Fast insulin secretion reflects exocytosis of docked granules in mouse pancreatic B-cells

Abstract

A readily releasable pool (RRP) of granules has been proposed to underlie the first phase of insulin secretion. In the present study we combined electron microscopy, insulin secretion measurements and recordings of cell capacitance in an attempt to define this pool ultrastructurally. Mouse pancreatic B-cells contain approximately 9,000 granules, of which 7% are docked below the plasma membrane. The number of docked granules was reduced by 30% (200 granules) during 10 min stimulation with high K+. This stimulus depolarized the cell to -10 mV, elevated cytosolic [Ca2+] ([Ca2+](i)) from a basal concentration of 130 nM to a peak of 1.3 microM and released 0.5 ng insulin/islet, corresponding to 200-300 granules/cell. The Ca2+ transient decayed towards the prestimulatory concentration within approximately 200 s, presumably reflecting Ca2+ channel inactivation. Renewed stimulation with high K+ failed to stimulate insulin secretion when applied in the absence of glucose. The size of the RRP, derived from the insulin measurements, is similar to that estimated from the increase in cell capacitance elicited by photolytic release of caged Ca2+. We propose that the RRP represents a subset of the docked pool of granules and that replenishment of RRP can be accounted for largely by chemical modification of granules already in place or situated close to the plasma membrane.

Mathematical modeling and statistical analysis of calcium-regulated insulin granule exocytosis in β-cells from mice and humans

“…Also, human β-cell-exocytosis does not plateau for depolarizations up to 500 ms (Braun et al, , 2009, again arguing against pool depletion. However, this does not means that depletion of the RRP is irrelevant for biphasic insulin secretion, indeed there is good evidence for a role of a finite pool of granules (Daniel et al, 1999;Henquin et al, 2002;Olofsson et al, 2002), but rather that short depolarizations of increasing lengths do not show evidence of a smaller IRP. Finally, our results confirmed that cAMP-raising agents such as forskolin increase the efficacy of Ca 2+ on exocytosis.…”

“…At the plasma membrane the granules are docked and primed through Ca 2+ , temperature-and ATPdependent processes (Proks et al, 1996;Renström et al, 1996;Eliasson et al, 1997). Ultrastructural analysis (Olofsson et al, 2002) in single mouse β-cells has revealed that ∼700 out of the total ∼10 000 granules are docked to the plasma membrane. Of these ∼200 are primed and readily releasable.…”

Syntaxin-3 regulates newcomer insulin granule exocytosis and compound fusion in pancreatic beta cells

“…Islet beta cells employ fusion of secretory granules [31,36], but to a much lesser extent than other secretory cells. The other mode is by recruitment of newcomer secretory granules to the PM, first described in islet beta cells [11,37], but which could be a mode of exocytosis in other neuroendocrine cells. Here, we show that SYN-3, particularly when produced in abundance to induce gain of function, can enhance both modes of exocytosis on newcomer SGs, without any effect on docked SGs.…”

Mechanisms of Exocytosis in Insulin‐Secreting B‐Cells and Glucagon‐Secreting A‐Cells

“…Electron microscopy on various neuronal and endocrine cells (von Gersdorff et al 1996;Plattner et al 1997), including B-cells (Olofsson et al 2002), has revealed a subset of secretory granules that is ''docked'' beneath the plasma membrane. These findings correlate with data obtained with capacitance measurements ( fig.…”

“…Despite many similarities of the exocytotic machinery in (neuro-) endocrine cells and that in neurotransmitter-releasing fast synapses, there are important differences: 1) synaptic vesicles are small (diameter Ͻ30 nm; von Gersdorff 1996) compared with peptide containing vesicles (hundreds of nm; Plattner et al 1997;Olofsson et al 2002); 2) synaptic exocytosis occurs without delay after the influx of Ca 2π and is at least one order-of magnitude faster than peptide secretion (Ämmälä et al 1993;Mennerick & Matthews 1996); 3) synaptic vesicles may stay largely intact during exocytosis and are refilled with new transmitter molecules immediately after endocytosis, whereas peptide-containing granules are assembled and filled with secretory peptides in the Golgi apparatus (Betz & Bewick 1992;Hutton 1994); 4) synaptic exocytosis generally requires higher concentrations of Ca 2π (Ämmälä et al 1993;Mennerick & Matthews 1996, Chow et al 1996Bollmann et al 2000); and 5) although most secretory cells contain more than one type of voltage-gated Ca 2π -channel, synaptic transmitter exocytosis depends on the faster N-or P-type (Hirning et al 1988;Uchitel et al 1992), whereas endocrine cells preferentially use the L-type (Lopez et al 1994;Barg et al 2001a). Taken together, the findings in neurones suggest that the site of Ca 2π -influx is within minimal distance of the Ca 2π -sensor at the synaptic vesicle and functionally associated within release sites.…”