Paracrine crosstalk between intestinal L- and D-cells controls secretion of glucagon-like peptide-1 in mice

Abstract

DPP-4 inhibitors, used for treatment of type 2 diabetes, act by increasing the concentrations of intact glucagon-like peptide-1 (GLP-1), but at the same time, they inhibit secretion of GLP-1, perhaps by a negative feedback mechanism. We hypothesized that GLP-1 secretion is feedback regulated by somatostatin (SS) from neighboring D-cells, and blocking this feedback circuit results in increased GLP-1 secretion. We used a wide range of experimental techniques, including gene expression analysis, immunohistochemical approaches, and the perfused mouse intestine to characterize the paracrine circuit controlling GLP-1 and SS. We show that 1) antagonizing the SS receptor (SSTr) 2 and SSTr5 led to increased GLP-1 and SS secretion in the mouse, 2) SS exhibits strong tonic inhibition of GLP-1 secretion preferentially through SSTr5, and 3) the secretion of S was GLP-1 receptor dependent. We conclude that SS is a tonic inhibitor of GLP-1 secretion, and interventions in the somatostain-GLP-1 paracrine loop lead to increased GLP-1 secretion.

Keywords: GLP-1; SSTr antagonist; paracrine loop; somatostatin; somatostatin receptors.

Fig. 1.

Mouse small intestinal mucosa: glucagon-like peptide (GLP)-1 and somatostatin (SS) cells are found in close proximity, GLP-1 receptors are present on D-cells, and both D- and L-cells show SS binding. A: representative images showing immunohistochemical staining of GLP-1 (green) and SS (red) positive cells in the proximal small intestine, female mice, n = 17. B: immunohistochemical staining of the GLP-1 receptor (black arrow) and immunohistochemical staining of SS-positive cells (white arrow) on the same slide, female mice, n = 8. C: left: autoradiography before immunohistochemical staining showing grains representing ¹²⁵I-labeled SS-28, male mice, n = 3. Right: immunohistochemical staining of GLP-1 (solid black arrow) in combination with autoradiography showing grains of ¹²⁵I-labeled SS-28 (punctuated black arrow). D: left: the same as C, but with immunohistochemical staining of SS-positive cells, male mice. Right: The same as C but with immunohistochemical staining of SS-positive cells. ab, antibody. n = 3. Bar = 50 µm.

Fig. 2.

The somatostatin receptor SSTr5 is the most expressed and enriched SSTr in the proglucagon-like positive cells of the mouse intestine. SSTr expression in proglucagon-positive cells (y-axis) compared with their surrounding proglucagon-negative cells (x-axis) from duodenum, jejunum, ileum, and colon. The dotted line represents fold change enrichment in the positive vs. the negative cells, and the gray box is defined as noise (threshold cycle level 35 < cycles). Male mice, n = 3, consisting of cells from 10 mice in each run pooled together (total 30 male mice).

Fig. 3.

Somatostatin (SS) receptor SSTr5 mRNA is more expressed in GLP-1 positive cells than SSTr2 mRNA, and none of them show high mRNA levels in the SS-positive cells. Dual in situ hybridization and immunohistochemical staining of SSTr2 and SSTr5 mRNA in small intestinal D- and L-cells. A: representative images of immunohistochemically stained GLP-1 cells (green) and in situ hybridization stained SSTr2 or SSTr5 mRNA (red dots) and a merge image (third picture) performed on mouse jejunum or proximal ileum sections. B: the same as above, however, with SS immunolabeled cells. Each red dot represents a single stained mRNA transcript. Dashed line outlines SS cell borders. Nuclei were visualized with DAPI counterstaining (blue). Ab, antibody; Pb, probe; bar = 5 µm. Male mice, n = 4.

Fig. 4.

Somatostatin (SS)-14 has a lower potency for SS receptor (SSTr) 5, but both SS-14 and SS-28 inhibit glucagon-like peptide-1 (GLP-1) secretion from the perfused mouse intestine. Mouse somatostatin receptor (mSSTr) subtypes 1 (A), 2 (B), 3 (C), 4 (D), and 5 (E) were transiently transfected in COS-7 cells, and activation of the receptors by SS-14 (●) and SS-28 (▲) was tested with increasing concentrations (100 pM to 1 μM) and analyzed with the inositol (1,4,5)-trisphosphate (IP₃) formation assay (A–E). Data are shown as means ± SE of independent experiments performed in duplicates, n ≥ 3. Statistical significance of the in vitro data are calculated by an unpaired t test based on percentage of activation by SS-14 or SS-28 comparing each concentration applied. Effect of SS-14 on GLP-1 output (F); data shown as means ± SE. Effect of SS-28 on GLP-1 output (G); data shown as means ± SE. Bombesin (BBS) was included as a positive control at the end of every experiment. One-way ANOVA for repeated measurements followed by Bonferroni post hoc analysis was used for statistical evaluation of the effect on GLP-1 secretion compared with baseline; *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.0001. SS-14 data n = 6; SS-28, male mice, n = 8–10.

Fig. 5.

Somatostatin (SS) receptor (SSTr) 5 agonist (SSTr5a) is a highly specific antagonist for the SSTr5, whereas the SSTr2 agonist (SSTr2a) is less specific. Both of the antagonists increase glucagon-like peptide-1 (GLP-1) and SS in the perfused mouse intestine. Mouse somatostatin receptor (mSSTr) subtypes 1 (A, F), 2 (B, G), 3 (C, H), 4 (D, I), and 5 (E, J) were transiently transfected in COS-7 cells, and the properties were analyzed with the inositol (1,4,5)-trisphosphate (IP₃) formation assay. To test the potential agonism of the receptors by the antagonists, increasing concentrations of the antagonists (1 nM to 10 μM) SSTr5 agonist (SSTr5a) (♦) and SSTr2a (■) were added (A–E). The antagonistic properties of SSTr2a and SSTr5a on the five receptors were tested by adding increasing concentrations (10 μM to 10 nM) together with a fixed concentration of SS-14 (F–J; ◊ for SSTr5a and □ for SSTr2a). Data are shown as means ± SE; independent experiments were performed in duplicates, n ≥ 3. Statistical significance of the in vitro data are calculated by unpaired t test based on percentage of activation by SSTr2a or SSTr5a comparing each concentration applied. Intra-arterial infusion of 100 nM, 1 μM, and 10 μM SSTr2a and the effect on GLP-1 and SS outputs shown as means ± SE (K, L). SSTr5a’s effect on GLP-1 and SS outputs shown as means ± SE (M, N). Bombesin (BBS) was included as a positive control at the end of each experiment. Statistical evaluation was carried out by one-way ANOVA followed by Bonferroni post hoc analysis for evaluation of the effect on GLP-1 secretion compared with baseline; *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.0001; male mice, n = 6–7.

Fig. 6.

Increased glucagon-like peptide-1 (GLP-1) levels increase somatostatin (SS) secretion. Enteroglucagon secretion (the sum of glicentin and oxyntomodulin) was used as a surrogate end point for secretion of proglucagon-derived products, including GLP-1. A: intra-arterial infusion of 500 pM GLP-1, 1 µM SS receptor (SSTr) 5 agonist (SSTr5a) or the two in combination shown as mean output ± SE. B: output of enteroglucagon during the three different stimulations. C: the same as A but showing the SS levels. D: the same as C but for SS output. E: GLP-1 output shown as mean ± SE after intra-arterial infusions of 1 µM SSTr5a, 1 µM exendin (9–39) (Ex9–39), or the two in combination. F: GLP-1 output during the three different stimulations based on output. G: the same as E but showing SS levels. H: the same as F but for SS levels. Statistical analysis of the output response from baseline was calculated by one-way ANOVA for repeated measurements followed by Bonferroni post hoc analysis. Bombesin (BBS) was included as a positive control at the end of each experiment. *P < 0.05, **P < 0.01; male mice, n = 5–6.