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The Function of Nuclear Protein 1 in adipogenesis

Tatiana Patricia Mendes Duarte 1,2,3 , Tae-Hyun Kim 1,3, † ,Bo Kyung b c
Yoon 1,2,3 , Yoseob Lee 1,2,3 , Nahee Hwang 1,2,3 , Kyu-Hye Chun 1,2,3 , a
1,3
Jae-Won Kim 1,2,3 ,Mi-Young Kim , Jae-Woo Kim 1,2,3
1 Biochemistry and Molecular Biology , Yonsei University College of Medicine , Seoul
03722, Korea, Brain Korea 21 PLUS Project for Medical Science, Yonsei University , d
2
Seoul 03722, Korea, Chronic Intractable Disease for Systems Medicine Research
3
Center, Yonsei University College of Medicine , Seoul 03722, Korea
† poster presentor
Abstract
Studying the biological networks controlling adipogenesis may facilitate
greater understanding of the pathophysiology of obesity. This study shows
that Nuclear protein 1 is present in the white adipose tissue and increases
its expression levels during adipogenesis. Nuclear protein 1 has been e f
extensively studied in cancer, however, its role in metabolism and
adipogenesis still has to be greatly explored. In early stage of adipogenesis,
when Nuclear protein 1 is knocked down in 3T3-L1 cells, adipogenesis is
shown to be impaired with Chop increase at day 0, which might constitute
a cause for the adipogenesis impairment due to C/EBPb activity inhibition.
In addition, adipogenesis showed to become impaired and, cell death was
also induced in treatment of Nuclear protein 1 inhibitor, ZZW-115. ZZW-
115 treated cells showed to have mitochondria superoxide increased g
presence in relation to the control, and it was correlated with apoptosis,
evidenced by pro-apoptotic BAX and PUMA increased expression in these
cells. Similar results were verified when Nuclear protein 1 was knocked
down at 2 days after induction of differentiation. Taken together, our
study demonstrated that Nuclear protein 1 might have a role in protecting
3T3-L1 cells from Chop expression in early adipogenesis and ROS excessive
production in late adipogenesis.
Result
a b
Np1
Figure 3. Nuclear protein 1 protects mature adipocytes from ROS production
18s
(a) 3T3-L1 cells were treated with ZZW-115, 4 days after induction of differentiation. The cells
were treated with 5uM, and 10uM. Oil red O staining was performed on day 7 of
c C/EBPa C/EBPb differentiation. (b) Nucleus and cytoplasm were extracted and Nuclear protein 1 localization
Np1
was checked via western blot. (c,d) Gene expression of related to mitochondria and autophagy
(e) Confocal microscopy image of MitoSOX Red mitochondrial superoxide indicator staining (f)
Oxygen consumption rate of 3T3-L1 cells control and ZZW-115 treated cells. (g) Electron
microscopic pictures of ZZW-115 treated 3T3-L1 cells. Red arrow (impaired mitochondria),
black arrow (autophagosome).
ADIPOQ PPARg
c
Np1 NRF1
a b
scramble siNp1
Figure 1. Nuclear protein 1 is mainly present in the white adipose tissue
(a) Expression of Nuclear protein 1 in mouse tissue. (b) Microarray data of 3T3-L1 cells supplied
by our team (c) Public data, Mouse Embryonic Fibroblasts (MEFs), adipogenic potential: A
Comprehensive Transcriptome Analysis-GSE152750. scramble siNp1
a b TFB1M TFAM
NP1
PPARg
C/EBPa
scramble siNp1
L32
Figure 4. Nuclear protein 1 depletion causes cell death in late adipogenesis
(a) Nuclear protein 1 knockdown via siRNA. Differentiation of 3T3-L1 cells was induced and 2
c C/EBPa PPARg AP2 days after siNp1 was transfected. Oil red O staining was performed on day 7. (b) Western blot
was performed to verify, BAX, phosphorylated DRP1-S616, Total DRP1 and LC3B. (c) siNp1
treated 3T3-L1 cells and control were harvested on day 5 and 6 and Np1, NRF1, TFAM and
TFB1M mRNA relative expressions were verified.
Conclusion

FASN Np1 CHOP 1. Nuclear protein 1 is present in the white adipose tissue
2. Nuclear protein 1 deficiency causes adipogenesis impairment
3. Nuclear protein 1 deficiency causes Chop increased expression in
early differentiation and consequently early adipogenesis
impairment
4. ZZW-115, Nuclear protein 1 inhibitor, treatment causes
Figure 2. Nuclear protein 1 expression in 3T3-L1 cells diffentiaion and Nuclear protein 1 differentiating adipocyte cell’s mitochondria impairment,
absence causes impairment of early adipogenesis (a) Nuclear protein 1 expression in 3T3-L1 autophagosome formation and, ultimately, cell death
after differentiation’s induction. (b) Oil red O staining of control (left) and knocked down
Nuclear protein 1 3T3-L1 cells (right), on day 6 of differentiation (c) Relative mRNA levels of 5. Nuclear protein 1 knockdown, similar to ZZW-115, causes
C/EBPa, PPARg, AP2, CHOP, FAS and Nuclear protein 1 of 3T3-L1 cells during adipogenesis. mitochondria impairment and cell death