Testosterone Inhibits Lipid Accumulation in Porcine Preadipocytes by Regulating ELOVL3

Abstract

Castration is commonly used to reduce stink during boar production. In porcine adipose tissue, castration reduces androgen levels resulting in metabolic disorders and excessive fat deposition. However, the underlying detailed mechanism remains unclear. In this study, we constructed porcine preadipocyte models with and without androgen by adding testosterone exogenously. The fluorescence intensity of lipid droplet (LD) staining and the fatty acid synthetase (FASN) mRNA levels were lower in the testosterone-treated cells than in the untreated control cells. In contrast, the mRNA levels of adipose triglycerides lipase (ATGL) and androgen receptor (AR) were higher than in the testosterone-treated cells than in the control cells. Subsequently, transcriptomic sequencing of porcine preadipocytes incubated with and without testosterone showed that the mRNA expression levels of very long-chain fatty acid elongase 3 (ELOVL3), a key enzyme involved in fatty acids synthesis and metabolism, were high in control cells. The siRNA-mediated knockdown of ELOVL3 reduced LD accumulation and the mRNA levels of FASN and increased the mRNA levels of ATGL. Next, we conducted dual-luciferase reporter assays using wild-type and mutant ELOVL3 promoter reporters, which showed that the ELOVL3 promoter contained an androgen response element (ARE); furthermore, its transcription was negatively regulated by AR overexpression. In conclusion, our study reveals that testosterone inhibits fat deposition in porcine preadipocytes by suppressing ELOVL3 expression. Moreover, our study provides a theoretical basis for further studies on the mechanisms of fat deposition caused by castration.

Keywords: ELOVL3; androgen receptor; lipid accumulation; pig; testosterone.

Figure 1

Testosterone treatment inhibited fat accumulation and altered the expression of lipid metabolism-related genes: (A) Oil Red O was used to stain porcine preadipocytes on the 6 d in the testosterone (T) and control groups (bar = 100 μm). (B) Quantification of LDs using Oil Red O analysis on the 6 d in the testosterone (T) and control groups. (C) The levels of FASN, ATGL, and AR mRNA in T group and control group on the 6 d of adipogenic differentiation of porcine preadipocytes were analyzed using quantitative RT-PCR. (D) LDs stained with BODIPY 493/503 in the T, testosterone + flutamide (T + F), and control groups (bar = 130 μm); (E) Quantitative RT-PCR analysis of the mRNA levels of FASN, ATGL, and AR on the 6 d in the T, T + F, and control groups. n = 3 per group. Statistical comparisons were performed with a t-test with one-way ANOVA. All data are expressed as means ± SEM. Results of the t-test are denoted by asterisks: * p < 0.05, *** p < 0.001 compared to the control (on the bars) or between the indicated groups. Results of one-way ANOVA are indicated by letters. The absence of significant differences is indicated by identical letters (p > 0.05), while different lowercase letters denote significant differences (p < 0.05), and different capital letters signify highly significant differences (p < 0.01).

Figure 2

Transcriptomic profiles of porcine preadipocytes incubated with testosterone: (A) Volcano plot of the statistically significant DEGs (adjusted p < 0.05 and |log2FoldChange| > 1) between the testosterone-treated and control preadipocytes. (B) GO analysis of the DEGs. (C) KEGG analysis of the down-regulated DEGs; (D) KEGG analysis of the up-regulated DEGs; (E) KEGG analysis of metabolism-related DEGs. n = 3 per group.

Figure 3

Knockdown of ELOVL3 expression inhibited porcine fat accumulation: (A) ELOVL3 mRNA levels in the different groups were tested with qRT-PCR. (B) Porcine preadipocytes were transfected with ELOVL3 siRNAs (si-713-ELOVL3, si-394-ELOVL3, and si-410-ELOVL3) or a negative control siRNA, and ELOVL3 expression interference efficiency was analyzed at 48 h after transfection. The relative expression of ELOVL3 was normalized, and the relative values were expressed as the fold of induction relative to the negative control. (C) LD accumulation in preadipocytes transfected with either si-713-ELOVL3 or a negative control siRNA was analyzed using Oil Red O staining at 6 d of induction (bar = 100 μm). (D) Quantification of LDs using Oil Red O analysis on the 6 d in the si-713-ELOVL3 and negative control groups. (E) The mRNA levels of ELOVL3, FASN, and ATGL in the si-713-ELOVL3 and negative control group were confirmed as measured using qRT-PCR. n = 3 per group. All data are expressed as means ± SEM. Results of the t-test are denoted by asterisks: * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the control (on the bars) or between the indicated groups. Results of one-way ANOVA are indicated by letters. The absence of significant differences is indicated by identical letters (p > 0.05), while different lowercase letters denote significant differences (p < 0.05), and different capital letters signify highly significant differences (p < 0.01).

Figure 4

AR targets the ELOVL3 promoter and inhibits its transcriptional activity: (A) Relative luciferase activity (Firefly: Renilla) at 48 h after transfection with different 5′ deletion ELOVL3 promoter constructs (−2000 bp/+100 bp, −1460 bp/+100 bp, −920 bp/+100 bp, −380 bp/+100 bp, −158 bp/+100 bp). (B) Effects of AR overexpression on wild-type and mutant ELOVL3 promoter activity. n = 3 per group. The luciferase activity was normalized, and the relative values were expressed as the fold of induction relative to the pcDNA3.1-EGFP vector activity. A one-way ANOVA test was used to assess the differences in luciferase activity. The absence of significant differences is denoted by the same letters (p > 0.05), while distinct lowercase letters indicate a significant difference (p < 0.05), and distinct capital letters signify a significant difference (p < 0.01).

Figure 5

Testosterone inhibits lipid accumulation in porcine preadipocytes by regulating ELOVL3.