Antagonizing somatostatin receptor subtype 2 and 5 reduces blood glucose in a gut- and GLP-1R-dependent manner

Abstract

Somatostatin (SS) inhibits glucagon-like peptide-1 (GLP-1) secretion in a paracrine manner. We hypothesized that blocking somatostatin subtype receptor 2 (SSTR2) and 5 (SSTR5) would improve glycemia by enhancing GLP-1 secretion. In the perfused mouse small intestine, the selective SSTR5 antagonist (SSTR5a) stimulated glucose-induced GLP-1 secretion to a larger degree than the SSTR2 antagonist (SSTR2a). In parallel, mice lacking the SSTR5R showed increased glucose-induced GLP-1 secretion. Both antagonists improved glycemia in vivo in a GLP-1 receptor-dependent (GLP-1R-dependent) manner, as the glycemic improvements were absent in mice with impaired GLP-1R signaling and in mice treated with a GLP-1R-specific antagonist. SSTR5a had no direct effect on insulin secretion in the perfused pancreas, whereas SSTR2a increased insulin secretion in a GLP-1R-independent manner. Adding a dipeptidyl peptidase 4 inhibitor (DPP-4i) in vivo resulted in additive effects on glycemia. However, when glucose was administered intraperitoneally, the antagonist was incapable of lowering blood glucose. Oral administration of SSTR5a, but not SSTR2a, lowered blood glucose in diet-induced obese mice. In summary, we demonstrate that selective SSTR antagonists can improve glucose control primarily through the intestinal GLP-1 system in mice.

Keywords: Diabetes; Endocrinology; G protein–coupled receptors; Glucose metabolism; Metabolism.

Figure 1. SSTR5a enhances glucose-induced GLP-1 secretion more than SSTR2a in the perfused mouse proximal small intestine.

GLP-1 and SS levels in the effluent from the perfused proximal small intestine of nonfasted C57BL/6JRj (A–H) or Sstr5^–/– and Sstr5^+/+ male mice (I–L). The intestine preparations were stimulated with luminal glucose (20 % w/v) alone or in combination with a simultaneous intra-arterial infusion of either 1 μM SSTR2a or SSTR5a, where after GLP-1 and SS were measured. (A–D) GLP-1 and SS output (fmol/min) or mean incremental output (fmol/20 min) in response to glucose and glucose + SSTR2a in C57BL/6JRj mice (GLP-1: n = 8, SS: n = 5). (E–H) GLP-1 and SS output (fmol/min) or mean incremental output (fmol/20 min) in response to glucose and glucose + SSTR5a in C57BL/6JRj mice (GLP-1 and SS: n = 6). (I–L) GLP-1 or SS output (fmol/min) or mean incremental output (fmol/20 min) after luminal infusion of glucose in male Sstr5^–/– (blue) or Sstr5^+/+ mice (black) (n = 5). Bombesin was used as the positive control. Data are presented as the mean ± SEM. Statistical significance was tested by paired t test (B, D, F, and H) or unpaired t test (J and L). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0001. The box plots show the median and 25th and 75th percentiles and the whiskers represent the smallest and highest value.

Figure 2. SSTR2a and SSTR5a lower blood glucose in vivo.

During hyperglycemia, in the perfused pancreas, SSTR2a increases insulin whereas SSTR5a does not. (A–C) Plasma blood glucose (mmol/L), insulin levels (pmol/L), and GLP-1 levels (pmol/L) after male C57BL/6JRj mice received vehicle (black), 4 mg/kg SSTR2a (red), or SSTR5a (blue) by s.c. injection 15 minutes before an oral glucose load (n = 8). (D–J) Insulin, glucagon, and insulin levels after the pancreas was perfused with a perfusion buffer at low-glucose concentration (3.5 mM) from 0 to 40 minutes, after which the buffer was exchanged to a high-glucose-containing buffer (15 mM) for the rest of the experiment (D and F–J) or at a constant concentration of 15 mM (E). 1 μM SSTR2a, SSTR5a, or Ex9–39 was added to the arterial perfusate via a side-arm. 10 mM arginine was used as positive control at the end of each perfusion experiment (n = 6). (K and L) Male mice received s.c. injections of vehicle (PBS), 4 mg/kg SSTR2a, or SSTR5a at time –15 minutes, and at time 0 minutes they received the i.p. injection of glucose or PBS. (K) Blood glucose (mmol/L) levels after the following injections: PBS s.c. at –15 minutes and i.p. PBS at 0 minutes (black line), s.c. PBS at time –15 minutes and i.p. glucose at 0 minutes (black dashed line), s.c. SSTR2a at –15 minutes and i.p. PBS at 0 minutes (red line), or s.c. SSTR2a at –15 minutes and i.p. glucose at 0 minutes (red dashed line) (n = 8). (L) The same as in K, but with SSTR5a, represented in blue. SSTR5a s.c. at –15 minutes and i.p. PBS (blue line), i.p. glucose at 0 minutes and s.c. SSTR5a at –15 minutes (blue dashed line) (n = 8). Data are presented as the mean ± SEM. Statistical significance at specific time points was assessed by 2-way ANOVA followed by Tukey post hoc analysis to correct for multiple testing in vivo and by paired t test in the perfusion experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3. The glucose-lowering effect of SSTR2a and SSTR5a is GLP-1R dependent.

(A) Blood glucose levels (mmol/L) in C57BL/6JRj mice receiving vehicle (black line), 4 mg/kg SSTR2a (red line), 4 mg/kg Ex9–39 (gray line), or SSTR2a + Ex9–39 (dashed red line) s.c. 15 minutes before an oral glucose load (n = 5–8). (B) The same as A but for 4 mg/kg SSTR5a (blue), 4 mg/kg SSTR5a + 4 mg/kg Ex9–39 (dashed blue line), n = 5–8. (C) Blood glucose levels (mmol/L) in Glp-1r^–/– or Glp-1r^+/+ mice after vehicle or 4 mg/kg SSTR2a. Glp-1r^+/+ receiving vehicle (black line), Glp-1r^–/– receiving vehicle (black dashed line), Glp-1r^+/+ receiving 4 mg/kg SSTR2a (red line), Glp-1r^–/– receiving 4 mg/kg SSTR2a (dashed red line), n = 8–13. (D) The same as in C but for 4 mg/kg SSTR5a indicated with blue. Glp-1r^+/+ receiving 4 mg/kg SSTR5a (blue line), Glp-1r^–/– receiving 4 mg/kg SSTR5a (dashed blue line), n = 10–13. (E) Blood glucose levels (mmol/L) in Sstr5^–/– or Sstr5^+/+ mice receiving vehicle or 4 mg/kg Ex9–39. Sstr5^+/+ receiving vehicle (black line), Sstr5^–/– receiving vehicle (black dashed line), Sstr5^+/+ receiving 4 mg/kg Ex9–39 (gray line), Sstr5^–/– receiving 4 mg/kg Ex9–39 (gray dashed line) (n = 4–5). (F) Blood glucose levels (mmol/L) after administration of vehicle (black line), 4 mg/kg SSTR2a (red line), 120 mg/kg DPP-4 (dashed black line), and a combination of SSTR2a and DPP-4i (red dashed line) (n = 8). (G) The same as in F but for 8 mg/kg SSTR5a (blue line) and SSTR5 + DPP-4i (blue dashed line) (n = 6–8). Data are shown as the mean ± SEM, and significance was evaluated based on iAUC by 1-way ANOVA followed by the Holm-Sidak post hoc analysis to correct for multiple testing.

Figure 4. In proximal perfused intestine of DIO and control mice, SSTR5a stimulates glucose-induced GLP-1 secretion more than SSTR2a, and SSTR5a improves glucose tolerance when applied orally in DIO mice.

(A–L) GLP-1 an SS output (fmol/min) or mean incremental output (fmol/20 min) in proximal intestinal perfusions of control and DIO mice. The intestine was stimulated with luminal glucose alone or in combination with simultaneous intra-arterial infusion of 1 μM SSTR2a or SSTR5a. (A) n = 7, (B) n = 5, (D) n = 7, (E) n = 6, (G) n = 7, (H) n = 5, (J) n = 7, and (K) n = 4. (M and N) In vivo studies in control and DIO mice undergoing an OGTT after oral administration of vehicle (black), 50 mg/kg SSTR2a (red), or SSTR5a (blue) 30 minutes before oral glucose. (M) Blood glucose levels (mmol/L) (n = 5–6, control mice). (N) Same as in M, but in DIO mice (n = 4). Data are presented as the mean ± SEM. Statistical significance at specific time points was assessed by 2-way ANOVA followed by Tukey post hoc analysis to correct for multiple testing (M) and by paired t test (C, F, I, and L), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The box plots show the median and 25th and 75th percentiles, and the whiskers represent the smallest and highest values.