Table 1_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。本团队前期研究证实，丙酮酸脱氢酶激酶4（Pyruvate Dehydrogenase Kinase 4, PDK4）在绵羊脂肪细胞分化过程中表达量升高，且与绵羊肌内脂肪含量呈正相关。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 本研究探究了PDK4过表达对绵羊脂肪细胞分化及脂肪沉积的影响。随后，通过脂质组学与转录组测序技术，筛选得到PDK4过表达所调控的关键脂质与差异表达基因。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，并在小鼠细胞模型中检测候选基因的表达情况。

结果 PDK4过表达可使绵羊脂肪细胞内甘油三酯沉积量显著提升2.32倍（p = 0.001），但对PPARγ的mRNA及蛋白表达水平无显著影响（p > 0.05）。过表达组（Over）与阴性对照组（NC）共鉴定得到80种差异表达脂质（DELs）与24个差异表达基因（DEGs），其中上调差异表达脂质20种、下调60种，上调差异表达基因21个、下调3个。此外，PDK4过表达可改变绵羊脂肪细胞内脂质的含量、组成、碳链长度及不饱和程度。差异表达基因TMEM273的表达受PDK4过表达负向调控，且与7种差异表达脂质显著相关（校正p < 0.01，|相关系数R| > 0.9）。在小鼠细胞模型中，PDK4敲除的NIH/3T3细胞（KO）的甘油三酯沉积量显著降低0.77倍（p < 0.01），但对BSCL2的表达无显著影响（p = 0.08）。预分化状态下的敲除细胞中，TMEM273的表达量显著上调2.78倍（p = 0.018）；而分化成熟的敲除细胞中，TMEM273的表达量则显著下调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，且在绵羊与小鼠体内均与脂质代谢密切相关。