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Histone deacetylase 6 inhibition restores leptin sensitivity and reduces obesity

Nature Metabolism volume 4, pages 44–59 (2022)Cite this article

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Abstract

The adipose tissue-derived hormone leptin can drive decreases in food intake while increasing energy expenditure. In diet-induced obesity, circulating leptin levels rise proportionally to adiposity. Despite this hyperleptinemia, rodents and humans with obesity maintain increased adiposity and are resistant to leptin’s actions. Here we show that inhibitors of the cytosolic enzyme histone deacetylase 6 (HDAC6) act as potent leptin sensitizers and anti-obesity agents in diet-induced obese mice. Specifically, HDAC6 inhibitors, such as tubastatin A, reduce food intake, fat mass, hepatic steatosis and improve systemic glucose homeostasis in an HDAC6-dependent manner. Mechanistically, peripheral, but not central, inhibition of HDAC6 confers central leptin sensitivity. Additionally, the anti-obesity effect of tubastatin A is attenuated in animals with a defective central leptin–melanocortin circuitry, including db/db and MC4R knockout mice. Our results suggest the existence of an HDAC6-regulated adipokine that serves as a leptin-sensitizing agent and reveals HDAC6 as a potential target for the treatment of obesity.

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Fig. 1: Inhibition of HDAC6 reverses DIO.
Fig. 2: Obesity induces HDAC6 activity in adipose tissue.
Fig. 3: Tubastatin treatment improves metabolic function in DIO mice.
Fig. 4: Tubastatin-induced weight loss requires leptin–melanocortin signaling.
Fig. 5: Inhibition of HDAC6 leads to increased leptin action.
Fig. 6: HDAC6 regulates body weight in a cell-nonautonomous manner.
Fig. 7: Tubastatin does not alter blood–brain barrier permeability of leptin.
Fig. 8: A non-hydroxamate HDAC6-specific inhibitor reverses DIO.

Data availability

Figures 2 and 6 and Extended Data Figs. 1, 2 and 6–10 have associated raw data provided as source data files. All raw data are also available upon request. 5edu.pdb was used for the docking reported in Fig. 8. RNA-seq results are deposited to the GEO database with accession no. GSE190156.Source data are provided with this paper.

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Acknowledgements

This work was supported by National Institutes of Health (NIH) R01DK070332 and 1R01DK125830 to R.D.C. and MDRC Pilot and Feasibility Grant (NIH grant P30-DK020572), the Klatskin–Sutker Discovery Fund and the American Diabetes Association grant no. 7-21-JDF-032 to I.C. T.A.M. was supported by the NIH (HL116848, HL127240, HL147558 and DK119594) and the American Heart Association (16SFRN31400013). R.A.B. received funding from the Canadian Institutes of Health Research (FRN-216927). The indirect calorimetry study was performed by the Vanderbilt Mouse Metabolic Phenotyping Center (DK059637). We thank V. Zachariou from the Icahn School of Medicine at Mount Sinai for providing the HDAC6flox mice, V. P. Krymskaya from the University of Pennsylvania for TSC2+/+ and TSC−/− mouse embryonic fibroblasts, O. McGuinness and D. Wasserman from Vanderbilt University and Cone laboratory members for useful discussions.

Author information

Author notes

  1. Pauline Lining Pan

    Present address: Department of Pharmacology, University of Michigan Medical Center, Ann Arbor, MI, USA

Authors and Affiliations

  1. Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA

    Işın Çakır, Colleen K. Hadley, Pauline Lining Pan, Danielle T. Porter, Qiuyu Wang, Jiandie D. Lin & Roger D. Cone

  2. College of Literature, Science and the Arts, University of Michigan, Ann Arbor, MI, USA

    Colleen K. Hadley

  3. Department of Medicine, Division of Cardiology and the Consortium for Fibrosis Research & Translation, University of Colorado Anschutz Medical Campus, Aurora, CO, USA

    Rushita A. Bagchi & Timothy A. McKinsey

  4. Department of Molecular Physiology & Biophysics, Vanderbilt University, Nashville, TN, USA

    Masoud Ghamari-Langroudi & Michael J. Litt

  5. Warren Center for Neuroscience Drug Discovery, Department of Pharmacology, Vanderbilt University, Nashville, TN, USA

    Masoud Ghamari-Langroudi

  6. Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA

    Michael J. Litt

  7. Chemical Synthesis Core, Vanderbilt Institute of Chemical Biology, Nashville, TN, USA

    Somnath Jana

  8. Vahlteich Medicinal Chemistry Core, College of Pharmacy, University of Michigan, Ann Arbor, MI, USA

    Susan Hagen, Pil Lee & Andrew White

  9. Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI, USA

    Andrew White

  10. Department of Cell & Developmental Biology, Michigan Medicine, University of Michigan, Ann Arbor, MI, USA

    Jiandie D. Lin

  11. Department of Molecular and Integrative Physiology, School of Medicine, University of Michigan, Ann Arbor, MI, USA

    Roger D. Cone

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Contributions

I.C. and R.D.C. conceived and designed the study. I.C., R.A.B., C.K.H., P.L.P., D.T.P., Q.W., M.G.L., M.L., S.J., S.H. and P.L. performed experiments. I.C., R.A.B., C.K.H., P.L.P., M.G.L., M.L., S.J., S.H., D.T.P., Q.W., P.L., A.W., J.D.L., T.A.M. and R.D.C. analyzed data. I.C. and R.D.C. wrote the manuscript.

Corresponding authors

Correspondence to Işın Çakır or Roger D. Cone.

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Competing interests

T.A.M. is on the Scientific Advisory Board of Artemes Bio and Eikonizo Therapeutics, received funding from Italfarmaco for an unrelated project and has a subcontract from Eikonizo Therapeutics for an SBIR grant from the NIH (HL154959). I.C., A.W., P.L. and R.D.C. have filed a provisional patent application on non-hydroxymate HDAC6 blockers. Other authors declare no competing interests.

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Nature Metabolism thanks Rexford Ahima and the other, anonymous, reviewers for their contribution to the peer review of this work. Primary handling editors Isabella Samuelson, Ashley Castellanos-Jankiewicz.

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Extended data

Extended Data Fig. 1 Effect of HDAC6 Inhibitors on DIO Mice.

a, b, Body weight change (Veh vs. 12.5 mg/kg TubA P = 2.8E−3, Veh vs. 25 mg/kg TubA P = 4.8E-12, Veh vs. 50 mg/kg TubA P = 7E-15) and cumulative food intake (Veh vs. 12.5 mg/kg TubA P = 0.012, Veh vs. 25 mg/kg TubA P = 7.7E-7, Veh vs. 50 mg/kg TubA P = 1.1E-10 by two-wat ANOVA with Tukey correction for a and b) of DIO wild-type mice treated with indicated doses of tubastatin or vehicle (n = 5 mice, Veh; n = 4 mice for drug groups). c, Wild-type DIO mice were treated with vehicle or the indicated doses of tubastatin. Ac-αtubulin and total αtubulin was analyzed in eWAT lysates (n = 3. Veh vs. 12.5 mg/kg TubA P = 0.045, Veh vs. 25 mg/kg TubA P = 3.2E-4, Veh vs. 50 mg/kg TubA P < E-15, by one-way ANOVA with Dunnett correction). d, Time-course food intake measurements of DIO wild-type mice following tubastatin administration (n = 4. 6 h P = 4.1E-3, 16 h P = 6E-6, 24 h P = 6.8E-8 by two-way ANOVA with Sidac correction). e, f, Cumulative food intake (1day P = 5.4E-3, 2day P = 2.9E-3, 3day P = 8.6E-, 4day P = 0.025 by multiple unpaired two-sample t-test) (e) and body weight change (P = 9.7E-3 by two-way ANOVA with Sidak correction for e and f) (f) of DIO wild-type mice treated twice with vehicle (n = 10) or tubastatin. (n = 8). g, h, Female DIO wild-type mice were treated with vehicle (n = 4) or tubastatin (n = 5) for two weeks. Change in body weight (P = 4.4E-6) (g), and cumulative food intake (P = 0.015 by two-way ANOVA with Sidak correction for g and h) (h) of the animals.

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Extended Data Fig. 2 HDAC6-specific Weight Loss response to HDAC6 inhibitors.

a, Growth curves of WT (n = 18 mice) and HDAC6 KO (n = 12 mice) mice on high fat diet (left) and their body composition (right). b, Body weight of daily vehicle (n = 5) or tubastatin (i.p., 12.5 mg/kg, n-6) treated DIO HDAC6 KO mice. c, Structure of tubastatin and BRD3067 (top). Immunoblots from 293 T lysates 24 hr after drug treatment. d, e, Body weight (BRD3067 vs. TubA P = 7.0E-3, Veh vs. TubA P = 2.0E-3) and food intake (day 1 BRD3067 vs. TubA P = 5.8E-4, Veh vs. TubA P = 9.7E-9, day 2 BRD3067 vs. TubA P = 2.6E-5, Veh vs. TubA P = 2.8E-7, day 3 BRD3067 vs. TubA P = 9.5E-3, Veh vs. TubA P = 2.6E-5, day 4 BRD3067 vs. TubA P = 1.7E-5, Veh vs. TubA P = 3.0E-8, day 5 BRD3067 vs. TubA P = 4.7E-5, Veh vs. TubA P = 6.2E-9, day 6 BRD3067 vs. TubA P = 2.0E-3, Veh vs. TubA P = 3.4E-9, Veh vs. BRD3067 P = 1.8E-3, day 7 BRD3067 vs. TubA P = 8.5E-4, Veh vs. TubA P = 1.4E-6, two-way ANOVA with Tukey post-hoc test) of vehicle, tubastatin, or BRD3067-treated DIO wild-type mice (n = 6). f, g, Body weight change of DIO wild-type mice treated daily with vehicle (n = 12), ricolinostat (25 mg/kg, i.p., n = 12, P = 2.7E-7 by two-way ANOVA with Sidak correction) (f) or CAY10603 (12.5 mg/kg, i.p., n = 4, P = 2.5E-9 by two-way ANOVA with Sidak correction) (g). h, i, Weight change (P = 9.2E-4) and cumulative food intake (P = 8.9E-6 by two-way ANOVA with Sidak correction for h and i) of wild-type and HDAC6 KO DIO mice treated daily with i.p. CAY10603 (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 as analyzed by two-way analysis of variance (ANOVA) with Sidak’s correction Tukey’s post-hoc test for multiple comparison. Data are represented as mean ± s.e.m.

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Extended Data Fig. 3 Tubastatin does not affect blood pressure or heart rate.

a, b, Heart rate and blood pressure of wild-type mice measured real-time during the first 4 days of the vehicle or tubastatin administrations (n = 3 mice per group). Data are represented as mean ± s.e.m.

Extended Data Fig. 4 Tubastatin improves metabolic function in diet-induced obese mice.

a, RER, b, EE, and c, linear regression analysis of EE versus lean body mass (LBM) by ANCOVA of DIO wild-type mice treated with vehicle (n = 6) or tubastatin (n = 5). Linear regression was plotted using https://www.mmpc.org/shared/regression.aspx. d, Energy expenditure (EE) of DIO wild-type mice placed into metabolic chambers where they were allowed to eat ad libitum (n = 5) or provided the proportion of food consumed by the TubA group compared to the vehicle group (Pair-Fed, n = 6) (Dark P = 0.02, Light P = 0.026 by two-way ANOVA with Sidac correction). e-h, DIO mice were placed into metabolic chambers and treated with vehicle (n = 17) or tubastatin (n = 15) for 5 consecutive days. Total distance travelled in the cage (e, f), mean pedestrian speed (g), and percentage of sleep of the animals during the treatment period (h). i, Body weight change of DIO wild-type mice treated with vehicle (Veh and Pair-fed groups) or tubastatin (n = 6 per group) for 12 consecutive days. Pair-fed group’s food intake was matched to the daily average food intake of the tubastatin group’s (Veh vs. Pair-fed P = 1.3E-6, Veh vs. TubA P = 4.7E-7). j, Change in fat mass (Veh vs. Pair-fed P = 6.1E-4, Veh vs. TubA P = 2.3E-6, Pair-fed vs. TubA P = 0.015 by one-way ANOVA with Tukey’s post-hoc test for i and j) of the mice in (i). *P < 0.05, **P < 0.01, ***P < 0.001 as analyzed by one-way ANOVA with Tukey’s post-hoc test or Sidak test for multiple comparison. Data are represented as mean ± s.e.m.

Extended Data Fig. 5 Tubastatin increases hypothalamic leptin signaling.

Wild-type DIO mice were treated with i.p. vehicle of tubastatin (n = 3 mice per group), and co-treated with i.p. leptin. Mice were perfused, and hypothalamic STAT3 phosphorylation was analyzed by immunofluorescent staining. Arcuate nucleus (ARC) (P = 0.0054) and dorsomedial hypothalamic (DMH) (P = 0.094) confocal images of p-STAT3Y705 stainings (left) and the quantification of the fluorescent intensities (right bar graphs) Scale bar: 20 µm. The experiment was conducted in two independent cohorts. *P < 0.05, **P < 0.01, ***P < 0.001 as analyzed by unpaired two-tailed t-test.

Extended Data Fig. 6 HDAC6-dependent regulation of obesity is peripherally mediated.

a, Brain HDAC6 activity of DIO mice following icv vehicle or TubA (25 µg) administration (n = 6, P = 2.8E-6 by unpaired two-tailed t-test). b, Immunoblots of acetylated αTubulin (Ac-αTubulin), total αTubulin or GAPDH in spleen, kidney, skeletal muscle, liver, and the hypothalamus. The results were confirmed in two independent cohorts. c, Ac-αTubulin and total αTubulin immunoblot of eWAT samples from DIO HDAC6 KO mice treated with vehicle or tubastatin. This experiment was done in cohort of animals. d, HDAC6 mRNA expression in the cortex (brain), the hypothalamus, adipose tissue, liver and skeletal muscle of the indicated genotypes (n = 3 per group; BAT P = 4.5E-4, iWAT P = 7.2E-3, eWAT P = 5.6E-5 by unpaired multiple t-test. The results were repeated in two independent experiments). e, f, Total (left panels) and time-course (right panels) energy expenditure (e) and respiratory exchange ratios (RER) (f) of AdipoCre (n = 5) and HDAC6Adipo∆ (n = 6) mice. g, Physical activity profile of the mice in (e) (n = 4 for AdipoCre, n = 6 for HDAC6Adipo∆; Dark P = 4.4E-3). Data are represented as mean ± s.e.m.

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Extended Data Fig. 7 Phenotype of the neuron-specific HDAC6 knockout mice.

a, b, Immunoblots of HDAC6 and total αTubulin in the hypothalamus (Hypoth), cortex, liver, and skeletal muscle of SynCre controls (n = 3) and HDAC6Syn∆ (n = 3) mice (a), and the quantification of HDAC6 band intensities normalized to tubulin (Cortex P = 4.6E-4, Hypothalamus P = 9.6E-3 by multiple unpaired t-test) (b). Immunoblot results were confirmed in two cohorts of mice. c, mRNA expression of HDAC6 in the indicated tissues (n = 4; Cortex P = 0.012, Hypothalamus P = 1.3E-3 by multiple t-test). d, Body weight of control and neuron-specific HDAC6 knockout (HDAC6Syn∆) mice on standard or high-fat diet (n = 18, HDAC6flox chow; n = 20, HDAC6flox HFD; n = 16, SynCre chow, n = 17, SynCre HFD; n = 18, HDAC6Syn∆ chow; n = 20, HDAC6Syn∆ HFD; HFD vs. Chow for all genotypes P = 1.2E-11, HDAC6flox HFD vs. SynCre HFD P = 6.3E-5, HDAC6flox HFD vs. HDAC6Syn∆ HFD P = 1E-6 by mixed-effect analysis with Tukey’s post-doc test.). *P < 0.05, **P < 0.01, ***P < 0.001. Data are represented as mean ± s.e.m.

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Extended Data Fig. 8 Metabolic phenotype of the liver specific HDAC6 KO mice.

Wild-type (n = 7 mice) and HDAC6flox/Y (n = 8) mice were treated with tail-vein injection of AAV8-TBG-iCre. Mice were placed on HFD four weeks after viral injection. a, Liver HDAC6 expression analyzed by qPCR (P = 2.5E-8 by two-tailed unpaired t-test). b, Body weight of the cohorts on HFD. c, Weekly food intake of the mice measured at indicated times. d, Body composition measured after 19-week of HFD exposure) Fat P = 0.049, Fluid P = 7.2E-3 by multiple unpaired t-test). e, Glucose tolerance test conducted after 20 weeks on HFD. f, Insulin tolerance test conducted after 23 weeks on HFD. g-I, Mice were treated with 25 mg/kg tubastatin by daily i.p. injections. Weight change (g), cumulative food intake (h), and body composition after tubastatin treatment (i). *P < 0.05, **P < 0.01, ***P < 0.001. Data are represented as mean ± s.e.m.

Extended Data Fig. 9 The anti-obesity effect of HDAC6 inhibition requires a potentially unidentified systemic factor.

a, Leptin dose–response curves of N1-LRb cells stably expressing the luciferase construct under a STAT3-responsive promoter (n = 32 per time point). b, Immunoblots for p-STAT3 and total STAT3 from the lysates of N1-LRb cells pretreated with leptin or PBS for 24 hr followed by leptin stimulation at the indicated doses. c, mRNA expression of leptin-responsive transcripts in N1-LRb cells treated with vehicle or leptin (100 ng/mL) for 4 h (n = 3 for Nav2; n = 4 for other groups, Bcl3 P = 3.9E-5, Elfn1 P = 4.5E-5, Nav2 P = 0.012, Socs3 P = 1.5E-7 by multiple unpaired t-test). d, mRNA expression of leptin responsive transcripts in N1-LRb cells treated with TubA at indicated doses for 2 hr, and stimulated with leptin (2 ng/mL) for 4 hr (n = 4). The results was confirmed in two independent experiments. e, Plasma from vehicle or TubA-treated ob/ob mice was deproteinized by proteinase K treatment followed by heat inactivation. Expression of the leptin responsive transcripts in N1-LRb cells pretreated with deproteinized plasma for 2 h, followed by leptin (2 ng/mL) stimulation for 4 h (n = 4). f, g, Food intake (Veh Saline vs. Veh Leptin; 3 h P = 3.6E-4, 6 h P = 7.2E-4, 16 h P = 8.7E-7, 24 h P = 1.3E-5; TubA Saline vs. TubA Leptin; 3 h P = 0.011, 6 h P = 9.4E-3, 16 h P = 9.4E-10, 24 h P = 7.1E-9; Veh Leptin vs. TubA Leptin: 3 h P = 0.34, 6 h P = 0.038, 16 h P = 0.032, 24 h P = 0.30) and body weight change (Veh Saline vs. Veh Leptin; 16 h P = 1.2E-6, 24 h P = 0.014; TubA Saline vs. TubA Leptin; 16 h P = 8.7E-9, 24 h P = 3.2E-6; Veh Leptin vs. TubA Leptin: 16 h P = 8E-4, 24 h P = 0.025 by two-way ANOVA with Tukey’s post-hoc test for f and g) of 24h-fasted lean HDAC6Adipo∆ mice upon treatment with vehicle, saline, TubA and/or leptin (n = 6 mice for Veh groups, n = 8 mice for TubA groups). h, N1-LepRb cells were treated with celastrol (500 nM) or tubastatin (1 µM) for 24 hr, and stimulated with leptin for 15 min. The level of STAT3 phosphorylation and αtubulin acetylation was analyzed by immunoblots, and confirmed in two independent experiments. i, Body weights of DIO wild-type mice treated with vehicle (n = 4), tubastatin (n = 3), IL6 neutralizing antibodies (anti-IL6, n = 4), or tubastatin+anti-IL6 (n = 4; Veh+Saline vs. TubA+Saline P = 3.2E-9, Veh+anti-IL6 vs. TubA+anti-IL6 P = 3.3E-5 by two-way ANOVA with Tukey’s post-hoc test). j, Body weight change of DIO IL1R1 KO mice treated with vehicle (n = 5) or tubastatin (n = 6, P = 1E-15 by two-way ANOVA with Sidak correction). *P < 0.05, **P < 0.01, ***P < 0.001 as analyzed Student’s t-test, two-way ANOVA with Tukey’s post-hoc test, or Sidak’s multiple comparison. Data are represented as mean ± s.e.m.

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Extended Data Fig. 10 HDAC6 inhibitors induce heat shock response.

a, b, HSP70 mRNA level in iWAT explants from DIO mice 24 hr after, vehicle (n = 41) vs. TubA (n = 42, P = 1.1E-3) (a) or Vehicle (n = 4) vs. CAY10603 (n = 4, P = 3.8E-4, by two-tailed unpaired t-test for a and b) (b) treatments. c, HSP25 and HSP70 mRNA expression in 293 T cells transfected with the indicated constructs (n = 6. HSP25: GFP vs. HDAC6CI P = 1.5E-9, HDAC6 WT vs. HDAC6CI P = 2.8E-9; HSP70: GFP vs. HDAC6CI P = 3E-5, HDAC6 WT vs. HDAC6CI P = 4.5E-9, GFP vs. HDAC6 WT P = 3.1E-5). d, TSC2 +/+ and TSC−/− mouse embryonic fibroblasts (MEFs) were treated with vehicle (DMSO) or celastrol for 24 hr. Cell lysates were analyzed by immunoblots. The results were confirmed in three independent experiemnts e, p-PERK and total PERK protein levels in liver homogenates from wild-type DIO mice treated with vehicle or celastrol. Th experiment was conducted in two independent cohorts of mice with similar outcomes. Data are represented as mean ± s.e.m.

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Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2

Reporting Summary

Supplementary Tables

Supplementary Table 1. Leptin-induced RNA-seq results from stably LepRb-expressing hypothalamic N1 cells. Supplementary Table 2. RNA-seq results from epididymal WAT of wild-type DIO mice treated with vehicle or tubastatin. Supplementary Table 3. RNA-seq results from inguinal WAT of wild-type DIO mice treated with vehicle or tubastatin. Supplementary Table 4. RNA-seq results from the epididymal WAT of AdipoCre (AdCre) and Adipo-HDAC6 KO (AdKO) DIO mice. Supplementary Table 5. RNA-seq results from the epididymal WAT of AdipoCre (AdCre) and global HDAC6 knockout (HDAC6_KO) DIO mice. Supplementary Table 6. RNA-seq results from the epididymal WAT of vehicle (Veh) or Tubastatin A HCl (TubA)-treated HDAC6 KO DIO mice. Supplementary Table 7. Log2(fold change) values for genes with expressions significantly altered by tubastatin treatment in DIO wild-type and HDAC6 KO mice. Supplementary Table 8. RT–qPCR primer sequences.

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Çakır, I., Hadley, C.K., Pan, P.L. et al. Histone deacetylase 6 inhibition restores leptin sensitivity and reduces obesity. Nat Metab 4, 44–59 (2022). https://doi.org/10.1038/s42255-021-00515-3

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