Role of leptin during perinatal metabolic programming and obesity.

The incidence of obesity is rapidly increasing all over the world in epidemic proportions.The epidemia now affects young children and accumulative evidences suggest that the origin of the disease may occur during foetal development and early life. This has introduced the concept of "developmental programming" supported by experimental studies in animal models and numerous epidemiological data. This concept supports the idea that nutritional and hormonal status during pregnancy and early life could interfere irreversibly on the development of the organs involved in the control of food intake and metabolism and particularly the hypothalamic structures responsible of the establishment of the ingestive behaviour and regulation of energy expenditure. The mechanisms responsible of this developmental programming remain poorly documented. However, recent research indicate that the adipokine leptin plays a critical role in this programming.

Study of hypothalamic leptin receptor expression in low-birth-weight piglets and effects of leptin supplementation on neonatal growth and development.

Low birth weight resulting from intrauterine growth retardation (IUGR) is a risk factor for further development of metabolic diseases. The pig appears to reproduce nearly all of the phenotypic pathological consequences of human IUGR and is likely to be more relevant than rodents in studies of neonatal development. In the present work, we characterized the model of low-birth-weight piglets with particular attention to the hypothalamic leptin-sensitive system, and we tested whether postnatal leptin supplementation can reverse the precocious signs of adverse metabolic programming. Our results demonstrated that 1) IUGR piglets present altered postnatal growth and increased adiposity; 2) IUGR piglets exhibit abnormal hypothalamic distribution of leptin receptors that may be linked to further disturbance in food-intake behavior; and 3) postnatal leptin administration can partially reverse the IUGR phenotype by correcting growth rate, body composition, and development of several organs involved in metabolic regulation. We conclude that IUGR may be characterized by altered leptin receptor distribution within the hypothalamic structures involved in metabolic regulation and that leptin supplementation can partially reverse the IUGR phenotype. These results open interesting therapeutic perspectives in physiopathology for the correction of defects observed in IUGR.

Early postnatal leptin blockage leads to a long-term leptin resistance and susceptibility to diet-induced obesity in rats.

OBJECTIVE: Using a recombinant rat leptin antagonist, we investigated the effects of early postnatal leptin disruption on long-term leptin sensitivity and metabolic phenotype. DESIGN: Three groups of 10 newborn female Wistar rats were injected subcutaneously with either saline (control) or leptin antagonist (at 2.5 or 7.5 microg g(-1) day(-1)) from postnatal day 2 to day 13. RESULTS: At weaning (day 28), antagonist-treated rats presented similar body weight (BW) compared to control animals. At 3 months of age, there was no significant change in BW, food intake and leptin or insulin levels between groups. Only a disturbed relationship between circulating insulin and glucose levels was observed in antagonist-treated animals. At 4 months of age, treated animals developed a leptin resistance appreciated by the lack of response to a 7-days leptin treatment (1 mg kg(-1) day(-1)) in term of decrease in food intake and BW. At 8 months of age, following 3 months of high-energy diet, rlepm7.5 animals presented higher BW gain associated with increased body fatness and striking hyperleptinaemia as compared to control animals. CONCLUSION: The blockage of leptin action during the critical period of early life in rodents has long-term consequences by altering the capacity to respond to leptin during adulthood, thus predisposing the animals to obesity. These findings clearly demonstrate the physiological importance of the postnatal leptin surge for the optimal onset of the metabolic regulation, at least in rodents, and its implication in the prevention of unfavourable developmental programming.

Lack of cross-desensitization between leptin and prolactin signaling pathways despite the induction of suppressor of cytokine signaling 3 and PTP-1B.

Hyperprolactinemia and hyperleptinemia occur during gestation and lactation with marked hyperphagia associated with leptin resistance. Prolactin (PRL) induces the expression of orexigenic neuropeptide Y (NPY) through the activation of JAK-2/STAT-3 signaling pathway in hypothalamic paraventricular nucleus (PVN) leading to hyperphagia. PRL may also act through the inhibition of anorexigenic effect of leptin via induction of suppressor of cytokine signaling 3 (SOCS-3). This paper aimed to co-localize PRL (PRL-R) and leptin (ObRb) receptors in the hypothalamus of female rats and investigate the possible cross-desensitization between PRL-R and ObRb. We showed that: 1) PRL-R and ObRb are expressed in the PVN and co-localized in the same neurons; 2) in lactating females leptin failed to activate JAK-2/STAT-3 signaling pathway; 3) in Chinese Hamster Ovary (CHO) stably co-expressing PRL-R and ObRb, overexposure to PRL did not affect leptin signaling but totally abolished PRL-dependent STAT-5 phosphorylation. The overexposure to leptin produces similar results with strong alteration of leptin-dependent STAT-3 phosphorylation, whereas PRL-dependent STAT-5 was not affected; and 4) CHO-ObRb/PRL-R cells overexposure to leptin or PRL induces the expression of negative regulators SOCS-3 and PTP-1B. Thus, we conclude that these negative regulators affect specifically the inducer signaling pathway; for instance, SOCS-3 induced by PRL will affect PRL-R signaling but not ObRb signaling and vice versa. Finally, the lack of cross-desensitization between PURL-R and ObRb suggests that hyperphagia observed during gestation and lactation may be attributed to a direct effect of PRL on NPYexpression, and is most likely exacerbated by the physiological leptin resistance state.

Interferon-tau upregulates prolactin receptor mRNA in the ovine endometrium during the peri-implantation period.

Our objective was to determine the effect of ovine interferon-tau (IFN-tau) on prolactin receptor (PRL-R) gene expression in the ovine endometrium. IFN-tau is an embryonic cytokine which, via its paracrine anti-luteolytic activity, plays a critical role in maternal recognition of pregnancy in ruminants. Using ribonuclease protection assay procedures, we compared endometrial PRL-R mRNA levels in ewes that were intrauterine injected with either 2 mg bovine serum albumin or 2 mg recombinant ovine IFN-tau on day 10 of the oestrous cycle (day 0 = day of oestrus). IFN treatment significantly increased the abundance of both the long and short forms of PRL-R mRNA in the ovine uterus, but had no effect on the long:short form ratio. In situ hybridization experiments revealed that the increase in abundance of PRL-R mRNA in the uterus was localized to the glandular compartment of the endometrium. In pregnant ewes, a similar increase in PRL-R mRNA abundance was found to occur in ovine endometrium on days 14-15 post conception. Collectively, these data provided strong evidence that IFN-tau modulates the level of lactogenic hormone receptor mRNA in the ovine uterus. Whether the effect of IFN-tau on PRL-R expression is mediated directly or influenced, at least in part, by progesterone remains to be elucidated.

Leptin in horses: tissue localization and relationship between peripheral concentrations of leptin and body condition.

Obesity has been a major concern in the horse industry for many years, and the recent discovery of leptin and leptin receptors in numerous nonequine species has provided a basis for new approaches to study this problem in equine. The objectives were to: 1) clone a partial sequence ofthe equine leptin and leptin receptor genes so as to enable the design of primers for RT-PCR determination of leptin and leptin receptor gene presence and distribution in tissues, 2) develop a radioimmunoassay to quantify peripheral concentrations of leptin in equine, 3) determine if peripheral concentrations of leptin correlate with body condition scores in equine, and 4) determine if changing body condition scores would influence peripheral concentrations of leptin in equine. In Experiment 1, equine leptin (GenBank accession number AF179275) and the long-form of the equine leptin receptor (GenBank accession number AF139663) genes were partially sequenced. Equine leptin receptor mRNA was detected in liver, lung, testis, ovary, choroid plexus, hypothalamus, and subcutaneous adipose tissues using RT-PCR. In Experiment 2, 71 horses were categorized by gender, age, and body condition score and blood samples were collected. Sera were assayed for leptin using a heterologous leptin radioimmunoassay developed for equine sera. Serum concentrations of leptin increased in horses with body condition score (1 = thin to 9 = fat; r = 0.64; P = 0.0001). Furthermore, serum concentrations of leptin were greater in geldings and stallions than in mares (P = 0.0002), and tended to increase with age of the animal (P = 0.08). In Experiment 3, blood samples, body weights, and body condition scores were collected every 14 d from 18 pony mares assigned to gain or lose weight over a 14-wk interval based on initial body condition score. Although statistical changes (P = 0.001) in body condition scores were achieved, congruent statistical changes in peripheral concentrations of leptin were not observed, likely due to the small range of change that occurred. Nonetheless, serum concentrations of leptin tended to be greater in fat-restricted mares than in thin-supplemented mares (P = 0.09). We conclude that leptin and leptin receptors are present in equine tissues and that peripheral concentrations of leptin reflect a significant influence of fat mass in equine.

Establishment of feeder-independent cloned caprine trophoblast cell line which expresses placental lactogen and interferon tau.

A feeder-independent cloned trophoblast cell line, HTS-1, was established from a mature placenta of Shiba goat (Capra hircus). During the growth phase, single HTS-1 cells exhibited ruffled membranes or lamellipodia often accompanied by elongated cell shape, indicating highly motile nature of the cells. At or near confluence, HTS-1 cells formed monolayers with few sign of cellular overlapping. Binucleate cells were found at a high frequency especially in the peripheral regions of monolayers. In small colonies and the monolayers, majority of HTS-1 cells assumed polygonally shaped cobble-stone like morphology characteristic to epithelial cells, although considerable variations in cellular morphology were observed despite of repeated cloning. Time-lapse video recordings of HTS-1 cells during culture revealed that not only the small colonies but also the monolayers near or at confluence were remarkably motile, often causing extreme elongation of the cells within them. The extremely plastic nature of HTS-1 cells in vitro is likely to be the reflection of the extraordinary capacity of caprine trophoblast cells to be stretched to extreme thinness in vivo as shown by electron microscopy. HTS-1 cells cultured on matrigel are highly invasive, and express MT1-MMP which, in the mouse, has been known to be expressed at the invasive edge of trophoblast both in vitro and in vivo. HTS-1 cells express placental lactogen (PL) and interferon-tau (IFNtau), as confirmed by immunocytochemistry, Western blotting and RT-PCR analysis. Both PL and IFNtau expression in the cells appeared to be down-regulated by cell-cell contact. In the medium conditioned by HTS-1 cells, the presence of secretory form of PL and IFNtau was confirmed by Western blotting. The HTS-1 cell line will serve as a useful in vitro model for the analysis of the molecular and/or cellular mechanisms underlying synepitheliochorial placentation in bovidae animals.

Up-regulation of polymeric immunoglobulin receptor mRNA in mammary epithelial cells by IFN-gamma.

As shown in previous in vivo experiment, the amount of polymeric immunoglobulin receptor (pIgR), which mediates the transcytosis of pIgA across epithelial cells, is regulated by lactogenic hormones (PRL and cortisol) during the development of the mammary gland. In the present in vitro study, it appeared that these hormones were insufficient to induce the strong expression of the gene that we observed in vivo. Several papers have shown that IFN-gamma is a strong stimulator of pIgR gene expression in different models. In contrast, nothing is known of the effects of IFN-gamma on pIgR gene expression in the mammary gland. We report here that IFN-gamma strongly increased pIgR mRNA levels through a direct effect on mammary epithelial cells. We show that IFN-gamma activated not only Stat1 but also Stat5 and that expression of the pIgR and IRF-1 genes was strongly correlated following IFN-gamma stimulation in mammary epithelial cells. In conclusion, these experiments enabled the analysis of different types of regulation of pIgR gene expression in the mammary gland and suggest possible co-operation between circulating hormones and locally produced cytokines, leading to pIgR gene expression in the mammary gland.

Use of somatic cells from goat milk for dynamic studies of gene expression in the mammary gland.

Somatic cells are present in the milk throughout lactation and consist of leukocytes and epithelial cells exfoliated from the mammary epithelium. Our objective was to determine the efficacy of using somatic cells from goat milk for dynamic studies of gene expression in the mammary gland. Over a 4-wk interval, cells were isolated from daily morning milk samples and samples taken 30 min after milking. They were characterized by direct cell counts and by flow cytometry analysis after immunostaining with antibodies directed against cytokeratin and CD45, a common leukocyte antigen. Epithelial cell counts within the morning milk ranged from 15 to 45% of total milk somatic cells. After-milking samples contained twice as many cells as did morning milk samples. The RNA was extracted from the somatic cells of both types of milk samples with equivalent efficiency (a mean of 1.2 microg RNA/mL of milk). Four mRNA variants of the alpha-S1 casein gene were detected by Northern blot analysis and the amount of each mRNA in milk cells was related to protein concentration in milk. The comparison between mRNA from the mammary gland and from congruently collected milk cells showed that relative amounts of mRNA for each milk-protein (alpha-S1 and kappa-casein and alactalbumin) were conserved. In a third experiment, daily milk cell RNA preparations were extracted to assess the effect of growth hormone (GH) on mammary gene expression; four goats were separated into two groups in order to perform a switch-back design consisting of three treatment weeks: Control, GH-Control or GH-Control-GH. In this study, treatment of goats with GH increased milk yields by 5%. Throughout the control and GH treatments, the expression of the three milk-protein genes studied were highly and significantly correlated (r = 0.949 and r = 0.958, P < 0.001 for, respectively, alpha-S1 and kappa-casein and for alpha-S1 casein and alpha-lactalbumin). During GH treatment, the three milk-protein mRNA abundances increased with the same pattern. In conclusion, the opportunity to use milk somatic cells for RNA preparation and analysis provides a significant improvement over the use of biopsy samples in assessing gene activity in the mammary gland and allows easy and repetitive sampling without damaging mammary tissue. Furthermore, we propose that this method could be used to investigate the transcriptional status of the mammary gland of an animal in relation to its genotype, nutritional and pathologic status, and under influence by hormonal factors.

Identification of leptin receptors in human breast cancer: functional activity in the T47-D breast cancer cell line.

To evaluate whether leptin plays a putative role in breast tumorigenesis, we studied the expression of its long and short receptor isoforms in various tumoral breast tissues. We applied semiquantitative RT-PCR method to RNA extracted from 20 breast cancer biopsies and two human breast cancer cell lines (T47-D and MCF-7). Our results showed the expression of both leptin receptor transcripts in all tumoral tissues examined. By in situ hybridization experiments, we localized leptin receptors in proliferating epithelial cells. Study of leptin effects on human breast cancer cells growth was performed by [3H]-thymidine incorporation method and colorimetric MTT assay. We demonstrated that leptin (50-100 ng/ml) significantly stimulates proliferation of the human breast cancer cell line T47-D (P<0.05). Western blot analysis indicated that leptin induces a time-dependent activation of mitogen-activated protein kinases (MAPKinase) 1 and 2 in T47-D cell line. Moreover, the specific MAPK-inhibitor PD 98059 blocked cell proliferation induced by leptin. In conclusion, we demonstrate that leptin receptors are expressed in breast cancer and that leptin induces proliferation in the T47-D cell line via activation of the MAPKinases pathway. These data suggest that leptin and its receptors may be implicated in mammary cell proliferation and in breast cancer pathogenesis.

Leptin expression in the ovine mammary gland: putative sequential involvement of adipose, epithelial, and myoepithelial cells during pregnancy and lactation.

We examined the ability of the ovine mammary gland to synthesize leptin throughout pregnancy and lactation. Leptin gene expression was assayed by real-time reverse transcription and polymerase chain reaction in mammary gland from ewes at 15, 80, 106, 112, or 141 d of pregnancy and at 0 (30 min after parturition), 3, 48, or 70 d of lactation. Leptin mRNA level was high at the beginning (the first 80 d) and at the end of pregnancy and was lower at mid-pregnancy and throughout lactation. Furthermore, during these periods of mammary leptin expression, the location of leptin protein, as determined by immunohistochemical analysis, changed within mammary tissue. It was located in adipose cells during early stages of pregnancy, in epithelial cells after full cell differentiation just before parturition, and in myoepithelial cells after parturition. These data, compared with published data on leptin receptor gene expression, provide evidence that leptin could be produced by different cell types of the mammary gland and could act as a paracrine factor on mammary cell growth and differentiation via adipose-epithelial cells and myoepithelial-epithelial cell interactions.

Differential roles for signal transducers and activators of transcription 5a and 5b in PRL stimulation of ERalpha and ERbeta transcription.

PRL has been shown to stimulate mRNA expression of both ERalpha and ERbeta in the rat corpus luteum and decidua of pregnancy. To investigate whether PRL may stimulate ER expression at the level of transcription and which transcription factors may mediate this stimulation, we have cloned the 5'-flanking regions of both rat ER genes. A constitutively active PRL receptor (PRL-R(CA)) stimulated both ERalpha and ERbeta promoter activity, indicating that PRL is acting to stimulate ER transcription. Putative signal transducer and activator of transcription (Stat)5 response elements were identified at -189 in the ERalpha promoter and at -330 in the ERbeta promoter. Mutation of these response elements or overexpression of dominant negative Stat5 prevented stimulation of ERalpha and ERbeta promoter activity, indicating that PRL regulation of ER expression requires both intact Stat5 binding sites as well as functional Stat5. Interestingly, either Stat5a or Stat5b could stimulate ERalpha transcription while stimulation of ERbeta occurred only in the presence of Stat5b. Through mutational analysis, a single nucleotide difference between the ERalpha and ERbeta Stat5 response elements was shown to be responsible for the lack of Stat5a-mediated stimulation of ERbeta. These findings indicate that PRL stimulation of ER expression occurs at the level of transcription and that PRL regulation of ERalpha can be mediated by either Stat5a or Stat5b, while regulation of ERbeta appears to be mediated only by Stat5b.

Expression of BRCA1 gene in ewe mammary epithelial cells during pregnancy: regulation by growth hormone and steroid hormones.

OBJECTIVE: Steroid hormones (estradiol and progesterone) in association with prolactin and growth hormone are involved in lobulo alveolar development of the mammary gland during pregnancy. We hypothesized that the BRCA1 gene may be induced by these different hormones. METHODS AND RESULTS: In this study, we have demonstrated by Northern blot and in situ hybridization, that the expression of ovine (o) BRCA1 mRNA in mammary epithelial cells increased dramatically during a short period in the second half of pregnancy (days 70 to 112) and decreased at the end of pregnancy. The increase in oBRCA1 mRNA expression is concomitant with rapid lobulo alveolar growth. Using an in vivo protocol to artificially induce mammary gland development, we demonstrated by the real-time RT-PCR method that growth hormone in association with estrogen, progesterone and hydrocortisone induces an increase of BRCA1 mRNA expression in the ewe mammary gland. Moreover, we showed that estradiol and progesterone induce oBRCA1 expression in primary cultures of ewe mammary gland. CONCLUSIONS: These results suggest that BRCA1 is a potential regulator of the effects of steroid hormones and growth hormone in the induction of mammary epithelial cell proliferation.

Tissue-specific induction of SOCS gene expression by PRL.

The mechanisms whereby tissue sensitivity to PRL is controlled are not well understood. Here we report that expression of mRNA and protein for members of the SOCS/CIS/JAB family of cytokine signaling inhibitors is increased by PRL administration in ovary and adrenal gland of the lactating rat deprived of circulating PRL and pups for 24 h but not in mammary gland. Moreover, suckling increases SOCS mRNA in the ovary but not in the mammary gland of pup-deprived rats. Deprivation of PRL and pups for 48 h allows the mammary gland to induce SOCS genes in response to PRL administration, and this is associated with a decrease in basal SOCS-3 mRNA and protein expression to the level seen in other tissues, suggesting that SOCS-3 induced refractoriness related to filling of the gland. In reporter assays, SOCS-1, SOCS-3, and CIS, but not SOCS-2, are able to inhibit transactivation of the STAT 5-responsive beta-lactoglobulin promoter in transient transfection assays. Moreover, suckling results in loss of ovarian and adrenal responsiveness to PRL administered 2 h after commencement of suckling, as determined by STAT 5 gel shift assay. Immunohistochemistry was used to localize the cellular sites of SOCS-3 and CIS protein expression in the ovary and adrenal gland. We propose that induced SOCS-1, SOCS-3, and CIS are actively involved in the cellular inhibitory feedback response to physiological PRL surges in the corpus luteum and adrenal cortex during lactation, but after pup withdrawal, the mammary gland is rendered unresponsive to PRL by increased levels of SOCS-3.

Mutations of ovine and bovine placental lactogens change, in different ways, the biological activity mediated through homologous and heterologous lactogenic receptors.

The biological activities of ovine (o) and bovine (b) placental lactogens (PLs) and their mutated analogues were compared using several binding and in vitro bioassays. In almost all cases, the biological activities of these analogues mediated through rat (r) prolactin receptor (PRLR) showed little or no change, despite a remarkable decrease in their capacity to bind to the extracellular domain of rPRLR and despite compromised stability of the 2:1 complexes. These results indicate that mutations impairing the ability of oPL or bPL to form stable complexes with lactogenic receptors do not necessarily lead to a decrease in the biological activity, because the transient existence of the homodimeric complex is still sufficient to initiate the signal transduction. In contrast, oPL and bPL analogues completely, or almost completely, lost their ability to activate homologous PRLRs, and some of them even acted as site-2 antagonists. To explain the difference between the activity transduced through homologous and that transduced through heterologous PRLRs, we propose the novel term 'minimal time of homodimer persistence'. This concept assumes that in order to initiate the signal transduction, the associated kinase JAK2 has to be transphosphorylated and this requires a 'minimal time' of homodimer existence. In the case of homologous interaction between ruminant PLs and homologous PRLRs, this 'minimal time' is met, though the interaction with homologous PRLRs has a shorter half-life than that with heterologous PRLRs. Therefore oPL or bPL are active in cells possessing both homologous and heterologous PRLRs. Mutations of oPL or bPL lead to reduced affinity and, consequently, the 'time of homodimer persistence' is shortened. Although in the case of heterologous interaction the 'minimal time' is still sufficient to initiate the biological activity, in homologous interactions, which are already weaker than heterologous interactions, further destabilization of the complex shortens its persistence to below the 'minimal time', leading to full or partial loss of biological activity.

Central infusion of leptin into well-fed and undernourished ewe lambs: effects on feed intake and serum concentrations of growth hormone and luteinizing hormone.

Leptin has been implicated in the regulation of feed intake, growth, and reproduction. The objective of this study was to determine if centrally administered leptin would affect feed intake and the secretion of growth hormone (GH) and luteinizing hormone (LH) in ewe lambs. Eighteen ewe lambs were ovariectomized and fitted with intracerebroventricular (i.c.v.) cannulae. Lambs were randomly assigned to receive either a maintenance diet (fed), or a diet that provided 38% of maintenance requirements (diet-restricted) for 14 weeks. Subsequently, recombinant ovine leptin or vehicle was continuously infused, via i.c.v. cannulae, in a linearly increasing dose for 8 days, reaching a maximum of 1.25 microg/kg per h. Feed intake was recorded on days -1 to 7. Blood was collected via jugular cannulae every 10 min for 4 h on days 0, 2, 4, 6 and 8 for the determination of serum leptin, insulin, LH and GH. Leptin suppressed feed intake in fed lambs on days 4 to 7 (P<0.001), but had no effect on feed intake in diet-restricted lambs (P>0.25). Fed lambs had greater serum concentrations of leptin than diet-restricted lambs (P=0.007). Also, although not different on day 0 (pretreatment), on day 8 serum leptin concentrations were greater in leptin-treated lambs than in saline-treated lambs (P=0.003). Insulin was lower in diet-restricted than in fed lambs (P=0.003), but was not affected by leptin treatment (P=0.82). LH pulse frequencies were lower in diet-restricted lambs than in fed lambs (P=0.038), but were not affected by leptin treatment (P=0.85). Mean serum GH was greater in diet-restricted than in fed lambs (P<0.01). In diet-restricted lambs treated with leptin or saline, mean GH did not differ on day 0, but increased in response to leptin treatment (P<0.006). Treatment of fed lambs with leptin did not affect serum GH (P>0.32). From this work, we propose that leptin represents an important functional link between adipose stores and hypothalamic function in ruminants. We demonstrate that leptin concentrations change in response to reduced nutritional status, and that leptin has the ability to regulate multiple physiological processes in lambs, including both feed intake and secretion of GH.

Leptin in ruminants. Gene expression in adipose tissue and mammary gland, and regulation of plasma concentration.

This paper reviews data on leptin gene expression in adipose tissue (AT) and mammary gland of adult ruminants, as well as on plasma leptin variations, according to genetic, physiological, nutritional and environmental factors. AT leptin mRNA level was higher in sheep and goat subcutaneous than visceral tissues, and the opposite was observed in cattle; it was higher in fat than in lean selection line in sheep; it was decreased by undernutrition and increased by refeeding in cattle and sheep, and not changed by adding soybeans to the diet of lactating goats; it was increased by injection of NPY to sheep, and by GH treatment of growing sheep and cattle. Insulin and glucocorticoids in vitro increased AT leptin mRNA in cattle, and leptin production in sheep. Long daylength increased AT lipogenic activities and leptin mRNA, as well as plasma leptin in sheep. Mammary tissue leptin mRNA level was high during early pregnancy and was lower but still expressed during late pregnancy and lactation in sheep. Leptin was present in sheep mammary adipocytes, epithelial and myoepithelial cells during early pregnancy, late pregnancy and lactation, respectively. Plasma leptin in cattle and sheep was first studied thanks to a commercial "multi-species" kit. It was positively related to body fatness and energy balance or feeding level, and decreased by beta-agonist injection. The recent development of specific RIA for ruminant leptin enabled more quantitative study of changes in plasma leptin concentration, which were explained for 35--50% by body fatness and for 15--20% by feeding level. The response of plasma leptin to meal intake was related positively to glycemia, and negatively to plasma 3-hydroxybutyrate. The putative physiological roles of changes in leptin gene expression are discussed in relation with published data on leptin receptors in several body tissues, and on in vivo or in vitro effects of leptin treatment.

Role of protein kinases in the prolactin-induced intracellular calcium rise in Chinese hamster ovary cells expressing the prolactin receptor.

There is still only limited understanding of the early steps of prolactin signal transduction in target cells. It has been shown that prolactin actions are associated with cell protein phosphorylation, Ca2+ increases, and so on. However, the link between the activation of kinases and calcium influx or intracellular Ca2+ mobilization has not yet been clearly established. Chinese hamster ovary (CHO) cells, stably transfected with the long form of rabbit mammary gland prolactin receptor (PRL-R) cDNA were used for PRL-R signal transduction studies. Spectrofluorimetric techniques were used to measure intracellular calcium ([Ca2+]i) in cell populations with Indo1 as a calcium fluorescent probe. We demonstrate that, although protein kinase C activation (PMA or DiC8) caused a calcium influx in CHO cells, prolactin-induced PKC activation was not responsible for the early effect of prolactin on [Ca2+]i. Activation of protein kinase A (PKA) or protein kinase G did not modify [Ca2+]i and inhibition of PKA pathway did not affect the prolactin response. In the same way, phosphatidylinositol-3 kinaseinhibition had no effect on the prolactin-induced Ca2+ increase. On the other hand, tyrosine kinase inhibitors (herbimycin A, lavendustin A, and genistein) completely blocked the effect of prolactin on [Ca2+]i (influx and release). W7, a calmodulin-antagonist, and a specific inhibitor of calmodulin kinases (KN-62), only blocked prolactin-induced Ca2+ influx but had no significant effect on Ca2+ release. Using pharmacological agents, we present new data concerning the involvement of protein phosphorylations in the early effects of prolactin on ionic channels in CHO cells expressing the long form of PRL-R. Our results suggest that, at least in the very early steps of prolactin signal transduction, serine-threonine phosphorylation does not participate in the prolactin-induced calcium increase. On the other hand, tyrosine phosphorylation is a crucial, very early step, since it controls K+ channel activation, calcium influx, and intracellular calcium mobilization. Calmodulin acts later, since its inhibition only blocks the prolactin-induced Ca2+ influx.

Recombinant prolactin receptor extracellular domain of rainbow trout (Oncorhynchus mykiss): subcloning, preparation, and characterization.

The cDNA of the extracellular domain of rainbow trout (Oncorhynchus mykiss) prolactin receptor (trPRLR-ECD) was cloned in the prokaryotic expression vector pMON to enable its expression in Escherichia coli after induction with nalidixic acid. The bacterially expressed trPRLR-ECD protein, contained within the refractile body pellet, was solubilized in 4.5 M urea, refolded, and purified on a Q-Sepharose column, pH 8, by stepwise elution with NaCl. The bioactive monomeric 26-kDa fraction was eluted in 0.2 M NaCl, yielding 20 mg/2.5 L of induced culture. The purified protein was over 98% homogeneous, as shown by SDS-PAGE in the presence or absence of reducing agent and by chromatography on a Superdex column. Binding experiments using [125I]ovine placental lactogen (oPL) as a ligand revealed that human growth hormone (hGH), oPL, and ovine prolactin (oPRL) were the most effective competitors, with respective IC50 values of 1.32, 2.27, and 2.70 nM. Chicken (ch) PRL did not compete at all, and homologous trPRL was much less effective, with a corresponding IC50 value of 1826 nM. Gel-filtration was used to determine the stoichiometry of trPRLR-ECD's interaction with oPL, hGH, and oPRL. Only oPL yielded a 2:1 complex, whereas hGH and oPRL formed only 1:1 complexes, with excess trPRLR-ECD being seen at the initial 2:1 trPRLR-ECD:hGH or trPRLR-ECD:oPRL ratios. No studies were performed with chPRL because of its inability to compete with [125I]oPL or with trPRL because of its low affinity toward trPRLR-ECD. The present results agree with previous findings indicating, as in mammals, that homologous PRL interacts transiently with its receptor and suggest that transient homologous PRL-induced homodimerization of the receptor is sufficient to initiate a biological signal, despite the fact that, in classical binding experiments, only low specific binding can be detected.

Recombinant extracellular domain of rabbit growth hormone receptor and biological activity of somatogenic hormones.

The cDNA of the extracellular domain of rabbit growth hormone receptor (rbGHR-ECD) was cloned in the prokaryotic expression vector pMON, to enable its expression in Escherichia coli after induction with nalidixic acid. The bacterially expressed rbPRLR-ECD protein, contained within the refractile-body pellet, was solubilized in 4.5 M urea, refolded, and purified on a Q-Sepharose column, pH 8, by stepwise elution with NaCl. The bioactive monomeric 28-kDa fraction was eluted in 0.15 M NaCl, yielding 50 mg/2.5 l of induced culture. The purified protein was over 98% homogeneous, as shown by SDS-PAGE in the presence or absence of reducing agent, and by chromatography on a Superdex column. Gel filtration was used to determine the stoichiometry of rbGHR-ECD's interaction with human (h), ovine (o), chicken (ch) and common carp (cc) GHs and with bovine (b) and caprine (c) placental lactogens (PLs). The formation of 2:1 complexes was indicated in all cases. Binding experiments using radiolabelled oGH as a ligand revealed it to be the most effective competitor, followed by bPL, cPL, hGH chGH and ccGH, with respective IC50 values of 0.27, 0.94, 1.55, 2.13, 41.9 and 51.2 nM. Rabbit GHR-ECD inhibited the bPL-inducible proliferation of FDC-P1 cells stably transfected with rbGHR and Nb2 cells possessing rat PRLR. The biological activity of oGH, hGH, cPL, bPL, chGH and ccGH was tested in the FDC-P1 cells stably transfected with rbGHR and yielded the respective EC50 values (in nM) of 0.024, 0.023, 0.021, 0.24, 4.71 and 0.49. These results indicate remarkable discrepancies between the binding capacities and biological activities: the possible reasons for these findings are discussed.