BCAA Catabolism-Gill 18Mar2016-Final 1
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Transcript of BCAA Catabolism-Gill 18Mar2016-Final 1
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