Umetsu Araya, Watanabe Megumi, Sato Tatsuya, Higashide Megumi, Nishikiori Nami, Furuhashi Masato, Ohguro Hiroshi
Department of Ophthalmology, Sapporo Medical University School of Medicine, Japan.
Department of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University School of Medicine, Japan.
FEBS Open Bio. 2024 Dec;14(12):2026-2037. doi: 10.1002/2211-5463.13890. Epub 2024 Oct 8.
The TGF-β superfamily plays a pivotal role in the regulation of adipogenesis, but little is known about the potential differential role of the three isoforms of TGF-β, TGF-β-1~3. To further elucidate their role, two-dimensionally (2D) and three-dimensionally (3D) cultured 3T3-L1 mouse preadipocytes were subjected to the following analyses: (a) qPCR analysis of adipogenesis-related factors and major extracellular matrix protein (2D and /or 3D), (b) lipid staining by Oil Red O (2D) or BODIPY (3D), (c) Seahorse cellular metabolic measurement (2D), and (d) size and stiffness measurements of 3D 3T3-L1 spheroids. In the 2D cultured 3T3-L1 cells, mRNA expression levels of adipogenesis-related genes and Oil Red O lipid staining intensity were significantly increased by adipogenesis and they were substantially decreased following treatment with 0.1 nm TGF-β isoforms, with TGF-β2 having the greater effects. Consistent with these results, treatment with TGF-β2 resulted in suppression of mitochondrial and glycolytic functions in 2D cultured 3T3-L1 cells. However, the inhibitory effect of TGF-β on adipogenesis decreased under 3D spheroid culture conditions and TGF-β isoforms did not affect adipogenesis-induced (a) enlargement and downsizing of 3T3-L1 spheroids, (b) increase in BODIPY lipid staining intensity, and (c) up-regulation of the mRNA expression of adipogenesis-related genes. The findings presented herein suggest that the three TGF-β isoforms have different suppressive effects on adipogenesis-related cellular properties of 2D cultured 3T3-L1 cells and that their effects decrease under 3D spheroid culture conditions.
转化生长因子-β（TGF-β）超家族在脂肪生成的调控中起关键作用，但对于TGF-β的三种亚型TGF-β-1至3的潜在差异作用知之甚少。为了进一步阐明它们的作用，对二维（2D）和三维（3D）培养的3T3-L1小鼠前脂肪细胞进行了以下分析：（a）对脂肪生成相关因子和主要细胞外基质蛋白进行定量聚合酶链反应（qPCR）分析（2D和/或3D），（b）用油红O进行脂质染色（2D）或用硼二吡咯甲烷（BODIPY）进行脂质染色（3D），（c）进行海马细胞代谢测量（2D），以及（d）测量3D 3T3-L1球体的大小和硬度。在2D培养的3T3-L1细胞中，脂肪生成相关基因的mRNA表达水平和油红O脂质染色强度随着脂肪生成而显著增加，在用0.1纳米TGF-β亚型处理后大幅降低，其中TGF-β2的作用更大。与这些结果一致，用TGF-β2处理导致2D培养的3T3-L1细胞的线粒体和糖酵解功能受到抑制。然而，在3D球体培养条件下，TGF-β对脂肪生成的抑制作用减弱，TGF-β亚型不影响脂肪生成诱导的以下变化：（a）3T3-L1球体的增大和缩小，（b）BODIPY脂质染色强度的增加，以及（c）脂肪生成相关基因mRNA表达的上调。本文提出的研究结果表明，三种TGF-β亚型对2D培养的3T3-L1细胞的脂肪生成相关细胞特性具有不同的抑制作用，并且在3D球体培养条件下它们的作用减弱。
