from arterial BCAAs. However, because stomach metabolism of lumen-derived free BCAAs is possible, it could affect conclusions about BCAA requirements and the proportion of BCAA metabolism that occurs in the digestive system.

Generally, liver is the primary site of catabolism of indispensable amino acids. It is also likely that liver is the primary site of BCAA oxidation in the rat (28, 53, 56). What sets BCAAs apart from other indispensable amino acids is that nitrogen transfer via transamination is largely extrahepatic. For example, in the rat, BCATm is not found in liver, but liver has the highest concentration and activity of BCKD (20, 21, 56). On the other hand, nonhuman primates and humans have measurable liver BCAT activity, but it is still <6% of total body capacity (17, 56), and BCATm protein is near the limits of detection in both human and monkey liver (Fig. 8). In the present study, BCATm is found in monkey liver in what appears to be the phagocytic Kupffer cells, which make up a small proportion of the cells in the liver. In contrast, BCKD is found in the far more numerous liver hepatocytes in monkey and rat (Fig. 7C). Thus monkey liver is an example of a tissue where BCKD is expressed in a cell without BCATm. Whether or not this is true in human liver remains to be determined. Nevertheless, it is still likely that, as in the rat, under most conditions liver is a primary site of BCAA oxidation in humans and nonhuman primates. This hypothesis is supported by a case report showing that, after transplantation of a normal liver to a patient with maple syrup urine disease (BCKD defect), the patient was able to tolerate a diet with a normal protein content (58).

For BCATc, the brain is the primary site of expression in human, monkey, sheep, and rat (14, 56; see also Fig. 8), and in rat, BCATc is found in neurons in culture (3, 25). However, the expression of BCATc in rat peripheral nerves was unexpected. There is evidence that BCATc has a more widespread distribution in other animals. By separating BCATc from BCATm activity by means of DEAE-cellulose chromatography of crude tissue homogenates, Goto et al. (17) concluded that BCATc activity represents a variable but significant proportion of total BCAT activity in most human tissues. In a more recent study involving BCATc, BCATc-specific antibodies were used to immunoprecipitate BCATc from sheep tissue homogenates (5). Significant activity attributed to BCATc was found in sheep muscle, along with BCATc mRNA (5). If the rat is representative of other animals, then the BCATc measured in these studies may be localized in nervous elements of peripheral tissues of these animals as well.

Although we have not yet investigated the types of nerves that express BCATc, in the rat this isozyme is found in neural elements supplying the salivary gland and pancreas, as well as in Auerbach's and Meissner's plexuses in the gut wall. These nerves are all parts of the autonomic nervous system, supplying motor neurons that innervate glands and smooth muscle in the digestive system. In the stomach and intestine, some of the BCATc immunolabeling may be associated with the interstitial cells of Cajal. These cells are closely associated with enteric ganglia and smooth muscle cells and have some neuronal characteristics (51). In the sciatic nerve, the nonuniform distribution of BCATc immunoreaction in the axons (see Fig. 6) may reflect the association of BCATc with a particular class of neurons or with a specific neuronal component that is unevenly distributed in the axoplasm. BCATc is a target of the neuroactive drug gabapentin (25), which is used widely to treat neuropathic pain (1). We have also found BCATc in spinal cord neurons (Sweatt AJ, Garcia-Espinosa MA, Wallin R, and Huston SM, unpublished observations). It is possible that inhibition of BCATc by gabapentin may contribute to its efficacy in the treatment of neuropathic pain.

In the central nervous system, the BCAT isozymes are thought to participate in a shuttle that provides amino nitrogen for de novo synthesis of excitatory neurotransmitter glutamate in brain and in the retina (25, 40, 64). In this scheme, nitrogen is shuttled between BCATm in astroglia and BCATc in glutamatergic and/or {gamma}-aminobutyric acid (GABAergic) neurons. In the peripheral nervous system, including the autonomic division innervating the gastrointestinal tract, the principal neurotransmitters are acetylcholine, the catecholamines (norepinephrine and epinephrine), and neuroactive peptides. For these neurotransmitter systems, the function of a nitrogen shuttle operating between neurons and surrounding glia is not clear, although {gamma}-aminobutyric acid is synthesized in tissues outside the brain. An alternative function for BCATc might be to regulate (or attenuate) the anabolic signal provided by leucine (12, 19, 42, 61). The apparent absence of BCKD in the peripheral nerves indicates that KIC produced from leucine by BCATc in neurons could not be metabolized and is likely released. Finally, it should be noted that, for both BCATm and BCATc, nonenzymatic roles in intracellular signaling may be possible. Recent work has shown that a splice variant of BCATm with an internal deletion of 12 amino acids is expressed in colon carcinoma cells (41). This isoform of BCATm binds to the thyroid hormone receptor and enhances its effects on nuclear transcription activity (41). Furthermore, BCATm has a redox-active CXXC center (9) and has been found to associate with BCKD and other proteins (27). Identifying the constituents of neurons with which BCATc might interact may shed light on its role in the peripheral nervous system.


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The work reported here was supported by Grants DK-34738 and NS-38641 from the US National Institutes of Health and Grant 98-35200-6067 from the US Department of Agriculture (S. M. Hutson).


    ACKNOWLEDGMENTS
 
Current address for Agus Suryawan: US Dept. of Agriculture/Agriculture Research Service, Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. J. Sweatt, Dept. of Biochemistry, Wake Forest Univ. School of Medicine, Medical Center Blvd., Winston-Salem NC 27157 (E-mail: asweatt{at}wfubmc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 RESULTS
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 REFERENCES
 

  1. Backonja M and Glanzman RL. Gabapentin dosing for neuropathic pain: evidence from randomized, placebo-controlled clinical trials. Clin Ther 25: 81-104, 2003.[CrossRef][ISI][Medline]
  2. Biolo G and Tessari P. Splanchnic versus whole-body production of alpha-ketoisocaproate from leucine in the fed state. Metabolism 46: 164-167, 1997.[ISI][Medline]
  3. Bixel M, Shimomura Y, Hutson S, and Hamprecht B. Distribution of key enzymes of branched-chain amino acid metabolism in glial and neuronal cells in culture. J Histochem Cytochem 49: 407-418, 2001.[Abstract/Free Full Text]
  4. Bixel MG, Hutson SM, and Hamprecht B. Cellular distribution of branched-chain amino acid aminotransferase isoenzymes among rat brain glial cells in culture. J Histochem Cytochem 45: 685-694, 1997.[Abstract/Free Full Text]
  5. Bonfils J, Faure M, Gibrat JF, Glomot F, and Papet I. Sheep cytosolic branched-chain amino acid aminotransferase: cDNA cloning, primary structure and molecular modelling and its unique expression in muscles. Biochim Biophys Acta 1494: 129-136, 2000.[ISI][Medline]
  6. Burrin DG, Stoll B, Chang X, van Goudever JB, Fujii H, Hutson S, and Reeds PJ. Parenteral nutrition results in impaired lactose digestion and hexose absorption when enteral feeding is initiated in infant pigs. Am J Clin Nutr 78: 461-470, 2003.[Abstract/Free Full Text]
  7. Chang TW and Goldberg AL. The metabolic fates of amino acids and the formation of glutamine in skeletal muscle. J Biol Chem 253: 3685-3693, 1978.[ISI][Medline]
  8. Chen H, Wong EA, and Webb KE Jr. Tissue distribution of a peptide transporter mRNA in sheep, dairy cows, pigs, and chickens. J Anim Sci 77: 1277-1283, 1999.[Abstract/Free Full Text]
  9. Conway ME, Yennawar N, Wallin R, Poole LB, and Hutson SM. Identification of a peroxide-sensitive redox switch at the CXXC motif in the human mitochondrial branched chain aminotransferase. Biochemistry 41: 9070-9078, 2002.[CrossRef][ISI][Medline]
  10. Cree TC, Hutson SM, and Harper AE. Gas-liquid chromatography of alpha-keto acids: quantification of the branched-chain-alpha-keto acids from physiological sources. Anal Biochem 92: 159-163, 1979.[Medline]
  11. Davoodi J, Drown PM, Bledsoe RK, Wallin R, Reinhart GD, and Hutson SM. Overexpression and characterization of the human mitochondrial and cytosolic branched-chain aminotransferases. J Biol Chem 273: 4982-4989, 1998.[Abstract/Free Full Text]
  12. Dumont FJ and Su Q. Mechanism of action of the immunosuppressant rapamycin. Life Sci 58: 373-395, 1996.[CrossRef][ISI][Medline]
  13. Elango R, Pencharz PB, and Ball RO. The branched-chain amino acid requirement of parenterally fed neonatal piglets is less than the enteral requirement. J Nutr 132: 3123-3129, 2002.[Abstract/Free Full Text]
  14. Faure M, Hayes H, Bledsoe RK, Hutson SM, and Papet I. Assignment of the gene of mitochondrial branched chain aminotransferase (BCAT2) to sheep chromosome band 14q24 and to cattle and goat chromosome bands 18q24 by in situ hybridization. Cytogenet Cell Genet 83: 96-97, 1998.[ISI][Medline]
  15. Flaim KE, Peavy DE, Everson WV, and Jefferson LS. The role of amino acids in the regulation of protein synthesis in perfused rat liver. I. Reduction in rates of synthesis resulting from amino acid deprivation and recovery during flow-through perfusion. J Biol Chem 257: 2932-2938, 1982.[Abstract/Free Full Text]
  16. Gelfand RA, Glickman MG, Jacob R, Sherwin RS, and DeFronzo RA. Removal of infused amino acids by splanchnic and leg tissues in humans. Am J Physiol Endocrinol Metab 250: E407-E413, 1986.[Abstract/Free Full Text]
  17. Goto M, Shinno H, and Ichihara A. Isozyme patterns of branched-chain amino acid transaminase in human tissues and tumors. GANN 68: 663-667, 1977.[ISI][Medline]
  18. Hall TR, Wallin R, Reinhart GD, and Hutson SM. Branched chain aminotransferase isoenzymes. Purification and characterization of the rat brain isoenzyme. J Biol Chem 268: 3092-3098, 1993.[Abstract/Free Full Text]
  19. Hara K, Yonezawa K, Weng QP, Kozlowski MT, Belham C, and Avruch J. Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. J Biol Chem 273: 14484-14494, 1998.[Abstract/Free Full Text]
  20. Harper AE. Thoughts on the role of branched-chain alpha-keto acid dehydrogenase complex in nitrogen metabolism. Ann NY Acad Sci 573: 267-273, 1989.[ISI][Medline]
  21. Harris RA, Hawes JW, Popov KM, Zhao Y, Shimomura Y, Sato J, Jaskiewicz J, and Hurley TD. Studies on the regulation of the mitochondrial alpha-ketoacid dehydrogenase complexes and their kinases. Adv Enzyme Regul 37: 271-293, 1997.[CrossRef][ISI][Medline]
  22. Humason GL. Animal Tissue Techniques. San Francisco: WH Freeman, 1979.
  23. Hutson S. Structure and function of branched chain aminotransferases. Prog Nucleic Acid Res Mol Biol 70: 175-206, 2001.[ISI][Medline]
  24. Hutson SM. Subcellular distribution of branched-chain aminotransferase activity in rat tissues. J Nutr 118: 1475-1481, 1988.[ISI][Medline]
  25. Hutson SM, Berkich D, Drown P, Xu B, Aschner M, and LaNoue KF. Role of branched-chain aminotransferase isoenzymes and gabapentin in neurotransmitter metabolism. J Neurochem 71: 863-874, 1998.[ISI][Medline]
  26. Hutson SM, Bledsoe RK, Hall TR, and Dawson PA. Cloning and expression of the mammalian cytosolic branched chain aminotransferase isoenzyme. J Biol Chem 270: 30344-30352, 1995.[Abstract/Free Full Text]
  27. Hutson SM, Conway ME, Fujii H, and Wallin R. Discovery of a regulated metabolon involving key enzymes of the leucine catabolic pathway. Experimental Biology 2003 FASEB Ann Mtng San Diego. (Abstract 524.8)
  28. Hutson SM, Cree TC, and Harper AE. Regulation of leucine and alpha-ketoisocaproate metabolism in skeletal muscle. J Biol Chem 253: 8126-8133, 1978.[ISI][Medline]
  29. Hutson SM and Hall TR. Identification of the mitochondrial branched chain aminotransferase as a branched chain alpha-keto acid transport protein. J Biol Chem 268: 3084-3091, 1993.[Abstract/Free Full Text]
  30. Hutson SM and Harper AE. Blood and tissue branched-chain amino and alpha-keto acid concentrations: effect of diet, starvation, and disease. Am J Clin Nutr 34: 173-183, 1981.[Abstract]
  31. Hutson SM, Lieth E, and LaNoue KF. Function of leucine in excitatory neurotransmitter metabolism in the central nervous system. J Nutr 131: 846S-850S, 2001.[Abstract/Free Full Text]
  32. Hutson SM, Wallin R, and Hall TR. Identification of mitochondrial branched chain aminotransferase and its isoforms in rat tissues. J Biol Chem 267: 15681-15686, 1992.[Abstract/Free Full Text]
  33. Hutson SM and Zapalowski C. Relationship of branched-chain amino acids to skeletal muscle gluconeogenic amino acids. In: Metabolism and Clinical Implications of Branched-Chain Amino and Keto-Acids, edited by Walser M and Williamson JR. New York: Elsevier/North Holland, 1981, p. 245-250.
  34. Hutson SM, Zapalowski C, Cree TC, and Harper AE. Regulation of leucine and alpha-ketoisocaproic acid metabolism in skeletal muscle. Effects of starvation and insulin. J Biol Chem 255: 2418-2426, 1980.[Free Full Text]
  35. Ichihara A. Aminotransferases of branched-chain amino acids. In: Transaminases, edited by Christen P and Metzler DE. New York: Wiley & Sons, 1985, p. 430-438.
  36. Ichihara A, Noda C, and Goto M. Transaminase of branched chain amino acids. X. High activity in stomach and pancreas. Biochem Biophys Res Commun 67: 1313-1318, 1975.[ISI][Medline]
  37. Kholodilov NG, Neystat M, Oo TF, Hutson SM, and Burke RE. Upregulation of cytosolic branched chain aminotransferase in substantia nigra following developmental striatal target injury. Brain Res Mol Brain Res 75: 281-286, 2000.[ISI][Medline]
  38. Labow BI and Souba WW. Glutamine. World J Surg 24: 1503-1513, 2000.[CrossRef][ISI][Medline]
  39. LaNoue KF, Berkich DA, Conway M, Barber AJ, Hu LY, Taylor C, and Hutson S. Role of specific aminotransferases in de novo glutamate synthesis and redox shuttling in the retina. J Neurosci Res 66: 914-922, 2001.[CrossRef][ISI][Medline]
  40. Lieth E, LaNoue KF, Berkich DA, Xu B, Ratz M, Taylor C, and Hutson SM. Nitrogen shuttling between neurons and glial cells during glutamate synthesis. J Neurochem 76: 1712-1723, 2001.[CrossRef][ISI][Medline]
  41. Lin HM, Kaneshige M, Zhao L, Zhang X, Hanover JA, and Cheng SY. An isoform of branched-chain aminotransferase is a novel co-repressor for thyroid hormone nuclear receptors. J Biol Chem 276: 48196-48205, 2001.[Abstract/Free Full Text]
  42. Lynch CJ, Patson BJ, Anthony J, Vaval A, Jefferson LS, and Vary TC. Leucine is a direct-acting nutrient signal that regulates protein synthesis in adipose tissue. Am J Physiol Endocrinol Metab 283: E503-E513, 2002.[Abstract/Free Full Text]
  43. Malaisse WJ, Hutton JC, Carpinelli AR, Herchuelz A, and Sener A. The stimulus-secretion coupling of amino acid-induced insulin release: metabolism and cationic effects of leucine. Diabetes 29: 431-437, 1980.[Abstract]
  44. Matthews DE, Harkin R, Battezzati A, and Brillon DJ. Splanchnic bed utilization of enteral alpha-ketoisocaproate in humans. Metabolism 48: 1555-1563, 1999.[ISI][Medline]
  45. Matthews DE, Marano MA, and Campbell RG. Splanchnic bed utilization of leucine and phenylalanine in humans. Am J Physiol Endocrinol Metab 264: E109-E118, 1993.[Abstract/Free Full Text]
  46. May ME and Buse MG. Effects of branched-chain amino acids on protein turnover. Diabetes Metab Rev 5: 227-245, 1989.[ISI][Medline]
  47. Metges CC, El-Khoury AE, Selvaraj AB, Tsay RH, Atkinson A, Regan MM, Bequette BJ, and Young VR. Kinetics of L-[1-13C]leucine when ingested with free amino acids, unlabeled or intrinsically labeled casein. Am J Physiol Endocrinol Metab 278: E1000-E1009, 2000.[Abstract/Free Full Text]
  48. Odessey R, Khairallah EA, and Goldberg AL. Origin and possible significance of alanine production by skeletal muscle. J Biol Chem 249: 7623-7629, 1974.[Abstract/Free Full Text]
  49. Reed LJ, Damuni Z, and Merryfield ML. Regulation of mammalian pyruvate and branched-chain alpha-keto acid dehydrogenase complexes by phosphorylation-dephosphorylation. Curr Top Cell Regul 27: 41-49, 1985.[ISI][Medline]
  50. Rudel L, Deckelman C, Wilson M, Scobey M, and Anderson R. Dietary cholesterol and downregulation of cholesterol 7 alpha-hydroxylase and cholesterol absorption in African Green monkeys. J Clin Invest 93: 2463-2472, 1994.[ISI][Medline]
  51. Sanders KM. A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract. Gastroenterology 111: 492-515, 1996.[ISI][Medline]
  52. Sener A, Somers G, Devis G, and Malaisse WJ. The stimulus-secretion coupling of amino acid-induced insulin release. Biosynthetic and secretory responses of rat pancreatic islet to L-leucine and L-glutamine. Diabetologia 21: 135-142, 1981.[ISI][Medline]
  53. Shinnick FL and Harper AE. Branched-chain amino acid oxidation by isolated rat tissue preparations. Biochim Biophys Acta 437: 477-486, 1976.[ISI][Medline]
  54. Sloan JL, Grubb BR, and Mager S. Expression of the amino acid transporter ATB0+ in lung: possible role in luminal protein removal. Am J Physiol Lung Cell Mol Physiol 284: L39-L49, 2003.[Abstract/Free Full Text]
  55. Sobrevia L, Medina V, Reinicke K, and Bravo I. Uptake of L-leucine and L-phenylalanine across the basolateral cell surface in isolated oxyntic glands. Biochim Biophys Acta 1106: 257-263, 1992.[ISI][Medline]
  56. Suryawan A, Hawes JW, Harris RA, Shimomura Y, Jenkins AE, and Hutson SM. A molecular model of human branched-chain amino acid metabolism. Am J Clin Nutr 68: 72-81, 1998.[Abstract]
  57. Wallin R, Hall TR, and Hutson SM. Purification of branched chain aminotransferase from rat heart mitochondria. J Biol Chem 265: 6019-6024, 1990.[Abstract/Free Full Text]
  58. Wendel U, Saudubray JM, Bodner A, and Schadewaldt P. Liver transplantation in maple syrup urine disease. Eur J Pediatr 158, Suppl 2: S60-S64, 1999.[ISI][Medline]
  59. Wohlhueter RM and Harper AE. Coinduction of rat liver branched chain alpha-keto acid dehydrogenase activities. J Biol Chem 245: 2391-2401, 1970.[Abstract/Free Full Text]
  60. Xu G, Kwon G, Cruz WS, Marshall CA, and McDaniel ML. Metabolic regulation by leucine of translation initiation through the mTOR-signaling pathway by pancreatic beta-cells. Diabetes 50: 353-360, 2001.[Abstract/Free Full Text]
  61. Xu G, Kwon G, Marshall CA, Lin TA, Lawrence JC Jr, and McDaniel ML. Branched-chain amino acids are essential in the regulation of PHAS-I and p70 S6 kinase by pancreatic beta-cells. A possible role in protein translation and mitogenic signaling. J Biol Chem 273: 28178-28184, 1998.[Abstract/Free Full Text]
  62. Yu YM, Burke JF, Vogt JA, Chambers L, and Young VR. Splanchnic and whole body L-[1-13C,15N]leucine kinetics in relation to enteral and parenteral amino acid supply. Am J Physiol Endocrinol Metab 262: E687-E694, 1992.[Abstract/Free Full Text]
  63. Yu YM, Wagner DA, Tredget EE, Walaszewski JA, Burke JF, and Young VR. Quantitative role of splanchnic region in leucine metabolism: L-[1-13C,15N]leucine and substrate balance studies. Am J Physiol Endocrinol Metab 259: E36-E51, 1990.[Abstract/Free Full Text]
  64. Yudkoff M, Daikhin Y, Grunstein L, Nissim I, Stern J, and Pleasure D. Astrocyte leucine metabolism: significance of branched-chain amino acid transamination. J Neurochem 66: 378-385, 1996.[ISI][Medline]
  65. Zielke HR, Huang Y, Baab PJ, Collins RM Jr, Zielke CL, and Tildon JT. Effect of alpha-ketoisocaproate and leucine on the in vivo oxidation of glutamate and glutamine in the rat brain. Neurochem Res 22: 1159-1164, 1997.[ISI][Medline]