Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, 310018, P. R. China; College of Animal Sciences, Zhejiang University, Hangzhou 310058, P. R. China.
College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, 712100, P. R. China.
J Dairy Sci. 2018 Mar;101(3):2641-2649. doi: 10.3168/jds.2017-13543. Epub 2018 Jan 10.
Even though recent evidence in goat mammary epithelial cells (GMEC) suggest a role of peroxisome proliferator-activated receptor delta (PPARD) in regulating lipid homeostasis, its role is not fully understood. Our hypothesis was that PPARD regulates lipid transport processes in GMEC and, thus, plays a crucial role in regulating fat formation. The PPARD was overexpressed using an adenovirus system (Ad-PPARD) with recombinant green fluorescent protein (Ad-GFP) as the control. Results revealed that overexpression of PPARD markedly upregulated the mRNA abundance of PPARD. Compared with the control (Ad-GFP+dimethyl sulfoxide), overexpression of PPARD alone had no effect on mRNA expression of CD36, SCD1, FABP4, ACSL1, and ADRP. The cultures overexpressing PPARD with the PPARD ligand GW0742 (GW) upregulated the expression of CD36, FABP3, FABP4, ACSL1, and ADRP. Overexpression of PPARD in GMEC plus GW increased the concentration of 16:1 and 18:1-trans and was associated with upregulation of SCD1. Compared with the control (Ad-GFP+dimethyl sulfoxide), the decrease of triacylglycerol concentration coupled with upregulation of genes related to lipid droplet secretion (e.g., ADRP and ACSL1) induced by PPARD overexpression suggests a role in lipid droplet (LD) secretion. Luciferase assay revealed that GW increased the ADRP promoter activity in a dose-dependent manner. Knockdown of PPARD impaired the increase of ADRP promoter activity induced by GW, whereas GW enhanced the activity of ADRP promoter in GMEC overexpressing PPARD. Data with the ADRP 5'-flanking truncated luciferase reporter suggest a core region (-1,444 to -990 bp) response element for the induction of GW. This core region contains a known PPARG response element (PPRE) at -1,003 to -990 bp. When the PPRE was mutated, the overexpression of PPARD had no effect on ADRP promoter activity. Collectively, these results reveal a novel role for PPARD in lipid homeostasis via promoting fatty acid transport and LD formation through a mechanism of direct binding to the promoter of key genes. Hence, PPARD activity may contribute to fatty acid transport and LD formation during lactation.
尽管最近在山羊乳腺上皮细胞（GMEC）中的证据表明过氧化物酶体增殖物激活受体δ（PPARD）在调节脂质稳态中起作用，但它的作用尚未完全阐明。我们的假设是，PPARD 调节 GMEC 中的脂质转运过程，因此在调节脂肪形成中起着至关重要的作用。使用腺病毒系统（Ad-PPARD）过表达 PPARD，用重组绿色荧光蛋白（Ad-GFP）作为对照。结果表明，PPARD 的过表达显著上调了 PPARD 的 mRNA 丰度。与对照（Ad-GFP+二甲基亚砜）相比，单独过表达 PPARD 对 CD36、SCD1、FABP4、ACSL1 和 ADRP 的 mRNA 表达没有影响。用 PPARD 配体 GW0742（GW）过表达 PPARD 的培养物上调了 CD36、FABP3、FABP4、ACSL1 和 ADRP 的表达。GMEC 中 PPARD 的过表达加上 GW 增加了 16:1 和 18:1-反式的浓度，并与 SCD1 的上调有关。与对照（Ad-GFP+二甲基亚砜）相比，PPARD 过表达导致三酰甘油浓度降低，同时与脂质滴分泌相关的基因（如 ADRP 和 ACSL1）上调，表明其在脂质滴（LD）分泌中起作用。荧光素酶测定表明，GW 呈剂量依赖性地增加 ADRP 启动子活性。PPARD 的敲低削弱了 GW 诱导的 ADRP 启动子活性的增加，而 GW 增强了 GMEC 中转染 PPARD 的 ADRP 启动子活性。带有 ADRP 5'-侧翼截断荧光素酶报告基因的数据表明，GW 诱导的核心区域（-1444 至-990 bp）反应元件。该核心区域在-1003 至-990 bp 处包含一个已知的 PPARG 反应元件（PPRE）。当 PPRE 发生突变时，PPARD 的过表达对 ADRP 启动子活性没有影响。总之，这些结果揭示了 PPARD 通过直接结合关键基因的启动子来促进脂肪酸转运和 LD 形成，从而在脂质稳态中发挥新的作用。因此，PPARD 活性可能有助于哺乳期脂肪酸转运和 LD 形成。
