Downregulation of macrophage Irs2 by hyperinsulinemia impairs IL-4-indeuced M2a-subtype macrophage activation in obesity

Abstract

M2a-subtype macrophage activation is known to be impaired in obesity, although the underlying mechanisms remain poorly understood. Herein, we demonstrate that, the IL-4/Irs2/Akt pathway is selectively impaired, along with decreased macrophage Irs2 expression, although IL-4/STAT6 pathway is maintained. Indeed, myeloid cell-specific Irs2-deficient mice show impairment of IL-4-induced M2a-subtype macrophage activation, as a result of stabilization of the FoxO1/HDAC3/NCoR1 corepressor complex, resulting in insulin resistance under the HF diet condition. Moreover, the reduction of macrophage Irs2 expression is mediated by hyperinsulinemia via the insulin receptor (IR). In myeloid cell-specific IR-deficient mice, the IL-4/Irs2 pathway is preserved in the macrophages, which results in a reduced degree of insulin resistance, because of the lack of IR-mediated downregulation of Irs2. We conclude that downregulation of Irs2 in macrophages caused by hyperinsulinemia is responsible for systemic insulin resistance via impairment of M2a-subtype macrophage activation in obesity.

Fig. 1

MΦ Irs2 deficiency caused inflammation and insulin resistance in the liver and WAT under a HF diet. a Phosphorylation and protein levels of STAT6 and Irs2 in peritoneal MΦs from NC and HF diet-fed mice (n = 4). b, c Expression levels of Irs2 mRNA in the siglecF^-CD11b⁺F4/80⁺cells of the SVF of the adipose tissue from ob/ob and HF diet-fed mice (n = 4). d Glucose tolerance test in the MIrs2KO mice (n = 7–14). e GIR, EGP and Rd in the MIrs2KO mice in the hyperinsulinemic-euglycemic clamp study (n = 9–10). f Phosphorylation levels of Akt (ser473) in the liver, WAT and skeletal muscle of the MIrs2KO mice after insulin infusion (n = 3–4). g Adipocyte cell size in the MIrs2KO mice (n = 6). h F4/80-positive CLS in the WAT of MIrs2KO mice (scale bar, 200 μm) (n = 6). i Quantitative RT-PCR analysis of the genes encoding inflammatory cytokines in the WAT of the MIrs2KO mice (n = 6). The data are mean ± SEM. followed by one-way ANOVA with a post hoc test or Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 2

IL-4-induced M2a-subtype MΦ activation was impaired in WAT of the HF diet-fed MIrs2KO mice. a The percentages of siglecF^-CD11b⁺F4/80⁺, siglecF^-CD11b⁺F4/80⁺CD11c⁺CD206^- (M1-type MΦs) and siglecF^-CD11b⁺F4/80⁺CD11c^-CD206⁺ (M2-type MΦs) cells in the SVF of the adipose tissue (n = 10–14). b Expression levels of M2-type and M1-type MΦ marker genes in the siglecF^-CD11b⁺F4/80⁺ cells in the SVF of the adipose tissue from the control and MIrs2KO mice (n = 10–14). c Expression levels of M2a-subtype MΦ marker genes in the BMDM of the control and MIrs2KO mice after IL-4 stimulation for 48 h (n = 6–8). d Arginase activity in the BMDM of the control and MIrs2KO mice after IL-4 stimulation for 24 h (n = 7–10). e Expression levels of the M1-type MΦ marker genes in the BMDM of the control and MIrs2KO mice after LPS stimulation for 48 h (n = 3). f MCP-1, CCR2, IL-18 and IL-6 expression levels in 3T3-L1 cells in co-culture with the BMDM of MIrs2KO mice (n = 12). g Irs2 phosphorylation and protein levels in the BMDM of the control and MIrs2KO mice after IL-4 stimulation (n = 3–4). h Akt phosphorylation and protein levels in the BMDM of the C57BL/6 mice after IL-4 stimulation with or without LY294002 treatment (n = 3). i IL-4-induced Akt phosphorylation in the BMDM of the MIrs2KO mice (n = 4). j Expression levels of the M2a-subtype MΦ marker genes in the BMDM of the C57BL/6 mice after IL-4 stimulation with or without LY294002 treatment (n = 4–6). k Expression levels of the M2a-subtype MΦ marker genes in the BMDM of the C57BL/6 mice after IL-4 stimulation with or without CA-FoxO1 treatment (n = 4–6). l FoxO1 phosphorylation and protein levels in the BMDM of the control and MIrs2KO mice after IL-4 stimulation (n = 3–4). m Expression levels of the M2a-subtype MΦ marker genes in the BMDM of the MIrs2KO mice after IL-4 stimulation with siFoxO1 transfection (n = 5–6). The data are mean ± SEM. followed by one-way ANOVA with a post hoc test or Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 3

FoxO1 bound to HDAC3 and NCoR1 to form a corepressor complex. a Arg1 promoter activity after IL-4 stimulation with or without CA-FoxO1 transfection (n = 4–5). b Mutant A, B, and A plus B Arg1 promoter activities after IL-4 stimulation with or without CA-FoxO1 transfection (n = 5–6). c The binding of DNA nucleotides of the B area of the Arg1 promoter and proteins, and the blocking of the DNA-protein complex formation in a dose-dependent manner by FoxO1, HDAC3 and NCoR1 antibody were determined by EMSA in RAW264.7 cells. d Chip-qPCR using FoxO1, HDAC3 and NCoR1 antibody in the BMDM of the C57BL/6 mice before and after IL-4 stimulation (n = 5–10). e Chip-qPCR using FoxO1, HDAC3 and NCoR1 antibody in the BMDM of the MIrs2KO mice before and after IL-4 stimulation (n = 3–16). f Co-immunoprecipitation (Co-IP) with FoxO1, HDAC3 and NCoR1 antibody without IL-4 stimulation in the Raw 264.7 cells and BMDM. g Expression levels of Arg1 in the BMDM of the C57BL/6 mice after IL-4 stimulation with siHDAC3 or siNCoR1 transfection (n = 3–8). The data are mean ± SEM. followed by one-way ANOVA with a post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 4

MIRKO mice exhibited improved insulin sensitivity and glucose tolerance. a Glucose tolerance test in the MIRKO mice (n = 7–13). b GIR, EGP and Rd in the MIRKO mice in the hyperinsulinemic-euglycemic clamp study (n = 8–9). c Phosphorylation levels of Akt (ser473) in the liver, WAT and skeletal muscle of the MIRKO mice after insulin infusion (n = 3–4). d Adipocyte cell size in the MIRKO mice (n = 4–6). e F4/80-positive CLS in the WAT of the MIRKO mice (scale bar, 200 μm) (n = 4–6). f Quantitative RT-PCR analysis of the genes encoding inflammatory cytokines in the WAT of the MIRKO mice (n = 4–6). g The percentages of siglecF^-CD11b⁺F4/80⁺, siglecF^-CD11b⁺F4/80⁺CD11c⁺CD206^- (M1-type MΦs) and siglecF^-CD11b⁺F4/80⁺CD11c^-CD206⁺ (M2-type MΦs) cells in the SVF of the adipose tissue from the MIRKO mice (n = 4–6). h Expression levels of M2-type and M1-type MΦ marker genes in the siglecF^-CD11b⁺F4/80⁺ cells in the SVF of the adipose tissue from the control and MIRKO mice (n = 4–5). Data are mean ± SEM. followed by one-way ANOVA with a post hoc test or Student’s t test. *P < 0.05; ***P < 0.001

Fig. 5

The Irs2 mRNA levels and number of M2a-subtype MΦs were elevated in HF diet-fed MIRKO mice. a Expression levels of IR, IL-4R, Irs2 and STAT6 in the siglecF^-CD11b⁺F4/80⁺ cells of the SVF of the adipose tissue from the control and MIRKO mice (n = 4–6). b IL-4-induced STAT6, Irs2 and Akt phosphorylation in peritoneal MΦs of HF diet-fed MIRKO mice (n = 3–6). c Irs2 expression levels in the BMDM of the control and MIRKO mice after 100 nM insulin stimulation for 3 h (n = 3–4). d IL-4-induced M2a-subtype marker genes in the BMDM after 100 nM insulin pretreatment for 8 h (n = 3–4). e IL-4-induced Arg1 expression levels in the BMDM pretreated with 100 nM insulin for 8 h after siFoxO1, siNCoR1, or siHDAC3 treatment (n = 4–16). f MCP-1, CCR2, IL-18 and IL-6 expression levels in 3T3-L1 cells in co-culture with BMDM of the C57BL/6 mice and 3T3-L1 cells after insulin stimulation for 24 h (n = 7). The data are mean ± SEM. followed by one-way ANOVA with a post hoc test or Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 6

IL-4-induced M2a-subtype MΦ activation was regulated by both the Irs2 and STAT6 pathways. a Akt phosphorylation and protein levels in the BMDM of the C57BL/6 mice after IL-4 or insulin stimulation (n = 3–4). b Expression levels of the M2a-subtype MΦ marker genes in the BMDM of the C57BL/6 mice after IL-4 or insulin stimulation (n = 4). c STAT6 phosphorylation and protein levels in the BMDM of the C57BL/6 mice after IL-4 or insulin stimulation (n = 3–4). d Expression levels of the M2a-subtype MΦ marker genes in the BMDM of the C57BL/6 mice after IL-4 stimulation following siSTAT6 transfection (n = 5–6). e STAT6 phosphorylation and protein levels in the BMDM of the control and MIrs2KO mice after IL-4 or insulin stimulation (n = 3–4). f STAT6 phosphorylation and protein levels in the BMDM of the C57BL/6 mice after IL-4 stimulation following LY294002 treatment (n = 4). N.D not-detected. The data are mean ± SEM. followed by one-way ANOVA with a post hoc test. *P < 0.05; ***P < 0.001

Fig. 7

Scheme illustrating the mechanism of impaired IL-4-induced M2a-subtype MΦ activation in obesity. IL-4/Irs2/Akt pathway was essential for activation of M2a-subtype MΦs, in addition to IL-4/STAT6 pathway. Both pathways were necessary for the full activation of M2a-subtype MΦs (left panel). In obesity, although IL-4/STAT6 pathway was maintained, IL-4/Irs2/Akt pathway was selectively impaired along with decreased Irs2 expression and stabilization of the FoxO1/HDAC3/NCoR1 corepressor complex in MΦs (right panel)