Non-homeostatic body weight regulation through a brainstem-restricted receptor for GDF15

An Erratum to this article was published on 08 November 2017

Abstract

Under homeostatic conditions, animals use well-defined hypothalamic neural circuits to help maintain stable body weight, by integrating metabolic and hormonal signals from the periphery to balance food consumption and energy expenditure1,2. In stressed or disease conditions, however, animals use alternative neuronal pathways to adapt to the metabolic challenges of altered energy demand3. Recent studies have identified brain areas outside the hypothalamus that are activated under these ‘non-homeostatic’ conditions4,5,6, but the molecular nature of the peripheral signals and brain-localized receptors that activate these circuits remains elusive. Here we identify glial cell-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) as a brainstem-restricted receptor for growth and differentiation factor 15 (GDF15). GDF15 regulates food intake, energy expenditure and body weight in response to metabolic and toxin-induced stresses; we show that Gfral knockout mice are hyperphagic under stressed conditions and are resistant to chemotherapy-induced anorexia and body weight loss. GDF15 activates GFRAL-expressing neurons localized exclusively in the area postrema and nucleus tractus solitarius of the mouse brainstem. It then triggers the activation of neurons localized within the parabrachial nucleus and central amygdala, which constitute part of the ‘emergency circuit’ that shapes feeding responses to stressful conditions7. GDF15 levels increase in response to tissue stress and injury, and elevated levels are associated with body weight loss in numerous chronic human diseases8,9. By isolating GFRAL as the receptor for GDF15-induced anorexia and weight loss, we identify a mechanistic basis for the non-homeostatic regulation of neural circuitry by a peripheral signal associated with tissue damage and stress. These findings provide opportunities to develop therapeutic agents for the treatment of disorders with altered energy demand.

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Figure 1: Identification of GFRAL as the GDF15 receptor.
Figure 2: Gfral knockout mice are resistant to GDF15.
Figure 3: Neuronal activation in GDF15-treated mice.
Figure 4: Emergency pathway activation is GFRAL-dependent.

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Acknowledgements

We thank N. Maddox, H. Tran, J. Oeffinger and R. Suriben for cloning, sequencing and genotyping, M. Bailey for purification of recombinant protein, R. Suto for help with solving the crystal structures and S. Talukdar, D. Kaplan, R. Suriben, S. Katewa and J.-L. Chen for critical reading of the manuscript.

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Authors

Contributions

J.-Y.H., D.A.L., J.T., W.D.S., Y.A.C., H.T. and B.B.A. directed the work. B.B.A., J.-Y.H., and D.A.L. designed experiments, analysed data and wrote the manuscript, with comments from all of the authors. S.C. and J.-Y.H. developed methods for the library screen. S.C., J.-Y.H., D.A.A., B.L. and J.C. developed and performed cell-based assays. W.J., M.L., M.M. and M.S. performed IHC and IF experiments in brain sections. M.C. designed, managed and performed mouse experiments along with Z.G., D.F. and C.T. GLP1R knockout experiments were done by S.-P.W., J.Ya. and J.Yi. Crystal structures were solved by D.L and A.W. A.Ke and H.M. created all expression constructs. M.W., T.L., G.H., J.H., and A.Ku expressed and purified all recombinant proteins under the guidance of R.H., T.P. and D.A.L. Surface plasmon resonance experiments were performed by K.M.

Corresponding authors

Correspondence to Jer-Yuan Hsu or Bernard B. Allan.

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Competing interests

D.L. and A.W. declare no direct competing financial interests. All other authors are or were employees of NGM Biopharmaceuticals or Merck Research Labs and may hold stock or stock options in these companies. NGM Biopharmaceuticals, Inc. has filed a non-provisional patent application entitled “Compositions and Methods for Modulating Body Weight” (PCT/US2017/020753), which discloses the GFRAL–GDF15 protein complex, and methods of identifying agents that may modulate the protein complex interaction(s) and potential uses of those agents to control body weight. J.-Y.H., S.C., J.H., J.T., W.D.S., Y.A.C. and H.T. are listed as inventors.

Additional information

Reviewer Information Nature thanks R. Palmiter, M. Saarma 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 GDF15 does not bind TGFβ receptors.

a, Phylogenetic tree of GDF15, TGFβ and GDNF superfamily members. b, Sequence conservation (%) of GDF15 versus individual superfamily members. c, Binding of 125I-labelled TGFβ or 125I-labelled GDF15 (30 pM) to COS7 cells transfected with combinations of type I and type II TGFβ family receptors. Data are mean ± s.d. Source data

Extended Data Figure 2 GDF15 apo-crystal structure.

a, Crystal structure of GDF15 dimer at 1.97 Å resolution. b, Intramolecular disulfides (pink and yellow) within the cysteine knot core shown on a backbone trace of the GDF15 monomer subunit. c, GDF15 sequence alignment and disulfide arrangement versus solved structures of 7-cysteine and 9-cysteine BMP, TGFβ, GDF and GDNF superfamily members (PDB accession codes indicated). GDF15 contains a novel (1→2, 3→7) disulfide arrangement (blue) not previously identified for other 9-cysteine family members (1→3, 2→7) (red).

Extended Data Figure 3 Sequence comparison of GFRAL with GFRα family members.

a, Mature GFRAL extracellular domain sequence alignment and disulfide arrangement against mature GFRα(1–4) family members. GFRAL (blue) maintains the conserved disulfide bonding network observed in all other family members (red) in the ligand recognition domain 2. GFRAL contains a novel arrangement of disulfide bonds in the crystal structure when compared with solved structures of GFRα1 and GFRα3 in domain 3, which may impact binding or activity of co-receptor RET. b, Phylogenetic tree of GFRAL versus GFRα family members. c, Sequence conservation (%) of GFRAL versus individual GFRα family members.

Extended Data Figure 4 GDF15 binds GFRAL exclusively and signals through RET.

a, GDF15 physically interacts with GFRAL and GFRAL–RET on the cell surface. HEK293T cells (transfected with Flag–GFRAL or Flag–GFRAL + RET) bound with125I-labelled GDF15 were crosslinked by BS3 and then immunoprecipitated using anti-Flag. Unlabelled GDF15 added at 100× molar excess of 125I-labelled GDF15 blocked radio-ligand-receptor complex formation. Asterisks mark unique bands present only in the 125I-labelled GDF15 crosslinked complex in Flag–GFRAL + RET cells. b, RET is required for GDF15-induced signalling. HEK293T cells transfected with indicated plasmids were treated with 10 nM GDF15 for 30 min at 37 °C. pERK or RAB11 (loading control) in whole-cell lysates were detected by western blotting. A signalling-defective RET mutant (Y1062F) failed to induce pERK activation by GDF15 (top). Co-expression of GFRAL and RET is required for GDF15-induced pERK activation (bottom). c, Specific binding of 125I-labelled GDF15 to HEK293T cells co-transfected with GFRAL and RET. d, GDF15 does not activate RET through other GFRα family receptors. HEK293T cells co-transfected with RET and GFRα1 (i), GFRα2 (ii), GFRα3 (iii) or GFRα4 (iv) were treated with GDF15, GDNF, neurturin, artemin or persephin, as indicated. e, GDF15 binds GFRAL, but not GFRα family receptors. HEK293T cells transfected with GFRAL, GFRα1, GFRα2, GFRα3 or GFRα4 were bound by Fc–GDF15 and detected by a fluorescent anti-Fc secondary antibody. f, Non-iodinated GDF15 (but not GDNF, neurturin, artemin or persephin) competes with the binding of 125I-labelled GDF15 (150 pM) to GFRAL-transfected HEK293T cells. g, RET-dependent reporter gene is activated by GDF15 but not by GDNF, neurturin, artemin or persephin in HEK293T cells co-transfected with GFRAL and RET. Data are mean ± s.d. Data are representative of two (b, c) or three (e) experiments. Source data

Extended Data Figure 5 Comparison of GDF15–GFRAL interactions with other GDNF–GFRα family members.

a, Ribbon comparison of co-crystal of GDF15–GFRAL (light blue and yellow) with solved family members GDNF–GFRα1 (green and magenta; PDB code 2E5V) and artemin–GFRα3 (blue and red; PDB code 2GH0) through superimposition of the ligands. In all cases, each receptor specifically contacts its cognate ligand along the finger domains of the ligand. b, Diagram showing residue-specific intermolecular contacts (≤ 4.5 Å) between ligand and receptor for solved structures of GDF15, GDNF, and artemin with cognate receptors. The conserved RRR motif common to all GFRα1–4 receptors corresponds to SKE in GFRAL (yellow). c, Close-up view of the hydrogen bonding network comprising the RRR motif common to all GFR receptors and ligands, demonstrating the divergent nature of GDF15–GFRAL interactions. d, Electrostatic surface rendering (superimposition of domains 2 and 3) of GFRAL, GFRα1 and GFRα3 with monomer subunit of GDF15, GDNF and artemin, respectively (green backbone), demonstrating the unique hydrophobic and electrostatic recognition of each ligand’s finger domain for each receptor.

Extended Data Figure 6 Gfral knockout mice on chow diet.

a, Schematic representation of genetic targeting used to create Gfral knockout mice (TF3754), and representative genotyping data identifying wild-type (+/+), heterozygous (+/−) and knockout (−/−) mice. b, Indirect calorimetry measurement of RER in 16-week-old Gfral+/+ and Gfral−/− mice on chow diet. n = 4 animals per group. c, Body weight of Gfral+/+ and Gfral−/− mice on chow diet at three months of age (n = 4 wild-type and n = 6 knockout animals) and six months of age (n = 5 animals per group). All data are ± s.e.m. Source data

Extended Data Figure 7 Neuronal activation by GDF15.

a, Gene expression profile of Gfral mRNA in mouse tissues measured by quantitative PCR with reverse transcription. Data are mean ± s.d. n = 6 individual dorsal medulla tissue samples. b, GFRAL immunofluorescence in area postrema and NTS in a Gfral wild-type mouse (left) but not in a Gfral knockout mouse (right). c, FOS in area postrema and NTS neurons 1 hour after administration of GDF15 to a Gfral wild-type mouse (left) but not in a Gfral knockout mouse (right). d, FOS in central amygdala neurons 1 hour after administration of GDF15 to wild-type mice. e, No increase in FOS in hypothalamic neurons 1 hour after administration of GDF15 to wild-type mice. Data are representative of two independent experiments with n = 3 mice per experiment (b, c), and four independent experiments with n = 3 (PBS, d) or n = 5 (GDF15, e) mice per experiment. Source data

Extended Data Figure 8 GDF15 and GLP1 activate independent pathways.

a, Body weight reduction after an injection of recombinant Fc–GDF15 or vehicle in wild-type (Glp1r+/+) or knockout (Glp1r−/−) mice. n = 6 animals. b, Body weight reduction after daily injections of GLP1 (Victoza) in wild-type (Gfral+/+) or knockout (Gfral−/−) mice. n = 9 animals per group. All data are mean ± s.e.m. Source data

Extended Data Figure 9 Gfral knockout mice are resistant to cisplatin-induced loss of tissue mass.

a, Changes in whole body lean mass in wild-type and Gfral knockout mice treated with cisplatin (4 mg kg−1) once per week for six weeks. n = 8 (wild-type), n = 6 (knockout + vehicle) n = 7 (knockout + cisplatin) animals. P values obtained by two-tailed Holm–Sidak t-test (α = 0.05). b, Changes in wet weight of tissues isolated from wild-type and Gfral knockout mice treated with cisplatin (4 mg kg−1) once per week for six weeks. n = 6 animals. P values were calculated using two-tailed Mann–Witney test (95% confidence interval). All data are mean ± s.e.m. Source data

Extended Data Table 1 Data collection, phasing and refinement statistics

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Hsu, JY., Crawley, S., Chen, M. et al. Non-homeostatic body weight regulation through a brainstem-restricted receptor for GDF15. Nature 550, 255–259 (2017). https://doi.org/10.1038/nature24042

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