title: Detection of insulin granule exocytosis by an electrophysiology method with high temporal resolution reveals enlarged insulin granule pool in BIG3-knockout mice.
abstract: We recently identified BIG3 as a negative regulator of insulin granule biogenesis and reported increased insulin secretion in BIG3-knockout (BKO) mice. To pinpoint the site of action for BIG3, we investigated whether BIG3 regulates quantal insulin granule exocytosis. We established an assay to detect insulin granule exocytosis by recording ATP-elicited currents at high temporal resolution by patch clamp. Similarly to insulin, ATP release was increased in BKO β-cells. Although the frequency of insulin granule exocytosis was increased in BKO β-cells, quantal size or release kinetics remained unchanged. Electron microscopy studies showed that the number of insulin granules was increased by >60% in BKO β-cells. However, the number of morphologically docked granules was unaltered. The number of insulin granules having significant distances away from plasma membrane was greatly increased in BKO β-cells. Thus, BIG3 negatively regulates insulin granule exocytosis by restricting insulin granule biogenesis without the release kinetics of individual granules at the final exocytotic steps being affected. Depletion of BIG3 leads to an enlarged releasable pool of insulin granules, which accounts for increased release frequency and consequently increased insulin secretion.
authors.affiliation: Singapore Bioimaging Consortium.
authors.forename: Tao
authors.initials: T
authors.lastname: Liu
authors.affiliation: Singapore Bioimaging Consortium, Institute of Molecular and Cell Biology, and.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Li
authors.affiliation: Institute of Molecular and Cell Biology, and Joint IMB-IMCB Electron Microscopy Suite, Agency for Science, Technology and Research (A*STAR), Singapore;
authors.forename: Natalia V
authors.initials: NV
authors.lastname: Gounko
authors.affiliation: Institute of Molecular Medicine, Peking University, Peking, China; and.
authors.forename: Zhuan
authors.initials: Z
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Pharmaceutical Biotechnology, Department of Medicine, and Pharmacology and Pharmacy, The University of Hong Kong, Hong Kong, China.
authors.forename: Aimin
authors.initials: A
authors.lastname: Xu
authors.affiliation: Institute of Molecular and Cell Biology, and.
authors.forename: Wanjin
authors.initials: W
authors.lastname: Hong
authors.affiliation: Singapore Bioimaging Consortium, weiping_han@sbic.a-star.edu.sg.
authors.forename: Weiping
authors.initials: W
authors.lastname: Han
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology*; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008564:Membrane Potentials / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D018408:Patch-Clamp Techniques; D011506:Proteins / Q000235:genetics / Q000502:physiology*
chemical_list: D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; C443118:Wdr5 protein, mouse
keywords: Ca2+; brefeldin A-inhibited guanine nucleotide exchange protein 3; diabetes; exocytosis; fusion; insulin