A recombinant proinsulin-transferrin fusion protein (ProINS-Tf) has been previously reported to be a novel long-lasting INS analog, acting specifically on the inhibition of hepatic glucose output. In this study, we investigated the biodistribution, activation and tissue retention of ProINS-Tf to elucidate its liver targeted anti-diabetic mechanism. The biodistribution study revealed that ProINS-Tf exhibited liver specific accumulation after a single intravenous injection, whereas transferrin (Tf) or insulin (INS) showed relatively even distribution among different organs. The conversion of inactive ProINS-Tf into an active immune-reactive INS-Tf form (irINS-Tf) via a Tf receptor (TfR) mediated process only occurred in the liver, but not in other organs. In addition, ProINS-Tf demonstrated a prolonged retention in the liver after an intravenous injection, suggesting the enhanced association of the bifunctional active form, irINS-Tf, within liver cells. Taken together, our results indicate that ProINS-Tf is a highly liver-targeted INS prodrug with a combination of 3 specific actions in liver cells: (1) TfR-mediated binding and uptake of the prodrug on the cell surface, (2) liver-specific, TfR-mediated conversion of the prodrug into its active form, and (3) the bifunctional binding of the active fusion protein to both Tf and INS receptors in the liver to achieve prolonged retention and thus enhanced anti-diabetic activities.

重组胰岛素原-转铁蛋白融合蛋白（ProINS-Tf）先前已被报道是一种新型的长效INS类似物，专门作用于抑制肝葡萄糖输出。在这项研究中，我们研究了ProINS-Tf的生物分布，活化和组织保留，以阐明其肝脏靶向的抗糖尿病机制。生物分布研究表明，单次静脉内注射后，ProINS-Tf表现出肝特异性积累，而转铁蛋白（Tf）或胰岛素（INS）在不同器官之间表现出相对均匀的分布。通过Tf受体（TfR）介导的过程将无活性的ProINS-Tf转化为有免疫活性的INS-Tf形式（irINS-Tf）仅发生在肝脏中，而没有发生在其他器官中。此外，ProINS-Tf在静脉内注射后显示在肝脏中的保留时间延长，表明肝细胞内双功能活性形式irINS-Tf的缔合增强。两者合计，我们的结果表明ProINS-Tf是高度靶向肝脏的INS前药，在肝细胞中具有以下3种特定作用的组合：（1）TfR介导的结合和前药在细胞表面的摄取，（2）肝特异性，TfR介导的前药转化为其活性形式，以及（3）活性融合蛋白与肝脏中的Tf和INS受体双功能结合，从而延长了保留时间，从而增强了抗糖尿病活性。