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TFEB–GDF15 axis protects against obesity and insulin resistance as a lysosomal stress response

Nature Metabolism volume 3, pages 410–427 (2021)Cite this article

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Abstract

TFEB, a key regulator of lysosomal biogenesis and autophagy, is induced not only by nutritional deficiency but also by organelle stress. Here, we find that Tfeb and its downstream genes are upregulated together with lipofuscin accumulation in adipose tissue macrophages (ATMs) of obese mice or humans, suggestive of obesity-associated lysosomal dysfunction/stress in ATMs. Macrophage-specific TFEB-overexpressing mice display complete abrogation of diet-induced obesity, adipose tissue inflammation and insulin resistance, which is independent of autophagy, but dependent on TFEB-induced GDF15 expression. Palmitic acid induces Gdf15 expression through lysosomal Ca2+-mediated TFEB nuclear translocation in response to lysosomal stress. In contrast, mice fed a high-fat diet with macrophage-specific Tfeb deletion show aggravated adipose tissue inflammation and insulin resistance, accompanied by reduced GDF15 level. Finally, we observe activation of TFEB–GDF15 in ATMs of obese humans as a consequence of lysosomal stress. These findings highlight the importance of the TFEB–GDF15 axis as a lysosomal stress response in obesity or metabolic syndrome and as a promising therapeutic target for treatment of these conditions.

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Fig. 1: Increased TFEB expression in adipose tissue macrophages from obese mice and humans.
Fig. 2: Protection against diet-induced obesity and insulin resistance in mice with macrophage TFEB overexpression.
Fig. 3: Reduced adipose tissue inflammation and enhanced lipid catabolism/thermogenesis in TFEBMac-Tg mice.
Fig. 4: Autophagy-independent metabolic improvement in TFEBMac-Tg mice.
Fig. 5: Identification of GDF15 as a TFEB target, and activation of the TFEB–GDF15 axis in ATMs of humans.
Fig. 6: TFEB-dependent GDF15 expression downstream of lysosomal stress.
Fig. 7: GDF15-dependent improvement of metabolic profile in TFEBMac-Tg mice.
Fig. 8: Aggravated adipose inflammation and insulin resistance in TfebΔMac obese mice.

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Data availability

The data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank S. J. Baek (Seoul National University) for human GDF15-promoter luciferase vectors, H. Xu (University of Michigan) for GCaMP3-ML1 plasmid and R. J. Youle (National Institute of Neurological Disorders and Stroke) for TFEB-KO and TFEB/TFE3/MITF triple-KO HeLa cell lines. This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (2013R1A6A3A04065825 to K.H.K.), a National Research Foundation of Korea grant funded by the Korean government (2019R1A2C3002924 to M.-S.L.), the Bio & Medical Technology Development Program (2017M3A9G7073521 to M.-S.L.), a grant from the Faculty Research Assistance Program of Yonsei University College of Medicine (6-2016-0055 to M.-S.L.), the Korea Mouse Phenotyping Project of the Ministry of Science, ICT and Future Planning through the National Research Foundation (NRF-2014M3A9D5A01073886 to C.S.C.) and the Basic Science Research Program, through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (2017R1A2B4008673 to Y.J.J.).

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Author notes

  1. These authors contributed equally: Jinyoung Kim, Seong Hun Kim.

Authors and Affiliations

  1. Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea

    Jinyoung Kim, Seong Hun Kim, Hyereen Kang, Soyeon Lee, Yu-Mi Lim, Kook Hwan Kim & Myung-Shik Lee

  2. Discovery 1 team, GI Innovation, Seoul, Korea

    Seong Hun Kim & Kook Hwan Kim

  3. Korea Mouse Metabolic Phenotyping Center, Lee Gil Ya Cancer and Diabetes Institute, Gachon University College of Medicine, Incheon, Korea

    Shi-Young Park, Yoonil Cho & Cheol Soo Choi

  4. Brain Korea 21 Project for Medical Science, Department of Physiology, Yonsei University College of Medicine, Seoul, Korea

    Ji Woong Ahn, Young-Hwan Kim & Seungsoo Chung

  5. Endocrinology, Internal Medicine, Gachon University Gil Medical Center, Incheon, Korea

    Cheol Soo Choi

  6. Department of Physiology, University of Ulsan College of Medicine, Seoul, Korea

    Yeon Jin Jang

  7. Department of Family Medicine, University of Ulsan College of Medicine, Seoul, Korea

    Hye Soon Park

  8. Department of General Surgery, Inha University, College of Medicine, Incheon, Korea

    Yoonseok Heo

  9. Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea

    Myung-Shik Lee

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Contributions

K.H.K. and M.-S.L. conceived the experiments. K.H.K., J.K. and S.H.K. designed and conducted all experiments except the portions indicated below. K.H.K., J.K., S.H.K. and M.-S.L. analysed/interpreted the data. S.-Y.P., Y.C. and C.S.C. measured and analysed body composition and indirect calorimetry. H.K. measured intracellular/lysosomal Ca2+ and mitochondrial ROS contents. J.W.A., Y.-H.K. and S.C. conducted electrophysiology experiments. S.L. conducted experiments with BMDMs. Y.-M.L. measured the lysosomal pH. Y.J.J., H.S.P. and Y.H. provided human adipose tissues and analysed the data. K.H.K., J.K. and M.-S.L. wrote the manuscript.

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Correspondence to Kook Hwan Kim or Myung-Shik Lee.

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Peer review information Nature Metabolism thanks Carmine Settembre and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Elena Bellafante; George Caputa.

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Extended data

Extended Data Fig. 1 Tfeb and Gdf15 expression in immune cells and adipose tissue.

a, A heatmap showing the relative expression of Tfeb and Gdf15 mRNA in various immune cell types of C57BL/6N mice. b, Tfeb mRNA expression in macrophage and non-macrophage fractions separated by magnetic-activated cell sorting (MACS). c, Tfeb mRNA expression in M1-like and M2-like macrophages estimated by real-time RT-PCR after FACS sorting. d, A heatmap showing relative expression of Tfeb and its target genes in ATM subsets of C57BL/6N mice fed chow diet or HFD (GSE126407). DP-ATM and VA-ATM indicate monocyte-derived CD11c+CD64+ double-positive ATM and vasculature-associated ATM, respectively. VA-ATM1 and VA-ATM2 are distinguished by expression level of MHCII. e, Correlation between VAT TFEB mRNA level and body weight, fasting glucose level or VAT adipocyte size in pooled human subjects (lean, obese and obese T2DM) (n=54). f, Correlation between TFEB mRNA level in SAT and body weight, fasting glucose level or adipocyte sizes of SAT in pooled human subjects (n=54). All data are shown as means ± SEM. *P < 0.05 and **P < 0.01 by one-way ANOVA with Tukey’s test (b,c) or Pearson’s correlation analysis (e,f).

Source data

Extended Data Fig. 2 Generation and characterization of TFEBMac-Tg mouse.

a, A schematic diagram of generation of macrophage-specific TFEB-Tg mice. Excision of LoxP sites by Cre recombinase expressed under the control of the LysM promoter leads to expression of constitutively active TFEB transgene in myeloid cells. b, Relative mRNA expression of TFEB/Tfeb in peritoneal macrophages from TFEBMac-Tg and control mice. c, Immunoblots of peritoneal macrophages from TFEBMac-Tg and control mice using antibodies specific for human TFEB (hTFEB), Flag, LC3, p62 or HSP90 (a loading control). Numbers below immunoblot bands represent densitometric values. d, Representative images of hTFEB immunofluorescence in peritoneal macrophages from TFEBMac-Tg and control mice. Scale bars, 20 μm. e, Immunofluorescence images showing TFEB colocalized with F4/80 in the brain, liver, pancreas and adipose tissues. Occasional F4/80+ cells without nuclear TFEB were seen because efficiency of Cre-mediated excision of LoxP sequences was not 100%. Scale bars, 20 μm. f, Fasting blood glucose and serum insulin level in TFEBMac-Tg and control mice on chow diet (n=11). g, Serum CHO, FFA and TG levels in TFEBMac-Tg and control mice on chow diet (n=11). All data are shown as means ± SEM. *P < 0.05 and ***P < 0.001 by two-tailed Student’s t-test.

Source data

Extended Data Fig. 3 Depletion of macrophages by clodronate administration.

a-e, GTT (a), fasting blood glucose (b), body weight (c), immunohistochemistry of adipose tissue using F4/80 antibody (d), and real-time RT-PCR analysis of F4/80 or Cd11b expression (e) in adipose tissue from HFD-fed TFEBMac-Tg and control mice with or without clodronate treatment (Clod) (n=4 each group). Scale bars, 200 μm. All data are shown as means ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 by two-way ANOVA with Bonferroni’s test (a) or one-way ANOVA with Tukey’s test (b,c,e). (In a, red asterisk, comparison between untreated TFEBMac-Tg and untreated TFEBMac-Con mice; black asterisk, comparison between treated TFEBMac-Con and untreated TFEBMac-Con mice). (n.s., not significant).

Source data

Extended Data Fig. 4 Characterization of HFD-fed TFEBMac-Tg mice.

a, Representative gross images of adipose tissues from TFEBMac-Tg and control mice fed HFD for 12 weeks. b,c, Distribution of adipocyte size (diameter) in eWAT (b) and iWAT (c) of TFEBMac-Tg and control mice fed HFD for 12 weeks (n=4-6). d,e, Quantification of CLS (d) and F4/80 immunohistochemistry (e) in eWAT of TFEBMac-Tg and control mice fed HFD for 12 weeks (n=4-6). f, Relative expression of inflammatory markers in iWAT of TFEBMac-Tg and control mice fed HFD for 12 weeks (n=4-6). g, Average EE during day and night time periods in TFEBMac-Tg and control mice fed HFD for 8 weeks (n=8-10). h,i, Average food intake (h) and locomotor activity (i) during day and night time periods in TFEBMac-Tg and control mice fed HFD for 8 weeks (n=8-10). j-n, T3 (j), T4 (k), fibroblast growth factor 21 (FGF21) (l), adiponectin (m) and leptin (n) levels in TFEBMac-Tg and control mice fed HFD for 12 weeks (n=8-15). o, serum GDF15 in TFEBMac-Con/Atg7W/W, TFEBMac-Tg/Atg7W/W, TFEBMac-Con/Atg7DMac and TFEBMac-Tg/Atg7DMac mice fed HFD for 12 weeks (n=7-11) p, Relative Gdf15 mRNA levels in adipose tissues from C57BL/6N mice fed chow diet or HFD for 12 weeks (left, n=3-6) and from ob/ob mice (right, n=5). q, Relative Gdf15 mRNA level in mature adipocytes and SVCs of eWAT from C57BL/6N mice fed chow diet or HFD for 12 weeks estimated by real-time RT-PCR (n=3) (left). Expression of Adipoq and Cd45 mRNA level showing successful fractionation of adipocytes and SVCs (middle and right). r, Relative Gdf15 mRNA level in macrophage and non-macrophage fractions of SVCs of eWAT estimated by real-time RT-PCR after MACS separation. All data are shown as means ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 by two-tailed Student’s t-test (d,e,f,g,n,p) or one-way ANOVA with Tukey’s test (o,q,r). (n.s., not significant).

Source data

Extended Data Fig. 5 Metabolic profile in thermoneutrality condition.

a-e, Body weight (a), body weight gain (b), adipose tissue weight (c), H&E staining of adipose tissues (d) and serum GDF15 (e) in HFD-fed TFEBMac-Tg and control mice housed at normal ambient (22 °C) or thermoneutral temperature (30 °C) (n=6-12). Scale bars, 200 μm. All data are shown as means ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 by two-way ANOVA with Bonferroni’s test (a) or one-way ANOVA with Tukey’s test (b,c,e). (n.s., not significant).

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Extended Data Fig. 6 Gdf15 mRNA expression in tissues with TFEB overexpression and metabolic profile of HFD-fed TFEBMac-Tg/Gdf15–/– mice.

a, Relative Gdf15 mRNA level in BMDMs of TFEBMac-Tg and control mice (n=3). b, Relative Gdf15 mRNA expression in skeletal muscle of mice injected intramuscularly with AAV2.1-TFEB (GSE62975) and in the liver of mice injected intravenously with HDAd-Pepck-TFEB (GSE41139). c,d, Relative Gdf15 mRNA levels in eWAT, iWAT and BAT of TFEBMac-Tg and control mice fed HFD (c, n=4-6) or chow diet (d, n=3-4) for 12 weeks. e,f, Real-time RT-PCR analysis of GDF15 expression (e) and serum GDF15 (f) in HFD-fed TFEBMac-Tg and control mice with or without clodronate treatment (Clod) (n=4 each group). g-i, Fasting blood glucose (g), fasting serum insulin (h) and serum CHO/FFA/TG levels (i) in TFEBMac-Con/Gdf15+/+, TFEBMac-Tg/Gdf15+/+, TFEBMac-Con/Gdf15–/– and TFEBMac-Tg/Gdf15–/– mice fed HFD for 12 weeks (n=7-12). All data are shown as means ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 by two-tailed Student’s t-test (a,b,c,d) or one-way ANOVA with Tukey’s test (e,f,g,h,i).

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Extended Data Fig. 7 Metabolic profile of chow- or HFD-fed GDF15-Tg mice.

a-c, Body weight (a), GTT (b) and ITT (insulin 1.5 U/kg) (c) in GDF15-Tg and control mice fed chow diet or HFD for 12 weeks (n=3-4). d-h, Food intake (d), tissue weight (e), representative H&E staining of eWAT, iWAT, BAT and the liver (f), serum ALT/AST (g) and serum CHO/FFA/TG levels (h) in GDF15-Tg and control mice fed chow diet or HFD for 12 weeks (n=3-4). Scale bars, 200 μm. i, Representative TH immunohistochemistry of BAT and iWAT in GDF15-Tg and control mice fed HFD for 12 weeks. Arrows indicate cells with positive TH staining. Scale bars, 50 μm for BAT and 200 μm for iWAT. All data are shown as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-way ANOVA with Bonferroni’s test (a,b,c) or one-way ANOVA with Tukey’s test (e,g,h). (In a-c, *P < 0.05, **P < 0.01 and ***P < 0.001 between GDF15-Tg and control mice fed chow diet. #P < 0.05, ##P < 0.01, and ###P < 0.001 between GDF15-Tg and control mice fed HFD). (n.s., not significant).

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Extended Data Fig. 8 GDF15 increases sympathetic tone.

a-b, Action potential (AP) firing number under various stimulus intensity (a), and relationship between AP firing number and stimulus intensity (b) in mice treated with vehicle (Veh) or GDF15. c-d, Voltage tracing under various stimulus intensity (c), and AP threshold stimuli (d) in mice treated with vehicle or GDF15 (n=6). All data are shown as means ± SEM. **P < 0.01 and ***P < 0.001 by two-tailed Student’s t-test.

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Extended Data Fig. 9 Gdf15 mRNA expression in cells treated with lysosomal stressors, and lysosomal stress in adipose tissues of obese mice and human subjects.

a, Quantification of nuclear TFEB in peritoneal macrophages treated with NH4Cl (20 μM), CQ (25 μM), BAF (50 nM) or ultrapure water (as vehicle) for 12 h (right). Representative immunofluorescence pictures are presented (left). Scale bars, 20 μm. b, Relative GDF15 (Gdf15) mRNA levels in BMDMs, HeLa, FaO and AML12 cells treated with CQ (25 μM) or NH4Cl (20 μM) for 12 h (n=3). c, Relative GDF15 mRNA levels in WT HeLa, TFEB-KO HeLa and TKO HeLa cells treated with NH4Cl (20 μM) for 12 h (n=3). d, Representative F4/80-GPNMB double immunofluorescence of eWAT from C57BL/6N mice fed chow diet or HFD. Scale bars, 20 μm. e, Relative GPNMB mRNA levels in VAT or SAT from lean subjects and obese subjects with or without T2DM (n=14-24). f, Representative CD68/GPNMB double immunohistochemistry in serial VAT or SAT sections from lean subjects and obese subjects with or without T2DM. Arrows indicate positive cells stained with each antibody. Scale bars, 100 μm. g, Relative Gpnmb mRNA levels in peritoneal macrophages treated with BSA or PA (400 μM) for 16 h (n=3). All data are shown as means ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 by one-way ANOVA with Tukey’s test (a,b,c,e) or two-tailed Student’s t-test (g).

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Extended Data Fig. 10 Cellular events associated with PA-induced TFEB activation.

a, Effect of ML-SI3, a TRPML1 inhibitor, on PA-induced TFEB nuclear translocation (right). Representative immunofluorescence pictures are presented (left). Scale bars, 5 μm. b, Mitochondrial ROS stained with MitoSOX in peritoneal macrophages treated with BSA or PA (400 μM) for 1 h in the presence or absence of Mito-Tempo (500 μM) pretreatment (lower). Gating strategy and representative scattergrams are presented (upper and middle). c, Macrophages were treated with PA or BSA control, and cell extract was subjected to immunoblot analysis to study phosphorylation of mTOR and its target, 4EBP1. Representative immunoblot (left). Densitometric ratio of phospho-mTOR or -4EBP1 band to HSP90 band (right). d, Macrophages were treated with PA or BSA control were subjected to immunofluorescence, and colocalization between mTOR and LAMP1 was examined (right). Representative immunofluorescence pictures are presented (left). Scale bars, 5 μm. e, Raw264.7 cells transfected with pcDNA 3.1-HA or HA-DCnA-H151Q were treatment with 400 μM PA or BSA. After immunostaining with anti-HA and TFEB antibodies, cells were analyzed by confocal microscopy (yellow arrow head, HA-DCnA-H151Q-transfected cells showing no TFEB translocation after PA treatment; red arrow head, untransfected cells showing TFEB translocation after PA treatment). Scale bars, 5 μm. All data are shown as means ± SEM. ***P < 0.001 by one-way ANOVA with Tukey’s test. (n.s., not significant).

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Kim, J., Kim, S.H., Kang, H. et al. TFEB–GDF15 axis protects against obesity and insulin resistance as a lysosomal stress response. Nat Metab 3, 410–427 (2021). https://doi.org/10.1038/s42255-021-00368-w

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