Neutralization of osteopontin inhibits obesity-induced inflammation and insulin resistance

Abstract

Objective: Obesity is associated with a state of chronic low-grade inflammation mediated by immune cells that are primarily located to adipose tissue and liver. The chronic inflammatory response appears to underlie obesity-induced metabolic deterioration including insulin resistance and type 2 diabetes. Osteopontin (OPN) is an inflammatory cytokine, the expression of which is strongly upregulated in adipose tissue and liver upon obesity. Here, we studied OPN effects in obesity-induced inflammation and insulin resistance by targeting OPN action in vivo.

Research design and methods: C57BL/6J mice were fed a high-fat diet to induce obesity and were then intravenously treated with an OPN-neutralizing or control antibody. Insulin sensitivity and inflammatory alterations in adipose tissue and liver were assessed.

Results: Interference with OPN action by a neutralizing antibody for 5 days significantly improved insulin sensitivity in diet-induced obese mice. Anti-OPN treatment attenuated liver and adipose tissue macrophage infiltration and inflammatory gene expression by increasing macrophage apoptosis and significantly reducing c-Jun NH(2)-terminal kinase activation. Moreover, we report OPN as a novel negative regulator for the activation of hepatic signal transducer and activator of transcription 3 (STAT3), which is essential for glucose homeostasis and insulin sensitivity. Consequently, OPN neutralization decreased expression of hepatic gluconeogenic markers, which are targets of STAT3-mediated downregulation.

Conclusions: These findings demonstrate that antibody-mediated neutralization of OPN action significantly reduces insulin resistance in obesity. OPN neutralization partially decreases obesity-associated inflammation in adipose tissue and liver and reverses signal transduction related to insulin resistance and glucose homeostasis. Hence, targeting OPN could provide a novel approach for the treatment of obesity-related metabolic disorders.

FIG. 1.

Insulin sensitivity is improved by OPN neutralization. Mice were fed a high-fat diet (HF) to induce obesity or normal chow (NC), respectively, for 24 weeks and were treated intravenously with an OPN-neutralizing (Anti-OPN) or control antibody three times during 5 days at the end of the feeding period. An ITT was performed in lean and obese OPN antibody-treated (dashed lines) and control antibody–treated (solid lines) mice 1 day after the last antibody application (n = 5 per group for NC and n = 8 per group for HF). A and B: Percent of basal glucose during ITT in mice on high-fat diet (A) and normal chow (B). C: Area under the curve. D: HOMA-IR was calculated. *P ≤ 0.05; **P ≤ 0.01; #P = 0.06.

FIG. 2.

Adipose tissue macrophage accumulation is reduced by OPN neutralization. Obese high-fat diet–fed (HF) and lean normal chow–fed (NC) mice were treated with OPN-neutralizing (Anti-OPN) or control antibody (n = 8 per group for HF and n = 5 per group for NC mice). A: mRNA expression of the macrophage marker F4/80 (encoded by Emr1 gene) was analyzed in GWAT by real-time RT-PCR. The mean of control HF was set to 100%. B: Percentage of macrophages (F4/80-positive cells) in the SVC fraction of GWAT as determined by flow cytometry. C: Adipose tissue macrophage accumulation was determined by immunofluorescence of F4/80⁺ cells (upper row) and immunohistochemical staining of Mac-2⁺ cells (bottom row) in GWAT isolated from high-fat diet–fed mice after anti-OPN or control antibody treatment. Representative pictures are given in 40-fold magnification. D: Adipose tissue macrophages as detected by F4/80 positivity in tissue sections were counted as F4/80⁺ cells relative to total number of cells. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

A: Apoptotic cells were determined by tunel staining (green), macrophages were stained red by immunoflourescence using anti-F4/80 monoclonal antibody on frozen sections. Representative pictures are given. B: Quantification of apoptotic macrophages (TUNEL and F4/80 double-positive cells per F4/80-positive cells). The mean of Control HF was set to 100%. *P ≤ 0.05. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

Adipose tissue inflammatory signaling and cytokine expression is attenuated by OPN neutralization in obese mice. Obese high-fat diet–fed (HF) and lean normal chow–fed (NC) mice were treated with OPN-neutralizing (α-OPN) or control antibody (n = 8 per group for HF and n = 5 per group for NC mice). A–D: Immunoblot analysis and quantification of JNK1 and JNK2 phosphorylation in GWAT. Representative blots are given for obese (A) and lean (C) adipose tissue. The diagrams show means of the chemiluminescence intensity ratios from phosphorylated versus total JNK protein for obese (B) and lean (D) anti-OPN– and control-treated mice. E and F: mRNA expression of the inflammatory genes for IL-6 (Il6) (E) and TNF-α (Tnf) (F) was analyzed in GWAT. The mean of Control HF was set to 100%. *P ≤ 0.05; **P ≤ 0.01.

FIG. 5.

OPN neutralization decreases hepatic inflammation. Obese high-fat diet–fed (HF) mice were treated with OPN-neutralizing (Anti-OPN) or control (n = 8 per group) antibody. Hepatic expression of genes for the inflammatory cytokines TNF-α (Tnf) (A) and TGF-β1 (Tgfb1) (B), the anti-inflammatory IL-10 (Il10) (C), and for the macrophage marker F4/80 (Emr1) (D) was analyzed by real-time RT-PCR. The mean of Control was set to 100%. E–G: Hepatic macrophage accumulation and apoptosis. E: Macrophages were stained red by immunoflourescence using Mac-2 monoclonal antibody on frozen sections of livers isolated from high-fat diet–fed mice after anti-OPN or control treatment. Apoptotic cells were determined by TUNEL staining (green). Representative pictures are given in 40-fold magnification. F: Hepatic macrophages were counted as Mac-2⁺ cells relative to total number of cells. G: Quantification of apoptotic macrophages (TUNEL and Mac-2 double-positive cells per Mac-2–positive cells). The mean of Control was set to 100%. *P ≤ 0.05; **P ≤ 0.01. (A high-quality color representation of this figure is available in the online issue.)

FIG. 6.

OPN interferes with hepatic STAT3 activation in obesity. A–C: Obese high-fat diet–fed (HF) mice were treated with OPN-neutralizing (Anti-OPN) or control (n = 8 per group) antibody. STAT phosphorylation was determined by immunhistochemical analysis of pSTAT(Tyr705) on paraffin sections of livers isolated from high-fat diet–fed mice after anti-OPN or control treatment. A: Representative immunohistochemical pictures are given in 80- and 200-fold magnification, respectively. The representative TissueFaxs scattergramm shows the percentage of pSTAT(Tyr705) positive cells in liver; (n = 5 per group). B: Quantification of pSTAT3-positive cells determined by TissueFaxs. C: Immunoblot analysis of STAT3 tyrosine 705 phosphorylation in HepG2 cells. Cells were stimulated or not with 0.5 μg/ml human recombinant OPN for 30 min and 2 days followed by immunoblotting for pSTAT3(Tyr705) and STAT3. A representative blot is given along with mean ratios. ***P ≤ 0.001. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 7.

OPN neutralization downregulates expression of hepatic gluconeogenic markers. Obese high-fat diet–fed (HF) mice were treated with OPN-neutralizing or control antibody (n = 8 per group). Hepatic mRNA expression of genes encoding GSK-3β (Gsk3b) (A) and the gluconeogenic enzymes PEPCK (Pck1) (B) and G6P (G6pc) (C) was analyzed. *P ≤ 0.05.