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

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 108; no. 31; pp. 12815 - 12820
• Main Authors: Reiner, Thomas, Thurber, Greg, Gaglia, Jason, Vinegoni, Claudio, Liew, Chong Wee, Upadhyay, Rabi, Kohler, Rainer H., Li, Li, Kulkarni, Rohit N., Benoist, Christophe, Mathis, Diane, Weissleder, Ralph
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 02-08-2011; National Acad Sciences
• Subjects: 3T3 cells; Amino Acid Sequence; Animals; Average linear density; Binding Sites - genetics; Biological Sciences; Cell Line, Tumor; Diabetes Mellitus, Type 1 - chemically induced; Diabetes Mellitus, Type 1 - genetics; Diabetes Mellitus, Type 1 - metabolism; Fluorescence; Fluorescent Dyes - chemistry; Glucagon-Like Peptide-1 Receptor; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HEK293 Cells; Histology; Humans; Imaging; Insulin-Secreting Cells - drug effects; Insulin-Secreting Cells - metabolism; Insulin-Secreting Cells - pathology; Islets of Langerhans; Laparoscopy - instrumentation; Laparoscopy - methods; Lysine - chemistry; Lysine - genetics; Lysine - metabolism; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Confocal; Microscopy, Fluorescence; Molecular Sequence Data; NIH 3T3 Cells; Pancreas; Pancreas - metabolism; Pancreas - pathology; Peptides - chemistry; Peptides - genetics; Peptides - metabolism; Receptors; Receptors, Glucagon - genetics; Receptors, Glucagon - metabolism; Streptozocin - toxicity; Type 1 diabetes mellitus; Venoms - chemistry; Venoms - genetics; Venoms - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1109859108

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 K12 position, which could subsequently be conjugated to fluorophores via bioorthogonal copper-catalyzed click-chemistry. Cell assays confirmed that the resulting fluorescent probe (E4X12-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, E4X12-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.