gene that is specific to ‘‘lean’’ animals. Alternatively, generating transgenic mice using for instance the aP2 promoter to direct over expression of the TRAP gene selectively to adipocyte lineage cells may provide another viable strategy to obtain mice with the obese phenotype. TRAP knock out mice [31] have not been reported to have an altered adipose tissue phenotype, although our work would suggest more detailed studies would be worthwhile. This does not necessarily imply that monomeric TRAP has no role in normal adipose development, since the role of TRAP in bone is also partly redundant (the knockout animals are only mildly osteopetrotic). An interesting feature of the TRAP-over expressing transgenic mice is that there is an increased macrophage content in adipose tissue, so TRAP is directly or indirectly proinflammatory. A systemic effect of TRAP on macrophages could also underlie the increase in relative spleen size in these animals. It may be that in adipose tissue, as in bone, there is a complex homeostatic interaction between TRAP-producing and TRAP-responsive cells. It is interesting to note that elevated levels of serum TRAP previously has been positively correlated with serum insulin levels in NIDDM rat models [32]. In osteoclasts the post-translational proteolytic processing of TRAP is partly involving the cysteine protease cathepsin K [33]. Cathepsin K has been shown to be a marker of adiposity [34] and cathepsin K mice gained less weight when put on high-fat diet and exhibited increased adipocyte lipolysis [35]. However, while TRAP seems to be mainly expressed in macrophages, cathepsin K seems to be expressed in the adipocyte itself [36] suggesting distinct roles for these proteins in adipose tissue. The clear relevance of the mouse model was demonstrated in human patient material, where an increased expression of monomeric TRAP, but not proteolytically processed TRAP, was apparent in adipose tissue of obese subjects. Human obesity is commonly characterized by a combination of hypertrophic and hyperplastic adipose tissue, although hypertrophy usually domi- nates [37,38], but some subjects exhibit predominantly hyperplas- tic (i.e. smaller adipocyte volume than expected) obesity [39]. When our obese subjects were divided according to fat cell size both TRAP mRNA and monomeric TRAP were showed a tendency to a further increase in those with hyperplastic obesity as compared to those with hypertrophic obesity. In both hMSC and pre-adipocytes from mouse and humans monomeric TRAP stimulated both proliferation and differentia- tion, measured as increase of GPDH enzyme activity. However, proteolytically processed TRAP had no effect on these processes. These data are somewhat unexpected, as usually a positive effect on proliferation is accompanied by a negative effect on differentiation and the opposite. Nevertheless, the findings are consistent with the hypothesis that monomeric TRAP could exert a dual effect on both proliferation and differentiation of pre- adipocytes which would increase the impact of monomeric TRAP although the effect was small in each step. It is, however, possible that the adipogenic effect of monomeric TRAP is stronger in vivo than in vitro. The effect on differentiation in vitro was observed in the presence or absence of roziglitazone suggesting that mono- meric TRAP can activate adipogenesis at variable levels of PPARc activity. Although TRAP has not been shown previously to affect the proliferation and differentiation of adipocytes, TRAP has been shown to induce differentiation of osteoblasts [9] and haemato- poietic cells [40]. Obesity is associated with a low-grade inflammation in adipose tissue [41,42], where a hall-mark is influx of macrophages into the tissue [21,22]. The latter has been hypothesized to be a consequence of adipocyte necrosis because of adipocyte hypertro- TRAP Induces Obesity phy [43] and/or increase of monocyte attractants such as MCP-1/ CCL2 [44–46]. TRAP is normally expressed in cells from the myeloid linage [11,47,48] and since the construct in the transgenic TRAP over expressing mouse used in this study is under the influence of the normal TRAP promoter [10] we expected that only cells normally competent to express TRAP would have an elevated expression of the enzyme. Confirming this idea, we found that in mouse adipose tissue, almost 90% of the TRAP transcripts originated from the myeloid specific transcript 1C and in human adipose tissue, TRAP were mainly expressed in CD68 positive macrophages. This indicates that the main source of TRAP in adipose tissue is macrophages. It should be noted, however, that CD68 is expressed in cell types other than macrophages, e.g. fibroblasts, although the antibody used in this study has been shown to recognize CD68 preferably in macrophages [49]. It is unlikely that monomeric TRAP is secreted by other cells than macrophages since the 1C transcript, which is the predominant transcript in adipose tissue, is regulated by PU.1 and MiTF which are myeloid specific transcription factors [50]. Furthermore, the TRAP staining in the original transgene study [10] does not support the idea that significant levels of TRAP can be expressed by non myeloid cells other than in liver and kidney. However, we cannot rule out that secretion of TRAP from cells other than macrophages occur within adipose tissue. In macrophages, TRAP has traditionally been considered a lysosomal constituent. However, recent studies in osteoclasts has shown that there is likely to be two populations of TRAP present, one intracellular which is mainly proteolytically processed and one secreted which is mainly monomeric [33]. Previously published data [16] as well as in vitro experiments of this study have shown that monomeric TRAP can be secreted also from macrophages in vitro. This is also in line with other studies showing that monomeric, rather than proteolytically processed TRAP seems to be the dominant secreted form also in other cell types [13,51]. Together these data suggest that also in macrophages different isoforms of TRAP might be present intracellularly or secreted. Once secreted from macrophages, monomeric TRAP seemingly acts on adipose tissue to induce adipogenesis. The molecular mechanism behind the action of monomeric TRAP on adipogen- esis is unknown, but it can be speculated that TRAP is recognized by a surface receptor on pre-adipocytes and that signals from this receptor regulate adipogenesis. Obesity is strongly linked to insulin resistance [52,53] and several studies have highlighted the correlation between adipocyte size rather than adipose tissue mass and insulin malfunction [53– 58]. The enlargement of adipocytes may not have pathophysio- logical significance by itself but rather be a manifestation of other pathogenetic factors leading to insulin resistance [58–60], such as increased adipocyte lipolysis resulting in elevated fatty acids, which in turn cause insulin resistance [61]. On the other hand, enlarged adipocytes may be pathogenic themselves by, for example, a change in production of adipokines and cytokines [58], the latter linking adipose inflammation to insulin resistance. Recently it was also shown that TNFa down regulates eNOS expression in metabolically active tissues which might sustain obesity [62]. Certain macrophage populations in adipose tissue might be associated with insulin resistance and obesity [26]. As judged by circulating insulin and glucose levels as well as HOMA index and by measurement of adipocyte lipolysis and lipogenesis, the obese TRAP+ mouse exhibited normal adipocyte lipid and glucose metabolism as well as insulin and catecholamine sensitivity, although it can not be excluded that some alterations of insulin action occurred in skeletal muscle or liver, which were not directly examined. Also, there was no evidence of major changes in PLoS ONE | www.plosone.org 9 March 2008 | Volume 3 | Issue 3 | e1713