In vitro and in vivo effect of acylated and unacylated ghrelin on neonatal glucose homeostasis.

Maintenance of normal glucose homeostasis is crucial for survival during the perinatal period. Acylated ghrelin (AG) but not unacylated ghrelin (UAG) inhibits insulin release from pancreatic islets in adult rats. Circulating AG concentrations are low in the fetus and progressively increase in the postnatal period. We tested the hypothesis that AG has insulinostatic effects in vitro and in vivo during the perinatal period. In vitro, AG (10(-10)-10(-8) M) caused a 25-53% decrease in insulin secretion by islets from 5-d-old rat pups under normo- and hyperglycemic conditions, an effect that was mediated through the growth hormone secretagogue receptor (GHSR- 1a). Ghrelin (1-5) amide, [Dap3]-octanoyl, a pentapeptide that is resistant to deacylation and binds the GHSR-1a, had similar effects at 10(-8) M. In vivo, AG, UAG, or GHRP-6 [D-Lys3], a GHSR-1a antagonist, did not affect insulin or glucagon concentrations during the first 3 h of life. In 6-d-old pups, AG, UAG, or ghrelin (1-5) amide, [Dap3]-octanoyl did not affect glucose-induced insulin or C-peptide concentrations. In summary, AG has insulinostatic effects in vitro as early as during the perinatal period. These effects could not be confirmed in vivo, possibly due to the short half-life of AG in rat neonates.

Ontogeny of acylated ghrelin degradation in the rat.

Ghrelin circulates as acylated (AG) and unacylated (or desacyl) ghrelin (UAG). We aimed at clarifying the effect of age and sex on plasma deacylation and degradation of AG in vivo and in vitro in the rat. In vivo, we compared AG and UAG concentrations following administration of 1 microg AG intraperitoneally in rat neonates during the first 3h of life. AG administration caused a 2-3 times increase in plasma AG concentrations contrasting with a approximately 1000 times increase in UAG concentrations suggesting rapid deacylation of AG into UAG. In vitro, we demonstrated that AG degradation was greater in the fetus (97% over 30 min) and decreased progressively to 57% in adult animals (P<0.001). Carboxylesterase and butyrylcholinesterase activities were determined during the fetal (day 21 of pregnancy) and postnatal period (days 1, 6, 13, 21 and 28) and in the adult rat and were found to increase with age (P<0.001). While inhibition of carboxylesterase and butyrylcholinesterase did not affect AG deacylation, serine protease inhibitors decreased AG degradation in the adult rat (from 59% to 23%) and, to a lesser extent, in the rat neonate (from 92% to 57%) by reducing both deacylation and degradation into non-UAG metabolites. Our data suggest that degradation of AG into UAG and non-UAG metabolites is much faster in the fetus and in the rat neonate compared to the adult. We speculate that this process allows for fine tuning of the physiological effects of both AG and UAG.

Ontogeny of ghrelin, obestatin, preproghrelin, and prohormone convertases in rat pancreas and stomach.

The processing of preproghrelin in the stomach by prohormone convertase (PC) 1/3 produces ghrelin and possibly obestatin. In the neonate, the pancreas is also a major source of ghrelin. We compared the ontogeny of preproghrelin, ghrelin, obestatin, and PCs in the stomach and pancreas from rat embryos (day 21) and neonates (days 1, 6, 13, 21, and 28) by immunohistochemistry. In stomach, preproghrelin positive cells were present from embryonic day 21 and were in excess of ghrelin cells. The number of ghrelin positive cells progressively increased with age. When preproghrelin cells were immunoreactive for ghrelin, they were also immunoreactive for obestatin and PC1/3. In pancreas, we only found 0 to 2 preproghrelin positive cells per islet and each of these cells was also positive for ghrelin and obestatin. None of the ghrelin positive cells stained for insulin, but we observed ghrelin positive/glucagon negative and ghrelin positive/glucagon positive cells. Ghrelin positive cells contained PC1/3 or PC2. In summary, in stomach, an excess of preproghrelin positive cells compared with ghrelin/PC1/3 positive cells suggests that PC1/3 determines preproghrelin processing to ghrelin. In pancreas, the colocalization of PC1/3 or PC2 in ghrelin positive cells points to a role for both PCs in preproghrelin processing.