Munc18-1 regulates first-phase insulin release by promoting granule docking to multiple syntaxin isoforms

Abstract

Attenuated levels of the Sec1/Munc18 (SM) protein Munc18-1 in human islet β-cells is coincident with type 2 diabetes, although how Munc18-1 facilitates insulin secretion remains enigmatic. Herein, using conventional Munc18-1(+/-) and β-cell specific Munc18-1(-/-) knock-out mice, we establish that Munc18-1 is required for the first phase of insulin secretion. Conversely, human islets expressing elevated levels of Munc18-1 elicited significant potentiation of only first-phase insulin release. Insulin secretory changes positively correlated with insulin granule number at the plasma membrane: Munc18-1-deficient cells lacked 35% of the normal component of pre-docked insulin secretory granules, whereas cells with elevated levels of Munc18-1 exhibited a ∼20% increase in pre-docked granule number. Pre-docked syntaxin 1-based SNARE complexes bound by Munc18-1 were detected in β-cell lysates but, surprisingly, were reduced by elevation of Munc18-1 levels. Paradoxically, elevated Munc18-1 levels coincided with increased binding of syntaxin 4 to VAMP2 at the plasma membrane. Accordingly, syntaxin 4 was a requisite for Munc18-1 potentiation of insulin release. Munc18c, the cognate SM isoform for syntaxin 4, failed to bind SNARE complexes. Given that Munc18-1 does not pair with syntaxin 4, these data suggest a novel indirect role for Munc18-1 in facilitating syntaxin 4-mediated granule pre-docking to support first-phase insulin exocytosis.

FIGURE 1.

Munc18-1^+/− mice show impaired glucose tolerance corresponding to impaired KCl-stimulated insulin release from the islets. A, islets from Munc18-1^+/+ and Munc18-1^+/− mice were perifused with 2.8 mm glucose followed by stimulation with 35 mm KCl. B, quantitation of the area under the curve (AUC) for insulin secretion from three independent islet batches isolated from Munc18-1^+/+ (black bars) and Munc18-1^+/− (gray bars), normalized to base line; *, p < 0.05 versus Munc18–1^+/+. C, islet insulin content from Munc18-1^+/+ and Munc18-1^+/− isolated mouse islets from B. D, islets isolated from Munc18-1^+/+ and Munc18-1^+/− mice in B were lysed for immunoblot detection of multiple Munc18 and syntaxin proteins. E, islet β-cell area from pancreatic sections from Munc18-1^+/+ and Munc18-1^+/− mice (bar = 50 μm), as determined by quantitation of insulin-stained pancreas sections from three pairs of mice. F, intraperitoneal glucose tolerance tests of Munc18-1^+/+ and Munc18-1^+/− mice was performed by intraperitoneal injection of d-glucose (2 g/kg of body weight) into 11 pairs of male mice (age 4–6 months) fasted for 18 h; *, p < 0.05 versus Munc18-1^+/+. G, insulin tolerance testing (ITT) of Munc18-1^+/+ and Munc18-1^+/− was performed by intraperitoneal injection of insulin (0.75 units/kg of body weight) into male mice (age 4–6 months) fasted for 6 h. Blood glucose levels were normalized to basal = 100% for each animal for calculation of the mean percent ± S.E.; *, p < 0.05 versus Munc18-1^+/+. IB, immunoblot.

FIGURE 2.

Impaired glucose tolerance and islet defects in β-cell-specific Munc18-1 knock-out mice. A, islet protein expression from tamoxifen-induced β-cell specific Cre^+/−-Munc18-1 knock-out (β-cell Munc18-1KO) mice and performed as described in the legend to Fig. 1. B, islets from tamoxifen-induced Cre^−/− and -Cre^+/− mice were perifused with 2.8 mm glucose followed by stimulation with 35 mm KCl. C, area under the curve (AUC) for insulin secretion from islets quantified, normalized to base line for each; *, p < 0.05 versus tamoxifen-Cre^−/−. Intraperitoneal glucose tolerance test (IPGTT; D) and insulin tolerance test (ITT) (E) of tamoxifen- or vehicle-induced β-cell specific Cre^−/−- and Cre^+/−-Munc18-1 (β-cell Munc18-1KO) mice (five pairs of vehicle-injected, six pairs of tamoxifen-induced male mice), performed as described in Fig. 1; *, p < 0.05 versus tamoxifen-induced Cre^−/−-Munc18-1. TM, tamoxifen; Veh, vehicle.

FIGURE 3.

Munc18-1^+/− islet β-cells display altered insulin granule distribution. A, transmission electron microscopy of Munc18-1^+/+ and Munc18-1^+/− islets. Islets were incubated overnight and then placed in glucose-free MKRBB for 2 h and left unstimulated for fixation for EM as described under “Experimental Procedures.” Arrows denote the plasma membrane; N denotes location of the nucleus. Sections are equivalent in size, bar = 500 nm. B, distribution of insulin granules in Munc18-1^+/+ and Munc18-1^+/− cells. Criteria for inclusion required clear demarcation of the plasma membrane and presence of nucleus in each field tabulated. Insulin granule distance from the plasma membrane was tabulated for Munc18-1^+/+ and Munc18-1^+/− electron micrographs from 10 sections/each of 12 mice, with a total of 2,235 Munc18-1^+/+ and 2,132 Munc18-1^+/− insulin granule distances measured and grouped into categories based upon distance from the PM; *, p < 0.05 versus Munc18-1^+/+. C, total granule number per field/section in Munc18-1^+/+ (black bar) and Munc18-1^+/− cells (gray bar) under each condition was quantified.

FIGURE 4.

Human islets overexpressing Munc18-1 show enhanced first-phase insulin release. Isolated human islets were transduced with control (LacZ) or Munc18-1 adenovirus (multiplicity of infection = 100) for 40 h for the following studies. A, solubilized for immunodetection of Munc18-1 overexpression as well as syntaxin proteins. B, perifused with 2.8 mm glucose followed by stimulation with 20 mm glucose, returned to 2.8 mm glucose for 20 min, and then stimulated with 35 mm KCl. C, quantified for the area under the curve (AUC) analysis for first (11–17 min) and second (18–45 min) phases as well as KCl-induced insulin secretion from human islets perifused in B, normalized to base line; *, p < 0.05 versus control adenovirus (Ad). Data represent three independent sets of donor islets (donor profiles listed in supplemental Table S3). IB, immunoblot; U, unit.

FIGURE 5.

Increased accumulation of VAMP2-bound insulin granules at the PM of Munc18-1 overexpressing MIN6 cells correlates with enhanced insulin secretion. MIN6 cells transduced to express Munc18-1 or control (LacZ) were subjected to 2 h of incubation in glucose-free MKRBB for subsequent analyses. A, KCl stimulation (35 mm, 2 min) and quantitation of insulin release. B, assessment of KCl-stimulated calcium influx using Fura-2 imaging. C, assessment of VAMP2 abundance in PM fractions by immunoblotting (IB; inset) and optical density scanning quantitation. Data in each panel represent three to four independent experiments. *, p < 0.05 versus control (Con) adenovirus (Control-Ad).

FIGURE 6.

Munc18-1 overexpression does not increase formation of syntaxin 1A-based SNARE complexes. MIN6 cells transduced to express Munc18-1-adenovirus or control (LacZ, control adenovirus; Con-Ad) were subjected to 2 h of incubation in glucose-free MKRBB (basal condition) for preparation of cleared detergent cell lysates for use in binding assays. A, GST-VAMP2 pulldown reactions entailed incubation of recombinant GST-VAMP2 protein linked to glutathione-Sepharose beads with detergent-solubilized MIN6 cell lysates, and coprecipitated proteins were detected by immunoblot (IB). Ponceau S staining of the GST-VAMP2 protein was used to gauge the equivalence of precipitation between reactions. Immunoprecipitation reactions with detergent-solubilized MIN6 cell lysates were performed using antibodies against the following: B, VAMP2; C, syntaxin 1A; or D, Munc18-1. Co-precipitated proteins were resolved on 10–12% SDS-PAGE for detection by immunoblot. Lysate lanes in B show the lack of effect of Munc18-1-Ad upon SNARE protein expression levels. Data are representative of at least three independent experiments for each data panel. Each set of experiments was quantified by optical density scanning, with bar graphs showing ratios of protein associations to the right of the corresponding set of immunoblots; *, p < 0.05 versus control-Ad transduced cells (Con-Ad). Ad, adenovirus.

FIGURE 7.

Syntaxin 4-based SNARE assembly is increased in Munc18-1 overexpressing β-cells. MIN6 cells transduced to express Munc18-1 or control (LacZ) were treated as described in the legend to Fig. 6. A, recombinant GST-VAMP2 protein was incubated with MIN6 cell lysates, and coprecipitated proteins were detected by immunoblot (IB). Ponceau S staining of the GST-VAMP2 protein showed equal precipitation between reactions. Immunoprecipitation reactions with detergent-solubilized MIN6 cell lysates were performed using antibodies against VAMP2 (B) or Munc18-1 (C). D, GST-VAMP2 pulldown reactions using non-transduced MIN6 detergent lysates were supplemented with recombinantly expressed and purified Munc18-1 protein (0 or 250 ng per reaction) and coprecipitated proteins detected by immunoblot. E, PM subcellular fractions prepared from Munc18-1 or control-transduced cells were used in anti-Munc18-1 immunoprecipitation reactions, and co-precipitated proteins were detected by immunoblot. Data are representative of at least three independent experiments for each data panel. Each set of experiments was quantified by optical density scanning, with bar graphs showing ratios of protein associations to the right of the corresponding set of immunoblots; *, p < 0.05 versus Con-Ad. Ad, adenovirus; Con, control adenovirus-transduced cells.

FIGURE 8.

Knockdown of syntaxin 4 abrogates the effect of Munc18-1 overexpression upon secretion from MIN6 β-cells. MIN6 cells were co-transfected to express either RNAi targeting syntaxin 4 (siSyn4) or a non-targeting control (siCon), Munc18-1, or control (pcDNA3.1 vector), all with human proinsulin (reporter of secretion selectively in transfectable cells). Following 48 h of incubation, cells were subsequently incubated for 2 h in glucose-free MKRBB (basal condition) and stimulated with KCl (50 mm, 20 min) for the following: preparation of cleared detergent cell lysates for detection of syntaxin 4 knockdown and Munc18-1 overexpression levels (A) and for quantitation of human C-peptide release, reporting insulin release (B). No significant differences in basal secretion were detected; data are presented as the average stimulation index (stimulated/basal) in four independent experiments; *, p < 0.05 versus Con/siCon; **, p < 0.05 versus Munc18-1/siCon.