I would like to thank Dr. Giban Ray for technical assistance and members of Kienesberger and Pulinilkunnil lab for all the support in this project. Abnormalities in BCAA metabolism generate toxic metabolic intermediates 1 . However the contribution of different tissue explaining the maladaptation of BCAA metabolism is unknown primarily because limited information is available on tissue specific expression of BCAA catabolizing enzymes. INTRODUCTION Tissue distribution of BCAA catabolizing enzymes in C57BL6J mice and their expression profile in muscle differentiation a University of New Brunswick, Saint John, New Brunswick, Canada, b Biochemistry and Molecular Biology, Dalhousie Medicine New Brunswick, Dalhousie University, Saint John, New Brunswick, Canada Gill Kazevman a , Andrew M. Cowie b , Kenneth D’Souza b , Petra C. Kienesberger a,b , PhD, Thomas Pulinilkunnil a,b , PhD RESEARCH OBJECTIVES- METHODS CONCLUSIONS FUTURE DIRECTIONS REFERENCES ACKNOWLEDGEMENTS Tissue specific expression of BCAA catabolizing enzymes Muscle cell differentiation, from Myoblasts to Myotubes, affects the distribution of BCAA catabolizing enzymes • BCAA catabolizing enzymes exhibit tissue specific expression depending on the type of substrate that is metabolically preferred by the tissue. • Liver, soleus and BAT had the highest BCKDH expression whereas brain showed lowest BCKDH content. Decreased BCKDH expression in brain corresponded with increased inhibitory phosphorylation of BCKDH • BCKDH phosphorylation decreased over the time course of myoblast differentiation to myotubes suggesting that BCAA oxidation is strongly associated with muscle differentiation Our objectives were; 1) To determine the tissue specific expression pattern of BCAA catabolizing enzymes across 10 different tissues and 2) To examine the regulation of BCAA catabolism in mouse myotube differentiation. 1. Courtney R Green, Martina Wallace, Ajit S Divakaruni, Susan A Phillips, Anne N Murphy, Theodore P Ciaraldi, Christian M Metallo. Branched-chain amino acid catabolism fuels adipocyte differentiation and lipogenesis. Nature Chemical Biology, 2015. 0 Hours 16 Hours 48 Hours 72 Hours RESULTS Figure 4. Adenoviral overexpression and silencing of BCKDH and BCKDK in differentiated C2C12 cells to ascertain the role of BCAA metabolizing enzymes in muscle differentiation and function. 96 Hours Figure 2. C57BI/6 mice were euthanized and ten different tissues were isolated for immunoblot analysis. B) BCKDK expression is high in brown adipose tissue. C) Phosphatase PPM1K expression is high brown adipose tissue. D) BCKDH expression is high in oxidative tissue such as soleus and brown adipose tissue compared to glycolytic tissue (extensor digitorum longus muscle and subcutaneous adipose tissue). E) Inactivated (phosphorylated) BCKDH was high in brain and low in liver. A B C D E Figure 3. C2C12 mouse myoblasts were differentiated in 0.2 % fetal bovine serum. A) Microscopic image depicting differentiation process of myotubes from myoblasts over 0, 16, 48, 72, 96 hours with media renewals every 48 hr. B) Immunoblot analysis of BCAA catabolizing enzymes in whole cell lysates collected over the course of differentiation. C) Myogenin expression increase at 36 hr and remained high till 72 hr, indicating completion of differentiation, D) Phosphorylation of BCKDH decreased with time, E) while BCKDK remains constant with time. F) Interestingly, mTOR is more active indicating possible activation of protein synthesis. A B C The Branched-chain amino acids (BCAAs) leucine, iso-leucine, and valine are essential amino acids which are acquired from the diet. These amino acids are not only energy substrates but also driver of protein synthesis and cellular growth. BCAAs are intracellularly catabolized by the concerted action of branched chain aminotransferase (BCAT2) resulting in the formation of branched chain keto-acids (BCKAs) that are further catabolized by branched chain keto-acid dehydrogenase (BCKDH) to generate intra- mitochondrial derivatives of acetyl CoA. BCKDH’s activity is inhibited by BCKDH kinase (BCKDK) phosphorylation of Serine 293 and this site is dephosphorylated by BCKDH phosphatase (BCKDHP also called PPM1K). Figure 1. BCAA catabolism pathway D E F * vs 0 hours P ≤ 0.05 ** vs 0 hours P ≤ 0.01 *** vs 0 hours P ≤ 0.001 **** vs 0 hours P ≤ 0.0001