Background: Ghrelin stimulates cell proliferation in a number of tissues including pituitary. AMPK, a heterotrimer kinase enzyme, is an important sensor and regulator of cellular energy balance. We have shown that ghrelin can change AMPK activity in various tissues and this mechanism could play a role in its metabolic effects. AMPK has recently been established to strongly inhibit cell proliferation and tumorigenesis. We therefore hypothesised that ghrelin stimulates cell proliferation via inhibition of AMPK activity in the pituitary.
Methods: The GH3 cell line was treated with ghrelin 10−6, 10−7 and 10−9 M and cells were harvested in lysis buffer at 30 min, 60 min, 90 min, 2 h, 3 h, 6 h and 24 h. The effect of ghrelin on AMPK activity was studied with a kinase assay using γ32P-ATP and with immunoblotting using phosphorylation-specific antibodies for alpha-AMPK.
Results: AMPK activity was significantly decreased in the ghrelin-treated cells compared to the media treated controls at 60 and 90 minutes for the 10−6 and 10−7 M, but also at the 6 h for the 10−9 M. The peak effect was at 60 minutes (control 21.0±0.7 pmol ATP/min/mg protein vs ghrelin 10−7 M 4.7±0.4 pmol ATP/min/mg protein; P<0.01). Immunoblotting for pAMPK showed a reduction in pAMPK content at 60 min after 10−6M ghrelin treatment (88% of control).
Conclusion: We propose that the pro-proliferative effects of ghrelin involves the inhibition of AMPK which could lead to upregulation of the Akt and mTOR-S6kinase pathway and downregulation of the p53-p21 pathway leading to increased protein synthesis and cell cycle progression.