Abstract
In obesity, inflammation of white adipose tissue (AT) is associated with diminished generation of beige adipocytes ('beige adipogenesis'), a thermogenic and energy-dissipating function mediated by beige adipocytes that express the uncoupling protein UCP1. Here we delineated an inflammation-driven inhibitory mechanism of beige adipogenesis in obesity that required direct adhesive interactions between macrophages and adipocytes mediated by the integrin α4 and its counter-receptor VCAM-1, respectively; expression of the latter was upregulated in obesity. This adhesive interaction reciprocally and concomitantly modulated inflammatory activation of macrophages and downregulation of UCP1 expression dependent on the kinase Erk in adipocytes. Genetic or pharmacological inactivation of the integrin α4 in mice resulted in elevated expression of UCP1 and beige adipogenesis of subcutaneous AT in obesity. Our findings, established in both mouse systems and human systems, reveal a self-sustained cycle of inflammation-driven impairment of beige adipogenesis in obesity.
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Acknowledgements
We thank S. Grossklaus, B. Gercken, M. Prucnal and K. Bär for technical assistance; T. Yednock for discussions; and C. Ballantyne (Baylor College of Medicine) for αL-integrin-deficient mice. Supported by the German Center for Diabetes Research (T.C.), Deutsche Forschungsgemeinschaft (CH279/5-1 to T.C.), the European Research Council (DEMETINL to T.C.) and the US National Institutes of Health (DE024716 to G.H.; and DE026152 to G.H. and T.C.).
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K.-J.C. designed and performed experiments, analyzed and interpreted data and wrote the paper; A.C. performed experiments, analyzed and interpreted data and wrote the paper; M.E., R.G.-M., V.I.A., I.M., M.N., J.G., J.P. and J.-H.L. performed experiments and analyzed data; T.Z., S.E.G., K.P.K. and T.P. participated in experimental design and discussion; T.P. provided mice with loxP-flanked Itga4; M.B. performed research, analyzed and interpreted data; G.H. participated in experimental design and edited the paper; and T.C. designed the study and wrote the paper.
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S.E.G. is a former employee of ELAN Pharmaceuticals and Biogen Idec. The ELND002 inhibitor of α4 integrin was produced by ELAN Pharmaceuticals and was provided by ELAN Pharmaceuticals and by Biogen Idec.
Integrated supplementary information
Supplementary Figure 1 α4 integrin expression in monocytes and macrophages from Cre+α4f/f and Cre−α4f/f mice.
a) Depicted is a representative histogram of flow cytometry analysis for α4 integrin expression in isolated bone marrow monocytes (CD11b+Ly6G−) from Cre−α4f/f and Cre+α4f/f mice, 2 weeks after poly-(I:C) injection. b) PBMC were isolated from blood of obese Cre+α4f/f (n=6 mice) and Cre−α4f/f mice (n=7 mice) and α4 integrin expression in monocytes (CD11b+Ly6G− cells) was analyzed by flow cytometry. The percentage of α4 integrin-positive monocytes is depicted. c) Stromal vascular fraction (SVF) cells from SAT of obese Cre+α4f/f (n=3 mice) and Cre−α4f/f (n=4 mice) mice were isolated and α4 integrin expression in macrophages (MΦ; defined as F4/80+CD11b+) was analyzed by flow cytometry. The percentage of α4 integrin-positive macrophages is depicted.
Data are presented as mean ± SEM. Mann-Whitney U-test in (b) and Student's t-test in (c); data in (b) are pooled from 2 experiments; data in (c) are from one experiment.
Supplementary Figure 2 Metabolic parameters of obese Cre+α4f/f and Cre−α4f/f mice.
a) The weights of SAT, VAT and liver of obese Cre+α4f/f and Cre−α4f/f mice (fed a HFD for 20 weeks) are shown (n=8 Cre+α4f/f mice and n=9 Cre−α4f/f mice). b) Quantification of adipocyte cell diameter of SAT (n=5 mice/group) and VAT (n=4 mice/group) and fitting curve of obese Cre+α4f/f (black line) and Cre−α4f/f mice (grey line). Left: quantification of adipocyte cell size and fitting curve is shown. Right: Mean adipocyte diameter is shown. c) Fasting blood levels of glucose (Glu), triglycerides (TG), and cholesterol (Chol) from obese Cre+α4f/f and Cre−α4f/f mice are shown (n=14 Cre+α4f/f mice and n=15 Cre−α4f/f mice). Data are presented as mean ± SEM. *P < 0.05. Student's t-test in (a) and (c), Mann-Whitney U-test in (b). Data in (a) are pooled from 3 experiments; data in (b) are from one experiment; data in (c) are pooled from 5 experiments.
Supplementary Figure 3 Crown like structures (CLSs), non–CLS-associated macrophages and macrophage–adipocyte contact area in the SAT of obese Cre+α4f/f and Cre−α4f/f mice.
a ) Representative sections demonstrating macrophage staining (F4/80 staining) in SAT from obese Cre+α4f/f and Cre−α4f/f mice are shown. Scale bar is 100μm. b) Quantification of CLS and non-CLS-associated macrophages in SAT from obese Cre+α4f/f (n=5 mice) and Cre−α4f/f (n=5 mice) mice is depicted. Shown are the number of CLS per 100 mm2 of tissue and the number of non-CLS-associated macrophages per 100 adipocytes. c) The surface area of macrophages in contact with adipocytes from the SAT of obese Cre+α4f/f (n=5 mice) and Cre−α4f/f (n=5 mice) mice was calculated. Data are expressed as μm2 of contact area per macrophage.
Data are presented as mean ± SEM. *P < 0.05 Student's t-test in (b), Mann-Whitney U in (c). In (a) representative histological images are from analysis performed on 5 mice per genotype. Data in (b)-(c) are from one experiment.
Supplementary Figure 4 α4-integrin-dependent adhesive interactions between macrophages and adipocytes in the obese SAT.
Immunofluorescence analysis for macrophages (F4/80, green), adipocytes (caveolin-1, red) and DAPI (blue) was performed in the SAT of obese Cre−α4f/f and obese Cre+α4f/f mice. (a) Representative 3D-reconstruction of a macrophage-adipocyte interaction in the SAT of an obese Cre−α4f/f mouse is shown. The white line shows the position of the sectional plane depicted in (b). Panel (b) depicts a 70μm long segment of the sectional plane shown in (a) containing the macrophage-adipocyte contact area. (c) Representative 3D-reconstruction of a macrophage-adipocyte interaction in the SAT of an obese Cre+α4f/f mouse is shown. The white line shows the position of the sectional plane depicted in (d). Panel (d) depicts a 70μm long segment of the sectional plane shown in (c) containing the macrophage-adipocyte contact area. Representative images are from analysis performed on 5 mice per genotype.
Supplementary Figure 5 Netrin-1 expression of SAT and VAT from obese Cre+α4f/f and Cre−α4f/f mice.
a-b) Ntn1 mRNA expression in (a) SAT and (b) VAT of obese Cre+α4f/f and Cre−α4f/f mice was evaluated by qPCR (n= 9 Cre+α4f/f mice and n=11 Cre−α4f/f mice in SAT, n= 8 Cre+α4f/f mice and n= 8 Cre−α4f/f mice in VAT). 18S expression was used for normalization; the Ntn1 expression of obese Cre−α4f/f mice was set as 1 in each case.
Data are presented as mean ± SEM. Mann-Whitney U-test in (a), (b). Data in (a), (b) are pooled from 3 experiments.
Supplementary Figure 6 Food intake and analysis of the VAT and BAT of obese Cre+α4f/f and Cre−α4f/f mice.
a) Food intake of obese Cre+α4f/f and Cre−α4f/f mice was assessed in metabolic cages over 72 h. Average food intake in the light or dark period per day is shown (n=3 Cre+α4f/f mice and n=5 Cre−α4f/f mice). b-c) Obese Cre+α4f/f or Cre−α4f/f mice were challenged with a temperature of 4°C for 12h. b) Gene expression in VAT and BAT upon cold exposure was evaluated by qPCR (n=4 Cre+α4f/f mice and n=6 Cre−α4f/f mice). 18S expression was used for normalization of mRNA expression and the respective gene expression of obese Cre−α4f/f mice was set as 1. c) Representative sections of UCP1 staining in BAT from obese Cre+α4f/f or Cre−α4f/f mice exposed to cold.
Data are presented as mean ± SEM. *P < 0.05. ANCOVA in (a) and Mann-Whitney U-test in (b). Data in (a)–(b) are from one experiment. In (c) representative images are from analysis performed on 5 Cre−α4f/f mice and 4 Cre+α4f/f mice.
Abbreviations: Ucp1, uncoupling protein 1; Cidea, cell death-inducing DNA fragmentation factor-like effector A; Ppargc1α, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; Prdm16, PR (PRD1-BF1-RIZ1 homologous)-domain containing 16; Cox8b, cytochrome c oxidase subunit 8b; Dio2, type 2 deiodinase; Elovl3, Elongation of very long chain fatty acids protein 3
Supplementary Figure 7 Blocking α4 integrin improves insulin sensitivity in ob/ob mice.
Ob/Ob mice were implanted with an Alzet osmotic pump including α4-inhibitor (α4-inh.) or PBS (Con). a) Insulin tolerance test (ITT) from control- or α4-inhibitor-treated mice 6 weeks after pump implantation is shown (Con, n=6 mice; α4-inh., n=5 mice). b) Core temperature of control- or α4-inhibitor-treated mice is shown (Con, n=6 mice; α4-inh., n=5 mice). c) Representative cropped blot images showing immunoblotting for UCP1 (and vinculin) in SAT of 2 control- and 2 α4-inhibitor-treated ob/ob mice. Densitometric analysis of UCP1 immunobloting from a total of 5 control- and 5 α4-inhibitor-treated mice is shown. The protein amounts of UCP1 were normalized against vinculin and the UCP1 amounts (normalized over vinculin) in SAT from control-treated mice were set as 1. d) The number of pro-inflammatory macrophages (defined as F4/80+CD11b+CD11c+CD206−) from SAT of control- or α4-inhibitor-treated ob/ob mice was analyzed by flow cytometry. Data are presented as relative to control; cells/gram tissue from control-treated mice was set as the 100% (n=5 mice per group).
Data are presented as mean ± SEM. *P < 0.05. Mann-Whitney U-test in (a), (d), Student's t-test in (b), (c). Data in (a), (b) are representative of 2 experiments; data in (c), (d) are from one experiment.
Supplementary Figure 8 T3- and norepinephrine-dependent upregulation of Ucp1.
Primary SAT adipocytes were treated in the absence (Con) or presence of T3 and norepinephrine (NE/T3) for 3 hours and the mRNA expression of Ucp1 was detected by qPCR. 18S expression was used for normalization and the gene expression of Ucp1 in the absence of NE/T3 was set as 1. Shown are data from n=4 separate primary cell isolations.
Data are mean ± SEM. *P < 0.05. Mann-Whitney U-test was used. Data are representative of 2 experiments.
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Chung, KJ., Chatzigeorgiou, A., Economopoulou, M. et al. A self-sustained loop of inflammation-driven inhibition of beige adipogenesis in obesity. Nat Immunol 18, 654–664 (2017). https://doi.org/10.1038/ni.3728
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DOI: https://doi.org/10.1038/ni.3728
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