Fazal Wahab

GnRH-Deficient Phenotypes in Humans and Mice with Heterozygous Variants in KISS1/Kiss1

CONTEXT KISS1 is a candidate gene for GnRH deficiency. OBJECTIVE Our objective was to identify deleterious mutations in KISS1. PATIENTS AND METHODS DNA sequencing and assessment of the effects of rare sequence variants (RSV) were conducted in 1025 probands with GnRH-deficient conditions. RESULTS Fifteen probands harbored 10 heterozygous RSV in KISS1 seen in less than 1% of control subjects. Of the variants that reside within the mature kisspeptin peptide, p.F117L (but not p.S77I, p.Q82K, p.H90D, or p.P110T) reduces inositol phosphate generation. Of the variants that lie within the coding region but outside the mature peptide, p.G35S and p.C53R (but not p.A129V) are predicted in silico to be deleterious. Of the variants that lie outside the coding region, one (g.1-3659C→T) impairs transcription in vitro, and another (c.1-7C→T) lies within the consensus Kozak sequence. Of five probands tested, four had abnormal baseline LH pulse patterns. In mice, testosterone decreases with heterozygous loss of Kiss1 and Kiss1r alleles (wild-type, 274 ± 99, to double heterozygotes, 69 ± 16 ng/dl; r(2) = 0.13; P = 0.03). Kiss1/Kiss1r double-heterozygote males have shorter anogenital distances (13.0 ± 0.2 vs. 15.6 ± 0.2 mm at P34, P < 0.001), females have longer estrous cycles (7.4 ± 0.2 vs. 5.6 ± 0.2 d, P < 0.01), and mating pairs have decreased litter frequency (0.59 ± 0.09 vs. 0.71 ± 0.06 litters/month, P < 0.04) and size (3.5 ± 0.2 vs. 5.4 ± 0.3 pups/litter, P < 0.001) compared with wild-type mice. CONCLUSIONS Deleterious, heterozygous RSV in KISS1 exist at a low frequency in GnRH-deficient patients as well as in the general population in presumably normal individuals. As in Kiss1(+/-)/Kiss1r(+/-) mice, heterozygous KISS1 variants in humans may work with other genetic and/or environmental factors to cause abnormal reproductive function.

Decrease in Hypothalamic Kiss1 and Kiss1r Expression: A Potential Mechanism for Fasting-induced Suppression of the HPG Axis in the Adult Male Rhesus Monkey (Macaca mulatta)

Fasting suppresses functioning of the hypothalamic-pituitary-gonadal (HPG) axis by mechanisms that are incompletely understood. In 2003, hypothalamic kisspeptin-Kiss1r signaling was discovered to play a significant role in regulating the HPG axis. We have recently shown that in adult male macaques, short-term fasting attenuates the response of the HPG axis to an exogenous kisspeptin challenge. In the present study, we explored the mechanism underlying this attenuated response by examining the modulation of the hypothalamic expression of KISS1 and KISS1R under short-term fasting and normal feeding conditions in the adult male macaques. Hypothalamic mRNA was extracted from normal fed (n=3) and 48-h fasted (n=3) monkeys. KISS1, KISS1R, and GNRH1 mRNA were quantified by reverse transcription followed by real-time polymerase chain reaction. In addition, blood samples were collected for measurement of plasma concentrations of glucose, cortisol, leptin, and testosterone. In contrast to fed animals, plasma glucose, leptin, and testosterone levels decreased and cortisol levels increased in fasted animals. The hypothalamic expression of KISS1 and KISS1R mRNA was significantly lower (p<0.05) in fasted monkeys compared to fed monkeys while hypothalamic GNRH1 mRNA expression was comparable between the 2 groups. Thus, our results demonstrate that expression of hypothalamic KISS1 and KISS1R decrease after a short-term fasting in monkeys. This decrease may contribute to the suppression of the HPG axis during fasting conditions in primates. In addition, our finding of lower expression of KISS1R in fasted monkeys provides an explanation for the attenuation in the HPG axis response to peripheral kisspeptin challenge during short-term fasting.

Kisspeptin as a link between metabolism and reproduction: Evidences from rodent and primate studies

Changes in metabolic status gate reproductive activity by still incompletely deciphered mechanisms. Many neuropeptides have been shown to be involved in restraining hypothalamic gonadotropin releasing hormone (GnRH) release under conditions of negative energy balance. Broadly, on the basis of their effect on feeding, these can be grouped as orexigenic and anorexigenic neuropeptides. Reciprocally correlated, in response to changes in systemic concentrations of metabolic hormones, the secretion of orexigenic neuropeptides increases while that of anorexigenic neuropeptides decreases during conditions of food restriction. Recently, kisspeptin signaling in hypothalamus has appeared as a pivotal regulator of the GnRH pulse generator. Kisspeptin apparently does not affect feeding, but in light of accumulating data, it has emerged as one of the major conduits in relaying body metabolic status information to GnRH neurons. The present review examines such data obtained from rodent and primate models, which suggest kisspeptin-Kiss1r signaling as a possible pathway providing a link between metabolism and reproduction.

Short-term fasting attenuates the response of the HPG axis to kisspeptin challenge in the adult male rhesus monkey (Macaca mulatta)

AIMS In primates, changes in nutritional status affect the hypothalamic-pituitary-gonadal (HPG) axis by still poorly understood mechanisms. Recently, hypothalamic kisspeptin-GPR54 signaling has emerged as a significant regulator of this neuroendocrine axis. The present study was designed to examine whether suppression of the reproductive function by acute food-restriction in a non-human primate is mediated by decreased responsiveness of the HPG axis to endogenous kisspeptin drive. MAIN METHODS Five intact adult male rhesus monkeys habituated to chair-restraint, received intravenous boli of human kisspeptin-10 (KP10, 50 microg), hCG (50 IU), and vehicle (1 ml) in both fed and 48-h fasting conditions. Plasma concentrations of glucose, cortisol and testosterone (T) were measured by using enzymatic and specific RIAs, respectively. KEY FINDINGS The acute 48-h fasting decreased plasma glucose (P<0.01) and T (P<0.005) levels, and increased cortisol levels (P<0.05). KP10 administration caused a robust stimulation of T secretion in both fed and fasted monkeys. However, mean T concentration and T AUC after KP10 administration were significantly (P<0.01-0.005) reduced in fasted monkeys. Likewise, the time of the first significant increase in post-KP10 T levels was also significantly (P<0.01) delayed. T response to hCG stimulation was similar in fed and fasted monkeys. SIGNIFICANCE The present results indicate that under fasting conditions the KP10 induced T response is delayed and suppressed. These data support the notion that fasting-induced suppression of the HPG axis in the adult male rhesus monkey may involve, at least in part, a reduction in the sensitivity of the GnRH neuronal network to endogenous kisspeptin stimulation.

The kisspeptin signaling pathway and its role in human isolated GnRH deficiency.

Amplification of the neurosecretory activity of the GnRH system is the defining neuroendocrine event for sexual maturation. The physiological mechanisms that drive GnRH secretion at puberty have been difficult to identify but the discovery in 2003 that the G protein coupled receptor KISS1R is a key regulator of pubertal development in mice and men has ushered in a new chapter in reproductive neuroendocrinology. KISS1R is activated by endogenous peptides derived from a precursor protein, kisspeptin. Despite kisspeptins importance in driving the reproductive cascade, relatively few patients with GnRH deficient states and mutations in the kisspeptin pathway have been described. Yet, these cases, coupled with loss-of-function mouse models, provide unique and complementary information into the biological role of this signaling system in the control of GnRH secretion. This article will examine some of the subtleties in genotype-phenotype correlations in both mice and men carrying disabling mutations in the kisspeptin pathway.

Effect of peripheral kisspeptin administration on adiponectin, leptin, and resistin secretion under fed and fasting conditions in the adult male rhesus monkey (Macaca mulatta).

In the last few years, kisspeptin-KISS1R signaling has appeared as a major regulator of the reproductive function in several vertebrate species. However, KISS1(encoding kisspeptin) and its putative receptor, KISS1R, are expressed in several other tissues. Adipose tissue, which secretes many peptides with diverse functions in normal physiology, expresses KISS1, which is modulated by gonadal steroids as well as by body nutritional status. Similarly, KISS1Rexpression is also found in adipose tissue, but the local role of kisspeptin in adipocyte function is currently unknown. Therefore, in the present study the effects of exogenous human kisspeptin-10 (KP10) were studied on three important adipokines, namely, adiponectin, leptin, and resistin in a set of four chair-restraint habituated intact adult male rhesus monkeys under; 1) normal fed conditions, 2) 24-h fasting conditions, and 3) 48-h fasting conditions. Plasma resistin and leptin levels decreased (p<0.01), whereas adiponectin levels increased (p<0.05) in fasted monkeys. Kisspeptin administration significantly increased (p<0.05) mean plasma adiponectin levels under fed and 24-h fasting conditions as compared to pretreatment or vehicle-treatment levels. A stimulatory effect was also observed on the 48-h fasting stimulated plasma adiponectin levels, but it lacked statistical significance. In contrast, no effect of kisspeptin was observed on mean plasma leptin and resistin levels. Thus, the present study demonstrated a stimulatory effect of peripheral kisspeptin administration on the plasma adiponectin levels under fed and 24-h fasting conditions in the adult male rhesus monkey. These findings, therefore, assign a novel role to kisspeptin, a regulator of adipocyte function in higher primate.

Study on the effect of peripheral kisspeptin administration on basal and glucose-induced insulin secretion under fed and fasting conditions in the adult male rhesus monkey (Macaca mulatta).

Kisspeptin (KP)-Kiss1r, a ligand-receptor pair, has recently been implicated as a pivotal regulator of the neuroendocrine reproductive axis. KISS1 (encoding KP) as well as KISS1R (encoding receptor for KP) are expressed in several peripheral tissues including the pancreas. But the specific role of KP in the physiology of pancreas is still incompletely deciphered. This study was designed to examine the effect of peripheral KP administration on basal and glucose-induced plasma insulin (an important pancreatic hormone) secretion under fed and fasting conditions in the adult male rhesus monkey. A set of 4 chair-restraint habituated intact adult male rhesus monkeys were assigned to receive intravenous bolus administration of human kisspeptin-10 (KP10, 50 μg), and vehicle (1 ml) in normal fed and fasting conditions without or with glucose infusions. Plasma concentrations of insulin were measured by using a specific IRMA. Glucose infusion significantly stimulated plasma insulin levels (p<0.005). Vehicle administration did not affect both basal and glucose stimulated insulin in fed as well as in fasting condition. KP10 administration had no effect on the basal insulin levels in both fed and fasting as compared to pretreatment or vehicle treatment levels, while it significantly heightened glucose stimulated insulin levels (p<0.05) in both fed and fasted monkeys. The present results show that KP administration does not affect the basal secretion of insulin under both fed and fasting condition while potentiated the glucose-induced insulin levels in the adult male rhesus monkey. Therefore, these findings suggest a potential role of KP in the physiology of pancreas.

Intratesticular action of kisspeptin in rhesus monkey (Macaca mulatta)

Kisspeptin‐Kiss1R signalling in mammals has been implicated as an integral part of the reproductive cascade. Kisspeptinergic neurons upstream of GnRH neurons are involved in the activation of the hypothalamic GnRH pulse generator during pubertal onset. Thus, the major research focus has been on the central effects of kisspeptin. The demonstration of the presence of KissR expression in human testes suggests additional unknown actions of kisspeptin‐KISS1R signalling at the distal component of the male reproductive axis. Here we explored the impact of kisspeptin at the testis in the adult male rhesus monkey. We employed the clamped monkey model to assess the intratesticular actions of kisspeptin. Plasma testosterone and LH levels were monitored in four adult male monkeys. The peripheral administration of human kisspeptin‐10 (50 μg, iv bolus) caused a single LH pulse, which was followed by a robust increase in plasma testosterone levels sustained for at least 180 min. This response was abolished when kisspeptin was administered to GnRH receptor antagonist (acyline) pre‐treated animals. However, kisspeptin administration significantly (P < 0.005) elevated hCG‐stimulated testosterone levels in acyline pre‐treated monkeys when compared with saline+ hCG treatment. These results revealed a novel peripheral facet of kisspeptin signalling.

Kisspeptin signalling in the physiology and pathophysiology of the urogenital system

Kisspeptin is a peptide hormone, which signals via the G-protein-coupled kisspeptin receptor (KISS1R). Kisspeptin–KISS1R signalling has been implicated in various physiological and pathophysiological processes in the urogenital system, including critical roles in ovarian function as a key player in the regulation of oocyte development. Kisspeptin also has roles in several different functions of the male reproductive tract, such as spermatogenesis and sperm capacitation, and is also thought to be involved in kidney physiology — studies in preclinical animal models have reported that expression of kisspeptin and/or KISS1R is altered in chronically impaired kidneys. The wider importance of kisspeptin action in the urogenital tract has been highlighted by the finding that it suppresses metastasis of urogenital carcinomas; besides the possible therapeutic potential of this finding, tissue and tumour-stage-specific alterations in kisspeptin and KISS1/KISS1R expression could potentially be used as biomarkers for the diagnosis and prognosis of urogenital carcinomas.

Study of the effect of 26RF- and 43RF-amides on testosterone and prolactin secretion in the adult male rhesus monkey (Macaca mulatta).

RF-amides (RFa), a superfamily of evolutionary-conserved neuropeptides, are expressed in both invertebrates and vertebrates. While some endocrine functions have been attributed to these peptides in lower vertebrates and few mammalian models, not much is known about their actions in primates. Therefore, the present study was designed to examine the effects of peripheral administration of two recently cloned human RFa peptides, 26RFa and 43RFa, on testosterone and prolactin secretion in the adult male adult male rhesus monkey (Macaca mulatta). For control purposes, a scrambled sequence of 26RFa (Sc-26RFa) and normal saline (1ml) were injected. Three different doses of 26RFa and 43RFa (19-nmol, 38-nmol and 76-nmol) and a single dose (38-nmol) of Sc-26RFa were tested. A set of four chair-restraint habituated monkeys was used. Comparison of post-treatment T levels with respective pre levels showed that none of the doses of both 26RFa and 43RFa changed T release. Similarly, Sc-26RFa and saline administration also did not affect T levels. In contrast, all doses of 26RFa and 43RFa significantly (P<0.05) stimulated prolactin secretion. 43RFa dose dependently increased prolactin secretion while dose dependency was not observed for 26RFa. Saline and Sc-26RFa injection had no effect on prolactin concentrations. Thus, present study demonstrated that peripheral administration of 26RFa and 43RFa, in the doses tested, have no effect on T secretion, suggesting possible selective lack of their neuroendocrine role in controlling hypothalamic-pituitary-gonadal axis in the adult male primates. The prominent stimulation of prolactin suggests a neuroendocrine role of RFa peptides in regulation of prolactin release in primates.