Exploring the contribution of mammary-derived serotonin on liver and pancreas metabolism during lactation

Abstract

During lactation, the murine mammary gland is responsible for a significant increase in circulating serotonin. However, the role of mammary-derived serotonin in energy homeostasis during lactation is unclear. To investigate this, we utilized C57/BL6J mice with a lactation and mammary-specific deletion of the gene coding for the rate-limiting enzyme in serotonin synthesis (TPH1, Wap-Cre x TPH1FL/FL) to understand the metabolic contributions of mammary-derived serotonin during lactation. Circulating serotonin was reduced by approximately 50% throughout lactation in Wap-Cre x TPH1FL/FL mice compared to wild-type mice (TPH1FL/FL), with mammary gland and liver serotonin content reduced on L21. The Wap-Cre x TPH1FL/FL mice had less serotonin and insulin immunostaining in the pancreatic islets on L21, resulting in reduced circulating insulin but no changes in glucose. The mammary glands of Wap-Cre x TPH1FL/FL mice had larger mammary alveolar areas, with fewer and smaller intra-lobular adipocytes, and increased expression of milk protein genes (e.g., WAP, CSN2, LALBA) compared to TPH1FL/FL mice. No changes in feed intake, body composition, or estimated milk yield were observed between groups. Taken together, mammary-derived serotonin appears to contribute to the pancreas-mammary cross-talk during lactation with potential implications in the regulation of insulin homeostasis.

Fig 1. Mammary-specific disruption of TPH1 reduces circulating and mammary serotonin by 1-fold.

Model validation of control dams (TPH1^FL/FL, n = 5) and dams with mammary gland deletion of TPH1 (Wap-Cre x TPH1^FL/FL, n = 5) first lactation mice had (A) serum collected on pregnancy day 0 (P0) and during lactation (L1, L10 and L21) to measure circulating serotonin concentrations. On L21, mice were euthanized to harvest organs to measure (B) mammary gland and (C) duodenum serotonin concentrations. Data is presented as least squares means (LSM) ± standard error of the mean (SEM). Significance declared at (*) P ≤ 0.05.

Fig 2. Mammary-specific disruption of TPH1 averts involution.

Control dams (TPH1^FL/FL, n = 5) and dams with mammary gland deletion of TPH1 (Wap-Cre x TPH1^FL/FL, n = 5) were euthanized on L21 for histological and transcriptional evaluation of the mammary gland. (A) Hematoxylin and eosin (H&E) staining at 20 × magnification to quantify (B) alveoli area (C) alveoli number (D) adipocyte number and (E) total adipocyte area. (F) Mammary gland gene expression of serotonin synthesis and metabolism (TPH1, SERT, 5-HTR1A, -1B, -1D, -1F, -2A, -2B, -2C, -3A, -3B, -4, -5A, -6, and -7), glucose transporters (GLUT1, -8 and 12) and lipid metabolism genes (INSIG1, SREBP1, CPT1 and LPL) were measured. Positive or negative relative mRNA expression (ΔΔCt) indicates gene upregulation and downregulation of TPH1^FL × Wap^Cre mice relative to TPH1^FL, respectively. Scale bar = 100 μm. Data is presented least squares means (LSM) ± standard error of the mean (SEM). Significance declared at (*) P ≤ 0.05 and (#) denotes a statistical tendency at 0.05 < P ≤ 0.10.

Fig 3. Mammary-specific disruption of TPH1 reduces pancreatic serotonin and insulin.

Control dams (TPH1^FL/FL, n = 5) and dams with mammary gland deletion of TPH1 (Wap-Cre x TPH1^FL/FL, n = 5) were euthanized on L21 for histological evaluation of the pancreas. (A) Hematoxylin and eosin (H&E) staining at 20 × magnification to quantify (B) islet of Langerhans area. (C-E) Immunofluorescence staining in pancreas tissue at 40 × magnification to quantify (F) serotonin (5-HT) and (G) insulin intensity. (H) Positive controls for staining 5-HT and insulin using mouse duodenum and pancreas, respectively. The negative control had no primary antibody applied. (I) Serum was collected on pregnancy day 0 (P0) and during lactation (L1, L10 and L21) to measure circulating insulin concentrations and (J) pancreas protein 5-HT concentrations were measured at L21. Scale bar = 100 μm. Data is presented least squares means (LSM) ± standard error of the mean (SEM). Significance declared at (*) P ≤ 0.05 and (**) P ≤ 0.001.

Fig 4. Mammary-specific disruption of TPH1 tends to reduce liver serotonin concentrations.

Control dams (TPH1^FL/FL, n = 5) and dams with mammary gland deletion of TPH1 (Wap-Cre x TPH1^FL/FL, n = 5) were euthanized on L21 for histological and transcriptional evaluation of the liver. (A) Hematoxylin and eosin (H&E) staining at 20 × magnification was performed to quantify (B) hepatocyte number. (C) Liver serotonin (5-HT) concentrations at L21 and (D) liver gene expression of serotonin synthesis and metabolism (TPH1, SERT, 5-HTR1A, -1B, -1D, -1F, -2A, -2B, -2C, -3A, -3B, -4, -5A, -6, and -7) and glucose metabolism (PFK1, PEPCK, PCK1, PC and PDK4) genes were measured. (E) Serum was collected on pregnancy day 0 (P0) and during lactation (L1, L10, and L21) to measure circulating glucose concentrations. Positive or negative relative mRNA expression (ΔΔCt) indicates gene upregulation and downregulation of Wap-Cre x TPH1^FL/FL mice relative to TPH1^FL/FL, respectively. Scale bar = 100 μm. Data is presented as least squares means (LSM) ± standard error of the mean (SEM). Significance is declared at (*) P ≤ 0.05, and (#) denotes a statistical tendency at 0.05 < P ≤ 0.10.