Circadian temporal profiling of MMH-D3 hepatocytes

The circadian clock generates daily rhythms in mammalian liver processes, such as glucose and lipid homeostasis, xenobiotic metabolism, and regeneration. The mechanisms governing these rhythms are not well understood, particularly the distinct contributions of the cell-autonomous clock and central pacemaker to rhythmic liver physiology. Through microarray expression profiling in MMH-D3 hepatocytes, we identified over 1,000 transcripts that exhibit circadian oscillations, demonstrating that many rhythms can be driven by the cell-autonomous clock and that MMH-D3 is a valid circadian model system. The genes represented by these circadian transcripts displayed both co-phasic and anti-phasic organization within a protein-protein interaction network, suggesting the existence of competition for binding sites or partners by genes of disparate transcriptional phases. Multiple pathways displayed enrichment in MMH-D3 circadian transcripts, including the polyamine synthesis module of the glutathione metabolic pathway. The polyamine synthesis module, which is highly associated with cell proliferation and whose products are required for initiation of liver regeneration, includes enzymes whose transcripts exhibit circadian oscillations, such as ornithine decarboxylase (Odc1) and spermidine synthase (Srm). Metabolic profiling revealed that the enzymatic product of SRM, spermidine, cycles as well. Thus, the cell-autonomous hepatocyte clock can drive a significant amount of transcriptional rhythms and orchestrate physiologically relevant modules such as polyamine synthesis. Samples were collected every 2 hours for a duration of 46 hours from differentiated MMH-D3 hepatocytes synchronized via serum shock. Cells were synchronized by serum shock and incubated for 12 hours, and then samples were collected every 2 hours from 12 hours post-serum shock to 58 hours post-serum shock, for a total of 24 samples.

哺乳动物肝脏的诸多生理过程（如葡萄糖与脂质稳态、异生物质代谢及组织再生）均由昼夜节律钟（circadian clock）调控产生每日节律。目前学界对这些节律的调控机制尚不完全明晰，尤其是细胞自主生物钟（cell-autonomous clock）与中枢起搏点（central pacemaker）对肝脏节律性生理功能的差异化贡献。研究团队通过对MMH-D3肝细胞开展微阵列表达谱分析（microarray expression profiling），鉴定出逾1000个呈现昼夜节律振荡（circadian oscillations）的转录本（transcripts），证实诸多节律可由细胞自主生物钟驱动，且MMH-D3细胞是一套可靠的昼夜节律模型系统。这些节律性转录本所对应的基因在蛋白质相互作用网络（protein-protein interaction network）中同时呈现同相（co-phasic）与反相（anti-phasic）的组织模式，提示转录时相迥异的基因之间可能存在结合位点或结合伴侣的竞争关系。多个通路在MMH-D3细胞的节律性转录本中显著富集，其中包括谷胱甘肽代谢通路（glutathione metabolic pathway）中的多胺合成模块（polyamine synthesis module）。该多胺合成模块与细胞增殖（cell proliferation）高度相关，其产物是肝脏再生（liver regeneration）启动所必需的，模块内的酶编码转录本（如鸟氨酸脱羧酶（Odc1）与亚精胺合酶（Srm））均呈现昼夜节律振荡。代谢组分析（metabolic profiling）进一步发现，Srm的酶促产物亚精胺（spermidine）同样存在节律性波动。综上，肝细胞的细胞自主生物钟可驱动大量转录节律，并协调多胺合成这类具有生理意义的功能模块。本研究通过血清休克法同步化分化型MMH-D3肝细胞，将细胞培养12小时后，自血清休克后12小时起，每2小时采集一次样本，直至休克后58小时，总采样时长为46小时，最终共获取24份样本。