Macrophage-Specific Progranulin Deficiency Prevents Diet-Induced Obesity through the Inhibition of Hypothalamic and Adipose Tissue Inflammation

Abstract

Backgruound: Chronic low-grade inflammation in multiple metabolic organs contributes to the development of insulin resistance induced by obesity. Progranulin (PGRN) is an evolutionarily-conserved secretory protein implicated in immune modulation. The generalized deletion of the PGRN-encoded Grn gene improves insulin resistance and glucose intolerance in obese mice fed a high-fat diet (HFD). However, it remains unclear which cells or organs are responsible for the beneficial metabolic effect of Grn depletion.

Methods: Considering the critical role of macrophages in HFD-induced obesity and inflammation, we generated mice with a macrophage-specific Grn depletion (Grn-MΦKO mice) by mating lysozyme M (LysM)-Cre and Grn-floxed mice. Body weight, food intake, energy expenditure, and glucose and insulin tolerance were compared between Grn-MΦKO mice and their wildtype (WT) controls under normal chow diet (NCD)- or HFD-fed conditions. We also examined macrophage activation and inflammation- related gene expression in the visceral adipose tissue and hypothalamus along with insulin and leptin signaling.

Results: Grn-MΦKO mice showed no alteration in metabolic phenotypes under NCD-fed conditions. However, upon HFD feeding, these mice exhibited less weight gain and improved glucose and insulin tolerance compared to WT mice. Moreover, HFD-induced macrophage activation and proinflammatory cytokine expression were significantly reduced in both the adipose tissue and hypothalamus of Grn-MΦKO mice, while HFD-induced impairments in leptin and insulin signaling showed improvement.

Conclusion: Macrophage-derived PGRN and possibly other Grn products play a critical role in the development of HFD-induced obesity, tissue inflammation, and impaired hormonal signaling in both central and peripheral metabolic organs.

Keywords: Adipose tissue; Hypothalamus; Inflammation; Macrophages; Metabolic diseases; Obesity; Progranulins.

Fig. 1.

Macrophage Grn deficiency does not cause alterations to energy or glucose metabolism under normal diet conditions. (A, B) Comparison of body weights and fat and lean masses between normal chow diet (NCD)-fed Grn-wild-type (WT) (Grn^f/f) mice and macrophage-specific Grn depletion (Grn-MΦKO) mice (n=4). (C) Average daily food intake values from 5 to 20 weeks of age (n=4). (D) Energy expenditure (EE) curves and average values during the day and night measured at 15 weeks of age (n=4). (E) Respiratory exchange ratio (RER) measured at 15 weeks of age (n=4). (F, G) Blood glucose levels and area under the curve (AUC) values during glucose and insulin tolerance tests (GTT and ITT) conducted at 14 weeks of age (n=4). Results are presented as a mean±standard error of the mean. NS, not significant.

Fig. 2.

Macrophage Grn deficiency prevents obesity and improves glucose metabolism under high-fat diet (HFD) conditions. (A, B) Body weights and fat and lean masses in HFD-fed Grn-wild-type (WT) (Grn^f/f) mice and macrophage-specific Grn depletion (Grn-MΦKO) mice (n=5). (C) Average daily food intake from 5 to 20 weeks of ages (n=5). (D) Energy expenditure (EE) in Grn-WT (Grn^f/f) mice and Grn-MΦKO mice fed an HFD for 15 weeks (n=4). (E, F) mRNA expression levels of thermogenic genes in the brown adipose tissue (BAT) and inguinal white adipose tissues (iWAT) of HFD-fed Grn-WT and Grn-MΦKO mice (n=4). (G, H) Glucose and insulin tolerance tests (GTT and ITT) conducted after 14 weeks of HFD feeding (n=5). Results are presented as a mean±standard error of the mean. NS, not significant; Ucp1, uncoupling protein 1; Prdm16, PR domain containing 16; Pgc1α, Pparg coactivator 1 alpha; Dio2, iodothyronine deiodinase 2; AUC, area under the curve. ^aP<0.05, ^bP<0.01, ^cP<0.001 between indicated groups.

Fig. 3.

High-fat diet (HFD)-induced changes in the adipose tissues are fully suppressed by macrophage Grn deficiency. (A) Histological analysis of the epididymal while adipose tissues of normal chow diet (NCD)-fed Grn-wild-type (WT) mice and HFD (10 weeks)-fed Grn-WT and macrophage-specific Grn depletion (Grn-MΦKO) mice. Measurements of adipocyte sizes and numbers of crown-like structure (CLS) formations (n=6). Scale bars, 200 μm. (B) Fluorescence-activated cell sorting analysis of stromal vascular fractions of the epididymal adipose tissues showing the M1 and M2 polarization of adipose tissue macrophages (ATMs) in NCDPGRN⁺or HFD (10 weeks)-fed Grn-WT and HFD-fed Grn-MΦKO mice (n=3–4). (C) Quantitative polymerase chain reaction analysis of inguinal white adipose tissues (n=6). (D) Enzyme-linked immunosorbent assay (ELISA) of plasma interleukin 6 (IL-6) levels (n=4–7). (E) Immunoblotting analysis of Akt phosphorylation in the adipose tissue in response to intraperitoneal insulin injection (n=3–4). Results are presented as a mean±standard error of the mean. C/ebpα, CCAAT enhancer binding protein α; Pparγ, peroxisome proliferator-activated receptor γ; Fabp4, fatty acid binding protein 4; Glut4, glucose transporter 4; Tnfα, tumor necrosis factor-α; Ccl2, C-C motif chemokine ligand 2; NS, not significant. ^aP<0.05, ^bP<0.01, ^cP<0.001 between indicated groups.

Fig. 4.

Reduced inflammation and improved leptin sensitivity in the hypothalamus of macrophage Grn-deficient mice. (A) Green fluorescence protein (GFP) immunostaining showing GFP-labeled lysozyme M (LysM)⁺ macrophages in the hypothalamic arcuate nucleus (ARC) of LysM-Cre;GFP mice on an normal chow diet (NCD) and LysM-Cre;GFP and macrophage-specific Grn depletion (Grn-MΦKO);GFP mice on an high-fat diet (HFD) for 10 weeks (n=4). Scale bars, 50 μm. (B) Ionized calcium-binding adaptor molecule 1 (Iba1) immunostaining showing changes in the hypothalamic microglia (n=4). Scale bars, 50 μm. (C) Glial fibrillary acidic protein (GFAP) immunostaining showing changes in the hypothalamic astrocytes (n=4). Scale bars, 50 μm. (D) Quantitative polymerase chain reaction analysis of proinflammatory cytokines (interleukin 1β [Il-1β], Il-6, tumor necrosis factor-α [Tnfα]) and anti-inflammatory cytokines (Il-4, Il-10) in the mediobasal hypothalamus under 10-week HFD-fed conditions (n=6). (E) Food intakes for 24 hours after the intracerebroventricular administration of leptin (1 μg) in 8-week HFD-fed mice (n=4–5). (F) Determination of hypothalamic leptin signaling via the immunoblotting of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and total STAT3 at 45 minutes following the intracerebroventricular injection of saline or leptin (n=3). Results are presented as a mean±standard error of the mean. NS, not significant. ^aP<0.05, ^bP<0.01, ^cP<0.001 between indicated groups.