Zhao Chen, Yao Xiangping, Chen Xiaochang, Wu Wenjing, Xi Fengxue, Yang Gongshe, Yu Taiyong
Laboratory of Animal Fat Deposition & Muscle Development, College of Animal Science and Technology, Northwest A&F University, Yangling Shaanxi, 712100, China.
Cell Prolif. 2018 Apr;51(2):e12401. doi: 10.1111/cpr.12401. Epub 2017 Nov 23.
Intramuscular fat (IMF) has a significant influence on porcine meat quality. Ubiquitin D (UBD) is involved in the management of diverse intracellular processes. However, its physiological functions in adipose cell differentiation and proliferation are still poorly defined.
Intramuscular and subcutaneous preadipocytes were isolated from the longissimus dorsi and neck subcutaneous deposits of Chinese native Guanzhong Black piglets (3-5 days old), respectively. Lentivirus with short hairpin RNA (shRNA) for UBD was applied to knockdown UBD expression. We used real-time PCR and Western blot analysis to detect gene expression. Lipid droplets were dyed with Oil Red O, and cell proliferation was assessed using flow cytometry, 5-ethynyl-2'-deoxyuridine incorporation and cell counting assays.
Lipogenesis through the Akt/mTOR pathway was inhibited when preadipocytes were transfected with UBD shRNA. The expression of adipogenic genes and the number of lipid droplets were obviously diminished. Moreover, repression of UBD attenuated cell proliferation. UBD downregulation resulted in cell cycle arrest because of a decreased proportion of S-phase cells, and the expression of positive cell proliferation markers was significantly decreased.
These observations illustrated that knockdown of UBD partially suppressed porcine intramuscular and subcutaneous preadipocyte adipogenesis through the Akt/mTOR signalling and inhibited cell proliferation, suggesting the essential role of UBD in the differentiation of preadipocytes.
肌内脂肪（IMF）对猪肉品质有显著影响。泛素D（UBD）参与多种细胞内过程的调控。然而，其在脂肪细胞分化和增殖中的生理功能仍不清楚。
分别从中国本土关中黑猪仔猪（3 - 5日龄）的背最长肌和颈部皮下脂肪中分离出肌内和皮下前体脂肪细胞。应用携带UBD短发夹RNA（shRNA）的慢病毒来敲低UBD表达。我们使用实时PCR和蛋白质印迹分析来检测基因表达。用油红O对脂滴进行染色，并使用流式细胞术、5-乙炔基-2'-脱氧尿苷掺入法和细胞计数法评估细胞增殖。
当用UBD shRNA转染前体脂肪细胞时，通过Akt/mTOR途径的脂肪生成受到抑制。脂肪生成相关基因的表达和脂滴数量明显减少。此外，UBD的抑制减弱了细胞增殖。UBD下调导致细胞周期停滞，因为S期细胞比例降低，且细胞增殖阳性标志物的表达显著下降。
这些观察结果表明，敲低UBD通过Akt/mTOR信号通路部分抑制了猪肌内和皮下前体脂肪细胞的脂肪生成，并抑制了细胞增殖，提示UBD在脂肪前体细胞分化中起重要作用。
