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Endocrinology Vol. 149, No. 3 1350-1357
Copyright © 2008 by The Endocrine Society

Osteopontin Expression in Human and Murine Obesity: Extensive Local Up-Regulation in Adipose Tissue but Minimal Systemic Alterations

Florian W. Kiefer1, Maximilian Zeyda1, Jelena Todoric, Joakim Huber, René Geyeregger, Thomas Weichhart, Oskar Aszmann, Bernhard Ludvik, Gerd R. Silberhumer, Gerhard Prager and Thomas M. Stulnig

Clinical Divisions of Endocrinology and Metabolism (F.W.K., M.Z., J.T., J.H., R.G., B.L., T.M.S.) and Nephrology and Dialysis (T.W.), Department of Internal Medicine III, and Clinical Divisions of Plastic and Reconstructive Surgery (O.A.) and General Surgery (G.R.S., G.P.), Department of Surgery, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria

Address all correspondence and requests for reprints to: Thomas M. Stulnig, Clinical Division of Endocrinology and Metabolism, Department of Internal Medicine III, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria. E-mail: thomas.stulnig{at}meduniwien.ac.at.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Obesity is associated with a chronic low-grade inflammation characterized by macrophage infiltration of adipose tissue (AT) that may underlie the development of insulin resistance and type 2 diabetes. Osteopontin (OPN) is a multifunctional protein involved in various inflammatory processes, cell migration, and tissue remodeling. Because these processes occur in the AT of obese patients, we studied in detail the regulation of OPN expression in human and murine obesity. The study included 20 morbidly obese patients and 20 age- and sex-matched control subjects, as well as two models (diet-induced and genetic) of murine obesity. In high-fat diet-induced and genetically obese mice, OPN expression was drastically up-regulated in AT (40 and 80-fold, respectively) but remained largely unaltered in liver (<2-fold). Moreover, OPN plasma concentrations remained unchanged in both murine models of obesity, suggesting a particular local but not systemic importance for OPN. OPN expression was strongly elevated also in the AT of obese patients compared with lean subjects in both omental and sc AT. In addition, we detected three OPN isoforms to be expressed in human AT and, strikingly, an obesity induced alteration of the OPN isoform expression pattern. Analysis of AT cellular fractions revealed that OPN is exceptionally highly expressed in AT macrophages in humans and mice. Moreover, OPN expression in AT macrophages was strongly up-regulated by obesity. In conclusion, our data point toward a specific local role of OPN in obese AT. Therefore, OPN could be a critical regulator in obesity induced AT inflammation and insulin resistance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
OBESITY IS A major risk factor for the development of insulin resistance, which is a fundamental step toward type 2 diabetes (1). The chronic low-grade inflammation associated with obesity as determined by increased systemic concentrations of inflammatory markers and cytokines in patients and animal models of obesity (2, 3) probably represents a crucial link between obesity and insulin resistance (4, 5). The main origin of this systemic inflammatory response is located in adipose tissue (AT) (6). AT produces a variety of inflammatory adipokines such as IL-1β, IL-6, TNF-{alpha}, and monocyte chemoattractant protein-1 (MCP-1), the serum concentrations of all of which are elevated in obesity (7, 8, 9). Inflammatory adipokines are predominantly produced by nonfat cells such as AT macrophages (ATMs) (8, 10), the abundance of which is markedly increased by obesity in patients and rodent models (7, 8, 11, 12, 13).

Osteopontin (OPN), also named secreted phosphoprotein-1 (SPP1) and sialoprotein-1, is encoded by the SPP1 gene, which is transcribed into three mRNA isoforms, OPN-a, -b, and -c. OPN-a constitutes the full-length variant, whereas isoforms b and c result from alternative splicing (14, 15). OPN is a multifunctional protein expressed in activated macrophages and T cells, osteoclasts, hepatocytes, smooth muscle, endothelial, and epithelial cells (16, 17). OPN was originally classified as a T helper type 1 cytokine that is involved in physiological and pathological mineralization in bone and kidney, cell survival, inflammation, and tumor biology (16, 18). OPN induces the expression of a variety of other proinflammatory cytokines and chemokines in peripheral blood mononuclear cells (19). Moreover, it functions in cell migration, particularly of monocytes/macrophages (17), and stimulates expression of matrix metalloproteases to induce matrix degradation and facilitate cell motility (20). Notably, OPN plays a role in various inflammatory disorders, such as rheumatoid arthritis (21), atherosclerosis (22), and cardiac fibrosis (23), which are linked to obesity (24, 25). Furthermore, OPN is implicated in diabetic macro- and microvascular diseases (26, 27).

Previous microarray data on AT inflammation from our group indicated considerable up-regulation of OPN gene expression in murine obesity (28). Here, we show in two different models of murine obesity, diet-induced and genetic, that OPN gene and protein expression is highly up-regulated in AT, and identify ATMs as the main source for OPN. In addition, OPN expression was markedly increased in both omental and sc AT of obese humans compared with lean controls. We detected that the OPN isoform expression pattern is significantly shifted toward isoform b in the AT of obese patients, indicating a pathophysiological impact of OPN isoforms. Notably, the elevation of hepatic OPN expression was by far less pronounced than in AT, and plasma concentrations were not affected by obesity in murine models and only moderately altered in morbidly obese patients. Therefore, we emphasize an important local role for OPN in obesity driven AT inflammation, which may thereby critically contribute to the development of diabetes and other inflammation-related complications of obesity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients
Omental and abdominal sc ATs were obtained from nondiabetic morbidly obese patients [body mass index (BMI) ≥ 40 kg/m2; n = 20] or a control group (BMI < 30 kg/m2; n = 20], referred to as "lean" (60% of those having a BMI > 25 kg/m2). Caucasian men (n = 12; six obese) and women (n = 28; 14 obese) aged 18–60 yr who underwent elective laparoscopic surgery were included in the study. Tissue specimens were immediately frozen in liquid nitrogen. For AT fractionation, sc AT was obtained from patients undergoing reductional plastic surgery (BMI = 29.8 ± 2.5 kg/m2, n = 5). These studies have been approved by the ethics committee of the Medical University of Vienna and the General Hospital Vienna (EK no. 275/2006 and 290/2006). Patients gave written informed consent before taking part in the study.

Animals
To stimulate diet-induced obesity, male C57BL/6J mice (Charles River Laboratories, Sulzfeld, Germany) aged 7 wk were placed for 20 wk on a high-fat (60 kcal percent fat, D12492; Research Diets Inc., New Brunswick, NJ) and low-fat (10 kcal percent fat, D12450B; Research Diets Inc.) diet to induce obesity and to serve as lean controls, respectively. Mice had free access to food and water. After 20 wk on the diet, mice were killed by cervical dislocation. As a genetic obesity model, male obese db/db mice (C57BL/KsJ-leprdb/leprdb) obese mice and their lean littermates (db/+) were purchased from Charles River Laboratories, kept on standard rodent diet, and killed at an age of 13 wk. Peritoneal macrophages were isolated by peritoneal lavage with 10 ml PBS. After killing, gonadal white AT was removed and immediately snap frozen in liquid nitrogen. All mice were maintained on a 12-h light, 12-h dark cycle. The study protocols were approved by the local ethics committee for animal experiments, and followed the guidelines on accommodation and care of animals formulated by the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes.

Gene expression and plasma protein concentration
AT was homogenized in TRIZOL reagent (Invitrogen Corp., Carlsbad, CA), and RNA was isolated according to the manufacturer’s protocol. One microgram of total RNA was treated with DNase I and reverse transcribed into cDNA using Superscript II and random hexamer primers (all Invitrogen). Gene expression normalized to 18S rRNA was analyzed by quantitative real-time RT-PCR on an ABI Prism 7000 cycler using commercial Assays-on-Demand kits (all Applied Biosystems, Foster City, CA). Alternatively, the expression of the following murine genes were quantified by use of given self-designed primer pairs and iTaq SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA): Spp1 (5'-CCATCTCAGAAGCAGAATCTCCTT-3', 5'-GGTCATGGCTTTCATTGGAATT-3'); AdipoQ (5'-GTCATGCCGAAGATGACGTTACT-3', 5'-TCACCCTTAGGACCAAGAAGACC-3'); Ccl2 (5'-AGGTCCCTGTCATGCTTCTGG-3', 5'-CTGCTGCTGGTGATCCTCTTG-3'); and Tnf (5'-CCAGACCCTCACACTCAGATCA-3', 5'-TGGTATGAGATAGCAAATCGGCT-3').

To analyze expression of SPP1 isoforms, the primer pair 5'-ACCTGTGCCATACCAGTTAAA-3' and 5'-CTTTCGTTGGACTTACTTGGAA-3' was designed to distinguish the three SPP1 isoforms, a, b, and c (16), giving products of 945, 903, and 864 bp, respectively. Sequences of products were verified. After PCR amplification, semiquantitative analysis of SPP1 isoforms was performed by 2.5% agarose gel electrophoresis, and subsequent densitometric analysis of ethidium bromide staining using a Lumi-imager F1 (28) and Lumia 32 software (both Roche, Mannheim, Germany). For quantitative real-time RT-PCR to selectively quantify OPN isoforms a and b, we designed the following primers: 5'-CAGAATGCTGTGTCCTCTGAAGA-3' and 5'-GTCAATGGAGTCCTGGCTGTC-3' (SPP1-a), and 5'-CTAGCCCCACAGACCCTTCC-3 and 5'-CAATGGAGTCCTGGCTGTCC-3 (SPP1-b).

Human and murine OPN plasma concentrations were detected by ELISA kits (Assay Designs, Ann Arbor, MI) according to the manufacturer’s instructions.

Immunohistochemistry
Frozen sections were prepared from murine gonadal and human omental AT. Murine sections were stained with goat antimouse OPN IgG (R&D Systems, Minneapolis, MN) detected by a donkey antigoat IgG AlexaFluor 488 conjugate (Molecular Probes, Eugene, OR). Human sections were stained with mouse monoclonal anti-OPN IgG (Santa Cruz Biotechnology, Santa Cruz, CA) and biotin-conjugated monoclonal mouse antihuman CD206 antibody (Abcam, Cambridge, UK). Primary antibodies were detected with TRITC-conjugated goat antimouse IgG (Jackson ImmunoResearch Inc., West Grove, PA) and an avidin AlexaFluor 488 conjugate (Molecular Probes). Nuclei were visualized by 4',6-diamidino-2'-phenylindole staining. Slides were mounted in Aquatex (Merck & Co., Inc., Whitehouse Station, NJ) and examined under a fluorescence microscope (Leica, Wetzlar, Germany).

AT fractionation
Murine gonadal white AT and sc human AT were cut into small pieces, washed in PBS, and 0.5 g tissue/ml was digested with 0.03 mg/ml Liberase Blendzyme 3 (Roche) and 50 U/ml DNase I (Sigma-Aldrich, St. Louis, MO) in RPMI-1640 (Invitrogen) for 60 min at 37 C. Digested tissues were passed through 200-µm mesh filters. Material retained by the filter is referred to as "matrix" according to Fain et al. (29). After centrifugation at 1000 x g for 10 min at 4 C, floating cells were transferred into a fresh tube, centrifuged once again, and, subsequently, RNA was extracted from the floating adipocyte suspension. The pellets of the first centrifugation comprised the stromal vascular cell (SVC) fraction. Red blood cells were lysed in hemolysis buffer and remaining cells passed through a 70-µm mesh filter. ATMs were isolated using F4/80 and CD14 magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) for mouse and human, respectively, as described previously (13). The macrophage-depleted flow through is referred to as depleted SVC (dSVC) in the remaining text. The purity of mouse and human ATMs, as determined by flow cytometric analysis of F4/80- and CD14-positive cells, respectively, was more than 90%. Murine and human dSVC contained less than 10% F4/80 and less than 5% CD14-positive cells, respectively.

Statistics
All data are given as mean ± SE. Comparisons between mice of different genotypes or diets were assessed by the unpaired two-tail Student’s t test. One-way repeated measures ANOVA was used in a factorial design to compare gene expression data between human AT of different groups (obese vs. lean) and origin (sc vs. omental) using {Delta}CT values from quantitative real-time PCR. Correlation was analyzed by Spearman’s {rho}. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Diet-induced obesity drastically increases OPN expression in the AT
To assess the relationship of obesity and OPN expression, we used a model of diet-induced murine obesity and analyzed the OPN gene (Spp1) expression in metabolically relevant organs. Therefore, C57BL/6J mice were fed a high-fat diet for 6 months, which resulted in an average weight of 50.6 ± 1.1 g, compared with 32.9 ± 1.2 g of low-fat diet fed controls. We found that diet-induced obesity caused a 42 ± 12-fold up-regulation of OPN mRNA expression in gonadal white AT (Fig. 1AGo). This increase considerably exceeded up-regulation of other inflammatory markers, such as the genes for F4/80 (Emr1), TNF-{alpha} (Tnf), and MCP-1 (Ccl2). Although statistically significant, high-fat diet-induced up-regulation of OPN gene expression was by far less pronounced (<2-fold) in the liver (Fig. 1BGo). Increased OPN gene expression was reflected by a marked increase of OPN protein expression as semiquantitatively detected by immunohistochemistry (Fig. 1CGo). OPN protein was detected in mononuclear cells but was also associated with adipocyte membranes. Muscle OPN gene expression was at the lower level of detection in all animals studied (data not shown).


Figure 1
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  		FIG. 1. Regulation of OPN expression in AT by diet-induced obesity. C57BL/6J mice fed a high-fat (HF) diet (black bars) for 6 months were compared with low-fat (LF) diet-fed animals (white bars). Diagrams show mean ± SE of expression of the genes for F4/80 (a macrophage marker), TNF, MCP-1 in gonadal AT (A), and of OPN mRNA in the liver (B) given in percentage of the LF group (n = 6 for each group). **, P < 0.01; ***, P < 0.01. C, Representative immunohistochemical stains of OPN protein expression in AT along with an isotype control (x40 objective).

 
Up-regulation of OPN expression in AT by genetically determined obesity
As a second model of murine obesity, we compared leptin-receptor deficient mice (db/db; weight 43.7 ± 1.7 g) to their lean littermates (db/+, 27.3 ± 1.4 g). In this model of genetic obesity, OPN gene expression in AT was increased by 82 ± 29-fold (range 40.6- to 195.0-fold), whereas mean up-regulation compared with lean db/+ mice of other inflammatory markers CD68, TNF-{alpha}, and MCP-1 was only 16.2 ± 3.1, 2.6 ± 0.4, and 19.7 ± 5.0, respectively (Fig. 2AGo). Hepatic OPN gene expression was unaltered in db/db vs. db/+ animals (Fig. 2BGo).


Figure 2
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  		FIG. 2. OPN gene expression in AT from genetically obese mice. Obese db/db (black bars) mice were compared with their lean littermates (db/+) (white bars). Diagrams show mean ± SE of expression of the genes for CD68 (a macrophage marker), TNF, MCP-1 in gonadal AT (A), and of OPN mRNA in the liver (B) given in percentage of the db/+ group (n = 5 for each group). *, P < 0.05; **, P < 0.01.

 
Elevated expression of OPN in AT of obese patients
To investigate OPN gene expression in human obesity, we analyzed its expression in omental and sc AT of morbidly obese patients (BMI = 53.0 ± 2.5 kg/m2) and a lean control group (BMI = 25.2 ± 0.7 kg/m2). Indeed, human obesity was associated with a marked increase in OPN expression in omental as well as sc AT (7.1 ± 2.0-fold and 3.3 ± 1.5-fold, respectively; Fig. 3Go), regardless of patient age and sex (data not shown). Expression in sc AT was higher than in omentum, but this difference just failed to reach statistical significance (P = 0.077 for ANOVA interaction term). Univariate correlation between OPN gene expression in omental AT and markers of obesity and insulin resistance revealed significance for BMI ({rho} = 0.382; P = 0.015), percent body fat ({rho} = 0.475; P = 0.006), waist circumference ({rho} = 0.400; P = 0.017), leptin ({rho} = 0.486; P = 0.003), insulin ({rho} = 0.410; P = 0.016), and homeostasis model assessment of insulin resistance ({rho} = 0.403; P = 0.018), but not for fasting glucose ({rho} = 0.201; P = 0.219). These correlations were not significant after adjustment for body fat.


Figure 3
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  		FIG. 3. Increased OPN expression in the AT of obese patients. Expression of OPN mRNA was assessed in omental and sc AT of lean and obese subjects (n = 20 for each group). OPN expression (mean ± SE) is expressed in percentage (%) of omental AT from lean subjects. Univariate ANOVA revealed in P < 0.0005 for obesity, P = 0.077 for location, and P = 0.73 for the interaction term. Results from post hoc analyses are indicated by asterisks. **, P < 0.01; ***, P < 0.001.

 
Obesity alters the OPN isoform expression pattern
The OPN gene (SPP1) contributes to three mRNA isoforms, OPN-a, -b, and -c. OPN-a constitutes the full-length variant, whereas isoforms b and c result from alternative splicing (14, 15). To complete the picture of OPN expression in human AT, we studied the relative expression of the three known isoforms of the OPN gene (SPP1) in omental AT and the impact of obesity on their expression pattern. In the AT of lean subjects, full-length OPN-a was the predominantly expressed isoform (67.7 ± 1.8%; Fig. 4Go) ahead of OPN-b (30.1 ± 1.7%). However, in obese patients, relative expression of OPN-a was significantly reduced (47.5 ± 5.5%), whereas OPN-b expression was increased to 48.1 ± 6.2% (Fig. 4Go). OPN-c was expressed at low levels in both groups and not significantly altered in obesity (2.1 ± 0.7% vs. 4.5 ± 1.5%; P = 0.15). In addition, we analyzed the obesity induced increase of the expression of the predominant isoforms a and b by selective quantitative real-time RT-PCR. Both isoforms were markedly up-regulated, and the increase of OPN-b expression was significantly more pronounced compared with OPN-a (7.9 ± 2.0 vs. 4.5 ± 1.2-fold).


Figure 4
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  		FIG. 4. Altered OPN isoform expression in obesity. OPN (Spp1) isoform expression was analyzed in omental AT of lean and obese subjects (n = 20 for each group). A, Representative agarose gel electrophoresis of PCR products of the known OPN isoforms (a–c, as indicated). B, Summary of the densitometric analysis. Diagram gives means ± SEM of the percentages of each isoform related to total OPN of lean (white bars) and obese (black bars) patients, respectively. **, P < 0.01; ***, P < 0.001. C, Selective quantitative real-time RT-PCR of OPN isoforms a and b. Diagram gives means ± SEM of indicated isoform expression in obese patients (black bars) related to the lean controls (white bars). *P < 0.05. mw, Molecular weight marker; ns, not significant; ntc, nontemplate control.

 
Weak association between obesity and systemic concentrations of OPN
Next, we were interested in whether the outstanding increase of AT OPN expression by obesity is reflected by OPN plasma concentrations. In the investigated murine models of obesity, neither diet-induced (Fig 5AGo) nor genetic obesity (db/db mice; Fig. 5BGo) altered OPN plasma concentrations. In morbidly obese patients, OPN plasma concentrations were 1.7-fold elevated compared with lean controls (Fig. 5CGo). However, there was no significant correlation between OPN plasma concentrations and OPN gene expression in omental and sc AT, respectively (data not shown).


Figure 5
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  		FIG. 5. OPN plasma concentrations in obesity. A, OPN plasma concentrations in diet-induced obesity. C57BL/6J mice were fed a high-fat (HF) or a low-fat diet (LF) for 6 months. B, OPN plasma concentrations in genetic obesity (db/db compared with lean db/+, n = 5). C, OPN plasma concentrations in obese patients and lean subjects. *P < 0.05. ns, Not significant.

 
ATMs express particularly high levels of OPN and strongly up-regulate OPN expression in obesity
Association of a secreted protein such as OPN with different cell types as seen with immunofluorescence analyses (Fig. 1CGo) cannot determine the cellular source of OPN expression. To analyze the cell types responsible for OPN expression in AT, we fractionated murine (db/+) gonadal and human sc AT into ATMs, macrophage-dSVC, adipocytes, and, for human AT, AT matrix. AT matrix comprises cells of the AT that are resistant to dissociation during collagenase digestion and was shown to be the main source of several adipokines (29). Macrophage markers (F4/80, CD68) and adipocyte-specific adiponectin were highly enriched in the ATM and adipocyte fractions, respectively, as expected (Figs. 6AGo and 7AGo). In human AT, OPN gene expression was highest in ATM, but substantial expression was also found in dSVC (Fig. 6AGo) despite few ATMs remaining in this fraction according to flow cytometric analysis (<5%; data not shown). High OPN expression in ATMs was also confirmed at the protein level by double-immunofluorescence staining of OPN and the ATM marker CD206 (30) in human omental AT (Fig. 6BGo). Some OPN could also be detected on the surface of adipocytes.


Figure 6
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  		FIG. 6. OPN expression in human ATMs. A, Human sc AT was fractionated into ATMs, macrophage-dSVCs, adipocytes (AC), and matrix (MX) as detailed in the Materials and Methods section. Expression of indicated genes was determined by quantitative real-time RT PCR. Expression in ATM of each donor was set to 100%, with the exception of adiponectin, which was set to 100% in adipocytes. Diagrams show mean ± SE (n = 5). B, Representative immunohistochemical staining of OPN (red) and the ATM marker CD206 (green) in omental AT (x40 objective).

 

Figure 7
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  		FIG. 7. The OPN gene is highly expressed in murine ATMs and up-regulated in obesity. A, OPN expression in AT cellular fractions and peritoneal macrophages. AT of db/db mice was fractionated into ATM, macrophage-dSVCs and adipocytes (AC), as detailed in the Materials and Methods section. Expression of indicated genes was determined by quantitative real-time RT PCR, and expression in ATM was set to 100%, with the exception of adiponectin, which was set 100% in adipocytes. For comparison, OPN gene expression (mean ± SE; n = 5) is also given for peritoneal macrophages (PM). B, Regulation of OPN gene expression in ATMs by obesity. OPN mRNA expression in isolated ATMs of obese (db/db) compared with lean mice (db/+). Diagram shows mean ± SE (n = 5) related to the db/+ group (100%). **P < 0.01.

 
In murine AT, OPN gene expression was by far highest in ATMs, and virtually absent in adipocytes and other fractions (Fig. 7AGo). Because the obesity induced increase of OPN gene expression in murine AT conspicuously exceeded that of the macrophage activation marker CD68 (82 vs. 16-fold; Fig. 2AGo), we analyzed whether OPN is up-regulated within ATMs. When comparing OPN gene expression in ATMs isolated from obese db/db mice with that from lean db/+ animals, we found a more than 20-fold OPN expression within these cells (Fig. 7BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Pathophysiology, biomarkers, and novel therapeutic approaches targeting obesity driven inflammation are the main goals of today’s obesity research. Here, we show that the multifunctional inflammatory protein OPN is extensively up-regulated in the AT of obese humans, as well as of diet-induced and genetically (db/db) obese mice. OPN up-regulation in metabolically relevant tissues in obesity appears to be specific for AT because hepatic expression differed only to a minor extent (Figs. 1BGo and 2BGo), and OPN is only marginally expressed in muscle. Unaltered hepatic OPN gene expression in db/db mice was corroborated on the protein level by Sahai et al. (31), who could not detect any difference between untreated db/db and lean littermates. Although OPN plasma concentrations were significantly elevated in obese patients (Fig. 5Go), similar to recently published data (32), this increase is rather small compared with its regulation in AT. Also in murine diet-induced obesity, a moderate increase in OPN plasma concentrations has been shown by a recent publication (33). The animals in that study gained somewhat more weight after high-fat feeding than those of our study, but this probably does not explain the difference with our result showing no diet effect on OPN plasma concentrations. However, our additional analysis of genetically obese mice confirms that OPN plasma concentrations are not affected by obesity (Fig. 5Go). Therefore, our data suggest a particularly local impact of OPN within obese AT, which is not reflected by systemic OPN concentrations.

The relative expression in omental AT of the two predominant isoforms OPN-a and -b differed significantly between lean and obese patients, leading to a shift toward isoform b, whereas OPN-c was only marginally expressed in both lean and obese AT (Fig. 4Go). Data on expression and functional relevance of different OPN isoforms are very rare and limited to a putative role in tumor cells (14, 34). Isoform b is lacking exon 5 in the N-terminal region as does isoform c that additionally lacks parts of exon 4. Multiple receptor binding sites for integrins and CD44 that are critical for OPN functions in cell migration have been mapped to regions C terminal to exons 4 and 5 (35). Isoform c that supports anchorage-independent tumor cell growth is of higher solubility compared with OPN-a (34). Therefore, it could be speculated that increased availability of more soluble OPN-b enhances macrophage infiltration and/or activation in obese AT. However, the pathophysiological impact of the obesity associated alterations of the OPN isoform expression pattern in AT still has to be elucidated in detail.

The main source of OPN in human (Fig. 6Go) and murine genetic (Fig. 7Go) and diet-induced (33) obesity is ATMs. Adipocytes stain positive for OPN in immunohistochemical analyses of human AT sections (Fig. 6BGo) (32). However, this cannot be considered as evidence for OPN expression in adipocytes, as proposed by Gomez-Ambrosi et al. (32), because OPN is primarily a secreted protein that could bind to adipocyte membranes. The negligible expression of the OPN gene in isolated adipocytes compared with ATMs (Figs. 6Go and 7Go) indicates that ATMs are the major source of OPN in obese AT, with some contribution by other not yet identified cells from the stromal vascular fraction. OPN is also expressed and functional in T cells, and thereby promotes autoimmunity and inflammation (36, 37). Of note, T cells have also been shown to accumulate in murine AT upon obesity (38). Therefore, OPN in T cells could be a putative link between obesity and autoimmune disorders. However, the role of T cells in obesity induced AT inflammation and a possible function of OPN in AT-infiltrating T cells need to be studied yet.

OPN was shown to be a critical mediator of macrophage infiltration into inflamed tissue (39) and inflammatory cell infiltration into arthritic joints (40) and in cutaneous contact hypersensitivity (41). Thus, OPN is involved in migration of macrophages and macrophage-like cells in a variety of inflammatory disorders. In addition, OPN has stimulated inflammatory responses of myeloid cells (19, 42). Therefore, ATMs may not only be the main source, but also a major target of OPN action in AT, e.g. by attracting more ATMs after being activated once. Notably, ATMs have recently been shown to express CD44 and, particularly, integrin {alpha}vβ5 on their surface (13), both well-known receptors for OPN. Therefore, an autocrine/paracrine activation of ATMs via OPN appears probable.

OPN induces matrix metalloproteinases in a variety of cells (20, 27). By this mechanism, OPN may rather be detrimentally involved in obesity related tissue remodeling (43, 44). Although pro-inflammatory by nature, OPN function does not necessarily have to be destructive for the surrounding tissue. In this sense OPN has protected resident cells from tissue damage such as that induced by activated macrophages through inhibiting inducible nitric oxide synthase (45). However, net effects of OPN on AT remodeling in obesity are still elusive.

Our data indicate that the extensive elevation of AT OPN expression in obesity results from an increased abundance of ATMs (8) accompanied by OPN up-regulation within ATMs as shown in genetically (Fig. 7BGo) and diet-induced obese mice (33). Due to the marked up-regulation in ATMs, OPN expression could qualify as a marker for activated, detrimental ATMs. Several mediators could be responsible for the induction of high OPN expression in ATMs. Proinflammatory cytokines such as IL-1β TNF-{alpha}, nitric oxide, and lipopolysaccharide, but also antiinflammatory IL-10, induced OPN expression in several experimental settings (19, 46, 47). All of these factors are related to obesity induced AT inflammation (3, 48, 49). Moreover, OPN expression can be up-regulated by hyperglycemia via the Rho/Mek1 pathway in rat aortic smooth muscle cells (50). Because OPN by itself induces expression of a variety of cytokines and chemokines (19), an upward spiral of aggravating AT inflammatory alterations involving OPN may exist that could provoke cytokine-mediated insulin resistance of adipocytes (51, 52, 53). Breaking this vicious circle by targeting OPN action could be a promising strategy for prevention of obesity induced insulin resistance, as recently indicated by improved insulin sensitivity in OPN knockout mice (33).

The role of OPN in the pathophysiology of human obesity awaits elucidation. As a preliminary hint on a potential pathophysiological impact of OPN in obesity associated AT inflammation, we performed correlation analyses. OPN gene expression in omental and sc AT highly positively correlated with waist circumference, BMI, percent body fat, serum leptin, and markers of glucose homeostasis (serum insulin, homeostasis model assessment of insulin resistance) in univariate correlation analyses in our study population, and similar results have recently been found in another study (32). Thus, OPN could mediate some effects of excess body fat on glucose homeostasis, even though these correlations of OPN gene expression were not significant after adjustment for body fat in our population.

In conclusion, obesity is associated with a striking increase of OPN expression selectively within AT. ATMs are the major source of OPN and also of its obesity induced up-regulation in the AT. These data point toward a specific pathophysiological role of OPN in obesity acting primarily within the AT. Therefore, OPN could be a key regulator of inflammatory processes linked to obesity induced AT inflammation and become a major target for treatment of AT inflammation-related disorders.


   Footnotes
 
This work was supported by the Austrian Science Fund project no. P18776-B11, and as part of Cell Communication in Health and Disease (CCHD) (W1205-B09; both to T.M.S.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online November 29, 2007

1 F.W.K. and M.Z. contributed equally to this study. Back

Abbreviations: AT, Adipose tissue; ATM, adipose tissue macrophage; BMI, body mass index; dSVC, depleted stromal vascular cell; MCP-1, monocyte chemoattractant protein-1; OPN, osteopontin; SSP1, secreted phosphoprotein-1; SVC, stromal vascular cell.

Received September 21, 2007.

Accepted for publication November 19, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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