title: Accurate measurement of pancreatic islet beta-cell mass using a second-generation fluorescent exendin-4 analog.
abstract: 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.
authors.affiliation: Center for Systems Biology, Massachusetts General Hospital, Boston, MA 02114, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Reiner
authors.forename: Greg
authors.initials: G
authors.lastname: Thurber
authors.forename: Jason
authors.initials: J
authors.lastname: Gaglia
authors.forename: Claudio
authors.initials: C
authors.lastname: Vinegoni
authors.forename: Chong Wee
authors.initials: CW
authors.lastname: Liew
authors.forename: Rabi
authors.initials: R
authors.lastname: Upadhyay
authors.forename: Rainer H
authors.initials: RH
authors.lastname: Kohler
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Rohit N
authors.initials: RN
authors.lastname: Kulkarni
authors.forename: Christophe
authors.initials: C
authors.lastname: Benoist
authors.forename: Diane
authors.initials: D
authors.lastname: Mathis
authors.forename: Ralph
authors.initials: R
authors.lastname: Weissleder
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D003922:Diabetes Mellitus, Type 1 / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism; D000077270:Exenatide; D005456:Fluorescent Dyes / Q000737:chemistry; D000067757:Glucagon-Like Peptide-1 Receptor; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D010535:Laparoscopy / Q000295:instrumentation / Q000379:methods; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D010179:Pancreas / Q000378:metabolism / Q000473:pathology; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018027:Receptors, Glucagon / Q000235:genetics / Q000378:metabolism; D013311:Streptozocin / Q000633:toxicity; D014688:Venoms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; C000593833:GLP1R protein, human; C000593834:Glp1r protein, mouse; D000067757:Glucagon-Like Peptide-1 Receptor; D010455:Peptides; D018027:Receptors, Glucagon; D014688:Venoms; D049452:Green Fluorescent Proteins; D013311:Streptozocin; D000077270:Exenatide; D008239:Lysine