18O-Tracer Metabolomics Reveals Protein Turnover and CDP-Choline Cycle Activity in Differentiating 3T3-L1 Pre-Adipocytes

The differentiation of precursor cells into mature adipocytes (adipogenesis) has been an area of increased focus, spurred by a rise in obesity rates. Though our understanding of adipogenesis and its regulation at the cellular level is growing, many questions remain, especially regarding the regulation of the metabolome. The 3T3-L1 cell line is the most well characterized cellular model of adipogenesis. Using a time course metabolomics approach, we show that the 3T3-L1 preadipocyte metabolome is greatly altered during the first 48 hours of differentiation, where cells go through about two rounds of cell division, a process known as mitotic clonal expansion. Short-chain peptides were among several small molecules that were increased during mitotic clonal expansion. Additional indicators of protein turnover were also increased, including bilirubin, a degradation product of heme-containing proteins, and 3-methylhistidine, a post-translationally modified amino acid that is not reutilized for protein synthesis. To study the origin of the peptides, we treated differentiating preadipocytes with 18O labeled water and found that 18O was incorporated into the short chain peptides, confirming them, at least in part, as products of hydrolysis. Inhibitors of the proteasome or matrix metalloproteinases affected the peptide levels during differentiation, but inhibitors of autophagy or peptidases did not. 18O was also incorporated into several choline metabolites including cytidine 5'-diphosphocholine (CDP-choline), glycerophosphocholine, and several phosphatidylcholine species, indicative of phosphatidylcholine synthesis/degradation and of flux through the CDP-choline cycle, a hallmark of proliferating cells. 18O-Tracer metabolomics further showed metabolic labeling of glutamate, suggestive of glutaminolysis, also characteristic of proliferating cells. Together, these results highlight the utility of 18O isotope labeling in combination with metabolomics to uncover changes in cellular metabolism that are not detectable by time-resolved metabolomics.脂肪生成（adipogenesis），即前体细胞向成熟脂肪细胞的分化过程，长期以来都是备受关注的研究领域，而肥胖患病率的攀升进一步提升了该领域的研究热度。尽管我们对脂肪生成及其细胞层面调控机制的认知正不断深化，但仍有诸多问题尚未解答，尤其是在代谢组的调控方面。3T3-L1细胞系是目前表征最为完善的脂肪生成细胞模型。本研究采用时间序列代谢组学方法，发现3T3-L1前体脂肪细胞的代谢组在分化初始的48小时内发生了显著改变——此阶段细胞会经历约两轮细胞分裂，即有丝分裂克隆扩增（mitotic clonal expansion）过程。短链肽是有丝分裂克隆扩增过程中上调的多种小分子之一。其他与蛋白质周转相关的标志物也出现上调，包括含血红素蛋白质的降解产物胆红素，以及无法被重新用于蛋白质合成的翻译后修饰氨基酸3-甲基组氨酸。为探究这些短链肽的来源，我们对分化中的前体脂肪细胞施加了氧18（¹⁸O）标记水，结果发现¹⁸O被整合到短链肽中，这至少在一定程度上证实了这些肽是水解产物。蛋白酶体或基质金属蛋白酶的抑制剂会影响分化过程中的肽水平，而自噬或肽酶的抑制剂则无此效果。¹⁸O还被整合到多种胆碱代谢物中，包括胞苷5'-二磷酸胆碱（CDP-choline）、甘油磷酸胆碱以及多种磷脂酰胆碱亚型，这表明磷脂酰胆碱的合成与降解以及通过CDP-胆碱循环的代谢流发生了改变，而该循环是增殖细胞的标志性特征之一。¹⁸O示踪代谢组学还显示谷氨酸出现了代谢标记，这提示存在谷氨酰胺分解途径，这也是增殖细胞的典型特征。综上，本研究结果表明，将¹⁸O同位素标记与代谢组学相结合，能够揭示时间分辨代谢组学无法检测到的细胞代谢变化，凸显了该方法的应用价值。