Controlled cortical impact injury and craniotomy result in divergent alterations of pyruvate metabolizing enzymes in rat brain ☆ Guoqiang Xing a, b, d, ⁎ , 1 , Ming Ren a, 1 , J. Timothy O'Neill c , Ajay Verma a , William D. Watson a a Department of Neurology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA b Department of Psychiatry, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA c Department of Pediatrics, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA d Friends Research Institute, Baltimore, MD 21201, USA abstract article info Article history: Received 13 October 2011 Accepted 4 December 2011 Available online 14 December 2011 Keywords: Head injury Pyruvate dehydrogenase Kinase Phosphatase mRNA Protein In situ hybridization Real-time PCR Western blotting Rat Dysregulated glucose metabolism and energy deficit is a characteristic of severe traumatic brain injury (TBI) but its mechanism remains to be fully elucidated. Phosphorylation of pyruvate dehydrogenase (PDH) is the rate-limiting mitochondria enzyme reaction coupling glycolysis to the tricarboxylic acid cycle. Phosphoryla- tion of PDH E1α1 subunit catalyzed by PDH kinase (PDK) inhibits PDH activity, effectively decoupling aerobic glycolysis whereas dephosphorylation of phosphorylated PDHE1α1 by PDH phosphatase (PDP) restores PDH activity. We recently reported altered expression and phosphorylation of pyruvate dehydrogenase (PDH) fol- lowing TBI. However, little is known about PDK and PDP involvement. We determined PDK (PDK1–4) and PDP isoenzyme (PDP1–2) mRNA and protein expression in rat brain using immunohistochemistry and in situ hy- bridization techniques. We also quantified PDK and PDP mRNA and protein levels in rat brain following TBI using quantitative real-time PCR and Western blot, respectively. Controlled cortical impact-induced TBI (CCI-TBI) and craniotomy significantly enhanced PDK1-2 isoenzyme mRNA expression level but significantly suppressed PDP1 and PDK4 mRNA expression after the injury (4 h to 7 days). CCI-TBI and craniotomy also significantly increased PDK1–4 isoenzyme protein expression but suppressed PDP1–2 protein expression in rat brain. In summary, the divergent changes between PDK and PDP expression indicate imbalance between PDK and PDP activities that would favor increased PDHE1α1 phosphorylation and enzyme inhibition contributing to impaired oxidative glu- cose metabolism in TBI as well as craniotomy. © 2011 Elsevier Inc. All rights reserved. Introduction Diffuse dysregulated glucose metabolism and energy failure is a metabolic characteristic and indicator of poor prognosis for patients with severe traumatic brain injury (TBI) (Hattori et al., 2003; Hillered et al., 2006; Lifshitz et al., 2003; Prins et al., 2004; Verweij et al., 2000; Vespa et al., 2005; Yoshino et al., 1991). Despite decades of research, the mechanism responsible remains poorly understood. Recently stud- ies suggest an important role of pyruvate dehydrogenase (PDH) in the altered brain energy metabolism following TBI (Opii et al., 2007; Robertson et al., 2007; Roche et al., 2001)(Xing et al., 2009). Pyruvate dehydrogenase complex (PDC) is the rate-limiting mito- chondrial enzyme catalyzing the irreversible oxidative decarboxylation of pyruvate to acetyl-CoA that links the utilization of glucose and lactate with the mitochondrial tricarboxylic acid cycle (TCA) to meet the constant energy demands of neural cells under physiological conditions (Patel and Korotchkina, 2006). PDC is a large complex containing multi- ple copies of each of three enzymes: PDH (E1), dihydrolipolyl transace- tylase (E2), and dihydrolipolyl dehydrogenase (E3). PDC activity is regulated by multiple factors including the concentrations of Ca 2+ and Mg 2+ , ATP/ADP ratio, and the phosphorylation state of PDH E1α1 sub- unit (PDHE1α1) (Patel and Korotchkina, 2006). Phosphorylation of any one of the 3 serine residues (S1, S2, and S3) of PDHE1α1 catalyzed by pyruvate dehydrogenase kinase (PDK) inhibits PDH activity, whereas dephosphorylation of the three serine residues catalyzed by pyruvate dehydrogenase phosphatase (PDP) activates PDH (Roche and Hiromasa, 2007). So far four PDK isoenzymes (PDK1–4) and two PDP isoenzymes (PDP1 and PDP2) have been reported in peripheral tissues. However, their CNS distribution and potential role in brain energy metabolism in traumatic brain injury remains unknown. We hypothesized that the tightly-regulated balance between PDK and PDP isoenzyme expression is disrupted following TBI which may, in turn, im- pair or shutdown PDH-regulated glucose metabolism. In this study, we examined the expression of PDK and PDP mRNA and protein levels in the brains of control rats and rats following con- trolled cortical impact (CCI)-induced TBI and craniotomy (sham CCI) Experimental Neurology 234 (2012) 31–38 ☆ Funding organizations: This work was supported, in part, by GEMI Fund and Henry Jackson Foundation TRP grant. ⁎ Corresponding author. E-mail address: gxing99@yahoo.com (G. Xing). 1 These authors contributed equally to this work. 0014-4886/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2011.12.007 Contents lists available at SciVerse ScienceDirect Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr