-Actin was used as loading control. DISCUSSION Our results showed that lack of REDD1 expression induces dWAT expansion fueled by the increased number and size of mature adipocytes and their elevated differentiation potential. adipocyte precursor cells (APCs) numbers were lower in KO skin. analysis revealed increased differentiation of skin-derived KO APCs as indicated by higher lipid accumulation and increased adipogenic marker expression. 3T3L1 cells overexpressing REDD1 had decreased sensitivity to differentiation. Overall, our findings indicate that REDD1 silencing induced expansion of dWAT through hypertrophy and hyperplasia. This not previously reported REDD1-dependent mechanism of adipogenesis could be used to preferentially target skin-associated adipose tissue for therapeutic purposes. KO mice appeared to be resistant to the development of obesity on a high-fat diet (Williamson et al., 2014). It was also shown that REDD1 regulates lipolysis and lipogenesis in cultured human and mouse adipocytes (Schupp et al., 2013). In our previous work, we showed that REDD1 plays an important role in skin where it is causatively involved in the development of glucocorticoid-induced AZD8330 skin atrophy: mice lacking REDD1 displayed cutaneous hyperplasia, and all skin compartments including dWAT appeared to be partially resistant to atrophy induced by chronic treatment with glucocorticoids (Baida et al., 2015). The important roles of Akt/mTOR and REDD1 in adipogenesis, lipid metabolism, and energy homeostasis prompted us to look closer at the fat depots in KO animals with a specific focus on AZD8330 dWAT and regulation of adipocyte differentiation by REDD1. AZD8330 Unexpectedly, KO mice appeared to be lighter than wild type (WT) mice in the same genetic background and had significantly reduced total fat mass. However, KO mice have increased dWAT and subcutaneous fat (sWAT) depots. We found that dWAT extension was established early during postnatal skin development, in part due to the increased generation of new mature adipocytes and an influence on adipocyte differentiation. This extension of dWAT established at the early stages of postnatal development persisted via whole life of KO animals and was independent on hair cycle. RESULTS REDD1 loss negatively regulates body weight and gonadal and interscapular adipose depots but induces expansion of the dermal adipose layer Akt/mTOR pathway plays an important role in adipocyte differentiation, and one of its major inhibitors, REDD1, is known to control energy homeostasis and lipogenesis. Thus, we assessed the AZD8330 effect of knockout on the different adipose tissue depots. First, we measured body weight, body composition, and the size of different fat depots in adult, 8 week old male and female WT and KO mice. We found a 15C20% decrease in total weight in KO mice of both sexes (Figure 1a). This was a consistent observation for numerous KO animal generations. KO animals also had significantly decreased total amount of fat and increased total body lean mass compared to WT animals as measured by EchoMRI? (Table 1). Analysis of the size of selected fat depots (measured by weight) revealed a significant decrease in fat accumulation in gonadal WAT depot and interscapular brown adipose depot in KO mice (Figure 1a). Interestingly, the depots associated with skin: inguinal subcutaneous WAT (sWAT) and dWAT were significantly increased in KO animals (8 week old and older) of both sexes (Figure 1aCb.) Open in a separate window Figure 1. Differential IKZF2 antibody effect of REDD1 loss on adipose depots: selective increase in dWAT and subcutaneous WAT.a. Body weight and size of fat depots (% to total body weight) were measured in 8 week old WT and REDD1 KO animals, and presented as mean SD (N=5, for each sex/genotype). b. Representative images of Perilipin1+ mature adipocytes in dWAT layer in WT and REDD1 KO females. Scale bar=100m. c, d. Quantification of dWAT area (c) and number of mature adipocytes (d) per mm of skin. e. Mature adipocyte size distribution in WT and REDD1 KO dWAT. At least 30 images of 3C5 individual pores and skin samples from WT or REDD1 KO animals were analyzed. Data is offered as mean +/? SD. Statistically significant difference between REDD1 KO and WT: * p 0.05 (t-test for any, c, d). Table 1. Decreased total excess fat mass in REDD1 KO animals. KO. Both adipocyte hypertrophy (improved adipocyte size) and hyperplasia (improved adipocyte numbers driven by pre-adipocyte proliferation and differentiation) travel physiological raises in dWAT thickness (Festa et al., 2011; Foster et al., 2018). Therefore, to further quantify the effect of REDD1 loss on dWAT, we assessed the size and quantity of Perilipin1+ adult dermal adipocytes. As demonstrated in Number 1b, there was a dramatic difference in the thickness of the dWAT in KO compared to WT pores and skin during the quiescent stage of the hair cycle (telogen, 8 week aged mice). Morphometric AZD8330 analysis of Perilipin1+ adipocytes exposed that both the size and quantity of adult.