(a) mRNA levels for Tph1 and Tph2 measured by real time RT-PCR in islet RNA at the dates of gestation (G) or postpartum (P) indicated are shown relative to the levels in islets from non-pregnant female mice. n = 3 6 mice per data point. (b) Western blots were performed with Tph1 and β-actin antisera on protein extracted from islets of non-pregnant female (NP), pregnant (G12) and postpartum day 23 (P23) female mice and MIN6 insulinoma cells. (c, d) Tissue 5-HT concentrations were assayed by HPLC in islets (c) and duodenum (d) isolated from 3 non-pregnant (NP) and 3 pregnant (G12) female mice. (e) The mRNA shown were amplified by RT-PCR from mouse islets, β-cells (GFP⁺), non-β islet cells (GFP⁻), brain, and the β-cell lines MIN6 and βTC3. (f) Immunofluorescent staining labels pancreata from non-pregnant female (Non-preg) and G12 pregnant mice with insulin and glucagon in green and 5-HT in red. (g) Immunoperoxidase staining labels 5-HT and TPH1 in pancreata from non-pregnant (Non-preg), pregnant (37 weeks gestation) and postpartum day 2 human autopsies. (h) Immunohistochemical co-staining labels insulin (red, fluorescent) and TPH1 (black, peroxidase) in pregnant human autopsy pancreas. (i, j) Secreted insulin (i) and 5-HT (j) was assayed by ELISA (i) and HPLC (j), following a 1 hour incubation of islets isolated from pregnant (G13 G15) mice in 2 mM or 20 mM glucose. n = 5 groups of 40 islets. All data are presented as mean ± standard error. Statistical significance vs. islets cultured in 2mM glucose (i and j) or vs. non-pregnant control (a and c) was analyzed by Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bar indicates 20 μm.

(a) The treatment schedules for mouse experiments are shown. (b d) Blood glucose levels were measured after intraperotoneal injection of 2 g kg⁻¹ glucose in pregnant mice (G13) fed a tryptophan-free (“Trp-free”, b), histidine-free (“His-free”, b) or low tryptophan (“Low Trp”, c) diet or treated with PCPA (300 mg kg⁻¹ day⁻¹)(c) or non-pregnant female mice (d) treated as shown. n = 5 18 mice per treatment group. (e) The Htr mRNAs shown were amplified by RT-PCR from mouse islet and brain RNA. (f) The mRNAs shown were amplified by RT-PCR from mouse islets, β-cells (GFP⁺), non-β islet cells (GFP⁻), brain, and β-cell lines MIN6 and βTC3. (g, h) mRNA levels for Htr2a and Htr2b (g) and Htr1b and Htr1d (h) measured by real time RT-PCR in islet RNA harvested at the gestational dates indicated are shown relative to the levels in islets from non-pregnant female mice. n = 3–6 mice per data point. (i) Blood glucose levels were measured after intraperitoneal injection of glucose (2 g kg⁻¹)in pregnant mice treated with SB204741 (1 mg kg⁻¹ day⁻¹), Methysergide (3 mg kg⁻¹ day⁻¹) or Ketanserin (1 mg⁻¹ kg⁻¹ day⁻¹). n = 5 18 mice per treatment group. (j) Pregnant mice were treated as in Fig. 2a with tryptophan-free diet, PCPA or SB204741 and fasted for 6 hours at G13 before measuring insulin in plasma collected before and 30 minutes after intraperitoneal injection of 2 g/kg glucose. n = 4 7 mice per group. All data are presented as mean ± standard error. Statistical significance vs. untreated control (b, c, i and j) or vs. non-pregnant control (g and h) was analyzed by Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

(a, b) Blood glucose levels were measured after intraperitoneal injection of glucose (2 g kg⁻¹) in pregnant (a) and non-pregnant (b) mice with the genotypes shown. n = 4 7 mice per group. (c, d) In non-pregnant and pregnant control mice, pregnant mice treated with SB204741 or PCPA, and pregnant Htr2b^−/− mice, β-cell proliferation (c) was quantified by counting the percent of insulin ⁺ cells labeled with BrdU, and relative β-cell mass (d) was calculated as the area of insulin ⁺ cells / total pancreatic area. n = 4 6 mice per group. (e) Islets isolated from Htr2b^+/+ (black) or Htr2b^−/− (red) mice were transplanted into the kidney capsule of Htr2b^+/+ mice. (f) Immunofluorescent staining for 5-HT (red) and insulin (green) was performed on donor islets with the genotypes shown transplanted under the kidney capsule in non-pregnant or G13 pregnant hosts. (g) Immunofluorescent staining for insulin (red) and BrdU (green) together with DNA staining with DAPI (blue) was performed on host pancreatic islets and on donor islets with the genotypes shown transplanted under the kidney capsule in non-pregnant and G13 pregnant mice. (h) β-Cell proliferation in transplanted islets with the genotypes shown was quantified by counting the percent of insulin ⁺ cells labeled with BrdU in non-pregnant (NP) and pregnant mice. n = 3 4 per group. All data are presented as mean ± standard error. Statistical significance vs. Htr2b^+/+ control (a), vs. pregnant untreated Htr2b^+/+ control (c and d) or vs. pregnant Htr2b^+/+ control (h) was analyzed by Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bar indicates 20 μm.

(a d) mRNA levels for Tph1 (a), Tph2 (b), Htr1d (c) and Htr2b (d) were measured by real-time RT-PCR in RNA from islets isolated from G3 pregnant mice and cultured with the concentration of mouse prolactin (PRL) indicated for 72 hours. n = 3 6 per group. (e) β-Cell proliferation was quantified by counting the percent of insulin ⁺ cells labeled with BrdU in mouse islets cultured with prolactin (1,000 ng ml⁻¹, PRL), 5-HT (10 μM) or no added hormone (control) for 4 days. n = 5 10 groups of islets per data point. (f, g) Levels of cyclin mRNAs were measured by real-time RT-PCR in RNA (f) from islets harvested at the dates indicated and shown relative to the levels in control islets from non-pregnant female mice or (g) from islets cultured with 5-HT (10 μM) for the time indicated and shown relative to the levels in control islets cultured without 5-HT for 24 hours. n = 4 10 groups of islets per data point. (h) A proposed model is shown for 5-HT regulation of β-cell proliferation during pregnancy. All data are presented as mean ± standard error. Statistical significance vs. untreated control (a, e and g) or vs. non-pregnant control (f) was analyzed by Student’s t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001.