Global Phosphoproteomic Analysis of Insulin/Akt/mTORC1/S6K Signaling in Rat Hepatocytes

Insulin resistance is a hallmark
of type 2 diabetes. Although multiple
genetic and physiological factors interact to cause insulin resistance,
deregulated signaling by phosphorylation is a common underlying mechanism.
In particular, the specific phosphorylation-dependent regulatory mechanisms
and signaling outputs of insulin are poorly understood in hepatocytes,
which represents one of the most important insulin-responsive cell
types. Using primary rat hepatocytes as a model system, we performed
reductive dimethylation (ReDi)-based quantitative mass spectrometric
analysis and characterized the phosphoproteome that is regulated by
insulin as well as its key downstream kinases including Akt, mTORC1,
and S6K. We identified a total of 12 294 unique, confidently
localized phosphorylation sites and 3805 phosphorylated proteins in
this single cell type. Detailed bioinformatic analysis on each individual
data set identified both known and previously unrecognized targets
of this key insulin downstream effector pathway. Furthermore, integrated
analysis of the hepatic Akt/mTORC1/S6K signaling axis allowed the
delineation of the substrate specificity of several close-related
kinases within the insulin signaling pathway. We expect that the data
sets will serve as an invaluable resource, providing the foundation
for future hypothesis-driven research that helps delineate the molecular
mechanisms that underlie the pathogenesis of type 2 diabetes and related
metabolic syndrome.

胰岛素抵抗是2型糖尿病的标志性病理特征。尽管多种遗传与生理因素相互作用可诱发胰岛素抵抗，但磷酸化信号通路失调是其普遍存在的核心潜在分子机制。具体而言，作为机体最重要的胰岛素响应细胞类型之一的肝细胞中，胰岛素介导的磷酸化特异性调控机制与信号输出模式仍未得到充分阐释。本研究以原代大鼠肝细胞为模型体系，采用基于还原二甲基化（reductive dimethylation, ReDi）的定量质谱分析技术，系统表征了胰岛素及其关键下游激酶（包括Akt、mTORC1与S6K）所调控的磷酸化蛋白质组（phosphoproteome）。本研究在该单一细胞类型中共鉴定得到12294个定位置信度可靠的独特磷酸化位点，以及3805个磷酸化蛋白质。对各独立数据集的详细生物信息学分析，不仅鉴定出该关键胰岛素下游效应通路的已知靶标，还发现了此前未被报道的全新靶标。此外，通过对肝脏Akt/mTORC1/S6K信号轴的整合分析，本研究明确了胰岛素信号通路中数种近缘激酶的底物特异性。本研究团队期望本数据集可作为极具价值的研究资源，为未来基于假说驱动的相关研究奠定基础，助力阐明2型糖尿病及相关代谢综合征的发病分子机制。