Research ArticleMetabolism

Lactation improves pancreatic β cell mass and function through serotonin production

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Science Translational Medicine  29 Apr 2020:
Vol. 12, Issue 541, eaay0455
DOI: 10.1126/scitranslmed.aay0455
  • Fig. 1 Lactation improves glucose homeostasis and β cell function in postpartum women.

    (A to C) Metabolic phenotypes in postpartum women by lactation status. A total of 174 women (lactated, n = 85; non-lactated, n = 99) were included in the analysis. (A and B) Plasma glucose concentrations were measured during the 75-g OGTT (A) at 2 months postpartum in lactating and non-lactating women and (B) at a mean of 3.6 years after delivery in previously lactated and non-lactated women. (C) Hyperbolic curves plotting the Matsuda index (insulin sensitivity) and insulinogenic index during and after lactation. Data are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 2 Lactation improves glucose homeostasis and β cell function in mice.

    (A) Scheme of the mouse model for lactation. Female C57BL/6J mice were randomized to either lactating or non-lactating groups, and the metabolic phenotypes were evaluated at 3, 6, and 12 weeks after delivery. (B) Body weight by lactation status. n = 3 or 4 mice per time point. (C to H) Metabolic phenotypes of (C to E) lactating mice at 3 weeks postpartum and (F to H) lactated mice at 6 weeks postpartum. n = 3 or 4 mice per group. (C and F) Intraperitoneal glucose tolerance test (2 g/kg) after an overnight fasting. (D and G) Intraperitoneal ITT (0.75 U/kg) after a 6-hour fasting. (E and H) Plasma insulin concentrations were measured after intraperitoneal glucose injection (2 g/kg). (I and J) β cell mass was quantified as the percentage of the insulin-positive area relative to the area of the whole pancreas at (I) 3 weeks and (J) 6 weeks postpartum (PP). n = 3 or 4 mice per group. (K and L) β Cell proliferation rate as the percentage of insulin and Ki-67 co-positive cells relative to all insulin positive cells at (K) 3 weeks and (L) 6 weeks postpartum. n = 3 mice per group. Data are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 3 5-HT improves β cell function during lactation.

    (A) Plasma prolactin concentrations were measured using ELISA and compared by lactation status. n = 3 or 4 mice per time point. (B and C) mRNA expressions of (B) Prlr and (C) Tph1 in lactating pancreatic islets were assessed by qRT-PCR. n = 3 or 4 mice per group. (D) mRNA expressions of Tph1 in βTC3 and MIN6 cells treated with prolactin (ng/ml) for 24 hours were assessed by qRT-PCR. n = 3 or 4 replicates. (E) Representative immunofluorescence images of pancreatic islets obtained from C57BL/6J, Prlr βiKO, and Tph1 βiKO mice during and after lactation and stained for insulin (green) and 5-HT (red). Islets from virgin mice were used as a negative control, and islets from pregnant mice were used as a positive control. Scale bar, 50 μm. (F) ELISA was used to measure the 5-HT content in islets isolated from non-lactating and lactating mice. 5-HT concentrations were normalized to the protein content. n = 3 mice per group. (G to P) The metabolic phenotypes of (G to K) Tph1 βiKO and (L to P) Htr2b βKO mice during lactation (3 weeks postpartum). n = 4 mice per group. (G and L) Blood glucose and (H and M) plasma insulin concentrations were measured after intraperitoneal glucose injection (2 g/kg) after an overnight fasting. (I and N) β cell mass was quantified as the percentage of the insulin-positive area relative to the area of the entire pancreas. n = 3 or 4 mice per group. (J and O) β Cell proliferation rate, taken as the percentage of insulin and Ki-67 co-positive cells relative to all insulin-positive cells. n = 3 or four mice per group. (K and P) Islets isolated from lactating mice at 3 weeks postpartum were incubated for 15 min with 2.8 or 16.8 mM glucose, and secreted insulin concentrations were measured with ELISA (n = 10 islets/well). Insulin secretion was normalized to the total insulin content extracted from the islets. n = 6 replicates per group. Data are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 4 5-HT protects β cells against oxidative stress.

    (A to F) Alloxan was administered to induce oxidative stress in β cells of non-lactating/lactating C57BL/6J, lactating Tph1 βiKO, and RIP-CreMgn mice (150 mg/kg mouse). n = 4 mice per group. (A and C) Random glucose concentrations and (B and D) immunohistochemistry for insulin (brown) before and 3 days after alloxan injection (150 mg/kg mouse) in (A and B) non-lactating/lactating C57BL/6J mice and (C and D) control/RIP-CreMgn mice. (E and F) Representative immunofluorescence images for (E) 8-oxo-dG (green) or (F) TUNEL (green) at 6 hours after injection of alloxan (150 mg/kg mouse) in non-lactating/lactating C57BL/6J, lactating Tph1 βiKO, and RIP-CreMgn mice (5-HT in red; insulin in gray). (G) Flow cytometry was used to measure intracellular ROS as the DHE fluorescence intensity in β cells of lactating Tph1 βiKO mice. Mean fluorescence intensity is depicted in the right panel for quantitative analysis. Islets from three or more mice were pooled for each data point. The experiment was repeated four times. (H and I) Cell survival was measured by MTT assay in Tph1-null MIN6 cells treated with (H) 5-HT and (I) 5-HTP in the presence or absence of H2O2 (200 μM). n = 4 to 6 replicates per group. (J to L) Flow cytometry was used to measure the intracellular ROS as the DCF-DA fluorescence intensity in Tph1-null MIN6 cells treated with 5-HT (500 μM) and 5-HTP (200 μM) (J) without and (K) with H2O2. N-acetylcysteine (NAC) (5 mM) was used as a control. n = 3 replicates per group. (L) Mean fluorescent intensity for (J and K) is depicted for quantitative analysis. n = 3 replicates per group. (M) In vitro free radical scavenging activity was measured by the ratio of DMPD radical inhibited by 5-HT and 5-HTP. n = 4 replicates per group. Data are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Table 1 Lactation improves insulin sensitivity and β cell function in postpartum women.

    The Matsuda index, insulinogenic index, and disposition index during and after lactation (at 2 months postpartum in lactating/non-lactating women and at a mean of 3.6 years after delivery in lactated/non-lactated women). A total of 174 women (lactated, n = 85; non-lactated, n = 99) were included in the analysis. Data are expressed as the means and SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.

    Initial follow-upLast follow-up
    Non-
    lactating
    SEMLactatingSEMPNon-
    lactated
    SEMLactatedSEMP
    Matsuda index
    (insulin
    sensitivity)
    5.3240.1776.0850.2570.015*4.2490.2185.5690.2870.000***
    Insulinogenic
    index
    0.4440.0310.4740.0280.5000.5050.0660.5350.0620.230
    Disposition
    index
    2.2290.1632.6420.1700.0872.2120.2613.1080.4980.020*

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/541/eaay0455/DC1

    Fig. S1. Metabolic phenotypes of lactating and lactated mice.

    Fig. S2. β Cell characteristics during and after lactation.

    Fig. S3. Ex vivo glucose-stimulated insulin secretion of isolated islets from lactating/lactated mice.

    Fig. S4. The specificity and efficiency of Cre lines.

    Fig. S5. Metabolic phenotypes of Tph1 βiKO mice.

    Fig. S6. Metabolic phenotypes of Htr2b βKO mice.

    Fig. S7. Establishment of Tph1-null MIN6 cell line and antioxidant properties of 5-HT.

    Fig. S8. Schematic summary of this study.

    Table S1. Baseline characteristics and glycemic profiles during gestation.

    Table S2. Glycemic profiles of the 75-g OGTT during and after lactation.

    Table S3. Primer sequences for genotyping.

    Table S4. Primer sequences for qRT-PCR and CRISPR KO.

    Data file S1. Raw data.

  • The PDF file includes:

    • Fig. S1. Metabolic phenotypes of lactating and lactated mice.
    • Fig. S2. β Cell characteristics during and after lactation.
    • Fig. S3. Ex vivo glucose-stimulated insulin secretion of isolated islets from lactating/lactated mice.
    • Fig. S4. The specificity and efficiency of Cre lines.
    • Fig. S5. Metabolic phenotypes of Tph1 βiKO mice.
    • Fig. S6. Metabolic phenotypes of Htr2b βKO mice.
    • Fig. S7. Establishment of Tph1-null MIN6 cell line and antioxidant properties of 5-HT.
    • Fig. S8. Schematic summary of this study.
    • Table S1. Baseline characteristics and glycemic profiles during gestation.
    • Table S2. Glycemic profiles of the 75-g OGTT during and after lactation.
    • Table S3. Primer sequences for genotyping.
    • Table S4. Primer sequences for qRT-PCR and CRISPR KO.

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    Other Supplementary Material for this manuscript includes the following:

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