A clue to the link between macrophage infiltration of adipose tissue and disordered adipocyte function came from the unex- pected biology of a TRAP over-expressing mouse transgenic line. To determine whether TRAP activity is limiting for bone turnover, a transgenic line was produced in which multiple additional copies of the complete gene were inserted in the germ line. The mice showed increased bone turnover, and over- expressed TRAP in the same locations as the endogenous genes [10]. The line was originally generated on a complex mixed genetic background. In the course of subsequent breeding and backcrossing to the FVB/N background, a sub-line of the transgenic mice was unexpectedly found to develop obesity. We therefore investigated the phenotype of the obese and a lean sub- line and also if TRAP can influence the proliferation and/or differentiation of adipocyte precursor cells. In this study, we present evidence that the obese phenotype in these animals is causally linked to over-production of TRAP. We demonstrate that certain macrophages in adipose tissue secrete monomeric TRAP that induces insulin-sensitive obesity by formation of new small adipocytes i.e. hyperplastic obesity. The results provide further evidence of the complex multifunctional roles of TRAP. Results Transgenic over expression of monomeric, but not proteolytically processed TRAP is associated with early onset spontaneous hyperplastic obesity in mice During breeding of a number of TRAP over expressing mouse sub-lines derived from one of the original high-expressing founders [10], we noted that one of these sub-lines (TRAP+) gained excessive body weight after weaning and conspicuously developed spontane- ous obesity (Figure 1A). The breeding of these lines was originally undertaken in an attempt to identify a more penetrant bone phenotype through association with other variables that affects bone density. Comparison of organ weights in TRAP+ versus non- transgenic littermate WT mice showed that two groups of organs were increased in TRAP+ mice; (1) lymphoid organs i.e. spleen by ,50% and (2) different adipose tissue depots i.e. mesenteric- and brown fat by ,60–120% depending on depot (Figure 1B and Table S2). On the other hand, the weights of other major internal organs e.g. liver, heart and kidneys, relative to total body mass were reduced in TRAP+ compared to WT mice. This increase in adipose tissue depots in TRAP+ mice was neither associated with increased food intake in adult males or females (Figure 1 C, D) nor increased adipocyte cell volume (Figure 1E). However, mRNA levels for several genes known to be associated with increased adipogenesis e.g. PPARc (Z = 2.03 p = 0.042), glycerol-3-phosphate dehydrogenase (GPDH; Z = 2.13 p = 0.033) and lipoprotein lipase (LPL; Z = 2.68 p = 0.01) were significantly up-regulated in adipose tissue from TRAP+ compared to WT mice (Figure 1F). The obese TRAP over expressing mouse sub-line (TRAP+) was next compared to an apparently lean transgenic sub-line from the same founder (TRAP+p). Comparison of growth curves of male and female WT, TRAP+p and TRAP+ mice showed that TRAP+ mice weighed 20–40% more than either the non-transgenic WT mice, or the lean transgenic TRAP+p mice (Figure 2 A, B and Table S3) throughout the first year of life. In male and female TRAP+ mice relative fat content was increased ,50% compared to WT mice while lean body mass was decreased by ,12–14% (Figure 2 C, D and Table S4). Neither relative lean nor fat mass differed in male or female TRAP+p mice compared to WT mice. Therefore, the obesity of the TRAP+ mice can be directly attributed to the TRAP transgene, which appears necessary but not sufficient to generate the phenotype. TRAP Induces Obesity The obese and lean TRAP over-expressing lines both expressed elevated levels of TRAP mRNA (WT vs TRAP+ Z = 2.94 p = 0.003) in adipose tissue (Figure 2 E), so the basis of differential adiposity is not due to differential expression of the transgene. The difference between the sub-lines emerged when the proteins were examined (Figure 2 F). The lean TRAP+p mice mainly expressed proteolytically processed TRAP with increased enzyme activity (WT vs TRAP+p Z = 3.80 p = 0.000143) (Figure 2E), whereas the obese TRAP+ mice expressed both monomeric and proteolytically processed TRAP in the adipose tissue (Figure 2F). We concluded therefore that the lean mice more effectively cleave TRAP, so that the obese mice, in effect, selectively over-express the monomeric form of the protein with low enzyme activity. Increased expression of monomeric, but not proteolytically processed TRAP in adipose tissue from obese humans Based upon the observations in mice, we asked whether monomeric TRAP is expressed in human adipose tissue, and whether it is over-expressed in obese patients. Both TRAP mRNA expression and monomeric TRAP protein expression were increased by 400% among the obese subjects compared to lean subjects (Figure 3A and Table S5). We also stratified the obese patient group into one group displaying a mainly hypertrophic phenotype (large fat cells) and one group displaying a more pronounced hyperplastic phenotype (small fat cells) in spite of similar body mass index. Although TRAP mRNA and monomeric TRAP protein were elevated in both obese groups, (Figure 3 B–D and Table S5), expression of proteolytically processed TRAP appeared not to be different (Figure 3C). Monomeric, but not proteolytically processed TRAP induces adipogenesis ex vivo in adipocyte precursor cells To determine whether expression of monomeric TRAP in adipose tissue might have a direct causal role in generating obesity, we used in vitro assays. Adipocyte progenitor cells were simultaneously cultured under three different conditions; (1) control (standard medium), (2) in the presence of monomeric TRAP or (3) in the presence of proteolytically processed (cleaved) TRAP. For assessing effects on proliferation, the TRAP isoforms were present for 3 days in sub confluent, proliferating cultures. In differentiating cultures, the TRAP isoforms were added to sub confluent cultures for 2–3 days until confluence, as well as during induction of adipocyte differentiation for an additional 4 days. Monomeric TRAP, at 10211–10212 M, caused a 30% enhance- ment of cell proliferation (Figure 4A and Table S6) and a 250% increase of terminal adipocyte differentiation (Figure 4B) in the mouse pre-adipocyte 3T3-L1 cell line. In contrast, proteolytically processed TRAP affected neither proliferation nor differentiation of this cell line (Figure 4A–B) in comparison to control cells. Similar differential effects of monomeric vs. proteolytically processed TRAP were also observed in adipocytes derived from human MSCs (Figure 4C–D) as well as human pre-adipocytes (Figure 4E–F), although at somewhat higher concentrations of monomeric TRAP. The stimulation of differentiation by monomeric TRAP was independent of the presence (Figure 4A, C, and E) or absence (data not shown) of the PPARc activator roziglitazone. Macrophages are a source of monomeric TRAP in adipose tissue In line with previous studies in animal models of obesity [21,22], mRNA for c-fms (Figure 5A; Z = 2.72 p = 0.01) as well as F4/80 staining (Figure 5B) were increased in adipose tissue from obese PLoS ONE | www.plosone.org 2 March 2008 | Volume 3 | Issue 3 | e1713