TRAP Induces Obesity Figure 4. Stimulation of adipocyte proliferation and differentiation by monomeric TRAP but not proteolytically processed TRAP. The following cells were used: Mouse 3T3-L1 preadipocytes (A, B), Human mesenchymal stem cells (hMSC) (C, D) and human pre-adipocytes (E, F). Proliferation (BrdU) was assessed after 1 day (3T3-L1), 2 days (3T3-L1, hMSC), 3 days (3T3-L1) or 5 days (pre-adipocytes) after seeding. Differentiation (GPDH-activity) was measured 4 days (3T3-L1) or 12 days (hMSC, pre-adipocytes) after start of differentiation. hMSC were treated with TRAP either before (Pro) or at (Post) confluence. mTRAP = monomeric TRAP, cTRAP = proteolytically processed TRAP. Data are expressed as percentage of control. Statistical analysis was performed using T-test, * p,0.05, comparing control cells with stimulated cells. doi:10.1371/journal.pone.0001713.g004 Discussion TRAP overexpressing mice were originally generated in order to study the skeletal phenotype and bone turnover [10]. Unexpectedly, in the course of breeding to a different genetic background we found one sub-strain of TRAP over expressing mice that presented an early onset obesity that did not seem to be due to apparent over-eating. The differences in body weight (,20–40%) was evident already at one month of age and up to 1 year of age the body weight curves in TRAP+ and WT mice were parallel. This indicates that other features such as decreased physical activity/energy expenditure could explain the obesity in TRAP+ animals. It is of interest to note that there was clear relative difference in organ size of TRAP+ animals, where most internal organs were relatively smaller in the TRAP+ mice compared to WT mice. This might suggest that adipose tissue expanded in TRAP+ mice leading to redistribution of energy expenditure on behalf of other organs which diminished in size. In the TRAP over expressing mice, adipocyte volume was normal and several genes known to increase adipogenesis (PPARc, GPDH and LPL) were up regulated. This suggested that the obesity was largely due to enhanced formation of new fat cells through differentiation and/or proliferation rather than lipid filling of pre-existing adipocytes, i.e. the TRAP+ mouse develops hyperplastic obesity. Only two previous models have demonstrated hyperplasia, rather than hypertrophy, as the major cause of obesity [29,30]. The results also suggested that the monomeric form of TRAP is specifically causing the effect of TRAP on adipogenesis. A second sub-strain (TRAP+p) without detectable expression of monomeric TRAP, but with elevated levels of the proteolytically processed form in adipose tissue was lean. Since the lines derive from the same founder, and over-express the mRNA and total protein to the same extent, we suggest that there is an independently segregating variant allele of a gene involved in the proteolytic processing of TRAP. Assuming this PLoS ONE | www.plosone.org 6 March 2008 | Volume 3 | Issue 3 | e1713