Background Ghrelin is a gut peptide that increases food intake, in part by activating NPY/Agrp neurons in the hypothalamic arcuate nucleus, an area crucial for appetite regulation. The intracellular mechanisms mediating ghrelin's orexigenic effects are not fully known, and elucidating these signaling pathways could identify new targets for anti-obesity medications. Because ghrelin and insulin exert opposing effects on feeding and hypothalamic neuronal activity, we hypothesized that these hormones antagonize one another's cell signaling pathways. We have previously shown that ghrelin does not affect insulin-mediated activation of hypothalamic protein kinase B, a plausible consequence of ghrelin signaling via phospholipase C. Another possible point of competitive convergence for ghrelin and insulin is the protein kinase A (PKA) pathway, which is activated by ghrelin in some non-neuronal tissues. If this also occurs in hypothalamus, it could induce npy gene expression and stimulate food intake. Moreover, insulin inhibits PKA in the hypothalamus via phosphodiesterase 3B and could therefore antagonize ghrelin signaling. In the current study, we addressed whether PKA signaling is required for ghrelin's orexigenic actions. We sought to determine if the feeding effects of centrally administered ghrelin are attenuated by pretreatment with the PKA inhibitor H89.
Methods Male Wistar rats were implanted with 3rd intracerebroventricular (ICV) cannulas 1 week prior to studies. Cannula placement was confirmed by angiotensin II testing. Animals were habituated to daily handling and food-intake measurement for 1 week. On the experimental day, ad libitum-fed rats were given a 1 μL ICV injection of either H89 (12.5 nmol) or DMSO vehicle; 30 minutes later, they received a 2 μL ICV injection of either ghrelin (60 pmol) or saline vehicle. Food intake was measured 1 and 2 h after the second injection.
Results Pretreatment with H89 decreased ghrelin-induced feeding (1 hr: 2.6 ± 0.4 g [ghrelin] vs 1.5 ± 0.6 g [H89 + ghrelin], p = .11; 2 hr: 3.6 ± 0.4 g [ghrelin] vs 1.5 ± 0.4 g [H89 + ghrelin], p = .03). This dose of H89 alone did not significantly affect food intake at any time point (1 hr: 0.4 ± 0.1 g [vehicle] vs 1.2 ± 0.7 g [H89], p = .41; 2 hr: 0.4 ± 0.1 g (vehicle) vs 1.8 ± 0.8 [H89], p = .22).
Conclusions Inhibition of central PKA signaling significantly attenuates ghrelin-induced feeding. This finding suggests that the ghrelin receptor signals via PKA in the hypothalamus and that this pathway is important for ghrelin's orexigenic actions. Although the downstream consequences of hypothalamic PKA signaling are not known, a likely target of this pathway is CREB phosphorylation, which could induce npy gene expression and stimulate food intake.
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