Oxytocin regulates body composition

Abstract

The primitive neurohypophyseal nonapeptide oxytocin (OXT) has established functions in parturition, lactation, appetite, and social behavior. We have shown that OXT has direct actions on the mammalian skeleton, stimulating bone formation by osteoblasts and modulating the genesis and function of bone-resorbing osteoclasts. We deleted OXT receptors (OXTRs) selectively in osteoblasts and osteoclasts using Col2.3Cre and Acp5Cre mice, respectively. Both male and female Col2.3Cre⁺:Oxtr^fl/fl mice recapitulate the low-bone mass phenotype of Oxtr^+/- mice, suggesting that OXT has a prominent osteoblastic action in vivo. Furthermore, abolishment of the anabolic effect of estrogen in Col2.3Cre⁺:Oxtr^fl/fl mice suggests that osteoblastic OXTRs are necessary for estrogen action. In addition, the high bone mass in Acp5Cre⁺:Oxtr^fl/fl mice indicates a prominent action of OXT in stimulating osteoclastogenesis. In contrast, we found that in pregnant and lactating Col2.3Cre⁺:Oxtr^fl/fl mice, elevated OXT inhibits bone resorption and rescues the bone loss otherwise noted during pregnancy and lactation. However, OXT does not contribute to ovariectomy-induced bone loss. Finally, we show that OXT acts directly on OXTRs on adipocytes to suppress the white-to-beige transition gene program. Despite this direct antibeiging action, injected OXT reduces total body fat, likely through an action on OXT-ergic neurons. Consistent with an antiobesity action of OXT, Oxt^-/- and Oxtr^-/- mice display increased total body fat. Overall, the actions of OXT on bone mass and body composition provide the framework for future therapies for osteoporosis and obesity.

Keywords: adipose tissue; bone phenotype; conditional knockout; pituitary hormone.

Fig. 1.

OXT deficiency reduces bone mass but does not affect ovariectomy-induced bone loss. Representative µCT images and trabecular bone microstructural parameters—BMD, BV/TV, Tb.N, Tb.Th, trabecular spacing (Tb.Sp), and Conn.D—of 10-mo-old male (A) and female (B) wild-type (+/+), heterozygotes (+/−), or homozygotes (−/−) for the oxytocin gene, Oxt (n = 4 to 8 mice per group). (C) Representative µCT images and microstructural parameters (% control) showing the effect of ovariectomy (OVX) or sham operation (Sham; control) on 2-mo-old Oxt^+/+ and Oxt^−/− mice (n = 3 to 9 per group). (D) µCT-based trabecular bone microstructural parameters in female wild-type and Oxtr-deficient (Oxtr^−/−) mice (n = 3 to 4 per group). (E) Representative cultures and quantitation of CFU-F stained for alkaline phosphatase and CFU-OB labeled with von Kossa stain. The colonies formed when bone marrow stromal cells isolated from Oxtr^+/+ or Oxtr^−/− mice were allowed to grow in differentiation media (β-glycerol phosphate, ascorbic acid, and dexamethasone) for 7 and 21 d, respectively. Colonies per well were counted in triplicate. Data are expressed as mean ± SEM; comparisons with control mice, or as shown; *P < 0.05, **P < 0.01, or showing a trend ^0.05 < P < 0.1, 2-tailed Student’s t test or one-way ANOVA with Holm–Sidak correction.

Fig. 2.

Cell-selective mutants for the oxytocin receptor display bone mass phenotypes. (A) Trabecular bone microstructural parameters—BMD, BV/TV, Tb.Th, Tb.N, trabecular spacing (Tb.Sp), and Conn.D—of 4-mo-old male mice lacking or haploinsufficient in the Oxtr gene in osteoblasts (Col2.3Cre⁺:Oxtr^fl/fl or Col2.3Cre⁺:Oxtr^fl/+ mice) or control littermates with the intact Oxtr gene (pooled, Col2.3Cre⁻:Oxtr^fl/fl and Col2.3Cre⁻:Oxtr^fl/+ mice) (n = 4 to 9 mice per group). (B) Reduced bone formation parameters—MAR, mineralizing surface (MS), and BFR—in mature female Col2.3Cre⁺:Oxtr^fl/fl mice compared with control Col2.3Cre⁻:Oxtr^fl/fl mice (n = 5 mice per group). (C) Total bone, lumbar spine (L4 to L6), femoral, and tibial areal BMD showing the effect of 17β-estradiol (50 µg/kg) or vehicle injected biweekly over 4 wk into Col2.3Cre⁺:Oxtr^fl/fl or pooled control Col2.3Cre⁻:Oxtr^fl/fl and Col2.3Cre⁻:Oxtr^fll+ mice (n = 4 to 10 mice per group). (D) Representative µCT images of trabecular and microstructural parameters of 3-mo-old male and female mice lacking the Oxtr gene in osteoclasts (Acp5Cre⁺:Oxtr^fl/fl mice) or control littermates (pooled Acp5Cre⁻:Oxtr^fl/fl and Acp5Cre⁻:Oxtr^fl/+ mice) (n = 3 to 12 mice per group). (E) Representative µCT images of trabecular bone and BMD at various times during pregnancy (day 19, P19), lactation (day 14, L14) and weaning (day 14, W14) (n = 3 to 6 mice per group). Data are expressed as mean ± SEM; comparisons with control mice, *P < 0.05, **P < 0.01, or showing a trend ^0.05 < P < 0.1, 2-tailed Student’s t test.

Fig. 3.

Oxytocin signaling directly affects body fat. Incremental enhancements in body fat (% of total body weight) in male and/or female mice deficient in OXT (Oxt^−/− mice) (A) or the OXT receptor (Oxtr^−/− mice) (B) compared with their respective wild-type littermates (Oxt^+/+ or Oxtr^+/+ mice) measured by the GE Lunar Piximus (n = 4 to 7 mice per group). (C) No such difference was noted in mice lacking OXTRs specifically in osteoblasts, namely in Col2.3Cre⁺:Oxtr^fl/fl vs. control Col2.3Cre⁻:Oxtr^fl/fl and Col2.3Cre⁻:Oxtr^fl/+ mice. This latter finding suggests that signals from the osteoblast did not modulate the proadiposity effects of OXTR deletion (n = 4 to 5 mice per group). (D) Effect of i.p. OXT injection on fat mass (g and %), lean mass (g), and total body weight (g) over 5 wk of treatment (n = 6 to 7 mice per group). Data are expressed as mean ± SEM; comparisons with control mice, *P < 0.05, **P < 0.01, or showing a trend ^0.05 < P < 0.1, 2-tailed Student’s t test.

Fig. 4.

Oxytocin acts on adipocyte oxytocin receptors to suppress beiging. (A) qPCR showing the expression of OXTR in mouse tissues of interest, including the uterus, ovary, testes, 3 brain regions, and visceral (v) and subcutaneous (s) WAT and BAT. (B) Western blot analysis using an anti-OXTR antibody (Abcam; ab181077) showed OXTR protein expression in female uterus, ovary, liver, BAT, and visceral, subcutaneous, and perigonadal WAT. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as a loading control. (C) Immunohistochemistry using the same anti-OXTR antibody confirming high expression levels in uterus, with a strong signal in subcutaneous and visceral WAT and limited expression in BAT. (Scale bar: 50 µm.) (D) Immunofluorescence images of OXTR expression in various brain regions, including the nucleus tractus solitarius, lateral reticular nucleus, inferior olive nucleus, and raphe pallidus. The whole brain section was composed from 12 images. (Scale bar: 100 µm.) (E) qPCR on RNA isolated from adipocytes derived from precursor 3T3.L1 cells following a 14-d incubation in differentiation medium and a 2-d incubation with OXT. There was a significant (*P < 0.05, **P < 0.01) reduction in or a trend toward reduced expression (^0.05 < P < 0.1) of certain genes involved in beiging, namely Cox7a, Cox8b, Cebpb, Retn, and Cidea. Certain genes involved in steroidogenesis and thrombosis, namely Hsd17b12 and Serpine1, respectively, were up-regulated. The qPCR data were normalized to housekeeping genes Actb (A) Gapdh (A and D), Rps11 (A and D), and Tuba1a (A and D). Three biological replicates with 3 technical replicates were used. Data are expressed as mean ± SEM; comparisons with vehicle treatment, 2-tailed Student’s t test.