Exocytosis of the hormone glucagon-like peptide 1 (GLP-1) by the intestinal L cell is essential for the incretin effect after nutrient ingestion and is critical for the actions of dipeptidyl peptidase 4 inhibitors that enhance GLP-1 levels in patients with type 2 diabetes. Two-photon microscopy revealed that exocytosis of GLP-1 is biphasic, with a first peak at 1–6 min and a second peak at 7–12 min after stimulation with forskolin. Approximately 75% of the exocytotic events were represented by compound granule fusion, and the remainder were accounted for by full fusion of single granules under basal and stimulated conditions. The core SNARE protein syntaxin-1a (syn1a) was expressed by murine ileal L cells. At the single L-cell level, first-phase forskolin-induced exocytosis was reduced to basal (P < 0.05) and second-phase exocytosis abolished (P < 0.05) by syn1a knockout. L cells from intestinal-epithelial syn1a–deficient mice demonstrated a 63% reduction in forskolin-induced GLP-1 release in vitro (P < 0.001) and a 23% reduction in oral glucose–stimulated GLP-1 secretion (P < 0.05) in association with impairments in glucose-stimulated insulin release (by 60%; P < 0.01) and glucose tolerance (by 20%; P < 0.01). The findings identify an exquisite mechanism of metered secretory output that precisely regulates release of the incretin hormone GLP-1 and hence insulin secretion after a meal.

The incretin hormone glucagon-like peptide 1 (GLP-1) plays an essential role in the maintenance of normoglycemia through enhancement of glucose-dependent insulin secretion and suppression of glucagon release, gastric emptying, and appetite (1,2). As a consequence of these beneficial actions, GLP-1 receptor agonists are now widely used to treat patients with type 2 diabetes (T2D) and obesity. In contrast to the actions of GLP-1 derivative drugs, therapeutic dipeptidyl peptidase 4 (DPP-4) inhibitors prevent the degradation of endogenously secreted GLP-1 as well as the other incretin hormone glucose-dependent insulinotropic polypeptide (GIP) (3,4). The possibility of using GLP-1 secretagogues, alone or in combination with DPP-4 inhibition, therefore has engendered considerable interest as a new approach to incretin therapy (57).

Hormone secretion by the intestinal L cell, which includes not only GLP-1 but also the related peptides GLP-2 and oxyntomodulin, demonstrates two peaks of release after nutrient ingestion (8,9). Rodent studies have demonstrated that part of the early peak of GLP-1 secretion is mediated indirectly through vagal pathways originating in the duodenum that activate muscarinic receptors on the distal L cell (1012). In contrast, the later peak of GLP-1 release is initiated by direct contact of luminal nutrients with the L cell, resulting in transporter- and ion channel–mediated depolarization as well as activation of multiple G-protein–coupled receptors (1316). Many of these receptors are expressed not only by the intestinal L cell but also by the pancreatic β-cell (17), consistent with the coordinated release of GLP-1 and insulin in vivo. However, in contrast to the β-cell for which the signaling pathways regulating insulin secretion are well-established (18), much less is known about the intestinal L cell.

Glucose-mediated depolarization of the β-cell opens voltage-gated calcium channels, thereby activating the calcium sensor synaptotagmin-7 (19). This relieves the clamping action of synaptotagmin-7 on the SNARE fusion machinery, which consists of the vesicle SNARE protein VAMP and the plasma membrane SNAREs syntaxin (syn) and SNAP23/25 (20), thus permitting insulin exocytosis. Although the β-cell expresses several SNARE isoforms, VAMP2 and syn1a are most important for the first phase of glucose-stimulated insulin secretion, with VAMP8 and syn3/4 contributing to both first and second phase release (2123). The SNARE proteins also mediate several types of exocytotic events in the β-cell. Thus, predocked secretory granules (SGs) contribute predominantly to first phase secretion, whereas newcomer granules recruited from an intracellular reserve pool account for some of first and practically all of second phase insulin exocytosis (24). Furthermore, these SGs undergo temporally and physically distinct types of fusion with the plasma membrane, which are termed full, sequential, compound, and kiss and run (2429).

Although one study demonstrated a role for synaptotagmin-7 in GLP-1 secretion (30), little is known about the role of the core SNARE proteins in the L cell. We have reported that the murine GLUTag L-cell line expresses multiple isoforms of VAMP and syn as well as SNAP25 (31). VAMP2 was demonstrated to play an important role in GLP-1 secretion by these cells and was found to be expressed in primary mouse intestinal L cells. Furthermore, VAMP2 coimmunoprecipitated with syn1a and SNAP25 in the GLUTag cells, suggesting that these SNARE proteins form a functional exocytotic complex in the L cell. Given the current interest in therapeutic approaches to enhance release of GLP-1 into the circulation (57), we have now used novel knockout (KO) mouse models in vivo and ex vivo in combination with single-cell visualization of exocytosis to interrogate GLP-1 release by the primary intestinal L cell. We show that GLP-1 release is biphasic, as mediated primarily by multigranular (i.e., compound) fusion under both basal and stimulated conditions, and that the SNARE protein syn1a plays an essential role in the exocytosis of GLP-1. These findings identify novel regulatory mechanisms that underlie secretion of the incretin hormone GLP-1 and have implications regarding the development of GLP-1 secretagogues for therapeutic use in the treatment of T2D and obesity.

Animals

Male C57BL/6 mice (7–12 weeks old) were from Charles River Laboratories. Female syn1afl/fl (32) were crossed with male villin-creERT2+/0 [B6-Tg(Vil-cre/ERT2)23Syr] (33,34) mice, and the resultant tamoxifen-inducible intestinal-epithelial syn1afl/fl;villin-creERT2+/0 (IE-syn1a KO) animals were crossed with proglucagon (Gcg)-Venus mice (35) to generate Venus-IE-syn1a KO animals (Supplementary Table 1 lists the primers). Age- (7–12 weeks) and sex-matched littermates were studied, and controls included vehicle- and tamoxifen (in 1 mg/100 μL sunflower oil i.p. for 5 days)-treated syn1afl/fl, villin-creERT2+/0, syn1afl/+;villin-creERT2+/0, and syn1afl/+;villin-creERT2+/0;Gcg-Venus mice as well as vehicle-treated syn1afl/fl;villin-creERT2+/0 and syn1afl/fl;villin-creERT2+/0;Gcg-Venus animals as appropriate for the KO model being studied. Two to 3 days after induction, mice were fasted overnight followed by an oral glucose tolerance test (OGTT) (5 g/kg glucose [we have previously shown that 6 g/kg but not 1.5 or 3 g/kg permits detection of bioactive GLP-1 release in mice (36)]) or intraperitoneal glucose tolerance test (IPGTT) (3 g/kg glucose as determined in pilot studies to match the glycemic response observed in the OGTT [data not shown]) and tail-vein blood collection. Glycemia was determined by using a glucose meter (OneTouch; LifeScan) and plasma total GLP-1 (Mesoscale Discovery), total GIP (Millipore), and insulin (Crystal Chem) by assay kit as previously reported (37). Plasma GLP-1 levels were normalized to the control values obtained for each individual plate used for analysis. Because of their high concentration of L cells (38), ileal segments were collected for primary culture and analyses. All animal protocols were approved by the Animal Care Committee of the University of Toronto (Toronto, Ontario, Canada).

Adult Mouse Ileal Crypt Cultures

Isolated crypts from 10-cm mouse ileum were plated overnight as previously reported (35). Two-hour secretion assays were followed by ELISA (Millipore) for active GLP-1 levels in media and cells (12,35). Secretion was calculated as the percentage of total culture content detected in the media. Experiments were conducted in quadruplicate wells to make n = 1 per mouse.

Two-photon microscopy was performed on adult mouse ileal crypt (AMIC) cultures perfused at 2 mL/min and 30°C. Sulforhodamine B (0.8 mmol/L) (25) was added 2–3 min before visualization with a Nikon A1R multiphoton microscope for up to 3 min (basal) followed by 12 min with 50 μmol/L forskolin. L cells were identified by Venus (yellow fluorescent protein) fluorescence, and the cell membrane was defined by the extracellular localization of sulforhodamine B. Data were analyzed with NIS-Elements software (Nikon). Focal events occurring for ≤10 s were classified as full fusion, whereas events of longer duration were considered to be compound fusion.

Morphometric and Immunometric Analyses

Crypt-villus height was measured in hematoxylin-eosin–stained ileal sections in a minimum of 20 well-oriented axes per mouse. Ileal sections and AMIC cultures were stained for syn1a and GLP-1 (Supplementary Table 2 lists the antibodies) followed by visualization with a Zeiss deconvolution microscope and analysis of fluorescent intensity with ImageJ software (39). Negative controls omitted the primary antisera.

Molecular Analyses

Ileal Venus-positive and -negative cells were collected from female Gcg-Venus mice (n = 3) by FACS. Barcode ligation and end repair were achieved by using the Ovation Rapid DR Multiplex System 1–96 (NuGEN). Combined barcoded libraries underwent SE50 sequencing with an Illumina HiSeq 2500 system (Genomics Core Facility, Cancer Research UK Cambridge Institute). Sequence reads were demultiplexed by using the CASAVA pipeline (Illumina) and aligned to the mouse genome (GRCm38) by using TopHat 2.1.0 software (http://ccb.jhu.edu/software/tophat/index.shtml). Differential gene expression was determined with Cufflinks 2.2.1 software (http://cole-trapnell-lab.github.io/cufflinks) as previously reported (35).

Total RNA from ileal mucosal scrapes and AMIC cultures was reverse transcribed and analyzed by PCR with primer/probe sets from Applied Biosystems (Supplementary Table 3). Histone 3A was used as the internal control for analysis by the ΔΔCt method.

Adenovirus Studies

Syn1afl/fl mice were anesthetized and laparotomized, and 0.75 mL of 0.9–1.8 × 108 infection units/mL adenovirus-red fluorescent protein (Adv-RFP)–improved Cre recombinase or Adv-RFP (control; Vector Biolabs) was injected into the distal ileal lumen. Animals recovered for 2 days and were then relaparotomized and 0.75 mL oleoylethanolamide (OEA) 15 μmol/L was injected into the ileum followed by sampling for analysis of glycemia, plasma hormone levels, and ileal gene expression as above. AMIC cultures were infected with 2.4 × 107 infectious units/mL Adv-RFP–improved Cre recombinase or Adv-RFP for 48 h followed by a 2-h GLP-1 secretion assay and analysis by ELISA and quantitative RT-PCR as above.

Statistical Analyses

Data are shown as mean ± SD. Statistical differences were determined by Student t test or one- or two-way ANOVA followed by Student t test or Dunnett's multiple comparison test, as appropriate.

Immunostaining of the ileum from normal mice revealed expression of syn1a in GLP-1–expressing cells as well as in other cells in the crypt and scattered through the villous epithelium (Fig. 1A). We also examined colocalization of GLP-1 and syn1a in AMIC cultures, a valuable ex vivo model for the study of GLP-1 secretion by primary L cells (Fig. 1B) (35). Of the 1.1 ± 0.1% of all GLP-1-positive AMIC cells (n = 1,670–5,095 cells each in six independent cultures), 100% coexpressed syn1a. Confirmation of specificity of the syn1a staining showed that AMIC cultures generated from IE-syn1a KO mice demonstrated a significant loss of syn1a staining in the L cells (P < 0.001) as well as a more global loss in other unidentified crypt cells (Fig. 1C and D).

Figure 1

Primary murine intestinal L cells express syn1a and secrete GLP-1 in vitro. AC: Mouse intestine (A) and AMIC cultures (B and C) were immunostained for GLP-1 (red) and syn1a (green); DAPI is blue, and negative controls are shown in the inset. Arrows indicate GLP-1–positive cells. Representative images are shown from six IE-syn1a control mice (A and B) and four IE-syn1a KO animals (C). D: Quantification of control and IE-syn1a KO AMIC L-cell syn1a immunofluorescence intensity (n = 4 cells from four control mice and 6 cells from five IE-syn1a KO mice). EH: AMIC cultures from C57BL/6 mice were treated for 2 h with forskolin/IBMX (F/I) (n = 10–11) or varying concentrations of GIP (n = 7–12) or OEA (n = 5–7), with F/I as the positive control. GLP-1 secretion as a percentage of total content (E) and total GLP-1 content (F) for the same cultures are shown. Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 1

Primary murine intestinal L cells express syn1a and secrete GLP-1 in vitro. AC: Mouse intestine (A) and AMIC cultures (B and C) were immunostained for GLP-1 (red) and syn1a (green); DAPI is blue, and negative controls are shown in the inset. Arrows indicate GLP-1–positive cells. Representative images are shown from six IE-syn1a control mice (A and B) and four IE-syn1a KO animals (C). D: Quantification of control and IE-syn1a KO AMIC L-cell syn1a immunofluorescence intensity (n = 4 cells from four control mice and 6 cells from five IE-syn1a KO mice). EH: AMIC cultures from C57BL/6 mice were treated for 2 h with forskolin/IBMX (F/I) (n = 10–11) or varying concentrations of GIP (n = 7–12) or OEA (n = 5–7), with F/I as the positive control. GLP-1 secretion as a percentage of total content (E) and total GLP-1 content (F) for the same cultures are shown. Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

GLP-1 secretion by AMIC cultures from normal mice was increased to 2.3-fold of control (P < 0.01) in response to treatment with forskolin plus 3-isobutyl-1-methylxanthine (IBMX) without any change in GLP-1 synthesis (Fig. 1E and F). Similarly, treatment with the physiological secretagogues GIP (Fig. 1G) and the GPR119 agonist OEA (Fig. 1H) enhanced GLP-1 release to 1.7- and 3.5-fold of control (P < 0.05–0.001), respectively. These findings supported the use of both the IE-syn1a KO mouse model and AMIC cultures in further studies to assess the role of syn1a in L-cell secretion.

Compared with control animals, IE-syn1a KO mice demonstrated increases in body (Fig. 2A) and intestinal (Fig. 2B) weight 7–12 days after the completion of daily tamoxifen injections (P < 0.05–0.001). However, upon normalization of intestinal weight to body weight, the observed increase in intestinal weight was maintained only in male animals lacking syn1a (Fig. 2C and Supplementary Fig. 1), suggesting sexual dimorphism. Morphological characterization of the intestinal epithelium further revealed that IE-syn1a KO animals had a small, but significant increase in crypt depth (P < 0.01) (Fig. 2D). Furthermore, the 39% reduction of syn1a (i.e., Stx1a) mRNA observed in intestinal mucosal scrapes from KO animals (P < 0.05) (Fig. 2E) was associated with increases in the expression of Stx1b and -2 (P < 0.05–0.01) (Fig. 2F). Although the function of syn1b in the intestinal epithelium is not known, it appears to play a role in mast cell degranulation (40). In contrast, Stx2 (or epimorphin) expression in mesenchymal cells abutting the epithelium has been shown to regulate the morphology of the crypt-villus axis (41), suggesting a role in the observed increase in crypt depth. Examination of two other syn isoforms that also localize to the plasma membrane (Stx3 and -4) (42) did not reveal any other adaptive changes (Fig. 2F).

Figure 2

IE-syn1a KO mice demonstrate intestinal adaptive responses. AC: Body weight, intestinal weight, and intestinal weight normalized to body weight in male and female control and IE-syn1a KO mice (n = 10–13). D: Crypt depth and villus height in control and IE-syn1a KO mice (n = 5–8). E and F: Stx1a and Stx isoform and Gcg transcript levels in ileal mucosal scrapes from control and IE-syn1a KO mice (n = 10–13). Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control mice.

Figure 2

IE-syn1a KO mice demonstrate intestinal adaptive responses. AC: Body weight, intestinal weight, and intestinal weight normalized to body weight in male and female control and IE-syn1a KO mice (n = 10–13). D: Crypt depth and villus height in control and IE-syn1a KO mice (n = 5–8). E and F: Stx1a and Stx isoform and Gcg transcript levels in ileal mucosal scrapes from control and IE-syn1a KO mice (n = 10–13). Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control mice.

To determine whether intestinal-epithelial syn1a is related to glucose tolerance, we performed an OGTT in control and IE-syn1a KO mice 2 days after completion of tamoxifen or vehicle injection. When considered together, male and female IE-syn1a KO animals displayed a significant impairment in glucose tolerance, with blood glucose levels reaching 1.3- and 1.4-fold of controls at 45 min (P < 0.05) and 60 min (P < 0.001), respectively (Fig. 3A). Accordingly, the 2-h glycemic area under the curve in IE-syn1a KO mice was also significantly increased to 1.2-fold of controls (P < 0.01); this phenotype was a result of differences in male mice only (Fig. 3B and Supplementary Fig. 2). IE-syn1a KO mice also showed a reduction in plasma insulin levels compared with controls, which reached statistical significance at 60 min (Fig. 3C) and appeared to be more apparent in male mice (Fig. 3C and D and Supplementary Fig. 2). Furthermore, basal GLP-1 levels were 17% lower in IE-syn1a KO mice than in controls (P < 0.05), with an even further reduction at 10 min post–oral glucose administration (by 23%; P < 0.05 vs. basal; P < 0.01 vs. the difference at 0 min) (Fig. 3E). However, plasma GLP-1 levels were reduced in both male and female IE-syn1a KO animals, although this change reached significance only in the males (P < 0.05) (Fig. 3F and Supplementary Fig. 3A). Gene expression analysis of the GLP-1 prohormone Gcg in the intestinal mucosa showed no difference between KO and control animals (Fig. 2F), indicating that the reduced GLP-1 levels in IE-syn1a KO mice are not due to a deficiency in GLP-1 production. Similar to the reduction in GLP-1 levels, both male and female IE-syn1a KO animals also displayed significantly lower plasma GIP concentrations by 31–53% at 0, 10, and 60 min (P < 0.05–0.01) (Fig. 3G and H and Supplementary Fig. 3). Furthermore, a requirement for the incretin hormones in the impaired glucose tolerance of KO animals was confirmed by the demonstration that male and female mice with loss of IE-syn1a had normal glucose tolerance in response to an IPGTT (Fig. 3I and J). Together, these data suggest that the compromised glucose homeostasis in IE-syn1a KO animals occurs, at least in part, as a result of reductions in both GLP-1 and GIP secretion.

Figure 3

IE-syn1a KO mice demonstrate reduced GLP-1 secretory responses during an OGTT in association with impaired glucose tolerance. AH: Male and female control and IE-syn1a KO mice (n = 8–22 combined; n = 3–13 for males; n = 3–14 for females) were administered an OGTT at 0 min followed by blood sampling at 0, 10, 60, 90, and 120 min; a second cohort of mice (n = 7–10 combined; n = 4–5 for males; n = 3–5 for females) also included sampling for blood glucose at 30 and 45 min. Blood glucose (A and B) and levels of plasma insulin (C and D), GLP-1 (E and F) (plasma GLP-1 levels were normalized to control levels at 0 min [20.8 ± 3.1 pg/mL; n = 25] to account for variance found between two different assay plates), and GIP (G and H) are shown. I and J: Separate cohorts of male and female mice (n = 6 combined; n = 3 each for males and females) were administered an IPGTT at 0 min followed by blood sampling for determination of blood glucose. Data are mean ± SD, with panels B, D, F, H, and J representing the area under the curve (AUC) separated for males (left) and females (right) for the data shown in panels A, C, E, G, and I, respectively. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control mice or as indicated for the Δ response. CON, controls.

Figure 3

IE-syn1a KO mice demonstrate reduced GLP-1 secretory responses during an OGTT in association with impaired glucose tolerance. AH: Male and female control and IE-syn1a KO mice (n = 8–22 combined; n = 3–13 for males; n = 3–14 for females) were administered an OGTT at 0 min followed by blood sampling at 0, 10, 60, 90, and 120 min; a second cohort of mice (n = 7–10 combined; n = 4–5 for males; n = 3–5 for females) also included sampling for blood glucose at 30 and 45 min. Blood glucose (A and B) and levels of plasma insulin (C and D), GLP-1 (E and F) (plasma GLP-1 levels were normalized to control levels at 0 min [20.8 ± 3.1 pg/mL; n = 25] to account for variance found between two different assay plates), and GIP (G and H) are shown. I and J: Separate cohorts of male and female mice (n = 6 combined; n = 3 each for males and females) were administered an IPGTT at 0 min followed by blood sampling for determination of blood glucose. Data are mean ± SD, with panels B, D, F, H, and J representing the area under the curve (AUC) separated for males (left) and females (right) for the data shown in panels A, C, E, G, and I, respectively. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control mice or as indicated for the Δ response. CON, controls.

To confirm that loss of syn1a impairs L-cell secretory function, AMIC cultures were generated from crypts isolated from IE-syn1a KO and control animals. Of note, IE-syn1a KO AMIC cultures had a 78% reduction in Stx1a expression (P < 0.01) (Fig. 4A). This more-profound knockdown in the crypt cultures compared with the mucosal scrapes is likely a consequence of epithelial cell enrichment in the cultures, whereas both villus epithelial and non–villin-expressing syn1a-positive cells contribute to the syn1a signal in the scrapes. We also examined the Stx1b, -2, -3, and -4 isoforms in the AMIC cultures and found no differences in expression (Fig. 4B), further suggesting that adaptive changes observed in the mucosa are independent of the intestinal-epithelial cells.

Figure 4

AMIC cultures from IE-syn1a KO mice demonstrate reduced GLP-1 secretory responses. A and B: Stx1a and Stx isoform and Gcg transcript levels in AMIC cultures from male and female control and IE-syn1a KO mice (n = 4–6). C and D: Control and IE-syn1a KO AMIC cultures were treated for 2 h with vehicle (basal) or forskolin plus IBMX (F/I) (n = 7–9) followed by determination of GLP-1 release into the media (control basal secretion was 5.9 ± 2.2% of total cell content) and GLP-1 content in the media plus cells. Data are mean ± SD. **P < 0.01, ***P < 0.001 vs. basal. ###P < 0.001 as indicated.

Figure 4

AMIC cultures from IE-syn1a KO mice demonstrate reduced GLP-1 secretory responses. A and B: Stx1a and Stx isoform and Gcg transcript levels in AMIC cultures from male and female control and IE-syn1a KO mice (n = 4–6). C and D: Control and IE-syn1a KO AMIC cultures were treated for 2 h with vehicle (basal) or forskolin plus IBMX (F/I) (n = 7–9) followed by determination of GLP-1 release into the media (control basal secretion was 5.9 ± 2.2% of total cell content) and GLP-1 content in the media plus cells. Data are mean ± SD. **P < 0.01, ***P < 0.001 vs. basal. ###P < 0.001 as indicated.

Secretion assays with AMIC cultures generated from IE-syn1a KO mice demonstrated no difference in basal GLP-1 secretion compared with control animals (Fig. 4C). However, IE-syn1a KO mice displayed a 2.6-fold reduction in forskolin-stimulated GLP-1 secretion to 37% of the response found in controls (P < 0.001) (Fig. 4C). Total GLP-1 content of the cultured cells did not differ between the genotypes or treatment groups (Fig. 4D). Gcg gene expression also did not differ as a result of loss of syn1a (Fig. 4B), further suggesting that the reduced GLP-1 secretion is a result of disrupted L-cell secretory function.

To examine more directly the role of syn1a in GLP-1 secretion by the primary L cell, we initially conducted in vivo and in vitro studies that used adenovirus-mediated knockdown of syn1a. However, application of the control adenovirus alone blunted the ability of the intestinal L cells to respond to known secretagogues, making this an inappropriate model for further study (Supplementary Fig. 4). We therefore crossed the IE-syn1a KO animals with mice expressing Venus under the control of the Gcg promoter (35) to permit identification of the L cell for exocytotic analysis. Although Venus-IE-syn1a KO mice did not show changes in body weight, they were found to have increased intestinal weight, which after normalization to body weight, was maintained only in male animals lacking syn1a (Fig. 5A–C and Supplementary Fig. 5). This pattern was similar to that observed in the IE-syn1a KO mice that did not express Gcg-Venus (Supplementary Fig. 1). Furthermore, although Stx1a expression was reduced by 58% (P < 0.05) in the intestinal mucosa (Fig. 5D), no significant changes were observed in expression of Stx1b and -2 (Fig. 5E). However, an OGTT revealed that Venus-IE-syn1a KO animals, like the IE-syn1a KO mice, had impaired glucose tolerance, with significantly elevated glycemia at 60 min after oral glucose administration compared with controls (1.3-fold of control values; P < 0.05) (Fig. 5F). Like the IE-syn1a KO mice, these animals also demonstrated sexual dimorphism, with significant changes found only in the male mice (Fig. 5G and Supplementary Fig. 6). Collectively, these findings suggest an impairment in GLP-1 secretion in the Venus-IE-syn1a KO animals, despite the finding of a 1.8-fold increase in intestinal mucosal Gcg gene expression (P < 0.05) (Fig. 5E).

Figure 5

Venus-IE-syn1a KO mice demonstrate impaired glucose tolerance during an OGTT. AC: Body weight, intestinal weight, and intestinal weight normalized to body weight in male and female control and IE-syn1a KO mice. D and E: mRNA transcript levels for Stx1a and Stx isoforms and Gcg in ileal mucosal scrapes from control and IE-syn1a KO mice. F: Control and IE-syn1a KO mice were administered an OGTT at 0 min followed by blood sampling for determination of blood glucose levels (n = 9–12 for AF). G: Area under the curve (AUC) separated for males (left) (n = 4–6) and females (right) (n = 3–5) for the data shown in panel F. H: RNA sequencing for syn isoforms in FACS-isolated Venus-positive vs. Venus-negative jejunal/ileal epithelial cells from female Gcg-Venus mice (inset shows expanded scale) (n = 3). Data are mean ± SD. *P < 0.05, **P < 0.01 vs. control mice. FPKM, fragments per kilobase of exon per million fragments mapped.

Figure 5

Venus-IE-syn1a KO mice demonstrate impaired glucose tolerance during an OGTT. AC: Body weight, intestinal weight, and intestinal weight normalized to body weight in male and female control and IE-syn1a KO mice. D and E: mRNA transcript levels for Stx1a and Stx isoforms and Gcg in ileal mucosal scrapes from control and IE-syn1a KO mice. F: Control and IE-syn1a KO mice were administered an OGTT at 0 min followed by blood sampling for determination of blood glucose levels (n = 9–12 for AF). G: Area under the curve (AUC) separated for males (left) (n = 4–6) and females (right) (n = 3–5) for the data shown in panel F. H: RNA sequencing for syn isoforms in FACS-isolated Venus-positive vs. Venus-negative jejunal/ileal epithelial cells from female Gcg-Venus mice (inset shows expanded scale) (n = 3). Data are mean ± SD. *P < 0.05, **P < 0.01 vs. control mice. FPKM, fragments per kilobase of exon per million fragments mapped.

Because the heterogeneous cell population of both the ileal mucosa and the isolated ileal crypt cultures limits our understanding of L-cell–specific gene expression, we compiled a syn isoform expression profile from Venus-positive L cells by RNA sequencing (Fig. 5H). Of the four syn isoforms known to be localized on the plasma membrane, only Stx1a appeared to be enriched (by 3.6-fold) in the Venus-positive L cells compared with Venus-negative intestinal-epithelial cells. Collectively, these data validate the use of Venus-IE-syn1a KO mice as a model for these studies of the role of syn1a in GLP-1 exocytosis.

Because exocytosis by the primary intestinal L cell has not been previously described, we used two-photon microscopy to visualize SG fusion events in Venus-positive L cells from both control and Venus-IE-syn1a KO mice under basal and 50 μmol/L forskolin-stimulated conditions (Fig. 6A and Supplementary Fig. 7). In the absence of forskolin, all the Venus-positive and some of the Venus-negative cells demonstrated periodic exocytotic events; an increase in exocytosis in both cell types was noted upon the addition of forskolin. However, some cells demonstrated a profound level of activity compared with the surrounding cells, with a large granule size consistent with the known characteristics of mast cells (Supplementary Video 1) (43). As has been reported for the β-cell (2429), distinct types of exocytotic events were observed in Venus-positive L cells from control animals. Thus, throughout the duration of the recordings, we identified both single (full) (Supplementary Video 2) and multigranular (compound) (Supplementary Video 3) SG fusions that differed in the size of the signal and the duration of the event (Fig. 6A).

Figure 6

Two-photon microscopy of primary ileal L cells demonstrates the role of syn1a in multiple forms of exocytosis. A: Representative images and fluorescent intensity tracings of single/full SG and multigranular/compound SG fusion events in Venus-positive L cells from control mice. BD: Quantification of the total number of fusion events collected over a 3-min basal period and a 12-min forskolin infusion period in control (B) and Venus-IE-syn1a KO (C) mice; the same data were binned into different phases of secretion (basal t = −3 to −1 min; first phase t = 1–6 min; second phase t = 7–12 min) and then classified as full vs. compound fusion (D). The dotted line in panel B indicates the best-fit curve for stimulated exocytosis (R2 = 0.82). *P < 0.05, ** P < 0.01, *** P < 0.001 vs. basal; #P < 0.05, ###P < 0.001 vs. the same phase in control mice for total fusion events. E: Cumulative total fusion events. *P < 0.05, **P < 0.01 for each time point at 4–12 min. n = 9 cells from six control mice, and n = 6 cells from three IE-Venus-syn1a KO. Data are mean ± SD.

Figure 6

Two-photon microscopy of primary ileal L cells demonstrates the role of syn1a in multiple forms of exocytosis. A: Representative images and fluorescent intensity tracings of single/full SG and multigranular/compound SG fusion events in Venus-positive L cells from control mice. BD: Quantification of the total number of fusion events collected over a 3-min basal period and a 12-min forskolin infusion period in control (B) and Venus-IE-syn1a KO (C) mice; the same data were binned into different phases of secretion (basal t = −3 to −1 min; first phase t = 1–6 min; second phase t = 7–12 min) and then classified as full vs. compound fusion (D). The dotted line in panel B indicates the best-fit curve for stimulated exocytosis (R2 = 0.82). *P < 0.05, ** P < 0.01, *** P < 0.001 vs. basal; #P < 0.05, ###P < 0.001 vs. the same phase in control mice for total fusion events. E: Cumulative total fusion events. *P < 0.05, **P < 0.01 for each time point at 4–12 min. n = 9 cells from six control mice, and n = 6 cells from three IE-Venus-syn1a KO. Data are mean ± SD.

The pattern of fusion events observed in L cells from Venus-control mice was strongly indicative of biphasic exocytosis. Hence, after the basal period, the addition of forskolin stimulated a first phase of exocytosis at 1–6 min followed by a second phase at 7–12 min (Fig. 6B, D, and E). Between single L cells from Venus-IE-syn1a KO and Venus-control mice, no obvious change was seen in the number of SG fusion events during the basal period (Fig. 6B–E). However, recordings from Venus-IE-syn1a KO L cells demonstrated that syn1a depletion dramatically reduced the number of SG fusion events under stimulating conditions such that first-phase exocytosis was reduced to basal levels (P < 0.05) and second phase was abolished (P < 0.05). Finally, determination of the contributions of full and compound fusion events to each phase of secretion revealed that L-cell exocytosis is largely mediated by compound fusion (∼75% of total events) under both basal and stimulated conditions (Fig. 6D). Of note, in the absence of syn1a, the reduced number of SG fusion events in both first- and second-phase secretion was attributed to the loss of both full and compound fusions. This result demonstrates the preservation of SG fusion competence under basal conditions but a requirement for syn1a in stimulated SG fusion in the primary L cell. Taken together, these data illuminate the spatiotemporal activity of the SG exocytosis that underlies GLP-1 secretion.

The actions of the incretin hormones GLP-1 and GIP account for ∼50% of the insulin response to nutrient ingestion (44,45). However, despite the importance of GLP-1 in the maintenance of glucose homeostasis, major gaps remain in our understanding of the molecular machinery that regulates GLP-1 release. Although studies on the primary L cell have been limited in the past largely because of the diffuse dispersion of these cells throughout the intestinal epithelium (38), recent advances now permit direct visualization of exocytosis by reporter-labeled L cells ex vivo after primary culture (35). From these approaches, the findings of the current study demonstrate the dynamics of SG fusion to the plasma membrane of the primary intestinal L cell and the essential role of the core SNARE protein syn1a in secretagogue-induced exocytosis of GLP-1.

Two-photon microscopy demonstrated that exocytosis by the primary L cell is mediated through various exocytotic events of which the majority are compound SG fusion rather than full fusion of single SGs, under both basal and stimulated conditions. These findings contrast those of the β-cell, for which different types of fusion events occur during different phases of secretion (2126,28,29). Hence, approximately one-half of first-phase insulin secretion is mediated by predocked SGs undergoing full fusion (46). In contrast, the sustained second phase of insulin exocytosis is largely determined by influx of newcomer SGs from the intracellular reserve pool, which then undergoes mostly full fusion, with only a small contribution attributed to compound exocytosis (24,28). Of note, the use of two-photon microscopy in the current study precluded determination of any contribution of kiss-and-run exocytosis to GLP-1 secretion as well as to whether the detected SGs were predocked or newcomer, which would be better observed by total internal reflection fluorescence and electron microscopy. Furthermore, deletion of syn1a from the plasma membrane prevented not only single but also multigranular fusion events. Indeed, compound fusion may require other isoforms of syn expressed in the L cell (31), as demonstrated for the β-cell (23,47). However, prevention of initial SG fusion with the plasma membrane is expected to preclude detection by two-photon microscopy of any subsequent SG-to-SG interactions, including not only compound but also sequential fusion events. Thus, the factors that determine the exact nature of exocytotic fusion events in the L cell remain to be fully defined. However, one signaling pathway that may be involved is Cdc42-dependent reorganization of the actin cytoskeleton that forms a permissive barrier to GLP-1 secretion (48) and, therefore, may decrease the ability of SGs to move toward the plasma membrane. Because a similar mechanism mediates glucose-stimulated exocytosis of newcomer SGs in the β-cell (49,50), future studies to interrogate the relationships among Cdc42, the actin cytoskeleton, and SG dynamics in the intestinal L cell are warranted. Furthermore, altered β-cell SNARE protein expression in response to gluco- and lipotoxicity is known to affect insulin secretion (51,52). However, although GLP-1 release also is dysregulated during feeding of a high-fat or Western diet and in association with hyperglycemia caused by circadian disruption (5355), whether this is due to changes in expression of syn1a and/or other L-cell SNARE proteins remains unknown.

Single-cell imaging indicated that GLP-1 secretion by the primary L cell is biphasic, with the first phase occurring 1–6 min after stimulation and a more sustained second phase at 7–12 min. Of note, the isolated rat ileum has previously been found to demonstrate biphasic release of GLP-1 in response to bethanechol and bombesin but not calcitonin gene-related peptide, with a first peak at 2–4 min and a second phase maintained for the duration of the vascular perfusion (10). Furthermore, re-examination of total internal reflection fluorescence microscopy data from the GLUTag L-cell line also suggests the existence of two phases of exocytosis. Hence, ∼0.4 fusions/100 μm2-30 s were detected under basal conditions, and this increased to ∼1.5 at 1–3 min after KCl-mediated depolarization followed by a second phase with ∼1 event/100 μm2-30 s over the ensuing 4–7 min (31). Thus, unlike the β-cell for which KCl-induced depolarization increases first-phase secretion, whereas glucose induces biphasic release (2126,28,29), the intestinal L cell demonstrates biphasic secretion after activation by KCl, forskolin, and some, but not all, physiological secretagogues.

Compared with the normal intestine wherein L cells constitute ∼0.5% of epithelial cells (56), 1.1% of the cells in the AMIC cultures expressed GLP-1, indicating a relative enrichment in this ileal crypt cell model. Furthermore, 100% of the AMIC L cells expressed syn1a and, of the syntaxin isoforms known to be expressed on the plasma membrane (Stx1a, -1b, -2, -3, and -4) (42), only Stx1a was found to be enriched. Consistent with an important role for this core SNARE protein in the L cell, KO of syn1a reduced forskolin-stimulated GLP-1 secretion at both the single-cell and the population level in AMIC cultures and reduced OGTT-induced GLP-1 release in the mouse in vivo. Although no profound effects of the KO were found on basal GLP-1 release in the ex vivo and single-cell models examined, basal GLP-1 levels were reduced in vivo as a potential consequence of villin-driven Cre expression resulting in altered release of or responsiveness to paracrine regulators of GLP-1 secretion. Of note, IE-syn1a KO animals demonstrated even greater reductions in basal and stimulated GIP secretion, likely contributing to the glucose intolerant phenotype through both the reduction of its own insulinotropic actions and reduced L-cell stimulation by GIP (11). Future studies examining exocytotic dynamics in the GIP-producing K cell are clearly warranted. Finally, the maintenance of basal L-cell exocytosis and only partial loss of the first phase of secretion in the absence of syn1a suggest contributions by another syn isoform in the L cell to mediate fusion of predocked SGs, with the almost complete loss of second-phase secretion explained by the loss of syn1a-mediated newcomer SG fusion comparable to findings in the β-cell (47). Nonetheless, our previous report of expression of VAMP2 in primary L cells, a role for VAMP2 in GLP-1 release, and an interaction among VAMP2, syn1a, and SNAP25 in GLUTag cells (31) is consistent with the notion that these proteins form a core SNARE complex in the primary intestinal L cell.

Although both the syn1a KO models used in this study were generated from villin-creERT2+/0 and syn1afl/fl mice, small differences were noted in the IE-syn1a KO mice compared with those crossed with Gcg-Venus animals. Hence, the IE-syn1a KO mice demonstrated increases in Stx1b and Stx2 expression. Conversely, the Venus-IE-syn1a KO animals exhibited an increase only in Gcg expression, which could reflect a potential effect of Gcg-driven Venus expression in the L cell. However, the subtle differences in gene expression between the KO models were presumed not to influence GLP-1 secretion because L cell secretion was impaired by loss of syn1a in both the ex vivo secretion assays (IE-syn1a KO) and the single-cell experiments (Venus-IE-syn1a KO). Furthermore, when taken with the rigorous inclusion of multiple genotypes as controls for each of these animal models, our finding of consistent effects of syn1a KO to reduce GLP-1 secretion suggests that these small differences in mouse models had no impact on the conclusions of this study.

Although both male and female animals displayed reductions in GLP-1 and GIP levels, IE-syn1a KO mice demonstrated sexual dimorphism in their metabolic responses to oral glucose administration; similar differences in glucose tolerance were also found in the Venus-IE-syn1a KO animals. The female animals, therefore, demonstrated an improved capacity to compensate for the reduction in the incretin hormones. However, whether these findings can be extrapolated to humans remains unclear because a previous study indicated that OGTT-induced changes in the levels of glucose, insulin, and both incretin hormones are all increased in women (57).

The mechanisms underlying GLP-1 release from the intestinal L cell are of increasing interest as a therapeutic approach to the treatment of hyperglycemia in patients with T2D either alone or in combination with DPP-4 inhibition (57). The current findings identify an exquisite mechanism of metered secretory output that precisely regulates release of the incretin hormone GLP-1 and hence, insulin secretion after a meal. Furthermore, because the intestinal L cell cosecretes a number of other biologically active peptide hormones, these findings may also have implications in other disease states. The demonstration of a role for syn1a in modulating stimulated SG fusion events provides impetus for further studies to elucidate the full complement of SNARE and cognate accessory proteins in the primary L cell that mediate distinct exocytotic events as well as their coupling to the diverse signaling pathways activated by nutrient ingestion. Elucidation of the exact mechanisms underlying GLP-1 release holds important implications for the development of GLP-1 secretagogues to treat patients with T2D or obesity.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db16-1403/-/DC1.

Funding. S.E.W. was supported by Novo Nordisk Banting & Best Diabetes Centre (BBDC) (University of Toronto) Graduate Studentships, S.E.W. and H.M.S. by Ontario Graduate Scholarships, Y.N. by a University of Toronto Research Opportunity Summer Studentship, and S.J.H. by a BBDC Summer Studentship. Research in the F.R./F.M.G. laboratory was funded by the Wellcome Trust (106262/Z/14/Z, 106263/Z/14/Z) and the Medical Research Council (MRC_MC_UU_12012/3, MRC_MC_UU_12012/5). P.L.B. was supported by the Canada Research Chairs program. These studies were supported by operating grants to P.L.B. from the Natural Sciences and Engineering Research Council of Canada (RGPIN418) and the Canadian Institutes of Health Research (PJT-15308). The Nikon A1R multiphoton microscope and the Mesoscale Discovery Sector 2400A used in this study was supported by the 3D (Diet, Digestive Tract and Disease) Centre funded by the Canadian Foundation for Innovation and Ontario Research Fund (project numbers 19442 and 30961).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. S.E.W., H.M.S., Y.N., S.J.H., A.B.H., F.R., F.M.G., and P.L. researched data. S.E.W. and P.L.B. wrote the manuscript. H.M.S., Y.N., S.J.H., A.B.H., F.R., F.M.G., and H.Y.G. reviewed and edited the manuscript. P.L.B. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented at the 75th Scientific Sessions of the American Diabetes Association, Boston, MA, 5–9 June 2015; 76th Scientific Sessions of the American Diabetes Association, New Orleans, LA, 10–14 June 2016; and 77th Scientific Sessions of the American Diabetes Association, San Diego, CA, 9–13 June 2017.

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