Pregnancy-induced adaptation of central sensitivity to leptin and insulin.

Pregnancy is a time of increased food intake and fat deposition in the mother, and adaptations of glucose homeostasis to meet the energy demands of the growing fetus. As part of these adaptations, leptin and insulin concentrations increase in the maternal circulation during pregnancy. Central effects of leptin and insulin, however, are counterproductive to pregnancy, as increased action of these hormones in the brain lead to suppression of food intake. To prevent this, it is well documented that pregnancy induces a state of leptin- and insulin-insensitivity in the brain, particularly the hypothalamus, in a range of species. While the mechanisms underlying leptin- or insulin-insensitivity during pregnancy vary between species, there is evidence of reduced transport into the brain, impaired activation of intracellular signalling pathways, including reduced leptin receptor expression, and attenuated activation of downstream neuronal pathways, especially for leptin insensitivity. Pregnancy-induced changes in prolactin, growth hormone and leptin are discussed in terms of their role in mediating this reduced response to leptin and insulin.

Energy homeostasis and running wheel activity during pregnancy in the mouse.

Pregnancy and lactation are metabolically challenging states, where the mother must supply all the energy requirements for the developing fetus and growing pups respectively. The aim of the current study was to characterize many aspects of energy homeostasis before and during pregnancy in the mouse, and to examine the role of voluntary activity on changes in energy expenditure during pregnancy. In a secondary aim, we evaluate measures of energy homeostasis during pregnancy in mice that successfully reared their litter or in mice that went on to abandon their litter, to determine if an impairment in pregnancy-induced adaptation of energy homeostasis might underlie the abandonment of pups soon after birth. During pregnancy, food intake was increased, characterized by increased meal size and duration but not number of meals per day. The duration of time spent inactive, predicted to indicate sleep behaviour, was increased both early and late in pregnancy compared to pre-pregnancy levels. Increased x + y beam breaks, as a measure of activity increased during pregnancy and this reflected an increase in ambulatory behaviour in mid pregnancy and an increase in non-ambulatory movement in late pregnancy. Energy expenditure, as measured by indirect calorimetry, increased across pregnancy, likely due to the growth and development of fetal tissue. There was also a dramatic reduction in voluntary wheel running as soon as the mice became pregnant. Compared with successful pregnancies and lactations, pregnancies where pups were abandoned soon after birth were associated with reduced body weight gain and an increase in running wheel activity at the end of pregnancy, but no difference in food intake or energy expenditure. Overall, during pregnancy there are multiple adaptations to change energy homeostasis, resulting in partitioning of provisions of energy to the developing fetus and storing energy for future metabolic demands.

Prolactin receptors in Rip-cre cells, but not in AgRP neurones, are involved in energy homeostasis.

Among its many functions, prolactin has been implicated in energy homeostasis, particularly during pregnancy and lactation. The arcuate nucleus is a key site in the regulation of energy balance. The present study aimed to examine whether arcuate nucleus neuronal populations involved in energy homeostasis are prolactin responsive and whether they can mediate the effects of prolactin on energy homeostasis. To determine whether Agrp neurones or Rip-Cre neurones are prolactin responsive, transgenic mice expressing the reporter td-tomato in Agrp neurones (td-tomato/Agrp-Cre) or Rip-Cre neurones (td-tomato/Rip-Cre) were treated with prolactin and perfused 45 minutes later. Brains were processed for double-labelled immunohistochemistry for pSTAT5, a marker of prolactin-induced intracellular signalling, and td-tomato. In addition, Agrp-Cre mice and Rip-Cre mice were crossed with mice in which the prolactin receptor gene (Prlr) was flanked with LoxP sites (Prlrlox/lox mice). The Prlrlox/lox construct was designed such that Cre-mediated recombination resulted in deletion of the Prlr and expression of green fluorescent protein (GFP) in its place. In td-tomato/Rip-Cre mice, prolactin-induced pSTAT5 was co-localised with td-tomato, indicating that there is a subpopulation of Rip-Cre neurones in the arcuate nucleus that respond to prolactin. Furthermore, mice with a specific deletion of Prlr in Rip-Cre neurones had lower body weights, increased oxygen consumption, increased running wheel activity and numerous cells in the arcuate nucleus had positive GFP staining indicating deletion of Prlr from Rip-Cre neurones. By contrast, no co-localisation of td-tomato and pSTAT5 was observed in td-tomato/Agrp-Cre mice after prolactin treatment. Moreover, Prlrlox/lox /Agrp-Cre mice had no positive GFP staining in the arcuate nucleus and did not differ in body weight compared to littermate controls. Overall, these results indicate that Rip-Cre neurones in the arcuate nucleus are responsive to prolactin and may play a role in the orexigenic effects of prolactin, whereas prolactin does not directly affect Agrp neurones.

Central Effects of Leptin on Glucose Homeostasis are Modified during Pregnancy in the Rat.

Despite increased leptin concentrations during pregnancy, fat mass and food intake are increased. The satiety response to central leptin is suppressed, indicating a state of leptin insensitivity in the hypothalamus. Although the regulation of food intake is a major function of leptin, this hormone also influences a wide range of functions within the body. These actions include the regulation of glucose homeostasis, which undergoes major adaptation in the maternal body to generate optimal conditions for foetal development and growth. The present study aimed to investigate the effects of central leptin treatment on glucose homeostasis in pregnant rats to determine whether pregnancy-induced leptin insensitivity is functionally specific, and to further investigate changes in glucose homeostasis during pregnancy. After an overnight fast, nonpregnant and day 14 pregnant rats received an i.c.v. injection of leptin (100 ng or 4 µg) or vehicle then underwent a glucose tolerance test (GTT). Further groups of nonpregnant and day 14 pregnant rats were killed 30 min after leptin (doses ranging from 40 ng-4 µg) or vehicle i.c.v. injections for western blot analysis of phospho-signal transducer and activator of transcription 3 (STAT3) and phospho-Akt in various hypothalamic nuclei. Central leptin injection prior to a GTT lead to lowered basal insulin concentrations and impaired glucose tolerance in nonpregnant female rats, whereas the same doses of leptin had no significant effect on glucose tolerance in day 14 pregnant rats, indicating that, similar to the satiety actions of leptin, the effects of leptin on glucose homeostasis are suppressed during pregnancy. Furthermore, in the arcuate nucleus and ventromedial and dorsomedial nuclei of the hypothalamus, comprising three leptin-sensitive areas, there was no evidence that leptin induced Akt phosphorylation despite significant increases in phospho-STAT3, suggesting that leptin does not act through phospho-Akt in these areas in female rats.

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.

Suppression of leptin-induced hypothalamic JAK/STAT signalling and feeding response during pregnancy in the mouse.

Hyperphagia during pregnancy, despite rising concentrations of the satiety hormone leptin, suggests that a state of leptin resistance develops. This study investigated the satiety response and hypothalamic responses to leptin during pregnancy in the mouse. Pregnant (day 13) and nonpregnant mice received an i.p. injection of either leptin or vehicle and then 24-h food intake was measured. Further groups of pregnant and nonpregnant mice were perfused 2 h after leptin or vehicle injections and brains were processed for pSTAT3 and pSTAT5 immunohistochemistry. Leptin treatment significantly decreased food intake in nonpregnant mice. In pregnant mice, however, leptin treatment did not suppress food intake, indicating a state of leptin resistance. In the arcuate nucleus, leptin treatment increased the number of cells positive for pSTAT3, a marker of leptin activity, to a similar degree in both nonpregnant and pregnant mice. In the ventromedial nucleus (VMN), the leptin-induced increase in pSTAT3-positive cell number was significantly reduced in pregnant mice compared to that in nonpregnant mice. In nonpregnant mice, leptin treatment had no effect on the number of pSTAT5-positive cells, suggesting that in this animal model, leptin does not act through STAT5. In pregnant mice, basal levels of pSTAT5 were higher than in nonpregnant mice, and leptin treatment led to a decrease in the number of pSTAT5-positive cells in the hypothalamus. Overall, these results demonstrate that during pregnancy in the mouse, a state of leptin resistance develops, and this is associated with a reduced sensitivity of the VMN to leptin.

Loss of acute satiety response to cholecystokinin in pregnant rats.

During pregnancy, food intake and fat mass are increased to meet the energy demands of the growing conceptus and to prepare for the subsequent demands of lactation. A state of leptin resistance develops during pregnancy in the rat, which can facilitate the increase in food intake despite pregnancy-induced increases in leptin concentrations. Cholecystokinin (CCK) is a satiety factor that is released from the gut during feeding and acts to terminate short-term food intake. Circulating leptin concentrations can modulate the anorexic response to CCK; low leptin concentrations decrease the potency of CCK to reduce food intake. Because rats are leptin resistant by day 14 of pregnancy, it was hypothesised that the feeding response to CCK would be attenuated at that time. Nonpregnant and day 14 pregnant rats received an i.p. injection of CCK-8 (3 µg/kg body weight) or vehicle directly before the start of the dark phase. Food intake was measured 30 min after lights out. Approximately 90 min after receiving either CCK-8 or vehicle, rats were transcardially perfused with 4% paraformaldehyde. Food intake was significantly decreased in CCK-treated nonpregnant rats, although similar treatment did not reduce food intake in day 14 pregnant rats. CCK treatment lead to significant increased in c-Fos expression in the nucleus of the solitary tract (NTS) in both nonpregnant and pregnant rats compared to vehicle treatment, although the number of CCK-induced c-Fos positive cells was significantly less in pregnant rat compared to nonpregnant rats. Although CCK treatment increased the number of c-Fos positive cells in the hypothalamic paraventricular nucleus and supraoptic nucleus in nonpregnant rats, no significant increase was observed in these areas during pregnancy. These results indicate that pregnant rats are no longer responsive to the actions of CCK on short-term food intake and that CCK action in the NTS is reduced during pregnancy.

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.

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.

Leptin resistance during pregnancy in the rat.

The adipose-derived hormone leptin primarily acts in the hypothalamus to decrease appetite and increase energy expenditure, thereby maintaining body fat levels around a set point. Pregnancy is a physiological state where this feedback mechanism is not beneficial. Successful reproductive efforts are highly demanding on the resources of the mother; thus, it is imperative that the maternal body can increase energy stores without restraint. Food intake, fat mass and serum leptin concentrations increase during pregnancy in the rat, suggesting that the feedback loop between adipose tissue and appetite is disrupted and a state of leptin resistance exists. In support of this, there is an attenuation of the satiety response to exogenous leptin administration in pregnant rats. This state of leptin resistance is associated with impaired activation of the leptin-induced Janus activating kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathway in the ventromedial nucleus of the hypothalamus (VMH) and arcuate nucleus, and reduced expression of leptin receptor mRNA in the VMH. Furthermore, pregnant rats do not show a satiety response to exogenous alpha-melanocyte stimulating hormone. This model offers the possibility of examining how hypothalamic leptin signalling can be modified in response to changes in physiological conditions.

Suppression of leptin receptor messenger ribonucleic acid and leptin responsiveness in the ventromedial nucleus of the hypothalamus during pregnancy in the rat.

Pregnancy in the rat is a state of leptin resistance associated with impaired leptin signal transduction in the hypothalamus. The aim of this study was to determine whether this leptin-resistant state is mediated by a change in the level of leptin receptors in the hypothalamus. Real-time RT-PCR was used to determine levels of mRNA for the various leptin receptor isoforms in a number of microdissected hypothalamic nuclei and the choroid plexus. To investigate the functional activation of the leptin receptor, immunohistochemistry for phosphorylated signal transducer and activator of transcription 3 (pSTAT3) was examined in the arcuate nucleus and the ventromedial nucleus of the hypothalamus (VMH) of fasted diestrous and d-14 pregnant rats after an intracerebroventricular (i.c.v.) injection of either leptin (4 mug) or vehicle. A significant reduction of Ob-Rb mRNA levels was observed in the VMH during pregnancy compared with the nonpregnant controls. Furthermore, in pregnant rats the number of cells positive for leptin-induced pSTAT3 in the VMH was greatly reduced during pregnancy compared with nonpregnant rats. There were no differences in the level of Ob-Rb mRNA or in the number of leptin-induced pSTAT3-positive cells in the arcuate nucleus of nonpregnant and pregnant rats. These data implicate the VMH as a key hypothalamic site involved in pregnancy-induced leptin resistance. There were also reduced levels of mRNA for Ob-Ra, a proposed leptin transporter molecule, in the choroid plexus on d 7 and 21 of pregnancy. Hence, diminished transport of leptin into the brain may also contribute to pregnancy-induced leptin resistance.

Region-specific reduction in leptin-induced phosphorylation of signal transducer and activator of transcription-3 (STAT3) in the rat hypothalamus is associated with leptin resistance during pregnancy.

Leptin concentrations increase during pregnancy, but this does not prevent the pregnancy-induced increase in food intake, suggesting a state of leptin resistance. This study investigated the response to intracerebroventricular leptin administration in pregnant rats. After fasting, nonpregnant, d-7 and d-14 pregnant rats received leptin (4 microg) or vehicle, then food intake was measured. Serial blood samples were collected in another group of rats to determine plasma leptin concentrations. Further groups of d-14 pregnant and nonpregnant rats were killed after leptin or vehicle treatment, and brains were collected. Hypothalamic nuclei were microdissected, and levels of signal transducer and activator of transcription (STAT)3 phosphorylation were measured using Western blot analysis. Fasting decreased leptin concentrations in both pregnant and nonpregnant rats. Leptin treatment significantly reduced food intake in nonpregnant and d-7 pregnant rats but not in d-14 pregnant rats. In addition, there was no postfasting hyperphagic response in the pregnant rats. In the pregnant rats, leptin-induced STAT3 phosphorylation was suppressed in the arcuate nucleus and, to a lesser extent, in the ventromedial hypothalamus (VMH), compared with nonpregnant rats. Unstimulated STAT3 levels were also decreased in the VMH during pregnancy. Leptin-induced phosphorylation of STAT3 in the dorsomedial and lateral hypothalamus was not different between pregnant and nonpregnant rats. These data indicate that pregnant rats become resistant to the satiety action of leptin. Furthermore, leptin-induced activation of the STAT3 is impaired during pregnancy, specifically in the arcuate nucleus and VMH. These data support the hypothesis that pregnancy is a state of hypothalamic leptin resistance.

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.

Suppression of pulsatile luteinizing hormone secretion but not luteinizing hormone surge in leptin resistant obese Zucker rats.

The adipose tissue-derived hormone leptin may be a primary mediator linking nutritional status and reproduction. The present study used the leptin-resistant obese female Zucker rat to investigate whether leptin signalling is required for normal pulsatile luteinizing hormone (LH) secretion and/or generation of the LH surge. For the pulsatile LH secretion study, an indwelling atrial catheter was implanted and a low dose of oestrogen given as a subcutaneous implant to lean and obese ovariectomized (OVX) Zucker rats. One week following OVX, blood samples were collected every 10 min for 3 h during the morning. Plasma LH concentrations were measured by radioimmunoassay. For the LH surge study, lean and obese OVX rats were given a high dose of oestrogen as a subcutaneous implant. Two days later, rats were given progesterone at 09.00 h to induce a proestrus-like LH surge. Blood samples were collected from an indwelling atrial catheter throughout that and the following day and plasma LH concentrations were measured by radioimmunoassay. LH pulse amplitude and mean LH secretion were profoundly attenuated in obese Zucker rats compared with lean littermates, whereas LH pulse frequency was not significantly different between phenotypes. The opioid receptor antagonist naloxone did not affect the pattern of pulsatile LH secretion in obese rats, suggesting that leptin does not exert its facilitatory effects on LH secretion through an opioidergic pathway. Both lean and obese rats showed characteristic steroid-induced LH surges. It therefore appears that a leptin signal is required for generation of a normal pattern of pulsatile LH secretion, but is not a necessary component of the steroid-induced LH surge.

Gastroenteritis of infants in two Canadian communities.

Enteroviruses were isolated from 11 of 1391 rectal swabs collected from 1103 infants aged less than 2 years who were hospitalized in Toronto, Ontario, and New Westminster, British Columbia, between April 1966 and March 1969. Viruses were recovered from 23 of 1231 rectal swabs obtained from 1076 age-matched control patients without gastroenteritis. The dominance of coxsackievirus and echovirus strains in Toronto patients with and without gastroenteritis contrasted sharply to their extremely low incidence in New Westminster where the Sabin attenuated strains of poliovirus composed the majority of isolates from both categories of patient. Although enteropathogenic bacteria were identified in 36 cases of gastroenteritis and 11 control subjects, no patient excreted a bacterial pathogen and a virus simultaneously.