Distribution of D-3-aminoisobutyrate-pyruvate aminotransferase in the rat brain
© Abe et al.; licensee BioMed Central Ltd. 2014
Received: 10 September 2013
Accepted: 4 April 2014
Published: 27 April 2014
D-3-aminoisobutyrate, an intermediary product of thymine, is converted to 2-methyl-3-oxopropanoate using pyruvate as an amino acceptor by D-3-aminoisobutyrate-pyruvate aminotransferase (D-AIB AT; EC 18.104.22.168). A large amount of D-AIB AT is distributed in the kidney and liver; however, small amounts are found in the brain. Recently, D-AIB AT was reported to metabolize asymmetric dimethylarginine (ADMA) in vivo and was suggested to be an important enzyme for nitric oxide metabolism because ADMA is a competitive inhibitor for nitric oxide synthase. In this study, we examined the distribution of D-AIB AT in the rat brain further to understand its role. We measured D-AIB AT mRNA and protein expression using quantitative RT-PCR and Western blotting, and monitored its distribution using immunohistochemical staining.
D-AIB AT was distributed throughout the brain, with high expression in the cortex and hippocampus. Immunohistochemical staining revealed that D-AIB AT was highly expressed in the retrosplenial cortex and in hippocampal neurons.
Our results suggest that D-AIB AT is distributed in the examined- just the regions and may play an important role there.
KeywordsD-3-aminoisobutyrate-pyruvate aminotransferase Asymmetric dimethylarginine RT-PCR Western blotting Immunohistochemistry
Male Wistar rats (CLEA Japan, Tokyo, Japan), 6–8 weeks old, were housed in air-conditioned rooms (temperature, 22 ± 2°C) with a 12-h light–dark cycle. After rats were anaesthetized with 30 ml of diethyl ether and decapitated, the brain was removed and tissues used for examination (frontal cortex, temporal cortex, cerebellum striatum, thalamus, hippocampus, midbrain, pons, and olfactory bulb) were dissected. All experiments were approved by the Ethics Review Committee for Animal Experimentation of Ehime University.
RNA extraction and quantitation of D-AIB AT mRNA expression
We examined ten rat (the ages of rat breakdown was that 6 weeks old is six, 7 weeks old is two and 8 weeks old is two) for RT-PCR. RNA was extracted from each brain region isolated according to the RNeasy Lipid Tissue Mini kit instructions (Qiagen, Valencia, CA, USA), which included DNase treatment (Qiagen). Following assessment of RNA quality and quantity with the NanoDrop (NanoDrop Technologies, DE, USA), 1 μg of total RNA was used for cDNA synthesis with random hexamers using Moloney murine leukemia virus reverse transcriptase (Applied Biosystems, Austin, TX, USA). The expression of the D-AIB AT gene transcript was quantified by real-time PCR with the TaqMan Gene Expression Assay (Applied Biosystems). TaqMan primer-probe sets for D-AIB AT (agxt2, Rn00582928_m1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Ss03375435_u1), used as an endogenous control, were purchased from Applied Biosystems. Quantitative RT-PCR was performed using the StepOnePlus System (Applied Biosystems). Changes in D-AIB AT mRNA expression were calculated after normalization with GAPDH expression. The cDNA from an arbitrarily selected control rat were used as a calibrator sample. The ΔΔCT method provided a relative quantification ratio according to the calibrator, which allowed statistical comparisons of gene expression among samples. Values of fold changes in the sample versus the frontal cortex samples represented averages from triplicate measurements. Statistical calculations were carried out using the SPSS Statistical Software Package 11.5 (SPSS, Tokyo, Japan). The mRNA expression differences among brain tissues were analyzed by analysis of variance with repeated measures followed by the Bonferroni post hoc test. A P value less than 0.05 was considered statistically significant.
We examined four rat (all ages of rat is 6 weeks old) for Western blotting. Brain tissue was homogenized in lysis buffer containing 8.1 mM Na2HPO4, 2.68 mM KCl, 1.47 mM KH2PO4, 137 mM NaCl, 1 mM EDTA, and 2 mM 2-mercaptoethanol. Homogenates were centrifuged at 16,000 g for 20 min at 4°C. The supernatant was decanted into a new centrifuge tube. Protein concentrations for each brain region were determined using the BCA Protein Assay Kit (Thermo Fisher Scientific K.K., Yokohama, Japan). Protein samples (10 μg) were suspended in Laemmli sample buffer, and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis was performed according to standard procedures. Total lysates were separated on 1% SDS-polyacrylamide gels, and were blotted onto polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocking with Blocking One (Nacalai Tesque, Kyoto, Japan), the membranes were incubated with either anti-D-AIB AT rabbit polyclonal primary antibody (specificity of antibody show Additional files 1 and 2) generated using the D-AIB-AT peptide ‘SPYTLGLTNVGIYKMEL’ (Japan Bioserum, Hiroshima, Japan), or anti-GAPDH rabbit primary antibody (internal control; Abcam, Cambridge, UK) diluted in 1% (v/v) Tween 20 at room temperature for 3 h. Membranes were treated with anti-rabbit goat IgG horseradish peroxidase-linked Whole Antibody (GE Healthcare, Buckinghamshire, UK) diluted in 1% (v/v) Tween 20 at room temperature for 1 h. Visualization using ChemiDoc MP (Bio Rad) was used for detection. Image J (http://rsb.info.nih.gov/ij/index.html) was used for Western blot analysis following digitization.
D-AIB AT immunohistochemistry
Wistar rat brains on postnatal day 7, 14, 28, and 56 were used in this study. Animals were anesthetized by intraperitoneal injection of chloral hydrate (10 mg/kg) and then perfused transcardially with physiological saline, followed by 4% (w/v) paraformaldehyde in 100 mM phosphate buffer (pH 7.4). Brains were removed and post-fixed in the same solution for 12 h at 4°C and embedded in paraffin wax. Frontal sections of rat brains were cut at 5 μm, dewaxed in xylene, and rehydrated with a graded series of ethanol solutions. Histochemistry was performed using the anti-D-AIB AT antibody (this antibody same as Western) in combination with the avidin-biotin peroxidase complex (ABC) method using the VECTASTAIN ABC kit (Vector Labs, Burlingame, CA, USA). Endogenous peroxidase was blocked in methanolic hydrogen peroxidase at 36°C for 30 min, and non-specific protein binding was suppressed by incubation with 1% (w/v) bovine serum albumin at 36°C for 30 min, followed by rinsing with 0.02 M phosphate-buffered saline (PBS; pH 7.4). Sections were incubated with the anti D-AIB AT antibody at 4°C overnight. After rinsing with PBS, the sections were further incubated with the VECTASTAIN ABC reagent at room temperature for 30 min. Finally, the sections were colorized with PBS containing 0.006% (w/v) 3-3-diaminobenzidine tetrahydrochloride and 0.003% (v/v) H2O2 at room temperature for 30 min, and lightly counterstained with hematoxylin.
To our knowledge, this is the first report to examine the distribution of D-AIB AT in the rat brain. D-AIB AT was distributed in examined- just the regions, with expression being greatest in deep layers of the cortex, hippocampus, and brain stem. Immunohistochemistry revealed that D-AIB AT expression was highest in the retrosplenial cortex, hippocampus, brain stem, and choroid plexus.
High amounts of D-AIB AT are distributed in the kidney and liver , and small amounts are observed in various regions of the brain . Although the only known role of D-AIB AT relates to the metabolism of D-AIB, its precise function in the central nervous system remains unclear. Recently, D-AIB AT was identified as an enzyme that not only catalyzes the metabolism of D-AIB but also catalyzes the degradation of ADMA, which is a competitive inhibitor of NOS . ADMA is present in the central nervous system  and is reported to be ubiquitously distributed in various regions of the brain . D-AIB AT inhibits NO production in vitro and actively degrades ADMA in vivo; however, ADMA is also degraded by dimethylarginine dimethylaminohydrolase (DDAH) 1, a member of the DDAH enzyme family . We hypothesize that D-AIB AT in the brain effectively metabolizes ADMA as an ADMA: pyruvate aminotransferase, an aminotransferase that catalyzes ADMA with pyruvate to form α-keto- δ-(NG,NG- dimethyl-guanidino) valeric acid and alanine in vivo (Figure 1), resulting in a decrease to ADMA brain levels, which helps NOS improve endothelial NO production in the brain.
In this study, we report that D-AIB AT is distributed in the examined- just the regions. Several studies support the idea that ADMA may be important regulator of the NO system, where elevated ADMA concentrations are associated with hypertension , congestive heart failure , progression of chronic kidney disease  and atherosclerosis . D-AIB AT may be related to the vulnerability for cerebro-vascular disease.
Our immunohistochemical studies revealed that D-AIB AT is widely expressed in the retrosplenial cortex and hippocampus. The rat retrosplenial cortex is similar to Brodmann areas 29 and 30 in primates, which has abundant reciprocal projections with the hippocampus directly and indirectly . Inactivation of the retrosplenial cortex is reported to impair active navigation in dark testing conditions in a rat model with tetracaine anesthetization of the retrosplenial cortex, suggesting that this region is important for spatial memory . Hippocampal atrophy is known to be one of the main symptoms of Alzheimer’s disease (AD), and disruption of fronto-hippocampal connections, not only directly but indirectly through damage of the retrosplenial posterior cingulate cortex, is observed in AD . Metabolic decline in the retrosplenial cortex is also reported in AD following positron emission tomography [23, 24]. Pathological changes in this region can also occur in schizophrenia, bipolar disorder, and post-traumatic stress disorder review . Therefore the expression of D-AIB AT in brain lesions formed following neurological disease progression.
In conclusion, D-AIB AT is widely distributed in the brain and may work as an aminotransferase not only related to degradation of D-AIB from thymine. Further studies will be needed to clarify the role of D-AIB in the central nervous system.
Real time-polymerase chain reaction
Nitric oxide synthase
Endothelial nitric oxide
Neuronal nitric oxide
Sodium dodecyl sulfate
Avidin-biotin peroxidase complex
The authors wish to thank Ms. Mayumi Doi for her technical assistance. This work was partially supported by Grants from the Ministry of Health, Labour and Welfare of the Japanese Government and from the Japanese Ministry of Education, Culture, Sports, Science and Technology.
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