title: COPII-Dependent ER Export: A Critical Component of Insulin Biogenesis and β-Cell ER Homeostasis.
abstract: Pancreatic β-cells possess a highly active protein synthetic and export machinery in the endoplasmic reticulum (ER) to accommodate the massive production of proinsulin. ER homeostasis is vital for β-cell functions and is maintained by the delicate balance between protein synthesis, folding, export, and degradation. Disruption of ER homeostasis by diabetes-causing factors leads to β-cell death. Among the 4 components to maintain ER homeostasis in β-cells, the role of ER export in insulin biogenesis is the least understood. To address this knowledge gap, the present study investigated the molecular mechanism of proinsulin ER export in MIN6 cells and primary islets. Two inhibitory mutants of the secretion-associated RAS-related protein (Sar)1 small GTPase, known to specifically block coat protein complex II (COPII)-dependent ER export, were overexpressed in β-cells using recombinant adenoviruses. Results from this approach, as well as small interfering RNA-mediated Sar1 knockdown, demonstrated that defective Sar1 function blocked proinsulin ER export and abolished its conversion to mature insulin in MIN6 cells, isolated mouse, and human islets. It is further revealed, using an in vitro vesicle formation assay, that proinsulin was packaged into COPII vesicles in a GTP- and Sar1-dependent manner. Blockage of COPII-dependent ER exit by Sar1 mutants strongly induced ER morphology change, ER stress response, and β-cell apoptosis. These responses were mediated by the PKR (double-stranded RNA-dependent kinase)-like ER kinase (PERK)/eukaryotic translation initiation factor 2α (p-eIF2α) and inositol-requiring protein 1 (IRE1)/x-box binding protein 1 (Xbp1) pathways but not via activating transcription factor 6 (ATF6). Collectively, results from the study demonstrate that COPII-dependent ER export plays a vital role in insulin biogenesis, ER homeostasis, and β-cell survival.
authors.affiliation: Department of Physiology (J.F., B.P.J., F.S., J.W., T.B., X.C.) and Center for Molecular Medicine and Genetics (X.Z., K.Z.), School of Medicine, Department of Physics and Astronomy (T.S.), College of Liberal Arts and Sciences, and Department of Pharmaceutical Sciences (A.K.), Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, and John D. Dingell VA Medical Center (A.K.), Detroit, Michigan 48201; Department of Internal Medicine (M.L.), University of Michigan, Ann Arbor, Michigan 48109; and Department of Pathology (D.J.T.), Microscopy Imaging Center, University of Vermont College of Medicine, Burlington, Vermont 05405.
authors.forename: Jingye
authors.initials: J
authors.lastname: Fang
authors.affiliation: Department of Physiology (J.F., B.P.J., F.S., J.W., T.B., X.C.) and Center for Molecular Medicine and Genetics (X.Z., K.Z.), School of Medicine, Department of Physics and Astronomy (T.S.), College of Liberal Arts and Sciences, and Department of Pharmaceutical Sciences (A.K.), Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, and John D. Dingell VA Medical Center (A.K.), Detroit, Michigan 48201; Department of Internal Medicine (M.L.), University of Michigan, Ann Arbor, Michigan 48109; and Department of Pathology (D.J.T.), Microscopy Imaging Center, University of Vermont College of Medicine, Burlington, Vermont 05405.
authors.forename: Ming
authors.initials: M
authors.lastname: Liu
authors.affiliation: Department of Physiology (J.F., B.P.J., F.S., J.W., T.B., X.C.) and Center for Molecular Medicine and Genetics (X.Z., K.Z.), School of Medicine, Department of Physics and Astronomy (T.S.), College of Liberal Arts and Sciences, and Department of Pharmaceutical Sciences (A.K.), Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, and John D. Dingell VA Medical Center (A.K.), Detroit, Michigan 48201; Department of Internal Medicine (M.L.), University of Michigan, Ann Arbor, Michigan 48109; and Department of Pathology (D.J.T.), Microscopy Imaging Center, University of Vermont College of Medicine, Burlington, Vermont 05405.
authors.forename: Xuebao
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Department of Physiology (J.F., B.P.J., F.S., J.W., T.B., X.C.) and Center for Molecular Medicine and Genetics (X.Z., K.Z.), School of Medicine, Department of Physics and Astronomy (T.S.), College of Liberal Arts and Sciences, and Department of Pharmaceutical Sciences (A.K.), Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, and John D. Dingell VA Medical Center (A.K.), Detroit, Michigan 48201; Department of Internal Medicine (M.L.), University of Michigan, Ann Arbor, Michigan 48109; and Department of Pathology (D.J.T.), Microscopy Imaging Center, University of Vermont College of Medicine, Burlington, Vermont 05405.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Sakamoto
authors.affiliation: Department of Physiology (J.F., B.P.J., F.S., J.W., T.B., X.C.) and Center for Molecular Medicine and Genetics (X.Z., K.Z.), School of Medicine, Department of Physics and Astronomy (T.S.), College of Liberal Arts and Sciences, and Department of Pharmaceutical Sciences (A.K.), Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, and John D. Dingell VA Medical Center (A.K.), Detroit, Michigan 48201; Department of Internal Medicine (M.L.), University of Michigan, Ann Arbor, Michigan 48109; and Department of Pathology (D.J.T.), Microscopy Imaging Center, University of Vermont College of Medicine, Burlington, Vermont 05405.
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Taatjes
authors.affiliation: Department of Physiology (J.F., B.P.J., F.S., J.W., T.B., X.C.) and Center for Molecular Medicine and Genetics (X.Z., K.Z.), School of Medicine, Department of Physics and Astronomy (T.S.), College of Liberal Arts and Sciences, and Department of Pharmaceutical Sciences (A.K.), Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, and John D. Dingell VA Medical Center (A.K.), Detroit, Michigan 48201; Department of Internal Medicine (M.L.), University of Michigan, Ann Arbor, Michigan 48109; and Department of Pathology (D.J.T.), Microscopy Imaging Center, University of Vermont College of Medicine, Burlington, Vermont 05405.
authors.forename: Bhanu P
authors.initials: BP
authors.lastname: Jena
authors.affiliation: Department of Physiology (J.F., B.P.J., F.S., J.W., T.B., X.C.) and Center for Molecular Medicine and Genetics (X.Z., K.Z.), School of Medicine, Department of Physics and Astronomy (T.S.), College of Liberal Arts and Sciences, and Department of Pharmaceutical Sciences (A.K.), Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, and John D. Dingell VA Medical Center (A.K.), Detroit, Michigan 48201; Department of Internal Medicine (M.L.), University of Michigan, Ann Arbor, Michigan 48109; and Department of Pathology (D.J.T.), Microscopy Imaging Center, University of Vermont College of Medicine, Burlington, Vermont 05405.
authors.forename: Fei
authors.initials: F
authors.lastname: Sun
authors.affiliation: Department of Physiology (J.F., B.P.J., F.S., J.W., T.B., X.C.) and Center for Molecular Medicine and Genetics (X.Z., K.Z.), School of Medicine, Department of Physics and Astronomy (T.S.), College of Liberal Arts and Sciences, and Department of Pharmaceutical Sciences (A.K.), Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, and John D. Dingell VA Medical Center (A.K.), Detroit, Michigan 48201; Department of Internal Medicine (M.L.), University of Michigan, Ann Arbor, Michigan 48109; and Department of Pathology (D.J.T.), Microscopy Imaging Center, University of Vermont College of Medicine, Burlington, Vermont 05405.
authors.forename: James
authors.initials: J
authors.lastname: Woods
authors.affiliation: Department of Physiology (J.F., B.P.J., F.S., J.W., T.B., X.C.) and Center for Molecular Medicine and Genetics (X.Z., K.Z.), School of Medicine, Department of Physics and Astronomy (T.S.), College of Liberal Arts and Sciences, and Department of Pharmaceutical Sciences (A.K.), Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, and John D. Dingell VA Medical Center (A.K.), Detroit, Michigan 48201; Department of Internal Medicine (M.L.), University of Michigan, Ann Arbor, Michigan 48109; and Department of Pathology (D.J.T.), Microscopy Imaging Center, University of Vermont College of Medicine, Burlington, Vermont 05405.
authors.forename: Tim
authors.initials: T
authors.lastname: Bryson
authors.affiliation: Department of Physiology (J.F., B.P.J., F.S., J.W., T.B., X.C.) and Center for Molecular Medicine and Genetics (X.Z., K.Z.), School of Medicine, Department of Physics and Astronomy (T.S.), College of Liberal Arts and Sciences, and Department of Pharmaceutical Sciences (A.K.), Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, and John D. Dingell VA Medical Center (A.K.), Detroit, Michigan 48201; Department of Internal Medicine (M.L.), University of Michigan, Ann Arbor, Michigan 48109; and Department of Pathology (D.J.T.), Microscopy Imaging Center, University of Vermont College of Medicine, Burlington, Vermont 05405.
authors.forename: Anjaneyulu
authors.initials: A
authors.lastname: Kowluru
authors.affiliation: Department of Physiology (J.F., B.P.J., F.S., J.W., T.B., X.C.) and Center for Molecular Medicine and Genetics (X.Z., K.Z.), School of Medicine, Department of Physics and Astronomy (T.S.), College of Liberal Arts and Sciences, and Department of Pharmaceutical Sciences (A.K.), Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, and John D. Dingell VA Medical Center (A.K.), Detroit, Michigan 48201; Department of Internal Medicine (M.L.), University of Michigan, Ann Arbor, Michigan 48109; and Department of Pathology (D.J.T.), Microscopy Imaging Center, University of Vermont College of Medicine, Burlington, Vermont 05405.
authors.forename: Kezhong
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Department of Physiology (J.F., B.P.J., F.S., J.W., T.B., X.C.) and Center for Molecular Medicine and Genetics (X.Z., K.Z.), School of Medicine, Department of Physics and Astronomy (T.S.), College of Liberal Arts and Sciences, and Department of Pharmaceutical Sciences (A.K.), Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State University, and John D. Dingell VA Medical Center (A.K.), Detroit, Michigan 48201; Department of Internal Medicine (M.L.), University of Michigan, Ann Arbor, Michigan 48109; and Department of Pathology (D.J.T.), Microscopy Imaging Center, University of Vermont College of Medicine, Burlington, Vermont 05405.
authors.forename: Xuequn
authors.initials: X
authors.lastname: Chen
mesh_terms: D000256:Adenoviridae / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D022181:COP-Coated Vesicles / Q000737:chemistry*; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005947:Glucose / Q000737:chemistry; D006706:Homeostasis; D006801:Humans; D007328:Insulin / Q000096:biosynthesis*; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism; D051379:Mice; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011384:Proinsulin / Q000378:metabolism; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism
chemical_list: D007328:Insulin; D034741:RNA, Small Interfering; D011384:Proinsulin; C493083:SAR1A protein, human; D020559:Monomeric GTP-Binding Proteins; D005947:Glucose