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The rs1421085 variant within FTO promotes brown fat thermogenesis

Nature Metabolism volume 5, pages 1337–1351 (2023)Cite this article

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Abstract

One lead genetic risk signal of obesity—the rs1421085 T>C variant within the FTO gene—is reported to be functional in vitro but lacks evidence at an organism level. Here we recapitulate the homologous human variant in mice with global and brown adipocyte-specific variant knock-in and reveal that mice carrying the C-allele show increased brown fat thermogenic capacity and resistance to high-fat diet-induced adiposity, whereas the obesity-related phenotypic changes are blunted at thermoneutrality. Both in vivo and in vitro data reveal that the C-allele in brown adipocytes enhances the transcription of the Fto gene, which is associated with stronger chromatin looping linking the enhancer region and Fto promoter. Moreover, FTO knockdown or inhibition effectively eliminates the increased thermogenic ability of brown adipocytes carrying the C-allele. Taken together, these findings identify rs1421085 T>C as a functional variant promoting brown fat thermogenesis.

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Fig. 1: The homologous rs1421085 T>C variant promotes energy expenditure and resists HFD-induced adiposity.
Fig. 2: The homologous rs1421085 T>C variant augments the thermogenic capacity of brown adipocytes.
Fig. 3: Brown adipocyte-specific knock-in of the homologous rs1421085 T>C variant enhances thermogenesis and resists HFD-induced adiposity.
Fig. 4: The homologous rs1421085 T>C variant increases thermogenesis via upregulating Fto expression.
Fig. 5: Thermoneutrality blunts the effects of the homologous rs1421085 T>C variant on energy expenditure and HFD-induced adiposity.

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Data availability

All data generated or analyzed during this study are included in this article and its supplementary information files. Abbreviations and corresponding full names used in this study are listed in Supplementary Table 4. Source data are provided with this paper.

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Acknowledgements

We thank all the participants for their involvement in this study and we thank J. Shen and Q. Liao from BGI-Shenzhen for their comments on the adaptive evolution of the FTO variant. This work was supported by grants from the National Key Research and Development Program of China (2022YFC2505201 to J.W. and 2021YFA1301103 to G.N.), the National Natural Science Foundation of China (92157204 to R.L., 91957124 to J.W., 82088102 to G.N., 82250901 to R.L. and 81930021 to G.N.), Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (20161306 and 20171903 Round 2 to R.L.), innovative research teams of high-level local universities in Shanghai (J.W. and G.N.), the Outstanding Academic Leader Program of Shanghai Municipal Health Commission (2018BR01 to J.W.), Science and Technology Commission of Shanghai Municipality (21JC1404400 to G.N.) and the Program of Shanghai Academic/Technology Research Leader (20XD1403200 to J.W. and 23XD1422400 to R.L.).

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Author notes

  1. These authors contributed equally: Zhiyin Zhang, Na Chen, Nan Yin, Ruixin Liu, Yang He.

Authors and Affiliations

  1. Department of Endocrine and Metabolic Diseases, Shanghai Institute of Endocrine and Metabolic Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Zhiyin Zhang, Na Chen, Nan Yin, Ruixin Liu, Yang He, Danjie Li, Muye Tong, Aibo Gao, Peng Lu, Yuxiao Zhao, Weiqiong Gu, Jie Hong, Weiqing Wang, Guang Ning & Jiqiu Wang

  2. Shanghai National Clinical Research Center for Metabolic Diseases, Key Laboratory for Endocrine and Metabolic Diseases of the National Health Commission of the PR China, Shanghai National Center for Translational Medicine, Shanghai, China

    Zhiyin Zhang, Na Chen, Nan Yin, Ruixin Liu, Yang He, Danjie Li, Muye Tong, Aibo Gao, Peng Lu, Yuxiao Zhao, Weiqiong Gu, Jie Hong, Weiqing Wang, Guang Ning & Jiqiu Wang

  3. Shanghai Institute of Immunology, State Key Laboratory of Oncogenes and Related Genes, Shanghai Jiao Tong University School of Medicine, Shanghai, China

    Huabing Li

  4. Laboratory of Aquacultural Resources and Utilization, Ministry of Education, College of Fishery and Life Science, Shanghai Ocean University, Shanghai, China

    Junfang Zhang

  5. Shengjing Hospital of China Medical University, Shenyang, China

    Dan Zhang

  6. Department of Epidemiology, School of Public Health and Tropical Medicine, Tulane University, New Orleans, LA, USA

    Lu Qi

  7. Department of Nutrition, Harvard TH Chan School of Public Health, Boston, MA, USA

    Lu Qi

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  1. Zhiyin Zhang
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Contributions

J.W. designed the experiments and supervised the study. Z.Z., N.C., N.Y. and Y.H. carried out animal and molecular experiments. J.W. and R.L. analyzed all the experimental data. D.L., M.T., A.G., P.L., Y.Z. and H.L. provided support in animal model tests. J.Z. provided support in 3C tests. D.Z. collected fetal BAT samples. W.G., J.H. and W.W. provided support for the genetic analysis. G.N. and L.Q. contributed to text revision and discussion. J.W., R.L., Z.Z., N.C. and N.Y. wrote the paper.

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Correspondence to Jiqiu Wang.

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Nature Metabolism thanks George Stratigopoulos and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Isabella Samuelson, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 The sequencing of the homologous rs1421085 T>C variant in three knock-in mice.

(A to B) Sanger sequence analysis of KIcas9 (A) and KIES (B) mice, respectively. (C) Loxp (left, top), Cre expression (left, bottom), BAT DNA sequence analysis of KIfl/fl and Ucp1-KIfl/fl mice (right). The data is representative of least two independent experiments.

Extended Data Fig. 2 The homologous rs1421085 T>C variant protests HFD-induced obesity but shows no change in body weight under chow diet in KIcas9 mice.

(A) Body weight curve of male KIcas9 mice and wild-type (WT) littermates under normal chow diet (NCD). n = 10 (WT) and 15 (KIcas9) mice per group. (B to K) IPGTT (B) and ITT (C), TG (D), TC (E), HDL-c (F), and LDL-c (G), H&E staining of iWAT, eWAT, and liver (H), the ratio of fat mass and lean mass to body weight (I), the tissue mass weight (BAT, iWAT, and eWAT) (J), and their ratio to body weight (K) in male KIcas9 mice and WT littermates under HFD. For B and C, n = 17 per group. For D to G, n = 10 mice per group. For H, images are representative of 4 mice per group; iWAT: n = 180 (WT) and 315 (KIcas9) LDs; eWAT: n = 201 (WT) and 223 (KIcas9) LDs; liver: n = 24 (WT) and 20 (KIcas9) regions; scale bar = 200 μm. For I, n = 11 (WT) and 16 (KIcas9) mice per group. For J, BAT: n = 11 (WT) and 15 (KIcas9); iWAT: n = 11 (WT) and 15 (KIcas9); eWAT: n = 10 (WT) and 14 (KIcas9). For K, BAT: n = 11 (WT) and 15 (KIcas9); iWAT: n = 11 (WT) and 15 (KIcas9); eWAT: n = 8 (WT) and 14 (KIcas9). (L) The validation of diet-induced adiposity phenotype of male KIcas9 mice (F3 generation) under HFD challenge initiated from 11 to 27 weeks old. For HFD week 0 to 13, n = 18 (WT) and 19 (KIcas9) mice per group, for HFD week 14 to 16, n = 17 (WT, one died before HFD week 14) and 19 (KIcas9) mice per group. P= 0.0224, 0.0275, 0.0307, 0.0217, 0.0125, 0.0247, 0.008, 0.0244, 0.0266 between groups, at each week of HFD week 8 to 16 (* P < 0.05). (M) Body weight curve of female KIcas9 and WT littermate mice under NCD. n = 21 (WT) and 20 (KIcas9) mice per group. (N to R) Body weight curve (N), body mass (O), the ratio of lean mass to body weight (P), the tissue weights (BAT, iWAT, and eWAT) (Q), and their ratio to body weight (R) in female KIcas9 mice and WT littermates under HFD. For N, n = 9 (WT) and 8 (KIcas9) mice per group, P = 0.0326, 0.0271, 0.0435, 0.0269, 0.0302, 0.0496, 0.0413, 0.0451, 0.0375, 0.0299 at week 1, 2, 3, 4, 6, 7, 8, 10, 11, 12 of HFD between groups (* P < 0.05). For O to P, n = 8 mice per group. For Q to R, n = 9 (WT) and 8 (KIcas9) mice per group. Statistical significance was assessed by unpaired two-sided Student’s t-test (A to R). Data are mean ± s.e.m. of biologically independent samples. Data are representative of at least two independent experiments (B to G).

Source data

Extended Data Fig. 3 The homologous rs1421085 T>C variant protests HFD-induced obesity but shows no change in body weight under chow diet in KIES mice.

(A) Body weight of male KIES mice and WT littermates aged 8- and 23-week-old under NCD. n = 8 mice per group. (B) Body weight curve of female KIES mice and WT littermates under NCD. n = 8 mice per group. (C) The ratio of fat mass and lean mass to body weight in male KIES and WT mice of HFD. n = 12 (WT) and 13 (KIES) mice per group. (D to F) The tissue weights (BAT, iWAT, and eWAT) (D) and their ratio to body weight (E), H&E staining (iWAT, eWAT, and liver) (F) in male KIES mice and WT littermates under HFD. For D and E, n = 12 (WT) and 13 (KIES) mice per group. For F, images are representative of 4 mice per group; iWAT: n = 1082 (WT) and 1019 (KIES) LDs; eWAT: n = 235 (WT) and 310 (KIES) LDs; liver: n = 16 regions per group; scale bar = 200 μm. (G to K) Body weight curve (G), the fat mass and lean mass (H) and their ratio to body weight (I), the tissue weights (BAT, iWAT, eWAT, and liver) (J) and their ratio to body weight (K) of female KIES and WT littermate mice fed HFD from 10- to 24-week-old. For G, n = 13 (WT) and 10 (KIES) mice per group, P = 0.0005, 0.0116, 0.0433, 0.0159, 0.0220, 0.0197, 0.0296, 0.0302, 0.0074, 0.0361, 0.0339, 0.0191, 0.0165, 0.0303, 0.0335 between groups, at each week of HFD week 0 to 14 (* P < 0.05). For H, J and K, n = 13 (WT) and 10 (KIES) mice per group. For I, fat mass: n = 12 (WT) and 10 (KIES); lean mass: n = 13 (WT) and 10 (KIES) mice per group. (L to M) Respiratory exchange ratio (RER) (L) and total physical activities (M) of male KIES and WT mice. Data were collected after the first week of HFD feeding, n = 8 mice per group. (N to O) H&E staining (BAT, iWAT, eWAT, and liver) (N) and transmission electron microscopy (TEM) morphology (BAT) (O) of female KIES and WT mice fed HFD from 10- to 24-week-old. For N, images are representative of 4 mice per group; BAT: n = 353 (WT) and 287 (KIES) LDs; iWAT: n = 199 (WT) and 199 (KIES) LDs; eWAT: n = 247 (WT) and 274 (KIES) LDs; liver: n = 4 regions per group; scale bar = 200 μm. For O, n = 7 (WT) and 6 (KIES); scale bar = 1 μm. Statistical significance was assessed by unpaired two-sided Student’s t-test (A to O). Data are mean ± s.e.m. of biologically independent samples, representative of at least two independent experiments.

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Extended Data Fig. 4 The brown adipocyte-specific rs1421085 T>C knock-in (Ucp1-KIfl/fl) mice resist HFD-induced adiposity.

(A to E) The mRNA expression of Fto (A), Irx3 (B), Irx5 (C), Irx6 (D), and Rpgrip1l (E) in the tissues related to energy homeostasis in 4–5 weeks old male homologous rs1421085fl/fl (KIfl/fl) and WT mice. For A, BAT: n = 11 (WT) and 9 (KIES); iWAT, eWAT, liver, muscle and hypothalamus: n = 11 (WT) and 10 (KIES). For B, BAT and iWAT: n = 11 (WT) and 10 (KIES); eWAT, liver, muscle and hypothalamus: n = 11 per group. For C, BAT: n = 10 (WT) and 11 (KIES); iWAT: n = 10 (WT) and 6 (KIES); eWAT: n = 11 per group; liver: n = 10 (WT) and 11 (KIES); muscle: n = 11 (WT) and 10 (KIES); hypothalamus: n = 11 per group. For D, BAT: n = 11 (WT) and 10 (KIES); iWAT, eWAT, liver, muscle and hypothalamus: n = 11 per group. For E, BAT: n = 11 (WT) and 10 (KIES); iWAT, eWAT, liver, muscle, hypothalamus: n = 11 per group. (F to I) Body weight curve (F), the ratio of fat mass and lean mass to body weight (G), the tissue mass (BAT, iWAT, and eWAT) and their ratio to body weight (H and I) in male Ucp1-KIfl/fl and KIfl/fl mice under NCD. For F, n = 10 mice per group. For G and I, n = 12 (KIfl/fl) and 7 (Ucp1-KIfl/fl) mice per group. For H, n = 11 (KIfl/fl) and 7 (Ucp1-KIfl/fl) mice per group. (J) The representative H&E images of iWAT, eWAT, and liver in male Ucp1-KIfl/fl and KIfl/fl mice under HFD; images are representative of 4 mice per group; iWAT: n = 194 (KIfl/fl) and 221 (Ucp1-KIfl/fl) LDs; eWAT: n= 274 (KIfl/fl) and 187 (Ucp1-KIfl/fl) LDs; liver: n = 16 regions per group; scale bar = 200 μm. (K to M) Cumulative food intake (K), RER (L), and total physical activities (M) of single-housed 10-week-old male KIES and WT mice. For K, n = 9 mice per group. For L and M, n = 10 mice per group. Data were collected after the first week of HFD feeding. Statistical significance was assessed by unpaired two-sided Student’s t-test (A to J and L to M) and two-way ANOVA followed by Bonferroni post tests (K). Data are mean ± s.e.m. of biologically independent samples, representative of at least two independent experiments. Exclusion criteria for outliers please see Methods.

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Extended Data Fig. 5 The mRNA expression of candidate target genes of the rs1421085 T>C variant in the knock-in mouse models.

(A to D) The mRNA expression of Irx3, Irx5, Irx6, and Rpgrip1l in male BAT tissues of three knock-in mouse models and induced mature brown adipocytes. For A, n = 6 (WT) and 7 (KIcas9) mice per group. For B, n = 12 (WT) and 13 (KIES) for Irx3; n = 7 (WT) and 13 (KIES) for Irx5, Irx6 and Rpgrip1l. For C, n = 15 (KIfl/fl) and 7 (Ucp1-KIfl/fl) for Irx3, Irx6 and Rpgrip1l; n = 15 (KIfl/fl) and 6 (Ucp1-KIfl/fl) for Irx5. For D, n = 4 (WT) and 3 (KIES) per group. Statistical significance was assessed by unpaired two-sided Student’s t-test (A to D). Data are mean ± s.e.m. of biologically independent samples, representative of at least two independent experiments.

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Extended Data Fig. 6 The expression and demethylase activity of FTO affects brown adipocyte thermogenesis.

(A) The mRNA expression of Fto and thermogenesis-related genes at different time points during brown adipocytes differentiation derived from male WT BAT SVFs, n = 6 (Day 2 to Day 6) or 5 (Day 8). (B) Correlation analysis of the mRNA levels of Fto and Ucp1, n = 24. (C and D) Oil Red O staining (C) and OCR measurement (D) of induced brown adipocytes derived from male WT BAT SVFs after Fto knockdown. For C, images are representative of 3 independent biological samples, scale bar = 100 μm; for D, n = 5 (shNS, shFTO-1 and shFTO-2) or 4 (shFTO-3) per group. shNS: control virus. (E) Oil Red O staining of induced brown adipocytes derived from BAT SVFs with Fto overexpression, images are representative of 3 independent biological samples, scale bar = 100 μm. EV: control virus. (F) The m6A abundance in induced mature brown adipocytes in the presence of Entacapone (ENT, 50 μM) or DMSO, n = 3 per group. (G) The mRNA expression of thermogenesis-related genes in induced mature brown adipocytes in the presence of ENT (50 μM) or DMSO, for DMSO, n = 3 (Fto) or 4 (Ucp1, Pgc-1α, Prdm16, Dio2, Cidea, Cox7a, Cox8b) per group; for ENT, n = 4 per group. (H) The protein levels of FTO and UCP1 in induced mature brown adipocytes in the presence of ENT (50 μM) or DMSO. n = 3 per group. For C to E, the SVFs were infected with lentivirus two days before induction (MOI = 50). Statistical significance was assessed by unpaired two-sided Student’s t-test (D, F to H). Data are mean ± s.e.m. of biologically independent samples, representative of at least two independent experiments.

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Extended Data Fig. 7 The Fto deficiency in BAT leads to HFD-induced adiposity.

(A) The mRNA expression of Fto in indicated adipose tissues in 4-week-old female Ftofl/fl; Myf5-cre (MFKO) mice and Ftofl/fl littermates. n = 3 per group for BAT; n = 3 (Ftofl/fl) and 4 (MFKO) for iWAT; n = 4 per group for eWAT. (B to E) Body weight and body weight gain curve (B), body mass (C) and their ratio to body weight (D), and average food intake (E) of male MFKO mice and littermate controls under HFD challenge initiating from 9 to 19 weeks old. For B, n = 8 (Ftofl/fl) and 12 (MFKO) mice per group. For C and D, n = 8 mice per group. For E, n = 4 (Ftofl/fl) and 3 (MFKO) mice per group. (F to I) Representative 24 h O2 consumption (F) and CO2 production (G), average RER (H), EE with body weight as a covariant (I) of single-housed 21-week-old male MFKO and Ftofl/fl mice under 12 weeks of HFD, n = 8 per group. (J and K) Representative images of H&E staining (J) and TEM images (K) in BAT of male MFKO mice and Ftofl/fl littermates. Images are representative of 4 mice per group, scale bar = 200 μm (J) and 1 μm (K). (L) The mRNA expression of myogenesis-related genes in quadriceps femoris of male MFKO mice and littermate controls under NCD. n = 10 (Ftofl/fl) and 7 (MFKO) for MyoD; n = 9 (Ftofl/fl) and 7 (MFKO) for MyoG . (M) The protein expression of FTO, UCP1, and PGC-1α in induced mature brown adipocytes derived from BAT SVFs of male MFKO and littermate control mice, n = 4 per group. Statistical significance was assessed by unpaired two-sided Student’s t-test (A, C to E, H, L and M), and two-way ANOVA followed by Bonferroni post tests (B, F and G) and two-sided ANCOVA (I). The data were presented as means ± s.e.m. of biologically independent samples, representative of at least two independent experiments.

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Extended Data Fig. 8 FTO protein stabilizes Ucp1 mRNA and increases UCP1 protein expression.

(A to D) RNA Immunoprecipitation (RIP)-qPCR analysis of the interaction of FTO protein and target mRNA in induced mature brown adipocytes. Representative immunoblots showing the products of IP by FTO antibody, with IgG as a negative control (A), agarose gel electrophoresis (AGE) (B), and qPCR analysis of Ucp1 (C) and C/ebpα (D) mRNA abundance of FTO-IP products, n = 4 per group. The enrichment of Ucp1 and C/ebpα mRNA was normalized to 7.7% input. (E and F) Half-life analysis of Ucp1 (E) and C/ebpα (F) mRNA in induced mature brown adipocytes with Fto knockdown. The male WT BAT SVFs were infected with lentivirus two days before induction (MOI = 75). (G and H) Half-life analysis of Ucp1 (G) and C/ebpα (H) mRNA in induced mature brown adipocytes derived from BAT SVFs of male KIES and WT mice. Actinomycin D (5 μg/ml) was added on the eighth day of induction; total RNA was isolated at indicated time points; calculated half-times t1/2 = In2/Kdecay. For E, n = 4 (0 h, 0.5 h, 2 h) or 3 (1 h) for shNC; n = 3 (0 h, 0.5 h, 2 h) or 4 (1 h) for shFTO. For F to H, n = 4 per group. (I) The protein expression and quantification analysis of FTO and UCP1 in induced mature brown adipocytes derived from BAT SVFs of male KIES and WT littermate mice in the presence of ENT (50 μM) or DMSO, n = 3 per group. Statistical significance was assessed by unpaired two-sided Student’s t-test. Data are mean ± s.e.m. of biologically independent samples, representative of at least two independent experiments.

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Extended Data Fig. 9 Thermoneutrality (30 °C) blunts the effects of the brown adipocyte-specific knock-in of the rs1421085 T>C variant on energy expenditure and HFD-induced adiposity under sub-thermoneutrality (22 °C).

(A to C) Average RER (A), total physical activities (B), and cumulative food intake (C) measured in single-housed 22-week-old male KIES and WT mice after 7-week room temperature (22 °C) or thermoneutrality (30 °C) housing under HFD. For A and B, n = 8 per group at 22 °C; n = 7 (WT) and 8 (KIES) at 30 °C. For C, n = 7 per group at 22 °C; n = 6 (WT) and 7 (KIES) at 30 °C. (D to F) Body weight gain curve (D), tissue weights (E), and the representative images of H&E staining (F) in male Ucp1-KIfl/fl mice and KIfl/fl littermates under HFD at 22 °C and 30 °C. For D, n = 4 (22 °C) and 7 (30 °C) mice per group, P = 0.0469, 0.0485 between groups at week 3 to 4 at 22 °C, respectively (* P < 0.05). For E, n = 4 (22 °C) and 6 (30 °C) mice per group. For F, images are representative of 4 mice per group; BAT: n = 375 (KIfl/fl) and 298 (Ucp1-KIfl/fl) LDs at 22 °C, n = 476 (KIfl/fl) and 452 (Ucp1-KIfl/fl) LDs at 30 °C; iWAT: n = 214 (KIfl/fl) and 477 (Ucp1-KIfl/fl) LDs at 22 °C, n = 495 (KIfl/fl) and 461 (Ucp1-KIfl/fl) LDs at 30 °C; eWAT: n = 123 (KIfl/fl) and 253 (Ucp1-KIfl/fl) LDs at 22 °C, n = 239 (KIfl/fl) and 227 (Ucp1-KIfl/fl) LDs at 30°C; liver: n = 8 regions per group; scale bar = 200 μm. Statistical significance was assessed by unpaired two-sided Student’s t-test (A, B, D to F) and two-way ANOVA followed by Bonferroni post tests (C). Data are mean ± s.e.m. of biologically independent samples, representative of at least two independent experiments.

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Extended Data Fig. 10 FTO and UCP1 proteins are co-localized in human fetal brown adipocytes.

Representative immunofluorescence (IF) images of FTO (green) and UCP1 (red) in human fetal brown adipocytes, in which the nuclei are stained with DAPI (blue) and LDs are marked with PLIN1 (pink). Left to right panels: FTO, UCP1, PLIN1, PLIN1/FTO/UCP1/DAPI merged (scale bar = 100 μm), and PLIN1/FTO/UCP1/DAPI merged (scale bar = 20 μm).

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Zhang, Z., Chen, N., Yin, N. et al. The rs1421085 variant within FTO promotes brown fat thermogenesis. Nat Metab 5, 1337–1351 (2023). https://doi.org/10.1038/s42255-023-00847-2

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