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. 2015 Jan;64(1):128-36.
doi: 10.2337/db13-1835. Epub 2014 Sep 4.

Adiponectin induces A20 expression in adipose tissue to confer metabolic benefit

Laura E Hand  1 , Paola Usan  2 , Garth J S Cooper  3 , Lance Y Xu  4 , Basil Ammori  5 , Peter S Cunningham  1 , Reza Aghamohammadzadeh  5 , Handrean Soran  5 , Adam Greenstein  5 , Andrew S I Loudon  1 , David A Bechtold  6 , David W Ray  7
Affiliations

Adiponectin induces A20 expression in adipose tissue to confer metabolic benefit

Laura E Hand et al. Diabetes. 2015 Jan.

Abstract

Obesity is a major risk factor for metabolic disease, with white adipose tissue (WAT) inflammation emerging as a key underlying pathology. We detail that mice lacking Reverbα exhibit enhanced fat storage without the predicted increased WAT inflammation or loss of insulin sensitivity. In contrast to most animal models of obesity and obese human patients, Reverbα(-/-) mice exhibit elevated serum adiponectin levels and increased adiponectin secretion from WAT explants in vitro, highlighting a potential anti-inflammatory role of this adipokine in hypertrophic WAT. Indeed, adiponectin was found to suppress primary macrophage responses to lipopolysaccharide and proinflammatory fatty acids, and this suppression depended on glycogen synthase kinase 3β activation and induction of A20. Attenuated inflammatory responses in Reverbα(-/-) WAT depots were associated with tonic elevation of A20 protein and ex vivo shown to depend on A20. We also demonstrate that adipose A20 expression in obese human subjects exhibits a negative correlation with measures of insulin sensitivity. Furthermore, bariatric surgery-induced weight loss was accompanied by enhanced WAT A20 expression, which is positively correlated with increased serum adiponectin and improved metabolic and inflammatory markers, including C-reactive protein. The findings identify A20 as a mediator of adiponectin anti-inflammatory action in WAT and a potential target for mitigating obesity-related pathology.

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Figures

Figure 1
Figure 1
Phenotype of Reverbα−/− mice. A and B: Body weight (A) and visceral WAT (B) mass of 12-week-old male Reverbα−/− mice and WT littermates maintained on an NC diet (n = 16–20/group). C: Histological sections of WAT from WT and Reverbα−/− mice fed NC and WT mice fed HFD to induce DIO. iNOS immunoreactivity demonstrates characteristic macrophage cuffs in WAT of WT DIO mice, which are virtually absent in Reverbα−/− mice despite their obese phenotype. D and E: Body weight (D) and intraperitoneal glucose tolerance test results (E) of WT and Reverbα−/− mice fed NC or HFD for 10 weeks (n = 5–6/group). F and G: Serum leptin (F) and adiponectin (G) concentrations of 12-week-old Reverbα−/− mice were significantly higher than in WT controls (n = 18–21/group) and positively correlated with adipose tissue mass (P < 0.05, Pearson correlation). H: ELISA analysis of serum adiponectin in WT and Reverbα−/− mice fed NC vs. HFD. I: Immunoblot analysis of monomeric (top) and multimeric (bottom) adiponectin in sera of WT and Reverbα−/− mice (black arrowhead, high molecular weight; white arrowhead, hexamer; gray arrowhead, trimer). J: ELISA analysis of high-molecular-weight adiponectin in sera of WT and Reverbα−/− mice expressed as a ratio of total adiponectin. Data are mean ± SEM; statistical significance was determined using Student t test or one-way ANOVA with Bonferroni post hoc test (C). *P < 0.05, **P < 0.01, ***P < 0.001. Adn, adiponectin; BW, body weight; HMW, high molecular weight. (A high-quality color representation of this figure is available in the online issue.)
Figure 2
Figure 2
Adiponectin-induced cross-tolerance to endotoxin is mediated by A20. A: Quantitative RT-PCR analysis of IL6 and TNFα induction in macrophages pretreated with adiponectin for the indicated times. B: Immunoblot analysis of A20 expression in murine primary macrophages cultured for the indicated times with 3 μg/mL adiponectin. C: Quantitative RT-PCR analysis of A20 expression in primary murine macrophages pretreated with adiponectin (3 μg/mL, 6 h) and challenged with LPS (100 ng/mL, 4 h). D: Quantitative RT-PCR and immunoblot analyses of A20 expression of primary murine macrophages transfected with control or A20-specific siRNA. E: Quantitative RT-PCR analysis of macrophages transfected with control or A20-specific siRNA and subject to adiponectin (3 μg/mL, 18 h) pretreatment, followed by LPS challenge (100 ng/mL, 4 h). F: Immunoblot analysis of cytosolic (left) and nuclear (right) extracts of primary macrophages treated with 3 μg/mL adiponectin for the indicated times. G–I: Quantitative RT-PCR analysis of primary murine macrophages treated with vehicle control [dimethyl sulfoxide] or 50 μmol/L SB216763 and stimulated with 3 μg/mL adiponectin (G and H) or pretreated with adiponectin (3 μg/mL, 18 h) before LPS challenge (100 ng/mL, 4 h) (I). Data are mean ± SEM from three independent experiments normalized to mouse 18S rRNA control and fold change relative to control untreated cells. Statistical significance was determined using the Student t test (D: ***P < 0.001), two-way ANOVA (E: *P < 0.05, **P < 0.01, ***P < 0.001 A20 siRNA vs. control siRNA; ###P < 0.001 adiponectin pretreatment vs. no adiponectin treatment), or one-way ANOVA (A,F and I: *P < 0.05, **P < 0.01, ***P < 0.001 adiponectin pretreatment vs. no adiponectin treatment) with Bonferroni post hoc test. Adn, adiponectin; Nucl., nuclear; SB, SB216763; siA20, A20 siRNA; siCtrl, control siRNA.
Figure 3
Figure 3
A20 expression is increased in Reverbα−/− WAT. A: Representative immunoblot of A20 in WAT from NC- and HFD-fed Reverbα−/− and WT control mice (n = 6/group). B: ELISA of adiponectin in culture supernatants of WAT explants from Reverbα−/− mice and WT controls. C: Quantitative RT-PCR analysis of IL6, TNFα, and A20 in WAT explants from Reverbα−/− mice and their WT controls in both an unstimulated and an LPS-stimulated (100 ng/mL, 4 h) state. D and E: Quantitative RT-PCR analysis of A20 expression in WAT explants treated with adiponectin (3 μg/mL, 2 h) (D) or IL6, TNFα, and A20 expression in WAT subject to adiponectin pretreatment (3 μg/mL, 18h) prior to LPS challenge (100 ng/mL, 4h) (E). Data are mean ± SEM from three independent experiments normalized to mouse 18S rRNA control and fold-change relative to WT control samples (C) or control treated (D and E). Statistical significance was determined using Student t test (B and D: *P < 0.05, ***P < 0.001), one-way ANOVA (E, ***P < 0.001 control vs. treatment) or two-way ANOVA (C: *P < 0.05, **P < 0.01, ***P < 0.001 control vs. LPS treatment; ##P < 0.01, ###P < 0.001 genotype difference ) with Bonferroni post hoc test. Adn, adiponectin; nd; not detected.
Figure 4
Figure 4
Weight loss following bariatric surgery is accompanied by increased expression of A20 in WAT. A: Average patient BMI pre- and postbariatric surgery (n = 12). B–I: Serum metabolic and inflammatory markers in patients pre- and postbariatric surgery (n = 12). J: Quantitative RT-PCR analysis of A20 expression in WAT biopsy specimens from patients pre- and postbariatric surgery (n = 12). K and L: Serum adiponectin and WAT A20 expressions correlated with HOMA-IR from patients prebariatric surgery (P < 0.05, Pearson correlation). M: WAT A20 expression correlated with serum adiponectin in patients both pre- and postbariatric surgery (P < 0.05, Pearson correlation). Data are mean ± SEM normalized to human 18S rRNA control and fold change relative to presurgery samples. Statistical significance was determined using the paired t test: *P < 0.05, **P < 0.01, ***P < 0.001.
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