Knockdown of LXRα Inhibits Goat Intramuscular Preadipocyte Differentiation

Abstract

Goat intramuscular fat (IMF) content is mainly determined by the processes of intramuscular preadipocytes adipogenic differentiation and mature adipocyte lipid accumulation. However, the underlying regulators of these biological processes remain largely unknown. Here, we report that the expression of Liver X receptor alpha (LXRα) reaches a peak at early stage and then gradually decreases during goat intramuscular adipogenesis. Knockdown of LXRα mediated by two independent siRNAs significantly inhibits intramuscular adipocytes lipid accumulation and upregulates preadipocytes marker- preadipocyte factor 1 (pref1) expression. Consistently, siRNA treatments robustly decrease mRNA level of adipogenic related genes, including CCAAT enhancer binding protein alpha (Cebpα), Peroxisome proliferator activated receptor gamma (Pparg), Sterol regulatory element binding protein isoform 1c (Srebp1c), Fatty acids binding protein (aP2) and Lipoprotein lipase (Lpl). Next, adenovirus overexpression of LXRα does not affect intramuscular adipocytes adipogenesis manifested by Oil Red O signal measurement and adipogenic specific genes detection. Mechanically, we found that both CCAAT enhancer binding protein beta (Cebpβ) and Kruppel like factor 8 (Klf8) are potential targets of LXRα, indicated by having putative binding sites of LXRα at the promoter of these genes and similar expression pattern during adipogenesis comparing to LXRα. Importantly, mRNA levels of Cebpβ and Klf8 are downregulated significantly in goat LXRα knockdown intramuscular adipocyte. These results demonstrate that loss function of LXRα inhibits intramuscular adipogenesis possibly through down-regulation of Cebpβ and Klf8. Our research will provide new insights into mechanical regulation of goat IMF deposition.

Keywords: Capra hircus; LXRα; goat; intramuscular adipocyte; intramuscular fat.

Figure 1

The LXRα gene expression pattern in various tissues and during intramuscular preadipocyte differentiation. (A) The LXRα mRNA level in heart (Hea), spleen (Spl), lung (Lun), longissimus dorsi (LD) muscle, subcutaneous white adipose tissue (SWAT) and visceral WAT (VWAT), n = 6. (B) The LXRα mRNA level on Days 0–7 in induced differentiation intramuscular adipocyte (n = 6). Data are shown as the means ± standard error of the mean (SEM). Different lowercase represents significant difference (p < 0.05).

Figure 2

Knockdown of LXRα inhibits goat intramuscular adipocyte lipid accumulation. (A,B) The knockdown efficiency of LXRα at mRNA (A) and protein (B) level (n = 6). (C,D) The Oil Red O staining (×100) and lipid accumulation between control and siRNAs treatment intramuscular adipocyte cells (n = 6). ** p < 0.01, *** p < 0.001, compared to that of negative control (NC). Data are shown as the means ± SEM.

Figure 3

Knockdown of LXRα upregulates negative- and downregulates positive-intramuscular adipogenic genes. The mRNA levels between control and siRNAs treatment intramuscular adipocyte cells (n = 6) of: Pref-1 (A); Cebpα (B); Pparg (C); Srebp1c (D); aP2 (E); and Lpl (F). * p < 0.05, ** p < 0.01, *** p < 0.001, compared to that of negative control (NC). Data are shown as the means ± SEM.

Figure 4

Overexpression of LXRα does not impact on goat intramuscular adipocyte differentiation. (A,B) The overexpression efficiency of LXRα at mRNA and protein level (n = 6). (C,D) The Oil Red O staining (×100) and lipid accumulation between control and overexpression treatment intramuscular adipocyte cells (n = 4). Data are shown as the means ± SEM.

Figure 5

Overexpression of LXRα does not influence intramuscular adipogenic genes. (A–F) The mRNA levels between control and siRNAs treatment intramuscular adipocyte cells (n = 6) of: Pref-1 (A) Cebpα (B); Pparg (C); Srebp1c (D); aP2 (E); and Lpl (F). Data are shown as the means ± SEM.

Figure 6

Knockdown of LXRα inhibits intramuscular adipogenesis trough downregulation of Cebpβ and Klf8: (A) the LXRα binding DNA motif; (B) the LXRα binding sites prediction at the promoters of Cebpβ and Klf8, blue circles represent the LXRα binding sites, gray boxes represent exons; (C) the Cebpβ and Klf8 expression trend during intramuscular adipogenesis (n = 4); and (D,E) the mRNA levels of Cebpβ and Klf8 in LXRα-siRNA treated cells (n = 6). Data are shown as the means ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to that of negative control (NC).