Rachael A. Augustine

Leptin Deficiency and Diet-Induced Obesity Reduce Hypothalamic Kisspeptin Expression in Mice

The hormone leptin modulates a diverse range of biological functions, including energy homeostasis and reproduction. Leptin promotes GnRH function via an indirect action on forebrain neurons. We tested whether leptin deficiency or leptin resistance due to a high-fat diet (HFD) can regulate the potent reproductive neuropeptide kisspeptin. In mice with normalized levels of estradiol, leptin deficiency markedly reduced kisspeptin gene expression, particularly in the arcuate nucleus (ARC), and kisspeptin immunoreactive cell numbers in the rostral periventricular region of the third ventricle (RP3V). The HFD model was used to determine the effects of diet-induced obesity and central leptin resistance on kisspeptin cell number and gene expression. DBA/2J mice, which are prone to HFD-induced infertility, showed a marked decrease in kisspeptin expression in both the RP3V and ARC and cell numbers in the RP3V after HFD. This is the first evidence that kisspeptin can be regulated by HFD and/or increased body weight. Next we demonstrated that leptin does not signal (via signal transducer and activator of transcription 3 or 5, or mammalian target of rapamycin) directly on kisspeptin-expressing neurons in the RP3V. Lastly, in leptin receptor-deficient mice, neither GnRH nor kisspeptin neurons were activated during a preovulatory-like GnRH/LH surge induction regime, indicating that leptins actions on GnRH may be upstream of kisspeptin neurons. These data provide evidence that leptins effects on reproductive function are regulated by kisspeptin neurons in both the ARC and RP3V, although in the latter site the effects are likely to be indirect.

RFamide-Related Peptide-3 Receptor Gene Expression in GnRH and Kisspeptin Neurons and GnRH-Dependent Mechanism of Action

RFamide-related peptide-3 (RFRP-3) is known to inhibit the activity of GnRH neurons. It is not yet clear whether its G protein-coupled receptors, GPR147 and GPR74, are present on GnRH neurons or on afferent inputs of the GnRH neuronal network or whether RFRP-3 can inhibit gonadotropin secretion independently of GnRH. We tested the following: 1) whether GnRH is essential for the effects of RFRP-3 on LH secretion; 2) whether RFRP-3 neurons project to GnRH and rostral periventricular kisspeptin neurons in mice, and 3) whether Gpr147 and Gpr74 are expressed by these neurons. Intravenous treatment with the GPR147 antagonist RF9 increased plasma LH concentration in castrated male rats but was unable to do so in the presence of the GnRH antagonist cetrorelix. Dual-label immunohistochemistry revealed that approximately 26% of GnRH neurons from male and diestrous female mice were apposed by RFRP-3 fibers, and 19% of kisspeptin neurons from proestrous female mice were apposed by RFRP-3 fibers. Using immunomagnetic purification of GnRH and kisspeptin cells, single-cell nested RT-PCR, and in situ hybridization, we showed that 33% of GnRH neurons and 9-16% of rostral periventricular kisspeptin neurons expressed Gpr147, whereas Gpr74 was not expressed in either population. These data reveal that RFRP-3 can act at two levels of the GnRH neuronal network (i.e. the GnRH neurons and the rostral periventricular kisspeptin neurons) to modulate reproduction but is unable to inhibit gonadotropin secretion independently of GnRH.

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.

Leptin Rapidly Improves Glucose Homeostasis in Obese Mice by Increasing Hypothalamic Insulin Sensitivity

Obesity is associated with resistance to the actions of both leptin and insulin via mechanisms that remain incompletely understood. To investigate whether leptin resistance per se contributes to insulin resistance and impaired glucose homeostasis, we investigated the effect of acute leptin administration on glucose homeostasis in normal as well as leptin- or leptin receptor-deficient mice. In hyperglycemic, leptin-deficient Lepob/ob mice, leptin acutely and potently improved glucose metabolism, before any change of body fat mass, via a mechanism involving the p110α and β isoforms of phosphatidylinositol-3-kinase (PI3K). Unlike insulin, however, the anti-diabetic effect of leptin occurred independently of phospho-AKT, a major downstream target of PI3K, and instead involved enhanced sensitivity of the hypothalamus to insulin action upstream of PI3K, through modulation of IRS1 (insulin receptor substrate 1) phosphorylation. These data suggest that leptin resistance, as occurs in obesity, reduces the hypothalamic response to insulin and thereby impairs peripheral glucose homeostasis, contributing to the development of type 2 diabetes.

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.

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.

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.

Both p110α and p110β 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 p110α and p110β. Total hypothalamic PI3K activity was inhibited 65% by a p110α inhibitor, and 35% by a p110β inhibitor, with a combination of inhibitors being equally effective as the broad‐spectrum PI3K inhibitor wortmannin. Individual i.c.v. administration of p110α and p110β 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 p110α and p110β 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 p110α and p110β inhibitors. The data suggest that p110α and p110β 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.

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

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.