Leptin receptor expression increases in placenta, but not hypothalamus, during gestation in Mus musculus and Myotis lucifugus.

In addition to effects on appetite and metabolism, the hormone leptin is required for reproduction in mammals. Maternal plasma leptin is increased above non-pregnant levels in all mammals thus far examined, including humans. The increase in plasma leptin appears to result in part from upregulation of adipose leptin secretion (e.g., in mice), or from production and secretion of leptin from the placenta (e.g., in humans and some bats). The placenta may also modulate maternal leptin levels via production of a plasma leptin-binding protein (mice, humans). Thus, the placenta plays a coordinating role in regulation of maternal leptin during pregnancy. In this study, the hypothesis that the placenta is also a target organ for leptin in diverse taxa was tested by examining the expression of leptin receptors (Ob-R) in placentae from species of distantly related mammalian taxa, Mus musculus (the laboratory mouse) and Myotis lucifugus (the little brown myotis, also called the little brown bat). A partial sequence of M. lucifugus Ob-R cDNA was first obtained and found to share approximately 78-88% homology at the nucleotide level with known mammalian Ob-R cDNAs. Using probes and primers designed from this sequence, receptor expression was detected in numerous tissues of M. lucifugus, including placenta, which expressed two major receptor isoforms as judged by molecular size. In both species, Ob-R mRNA expression in placenta significantly increased from early to late gestation. Expression of Ob-R mRNA was not affected by cAMP treatment in vitro. The increase in Ob-R mRNA expression in placenta was specific, since Ob-R mRNA expression did not change during gestation in either species in hypothalamus, the major site of the central actions of leptin. Thus, Ob-R is expressed in placenta throughout gestation in mice and bats, and its expression increases over the course of gestation, which raises the possibility that leptin may exert temporally distinct effects on placental growth or function throughout gestation. Because similar placenta-specific changes in leptin receptor expression occurred in species from distantly related mammalian taxa which collectively comprise approximately 70% of all known mammalian species, it is possible that placental actions of leptin are conserved across mammals, even in those species (such as the Swiss-Webster strain of mouse) in which the placenta does not itself produce leptin.

In vivo studies on the role of the peripheral benzodiazepine receptor (PBR) in steroidogenesis.

In various steroidogenic cell models, mitochondrial preparations and submitochondrial fractions, the expression of the mitochondrial 18 kDa peripheral-type benzodiazepine receptor (PBR) protein confers the ability to take up and release, upon ligand activation, cholesterol. Thus, cholesterol becomes available to P450scc on the inner mitochondrial membrane. These in vitro studies were validated by in vivo experiments. Treatment of rats with ginkgolide B (GKB), specifically reduced the ligand binding capacity, protein, and mRNA expression of the adrenocortical PBR and circulating glucocorticoid levels. Treatment with GKB also resulted in inhibition of PBR protein synthesis and corticosterone production by isolated adrenocortical cells in response to ACTH. The ontogeny of both PBR binding capacity and protein directly paralleled that of ACTH-inducible steroidogenesis in rat adrenal cells and in rats injected with ACTH. In addition, the previously described suppression of luteal progesterone synthesis in the pregnant rat by continuous in vivo administration of a gonadotropin-releasing hormone agonist may be due to decreased luteal PBR ligand binding and mRNA. These results suggest that (i) PBR is an absolute prerequisite for adrenocortical and luteal steroidogenesis, (ii) regulation of adrenal PBR expression may be used as a tool to control circulating glucocorticoid levels and (iii) the stress hypo-responsive period of neonatal rats may result from decreased adrenal cortical PBR expression.

Leptin, corticotropin-releasing hormone (CRH), and neuropeptide Y (NPY)in free-ranging pregnant bats.

Leptin, the product of the obese gene first identified in mice, restores fertility in obese mice, and accelerates puberty in mice. We hypothesized that leptin's putative role in reproduction may extend to pregnancy and lactation. Leptin levels were determined in Myotis lucifugus, the little brown bat, a free-ranging mammal with a seasonal breeding cycle. The present study shows that plasma levels of leptin progressively rise during pregnancy, supporting a potential role for leptin in the maintenancy of pregnancy. In contrast, leptin was significantly lower during lactation, a time when most mammals, including bats, demonstrate reduced fertility. In addition to its possible roles in reproduction, leptin appears important in regulation of energy balance. M. lucifugus spontaneously fasts for up to 16 h each day during the active season, which allowed us to test the hypothesis that acute fasting was associated with decreased leptin. Leptin was significantly lower in fasted (lactating) bats, compared to those that recently returned from nightly foraging. Although postprandial lactating bats had a significantly higher fat index than fasted bats, plasma leptin and body fat were not significantly correlated, and were only weakly correlated (r2 = 0.26) when both pregnant and lactating females were included in the analysis. Similar changes during pregnancy, lactation, and the daily feeding cycle were observed in the hypothalamic neuropeptide, corticotropin-releasing hormone (CRH), which is believed to play an important role in energy balance and reproduction. By contrast, neuropeptide Y (NPY) increased during pregnancy but did not change during fasting. These results suggest that leptin's putative role in reproduction may extend to pregnancy and lactation, and that spontaneous, acute fasting results in decreased circulating levels of leptin in M. lucifugus.

Changes in responsiveness of the hypothalamic-pituitary-adrenocortical axis to 2-deoxy-D-glucose in developing rats.

During the first 2-3 weeks of postnatal life, the hypothalamic-pituitary-adrenocortical axis in rats exists in a relatively dormant state, termed the stress-hyporesponsive period. The development of the hypothalamic-pituitary-adrenal axis in young rats was examined by testing the ability of the nonmetabolizable glucose analog 2-deoxy-D-glucose (2-DG) to stimulate CRF in vitro and ACTH in vivo. Intraperitoneal injection of 2-DG into rats 11-12 days of age or into adult rats resulted in significant hyperglycemia by 60 min that was greater in magnitude in the adults. This response was accompanied by a significant increase in plasma ACTH to levels more than 500% of the noninjected or saline-injected control values in adults. A much smaller (approximately 200%) but still significant ACTH response was observed 60 min after 2-DG injection in the neonates. The drug had no effect on the ACTH response to exogenous CRF in the neonates. The pattern of corticosterone secretion paralleled that of ACTH, with a very moderate rise (from less than 1 to 2 micrograms/dl) seen in the neonate. To test the hypothesis that CRF was driving the ACTH response to glucoprivation induced by 2-DG in the neonate and to determine the ontogeny of hypothalamic responsiveness to this stressor, complete hypothalami were existed from rats 10-35 days of age and incubated in a defined buffer containing 5.5 mM glucose with or without 22 mM 2-DG. There was no effect of the analog on CRF secretion until day 35, at which time the magnitude of the response resembled that previously reported to occur in adult tissue. To determine if the failure to observe a CRF response was due to heightened sensitivity to the negative feedback effects of glucocorticoids, 8- to 10-day-old pups were adrenalectomized and returned to their mothers for 3 days, at which time the hypothalami were removed and tested for CRF secretion. No difference was observed between basal CRF secretory rates in the control or adrenalectomized groups, and there was still no significant response to 2-DG. Moreover, adrenalectomy did not potentiate the ACTH response to injection of 2-DG in vivo. The results suggest that during neonatal life in the rat, the hypothalamic glucostat/CRF cell mechanism is incapable of promoting a normal secretory response to glucoprivation. This deficit is probably not related to the increased sensitivity to negative feedback that has been proposed to account in part for the attenuated ACTH responses to stress in the neonatal animal.(ABSTRACT TRUNCATED AT 400 WORDS)

Development in rats of the brain-pituitary-adrenal response to hypoglycemia in vivo and in vitro.

To clarify the nature of the stress hyporesponsive period that occurs in neonatal rats, the development of the response of the brain-pituitary-adrenal axis to hypoglycemia stress in rats was assessed in vivo and in vitro. Hypothalami were removed from the brains of neonatal (9-35 days postnatal) or adult rats and incubated in vitro for sequential 30-min periods in Krebs buffer for determination of corticotropin-releasing factor (CRF) secretion under conditions of altered glucose concentrations. As expected from previous studies, CRF secretion from adult hypothalami was significantly increased in severely hypoglycemic conditions (0.55 mM glucose) by approximately 50% above base-line values (in 5.5 mM glucose). However, lowering glucose did not elicit an increase in CRF release from hypothalami of rats less than 35 days of age. Hypothalami obtained from rats less than or equal to 24 days old also failed to show consistent secretory responses to potassium depolarization. At 35 days postnatal the response to hypoglycemia was significant and similar to the adult response. To determine if the lack of hypothalamic response to hypoglycemia in vitro could be correlated with the in vivo responses to hypoglycemia, rats aged 4 days to adult were injected intraperitoneally with porcine insulin and killed at different times after injection. Insulin injections lowered plasma glucose levels in fasted 4-day-old rats in a dose-dependent fashion, but a nadir in glucose (approximately 40 mg/dl) was not reached until 90 min; the same treatment produced a nadir in glucose within 30 min in fasted rats 10 days old and older, suggesting that the 4-day-old rats are relatively insulin insensitive.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of corticotropin-releasing factor from cultured rat hypothalamic cells: effects of catecholamines.

An understanding of the regulation of CRF secretion in rats is currently incomplete, in part due to the lack of sensitive in vitro models available for studying this neuropeptide. In particular, the effects of catecholamines on CRF secretion, and the receptor subtypes mediating these actions have long been the subject of much debate. A cultured cell model has been adapted for studying secretory responses of hypothalamic cells of 1-week-old rats. Between 7-16 days in monolayer culture the cells secreted detectable levels of immunoreactive CRF, and this release was paralleled by the appearance of punctate bead-like regions of immunoreactivity along fine cellular processes. CRF secretion was increased up to 4-fold by norepinephrine (EC50, approximately 0.5 microM). The increase in CRF secretion produced by norepinephrine was blocked by the beta-receptor antagonist propranolol, but not by the alpha-antagonist prazosin. Moreover, the beta-receptor agonist isoproterenol significantly elevated CRF secretion, whereas the alpha-agonist phenylephrine was without effect, except at high concentrations. Addition of phenylephrine, however, potentiated the effect of isoproterenol, but this response was still significantly less than that produced by norepinephrine. Forskolin (EC50, approximately 0.7 microM) and the active phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (EC50, approximately 40 nM) also increased CRF secretion by 3- to 4-fold. Inactive phorbol derivatives had no effect on CRF release from these cultures. The results indicate that cultured neonatal rat hypothalamic cells are a powerful model for the study of CRF release in vitro, and that norepinephrine acts directly at the isolated cell level to stimulate secretion of this peptide, primarily by activating beta-adrenoceptors. The results also suggest that at least two functional second messenger systems (adenylate cyclase and protein kinase-C) are involved in CRF secretion and are already functional in the neonatal hypothalamus.

Regulation of corticotropin-releasing factor secretion in vitro by glucose.

The neurosecretory responses of the isolated rat hypothalamus were assessed in vitro. Rat hypothalamic blocks were incubated for 30 min in a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered salt solution with 5.5 mM glucose (base-line collection period). The blocks were transferred to fresh buffer with a new concentration of glucose with or without various additions (test period); corticotropin-releasing factor (CRF) and other hormones in the media were determined by radioimmunoassay. CRF secretion was maximally increased to approximately 200% of base line at glucose concentrations less than 4 mM and decreased to 65% of base line at higher glucose concentrations. The increase in CRF secretion at low glucose (0.55 or 1.38 mM) was Ca2+ dependent and completely reversible. Hexamethonium, cyproheptadine, and atropine partially blocked the CRF response to 0.55 mM glucose. Glucose concentrations from 0 to 11 mM had no effect on the CRF response to 47.5 mM KCl. The inhibitory effects of high glucose were completely reversed by the addition of 2-deoxy-D-glucose (3-49 mM). Glucose levels did not alter secretion of either gonadotropin-releasing hormone or arginine vasopressin from hypothalamic blocks. The results suggest that the isolated rat hypothalamus is extremely sensitive to the level of glucose and that CRF is rapidly and reversibly secreted in response to slight reductions in glucose concentrations. These concentrations are consistent with those observed during moderate to severe hypoglycemia in vivo. The rise in glucocorticoids observed in vivo during hypoglycemia may result at least in part from the ability of the hypothalamus to directly sense glucose levels and promote secretion of CRF.