A fluorescent timer reporter enables sorting of insulin secretory granules by age.
Animals
Cell Line
Exocytosis
/ physiology
Fluorescent Dyes
/ chemistry
Glucose
/ metabolism
Humans
Insulin
/ metabolism
Insulin Secretion
/ physiology
Insulin-Secreting Cells
/ metabolism
Islets of Langerhans
/ metabolism
Male
Membrane Proteins
/ metabolism
Mice
Mice, Inbred C57BL
Microscopy, Fluorescence
/ methods
Secretory Vesicles
/ metabolism
Time Factors
dsRed-E5
exocytosis
fluorescent timer
granule aging
insulin
insulin secretion
insulin secretory granule (SG)
type 2 diabetes
vesicles
β-cell
Journal
The Journal of biological chemistry
ISSN: 1083-351X
Titre abrégé: J Biol Chem
Pays: United States
ID NLM: 2985121R
Informations de publication
Date de publication:
03 07 2020
03 07 2020
Historique:
received:
05
01
2020
revised:
21
03
2020
pubmed:
29
4
2020
medline:
12
1
2021
entrez:
29
4
2020
Statut:
ppublish
Résumé
Within the pancreatic β-cells, insulin secretory granules (SGs) exist in functionally distinct pools, displaying variations in motility as well as docking and fusion capability. Current therapies that increase insulin secretion do not consider the existence of these distinct SG pools. Accordingly, these approaches are effective only for a short period, with a worsening of glycemia associated with continued decline in β-cell function. Insulin granule age is underappreciated as a determinant for why an insulin granule is selected for secretion and may explain why newly synthesized insulin is preferentially secreted from β-cells. Here, using a novel fluorescent timer protein, we aimed to investigate the preferential secretion model of insulin secretion and identify how granule aging is affected by variation in the β-cell environment, such as hyperglycemia. We demonstrate the use of a fluorescent timer construct, syncollin-dsRedE5TIMER, which changes its fluorescence from green to red over 18 h, in both microscopy and fluorescence-assisted organelle-sorting techniques. We confirm that the SG-targeting construct localizes to insulin granules in β-cells and does not interfere with normal insulin SG behavior. We visualize insulin SG aging behavior in MIN6 and INS1 β-cell lines and in primary C57BL/6J mouse and nondiabetic human islet cells. Finally, we separated young and old insulin SGs, revealing that preferential secretion of younger granules occurs in glucose-stimulated insulin secretion. We also show that SG population age is modulated by the β-cell environment
Identifiants
pubmed: 32341128
pii: S0021-9258(17)50315-4
doi: 10.1074/jbc.RA120.012432
pmc: PMC7335792
pii:
doi:
Substances chimiques
Fluorescent Dyes
0
Insulin
0
Membrane Proteins
0
Sycn protein, rat
0
Glucose
IY9XDZ35W2
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
8901-8911Commentaires et corrections
Type : CommentIn
Informations de copyright
© 2020 Yau et al.
Déclaration de conflit d'intérêts
Conflict of interest—CJR is a current employee of AstraZeneca and owns stock in the company.
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