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. 2024 Feb 9;14(4):585.
doi: 10.3390/ani14040585.

NOTCH1 as a Negative Regulator of Avian Adipocyte Differentiation: Implications for Fat Deposition

Zheng Wang  1 , Yue Su  1 , Mingyu Zhao  1 , Zhenhua Ma  1 , Jianhui Li  2 , Zhuocheng Hou  3 , Huifeng Li  1
Affiliations

NOTCH1 as a Negative Regulator of Avian Adipocyte Differentiation: Implications for Fat Deposition

Zheng Wang et al. Animals (Basel). .

Abstract

The NOTCH signaling pathway plays a pivotal role in diverse developmental processes, including cell proliferation and differentiation. In this study, we investigated whether this signaling molecules also contribute to avian adipogenesis. Using previous mRNA-seq datasets, we examined the expression of 11 signaling members during avian adipocyte differentiation. We found most members are down-regulated throughout differentiation (p < 0.05). As a representative, NOTCH1 was decreased in cultured chicken abdominal adipocytes during adipogenesis at mRNA and protein levels (p < 0.05). Moreover, using an overexpression plasmid for NOTCH1's intracellular domain (NICD1), as well as siRNA and DAPT to activate or deplete NOTCH1 in cells, we investigated the role of NOTCH1 in avian adipogenesis. Our findings illuminate that NOTCH1 activates the expression of HES1 and SOCS3 while it decreases NR2F2 and NUMB (p < 0.05), as well as inhibits oleic acid-induced adipocyte differentiation (p < 0.01). We further demonstrate that HES1, a downstream transcription factor activated by NOTCH1, also significantly inhibits adipogenesis by suppressing PPARγ and C/EBPα (p < 0.01). Collectively, these findings establish NOTCH1 as a negative regulator of avian adipocyte differentiation, unveiling NOTCH signaling as a potential target for regulating avian fat deposition.

Keywords: HES1; NOTCH1; adipocyte differentiation; avian; metabolic regulation.

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Conflict of interest statement

The research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Figures

Figure 1
Figure 1
The expression changes in NOTCH signaling during avian adipocyte differentiation. (A) Heatmap analysis of expression patterns for NOTCH signaling–related genes identified with mRNA–seq analysis of gene expression data from chicken abdominal adipocytes at 0 h, 72 h, 120 h, 168 h post induction (n = 4, Raw in Z–Score). (B) Heatmap analysis of expression patterns for NOTCH signaling–related genes identified with mRNA–seq analysis of gene expression data from Pekin duck subcutaneous adipocytes at 0 h, 24 h, 48 h, 72 h post induction (n = 4, Raw in Z–Score). (C) Comparison between mRNA–seq and RT–qPCR expression measurements for the chosen genes. The graph illustrates the log2 fold changes for both mRNA–seq and RT–qPCR data.
Figure 2
Figure 2
NOTCH1 is inhibited during adipocyte differentiation. (A) ICP1 cells were induced to differentiate with 160 μM oleic acid and total RNA was extracted at specified time points (0 h corresponds to the time of oleic acid supplementation). Subsequently, RT−qPCR was conducted to assess NOTCH1 expression throughout adipocyte differentiation (n = 3). The mRNA expression was normalized to GAPDH mRNA, and the results are presented relative to the mRNA level of NOTCH1 at 0 h, which is set as 1. (B) Western blot analysis of protein abundance of NOTCH1 during the ICP1 cells differentiation; GAPDH was utilized as an internal control. The protein abundance of NOTCH1 and GAPDH were quantitatively compared (n = 3). The data are presented as mean ± SD, and statistical significance was determined through one−way ANOVA. * p < 0.05; ** p < 0.01.
Figure 3
Figure 3
Effect of NOTCH1 on signaling−related genes in ICP1 cells. (A) Western blot analysis of protein abundance of NOTCH1, NUMB, SOCS3, and GAPDH in NICD1 overexpression ICP1 cells and control cells; GAPDH is an internal control. The protein abundance of NOTCH1, NUMB, SOCS3, and GAPDH was quantitatively compared (n = 3). (B) The relative mRNA expression of NOTCH1, HES1, HEY1, HEY2, NR2F2, NUMB, and SOCS3 in NICD1 overexpression ICP1 cells and control cells was determined using RT−qPCR; GAPDH served as an internal control (n = 3). (C) Western blot analysis of protein abundance of NOTCH1, NUMB, SOCS3, and GAPDH in NOTCH1 knockdown ICP1 cells and control cells. The abundance of each protein was quantitatively compared (n = 3). (D) Relative mRNA expression of NOTCH1, HES1, HEY1, HEY2, NR2F2, NUMB, and SOCS3 in NOTCH1 knockdown ICP1 cells and control cells was determined using RT−qPCR (n = 3). Data are shown as mean ± SD, and significance was determined using Student’s t−tests. * p < 0.05; ** p < 0.01.
Figure 4
Figure 4
NOTCH1 inhibits fat accumulation in differentiating adipocyte. (A) Cultured ICP1 cells expressing pcDNA3.1 or pcDNA–NICD1 was induced to differentiate into adipocytes with 160 μM oleic acid. At the end of induction (72 h), cells were subjected to Oil Red O staining to evaluate lipid accumulation. All micrographs were captured at the same magnification, and a 20 μm scale bar is provided in the lower right panel for reference. (B) Relative mRNA expression of mature adipocyte marker genes in NICD1 overexpression ICP1 cells and control cells was determined using RT–qPCR; GAPDH served as an internal control (n = 3). (C) Representative images of NOTCH1 knockdown promoted the lipid accumulation by ORO staining on 72 h post induction. (D) Relative mRNA expression of mature adipocyte marker genes in NOTCH1 knockdown ICP1 cells and control cells was determined using RT–qPCR (n = 3). Data are shown as mean ± SD and significance was determined using Student’s t–tests. * p < 0.05; ** p < 0.01.
Figure 5
Figure 5
DAPT inhibits NOTCH1 and HES1 and promotes adipogenic differentiation. (A) Western blot analysis of protein abundance of NICD1 (nuclear), LMNB1 (nuclear), NOTCH1 (cytoplasm), and GAPDH (cytoplasm) in ICP1 cells treated with various concentrations of DAPT for 48 h; LMNB1 and GAPDH as internal control of nuclear protein and cytoplasmic protein, respectively (n = 3). (B) The protein abundance of NICD1 (nuclear) and LMNB1 (nuclear) was quantitatively compared (n = 3). (C) The protein abundance of NOTCH1 (cytoplasm) and GAPDH (cytoplasm) was quantitatively compared (n = 3). (D) The mRNA level of HES1 in ICP1 cells treated with various concentrations of DAPT for 48 h was determined using RT-qPCR (n = 3). (E) Representative images of 5 μM DAPT promoted the lipid droplet formation in ICP1 cells by ORO staining on 72 h post induction. All micrographs are the same magnification, and a 20 μm scale bar is provided in the lower right panel for reference. (F) Relative mRNA expression of mature adipocyte marker genes in 5 μM DAPT treated with ICP1 cells and alone DMSO treated with cells on 72 h post induction was determined using RT–qPCR (n = 3). Data are shown as mean ± SD and significance was determined using One–way ANOVA or Student’s t–tests. * p < 0.05; ** p < 0.01.
Figure 6
Figure 6
HES1 overexpression inhibits PPARγ and C/EBPα and promotes adipogenic differentiation. (A) Cultured ICP1 cells expressing pcDNA3.1 or pcDNA–HES1 was induced to differentiate into adipocytes with 160 μM oleic acid. At the end of differentiation (72 h), cells were subjected to Oil Red O staining to evaluate lipid accumulation. All micrographs were captured at the same magnification, and a 20 μm scale bar is provided in the lower right panel for reference. (B) Relative mRNA expression of HES1 and mature adipocyte marker genes in HES1 overexpression ICP1 cells and control cells was determined using RT–qPCR; GAPDH served as an internal control (n = 3). (C) Predicted binding sites and putative binding sequence for the HES1 transcription factor in the PPARγ and C/EBPα promoter as identified in the JASPAR database. (D) The cells were co–transfected with pGL3–PPARγ–WT or pGL3–PPARγ–MUT and pcDNA3.1–HES1 or pcDNA3.1, and pRL–TK plasmid (24:24:1). After 72 h of co–transfection, relative luciferase activity was measured. The effect of HES1 overexpression on the luciferase activity of pGL3–PPARγ–WT and pGL3–PPARγ–MUT in ICP1 cells (n = 3). (E) The effect of HES1 overexpression on the luciferase activity of wild type reporter pGL3–C/EBPα–WT and mutation reporter pGL3–C/EBPα–MUT in ICP1 cells (n = 3). Data are shown as mean ± SD and significance was determined using Student’s t–tests. * p < 0.05; ** p < 0.01.
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References

    1. Baeza E., Guillier L., Petracci M. Review: Production factors affecting poultry carcass and meat quality attributes. Animal. 2018;16((Suppl. 1)):100331. doi: 10.1016/j.animal.2021.100331. - DOI - PubMed
    1. Mathew H., Castracane V.D., Mantzoros C. Adipose tissue and reproductive health. Metabolism. 2018;86:18–32. doi: 10.1016/j.metabol.2017.11.006. - DOI - PubMed
    1. Wang G., Kim W.K., Cline M.A., Gilbert E.R. Factors affecting adipose tissue development in chickens: A review. Poult. Sci. 2017;96:3687–3699. doi: 10.3382/ps/pex184. - DOI - PubMed
    1. Ghaben A.L., Scherer P.E. Adipogenesis and metabolic health. Nat. Rev. Mol. Cell Biol. 2019;20:242–258. doi: 10.1038/s41580-018-0093-z. - DOI - PubMed
    1. Schwalie P.C., Dong H., Zachara M., Russeil J., Alpern D., Akchiche N., Caprara C., Sun W., Schlaudraff K.U., Soldati G., et al. A stromal cell population that inhibits adipogenesis in mammalian fat depots. Nature. 2018;559:103–108. doi: 10.1038/s41586-018-0226-8. - DOI - PubMed
-