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 intermediates1.

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

aUniversity of New Brunswick, Saint John, New Brunswick, Canada, bBiochemistry and Molecular Biology, Dalhousie Medicine New Brunswick, Dalhousie University, Saint John, New Brunswick, Canada

Gill Kazevmana, Andrew M. Cowieb, Kenneth D’Souzab, Petra C. Kienesbergera,b, PhD, Thomas Pulinilkunnila,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