Lipopolysaccharide-Mediated Effects of the Microbiota on Sleep and Body Temperature.

Background: Recent research suggests that microbial molecules translocated from the intestinal lumen into the host's internal environment may play a role in various physiological functions, including sleep. Previously, we identified that butyrate, a short-chain fatty acid, produced by intestinal bacteria, and lipoteichoic acid, a cell wall component of gram-positive bacteria induce sleep when their naturally occurring translocation is mimicked by direct delivery into the portal vein. Building upon these findings, we aimed to explore the sleep signaling potential of intraportally administered lipopolysaccharide, a primary component of gram-negative bacterial cell walls, in rats. Results: Low dose of lipopolysaccharide (1 µg/kg) increased sleep duration and prolonged fever, without affecting systemic lipopolysaccharide levels. Interestingly, administering LPS systemically outside the portal region at a dose 20 times higher did not affect sleep, indicating a localized sensitivity within the hepatoportal region, encompassing the portal vein and liver, for the sleep and febrile effects of lipopolysaccharide. Furthermore, both the sleep- and fever-inducing effects of LPS were inhibited by indomethacin, a prostaglandin synthesis inhibitor, and replicated by intraportal administration of prostaglandin E2 or arachidonic acid, suggesting the involvement of the prostaglandin system in mediating these actions. Conclusions: These findings underscore the dynamic influence of lipopolysaccharide in the hepatoportal region on sleep and fever mechanisms, contributing to a complex microbial molecular assembly that orchestrates communication between the intestinal microbiota and brain. Lipopolysaccharide is a physiological component of plasma in both the portal and extra-portal circulation, with its levels rising in response to everyday challenges like high-fat meals, moderate alcohol intake, sleep loss and psychological stress. The increased translocation of lipopolysaccharide under such conditions may account for their physiological impact in daily life, highlighting the intricate interplay between microbial molecules and host physiology.

Lipoteichoic acid, a cell wall component of Gram-positive bacteria, induces sleep and fever and suppresses feeding.

Fragments of the bacterial cell wall are bioactive microbial molecules that have profound effects on the function of the brain. Some of the cell wall constituents are common to both Gram-positive and Gram-negative bacteria, e.g., peptidoglycans, while other cell wall components are specific to either Gram-positive or Gram-negative microbes. Lipopolysaccharide (LPS), also called endotoxin, is found exclusively in Gram-negative bacteria, while lipoteichoic acid (LTA) is specific to Gram-positive bacteria. The effects of peptidoglycans, their fragments, and LPS are well characterized, they induce sleep, fever and anorexia. In the present study, we investigated the sleep, body temperature and food intake modulating effects of LTA. We found that intraperitoneal injection of 100 and 250 µg LTA from B. subtilis and S. aureus increases non-rapid-eye movement sleep (NREMS) in mice. The effects were dose-dependent, and the changes were accompanied by decreased motor activity and feeding as well as febrile responses. Intraperitoneal injection of 10 µg LTA induced monophasic increases in body temperature, while 100 and 250 µg LTA from B. subtilis induced initial hypothermia followed by fever. Treatment with 250 µg LTA from S. aureus elicited monophasic hypothermia. Administration of 300 µg/kg LTA from S. aureus directly into the portal vein elicited similar sleep responses in rats but did not affect body temperature. The sleep-modulating effects of LTA were similar to that of LPS in mice, although LTA appears to be less potent. These findings suggest that the role of LTA in signaling by Gram-positive bacteria in the host body is analogous to the role of LPS/endotoxin in signaling by Gram-negative microbes. LTA may play a role in the development of sickness response in clinically manifest Gram-positive bacterial infections and may contribute to sleep signaling by the commensal intestinal microbiota.

Perfect timing: circadian rhythms, sleep, and immunity - an NIH workshop summary.

Recent discoveries demonstrate a critical role for circadian rhythms and sleep in immune system homeostasis. Both innate and adaptive immune responses - ranging from leukocyte mobilization, trafficking, and chemotaxis to cytokine release and T cell differentiation -are mediated in a time of day-dependent manner. The National Institutes of Health (NIH) recently sponsored an interdisciplinary workshop, "Sleep Insufficiency, Circadian Misalignment, and the Immune Response," to highlight new research linking sleep and circadian biology to immune function and to identify areas of high translational potential. This Review summarizes topics discussed and highlights immediate opportunities for delineating clinically relevant connections among biological rhythms, sleep, and immune regulation.

Nicotinic acid promotes sleep through prostaglandin synthesis in mice.

Nicotinic acid has been used for decades for its antiatherogenic properties in humans. Its actions on lipid metabolism intersect with multiple sleep regulatory mechanisms, but its effects on sleep have never been documented. For the first time, we investigated the effects of acute systemic administration of nicotinic acid on sleep in mice. Intraperitoneal and oral gavage administration of nicotinic acid elicited robust increases in non-rapid-eye movement sleep (NREMS) and decreases in body temperature, energy expenditure and food intake. Preventing hypothermia did not affect its sleep-inducing actions suggesting that altered sleep is not secondary to decreased body temperature. Systemic administration of nicotinamide, a conversion product of nicotinic acid, did not affect sleep amounts and body temperature, indicating that it is not nicotinamide that underlies these actions. Systemic administration of monomethyl fumarate, another agonist of the nicotinic acid receptor GPR109A, fully recapitulated the somnogenic and thermoregulatory effects of nicotinic acid suggesting that they are mediated by the GPR109A receptor. The cyclooxygenase inhibitor indomethacin completely abolished the effects of nicotinic acid indicating that prostaglandins play a key role in mediating the sleep and thermoregulatory responses of nicotinic acid.

Sleep and body temperature in TNFα knockout mice: The effects of sleep deprivation, ß3-AR stimulation and exogenous TNFα.

Increased production of pro-inflammatory cytokines is assumed to mediate increased sleep under inflammatory conditions, such as systemic infections or recovery from sleep loss. The role of cytokines in sleep regulation under normal conditions is less clear. In the present study, we investigated the role of endogenous tumor necrosis factor alpha (TNFα) in sleep regulation using TNFα knockout (KO) mice. Under control conditions at thermoneutral ambient temperature, total sleep time did not differ between TNFα KO and wild-type (WT) mice, but TNFα KO mice had increased rapid-eye-movement sleep (REMS), accompanied by decreased motor activity and body temperature. Exposure to 17 °C induced decreases in total sleep time similarly in both genotypes. Sleep deprivation by gentle handling elicited robust rebound increases in non-rapid-eye movement sleep (NREMS), REMS and electroencephalographic (EEG) slow-wave activity (SWA), accompanied by suppressed motor activity and decreased body temperature; there was no significant difference between the responses of WT and KO mice. Systemic injection of the beta3-adrenergic receptor (ß3-AR) agonist CL-316,243 induced increases in NREMS and body temperature. The temperature response, but not the sleep effect, was attenuated in the KO animals. Systemic injection of TNFα induced increased NREMS, reduced REMS and biphasic temperature responses in both genotypes. In the KO mice, the NREMS-promoting effects of exogenously administered TNFα was decreased, while REMS suppression was enhanced, and the first, hypothermic, phase of temperature response was attenuated. Overall, TNFα KO mice did not show any deficiency in sleep regulation which suggests that the role of endogenous TNFα in sleep regulation is less pronounced than previously suggested.

Butyrate, a metabolite of intestinal bacteria, enhances sleep.

Emerging evidence suggests that the intestinal microbiota is a source of sleep-promoting signals. Bacterial metabolites and components of the bacterial cell wall are likely to provide important links between the intestinal commensal flora and sleep-generating mechanisms in the brain. Butyrate is a short-chain fatty acid produced by the intestinal bacteria by the fermentation of nondigestible polysaccharides. We tested the hypothesis that butyrate may serve as a bacterial-derived sleep-promoting signal. Oral gavage administration of tributyrin, a butyrate pro-drug, elicited an almost 50% increase in non-rapid-eye movement sleep (NREMS) in mice for 4 hours after the treatment. Similarly, intraportal injection of butyrate led to prompt and robust increases in NREMS in rats. In the first 6 hours after the butyrate injection, NREMS increased by 70%. Both the oral and intraportal administration of butyrate led to a significant drop in body temperature. Systemic subcutaneous or intraperitoneal injection of butyrate did not have any significant effect on sleep or body temperature. The results suggest that the sleep-inducing effects of butyrate are mediated by a sensory mechanism located in the liver and/or in the portal vein wall. Hepatoportal butyrate-sensitive mechanisms may play a role in sleep modulation by the intestinal microbiota.

Mice Lacking Alternatively Activated (M2) Macrophages Show Impairments in Restorative Sleep after Sleep Loss and in Cold Environment.

The relationship between sleep, metabolism and immune functions has been described, but the cellular components of the interaction are incompletely identified. We previously reported that systemic macrophage depletion results in sleep impairment after sleep loss and in cold environment. These findings point to the role of macrophage-derived signals in maintaining normal sleep. Macrophages exist either in resting form, classically activated, pro-inflammatory (M1) or alternatively activated, anti-inflammatory (M2) phenotypes. In the present study we determined the contribution of M2 macrophages to sleep signaling by using IL-4 receptor α-chain-deficient [IL-4Rα knockout (KO)] mice, which are unable to produce M2 macrophages. Sleep deprivation induced robust increases in non-rapid-eye-movement sleep (NREMS) and slow-wave activity in wild-type (WT) animals. NREMS rebound after sleep deprivation was ~50% less in IL-4Rα KO mice. Cold exposure induced reductions in rapid-eye-movement sleep (REMS) and NREMS in both WT and KO mice. These differences were augmented in IL-4Rα KO mice, which lost ~100% more NREMS and ~25% more REMS compared to WTs. Our finding that M2 macrophage-deficient mice have the same sleep phenotype as mice with global macrophage depletion reconfirms the significance of macrophages in sleep regulation and suggests that the main contributors are the alternatively activated M2 cells.

Brown adipose tissue plays a central role in systemic inflammation-induced sleep responses.

We previously identified brown adipose tissue (BAT) as a source of sleep-inducing signals. Pharmacological activation of BAT enhances sleep while sleep loss leads to increased BAT thermogenesis. Recovery sleep after sleep loss is diminished in mice that lack uncoupling protein 1 (UCP-1), and also in wild-type (WT) mice after sensory denervation of the BAT. Systemic inflammation greatly affects metabolism and the function of adipose tissue, and also induces characteristic sleep responses. We hypothesized that sleep responses to acute inflammation are mediated by BAT-derived signals. To test this, we determined the effects of systemic inflammation on sleep and body temperature in UCP-1 knockout (KO) and WT mice. Intraperitoneal injections of lipopolysaccharide, tumor necrosis factor-α, interleukin-1 beta and clodronate containing liposomes were used to induce systemic inflammation. In WT animals, non-rapid-eye movement sleep (NREMS) was elevated in all four inflammatory models. All NREMS responses were completely abolished in UCP-1 KO animals. Systemic inflammation elicited an initial hypothermia followed by fever in WT mice. The hypothermic phase, but not the fever, was abolished in UCP-1 KO mice. The only recognized function of UCP-1 is to promote thermogenesis in brown adipocytes. Present results indicate that the presence of UCP-1 is necessary for increased NREMS but does not contribute to the development of fever in systemic inflammation.

The role of the brown adipose tissue in ß3-adrenergic receptor activation-induced sleep, metabolic and feeding responses.

Brown adipose tissue (BAT) is regulated by the sympathetic nervous system via ß3-adrenergic receptors (ß3-AR). Here we tested the hypothesis that pharmacological stimulation of ß3-ARs leads to increased sleep in mice and if this change is BAT dependent. In wild-type (WT) animals, administration of CL-316,243, a selective ß3-AR agonist, induced significant increases in non-rapid-eye movement sleep (NREMS) lasting for 4-10 h. Simultaneously, electroencephalographic slow-wave activity (SWA) was significantly decreased and body temperature was increased with a delay of 5-6 h. In uncoupling protein 1 (UCP-1) knockout mice, the middle and highest doses of the ß3-AR agonist increased sleep and suppressed SWA, however, these effects were significantly attenuated and shorter-lasting as compared to WT animals. To determine if somnogenic signals arising from BAT in response to ß3-AR stimulation are mediated by the sensory afferents of BAT, we tested the effects of CL-316,243 in mice with the chemical deafferentation of the intra-scapular BAT pads. Sleep responses to CL-316,243 were attenuated by ~50% in intra-BAT capsaicin-treated mice. Present findings indicate that the activation of BAT via ß3-AR leads to increased sleep in mice and that this effect is dependent on the presence of UCP-1 protein and sleep responses require the intact sensory innervation of BAT.

Effects of Macrophage Depletion on Sleep in Mice.

The reciprocal interaction between the immune system and sleep regulation has been widely acknowledged but the cellular mechanisms that underpin this interaction are not completely understood. In the present study, we investigated the role of macrophages in sleep loss- and cold exposure-induced sleep and body temperature responses. Macrophage apoptosis was induced in mice by systemic injection of clodronate-containing liposomes (CCL). We report that CCL treatment induced an immediate and transient increase in non-rapid-eye movement sleep (NREMS) and fever accompanied by decrease in rapid-eye movement sleep, motor activity and NREMS delta power. Chronically macrophage-depleted mice had attenuated NREMS rebound after sleep deprivation compared to normal mice. Cold-induced increase in wakefulness and decrease in NREMS, rapid-eye movement sleep and body temperature were significantly enhanced in macrophage-depleted mice indicating increased cold sensitivity. These findings provide further evidence for the reciprocal interaction among the immune system, sleep and metabolism, and identify macrophages as one of the key cellular elements in this interplay.

Sickness behaviour after lipopolysaccharide treatment in ghrelin deficient mice.

Ghrelin is an orexigenic hormone produced mainly by the gastrointestinal system and the brain. Much evidence also indicates a role for ghrelin in sleep and thermoregulation. Further, ghrelin was recently implicated in immune system modulation. Administration of bacterial lipopolysaccharide (LPS) induces fever, anorexia, and increased non-rapid-eye movement sleep (NREMS) and these actions are mediated primarily by proinflammatory cytokines. Ghrelin reduces LPS-induced fever, suppresses circulating levels of proinflammatory cytokines and reduces the severity and mortality of various models of experimental endotoxemia. In the present study, we determined the role of intact ghrelin signaling in LPS-induced sleep, feeding, and thermoregulatory responses in mice. Sleep-wake activity was determined after intraperitoneal, dark onset administration of 0.4, 2 and 10 µg LPS in preproghrelin knockout (KO) and wild-type (WT) mice. In addition, body temperature, motor activity and changes in 24-h food intake and body weight were measured. LPS induced dose-dependent increases in NREMS, and suppressed rapid-eye movement sleep, electroencephalographic slow-wave activity, motor activity, food intake and body weight in both Ppg KO and WT mice. Body temperature changes showed a biphasic pattern with a decrease during the dark period followed by an increase in the light phase. The effects of the low and middle doses of LPS were indistinguishable between the two genotypes. Administration of 10 µg LPS, however, induced significantly larger changes in NREMS and wakefulness amounts, body temperature, food intake and body weight in the Ppg KO mice. These findings support a role for ghrelin as an endogenous modulator of inflammatory responses and a central component of arousal and feeding circuits.

Brown adipose tissue at the intersection of sleep and temperature regulation.

The article demonstrates the importance of brown fat in creating and maintaining a metabolic environment which is permissive for optimal restorative sleep after sleep loss. The authors propose that impaired brown fat function could be a common underlying cause of poor sleep and metabolic disorders.

Intact brown adipose tissue thermogenesis is required for restorative sleep responses after sleep loss.

Metabolic signals related to feeding and body temperature regulation have profound effects on vigilance. Brown adipose tissue (BAT) is a key effector organ in the regulation of metabolism in several species, including rats and mice. Significant amounts of active BAT are also present throughout adulthood in humans. The metabolic activity of BAT is due to the tissue-specific presence of the uncoupling protein-1 (UCP-1). To test the involvement of BAT thermogenesis in sleep regulation, we investigated the effects of two sleep-promoting stimuli in UCP-1-deficient mice. Sleep deprivation by gentle handling increased UCP-1 mRNA expression in BAT and elicited rebound increases in non-rapid-eye-movement sleep and rapid-eye-movement sleep accompanied by elevated slow-wave activity of the electroencephalogram. The rebound sleep increases were significantly attenuated, by ~ 35-45%, in UCP-1-knockout (KO) mice. Wild-type (WT) mice with capsaicin-induced sensory denervation of the interscapular BAT pads showed similar impairments in restorative sleep responses after sleep deprivation, suggesting a role of neuronal sleep-promoting signaling from the BAT. Exposure of WT mice to 35 °C ambient temperature for 5 days led to increased sleep and body temperature and suppressed feeding and energy expenditure. Sleep increases in the warm environment were significantly suppressed, by ~ 50%, in UCP-1-KO animals while their food intake and energy expenditure did not differ from those of the WTs. These results suggest that the metabolic activity of the BAT plays a role in generating a metabolic environment that is permissive for optimal sleep. Impaired BAT function may be a common underlying cause of sleep insufficiency and metabolic disorders.

Central but not systemic administration of ghrelin induces wakefulness in mice.

Ghrelin is a brain-gut peptide hormone widely known for its orexigenic and growth hormone-releasing activities. Findings from our and other laboratories indicate a role of ghrelin in sleep regulation. The effects of exogenous ghrelin on sleep-wake activity in mice are, however, unknown. The aim of the present study was to determine the sleep-modulating effects of ghrelin after central and systemic administrations in mice. Sleep-wake activity after intracerebroventricular (i.c.v.) administration of 0.2, 1 and 5 µg ghrelin and intraperitoneal injections of 40, 100, and 400 µg/kg ghrelin prior to light onset were determined in C57BL/6 mice. In addition, body temperature, motor activity and 1-hour food intake was measured after the systemic injections. Sleep effects of systemic ghrelin (40 and 400 µg/kg) injected before dark onset were also determined. I.c.v. injection of ghrelin increased wakefulness and suppressed non-rapid-eye-movement sleep and electroencephalographic slow-wave activity in the first hour after injections. Rapid-eye-movement sleep was decreased for 2-4 hours after each dose of ghrelin. Sytemic administration of ghrelin did not induce changes in sleep-wake activity in mice at dark or light onset. Motor activity and body temperature remained unaltered and food intake was significantly increased after systemic injections of ghrelin given prior the light period. These findings indicate that the activation of central, but not peripheral, ghrelin-sensitive mechanisms elicits arousal in mice. The results are consistent with the hypothesis that the activation of the hypothalamic neuronal circuit formed by ghrelin, orexin, and neuropeptide Y neurons triggers behavioral sequence characterized by increased wakefulness, motor activity and feeding in nocturnal rodents.

The effects of C75, an inhibitor of fatty acid synthase, on sleep and metabolism in mice.

Sleep is greatly affected by changes in metabolic state. A possible mechanism where energy-sensing and sleep-regulatory functions overlap is related to lipid metabolism. Fatty acid synthase (FAS) plays a central role in lipid metabolism as a key enzyme in the formation of long-chain fatty acids. We studied the effects of systemic administration of C75, an inhibitor of FAS, on sleep, behavioral activity and metabolic parameters in mice. Since the effects of C75 on feeding and metabolism are the opposite of ghrelin's and C75 suppresses ghrelin production, we also tested the role of ghrelin signaling in the actions of C75 by using ghrelin receptor knockout (KO) mice. After a transient increase in wakefulness, C75 elicited dose-dependent and long lasting inhibition of REMS, motor activity and feeding. Simultaneously, C75 significantly attenuated slow-wave activity of the electroencephalogram. Energy expenditure, body temperature and respiratory exchange ratio were suppressed. The diurnal rhythm of feeding was completely abolished by C75. There was significant correlation between the anorectic effects, the decrease in motor activity and the diminished energy expenditure after C75 injection. We found no significant difference between wild-type and ghrelin receptor KO mice in their sleep and metabolic responses to C75. The effects of C75 resemble to what was previously reported in association with visceral illness. Our findings suggest that sleep and metabolic effects of C75 in mice are independent of the ghrelin system and may be due to its aversive actions in mice.

Impaired wake-promoting mechanisms in ghrelin receptor-deficient mice.

Ghrelin receptors are expressed by key components of the arousal system. Exogenous ghrelin induces behavioral activation, promotes wakefulness and stimulates eating. We hypothesized that ghrelin-sensitive mechanisms play a role in the arousal system. To test this, we investigated the responsiveness of ghrelin receptor knockout (KO) mice to two natural wake-promoting stimuli. Additionally, we assessed the integrity of their homeostatic sleep-promoting system using sleep deprivation. There was no significant difference in the spontaneous sleep-wake activity between ghrelin receptor KO and wild-type (WT) mice. WT mice mounted robust arousal responses to a novel environment and food deprivation. Wakefulness increased for 6 h after cage change accompanied by increases in body temperature and locomotor activity. Ghrelin receptor KO mice completely lacked the wake and body temperature responses to new environment. When subjected to 48 h food deprivation, WT mice showed marked increases in their waking time during the dark periods of both days. Ghrelin receptor KO mice failed to mount an arousal response on the first night and wake increases were attenuated on the second day. The responsiveness to sleep deprivation did not differ between the two genotypes. These results indicate that the ghrelin-receptive mechanisms play an essential role in the function of the arousal system but not in homeostatic sleep-promoting mechanisms.

Energy homeostasis regulatory peptides in hibernating grizzly bears.

Grizzly bears (Ursus arctos horribilis) are inactive for up to 6 months during hibernation. They undergo profound seasonal changes in food intake, body mass, and energy expenditure. The circa-annual regulation of metabolism is poorly understood. In this study, we measured plasma ghrelin, leptin, obestatin, and neuropeptide-Y (NPY) levels, hormones known to be involved in the regulation of energy homeostasis, in ten grizzly bears. Blood samples were collected during the active summer period, early hibernation and late hibernation. Plasma levels of leptin, obestatin, and NPY did not change between the active and the hibernation periods. Plasma total ghrelin and desacyl-ghrelin concentrations significantly decreased during the inactive winter period compared to summer levels. The elevated ghrelin levels may help enhance body mass during pre-hibernation, while the low plasma ghrelin concentrations during hibernation season may contribute to the maintenance of hypophagia, low energy utilization and behavioral inactivity. Our results suggest that ghrelin plays a potential role in the regulation of metabolic changes and energy homeostasis during hibernation in grizzly bears.

ATP and the purine type 2 X7 receptor affect sleep.

Sleep is dependent upon prior brain activities, e.g., after prolonged wakefulness sleep rebound occurs. These effects are mediated, in part, by humoral sleep regulatory substances such as cytokines. However, the property of wakefulness activity that initiates production and release of such substances and thereby provides a signal for indexing prior waking activity is unknown. We propose that extracellular ATP, released during neuro- and gliotransmission and acting via purine type 2 (P2) receptors, is such a signal. ATP induces cytokine release from glia. Cytokines in turn affect sleep. We show here that a P2 receptor agonist, 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP), increased non-rapid eye movement sleep (NREMS) and electroencephalographic (EEG) delta power while two different P2 receptor antagonists, acting by different inhibitory mechanisms, reduced spontaneous NREMS in rats. Rat P2X7 receptor protein varied in the somatosensory cortex with time of day, and P2X7 mRNA was altered by interleukin-1 treatment, by sleep deprivation, and with time of day in the hypothalamus and somatosensory cortex. Mice lacking functional P2X7 receptors had attenuated NREMS and EEG delta power responses to sleep deprivation but not to interleukin-1 treatment compared with wild-type mice. Data are consistent with the hypothesis that extracellular ATP, released as a consequence of cell activity and acting via P2 receptors to release cytokines and other sleep regulatory substances, provides a mechanism by which the brain could monitor prior activity and translate it into sleep.

Restricted feeding-induced sleep, activity, and body temperature changes in normal and preproghrelin-deficient mice.

Behavioral and physiological rhythms can be entrained by daily restricted feeding (RF), indicating the existence of a food-entrainable oscillator (FEO). One manifestation of the presence of FEO is anticipatory activity to regularly scheduled feeding. In the present study, we tested if intact ghrelin signaling is required for FEO function by studying food anticipatory activity (FAA) in preproghrelin knockout (KO) and wild-type (WT) mice. Sleep-wake activity, locomotor activity, body temperature, food intake, and body weight were measured for 12 days in mice on a RF paradigm with food available only for 4 h daily during the light phase. On RF days 1-3, increases in arousal occurred. This response was significantly attenuated in preproghrelin KO mice. There were progressive changes in sleep architecture and body temperature during the subsequent nine RF days. Sleep increased at night and decreased during the light periods while the total daily amount of sleep remained at baseline levels in both KO and WT mice. Body temperature fell during the dark but was elevated during and after feeding in the light. In the premeal hours, anticipatory increases in body temperature, locomotor activity, and wakefulness were present from RF day 6 in both groups. Results indicate that the preproghrelin gene is not required for the manifestation of FAA but suggest a role for ghrelinergic mechanisms in food deprivation-induced arousal in mice.

The preproghrelin gene is required for the normal integration of thermoregulation and sleep in mice.

Peptidergic mechanisms controlling feeding, metabolism, thermoregulation, and sleep overlap in the hypothalamus. Low ambient temperatures and food restriction induce hypothermic (torpor) bouts and characteristic metabolic and sleep changes in mice. We report that mice lacking the preproghrelin gene, but not those lacking the ghrelin receptor, have impaired abilities to manifest and integrate normal sleep and thermoregulatory responses to metabolic challenges. In response to fasting at 17 degrees C (a subthermoneutral ambient temperature), preproghrelin knockout mice enter hypothermic bouts associated with reduced sleep, culminating in a marked drop in body temperature to near-ambient levels. Prior treatment with obestatin, another preproghrelin gene product, attenuates the hypothermic response of preproghrelin knockout mice. Results suggest that obestatin is a component in the coordinated regulation of metabolism and sleep during torpor.