Laboratory of Experimental Cardiology, Department of Cardiology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, the Netherlands; Department of Medical Cell Biology and Genetics, Guangdong Key Laboratory of Genome Stability & Disease Prevention, Shenzhen Key Laboratory for Anti-ageing and Regenerative Medicine, Health Science Center, Shenzhen University, Nanhai Ave, 3688, Shenzhen, China.
Department of Medicine, Division of Endocrinology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, the Netherlands; Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, the Netherlands.
Biochim Biophys Acta Mol Cell Biol Lipids. 2019 Dec;1864(12):158511. doi: 10.1016/j.bbalip.2019.08.007. Epub 2019 Aug 26.
Brown adipose tissue (BAT) is a potential target to treat cardiometabolic disorders because of its capacity to combust glucose and fatty acids for thermoregulation. Its cellular and molecular investigation in humans is hampered by the limited availability of cell material and the heterogeneity of BAT between and within individuals. In this study, monoclonal lines of conditionally immortalized brown preadipocytes (iBPAs) of mouse and human origin were generated. Conditional immortalization was achieved by doxycycline-controlled expression of simian virus 40 large tumor antigen (LT) with a repressor-based Tet-On system. In the presence of doxycycline, both the murine and human cell lines showed long-term proliferation capacity with a population doubling time of ~28 h. After switching off LT expression by doxycycline removal and exposure to adipogenic differentiation medium, cells from both species acquired brown adipocyte properties. This was evidenced by the accumulation of multilocular lipid droplets, the upregulation of brown adipocyte markers including uncoupling protein 1 and an increase in lipolysis and oxygen consumption following adrenergic stimulation. Switching off LT expression before the onset of adipogenic differentiation was only critical for inducing adipogenesis in the human iBPAs, while their murine counterparts showed adipogenesis upon exposure to the adipogenic differentiation cocktail regardless of LT expression. When switched to proliferation medium, cultures of adipogenically differentiated human iBPAs de-differentiated and resumed cell division without losing their adipogenic capacity. We suggest that iBPAs represent an easy-to-use model for fundamental and applied research into BAT offering unique experimental opportunities due to their capacity to switch between proliferative and differentiated states.
棕色脂肪组织（BAT）是治疗代谢综合征的潜在靶点，因为它具有燃烧葡萄糖和脂肪酸以进行体温调节的能力。由于细胞材料的有限可用性以及个体之间和个体内部 BAT 的异质性，其在人类中的细胞和分子研究受到阻碍。在这项研究中，生成了源自小鼠和人类的条件永生化棕色前脂肪细胞（iBPA）的单克隆系。通过四环素控制的猴病毒 40 大肿瘤抗原（LT）表达，利用基于阻遏物的 Tet-On 系统实现条件永生化。在存在多西环素的情况下，两种细胞系均表现出长期增殖能力，倍增时间约为 28 小时。在去除多西环素并暴露于脂肪生成分化培养基后，关闭 LT 表达后，两种物种的细胞均获得了棕色脂肪细胞特性。这表现在多房脂质滴的积累、棕色脂肪细胞标志物包括解偶联蛋白 1 的上调以及肾上腺素能刺激后的脂解和耗氧量增加。在脂肪生成分化开始之前关闭 LT 表达对于诱导人类 iBPA 的脂肪生成至关重要，而其小鼠对应物在暴露于脂肪生成分化鸡尾酒时无论 LT 表达如何都会发生脂肪生成。当切换到增殖培养基时，分化的人 iBPA 的培养物去分化并重新开始细胞分裂，而不会失去其脂肪生成能力。我们建议 iBPA 代表一种易于使用的 BAT 基础和应用研究模型，由于其在增殖和分化状态之间切换的能力，提供了独特的实验机会。
