Obestatin partially affects ghrelin stimulation of food intake and growth hormone secretion in rodents

Abstract

Administration of ghrelin, an endogenous ligand for the GH secretagogue receptor 1a (GHSR 1a), induces potent stimulating effects on GH secretion and food intake. However, more than 7 yr after its discovery, the role of endogenous ghrelin remains elusive. Recently, a second peptide, obestatin, also generated from proteolytic cleavage of preproghrelin has been identified. This peptide inhibits food intake and gastrointestinal motility but does not modify in vitro GH release from pituitary cells. In this study, we have reinvestigated obestatin functions by measuring plasma ghrelin and obestatin levels in a period of spontaneous feeding in ad libitum-fed and 24-h fasted mice. Whereas fasting resulted in elevated ghrelin levels, obestatin levels were significantly reduced. Exogenous obestatin per se did not modify food intake in fasted and fed mice. However, it inhibited ghrelin orexigenic effect that were evident in fed mice only. The effects of obestatin on GH secretion were monitored in superfused pituitary explants and in freely moving rats. Obestatin was only effective in vivo to inhibit ghrelin stimulation of GH levels. Finally, the relationship between octanoylated ghrelin, obestatin, and GH secretions was evaluated by iterative blood sampling every 20 min during 6 h in freely moving adult male rats. The half-life of exogenous obestatin (10 microg iv) in plasma was about 22 min. Plasma obestatin levels exhibited an ultradian pulsatility with a frequency slightly lower than octanoylated ghrelin and GH. Ghrelin and obestatin levels were not strictly correlated. In conclusion, these results show that obestatin, like ghrelin, is secreted in a pulsatile manner and that in some conditions; obestatin can modulate exogenous ghrelin action. It remains to be determined whether obestatin modulates endogenous ghrelin actions.

Figure 1. Obestatin effects on spontaneous and ghrelin-induced food intake, in fed and 24h fasted mice, during the dark period

Cumulative food intake 1, 3, 5 hours after injection of saline (blue square), 1μmol/kg ghrelin (green square), 1μmol/kg obestatin (dark triangle) or 1μmol/kg obestatin + ghrelin (red triangle) : Panel A : in 24 h refeeding fasted mice (n= 20, 5 in each group) ; Panel B : in ad libitum fed mice (n= 20, 5 in each group) a: ghrelin vs saline, P<0.01 ; b : ghrelin vs obestatin, P<0.01 ; c : ghrelin vs obestatin + ghrelin, P<0.05 ; d : ghrelin vs obestatin, P<0.05 Panel C : Cumulative food intake 18 h post injection in 24 h fasted (left) and ad libitum fed (right) mice receiving saline (blue), 1μmol/kg ghrelin (green), 1μmol/kg obestatin (dark) or 1μmol/kg obestatin + ghrelin (red) a : ghrelin vs saline, P<0.05 ; b : ghrelin vs obestatin, P=0.06

Figure 2. Obestatin effects on spontaneous or ghrelin induced GH secretion in the rat

Panel A : Mean profile of GH release from superfused rat pituitaries in basal condition and after infusion of obestatin 10⁻⁶M (dark triangle), ghrelin 10⁻⁷M (green square), obestatin 10⁻⁷M + ghrelin 10⁻⁷M (pink triangle), obestatin 10⁻⁶M + ghrelin 10⁻⁷M (red triangle). The different peptides were infused during 15 minutes (materialised by dotted line). Each point and vertical bar indicates mean +/− sem of 4 chambers. Panel B : Effect of 10 μg obestatin (dark triangle), 10 μg ghrelin (green square) or 10 μg obestatin + ghrelin (red triangle) on GH secretion in freely moving rats. The peptides were injected at t=0, and blood were collected pre-injection and 5, 10, 20, 30, 45, 60 minutes post injection. Data are means +/− sem. Number of animals are indicated in parentheses. *: Ghrelin vs Obestatin + Ghrelin, P<0.05; #: Ghrelin vs Obestatin, P<0.05

Figure 3. Determination of exogenous obestatin half-life in rat plasma

Obestatin immunoreactivity was detected by RIA in rat plasma after iv injection of 10 μg synthetic peptide. Data are expressed as % obestatin concentrations measured immediately after the injection. Values are the means±sem of six determinations. A semilogarithmic plot of the data is given in the inset.

Figure 4. Ghrelin, obestatin and GH profiles in freely moving rats

Representative octanoylated ghrelin (triangle), obestatin (red square) and GH (diamond-shaped) secretory patterns during a 6-h sampling period (3-h during the light period and 3-h during the dark period) in four freely moving male rats.