Accurate measurement of pancreatic islet p-cell mass using a second-generation fluorescent exendin-4 analog

摘要

The hallmark of type 1 diabetes is autoimmune destruction of the insulin-producing p-cells of the pancreatic islets. Autoimmune diabetes has been difficult to study or treat because it is not usually diagnosed until substantial p-cell loss has already occurred. Imaging agents that permit noninvasive visualization of changes in p-cell mass remain a high-priority goal. We report on the development and testing of a near-infrared fluorescent p-cell imaging agent. Based on the amino acid sequence of exendin-4, we created a neo-peptide via introduction of an unnatural amino acid at the K_(12) position, which could subsequently be conjugated to f luorophores via bioorthogonal copper-catalyzed click-chemistry. Cell assays confirmed that the resulting fluorescent probe (E4_(x12)-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 p-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 p-cell mass in mice treated with strep-tozotocin, a β-cell toxin. Individual islets could also be visualized by endoscopic imaging. In short, E4_(x12)-VT750 showed strong and selective binding to glucose-like peptide-1 receptors and permitted accurate measurement of p-cell mass in both diabetic and nondia-betic 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.