Abstract
Oxytocin (OXT), a nine-amino-acid peptide produced in the hypothalamus and released by the posterior pituitary, has well-known actions in parturition, lactation and social behaviour1, and has become an intriguing therapeutic target for conditions such as autism and schizophrenia2. Exogenous OXT has also been shown to have effects on body weight, lipid levels and glucose homeostasis1,3, suggesting that it may also have therapeutic potential for metabolic disease1,4. It is unclear, however, whether endogenous OXT participates in metabolic homeostasis. Here we show that OXT is a critical regulator of adipose tissue lipolysis in both mice and humans. In addition, OXT serves to facilitate the ability of β-adrenergic agonists to fully promote lipolysis. Most surprisingly, the relevant source of OXT in these metabolic actions is a previously unidentified subpopulation of tyrosine hydroxylase-positive sympathetic neurons. Our data reveal that OXT from the peripheral nervous system is an endogenous regulator of adipose and systemic metabolism.
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Data availability
The RNA-seq data used in Extended Data Fig. 1c are available on the Gene Expression Omnibus under the accession number GSE108077. The single-nucleus RNA-seq data used in Extended Data Fig. 7a,b are available on the Gene Expression Omnibus under the accession number GSE176171. All unique materials are available from the authors on request. Other materials are available from commercial sources as described in the Methods. Source data are provided with this paper.
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Acknowledgements
This work was supported by US National Institutes of Health grants R01 DK133223 and RC2 DK116691 to E.D.R., P30 DK056341 to S.K., and R01 DK120649 and RC2 DK129961 to P.C. We thank C. Usher for artistic support; H. Muenzberg-Gruening for helpful discussions; and the study participants for their participation.
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Contributions
E.L. and E.D.R. conceived of the project. E.L., L.W. and D.W. performed the experiments. J.C. and Z.L. performed the Adipo-Clear experiments under the supervision of P.C. S.K. and G.I.S. provided human samples. E.L. and E.D.R. wrote the manuscript. All authors reviewed the manuscript before submission.
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E.D.R. receives consulting fees from Novartis. All other authors declare no competing interests.
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Nature thanks James Blevins and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
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Extended data figures and tables
Extended Data Fig. 1 OXTR expression in adipocytes.
a. Oxtr mRNA expression in mouse eWAT, iWAT and BAT; n = 4. eWAT vs. iWAT P = 0.0062; iWAT vs. BAT P = 0.0242. b. OXTR protein expression in mouse eWAT, iWAT and BAT. Representative image from two western blots. c. Expression of Oxtr in TRAP-seq data from isolated white, beige and brown adipocytes; n = 3–5 animals per group. d. Oxtr mRNA expression in the stromal vascular fraction (SVF) and adipocytes of mouse iWAT and eWAT; n = 4. iWAT P < 0.0001, eWAT P < 0.0001. e. OXTR protein expression in the SVF and adipocytes of mouse iWAT and eWAT. Representative image from two western blots. f. Representative images of cleared eWAT and iWAT of WT mice immunostained for OXTR and PLIN1. Scale bars, 80 um for low magnification images and 20 um for high magnification images. Representative images are shown from two independent experiments. g. Representative image of isolated adipocytes from eWAT of WT mice. Red represents OXTR antibody staining, and green represents BODIPY staining. Scale bar, 20 um. h. Oxtr mRNA expression in SVF and adipocytes differentiated from SVF ex vivo; n = 4 biologically independent samples per group. P < 0.0001. i. OXTR protein levels in SVF and adipocytes differentiated from SVF ex vivo. Representative image from two western blots. j. Oxtr mRNA expression by qPCR in isolated adipocytes of iWAT and eWAT, harvested from mice housed at 30 °C or 4 °C for 1 week; warm n = 5, cold n = 6. iWAT P = 0.0083 and eWAT P = 0.0019. k. OXTR protein expression in isolated adipocytes of iWAT and eWAT, harvested from mice housed at 30 °C or 4 °C for 1 week. Representative image from two western blots. Data are presented as mean ± s.e.m. Statistical comparisons were made using 2-tailed Student’s t test. * denotes P < 0.05; **P < 0.01 and ****P < 0.0001. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 2 OXT induces lipolysis through ERK signaling.
a. Glycerol release from cultured mouse adipocytes treated with different doses of OXT for 3 h; n = 3. b. cAMP levels in cultured mouse adipocytes treated with vehicle, 1uM or 10 uM OXT for 15 min; n = 3. c. Western blotting of PKA substrate from cultured adipocytes treated with vehicle or 10uM OXT for 15 min; n = 3. d. Glycerol release from cultured mouse adipocytes treated with vehicle or ISO in the presence of DMSO, PKG inhibitor KT5823 or PKA inhibitor H89; n = 3. Vehicle, ISO vs. control, P < 0.0001; KT5823, ISO vs. Control, P = 0.0001; Vehicle vs. KT5823 after ISO, P = 0.0007; Vehicle vs. H89 after ISO, P < 0.0001. e. Glycerol release from cultured mouse adipocytes treated with vehicle or OXT in the presence of DMSO, PKG inhibitor KT5823 or PKA inhibitor H89; n = 3. Vehicle, OXT vs. control, P = 0.0005; KT5823, OXT vs. control, P = 0.0001; and H89, OXT vs. control, P = 0.0021. f. Dose response for pERK1/2 induced by OXT in cultured mouse adipocytes. Representative image from two western blots. g. Time course of OXT-induced (10 uM) ERK activation and PKA activity in cultured mouse adipocytes. Representative image from two western blots. h. Glycerol release from cultured mouse adipocytes treated with OXT (10 uM), OXT + the MEK inhibitor Trametinib (Tra, 5 nM) or OXT + the ERK inhibitor Temuterkib (Tem, 2 uM); n = 3. Vehicle vs. OXT, P < 0.0001; OXT+Tra vs, OXT alone, P < 0.0001; and OXT+Tem vs. OXT alone, P < 0.0001. Data are presented as mean ± s.e.m. Statistical comparisons were made using 2-tailed Student’s t test. ** denotes P < 0.01; ***P < 0.001, and ****P < 0.0001. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 3 Knockout of OXTR in adipocytes has minimal effect on body weight.
a. Scheme of the floxed Oxtr allele. b. Oxtr mRNA levels in adipocytes of eWAT and iWAT from Oxtrflox and OxtrΔAd mice housed at 4 °C for 1 week; n = 5. eWAT, P < 0.0001; iWAT, P = 0.0026. c. OXTR protein levels in adipocytes of eWAT from Oxtrflox and OxtrΔAd mice housed at 4 °C for 1 week; n = 3. d. Representative images of cleared eWAT and iWAT of Oxtrflox and OxtrΔAd mice immunostained for OXTR and PLIN1. Scale bars, 80 um for low magnification images and 20 um for high magnification images. Representative images are shown from two independent experiments. e. Representative images of isolated adipocytes from eWAT of Oxtrflox and OxtrΔAd mice. Red represents OXTR antibody staining, and green represents BODIPY staining. Scale bar, 20 um. f. Body weight of Oxtrflox and OxtrΔAd mice on chow (RD) and HFD; Chow Oxtrflox n = 10, Chow OxtrΔAd n = 8, HFD Oxtrflox n = 14, HFD OxtrΔAd n = 15. g. Lean and fat mass of Oxtrflox and OxtrΔAd mice on chow (RD); Oxtrflox n = 10, OxtrΔAd n = 8. h. Tissue weight of liver, BAT, iWAT, and eWAT of Oxtrflox and OxtrΔAd mice on HFD for 16 weeks; n = 7. Liver, Oxtrflox vs. OxtrΔAd, P = 0.0466; eWAT, Oxtrflox vs. OxtrΔAd, P = 0.0465. i. H&E staining of liver, BAT, iWAT, and eWAT of Oxtrflox and OxtrΔAd mice on HFD for 16 weeks. Scale bar, 200 um. j. Analysis of adipocyte size of iWAT and eWAT in mice as described in f; n = 5. iWAT 2-3, P = 0.0082, iWAT 7-8, P = 0.0205, eWAT 6-7, P = 0.0039 and eWAT 10–20, P = 0.0291. k. Serum FFA levels from female Oxtrflox and OxtrΔAd mice housed at 4 °C at the indicated times; n = 5. 0.5 h, Oxtrflox vs. OxtrΔAd, P = 0.012; 2 h, Oxtrflox vs. OxtrΔAd, P = 0.0462. Data are presented as mean ± s.e.m. Statistical comparisons were made using 2-tailed Student’s t test (b, g, h and k) or 2-way ANOVA (f and j). * denotes P < 0.05; **P < 0.01 and ****P < 0.0001. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 4 OxtrΔAd mice are more sensitive to cold challenge due to decreased pPLIN1 and pHSL levels.
a. Rectal temperature of Oxtrflox and OxtrΔAd mice exposed to 4 °C; Oxtrflox n = 5, OxtrΔAd n = 6. 3 h, P = 0.0002. b. Western blotting of UCP-1 in BAT and iWAT of chow-fed Oxtrflox and OxtrΔAd mice housed at 4 °C for 1 week; n = 4. c. mRNA levels of thermogenic genes in cultured adipocytes treated with vehicle or OXT (10uM) for 24 h; n = 3 biologically independent samples per group. d. Western blotting of UCP-1, HSL and ATGL in cultured adipocytes treated with vehicle or OXT (10 uM) for 24 h. Representative image from two western blots. e. Lipe and Pnpla2 mRNA levels in eWAT of chow-fed Oxtrflox and OxtrΔAd mice housed at 4 °C for 1 week; n = 5. f. Lipe and Pnpla2 mRNA levels in iWAT of chow-fed Oxtrflox and OxtrΔAd mice housed at 4 °C for 1 week; Oxtrflox n = 5, OxtrΔAd n = 6. g. Western blotting of pPLIN1, PLIN1, pHSL, HSl, and ATGL of eWAT of Oxtrflox and OxtrΔAd mice housed at 4 °C for 1 week; n = 5. h. Western blotting of pPLIN1, PLIN1, pHSL, HSl, and ATGL of iWAT of Oxtrflox and OxtrΔAd mice housed at 4 °C for 1 week; n = 5. Data are presented as mean ± s.e.m. Statistical comparisons were made using 2-tailed Student’s t test (c, e, and f) or 2-way ANOVA (a). *** denotes P < 0.001. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 5 Effect of OXT on ISO-induced lipolysis.
a. Glycerol release from cultured adipocytes treated with OXT (100 nM), isoproterenol (ISO; 1 uM), or both OXT and ISO; n = 4 biologically independent samples per group. ISO + OXT vs. ISO alone, P < 0.0001. b. Glycerol release from cultured adipocytes treated with varying doses of OXT in the presence or absence of ISO (1uM); n = 3 biologically independent samples per group. P < 0.0001 for all comparisons shown. c. Glycerol release from human adipose explants treated with OXT (10 uM), isoproterenol (ISO; 10 uM), or both in the presence or absence of the MEK inhibitor trametinib (Tra) (5 nM); n = 4. OXT vs. ISO + OXT, P = 0.0002; ISO vs. ISO + OXT, P = 0.0042; ISO + OXT vs. ISO + OXT+Tra, P = 0.0004. d. Glycerol release from human adipose explants treated with different doses of ISO with or without OXT (10 uM); n = 3. ISO 1 uM, P = 0.0054; ISO 10 uM, P < 0.0001. e. Serum FFA levels of female Oxtrflox and OxtrΔAd mice after ISO treatment; n = 5. ISO, P = 0.0044. f. Western blotting of pPLIN1, pHSL, pERK, and PKA activity from the same cells shown in Fig. 1g. Representative image from two western blots. g. Western blotting of pPLIN1, pHSL, pERK, and PKA activity from cultured adipocytes treated with OXT (1 uM), isoproterenol (ISO; 1 uM), or both OXT and ISO, in the presence or absence of the MEK inhibitor Trametinib (Tra; 5 nM) or the ERK inhibitor Temuterkib (Tem; 2 uM). Representative image from two western blots. h. cAMP levels in the same cells shown in Fig. 1g; n = 3 biologically independent samples per group. Data are presented as mean ± s.e.m. Statistical comparisons were made using 2-tailed Student’s t test (a-c,e and h) or 2-way ANOVA (d). ** denotes P < 0.01; ***P < 0.001 and ****P < 0.0001. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 6 Effect of OXT on HSL, PLIN1 and ATGL.
a. Representative images showing the distribution of HSL in cultured adipocytes treated with vehicle or OXT (10uM) in the presence or absence of ISO (10 uM). Scale bar, 10 um. b. Representative images showing the distribution of PLIN1 in lipid droplets of cultured adipocytes treated with vehicle or OXT (10 uM) in the presence or absence of ISO (10 uM). Scale bar, 20 um. c. Western blotting of pPLIN1 and PLIN1 in cultured adipocytes treated with OXT (10 uM), ISO (10 uM), or both for 3 h. Representative image from two western blots. d. Glycerol release from cultured adipocytes treated with OXT (10 uM), ISO (10 uM), or both in the presence or absence of the ATGL inhibitor Atglistatin (100 uM) for 3 h; n = 4 biologically independent samples per group. OXT vs. OXT + ISO, P < 0.0001; ISO vs. OXT + ISO, P < 0.0001; OXT + Atglistatin vs. OXT + ISO + Atglistatin, P < 0.0001; ISO+ Atglistatin vs. ISO + OXT + Atglistatin, P < 0.0001. e. Fold change of glycerol release in d (normalized to control); n = 4 biologically independent samples per group. Data are presented as mean ± s.e.m. Statistical comparisons were made using 2-tailed Student’s t test. **** denotes P < 0.0001. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 7 Sympathetic nerves are the source of OXT in fat.
a. Single nucleus RNA-seq of mouse adipose tissue (epididymal and inguinal) shows undetectable Oxt expression. b. Single nucleus RNA-seq of human white adipose tissue (subcutaneous and visceral) shows undetectable OXT expression. c. Gating strategy to sort tdTomato+ cells from Ai9 mice and Oxt-Cre::Ai9 mice presented in d. d. Representative flow cytometric analysis of tdTomato-positive cells from iWAT and eWAT SVF of Oxt-Cre::Ai9 mice. e. Sympathetic nerves from cleared iWAT of Oxt-Cre::Ai9 mice. RFP (red) marks expression of Oxt-Cre, with counterstaining with anti-TH (green). Scale bar, 200 um. f. Sympathetic nerves from cleared eWAT of Oxt-Cre::NuTRAP mice. GFP (green) marks expression of Oxt-Cre, with counterstaining with anti-TH (red). Scale bar, 100 um. g. Aorticorenal ganglion (ARG) of Oxt-Cre::Ai9 mice perfused with PBS. tdTomato (red) marks expression of Oxt-Cre. Arrowhead indicates autofluorescent blood vessel debris. Scale bar, 200 um. h. Co-immunostaining of RFP and TH in the L1 ganglion of Oxt-Cre::Ai9 mice. Scale bar, 50 um. i. mCherry-positive cells in the L1 ganglion of Oxt-Cre mice injected with AAV2/Retro-hsyn-Flex-mCherry in iWAT. Scale bar, 200 um. j. Co-immunostaining of mCherry and TH in the L1 ganglion of mice in e. Scale bars, 50 um (region 1) and 20 um (region 2). For e-j, representative images are shown from two independent experiments.
Extended Data Fig. 8 Percentage of OXT+ sympathetic neurons among all adipose-innervating sympathetic neurons.
a. Representative images of ARG from 4 WT mice co-immunolabeled by OXT and TH. Scale bars, 50 um. b. Representative images of L1 sympathetic ganglia from 3 WT mice immunostained with OXT and TH. Scale bars, 50 um. c. Representative images of thoracic sympathetic ganglia T1-T3 from WT mice immunostained with OXT and TH. Scale bars, 50 um. d. Representative three-dimensional projection and two-dimensional section of cleared iWAT of WT mice immunostained with OXT (Red) and TH (Green). Scale bars, 200 um for 3D projection and 80 um for 2D section. e. Quantification of the percentage of OXT+ neurons among TH+ sympathetic neurons in ARG and L1 ganglia from mice in a, b and Fig. 2h, calculated as # of OXT+ neurons/ # of TH+ neurons *100%; for L1, n = 3 biologically independent samples, for ARG, n = 5 biologically independent samples. f. Upper panel: schematic of experiment assessing the percentage of OXT+ sympathetic neurons among adipose-innervating sympathetic neurons. AAV2/Retro-hSyn-Flp was injected into the iWAT or eWAT of Oxt-Cre_RC::FL-hM3Dq mice. Adipose tissue-innervating sympathetic neurons expressed either EGFP (Flp + /Cre-) or mCherry (Flp +/Cre +), while adipose-innervating OXT+ neurons expressed mCherry because they were Oxt-Cre +/Flp +. Schematic in upper panel of f, credit: C. Usher. Lower panel: Scheme of RC::FL-hM3Dq mouse. EGFP protein is expressed after Flp-mediated excision of the FRT-flanked stop cassette (STOP). hM3Dq-mCherry fusion protein is expressed after excision of the stop cassette and Cre-dependent recombination of lox2272 and loxP sites. g. Representative immunostaining images of ARG and L1 ganglia from mice in f. Scale bars, 50 um. h. Quantification of the percentage of OXT+ sympathetic neurons among adipose-innervating sympathetic neurons in ARG and L1 ganglia from mice in f, equal to # of mCherry+ neurons/ # of neurons either GFP+ or mCherry + *100%; n = 4. i. RPa from mice in Fig. 3a, showing mCherry in all mice, but c-Fos activation (green) in mice that received both DREADD and CNO. Scale bar, 50 um. For c, d and i, representative images are shown from two independent experiments. Data are presented as mean ± s.e.m in e and h.
Extended Data Fig. 9 Loss of OXT in TH+ neurons reduces lipolysis.
a. Scheme of the floxed Oxt allele. b. Co-immunostaining of OXT and TH in L1 sympathetic ganglia of Oxtflox and OxtΔTH mice. Scale bars, 50 um. c. Representative three-dimensional projection of cleared iWAT of Oxtflox and OxtΔTH mice co-immunolabeled by OXT (red) and TH (green). Scale bars, 200 um. d. Daily food intake of chow-fed Oxtflox and OxtΔTH mice; n = 7. e. Body weight of Oxtflox and OxtΔTH mice on chow and HFD; chow Oxtflox n = 7, chow OxtΔTH n = 7, HFD Oxtflox n = 9 and HFD OxtΔTH n = 10. f. Lean and fat mass of the Oxtflox (n = 6) and OxtΔTH (n = 7) mice on chow. g. Lean and fat mass of the Oxtflox (n = 5) and OxtΔTH mice (n = 7) on HFD. h. Tissue weight of liver, BAT, iWAT, and eWAT of Oxtflox (n = 5) and OxtΔTH (n = 7) mice on HFD for 16 weeks. Exact P value: eWAT, Oxtflox vs. OxtΔTH, P = 0.0448. i. Serum FFA levels from female Oxtflox and OxtΔTH mice housed at 4 °C at the indicated times; OxtΔTH at time point 0 n = 5, for the rest n = 6. Oxtflox vs. OxtΔTH at 0.5 h, P = 0.033; at 1 h, P = 0.0228. j. Serum FFA levels of female Oxtflox and OxtΔTH mice after ISO treatment; n = 6. ISO, Oxtflox vs. OxtΔTH, P = 0.0182. k. Western blotting of pPLIN1, PLIN1, pHSL and HSl of eWAT of Oxtflox and OxtΔTH mice housed at 4 °C for 1 week; n = 4. l. Norepinephrine levels in eWAT of Oxtflox and OxtΔTH mice housed at 4 °C for 1 week; n = 4. For d-l, data are from biologically independent samples. Data are presented as mean ± s.e.m. Statistical comparisons were made using 2-tailed Student’s t test (d, f-j and l) or 2-way ANOVA (e). *P < 0.05. For gel source data, see Supplementary Fig. 1.
Extended Data Fig. 10 Ablation of Oxt specifically in sympathetic neurons innervating eWAT leads to impaired lipolysis.
a. Co-immunostaining of OXT and TH in PVN and SON of WT mice. Scale bar, 200 um. Representative images are shown from two independent experiments. b. Representative immunostaining images showing OXT expression in PVN at three rostral-caudal positions of Oxtflox and OxtΔTH mice. Scale bar, 100 um. c. The number of OXT-positive neurons in PVH as described in b; n = 5. d. Serum OXT levels in Oxtflox mice injected with AAV2/Retro-hSyn-Cre (n = 6) or AAV2/Retro-hSyn-mCherry (n = 5) in eWAT. e. OXT levels in eWAT of mice described in a housed at 4 °C for 1 week; AAV2/Retro-hSyn-Cre n = 6, AAV2/Retro-hSyn-mCherry n = 5. P = 0.019. f. Serum FFA levels in mice described in a housed at 4 °C at the indicated times; AAV2/Retro-hSyn-Cre n = 5 mice at time point 0 and n = 6 mice at time points 0.5 h and 2 h, AAV2/Retro-hSyn-mCherry n = 6 mice. AAV2/Retro-hSyn-Cre vs. AAV2/Retro-hSyn-mCherry at 0.5 h, P = 0.0461; at 2 h, P = 0.0288. g. Serum FFA levels in mice described in a after overnight fasting; AAV2/Retro-hSyn-Cre n = 6, AAV2/Retro-hSyn-mCherry n = 7. P = 0.0141. h. Serum FFA levels in mice described in a after ISO injection; n = 6. P = 0.0119. Data are presented as mean ± s.e.m. Statistical comparisons were made using 2-tailed Student’s t test. * denotes P < 0.05.
Supplementary information
Supplementary Figure 1
This figure provides uncropped gels and blots for data shown in main figures and extended data figures.
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Li, E., Wang, L., Wang, D. et al. Control of lipolysis by a population of oxytocinergic sympathetic neurons. Nature 625, 175–180 (2024). https://doi.org/10.1038/s41586-023-06830-x
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DOI: https://doi.org/10.1038/s41586-023-06830-x
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