Zfp423 Maintains White Adipocyte Identity through Suppression of the Beige Cell Thermogenic Gene Program

Abstract

The transcriptional regulators Ebf2 and Prdm16 establish and maintain the brown and/or beige fat cell identity. However, the mechanisms operating in white adipocytes to suppress the thermogenic gene program and maintain an energy-storing phenotype are less understood. Here, we report that the transcriptional regulator Zfp423 is critical for maintaining white adipocyte identity through suppression of the thermogenic gene program. Zfp423 expression is enriched in white versus brown adipocytes and suppressed upon cold exposure. Doxycycline-inducible inactivation of Zfp423 in mature adipocytes, combined with β-adrenergic stimulation, triggers a conversion of differentiated adiponectin-expressing inguinal and gonadal adipocytes into beige-like adipocytes; this reprogramming event is sufficient to prevent and reverse diet-induced obesity and insulin resistance. Mechanistically, Zfp423 acts in adipocytes to inhibit the activity of Ebf2 and suppress Prdm16 activation. These data identify Zfp423 as a molecular brake on adipocyte thermogenesis and suggest a therapeutic strategy to unlock the thermogenic potential of white adipocytes in obesity.

Figure 1. Widespread accumulation of beige-like adipocytes in WAT depots of inducible adipocyte-specific Zfp423 knockout mice

(A) mRNA levels of Zfp423 in fractionated adipocytes isolated from anatomically distinct fat pads of adult C57BL/6 mice. * denotes p<0.05 from one-way ANOVA. n=6. (B) Inducible inactivation of Zfp423 in mature adipocytes (Zfp423-iAKO mice) is achieved by breeding the Adiponectin^rtTA transgenic mice to animals expressing Cre recombinase under the control of the tet-reponse element (TRE-Cre) and carrying floxed Zfp423 alleles (Zfp423^loxP/loxP). Littermates carrying only Adiponectin^rtTA and Zfp423^loxP/loxP alleles (i.e. Cre-) were used as the control animals. Mice were kept at room temperature and fed on normal chow until 8 weeks of age before switching to doxycycline (dox)-containing chow diet for another 28 days. (C) Relative mRNA levels of Zfp423 in fractionated adipocytes isolated from inguinal white adipose tissue (iWAT) and gonadal white adipose tissue (gWAT) of control and Zfp423-iAKO mice after dox feeding. n=4. (D) Representative photograph of iWAT from control and Zfp423-iAKO mice after dox feeding. (E) Representative hematoxylin and eosin (H&E) staining of iWAT section obtained from control mouse after dox feeding. Scale bar=200μM. (F) Representative H&E staining of iWAT section obtained from Zfp423-iAKO mouse after dox feeding (G) Relative mRNA levels of common adipocyte genes in the iWAT of control and Zfp423-iAKO mice after dox feeding. * denotes p<0.05 from Student’s t-test. n=4. (H), Relative mRNA levels of brown/beige-selective thermogenic genes in the iWAT of control and Zfp423-iAKO mice after dox feeding. * denotes p<0.05 from Student’s t-test. n=4. (I), Relative mRNA levels of white adipocyte-selective genes in the iWAT of control and Zfp423-iAKO mice after dox feeding. * denotes p<0.05 from Student’s t-test. n=4. (J) Relative mRNA levels of brown adipocyte- and beige adipocyte-selective markers in the inguinal WAT depots of control mice (iWAT), Zfp423-iAKO mice (Zfp423^−/− iWAT), cold-exposed control mice (Beige), and brown adipose tissue (BAT) of control mice after dox feeding. * denotes p<0.05 from one-way ANOVA. n=4–6. (K) Western blot of Ucp1 protein levels in the iWAT of control and Zfp423-iAKO mice after dox feeding. (L) Basal and maximal (FCCP) oxygen consumption rate (OCR) of diced iWAT isolated from control and Zfp423-iAKO mice after dox feeding. * denotes p<0.05 from Student’s t-test. n=5.

Figure 2. Beige adipocytes in Zfp423-iAKO mice arise through a conversion of adiponectin+ Zfp423-deficient adipocytes

(A) The Cre-dependent Rosa26R^mT/mG reporter allele was reconstituted to the Zfp423-iAKO background, allowing for indelible GFP labeling of Zfp423-deficient adipocytes. Mice were kept on normal chow until 8 weeks of age before switching to dox-containing chow for 7 days (“Pulse”). After the labeling period, the mice were switched back to a standard chow diet (devoid of Dox) for another 21 days (“Chase”). After the 21-day period, the presence of GFP- multilocular adipocytes indicates beige cells were derived from GFP- precursors. The presence of GFP+ multilocular adipocytes indicates beige cells that arise directly from mature adipocytes targeted during the pulse-labeling period. (B) mRNA levels of Cre in purified adipocytes and stromal vascular fraction (SVF) from iWAT of control and Zfp423-iAKO mice after 7 days of dox feeding. * denotes p<0.05 from Student’s t-test. n=4. (C) mRNA levels of Zfp423 in purified adipocytes and SVF from iWAT of control and Zfp423-iAKO mice after 7 days of dox feeding. * denotes p<0.05 from Student’s t-test. n=4. (D) Representative immunofluorescence staining of Perilipin (red) and GFP (green) in iWAT sections obtained from control (Adpn-rtTA; TRE-Cre; Rosa26R^mT/mG) mice after pulse-labeling (“Pulse”). (E) Same as in (D) but from Zfp423-iAKO mice (Adpn-rtTA; TRE-Cre; Zfp423^loxP/loxP;Rosa26R^mT/mG). (F) Same as in (D) but from additional control mice (Adpn-rtTA; Rosa26R^mT/mG). (G) Representative immunofluorescence staining of Perilipin (red) and GFP (green) in iWAT sections obtained from control (Adpn-rtTA; TRE-Cre; Rosa26R^mT/mG) mice held at room temperature (RT) for 21 days after the removal of doxycycline (“Chase”). (H) Same as in (G) but from Zfp423-iAKO mice (Adpn-rtTA; TRE-Cre; Zfp423^loxP/loxP;Rosa26R^mT/mG). White arrows denote GFP⁺ adipocytes. (I) Same as in (G) but from control mice (Adpn-rtTA; Rosa26R^mT/mG). (J) Representative immunofluorescence staining of Perilipin (red) and GFP (green) in iWAT sections obtained from control (Adpn-rtTA; TRE-Cre; Rosa26R^mT/mG) mice exposed to cold temperatures (6 °C) for 7 days after the removal of doxycycline (“Chase”). White arrows denote GFP⁺ adipocytes. Yellow arrows denote GFP⁻ adipocytes. (K) iWAT SVF isolated from control and Zfp423-iAKO mice were induced to differentiate in vitro. After differentiation, the adipocytes were treated with 5μM dox for 12 hours. Relative mRNA levels of common adipocyte genes and thermogenic genes were measured by qPCR in the differentiated adipocytes 2 days after dox treatment. * denotes p<0.05 from Student’s t-test. n=3

Figure 3. Mice lacking adipocyte Zfp423 are resistant to diet-induced obesity

(A) Control and Zfp423-iAKO mice were fed a standard chow diet until 8 weeks of age before switching to dox-containing chow or dox-containing high fat diet (HFD). Body weights of control and Zfp423-iAKO mice were measured weekly. * denotes control-HFD vs. Zfp423-iAKO-HFD p<0.05 from two-way ANOVA. n=6–8. (B) Fat pad weight (normalized to body weight) of control and Zfp423-iAKO mice after 16 weeks of dox-HFD feeding. * denotes p<0.05 from Student’s t-test. n=6. (C) Total fat mass and lean mass (normalized to body weight) of control and Zfp423-iAKO mice after 16 weeks of dox-HFD feeding. * denotes p<0.05 from Student’s t-test. n=12–13. (D) Body length of control and Zfp423-iAKO mice after 16 weeks of dox-HFD feeding. n=6. (E) Glucose tolerance test of control and Zfp423-iAKO mice after 16 weeks of dox feeding. * denotes control-HFD vs. Zfp423-iAKO-HFD p<0.05 from two-way ANOVA. n=6–8. (F) Insulin tolerance test of control and Zfp423-iAKO mice after 16 weeks of dox feeding. * denotes control-HFD vs. Zfp423-iAKO-HFD p<0.05 from two-way ANOVA. n=6–8. (G) Representative H&E staining of the liver of control mice after 16 weeks of dox-HFD feeding. Scale bar=200μM. (H) Same as in (G) but from Zfp423-iAKO mice. (I) Representative H&E staining of the iWAT from control mice after 16 weeks of dox-HFD feeding. Scale bar=200μM. (J) Same as in (J) but from Zfp423-iAKO mice. (K) Western blot of Ucp1 protein levels in the iWAT of control and Zfp423-iAKO mice after 16 weeks of dox-HFD feeding. (L) O₂ consumption in control and Zfp423-iAKO mice during two complete 12 hr light and dark cycles following 3 weeks of dox-HFD feeding. n=6. (M) Heat production in control and Zfp423-iAKO mice during two complete 12 hr light and dark cycles following 3 weeks of dox-HFD feeding. n=6. (N) Average O₂ consumption of control and Zfp423-iAKO mice during the 5-day measurement. * denotes p<0.05 from Student’s t-test. n=6. (O) Average heat production of control and Zfp423-iAKO mice during the 5-day measurement. * denotes p<0.05 from Student’s t-test. n=6.

Figure 4. Reversal of weight gain and glucose intolerance by inducible inactivation of adipocyte Zfp423 in obese mice

(A) Obesity was induced in 5 week-old control and Zfp423-iAKO mice by administering a high-fat diet (HFD) for 8 weeks. Then animals were switched to a dox-containing HFD (dox-HFD) for another 14 weeks. After 5 weeks of dox-HFD, the mice were treated daily with CL316243 (1mg/kg/24hr) or Vehicle (PBS) for 4 weeks via osmotic pumps. (B) Body weights of control and Zfp423-iAKO mice prior to CL316243 administration. n=12–13. (C) Glucose tolerance test (GTT) of control and Zfp423-iAKO mice immediately prior to CL316243 administration. n=6–7. (D) Relative mRNA levels of indicated genes in gWAT from dox-HFD fed control and Zfp423-iAKO mice prior to CL316243 administration. * denotes p<0.05 from Student’s t-test. n=4–5. (E) Same as in (D) but in iWAT. (F) Body weights of control and Zfp423-iAKO mice after vehicle or CL316243 administration. * denotes control-CL316243 vs. Zfp423-iAKO-CL316243 p<0.05 from two-way ANOVA. n=5–9. (G) Glucose tolerance test (GTT) of control and Zfp423-iAKO mice after vehicle or CL316243 administration. * denotes control-CL316243 vs. Zfp423-iAKO-CL316243 p<0.05 from two-way ANOVA. n=5. (H) Representative H&E staining of the liver from control mice receiving vehicle treatment (Week 18 of HFD as indicated in (A)). Scale bar=200μM. (I) Same as in (H) but from control mice receiving CL316243. (J) Same as in (H) but from Zfp423-iAKO mice receiving vehicle. (K) Same as in (H) but from Zfp423-iAKO mice receiving CL316243. (L) Representative H&E staining of iWAT from control mice receiving vehicle treatment (Week 18 of HFD as indicated in (A)). Scale bar=200μM. (M) Same as in (L) but from control mice receiving CL316243. (N) Same as in (L) but from Zfp423-iAKO mice receiving vehicle. (O) Same as in (L) but from Zfp423-iAKO mice receiving CL316243. (P) Relative mRNA levels of indicated genes in iWAT from control and Zfp423-iAKO mice after vehicle or CL316243 administration. (Week 18 of HFD as indicated in (A)) * denotes p<0.05 from two-way ANOVA. n=5. (Q) Same as in (P) but from gWAT.

Figure 5. Physiological regulation of brown and white adipocyte Zfp423 expression

(A) Western blot of endogenous Zfp423 proteins in adipose depots isolated from C57BL/6 mice held at room temperature or exposed to cold (6°C) for 3 days. (B) Relative mRNA levels of Zfp423 in fractionated iWAT adipocytes, Pdgfrα+; Lin-(CD31- and CD45-) cells and Pdgfrα-; Lin+ (CD31+ and CD45+) cells isolated from C57BL/6 mice held at room temperature or exposed to cold (6°C) for 3 days. Relative mRNA levels of Zfp423 in iWAT isolated from C57BL/6 mice held at thermoneutrality, exposed to cold (6°C) for 1 day or injected with CL 316243 (1mg/kg/day) for 3 days. * denotes p<0.05 from Student’s t-test or one-way ANOVA. n=4–6. (C) Relative mRNA levels of Zfp423 and thermogenic genes in iWAT of control and Zfp423-iAKO mice held at thermoneutrality (TN) or exposed to cold (CE) (6°C) for 1 day. * denotes p<0.05 from two-way ANOVA. n=5. (D) Representative H&E staining of interscapular BAT from 2 months-old and 12 months-old C57BL/6 mice. Scale bar=200μM. (E) Relative mRNA levels of Zfp423 and Ucp1 in interscapular BAT from 2 months-old and 12 months-old C57BL/6 mice. * denotes p<0.05 from Student’s t-test. n=6. (F) Representative H&E staining of interscapular BAT from C57BL/6 mice held at room temperature (RT) or thermoneutrality (TN). Scale bar=200μM. (G) Relative mRNA levels of Zfp423 and Ucp1 in interscapular BAT from C57BL/6 mice held at room temperature (RT) or thermoneutrality (TN). * denotes p<0.05 from Student’s t-test. n=6. (H) Animals conferring adipocyte-specific Zfp423 overexpression (Zfp423-AOE mice) were derived by breeding Adiponectin^rtTA transgenic mice with transgenic mice expressing full-length murine Zfp423 under the control of a promoter containing the tet-response element (TRE-Zfp423). Littermates carrying only the Adiponectin^rtTA allele were used as control animals. Mice were kept at room temperature and fed on standard chow until 8 weeks of age before switching to dox-containing chow diet for another 28 days. (I) mRNA levels of Zfp423 in the BAT of C57BL/6 mice held at room temperature (RT) or thermoneutrality (TN), and BAT of Zfp423-AOE mice held at room temperature. n=4–6. (J) Representative H&E staining of the BAT from control mice after dox feeding. Scale bar=200μM. (K) Same as in (J) but from Zfp423-AOE mice. (L) Relative mRNA levels of common adipocyte genes, thermogenic genes, and white adipocyte-selective genes in the BAT of control and Zfp423-AOE mice after dox feeding. * denotes p<0.05 from Student’s t-test. n=4.

Figure 6. Zfp423 suppresses the thermogenic gene program in white adipocytes through repression of Ebf2 transcriptional activity

(A) Schematic illustration of the zinc finger (ZF) domains of full-length Zfp423 (ZF 1–30), Zfp423 smad-binding mutant (ΔSBD), and Zfp423 C-terminal mutant (ZF 1–25). Western blots of FLAG-tagged Zfp423 protein levels in in vitro differentiated primary brown adipocytes expressing full-length Zfp423 and Zfp423 mutants. (B) Oil red O staining of in vitro differentiated brown adipocytes expressing full-length Zfp423 and Zfp423 mutants. (C) Relative mRNA levels of common adipocyte genes in in vitro differentiated brown adipocytes expressing full-length Zfp423 and Zfp423 mutants. n=3. (D) Relative mRNA levels of thermogenic genes in in vitro differentiated brown adipocytes expressing full-length Zfp423 and Zfp423 mutants. * denotes Vector vs. ZF 1–30 p<0.05 from one-way ANOVA; # denotes Vector vs. ZF 1-ΔSBD p<0.05 from one-way ANOVA; N.S= not significant. n=3. (E) Relative mRNA levels of common adipocyte genes and thermogenic genes in in vitro differentiated iWAT adipocytes virally expressing Zfp423, Ebf2, or Zfp423 and Ebf2. * denotes p<0.05 from one-way ANOVA. n=3. (F) Firefly luciferase activity (normalized to Renilla activity) in cells transfected with the Prdm16 enhancer-driven firefly luciferase reporter construct and Ebf2 or Ebf2/Pparγ/Rxrα vectors, with or without Zfp423 co-expression. * denotes p<0.05 from Student’s t-test. n=3. (G) iWAT SVF cells were isolated from control and Zfp423-iAKO mice and transduced with the indicated CRISPR lentivirus before in vitro differentiation. Ebf2 protein levels in the differentiated adipocyte cultures were detected by western blotting. (H) Relative mRNA levels of common adipocyte genes and thermogenic genes in in vitro differentiated iWAT adipocytes with indicated genotype and CRISPR lentivirus transduction. * denotes p<0.05 from two-way ANOVA. n=3.

Figure 7. BMP7 signaling disrupts the Zfp423-Ebf2 protein complex

(A) iWAT SVF cultures from C57BL/6 mice were co-transduced with retroviruses expressing either FLAG-tagged full-length (ZF 1–30) Zfp423 or mutant Zfp423 (ZF 1–25), along with full-length FLAG-Ebf2. After adipocyte differentiation, protein-protein interactions were analyzed by Ebf2 immunoblotting (anti-FLAG antibody) of Zfp423 immunoprecipitates (anti-Zfp423 antibody immunoprecipitation). (B) iWAT SVF cells from C57BL/6 mice were transduced with retrovirus expressing FLAG-tagged Zfp423 and Ebf2 before in vitro differentiation. After differentiation, the adipocytes were treated with vehicle or browning agents (10 μM rosiglitazone, 10 μM isoproterenol, or 10 nM BMPs) for 6 hr. Protein-protein interaction was analyzed by immunoblotting of Zfp423 immunoprecipitates. (C) iWAT SVF cells from C57BL/6 were transduced with retrovirus expressing FLAG-tagged full-length Zfp423 (ZF 1–30) or Zfp423 smad-binding mutant (ΔSBD) together with retrovirus expressing Ebf2 before in vitro differentiation. After differentiation, the differentiated adipocytes were treated with vehicle or 10 nM BMP7 for 6 hr. Protein-protein interaction was analyzed by immunoblotting of Zfp423 immunoprecipitates. (D) Proposed model for the inhibition of the thermogenic gene program in white adipocytes by Zfp423. Zfp423 binds to Ebf2 and serves as a transcriptional co-repressor at key Ebf2-target genes, such as Prdm16. This leads to a suppression of the overall thermogenic gene program in white adipocytes. (E) Proposed model for the interaction between BMP signaling and Ebf2 signaling in brown adipocyte differentiation. BMP7 triggers a Smad1/4 interaction with Zfp423 that leads to activation of Pparγ expression and adipogenesis. This sequesters Zfp423 from Ebf2, thereby allowing Ebf2 to drive the thermogenic gene program of brown adipose tissue.