Research ArticleHEALTH AND MEDICINE

The obesity-induced adipokine sST2 exacerbates adipose Treg and ILC2 depletion and promotes insulin resistance

See allHide authors and affiliations

Science Advances  13 May 2020:
Vol. 6, no. 20, eaay6191
DOI: 10.1126/sciadv.aay6191
  • Fig. 1 Obesity-associated reduction of adipose Tregs is linked to elevated expression and secretion of sST2.

    (A) Correlation between eWAT Foxp3 and IL-33 mRNA levels and body weight in a cohort of C57BL/6J mice fed HFD for 8 weeks. (B) Schematic diagrams of the ST2 isoforms. Arrows indicate the location of isoform-specific qPCR primers. (C) qPCR analysis of sST2 and ST2L expression in a panel of tissues from mice fed chow (filled, n = 4) and HFD (open, n = 4). Data in (C) represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, lean versus obese, two-tailed unpaired Student’s t test. (D) qPCR analysis of gene expression in stromal vascular fraction (SVF) and adipocytes (Adp) isolated from eWAT from lean (filled, n = 3) and HFD-fed (open, n = 3) mice. Data represent means ± SEM. **P < 0.01, SVF versus Adp, two-way ANOVA. (E) Concentrations of sST2 in conditioned media (CM) from eWAT explant culture from chow- and HFD-fed mice (n = 3). (F) qPCR analysis of gene expression in eWAT explants from HFD-fed mice (n = 3) treated with IL-33 (10 ng/ml) without or with recombinant sST2 (100 ng/ml). Data in (E) and (F) represent means ± SEM. *P < 0.05, ***P < 0.001, chow versus HFD (E) and rST2 + IL-33 versus IL-33 (F), two-tailed unpaired Student’s t test. Data in (C) to (F) are representative of three independent experiments.

  • Fig. 2 Overexpression of sST2 exacerbates HFD-induced insulin resistance.

    (A) Body weight of mice transduced with AAV-GFP (open, n = 5) and AAV-sST2 (filled, n = 6) fed HFD for 10 weeks. Data represent means ± SEM, two-way ANOVA with multiple comparisons. (B) Blood glucose and plasma insulin concentrations. Data represent means ± SEM. *P < 0.05, GFP versus sST2, two-tailed unpaired Student’s t test. (C) GTT (left) and ITT (right) in mice transduced with AAV-GFP (open, n = 6) or AAV-sST2 (filled, n = 7) after 9 and 11 weeks of HFD feeding, respectively. Data represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, GFP versus sST2, two-way ANOVA with multiple comparisons. Data in (A) to (C) are representative of three independent experiments. (D) Hematoxylin and eosin (H&E) and Sirius red staining of eWAT sections (top) and quantitation of CLS- and fibrosis-positive area (bottom). (E) qPCR analysis of eWAT gene expression. (F) Representative gating for resident Tregs and ILC2s in eWAT from transduced mice. (G) Percentage of immune cells in CD45+ SVF cells. Data in (E) and (G) represent means ± SEM. *P < 0.05, **P < 0.01, GFP versus sST2, two-tailed unpaired Student’s t test. Data in (D) to (G) are representative of three independent experiments. Scale bar = 100 μm.

  • Fig. 3 Adipocyte sST2 expression and secretion are stimulated by TNFα, which is antagonized by Zbtb7b.

    (A) Correlation between eWAT sST2 gene expression and body weight (left) and Ccl2 expression (right). (B) qPCR analysis of sST2 mRNA expression in differentiated C3H10T1/2 adipocytes treated with vehicle (Veh), IL-1β (10 ng/ml), TNFα (10 ng/ml), or IFN-γ (10 ng/ml) for 24 hours (left) or indicated doses of TNFα (right). Data represent means ± SD (n = 3). **P < 0.01, ***P < 0.001, versus Veh, one-way ANOVA. (C) The concentrations of sST2 in CM from C3H10T1/2 adipocytes (left) or eWAT explant culture (right) treated with Veh or TNFα (10 ng/ml) for 24 hours. Data represent means ± SD (n = 3). ***P < 0.001, Veh versus TNFα, two-tailed unpaired Student’s t test. (D) sST2 mRNA expression (left) and concentrations (right) in adipocytes treated with TNFα (10 ng/ml) with or without NF-κB inhibitor VIII (VIII; 4 μM) for 24 hours. Data represent means ± SD (n = 3). ***P < 0.001, Veh versus VIII, two-way ANOVA. Data in (B) to (D) are representative of three independent experiments. (E) Correlation between eWAT mRNA expression of Zbtb7b and bodyweight (left) and sST2 (right) in HFD-fed mice. (F) qPCR analysis of Zbtb7b expression in SVF and adipocytes isolated from chow-fed (filled, n = 3) or HFD-fed (open, n = 3) mouse eWAT. Data represent means ± SEM. ***P < 0.001, SVF versus Adp, two-way ANOVA. (G) qPCR analysis of Zbtb7b expression in eWAT from WT (filled, n = 5) and ob/ob (open, n = 3) or WT (filled, n = 4) and db/db (open, n = 4) mice. Data represent means ± SEM. *P < 0.05, **P < 0.01, lean versus obese, two-tailed unpaired Student’s t test. (H and I) qPCR analysis of Zbtb7b expression in differentiated C3H10T1/2 adipocytes (H) and explant fat culture (I) treated with 10 ng/ml TNFα for 24 hours. Data in (H) and (I) represent means ± SEM. *P < 0.05, Veh versus TNFα, two-tailed unpaired Student’s t test. (J) sST2 mRNA expression (left) and concentrations in CM (right) from C3H10T1/2 adipocytes expressing vector (Vec) or Zbtb7b following treatment with TNFα (10 ng/ml) for 24 hours. Data represent means ± SD (n = 3). ***P < 0.001, Vec versus Zbtb7b, two-way ANOVA. Data in (F) to (J) are representative of three independent experiments.

  • Fig. 4 Adipocyte-specific inactivation of Zbtb7b exacerbates insulin resistance.

    (A) Blood glucose and plasma insulin concentrations in HFD-fed control (Flox; n = 12) and ZAKO (n = 9) mice. Data represent means ± SEM. *P < 0.05, Flox versus ZAKO, two-tailed unpaired Student’s t test. (B) GTT (left) and ITT (right) in Flox (open, n = 12) and ZAKO (filled, n = 9) mice fed HFD for 8 and 11 weeks, respectively. Data represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Flox versus ZAKO, two-way ANOVA with multiple comparisons. Data in (A) and (B) are representative of three independent experiments. (C) Immunoblots of eWAT lysates from Flox and ZAKO mice fed HFD for 13 weeks following intravenous injection of saline (Sal; Flox, open, n = 7; and ZAKO, filled, n = 3) and insulin (Ins; Flox, open, n = 5; and ZAKO, filled, n = 5) for 10 min. (D) Plasma nonesterified fatty acid (NEFA) and β-hydroxybutyrate concentrations in mice treated with saline or insulin. Data represent means ± SEM. *P < 0.05, Flox versus ZAKO, two-way ANOVA. (E) H&E and Sirius red staining of eWAT and liver sections in HFD-fed mice. Quantitation of CLS- and fibrosis-positive area is shown on the right. (F) Liver triglyceride (TAG) content. (G) mRNA expression (left) and secretion (right) of sST2 in explant fat culture from Flox and ZAKO mice treated with TNFα (10 ng/ml) for 24 hours. Data represent means ± SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, Flox versus ZAKO, two-way ANOVA. Data in (E) to (G) are representative of three independent experiments. Scale bar = 100 μm.

  • Fig. 5 Adipocyte-specific inactivation of Zbtb7b diminishes fat-resident Tregs and ILC2s.

    (A) sST2 concentration in CM from explant fat culture and plasma of HFD-fed Flox (n = 12) and ZAKO (n = 9) mice. (B) Representative gating for fat-resident Treg and ILC2 cells from Flox and ZAKO mice. (C) Percentage of immune cells in CD45+ SVF cells from HFD-fed Flox and ZAKO mice. (D) qPCR analysis of eWAT gene expression from Flox and ZAKO mice. Data in (A), (C), and (D) represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; Flox versus ZAKO, two-tailed unpaired Student’s t test. (E) qPCR analysis of gene expression in explant fat culture from HFD-fed Flox and ZAKO mice treated with IL-33 (40 ng/ml) for 24 hours. Data represent means ± SEM (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, Flox versus ZAKO, two-way ANOVA. Data in (A) to (E) are representative of three independent experiments.

  • Fig. 6 Zbtb7b stabilizes IκBα by competing with β-TrCP for hnRNPU binding.

    (A) Immunoblots of total eWAT lysates from HFD-fed Flox and ZAKO mice. (B) Immunoblots of total lysates from C3H10T1/2 adipocytes expressing vector (vec) or Zbtb7b (ZB) treated with vehicle or TNFα (10 ng/ml) for 15 min. (C) Immunoblots of total lysates and α-Myc immunocomplexes from transiently transfected HEK293T cells. HA, hemagglutinin. (D) Immunoblots of total lysates from differentiated 10T1/2 adipocytes expressing Vec or Zbtb7b treated with cycloheximide (CHX; 100 μg/ml) for the indicated times. (E) A schematic model depicting the role of the Zbtb7b-sST2–IL-33 signaling pathway in the regulation of adipose tissue Treg/ILC2 homeostasis and insulin sensitivity. Data in (A) to (D) are representative of three independent experiments.

Supplementary Materials

  • Supplementary Materials

    The obesity-induced adipokine sST2 exacerbates adipose Treg and ILC2 depletion and promotes insulin resistance

    Xu-Yun Zhao, Linkang Zhou, Zhimin Chen, Yewei Ji, Xiaoling Peng, Ling Qi, Siming Li, Jiandie D. Lin

    Download Supplement

    This PDF file includes:

    • Figs. S1 to S5
    • Table S1

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article