Identification of kisspeptin (Kiss1) and its G protein‐coupled receptor 54 (Kiss1r) as an essential component of the hypothalamic‐pituitary‐gonadal (HPG) axis controlling gonadotrophin secretion raises the possibility that kisspeptin‐Kiss1r signalling may play a critical role in the transduction of stress‐induced suppression of reproduction. We examined the effects of: (i) three different stressors, known to suppress pulsatile luteinising hormone (LH) secretion; (ii) corticotrophin‐releasing factor (CRF); and (iii) corticosterone on Kiss1 and Kiss1r expression in key hypothalamic sites regulating gonadotrophin secretion: the medial preoptic area (mPOA) and arcuate nucleus (ARC). Ovariectomised oestrogen‐replaced rats were implanted with i.v., subcutaneous or i.c.v. cannulae. Blood samples were collected at 5‐min intervals for 5–6 h for detection of LH. Quantitative reverse transcriptase‐polymerase chain reaction was used to determine Kiss1 and Kiss1r mRNA levels in brain punches of the mPOA and ARC collected 6 h after restraint, insulin‐induced hypoglycaemia or lipopolysaccharide stress, or after i.c.v. administration of CRF, or acute or chronic subcutaneous administration of corticosterone. We observed down‐regulation of at least one component of the kisspeptin‐Kiss1r signalling system by each of the stress paradigms within the mPOA and ARC. CRF decreased Kiss1 and Kiss1r expression in both the mPOA and ARC. Both acute and chronic stress levels of corticosterone resulted in a concomitant decrease in Kiss1 and an increase in kiss1r mRNA expression in the mPOA and ARC. This differential regulation of Kiss1 and Kiss1r might account for the lack of effect corticosterone has on pulsatile LH secretion. Considering the pivotal role for kisspeptin‐Kiss1r signalling in the control of the HPG axis, these results suggest that the reduced Kiss1‐Kiss1r expression may be a contributing factor in stress‐related suppression of LH secretion.

Aims/hypothesis Kisspeptin is a novel peptide identified as an endogenous ligand of the G-protein-coupled receptor 54 (GPR-54), which plays a crucial role in puberty and reproductive function. High levels of GPR-54 and kisspeptin have been reported in the pancreas and we have previously shown that kisspeptin potentiates glucose-induced insulin release from isolated islets, although the mechanisms underlying this effect were unclear. Methods Insulin secretion from isolated mouse islets was measured to characterise the effects of kisspeptin. The effects of kisspeptin on both p42/44 mitogen-activated protein kinase (MAPK) phosphorylation and intracellular Ca 2+ ([Ca 2+ ] i ) in mouse islets were also investigated. Furthermore, kisspeptin was administered to rats in vivo and effects on plasma insulin levels measured. Results In the current study, kisspeptin induced a concentration-dependent potentiation of glucose-induced (20 mmol/l) insulin secretion from mouse islets, with maximal effects at 1 µmol/l, but had no effect on insulin secretion at a substimulatory concentration of glucose (2 mmol/l). Activation of GPR-54 by kisspeptin also caused reversible increases in [Ca 2+ ] i in Fura-2 loaded dispersed islet cells.The kisspeptin-induced potentiation of glucose-induced insulin secretion was completely abolished by inhibitors of phospholipase C and p42/44 MAPK, but not by inhibitors of protein kinase C or p38 MAPK. Intravenous administration of kisspeptin into conscious, unrestrained rats caused an increase in circulating insulin levels, whilst central administration of kisspeptin had no effect, indicating a peripheral site of action. Conclusions/interpretation These observations suggest that neither typical protein kinase C isoforms nor p38 MAPK are involved in the potentiation of glucose-induced insulin release by kisspeptin, but intracellular signalling pathways involving phospholipase C, p42/44 MAPK and increased [Ca 2+ ] i are required for the stimulatory effects on insulin secretion. The observation that kisspeptin is also capable of stimulating insulin release in vivo supports the conclusion that kisspeptin is a regulator of beta cell function.

Kisspeptin neurons in the arcuate nucleus of the hypothalamus generate gonadotrophinreleasing hormone (GnRH) pulses, and act as critical initiators of functional gonadotrophin secretion and reproductive competency. However, kisspeptin in other brain regions, most notably the posterodorsal subnucleus of the medial amygdala (MePD), plays a significant modulatory role over the hypothalamic kisspeptin population; our recent studies using optogenetics have shown that low-frequency light stimulation of MePD kisspeptin results in increased luteinsing hormone pulse frequency. Nonetheless, the neurochemical pathways that underpin this regulatory function remain unknown. To study this, we have utilised an optofluid technology, precisely combining optogenetic stimulation with intranuclear pharmacological receptor antagonism, to investigate the neurotransmission involved in this circuitry. We have shown experimentally and verified using a mathematical model that functional neurotransmission of both GABA and glutamate is a requirement for effective modulation of the GnRH pulse generator by amygdala kisspeptin neurons.

The bed nucleus of the stria terminalis (BNST) occupies a central position in the neural circuitry regulating the hypothalamic-pituitary-adrenocortical axis response to stress. The potential role of the BNST in stress-induced suppression of the gondotrophin-releasing hormone (GnRH) pulse generator, the central regulator of the reproductive system, was assessed by examining the effects of micro-infusion of corticotrophin-releasing factor (CRF) or its antagonist into the BNST on pulsatile luteinising hormone (LH) secretion or stress-induced inhibition of LH pulses, respectively. Ovariectomised oestrogen-treated rats were implanted chronically with bilateral cannulae in the dorsolateral BNST and i.v. catheters. CRF (25, 50 or 100 pmol in 200 nl of artificial cerebrospinal fluid) administered bilaterally into the BNST resulted in a dose-dependent decrease in LH pulse frequency, and induced Fos expression in glutamic acid decarboxylase immunostained neurones in the medial preoptic area. These results suggest that the activation of hypothalamic GABAergic neurones in response to intra-BNST administration of CRF may be involved in the suppression of LH pulses. Furthermore, administration of CRF antagonist (280 pmol astressin-B, three times at 20-min intervals) into the BNST effectively blocked the suppression of pulsatile LH secretion in response to restraint (1 h) but not hypoglycaemic (0.25 U insulin/kg, i.v.) stress. These data suggest that CRF innervation of the dorsolateral BNST plays a key, but differential, role in stress-induced suppression of the GnRH pulse generator.