Branched-chain amino acids govern the high learning ability phenotype in Tokai high avoider (THA) rats

Abstract

To fully understand the mechanisms governing learning and memory, animal models with minor interindividual variability and higher cognitive function are required. THA rats established by crossing those with high learning capacity exhibit excellent learning and memory abilities, but the factors underlying their phenotype are completely unknown. In the current study, we compare the hippocampi of parental strain Wistar rats to those of THA rats via metabolomic analysis in order to identify molecules specific to the THA rat hippocampus. Higher branched-chain amino acid (BCAA) levels and enhanced activation of BCAA metabolism-associated enzymes were observed in THA rats, suggesting that acetyl-CoA and acetylcholine are synthesized through BCAA catabolism. THA rats maintained high blood BCAA levels via uptake of BCAAs in the small intestine and suppression of BCAA catabolism in the liver. Feeding THA rats with a BCAA-reduced diet decreased acetylcholine levels and learning ability, thus, maintaining high BCAA levels while their proper metabolism in the hippocampus is the mechanisms underlying the high learning ability in THA rats. Identifying appropriate BCAA nutritional supplements and activation methods may thus hold potential for the prevention and amelioration of higher brain dysfunction, including learning disabilities and dementia.

Figure 1

Determination of metabolites involved in THA rat-specific high learning ability via metabolome analysis. (a) Schematic representation of the experimental protocol. The animals used in the experiment were bred in the same breeding environment. After weaning at 4 weeks of age, a behavioral avoidance test was conducted at 5 weeks of age. Sampling and experiments were carried out at the times described. (b) Comparison of avoidance rate during the lever-pressing test between Wistar (n = 6) and THA rats (n = 4). (c) Avoidance rates of THA rats, Wistar rats (Wistar-H, n = 3) showing high avoidance rate, and other Wistar rats (Wistar-L, n = 3). Data are expressed as the mean ± SD values. *P < 0.05. (d) Principal component analysis (PCA) of results obtained from metabolome analysis of the hippocampus in Wistar-L, Wistar-H, and THA rats. PCA was conducted using SampleStat software. Blue enclosure; Wistar-L, red enclosure; Wistar-H, and green enclosure; THA rats. (e) Hierarchical clustering analysis (HCA) of the metabolites in Wistar-L, Wistar-H, and THA rats. HCA was conducted using PeakStat software. Red indicates an increase of metabolites, and green indicates a decrease, respectively. The part surrounded by the red dotted line indicates BCAAs.

Figure 2

BCAAs are oxidized in the hippocampus of THA rats in response to the learning test. (a) Schematic presentation of the BCAA oxidation pathway. BCAAs are transaminated by BCAT1 or BCAT2 to generate BCKAs, which are subsequently oxidized by BCKDC. α-KG is the α-keto acid acceptor of the BCAA nitrogen group, and Glu is the product, which is utilized as a substrate for GABA synthesis. BCKDC is phosphorylated and inactivated by BCKDK. BCAA-derived intermediates are trapped in the mitochondria by CoA. The end products of the BCAA catabolic pathway, succinyl CoA and acetyl CoA act as TCA cycle intermediates for ATP production. R-CoA: acyl CoA. (b) Western blot analyses of BCAT1, BCKDK, phosphorylated BCKDHA (P-BCKDHA), and total BCKDHA (T-BCKDHA) as well as their quantified ratios in the hippocampi of Wistar rats (n = 4) and THA rats (n = 4) after avoidance tests. β-actin served as a loading control. (c) Acetyl-CoA and (d) choline concentrations in the hippocampus after the avoidance test (Wistar rats n = 6, THA rats n = 4). (e) ChAT expression in protein extracts from the hippocampus. β-actin served as a loading control. The expression level was determined via densitometric analysis. (f) Hippocampal acetylcholine levels in the above tissues (Wistar rats n = 6, THA rats n = 4). (g) Western blot analyses of phosphorylated 4E-BP1 (P-4E-BP1), total 4E-BP1 (T-4E-BP1), phosphorylated eIF4E (P-eIF4E), total eIF4E (T-eIF4E), phosphorylated S6 (P-S6), and total S6 (T-S6) expression in the above tissues. β-actin served as a loading control. The expression level was determined via densitometric analysis. (h) Western blot analyses of P-BCKDHA, T-BCKDHA, P-4E-BP1, T-4E-BP1, P-eIF4E, T-eIF4E, and ChAT and their quantified ratios in the hippocampi of Wistar rats and THA rats before the avoidance test. (i) Comparison of P-BCKDHA, T-BCKDHA, and ChAT expression before (BF) and after (AF) avoidance tests via western blot analyses of hippocampi from Wister (n = 4) or THA rats (n = 4), respectively. β-actin served as a loading control. The expression level was determined via densitometric analysis. Data are expressed as the mean ± SD values. *P < 0.05; **P < 0.01. Uncropped blots are presented in Supplementary Fig. S6.

Figure 2

BCAAs are oxidized in the hippocampus of THA rats in response to the learning test. (a) Schematic presentation of the BCAA oxidation pathway. BCAAs are transaminated by BCAT1 or BCAT2 to generate BCKAs, which are subsequently oxidized by BCKDC. α-KG is the α-keto acid acceptor of the BCAA nitrogen group, and Glu is the product, which is utilized as a substrate for GABA synthesis. BCKDC is phosphorylated and inactivated by BCKDK. BCAA-derived intermediates are trapped in the mitochondria by CoA. The end products of the BCAA catabolic pathway, succinyl CoA and acetyl CoA act as TCA cycle intermediates for ATP production. R-CoA: acyl CoA. (b) Western blot analyses of BCAT1, BCKDK, phosphorylated BCKDHA (P-BCKDHA), and total BCKDHA (T-BCKDHA) as well as their quantified ratios in the hippocampi of Wistar rats (n = 4) and THA rats (n = 4) after avoidance tests. β-actin served as a loading control. (c) Acetyl-CoA and (d) choline concentrations in the hippocampus after the avoidance test (Wistar rats n = 6, THA rats n = 4). (e) ChAT expression in protein extracts from the hippocampus. β-actin served as a loading control. The expression level was determined via densitometric analysis. (f) Hippocampal acetylcholine levels in the above tissues (Wistar rats n = 6, THA rats n = 4). (g) Western blot analyses of phosphorylated 4E-BP1 (P-4E-BP1), total 4E-BP1 (T-4E-BP1), phosphorylated eIF4E (P-eIF4E), total eIF4E (T-eIF4E), phosphorylated S6 (P-S6), and total S6 (T-S6) expression in the above tissues. β-actin served as a loading control. The expression level was determined via densitometric analysis. (h) Western blot analyses of P-BCKDHA, T-BCKDHA, P-4E-BP1, T-4E-BP1, P-eIF4E, T-eIF4E, and ChAT and their quantified ratios in the hippocampi of Wistar rats and THA rats before the avoidance test. (i) Comparison of P-BCKDHA, T-BCKDHA, and ChAT expression before (BF) and after (AF) avoidance tests via western blot analyses of hippocampi from Wister (n = 4) or THA rats (n = 4), respectively. β-actin served as a loading control. The expression level was determined via densitometric analysis. Data are expressed as the mean ± SD values. *P < 0.05; **P < 0.01. Uncropped blots are presented in Supplementary Fig. S6.

Figure 3

Mechanism of blood BCAA maintenance by molecules involved in absorption and oxidation. Mechanism of blood BCAA maintenance by molecules involved in absorption and oxidation. (a) Serum BCAA concentration in Wistar (n = 4) and THA (n = 4) rats after avoidance tests. (b) Bodyweight (Wistar rats n = 4, THA rats n = 5), (c) dietary intake during 24-h period (Wistar rats n = 4, THA rats n = 5), (d) BCAA concentration in the hepatic portal vein, and (e) western blot analysis of small intestine tissue probed with antibodies against B⁰AT1 with β-actin as a loading control (Wistar rats n = 3, THA rats n = 3). A relative ratio comparison is shown. (f) Comparison of indicated tissues between Wistar (n = 4) and THA (n = 4) rats before (BF test) or after (AF test) the avoidance test. Western blot analyses of BCAT2, P-BCKDHA, and T-BCKDHA and their quantified ratios. GAPDH or β-actin was used as a loading control. Data are expressed as the mean ± SD values. *P < 0.05; **P < 0.01. Uncropped blots are presented in Supplementary Fig. S6.

Figure 4

Effects of BCAA-restricted diet on learning ability and metabolism. (a) Schematic representation of the experiments with a BCAA-reduced diet. BCAA100: control (n = 6), BCAA20: one-fifth BCAA content of the control (n = 8). (b) Serum BCAA concentration in BCAA100 and BCAA20 groups after avoidance tests. (c) Comparison of the avoidance rate during the lever-pressing avoidance behavior test in BCAA100 and BCAA20 groups. (d) Hippocampal BCAA concentration in BCAA100 and BCAA20 groups after avoidance tests. (e) Western blot analyses of BCAT1, BCKDK, P-BCKDHA, and T-BCKDHA expression levels and their quantified ratios in the above tissues with β-actin as a loading control. (f) Hippocampal acetylcholine levels in the above tissues. Western blot analyses of (g) ChAT, (h) P-eIF4E, T-eIF4E, P-S6, and T-S6 and their quantified ratios in the above tissues with β-actin as a loading control. Data are expressed as the mean ± SD values. *P < 0.05; **P < 0.01. Uncropped blots are presented in Supplementary Fig. S6.

Figure 5

A schematic diagram explaining the high learning ability of THA rats. High levels of BCAAs in the blood of THA rats are due to increased absorption via the small intestine and suppression of BCAA metabolism in the liver. BCAAs are thus metabolized in the hippocampus and involved in the synthesis of acetylcholine, maintaining the high learning ability phenotype in THA rats.