expression of adipokines or cytokines involved in obesity mediated insulin resistance except for an increased mRNA expression of TNFa. Thus, the absence of fat cell hypertrophy and the modest change in expression of cytokines associated with an innate immune response may explain why obese TRAP+ mice had nor or little evidence of insulin resistance or altered fat cell metabolism in spite of macrophage infiltration. The enhanced number of adipocytes in the enlarged adipose tissue might cause the increased level of circulating leptin in TRAP+ mice. In light of these data, we propose a hypothesis of a paracrine effect of monomeric TRAP on adipogenesis. In this model, monomeric TRAP, likely secreted predominantly by macrophag- es, influences the development of obesity by inducing adipogenesis in a situation where there is an influx of macrophages into the adipose tissue or an up regulation of monomeric TRAP in pre- existing macrophages. This could increase the formation of new, normally sized adipocytes with normal insulin sensitivity and metabolism. This hypothesis is not apparently consistent with the proposed function of macrophages in adipose tissue, since they previously have been reported to secrete factors that increases expression of inflammatory genes in adipocytes [23,24] and inhibits adipogenesis [24,25]. However, macrophages consist of a heterogeneous population of cells and can be activated differently [63] and evidence was recently presented for the presence of distinct macrophage populations in adipose tissue some being protective against insulin resistance [64]. With respect to expression of monomeric and proteolytically processed TRAP in macrophages it has been shown that monomeric TRAP has a more restricted distribution than proteolytically processed TRAP [11]. However, the regulation of the different forms of TRAP in macrophages remains to be elucidated. Thus, macrophages, depending on their progeny and/or activation state, might secrete factors, for example monomeric TRAP, which could have variable effects on adipose tissue. In summary, certain macrophages in adipose tissue seem to secrete monomeric TRAP that induces obesity; at least partly due to adipogenic affects causing increased formation of normally sized adipocytes with normal insulin sensitivity, lipid and carbohydrate metabolism and adipokine/chemokine production. Materials and Methods Experimental animals Age-matched male and female mice from different litters (WT , TRAP+p and TRAP+) were kept under controlled light/dark conditions with food and water available ad libitum. The study was approved by the Stockholm South Animal Ethical Committee (S159/01 and S235/04) and maintained according to the guidelines of the Animal Welfare Board at Karolinska Institutet. Statistical analysis For statistical method and number of animals or subjects for each experiment see specific analysis. Values are given as mean6SD. Symbols for statistical comparison are * p,0.05; **p,0.01; ***p,0.005. NS; not significant Generation and genotyping of TRAP over expressing transgenic FVB/N mice TRAP over expressing transgenic FVB/N mice [10] (TRAP+ or TRAP+p) containing .30 copies of the TRAP gene were used. Genomic DNA was purified using Puregene (Gentra, Minneapolis, MN) from mouse-tails. Primers (Invitrogen, Carlsbad, CA)/probes (Biosearch Technologies, Novato, CA) for SV40 and TRAP were as follows; (A) SV40 primers 59CACCTGGTTGCTG ACTAAT TRAP Induces Obesity TGAGA 39/59GTGTGTCAGTTAGGGTGTGGAAA 39 and HEX labeled probe 59CCCCAGGCTCCCCAGCAGGCAG 39 annealing temperature 62uC. (B) TRAP primers 59 TGGTCATTT- CTTTGGGG CTTATCT 39/59GCTACTTGCGGTTTCAC- TATGGA 39 and FAM labeled probe 59TGTGA AGCCGCC- CAGGGAGTCCTC 39 annealing temperature 62uC. qPCR was run as stated under ‘‘Total RNA purification and RT-qPCR’’ with the exception that iQ Supermix (Bio-Rad, Hercules, CA) was used. Determination of organ weight index Organs were dissected out and the wet weights were determined. For n see Table S2. Statistical analysis was carried out using Mann-Whitney U test. Determination of body weight–growth curves Male (WT; n = 50, TRAP+p; n = 21, TRAP+; n = 56; for n at specific ages see Table S3) and female (WT; n = 68, TRAP+p; n = 22, TRAP+; n = 52; for n at specific ages see Table S3) were weighed at different time points. Statistical analyses were carried out using Kruskal Wallis followed by Mann Whitney U test. Measurement of lean mass and body fat content using DXA Dual-energy X-ray absorptiometry (DXA) was performed as described [65]. Male (WT; n = 24, TRAP+p; n = 17, TRAP+; n = 5) and female (WT; n = 28, TRAP+p; n = 14 , TRAP+; n = 9 ), 2–12 month mice were used (Supplementary Table IV). Statistical analysis was carried out using Kruskal Wallis test followed by Mann-Whitney U test. Calculation of adipocyte volume Adipocyte size was determined as described in [66]. Adipose tissue was obtained from the animals (WT; n = 8, TRAP+; n = 8) and adipocytes were isolated by collagenase treatment. Using direct microscopy, the diameter of 100 cells was determined and the mean fat cell volume was calculated. Statistical analysis was carried out using ANOVA. Measurement of food intake Animals were given a specific amount of food and the consumption was measured by weighing the remaining food every other day for two weeks. Food consumption was then calculated as intake of gram food/gram body weight over two weeks for male (WT; n = 3, TRAP+; n = 6) and female (WT; n = 3, TRAP+; n = 5) mice. Statistical analysis was carried out using Mann-Whitney U test. Total RNA purification and RT-qPCR on mouse and human adipose tissue Total RNA was extracted from gonadal or mesenteric adipose tissue of male and female mice (WT; n = 11, TRAP+p; n = 7, TRAP+; n = 9) using RNeasy Lipid Tissue Mini Kit (QIAGEN, Hilden, Germany), treated with DNase (Invitrogen, Carlsbad, CA), quantified using Ribogreen (Invitrogen, Carlsbad, CA) and then transcribed using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). From human subjects, total RNA was extracted from 300 mg subcutaneous fat tissue (n = 28) using RNeasy Mini Kit (QIAGEN, Hilden, Germany), determination of RNA purity and reverse transcription was performed as described [67]. Real- Time PCR was carried out on an iCycler iQ Real Time PCR Detection System (Bio-Rad, Hercules, CA) in triplets using, for mouse cDNA, PlatinumH SYBRH Green qPCR SuperMix UDG (Invitrogen, Carlsbad, CA) and, for human cDNA, iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) both with the addition PLoS ONE | www.plosone.org 10 March 2008 | Volume 3 | Issue 3 | e1713