Defective prolactin signaling impairs pancreatic β-cell development during the perinatal period

Abstract

Prolactin (PRL) and placental lactogens stimulate β-cell replication and insulin production in pancreatic islets and insulinoma cells through binding to the PRL receptor (PRLR). However, the contribution of PRLR signaling to β-cell ontogeny and function in perinatal life and the effects of the lactogens on adaptive islet growth are poorly understood. We provide evidence that expansion of β-cell mass during both embryogenesis and the postnatal period is impaired in the PRLR(-/-) mouse model. PRLR(-/-) newborns display a 30% reduction of β-cell mass, consistent with reduced proliferation index at E18.5. PRL stimulates leucine incorporation and S6 kinase phosphorylation in INS-1 cells, supporting a role for β-cell mTOR signaling in PRL action. Interestingly, a defect in the development of acini is also observed in absence of PRLR signaling, with a sharp decline in cellular size in both endocrine and exocrine compartments. Of note, a decrease in levels of IGF-II, a PRL target, in the Goto-Kakizaki (GK) rat, a spontaneous model of type 2 diabetes, is associated with a lack of PRL-mediated β-cell proliferation in embryonic pancreatic buds. Reduced pancreatic IGF-II expression in both rat and mouse models suggests that this factor may constitute a molecular link between PRL signaling and cell ontogenesis. Together, these results provide evidence that PRL signaling is essential for pancreas ontogenesis during the critical perinatal window responsible for establishing functional β-cell reserve.

Keywords: IGF-II; apoptosis; diabetes; insulin; placental lactogens; proliferation.

Fig. 1.

Reduction of pancreatic Igf2 expression along perinatal development and in PRLR^−/− mice. Igf2 gene expression was determined by qPCR in whole pancreases from PRLR^+/+ and PRLR^−/− mice at embryonic day €18.5, postnatal day (D)0.5, and D6.5 developmental stages (n = 6–23/group). Relative expression was calculated as a ratio (amoles of Igf2/pmoles of 18S). Data are represented as means ± SE. NS, not significant. Nonparametric statistical analyses were performed between the 2 genotypes *P <0.05.

Fig. 2.

Lack of prolactin receptor (PRLR) reduces pancreatic β-cell mass during perinatal development. A: pancreas sections from PRLR^+/+ and PRLR ^−/− at D6.5 after insulin immunostaining (brown). Scale bars, 200 μm. B: sections from PRLR^+/+ and PRLR ^−/− pancreases at D6.5 after double BrdU (brown) and insulin (pink) immunostaining. Scale bars, 50 μm.

Fig. 3.

Lack of PRLR reduces total pancreatic β-cell mass, proliferation, and size during perinatal development. A: β-cell surface was normalized to total pancreas surface of PRLR^+/+ and PRLR^−/− mice at E18.5 and D6.5 (6–8 pancreas sections per animal). Results are means ± SE (n = 3–6/group) and expressed as percentage of total pancreatic surface. *P < 0.05. B: determination of β-cell mass was calculated according to values at left normalized to pancreas weight. *P < 0.05, **P < 0.01. C: percentage of β-cell apoptosis of PRLR^+/+ and PRLR^−/− pancreases was quantified from insulin-TUNEL assay-immunostained sections and counterstaining with hematoxylin at E18.5 and D6.5. Approximately 3,000 cells were counted for each animal. D: β-cell proliferation was quantified by means of BrdU/insulin-immunostained and hematoxylin-counterstained pancreas sections. At least 2,000 cells were counted for each mouse (n = 5–7). *P < 0.05. E: determination of β-cell size was calculated (μm²) after quantification of total β-cell surface by ImageJ software divided by total number of nuclei [≥750 cells counted for each mouse (n = 3–6/group)]. *P < 0.05, **P < 0.01. F: insulin gene expression was quantified by qPCR in whole pancreases of PRLR^+/+ and PRLR^−/− mice at D6.5 (n = 10–22/group). ***P < 0.001.

Fig. 4.

PRL stimulates leucine incorporation and p70S6K phosphorylation in rat INS-1 cells. A: [¹⁴C]leucine uptake and incorporation into protein was measured in INS-1 cells incubated for 20 h in serum or in serum-free medium containing rat PRL (20 nM), rapamycin (rapa, 100 nM), or a combination of PRL + rapa. Cells treated with diluent represent negative controls (C). [¹⁴C]leucine was added for the final 16 h. Similar results (but with lower overall counts) were obtained when [¹⁴C]leucine was added for the final hour of incubation; in that case, PRL stimulated a 28% increase in leucine uptake relative to controls. *P < 0.05, ***P < 0.001. B: phosphorylated (p-)p70S6K was estimated by Western analysis of whole cell extracts of INS-1 cells incubated for 30 min, 120 min, or 20 h in RPMI containing 10% FBS or in basal serum-free medium containing diluent (C), rat PRL (20 nM, PRL), or insulin (1 μM, Ins). Immunoblots with anti-S6K and anti-tubulin antibodies were used as loading control. C: ratio of p-S6K to total protein of the densitometric assay is presented. Data are means ± SE. *P < 0.05.

Fig. 5.

PRLR impacts α- and δ-cells ontogenesis. A: pancreas sections from PRLR^+/+ and PRLR^−/− at D6.5 after glucagon and somatostatin (brown) immunostaining. Scale bars, 100 μm. B: quantification of average α- and δ-cell surface from pancreas sections of PRLR^+/+ and PRLR^−/− animals. Ppancreas sections (6–8 per mouse) were used to quantify percentage of α- and δ-cells. C: α- and δ-cell mass was calculated after quantification of α- and δ-cell surface by ImageJ, taking into account pancreas weights of PRLR^+/+ and PRLR^−/− mice. Values represent means ± SE (n = 3–6/group). Statistical differences are indicated: *P < 0.05, **P < 0.01.

Fig. 6.

Lack of PRLR modifies pancreatic acinar development. A: pancreas sections at D6.5 after double BrdU (brown) and insulin (pink) immunostaining. Scale bars, 150 μm. B: quantification of BrdU-positive cells in acini of PRLR^+/+ and PRLR^−/− mice at E18.5 and D6.5. Results are expressed as percentage of positive cells vs. total counted cells (≥2,000 cells/animal). *P < 0.05, **P < 0.01. C: hematoxylin-stained pancreas sections from PRLR^+/+ and PRLR^−/− mice at D6.5. Scale bars, 300 μm. D: determination of acinar cell size of PRLR^+/+ and PRLR^−/− mice at E18.5 and D6.5 using ImageJ software. Cell surface (mm²) was calculated for ≥750 cells per animal. Results are means ± SE (n = 3–6). *P < 0.05, **P < 0.01. E: lipase gene expression was determined by qPCR in whole pancreases from PRLR^+/+ and PRLR^−/− mice at D6.5 (n = 3–13/group). *P < 0.05.

Fig. 7.

Effect of PRL on pancreatic β-cell rudiments from Wistar and GK rats. A: average β-cell surface on total pancreas surface was measured on pancreatic buds from Wistar (white) and GK (black) fetuses at E13.5 incubated in the presence or absence of PRL for 7 days. B: β-cell proliferation index was determined by double insulin-BrdU immunostaining on pancreas buds of GK and Wistar fetus rats after PRL exposure (hatched). Results are expressed as fold increase compared with β-cell proliferation index measured in GK and Wistar buds in the absence of PRL.

Fig. 8.

Graphic summary of factors regulating β-cell proliferation and size as a function of pancreas developmental stages. At E13.5, PRLR is already expressed in fully differentiated β-cells and only activated by placental lactogen hormones (PL), since PRL levels are undetectable at this stage. However, PRL exposure stimulates embryonic pancreatic rudiment growth and β-cell proliferation (Fig. 7). PRLR activation leads to stimulation of insulin growth factor II (Igf2) expression, notably before birth, and presumably its secretion. At E18.5, PRLR is strongly activated by both PL and PRL inducing at least through S6K pathway (Fig. 4). Drastic IGF-II production was observed, which may act in an autocrine/paracrine manner through IGF receptor (IGF-R) signaling. Other sources of IGF-II could also account for enhanced β-cell proliferation (see Fig. 3D) and increased β-cell size (see Fig. 3E). After birth (D6.5), IGF-II was almost undetectable; PRL alone induces other target genes, whereas proliferation rate is largely reduced. Even though a second wave of β-cell proliferation will take place later (D20) in rodents.