Adipocyte/macrophage fatty acid-binding proteins contribute to metabolic deterioration through actions in both macrophages and adipocytes in mice

Abstract

Adipose tissue inflammation is a characteristic of obesity. However, the mechanisms that regulate this inflammatory response and link adipose inflammation to systemic metabolic consequences are not fully understood. In this study, we have taken advantage of the highly restricted coexpression of adipocyte/macrophage fatty acid-binding proteins (FABPs) aP2 (FABP4) and mal1 (FABP5) to examine the contribution of these lipid chaperones in macrophages and adipocytes to local and systemic inflammation and metabolic homeostasis in mice. Deletion of FABPs in adipocytes resulted in reduced expression of inflammatory cytokines in macrophages, whereas the same deletion in macrophages led to enhanced insulin signaling and glucose uptake in adipocytes. Using radiation chimerism through bone marrow transplantation, we generated mice with FABP deficiency in bone marrow and stroma-derived elements in vivo and studied the impact of each cellular target on local and systemic insulin action and glucose metabolism in dietary obesity. The results of these experiments indicated that neither macrophages nor adipocytes individually could account for the total impact of FABPs on systemic metabolism and suggest that interactions between these 2 cell types, particularly in adipose tissue, are critical for the inflammatory basis of metabolic deterioration.

Figure 1. Coculture experiments.

(A) Protein expression of aP2 and mal1 in adipocyte cell lines (WT-Ad, KO-Ad, KO+aP2-Ad, KO+GFP-Ad, and 3T3-L1), mouse macrophage cell lines (WT-Mac and KO-Mac), and thioglycollate-elicited primary macrophages from Ap2^+/+Mal1^+/+ (WT-pMac) and Ap2^–/–Mal1^–/– (KO-pMac) mice. (B) Dibutyryl cAMP–stimulated lipolysis (4-hour stimulated FFA release) in adipocyte cell lines. (C) Basal FFA release into the conditioned medium (CM) in adipocytes examined under the same conditions of coculture experiments for 16 hours. (D) Expression of Mcp1 in macrophages, WT-Mac or KO-Mac, incubated with conditioned medium from WT-Ad or KO-Ad adipocytes. Data were normalized to those in untreated macrophages. (E) Expression of Mcp1 in primary macrophages, WT-pMac or KO-pMac, incubated with conditioned medium from KO+GFP-Ad (FABP-deficient) or KO+aP2-Ad (FABP-reconstituted) adipocytes. Data were normalized to those in macrophages incubated with the conditioned medium from KO+GFP-Ad adipocytes. (F) Insulin-stimulated glucose uptake in 3T3-L1 adipocytes incubated in contact with immortalized macrophages, WT-Mac or KO-Mac. (G) Insulin-stimulated phosphorylation of Akt in 3T3-L1 adipocytes incubated in contact with immortalized macrophages. The graph on the right shows the quantification. (H) Insulin-stimulated phosphorylation of Akt in adipocyte cell lines, KO+GFP-Ad and KO+aP2-Ad, incubated in contact with primary macrophages, WT-pMac or KO-pMac. Data are shown as mean ± SEM. *P < 0.05, **P < 0.01.

Figure 2. Adipocyte and SV fractions in BMT (GFP-Tg→WT) mice.

GFP-labeled donor cells were used to track the destiny of bone marrow–derived cells. (A) Confirmation of the adipocyte (Ad) and SV fractions from WT, GFP-transgenic (GFP-Tg), and BMT (GFP-Tg→WT) mice by differential expression of adiponectin as an adipocyte and Pecam1 as a non-adipocyte SV marker. Data are shown as mean ± SEM. (B) Detection of Gfp mRNA by PCR in the same fractions shown in A. (C) Detection of GFP protein by Western blotting in the same fractions. Perilipin and PECAM-1 were controls of the Ad and SV fractions, respectively. (D) Fluorescence microscopic analysis of the adipose tissue fractions. In the adipocyte fraction, images were taken in both the centrifuged fat pad after digestion or in floating fat cells in DMEM with 10% cosmic calf serum (magnification, ×200). In the SV fraction, cells were observed at both the low and high magnifications (LM, ×100; and HM, ×400).

Figure 3. BMT experiments in FABP-deficient mice.

(A) Experimental design of the BMT studies using WT (Ap2^+/+Mal1^+/+) and FABP-deficient (Ap2^–/–Mal1^–/–) mice. The numbers in parentheses indicate the timing (week) of performed items. (B) Description and nomenclature for the groups of BMT mice. (C) Genotyping using DNA samples from blood and tail in BMT mice. The graphs on the right show the quantification for the percentages of WT and KO alleles in blood samples. Data are shown as mean ± SEM. (D) Genotyping using DNA samples from white adipose tissue (WAT), liver, and skeletal muscle (soleus) in BMT mice for the donor and recipient alleles. GTT, glucose tolerance test; ITT, insulin tolerance test; Met Cage, metabolic cage study; DEXA, dual energy x-ray absorptiometry; Clamp, hyperinsulinemic-euglycemic clamp study.

Figure 4. Glucose and lipid metabolism in BMT mice.

(A–C) Levels of fasting blood glucose (A), insulin (B), and the lipid variables cholesterol and triglycerides (C) in BMT mice (W→W, n = 9; K→W, n = 10; W→K, n = 9; and K→K, n = 8) at 22 weeks of age. (D–F) Glucose tolerance tests performed in WT (D) and FABP-deficient (E) recipients at 19 weeks of age. (F) The AUC of glucose levels during tolerance tests is shown. (G–I) Insulin tolerance tests were performed in WT (G) and FABP-deficient (H) recipients at 20 weeks of age. The AUC of glucose levels during tolerance tests is shown (I). Data are shown as mean ± SEM. *P < 0.05.

Figure 5. Hyperinsulinemic-euglycemic clamp in BMT mice.

(A–F) Hyperinsulinemic-euglycemic clamp studies were performed in all groups of BMT mice (W→W, n = 7; K→W, n = 5; W→K, n = 5; and K→K, n = 7) at 20 weeks of age. Glucose disposal rate (Rd) (A), glucose infusion rate (GIR) (B), hepatic glucose production (HGP) at the basal (C) and clamp (D) states, and tissue glucose uptake in epididymal fat (E) and gastrocnemius muscle (F). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01.

Figure 6. Adipose tissue characteristics and insulin action in BMT mice.

(A) H&E staining of the adipose tissue in BMT mice. Scale bars: 200 μm. (B) Adipose tissue macrophage content expressed as percentage of crown-like structure (CLS) detected by F4/80 staining per adipocyte. (C) Expression of F4/80, Mcp1, and Tnfa in the adipose tissue of BMT mice. (D) Insulin-stimulated IRβ tyrosine 1162/1163 and Akt serine 473 phosphorylation in the adipose tissues of BMT mice. The graphs to the right of the blots show the quantification from experiments performed at least in duplicate. Data are shown as mean ± SEM. *P < 0.05.

Figure 7. Liver tissue properties and insulin action in BMT mice.

(A) H&E staining of the liver sections in all groups of BMT mice. Scale bars: 200 μm. (B) Expression of F4/80, Mcp1, Tnfa, Il1b, Il6, and Il10 in the liver of BMT mice. (C) Insulin-stimulated IRβ tyrosine 1162/1163 and Akt serine 473 phosphorylation in the liver tissues of BMT mice. The graphs to the right of the blots show the quantification from experiments performed at least in duplicate. Data are shown as mean ± SEM. *P < 0.05.