title: Fluorescent protein vectors for pancreatic islet cell identification in live-cell imaging.
abstract: The islets of Langerhans contain different types of endocrine cells, which are crucial for glucose homeostasis. β- and α-cells that release insulin and glucagon, respectively, are most abundant, whereas somatostatin-producing δ-cells and particularly pancreatic polypeptide-releasing PP-cells are more scarce. Studies of islet cell function are hampered by difficulties to identify the different cell types, especially in live-cell imaging experiments when immunostaining is unsuitable. The aim of the present study was to create a set of vectors for fluorescent protein expression with cell-type-specific promoters and evaluate their applicability in functional islet imaging. We constructed six adenoviral vectors for expression of red and green fluorescent proteins controlled by the insulin, preproglucagon, somatostatin, or pancreatic polypeptide promoters. After transduction of mouse and human islets or dispersed islet cells, a majority of the fluorescent cells also immunostained for the appropriate hormone. Recordings of the sub-plasma membrane Ca(2+) and cAMP concentrations with a fluorescent indicator and a protein biosensor, respectively, showed that labeled cells respond to glucose and other modulators of secretion and revealed a striking variability in Ca(2+) signaling among α-cells. The measurements allowed comparison of the phase relationship of Ca(2+) oscillations between different types of cells within intact islets. We conclude that the fluorescent protein vectors allow easy identification of specific islet cell types and can be used in live-cell imaging together with organic dyes and genetically encoded biosensors. This approach will facilitate studies of normal islet physiology and help to clarify molecular defects and disturbed cell interactions in diabetic islets.
authors.affiliation: Department of Medical Cell Biology, Uppsala University, Biomedical Centre, Box 571, SE-751 23, Uppsala, Sweden.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Shuai
authors.affiliation: Department of Medical Cell Biology, Uppsala University, Biomedical Centre, Box 571, SE-751 23, Uppsala, Sweden.
authors.forename: Yunjian
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Department of Medical Cell Biology, Uppsala University, Biomedical Centre, Box 571, SE-751 23, Uppsala, Sweden.
authors.forename: Qian
authors.initials: Q
authors.lastname: Yu
authors.affiliation: Department of Medical Cell Biology, Uppsala University, Biomedical Centre, Box 571, SE-751 23, Uppsala, Sweden.
authors.forename: Erik
authors.initials: E
authors.lastname: Gylfe
authors.affiliation: Department of Medical Cell Biology, Uppsala University, Biomedical Centre, Box 571, SE-751 23, Uppsala, Sweden. Anders.Tengholm@mcb.uu.se.
authors.forename: Anders
authors.identifier: 0000-0003-4508-0836
authors.initials: A
authors.lastname: Tengholm
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D020013:Calcium Signaling; D005260:Female; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence / Q000379:methods; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein
keywords: Ca2+; Glucagon; Insulin; Islets; PP-cell; Pancreatic polypeptide; Somatostatin; cAMP; α-cell; β-cell; δ-cell