A Novel Insulin Receptor-Binding Protein from Momordica charantia Enhances Glucose Uptake and Glucose Clearance in Vitro and in Vivo through Triggering Insulin Receptor Signaling Pathway

Diabetes, a common metabolic disorder, is characterized by hyperglycemia. Insulin is the principal mediator of glucose homeostasis. In a previous study, we identified a trypsin inhibitor, named Momordica charantia insulin receptor (IR)-binding protein (mcIRBP) in this study, that might interact with IR. The physical and functional interactions between mcIRBP and IR were clearly analyzed in the present study. Photo-cross-linking coupled with mass spectrometry showed that three regions (17–21, 34–40, and 59–66 residues) located on mcIRBP physically interacted with leucine-rich repeat domain and cysteine-rich region of IR. IR-binding assay showed that the binding behavior of mcIRBP and insulin displayed a cooperative manner. After binding to IR, mcIRBP activated the kinase activity of IR by (5.87 ± 0.45)-fold, increased the amount of phospho-IR protein by (1.31 ± 0.03)-fold, affected phosphoinositide-3-kinase/Akt pathways, and consequently stimulated the uptake of glucose in 3T3-L1 cells by (1.36 ± 0.12)-fold. Intraperitoneal injection of 2.5 nmol/kg mcIRBP significantly decreased the blood glucose levels by 20.9 ± 3.2% and 10.8 ± 3.6% in normal and diabetic mice, respectively. Microarray analysis showed that mcIRBP affected genes involved in insulin signaling transduction pathway in mice. In conclusion, our findings suggest that mcIRBP is a novel IRBP that binds to sites different from the insulin-binding sites on IR and stimulates both the glucose uptake in cells and the glucose clearance in mice.糖尿病作为一种常见的代谢紊乱性疾病，以高血糖为核心特征。胰岛素是维持血糖稳态的关键介质。在前期研究中，我们鉴定出一种胰蛋白酶抑制剂，本研究将其命名为苦瓜胰岛素受体（insulin receptor, IR）结合蛋白（Momordica charantia insulin receptor-binding protein, mcIRBP），该蛋白可能与IR存在相互作用。本研究对mcIRBP与IR的物理相互作用及功能相互作用进行了系统解析。光交联（photo-cross-linking）耦合质谱（mass spectrometry）分析显示，mcIRBP上的三个区域（氨基酸残基17–21、34–40及59–66）可与IR的富亮氨酸重复结构域（leucine-rich repeat domain）及富半胱氨酸区域（cysteine-rich region）发生物理结合。IR结合实验结果表明，mcIRBP与胰岛素的结合行为呈现协同效应。mcIRBP与IR结合后，可将IR的激酶活性提升至原水平的(5.87 ± 0.45)倍，使磷酸化IR蛋白的表达量提升至(1.31 ± 0.03)倍，同时调控磷脂酰肌醇3-激酶（phosphoinositide-3-kinase, PI3K）/Akt通路，最终将3T3-L1细胞的葡萄糖摄取量提升至(1.36 ± 0.12)倍。以2.5 nmol/kg的剂量对正常小鼠及糖尿病小鼠进行腹腔注射mcIRBP后，二者的血糖水平分别显著降低了(20.9 ± 3.2)%与(10.8 ± 3.6)%。基因芯片（microarray）分析结果显示，mcIRBP可调控小鼠体内参与胰岛素信号转导通路的相关基因表达。综上，本研究结果表明，mcIRBP是一种新型IR结合蛋白，其在IR上的结合位点与胰岛素的结合位点存在差异，既可促进细胞摄取葡萄糖，又能增强小鼠体内的葡萄糖清除能力。