exerted throughout development, but the body temperature of PRLR KO adult mice was reported significantly lower than in WT animals [35]. To assess the impact of lactogen action on brown adipocyte physiology, we examined the effects of PRLR deletion on brown adipocyte differentiation in vitro. We showed that immortalized brown preadipocytes from WT newborn mice differentiated during exposure to insulin and T3 and expressed the adipogenic genes PPARc2 and UCP1. In striking contrast, PRLR-deficient cells failed to differentiate or to express these adipogenic genes despite long-term treatment with insulin and T3. This finding indicates that PRLR signaling, like insulin/IGF-1 signaling, is required for BAT differentiation. Whether or not lactogen action is also required for white adipose tissue differentiation is unclear, but PRL induces PPARc and C/EBPb mRNA levels in NIH 3T3 cells [36] and adult female PRLR KO mice have reductions in abdominal fat mass and decrease in adipocyte numbers [37–39]. This illustrates that beyond the role of PRLR signaling in adaptive thermogenesis at birth, this pathway may also affect later, white adipose tissue development and metabolism. A novel finding of our study is the first demonstration that IGF- 2 serves as a downstream target of lactogen action in the differentiation of brown adipocytes. We showed that PRLR deletion is accompanied by reductions in BAT IGF-2 mRNA levels in vivo and in vitro and that PRL stimulates IGF-2 expression in brown adipocytes. The latter effect appears to be mediated through JAK/STAT induction of IGF-2 transcription. We then showed that IGF-2 stimulates the proliferation and differentiation of brown adipocytes and reverses partially the defects in brown adipogenesis observed in PRLR-deficient cells. Even though IGF- 2 only represents one of the molecular PRL targets involved in BAT growth and differentiation, this illustrates that it can bypass the absence of PRLR and thereby acts downstream of lactogen signaling. The molecular mechanisms by which IGF-2 exerts its actions are not entirely clear, but the receptors that mediate its effects on placental and fetal growth, namely, the IGF-1R and IR- A, are expressed in the BAT of newborn mice. It is of considerable interest that plasma IGF-2 levels were reduced significantly in PRLR KO mice on the day of birth. This new observation suggests that lactogenic hormones that circulate in the fetus in late gestation (in rodents, PL, in humans, PL, PRL, and growth hormone) may also induce IGF-2 expression in tissues other than BAT; in theory, these could include the placenta and fetal liver, adrenal, pancreatic beta cells, and mesenchymal tissues, all of which express PRLR as well as IGF-2 [4–6,21]. In rodents, the expression of IGF-2 ceases a few weeks after birth and postnatal murine growth is IGF-2 independent [21]. In contrast, IGF-2 levels are maintained throughout development in humans, and IGF-2 levels are variably decreased in children with growth hormone deficiency [40]. The reductions in BAT IGF-2 expression and plasma IGF-2 concentrations in PRLR KO neonates suggest a model whereby lactogen action in BAT may be mediated in part through systemic and/or local IGF-2 production. This model is illustrated in Fig. 6. Prolactin and/or PL bind to the PRLR of the brown adipocyte leading to the activation of various signaling pathways including the MAP kinase (MAPK), phosphatidyl-inositol 3-kinase (PI-3K), and JAK2/STAT5 pathways and potentiation of IRS signaling [18]. This results in transcriptional activation of genes including IGF-2, which is secreted and exerts autocrine/paracrine/endo- crine effects by binding to IR-A and IGF-1R. Lactogen signaling thereby promotes cell proliferation, brown adipocyte differentia- tion and UCP1-mediated thermogenesis. The finding that IGF-2 reverses only partially the effects of PRLR deletion suggests that PRL target genes other than IGF-2 Prolactin and Brown Adipocytes might be of major pathophysiological importance. Interestingly, direct comparison between the predictive anti-adipogenic genes [41] and the putative PRL target genes identified by microarrays [24] reveals a strikingly homologous list of candidates. They include genes related to cell adhesion and extracellular matrix [42], Wnt signaling pathway [43], cell growth and maintenance which have been already described to play roles in adipocyte biology. Additional experiments must be undertaken to ascertain the involvement of the gene candidates linking PRLR signaling and adipocyte differentiation. In summary, our findings reveal the critical role played by PRLR signaling in brown adipocyte growth and differentiation, thermogenesis, and the response to cold stress. The mediation of lactogen action by induction of IGF-2 expression provides a novel mechanism subserving perinatal adaptation and energy homeo- stasis. Given the importance of intrauterine programming and BAT in energy balance, our findings implicate new roles for the lactogens in the interface between fetal and perinatal development and metabolic disease. MATERIALS AND METHODS PRLR KO mice All experimental designs and procedures are in agreement with the guidelines of the animal ethics committee of the Ministe`re de l’Agriculture (France). WT, and PRLR KO mice on a pure 129 genetic background were generated by heterozygous crossbreeding [19]. PCR analysis of tail DNA determined the genotypes of the offspring as described previously [44]. Histological analyses BAT was isolated from the interscapular space of newborn WT and PRLR KO mice, fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin. Sections (4 mm) were deparaffinized in xylene, rehydrated and stained with hematoxylin eosin. The adipocyte surfaces were analyzed using a Lucia software on 200 sections per genotype. Data are mean6SE, for 4 mice per genotype. Cell extracts, biochemical determinations, and enzyme assays BAT was homogenized in 25 mM Tris, pH 7.5, 1 mM EDTA. A fraction of the homogenate was stored at 280uC. The remaining fraction was centrifuged at 10,0006g for 10 min at 4uC, and the supernatant was kept at 280uC until use. Aliquots of homogenates and supernatants were used to determine protein content. Triglyceride content was determined on homogenates with PAP150 triglyceride kit (Biome´rieux, Marcy L’Etoile, France). Electron-microscopic analyses BAT were washed with PBS and fixed for 4 h with 2% glutaraldehyde (Merck)-2% paraformaldehyde (Sigma) in 0.1 M phosphate buffer, pH 7.4 (Merck) at room temperature. Tissues were rinsed in PBS, postfixed with 2% osmium tetroxyde (Uptima, Montlucon, France) for 30 min, dehydrated in graded ethanol, and embedded in Durcupan (Fluka, St Quentin, France). Ultrathin sections were realized and examined with a JEOL 1010 electron microscope. RT-PCR Total RNA was extracted from cells with TRIZOL reagent (InVitrogen, Cergy Pontoise, France) according to the manufacturer’s recommendations and RNA were thereafter processed for RT-PCR, PLoS ONE | www.plosone.org 9 February 2008 | Issue 2 | e1535