Abstracts

The branched-chain [alpha]-ketoacid dehydrogenase complex (BCKDC) catalyzes the rate limiting step in the oxidation of branched-chain amino acids (BCAAs), essential amino acids provided by dietary proteins. Activity of the complex is regulated by phosphorylation by a specific kinase (BDK) that causes inactivation and dephosphorylation by a specific phosphatase that causes activation. Lack of BDK expression results in complete activation of BCKDC and a decrease in BCAA[apos]s especially leucine (leu) in the brain. Leu plays a significant role in both glial and neuronal glutamate homeostasis. This homeostasis is regulated in part by transamination with [alpha]-ketoisocaproate (KIC) and glutamate. By facilitating the rapid consumption of glutamate to produce leu, the reamination of KIC by glutamate would in effect [ldquo]buffer[rdquo] the intraneuronal glutamate concentration. We hypothesize that proper regulation of the activity state of BCKDC by its kinase (BDK) is critically important for leu mediated neurotransmitter balance. Mice were starved for 10 hr before the determination of BCAAs concentrations in serum and brain. BCAAs were extracted from whole brain by using perchloric acid. Amino acid analysis was done by ion exchange chromatography with a Beckman 6300 automated amino acid analyzer. Sheep antiserum against purified rat BCKDC was used for the measurement of BCKDC E1a by Western blot in tissue extracts. Tissue wts were determined in 16 wk old mice. Seizures were evoked by handling. Concentrations of BCAAs were significantly reduced in blood (leu: 149 [plusmn] 12 mM vs. 71 [plusmn] 8.4 mM, P[lt] 0.01) and whole brain (leucine: 62 [plusmn] 6 mmol/g wet wt vs. 20 [plusmn] 2 mmol/g wet wt, P [lt] 0.0001). There was a paradoxical increase in expression of BCKDC E1 proteins in some tissues. Body wts of BDK^-/- mice were reduced (male: 25 [plusmn] 1 g vs. 29 [plusmn] 1 g, P[lt] 0.001) and their fur lacked normal luster. Actual wets of brain (448 [plusmn] 9 mg vs. 267 [plusmn] 21 mg, P[lt] 0.001), muscle (254 [plusmn] 6 mg vs. 190 [plusmn] 16 mg, P[lt] 0.05), and adipose tissue (epididimal fat: 501 [plusmn] 39 mg vs. 266 [plusmn] 13 mg, P[lt] 0.001) were decreased. Weights of liver (4.0 [plusmn] 0.1% vs. 4.4 [plusmn] 0.1%, P[lt] 0.05) and kidney (1.29 [plusmn] 0.02% vs. 1.46 [plusmn] 0.03%, P[lt] 0.001) relative to total body wt were increased in BDK^-/- mice. Neurological signs in BDK^-/- mice were hind limb flexion throughout life and tonic/clonic seizures after 6-7 months of age. Disruption of the BDK gene strongly suggests that regulation of BCKDC by phosphorylation is critically important for the conservation of BCAAs. The BDK^-/- mouse may prove useful model for studies on the regulatory role of BCAAs in seizureogenesis.