Adipocyte HDAC4 activation leads to beige adipocyte expansion and reduced adiposity

in Journal of Endocrinology
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Numerous studies have suggested that beige adipocyte abundance is correlated with improved metabolic performance, but direct evidence showing that beige adipocyte expansion protects animals from the development of obesity is missing. Previously, we have described that the liver kinase b1 (LKB1) regulates beige adipocyte renaissance in subcutaneous inguinal white adipose tissue (iWAT) through a class IIa histone deacetylase 4 (HDAC4)-dependent mechanism. This study investigates the physiological impact of persistent beige adipocyte renaissance in energy homeostasis in mice. Here we present that the transgenic mice H4-TG, overexpressing constitutively active HDAC4 in adipocytes, showed beige adipocyte expansion in iWAT at room temperature. H4-TG mice exhibited increased energy expenditure due to beige adipocyte expansion. They also exhibited reduced adiposity under both normal chow and high-fat diet (HFD) feeding conditions. Specific ablation of beige adipocytes reversed the protection against HFD-induced obesity in H4-TG mice. Taken together, our results directly demonstrate that beige adipocyte expansion regulates adiposity in mice and targeting beige adipocyte renaissance may present a novel strategy to tackle obesity in humans.

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  • Supplementary Figure 1. BAT thermogenesis in H4-TG mice. A, Immunoblots showing amounts of GFP-HDAC4.3A and Hsp90 in mature adipocytes and stromal vascular fraction (SVF) from iBAT, iWAT and eWAT of 8-week-old WT and H4-TG#D mice. B, q-PCR analysis of Ucp1 mRNA levels in iBAT from 8-week-old male C57bl/6J (WT) and ap2-GFP-HDAC4.3A (H4-TG#D) mice. Sample sizes: WT (n=5) and H4-TG#D (n=5). C, Immunoblots of Ucp1 and Hsp90 in the iBAT of 8-week old WT and H4-TG#D mice. D, Representative H&E staining of iBAT from 8-10-week-old WT and H4-TG#D mice. Scale bar: 100m. E, Core temperature of 8-week-old WT and H4-TG#D mice upon 4C cold challenge. Sample sizes: WT (n=4) and H4-TG#D (n=5).
  • Supplementary Figure 2. Beige adipocyte expansion in female H4-TG#D mice. A. q-PCR analysis of Ucp1 , Pgc1 and Cox8b mRNA levels in iWAT from ~8-week-old female WT and H4-TG#D mice. Sample sizes: WT (n=5) and H4-TG#D (n=4). B. Representative H&E staining of iWAT from ~8-week-old female WT and H4-TG#D mice. Scale bar: 50M. Arrows: multilocular beige adipocytes.
  • Supplementary Figure 3. Beige adipocyte expansion in male H4-TG#C mice. A. Immunoblots showing amounts of GFP-HDAC4.3A in WAT, iBAT, muscle and liver from WT and H4-TG#C mice. B. Heatmap showing Log2 fold change (FC) of thermogenic genes (Ucp1, Cidea, Cox8b, Dio2 and Plin5) in the iWAT of ~8-week-old male H4-TG#C and H4-TG#D mice. C. Immunoblots showing amounts of Ucp1 and Hsp90 in the iWAT of ~8-week-old male H4-TG#C mice.
  • Supplementary Figure 4. Inflammation marker expression and ex vivo thermogenic activity of iWAT of H4-TG mice. A, q-PCR analysis mRNA levels of macrophage markers in iWAT from ~8-week-old WT and H4-TG#D mice. Sample sizes: WT (n=5) and H4-TG#D (n=4). B, Ex vivo oxygen consumption of iWAT from 6-8 week-old WT and H4-TG#D mice. Sample sizes: WT (n=6) and H4-TG#D (n=7).
  • Supplementary Figure 5. H4-TG mice did not show increased energy expenditure after CL injection. Recordings of total energy expenditure (TEE) increases in response to CL in ~8-10-week old male WT and H4-TG#D mice. CL injected at 0 hour. Sample size: n=4 for each genotype.
  • Supplementary Figure 6. H4-TG mice did not show increased total energy expenditure (EE) at thermoneutrality. A, Immunoblots of Ucp1 and Hsp90 in iBAT and iWAT from WT and H4-TG#D mice housed at RT and thermoneutrality (30ºC). The samples at RT also showed in Supplementary Fig. 1C. B, q-PCR analysis of Ucp1 in iBAT from WT and H4-TG#D mice at RT and 30ºC. Sample sizes: WT/RT (n=4), H4-TG#D/RT (n=4), WT/30ºC (n=5), H4-TG#D/30ºC (n=5). C, Representative H&E staining of iBAT from ~12-week-old WT and H4-TG#D mice. Scale bar: 50M. D, Average TEE at night and day in ~12-week old male WT and H4-TG#D mice at thermoneutrality. E, Scatter plots of TEE at night and day over body weight in above mice. Averages of RER (F), food intake (G), and activity (H) at night and day in above mice. I, TEE partitioning of physical activity (PAEE) and food (TEF) in above mice. Sample size: n=5 for each genotype.
  • Supplementary Figure 7. H4-TG mice maintained beige adipocyte expansion after 4-week HFD. A, Immunoblots showing Ucp1 and Hsp90 in iWAT from 12-week-old male WT and H4-TG#D mice under normal chow and 4-week-HFD conditions. B, Representative H&E staining of iWAT from 12-week-old male WT and H4-TG#D mice after 4-week HFD. Scale bar: 50M. Arrows: multilocular beige adipocytes.
  • Supplementary Figure 8. H4-TG#C mice also showed reduced body weight at HFD. Body weight for male WT, H4-TG#D and H4-TG#C mice after ~10-week HFD. Sample sizes: WT (n=10), H4-TG#D (n=11) and H4-TG#C (n=4).
  • Supplementary Figure 9. H4-TG mice did not show elevated beige adipocyte markers after 10-week HFD. A, q-PCR analysis of selective beige adipocyte genes in iWAT from WT and H4-TG#D mice after 4- and ~10-week HFD. Sample sizes: WT/4w HFD (n=5), H4-TG#D /4w HFD (n=10), WT/10w HFD (n=8), H4-TG#D /10w HFD (n=5). B, Immunoblots of Ucp1 and Hsp90 in iWAT of above mice.
  • Supplementary Figure 10. Expression of adipogenesis markers was normal in the eWAT of H4-TG mice at NC and HFD. q-PCR analysis of key adipogenesis transcription factors, C/ebp, C/ebp and Ppar, in the eWAT from WT and H4-TG#D mice at ~26-week of age on NC (A) or after ~10-week HFD (B). Sample sizes: WT/NC (n=6), H4-TG#D/NC (n=5), WT/HFD (n=5) and H4-TG#D /HFD (n=5).
  • Supplementary Figure 11. H4-TG mice remained lean under HFD independently of Ucp1-mediated thermogenesis. A, q-PCR analysis of thermogenic genes (Ucp1, Dio2, Pgc1, Cidea, and Cox8b) in iWAT from WT, H4-TG, Ucp1 KO and H4-TG;Ucp1 KO mice. Sample sizes: WT (n=10), H4-TG (n=8), Ucp1 KO (n=7) and H4-TG;Ucp1 KO (n=8). B, Representative H&E staining of iWAT from Ucp1 KO and H4-TG;Ucp1 KO mice. Scale bar: 100M. Arrows: multilocular beige adipocytes. C, Body weight of WT, H4-TG, Ucp1 KO and H4-TG;Ucp1 KO mice under 4-week HFD. Sample sizes: WT (n=15), H4-TG (n=4), Ucp1 KO (n=11) and H4-TG;Ucp1 KO (n=4).
  • Supplementary Figure 12. H4-TG mice showed normal BAT thermogenic activity after ablation. A, q-PCR analysis of mRNA levels of Ucp1, Cox8b and Cidea in BAT from ~8-week-old male H4-TG#D;Cre- and H4-TG#D;Cre+ mice. All mice were injected with DT at 3-week of age. Sample sizes: n=4 for each genotype. B, Representative H&E staining of iBAT from H4-TG#D;Cre- and H4-TG#D;Cre+ mice. Scale bar: 100m. C, Core temperature of 8-week-old Cre-, Cre+, H4-TG#D;Cre- and H4-TG#D;Cre+ mice upon 4C cold challenge. All mice were injected with DT at 3-week of age. Sample sizes: Cre- (n=8), Cre+ (n=4), H4-TG#D;Cre- (n=9) and H4-TG#D;Cre+ (n=5).
  • Supplementary table 1: List of primer sequences for q-PCR

 

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    H4-TG mice exhibited beige adipocyte expansion in iWAT at room temperature. (A) Top: Schematic illustration of the ap2-GFP-HDAC4.3A (H4-TG) transgenic mice. ap2: fabp4 promoter; GFP: green fluorescent protein; HDAC4.3A: human HDAC4 with three SIK phosphorylation sites (Ser246, Ser467 and Ser632) Ser→Ala mutations (Red asterisks). Bottom: Immunoblot showing amounts of GFP-HDAC4.3A and HSP90 in WAT, iBAT, muscle and liver from C57bl/6J (WT) and ap2-GFP-HDAC4.3A (H4-TG#D) mice. (B) q-PCR analysis of Ucp1 mRNA levels in iWAT from 3-week and 8-week-old WT and H4-TG#D mice at room temperature (RT) and 8-week-old at thermoneutrality (30°C). Sample sizes: 3 week WT/RT (n = 9), 3 week H4-TG#D/RT (n = 9), 8 week WT/RT (n = 7), 8 week H4-TG#D/RT (n = 8), 8 week WT/30°C (n = 4), and 8 week H4-TG#D/30°C (n = 8). (C) Representative H&E staining of iWAT from 8-week-old WT and H4-TG#D mice at normal chow. Scale bar: 50 µM. Arrows: multilocular beige adipocytes. (D) Immunoblots showing UCP1 and HSP90 in iWAT from 8-week-old WT and H4-TG#D mice treated with PBS or CL. A full color version of this figure is available at https://doi.org/10.1530/JOE-18-0173.

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    HDAC4 activation promoted thermogenic gene expression in iWAT. (A) Molecular mechanism of beige adipocyte renaissance. Under cold stimulation, adipocyte HDAC4 activation could promote beige adipocyte renaissance non-cell autonomously. Liver kinase b1 (LKB1) activates SIKs to suppress HDAC4 activation, while cold stimulates HDAC4 activation through βAR-mediated inhibition of SIKs. (B) Pie chart analysis of HDAC4 dependency of upregulated genes identified in Lkb1AKO mice. (C) List of gene ontology (GO) number, description, count, percentage and Log10(p) value of the top four processes enriched in HDAC4-dependent genes that were upregulated in the iWAT of both Lkb1AKO and H4-TG#D mice. (D) q-PCR analysis of selective beige adipocyte genes in iWAT from WT and H4-TG#D mice under normal chow. Sample sizes: n = 5 for each genotype. A full color version of this figure is available at https://doi.org/10.1530/JOE-18-0173.

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    HDAC4 activation promoted beige adipocyte renaissance in iWAT. (A) Diagram showing beige adipocyte ablation strategy. In Ucp1-Cre;Rosa-iDTR mice at room temperature, DT injection (at 3 weeks of age) induces cell death of Ucp1+ brown adipocytes in iBAT and beige adipocytes in iWAT within 3 days. Brown adipocytes gradually repopulate the BAT in 3–5 weeks, while Ucp1+ beige adipocytes remain absent in iWAT. (B) Experimental design. Three-week-old H4-TG#D;Cre− (genotype: H4-TG#D;Rosa-iDTR) or H4-TG#D;Cre+ (genotype: H4-TG#D;Ucp1-Cre;Rosa-iDTR) mice were injected with DT once. Five weeks later, they were injected with either PBS or CL for 7-consective days. (C) Representative H&E staining of iWAT from 8-week-old H4-TG#D;Cre+ mice at normal chow, with or without DT injection at 3-week of age. Scale bar: 50 µM. Arrows: multilocular beige adipocytes. (D) q-PCR analysis of Ucp1 mRNA levels in iWAT from ~8-week-old H4-TG#D;Cre− and H4-TG#D;Cre+ mice treated with PBS or CL. DT was injected at 3-week of age. Sample size: H4-TG#D;Cre−/PBS (n = 4), H4-TG#D;Cre+/PBS (n = 3), H4-TG#D;Cre−/CL (n = 4) and H4-TG#D;Cre+/CL (n = 5). A full color version of this figure is available at https://doi.org/10.1530/JOE-18-0173.

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    H4-TG mice showed increased total energy expenditure (TEE). (A) Average TEE at night and day in ~8–10-week-old male WT and H4-TG#D mice at room temperature. (B) Scatter plots of TEE at night and day over body weight in above mice. Averages of RER (C), food intake (D), and activity (E) at night and day in above mice. (F) TEE partitioning of physical activity (PAEE) and food (TEF) in above mice. Sample size: n = 4 for each genotype.

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    H4-TG mice showed reduced adiposity and improved metabolic performance under normal chow. (A) Body weight in male WT and H4-TG#D mice on normal chow. Sample sizes: n = 24 for each genotype. Fat mass (B), lean mass (C) and food intake (D) of ~26-week-old WT and H4-TG#D mice. Sample sizes: n = 6 for each genotype. Representative H&E staining of eWAT (E) and adipocyte size distribution (F) in above mice. Scale bar: 50 µM. Total adipocytes counted: WT (n = 878) and H4-TG#D (n = 1319). (G) q-PCR analysis of mRNA levels of macrophage marker Cd68 and F4/80 in eWAT of above mice. Serum TAG (H), insulin (I) and glucose (J) levels in ~26-week-old WT and H4-TG#D mice after 4-h fasting. Sample sizes: n = 6 for each genotype. A full color version of this figure is available at https://doi.org/10.1530/JOE-18-0173.

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    H4-TG mice were lean and insulin sensitive under HFD. (A) Body weight for male WT and H4-TG#D mice under ~10-week HFD. Sample sizes: WT (n = 9) and H4-TG#D (n = 11). Fat mass (B), lean mass (C) and food intake (D) of male WT and H4-TG#D mice before and after 9-week HFD. Representative H&E staining of eWAT (E) and adipocyte size distribution (F) in above mice. Scale bar: 50 µM. Brown arrow: Crown-like structure. Total adipocytes counted: WT (n = 2293) and H4-TG#D (n = 2883). (G) q-PCR analysis of mRNA levels of macrophage marker Cd68 and F4/80 in eWAT of above mice. (H) Serum glucose levels (per fold of basal) in WT and H4-TG#D mice after 9-week HFD. AUC (area under curve) of ITT also shown. (I) Serum glucose levels (per mg/dL) in WT and H4-TG#D mice after 9-week HFD. AUC (area under curve) of ITT also shown. A.U. Arbitrary Unit. Sample sizes: WT (n = 9) and H4-TG#D (n = 10). A full color version of this figure is available at https://doi.org/10.1530/JOE-18-0173.

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    H4-TG mice with beige adipocyte ablation were not protected against HFD-induced obesity. (A) H4-TG#D mice were crossed to Ucp1-Cre;Rosa-iDTRHOM mice to generate the following four groups of mice: Cre− (genotype: Rosa-iDTR), Cre+ (genotype: Ucp1-Cre;Rosa-iDTR), H4-TG#D;Cre− (genotype: H4-TG#D;Rosa-iDTR), and H4-TG#D;Cre+ (genotype: H4-TG#D;Ucp1-Cre;Rosa-iDTR). (B) Experimental scheme. The four groups of mice were injected with DT at three-week of age to ablate UCP1+ brown and beige adipocytes. Four to five weeks later, the brown adipocytes were fully regenerated in iBAT, while beige adipocytes remained ablated in iWAT. Then the mice were fed with HFD for additional 4–5 weeks for metabolic measurements. (C) q-PCR analysis of Ucp1 mRNA levels in iWAT from Cre+ and H4-TG#D;Cre+ adult mice before, 3 days to 1 week, and more than 2 weeks after DT injection. Sample sizes: 0 days Cre+ (n = 18), 0 days H4-TG#D;Cre+ (n = 19), 3 days to 1 week Cre+ (n = 4), 3 days to 1 week H4-TG#D;Cre+ (n = 7), >2 week Cre+ (n = 5) and >2 week H4-TG;Cre+ (n = 8). (D) Representative H&E staining of iWAT from H4-TG#D;Cre− and H4-TG#D;Cre+ mice at different time points before and after DT injection. Scale bar: 50 µM. Arrows: multilocular beige adipocytes. (E and F) Body weight and weight gain of Cre−, Cre+, H4-TG#D;Cre− and H4-TG#D;Cre+ mice under HFD. Sample sizes: Cre− (n = 18), Cre+ (n = 18), H4-TG#D;Cre− (n = 17) and H4-TG#D;Cre+ (n = 13). (G) Serum glucose levels (per fold of basal) in Cre−, Cre+, H4-TG#D;Cre− and H4-TG#D;Cre+ mice after 5-week HFD. AUC (area under curve) of ITT also shown. (H) Serum glucose levels (per mg/dL) in Cre−, Cre+, H4-TG#D;Cre− and H4-TG#D;Cre+ mice after 5-week HFD. AUC (area under curve) of ITT also shown. AU: Arbitrary Unit. Sample sizes: Cre− (n = 18), Cre+ (n = 18), H4-TG#D;Cre− (n = 17) and H4-TG#D;Cre+ (n = 13). A full color version of this figure is available at https://doi.org/10.1530/JOE-18-0173.

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