[Brown, white, beige: the color of fat and new therapeutic perspectives for obesity...]. / Brun, blanc, beige: la couleur du gras et nouvelles perspectives thérapeutiques de l'obésité

Three articles published in the NEJM in 2009 have renewed the interest for brown adipose tissue (BAT) in humans. This review reports interesting new findings on adipocyte cell types that have been presented at the last meeting of the Endocrine Society in Houston, TX, in June 2012. Many studies have focused on identifying factors involved in brown adipocyte lineage, the site of adaptive thermogenesis. Indeed, the role of the transcription factors, such as PRDM16, in brown adipocyte differentiation has been unambiguously established. Very recently, the concept of "beigeing", defined as the occurrence of thermogenic brown adipocytes in white adipose tissue, has emerged, leading to the identification, by Bruce Spiegelman's group, of a new muscular hormone, called irisin, which is able to stimulate the "beigeing". This finding should convey toward the discovery of new mutations involved in the pathogenesis of obesity and lipodystrophies, and should be translated into innovative therapeutic perspectives. Finally, the nature of BAT innervation has been clarified and the presence of an autoregulatory loop between BAT and notably hypothalamic paraventricular nuclei via the sensory and sympathetic nervous systems has been delineated. These feedback circuits appear to be crucial to control BAT thermogenic activity.

Placental lactogens induce serotonin biosynthesis in a subset of mouse beta cells during pregnancy.

AIMS/HYPOTHESIS: Upregulation of the functional beta cell mass is required to match the physiological demands of mother and fetus during pregnancy. This increase is dependent on placental lactogens (PLs) and prolactin receptors, but the mechanisms underlying these events are only partially understood. We studied the mRNA expression profile of mouse islets during pregnancy to gain a better insight into these changes. METHODS: RNA expression was measured ex vivo via microarrays and quantitative RT-PCR. In vivo observations were extended by in vitro models in which ovine PL was added to cultured mouse islets and MIN6 cells. RESULTS: mRNA encoding both isoforms of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase (TPH), i.e. Tph1 and Tph2, were strongly induced (fold change 25- to 200-fold) during pregnancy. This induction was mimicked by exposing islets or MIN6 cells to ovine PLs for 24 h and was dependent on janus kinase 2 and signal transducer and activator of transcription 5. Parallel to Tph1 mRNA and protein induction, islet serotonin content increased to a peak level that was 200-fold higher than basal. Interestingly, only a subpopulation of the beta cells was serotonin-positive in vitro and in vivo. The stored serotonin pool in pregnant islets and PL-treated MIN6 cells was rapidly released (turnover once every 2 h). CONCLUSIONS/INTERPRETATION: A very strong lactogen-dependent upregulation of serotonin biosynthesis occurs in a subpopulation of mouse islet beta cells during pregnancy. Since the newly formed serotonin is rapidly released, this lactogen-induced beta cell function may serve local or endocrine tasks, the nature of which remains to be identified.

[Fertility preservation before puberty: from mice to men]. / Préservation de la fertilité avant la puberté : de la souris à l'homme.

Malignant tumors account for 1% of childhood cancers. The incidence is to the order of 122 cases per million children. The five-year survival after cancer before the age of 16years has improved from 50 to 80% in 40years. Assessment of potential for preservation of fertility should thus be a systematic element of care for children treated for a malignant tumor (high-dose chemotherapy with alkylizing agents, radiation therapy including the gonads) or those receiving hematopoietic stem cell grafts for malignant or benign disease (sickle-cell anemia, immune deficit). Among the techniques proposed, cryopreservation of ovarian tissue appears to be the most promising, or perhaps the only one available before puberty with encouraging result. Nevertheless the uncertainties, or even risks, related to these treatments, should not be neglected.

Preserving fertility in prepubertal children.

BACKGROUND: As a result of advances in treatment, almost 80% of children and adolescents who currently receive a diagnosis of cancer become long-term survivors. Potential adverse consequences of treatment include impaired puberty and fertility due to gonadal removal, genital tract injury or damage to germ cells from adjuvant therapy. In recent years, treatment of solid tumors and hematological malignancies has been modified in an attempt to minimize damage to the reproductive system. Simultaneously, advances in assisted reproductive technologies have led to new possibilities for the prevention and treatment of infertility. We review experimental data in animal models and clinical experience in adults and discuss strategies to preserve fertility in prepubertal children. CONCLUSIONS: Fertility preservation should now be considered in children facing cancer treatment that has a high risk of gonadal toxicity including high-dose chemotherapy and bilateral irradiation of the gonads at toxic doses.

Hypothesis paper Brain talks with fat--evidence for a hypothalamic-pituitary-adipose axis?

The adipose tissue signals to the brain via its secretory products. However, it is unknown whether the brain itself can directly contact the fat tissue. In order to test this hypothesis, the adipocytic expression of receptors for pituitary hormones and hypothalamic peptides was investigated. Besides FSH- and LH-receptors, adipocytes do express the specific receptors for ACTH, TSH, GH, prolactin, oxytocin and the three receptor subtypes for vasopressin. Thus, the adipose tissue might no longer be regarded as an inert and steady tissue but as a fast acting player downstream of and under the control of the brain. Based on this, the potential existence and clinical impact of a hypothalamic-pituitary-adipose axis should further be investigated.

Prolactin potentiates insulin-stimulated leptin expression and release from differentiated brown adipocytes.

The pituitary hormone prolactin (PRL) exerts pleiotropic effects, which are mediated by a membrane receptor (PRLR) present in numerous cell types including adipocytes. Brown adipose tissue (BAT) expresses uncoupling proteins (UCPs), involved in thermogenesis, but also secretes leptin, a key hormone involved in the control of body weight. To investigate PRL effects on BAT, we used the T37i brown adipose cell line, and demonstrated that PRLRs are expressed as a function of cell differentiation. Addition of PRL leads to activation of the JAK/STAT and MAP kinase signaling pathways, demonstrating that PRLRs are functional in these cells. Basal and catecholamine-induced UCP1 expression were not affected by PRL. However, PRL combined with insulin significantly increases leptin expression and release, indicating that PRL potentiates the stimulatory effect of insulin as revealed by the recruitment of insulin receptor substrates and the activation of phosphatidylinositol 3-kinase. To explore the in vivo physiological relevance of PRL action in BAT, we showed that leptin content was significantly increased in BAT of PRLR-null mice compared with wild-type mice, highlighting the involvement of PRL in the leptin secretion process. This study provides the first evidence for a functional link between PRL and energy balance via a cross-talk between insulin and PRL signaling pathways in brown adipocytes.

Growth hormone receptor signalling and actions in bone growth.

Growth hormone (GH) acts by binding to a membrane receptor that is part of the cytokine receptor superfamily. Ligand binding induces receptor dimerization leading to activation of the associated tyrosine kinase, Janus kinase (Jak) 2. Transphosphorylation of Jak2 occurs followed by tyrosine phosphorylation of the receptor, and numerous cytoplasmic proteins. Among these are the signal transducers and activators of transcription (Stat) proteins, as well as adaptor proteins leading to the activation of the Ras/mitogen-activated protein (MAP) kinase and the phosphatidyl-inositol-3'-kinase (PI 3-kinase) pathways. Activation of the GH receptor system is relatively transient, with several mechanisms being involved in down-regulation: internalization and degradation of the receptor and recruitment of phosphatases or specific inhibitors of the Jak-Stat pathway, the suppressors of cytokine signalling (SOCS) proteins. Finally, the use of the GH receptor knock-out mouse model has allowed us to dissect the role of this hormone in post-natal body growth and homeostasis.

Prolactin receptor expression in the epithelia and stroma of the rat mammary gland.

The importance of prolactin (PRL) in regulating growth and differentiation of the mammary gland is well known. However, it is not well established whether PRL acts solely on the mammary epithelia or if it can also directly affect the mammary stroma. To determine where PRL could exert its effects within the mammary gland, we investigated the levels of expression and the localization of the PRL receptor (PRLR) in the epithelia and stroma of the rat mammary gland at different physiological stages. For these studies, we isolated parenchymal-free 'cleared' fat pads and intact mammary glands from virgin, 18-day-pregnant and 6-day-lactating rats. In addition, intact mammary tissues were enzymatically digested to obtain epithelial cells, free of stroma. The mammary tissues, intact gland, stroma and isolated epithelia, were then used for immunocytochemistry, protein extraction and isolation of total RNA. PRLR protein was detected in tissues using specific polyclonal antisera (PRLR-l) by immunocytochemistry and Western blot analysis. Messenger RNA for PRLR was measured by ribonuclease protection assay. Immunocytochemistry and Western blots with the PRLR-1 antisera detected PRLR in wild-type rat and mouse tissues, whereas the receptor protein was absent in tissues from PRLR gene-deficient mice. PRLR was found to be present both in the epithelia and stroma of mammary glands from virgin, pregnant and lactating rats, as determined by immunocytochemistry and Western blotting. Western blots revealed the predominance of three bands migrating at 88, 90 and 92 kDa in each of the rat mammary samples. These represent the long form of the PRLR. During pregnancy and lactation, PRLR protein increased in the epithelial compartment of the mammary gland but did not change within the stromal compartment at any physiological stage examined. We also found PRLR mRNA in both the epithelia and stroma of the mammary gland. Again, the stroma contained lower levels of PRLR mRNA compared with the epithelia at all physiological stages examined. Also, the PRLR mRNA levels within the stroma did not change significantly during pregnancy or lactation, whereas PRLR mRNA within the epithelia increased twofold during pregnancy and fourfold during lactation when compared with virgin rats. We conclude from this study that PRLR is expressed both in the stromal and epithelial compartment of the mammary gland. This finding suggests PRL may have a direct affect on the mammary stroma and by that route affect mammary gland development.

Prolactin receptor signal transduction pathways and actions determined in prolactin receptor knockout mice.

Prolactin-receptor-deficient mice are a good model in which to study the various actions of prolactin. Female homozygous knockout mice are completely infertile and show a lack of mammary development, while hemizogotes are unable to lactate following their first pregnancy. Male and female homozygotes have markedly elevated serum prolactin levels, and in some instances pituitary hyperplasia is present. Maternal behaviour is severely affected in both hemizygous and homozygous animals. Bone formation is reduced in young animals and in adults (males and females). Finally, older males and females show a slight reduction in body weight, which seems to be due to reduced abdominal fat deposition in the knockout animals.

Implications of multiple phenotypes observed in prolactin receptor knockout mice.

The development of a mouse line deficient in the PRL receptor (PRLR) would be an ideal means to better understand the multiple functions of prolactin. We were worried initially that removal of the PRLR from the mouse genome might be lethal and were surprised to find this not to be the case. We identified numerous deficiencies in PRLR knockout (KO) animals. Female homozygous mice are completely infertile and lack normal mammary development, while hemizygotes are unable to lactate following their first pregnancy. PRLR KO males and females have markedly elevated (30- to 100-fold) serum prolactin levels and in some instances pituitary hyperplasia is present. Maternal behavior is severely affected in both hemizygous and heterozygous animals. Bone formation is reduced in young animals and adults (males and females). Recently, we noticed that older KO animals show a slight reduction in body weight which appears to be due to reduced abdominal fat deposition.

Body weight and fat deposition in prolactin receptor-deficient mice.

To explore the roles of the lactogens in adipose tissue development and function, we measured body weight, abdominal fat content, and plasma leptin concentrations in a unique model of lactogen resistance: the PRL receptor (PRLR)-deficient mouse. The absence of PRLRs in knockout mice was accompanied by a small (5-12%), but progressive, reduction in body weight after 16 weeks of age. Females were affected to a greater degree than males. The reduction in weight in female PRLR-deficient mice (age 8-9 months) was associated with a 49% reduction in total abdominal fat mass and a 29% reduction in fat mass expressed as a percentage of body weight. Lesser reductions were noted in male mice. Plasma leptin concentrations were reduced in females but not in males. That the reductions in abdominal fat may reflect in part the absence of lactogen action in the adipocyte is suggested by the demonstration of PRLR messenger RNA in normal mouse white adipose tissue. Nevertheless, steady state levels of PRLR messenger RNA in mature adipocytes are very low, suggesting that the effects of lactogens might be mediated by other hormones or cellular growth factors. Our observations suggest roles for the lactogens in adipose tissue growth and metabolism in pregnancy and postnatal life.

Prolactin, growth hormone, and epidermal growth factor activate Stat5 in different compartments of mammary tissue and exert different and overlapping developmental effects.

Prolactin (Prl)-induced phosphorylation of Stat (signal transducer and activator of transcription) 5 is considered a key event in functional mammary development and differentiation. We now demonstrate that not only Prl, but also growth hormone (GH) and epidermal growth factor (EGF), can activate Stat5 in mammary tissue. We investigated the roles of these hormones in mammary development using mice in which the respective receptors had been inactivated. Although Prl receptor (PrlR)-null mice are infertile, we were able to maintain pregnancies in a few mice by treatment with progesterone. Mammary tissue in these mice was severely underdeveloped and exhibited limited differentiation as assessed by the phosphorylation status of Stat5 and the expression of milk protein genes. PrlR +/- mice showed impaired mammary development and alveolar differentiation during pregnancy, which corresponded with reduced phosphorylation levels of Stat5a and 5b, and impaired expression of milk protein genes. Development of the glands in these mice was arrested at around day 13 of pregnancy. While Prl activated Stat5 only in the epithelium, GH and EGF activated Stat5 preferentially in the stroma. To assess the relevance of the GH receptor (GHR) in the mammary gland, we transplanted GHR-null epithelium into cleared fat pads of wild-type mice. These experiments demonstrated that the GHR in the epithelium is not required for functional mammary development. Similarly, the EGFR in the epithelium is not required for alveolar development. In contrast, epithelial PrlR is required for mammary development and milk protein gene expression during pregnancy. Although GH is not required for alveolar development, we were able to demonstrate its lactogenic function in cultured mammary epithelium from PrlR-null mice. However, ductal development in GHR-null mice was impaired, supporting the notion that GH signals through the stromal compartment. Our findings demonstrate that GH, Prl, and EGF activate Stat5 in separate compartments, which in turn reflects their specific roles in ductal and alveolar development and differentiation.

Bone homeostasis in growth hormone receptor-null mice is restored by IGF-I but independent of Stat5.

Growth hormone (GH) regulates both bone growth and remodeling, but it is unclear whether these actions are mediated directly by the GH receptor (GHR) and/or IGF-I signaling. The actions of GH are transduced by the Jak/Stat signaling pathway via Stat5, which is thought to regulate IGF-I expression. To determine the respective roles of GHR and IGF-I in bone growth and remodeling, we examined bones of wild-type, GHR knockout (GHR(-/-)), Stat5ab(-/-), and GHR(-/-) mice treated with IGF-I. Reduced bone growth in GHR(-/-) mice, due to a premature reduction in chondrocyte proliferation and cortical bone growth, was detected after 2 weeks of age. Additionally, although trabecular bone volume was unchanged, bone turnover was significantly reduced in GHR(-/-) mice, indicating GH involvement in the high bone-turnover level during growth. IGF-I treatment almost completely rescued all effects of the GHR(-/-) on both bone growth and remodeling, supporting a direct effect of IGF-I on both osteoblasts and chondrocytes. Whereas bone length was reduced in Stat5ab(-/-) mice, there was no reduction in trabecular bone remodeling or growth-plate width as observed in GHR(-/-) mice, indicating that the effects of GH in bone may not involve Stat5 activation.

Mammary gland development and the prolactin receptor.

Prolactin (PRL), synthesized by the anterior pituitary and to a lesser extent by numerous extrapituitary tissues, affects more physiological processes than all other pituitary hormones combined. This hormone is involved in > 300 separate effects in various vertebrate species where its role has been well documented. The initial step in its action is the binding to a specific membrane receptor which belongs to the superfamily of class 1 cytokine receptors. The function of this receptor is mediated, at least in part, by two families of signaling molecules: Janus kinases and signal transducers and activators of transcription. PRL-binding sites have been identified in a number of cells and tissues of adult animals. Disruption of the gene for the PRL receptor has provided a new animal model with which to better understand the actions of PRL on mammary morphogenesis and mammary gland gene expression. The recent availability of genetic mouse models provides new insights into mammary developmental biology and how the action of a hormone at specific stages of development can have effects later in life on processes such as mammary development and breast cancer initiation and progression.

Rescue of preimplantatory egg development and embryo implantation in prolactin receptor-deficient mice after progesterone administration.

PRL, a hormone secreted essentially by the pituitary and other extrapituitary sources such as decidua, has been attributed regulatory roles in reproduction and cell growth in mammals. These effects are mediated by a membrane PRL receptor belonging to the cytokine receptor superfamily. Null mutation of the PRL receptor gene leads to female sterility due to a severely compromised preimplantation development and a complete failure of the implantation of the few embryos reaching the blastocyst stage, strongly implicating PRL in the maternal control of implantation. We measured the hormonal status of -/- mice, which confirmed that the corpus luteum is unable to produce progesterone. Progesterone administration to -/- mice completely rescued the development of preimplantatory eggs and embryo implantation. Pregnancy could be maintained to 19.5 days postcoitum, with about 22% of resulting embryos reaching adulthood. Although progesterone and perhaps PRL appear to facilitate mouse preembryo development throughout the preimplantation stages, other factors as well as a possible direct effect of PRL on the uterus are probably necessary to fully maintain pregnancy. Finally, reduced ductal side-branching in the mammary gland can be rescued by progesterone treatment, but females exhibit reduced alveolar formation. Our model establishes the PRL receptor as a key regulator of reproduction and provides novel insights into the function of lactogenic hormones and their receptor.

Implantation and decidualization defects in prolactin receptor (PRLR)-deficient mice are mediated by ovarian but not uterine PRLR.

PRL and its homologs accomplish their biological effects through the PRL receptor (PRLR). We evaluated the expression and function of PRLR in the embryo and uterus during the periimplantation period because PRLR deficiency results in implantation failure. In wild-type mice, PRLR expression was localized to undecidualized stromal cells in the antimesometrial border on days 6-8 of pregnancy. A small population of PRLR-expressing cells was observed adjacent to the ectoplacental cone in the mesometrial stroma. Low levels of PRLR expression were also detected in the developing embryo on days 6-8. To determine the significance of PRLR expression in this distribution, we examined implantation and decidualization in PRLR-/- mice. Progesterone (P4) administration rescued infertility in PRLR-/- mice from the periimplantation period to midgestation. Artificially induced decidualization was absent in pseudopregnant PRLR-/- mice but was identical to wild-type in P4-treated PRLR-/- mice. Furthermore, wild-type and P4-treated PRLR-/- mice had similar expression of the implantation-specific genes, LIF, amphiregulin, HB-EGF, COX-1, COX-2, PPARdelta, Hoxa-10, cyclin-D3, VEGF, and its receptors, Flk-1 and neuropilin-1. Together, these results show that luteal P4 production via ovarian PRLR signaling is required for implantation and early pregnancy. The function of uterine PRLR remains unclear. However, the eventual loss of pregnancy in P4-treated PRLR-/- mice suggests that uterine PRLR may be essential for the support of late gestation.

From the molecular biology of prolactin and its receptor to the lessons learned from knockout mice models.

Prolactin (PRL), a polypeptide hormone secreted mainly by the pituitary and, to a lesser extent, by peripheral tissues, affects more physiological processes than all other pituitary hormones combined since it is involved in > 300 separate functions in vertebrates. Its main actions are related to lactation and reproduction. The initial step of PRL action is the binding to a specific membrane receptor, the PRLR, which belongs to the class 1 cytokine receptor superfamily. PRL-binding sites have been identified in a number of tissues and cell types in adult animals. Signal transduction by this receptor is mediated, at least in part, by two families of signaling molecules: Janus tyrosine kinases and signal transducers and activators of transcription (STATs). Disruption of the PRLR gene has provided a new mouse model with which to identify actions directly associated with PRL or any other PRLR ligands, such as placental lactogens. To date, several different phenotypes have been analyzed and are briefly described in this review. Coupled with the SAGE technique, this PRLR knockout model is being used to qualitatively and quantitatively evaluate the expression pattern of hepatic genes in two physiological situations: transcriptomes corresponding to livers from both wild type and PRLR KO mice are being compared, and following statistical analyses, candidate genes presenting a differential profile will be further characterized. Such a new approach will undoubtedly open future avenues of research for PRL targets. To date, no pathology linked to any mutation in the genes encoding PRL or its receptor have been identified. The development of genetic models provides new opportunities to understand how PRL can participate to the development of pathologies throughout life, as for example the initiation and progression of breast cancer.

Prolactin controls mammary gland development via direct and indirect mechanisms.

The inactivation of the prolactin receptor gene by homologous recombination has made it possible to investigate the role of prolactin signaling in mammary gland development without resort to ablative surgery of the endocrine glands. In knockout mice lacking the prolactin receptor, mammary development is normal up to puberty. Subsequently, the ducts branch less frequently than those of wild-type animals. While terminal end buds differentiate to alveolar buds in wild-type females by the end of puberty, in knockout females terminal end bud-like structures persist at the ductal ends. To distinguish between the developmental defects that are intrinsic to the epithelium and those that result from systemic endocrine alterations in prolactin receptor knockout mice, mammary epithelium from prolactin receptor knockouts was transplanted into mammary fat pads of wild-type mice. In virgin mice, the knockout epithelial transplants developed normally at puberty, indicating an indirect effect of prolactin on ductal development. Prolactin receptor knockout females are infertile due to multiple reproductive defects, but epithelial transplants allowed us to assess the extent to which the absence of prolactin receptor is limiting, under systemic conditions that allow full mammary gland development. During pregnancy, the prolactin receptor knockout transplants showed normal side branching and the formation of alveolar buds, but no lobuloalveolar development. Thus, prolactin affects mammary morphogenesis in two different ways: it controls ductal side branching and terminal end bud regression in virgin animals via indirect mechanisms, but acts directly on the mammary epithelium to produce lobuloalveolar development during pregnancy.

Osteoblasts are a new target for prolactin: analysis of bone formation in prolactin receptor knockout mice.

Bone development is a multistep process that includes patterning of skeletal elements, commitment of hematopoietic and/or mesenchymental cells to chondrogenic and osteogenic lineages, and further differentiation into three specialized cell types: chondrocytes in cartilage and osteoblasts and osteoclasts in bone. Although PRL has a multitude of biological actions in addition to its role in the mammary gland, very little is known about its effect on bone. Mice carrying a germline null mutation for the PRL receptor gene have been produced in our laboratory and used to study the role of PRL in bone formation. In -/- embryos, we observed an alteration in bone development of calvaria. In adults, histomorphometric analysis showed that the absence of PRL receptors leads to a decrease in bone formation rate using double calcein labeling and a reduction of bone mineral density, measured by dual energy x-ray absorptiometry. In addition, serum estradiol, progesterone, testosterone, and PTH levels were analyzed. We also established that osteoblasts, but not osteoclasts, express PRL receptors. This suggests that an effect of PRL on osteoblasts could be required for normal bone formation and maintenance of bone mass. Thus, the PRL receptor knockout mouse model provides a new tool to investigate the involvement of PRL in bone metabolism.

Mouse prolactin receptor gene: genomic organization reveals alternative promoter usage and generation of isoforms via alternative 3'-exon splicing.

In rodents, the prolactin receptor is expressed as multiple isoforms with identical extracellular and membrane-proximal region sequences but with different 3' sequences, encoding different cytoplasmic regions, and different 5' untranslated region (UTR) sequences. These divergent sequences could be the result of multiple prolactin receptor genes or of a single gene which displays alternative promoter usage and 3'-exon splicing. To investigate the molecular basis for these observations, we have cloned and determined the organization of the mouse prolactin receptor gene. Genomic DNA cloning allowed the arrangement of promoters 1A, 1B, and 1C to be determined. 5'-RACE-PCR from mouse liver identified two novel 5' prolactin receptor sequences, indicating that the gene has at least five different promoters, four of which are active in liver. The remaining nonvariable 5' UTR is encoded by a separate exon (exon 2), while a further 11 coding exons follow, the last 4 of which are alternatively spliced to produce the four isoforms of the receptor. Functional units were found to be exon specific. Thus, the multiple prolactin receptor isoforms are the product of a single gene of >120 kb which displays multiple promoter usage and 3'-exon splicing.