Osteopontin mediates obesity-induced adipose tissue macrophage infiltration and insulin resistance in mice

Abstract

Obesity is associated with a state of chronic, low-grade inflammation characterized by abnormal cytokine production and macrophage infiltration into adipose tissue, which may contribute to the development of insulin resistance. During immune responses, tissue infiltration by macrophages is dependent on the expression of osteopontin, an extracellular matrix protein and proinflammatory cytokine that promotes monocyte chemotaxis and cell motility. In the present study, we used a murine model of diet-induced obesity to examine the role of osteopontin in the accumulation of adipose tissue macrophages and the development of insulin resistance during obesity. Mice exposed to a high-fat diet exhibited increased plasma osteopontin levels, with elevated expression in macrophages recruited into adipose tissue. Obese mice lacking osteopontin displayed improved insulin sensitivity in the absence of an effect on diet-induced obesity, body composition, or energy expenditure. These mice further demonstrated decreased macrophage infiltration into adipose tissue, which may reflect both impaired macrophage motility and attenuated monocyte recruitment by stromal vascular cells. Finally, obese osteopontin-deficient mice exhibited decreased markers of inflammation, both in adipose tissue and systemically. Taken together, these results suggest that osteopontin may play a key role in linking obesity to the development of insulin resistance by promoting inflammation and the accumulation of macrophages in adipose tissue.

Figure 1. OPN plasma levels in DIO and expression of OPN in adipose tissue.

C57BL/6 mice (n = 10/group) were fed a LFD or HFD for 20 weeks. (A) OPN plasma levels were analyzed by ELISA and data expressed as mean ± SEM. ^#P < 0.005. (B) Adipose tissues were isolated and OPN mRNA expression was analyzed in whole EWAT, the AF, and the SVF. Data are presented as relative OPN mRNA expression normalized to TFIIB mRNA expression and are expressed as mean ± SEM. *P < 0.05, compared with LFD. (C) SVFs isolated from EWATs of obese wild-type mice (n = 6) were pooled and separated into macrophages, endothelial cells, and preadipocytes using magnetic immunoaffinity isolation. Cell fractions were analyzed for OPN (black bars, left y axis) and CD68 (white bars, right y axis) mRNA expression. Data are presented as mRNA expression relative to TFIIB mRNA expression and are expressed as mean ± SEM. **P < 0.01 compared with preadipocyte or endothelial cell fraction. (D) Paraffin-embedded EWAT was analyzed for OPN immunoreactivity and F4/80-positive macrophage content. Sections were counterstained with hematoxylin and images obtained at the indicated magnifications.

Figure 2. DIO and body composition in wild-type OPN^+/+ and OPN^–/– mice.

(A) Wild-type OPN^+/+ (black symbols, n = 12) and OPN^–/– (white symbols, n = 12) mice were fed a LFD (circles) or HFD (squares). Weight gain was followed for 25 weeks and data expressed as mean ± SEM. Body composition (fat mass, lean mass, and body mass) before (B) and after (C) feeding a HFD (n = 7–8/group) was analyzed in OPN^+/+ (black bars) and OPN^–/– (white bars) mice by quantitative NMR. Data are presented as mean ± SEM. ^#P < 0.05, compared with OPN^+/+ mice.

Figure 3. Insulin sensitivity in OPN^+/+ and OPN^–/– mice.

OPN^+/+ (black symbols) and OPN^–/– (white symbols) mice were fed a LFD (dashed lines) or HFD (solid lines) for 25 weeks. (A) Insulin sensitivity in these mice (n = 6/group) was determined at the end of the feeding period following an intraperitoneal injection of insulin (1 U/kg body weight). (B) Glucose clearance (n = 6/group) was analyzed following an intraperitoneal challenge of 1 g/kg body weight glucose. Data are presented as mean blood glucose concentration ± SEM. ^#P < 0.05, OPN^–/– compared with OPN^+/+ mice fed HFD.

Figure 4. OPN deficiency decreases ATM content in obese mice.

(A) ATM content was determined by immunohistochemical analysis of epididymal adipose tissues isolated from OPN^+/+ and OPN^–/– mice fed a LFD or HFD. Adipose tissues were stained using an absorbed rabbit anti-mouse macrophage antiserum (original magnification, ×100). (B) Epididymal adipose tissues from obese OPN^+/+ and OPN^–/– mice were analyzed for macrophage content using an F4/80 antibody (magnified as indicated). (C) Macrophage content was quantified by analyzing the fraction of F4/80-stained cells relative to total number of cells in epididymal adipose tissue from OPN^+/+ (black bars) and OPN^–/– (white bars) mice fed a LFD or HFD (n = 8/group). Values are expressed as mean ± SEM. (D) Macrophage content was quantitatively assessed by real-time RT-PCR for CD68 mRNA expression in EWAT, the AF, and the SVF isolated from OPN^+/+ (black bars) and OPN^–/– (white bars) mice (n = 12/group) fed a LFD or HFD for 25 weeks. Data are presented as relative CD68 mRNA expression normalized to TFIIB mRNA expression and are expressed as mean ± SEM. *P < 0.05, HFD compared with LFD; ^#P < 0.05, OPN^–/– mice compared with OPN^+/+ mice fed HFD.

Figure 5. Chemotaxis of macrophages isolated from OPN^+/+ and OPN^–/– mice.

(A) Peritoneal macrophages from OPN^+/+ and OPN^–/– mice were subjected to chemotaxis assays in modified Boyden chambers. Membranes of the transwell chambers were coated either with the substrate poly-D-lysine (PDL) as control or with recombinant OPN (5 ng/ml). Following attachment of the macrophages to the membrane, vehicle or MCP-1 (50 ng/ml) was added to the media in the lower chamber. Transwell migration was analyzed after 2 hours and expressed as cell numbers per HPF (×200). Experiments were repeated 4 times in triplicate. Data are expressed as mean ± SEM. *P < 0.05 compared with PDL alone; ^#P < 0.05 compared with OPN alone; ^ΧP < 0.05 compared with OPN^+/+. (B) Stromal vascular cells were isolated from epididymal adipose tissues harvested from OPN^+/+ (black bars) and OPN^–/– (white bars) mice fed a LFD or HFD for 25 weeks (n = 6/group). Cells were cultured in the bottom chambers, and peritoneal macrophages from wild-type mice were added to the insert. Migration was analyzed in triplicate after 2 hours as described in A. Data are expressed as mean ± SEM. ^†P < 0.05, compared with LFD; ^‡P < 0.05, compared with OPN^+/+ mice fed HFD.

Figure 6. Inflammatory gene expression in adipose tissues from OPN^+/+ and OPN^–/– mice.

OPN^+/+ and OPN^–/– mice were fed a LFD or HFD for 25 weeks. mRNA expression levels of the indicated inflammatory genes were analyzed in EWATs isolated from OPN^+/+ (black bars) and OPN^–/– (white bars) mice fed a LFD or OPN^+/+ (dark gray) and OPN^–/– mice (light gray) fed a HFD (n = 6/group). Data are presented as relative mRNA normalized to TFIIB mRNA and expressed as mean ± SEM. *P < 0.05, HFD compared with LFD; ^#P < 0.05, OPN^–/– mice compared with OPN^+/+ mice fed HFD.

Figure 7. Plasma cytokine and adipokine levels in OPN^+/+ and OPN^–/– mice.

(A) IL-6, MCP-1, and PAI-1 plasma levels were analyzed in OPN^+/+ (black bars) and OPN^–/– (white bars) mice (n = 10/group) fed either a LFD or a HFD. (B) Plasma adiponectin, leptin, and resistin levels were analyzed in plasma obtained from mice described in A. Data are presented as mean ± SEM. *P < 0.05, compared with LFD; ^#P < 0.05 compared with OPN^+/+ mice fed HFD.