Proline exacerbates hepatic gluconeogenesis via paraspeckle-dependent mRNA retention [RIP-Seq]

Type 2 Diabetes (T2D) is a global health issue characterized by abnormal blood glucose levels and often associated with excessive hepatic gluconeogenesis. Increased circulating non-essential amino acids (NEAAs) are consistently observed in T2D individuals; however, the specific contribution of each amino acid to T2D pathogenesis remains less understood. Here, we reported an unexpected role of the NEAA proline in coordinating hepatic glucose metabolism by modulating paraspeckle, a nuclear structure scaffolded by the long noncoding RNA Neat1. Mechanistically, proline diminished paraspeckles in hepatocytes, liberating the retained mRNA species into cytoplasm for translation, including the mRNAs of Ppargc1a and Foxo1, contributing to enhanced gluconeogenesis and hyperglycemia. We further demonstrated that the proline-paraspeckle-mRNA retention axis existed in diabetic liver samples, and intervening this axis via paraspeckle restoration significantly alleviated hyperglycemia in both female and male diabetic mouse models. Collectively, our results not only delineated a previously unappreciated proline-instigated, paraspeckle-dependent mRNA retention mechanism regulating gluconeogenesis, but also spotlighted proline and paraspeckle as potential targets for managing hyperglycemia. Overall design: To investigate whether proline stimulation influences mRNA dynamics of paraspeckle in mouse primary hepatocytes (MPH), and the MPH are treated with or without proline treatment. We than used RNA-immunoprecipitation coupled with high-throughput sequencing (RIP-seq) to profile the paraspeckle-retained mRNAs using NONO as a surrogate for paraspeckles.

2型糖尿病（Type 2 Diabetes, T2D）是一类全球性健康难题，以血糖水平异常为核心特征，常伴随肝脏过度糖异生现象。临床中持续观测到，2型糖尿病患者的循环非必需氨基酸（non-essential amino acids, NEAAs）水平升高，但各类氨基酸对2型糖尿病发病机制的具体贡献仍有待深入解析。本研究揭示了非必需氨基酸脯氨酸通过调控由长链非编码RNA（long noncoding RNA）Neat1搭建支架的核结构——旁斑（paraspeckle），进而协调肝脏葡萄糖代谢的全新功能。机制层面，脯氨酸可减少肝细胞内的旁斑数量，将滞留于核内的多种mRNA释放至细胞质进行翻译，其中包括Ppargc1a与Foxo1的mRNA，最终促成糖异生增强与高血糖的发生。研究团队进一步证实，在糖尿病患者的肝脏样本中存在“脯氨酸-旁斑-mRNA滞留”调控轴；通过恢复旁斑功能干预该调控轴，可显著缓解雌性与雄性糖尿病小鼠模型的高血糖症状。综上，本研究不仅阐明了一条此前未被认知的、由脯氨酸触发且依赖旁斑的mRNA滞留调控通路，该通路可调节肝脏糖异生过程，同时也明确了脯氨酸与旁斑可作为干预高血糖的潜在治疗靶点。总体实验设计：为探究脯氨酸刺激是否会影响小鼠原代肝细胞（mouse primary hepatocytes, MPH）内旁斑相关的mRNA动态变化，我们将小鼠原代肝细胞分为脯氨酸处理组与空白对照组。随后，以NONO作为旁斑的标志物，采用RNA免疫共沉淀结合高通量测序（RNA-immunoprecipitation coupled with high-throughput sequencing, RIP-seq）技术，对旁斑滞留的mRNA进行转录组谱分析。