目的:血清/糖皮质激素诱导激酶1(SGK1)基因编码丝氨酸/苏氨酸蛋白激酶,在细胞应激反应和多种代谢过程的调节中起重要作用。然而,它在牛脂肪形成中的作用仍然未知。在这项研究中,我们旨在阐明SGK1在牛脂积累和改善肉质中的作用。
方法:诱导前脂肪细胞分化以检测SGK1的时间表达模式。心,肝脏,肺,脾,脾肾,收集肌肉和脂肪组织以检测其组织表达谱。重组腺病毒和慢病毒被包装用于过表达和敲低。油红O染色,实时定量PCR,蛋白质印迹分析,酵母双杂交试验,通过荧光素酶分析和RNA-seq研究SGK1的调控机制。
结果:SGK1在脂肪细胞中表达显著增高,在分化的脂肪细胞中表达显著诱导。此外,SGK1的过表达极大地促进了脂肪形成并抑制了增殖,这可以通过脂滴的显着增加来显示,以及成脂标记基因和细胞周期相关基因的表达水平。相反,其敲除抑制脂肪生成并促进增殖。机械上,SGK1调控FoxO家族两个关键蛋白的磷酸化和表达,FOXO1/FOXO3。重要的是,SGK1通过磷酸化位点S256减弱FOXO1对PPARγ的转录抑制作用,然后促进牛脂肪沉积。
结论:SGK1是牛前脂肪细胞增殖和分化所必需的表观遗传调节因子,这有助于更好地了解牛的脂肪沉积和肉质改善。
OBJECTIVE: Serum/glucocorticoid-inducible kinase 1 (SGK1) gene encodes a serine/threonine protein kinase that plays an essential role in cellular stress response and regulation of multiple metabolic processes. However, its role in bovine adipogenesis remains unknown. In this study, we aimed to clarify the role of SGK1 in bovine lipid accumulation and improvement of meat quality.
METHODS: Preadipocytes were induced to differentiation to detect the temporal expression pattern of SGK1. Heart, liver, lung, spleen, kidney, muscle and fat tissues were collected to detect its tissue expression profile. Recombinant adenovirus and the lentivirus were packaged for overexpression and knockdown. Oil Red O staining, quantitative real-time PCR, Western blot analysis, Yeast two-hybrid assay, luciferase assay and RNA-seq were performed to study the regulatory mechanism of SGK1.
RESULTS: SGK1 showed significantly higher expression in adipose and significantly induced expression in differentiated adipocytes. Furthermore, overexpression of SGK1 greatly promoted adipogenesis and inhibited proliferation, which could be shown by the remarkable increasement of lipid droplet, and the expression levels of adipogenic marker genes and cell cycle-related genes. Inversely, its knockdown inhibited adipogenesis and facilitated proliferation. Mechanistically, SGK1 regulates the phosphorylation and expression of two critical proteins of FoxO family, FOXO1/FOXO3. Importantly, SGK1 attenuates the transcriptional repression role of FOXO1 for PPARγ via phosphorylating the site S256, then promoting the bovine fat deposition.
CONCLUSIONS: SGK1 is a required epigenetic regulatory factor for bovine preadipocyte proliferation and differentiation, which contributes to a better understanding of fat deposition and meat quality improvement in cattle.