Munc18b Is a Major Mediator of Insulin Exocytosis in Rat Pancreatic β-Cells

Abstract

Sec1/Munc18 proteins facilitate the formation of trans-SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor) complexes that mediate fusion of secretory granule (SG) with plasma membrane (PM). The capacity of pancreatic β-cells to exocytose insulin becomes compromised in diabetes. β-Cells express three Munc18 isoforms of which the role of Munc18b is unknown. We found that Munc18b depletion in rat islets disabled SNARE complex formation formed by syntaxin (Syn)-2 and Syn-3. Two-photon imaging analysis revealed in Munc18b-depleted β-cells a 40% reduction in primary exocytosis (SG-PM fusion) and abrogation of almost all sequential SG-SG fusion, together accounting for a 50% reduction in glucose-stimulated insulin secretion (GSIS). In contrast, gain-of-function expression of Munc18b wild-type and, more so, dominant-positive K314L/R315L mutant promoted the assembly of cognate SNARE complexes, which caused potentiation of biphasic GSIS. We found that this was attributed to a more than threefold enhancement of both primary exocytosis and sequential SG-SG fusion, including long-chain fusion (6–8 SGs) not normally (2–3 SG fusion) observed. Thus, Munc18b-mediated exocytosis may be deployed to increase secretory efficiency of SGs in deeper cytosolic layers of β-cells as well as additional primary exocytosis, which may open new avenues of therapy development for diabetes.

Syntaxin-4 mediates exocytosis of pre-docked and newcomer insulin granules underlying biphasic glucose-stimulated insulin secretion in human pancreatic beta cells

“…Remarkably, these newcomer SGs are recruited to the PM to undergo exocytosis with minimal to no residence time on the PM [4,5]. We had identified the exocytotic machinery mediating newcomer SGs [10,[16][17][18][19]]-a second SM-SNARE complex comprised of Munc18b, Syn-3, VAMP8 and SNAP25. In that body of work, we showed that depletion of endogenous Syn-3 [10] or VAMP8 [17] reduced newcomer SGs and that when exogenously expressed they restored the deficient newcomer SGs without affecting pre-docked SGs.…”

“…We previously reported that pancreatic acini employed Munc18c-Syn-4-VAMP8-SNAP23 as the putative SM-SNARE complex mediating basolateral exocytosis that causes pancreatitis [25][26][27]. We showed that all beta cell Syns displayed promiscuous binding to VAMP2 and VAMP8 and to SNAP25 and SNAP23, although each Syn has a preference for a particular VAMP [16,17]. To determine whether beta cell Syn-4-Munc18c complexes bind to VAMPs and SNAP25 isoform SNAP23, we performed immunoprecipitation experiments in INS-1 cells (Fig.…”

“…All of the antibodies used in the current work have been validated and used in similar experiments [16,17].…”

“…Immunoprecipitation This was performed with INS-1 cells as previously reported [16,17] and described in detail in ESM Methods. Cells intended for stimulation were pre-incubated for 30 min with 10 nmol/l GLP-1 at 0.8 mmol/l glucose, then stimulated with 16.7 mmol/l glucose (containing GLP-1) for 30 min.…”

Imaging Analysis of Insulin Secretion with Two-Photon Microscopy

“…Munc18b is one of the Munc18 isoforms that is expressed in pancreatic islets, and the deletion of Munc18b by shRNA reduced insulin secretion. 37) Interestingly, a Munc18b mutant, Munc18b-K314L/R315L (Munc18b-KR), is a mutant that facilitates SNARE complex assembly. The laboratory of Lam and Gaisano showed that Munc18b-KR facilitated the complex formation between Syntaxin1/2-VAMP2-SNAP25 and Syntaxin3-VAMP8-SNAP25 in islets that were stimulated by glucose, GLP-1, and IBMX.…”