Functional characterisation and translational applications of kisspeptin-10.
George, Jyothis Thomas
Background: Kisspeptins, recently discovered hypothalamic neuropeptides encoded by the KISS1 gene, are essential for normal pubertal development and are modulated by diverse endocrine, metabolic and environmental signals. Exogenous kisspeptin administration potently stimulates LH secretion - by direct action on GnRH neurons while kisspeptin antagonists inhibit pulsatile LH secretion. Human studies of kisspeptin had hitherto used kisspeptin-54 that is cleaved further and the smallest bioactive form is a decapeptide (kisspeptin-10) with a shorter half-life. Kisspeptin-10 is thus putatively more attractive in studies assessing LH pulsatility and is also the basis for the development of antagonists. Unmet clinical needs: Decreased LH pulse frequency is the central pathology in pubertal delay, late-onset male hypogonadism and hypothalamic amenorrhoea. Manipulation of LH pulse frequency also has therapeutic potential in contraception, PCOS and sex-steroid dependant diseases such as endometriosis and prostatic hyperplasia. Hypothesis: That exogenous kisspeptin-10 enhances pulsatile LH secretion in healthy men and in patients with reproductive disorders associated with decreased pulse frequency. Research strategy: A first-in-human dose escalation study of kisspeptin-10 was performed in men and subsequently replicated in women. An intravenous infusion regime was optimised in healthy men and subsequently applied to hypogonadal patients. Specific questions were addressed sequentially as summarised below with key results. Dose escalation study: Question: Does kisspeptin-10 stimulate LH secretion in men? Findings: Six iv bolus doses (0.01 to 3 μg/kg) of GMP kisspeptin-10 and vehicle were administered at least a week apart to six healthy men. Rapid increase in LH, with peak concentrations was seen by 45 min post injection in all volunteers. There was a clear dose-dependent increase in LH concentrations in response to kisspeptin- 10 (P <0.0001). Area-Under-Curve analysis over 60 min following kisspeptin-10 administration showed 0.3 and 1μg/kg doses to be maximally stimulatory (P <0.01) with a reduced response at 3 μg/kg. Assessing the effect of steroid milieu: Question: Steroid feedback is central to the regulation of LH secretion: what effect does the steroid milieu have on LH responses to kisspeptin-10? Findings: The response to iv kisspeptin-10 (0.3μg/kg,) in the normal follicular phase (n=10) was compared with that in the presence of low endogenous sex steroids/high LH secretion (6 postmenopausal women) and in women taking combined contraceptive therapy (n=8) with suppressed LH secretion. Despite widely varying baseline secretion, LH increased significantly following kisspeptin-10 administration in the follicular phase (6.3±1.2 to 9.4±1.3 IU/L P=0.006), postmenopausal (35.3±2.8 to 44.7±3.4 IU/L P=0.005), etonogestrel (4.6±0.2 to 7.5±0.9 IU/L, P=0.02), and COCP groups (2.2±0.9 to 3.7±1.4 IU/L P<0.001). Pulse frequency study: Question: GnRH and LH secretion are pulsatile: can kisspeptin-10 enhance LH pulsatility? Findings: Four healthy men attended our clinical research facility for two visits five days apart for 10-min blood sampling. At the first visit, baseline LH pulsatility was assessed over a 9-hour period. During the second visit, an infusion of kisspeptin-10 was administered for 9 hours at 1.5μg/kg/hr after an hour of baseline sampling. LH pulse frequency increased in all subjects, with a mean increase from 0.7±0.1 to 1.0±0.2 pulses/hr (P = 0.01), with resultant increase in mean LH from 5.2±0.8 IU/L at baseline to 14.1±1.7 IU/L (P <0.01). High dose, longer duration infusion study: Question: Can kisspeptin-10 enhance testosterone secretion? Findings: Four healthy men attended our clinical research facility for a 34-hour supervised stay. Blood samples were collected at 10 min intervals for two 12 hour periods on consecutive days and hourly overnight. After 10.5 hours of baseline sampling a continuous intravenous infusion of kisspeptin-10 (4μg/kg/hr) was maintained for 22.5 hrs. Mean LH increased from 5.5±0.8 at baseline to 20.9±4.9 IU/L (P <0.05) and serum testosterone increased from 16.6±2.4 to 24.0±2.5 nmol/L (P <0.001). Translational studies in hypogonadal men with type 2 diabetes Question: Can kisspeptin-10 normalise testosterone secretion in hypogonadal men? Findings: Five hypogonadal men with T2DM (age 33.6±3 yrs, BMI 40.6±6.3, testosterone 8.5±1.0 nmol/L, LH 4.7±0.7 IU/L, HbA1c <8 %, duration of diabetes <5 yrs) and seven age matched healthy men were studied. Kisspeptin-10 was administered intravenous (0.3 μg/kg) with frequent (10-min) blood sampling. Mean LH increased in controls (5.5±0.8 to 13.9±1.7 IU/L P <0.001) and in T2DM (4.7±0.7 to 10.7±1.2 IU/L P=0.02) with comparable ΔLH (P=0.18). Baseline serum sampling for LH at 10-min intervals and hourly testosterone measurements were performed subsequently in four T2DM men for 12 hours. An intravenous infusion of kisspeptin-10 (4 μg/kg/hr) was administered 5 days later for 11 hours, with increases in serum LH (3.9±0.1 IU/L to 20.7±1.1 IU/L (P=0.03,) and testosterone (8.5±1.0 to 11.4±0.9 nmol/L, P=0.002). LH pulse frequency at baseline was lower in hypogonadal men with diabetes (0.6±0.1 vs. 0.8±0.1 pulses/hr, P=0.03) and increased to 0.9±0 pulses/hr (P=0.05). Translational studies in pubertal delay: Question: Defective Neurokinin B activity is associated with pubertal delay and the hierarchical interactions between kisspeptins and Neurokinin B remain to be elucidated: can kisspeptin-10 stimulate LH secretion with impaired Neurokinin B signalling? Findings: Four patients with TAC3 or TACR3 inactivating mutations presenting with delayed puberty were admitted for two 12 hr blocks of blood sampling every 10 min with vehicle (saline) or kisspeptin-10 (1.5 μg/kg/hour) infused intravenously. Mean LH and LH pulses frequency increased with kisspeptin-10 (P<0.05). However, four patients with Kallmann syndrome (with defective GnRH neuron migration), studied in parallel, did not respond, suggesting a potential diagnostic application for kisspeptin-10 in pubertal dysfunction. Conclusions In first-in-man studies of kisspeptin-10, it was demonstrated that endogenous LH pulse frequency can be enhanced in healthy men. The therapeutic potential of this finding in common reproductive endocrine disorders associated with decreased LH pulse frequency, i.e., late-onset male hypogonadism and pubertal dysfunction, was suggested in subsequent studies. Furthermore, kisspeptin signalling occurs upstream of GnRH neurons and is independent of Neurokinin B signalling in the central regulation of the hypothalamic-pituitary-gonadal axis.