One of the most profound hallmarks of mammalian hibernation is the dramatic reduction in food intake during the winter months. Several species of hibernator completely cease food intake (aphagia) for nearly 7 months regardless of ambient temperature and in many cases, whether or not food is available to them. Food intake regulation has been studied in mammals that hibernate for over 50 years and still little is known about the physiological mechanisms that control this important behavior in hibernators. It is well known from lesion experiments in non-hibernators that the hypothalamus is the main brain region controlling food intake and therefore body mass. In hibernators, the regulation of food intake and body mass is presumably governed by a circannual rhythm since there is a clear seasonal rhythm to food intake: animals increase food intake in the summer and early autumn, food intake declines in autumn and actually ceases in winter in many species, and resumes again in spring as food becomes available in the environment. Changes in circulating hormones (e.g., leptin, insulin, and ghrelin), nutrients (glucose, and free fatty acids), and cellular enzymes such as AMP-activated protein kinase (AMPK) have been shown to determine the activity of neurons involved in the food intake pathway. Thus, it appears likely that the food intake pathway is controlled by a variety of inputs, but is also acted upon by upstream regulators that are presumably rhythmic in nature. Current research examining the molecular mechanisms and integration of environmental signals (e.g., temperature and light) with these molecular mechanisms will hopefully shed light on how animals can turn off food intake and survive without eating for months on end.

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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.

SUMMARYMammals that hibernate (hibernators) exhibit a circannual rhythm of food intake and body mass. In the laboratory during the winter hibernation period, many hibernators enter a series of multi-day torpor bouts, dropping their body temperature to near ambient, and cease to feed even if food is present in their cage. The mechanism(s) that regulates food intake in hibernators is unclear. Recently, AMP-activated protein kinase (AMPK) has been shown to play a key role in the central regulation of food intake in mammals. We hypothesized that infusing an AMPK activator, 5-aminoimidazole-4-carboxamide 1 B-D-ribofuranoside (AICAR), intracerebroventricularly (ICV) into the third ventricle of the hypothalamus would stimulate yellow-bellied marmots (Marmota flaviventris) to feed during their hibernation season. Infusion of AICAR ICV into marmots at an ambient temperature of 22°C caused a significant (P<0.05) increase in food intake. In addition, animals stimulated to feed did not enter torpor during the infusion period. Marmots ICV infused with saline did not increase food intake and these animals continued to undergo torpor at an ambient temperature of 22°C. Our results suggest that AICAR stimulated the food intake pathway, presumably by activating AMPK. These results support the hypothesis that AMPK may be involved in regulating food intake in hibernators and that there may be common neural pathways involved in regulating feeding and eliciting torpor.

Ghrelin is a recently discovered hormone which has profound effects on food intake and lipogenesis in mammals. In all mammals studied thus far, plasma ghrelin concentrations are increased before a meal and decrease immediately following a meal; ghrelin levels increase with fasting. The goldenmantled ground squirrel Spermophilus lateralis (also known as Callospermophilus lateralis (see Helgen et al., 2009)) is a diurnal hibernator which has a robust annual cycle of body mass gain and loss that is primarily controlled by food intake. We hypothesized that in spring, summer, and autumn, the endogenous ghrelin concentrations of hibernators would be similar to those of non-hibernators, but that during the winter hibernation season, plasma ghrelin concentrations would be low or undetectable. We found that peripherally injected ghrelin significantly increased food intake in June. Plasma ghrelin concentrations were significantly increased through 5 days of fasting during a shortterm fast in summer. Over a 24 hour period, ghrelin concentrations increased at night and decreased during the day with drops corresponding to times when squirrels were eating. In January, ghrelin concentrations are low but measurable even while animals are at low body temperature (Tb). The reason for the persistence of ghrelin in plasma at this time is unclear, but circulating ghrelin in hibernators may be involved with the control of sleep in these animals. This is the first report of ghrelin concentrations in a non-photoperiodic hibernator. We suggest that ghrelin may be important for the regulation of food intake and the body mass cycle in mammals that hibernate.

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.

Atrazine (ATR) is a commonly used pre-emergence and early postemergence herbicide. Rats gavaged with ATR and its chlorometabolites desethylatrazine (DEA) and deisopropylatrazine (DIA) respond with a rapid and dose-dependent rise in plasma corticosterone, whereas the major chlorometabolite, diaminochlorotriazine (DACT), has little or no effect on corticosterone levels. In this study, we investigated the possible sites of ATR activation of the hypothalamic-pituitary-adrenal (HPA) axis. ATR treatment had no effect on adrenal weights but altered adrenal morphology. Hypophysectomized rats or rats under dexamethasone suppression did not respond to ATR treatment, suggesting that ATR does not directly stimulate the adrenal gland to induce corticosterone synthesis. Immortalized mouse corticotrophs (AtT-20) and primary rat pituitary cultures were treated with ATR, DEA, DIA, or DACT. None of the compounds induced an increase in ACTH secretion or potentiated ACTH release in conjunction with CRH on ACTH release. In female rats gavaged with ATR, pretreatment with the CRH receptor antagonist astressin completely blocked the ATR-induced rise in corticosterone concentrations, implicating CRH release in ATR-induced HPA activation. Intracerebroventricular infusion of ATR, DEA, and DIA but not DACT at concentrations equivalent to peak plasma concentrations after gavage dosing resulted in an elevation of plasma corticosterone concentrations. However, ATR did not induce c-Fos immunoreactivity in the paraventricular nucleus of the hypothalamus. These results indicate that ATR activates the HPA axis centrally and requires CRH receptor activation, but it does not stimulate cellular pathways associated with CRH neuronal excitation.

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