Identification of IGF-2 as a target of lactogen signaling in BAT BAT differentiation is under the control of many hormones and growth factors including insulin and IGF-1. Since IGF-2 has been previously shown to mediate PRL signaling during mammary gland morphogenesis [24], we hypothesized that this growth factor might be a mediator of PRL-induced adipocyte differentiation. To test this possibility, we first compared the levels of IGF-2 mRNA in BAT of newborn PRLR KO mice with those in WT mice. IGF-2 transcript levels were 70% lower in BAT of PRLR KO mice than in BAT of WT littermates (Fig. 4A), indicating that endogenous IGF-2 gene expression might be under the direct control of PRLR signaling in brown adipocytes. There was also a 35% reduction in IGF-2 plasma levels in PRLR KO neonates (Fig. 4B), providing further evidence that lactogens regulate IGF-2 production during the fetal and perinatal periods. Support for a role for PRLR signaling in IGF-2 production was provided by studies of the effects of PRL on IGF-2 expression in brown adipocytes in vitro. We first examined the changes in IGF-2 mRNA levels during insulin/T3-induced differentiation of im- mortalized brown preadipocytes from newborn WT mice. As shown in Fig. 4C, IGF-2 mRNA levels were significantly higher in differentiated than in undifferentiated cells; thus brown adipocyte differentiation is accompanied by a sharp induction of IGF-2 expression. We next compared the levels of IGF-2 mRNA in immortalized PRLR KO cells and KO+PRLR cells with those in WT cells. IGF-2 mRNA levels were 75% lower in insulin/T3- treated PRLR-KO cells than in WT cells on day 6. IGF-2 expression was restored by re-introduction of PRLR into PRLR KO cells. Finally, we showed that a 3 h-PRL exposure provokes a 2-fold increase in the levels of IGF-2 mRNA in T37i brown adipocytes (Fig. 4D). Collectively, these findings establish a critical role for lactogen signaling in brown adipocyte IGF-2 expression. The mechanisms by which lactogens regulate IGF-2 are currently unknown. Since PRL is known to mediate most of its effects through activation of the JAK2/STAT5 pathway, we determined if the specific JAK2 inhibitor AG490 could blunt the effect of PRL on IGF-2 expression in T37i cells. As illustrated in Fig. 4D, we showed that the induction by PRL of IGF-2 mRNA levels was abolished in the presence of AG490. We next showed that PRL treatment of differentiated T37i cells induced phos- phorylation of STAT5 within 15 min (Fig. 4E). Since the IGF-2 gene promoter contains multiple consensus STAT binding sites, we performed electromobility shift assays with nuclear extracts of untreated or PRL-treated T37i cells and the STAT response element located 23184 bp upstream of the transcription initiation start site of IGF-2 gene (exon 1) (Fig. 4F). Specific oligonucleotide- protein complexes were detected in PRL-stimulated extracts, a finding compatible with a model in which PRL regulates IGF-2 transcription through STAT5 activation. IGF-2 induces brown adipocyte proliferation and differentiation Given the effects of lactogen signaling on IGF-2 expression in BAT, we addressed the role of IGF-2 in preadipocyte proliferation and adipogenesis. To test whether IGF-2 was able to stimulate mitogenesis of brown adipocytes, we exposed postconfluent (day 2) and differentiated (day 7) T37i cells to 20 nM IGF-2 or insulin (which served as a positive control) and measured 3H-thymidine incorporation (Fig. 5A). Treatment of proliferating cells with nanomolar concentrations of IGF-2 induced a 3-fold increase in 3H-thymidine incorporation. IGF-2 stimulated a 2-fold increase in 3H-thymidine incorporation even in differentiated cells, which as Prolactin and Brown Adipocytes expected had lower basal rates of DNA synthesis than undiffer- entiated cells. The potency of IGF-2 in these respects was comparable to that of insulin. Longer exposure to IGF-2 stimulation induced morphological changes and the appearance of intracyto- plasmic lipid droplets in T37i cells, demonstrating a major impact of IGF-2 on brown adipocyte differentiation (Fig. 5B). Quantification of Oil Red O staining and measurement of PPARc2 mRNA levels established that IGF-2 was as or more potent than insulin in induction of brown adipocyte differentiation (Fig. 5B). The mechanisms by which IGF-2 stimulates adipogenesis are currently unknown, but the receptors that mediate most IGF-2 effects in the fetus and placenta, namely insulin receptor form A (IR-A) and IGF-1 receptor (IGF-1R) [25] were expressed in brown adipocytes (Supplemental data Fig. S3 and Text S1). In addition, IGF-2 receptor (IGF-2R), which mediates IGF-2 turnover by endocytosis, was also detected in brown adipocytes. Since IGF-2 and insulin have been shown to differentially regulate gene expression through IR-A [26], the respective contributions of insulin and IGF-2 signaling in brown adipocyte proliferation and differentiation remain to be established. IGF-2 can partially reverse the effect of PRLR- deletion on BAT differentiation Given the adipogenic effects of IGF-2 in T37i cells and the induction by PRL of IGF-2 expression, we next determined if incubation with exogenous IGF-2 could reverse the defect in brown adipocyte differentiation observed in PRLR KO cells. As shown in Fig. 5C, IGF-2 promoted partial differentiation of PRLR KO cells, with appearance of foci of mature adipocytes. This finding suggests that IGF-2 can bypass the absence of PRLR and exert its actions downstream of PRL signaling. Our findings provide strong evidence that IGF-2 constitutes one of the major PRL targets in brown adipocytes and are compatible with an autocrine regulatory loop in which PRL stimulates endogenous IGF-2 production, which in turn facilitates activation of adipogenesis in BAT. DISCUSSION The lactogens play important roles in carbohydrate metabolism through effects on pancreatic beta cell mass and insulin production [27,28] and peripheral insulin sensitivity [29], however their roles in energy homeostasis and lipid metabolism are poorly understood [30]. In this study, we demonstrate that lactogen signaling is essential for brown adipocyte growth and differentiation, thermo- genic gene expression, and the adaptation to cold stress. Newborn PRLR KO mice have impaired BAT development as illustrated by sharp decreases in BAT weight, adipocyte size, and triglyceride content. The impaired development of BAT is underscored by a significant decrease in the expression of UCP1, which could be the consequence of altered BAT development, as illustrated by the decrease of PPARc2 and its coactivator PGC-1a. It is well established that PPARc2 is important for the development of BAT since mouse models with adipose-specific PPARc ablation have reduced or absent interscapular BAT [31]. Maladaptive responses to cold have been described in mouse models with deletions in PGC-1a [32] and PPARc [33]. Moreover, the cold sensitivity of PRLR KO neonates is also amplified by the reduced expression of b3Adr, a key effector for UCP1 expression in rodents [23]. Importantly, we observed a major thermoregulatory defect in PRLR KO animals at birth, highlighting the physiological importance of PRL signaling in adaptive thermoregulatory responses to cold challenge. Previous studies [34] demonstrating a thermogenic effect of PRL in sheep are consistent with our findings. It is currently unclear if the thermogenic effect of PRL is PLoS ONE | www.plosone.org 6 February 2008 | Issue 2 | e1535