Spatial hepatocyte plasticity of gluconeogenic gene expression and gluconeogenic activity during the metabolic transitions between fed, fasted and starvation states [targeted scRNA-seq]. Spatial hepatocyte plasticity of gluconeogenic gene expression and gluconeogenic activity during the metabolic transitions between fed, fasted and starvation states [targeted scRNA-seq]

Single cell analyses of hepatocytes across the liver lobule demonstrated that gluconeogenic gene expression (Pck1 and G6pc) is relatively low in the fed state and gradually increase first in the periportal hepatocytes during the initial fasting period. As the time of fasting progresses, pericentral hepatocyte gluconeogenic gene expression increases and following entry into the starvation state the pericentral hepatocytes are not significantly different than the periportal hepatocytes. Similarly, pyruvate-dependent gluconeogenic activity is approximately 10-fold higher in the periportal hepatocytes during the initial fasting states but with only 1.5-fold different between the pericentral and periportal hepatocytes in the starvation state. In parallel, starvation induced a reduction of canonical beta-catenin signaling and redistribution of pericentral and periportal glutamine synthetase and glutaminase resulting in an enhanced pericentral glutamine-dependent gluconeogenesis. These data demonstrate that hepatocyte gluconeogenic gene expression and gluconeogenic activity are highly spatially and temporally plastic across the liver lobule and underscore the critical importance of using well-defined feeding and fasting times to define the basis of hepatic insulin resistance and glucose production. Overall design: Mice were trained for 3 days by removing food at 5PM and feeding at 9:00 the next day (16h fasting overnight and 8h feeding daytime). On the day of experiment, mice were provided food with drinking water that contained 20% of sucrose. The fed (0h) mice were sacrificed at 13:00 equal to 4h of feeding (0h). For fasted mice, the food and sucrose water were taken out at 13:00, then sacrificed at 21:00 for fast 8h (8h), 5:00 the next day for fast16h (16h), 13:00 the next day for fast 24h (24h), and 19:00 the next day for fast 30h (30h).

针对全肝小叶（liver lobule）内的肝细胞（hepatocytes）进行的单细胞分析（single cell analyses）显示，糖异生基因表达（gluconeogenic gene expression，Pck1与G6pc）在进食状态下相对较低，并在空腹初期率先在门周肝细胞（periportal hepatocytes）中逐渐上调。随着空腹时长延长，中央周肝细胞（pericentral hepatocytes）的糖异生基因表达逐步升高；当进入饥饿状态后，中央周与门周肝细胞的糖异生基因表达已无显著差异。
类似地，在空腹初期，门周肝细胞的丙酮酸依赖性糖异生活性（pyruvate-dependent gluconeogenic activity）约为中央周肝细胞的10倍；而在饥饿状态下，两者的活性差异仅为1.5倍左右。
与此同时，饥饿状态可抑制经典β-连环蛋白（beta-catenin）信号通路，并使中央周与门周区域的谷氨酰胺合成酶（glutamine synthetase）及谷氨酰胺酶（glutaminase）发生重新分布，进而增强中央周肝细胞的谷氨酰胺依赖性糖异生过程。
上述数据表明，肝小叶内肝细胞的糖异生基因表达与糖异生活性具有显著的时空可塑性；同时也凸显了采用明确的进食与空腹时长，来解析肝脏胰岛素抵抗（hepatic insulin resistance）与葡萄糖生成机制的关键重要性。
整体实验设计：
研究人员对小鼠进行3天适应性训练：每日下午5点移除食物，次日上午9点进行喂食（夜间空腹16小时，日间进食8小时）。实验当日，小鼠可自由进食并饮用含20%蔗糖的饮用水。进食组（0h）小鼠于当日13:00处死，此时已进食4小时（对应0h组）。
对于空腹组小鼠，于当日13:00移除食物与蔗糖饮用水，分别于以下时间点处死：当日21:00（空腹8小时，即8h组）、次日5:00（空腹16小时，即16h组）、次日13:00（空腹24小时，即24h组）以及次日19:00（空腹30小时，即30h组）。