The Mutation Effect of PRDM16
To determine the biochemical function of the substitution inPRDM16 , 3T3-L1 cells (preadipocyte cell line) that ectopically
expressed cattle PRDM16 and PRDM16 MU (L779P mutation ofPRDM16 ) coding sequences were generated and induced to
differentiate towards beige adipocytes (Additional file 1: Fig.
S8 ). The overexpression efficiency was kept at equivalent levels
(Fig. 3a ). After full differentiation, no differences in
morphological characteristics between the PRDM16 and PRDM16 MU groups
was observed
(Additional
file 1: Fig. S9 ). In addition, no significant differences in the mRNA
and protein expression levels of PPARγ, a key adipogenesis-regulating
gene (Fig. 3b and 3c ). Notably, the differentiation
efficiency was lower for the control group (cells infected with an empty
vector) than the two other groups, supporting the idea that a lack of
PRDM16 significantly impeded the differentiation of brown adipocytes,
and the overexpression of PRDM16 would significantly increase the number
of brown adipocytes [17]. Despite the similar efficiency
in differentiation between the two ectopic PRDM16 overexpressing
groups, the mRNA expression levels of four BAT-selective genes
(UCP1 , C/EBPβ , PGC1-α and CIDEΑ ) were
significantly lower in the PRDM16 MU group than in the PRDM16 group
(Fig. 3d ). Moreover, overexpression of PRDM16 MU largely
increased UCP1 expression, which was far inferior to PRDM16
overexpression (Fig. 3d , 3e ). The mRNA levels of other
BAT-selective genes
(C/EBPβ ,PGC1-α and CIDEΑ ) were significantly lower in PRDM16
MU-overexpressing adipocytes than in PRDM16 -overexpressing
adipocytes (Fig. 3d ). The above results indicate that L779P
mutation significantly impaired the normal function
of PRDM16 in the formation of
brown adipocytes in southern cattle, which live in a warmer area
relative to northern cattle.