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A fluorescent timer reporter enables sorting of insulin secretory granules by age

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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 in vivo in the db/db mouse islets and ex vivo in C57BL/6J islets exposed to different glucose environments.

insulin
β-cell
vesicles
insulin secretion
type 2 diabetes
dsRed-E5
exocytosis
fluorescent timer
granule aging
insulin secretory granule (SG)

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Author contributions—B. Y., L. H., C. L., and D. R. L. data curation; B. Y., L. H., C. L., D. R. L., and M. A. K. formal analysis; B. Y., L. H., P. T., C. J. R., and M. A. K. methodology; B. Y. writing-original draft; B. Y. and M. A. K. project administration; L. H. and M. A. K. investigation; H. E. T., J. E. G., L. W., W. J. H., C. J. R., and M. A. K. resources; P. T. and M. A. K. supervision; P. T. and M. A. K. funding acquisition; M. A. K. conceptualization; M. A. K. writing-review and editing.

Funding and additional information—The work was supported by National Health and Medical Research Council (NHMRC) of Australia Grant GNT1139828. M. A. K. is supported by a Jennie Mackenzie Philanthropic Fellowship, University of Sydney.

Conflict of interest—CJR is a current employee of AstraZeneca and owns stock in the company.

Abbreviations—The abbreviations used are:

    GSIS

    glucose-stimulated insulin secretion

    SG

    secretory granule

    ISG

    insulin SG

    HTRF

    homogeneous time-resolved fluorescence

    FAOS

    flow cytometer–assisted organelle sorting

    IBMX

    3-isobutyl-1-methylxanthine

    DAPI

    4′,6-diamidino-2-phenylindole

    TIRF

    total internal reflection fluorescence

    ANOVA

    analysis of variance.