MC4R-dependent suppression of appetite by bone-derived lipocalin 2

Abstract

Bone has recently emerged as a pleiotropic endocrine organ that secretes at least two hormones, FGF23 and osteocalcin, which regulate kidney function and glucose homeostasis, respectively. These findings have raised the question of whether other bone-derived hormones exist and what their potential functions are. Here we identify, through molecular and genetic analyses in mice, lipocalin 2 (LCN2) as an osteoblast-enriched, secreted protein. Loss- and gain-of-function experiments in mice demonstrate that osteoblast-derived LCN2 maintains glucose homeostasis by inducing insulin secretion and improves glucose tolerance and insulin sensitivity. In addition, osteoblast-derived LCN2 inhibits food intake. LCN2 crosses the blood-brain barrier, binds to the melanocortin 4 receptor (MC4R) in the paraventricular and ventromedial neurons of the hypothalamus and activates an MC4R-dependent anorexigenic (appetite-suppressing) pathway. These results identify LCN2 as a bone-derived hormone with metabolic regulatory effects, which suppresses appetite in a MC4R-dependent manner, and show that the control of appetite is an endocrine function of bone.

Extended Data Figure 1. Identification of Lcn2 as an osteoblast-enriched gene and generation of mice lacking Lcn2 in osteoblasts (Lcn2_osb^−/−) and adipocytes (Lcn2_fat^−/−)

a, Real-time PCR analysis of Lcn2 expression levels in the indicated tissues from wild-type mice. b, Lcn2, Bglap and Alp expression levels in differentiating osteoblasts (day 0–day 15). c, Lack of Lcn2 expression in indicated tissues of the Lcn2–mCherry-reporter mouse. Scale bars, 40 μm. d, Targeting strategy used to generate a floxed allele of Lcn2. The targeting vector, which contains loxP sites within introns 2 and 6, is designed to delete a 1.9 kb genomic fragment containing Lcn2 exons 3–6. Location of probes used for Southern blotting (5′ and 3′) and primers used for PCR to detect the floxed (a and b) and mutant allele (c and d) are indicated. e, Southern-blot analysis on DNA from targeted embryonic stem (ES) cells and mice from F1 and F2 generation showing germline transmission of the mutated allele. f, i, Detection of Lcn2 floxed (primers a–b) and mutant allele, Δflox, (primers c–d) in genomic DNA isolated from tissues of Lcn2_osb^−/− (f) and Lcn2_fat^−/− (i) mice. Lcn2_osb^−/− mice showed 60% recombination efficiency and 55% decrease in Lcn2 expression specifically in bone, but not in other tissues. Lcn2_fat^−/− mice showed 76% recombination efficiency in fat, which led to 65% and 50% decrease in Lcn2 expression in white and brown fat, respectively. g, j, Serum levels of LCN2 are decreased by 67% in Lcn2_osb^−/− mice compared to their Lcn2^fl/fl littermates (g), whereas levels are decreased by 27% in Lcn2_fat^−/− mice compared to their Lcn2^fl/fl littermates (j). h, k, Tissue expression of Lcn2 in Lcn2_osb^−/− and their Lcn2^fl/fl littermates (h) and Lcn2_fat^−/− and their Lcn2^fl/fl littermates (k). Data are mean ±s.e.m.; n = 12 (a) and n = 9 (g, h, j, k) mice per group. Results are representative of three independent experiments. *P < 0.05 (Student’s t-test).

Extended Data Figure 2. Osteoblast-specific activation of Cre in Col1a1-Cre and Bglap-Cre mice

a, b, Tandem mass spectrum of an eluted peptide fragment of bone-derived (a) and fat-derived (b) LCN2. c, Specific expression of Cre in osteoblasts, but not any other tissues in mT/mG::Col1a1-Cre mice and in mT/mG::Bglap-Cre mice. Scale bars, 100 μm. Results are representative of three independent experiments.

Extended Data Figure 3. LCN2 regulates glucose homeostasis through its expression in osteoblasts

a, b, Glucose infusion rate (a) and serum insulin levels (b) during hyperglycaemic clamp. c, Insulin staining and islet histomorphometry of pancreatic sections. d, Ki67 immunostaining and histomorphometric analysis of Ki67-immunoreactive cells in pancreatic islets (arrows indicate Ki67-positive cells). e, Histomorphometric analysis of TUNEL-positive β-cells in the pancreas. f, Expression of insulin genes, cell cycle genes and β-cell dedifferentiation markers in pancreatic islets. g, Insulin and glucagon immunostaining and histomorphometric analysis of insulin- and glucagon-immunoreactive cells in pancreatic islets of Lcn2_osb^−/− mice and their Lcn2^fl/fl littermates. h, Insulin secretion in pancreatic islets treated with increasing doses of LCN2. i–p, Glucose tolerance (i), insulin tolerance (j) and glucose-stimulated insulin secretion (k), random-fed serum-insulin levels (l), fat pad mass (m), fat mass (n), food intake (o) and body weight (p) are not altered in Col1a1-Cre mice compared to their wild-type littermates. q, Expression levels of Cebpa, Pparg, Tgl (also known as Pnpla2), Plin1 (also known as Plin) and Lpl in white adipose tissue. r, s, Urinary levels of norepinephrine (r) and epinephrine (s) in Lcn2_osb^−/− mice and Lcn2^fl/fl littermates. t, u, Indirect calorimetry measurements (t) and daily food intake normalized to body weight (u) in Lcn2_osb^−/− mice and Lcn2^fl/fl littermates. Scale bars, 200 μm (c), 100 μm (d) and 50 μm (g). Data are mean ±s.e.m.; n = 5 (a, b, e, r–t), n = 8 (c, d, q), n = 7 (i–p), n = 3 (g) and n = 9 (u) mice per group. Results are representative of three independent experiments. *P < 0.05 (Student’s t-test).

Extended Data Figure 4. The anorexigenic function of LCN2 influences fat mass, body weight and insulin sensitivity identically in Lcn2_osb^−/− and Lcn2_(OC)osb^−/− mice

a–f, Body weight (a), fat mass (b), insulin tolerance (c), serum insulin levels (d), glucose-stimulated insulin secretion (e) and glucose tolerance (f) in pair-fed Lcn2_osb^−/− and Lcn2^fl/fl littermates. Data from Figs 2l, 2d, 1h, 2a, 1j and 1f, respectively, for fed Lcn2_osb^−/− mice are included for comparison. g, Detection of the Lcn2 floxed (primers a–b) and mutant allele, Δflox, (primers c–d) in genomic DNA isolated from tissues of Lcn2_(oc)osb^−/− mice. h, i, Serum levels of LCN2 (h) and tissue expression of Lcn2 (i) in Lcn2_(oc)osb^−/− mice and their Lcn2^fl/fl littermates. j–q, glucose tolerance (j), insulin tolerance (k), glucose-stimulated insulin secretion (l), random-fed serum-insulin levels (m), fat pad mass (n), fat mass (o), food intake (p) and body weight (q) in Lcn2_(oc)osb^−/− mice and their Lcn2^fl/fl littermates. r, Histomorphometric analysis of bone mass and Von Kossa staining of vertebral sections. BV/TV, bone volume over tissue volume; Ob.N./T.Ar, osteoblast numbers per trabecular area; OcS/BS, osteoclast surface per bone surface. s, Expression levels of osteoblastogenic and osteoclastogenic genes in bone in Lcn2_osb^−/− mice and their Lcn2^fl/fl littermates. t, Serum osteocalcin levels in Lcn2_osb^−/−mice and their Lcn2^fl/fl littermates. u, Lcn2 expression levels in indicated tissues following fasting–refeeding of wild-type mice. Data are mean ±s.e.m.; n = 6 (a–f, r), n = 3 (s), n = 10 (t) and n = 5 (u) mice per group; n = 3 wild-type, n = 7 Bglap-Cre, n = 7 Lcn2_(OC)osb^−/− and n = 6 Lcn2^fl/fl mice (h–q). Results are representative of three independent experiments. *P < 0.05 (Student’s t-test).

Extended Data Figure 5. Exogenous LCN2 decreases body-weight gain and increases energy expenditure in lean and obese mice

a–f, Wild-type mice were treated with LCN2 (150 ng g⁻¹ per day) or vehicle for 16 weeks. a, Serum levels of LCN2. b, Body-weight gain. c, Expression levels of Cebpa and Pparg in white adipose tissue. d, Serum insulin levels. e, Pancreas insulin staining and islet histomorphometry. Scale bar, 200 μm. f, Indirect calorimetry measurements. g, h, Coomassie blue staining (g) and mass spectrometry analysis (h) of recombinant mouse LCN2. Body-weight gain (i), insulin tolerance (j) and indirect calorimetry (k) measurements of Lepr^db/db mice treated with LCN2 (150 ng g ⁻¹ per day) or vehicle for 16 weeks. n = 8 mice wild-type and n = 6 Lepr^db/db mice per group. Results are representative of three independent experiments. Data are mean ±s.e.m.; *P < 0.05 (a–f; Student’s t-test). ^#P < 0.05 (i–k) for Lepr^db/db (vehicle or LCN2 treated) versus Lepr^db/+ (vehicle) groups (Student’s t-test). *P < 0.05 (i–k) Lepr^db/db (LCN2) versus Lepr^db/+ (vehicle) and Lepr^db/db (vehicle) groups (ANOVA).

Extended Data Figure 6. Increased food intake, compromised glucose metabolism and normal bone mass in Lcn2^−/− mice

a, Serum levels of leptin in Lcn2_osb^−/− mice and their Lcn2^fl/fl littermates. b, Expression levels of leptin in white adipose tissue. c–g, Expression levels of the gastrointestinal hormones that regulate appetite Cck (c), Pyy (d), Glp1 (also known as Zglp1; e), Gip (f) and ghrelin (Ghrl; g) under fed (F), 16-h fasted (FD) and re-fed (RF) (2 h after 16-h fasting) conditions for Lcn2_osb^−/− mice and their Lcn2^fl/fl littermates. h, Detection of the Lcn2 mutant allele, Δflox, (primers c–d) in genomic DNA isolated from tissues of Lcn2^−/− mice. i, j, Serum levels of LCN2 (i) and tissue expression of Lcn2 (j) in Lcn2^−/− mice and their Lcn2^+/+ littermates. k–s, Analysis of Lcn2 ^−/− mice and their Lcn2^+/+ littermates. k, Glucose tolerance test. l, Insulin tolerance test. m, Glucose-stimulated insulin secretion. n, Fat pad mass. o, Fat mass. p, Food intake. q, Body weight. r, Bone histomorphometric analysis. s, Expression levels of osteoblastogenic and osteoclastogenic genes in bone. t, u, LCN2 crosses the blood–brain barrier to regulate food intake. t, LCN2 levels in the brain and serum of Lcn2^−/− and wild-type littermates 2 h after i.p. administration of LCN2. u, LCN2 levels in the brain of wild-type mice 3 h after refeeding. v, LCN2 levels in the brain and serum of Lcn2^−/− mice following ICV administration of 0.02 ng h ⁻¹ LCN2 or vehicle for 8 days. Data are mean ±s.e.m.; n = 10 (a, b, i), n = 5 (c–g, k, l, p, r, s), n = 7 (m–o, q), n = 4 (j, t) and n = 3 (u, v) mice per group. Results are representative of three independent experiments. *P < 0.05 (Student’s t-test).

Extended Data Figure 7. MC4R, but not SLC22A17, is required for the stimulation of cAMP activity and anorexigenic gene expression induced by LCN2

a–d, Expression levels of neuropeptides regulating appetite in the hypothalamus of Lcn2^fl/fl and Lcn2_osb^−/− mice (a, c) and wild-type mice injected daily i.p. with LCN2 (150 ng g⁻¹ per day) or vehicle for 16 weeks (b, d). e, o, Mc4r (e) or Slc22a17 (o) expression levels in GT1-7 hypothalamic cells following transfection with Mc4r or Slc22a17 siRNA. si-scramble, Scramble siRNA. f, g, p, cAMP production in GT1-7 cells treated with LCN2 following pretreatment with the SHU9119 inhibitor (g) or silencing of Mc4r (f) or Slc22a17 (p) expression. h, Expression levels of melanocortin receptors and LCN2 receptors in HEK293T cells transfected with an MC4R expression vector or empty vector. ND, not detected. i, j, Western blot analysis of CREB phosphorylation (i) and FOS induction (j) in GT1-7 cells treated with LCN2, vehicle or α-MSH. k–t, Expression levels of Mc4r and targets in GT1-7 cells treated with LCN2 or α-MSH for 4h (k–n) and after silencing of Slc22a17 (q–t). u, Binding of biotinylated LCN2 and α-MSH in GT1-7 cells transiently transfected with Mc4r siRNA or scramble siRNA. v, Quantification of Fos-expressing neurons in the PVH of wild-type and Mc4r^−/− mice injected with LCN2 or MT-II. Data are mean ± s.e.m. of triplicates. n = 9 (a) and n = 7 (b–d) mice per group. Results are representative of three independent experiments. *P < 0.05 (Student’s t-test).

Extended Data Figure 8. Binding affinity and activation potency of LCN2 on MC3R and MC1R

a–f, Saturation (a, d) and competition binding (b, c, e, f) assay curves of LCN2 and α-MSH in HEK293T cells transfected with MC3R (a–c) or MC1R (d–f). Displacement of biotinylated α-MSH (b, e) or biotinylated LCN2 (c, f) by the indicated proteins. g, h, cAMP production in HEK293T cells transfected with MC3R (g) or MC1R (h) along with a cAMP/luciferase reporter and treated for 15 min as indicated. Results are representative of three independent experiments. Data are mean ±s.e.m.; i, EC₅₀, K_d and K_i of LCN2, α-MSH and AGRP on melanocortin receptors expressed in HEK293T cells.

Extended Data Figure 9. Binding and activation of PVH neurons by LCN2

a, Binding of biotinylated LCN2 to PVH and VMH neurons, but not neurons in the arcuate nucleus (ARC) in hypothalamic sections of Lcn2^−/− and Mc4r^−/− mice. Competition with unlabelled LCN2 or GST. b–d, Bright-field images of a targeted (as indicated by the arrows) neurons. b, A Sim-1 neuron from a Sim-1 cre::tdTomato mouse. c, A Pomc neuron from Pomc–hrGFP mouse. d, A Npy neuron from Npy–hrGFP mouse. The same neuron is shown in red (Alexa Fluor 594, tdTomato), green (FITC, hrGFP) and blue (Alexa Fluor 350, DAPI), as well the merged image. Scale bars, 100 μm (a) and 50 μm (b–d).

Extended Data Figure 10. MC4R is required for the improvement in glucose metabolism and energy expenditure by LCN2

a–d, Mc4r^−/− and wild-type littermates were treated with LCN2 (150 ng/g/day) or vehicle for 8 weeks. a, Indirect calorimetry measurements. b, Serum insulin levels. c, Glucose tolerance test. d, Insulin tolerance test. e–k, Serum levels of LCN2 (e), indirect calorimetry measurements (f–h), glucose tolerance test (i), insulin tolerance test (j) and glucose-stimulated insulin secretion (k) in Mc4r ^+/−, Lcn2_osb^+/−, Mc4r^+/−::Lcn2_osb^+/− and wild-type littermates. Data are mean ±s.e.m.; n = 5 wild-type and n = 7 Mc4r^−/− mice (a–d). l, LCN2 plasma levels in patients with mutated and normal MC4R sequences. n = 6 wild-type, Mc4r^+/− and Mc4r^+/−::Lcn2_osb^+/−mice and n = 5 Lcn2_osb^+/−mice (e–k). Results are representative of three independent experiments. *P < 0.05 indicated genotypes or treatments versus wild-type mice (Student’s t-test). ^#P < 0.05 when comparing either Mc4r^−/− (vehicle-treated) and Mc4r^+/− (LCN2-treated) groups (a) or Mc4r^+/−::Lcn2_osb^+/− mice versus wild-type, Mc4r^+/− and Lcn2_osb^+/− mice (f–k) (ANOVA).

Figure 1. LCN2 regulates glucose homeostasis through its expression in osteoblasts

a, mRNA expression of Lcn2 in bone. b, Serum levels of LCN2 in mice. c, d, mRNA and protein expression of LCN2 in wild-type (WT) tissues. BAT, brown adipose tissue; subc. fat, subcutaneous fat; WAT, white adipose tissue. c, Northern blot analysis of Lcn2 levels. Actb was used as a loading control. d, Western blot analysis of LCN2 levels. GAPDH was used as a loading control. e, LCN2 in bone and fat tissues of Lcn2–mCherry mice. Scale bar, 40 μm. f, g, Glucose-tolerance test of osteoblast-specific (f) or adipocyte-specific (g) Lcn2-knockout mice compared to wild-type (Lcn^fl/fl) littermates. h, i, Insulin-tolerance test of osteoblast-specific (h) or adipocyte-specific (i) Lcn2-knockout mice compared to wild-type (Lcn^fl/fl) littermates. j, k, Glucose-stimulated insulin secretion in osteoblast-specific (j) or adipocyte-specific (k) Lcn2-knockout mice or wild-type littermates. Data are mean ±s.e.m.; n = 10 (a, b), n = 12 (f), n = 7 (g, k), n = 6 (h) and n = 8 (i, j) mice per group; representative of three independent experiments. In f, h, j, the effect size (d) > 0.84; *P < 0.05 (Student’s t-test).

Figure 2. Inactivation of Lcn2 in osteoblasts increases food intake

a, b, Serum insulin levels of osteoblast-specific (a) or adipocyte-specific (b) Lcn2-knockout mice compared to wild-type (Lcn^fl/fl) littermates. c–l, Analysis of fat pad mass (c, f), body fat mass (d, g), body weight (BW; e, h, l) and cumulative food intake (i, j, k) of Lcn2_osb^−/− (c–e, i, k, l) and Lcn2_fat^−/− (f–h, j) mice. m, n Body weight (m) and HbA_1c (n) inversely correlate with serum LCN2 levels in patients with type 2 diabetes. o, p, LCN2 serum levels (o) and food intake (p) in wild-type mice that were fast (FD) or fasted and refed (RF). q, Food intake in Lcn2_osb^−/− mice that were fasted and refed after intraperitoneal LCN2 administration. Data are mean ±s.e.m.; n = 11 (a, c, o), n = 10 (b, d), n = 13 (e), n = 8 (f, h), n = 7 (g, j), n = 9 (i, l, p), n = 5 (k) and n = 4 (q) mice per group; representative of three independent experiments; d > 0.96 (a, c, d, i, k) and d > 0.74 (e, l). a, c, d, e, i, k, l, o, *P < 0.05 (Student’s t-test). q, *P < 0.05, vehicle-treated Lcn2_osb^−/− versus vehicle-treated wild-type mice and LCN2-treated Lcn2_osb^−/− mice (ANOVA); and #P < 0.05, LCN2-treated wild-type mice versus vehicle-treated wild-type mice (Student’s t-test).

Figure 3. LCN2 suppresses food intake in lean and obese mice

a, Daily food intake of wild-type mice treated with vehicle or LCN2. b, c, h, i, Analysis of fat mass (b, h) and body weight (c, i) of wild-type or Lepr^db/db mice treated with vehicle or LCN2. g, Cumulative (for the two phases, dark and light) daily food intake of wild-type or Lepr^db/db mice treated with vehicle or LCN2 during different light phases of the day. d–f, j, Glucose-tolerance test (d, j), glucose-stimulated insulin secretion (e), insulin-tolerance test (f) of wild-type or Lepr^db/db mice treated with LCN2. Data are mean ±s.e.m.; n = 8 wild-type and n = 6 Lepr^db/db mice; representative of three independent experiments. a–g, *P < 0.05 (Student’s t-test). h–j, #P < 0.05, Lepr^db/db versus Lepr^db/+ mice treated with vehicle (Student’s t-test). *P < 0.05, Lepr^db/db vehicle versus Lepr^db/+ (vehicle) and Lepr^db/db (LCN2) groups (ANOVA).

Figure 4. LCN2 signals directly in the brain to suppress food intake

a–f, i, Change in food intake (a, c, e, i) and body weight (b, d, f) in Lcn2^−/− or wild-type mice during ICV administration of LCN2 (a–d, i), leptin or MT-II (e, f). AUC, area under the curve. g, Western blot analysis of phosphorylated (p-) or total (t-) AMPK, ERK1/ERK2 and tyrosine kinase (tyr). β-actin was used as a loading control. Veh, vehicle; FBS, fetal bovine serum. h, cAMP production in GT1-7 cells after treatment with LCN2 or α-MSH. In i, data from c for vehicle and low-LCN2 are included. Data are mean ±s.e.m.; n = 6 (a, b), n = 10 (c, d, i) and n = 5 (e, f) mice per group; n = 3 (h); representative of three independent experiments. *P < 0.05 (Student’s t-test).

Figure 5. LCN2 binds to and signals through MC4R in the hypothalamus

a, cAMP production of HEK293T cells overexpressing MC4R after treatment with LCN2, leptin or α-MSH b, Binding of biotinylated LCN2 or GST (control) to hypothalamic sections from Lcn2^−/− or Mc4r ^−/− mice in the presence or absence of 100-fold excess of non-biotinylated LCN2 or GST. PVH, paraventricular nucleus of the hypothalamus; 3V, third ventricle. c–e, Saturation (c) and competitive binding (d, e) assay curves of LCN2 and α-MSH in Mc4r-transfected HEK293T cells. Leptin and AGRP were used as a negative and additional positive control, respectively. f, Fos expression in the PVH of wild-type and Mc4r ^−/− mice treated with LCN2 or MT-II. g–j, Representative traces (g–i) and change in resting membrane potential (j) of neurons after LCN2 treatment. LCN2 treatment induced depolarization in a Sim1-cre::tdTomato (g) but not Npy–hrGFP (humanized renilla green fluorescent protein) (h) or Pomc–hrGFP (i) neuron. Scale bars, 100 μm. Data are mean ±s.e.m.; representative of three independent experiments. *P < 0.05 (Student’s t-test).

Figure 6. MC4R mediates suppression of appetite by osteoblast-derived LCN2

a–f, Analysis of food intake (a, d), body weight (b, e), fat pad mass (c, g) and body fat mass (f) in Mc4r^−/− mice treated with LCN2 for 8 weeks(a–c) or in Mc4r^+/−, Lcn2_osb^+/−, Mc4r^+/−::Lcn2_osb^+/− and wild-type littermates (d–g). h, i, LCN2 plasma levels in patients with mutated and normal MC4R at different ages (h) and body-mass indexes (i). Data are mean ±s.e.m.; n = 5wild-type and n = 8 Mc4r ^−/− mice (a–c), n = 7 wild-type, Mc4r^+/− and Mc4r ^+/−::Lcn2_osb^+/− mice and n = 5 Lcn2_osb^+/− mice (d–g); representative of three independent experiments. a–c, *P < 0.05, vehicle-treated wild-type mice compared to the other groups; d–g, *P < 0.05, Mc4r^+/−::Lcn2_osb^+/− mice versus wild-type, Mc4r^+/− and Lcn2_osb^+/− mice (ANOVA); ^#P < 0.05, Mc4r^+/− versus wild-type mice (Student’s t-test).