Ghrelin, a novel gastric hormone, regulates food intake and energy metabolism via central mechanisms. The peripheral effect of ghrelin on adiposity is poorly understood. We established a stable 3T3-L1 cell line expressing ghrelin to study the direct effect of ghrelin on adipogenesis. Cells overexpressing ghrelin demonstrate significantly attenuated differentiation of preadipocytes into adipocytes. Expression of peroxisome proliferator-activator receptor-gamma is significantly inhibited as demonstrated by decrease of peroxisome proliferator-activator receptor-gamma mRNA and protein. Both ghrelin overexpression and exogenous ghrelin stimulate cell proliferation. Phosphorylation of mitogen-activated protein kinase is increased after treatment of cells with ghrelin. Ghrelin binding activity is demonstrated in both native and ghrelin-overexpressing 3T3-L1 cells by radiolabeled ghrelin, although reverse transcription-polymerase chain reaction with the primer sequence of the previously identified ghrelin receptor subtypes detected no signal. Our results demonstrate that ghrelin inhibits adipogenesis by stimulation of cell proliferation via the mediation of a ghrelin receptor, likely a novel unidentified subtype.

Enteric glia are important participants in information processing in the enteric nervous system. However, intercellular signaling mechanisms in enteric glia remain largely unknown. We postulated that intercellular calcium waves exist in enteric glia. Primary cultures of enteric glia were isolated from neonatal guinea pig taenia coli. Intracellular calcium in individual cells was quantified with fura-2 AM microfluorimetry. Single-cell stimulation was performed with a micromanipulator-driven glass pipette. Data were expressed as mean +/- SEM and analyzed by Student's t-test. Mechanical stimulation of a single enteric glial cell resulted in an increase in intracellular calcium, followed by concentric propagation to 36% +/- 3% of neighboring cells. Intercellular calcium waves were blocked by depletion of intracellular calcium stores with thapsigargin (1 microM). Pretreatment of enteric glia with the phospholipase C inhibitor U73122 (1 microM) significantly decreased the percentage of cells responding to mechanical stimulation (6% +/- 4%), but had no effect on waves induced by microinjection of the inositol trisphosphate (67% +/- 13% vs. 60% +/- 4% for control). Antagonism of inositol trisphosphate receptor attenuated intercellular calcium waves induced by both mechanical stimulation and microinjection of inositol trisphosphate. Uncoupling of gap junctions with octanol or heptanol significantly inhibited intercellular calcium wave propagation. Pretreatment of enteric glia with apyrase partially attenuated intercellular calcium waves. Our data demonstrate that enteric glial cells are capable of transmitting increases in intracellular calcium to surrounding cells, and that intercellular calcium waves involve a sequence of intracellular and extracellular steps in which phospholipase C, inositol trisphosphate, and ATP play roles.

Ghrelin, a gastric peptide hormone, has been reported to regulate growth hormone secretion and energy homeostasis. Here we show that ghrelin promotes neural proliferation in vivo and in vitro in the rat dorsal motor nucleus of the vagus (DMNV). Ghrelin receptor mRNA and immunoreactivity were detected in tissues from DMNV. Systemic administration of ghrelin (130 nmol kg −1 ) significantly increased 5-bromo-2 -deoxyuridine (BrdU) incorporation in the DMNV in adult rats with cervical vagotomy (BrdU positive cells; from 27 ± 4 to 69 ± 14 n = 5, P < 0.05). In vitro, exposure of cultured DMNV neurones to ghrelin significantly increased the percentage of BrdU incorporation into cells in both dose-dependent (10 −9 -10and time-dependent (6 h to 48 h) manners. Ghrelin significantly increased voltage-activated calcium currents in isolated single DMNV neurones from a mean maximal change of 141 ± 26 pA to 227 ± 37 pA. Upon removal of ghrelin, calcium currents slowly returned to baseline. Blocking L-type calcium channels by diltiazem (10 µM) significantly attenuated ghrelin-mediated increments in BrdU incorporation (n = 5, P < 0.05). Ghrelin acts directly on DMNV neurones to stimulate neurogenesis.

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