deletion of the thyroid hormone receptors, the insulin receptor, or insulin receptor substrates 1, 2, 3 or 4, which mediate many of the metabolic effects of insulin and IGF-1, have no effects on BAT mass in newborn mice [12,15–17]. These findings suggest that other hormones or growth factors might contribute to BAT growth and function during the fetal or perinatal periods. Given their presence in fetal and postnatal plasma and their binding to brown adipocytes in vivo and in vitro [4,5,18], we hypothesized that the lactogenic hormones may play a physiolog- ical role in the regulation of BAT development and thermogenesis during the perinatal period. To test that hypothesis, we employed three novel experimental models of lactogen action. The PRLR knockout (KO) mouse [19], which cannot respond to either PL (in utero) or PRL (perinatal and postnatal periods), was used to assess the effects of lactogen resistance in vivo on BAT mass, mitochondrial structure, gene expression, and the adaptation to cold. These studies were performed on the day of birth to establish their physiological relevance. Immortalized preadipocyte cell lines derived from the interscapular brown adipose depots of PRLR KO mice were employed to assess the direct effects of PRL signaling on adipocyte differentiation and function in vitro and to determine if reintroduction of PRLR can restore brown adipocyte commitment. Finally, the PRL-responsive T37i cell line [18], derived from a BAT hibernoma developed in a transgenic mouse [20], was used to assess the molecular mechanisms by which PRLR signaling controls BAT differentiation. We discovered that absence of PRLR signaling compromised the growth and differentiation of BAT in vivo and in vitro and reduced plasma concentrations and BAT expression of insulin-like growth factor-2 (IGF-2), which plays a central role in the control of placental and fetal growth [21,22]. We therefore examined the regulation of IGF-2 mRNA levels by PRL in isolated brown adipocytes and the effects of IGF-2 on brown adipocyte differentiation and growth; we then determined if exogenous IGF-2 could reverse the effects of PRLR-deficiency on BAT development in immortalized preadipocyte cell lines. Our findings suggest that the lactogens act in concert with IGF-2 to control brown adipocyte differentiation and growth. RESULTS Brown adipose tissue development is altered in PRLR KO mice The demonstration that prolactin receptors (PRLR) are expressed at high levels in brown adipocytes of fetal and newborn rodents [5] and are functional in brown adipocytes [18] led us to explore the physiological relevance of PRL action in BAT in vivo. To that end, we characterized BAT development in newborn PRLR KO mice and their wild type (WT) littermates. The body weights of newborn PRLR KO mice were not different from those of WT pups; however the absolute interscapular BAT mass, evaluated by its weight and the BAT/body weight ratio, was significantly reduced in PRLR KO mice as compared with WT littermates (Fig. 1A). These findings suggested that lactogens function as growth factors in BAT during fetal development, since the reduction in PRLR KO BAT mass was detected at birth. To assess further the impact of PRLR deletion on BAT development in vivo, we analyzed the histological appearance of brown adipocytes in newborn PRLR KO and WT mice (Fig. 1B and C). WT brown adipocytes were significantly larger than PRLR KO cells; there was a 35% decrease in mean cell surface of PRLR KO brown adipocytes (Fig. 1D), corresponding to a 50% reduction of cell volume. To determine if the reduction in cell size reflected a decrease in cellular lipids, we compared the triglyceride Prolactin and Brown Adipocytes content of PRLR KO BAT depots with that of WT depots. Triglyceride content was decreased by 40% in the absence of PRLR signaling, highlighting the importance of the lactogens in brown adipocyte function (Fig. 1E). However, the activities of citrate synthase (CS) and of glycerol-3 phosphate dehydrogenase (G3PDH), one of the enzymatic steps of the tricarboxylic acid cycle also implicated in the initiation of lipogenesis, did not differ between WT and KO BAT homogenates (Supplemental data Fig. S1 and Text S1). These observations suggest that the reduction of triglyceride content in PRLR KO BAT results from a defect in adipocyte differentiation rather than a defect in lipogenesis per se. We next examined the expression of adipogenic markers in order to evaluate the impact of PRLR on brown adipocyte differentiation in vivo. PPARc2 as well as SREBP1 expression in BAT were decreased in PRLR KO mice, consistent with a compromised adipocyte differentiation (Fig. 1F and G). In addition, there was a striking reduction in PGC-1a mRNA levels. Since PGC-1a regulates mitochondrial biogenesis and energy production through induction of UCP1 expression and through functional interaction with PPARc2 and the retinoic acid receptor (RXRa), the thermometa- bolic capacity of PRLR KO mice was subsequently evaluated. Alterations of thermogenesis and BAT mitochondria in PRLR KO animals To assess the effects of lactogen resistance (PRLR deletion) on thermogenesis, we subjected newborn mice to a cold challenge and calculated their survival rates during the 400-min period, since alternative methods such as measurement of rectal temperature or indirect calorimetry could not be performed on newborn animals. Newborn PRLR KO pups were more sensitive to cold exposure than their WT littermates; 50% mortality was observed at 125 and 200 min, in PRLR KO and WT mice, respectively, and 30% of the WT mice, but none of the PRLR KO mice, survived more than 300 min (Fig. 2A). The key effector of adaptive thermogenesis and cold adaptation is UCP1. To determine if UCP1 expression is altered in PRLR KO animals, we quantified UCP1 expression in interscapular BAT. Both UCP1 mRNA and protein levels were significantly reduced in BAT of newborn PRLR KO mice (Fig. 2B, 2C), suggesting that UCP1 deficiency accounts for their thermogenic defect. Given the prominent role played by the b3 adrenoceptor (b3Adr) in mediating catecholamines effects on thermogenesis in rodents [23], b3Adr transcripts were quantified in WT and PRLR KO neonates (Fig. 2D); its significant decrease strongly supports the thermogenic defect, and therefore the physiological relevance of PRLR signaling BAT function. BAT differs from white fat by its high degree of vascularization and mitochondrial density. Electron microscopic analysis of BAT demonstrated striking differences in mitochondrial morphology of PRLR KO mice compared to WT littermates (Fig. 2E). Smaller, sparse and often elongated mitochondria were present in the cytoplasm of PRLR KO brown adipocytes. This was associated with reduced fat droplets, in accordance with morphological and functional BAT abnormalities. Of note, the number of mitochon- drial DNA copies in brown adipocytes of PRLR KO mice did not differ from those in WT mice (supplemental data Fig. S2A). Accordingly, cytochrome c oxidase (COX) activity, the fourth complex of the respiratory chain selected as representative of the mtDNA-dependent mitochondrial activities, did not vary between KO and WT BAT (supplemental data Fig. S2B and Text S1). Thus, the respiratory chain activity of lactogen-resistant brown adipocytes was preserved while their thermogenic capacity was clearly impaired. Taken together, these observations strongly PLoS ONE | www.plosone.org 2 February 2008 | Issue 2 | e1535