Age-Dependent Labeling and Imaging of Insulin Secretory Granules

Abstract

Insulin is stored within the secretory granules of pancreatic β-cells, and impairment of its release is the hallmark of type 2 diabetes. Preferential exocytosis of newly synthesized insulin suggests that granule aging is a key factor influencing insulin secretion. Here, we illustrate a technology that enables the study of granule aging in insulinoma cells and β-cells of knock-in mice through the conditional and unequivocal labeling of insulin fused to the SNAP tag. This approach, which overcomes the limits encountered with previous strategies based on radiolabeling or fluorescence timer proteins, allowed us to formally demonstrate the preferential release of newly synthesized insulin and reveal that the motility of cortical granules significantly changes over time. Exploitation of this approach may enable the identification of molecular signatures associated with granule aging and unravel possible alterations of granule turnover in diabetic β-cells. Furthermore, the method is of general interest for the study of membrane traffic and aging.

Aged insulin granules display reduced microtubule-dependent mobility and are disposed within actin-positive multigranular bodies

“…1A and Movie S1). These time intervals were close to those applied in previous studies on insulin turnover (31,37). Our initial analyses indicated that young and old SGs did not significantly differ in regards to their collective diffusion coefficient (Fig.…”

“…The availability of cell-permeable nonfluorescent and fluorescent SNAP substrates, such as TMR-Star, allows for a broad variety of applications. Ins-SNAP labeled with TMRStar (Ins-SNAP TMR-Star ) is properly sorted into SGs and secreted upon glucose stimulation (31). Importantly, high-content image analyses formally demonstrated that the preferential release of young Ins-SNAP TMR-Star+ SGs is a homogenous response to stimulation rather than being primarily sustained by a more biosynthetically and secretory active subpopulation of cells.…”

“…SNAP (Ins-SNAP) (31). SNAP is derived from alkyl guanine DNA alkyl transferase, a DNA repair enzyme that irreversibly transfers an alkyl group from its substrate to a reactive cysteine residue (32)(33)(34)(35)(36).…”

SORCS1 is necessary for normal insulin secretory granule biogenesis in metabolically stressed β cells

“…Recent studies show that newly formed SGs are especially favored for recruitment into the readily releasable vesicle pool (40)(41)(42)(43)(44). Thus, a defect in the biogenesis of new SGs should result in a limited pool of these new vesicles.…”

“…When the granules are younger (approximately 12-18 hours) they appear green, and after 24-36 hours the granules are red, and thus the reporter serves as a live cell timer that enabled us to distinguish new versus old SGs. It is well documented that in insulin-secreting cells, there is preferential release of newly formed granules in response to a stimulus (40)(41)(42)(43)(44), which predicts that we should see selective depletion of green granules.…”