NFATc3 deficiency reduces the classical activation of adipose tissue macrophages

in Journal of Molecular Endocrinology
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Nuclear factors of activated T cells (NFAT) c3 have a prominent role in the regulation of proinflammatory factors in immune cells. The classically activated M1 macrophages are key players in the initiation and maintenance of adipose tissue (AT) inflammation. The role of NFATc3 in obesity and AT inflammation is unknown. We set out to determine how deficiency of NFATc3 effected macrophage polarization, inflammation and insulin resistance in visceral AT of high-fat diet (HFD)-fed mice. Nfatc3−/− and WT mice were fed a HFD for 8–17 weeks. Epididymal white AT (eWAT) F4/80(+) cells were characterized by fluorescence-activated cell sorting and quantitative RT-PCR. Results showed that Nfatc3−/− mice developed HFD-induced obesity similar to WT mice, but insulin sensitivity and glucose tolerance were improved, and liver fat accumulation was reduced in Nfatc3−/− mice compared to WT control mice. Moreover, M1 macrophage content and proinflammatory factors were reduced, whereas the alternatively activated M2 macrophage content was increased in eWAT of HFD-fed Nfatc3−/− mice compared to that of WT mice. In addition, eWAT insulin signaling was improved in HFD-fed Nfatc3−/− mice. Importantly, after bone-marrow-derived macrophages had been isolated from Nfatc3−/− mice and cultured in vitro, treatment of these cells with interferon-γ and lipopolysaccharide resulted in reduction of M1 inflammatory markers, suggesting that NFATc3 promoted M1 polarization by a cell-autonomous mechanism. The results demonstrated that NFATc3 played an important role in M1 macrophage polarization, AT inflammation and insulin resistance in response to obesity through transcriptional activation of proinflammatory genes.

 

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    Nfatc3−/− mice were protected from HFD-induced insulin resistance. (A and C) Nfatc3−/− mice and WT counterparts were fed a HFD starting at an age of 6 weeks. Body weights were measured every week; the gained body weights were calculated, which were not significant different between WT and Nfatc3−/− mice. (B) Representative images of mice after a HFD feeding for 16 weeks. (D) At 16 weeks of HFD, body fat accumulation was measured by nuclear magnetic resonance and shown as ratio of total body weight, and there were no significant differences between WT and Nfatc3−/− mice. (E) Glucose tolerance test. Glucose levels were measured after i.p. injection of 2 g/kg glucose. **P < 0.01 vs Nfatc3+/+. n = 6–8 per group from two independent experiments. (F) Insulin tolerance test. Glucose levels were measured after i.p. injection of 0.75 U/kg insulin. *P < 0.05 or **P < 0.01 vs Nfatc3+/+. n = 6-8 per group from two independent experiments.

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    Reduced adipose tissue inflammation and M1 macrophage content in Nfatc3−/− mice on HFD. (A) Representative images of epididymal fat pad (eWAT) from WT and Nfatc3−/− mice on HFD for 16 weeks were shown. (B) eWAT was weighed and the fat-to-body weight ratio was not significantly different between WT and Nfatc3−/− mice. (C, D and E) Gene expressions of MCP-1, IL-1β and TNFα in adipose tissue from HFD-fed Nfatc3−/− and Nfatc3+/+ mice were analyzed by real-time quantitative RT-PCR. *P < 0.05 vs WT mice. n = 3 per group. (F) The IFNγ concentrations in cultured eWAT supernatants were measured with a mouse IFNγ ELISA kit. (G) Insulin-stimulated phosphorylation of AKT in the adipose tissue of Nfatc3−/− and WT mice was determined by Western blot analysis. (H) The relative phosphorylation levels of AKT shown in (G) were quantified. *P < 0.05 or **P < 0.01 vs WT mice. n = 3 per group. A full color version of this figure is available at https://doi.org/10.1530/JME-18-0070.

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    Reduced M1 ATMs and increased M2 ATMs in Nfatc3−/− mice fed a HFD. (A and B) ATMs contents were assessed by flow cytometry in SVF cells obtained from eWAT of HFD-fed Nfatc3−/− and WT mice. Cells were gated for F4/80+ ATMs and analyzed for expression of markers of M1 (CD11c) and M2 (CD206) ATMs. (C and D) Data shown as percentage of CD45+ cells or total live SVF cells. *P < 0.05 or **P < 0.01 vs WT mice. n = 3–4 per group. (E) Expression analysis of inflammatory genes in adipose tissue from Nfatc3−/− and WT mice both on HFD. SVF cells obtained from eWAT of HFD-fed mice were analyzed by real-time quantitative RT-PCR. *P < 0.05 vs Nfatc3+/+. n = 3 per group. (F and G) Cells were gated for CD4+ T cells and analyzed for the expression of IFNγ for CD4+ Th1 cells (F) and for the expression of IL-4 for CD4+ Th2 cells (G).

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    Nfatc3−/− mice were protected from obesity-induced hepatic steatosis and inflammation. (A) Representative images of the livers from WT and Nfatc3−/− mice on HFD for 16 weeks were shown. Hematoxylin and eosin (H&E)–stained sections (B) and oil red O staining (C) from the liver of HFD-fed Nfatc3−/− and HFD-fed WT mice. Representative images were shown at 20× magnification. Similar results were seen in four independent samples. (D and E) Gene expressions of MCP-1 and TLR4 in the liver of HFD-fed Nfatc3−/− and HFD-fed WT mice were analyzed by real-time quantitative RT-PCR. *P < 0.05 vs Nfatc3+/+. n = 3 per group.

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    BMDMs from Nfatc3−/− mice were more inclined to differentiate into M2 macrophages. Bone-marrow-derived macrophages (BMDMs) from normal diet-fed Nfatc3−/− and WT mice were differentiated into M1 or M2 lineage. Markers of M1 (A) or M2 (B) macrophages were analyzed by real-time quantitative RT-PCR. *P < 0.05 or **P < 0.01 vs Nfatc3+/+. n = 3 per group.

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