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Blocking FSH induces thermogenic adipose tissue and reduces body fat

Abstract

Menopause is associated with bone loss and enhanced visceral adiposity. A polyclonal antibody that targets the β-subunit of the pituitary hormone follicle-stimulating hormone (Fsh) increases bone mass in mice. Here, we report that this antibody sharply reduces adipose tissue in wild-type mice, phenocopying genetic haploinsufficiency for the Fsh receptor gene Fshr. The antibody also causes profound beiging, increases cellular mitochondrial density, activates brown adipose tissue and enhances thermogenesis. These actions result from the specific binding of the antibody to the β-subunit of Fsh to block its action. Our studies uncover opportunities for simultaneously treating obesity and osteoporosis.

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Figure 1: Fsh antibody reduces obesity in mice on a high-fat diet.
Figure 2: Fsh antibody reduces adiposity in ovariectomized mice.
Figure 3: Antibody treatment blocks Fsh–Fshr interaction to activate Ucp1.
Figure 4: Fsh antibody induces beige-like adipose tissue.
Figure 5: Fsh antibody triggers Ucp1 activation and enhances mitochondrial biogenesis.

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Acknowledgements

Work at Mount Sinai was supported by the NIH (R01 DK80459 and DK113627 (M.Z., T.F.D and L.S.), AG40132, AG23176, AR065932 (M.Z.), and AR067066 (M.Z. and N.G.A.)). Work at Maine Medical Center Research Institute was supported by: NIGMS P30 GM106391, P30 GM103392 and the NIDDK R24 DK092759-06 to C.J.R; Physiology Core Facility P20 GM103465, NIGMS COBRE in Stem Cell Biology and Regenerative Medicine. We are grateful to J. Cao for advice on micro-CT imaging. T.R.K. acknowledges the NIH (P01 AG029531).

Author information

Affiliations

Authors

Contributions

M.Z., L.S. and C.J.R. conceived and supervised the experiments; P. Liu, E.R.-R., V.E.D., W.A.-A., J.L. and P. Lu examined the effects of Fsh antibody on adiposity; E.R.-R., V.E.D., J.B.v.K. and C.J.R. did metabolic cage testing and analysis; B.Z., S.T.R., Y.E.Y., X.Z., F.Y., E.X.G., E.R.-R., V.E.D., P. Liu and C.J.R. generated micro-CT data; A.C.S., V.S., D.J.O., C.B. and P. Liu generated quantitative NMR data; W.A.-A., Y.Z., N.G.A. and P. Liu designed and performed studies with Thermo and PhAMexcised mice; Y.J., T.Y., P. Liu, S.I., L.-L.Z., Z.B. and L.S. documented functional adipocyte Fshrs; Y.J., S.D., P.T., A.G. and T.Y. carried out signalling experiments; T.Y. and L.S. performed ELISAs; S.H. conducted computational modelling; Y.J., T.Y. and H.M. carried out mass spectrometric studies; T.R.K. provided fully glycosylated Fsh; J.I., S.E., A.B., R.L., T.F.D., N.G.A., M.R.R. and M.I.N. helped design experiments and evaluate data; P. Liu, R.L., T.Y., L.S., C.J.R. and M.Z. checked the datasets; P. Liu, T.Y., L.S., C.J.R. and M.Z. wrote the paper.

Corresponding author

Correspondence to Mone Zaidi.

Ethics declarations

Competing interests

Icahn School of Medicine at Mount Sinai has filed a provisional patent application that covers the application of FSH inhibition to decrease body fat (Provisional US Patent # 62/368,651). M.Z. is listed as inventor. M.Z. will receive royalties and/or licensing fees, according to Mount Sinai policies, if the aforementioned patent is commercialized. M.Z. also consults for Merck, Roche, Novartis, and a number of financial consulting platforms.

Additional information

Reviewer Information Nature thanks S. Kajimura, S. Teitelbaum and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Figure 1 Antibody blocks Fsh–Fshr interaction at physiological Fsh concentrations in plasma.

a, Recombinant mouse Fsh (Fshα–Fshβ chimaera, 2 μg) was passed through resin with immobilized polyclonal Fsh antibody or goat IgG. Elution (Eluate), flow-through (Flow), and consecutive wash fractions (Wash) were collected and immunoblotted, as shown, with a mouse monoclonal Fsh antibody (Hf2). b, The sequence of the Fshα–Fshβ chimaera. Peptides from the trypsinized eluate matched by mass spectrometry are marked in red, with the linker peptide shown as red solid circles. The antibody was raised against LVYKDPARPNTQK (green-filled circles). c, The eluted fraction was trypsinized and analysed by LC–MS/MS. Data were extracted and queried against a protein database containing the Fshα–Fshβ chimaera sequence using Proteome Discoverer 1.4 and MASCOT 2.5.1. Oxidated methionine and carbamidomethylated cysteine residues are shown in lower-cased letters (m and c, respectively; see Methods). d, Crystal structure of the human FSH–FSHR complex (PDB code 4AY9; FSHα not shown for clarity) indicates that the loop from the FSHβ subunit (yellow), containing the sequence LVYKDPARPKIQK (highlighted as sticks), tucks into a small groove generated by the FSHR (i). Computational modelling of Fsh bearing the peptide sequence LVYKDPARPNTQK shows an identical binding mode (ii). Positively charged residues (blue) of the peptide surface complement the negatively charged residues (red) of the Fshr binding site to generate strong electrostatic interactions at the binding surface (arrow) (iii). Given the small size of the groove (iv), binding of antibody to the peptide sequence will completely shield Fshβ from entering the Fshr binding pocket. e, We confirmed experimentally that the antibody blocked Fsh action using Thermo cells10, which have a Luc2-T2A-tdTomato transgene inserted at the initiation codon of the Ucp1 gene10. Thermo cells retain BAT capacity and report Ucp1 activation using Luc2 as reporter. The effect of Fsh (30 ng ml−1) and Fsh antibody (concentrations as noted) on Ucp1 expression was tested without fetal bovine serum (no endogenous Fsh) with the Arb3 agonist CL-316,243 (10−7 M). Notably, 1 μg ml−1 Fsh antibody abolished the inhibitory effect of near-circulating levels of Fsh on Ucp1 expression (also see Fig. 3c). Mean ± s.d.; *P ≤ 0.05, **P ≤ 0.01; in triplicate. f, Fsh antibody measured as goat IgG in mouse serum (ELISA) following single injection of antibody (100 μg, intraperitoneally) yielded serum goat IgG (antibody) concentrations that were 10–20-fold higher than those required to inhibit Fsh action in vitro (t1/2 = 25.6 h) n = 3 per group (f). Two-tailed Student’s t-test, mean ± s.e.m.

Source data

Extended Data Figure 2 Effect of Fsh antibody on body fat and energy homeostasis in mice fed on a high-fat diet.

ac, Results in Fig. 1 were confirmed independently by the C.J.R. laboratory using quantitative NMR (qNMR) (n = 12 per group) (a), dual energy X-ray absorptiometry (DXA) (n = 12 or 11 per group) (b), and tissue weight measurements for iWAT, gonadal WAT (gWAT) and BAT (n = 9 or 10 per group) (c). d, Effects of Fsh antibody on body fat under thermoneutral (30 °C) conditions. Three-month-old male mice were housed at 30 °C, fed on a high-fat diet, and injected with antibody or IgG (200 μg per mouse per day, intraperitoneally) for 3 weeks for measurements of body weight and fat and lean mass (by qNMR) (n = 3 or 4 per group). e, Indirect calorimetry (metabolic cages) showing 24-h, day and night parameters of thermogenesis, namely O2 consumption (VO2), energy expenditure (EE), resting EE (R-EE), active EE (A-EE), and respiratory quotient (RQ), as well as physical activity parameters, including X-, Y- and Z-breaks, running distance (Wheel Meters), running speed (Wheel Speed), walking distance (Ped Meters) and walking speed (Ped Speed), and food intake (e). The data were independently analysed by J.B.v.K. using penalized Spline (p-Spline) regression, with EE, RMR and A-EE (PA) shown (see Methods for details) (n = 4 per group). f, Plasma noradrenaline levels (HPLC, courtesy R. Jacobs, Yale School of Medicine) measured in samples from groups of 3-month-old female mice treated with antibody or goat IgG (200 μg per mouse per day, intraperitoneally) for 7 weeks, following which half the animals within the respective groups were killed, and the other half were treated with the tyrosine hydroxylase inhibitor α-methyl-p-tyrosine (AMPT, 250 mg kg−1, injection repeated after 2 h with 125 mg kg−1, intraperitoneally) (n = 7 or 8 per group). Blood was drawn 2 h after the last AMPT injection. g, Plasma irisin levels (ELISA kit, Phoenix, EK-067-29) were measured following treatment of 3-month-old mice with antibody or goat IgG (200 μg per mouse per day, intraperitoneally, 5 and 7 per group for IgG and antibody, respectively). We also measured serum meteorin-like (metrnl) (ELISA kit, R&D, DY7867); all samples were below assay detection. h, i, GTT (n = 12 per group) (h) or ITT (3 and 4 per group for IgG and antibody, respectively) (i) showed no significant difference between mice receiving Fsh antibody or IgG (AUC: area under curve). jl, Effect of Fsh antibody or IgG on plasma C-peptide (n = 3 or 4 per group), adiponectin (n = 5 per group) or leptin levels (n = 5 per group); on total cholesterol, triglycerides or free fatty acids (n = 5 per group) (k); and on oestradiol (E2) (n = 5 or 4 per group) (l). Two-tailed Student’s t-test; *P ≤ 0.05, **P ≤ 0.01, ^P = 0.0588, ^^P = 0.065, or as shown; mean ± s.e.m. m, Decomposition of TEE with p-Spline regression (e). Continuous time estimates of the RMR and AEE (PA) (shown as AEE) are shown for a typical calorimetry and activity dataset. The p-Spline regression model estimates the RMR from the correlation in time between the activity and energy expenditure data by minimizing the difference between the actual and predicted energy expenditure (AEE + RMR). By using a spline function instead of a constant intercept in the regression model, natural time variations that occur in the RMR can be determined and accurate estimates of the average RMR and AEE are obtained (for details, see Methods).

Source data

Extended Data Figure 3 Fsh antibody markedly reduces high-fat diet-induced obesity in 8-month-old mice.

Effect of Fsh antibody or goat IgG (200 μg per day per mouse, intraperitoneally) injected daily into 8-month-old C57BL/6 male mice (Charles Rovers) pair-fed on high-fat diet (n = 2 or 3 mice per group) (HFD, see Methods). ac, Food intake and body weight (a), fat mass, FM/TM and LM/TM (b), and TFV, SFV and VFV (coronal and transverse sections; visceral, red; subcutaneous, yellow) (c). Two-tailed Student’s t-test; *P ≤ 0.05; ^P = 0.06, mean ± s.e.m.

Source data

Extended Data Figure 4 Fshr-haploinsufficient male mice phenocopy the anti-adiposity action of Fsh antibody and fail to respond to antibody, confirming in vivo antibody specificity.

Effect of Fsh antibody or goat IgG (200 μg per day per mouse intraperitoneally) in male wild-type (Fshr+/+) or Fshr-haploinsufficient (Fshr+/−) mice that were pair-fed on high-fat diet (HFD, see Methods) (n = 3 or 5 mice per group). a, b, Total mass (TM), fat mass (FM), and FM/TM (quantitative NMR) (a), and TFV, SFV and VFV (micro-CT, coronal and transverse sections; visceral, red; subcutaneous, yellow) (b). Ucp1 immunostaining of sWAT sections showed smaller and densely staining beige-like cells in Fshr+/− mice and in antibody-treated wild-type mice. Two-tailed Student’s t-test; *P ≤ 0.05, **P ≤ 0.01; mean ± s.e.m.

Source data

Extended Data Figure 5 Effects of Fsh antibody in ovariectomized mice.

Ovariectomized or sham-operated mice on normal chow were injected with Fsh antibody or goat IgG (200 and 400 μg per mouse per day to sham-operated and ovariectomized mice, respectively) (see Methods and Fig. 2). a, Food intake and body weight (n = 5 per group). b, Plasma Fsh and oestrogen (E2) levels (plasma E2 mostly undetectable after ovariectomy) (n = 4 or 5 per group). c, Indirect calorimetry (metabolic cages) showing 24-h RQ, R-EE, A-EE, running distance (Wheel Meters), running speed (Wheel Speed), walking distance (Ped Meters), walking speed (Ped Speed) and food intake (n = 4 per group). d, Fsh antibody or IgG did not alter plasma glucose, total cholesterol, triglycerides or free fatty acids (n = 4 or 5 mice per group). Two-tailed Student’s t-test; **P ≤ 0.01, or as shown.

Source data

Extended Data Figure 6 Fsh antibody reduces body fat in mice fed on normal chow.

af, Three-month-old C56BL/6J female mice were either pair-fed (ac) or fed ad libitum (df) with normal chow and injected with Fsh antibody or IgG (100 μg per mouse per day) for 7 and 5 weeks, respectively. For pair-feeding, the amount of chow consumed ad libitum by the IgG group was given to the antibody-treated group. For ad libitum feeding, the antibody-treated group was allowed ad libitum access to food and the same amount of chow was given to the IgG group, with the leftover chow measured to determine food intake of the IgG group (see Methods). A significant increase in food intake by antibody-treated mice was noted in the ad libitum feeding protocol (d). Nonetheless, as with mice on a high-fat diet, in either feeding protocol (compare with Fig. 1 and Extended Data Fig. 2), antibody treatment caused a substantial decrease in total mass (TM), fat mass (FM) and FM/TM and increase in LM/TM on quantitative NMR (b) in mice that were pair-fed. Body weight (a) was also reduced (see Source Data for P values). However, whereas antibody-treated mice consumed substantially more chow than IgG-injected mice, they showed decreases in FM and FM/TM, but did not show a reduction in TM (e) or body weight (d) (also see Source Data for P values). Micro-CT showed profound decreases in thoracoabdominal fat, visualized in representative coronal and transverse sections (red, visceral fat; yellow, subcutaneous fat), and upon quantification of TFV, SFV and VFV (c, f) in both groups (n = 4, 5 or 6 per group for af). g, Indirect calorimetry (metabolic cages) showing A-EE, running distance (Wheel Meters), running speed (Wheel Speed), walking distance (Ped Meters), walking speed (Ped Speed) and food intake (n = 4 per group). Two-tailed Student’s t-test; *P ≤ 0.05, **P ≤ 0.01, ^P = 0.069, or as shown.

Source data

Extended Data Figure 7 Sanger sequencing confirms expression of Fshr on adipocytes.

a, Total RNA was extracted from adipocytes derived from mesenchymal stem cells (MSC-ad) that were isolated from mouse ear lobes or 3T3.L1 cells and cultured in differentiation medium (MDI, containing IBMX, dexamethasone, and insulin). Total RNA was reverse transcribed and PCR performed using overlapping primer sets (bold lines) to amplify three large cDNA fragments. Overlapping regions covered by primers for Sanger sequencing are shown by arrows. The sequence of the Fshr in MSC-ad cells was identical to mouse ovarian Fshr (GenBank ID: NM_013523.3). The 3T3.L1 cell Fshr lacked exon 2 (red box) and displayed three amino acid variations (H158Y, F190L and K243E), but was fully functional in terms of its ability to reduce cAMP levels and Ucp1 expression in response to Fsh (compare with Fig. 3b, c). b, Fsh also triggered upregulation of the lipogenic genes Fas and Lpl with a marginal increase in Pparg (**P ≤ 0.01, fold-change; qPCR, three biological replicates, in triplicate). The presence of a signalling-efficient Fshr in adipocytes is consistent with FSHR gene expression in human adipose tissue in GTex and GeneCard databases (http://www.gtexportal.org/home/gene/FSHR; http://www.genecards.org/cgi-bin/carddisp.pl?gene=FSHR&keywords=fshr). We likewise find in the mouse that Fshr expression in WAT is lower than in the ovary: 1.00 ± 0.47 versus 13.8 ± 1.31 (fold-change, qPCR, P ≤ 0.01, n = 3 or 4 mice per group, measurements in triplicate). Two-tailed Student’s t-test, mean ± s.e.m.

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Extended Data Figure 8 Fsh antibody alters gene expression in WAT and BAT.

Three-month-old female C57BL/6J mice pair-fed on high-fat diet were injected daily for 8 weeks with Fsh antibody or goat IgG (200 μg per mouse per day, intraperitoneally). a, Relative expression of genes (names noted) in BAT versus WAT (normalized to housekeeping genes and iWAT). b, Consistent with adipocyte beiging was enhanced brown gene expression (qPCR) in iWAT and BAT at 1 or 3 months (normalized to housekeeping genes and to IgG). Two-tailed Student’s t-test; mean ± s.e.m.; *P ≤ 0.05 (qPCR, n = 3 or 6 biological replicates, measured in triplicate). Also see Fig. 4d.

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Extended Data Figure 9 Monoclonal antibody Hf2 against a human FSHβ epitope markedly reduces body weight and WAT and induces beiging in mice fed on a high-fat diet.

The monoclonal antibody Hf2 was raised against a motif with the 13-amino-acid-long human FSHβ sequence (LVYKDPARPKIQK), which corresponds to the mouse Fshβ sequence (LVYKDPARPNTQK) against which the polyclonal antibody was raised (Extended Data Fig. 1). Hf2 specifically binds human FSHβ in an ELISA. We have also sequenced Hf2 (data available on request). ad, Effects of ~9-week exposure to Hf2 or mouse IgG (200 μg per day per mouse, intraperitoneally), injected daily into 6-month-old male C57BL/6J mice pair-fed on high-fat diet (see Methods). a, Food intake; b, body weight; c, fat mass, FM/TM and LM/TM (quantitative NMR); d, TFV, SFV and VFV (micro-CT, coronal and transverse sections; visceral, red; subcutaneous, yellow) (n = 5 per group for ad). e, Fluorescence and bright-field micrographs showing Ucp1 immunostaining in frozen sections of vWAT. DAPI: nuclear staining. Negative control: irrelevant IgG in place of first antibody in Hf2-treated mice. There is a marked increase in Ucp1 immunostaining with Hf2 treatment in vWAT, together with cell condensation, reminiscent of beiging. Two-tailed Student’s t-test, **P ≤ 0.01; mean ± s.e.m. For body weight changes, see Source Data. The proof-of-concept study shows that a profound anti-obesity action can result from targeting FSHβ with a monoclonal antibody, a prelude to translational efforts towards the future use of a humanized Hf2 or its equivalent in people.

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Extended Data Table 1 Strategy to confirm datasets at the laboratories of Mount Sinai Bone Program and Maine Medical Center Research Institute

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Liu, P., Ji, Y., Yuen, T. et al. Blocking FSH induces thermogenic adipose tissue and reduces body fat. Nature 546, 107–112 (2017). https://doi.org/10.1038/nature22342

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