Primary pituitary cell culture

The pituitary dispersion into single cells was made by enzymatic and mechanical disruption, as previously described^49,56,65. Then, cells were plated onto 24-well tissue culture plates at 200,000 cells/well with 0.5 ml (for expression and secretion analyses) or onto 48-well tissues plates at 50,000 cells/wells with 0.2 ml (for secretion analyses) of basic medium containing 10% horse serum. Cells were incubated for 36–48 h (37 °C) and after that, medium was removed and cells were pre-incubated for 1 h in fresh, warm (37 °C), serum-free medium to stabilize the cells. Then, medium was replaced with serum free medium containing different treatments in order to perform the following experiments: (1) “dose-response experiment” of leptin (10–100 ng/ml), adiponectin (10–1000 nM) and resistin (0.1–1000 nM) alone (4h-incubation); (2) “time-course experiment” of leptin (10 ng/ml), adiponectin (10 nM) or resistin (0.1 nM) alone for 4- and 24-h; (3) “functional interaction experiment” between leptin (10 ng/ml), adiponectin (10 nM) or resistin (0.1 nM) with primary regulator of GH release [i.e. GH-releasing hormone (GHRH; 10 nM), acylated-ghrelin (10 nM) or somatostatin (SST; 10 nM); 4h-incubation]. (4) “signaling pathway experiment” in order to study the intracellular signaling routes involved in the effects of leptin, adiponectin and resistin on pituitary cells function, medium containing inhibitors of key intracellular signaling pathways was added to the cell cultures (medium alone was used in the vehicle treated controls). After 90 minutes of stabilization, medium was replaced with medium alone (vehicle) or containing the selected inhibitor combined with leptin (10 ng/ml), adiponectin (10 nM) and resistin (0.1 nM) and incubated for 4 h. Specifically, inhibitors of the following signaling pathways were used: adenylyl cyclase (AC; MDL-12,330 A; 10 μM), protein kinase-A (PKA; H-89; 15 μM), phospholipase C (PLC; U73122; 50 μM), protein kinase C (PKC; Go6983; 20 μM), plasma membrane L-type voltage-sensitive Ca²⁺ channels (extracellular Ca²⁺; nifedipine; 1 μM), Ca²⁺ release from intracellular pools (intracellular Ca²⁺; thapsigargin; 10 μM), mitogen-activated protein kinase activity (MAPK; PD-98,059; 10 μM), phosphatidylinositol 3-kinase activity (PI3K; wortmannin; 1 μM), and mammalian target of rapamycin (mTOR; rapamycin; 10 μM). It should be mentioned that doses for leptin, adiponectin, resistin, GHRH, ghrelin, SST, or inhibitors of intracellular signaling pathways were selected based on previous studies^{19,32,33,35,36,49,50,52,79,80} that administration of these inhibitors alone did not modify basal hormonal secretions (data not shown).

In all experiments, after the corresponding incubation period (4 h and 24 h), medium was collected for hormone analysis (see below). Total RNA was extracted from selected cultures treated with leptin (10 ng/ml), adiponectin (10 nM) and resistin (0.1 nM) for gene expression analysis of pituitary hormone transcripts, receptors and other transcription factors important for the pituitary cells function. Controls consisted of serum-free media alone without any treatment. Each treatment was repeated at least three times on independent pituitary cell preparations (3–4 wells/treatment per experiment). It should be noted that, given the limited source of macaque cell preparations (n = 3) and of amount of cells obtained after dispersion of the pituitary gland, we were able to study only some selected endpoints (i.e. the effects of leptin, adiponectin and resistin at a single dose on the secretion of all the pituitary hormones at 4- and 24-h of incubation).