Imaging analysis reveals mechanistic differences between first- and second-phase insulin exocytosis

Abstract

The mechanism of glucose-induced biphasic insulin release is unknown. We used total internal reflection fluorescence (TIRF) imaging analysis to reveal the process of first- and second-phase insulin exocytosis in pancreatic beta cells. This analysis showed that previously docked insulin granules fused at the site of syntaxin (Synt)1A clusters during the first phase; however, the newcomers fused during the second phase external to the Synt1A clusters. To reveal the function of Synt1A in phasic insulin exocytosis, we generated Synt1A-knockout (Synt1A(-/-)) mice. Synt1A(-/-) beta cells showed fewer previously docked granules with no fusion during the first phase; second-phase fusion from newcomers was preserved. Rescue experiments restoring Synt1A expression demonstrated restoration of granule docking status and fusion events. Inhibition of other syntaxins, Synt3 and Synt4, did not affect second-phase insulin exocytosis. We conclude that the first phase is Synt1A dependent but the second phase is not. This indicates that the two phases of insulin exocytosis differ spatially and mechanistically.

Figure 1.

Dual-color TIRFM of dynamic interaction between docking and fusing GFP-tagged insulin granules and Cy3-labeled Synt1A clusters in glucose-induced insulin release in control mouse β cells. 2 d after β cells were infected with the vector expressing insulin-GFP (green), cells were treated with TAT-conjugated Cy3-labeled anti-Synt1A antibody (red) for 50 min. Images were recorded for GFP-tagged insulin granules (green) and Cy3-labeled Synt1A clusters (red) simultaneously (300-ms intervals). (A) Sites of insulin granule fusion events during first-phase release under 22 mM glucose stimulation (0–4 min after glucose stimulation). Solid boxes (1 × 1 μm) represent the sites of fusion events at Synt1A clusters. Dashed boxes indicate the sites of fusion events not at Synt1A clusters (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200608132/DC1). (B) Analysis of fusion events during first-phase release (n = 5 cells). The fused granules are divided into two categories, fusion from previously docked granules (red) and newcomers (green). Previously docked granule indicates that the morphologically previously docked granule is fused with the plasma membrane. Newcomer indicates that the granule approaches from the inside (being absent before stimulation), reaches the plasma membrane, and quickly fuses. As previously reported (Ohara-Imaizumi et al., 2004b), ∼75% of insulin granule fusion during the first phase was from previously docked granules, and the remaining was from newcomers. Most fusion from previously docked granules occurred at Synt1A clusters (78.1 ± 4.0% of all fusion from previously docked granules); fusion from newcomers occurred external to Synt1A clusters (85.0 ± 2.9% of all fusion from newcomers) during the first phase. Data are mean ± SEM. (C) Sequential images (1 × 1 μm, 300-ms intervals; A, box indicated by arrow) of fusion from previously docked granules (green) at the Synt1A cluster (red) during the first phase. (D) Sites of insulin granule fusion during second-phase insulin release (>5 min after glucose stimulation; Video 2). Solid and dashed boxes are as described above. (E) Analysis of fusion events during second-phase release, with fusion occurring mostly from newcomers (Ohara-Imaizumi et al., 2004b), and at sites distinct from Synt1A clusters (86.1 ± 1.2% of all fusion from newcomers; n = 5 cells). Data are mean ± SEM. (F) Sequential images (1 × 1 μm, 300-ms intervals; D, box indicated by arrow) of fusion from newcomers (green) external to Synt1A clusters (red) during the second phase.

Figure 2.

Synt1A deficiency impairs docking of insulin granules to the plasma membrane in pancreatic β cells. (A) TIRFM of insulin granules morphologically docked to the plasma membrane. (left) Typical TIRF images of docked insulin granules in WT or Synt1A^−/− β cells. The surrounding lines represent the outline of cells that attached to the cover glass. Bar, 5 μm. Pancreatic β cells were prepared from WT and Synt1A^−/− mice, fixed, and immunostained for insulin. (right) Number of insulin granules morphologically docked to the plasma membrane. Individual fluorescent spots shown in TIRF images were manually counted per 200 μm²; n = 15 cells. (B) Electron micrograph of β cell sections. (top) Typical EM images of the plasma membrane area facing the blood capillary (C) of WT and Synt1A^−/− β cells (B). Bar, 500 nm. (bottom) Number of morphologically docked insulin granules per 10 μm of plasma membrane. Granules at their shortest distance of <10 nm from the plasma membrane were qualified as morphologically docked granules (red arrowheads). Results are provided as the mean ± SEM.

Figure 3.

Effects of Synt1A deficiency on glucose-induced biphasic insulin release. (A) TIRFM of single insulin granule motion in WT β cells under 22 mM high glucose stimulation (Video 3, available at http://www.jcb.org/cgi/content/full/jcb.200608132/DC1). Red and green boxes indicate that the granules to be fused with the plasma membrane originated from previously docked (red) or newcomer (green) granules. Sequential images (1 μm × 1 μm; 300-ms intervals) of docking and fusion from the previously docked granules (red box) and the newcomers (green box) are presented. (B) Histogram of the number of fusion events (per 200 μm²) in WT β cells at 60-s intervals after stimulation (n =10 cells). The red column shows fusion from previously docked granules, and the green column shows fusion from newcomers. During the first phase, fusion occurred mostly from previously docked granules. The fusing granules during the second phase originated mostly from newcomers. (C) TIRFM during glucose stimulation in Synt1A^−/− β cells (Video 4) and sequential images of a newcomer granule docking and fusing (green box) under glucose stimulation. (D) Histogram of the number of fusion events (per 200 μm²) in the Synt1A^−/− cells at 60-s intervals after stimulation (n =10 cells). (E) Glucose-induced insulin release from perfused WT and Synt1A^−/− β cells stimulated with 22 mM glucose. The cells in the cell chamber (∼5 × 10⁵ cells per chamber) were perfused with KRB (0.5 ml/min) at 37°C, and the perfusate was analyzed for insulin by ELISA. (F) 22 mM glucose-induced changes in [Ca²⁺]_i in WT and Synt1A^−/− β cells. Changes in [Ca²⁺]_i were measured by 2 μM Fura-2 AM. Time 0 indicates when the high glucose was added. The fluorescence ratio (340/360) at time 0 was taken as 1. Results are provided as the mean ± SEM.

Figure 4.

Rescue of the number of docked insulin granules and fusion events by restoring Synt1A clusters to normal levels in Synt1A^−/− β cells. (A–C) TIRF images and the quantitation of Synt1A clusters (A), SNAP-25 clusters (B), and docked insulin granules (C) on the plasma membrane in WT or Synt1A^−/− β cells. Synt1A^−/− cells were infected with empty virus Adex1w (Ax-Cont) or with Adex1CA Synt1A (Ax-Synt1A). Cells were fixed and immunostained for Synt1A (A), SNAP-25 (B), and insulin (C). (top) Typical TIRF images. Surrounding lines represent the outline of cells attached to the cover glass. Bars, 5 μm. (bottom) Number of Synt1A (A) and SNAP-25 (B) clusters and docked insulin granules (C) on the plasma membrane. Individual fluorescent spots in TIRF images were manually counted per 200 μm². Data are mean ± SEM (*, P < 0.05; **, P < 0.0001; n = 15 cells). (D) Rescue of fusion events in Synt1A^−/− β cells. Synt1A^−/− cells were infected with Adex1CA insulin GFP and then Ax-Cont or Ax-Synt1A. The histogram shows the number of fusion events (per 200 μm²) at 60-s intervals after high glucose stimulation. A marked increase in fusion events from previously docked granules was observed in the Synt1A^−/− cells infected with Ax-Synt1A relative to Ax-Cont–infected cells. (E) Dual-color TIRFM of docking and fusing GFP-tagged insulin granules (green) and Synt1A clusters labeled with TAT-conjugated Cy3-labeled anti-Synt1A antibody (red) in glucose-induced release from Ax-Synt1A–infected Synt1A^−/− cells. Most fusion from previously docked granules occurred at Synt1A clusters (76% of all fusion from previously docked granules); fusion from newcomers occurred external to Synt1A clusters (81% of all fusion from newcomers) during the first phase. Data are mean ± SEM (n = 5 cells). Solid boxes (1 μm × 1 μm) represent the sites of fusion events at the Synt1A clusters. Dashed boxes indicate the sites of fusion events external to the Synt1A clusters. (F) Sites of insulin granule fusion during second-phase insulin release (>4 min after glucose stimulation). Fusion events during second-phase release occurred mostly from newcomers and at sites distinct from Synt1A clusters (82% of all fusion from newcomers; n = 5 cells). Data are mean ± SEM. Solid and dashed boxes are as described above.

Figure 5.

TIRFM of fusion of GFP-tagged insulin granules in biphasic insulin release from WT β cells treated with TAT-syntaxin-H3. WT cells expressing GFP-tagged insulin were treated with or without 70 mg/ml of TAT-Cont (A), TAT-Synt1A-H3 (B), TAT-Synt3-H3 (C), TAT-Synt4-H3 (D), or TAT-Synt1B-H3 (E) fusion protein for 50 min, and TIRF images were acquired every 300 ms by 22 mM glucose stimulation. The histogram shows the number of fusion events (n = 10 cells each) at 1-min intervals after high glucose stimulation in the TAT fusion protein–treated cells. The red column shows the fusion from previously docked granules, and the green column shows fusion from newcomers. Data are mean ± SEM.

Figure 6.

Glucose tolerance test in WT and Synt1A^−/− mice. (A) Oral glucose tolerance was tested in WT (n = 11) and Synt1A^−/− (n = 7) mice. Blood glucose levels were measured at the indicated times after glucose challenge at 2 g glucose/kg body weight (n = 6 each; *, P < 0.005; **, P < 0.0001). (B) Plasma insulin levels were measured in WT and Synt1A^−/− mice during the oral glucose tolerance test at the indicated times. Results are provided as the mean ± SEM.

Figure 7.

Schematic drawing of the role of Synt1A in biphasic insulin release. During first-phase insulin release, granules dock to Synt1A clusters and fuse at the site of Synt1A clusters. During second-phase release, granules move to the plasma membrane from the intracellular store and then fuse with the plasma membrane without any interaction with Synt1A clusters.