Myeloid-specific estrogen receptor alpha deficiency impairs metabolic homeostasis and accelerates atherosclerotic lesion development

Abstract

ERα is expressed in macrophages and other immune cells known to exert dramatic effects on glucose homeostasis. We investigated the impact of ERα expression on macrophage function to determine whether hematopoietic or myeloid-specific ERα deletion manifests obesity-induced insulin resistance in mice. Indeed, altered plasma adipokine and cytokine levels, glucose intolerance, insulin resistance, and increased adipose tissue mass were observed in animals harboring a hematopoietic or myeloid-specific deletion of ERα. A similar obese phenotype and increased atherosclerotic lesion area was displayed in LDL receptor-KO mice transplanted with ERα(-/-) bone marrow. In isolated macrophages, ERα was necessary for repression of inflammation, maintenance of oxidative metabolism, IL-4-mediated induction of alternative activation, full phagocytic capacity in response to LPS, and oxidized LDL-induced expression of ApoE and Abca1. Furthermore, we identified ERα as a direct regulator of macrophage transglutaminase 2 expression, a multifunctional atheroprotective enzyme. Our findings suggest that diminished ERα expression in hematopoietic/myeloid cells promotes aspects of the metabolic syndrome and accelerates atherosclerosis in female mice.

Fig. 1.

Hematopoietic/myeloid-specific ERα deletion causes glucose intolerance and insulin resistance in female mice. (A and B) Glucose intolerance in normal chow (NC)-fed BMT-KO vs. BMT-WT and MACER vs. f/f Control mice (n = 8 per group; area under the glucose curve, AUC). (C and D) Reduced insulin-stimulated glucose disposal rate (IS-GDR) in BMT-KO and MACER mice vs. BMT-WT and f/f Control, respectively (n = 6–8 per group). (E) Impaired insulin-stimulated 2-deoxyglucose uptake into soleus muscle in MACER compared with f/f Control (n = 8 per genotype). (F and G) Akt total protein and phosphorylation in insulin-stimulated soleus muscle from f/f Control and MACER, and C2C12 myotubes pretreated with conditioned media from WT or ERαKO BM-Mφ (n = 6 per condition). (H) JNK 1/2 total protein and phosphorylation in quadriceps muscle from BMT-KO vs. BMT-WT (n = 6 per genotype). (I–K) Blunted suppression of HGP (HGP % suppression) by insulin and hepatic steatosis in MACER and BMT-KO compared with f/f Control and BMT-WT mice (n = 6–8 per group). (L and M) JNK1/2 and IKKα/β total protein and phosphorylation in liver samples from BMT-KO and BMT-WT (n = 6 per genotype). *P < 0.05, between genotype difference.

Fig. 2.

Altered circulating adipokines, obesity, and increased atherosclerotic lesion area in female mice with a hematopoietic/myeloid-specific ERα deletion. (A and B) Plasma leptin and PAI-1 levels, (C) Gonadal adipose tissue mass in f/f Control, MACER, BMT-WT, and BMT-KO. (D) Total fat mass for f/f Control vs. MACER. (E) Emr1, CD68, Cd3e, Ccl5, and Ifng expression (n = 6–12 animals per genotype). (F) H&E (10×), F4/80 staining (40×), and adipocyte size in gonadal adipose tissue from NC-fed MACER vs. f/f Control mice (size marker = 100 μm). (G) Insulin-stimulated 2-deoxyglucose uptake and Akt phosphorylation in adipocytes after preconditioning with media from ERα WT or KO BM-Mφ (H) (n = 6). (I) Sudan IV staining of aortas harvested from BMT-WT and BMT-KO mice on LDLR-KO background (n = 12 mice per group). *P < 0.05, between genotype difference.

Fig. 3.

Impaired macrophage function in female mice with hematopoietic/myeloid-specific ERα deletion. Impaired LPS- (A) and OxLDL-induced (B) phagocytosis in BM-Mφ from MACER and BMT-KO vs. WT (n = 4–6 per genotype). (C) Increased basal cholesterol uptake in BM-Mφ from KO vs. WT. (D) Impaired cholesterol efflux to HDL particles in BM-Mφ from KO vs. WT. (E) Estradiol-induced Apoe, Abca1, and Tgm2 expression (relative to basal vehicle-treated) in KO vs. WT BM-Mφ (n = 4–6 per genotype). (F Upper) Schematic of full-length and truncated, 2-kb (ERE replete) and 1-kb (ERE deleted), Tgm2 promoters. (F Lower) Tgm2 promoter activation by estradiol is blunted in the 1-kb promoter lacking the consensus ERE (n = 3 per condition). (G) Estradiol and IL-4–induced Esr1 expression in WT BM-Mφ (n = 6 per condition). (H) Estradiol, IL-4, and combined estradiol + IL-4 induction of IL-4rα expression in WT and KO BM-Mφ (n = 6 per condition). (I) Reduced IL-4 receptor protein and IL-4–induced STAT6 phosphorylation in KO vs. WT BM-Mφ. (J) IL-4–induced expression of transcription factors (Ppard, Pparg, and Ppargc1; arbitrary units, AU), markers of alternative activation (Arg1, Chi3l3, Retnla, and Tgfb1). (K) Tgm2 (expression above basal) in KO vs. WT BM-Mφ assessed by q-PCR (n = 6 per genotype, normalized to 1.0). (L) FACS analysis of cell surface marker fluorescence for MHCII and CD11c in adipose tissue F4/80+ Mφ from vehicle and IL-4–treated f/f Control vs. MACER mice (n = 3 observations per genotype and treatment condition). (M) C¹⁴-labeled palmitate oxidation for KO vs. WT BM-Mφ (n = 6 per genotype). (N–P) Palmitate-induced Ifng, IL-6, and IL-1β gene expression in KO vs. WT BM-Mφ assessed by quantitative PCR (n = 6 per genotype). *P < 0.05, between genotype difference; ¹P < 0.05, difference between treatment condition, within genotype.