AMP-activated protein kinase (AMPK) is a cellular energy sensor that responds to low endogenous energy by stimulating fatty acid oxidation (through inactivation of acetyl-CoA carboxylase (ACC)) and food intake. Fasting generally stimulates phosphorylation of AMPK (pAMPK) and ACC (pACC), but it is unclear how AMPK and ACC react to a long-term fast (i.e. hibernation). We performed Western blots for total and pAMPK and pACC on tissues from a species of hibernator (Callospermophilus lateralis) after short-term summer fasting (1–5 days) and long-term winter fasting (3 months). Winter animals were sacrificed during hibernation at low body temperature (torpid, Tb~5°C) or at normal high Tb(euthermic, Tb~37°C). We found a general increase in pAMPK in most tissues (liver, muscle, white adipose tissue (WAT), but not hypothalamus) and pACC in all tissues after a short-term summer fast. Response of AMPK and ACC to a long-term winter fast differed by tissue—in liver, there was no difference in total or pAMPK or pACC between groups, but in muscle, WAT and BAT, euthermic GMGS had lower relative abundance of pAMPK and pACC than torpid animals. Therefore, AMPK may be an important energy sensor at all points in hibernator’s circannual cycles of food intake and Tb.

Hibernators exhibit a robust circannual cycle of body mass gain and loss primarily mediated by food intake, but the pathways controlling food intake in these animals have not been fully elucidated. Ghrelin is an orexigenic hormone that increases feeding in all mammals studied so far, but has not until recently been studied in hibernators. In other mammals, ghrelin stimulates feeding through phosphorylation and activation of AMP-activated protein kinase (AMPK). Activation of AMPK phosphorylates and deactivates acetyl Co-A carboxylase (ACC), a committed step in fatty acid synthesis. In order to determine the effects of exogenous ghrelin on food intake and metabolic factors (i.e. non-esterified fatty acids (NEFAs), and hypothalamic AMPK and ACC) in hibernators, ghrelin was peripherally injected into ground squirrels in all four seasons. Changes in food intake and body mass were recorded over a 2-6 hour period post injections, and squirrels were euthanized. Brains and blood were removed, and Western blots were performed to determine changes in phosphorylation of hypothalamic AMPK and ACC. A colorimetric assay was used to determine changes in concentration of serum NEFAs. We found that food intake, body mass, and locomotor activity significantly increased with ghrelin injections versus saline-injected controls, even in animals injected during their aphagic winter season. Injected ghrelin was correlated with increased phosphorylation of AMPK, but didn't have an effect on ACC in winter. Ghrelin-injected animals also had increased levels of serum NEFAs compared with saline controls. This study is the first to show an effect of injected ghrelin on a hibernator.