Inflammation and adipose tissue macrophages in lipodystrophic mice

Abstract

Lipodystrophy and obesity are opposites in terms of a deficiency versus excess of adipose tissue mass, yet these conditions are accompanied by similar metabolic consequences, including insulin resistance, dyslipidemia, hepatic steatosis, and increased risk for diabetes and atherosclerosis. Hepatic and myocellular steatosis likely contribute to metabolic dysregulation in both states. Inflammation and macrophage infiltration into adipose tissue also appear to participate in the pathogenesis of obesity-induced insulin resistance, but their contributions to lipodystrophy-induced insulin resistance have not been evaluated. We used aP2-nSREBP-1c transgenic (Tg) mice, an established model of lipodystrophy, to ask this question. Circulating cytokine elevations suggested systemic inflammation but even more dramatic was the number of infiltrating macrophages in all white and brown adipose tissue depots of the Tg mice; in contrast, there was no evidence of inflammatory infiltrates or responses in any other tissue including liver. Despite there being overt evidence of adipose tissue inflammation, antiinflammatory strategies including salicylate treatment and genetic suppression of myeloid NF-kappaB signaling that correct insulin resistance in obesity were ineffective in the lipodystrophic mice. We further showed that adipose tissue macrophages (ATMs) in lipodystrophy and obesity are very different in terms of activation state, gene expression patterns, and response to lipopolysaccharide. Although ATMs are even more abundant in lipodystrophy than in obesity, they have distinct phenotypes and likely roles in tissue remodeling, but do not appear to be involved in the pathogenesis of insulin resistance.

Fig. 1.

Circulating cytokines and gene expression. (A) Circulating cytokine and chemokine concentrations WT control (open bars) vs. Tg (filled bars) littermates. (B, C) mRNA expression in epididymal (epi) WAT; s.c. (sc) WAT; interscapular BAT, liver, muscle, and spleen. (n = 6) *P < 0.05 WT vs. Tg.

Fig. 2.

Adipose tissue histology and FACS analysis. (A–F) Histological sections from epi (A, C) and s.c. (B, D) WAT, and BAT (E, F) from representative 24-week-old WT control and Tg littermates stained with H&E. (G–I) mRNA expression from epi (G) and sc (H) WAT, and BAT (I) from WT control (open bars) vs. Tg (filled bars) littermates (n = 6). (J–O) Immunohistochemical localization of macrophages in epi (J, L), sc (K, M), and BAT (N, O) of a representative WT control vs. Tg littermates. (P) Counting of crown-like structures (CLS) per high power field (HPF) from the above male WT control and Tg littermates (10 HPF counted per mouse, n = 5 mice per genotype). (Q) Flow cytometry analyses of macrophage number from epi, sc, BAT, and spleen WT controls vs. Tg littermates. n = 4, *P < 0.05 WT vs. Tg.

Fig. 3.

Antiinflammatory strategies. (A–D) Sodium salicylate treatment of lipodystrophic mice. WT control (open bars) and Tg (filled bars) littermates were fed normal chow (control) or chow containing 4 g/kg sodium salicylate for 5 weeks. (E–H) C57BL/6J male mice treated with chow (open bars) and HFD (gray bars) for 10 weeks or HFD for 5 weeks followed by HFD plus salicylate for another 5 weeks (black bars). (I–L) Myeloid-selective deletion of IKKβ in Tg male mice. WT control (open bars), Tg (gray bars), and Tg/Ikkβ^Δmye (black bars) littermates were fed normal chow. (M–P) WT control male mice treated with chow (open bars) or with HFD (gray bars) and Ikkβ^Δmye mice treated with HFD (black bars) for 6 weeks. Body weights (A, E, I, M), fasting blood glucose (B, F, J, N), fasting insulin (C, G, K, O), and the resistance index, HOMA-IR (D, H, L, P) (homeostasis model assessment-insulin resistance: fasting glucose (mmol/L) × fasting insulin (mU/L)/22.5) were measured. n = 7, *P < 0.05.

Fig. 4.

Fat transplantation and adipose tissue apoptosis. (A–M) Normal WT fat was transplanted into Tg female littermate recipients. (A–C) Fasting insulin, liver triglyceride (TG) content, and brown adipocyte area were measured four weeks after the surgery for sham-operated WT controls (open bars), sham-operated Tg mice (black bars), and Tg mice transplanted with WT fat (Tg Tx, gray bars) (n = 6). *P < 0.05. (D–L) Histological sections (H&E staining) from parametrial (pm) WAT (D–F), sc (G–I) and BAT (J–L) of representative WT sham-operated, Tg sham-operated, and Tg transplanted littermates. (M) Histological section (H&E) of transplanted fat pad removed from the recipient 4 weeks after the surgery. (N–O) Histological sections stained for apoptosis of sc WAT from a representative WT control and Tg littermates. (P) Cell counting per HPF from the above WT control (open bars) vs. Tg (filled bars) littermates epi, sc and BAT (10 HPF counted per mice, n = 5 mice per genotype). *P < 0.05 WT vs. Tg.

Fig. 5.

Analyses of macrophage gene expression and activation. (A–B) mRNA gene expression from epi WAT sorted macrophages (CD11b+F4/80+) from Tg (white bars), HFD (gray bars) and ob/ob (black bars) and their respective littermate controls. Fold change in gene expression over the appropriate control is plotted on a log₂ scale (n = 5). P < 0.05 *Tg, HFD or ob/ob vs. the respective control, ^#Tg vs. HFD, ^¶Tg vs. ob/ob and ^§HFD vs. ob/ob. (C) CD11c expression (CD11b+F4/80+CD11c+) in WT mice (open bars) vs. Tg littermates (filled bars; n = 3–4). *P < 0.05 WT vs. Tg. (D) Adipose tissue macrophage activation based on CD40 expression (CD11b+F4/80+CD40+) in control mice (open bars) vs. Tg, HFD or ob/ob littermates respectively (filled bars; n = 3–4). *P < 0.05 control vs. ob/ob or HFD. (E–G) Epididymal WAT mRNA expression from WT control and Tg littermates injected with saline (open bars), LPS (black bars) or treated with sodium salicylate before injection with LPS (striped bars) (n = 5; *P < 0.05).