Prolactin and Brown Adipocytes Figure 3. PRLR is crucial for brown adipocyte differentiation. A) Schematic representation of the mutation of the PRLR gene by homologous recombination and gene targeting techniques. Black boxes represent exon 4, 5 and 6. NEO: neomycin gene ; arrows indicate the position of primers used for PCR. B) PCR genotyping of immortalized brown adipocyte cell lines. C) Morphologic features of wild-type (WT), PRLR KO (KO) and rescued- PRLR (KO+PRLR) brown adipocytes on day 0 and day 6 of the differentiation program. All cell lines presented fibroblast-like morphology (day 0). Inset: immunodetection of the SV40 large T antigen in the nucleus of WT and PRLR KO brown adipocytes. WT, KO and KO+PRLR cell lines were cultured in the presence of 20 nM insulin and 2 nM T3 for 6 days. Light microscopic examination revealed the presence of numerous intracytoplasmic lipid droplets in the WT brown adipocyte cell line whereas PRLR KO brown adipocytes were unable to accumulate lipid droplets in their cytoplasm. Overexpression of PRLR in PRLR KO brown adipocytes (KO+PRLR) by stable transfection of a murine PRLR expression vector restores their ability to differentiate into mature brown adipocytes. D) RT-PCR analyses of PRLR, PPARc2, UCP1 mRNA expression and 18S RNA used as internal control in WT, KO and rescued-PRLR (KO+PRLR) brown adipocytes. Note the reappearance of a strong PPARc2 expression in rescued-PRLR differentiated brown adipocytes. doi:10.1371/journal.pone.0001535.g003 suggest that lactogen signaling plays a pivotal role in the setting and/or maintenance of thermogenic function in BAT. Differentiation of immortalized PRLR deficient preadipocytes is impaired To investigate whether PRLR deficiency limits the ability of brown preadipocytes to undergo adipogenic conversion, we isolated cells from the stromal vascular fraction of interscapular BAT of PRLR KO and WT newborns. Several brown preadipocyte cell lines were established after stable transfection of the P1-TAg expression vector, which contains the immortal- izing SV40 large T Antigen (TAg) under the control of the proximal promoter of the human mineralocorticoid receptor gene. This transgene was previously used to generate the T37i brown adipocyte cell line [20] (Fig. 3A and B). The differentiation capacities of PRLR KO and WT cell lines were assessed in order to decipher the molecular events by which PRLR signaling contributes to the commitment to the brown adipose phenotype. Fig. 3C presents results obtained with representative clones of WT and PRLR KO preadipocytes. At post confluence (day 0) both cell lines exhibited fibroblast-like morphology and expressed the SV40 TAg, as demonstrated by its nuclear immunodetection (inset). After 6 days of exposure to insulin and T3, the WT preadipocytes differentiated and accumulated triglycerides within multilocular fat droplets (Fig. 3C, upper panel). In contrast, PRLR KO preadipocytes failed to accumulate cytoplasmic lipid droplets (Fig. 3C, middle panel), suggesting that PRLR signaling plays a crucial role in brown adipocyte differentiation. Similar results were obtained in all other independent cell lines derived from PRLR KO animals (data not shown). To assess further the role of PRLR signaling in response to PRL and PL which are present in the fetal calf serum, we stably transfected a murine PRLR expression vector into PRLR KO preadipocytes (KO+PRLR) and examined the adipogenic activa- tion cascade. After 6 days exposure to the differentiation mixture (insulin + T3), PRLR-rescued cells accumulated triglycerides and acquired the morphologic appearance of mature brown adipocytes (Fig. 3C, lower panel). RT-PCR analysis performed on WT, PRLR KO and PRLR-rescued cell lines confirmed that expression of PRLR correlated with that of PPARc2, a marker of adipocyte differentiation, and of UCP1, a marker of brown adipocyte lineage (Fig. 3D). These findings establish conclusively a direct role for lactogen signaling in brown adipocyte differentiation. PLoS ONE | www.plosone.org 5 February 2008 | Issue 2 | e1535