Sharon R. Ladyman

From feeding one to feeding many: hormone‐induced changes in bodyweight homeostasis during pregnancy

Pregnancy is associated with hyperphagia, increased fat mass, hyperleptinaemia and hyperprolactinaemia. The neuroendocrine control of bodyweight involves appetite‐regulating centres in the hypothalamus, containing both orexigenic and anorexigenic neurons that express leptin receptors (LepR). In the rat, central leptin resistance develops during mid pregnancy, well after hyperphagia becomes apparent, to negate the appetite suppressing effects of leptin. We have investigated the hypothalamic response to leptin during pregnancy and examined the role of pregnancy hormones in inducing these changes. We have shown that there are multiple levels of leptin resistance during pregnancy. Despite elevated serum leptin, neuropeptide Y and agouti related peptide mRNA in the arcuate nucleus are not suppressed and may even be increased during pregnancy. LepR mRNA and leptin‐induced pSTAT3 expression, however, are relatively normal in the arcuate nucleus. In contrast, both LepR and leptin‐induced pSTAT3 are reduced in the ventromedial hypothalamic nucleus. Injecting α‐melanocyte‐stimulating hormone (α‐MSH) into the brain, to bypass the first‐order leptin‐responsive neurons in the arcuate nucleus, also fails to suppress food intake during pregnancy, suggesting that pregnancy is also a melanocortin‐resistant state. Using a pseudopregnant rat model, we have demonstrated that in addition to the changes in maternal ovarian steroid secretion, placental lactogen production is essential for the induction of leptin resistance in pregnancy. Thus, hormonal changes associated with pregnancy induce adaptive changes in the maternal hypothalamus, stimulating food intake and then allowing elevated food intake to be maintained in the face of elevated leptin levels, resulting in fat deposition to provide energy stores in preparation for the high metabolic demands of late pregnancy 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 α‐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.

Hormonal induction of leptin resistance during pregnancy.

Despite elevated plasma leptin, food intake is increased during pregnancy leading to fat deposition. We have demonstrated that intracerebroventricular (icv) leptin is unable to suppress food intake in pregnant rats, as it does in non-pregnant animals. Hence, central leptin resistance develops during pregnancy. These changes are physiologically appropriate, providing increased energy reserves to help meet the high metabolic demands of fetal development and lactation. To characterise this central leptin resistance, we have measured levels of leptin receptor (Ob-Rb) mRNA in the hypothalamus, and examined leptin-induced phosphorylation of STAT3 (pSTAT3) in specific regions of the hypothalamus. In addition, to investigate the mechanism underlying pregnancy-induced leptin resistance, we have investigated effects of hormone treatments on hypothalamic responses to leptin in a pseudopregnant rat model. We observed a significant reduction of Ob-Rb mRNA levels in the ventromedial hypothalamic nucleus (VMH) during pregnancy, with no changes detected in other hypothalamic nuclei. Levels of leptin-induced pSTAT3 were specifically suppressed in the VMH and arcuate nucleus of pregnant rats compared to non-pregnant rats. Pseudopregnant rats were hyperphagic but did not become leptin resistant, suggesting that fetal or placental factors are required for the induction of leptin resistance. These data implicate the VMH as a key hypothalamic site involved in hormone-induced leptin resistance during pregnancy, and suggest that placental hormone secretion may mediate the hormone-induced loss of response to leptin.

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-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.

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.

Prolactin transport into mouse brain is independent of prolactin receptor

The anterior pituitary hormone prolactin exerts important physiologic actions in the brain. However, the mechanism by which prolactin crosses the blood‐brain barrier and enters the brain is not completely understood. On the basis of high expression of the prolactin receptor in the choroid plexus, it has been hypothesized that the receptor may bind to prolactin in the blood and translocate it into the cerebrospinal fluid (CSF). This study aimed to test this hypothesis by investigating transport of 125I‐labeled prolactin (125I‐prolactin) into the brain of female mice in the presence and absence of the prolactin receptor (PRLR–/–). Peripherally administered prolactin rapidly activates brain neurons, as evidenced by prolactin‐induced phosphorylation of signal transducer and activator of transcription 5 (pSTAT5) in neurons within 30 min of administration. The transport of prolactin into the brain was saturable, with transport effectively blocked only by a very high dose of unlabeled ovine prolactin. Transport was regulated, as in lactating mice with chronically elevated levels of prolactin, the rate of 125I‐prolactin transport into the brain was significantly increased compared to non‐lactating controls. There was no change in the rate of 125I‐prolactin transport into the brain in PRLR–/– mice lacking functional prolactin receptors compared to control mice, indicating transport is independent of the prolactin receptor. These data suggest that prolactin transport into the brain involves another as yet unidentified transporter molecule. Because CSF levels of 125I‐prolactin were very low, even up to 90 min after administration, the data suggest that CSF is not the major route by which blood prolactin gains access to neurons in the brain.—Brown, R. S. E., Wyatt, A. K., Herbison, R. E., Knowles, P. J., Ladyman, S. R., Binart, N., Banks, W. A., Grattan, D. R. Prolactin transport into mouse brain is independent of prolactin receptor. FASEB J. 30, 1002–1010 (2016). www.fasebj.org

JAK-STAT and feeding

The regulation of energy balance requires a complex system to homeostatically maintain the adult body at a precise set point. The central nervous system, particularly the hypothalamus, plays a key role in integrating a variety of signals that can relay information about the body’s energy stores. As part of this system, numerous cytokines and hormones contribute to the regulation of food intake and energy homeostasis. Cytokines, and some hormones, are known to act through JAK-STAT intracellular signaling pathways. The hormone leptin, which plays a vital role in appetite regulation, signals through the JAK-STAT pathway, and it is through this involvement that the JAK-STAT pathway has become an established component in the mechanisms regulating food intake within the body. Emerging research, however, is now showing that this involvement of JAK-STAT is not limited to its activation by leptin. Furthermore, while the JAK-STAT pathway may simply act to transmit the anorectic signal of circulating factors, this intracellular signaling pathway may also become impaired when normal regulation of energy balance is disrupted. Thus, altered JAK-STAT signaling may contribute to the breakdown of the normal homeostatic mechanisms maintaining body weight in obesity.

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.