Leptin regulates neuropeptides associated with food intake and GnRH secretion

The present review focused on the most important effects of leptin on the hypothalamus and on how leptin regulates neuropeptides associated with food intake and GnRH secretion. This review of the literature suggests that a reduction in leptin serum concentrations results from lower body energy reserves or poor energy availability, leading to hypothalamic secretion of neuropeptides such as NPY/AgRP and QRFP to stimulate food intake. Under these negative metabolic conditions, GnRH secretion is reduced, impairing reproductive functions. In contrast, when metabolic status is inversed by an increase in food availability, energy reserves or both, leptin serum concentrations increase to an action threshold reversing the pattern of secretion: i.e., reducing NPY/AgRP and QRFP and increasing POMC and Kisspeptin, and thereby reducing food intake and stimulating GnRH secretion to promote reproductive function.     

Regulation of hypothalamic expression of KiSS-1 and GPR54 genes by metabolic factors: analyses using mouse models and a cell line

It is well established that reproductive function is metabolically gated. However, the mechanisms whereby energy stores and metabolic cues influence fertility are yet to be completely deciphered. Recently, the hypothalamic KiSS-1/GPR54 system has emerged as a fundamental regulator of the gonadotropic axis, which conveys the modulatory actions of sex steroids to GnRH neurons. Evidence is also mounting that KiSS-1 neurons may also represent the link between systemic metabolic signals and central control of reproduction. To further explore this possibility, we examined the impact of changes in energy status and key metabolic regulators on the hypothalamic expression of KiSS-1 and GPR54 genes, using different mouse models and the hypothalamic cell line N6. Time-course analysis of the effects of short-term fasting revealed a rapid (12- and 24-h) decline in KiSS-1 and GPR54 mRNA levels, which preceded that of GnRH (48 h). In contrast, diet-induced obesity or obesity associated with leptin deficiency (ob/ob vs. wild-type mice) failed to induce overt changes in hypothalamic expression of KiSS-1 and GPR54 genes. However, leptin infusion of ob/ob mice evoked a significant increase in KiSS-1 and GPR54 mRNA levels compared with pair-fed controls. Moreover, leptin, but not insulin or IGF-I, stimulated KiSS-1 mRNA expression in the mouse hypothalamic cell line N6. In addition, neuropeptide Y (NPY) null mice showed decreased KiSS-1 mRNA levels at the hypothalamus, whereas exposure to NPY increased expression of KiSS-1 in hypothalamic N6 cells. In sum, our present data further characterize the functional relevance and putative key mediators (such as leptin and NPY) of the metabolic regulation of the hypothalamic KiSS-1 system in the mouse.     

Molecular mapping of the neural pathways linking leptin to the neuroendocrine reproductive axis

Negative energy balance and insufficient adipose energy stores decrease the production of leptin, thereby diminishing the leptin-supported secretion of GnRH from the hypothalamus and promoting decreased reproductive function. Leptin acts via its receptor (LepRb) to support the neuroendocrine reproductive axis, but the nature and location of the relevant LepRb neurons remain poorly understood. Possibilities include the direct or indirect action of leptin on hypothalamic GnRH neurons, or on kisspeptin (Kiss1) neurons that are major regulators of GnRH neurons. To evaluate these potential mechanisms, we employed immunohistochemical analysis of the female brain from various molecular mouse models and sheep. Our analysis revealed no LepRb in GnRH neurons or in anteroventral periventricular Kiss1 neurons, and very limited (0-6%) colocalization with arcuate nucleus Kiss1 cells, suggesting that leptin does not modulate reproduction by direct action on any of these neural populations. LepRb neurons, primarily in the hypothalamic ventral premammillary nucleus and a subregion of the preoptic area, lie in close contact with GnRH neurons, however. Furthermore, an unidentified population or populations of LepRb neurons lie in close contact with arcuate nucleus and anteroventral periventricular Kiss1 neurons. Taken together, these findings suggest that leptin communicates with the neuroendocrine reproductive axis via multiple populations of LepRb neurons that lie afferent to both Kiss1 and GnRH neurons.     

Expression of a leptin receptor in immortalized gonadotropin-releasing hormone-secreting neurons

Leptin is secreted by adipocytes and regulates food intake and energy balance through the activation of specific receptors (OB-R). Recent evidence suggests that it is also involved in the control of reproductive processes, by possibly acting on central and peripheral targets. In particular, it has been shown that leptin may indirectly stimulate GnRH release from hypothalamic fragments by acting on interneurons impinging on GnRH-secreting neurons. The possibility that leptin might additionally modulate the activity of GnRH-secreting neurons in a direct way has been addressed in the present study, by using the immortalized GnRH-secreting cell line GT1-7. The presence of OB-R messenger RNA (mRNA) (long form) was detected by RT-PCR analysis of total RNA from GT1-7 cells. An OB-R protein is also expressed in these cells, as shown by immunocytochemistry and by Western blot analysis. The latter has revealed the presence of a single immunoreactive OB-R with an approximate size of 130 kDa. To study the functionality of these receptors, the effect of leptin treatment on GnRH secretion and gene expression in GT1-7 cells were evaluated. Under static conditions, GnRH release was stimulated by exposure to low concentrations of leptin (10(-12) M after 30 min; 10(-10) M after 60 min). The 10(-12) M dose was selected for studying the effect of leptin on GnRH secretion under dynamic conditions. To this purpose, GT1-7 cells were placed in a perifusion system; treatment with leptin (10(-12) M) for 60 min stimulated GnRH release with no changes of pulse frequency. On the contrary, exposure to leptin (10(-12)-10(-10) M) for 1, 3, 6, and 24 h did not affect GnRH gene expression in GT1-7 cells. The present results indicate that GT1-7 cells possess OB-Rs and that leptin may directly affect their function. Taken together with the available reports, these findings suggest that leptin might participate in the regulation of reproductive processes by acting at multiple levels, both centrally and peripherally.     

Galanin-like peptide stimulates the release of gonadotropin-releasing hormone in vitro and may mediate the effects of leptin on the hypothalamo-pituitary-gonadal axis

Leptin regulates the hypothalamo-pituitary-gonadal axis in relation to nutritional status. The mechanism through which leptin mediates its effects on neuroendocrine reproductive circuits remains unclear. Galanin-like peptide (GALP) is a recently identified hypothalamic peptide, localized in the arcuate nucleus, which seems to be regulated by leptin and stimulates LH when administered centrally. Here, we demonstrate that leptin stimulates the release of GALP and GnRH in vitro from hypothalamic explants harvested from male rats. In addition, we show that GALP stimulates the release of GnRH from hypothalamic explants and GT1-7 cells. Furthermore, we demonstrate that GALP antiserum blocks the stimulatory action of leptin on GnRH release from hypothalamic explants. GALP is a ligand of the galanin receptors. We therefore investigated whether the effect of GALP on GnRH release may be mediated via a known galanin receptor. GALP-stimulated GnRH release from hypothalamic explants was attenuated (but not abolished) by the galanin receptor antagonist galantide. However, GALP-stimulated GnRH release from GT1-7 cells was not diminished by the coadministration of galantide. In addition, none of the cloned galanin receptors were expressed in GT1-7 cells by RT-PCR. These observations suggest that GALP may stimulate GnRH release through an indirect pathway involving a galanin receptor and via a direct action on GnRH neurons, possibly through a novel receptor. These findings suggest that GALP may mediate the actions of leptin on the reproductive axis and provide a link between nutrition and fertility.     

Adipose tissue and reproductive health

The understanding of adipose tissue role has evolved from that of a depot energy storage organ to a dynamic endocrine organ. While genetics, sexual phenotype and sex steroids can impact the mass and distribution of adipose tissue, there is a counter-influence of white adipocytes on reproduction. This primarily occurs via the secretion of adipokines, the most studied of which- leptin and adiponectin- are highlighted in this article. Leptin, the “satiety hormone” primarily acts on the hypothalamus via pro-opiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AgRP) neurons to translate acute changes in nutrition and energy expenditure, as well as chronic adipose accumulation into changes in appetite and potentially mediate insulin resistance via shared pathway and notably impacting reproductive health via influence on GnRH secreting neurons. Meanwhile, adiponectin is notable for its action in mediating insulin sensitivity, with receptors found at every level of the reproductive axis. Both have been examined in the context of physiologic and pathologic reproductive conditions. Leptin has been shown to influence puberty, pregnancy, hypothalamic amenorrhea, and lipodystrophy, and with a potential therapeutic role for both metabolic and reproductive health. Adiponectin mediates the relative state of insulin resistance in pregnancy, and has been implicated in conditions such as polycystic ovary syndrome and reproductive malignancies. There are numerous other adipokines, including resistin, visfatin, chemerin and retinol binding protein-4, which may also play roles in reproductive health and disease states. The continued examination of these and other adipokines in both normal reproduction and reproductive pathologies represents an important avenue for continued study. Here, we seek to provide a broad, yet comprehensive overview of many facets of these relationships and highlight areas of consideration for clinicians and future study.     

Decreased gonadotropin-releasing hormone neuronal activity is associated with decreased fertility and dysregulation of food intake in the female GPR-4 transgenic rat

Expression of a cAMP-specific phosphodiesterase in GnRH neurons in the GPR-4 transgenic rat resulted in decreased LH levels and pulse frequency and diminished fertility. We have characterized changes in fertility, adiposity, and reproductive and metabolic hormones with age. Although LH levels were decreased in 3-, 6-, and 9-month-old GPR-4 females relative to wild-type (WT) controls, GPR-4 females did not become anovulatory until 6 months of age. No differences were observed in FSH, estradiol, or androstenedione levels in 3-, 6-, or 9-month-old GPR-4 and WT females. At 9 months of age, GPR-4 females had significantly increased abdominal and sc fat depot weights that were associated with increased leptin and insulin levels not observed in WT females. We tested the hypothesis that metabolic changes observed at 9 months of age were the result of dysregulation of the mechanisms controlling energy balance. Two-month-old female GPR-4 rats placed on a high-energy diet gained weight at a rate significantly greater than WT females and, after 24 d, developed the same metabolic phenotype observed in 9-month-old GRP-4 females (increased abdominal and sc fat associated with elevated leptin and insulin concentrations). Overeating did not correlate with changes in estradiol or androstenedione levels. We conclude that decreased GnRH neuronal activity is closely associated with decreased reproductive function and dysregulation of food intake.     

Insulin regulation of GnRH gene expression through MAP kinase signaling pathways

In mammals, reproduction is acutely regulated by metabolic status. Insulin is an important nutritional signal from the periphery that may regulate the reproductive axis. To determine whether insulin acts directly on the GnRH neuron, we performed studies in mouse-derived GnRH-expressing cell lines. Both insulin receptor protein and mRNA were detected in these cells. A saturation radioligand binding assay revealed high affinity, low capacity binding sites for insulin in GnRH neurons. Insulin also stimulated GnRH promoter activity in GnRH neurons. This effect was blocked by pretreatment with the MEK inhibitor, PD98059, indicating a role for MAP kinase signaling. In transient transfection studies, insulin treatment stimulated expression of a 1250 bp mouse GnRH gene promoter fragment four-fold when compared to promoter activity in untreated cells. In contrast, insulin did not stimulate activity of a 587 bp fragment of the mGnRH gene promoter, indicating that the promoter elements mediating insulin stimulation of the GnRH promoter are located between -1250 and -587 bp. Our studies suggest that insulin may regulate reproductive function by direct effects on the GnRH neurons and specifically by stimulating GnRH gene expression.     

Galanin-like peptide as a link in the integration of metabolism and reproduction.

The arcuate nucleus is a hypothalamic center that couples energetics and reproduction. Peptide-releasing neurons in the arcuate nucleus receive and process humoral signals from the periphery and relay this information to other nuclei in the hypothalamus and preoptic area. Galanin-like peptide (GALP) is expressed in the arcuate nucleus, and GALP-containing neurons are targets for the action of leptin. GALP-containing neurons are closely apposed to gonadotropin-releasing hormone (GnRH) neurons in the preoptic area, and CNS injections of GALP stimulate GnRH-mediated secretion of luteinizing hormone. These observations indicate that GALP is a molecular signal that couples circulating indices of metabolism to the neuroendocrine reproductive system and, thus, regulates reproductive activity as a function of the energy state. In this article, we describe the involvement of GALP in metabolism and reproduction, and in the coupling between these two processes.     

Gonadotropin-releasing hormone-expressing neurons immortalized conditionally are activated by insulin: implication of the mitogen-activated protein kinase pathway

Energy balance exerts a critical influence on reproduction via changes in the circulating levels of hormones such as insulin. This modulation of the neuroendocrine reproductive axis ultimately involves variations in the activity of hypothalamic neurons expressing GnRH. Here we studied the effects of insulin in primary hypothalamic cell cultures as well as a GnRH neuronal cell line that we generated by conditional immortalization of adult hypothalamic neurons. These cells, which represent the first successful conditional immortalization of GnRH neurons, retain many of their mature phenotypic characteristics. In addition, we show that they express the insulin receptor. Consistently, their stimulation with insulin activates both the phosphatidylinositol 3-kinase and the Erk1/2 MAPK signaling pathways and stimulates a rapid increase in the expression of c-fos, demonstrating their responsiveness to this hormone. Further work performed in parallel in immortalized GnRH-expressing cells and primary neuronal cultures containing non-GnRH-expressing neurons shows that insulin induces the expression of GnRH in both models. In primary cultures, inhibition of the Erk1/2 pathway abolishes the stimulation of GnRH expression by insulin, whereas blockade of the phosphatidylinositol 3-kinase pathway has no effect. In conclusion, these data strongly suggest that GnRH neurons are directly sensitive to insulin and implicate for the first time the MAPK Erk1/2 signaling pathway in the central effects of insulin on the neuroendocrine reproductive axis.     

Gonadotropin-releasing hormone neurons coexpress orexin 1 receptor immunoreactivity and receive direct contacts by orexin fibers

The orexins are produced in neurons of the lateral hypothalamic area and implicated in the regulation of both feeding and reproductive function. Orexins stimulate LH secretion in steroid-primed ovariectomized female rats and suppress LH secretion in nonprimed ovariectomized rats. The aim of the present study was to characterize the neuroanatomical pathway by which orexin might modulate LH secretion in the rat. Using double- and triple-label immunofluorescence coupled with confocal microscopy, we found that 75-85% of GnRH neurons were contacted by orexin fibers, and triple labeling with synaptophysin provided additional confirmation of close contacts. Furthermore, about 85% of GnRH neurons were colocalized with the orexin receptor 1 (OX-R1), and the OX-R1-expressing GnRH neurons were contacted by orexin terminals, providing the basis for a functional neuroanatomical pathway. GnRH nerve terminals in the median eminence, however, do not express OX-R1. An additional study investigated the coexpression of neuropeptide Y Y4-like receptors and orexin fibers in relation to GnRH neurons. There is evidence that Y4 receptor stimulation results in LH release, and studies from our laboratory show Y4-like immunoreactivity in the majority of orexin cell bodies in the lateral hypothalamic area and some orexin fibers scattered throughout the hypothalamus. The present study found that, although Y4-positive orexin fibers are in present in the area of GnRH neurons, they never come in close contact with GnRH neurons. Together, these data suggest that Y4 receptor modulation of LH release is likely to be indirect through orexin cell bodies and that orexin modulates GnRH neurons directly via OX-R1.     

Neuroendocrine control by kisspeptins: role in metabolic regulation of fertility

The neurohormonal control of reproduction involves a hierarchical network of central and peripheral signals in the hypothalamic-pituitary-gonadal (HPG) axis. Development and function of this neuroendocrine system is the result of a lifelong delicate balance between endogenous regulators and environmental cues, including nutritional and metabolic factors. Kisspeptins are the peptide products of KISS1, which operate via the G-protein-coupled receptor GPR54 (also known as Kiss1R). These peptides have emerged as essential upstream regulators of neurons secreting gonadotropin-releasing hormone (GnRH), the major hypothalamic node for the stimulatory control of the HPG axis. They are potent elicitors of gonadotropin secretion in various species and physiological settings. Moreover, Kiss1 neurons in the hypothalamus participate in crucial features of reproductive maturation and function, such as brain-level sex differentiation, puberty onset and the neuroendocrine regulation of gonadotropin secretion and ovulation. Cotransmitters of Kiss1 neurons, such as neurokinin B, with roles in controlling the HPG axis have been identified by genetic, neuroanatomical and physiological studies. In addition, a putative role has been proposed for Kiss1 neurons in transmitting metabolic information to GnRH neurons, although the precise mechanisms are as yet unclear. In this Review, we present the major reproductive features of kisspeptins, especially their interplay with neurokinin B and potential roles in the metabolic control of puberty and fertility, and suggest new avenues for research.     

Gamma-aminobutyric acid neurons integrate and rapidly transmit permissive and inhibitory metabolic cues to gonadotropin-releasing hormone neurons

Negative energy balance inhibits fertility by decreasing GnRH release; however, the mechanisms are not well understood. GnRH neurons can be excited by activation of gamma-aminobutyric acid (GABA)(A) receptors, and GABAergic neurons provide a major synaptic input. We hypothesized that permissive metabolic signals mediated by leptin and inhibitory signals conveyed by neuropeptide Y (NPY) and opiates rapidly alter GABA(A) receptor-mediated drive to GnRH neurons. In fed and fasted female mice, GABAergic postsynaptic currents (PSCs) were recorded from GnRH neurons before and after in vitro treatment with leptin, NPY, or met-enkephalin. Leptin increased PSC frequency in fed and fasted mice, indicating that it increased presynaptic activity. Leptin also increased PSC size. Inhibiting leptin receptor signaling pathways within GnRH neurons abolished the latter effect, indicating a direct action on these cells. In fed, but not fasted, mice, NPY and met-enkephalin decreased PSC frequency in an antagonist-reversible manner, but did not alter PSC size. NPY-1 receptor antagonists alone increased frequency in fed and fasted mice, as did opiate receptor blockade in fasted animals, suggesting that endogenous NPY and opiates modulate GABAergic drive to GnRH neurons. These data suggest that GABAergic afferents integrate metabolic signals for delivery to GnRH neurons. Decreased sensitivity to NPY and opiates in fasted mice indicate that these peptides send physiologically relevant signals regarding energy balance to GnRH neurons.     

Neuropeptide signaling in the integration of metabolism and reproduction

Fertility is gated by nutrition and the availability of stored energy reserves, but the cellular and molecular mechanisms that link energy stores and reproduction are not well understood. Neuropeptides including galanin-like peptide (GALP), neuropeptide Y (NPY), products of the proopiomelanocortin (POMC; e.g., alpha-MSH and beta-endorphin), and kisspeptin are thought to be involved in this process for several reasons. First, the neurons that express these neuropeptides all reside in the hypothalamic arcuate nucleus, a critical site for the regulation of both metabolism and reproduction. Second, these neuropeptides are all targets for regulation by metabolic hormones, such as leptin and insulin. And third, these neuropeptides have either direct or indirect effects on feeding and metabolism, as well as on the secretion of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH). As the target for the action of metabolic hormones and sex steroids, these neuropeptides serve as molecular motifs integrating the control of metabolism and reproduction.     

Pancreatic neuronal melanocortin-4 receptor modulates serum insulin levels independent of leptin receptor

The leptin-regulated melanocortin (MC) system modulates energy homeostasis and hypothalamic MC neuronal circuits regulate insulin secretion. We therefore hypothesized that MC system components were present in the pancreas. In order to determine the veracity of the hypothesis, we examined c-Fos, melanocortin-4 receptor (Mc4r), and alpha-melanocyte-stimulating hormone (α-MSH) expression levels in nondiabetic (intact leptin receptor signaling) and Zucker diabetic fatty (ZDF; leptin receptor deficiency) rats. We infused rats via the third ventricle with the α-MSH analog Nle4, D-Phe7-α-MSH (NDP-MSH), a Mc4r agonist. Subsequently, both hypothalamic and pancreatic c-Fos and Mc4r mRNAs were upregulated. Likewise, immunohistochemical analysis showed that an increased Mc4r and α-MSH expression in nerves surrounding the pancreatic vasculature and islets. Increases in c-Fos, α-MSH, and Mc4r expression were independent of leptin receptor function. Conversely, serum insulin was significantly reduced by NDP-MSH treatment, an effect which was reversed by the Mc4r specific blocker HS014. Finally, proopiomelanocortin (POMC) mRNA, the precursor of α-MSH, was detected by RT-PCR in pancreatic tissue homogenates. These findings suggest that pancreatic Mc4r and autonomic neurons participate in a communication pathway between the central MC system and pancreatic islets to regulate insulin secretion.     

Glucokinase regulates reproductive function, glucocorticoid secretion, food intake, and hypothalamic gene expression

Because appetite, hypothalamic gene expression, reproductive function, and adrenal function are highly sensitive to acute changes in plasma glucose levels, it has been hypothesized hypothalamic neurons sensitive to glucose play a role in regulating these functions. To assess this hypothesis, we examined these neuronendocrine functions in mice in which the glucokinase gene, which plays an essential role in neuroendocrine glucose sensing, has been ablated. Haploinsufficiency in heterozygous glucokinase knockout mice produced effects similar to those produced by hypoglycemia: impaired reproductive function, elevated plasma corticosterone, increased food intake, and hypothalamic gene expression similar to that observed in fasted or leptin-deficient obese mice (increased hypothalamic neuropeptide Y mRNA and reduced hypothalamic proopiomelanocortin mRNA). Plasma glucose was elevated 2-fold in glucokinase knockout mice, consistent with a maturity-onset diabetes of the young phenotype, but plasma insulin and leptin levels were normal. These data support the hypothesis that glucokinase plays a key role in the neuroendocrine regulation of metabolic economy.