The PDC (pyruvate dehydrogenase complex) is strongly inhibited by phosphorylation during starvation to conserve substrates for gluconeogenesis. The role of PDHK4 (pyruvate dehydrogenase kinase isoenzyme 4) in regulation of PDC by this mechanism was investigated with PDHK4-/- mice (homozygous PDHK4 knockout mice). Starvation lowers blood glucose more in mice lacking PDHK4 than in wild-type mice. The activity state of PDC (percentage dephosphorylated and active) is greater in kidney, gastrocnemius muscle, diaphragm and heart but not in the liver of starved PDHK4-/- mice. Intermediates of the gluconeogenic pathway are lower in concentration in the liver of starved PDHK4-/- mice, consistent with a lower rate of gluconeogenesis due to a substrate supply limitation. The concentration of gluconeogenic substrates is lower in the blood of starved PDHK4-/- mice, consistent with reduced formation in peripheral tissues. Isolated diaphragms from starved PDHK4-/- mice accumulate less lactate and pyruvate because of a faster rate of pyruvate oxidation and a reduced rate of glycolysis. BCAAs (branched chain amino acids) are higher in the blood in starved PDHK4-/- mice, consistent with lower blood alanine levels and the importance of BCAAs as a source of amino groups for alanine formation. Non-esterified fatty acids are also elevated more in the blood of starved PDHK4-/- mice, consistent with lower rates of fatty acid oxidation due to increased rates of glucose and pyruvate oxidation due to greater PDC activity. Up-regulation of PDHK4 in tissues other than the liver is clearly important during starvation for regulation of PDC activity and glucose homoeostasis.
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.
The branched-chain amino acids (BCAAs) are required for protein synthesis and neurotransmitter synthesis. The branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) is the most important regulatory enzyme in the catabolic pathways of the BCAAs. Activity of the complex is controlled by covalent modification with phosphorylation of its branched-chain alpha-ketoacid dehydrogenase subunits by a specific kinase [branched-chain kinase (BDK)] causing inactivation and dephosphorylation by a specific phosphatase [branched-chain phosphatase (BDP)] causing activation. Tight control of BCKDC activity is important for conserving as well as disposing of BCAAs. Phosphorylation of the complex occurs when there is a need to conserve BCAAs for protein synthesis; dephosphorylation occurs when BCAAs are present in excess. The relative activities of BDK and BDP set the activity state of BCKDC. BDK activity is regulated by alpha-ketoisocaproate inhibition and altered level of expression. Less is known about BDP but a novel mitochondrial phosphatase was identified recently that may contribute to the regulation of BCKDC. Reduced capacity to oxidize BCAAs, as in maple syrup urine disease, results in excess BCAAs in the blood and profound neurological dysfunction and brain damage. In contrast, loss of control of BCAA oxidation results in growth impairment and epileptic-like seizures. These findings emphasize the importance of control of BCAA catabolism for normal neurological function. It is proposed that the safe upper limit of dietary BCAA intake could be established with a BCAA tolerance test and clamp protocol.
A novel phosphatase has been cloned and partially characterized. It has a mitochondrial leader sequence and its amino acid sequence places it in the PP2C family like two known mitochondrial phosphatases. Western blot analysis of subcellular fractions and confocal microscopy of 3T3L1 preadipocytes expressing the GFP-tagged protein confirm its mitochondrial localization. Western blot analysis indicates that the protein is expressed in several mouse tissues, with highest expression in brain, heart, liver, and kidney. The recombinant protein exhibits Mn 2+-dependent phosphoserine phosphatase activity against the branched-chain α-keto acid dehydrogenase complex, suggesting the enzyme may play a role in regulation of branched chain amino acid catabolism. Whether there are other mitochondrial substrates for the enzyme is not known.
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