Center for Systems Biology, Massachusetts General Hospital, Boston, MA 02114, USA.
Proc Natl Acad Sci U S A. 2011 Aug 2;108(31):12815-20. doi: 10.1073/pnas.1109859108. Epub 2011 Jul 18.
The hallmark of type 1 diabetes is autoimmune destruction of the insulin-producing β-cells of the pancreatic islets. Autoimmune diabetes has been difficult to study or treat because it is not usually diagnosed until substantial β-cell loss has already occurred. Imaging agents that permit noninvasive visualization of changes in β-cell mass remain a high-priority goal. We report on the development and testing of a near-infrared fluorescent β-cell imaging agent. Based on the amino acid sequence of exendin-4, we created a neopeptide via introduction of an unnatural amino acid at the K(12) position, which could subsequently be conjugated to fluorophores via bioorthogonal copper-catalyzed click-chemistry. Cell assays confirmed that the resulting fluorescent probe (E4(×12)-VT750) had a high binding affinity (~3 nM). Its in vivo properties were evaluated using high-resolution intravital imaging, histology, whole-pancreas visualization, and endoscopic imaging. According to intravital microscopy, the probe rapidly bound to β-cells and, as demonstrated by confocal microscopy, it was internalized. Histology of the whole pancreas showed a close correspondence between fluorescence and insulin staining, and there was an excellent correlation between imaging signals and β-cell mass in mice treated with streptozotocin, a β-cell toxin. Individual islets could also be visualized by endoscopic imaging. In short, E4(×12)-VT750 showed strong and selective binding to glucose-like peptide-1 receptors and permitted accurate measurement of β-cell mass in both diabetic and nondiabetic mice. This near-infrared imaging probe, as well as future radioisotope-labeled versions of it, should prove to be important tools for monitoring diabetes, progression, and treatment in both experimental and clinical contexts.
1 型糖尿病的标志是胰岛中产生胰岛素的β细胞的自身免疫性破坏。由于自身免疫性糖尿病通常在发生大量β细胞损失后才被诊断出来，因此一直难以研究或治疗。仍需要开发可用于非侵入性可视化β细胞量变化的成像剂。我们报告了一种近红外荧光β细胞成像剂的开发和测试。基于 exendin-4 的氨基酸序列，我们通过在 K(12)位置引入非天然氨基酸来创建一个新肽，然后可以通过生物正交铜催化点击化学将其与荧光团缀合。细胞测定证实，所得荧光探针（E4(×12)-VT750）具有高结合亲和力（约 3 nM）。其体内特性通过高分辨率活体成像、组织学、全胰腺可视化和内镜成像进行了评估。根据活体显微镜检查，该探针迅速与β细胞结合，并且如共聚焦显微镜所示，它被内化。整个胰腺的组织学显示荧光与胰岛素染色之间存在密切对应关系，并且在用β细胞毒素链脲佐菌素处理的小鼠中，成像信号与β细胞量之间存在极好的相关性。通过内镜成像也可以观察到单个胰岛。总之，E4(×12)-VT750 对葡萄糖样肽-1 受体表现出强烈且选择性的结合，并允许在糖尿病和非糖尿病小鼠中准确测量β细胞量。这种近红外成像探针以及未来的放射性同位素标记版本，应该在实验和临床环境中成为监测糖尿病进展和治疗的重要工具。
