Download expression and function of receptors for leptin and ghrelin in sh

LEPTIN AND GHRELIN REGULATE NEUROPEPTIDE Y GENE EXPRESSION AND
SECRETION IN THE SH-SY5Y HUMAN NEUROBLASTOMA CELL LINE
Ilaria Brivio, Elena Dozio, Massimiliano Ruscica, Marcella Motta, Paolo Magni
Department of Endocrinology, University of Milano, Milano, Italy.
Food intake and energy expenditure are regulated by a network which integrates central and
peripheral signals. Some hypothalamic peptidergic neurons are specific targets of hormones
inducing appetite, like ghrelin, mainly produced by the stomach, or satiety, like the adipocytesecreted peptide, leptin. These agents appear to specifically regulate hypothalamic neurons
producing the potent orexigenic peptide neuropeptide Y (NPY). In the rat, leptin, acting via OBRb, the long leptin receptor isoform, has been shown to induce satiety in parallel with a marked
reduction of the hypothalamic expression of NPY. Ghrelin, a hunger hormone predominantly
produced by the stomach, has an important appetite-stimulating activity, and it is believed to
antagonize leptin actions through the activation of NPY neurons in the hypothalamus. At present,
no information is available about the possible presence of such mechanisms in the human
species.
In the present study, we have evaluated in the SH-SY5Y human neuroblastoma cell line, which
expresses NPY : 1) the expression of leptin receptors (OB-Rs) by RT-PCR and Western blot and
the gene expression of leptin, ghrelin, and ghrelin receptors (GHS-R 1a and 1b) by RT-PCR; 2)
the activation of MAPK and JAK2-STAT3-SOCS3 signalling pathways by leptin (Western blot);
3) the effect of treatment with leptin and ghrelin on NPY gene expression (Northern blot) and
release (radioimmunoassay). The results obtained show that: 1) OB-Rs are expressed in SHSY5Y cells and, according to gene expression analysis, these receptors correspond, at least in
part, to the long isoform OB-Rb. Moreover, these cells express also GHS-R 1a and 1b mRNAs,
show a weak expression of ghrelin mRNA, and no expression of leptin gene. 2) Treatment with
10-8 M leptin resulted in reciprocal changes of pSTAT3 and SOCS3, with downregulation of the
former and upregulation of the latter, and a short-lived activation of the MAPK signalling
pathways. 3) Exposure for 3-6 h to 10-12 M/10-8 M leptin led to a reduction of NPY mRNA
levels, with a maximal efficacy at the lowest concentrations tested. Leptin treatment for a short
time (5-60 min) was associated with a significant increase of NPY release, probably due to
membrane depolarization. Exposure to 10-10 and 10-8 M ghrelin (30 and 60 min) stimulated NPY
release with a maximal efficacy at the lowest concentration tested. This study indicates that
functional OB-Rs and GHS-Rs are present in SH-SY5Y cells and their activation modulates
NPY expression and release. These findings suggest that NPY may be a target of leptin and
ghrelin action also in human neural cells.
(Supported by grants from MIUR and University of Milano)