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PEX13 is required for thermogenesis of white adipose tissue

in cold-exposed mice

Woo Yong Park 1, # , Gahee Song 1, # , Ja Yeon Park , Jae-Young Um 1, 3*
1, #
A-35 1 Department of Science in Korean Medicine, Graduate School, Kyung Hee University, Seoul 02447, Korea
2 Department of Pharmacology, College of Korean Medicine, Kyung Hee University, Seoul 02447, Korea
** Presenting Author : Woo Yong Park ¹# , # First Author, * Corresponding Author
1. Highlights Fig. 2. Rosiglitazone induces beige-differentiation and peroxisomal
biogenesis in 3T3-L1 adipocyte
■ PEX13 is upregulated in cold-exposed inguinal WAT and beige-induced adipocytes.
■ PEX13 regulates UCP1 and the subsequent beige development in white adipocytes.
■ PEX13 is required for thermogenesis of white adipocytes.
2. Introduction
In humans, 14 distinct PEX genes have been identified so far. PEX proteins produced by these
genes are involved in a variety of peroxisome biogenesis processes, including membrane
formation and peroxisomal protein import [1]. Peroxins (PEXs) such as PEX11 [2], PEX14
[3], and PEX16 [4] have been shown to be significant in the formation of adipogenesis.
Peroxisomes play an important role in BAT thermogenesis, as previously indicated [5, 6, 7].
Cold exposure also stimulates peroxisome biogenesis and activity [9, 10]. PEX5 [11] and
PEX16 [8] are two PEXs that have been elevated to play a substantial role during NST.
PEX13 has sparked early interest, as was the case with other PEXs, because its absence causes
Zellweger syndrome [12]. The peroxisomal protein translocon, which includes PEX13, PEX14,
and a few additional PEXs, is a transmembrane protein complex via which freshly produced
proteins are transported into the organelle matrix [13]. Despite the fact that PEX13 plays a
critical role in the biogenesis and function of peroxisomes, nothing is known about its role in
NST activation. We investigated whether PEX13 is engaged in the browning of adipocytes in
conjunction with other variables involved in the activation of beige adipocytes in this study.
3. Experimental procedures
3.1. Immunofluorescence (IF) staining Cells and tissues were fixed using 10% formalin and
blocked with 5% BSA for 1 h. After that, the cells and tissues were incubated with the
indicated primary antibodies (1:50 in 5% BSA) overnight at 4 ℃. After washing, the cells and
tissues were incubated with Alexa Flour (AF) 488- or 633-conjugated secondary antibody
(1:1000), and the fluorescence was detected using an EVOSR Cell Imaging systems (Thermo
Scientific, Carlsbad, CA, USA). The intensity of green (AF-488) or red (AF-633) in each
image was quantified by using ImageJ software and visualized by GraphPad Prism version 8
(GraphPad Software, San Diego, CA, USA).
3.2. Animal experiments Male C57BL/6 mice (7-week-old) were purchased from Deahan
Biolink Co. (Eumsung, Korea) and kept for 1 week prior to the experiments, then were
randomly divided to 25°C group (n = 5) or 4°C group (n = 10). The mice were kept at 4 ℃ for
300 min followed by resting at RT for 30 min, as previously described [27]. Following the
300-min-experiment, mice were sacrificed by cervical dislocation under CO asphyxiation.
2
After euthanasia, inguinal white adipose tissue (iWAT), epididymal white adipose tissue
(eWAT) and BAT were collected and placed in -80 ℃ until further use. All animal
experiments were performed according to the Guide for the Care and Use of Laboratory
Animals and were approved by the Institutional Review Board of Kyung Hee University Fig. 3. PEX13 knock-down impairs browning of SVF cells by rosiglitazone.
(confirmation number: KHUASP (SE)-13-012).
3.3. siRNA treatment siRNA (Pex13 Mouse siRNA Oligo Duplex, Locus ID 72129, CAT#
SR413481 and PEX13 Human siRNA Oligo Duplex, Locus ID 5194, CAT#: SR303451)
(OriGene Technologies Inc., Rockville, MD) was applied according to the manufacturer’s
instructions. Final concentration of 10 nM of siRNA was added to the cell culture media 1)
when the cells reached 70% confluence, 2) at Day 0 and 3) at Day 2.
3.4. Statistical analysis Data were expressed as the means ± standard error mean (SEM) of
three or more independent experiments. Statistical differences were calculated by one-way
ANOVA and a subsequent post-hoc one-tailed Mann-Whitney U test using Prism 8 (GraphPad
Software, San Diego, CA, USA). Multiple comparisons versus control group were done by
individual t-tests. Values of p < 0.05 were determined as statistical significance.
4. Results
Fig. 1. Cold stimulation increases peroxisomal biogenesis in WATs of C57BL/6 mice.

5. Conclusion
In this study, we showed the involvement of PEX13 in the development of beige
adipocytes. Cold-stimulated NST activation in mice was associated with the increase of
peroxisomal proteins including PEX13, PEX14 and PEX16 in iWAT but not eWAT. In
three different models of beige adipocyte differentiation, two by PPARγ activation by
rosiglitazone and one by β3AR agonist CL316,243 treatment, we could observe
mitochondrial UCP1 activation was accompanied by these peroxisomal proteins. Moreover,
when PEX13 synthesis was blocked by siPex13 pre-treatment, both in vitro models failed
to successfully differentiate into functional beige adipocytes; thus, the impaired increase in
NST-related markers was shown by either rosiglitazone or CL316,243. Overall, we suggest
PEX13 possesses a role during the browning process of adipocytes and thus contributes to
NST regulation.
6. References
[1] Y. Fujiki, K et al, Front Physiol, 5 (2014) 307. [7] M. Pavelka, H et al, Histochemistry, 50 (1976) 47-55. [13] Y. Liu, J et al, Am J Hum Genet, 65 (1999) 621-634.
[2] M. Shimizu et al, J Biochem, 139 (2006) 563-573. [8] A. Bagattin, L et al,. Proc Natl Acad Sci U S A, 107 (2010) 20376-20381.
[3] S. Zhu, G et al, J Pediatr Endocrinol Metab, 28 (2015) 93-99. [9] M. Pavelka, H et al, Histochemistry, 50 (1976) 47-55.
[4] D.C. Hofer et al, Biochim Biophys Acta Mol Cell Biol Lipids, 1862 (2017) 358-368. [10] H.M. Guardiola-Diaz et al, J Biol Chem, 274 (1999) 23368-23377.
[5] I. Ahlabo, T, J Histochem Cytochem, 19 (1971) 670-675. [11] K. Martens, A et al, Mol Genet Metab, 107 (2012) 735-747.
[6] R. Kramar, M et al, Biochim Biophys Acta, 531 (1978) 353-356. [12] H. Park et al. J Clin Invest, 129 (2019) 694-711.