Table 2_PDK4 gene positively regulates fat deposition in ovine adipocytes.xlsx

IntroductionIntramuscular fat (IMF) content is a crucial factor affecting meat quality and flavor in sheep. Our previous studies demonstrated that the pyruvate dehydrogenase kinase 4 (PDK4) expression increases during adipocyte differentiation and is positively correlated with IMF content in sheep. However, the effects of the PDK4 gene on lipid metabolism in ovine adipocytes remain unclear.
MethodsThe effects of PDK4 overexpression on ovine adipocyte differentiation and fat deposition were investigated. Subsequently, the key lipids and genes affected by PDK4 overexpression were identified using lipidomic and transcriptomic sequencing. Furthermore, a PDK4-knockout NIH/3T3 cell line was generated to verify the evolutionarily conserved functions of PDK4 and to examine the expression of candidate genes in a murine model.
ResultsPDK4 overexpression significantly increased triglyceride deposition by 2.32-fold in ovine adipocytes (p = 0.001) but did not affect PPARγ mRNA or protein expression levels (p > 0.05). A total of 80 differentially expressed lipids (DELs) and 24 differentially expressed genes (DEGs) were identified between the overexpression (Over) and negative control (NC) groups, including 20 upregulated DELs, 60 downregulated DELs, 21 upregulated DEGs, and 3 downregulated DEGs. Additionally, PDK4 overexpression altered lipid content, composition, carbon chain length, and degree of unsaturation in ovine adipocytes. TMEM273, one of the DEGs, was negatively affected by PDK4 overexpression and closely correlated with 7 DELs (adjusted p < 0.01, |R| > 0.9). In the murine cell model, PDK4 knockout of NIH/3T3 (KO) cells significantly decreased triglyceride deposition by 0.77-fold (p < 0.01) but did not affect BSCL2 expression (p = 0.08). TMEM273 expression was significantly increased by 2.78-fold in pre-differentiated KO cells (p = 0.018) and significantly decreased by 0.83-fold in differentiated KO cells (p = 0.005).
ConclusionPDK4 positively regulates fat deposition in both ovine and murine adipocytes. TMEM273 expression is negatively affected by PDK4 and correlated with lipid metabolism in sheep and mice.

引言：肌内脂肪（intramuscular fat, IMF）含量是影响绵羊肉质与风味的关键因素。本课题组前期研究证实，丙酮酸脱氢酶激酶4（pyruvate dehydrogenase kinase 4, PDK4）的表达水平在绵羊脂肪细胞分化过程中上调，且与IMF含量呈正相关。然而，PDK4基因对绵羊脂肪细胞脂质代谢的调控作用仍不明确。
方法：本研究解析了PDK4过表达对绵羊脂肪细胞分化及脂肪沉积的调控作用。随后，通过脂质组学与转录组测序，鉴定出PDK4过表达所调控的核心脂质与差异基因。此外，本研究构建了PDK4敲除的NIH/3T3细胞系，以验证PDK4功能的进化保守性，并在小鼠模型中检测候选基因的表达水平。
结果：PDK4过表达可显著提升绵羊脂肪细胞内甘油三酯沉积水平，增幅达2.32倍（p=0.001），但对过氧化物酶体增殖物激活受体γ（Peroxisome proliferator-activated receptor gamma, PPARγ）的mRNA及蛋白表达水平无显著影响（p>0.05）。过表达组（overexpression, Over）与阴性对照组（negative control, NC）间共鉴定出80种差异脂质（differentially expressed lipids, DELs）与24个差异基因（differentially expressed genes, DEGs），其中上调差异脂质20种、下调差异脂质60种，上调差异基因21个、下调差异基因3个。此外，PDK4过表达可改变绵羊脂肪细胞内的脂质含量、组成、碳链长度及不饱和度。差异基因跨膜蛋白273（transmembrane protein 273, TMEM273）的表达受PDK4过表达负向调控，且与7种差异脂质显著相关（校正后p<0.01，|R|>0.9）。在小鼠细胞模型中，PDK4敲除（knockout, KO）的NIH/3T3细胞的甘油三酯沉积量显著降低，降幅达0.77倍（p<0.01），但对伯纳德利-塞普先天性脂肪营养不良蛋白2（Berardinelli-Seip congenital lipodystrophy 2, BSCL2）的表达无显著影响（p=0.08）。预分化的敲除细胞中，TMEM273的表达量显著上调2.78倍（p=0.018）；而分化后的敲除细胞中，TMEM273的表达量显著下调0.83倍（p=0.005）。
结论：PDK4可正向调控绵羊与小鼠脂肪细胞的脂肪沉积过程。TMEM273的表达受PDK4负向调控，且在绵羊与小鼠中均与脂质代谢密切相关。