Histone deacetylase 6 inhibition restores leptin sensitivity and reduces obesity

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.

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. 6h P=4.1E-3, 16h P=6E-6, 24h 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.

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 293T lysates 24hr 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.

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-STAT3^Y705 stainings (left) and the quantification of the fluorescent intensities (right bar graphs) Scale bar: 20μm. 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 (25μg) 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 HDAC6^AdipoΔ (n=6) mice. g, Physical activity profile of the mice in (e) (n=4 for AdipoCre, n=6 for HDAC6^AdipoΔ; Dark P=4.4E-3). Data are represented as mean ± s.e.m.

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 HDAC6^SynΔ (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 (HDAC6SynΔ) mice on standard or high-fat diet (n=18, HDAC6 flox chow; n=20, HDAC6 flox HFD; n=16, SynCre chow, n=17, SynCre HFD; n=18, HDAC6^SynΔ chow; n=20, HDAC6^SynΔ HFD; HFD vs. Chow for all genotypes P=1.2E-11, HDAC6flox HFD vs. SynCre HFD P=6.3E-5, HDAC6flox HFD vs. HDAC6^SynΔ 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.

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

Wild-type (n=7 mice) and HDAC6^flox/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 25mg/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 pSTAT3 and total STAT3 from the lysates of N1-LRb cells pre-treated with leptin or PBS for 24hr 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 4h (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 2hr, and stimulated with leptin (2 ng/mL) for 4hr (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 pre-treated with deproteinized plasma for 2h, followed by leptin (2 ng/mL) stimulation for 4h (n=4). f, g, Food intake (Veh Saline vs. Veh Leptin; 3h P=3.6E-4, 6h P=7.2E-4, 16h P=8.7E-7, 24h P=1.3E-5; TubA Saline vs. TubA Leptin; 3h P=0.011, 6h P=9.4E-3, 16h P=9.4E-10, 24h P=7.1E-9; Veh Leptin vs. TubA Leptin: 3h P=0.34, 6h P=0.038, 16h P=0.032, 24h P=0.30) and body weight change (Veh Saline vs. Veh Leptin; 16h P=1.2E-6, 24h P=0.014; TubA Saline vs. TubA Leptin; 16h P=8.7E-9, 24h P=3.2E-6; Veh Leptin vs. TubA Leptin: 16h P=8E-4, 24h P=0.025 by two-way ANOVA with Tukey’s post-hoc test for f and g) of 24h-fasted lean HDAC6^AdipoΔ 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 (500nM) or tubastatin (1μM) for 24hr, and stimulated with leptin for 15min. 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.

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 293T cells transfected with the indicated constructs (n=6. HSP25: GFP vs. HDAC6^CI P=1.5E-9, HDAC6 WT vs. HDAC6^CI P=2.8E-9; HSP70: GFP vs. HDAC6^CI P=3E-5, HDAC6 WT vs. HDAC6^CI 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.

Fig. 1.. Inhibition of HDAC6 Reverses Diet-Induced Obesity.

a, b, Effect of daily intraperitoneal (i.p.) Tubastatin (TubA, 25 mg/kg, n= 6) or vehicle (Veh, n= 6) administration on wild-type DIO mice: a, Body weight (P=2.6E-9), b, cumulative food intake of the animals (P=1.7E-5). c, Body weight (P=1E-4) and d, food intake (week 1 P=0.0012, week 2 P=7E-6, week 3 P=0.00097) of TubA-treated WT (n=17) and HDAC6 KO (n= 14) DIO mice. e, Lean and fat mass of DIO mice determined by nuclear magnetic resonance (NMR) before and 32 days after TubA or Veh treatment (n= 6 per group, Fat mass at 32 days P= 1.4E-7). f, Plasma leptin concentration of DIO mice before and 32 days after tubastatin treatment (n= 6, P=0.0016). g-j, Food intake and growth curves of wild-type mice on regular diet (chow) or HFD treated by daily i.p. vehicle or tubastatin (25 mg/kg) (n=9 mice per group) For i, week1 P=0.0014, week2 P=0.00043, week 3 P=0.042, week4 P=0.037); j, P=1E-10). *P<0.05, **P<0.01, ***P<0.001 as analyzed by one-way or two-way analysis of variance (ANOVA) or mixed-effect analysis with Tukey’s post-hoc test for multiple comparison, or two-tailed Student’s t-test. Data are represented as mean ± s.e.m.

Fig. 2.. Obesity induces HDAC6 activity in the adipose tissue.

a, HDAC6 mRNA expression at the indicated tissues collected from lean and DIO wild-type mice fed ad lib or fasted overnight (n=5 for liver and hypothalamus of lean fed and muscle of lean fasted; n=6 for all other groups). *P<0.05, **P<0.01 as analyzed by one-way ANOVA. b-d, Representative immunoblots of tissue lysates from the white adipose tissue (WAT), liver and the hypothalamus of lean and DIO wild-type or HDAC6 KO mice. The western blots were reproduced in at least two cohorts. Quantifications are graphed on the right of western blots (n=20, c P=8.1E-11; n=6, d and e, by two-tailed unpaired t-test) ***P<0.001 as analyzed by two-tailed Student’s t-test. Data are represented as mean ± s.e.m.

Fig. 3.. Tubastatin Treatment Improves Metabolic Function in Diet-Induced Obese Mice.

a, 6 hr day-time fasting blood glucose measured 10 weeks after treatments (n=7, chow; n=9 HFD; n=8, HFD+TubA; chow vs. HFD P= 3.1E-8, HFD vs. HFD+TubA P=9.6E-7). b, c, Glucose tolerance tests performed after vehicle or TubA treatment of DIO wild-type mice (n=6 vehicle, n=6 TubA; 30min P=0.0076, 60min P=2.3E-4, 90 min P=0.023, 120min P=0.035) (b) or lean wild-type mice (n=8 vehicle, n=8 TubA; P=5.8E-4) (c). d, Hematoxylin and eosin (H&E) staining of liver sections of DIO mice after one month of vehicle or TubA treatment. Scale bar 50μm e, f, Expression of hepatic lipid (e) and glucose (f) metabolism genes analyzed by RT-qPCR 5 days of vehicle or tubastatin treatment of DIO wild-type mice (n=6; Me1 P=0.0047, Acaca P=0.011, Fasn P=0.021, Pck1 P=0.0024, GCK P=0.0041)). g, h, qPCR analysis of eWAT from DIO mice after 5 days of vehicle or TubA treatments (n= 6; Ppara P=0.0091, Me1 P=0.013, Fabp4 P=0.022, Fasn P=0.026, Leptin P=0.029, Pparg P=0.030, Arg1 P=0.0070, Chil3 P=0.032). i-l, DIO mice were placed into metabolic chambers and treated with vehicle or TubA for 5 consecutive days. i, Average daily food intake (P=3.9E-4), j, change in body weight (P=5.9E-4), k, respiratory quotient (RQ, Dark P=0.041), and l, energy expenditure (EE) during the treatment period (n=6, Veh; n=5, TubA). *P<0.05, **P<0.01, ***P<0.001 as analyzed by two-way analysis of variance (ANOVA) with Sidak’s correction for multiple comparison or two-tailed Student’s t-test. Data are represented as mean ± s.e.m.

Fig. 4.. Tubastatin-induced weight loss requires leptin-melanocortin signaling.

a, b, Body weight curves and food intake of daily vehicle or tubastatin-treated db/db mice (n=13 mice, Veh; n=12 mice, TubA; week 1 food intake P=1.2E-4). c, Body fat percentage of the db/db mice after three-week treatment (n=7, Veh; n=6 mice, TubA). d, Plasma leptin concentration of db/db mice measured by ELISA before and three-week after vehicle (n=7) or tubastatin (n=6) treatments. e, f, Body weight curves and food intake of daily vehicle or tubastatin-treated ob/ob mice (n=8, body weight P=0.015, week 1 of food intake 1.7E-3). g, h, Body weight curves and food intake of vehicle or tubastatin-treated MC4R KO mice (n=7, week 1 of food intake P=5.9E-5). *P<0.05, **P<0.01, ***P<0.001 as analyzed by mixed-effect analysis a, two-way ANOVA (b, d-h) with Sidak’s correction for multiple comparison or student’s t-test (c). Data are represented as mean ± s.e.m.

Fig. 5.. Inhibition of HDAC6 leads to increased leptin action.

a, b, Food intake (TubA+Sal vs. TubA+Lep 16h P=5.1E-6, 24h P=8.6E-5) and body weight change (6h Veh+Lep vs. TubA+Lep P=0.044, TubA+Sal vs. TubA+Lep P=7.1E-4; 16h Veh+Lep vs. TubA+Lep P=3.8E-5, TubA+Sal vs. TubA+Lep P=1.1E-10; 24h Veh+Lep vs. TubA+Lep P=3.E-6, TubA+Sal vs. TubA+Lep P=2.1E-11) of 24h-fasted lean wild-type mice upon treatment with vehicle, saline, TubA (25 mg/kg) and/or leptin (2.5 mg/kg) (n= 8 per group). c, d, Cumulative food intake (Veh+Lep vs. TubA+Lep P=2.1E-4, TubA+PBS vs. TubA+Lep P=1.9E-5) and body weight change intake (Veh+Lep vs. TubA+Lep P=2.0E-13, TubA+PBS vs. TubA+Lep P=3.5E-11) of ob/ob mice (n= 4–6) treated with vehicle+saline (n=6 mice), Vehicle+leptin (0.2 mg/kg) (n=6), TubA (25 mg/kg)+saline (n=5) or TubA+leptin (n=6) following 5 day vehicle or tubastatin pre-treatments. *P<0.05, **P<0.01, ***P<0.001 as analyzed by two-way ANOVA (a-c) or mixed-effect analysis (d) with Sidak’s correction or Tukey’s post-hoc test for multiple comparison. e, f, Food intake (3h Veh/Saline vs. Veh/Leptin P=0.048; 16h TubA/Saline vs. TubA/Leptin P=2.0E-4, 24h TubA/Saline vs. TubA/Leptin P=6.9E-9) and body weight change (for TubA/Saline vs. TubA/Leptin: 16h P=6.8E-3, 24h P=1.6E-6) of 24h-fasted DIO wild-type mice upon treatment with vehicle, saline, TubA (25 mg/kg) and/or leptin (5 mg/kg) (n= 8 mice for Veh/Saline, Veh/Leptin, and TubA/Leptin, n=12 mice for TubA/Saline). g, Hypothalamic mRNA expression of the indicated genes in lean wild-type mice 4h after i.p. vehicle (n=6) or leptin (5 mg/kg, n=5) administration (Asb4 P=3.8E-4, Atf3 P=4.1E-5, Bcl3 P=8.9E-5, Bdkrb1 P=0.073, Gbp7 P=3.0E-3, Has2 P=0.023, Irak3 P=7.8E-4, Irf9 P=5.1E-4, Muc1 P=3.3E-3, Nav2 P=0.014, Nlrc5 P=3E-8, Osmr P=6.7E-3, Ppaap2b P=5.9E-3, Serpina3g P=6.7E-3, Serpina3h P=4.2E-3, Slc16a2 P=3E-3, SOCS3 P=2.7E-7, Vwa5a P=3.6E-5). h, Hypothalamic mRNA expression of the leptin-regulated genes in DIO wild-type mice 4h after vehicle or tubastatin administration (n=4; (Atf3 P=0.031, Bcl3 P=1.7E-3, Gbp7 P=0.062, Irak3 P=0.023, Irf9 P=4.2E-3, Nav2 P=0.042, Osmr P=7.5E-6, Serpina3g P=0.047, Slc16a2 P=0.053, SOCS3 P=5.7E-4, Asb4 P=0.18, Muc1 P=0.17, Nlrc5 P=0.41). *P<0.05, **P<0.01, ***P<0.001 as analyzed by Student’s t-test. Data are represented as mean ± s.e.m.

Fig. 6.. HDAC6 regulates body weight in a cell non-autonomous manner.

a, b, Daily food intake and body weight change of DIO wild-type mice treated with the indicated doses of tubastatin (n= 7) or vehicle (DMSO, n= 7) by daily infusion into the lateral ventricle. c, Biodistribution of TubA in indicated tissues and serum collected 2 h post i.p. drug injection (n= 3). WAT is epididymal white adipose tissue. d, Immunoblots of acetylated αTubulin (Ac-αTubulin) and total αTubulin in the inguinal white adipose tissue (iWAT), epididymal WAT (eWAT) and intrascapular brown adipose tissue (BAT) of vehicle or tubastatin-treated wild-type DIO mice. The results were repeated in two independent experiments. e, Growth curves of regular diet-fed control (HDAC6flox (n=20 chow, n=20 HFD and AdipoCre (n=18 chow, n=18 HFD) and HDAC6^AdipoΔ mice (n=14 chow, n=19 HFD; HDAC6^AdipoΔ HFD vs. AdipoCre HFD P=1.0E-4; HDAC6^AdipoΔ HFD vs. HDAC6^AdipoΔ Chow P=3.3E-10). f, Body fat composition of control (n=38) and HDAC6^AdipoΔ (n=19) mice on HFD (P=0.011, unpaired, two-tailed t-test). g, Day-time fasting (6h) blood glucose of HFD-fed control (n=38) and HDAC6^AdipoΔ (n=19) mice (P=0.0047 by two-tailed t-test. *P<0.05, **P<0.01, ***P<0.001 as analyzed by Student’s t-test. h-j, Change in body weight (HDAC6^AdipoΔ Veh vs. HDAC6^AdipoΔ TubA P=4.9E-10; HDAC6flox Veh vs. HDAC6flox TubA P=2.6E-10, AdipoCre Veh vs. AdipoCre TubA P=2.6E-10, HDAC6^AdipoΔ TubA vs. AdipoCre/HDAC6flox TubA P=2.6E-10) and (h) cumulative food intake (HDAC6^AdipoΔ Veh vs. HDAC6^AdipoΔ TubA P=0.73; HDAC6flox Veh vs. HDAC6flox TubA P=1.3E-5, AdipoCre Veh vs. AdipoCre TubA P=4.2E-4, HDAC6^AdipoΔ TubA vs. AdipoCre/HDAC6flox TubA P=0.04) (i) of HDAC6^AdipoΔ mice (n=10 mice. HDAC6flox Veh; n=9 mice HDAC6 flox TubA; n=9 mice, AdipoCre Veh; n=9 mice, AdipoCre TubA;;n=8 mice, HDAC6^AdipoΔ Veh; n=10 mice, HDAC6^AdipoΔ TubA) (h), and weight change of HDAC6^SynΔ mice (n=10 mice, HDAC6 flox Veh; n=10 mice, HDAC6flox TubA; n=8 mice, SynCre Veh; n=7 mice, SynCre TubA, n=8 mice, HDAC6^SynΔ Veh, n=8 mice, HDAC6^SynΔ TubA; for all genotypes Veh vs TubA P=2.7E-10) (j) and their respective controls during daily vehicle or tubastatin treatments. k, Expression of the leptin responsive transcripts in N1-LRb cells pre-treated with plasma from vehicle or TubA-treated ob/ob mice for 2h, followed by leptin (2 ng/mL) stimulation for 4h (n=6; TubA^Plasma+PBS vs. TubA^Plasma+Lep: Bcl3 P=9.4E-4, Elfn1 P=0.023, Nav2 P=0.056, Socs3 P=0.0052). l, 24hr fasted lean wild-type or HDAC6^AdipoΔ mice were treated with leptin or saline alone or in combination with TubA. The food intake during refeeding were plotted as percent intake of the saline groups (n=4 for all groups except HDAC6^AdipoΔ Veh, where n=3. wt Veh vs. wt TubA: 3h P=2.2E-3, 6h P=1.3E-5, 16h P=2.2E-4, 24h P=5.0E-4; wt TubA vs. HDAC6^AdipoΔ Tub: 3h P=0.025, 6h P=7.3E-7, 16h P=2.8E-3, 24h P=2.6E-4). See also Extended Data Fig. 9f, g. m, Illustration summary: HFD induces and tubastatin suppresses HDAC6 activity in the adipose tissue. Inhibition of HDAC6 activity leads to central leptin sensitization through a potentially novel adipokine (Factor X). *P<0.05, **P<0.01, ***P<0.001 as analyzed by mixed-effect analysis (e-i) or one-way (j) or two-way ANOVA (k, l) with Tukey’s post-hoc test for multiple comparison. Data are represented as mean ± s.e.m.

Fig. 7.. Tubastatin does not alter blood-brain barrier permeability of leptin.

Wild-type mice were treated with vehicle or tubastatin (i.p.) followed by iv. Cy3-leptin. Mice were subsequently perfused and sections were prepared for imaging. a, Confocal images of Cy3 fluorescence (left) and the quantification of the fluorescence intensity (right) in the mediobasal hypothalamus (MBH) and the arcuate nucleus alone (ARC) (n=6). Arrowheads indicate Cy3-leptin. Scale bar: 20μm. b, c, Wild-type lean mice were implanted with a cannula in the lateral ventricle. Following 24-hr fasting, mice were treated with i.p. vehicle (n=6) or tubastatin (25mg/kg, n=6) and icv leptin (0.5μg per mouse). Food intake (16h P=0.027, 24h P=9.5E-3 by two-way ANOVA with Sidak correction (b) and 24hr body weight change (P=0.049 by unpaired two-tailed t-test) (c) was recorded. *P<0.05, **P<0.01, ***P<0.001. Data are represented as mean ± s.e.m.

Fig. 8.. A non-hydroxamate HDAC6-specific inhibitor reverses diet-induced obesity.

a, Chemical structures of tubastatin (top) and SE-7552 (bottom). The hydroxamate moiety of tubastatin is highlighted in blue. b, Docking of SE-7552 into the inhibitor binding site of HDAC6. The A chain of the 5edu.pdb was used for docking. The Trichostatin A in the 5edu.pdb is shown in green and the docked pose of the SE-7552 in magenta. One nitrogen of oxadiazole in SE-7552 is binding to Zinc as the carbonyl of hydroxamate in Trichostatin A, one of the fluorine in the difluoromethyl group is interacting with His610, the pyrimidine ring is sandwiched between Phe680 at the top and Phe620 at the bottom making pi-pi interactions with them. The amide is hydrogen bonding with Ser568, and the fluorobenzene ring is also interacting with Phe620 by T-shaped pi-pi stacking. The docking experiment was carried out using MOE software . The same results were obtained by gold docking program . c, Dose response curves of HDAC6 inhibition for the HDAC6-specific inhibitors CAY10603, tubastatin, and SE-7552 (n=2 per dose per compound, n=6 for no compound). d, Immunoblots from lysates of N1 cells treated with the indicated compounds for 24 hr. e, f, Body weight change of SE-7552 (50 mg/kg, i.p., n=12) treated wild-type DIO mice (n=13, P=3E-10 by mixed-effect analysis with Sidak correction) (e) or db/db mice (n=6) (f). *P<0.05, **P<0.01, ***P<0.001. Data are represented as mean ± s.e.m.