Serotonin Regulates Adult β-Cell Mass by Stimulating Perinatal β-Cell Proliferation

Abstract

A sufficient β-cell mass is crucial for preventing diabetes, and perinatal β-cell proliferation is important in determining the adult β-cell mass. However, it is not yet known how perinatal β-cell proliferation is regulated. Here, we report that serotonin regulates β-cell proliferation through serotonin receptor 2B (HTR2B) in an autocrine/paracrine manner during the perinatal period. In β-cell-specific Tph1 knockout (Tph1 βKO) mice, perinatal β-cell proliferation was reduced along with the loss of serotonin production in β-cells. Adult Tph1 βKO mice exhibited glucose intolerance with decreased β-cell mass. Disruption of Htr2b in β-cells also resulted in decreased perinatal β-cell proliferation and glucose intolerance in adulthood. Growth hormone (GH) was found to induce serotonin production in β-cells through activation of STAT5 during the perinatal period. Thus, our results indicate that GH-GH receptor-STAT5-serotonin-HTR2B signaling plays a critical role in determining the β-cell mass by regulating perinatal β-cell proliferation, and defects in this pathway affect metabolic phenotypes in adults.

Figure 1

5-HT regulates β-cell proliferation during the perinatal period. A: Immunohistochemical staining (5-HT in brown) and H&E staining in perinatal human pancreata. Fetal (gestational age 24 weeks), postnatal 8-day-old, and 3-month-old pancreata were stained. Pancreas samples from a 35-year-old pregnant woman (gestational age 37 weeks) and a 72-year-old man were used as a positive and negative control, respectively. B: Immunofluorescent (IF) staining of perinatal pancreas from C57BL6/J mice labeled insulin (green) and 5-HT (red). C–F: Pancreata of control (Cre) and Tph1 βKO mice were assessed at P0. C: Representative IF staining labeled insulin (green) and amylase (AMYL) (red) at P0. Images of a whole-slide section were obtained using a slide scanner. D: β-Cell mass was quantified as the percentage of insulin-positive area over whole pancreas area at P0 (n = 4 mice/group). E: IF staining labeled insulin (green) and phospho-Histone H3 (PHH3) (red). Arrows indicate insulin and PHH3 copositive cells. β-Cell proliferation was quantified by counting the percentage of insulin-positive cells labeled with PHH3 at P0 (n = 4 mice/group). F: mRNA levels of cyclin families were measured by qRT-PCR in RNAs extracted from pancreata at P0 (n = 4–5 mice/group). Scale bars = 50 μm. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. INS, insulin.

Figure 2

5-HT regulates adult β-cell mass and is necessary to maintain glucose homeostasis. Metabolic phenotypes were evaluated in 9-week-old male control and Tph1 βKO mice. A and B: Blood glucose and plasma insulin levels were measured after intraperitoneal glucose injection (2 g/kg) after a 16-h fast. Both Cre-only and floxed-only mice were used as controls for the glucose tolerance test. Because glucose levels were comparable between the two groups, Cre-only mice were used as the control in further experiments (n = 4–8 mice/group). C: Secreted insulin was measured with ELISA after a 15-min incubation of islets isolated from 9-week-old control (Cre) and Tph1 βKO mice in 2.8 or 16.8 mmol/L glucose. Insulin secretion was normalized to the total insulin content extracted from the islets (n = 5/group). D: β-Cell mass was quantified as the percentage of insulin-positive area over whole-pancreas area of 9-week-old mice (n = 4 mice/group). E: Immunofluorescent staining labeled insulin (green) and Ki-67 (red), with arrows indicating insulin and Ki-67 copositive cells. β-Cell proliferation was quantified by counting the percentage of insulin-positive cells labeled with Ki-67 in 9-week-old mice (n = 4 mice/group). F: Pancreas weight was measured after harvest in 9-week-old mice (n = 4 mice/group). Scale bar = 50 μm. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, control (Cre) vs. KO; ##P < 0.01, control (floxed) vs. KO. INS, insulin.

Figure 3

HTR2B is the downstream target of 5-HT in perinatal β-cells. A: mRNA expression levels of indicated HTRs were assessed by qRT-PCR in RNAs extracted from pancreata during embryonic development (n = 4 mice/group). B–E: Pancreata of control (Cre) and Htr2b βKO at P0 were evaluated. B: H&E staining was performed in control (Cre) and Htr2b βKO pancreas at P0. C: β-Cell mass was quantified as the percentage of insulin-positive area over whole-pancreas area at P0 (n = 4 mice/group). D: Pancreas weight was measured after harvest at P0 (n = 4–5 mice/group). E: Immunofluorescent staining labeled insulin (green) and phospho-Histone H3 (PHH3) (red), with arrows indicating insulin and PHH3 copositive cells. β-Cell proliferation was quantified by counting the percentage of insulin-positive cells labeled with PHH3 at P0 (n = 4 mice/group). F–J: Metabolic phenotypes were evaluated in 9-week-old male control and Htr2b βKO mice. F and G: Blood glucose and plasma insulin levels were measured after intraperitoneal glucose injection (2 g/kg) after a 16-h fast. Both Cre-only and floxed-only mice were used as controls for glucose tolerance tests. Because glucose levels were comparable between the two, Cre-only mice were used as the control in further experiments (n = 4 mice/group). H: Secreted insulin was measured with ELISA after a 15-min incubation of islets isolated from 9-week-old control (Cre) and Htr2b βKO mice in 2.8 or 16.8 mmol/L glucose. Insulin secretion was normalized to the total insulin content extracted from the islets (n = 3/group). I: β-Cell mass was quantified as the percentage of insulin-positive area over whole-pancreas area of 9-week-old mice (n = 4 mice/group). J: β-Cell proliferation was quantified by counting the percentage of insulin-positive cells labeled with Ki-67 in 9-week-old mice (n = 4 mice/group). K: Pancreas weight was measured after harvest in 9-week-old mice (n = 4 mice/group). Scale bar = 50 μm. Data are mean ± SEM. *P < 0.05, **P < 0.01 control (Cre) vs. KO; #P < 0.05, control (floxed) vs. KO. INS, insulin.

Figure 4

5-HT is not involved in β-cell proliferation induced by metabolic stress. HFD was fed to 12-week-old mice for 8 weeks. A: Blood glucose levels were measured after intraperitoneal glucose injection (2 g/kg) after a 16-h fast (n = 4–5 mice/group). B: β-Cell proliferation was quantified by counting the percentage of insulin-positive cells labeled with Ki-67 (n = 4 mice/group). C: β-Cell proliferation upon S-961 administration (100 nmol/kg mouse twice a day) for 7 days was quantified by counting the percentage of insulin-positive cells labeled with Ki-67 (n = 4 mice/group). Data are mean ± SEM. *P < 0.05, **P < 0.01.

Figure 5

GH regulates 5-HT production in β-cells during the perinatal period. Pancreas of control (Cre), Prlr βKO, Stat5 βKO, and Ghr βKO mice at P0 were examined. A: 5-HT production in β-cells at P0 were assessed by immunofluorescent (IF) staining, which labeled 5-HT (red) and insulin (green). B: Representative H&E staining and IF staining, labeled insulin (green) and phospho-Histone H3 (PHH3) (red) at P0. Arrows indicate insulin and PHH3 copositive cells. C–E: β-Cell proliferation of Prlr βKO, Stat5 βKO, and Ghr βKO mice at P0 was quantified by counting the percentage of insulin-positive cells labeled with PHH3 (n = 4–6 mice/group). F–H: β-Cell mass was quantified as the percentage of insulin-positive area over whole-pancreas area in Prlr βKO, Stat5 βKO, and Ghr βKO mice at P0 (n = 4–6 mice/group). I and J: Induction of Tph1 or Mki67 in islets isolated from 3-week-old control (Cre) and Tph1 βKO mice was measured by qRT-PCR. Islets were incubated with recombinant mouse GH (1,000 ng/mL) for 24 h (n = 3–5/group). K: Schematic describing the GHR-STAT5-TPH1-HTR2B axis for β-cell proliferation during the perinatal period. Scale bars = 50 μm. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. INS, insulin.