SNAP23 depletion enables more SNAP25/calcium channel excitosome formation to increase insulin exocytosis in type 2 diabetes
Tao Liang, … , Jeffrey E. Pessin, Herbert Y. Gaisano
Tao Liang, … , Jeffrey E. Pessin, Herbert Y. Gaisano
Published February 13, 2020
Citation Information: JCI Insight. 2020;5(3):e129694. https://doi.org/10.1172/jci.insight.129694.
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Research Article Endocrinology Metabolism

SNAP23 depletion enables more SNAP25/calcium channel excitosome formation to increase insulin exocytosis in type 2 diabetes

Abstract

SNAP23 is the ubiquitous SNAP25 isoform that mediates secretion in non-neuronal cells, similar to SNAP25 in neurons. However, some secretory cells like pancreatic islet β cells contain an abundance of both SNAP25 and SNAP23, where SNAP23 is believed to play a redundant role to SNAP25. We show that SNAP23, when depleted in mouse β cells in vivo and human β cells (normal and type 2 diabetes [T2D] patients) in vitro, paradoxically increased biphasic glucose-stimulated insulin secretion corresponding to increased exocytosis of predocked and newcomer insulin granules. Such effects on T2D Goto-Kakizaki rats improved glucose homeostasis that was superior to conventional treatment with sulfonylurea glybenclamide. SNAP23, although fusion competent in slower secretory cells, in the context of β cells acts as a weak partial fusion agonist or inhibitory SNARE. Here, SNAP23 depletion promotes SNAP25 to bind calcium channels more quickly and longer where granule fusion occurs to increase exocytosis efficiency. β Cell SNAP23 antagonism is a strategy to treat diabetes.

Authors

Tao Liang, Tairan Qin, Fei Kang, Youhou Kang, Li Xie, Dan Zhu, Subhankar Dolai, Dafna Greitzer-Antes, Robert K. Baker, Daorong Feng, Eva Tuduri, Claes-Goran Ostenson, Timothy J. Kieffer, Kate Banks, Jeffrey E. Pessin, Herbert Y. Gaisano

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Figure 8

SNAP23 acts as inhibitory SNARE that reduces SNAP25 binding to Stx-1A and Stx-3 from forming SNARE complexes, whereby these SNAP25 but not SNAP23 SNARE complexes could form more fusion-competent machineries with Cav, priming proteins and Ca2+ sensors.

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SNAP23 acts as inhibitory SNARE that reduces SNAP25 binding to Stx-1A an...
Coimmunoprecipitation (Co-IP) studies were performed on INS-832/13 cells before stimulation (basal) and after maximal stimulation with 16.7 mM glucose plus 10 nM GLP-1. (A) SNAP23 forms similar SM/SNARE complexes as those previously reported with SNAP25. Co-IP was performed with SNAP23 antibody. Shown are representative of 3 experiments with analysis of the 3 experiments shown in Supplemental Figure 7A. (B) SNAP23 depletion in INS-832/13 cells enables endogenous SNAP25 to promote formation of more SM/SNARE complexes to mediate the increased exocytosis of predocked SGs (Munc18a, Stx-1A, VAMP2) and newcomer SGs (Munc18b, Stx-3, VAMP8) that underlie the increased first- and second-phase GSIS shown in Supplemental Figures 2–4. INS-832/13 cells were first treated with Ad-SNAP23 shRNA/mCherry to deplete SNAP23 versus Ad-scrambled shRNA/mCherry (Control). Co-IP was performed with SNAP25 antibody. Shown are representative of 3 experiments, with analysis of the 3 experiments shown in Supplemental Figure 7B. (C) SNAP23 blocks SNAP25 (and vice versa) binding to Stx-1A and Stx-3. HEK293 cells transfected with Stx-1A, Stx-3, SNAP23, or SNAP25 were subjected to pulldown with GST-SNAP23 (in the presence or absence of expressed SNAP25) or GST-SNAP25 (in the presence or absence of expressed SNAP23) to assess how much Stx-1A (top) or Stx-3 (bottom) could be precipitated. GST was used as control. Shown are representative of 4 experiments, with analysis of the 4 experiments shown in Supplemental Figure 7C. SNAP23 SNARE complexes (D) are less able than SNAP25 complexes (E) to pull down Cavs (Cav1.2, -1.3, and -2.3) synaptotagmins (Syt-1 and -7) and priming proteins (RIM2, Munc13-1). Note that 16.7 mM glucose plus GLP-1 increased the binding of some of these proteins in SNAP25 but not SNAP23 complexes. This is a representative of 3 separate experiments with analysis shown in Supplemental Figure 8. (F) Control (Ad-scrambled shRNA/mCherry) or SNAP23-knockdown (Ad-SNAP23 shRNA/mCherry) human β cells were stimulated with 16.7 mM glucose plus 10 nM GLP-1, immunolabeled with SNAP25 and Stx-1A or Stx-3 antibodies, and then assessed by structured illumination microscopy imaging. SNAP23 depletion increased SNAP25 colocalization with Stx-1A (top; Pearson’s correlation coefficient [PCC], Control: –0.42 ± 0.09; KD: 0.36 ± 0.04, n = 11 from 2 independent experiments) and Stx-3 (bottom; PCC, Control: –0.74 ± 0.06: KD: 0.14 ± 0.02, n = 12 from 2 independent experiments). Scale bars: 2 μm. ***P < 0.001. Statistical significance was assessed by 2-tailed Student’s t test.