Imaging dynamic insulin release using a fluorescent zinc indicator for monitoring induced exocytotic release (ZIMIR)

Abstract

Current methods of monitoring insulin secretion lack the required spatial and temporal resolution to adequately map the dynamics of exocytosis of native insulin granules in intact cell populations in three dimensions. Exploiting the fact that insulin granules contain a high level of Zn 2+ , and that Zn 2+ is coreleased with insulin during secretion, we have developed a fluorescent, cell surface-targeted zinc indicator for monitoring induced exocytotic release (ZIMIR). ZIMIR displayed a robust fluorescence enhancement on Zn 2+ chelation and bound Zn 2+ with high selectivity against Ca 2+ and Mg 2+ . When added to cultured β cells or intact pancreatic islets at low micromolar concentrations, ZIMIR labeled cells rapidly, noninvasively, and stably, and it reliably reported changes in Zn 2+ concentration near the sites of granule fusion with high sensitivity that correlated well with membrane capacitance measurement. Fluorescence imaging of ZIMIR-labeled β cells followed the dynamics of exocytotic activity at subcellular resolution, even when using simple epifluorescence microscopy, and located the chief sites of insulin release to intercellular junctions. Moreover, ZIMIR imaging of intact rat islets revealed that Zn 2+ /insulin release occurred largely in small groups of adjacent β cells, with each forming a "secretory unit." Concurrent imaging of ZIMIR and Fura-2 showed that the amplitude of cytosolic Ca 2+ elevation did not necessarily correlate with insulin secretion activity, suggesting that events downstream of Ca 2+ signaling underlie the cell-cell heterogeneity in insulin release. In addition to studying stimulation-secretion coupling in cells with Zn 2+ -containing granules, ZIMIR may find applications in β-cell engineering and screening for molecules regulating insulin secretion on high-throughput platforms.probe development | zinc imaging | hormone secretion assay

Zinc and diabetes

“…These probes display a robust fluorescence enhancement on Zn 2+ chelation and have been successfully used to detect insulin secretion in real time with subcellular resolution [94][95][96].…”

Intracellular zinc in insulin secretion and action: a determinant of diabetes risk?

“…As Zn 2+ does not affect the uptake of C-peptide or proinsulin, this mechanism could potentially explain the impairments in both circulating insulin and the increased proinsulin:insulin ratio seen in risk allele carriers (82) . Interestingly, cytosolic free Zn 2+ , as measured with the eCALWY4 probe, was reduced in the global knockout (KO) mouse for ZnT8 (77) , alongside granule zinc concentrations as estimated by the release of zinc during exocytosis (26) . These findings imply a more complex role for this transporter in the regulation of zinc fluxes than has previously been appreciated.…”

“…to mitochondria with the addition of a thiamine pyrophosphate group (25) . Attachment to the cell surface to allow sampling of extracellular Zn 2+ has also been achieved, and used to measure co-release of Zn 2+ alongside insulin (26,27) . The latter 'click-SnAr-click' strategy was also used to add targeting moieties to other intracellular localisations (e.g.…”

Lipotoxicity disrupts incretin-regulated human β cell connectivity

“…The GJ blocker appeared to be relatively specific, since preincubation of islets with AGA: (a) reduced the distance between correlated cell pairs from 30 μm to 20 μm, consistent with an effect on GJ-coupling conduction and impedance (38) (Gaussian curve fitted to histogram of n = 3 donors; Figure 2D); (b) did not affect the percentage of GLP-1-responsive cells ( Figure 2E); (c) was unable to significantly alter intracellular Ca 2+ rises in response to 11 mM glucose or 20 mM KCl (Supplemental Figure 3, A and B, respectively); and (d) yielded identical results to experiments in which gene expression was silenced in β cells by greater than 80% using shRNA against human Cx36 (Supplemental Figure 4). GJ blockade severely diminished incretin-stimulated ( Figure 2F), but not glucose-stimulated, insulin secretion (117.3 ± 12.7 versus 132.7 ± 18.6 AU, BGA versus AGA, respectively; n = 4 recordings, NS) ( Figure 2G) measured using the sensitive membrane-tethered zinc (Zn 2+ ) probe ZIMIR (39), suggesting that GLP-1 may drive insulin release by improving correlated activity within a subpopulation of β cells (Supplemental Videos 2 and 3).…”

“…Insulin release was monitored using ZIMIR as previously described (39). Briefly, before imaging, islets were incubated for 2 hours in ZIMIR (1 mM), a membrane-bound probe that fluoresces upon binding of zinc (Zn 2+ ) coreleased with insulin from granules.…”