In vitro stimulation of the prepubertal rat gonadotropin-releasing hormonepulse generator by leptin and neuropeptide Y through distinct mechanisms

Leptin may act as a negative feedback signal to the brain in the control of appetite through suppression of neuropeptide Y (NPY) secretion and stimula tion of cocaine- and amphetamine-regulated transcript (CART), a new anorect ic peptide. We aimed at studying whether leptin, NPY, and CART have related effects on the hypothalamic control of the pituitary-gonadal system and th e developmental changes in NPY and CART effects. Using retrochiasmatic hypo thalamic explants from prepubertal 15-day-old male rats, the GnRH interpuls e interval (mean +/- so : 62 +/- 5 min) was significantly reduced by 10(-7) M of leptin (46 +/- 3.3 min) as well as 10(-7) M of NPY (47 +/- 4.4 min) a nd 10(-6) M of CART (46 +/- 2.7 min), whereas the GnRH pulse amplitude was not affected. The stimulatory effects of different NPY receptor agonists [h uman PYY3-36, porcine NPY13-36, human (D-Trp(32)) NPY, porcine (Leu(31) Pro (34)) NPY, human pancreatic polypeptide (PP)], as well as the absent effect s of rat PP were consistent with the involvement of the W-receptor subtype in mediation of NPY effects. Incubation with 10-7 M of a Y5-receptor select ive antagonist prevented the effect of NPY (61 +/- 4 us. 46 +/- 2 min), whe reas leptin and CART effects were not (47 +/- 3 us. 46 +/- 3 min and 46 +/- 3 us. 46 +/- 2 min, respectively), suggesting that NPY was not involved in leptin and CART effects. Using an anti-CART antiserum (1:1000), the reduct ion of GnRW interpulse interval caused by leptin was partially prevented (5 6.2 +/- 4 vs. 47.9 +/- 3.8 min), whereas the reduction of GnRH interval cau sed by NPY was not affected (45.9 +/- 2.5 us. 47.8 +/- 3.7). The GnRN inter pulse interval was decreased by 10(-7) M of NPY at 5 days (72 +/- 3.8 us. 9 1.9 +/- 3.5) as well as at 15 days, whereas such an effect was not observed anymore at 25 and 50 days. Similar effects were observed using 10(-6) M of CART-peptide. Using 10(-6) an of the YS-receptor antagonist, the GnRW inte rpulse interval was significantly increased at 15 days (66.6 1 2.7 min), 25 days (56.5 +/- 39.9 min), and 50 days (52.5 vs. 38.2 min), whereas no chan ge was observed at 5 days. Using the anti-CART antiserum, a significant inc rease of GnRH interpulse interval was observed at; 25 days only. In conclus ion, the stimulatory effects of leptin and NPY on the frequency of pulsatil e GnRH secretion before puberty involve two distinct mechanisms. NPY causes acceleration of GnRH pulsatility via the YS-receptor subtype, which is not involved in leptin effects while the CART is involved in leptin effects on GnRW secretion but not in NPY effects. The reduction of pulsatility by the Y5 antagonist provides evidence of endogenous NPY involvement in the contr ol of GnRW secretion from the time of onset of puberty.