Obestatin plays an opposite role in the regulation of pituitary somatotrope and corticotrope function in female primates and male/female mice

Abstract

Obestatin is a 23-amino-acid amidated peptide that is encoded by the ghrelin gene. Previous studies have shown obestatin can modulate the hypothalamic neuronal circuitry that regulates pituitary function, perhaps by modulating the actions of ghrelin. However, the direct actions of obestatin on pituitary function remain controversial. Here, primary pituitary cell cultures from a nonhuman primate (baboon) and mice were used to test the effects of obestatin on pituitary hormone expression and secretion. In pituitary cultures from both species, obestatin had no effect on prolactin, LH, FSH, or TSH expression/release. Conversely, obestatin stimulated proopiomelanocortin expression and ACTH release and inhibited GH expression/release in vitro, actions that were also observed in vivo in mice treated with obestatin. In vitro, obestatin inhibited the stimulatory actions of ghrelin on GH but not ACTH release. The inhibitory effect of obestatin on somatotrope function was associated with an overall reduction in pituitary transcription factor-1 and GHRH receptor mRNA levels in vitro and in vivo as well as a reduction in hypothalamic GHRH and ghrelin expression in vivo. The stimulatory effect of obestatin on ACTH was associated with an increase in pituitary CRF receptors. Obestatin also reduced the expression of pituitary somatostatin receptors (sst1/sst2), which could serve to modify its impact on hormone secretion. The in vitro actions of obestatin on both GH and ACTH release required the adenylyl cyclase and MAPK routes. Taken together, our results provide evidence that obestatin can act directly at the pituitary to control somatotrope and corticotrope function, and these effects are conserved across species.

Figure 1.

Effects of obestatin (Ob; 10nM) on GH and POMC/ACTH release and expression in primary pituitary cell cultures from baboons (4 and 24 hours) and mice (24 hours) (panel A) as well as in mice treated with obestatin in vivo (panel B). Data of hormonal release in vitro (primary cell cultures) and of GH/POMC expression in vitro and in vivo are expressed as percentage of vehicle-treated mice (control [C] or vehicle [Veh]; set at 100% within each experiment), whereas circulating GH and ACTH levels in mice treated with obestatin in vivo and their vehicle-treated controls are expressed as raw values. Values represent the mean ± SE (n = 3–5 individual in vitro experiments (3–4 wells per treatment per experiment [panel A] or 5–10 mice per group [panel B]). Asterisks indicate values that significantly differ from their respective control values: *, P ≤ .05; **, P ≤ .01. Control values for GH release are 38 ± 21 and 186 ± 94 ng/mL for baboon cultures at 4 and 24 hours, respectively, and 356 ± 147 ng/mL for mouse cultures at 24 hours. Control values for ACTH release are 4.6 ± 2.8 and 7.2 ± 4.5 ng/mL ACTH for baboon cultures at 4 and 24 hours, respectively, and 11.6 ± 7.8 ng/mL for mouse cultures at 24 hours.

Figure 2.

Effect of obestatin treatment (Ob; 10nM) on cell viability (24 hours) of mouse primary pituitary cell cultures from males and females (n = 3 from each gender; 8–10 weeks-old; 100,000 cells/well: 4 wells/treatment), assessed by trypan-blue (panel A) and alamarBlue (panel B) assays. Results are expressed as percent of vehicle-treated controls (C) and are the mean ± SE (set at 100%).

Figure 3.

Interaction of Obestatin (Ob; 10nM) with common regulators of somatotrope and corticotrope function in primary pituitary cell cultures from baboons and mice. Panels A–D, Effect of 24 hours treatment of obestatin alone or in combination with ghrelin (Ghl; 10nM) or somatostatin (SST; 100nM) on the secretion of GH (baboons [panel A] and mice [panel B]) and ACTH (baboons [panel C] and mice [panel D]). Values are expressed as percentage of controls (C; set at 100% within each experiment) and represent the mean ± SE of 3 to 5 independent experiments (3–4 wells per experiment). Values that do not share a common letter are statistically different (P ≤ .05).

Figure 4.

Intracellular signaling pathways of obestatin-regulated baboon GH and ACTH release. Effect of the inhibition of adenylyl cyclase (AC; MDL-12,330A; 10 μM), phospholipase C (PLC; U73122; 50 μM) or MAPK (PD-98,059; 10 μM) on obestatin(Ob)-inhibited GH release (panel A), and obestatin(Ob)-stimulated ACTH secretion (panel B). Values are expressed as percentage of vehicle-treated controls (C) without inhibitor (set at 100% within each experiment), and represent the mean ± SE of three independent experiments (three to four wells per experiment). Values that do not share a common letter are statistically different (P ≤ .05).

Figure 5.

Key regulatory components involved in obestatin-induced alterations on somatotrope and corticotrope function in primary pituitary cell cultures from baboons and mice (24 hours incubation) and/or in mice treated with obestatin in vivo. Effects of obestatin (Ob) on Pit-1expression (panel A), hypothalamic GHRH, ghrelin, CRF, and somatostatin (SST) expression (panel B; mice treated with obestatin in vivo); pituitary GHRH-receptor expression (GHRH-R; panel C); circulating IGF-1 levels (panel D; mice treated with obestatin in vivo); pituitary CRF receptor subtypes 1 and 2 expression (CRF-R1/2; panel E); pituitary somatostatin receptors subtypes 1, 2, and 5 expression (sst1/2/5; panel F); and ghrelin receptor expression (GHSR; panel G). All the data in panels A–C and E–G are expressed as percentage of vehicle-treated controls (shown by the dotted line set at 100%), whereas circulating IGF-1 levels are expressed as raw values. Values represent the mean ± SE (n = 3–5 individual in vitro experiments [3–4 wells per treatment per experiment or 5–10 mice per group]). Asterisks indicate values that significantly differ from their respective control values: *, P ≤ .05; **, P ≤ .01.

Figure 6.

Presence of obestatin as well as GPR39 and GLP1R in the pituitary. Panels A and B, Obestatin protein expression levels in stomachs and pituitaries of mice (panel A; n = 3 tissues per group). Absolute mRNA copy numbers (adjusted by a normalization factor [NF]) of GLP1R and GPR39 in the pituitary of baboons and mice (panel B; n = 5). Panel C, Effects of obestatin (24 hours) on the expression of GLP1R and GPR39 in primary pituitary cell cultures from baboons and mice. Data in panel C are expressed as percentage of vehicle-treated controls (shown by the dotted line set at 100%) and represent the mean ± SE (n = 3–5 individual in vitro experiments [3–4 wells per treatment per experiment]). Asterisks indicate values that significantly differ from their respective control values: *, P ≤ .05; **, P ≤ .01.