Liver BCATm transgenic mouse model reveals the important role of the liver in maintaining BCAA homeostasis

Abstract

Unlike other amino acids, the branched-chain amino acids (BCAAs) largely bypass first-pass liver degradation due to a lack of hepatocyte expression of the mitochondrial branched-chain aminotransferase (BCATm). This sets up interorgan shuttling of BCAAs and liver-skeletal muscle cooperation in BCAA catabolism. To explore whether complete liver catabolism of BCAAs may impact BCAA shuttling in peripheral tissues, the BCATm gene was stably introduced into mouse liver. Two transgenic mouse lines with low and high hepatocyte expression of the BCATm transgene (LivTg-LE and LivTg-HE) were created and used to measure liver and plasma amino acid concentrations and determine whether the first two BCAA enzymatic steps in liver, skeletal muscle, heart and kidney were impacted. Expression of the hepatic BCATm transgene lowered the concentrations of hepatic BCAAs while enhancing the concentrations of some nonessential amino acids. Extrahepatic BCAA metabolic enzymes and plasma amino acids were largely unaffected, and no growth rate or body composition differences were observed in the transgenic animals as compared to wild-type mice. Feeding the transgenic animals a high-fat diet did not reverse the effect of the BCATm transgene on the hepatic BCAA catabolism, nor did the high-fat diet cause elevation in plasma BCAAs. However, the high-fat-diet-fed BCATm transgenic animals experienced attenuation in the mammalian target of rapamycin (mTOR) pathway in the liver and had impaired blood glucose tolerance. These results suggest that complete liver BCAA metabolism influences the regulation of glucose utilization during diet-induced obesity.

Keywords: Amino acids; BCAA metabolism; BCATm; High-fat diet; Liver transgenic mouse.

Fig. 1

Tissue expression of BCATm in mice with low and high expression of the BCATm transgene. (A) Genomic PCR analysis of Bcat2 gene, which encodes BCATm, from ear DNA of either LivTg-LE (Bcat2-LE) or LivTg-HE (Bcat2-HE) mice. The transgene allele is about 560 bp, Vldlr was used as a loading control, while WT did not produce Bcat2. (B) Liver BCATm activity (U/mg = 1 µmol Val formed/min/mg protein at 37oC) in WT (white bar), LivTg-LE (gray bar) and LivTg-HE (black bar) mice was measured as described in Methods. (C–F) Western blotting of total BCATm protein (transgenic plus endogenous BCATm) from liver (C), kidney (D), muscle (E), and heart (F) tissues of WT, LivTg-LE, and LivTg-HE mice consuming a standard rodent chow. Pan-actin or β-tubulin was used as a loading control. Data represent mean ± SEM, n=6–12, mixed gender (A, B) or male mice (C–F), average age 30 weeks, ^aP≤ 0.05 as compared to WT; ^bP≤ 0.05 as compared to LivTg-LE.

Fig. 2

Expression of liver BCATm results in activation of hepatic but not extrahepatic BCKDC (E1α). E1α and P-E1α were determined by Western Blotting in liver (A), kidney (B), muscle (C), and heart (D) tissues of WT (white bar), LivTg-LE (gray bar), and LivTg-HE (black bar) mice consuming a standard rodent chow. Each graph shows the relative ratio between the phosphorylated and total form of E1α. Data represent mean ± SEM, n=6 male mice, average age 30 weeks. *P≤ 0.05 as compared to WT.

Fig. 3

BCKDC (E2) is not altered in the transgenic animals when fed a standard rodent chow. E2 was determined by Western Blotting in liver (A), kidney (B), muscle (C), and heart (D) tissues of WT (white bar), LivTg-LE (gray bar), and LivTg-HE (black bar) mice consuming a standard rodent chow. Each graph shows the relative E2 band intensity normalized to pan-actin. Data represent mean ± SEM, n=6 male mice, average age = 30 weeks.

Fig. 4

Expression of hepatic BCATm does not alter the growth rate or organ weights of the transgenic mice. (A,B) Growth curve (A) and organ weights (B) of WT (white circle/bar), LivTg-LE (gray circle/ bar), and LivTg-HE (black circle/ bar) consuming a standard rodent chow diet. (C,D) Growth curve (C) and organ weights (D) of WT (white circle/bar), and LivTg-HE (black circle/ bar) consuming a high fat diet (HFD). Data represent mean ± SEM, n=8–16 (A,B) and n=7–8 (C,D). Weekly weights were measured starting at 6 weeks, however the organ weights were measured at week 29 for mice on standard rodent chow diet and at week 24 for mice on HFD. Organ weights of LivTg-HE mice were expressed as a percent of WT organ weights. Organ weight abbreviations: epididymal fat (EF), gastrocnemius/soleus muscle (G/S), heart (H), kidney (K), and liver (L).

Fig. 5

Expression of BCATm raises BCKDC activity in the mouse liver under high fat diet feeding. BCATm (A,D,G), E1α and P-E1α (B,E,H), and E2 (C,F,I) were determined by Western Blotting in liver (A,B,C), muscle (D,E,F), and heart (G,H,I) tissues of WT (white bar) and LivTg-HE (black bar) consuming a high fat diet (HFD). For E1α, each graph shows the relative ratio between the phosphorylated and total E1α protein. For BCATm and E2, each graph shows the relative band intensity normalized to pan-actin. Data represent mean ± SEM, n=6 male mice, age 24 weeks. *P≤ 0.05 as compared to WT.

Fig. 6

High fat diet feeding results in higher blood glucose and impaired glucose tolerance in LivTg-HE mice compared to WT mice fed the same diet. (A, B) GTT. Blood glucose of WT and LivTg-HE mice was measured at 0 weeks (A) or after 14 weeks of a high fat diet feeding (HFD) (B). Data represent mean ± SEM, n=6–12, male mice, age 20 weeks, *P≤ 0.05 as compared to WT-HFD.

Fig. 7

Phosphorylation of S6K and 4E-BP1 is lower in the liver of LivTg-HE mice fed a high fat diet (HFD) compared to WT mice fed the same diet. P-S6K, S6K, P-4E-BP1, and 4E-BP1 were determined by Western Blotting in liver, muscle, and heart of Leu-gavaged WT-HFD and LivTg-HE-HFD mice. Animals were sacrificed 30 min after Leu gavage. Each graph shows the relative ratio between the phosphorylated and total form of S6K (A) and 4E-BP1(B). Data represent mean ± SEM, n=5–6 male mice, age 24 weeks. *P≤ 0.05 as compared to WT-HFD.