Measurement of AMP-activated protein kinase activity and expression in response to ghrelin.

Ghrelin is a circulating brain-gut peptide that is known to exert several metabolic effects such as stimulating appetite, inducing adiposity, increasing bone formation, and influencing the cardiovascular functions. AMP-activated protein kinase (AMPK), a highly conserved heterotrimeric protein that plays a key role in energy homeostasis, has been shown to mediate many of these metabolic effects of ghrelin. Ghrelin is shown to stimulate hypothalamic AMPK activity and inhibit liver and adipose tissue AMPK activity. The effects of ghrelin on AMPK activity can be studied using an elegant kinase assay, which involves immunoprecipitating AMPK protein from the tissue of interest followed by quantifying its enzymatic activity using radiolabeled adenosine triphosphate (ATP) in the presence of a suitable substrate. As a surrogate marker of AMPK activity, AMPK Thr(172) phosphorylation can be measured by Western blotting. Information about the AMPK pathway can also be gained by studying the mRNA expression of various AMPK subunits and by Western blotting for phosphorylated acetyl-CoA carboxylase, a key AMPK target. These methods have been widely used and published for investigating the effects of ghrelin on AMPK activity. In this chapter, we look into these experiments' methodology in detail.