Impaired growth and neurological abnormalities in branched-chain alpha-keto acid dehydrogenase kinase-deficient mice

Abstract

The BCKDH (branched-chain alpha-keto acid dehydrogenase complex) catalyses the rate-limiting step in the oxidation of BCAAs (branched-chain amino acids). Activity of the complex is regulated by a specific kinase, BDK (BCKDH kinase), which causes inactivation, and a phosphatase, BDP (BCKDH phosphatase), which causes activation. In the present study, the effect of the disruption of the BDK gene on growth and development of mice was investigated. BCKDH activity was much greater in most tissues of BDK-/- mice. This occurred in part because the E1 component of the complex cannot be phosphorylated due to the absence of BDK and also because greater than normal amounts of the E1 component were present in tissues of BDK-/- mice. Lack of control of BCKDH activity resulted in markedly lower blood and tissue levels of the BCAAs in BDK-/- mice. At 12 weeks of age, BDK-/- mice were 15% smaller than wild-type mice and their fur lacked normal lustre. Brain, muscle and adipose tissue weights were reduced, whereas weights of the liver and kidney were greater. Neurological abnormalities were apparent by hind limb flexion throughout life and epileptic seizures after 6-7 months of age. Inhibition of protein synthesis in the brain due to hyperphosphorylation of eIF2alpha (eukaryotic translation initiation factor 2alpha) might contribute to the neurological abnormalities seen in BDK-/- mice. BDK-/- mice show significant improvement in growth and appearance when fed a high protein diet, suggesting that higher amounts of dietary BCAA can partially compensate for increased oxidation in BDK-/- mice. Disruption of the BDK gene establishes that regulation of BCKDH by phosphorylation is critically important for the regulation of oxidative disposal of BCAAs. The phenotype of the BDK-/- mice demonstrates the importance of tight regulation of oxidative disposal of BCAAs for normal growth and neurological function.

Figure 1. Disruption of the BDK gene and identification of BDK^−/− mice

(A) Method used to disrupt the BDK gene by Lexicon Genetics [11]. BTK, Bruton's tyrosine kinase exon; LTR, viral long terminal repeat; NEO, neomycin gene; pA, SV40 polyadenylation sequence; PGK, phosphoglycerate kinase 1 promoter; SA, splice acceptor sequence; SD, splice donor sequence. (B) Genotyping of BDK^−/− mice by PCR analysis using genomic DNA isolated from BDK^+/+, BDK^+/− and BDK^−/− mice. (C) Western blot analyses for BDK using protein extracts prepared from BDK^+/+, BDK^+/− and BDK^−/− mice.

Figure 2. Increase in the amounts of BCKDH E1α subunit in various tissues of BDK^−/− mice

Western blot analysis of E2 and E1α subunits of BCKDH in the liver, brain, heart, kidney and muscle of BDK^+/+ and BDK^−/− mice and quantification of relative amounts of the E1α subunit of BCKDH in various tissues of BDK^−/− (closed bars) mice compared with that of BDK^+/+ (open bars). Extracts from an indicated tissue of all BDK^+/+ and BDK^−/− mice were run on one gel and subjected to Western blot analysis for determination of relative amounts of E1α in that tissue of BDK^−/− mice compared with that of BDK^+/+ mice. Images for each tissue of one BDK^+/+ mouse and one BDK^−/− mice were cropped from the resulting blots to make the composite figure given at the top. Comparisons of relative amounts of the proteins can be made for a given tissue but not across tissues. Results are means±S.E.M., n=3 (*P<0.05).

Figure 3. Alterations in the pattern of the growth curve in BDK^−/− mice

(A) Growth curves of male BDK^+/+ (□) and BDK^−/− (■) mice fed on a chow diet. (B) Growth rates of male BDK^+/+ (□) and BDK^−/− (■) mice fed on a chow diet. (C) Growth curves of male BDK^+/+ (□) and BDK^−/− (■) mice fed on a high protein diet. (D) Growth rates of male BDK^+/+ (□) and BDK^−/− (■) mice fed on a high protein diet. Values are means±S.E.M., n=11 for BDK^+/+ mice fed on a chow diet, n=7 for BDK^−/− mice fed on a chow diet, n=4 for both BDK^+/+ and BDK^−/− mice fed on a high protein diet (*P<0.05).

Figure 4. Differences in the physical appearance of BDK^−/− mice

(A) Appearance of 5-week-old BDK^−/− mice compared with that of 5 week old wild-type mice. Note the splayed hind limbs of the BDK^−/− mice. (B) BDK^−/− mice showing clinching of the hind limbs to the body. (C) BDK^−/− mouse having an epileptic seizure.

Figure 5. Hyperphosphorylation of eIF2α protein in the brains of BDK^−/− mice

Phosphorylation (P-) of eIF2α on the inhibitory Ser⁵¹ is significantly increased whereas the total amount of eIF2α is not changed in the brains of 3-week-old BDK^+/+ (open bars) and BDK^−/− (closed bars) mice. Values are means±S.E.M., n=3 (*P<0.05).