Attenuated hypothalamic responses to α-melanocyte stimulating hormone during pregnancy in the rat.

KEY POINTS: Increased appetite and weight gain occurs during pregnancy, associated with development of leptin resistance, and satiety responses to the anorectic peptide α-melanocyte stimulating hormone (α-MSH) are suppressed. This study investigated hypothalamic responses to α-MSH during pregnancy, using c-fos expression in specific hypothalamic nuclei as a marker of neuronal signalling, and in vivo electrophysiology in supraoptic nucleus (SON) oxytocin neurons, as a representative α-MSH-responsive neuronal population that shows a well-characterised α-MSH-induced inhibition of firing. While icv injection of α-MSH significantly increased the number of c-fos-positive cells in the paraventricular, supraoptic, arcuate and ventromedial hypothalamic nuclei in non-pregnant rats, this response was suppressed in pregnant rats. Similarly, SON oxytocin neurons in pregnant rats did not demonstrate characteristic α-MSH-induced inhibition of firing that was observed in non-pregnant animals. Given the known functions of α-MSH in the hypothalamus, the attenuated responses are likely to facilitate adaptive changes in appetite regulation and oxytocin secretion during pregnancy. ABSTRACT: During pregnancy, a state of positive energy balance develops to support the growing fetus and to deposit fat in preparation for the subsequent metabolic demands of lactation. As part of this maternal adaptation, the satiety response to the anorectic peptide α-melanocyte stimulating hormone (α-MSH) is suppressed. To investigate whether pregnancy is associated with changes in the response of hypothalamic α-MSH target neurons, non-pregnant and pregnant rats were treated with α-MSH or vehicle and c-fos expression in hypothalamic nuclei was then examined. Furthermore, the firing rate of supraoptic nucleus (SON) oxytocin neurons, a known α-MSH responsive neuronal population, was examined in non-pregnant and pregnant rats following α-MSH treatment. Intracerebroventricular injection of α-MSH significantly increased the number of c-fos-positive cells in the paraventricular, arcuate and ventromedial hypothalamic nuclei in non-pregnant rats, but no significant increase was observed in any of these regions in pregnant rats. In the SON, α-MSH did induce expression of c-fos during pregnancy, but this was significantly reduced compared to that observed in the non-pregnant group. Furthermore, during pregnancy, SON oxytocin neurons did not demonstrate the characteristic α-MSH-induced inhibition of firing rate that was observed in non-pregnant animals. Melanocortin receptor mRNA levels during pregnancy were similar to non-pregnant animals, suggesting that receptor down-regulation is unlikely to be a mechanism underlying the attenuated responses to α-MSH during pregnancy. Given the known functions of α-MSH in the hypothalamus, the attenuated responses will facilitate adaptive changes in appetite regulation and oxytocin secretion during pregnancy.

Reproductive Regulation of Gene Expression in the Hypothalamic Supraoptic and Paraventricular Nuclei.

Oxytocin secretion is required for successful reproduction. Oxytocin is synthesised by magnocellular neurones of the hypothalamic supraoptic and paraventricular nuclei and the physiological demand for oxytocin synthesis and secretion is increased for birth and lactation. Therefore, we used a polymerase chain reaction (PCR) array screen to determine whether genes that might be important for synthesis and/or secretion of oxytocin are up- or down-regulated in the supraoptic and paraventricular nuclei of late-pregnant and lactating rats, compared to virgin rats. We then validated the genes that were most highly regulated using real time-quantitative PCR. Among the most highly regulated genes were those that encode for suppressors of cytokine signalling, which are intracellular inhibitors of prolactin signalling. Prolactin receptor activation changes gene expression via phosphorylation of signal transducer and activator of transcription 5 (STAT5). Using double-label immunohistochemistry, we found that phosphorylated STAT5 was expressed in almost all oxytocin neurones of late-pregnant and lactating rats but was almost absent from oxytocin neurones of virgin rats. We conclude that increased prolactin activation of oxytocin neurones might contribute to the changes in gene expression by oxytocin neurones required for normal birth and lactation.

Hormone interactions regulating energy balance during pregnancy.

Appetite and food intake are increased during pregnancy, comprising an adaptive response that facilitates energy storage in preparation for the high metabolic demands of pregnancy and subsequent lactation. To maintain the increased energy intake in the face of increased adiposity and rising leptin levels, pregnant females become resistant to the central anorectic actions of leptin. In rats, pregnancy-induced leptin resistance is characterised by elevated neuropeptide Y and reduced pro-opiomelanocortin expression in the arcuate nucleus, reduced leptin receptor mRNA levels and suppression of leptin-induced phosphorylated signal transducer and activator of transcription-3 protein in the ventromedial hypothalamic nucleus, as well as a loss of anorectic responses to both leptin and alpha-melantocyte-stimulating hormone. Our recent data suggest that this leptin-resistance may also cause central insulin resistance and an altered peripheral glucose homeostasis. The specific hormone changes during pregnancy that might mediate these effects on leptin signalling are a current focus of investigation. In pseudopregnant rats, chronic i.c.v. infusion of ovine prolactin to mimic patterns of placental lactogen secretion that occur during pregnancy completely blocked the ability of leptin to suppress food intake. These data suggest that placental lactogen secretion may mediate the hormone-induced loss of response to leptin during pregnancy. This action of prolactin/placental lactogen appears to be mediated downstream of the primary leptin-responsive neurones in the mediobasal hypothalamus, possibly in the paraventricular nucleus. Our studies show complex hormone-induced adaptations in the normal hypothalamic pathways regulating body weight homeostasis during pregnancy.

Both p110alpha and p110beta isoforms of phosphatidylinositol 3-OH-kinase are required for insulin signalling in the hypothalamus.

Both insulin and leptin action in the brain are considered to involve activation of phosphoinositide 3-kinase (PI3K), although the roles of different PI3K isoforms in insulin signalling in the hypothalamus are unknown. In the present study, we characterised the roles of these isoforms in hypothalamic insulin and leptin signalling and investigated the cross-talk of both hormones. To evaluate PI3K levels in the hypothalamus, PI3K was immunoprecipitated using an antibody directed against the p85 subunit, and then total PI3K activity was measured in the presence of novel isoform-selective pharmacological inhibitors of each isoform of PI3K. Subsequently, these inhibitors were administered into the lateral ventricle of male Sprague-Dawley rats, followed by vehicle, insulin, leptin or both hormones 45 min later. PI3K activity was determined by immunohistochemical detection of phosphorylated AKT (S473). In a separate study, the effects of the inhibitors on the anorexigenic action of insulin and leptin were determined. Hypothalamic insulin signalling was specifically mediated by the combined actions of the class Ia isoforms p110alpha and p110beta. Total hypothalamic PI3K activity was inhibited 65% by a p110alpha inhibitor, and 35% by a p110beta inhibitor, with a combination of inhibitors being equally effective as the broad-spectrum PI3K inhibitor wortmannin. Individual i.c.v. administration of p110alpha and p110beta inhibitors partly prevented insulin-induced phosphorylated AKT (S473) in the arcuate nucleus, whereas simultaneous application completely blocked insulin action. Unlike insulin, leptin did not induce phosphorylated AKT in the hypothalamus, as detected by immunohistochemistry, and the anorectic effects of leptin were not affected by pre-treatment with a combination of p110alpha and p110beta inhibitors. The enhanced anorectic effect of a combined i.c.v. application of both insulin and leptin could be prevented by pre-treatment with the combination of p110alpha and p110beta inhibitors. The data suggest that p110alpha and p110beta isoforms of PI3K are necessary to mediate insulin action in the hypothalamus. The role of PI3K in leptin action is less clear, but it may be involved by means of an insulin-dependent sensitisation of leptin action.

Loss of hypothalamic response to leptin during pregnancy associated with development of melanocortin resistance.

Hypothalamic leptin resistance during pregnancy is an important adaptation that facilitates the state of positive energy balance required for fat deposition in preparation for lactation. Within the arcuate nucleus, pro-opiomelanocortin (POMC) neurones and neuropeptide Y (NPY)/agouti-related gene protein (AgRP) neurones are first-order leptin responsive neurones involved in the regulation of energy balance. The present study aimed to investigate whether the regulation of these neuropeptides is disrupted during pregnancy in association with the development of leptin resistance. As measured by quantitative in situ hybridisation, POMC and AgRP mRNA levels were not significantly different during pregnancy, whereas NPY mRNA levels increased such that, by day 21 of pregnancy, levels were significantly higher than in nonpregnant, animals. These data suggest that these neurones were not responding normally to the elevated leptin found during pregnancy. To further characterise the melanocortin system during pregnancy, double-label immunohistochemistry was used to quantify leptin-induced phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) in POMC neurones, using α-melanocyte-stimulating hormone (MSH) as a marker. The percentage of α-MSH neurones containing leptin-induced pSTAT3 did not significantly differ from nonpregnant animals, indicating that there was no change in the number of POMC neurones that respond to leptin during pregnancy. Treatment with α-MSH significantly reduced food intake in nonpregnant rats, but not in pregnant rats, indicating resistance to the satiety actions of α-MSH during pregnancy. The data suggest that multiple mechanisms contribute to leptin resistance during pregnancy. As well as a loss of responses in first-order leptin-responsive neurones in the arcuate nucleus, there is also a downstream disruption in the melanocortin system.

Quantitation of prolactin receptor mRNA in the maternal rat brain during pregnancy and lactation.

Prolactin receptor (PRL-R) expression in the brain is increased in lactating rats compared with non-pregnant animals. The aim of the present study was to determine the time-course of changes in PRL-R mRNA levels during pregnancy and/or lactation, and to determine relative levels of the two forms (short and/or long form) of receptor mRNA in specific brain regions. Brains were collected from female rats on dioestrus, days 7, 14 or 21 of pregnancy, day 7 of lactation or day 7 post-weaning. Frozen, coronal sections were cut (300 microm) and specific hypothalamic nuclei and the choroid plexus were microdissected using a punch technique. Total RNA was extracted and reverse transcribed, then first strand cDNA was amplified using quantitative real-time PCR. Results showed an up-regulation of long-form PRL-R mRNA in the choroid plexus by day 7 of pregnancy compared with dioestrus, which further increased on days 14 and 21 of pregnancy and day 7 of lactation, and then decreased to dioestrous levels on day 7 post-weaning. Short-form PRL-R mRNA levels increased on day 14 of pregnancy relative to dioestrus, increased further on day 7 of lactation and decreased on day 7 post-weaning. Changes in mRNA were reflected in increased levels of PRL-R immunoreactivity in the choroid plexus during pregnancy and lactation, compared with dioestrus. In the arcuate nucleus, long-form PRL-R mRNA was increased during pregnancy. In contrast to earlier work, no significant changes in short- or long-form PRL-R mRNA expression were detected in several other hypothalamic nuclei, suggesting that changes in hypothalamic mRNA levels may not be as marked as previously thought. The up-regulation of PRL-R mRNA and protein expression in the choroid plexus during pregnancy and lactation suggest a possible mechanism whereby increasing levels of peripheral prolactin during pregnancy may have access to the central nervous system. Together with expression of long-form PRL-R mRNA in specific hypothalamic nuclei, these results support a role for prolactin in regulating neuroendocrine and behavioural adaptations in the maternal brain.

Prolactin receptors in the brain during pregnancy and lactation: implications for behavior.

Numerous studies have documented prolactin regulation of a variety of brain functions, including maternal behavior, regulation of oxytocin neurons, regulation of feeding and appetite, suppression of ACTH secretion in response to stress, and suppression of fertility. We have observed marked changes in expression of prolactin receptors in specific hypothalamic nuclei during pregnancy and lactation. This has important implications for neuronal functions regulated by prolactin. In light of the high circulating levels of prolactin during pregnancy and lactation and the increased expression of prolactin receptors in the hypothalamus, many of these functions may be enhanced or exaggerated in the maternal brain. The adaptations of the maternal brain allow the female to exhibit the appropriate behavior to feed and nurture her offspring, to adjust to the nutritional and metabolic demands of milk production, and to maintain appropriate hormone secretion to allow milk synthesis, secretion, and ejection. This review aims to summarize the evidence that prolactin plays a key role in regulating hypothalamic function during lactation and to discuss the hypothesis that the overall role of prolactin is to organize and coordinate this wide range of behavioral and neuroendocrine adaptations during pregnancy and lactation.

A comparison of native and modified intravenous immunoglobulin for the management of hypogammaglobulinemia.

Ten patients with severe hypogammaglobulinemia received 6 monthly infusions of either native or modified intravenous immunoglobulin (IVIG) followed by 6 monthly infusions of the other product in a double-blind, crossover protocol. Clinical parameters were monitored on a daily basis and serum was obtained at 24 hours, 3 weeks, and 4 weeks after each infusion for measurement of total IgG, specific antibodies, and opsonizing antibodies against Streptococcus pneumoniae types 5, 12F, and 14. No differences between the products were seen for total IgG or antibodies against herpes simplex virus types 1 and 2, rubella, toxoplasma cytomegalovirus, diphtheria, or tetanus. Greater opsonizing antibody to the three strains of pneumococci were apparent for native IVIG until the third infusion, after which time products were equal. Clinical parameters (febrile or symptomatic days, missed work/school, time on antibiotics, culture positive infection, and hospitalizations) were equivalent during the treatment period with each preparation. This study showed equivalent efficacy of native IVIG as compared with reduced and alkylated IVIG during maintenance therapy for hypogammaglobulinemia.

Timentin therapy for bone, joint, and deep soft tissue infections in children.

Timentin, a combination of clavulanic acid (0.1 g) and ticarcillin (3.0 g), has proved effective in vitro against bacterial pathogens that produce beta-lactamases. The usual etiologic bacteria of osteochondritis of the foot (Pseudomonas species) and osteomyelitis/septic arthritis (Staphylococcus aureus) are commonly resistant to penicillins. To date, we have used Timentin to treat 30 children with bone, joint, and deep soft tissue infections. Timentin was administered intravenously at an average dosage of 207 mg/kg per day for mild to moderate infection and 310 mg/kg per day for bone and joint infections with systemic signs (sepsis). The lower dose was used in 24 patients and the other six patients, who had signs of sepsis, received the higher dose. All patients received Timentin intravenously over 30 minutes every four to six hours for a minimum of five days (mean 6.6 +/- 2.6 days, range five to 14 days). The mean time to defervescence and/or reduction in clinical symptoms was 1.6 +/- 1.3 days (range zero to four days). Osteochondritis due to P. aeruginosa was diagnosed in six patients, and septic bursitis, osteomyelitis, or septic arthritis due to S. aureus (13 patients) or Staphylococcus species and group A streptococci (four patients) was diagnosed in 17 patients. All isolates were susceptible to Timentin in vitro by disk-diffusion analysis. All patients showed a response to therapy with Timentin, with or without surgical intervention. All patients had clinical and microbiologic cures; no adverse reactions or side effects were observed. There have been no clinical or microbiologic relapses to date. Timentin may prove to be useful in specific bone and joint infections in children.

Intravenous immune globulin for hypogammaglobulinemia: a comparison of opsonizing capacity in recipient sera.

Twelve severely hypogammaglobulinemic patients received infusions of alkylated immune globulin and two other native nonalkylated products. Administration was separated by an interval of 3 weeks. Serum was obtained prior to and at 24 hr and 3 weeks after each infusion for measurement of total IgG, specific and opsonizing antibodies. The latter was accomplished against Streptococcus pneumoniae types 5, 12F and 14 and zymosan using chemiluminescence methodology. Changes in total IgG concentrations were comparable for the three products. Prior to enrollment, IgG levels averaged 115 +/- 72 mg/dl, increasing to 779 +/- 399 at 24 hr postinfusion, and were 337 +/- 200 after 3 weeks. No differences among the products were seen in their ability to produce antibodies against Herpes simplex virus types 1 and 2, rubella, toxoplasma, cytomegalovirus, or tetanus. However, differences in opsonizing antibody were observed between alkylated and native IgG preparations. Peak chemiluminescence responses of neutrophils following opsonization of S. pneumoniae with native immune globulin were significantly higher than with alkylated IgG, indicating greater functional capacity. These studies suggest that native immune serum globulin provides a greater potential for augmenting host defense mechanisms against pneumococcal infection in hypogammaglobulinemic patients.

Bacterial meningitis in older neonates.

During a five-year period, 24 patients' conditions (age range, 2 to 6 weeks) were diagnosed, and they were treated for bacterial meningitis. Organisms recovered from the CSF included group B Streptococcus (n = 6), Escherichia coli (n = 5), Listeria monocytogenes (n = 5), Hemophilus influenzae (n = 4), Streptococcus pneumoniae (n = 2), and group D and group A Streptococcus (one each). Initial antimicrobial therapy must include antibiotics that are effective across this spectrum of potential pathogens. Symptoms and signs were often subtle. Six children (25%) experienced major neurologic residua, including five patients (21%) in whom hydrocephalus developed. Ultrasound examination of the head at the end of therapy was an effective technique for early assessment of neurologic sequelae.