Effect of the ACAA1 Gene on Preadipocyte Differentiation in Sheep

Abstract

Acetyl-CoA acyltransferase 1 (ACAA1) functions as a key regulator of fatty acid β-oxidation in peroxisomes by catalyzing the cleavage of 3-ketoacyl-CoA to acetyl-CoA and acyl-CoA, which participate in the extension and degradation of fatty acids. Thus, ACAA1 is an important regulator of lipid metabolism and plays an essential role in fatty acid oxidation and lipid metabolism. Our previous study findings revealed that ACAA1 is closely associated with the peroxisome proliferator-activated receptor (PPAR) signaling and fatty acid metabolism pathways, which are involved in fat deposition in sheep, leading to our hypothesis that ACAA1 may be involved in fat deposition by regulating lipid metabolism. However, the associated molecular mechanism remains unclear. In the present study, to assess the potential function of ACAA1 in sheep preadipocyte differentiation, we knocked down and overexpressed ACAA1 in sheep preadipocytes and evaluated the pattern of ACAA1 gene expression during preadipocyte differentiation by qRT-PCR. ACAA1 was significantly expressed in the early stage of adipocyte differentiation, and then its expression decreased. ACAA1 deficiency increased lipid accumulation and the triglyceride content and promoted sheep preadipocyte differentiation, whereas ACAA1 overexpression inhibited adipogenesis and decreased lipid accumulation and the triglyceride content. Simultaneously, we demonstrated that ACAA1 deficiency upregulated the expressions of the adipogenic marker genes PPARγ and C/EBPα in sheep preadipocytes, but ACAA1 overexpression inhibited the expressions of these markers, indicating that ACAA1 affects lipid metabolism by regulating adipogenic marker genes. Our results may promote a better understanding of the regulation of adipogenesis by ACAA1.

Keywords: ACAA1 gene; adipogenesis; lipid metabolism; preadipocyte differentiation; sheep.

FIGURE 1

ACAA1 and adipogenic marker gene expression during sheep preadipocyte differentiation. (A) Sheep preadipocytes, mature adipocytes, and oil red O staining (200×). (B) Expression patterns of adipogenic marker genes (PPARγ and C/EBPα) in differentiating sheep adipocytes. (C) ACAA1 protein expression in differentiating sheep adipocytes was determined by Western blot analysis. (D,E) The protein expression levels of PPARγ and C/EBPα during adipocyte differentiation were evaluated on days 0, 2, 4, 6, and 8. Densitometric analyses of the Western blots. With the differentiation of cells, the expression levels of adipogenic genes gradually increased (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). ACAA1, acetyl-CoA acyltransferase 1; PPARγ, peroxisome proliferator-activated receptor γ; C/EBPα, CCAAT/enhancer binding protein α.

FIGURE 2

ACAA1 knockdown increases lipid accumulation and the triglyceride content during sheep preadipocyte differentiation. (A) Determination of the transfection efficiency of siRNA-ACAA1 (200×). (B) After 8 days of differentiation, the cellular lipid droplets were stained with oil red O (200×). (C) ACAA1 mRNA expression was detected after siRNA-ACAA1 or NC transfection on days 0, 4, and 8 of adipogenic differentiation. The siRNA-ACAA1 groups all showed significantly lower expressions than the control group (**p < 0.01). (D) The triglyceride content after 8 days of induction in the siRNA-ACAA1 group was significantly higher than that in the control group (**p < 0.01). (E) The lipid droplet content measured at 490 nm after 8 days of induction was significantly higher in the siRNA-ACAA1 group than that in the control group (**p < 0.01). siRNA, small interfering RNA; NC, negative control.

FIGURE 3

ACAA1 knockdown regulates adipogenic marker gene expression. (A) Sheep preadipocytes were transfected with siRNA-ACAA1 or siRNA-NC and differentiated into mature adipocytes. The mRNA expression levels of adipogenic marker genes (PPARγ and C/EBPα) were evaluated on days 0 (after cell amalgamation), 4, and 8 of adipogenic differentiation. On day 0 of adipogenic differentiation, the mRNA expression levels of the marker genes were higher than those of the control group, but only PPARγ reached a significant level (**p < 0.01). On days 4 and 8 of adipogenic differentiation, the mRNA expression levels of PPARγ and C/EBPα were significantly higher than those of the control group (*p < 0.05 and **p < 0.01). (B) Protein expression levels of ACAA1 and adipogenic marker genes on days 4 and 8 of adipogenic differentiation. Columns 1 and 2 are the siRNA-ACAA1 processing group and the siRNA-NC processing group, respectively (day 4). Columns 3 and 4 are the siRNA-ACAA1 processing group and the siRNA-NC processing group, respectively (day 8). β-actin is the internal reference gene. (C) The protein expression levels of ACAA1 and adipogenic marker genes were assessed on days 4 and 8 of adipogenic differentiation. The protein expression levels of ACAA1 were significantly lower than those of the control group (**p < 0.01), and the protein expression levels of PPARγ and C/EBPα were significantly higher than those of the control group (*p < 0.05, **p < 0.01, and ***p < 0.001). NC, negative control.

FIGURE 4

ACAA1 overexpression decreases lipid accumulation and the triglyceride content in preadipocytes. (A) Determination of the transfection efficiency of the ACAA1 overexpression plasmid (200×). (B) After 8 days of differentiation, the cellular lipid droplets were stained with oil red O (200×). (C) ACAA1 mRNA expression was assessed after cells were transfected with the ACAA1 overexpression or the NC plasmids on days 0, 4, and 8 of adipogenic differentiation. The expression in the over-ACAA1 group was significantly higher than that in the control group (****p < 0.01 and ***p < 0.001). The difference was largest in the early stage of cell differentiation. (D) After 8 days of induction, the triglyceride content in the over-ACAA1 group was significantly lower than that in the control group (**p < 0.01). (E) The lipid droplet content measured at 490 nm after 8 days of induction in the over-ACAA1 group was significantly lower than that in the control group (**p < 0.01).

FIGURE 5

ACAA1 overexpression regulates adipogenic marker gene expression. (A) Sheep preadipocytes were transfected with ACAA1 overexpression or negative control (NC) plasmids and differentiated into mature adipocytes. The mRNA expression levels of adipogenic marker genes (PPARγ and C/EBPα) were measured on days 0, 4, and 8 of adipogenic differentiation. Except day 0, the mRNA expression levels of PPARγ and C/EBPα were significantly lower than those of the control group (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). (B) Protein expression levels of ACAA1 and adipogenic marker genes on days 4 and 8 of adipogenic differentiation. Columns 1 and 2 are the over-ACAA1 processing and over-NC processing groups, respectively (day 4). Columns 3 and 4 are the over-ACAA1 processing and over-NC processing groups, respectively (day 8). β-actin is the internal reference gene. (C) The protein expression levels of ACAA1 and adipogenic marker genes were assessed on days 4 and 8 of adipogenic differentiation. The protein expression levels of ACAA1 were significantly higher than those of the control group (****p < 0.0001 and **p < 0.01), and the protein expression levels of PPARγ and C/EBPα were significantly lower than those of the control group (**p < 0.01 and ***p < 0.001).