Stearoyl-CoA Desaturase is Essential for Porcine Adipocyte Differentiation

Abstract

Fat deposition, which influences pork production, meat quality and growth efficiency, is an economically important trait in pigs. Numerous studies have demonstrated that stearoyl-CoA desaturase (SCD), a key enzyme that catalyzes the conversion of saturated fatty acids into monounsaturated fatty acids, is associated with fatty acid composition in pigs. As SCD was observed to be significantly induced in 3T3-L1 preadipocytes differentiation, we hypothesized that it plays a role in porcine adipocyte differentiation and fat deposition. In this study, we revealed that SCD is highly expressed in adipose tissues from seven-day-old piglets, compared to its expression in tissues from four-month-old adult pigs. Moreover, we found that SCD and lipogenesis-related genes were induced significantly in differentiated porcine adipocytes. Using CRISPR/Cas9 technology, we generated SCD^-/- porcine embryonic fibroblasts (PEFs) and found that the loss of SCD led to dramatically decreased transdifferentiation efficiency, as evidenced by the decreased expression of known lipid synthesis-related genes, lower levels of oil red O staining and significantly lower levels of triglyceride content. Our study demonstrates the critical role of SCD expression in porcine adipocyte differentiation and paves the way for identifying it as the promising candidate gene for less fat deposition in pigs.

Keywords: SCD; adipocytes; adipogenesis; transdifferentiation.

Figure 1

The expression level of stearoyl-CoA desaturase (SCD) and three transcriptional factors in various adipose depots from piglets and adult pigs. Subcutaneous adipose tissue from the dorsal and leg, perirenal adipose tissue, and leaf adipose tissue were collected from seven-day-old and four-month-old Bama pigs (n = 3/group). Real-time PCR results showed the expression levels of SCD (A), PPARG (B), C/EBPA (C) and SREBP-1C (D) in these adipose tissues. * p < 0.05, ** p < 0.01 and *** p < 0.001 were considered significant. (E) Western blot analysis results of SCD expression in the adipose tissues from seven-day-old piglets and four-month-old adult pigs.

Figure 2

SCD was significantly up-regulated in differentiated porcine adipocytes. Porcine preadipocytes were primarily cultured and differentiated into mature adipocytes in vitro. (A) Left: nondifferentiated preadipocytes; right: oil red O staining of the differentiated cells. Positive staining indicated lipid droplet formation. Scale bar, 100 μm. (B) The expression levels of SCD and the adipocyte differentiation marker genes PPARG and C/EBPA in preadipocytes and differentiated adipocytes. TUBULIN was used as a loading control. (C) The expression levels of genes related to lipid synthesis were measured in preadipocytes and differentiated adipocytes (n = 3/group), * p < 0.05.

Figure 3

Generation of SCD^-/- porcine embryo fibroblasts. (A) Gene structure of the porcine SCD gene and the location of the four sgRNAs that target exon 2 (highlighted in blue). Primers designed to estimate the target efficiency are also indicated, and the predicted PCR size is 403 bp (highlighted in red). (B) Different pairs of vectors containing sgRNA, including sgRNA1-sgRNA2, sgRNA1-sgRNA4 and sgRNA2-sgRNA3, were selected for co-transfecting cells, because of a potential frame shift effect. A template with H₂O was used as a negative control, and the DNA in the transfected empty vector was used as a positive control. Notably, the short band was observed in cells co-transfected with sgRNA1 and sgRNA4, and this pair of sgRNAs was then used for the remaining studies. (C) Plasmids containing sgRNA1 and sgRNA4 were co-transfected in porcine embryonic fibroblasts (PEFs), and single cells were sorted by flow cytometry and seeded into 96-well plates. A total of 20 single-cell colonies were selected for PCR and sequencing analysis. (D) Estimation of targeting efficiency. Notably, 5 biallelic mutant clones were identified, and the targeting efficiency was 25%. (E) Targeted sequence information of #9 colony. (F) Knockout efficiency of SCD in the #9 colony was examined by real-time PCR; ** p < 0.05.

Figure 4

SCD knockout inhibited PEF transdifferentiation into mature adipocytes. (A) SCD^+/+ and SCD^-/- were differentiated into adipocytes in vitro. Cells were subjected to oil red O staining after differentiation. Scale bar, 100 μm. (B) Western blot analysis of SCD in nondifferentiated and differentiated SCD^+/+ and SCD^-/- cells. TUBULIN was used as the loading control. Notably, SCD was undetectable in differentiated SCD^-/- cells. (C) The expression levels of the genes related to lipid synthesis, in both the SCD^+/+ and SCD^-/- cells. Notably, all of these genes were significantly lower in the SCD^-/- cells (n = 3/group). (D,E) TG (n = 7/group) and NEFA (n = 6/group) content levels in the SCD^+/+ and SCD^-/- cells. (F) The expression levels of the genes related to fatty acid β-oxidation in both the SCD^+/+ and SCD^-/- cells (n = 3/group); * p < 0.05, ** p < 0.01 and *** p < 0.001.