Wu Xiaohui, Li Qing, Wei Wanli, Xu Yuqiao
Department of Pathology in Basic Medical College & Department of Pathology in First Affiliated Hospital, Air Force Medical University, Xi'an 710032.
Institute of Obesity and Metabolism, Xi'an Medical University, Xi'an 710021, China.
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2020 Nov 28;45(11):1298-1307. doi: 10.11817/j.issn.1672-7347.2020.190364.
Jmjd3 can promote the differentiation of brown adipocytes, but its role in the ultrastructure of lipid droplets and mitochondria, the two key thermogenic organelles, is still unclear. The aim of this study is to investigate the effects of histone H3K27me3 demethylase Jmjd3 deletion on lipid droplets and mitochondria in brown adipocytes in mice.
Jmjd3 general knockout (Jmjd3) mice and adipose tissue specific conditional knockout (Jmjd3Fabp4-Cre) mice were constructed.Brown adipose tissue (BAT) was taken from the back of Jmjd3 and their littermates (Jmjd3) at the 19.5 days of embryo (E19.5). BAT was also taken from the back of 6-week-old Jmjd3Fabp4-Cre and their littermates (Jmjd3or Jmjd3Fabp4-Cre). The morphology of brown adipocytes was observed by light microscopy after HE staining. The ultrastructure of lipid droplets and mitochondria was observed by electron microscopy. Western blotting was used to detect the expression of H3K27me3, Jmjd3, and uncouple protein 1 (UCP1) in BAT. RT-qPCR was used to detect the expression of Jmjd3, peroxisome proliferator-activated receptor γ (PPARγ), fatty acid binding protein 4 (Fabp4), UCP1, PR domain-containing 16 (PRDM16), cell death-inducing DFFA-like effector a (CIDEa), mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1α), mitochondrial cytochrome c oxidase subunit I (COX1), and cytochrome c (Cyt c). ATP content in the BAT was tested. The body temperature of Jmjd3Fabp4-Cre and their control mice during cold stimulation was detected.
Two types of Jmjd3 knockout (Jmjd3 and Jmjd3Fabp4-Cre) mice were smaller than their littermates. The expression of Jmjd3 mRNA (<0.05) and protein in BAT was significantly decreased, and the protein expression of H3K27me3 was increased, indicating that the knockout mice were successfully constructed. HE staining showed that lipid droplets in BAT of Jmjd3 mice were significantly less than those in the control group. The results of electron microscopy showed that the area of brown adipocytes in Jmjd3 mice was smaller (<0.05), the number of lipid droplets was less (<0.01), and the size of lipid droplets was not significantly different (>0.05). Mitochondrial edema and cristae rupture were observed in the Jmjd3 group.The number and area of mitochondria were not affected (both >0.05), the number of mitochondrial cristae was significantly less than that of the control group (<0.05). The protein expression of UCP1 of Jmjd3 mice was decreased. The mRNA expression of UCP1, CIDEa, and PGC-1α in BAT of Jmjd3 mice was significantly less than that of the control group (all <0.05). ATP content in BAT of Jmjd3 mice was decreased (<0.05). HE staining showed that lipid droplets in BAT of Jmjd3Fabp4-Cre mice were larger than those in the control group. Electron microscopy showed that there was no significant difference in size of adipocytes between the 2 groups (>0.05). In the Jmjd3Fabp4-Cre mice, lipid droplets were less (<0.05), but their diameters were larger (<0.001). Mitochondrial edema and cristae rupture were observed in the Jmjd3Fabp4-Cre mice. The number of mitochondrial cristae in the Jmjd3Fabp4-Cre mice was significantly less than that in the control group (<0.05). The protein expression of UCP1 in Jmjd3Fabp4-Cre mice was decreased. The mRNA expression of UCP1, PRDM16, CIDEa, COX1, PGC-1α in BAT of Jmjd3Fabp4-Cre mice was significantly less than that of the control group (all <0.05). ATP content in BAT of Jmjd3Fabp4-Cre mice was decreased (<0.05). In the Jmjd3Fabp4-Cre mice, the body temperature was decreased more and the cold tolerance was poor (<0.05).
Jmjd3 promotes the differentiation of brown adipocytes by enhancing the formation of lipid droplets in mouse brown adipocytes and maintaining the normal morphology and function of mitochondria.
Jmjd3可促进棕色脂肪细胞分化，但其在脂质滴和线粒体这两个关键产热细胞器超微结构中的作用仍不清楚。本研究旨在探讨组蛋白H3K27me3去甲基化酶Jmjd3缺失对小鼠棕色脂肪细胞中脂质滴和线粒体的影响。
构建Jmjd3全身敲除（Jmjd3）小鼠和脂肪组织特异性条件敲除（Jmjd3Fabp4-Cre）小鼠。在胚胎第19.5天（E19.5）从Jmjd3及其同窝小鼠（Jmjd3）背部取棕色脂肪组织（BAT）。也从6周龄的Jmjd3Fabp4-Cre及其同窝小鼠（Jmjd3或Jmjd3Fabp4-Cre）背部取BAT。HE染色后通过光学显微镜观察棕色脂肪细胞的形态。通过电子显微镜观察脂质滴和线粒体的超微结构。采用蛋白质免疫印迹法检测BAT中H3K27me₃、Jmjd3和解偶联蛋白1（UCP1）的表达。采用RT-qPCR检测Jmjd3、过氧化物酶体增殖物激活受体γ（PPARγ）、脂肪酸结合蛋白4（Fabp4）、UCP1、含PR结构域蛋白16（PRDM16）、细胞死亡诱导DFFA样效应因子a（CIDEa）、线粒体转录因子A（TFAM）、过氧化物酶体增殖物激活受体γ共激活因子1α（PGC-1α）、线粒体细胞色素c氧化酶亚基I（COX1）和细胞色素c（Cyt c）的表达。检测BAT中的ATP含量。检测Jmjd3Fabp4-Cre及其对照小鼠在冷刺激期间的体温。
两种类型的Jmjd3敲除（Jmjd3和Jmjd3Fabp4-Cre）小鼠比其同窝小鼠体型小。BAT中Jmjd3 mRNA（<0.05）和蛋白表达显著降低，H3K27me3蛋白表达增加，表明成功构建了敲除小鼠。HE染色显示，Jmjd3小鼠BAT中的脂质滴明显少于对照组。电子显微镜结果显示，Jmjd3小鼠棕色脂肪细胞面积较小（<0.05），脂质滴数量较少（<0.01），脂质滴大小无显著差异（>0.05）。Jmjd3组观察到线粒体水肿和嵴断裂。线粒体数量和面积未受影响（均>0.05），线粒体嵴数量明显少于对照组（<0.05）。Jmjd3小鼠UCP1蛋白表达降低。Jmjd3小鼠BAT中UCP1、CIDEa和PGC-1α的mRNA表达明显低于对照组（均<0.05）。Jmjd3小鼠BAT中的ATP含量降低（<0.05）。HE染色显示，Jmjd3Fabp4-Cre小鼠BAT中的脂质滴大于对照组。电子显微镜显示，两组脂肪细胞大小无显著差异（>0.05）。在Jmjd3Fabp4-Cre小鼠中，脂质滴较少（<0.05），但其直径较大（<0.001）。Jmjd3Fabp4-Cre小鼠中观察到线粒体水肿和嵴断裂。Jmjd3Fabp4-Cre小鼠中线粒体嵴数量明显少于对照组（<0.05）。Jmjd3Fabp4-Cre小鼠中UCP1蛋白表达降低。Jmjd3Fabp4-Cre小鼠BAT中UCP1、PRDM16、CIDEa、COX1、PGC-1α的mRNA表达明显低于对照组（均<0.05）。Jmjd3Fabp4-Cre小鼠BAT中的ATP含量降低（<0.05）。在Jmjd3Fabp4-Cre小鼠中，体温下降更多，耐寒性较差（<0.05）。
Jmjd3通过增强小鼠棕色脂肪细胞中脂质滴的形成并维持线粒体的正常形态和功能来促进棕色脂肪细胞分化。
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