Suppression of hypothalamic oestrogenic signal sustains hyperprolactinemia and metabolic adaptation in lactating mice

ABSTRACT 17β-oestradiol (E2) inhibits overeating and promotes brown adipose tissue (BAT) thermogenesis, whereas prolactin (PRL) does the opposite. During lactation, the simultaneous decline

in E2 and surge in PRL contribute to maternal metabolic adaptations, including hyperphagia and suppressed BAT thermogenesis. However, the underlying neuroendocrine mechanisms remain unclear.

Here, we find that oestrogen receptor alpha (ERα)-expressing neurons in the medial basal hypothalamus (MBH), specifically the arcuate nucleus of the hypothalamus and the ventrolateral

subdivision of the ventromedial hypothalamus (vlVMH), are suppressed during lactation. Deletion of ERα from MBH neurons in virgin female mice induces metabolic phenotypes characteristic of

lactation, including hyperprolactinemia, hyperphagia and suppressed BAT thermogenesis. By contrast, activation of ERαvlVMH neurons in lactating mice attenuates these phenotypes. Overall, our

study reveals an inhibitory effect of E2–ERαvlVMH signalling on PRL production, which is suppressed during lactation to sustain hyperprolactinemia and metabolic adaptations. Access through

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OTHERS OXYTOCIN RECEPTOR INDUCES MAMMARY TUMORIGENESIS THROUGH PROLACTIN/P-STAT5 PATHWAY Article Open access 07 June 2021 OESTROGEN ENGAGES BRAIN MC4R SIGNALLING TO DRIVE PHYSICAL ACTIVITY

IN FEMALE MICE Article 13 October 2021 BETA CELL ADAPTATION TO PREGNANCY REQUIRES PROLACTIN ACTION ON BOTH BETA AND NON-BETA CELLS Article Open access 14 May 2021 DATA AVAILABILITY All data

supporting this publication are available in the Source Data published alongside the paper. A reporting summary for this article is available as a Supplementary Information file. Patch–seq

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_Nat. Commun._ 9, 1544 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS Investigators involved in this work were supported by grants from

the NIH (R01DK129548 and R56DK133776 to Yanlin He, R00DK107008, P30 DK020595 and R01DK123098 to P.X., R01 DK136627 and K01DK119471 to C.W., F32DK134121 to K.M.C.), USDA/CRIS

(3092-51000-062-04(B)S to C.W. and 3092-51000-056 to D.L.H), TCH-2023 Pediatric Pilot award to C.W., American Heart Association awards (20POST35120600 to Yang He and 20POST000204188 to L.T.)

and the DOD (Innovative Grant W81XWH-20-1-0075 to P.X.). E2 and progesterone were measured using liquid chromatography hyphenated with mass spectrometry at the Metabolomics Core at Baylor

College of Medicine. Patch–seq samples were processed and sequenced in the genetic core at Pennington Biomedical Research Center, which is funded by the COBRE grant P20 GM135002 from NIGMS.

H129-ΔTK-TT-dTomato virus was obtained from the Center for Neuroanatomy with Neurotropic Viruses, supported by NIH Virus Center grant no. P40 OD010996. AUTHOR INFORMATION Author notes *

These authors contributed equally: Meng Yu, Bing Feng, Jonathan C. Bean. AUTHORS AND AFFILIATIONS * Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of

Medicine, Houston, TX, USA Meng Yu, Jonathan C. Bean, Yongjie Yang, Hailan Liu, Yongxiang Li, Benjamin P. Eappen, Hesong Liu, Longlong Tu, Kristine M. Conde, Mengjie Wang, Xi Chen, Na Yin, 

Darah Ave Threat, Nathan Xu, Junying Han, Yi Zhu, Darryl L. Hadsell, Yang He & Chunmei Wang * Pennington Biomedical Research Center, Brain Glycemic and Metabolism Control Department,

Louisiana State University, Baton Rouge, LA, USA Bing Feng, Qianru Zhao, Peiyu Gao & Yanlin He * Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, The

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Scholar CONTRIBUTIONS M.Y. performed surgeries and monitored metabolic phenotypes. B.F., Q.Z. and Yang He performed electrophysiological studies and patch–seq sample collection. J.B.

performed the data analysis of patch–seq and secondary analysis of scRNA-seq results. Y.Y., Hailan Liu, Y.L. and B.E. assisted with data collection. Hesong Liu, L.T., K.C., M.W., X.C., N.Y.,

D.T., N.X., J.H., Q.Z. and P.G. assisted with the experiments. D.H. performed mammary gland whole-mount analysis. D.H., Y.Z., Yang He, P.X. and Yanlin He assisted with the manuscript

writing. Yanlin He supervised electrophysiological studies. C.W. performed the stereotaxic injection of the virus and supervised the metabolic studies. Yanlin He and C.W. conceived, designed

and supervised the project. CORRESPONDING AUTHORS Correspondence to Yanlin He or Chunmei Wang. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER

REVIEW PEER REVIEW INFORMATION _Nature Metabolism_ thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editors: Jean Nakhle and Ashley

Castellanos-Jankiewicz, in collaboration with the _Nature Metabolism_ team. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in

published maps and institutional affiliations. EXTENDED DATA EXTENDED DATA FIG. 1 LACTATIONAL HYPERPHAGIA AND HYPERPROLACTINEMIA. (A) Serum levels of PRL in virgin and lactating female mice

(n = 6). (B-E) The correlation between the number of pups and food intake (B), PRL levels (C), or BAT size (D), or UCP1 (E) in the BAT in lactating dams (B, n = 44; C, n = 7; D, n = 5; E, n 

= 5). Data are presented as mean ± SEM and/or individual data points. A, two-tailed unpaired t-tests. B-E, linear regression with 95% confidence. P values are shown in figures. Source data

EXTENDED DATA FIG. 2 LACTATIONAL SUPPRESSION OF ERΑMBH NEURONS. (A) Spontaneous firing frequency and resting membrane potential of ERαvlVMH neurons in virgin (n = 37), pregnancy (gestation

day 16-18, n = 18) and lactating mice on PPD1 (n = 37), PPD 5 (n = 31), PPD 10 (n = 27) and PPD 20 (n = 22). Neurons from 3 mice in each group. (B-E) Individual ERαvlVMH neurons (B), ERαARH

neurons (C), ERαPOA neurons (D), ERαPVN neurons (E), responses to PPT as indicated by the increased firing frequency and resting membrane potential in virgin, late pregnancy (G18) and

lactating mice on PPD2 and 5. Fractions indicate the responsive neuron number out of total recorded neurons. Neurons from 3 mice in each group. (F) Percentage of spontaneous

firing/PPT-responsive, firing/PPT-irresponsive, silent/PPT-responsive and silent/PPT-irresponsive ERαvlVMH subpopulations from virgin, gestation day 18 and PPD5 mice. (G) Representative

traces of ERαvlVMH neurons excited by PPT in virgin and lactating female mice. (H) The percentage of ERα, ERβ, and GPER neurons in total VMH neurons from female mice indicated by secondary

analysis of scRNA-Seq data60,67. (I) Serum levels of E2 in females during peri-parturition and lactation (peri-parturition, n = 5; PPD2, n = 4; PPD5, n = 4; PPD9, n = 4; virgin, n = 3). Data

are presented as mean ± SEM and/or individual data points (A-E and I), or as part of whole bar (F) or Pie graphs (H). A, groups vs Virgin in ordinary one-way ANOVA followed by Dunnett’s

multiple comparisons test. I, ordinary one-way ANOVA followed by Šídák’s multiple comparisons test. B-E, “n” is indicated in figures. P values are shown in figures. Source data EXTENDED DATA

FIG. 3 DELETION OF ERΑ FROM MBH NEURONS CAUSES HYPERPROLACTINEMIA AND METABOLIC CHANGES. (A) Immunostaining of ERα in virgin female control and ERαMBH-KO mice with deletion of ERα from the

entire vlVMH but not the ARH. (B–C) Body size (B) and food intake (C) of virgin female control (n = 6) and ERαMBH-KO mice (n = 7). (D) Food efficiency (body weight gain/accumulated food

intake) of virgin female control and ERαMBH-KO mice by 16 days after virus injection (n = 6). (E-F) Body weight (E) and food intake (F) in virgin female control and ERαMBH-KO mice before

they entered the TSE metabolic cages, 2 weeks after virus injection (control, n = 8; ERαMBH-KO, n = 7). (G-I) Temporal levels of O2 consumption during the dark cycle and light cycle within 3

days (G), ANCOVA analysis of O2 consumption body weight as a covariant66,70 (H) and predicted O2 consumption at a presumed body weight (I) in virgin female control and ERαMBH-KO mice

measured by the TSE PhenoMaster metabolic cages (control, n = 8; ERαMBH-KO, n = 6). (J-L) Temporal levels of CO2 consumption during the dark cycle and light cycle within 3 days (J), ANCOVA

analysis of CO2 consumption body weight as a covariant (K) and predicted CO2 consumption at a presumed body weight (L) in virgin female control and ERαMBH-KO mice measured by the TSE

PhenoMaster metabolic cages66,70 (control, n = 8; ERαMBH-KO, n = 6). (M-O) Serum PRL levels (M), body weight (N) and food intake (O) of control WT mice receiving AAV-Cre or AAV-GFP virus

into the vlVMH (n = 5). Data are presented as mean ± SEM and/or individual data points. D, E, I and L, two-tailed unpaired t-tests. H and K, ANCOVA analysis by CalR. P values are shown in

figures. Source data EXTENDED DATA FIG. 4 DELETION OF ERΑ FROM MBH NEURONS CAUSES HYPERPROLACTINEMIA AND NEUROENDOCRINOLOGY CHANGES. (A) Immunohistochemistry staining of pSTAT5 in the

hypothalamus of virgin female control and ERαMBH-KO mice. 3 V, third ventricle. (B) H&E staining of the pituitary and immunofluorescence of PRL in the pituitary of virgin female control

and ERαMBH-KO mice. The scale bar is 200 μm. (C) Boxed areas of (B) are magnified as indicated. The scale bar is 50 μm. (D) Immunohistochemistry staining of ERα in the hypothalamus of virgin

female control and ERαMBH-KO mice 4 months (2 months chow diet followed by 2 months HFD) after surgery. The scale bar is 200 μm. (E-H) Serum levels of GH (E), IGF1 (F), ACTH (G) and TSH (H)

in virgin female control (n = 6) and ERαMBH-KO (E, n = 8; F, G, H, n = 7) mice 4 months (2 months chow diet followed by 2 months HFD) after surgery. (I) Serum levels of TSH in virgin female

control and ERαMBH-KO mice on chow diet 6 weeks after surgery (n = 8). (J) H&E staining of iWAT in virgin female control and ERαMBH-KO mice by 4 months after surgery. The scale bar is

500 or 100 μm. (K-M) Serum levels of P4 (K), LH (L) and FSH (M) in virgin female control (K, n = 4; L, n = 6; M, n = 6) and ERαMBH-KO (K, n = 6; L, n = 7; M, n = 7) mice 4 months (2 months

chow diet followed by 2 months HFD) after surgery. Data are presented as mean ± SEM and/or individual data points. H, two-tailed unpaired t-tests. P value is shown in the figure. Source data

EXTENDED DATA FIG. 5 DELETION OF ERΑ FROM VLVMH NEURONS INCREASES PRL LEVELS. (A) Immunofluorescence staining of ERα in the hypothalamus of control side and ¼ ERαvlVMH-KO side from one

virgin female mouse. The scale bar is 200 μm. (B) Quantification of ERα deletion in the half vlVMH KO side compared to the control side (n = 5). (C) The appearance of mammary glands in

female virgin control and virgin ¼ ERαvlVMH-KO mice. (D) Body weight at 12 weeks after virus injection and (E) average of 4 days-food intake of virgin control and ¼ ERαvlVMH-KO mice by 12

weeks after virus injection (n = 5). (F) Serum PRL level of virgin control and ¼ ERαvlVMH-KO mice at 6 weeks and 12 weeks after virus injection (n = 5). Data are presented as mean ± SEM and

individual data points. F, ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test. P values are shown in the figure. Source data EXTENDED DATA FIG. 6 ACTIVATION OF ERΑVLVMH

NEURONS INHIBITS LACTATIONAL HYPERPROLACTINEMIA AND HYPERPHAGIA. (A) Bright-field illumination and fluorescence for mCherry of a recorded ERαvlVMH neuron in a brain slice of ERαvlVMH-hM3Dq

mouse (up), and a representative trace of ERαvlVMH neuron in ERαvlVMH-hM3Dq mouse treated with CNO (down). The scale bar is 20 μm. (B) The spontaneous firing frequency (n = 6) and resting

membrane potential (n = 8) of ERαvlVMH neurons before and after treatment of CNO, and after the wash out of CNO. (C) c-fos expression in the MBH of a ERα-Cre mouse that received the

injection AAV-DIO-hM3Dq-mCherry virus into one side of the vlVMH (hM3Dq-mCherry) and the injection of control AAV-DIO-mCherry virus into the other side of the vlVMH. Increased c-fos

expression in the hM3Dq-mCherry side indicates activation of ERαvlVMH neurons, compared to the control mCherry side 2 hours after injection of CNO. Black arrows indicate magnified images.

White arrows indicate neurons that co-express hM3Dq-mCherry and c-fos in the magnified picture of the white boxed area. Scale bar is 200 or 100 μm. (D) The c-fos quantification results of

(C) (n = 4). (E) Serum PRL levels of control (n = 12) and ERαvlVMH-hM3Dq (n = 14) lactating dams on PPD2. (F) Serum PRL levels of control (n = 12) and ERαvlVMH-hM3Dq (n = 10) lactating dams

on PPD9. (G-I) Body weight (G), daily food intake (H) and BAT temperature (I) of control (G, n = 15; H, n = 12; I, n = 5) and ERαvlVMH-hM3Dq (G and H, n = 11; I, n = 5) mice during pregnancy

before CNO injection. (J-M) Body weight (J), daily food intake (K), BAT temperature (L) and averaged body weight of pups (M) in control (J, n = 15; K, n = 14; L, n = 10; M, n = 8) and

ERαvlVMH-hM3Dq (J, n = 21; K, n = 15; L, n = 7; M, n = 9) mice following the injection of CNO during lactation (excluding those with loss of all pups). (N-P) Maternal care time spent on

nursing (N), grooming (O) and nest building (P) of control (n = 5) and ERαvlVMH-hM3Dq lactating dams (n = 4) on PPD5. Data are presented as mean ± SEM and/or individual data points. B,

two-tailed paired t-tests. D, two-tailed unpaired t-tests. E and F, two-way RM ANOVA by multiple comparisons test with uncorrected Fisher’s LSD. K, two-way ANOVA followed by Šídák’s multiple

comparisons test with Geisser-Greenhouse correction. P values are shown in figures. Source data EXTENDED DATA FIG. 7 METABOLIC CHANGES INDUCED BY CENTRAL OR SYSTEMIC SUPPLEMENT OF E2 DURING

LACTATION. (A-B) The number of pups survived with systemic (A) or central supplement (B) of E2. (C-D) Body weight of lactating dams with systemic (C) or central (D) supplement of E2. (E-F)

Daily food intake of lactating dams with systemic (E) or central (F) supplement of E2. (G-H) The average body weight of pups raised by lactating dams with systemic (G) or central (H)

supplement of E2. (systemic: vehicle, n = 15; E2 0.5 ug/day, n = 7; E2 2.4 ug/day, n = 10. central: n = 5). (I-J) H&E staining of mammary glands enmeshed in the iWAT of lactating dams

with systemic supplement of vehicle (I) or E2 (J). Data are presented as mean ± SEM. A, E and G, two-way ANOVA followed by Šídák’s multiple comparisons test with Geisser-Greenhouse

correction. P values are shown in figures. Source data EXTENDED DATA FIG. 8 LACTATIONAL REMODULATION OF GENE PROFILES OF ERΑVLVMH NEURONS. (A) Electrophysiology recordings of the three

ERαvlVMH subpopulations (virgin firing vs PPD5 firing vs PPD5 silent) subjected to Patch-Seq. Resting membrane potential (left) and firing frequency (right) of these three ERαvlVMH

subpopulations and their responsiveness to PPT (n = 10, neurons from 3 mice per subpopulation). (B-C) Volcano plot and (B) GO analysis (C) of the differentially expressed genes between

virgin firing ERαvlVMH neurons and PPD5 firing ERαvlVMH subpopulation. (D-E) Volcano plot and (D) GO analysis (E) of the differentially expressed genes between PPD5 firing subpopulation and

PPD5 silent ERαvlVMH subpopulation. (F) Heatmap of Patch-Seq-identified genes that potentially mediate ERα downstream signal pathways. (G) Secondary analysis of published scRNA-Seq data of

VMH neurons60 identified 4 potassium channels that displayed the same changes as Patch-Seq data. These 4 genes were expressed at higher levels in lactating ERαVMH neurons compared to virgin

ERαVMH neurons or lactating non-ERαVMH neurons (virgin ERα+, n = 3932; lactating ERα+, n = 5462; virgin ERα-, n = 9373; lactating ERα-, n = 9776; all information in the SUPPLEMENTARY TABLE

1). (H) Percentage of ERαVMH neurons out of the whole VMH neurons in virgin and lactating female mice based on the secondary analysis of published scRNA-Seq data60(n = 7). (I) Heatmap of

Patch-Seq-identified inhibitory channels between OVX-V and OVX-E mice by reanalyzing published RNA-Seq data from the VMH of OVX-V and OVX-E mice18. Data are presented as mean ± SEM and/or

individual data points. A-bar graphs, one-way ANOVA followed by Dunnett’s multiple comparisons test or two-tailed unpaired t-tests. G, Wald test followed by Bonferroni correction. P values

are shown in figures. Source data EXTENDED DATA FIG. 9 LACTATIONAL REMODULATION OF PRL SIGNALING IN ERΑVLVMH NEURONS. (A-B) Secondary analysis of the population (A) and expression (B) of

Prlr in the VMH60(n = 7). CPM, counts per million. (C) Secondary analysis of the expression of Prlr in OVX-V (n = 3) vs OVX-E2 (n = 4) female mice18. Data are presented as mean ± SEM and

individual data points. (D) The expression of Prlr, Stat5a, Stat5b and receptors of other sex hormones in ERαVMH neurons from our Patch-Seq data. (E) Graphic summary. Lactational suppression

of ERαvlVMH neurons suppresses BAT thermogenesis, promotes feeding and maintains lactational hyperprolactinemia. Created in BioRender. Wang, C. (2025) https://BioRender.com/i72c521. Source

data SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Table 1. REPORTING SUMMARY SOURCE DATA SOURCE DATA FIG. 1 Statistical Source Data. SOURCE DATA FIG. 1 Unprocessed

western blot image. SOURCE DATA FIG. 2 Statistical Source Data. SOURCE DATA FIG. 3 Statistical Source Data. SOURCE DATA FIG. 4 Statistical Source Data. SOURCE DATA FIG. 5 Statistical Source

Data. SOURCE DATA FIG. 6 Statistical Source Data. SOURCE DATA FIG. 8 Statistical Source Data. SOURCE DATA EXTENDED DATA FIG. 1 Statistical Source Data. SOURCE DATA EXTENDED DATA FIG. 2

Statistical Source Data. SOURCE DATA EXTENDED DATA FIG. 3 Statistical Source Data. SOURCE DATA EXTENDED DATA FIG. 4 Statistical Source Data. SOURCE DATA EXTENDED DATA FIG. 5 Statistical

Source Data. SOURCE DATA EXTENDED DATA FIG. 6 Statistical Source Data. SOURCE DATA EXTENDED DATA FIG. 7 Statistical Source Data. SOURCE DATA EXTENDED DATA FIG. 8 Statistical Source Data.

SOURCE DATA EXTENDED DATA FIG. 9 Statistical Source Data. RIGHTS AND PERMISSIONS Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article

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publishing agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Yu, M., Feng, B., Bean, J.C. _et al._ Suppression of hypothalamic oestrogenic signal

sustains hyperprolactinemia and metabolic adaptation in lactating mice. _Nat Metab_ 7, 759–777 (2025). https://doi.org/10.1038/s42255-025-01268-z Download citation * Received: 28 June 2024 *

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