Scientia Agricultura Sinica ›› 2025, Vol. 58 ›› Issue (8): 1638-1649.doi: 10.3864/j.issn.0578-1752.2025.08.014

• ANIMAL SCIENCE·VETERINARY SCIENCE • Previous Articles     Next Articles

Mechanistic Study of SCD5 Regulation of Lipid Droplet Quantity via TRIM15

BAI WenZhe1,2(), LI JiHao1, FANG QianHai1,2, ZHANG Fan1,2, HU RuiQi1, CHEN HongBo2, BI YanZhen1(), WANG Rui2()   

  1. 1 Key Laboratory of Animal Embryo Engineering and Molecular Breeding in Hubei Province, Institute of Animal Husbandry and Veterinary Medicine, Hubei Academy of Agricultural Sciences, Wuhan 430064
    2 Hubei Provincial Livestock Seed Industry Technology Innovation Center, Schoool of Animal Science and Nutritional Engineering, Wuhan Polytechnic University, Wuhan 430023
  • Received:2024-10-15 Accepted:2025-02-28 Online:2025-04-16 Published:2025-04-21
  • Contact: BI YanZhen, WANG Rui

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Abstract:

【Background】 Fat deposition plays a critical role in determining pork quality traits such as flavor, tenderness, and color. Stearoyl-CoA desaturase (SCD) is a key enzyme involved in the regulation of lipid metabolism. Among its family members, stearoyl-CoA desaturase 5 (SCD5) functions as a rate-limiting enzyme for the synthesis of monounsaturated fatty acids (MUFAs), thereby influencing fatty acid composition and triacylglycerol (TAG) content. However, the specific role of SCD5 in the biogenesis of lipid droplets (LD) remains unclear. Tripartite motif-containing 15 (TRIM15), a member of the tripartite motif protein family, has been shown to affect LD biogenesis by modulating TAG metabolism. Notably, the expression of TRIM15 is significantly downregulated upon SCD5 deletion, suggesting a potential regulatory relationship between the two. Nevertheless, the exact mechanism of their interaction during LD formation remains to be elucidated.【Objective】This study aims to investigate the regulatory role of SCD5 in LD biogenesis and to determine whether SCD5 mediates LD formation and accumulation through TRIM15. The goal is to further elucidate the molecular mechanisms by which SCD5 regulates lipid metabolism, thereby providing a theoretical basis for the genetic improvement of pork quality traits.【Method】In this study, we utilized CRISPR/Cas9 technology to construct an SCD5 knockout cell line from porcine kidney cells (PK-15). We also established an SCD5-overexpressing PK-15 cell line by transfecting an SCD5 overexpression vector and overexpressed SCD5 in mouse adult myoblasts (C2C12). Lipid droplet content was analyzed via flow cytometry with Bodipy staining, and the number of lipid droplets was assessed using Bodipy staining, confocal microscopy, and oil red O staining to study SCD5's role in lipid droplet biogenesis. RNA-seq analysis of PK-15 SCD5-deficient cells showed significant downregulation of TRIM15. The expression of TRIM15 was confirmed by RT-qPCR and Western blot. To further investigate the roles of SCD5 and TRIM15 in lipid droplet biogenesis, a TRIM15 overexpression vector was constructed and introduced into PK-15 SCD5-deficient cells, exploring the role of SCD5 and TRIM15 in LD biogenesis.【Result】Flow cytometry analysis with Bodipy staining demonstrated that SCD5 deletion significantly reduced lipid droplet content, whereas SCD5 overexpression significantly increased it in PK-15 cells. Similar results were observed with SCD5 overexpression in C2C12 cells. Confocal imaging with Bodipy staining and oil red O staining revealed that SCD5 deletion significantly reduced the number of lipid droplets (LDs) in PK-15 cells. Overexpression of SCD5 in C2C12 cells produced similar results. RNA-seq analysis of PK-15 SCD5-deficient cells, combined with GO and KEGG enrichment analyses, indicated that SCD5 is involved in multicellular development and lipid metabolism pathways, suggesting its regulatory role in lipid metabolism. Gene analysis related to lipid metabolism identified TRIM15 as the most significantly downregulated gene, suggesting that SCD5 may regulate lipid metabolism via TRIM15. Overexpression of TRIM15 in SCD5-deficient PK-15 cells was assessed using Bodipy-stained flow cytometry, confocal imaging, and oil red O staining. The results showed that TRIM15 overexpression restored lipid droplet content and significantly increased the number of LDs in SCD5-deficient cells.【Conclusion】In summary, this study demonstrated that SCD5 mediates LD biogenesis and regulates LD quantity by modulating TRIM15 expression, thereby controlling fat deposition. These findings provide theoretical support for improving pork quality.

Key words: SCD5, TRIM15, Lipid droplets, Lipid metabolism

Table 1

Information of primers"

基因
Gene		 引物序列
Primer sequences (5′ to 3′)		 产物大小
Product size (bp)		 Tm值
Tm
value
SCD5-F CTGCTCTGGGCCTACTTCTG 195 60
SCD5-R CCTGGACACTCGAAGATGT
GAPDH-F TCGGAGTGAACGGATTTGGC 189 60
GAPDH-R TGACAAGCTTCCCGTTCTCC
TRIM15-F GGCATTCTCAGAAAACCTGGTG 69 60
TRIM15-R GGTCAGAGGGTCCAGAGTGA

Fig. 1

SCD5 deletion down-regulates the number of lipid droplets A: PK-15 SCD5-/- monoclonal cells SCD5 mRNA expression level; B: PK-15 SCD5-/- monoclonal cells SCD5 protein expression level; C: Fluorescence intensity detected by flow cytometry (the number of cells detected was 20,000); D: Laser confocal imaging (Bodipy staining); E: Image J analysis of the number of lipid droplets in Figure D; F: Oil red O staining; **: P<0.01"

Fig. 2

SCD5 overexpression upregulates the number of lipid droplets A: SCD5 mRNA expression level in PK-15 SCD5 overexpressing monoclonal cells; B: SCD5 protein expression level in PK-15 SCD5 overexpressing monoclonal cells; C: Fluorescence intensity detected by flow cytometry (the number of cells detected was 20,000); D: Laser confocal imaging (Bodipy staining); E: ImageJ analysis of fluorescence quantity in Figure D; F: Oil red O staining; **: P<0.01"

Fig. 3

Ectopic expression of SCD5 in C2C12 upregulates the number of lipid droplets A: SCD5 mRNA expression level in C2C12; B: Overexpression of SCD5 protein expression level in C2C12; C: Fluorescence intensity detected by flow cytometry (the number of cells detected was 20,000); D: Laser confocal imaging (Bodipy staining); E: ImageJ analysis of the number of lipid droplet in Figure D；**: P<0.01"

Fig. 4

SCD5 deletion downregulates TRIM15 expression A: Histogram of GO function analysis; B: Dot plot of KEGG pathway enrichment analysis; C: Heat map of genes related to lipid metabolism; D: TRIM15 mRNA expression level in SCD5-/- cell lines; E: TRIM15 protein expression level in SCD5-/- cell lines; *: P<0.05"

Fig. 5

SCD5 regulates the number of lipid droplets through TRIM15 A: TRIM15 mRNA expression level; B: TRIM15 protein expression level; C: Fluorescence intensity detected by flow cytometry (the number of cells detected was 20000); D: ImageJ analysis of the number of lipid droplet in Figure D; E: Laser confocal imaging (Bodipy staining); F: Oil red O staining; **: P<0.01"

Fig. 6

Mechanism of SCD5 regulating the number of lipid droplets through TRIM15"

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