Obestatin improves memory performance and causes anxiolytic effects in rats

Obestatin is a peptide hormone that is derived from the same polypeptide precursor (preprogrelin) as ghrelin, but it acts in opposing way on ingestive behavior. Our previous studies showed that ghrelin affects memory and anxiety. Here, we studied the possible effects of icv obestatin injection in rats upon memory retention (using two different paradigms), anxiety like behavior (plus maze test), and food intake. Obestatin induces an increase in the percentage of open arms entries (Obestatin 3.0nmol/rat: 61.74+/-1.81), and percentage of time spent in open arms (Obestatin 3.0nmol/rat: 72.07+/-4.21) in relation to the control (33.31+/-1.54; 25.82+/-1.68), indicating an anxiolytic effect. The two doses of obestatin increased latency time in a step down test and the percentage time of novel object exploration, suggesting that the peptide influences learning and memory processes that involve the hippocampus and the amygdala. This report provides evidence indicating that obestatin effects are functionally opposite on anxiety and hunger to the ghrelin effects, while both these related peptides increase memory retention.     

Ghrelin increases anxiety-like behavior and memory retention in rats

Ghrelin is a peptide found in the hypothalamus and stomach that stimulates food intake and whose circulating concentrations are affected by nutritional state. Very little is known about other central behavioral effects of ghrelin, and thus, we investigated the effects of ghrelin on anxiety and memory retention. The peptide was injected intracerebroventricularly in rats and we performed open-field, plus-maze, and step-down tests (inhibitory avoidance). The administration of ghrelin increased freezing in the open field and decreased the number of entries into the open spaces and the time spent on the open arms in the plus-maze, indicating an anxiogenic effect. Moreover, the peptide increased in a dose-dependent manner the latency time in the step-down test. A rapid and prolonged increase in food intake was also observed. Our results indicate that ghrelin induces anxiogenesis in rats. Moreover, we show for the first time that ghrelin increases memory retention, suggesting that the peptide may influence processes in the hippocampus.     

Peripheral ghrelin injections stimulate food intake, foraging, and food hoarding in Siberian hamsters

Fasting triggers many effects, including increases in circulating concentrations of ghrelin, a primarily stomach-derived orexigenic hormone. Exogenous ghrelin treatment stimulates food intake, implicating it in fasting-induced increases in feeding, a consummatory ingestive behavior. In Siberian hamsters, fasting also stimulates appetitive ingestive behaviors such as foraging and food hoarding. Therefore, we tested whether systemic ghrelin injections (3, 30, and 200 mg/kg) would stimulate these appetitive behaviors using a running wheel-based food delivery system coupled with simulated burrow housing. We also measured active ghrelin plasma concentrations after exogenous ghrelin treatment and compared them to those associated with fasting. Hamsters had the following: 1) no running wheel access, free food; 2) running wheel access, free food; or 3) foraging requirement (10 revolutions/pellet), no free food. Ghrelin stimulated foraging at 0-1, 2-4, and 4-24 h postinjection but failed to affect wheel running activity not coupled to food. Ghrelin stimulated food intake initially (200-350%, first 4 h) across all groups; however, in hamsters with a foraging requirement, ghrelin also stimulated food intake 4-24 h postinjection (200-250%). Ghrelin stimulated food hoarding 2-72 h postinjection (100-300%), most markedly 2-4 h postinjection in animals lacking a foraging requirement (635%). Fasting increased plasma active ghrelin concentrations in a time-dependent fashion, with the 3- and 30-mg/kg dose creating concentrations of the peptide comparable to those induced by 24-48 h of fasting. Collectively, these data suggest that exogenous ghrelin, similar to fasting, increases appetitive behaviors (foraging, hoarding) by Siberian hamsters, but dissimilar to fasting in this species, stimulates food intake.     

Lateral ventricular ghrelin and fourth ventricular ghrelin induce similar increases in food intake and patterns of hypothalamic gene expression

The gut peptide ghrelin has been shown to stimulate food intake after both peripheral and central administration, and the hypothalamic arcuate nucleus has been proposed to be the major site for mediating this feeding stimulatory action. Ghrelin receptors are widely distributed in the brain, and hindbrain ghrelin administration has been shown to potently stimulate feeding, suggesting that there may be other sites for ghrelin action. In the present study, we have further assessed potential sites for ghrelin action by comparing the ability of lateral and fourth ventricular ghrelin administration to stimulate food intake and alter patterns of hypothalamic gene expression. Ghrelin (0.32, 1, or 3.2 nmol) in the lateral or fourth ventricle significantly increased food intake in the first 4 h after injection, with no ventricle-dependent differences in degree or time course of hyperphagia. One nanomole of ghrelin into either the lateral or fourth ventricle resulted in similar increases in arcuate nucleus neuropeptide Y mRNA expression. Expression levels of agouti-related peptide or proopiomelanocortin mRNA were not affected by ghrelin administration. These data demonstrate that ghrelin can affect food intake and hypothalamic gene expression through interactions at multiple brain sites.     

Ghrelin microinjection into forebrain sites induces wakefulness and feeding in rats

Ghrelin, a gut-brain peptide, is best known for its role in the stimulation of feeding and growth hormone release. In the brain, orexin, neuropeptide Y (NPY), and ghrelin are parts of a food intake regulatory circuit. Orexin and NPY are also implicated in maintaining wakefulness. Previous experiments in our laboratory revealed that intracerebroventricular injections of ghrelin induce wakefulness in rats. To further elucidate the possible role of ghrelin in the regulation of arousal, we studied the effects of microinjections of ghrelin into hypothalamic sites, which are implicated in the regulation of feeding and sleep, such as the lateral hypothalamus (LH), medial preoptic area (MPA), and paraventricular nucleus (PVN) on sleep in rats. Sleep responses, motor activity, and food intake after central administration of 0.04, 0.2, or 1 mug (12, 60, or 300 pmol) ghrelin were recorded. Microinjections of ghrelin into the LH had strong wakefulness-promoting effects lasting for 2 h. Wakefulness was also stimulated by ghrelin injection into the MPA and PVN; the effects were confined to the first hour after the injection. Ghrelin's non-rapid-eye-movement sleep-suppressive effect was accompanied by attenuation in the electroencephalographic (EEG) slow-wave activity and changes in the EEG power spectrum. Food consumption was significantly stimulated after microinjections of ghrelin into each hypothalamic site. Together, these results are consistent with the hypothesis that forebrain ghrelinergic mechanisms play a role in the regulation of vigilance, possibly through activating the components of the food intake- and arousal-promoting network formed by orexin and NPY.     

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.     

Selective serotonin reuptake inhibitor (fluoxetine) decreases the effects of ghrelin on memory retention and food intake

Ghrelin (Ghr) is an appetite stimulating hormone that is produced peripherally, by the stomach, and centrally as well. Previous investigations show that Ghr increases food intake and memory retention in rats, and that extra-hypothalamic structures, such as the hippocampus, participate in these effects. In the present work we analyzed the effect on food intake and memory retention induced by Ghr after serotonin (5-HT) availability modification at the serotoninergic synapses. Animals only treated with a selective serotonin reuptake inhibitor (SSRI), fluoxetine (FLU) 5 mg/kg or clomipramine (CLO) 2.5 and 5 mg/kg, showed a significant reduction in both food intake and memory retention. On the contrary, Ghr administration induces a significant increase in food intake and a dose-dependent increase in short and long term memory retention. When the animals were treated with FLU prior to Ghr injection, the food intake induced, as well as the expression of short and long term memory retention, was decreased. In conclusion, evidence presented in this paper suggests that the effects of Ghr on both feeding and memory retention in extra-hypothalamic structures such as the hippocampus, could depend on the availability of 5-HT.     

The Amygdala as a Neurobiological Target for Ghrelin in Rats: Neuroanatomical,Electrophysiological and Behavioral Evidence

Here, we sought to demonstrate that the orexigenic circulating hormone, ghrelin, is able to exert neurobiological effects (including those linked to feeding control) at the level of the amygdala, involving neuroanatomical, electrophysiological and behavioural studies. We found that ghrelin receptors (GHS-R) are densely expressed in several subnuclei of the amygdala, notably in ventrolateral (LaVL) and ventromedial (LaVM) parts of the lateral amygdaloid nucleus. Using whole-cell patch clamp electrophysiology to record from cells in the lateral amygdaloid nucleus, we found that ghrelin reduced the frequency of mEPSCs recorded from large pyramidal-like neurons, an effect that could be blocked by co-application of a ghrelin receptor antagonist. In ad libitum fed rats, intra-amygdala administration of ghrelin produced a large orexigenic response that lasted throughout the 4 hr of testing. Conversely, in hungry, fasted rats ghrelin receptor blockade in the amygdala significantly reduced food intake. Finally, we investigated a possible interaction between ghrelin''s effects on feeding control and emotional reactivity exerted at the level of the amygdala. In rats allowed to feed during a 1-hour period between ghrelin injection and anxiety testing (elevated plus maze and open field), intra-amygdala ghrelin had no effect on anxiety-like behavior. By contrast, if the rats were not given access to food during this 1-hour period, a decrease in anxiety-like behavior was observed in both tests. Collectively, these data indicate that the amygdala is a valid target brain area for ghrelin where its neurobiological effects are important for food intake and for the suppression of emotional (anxiety-like) behaviors if food is not available.     

Leptin downregulates ghrelin levels in streptozotocin-induced diabetic mice

Ghrelin, an orexigenic peptide produced in the stomach, is increased in streptozotocin (STZ)-induced diabetic (DM) mice. This study clarifies the regulation of ghrelin levels by leptin in STZ-DM mice. STZ-DM mice had higher plasma ghrelin concentrations and greater ghrelin mRNA expression than control mice. Changes in ghrelin levels were dose dependently attenuated by the subcutaneous injection of leptin (0-27 nmol x kg(-1) x day(-1) over 7 days). Leptin treatment also partially reversed the hyperphagia and hyperglycemia observed in STZ-DM mice, but not the hypoinsulinemia, and there was a decrease in plasma ghrelin concentrations and ghrelin mRNA levels compared with STZ-LEP pair-fed mice. These results indicate that leptin treatment partially reverses elevated plasma ghrelin levels in STZ-DM mice independent of food intake and insulin, and suggest that hypoleptinemia in STZ-DM mice upregulates ghrelin.     

Stimulatory effect of n-octanoylated ghrelin on locomotor activity in the goldfish, Carassius auratus

Ghrelin is implicated in growth and feeding regulation in fish. The influence of ghrelin on behavior has not been well studied and the physiological role of des-fatty acid modification of this peptide is unclear. Therefore, the effects of intracerebroventricular (ICV) and intraperitoneal (IP) administration of synthetic n-octanoylated (acyl) goldfish ghrelin and des-n-octanoylated (des-acyl) ghrelin on locomotor and orexigenic activity in the goldfish were examined. ICV administration of acyl ghrelin at doses of 1 and 2 pmol/g body weight (BW) and IP administration at 16 pmol/g BW both induced significant increases in locomotor activity during for 45-60 min after treatment. Cumulative food intake was significantly increased by ICV injection of acyl ghrelin at doses of 1 and 2 pmol/g BW and IP injection at 8 and 16 pmol/g BW during the 60-min post-treatment observation period. In contrast, ICV and IP administration of des-acyl ghrelin produced no changes in locomotor and orexigenic activity. We also analyzed fasting-induced changes in the expression of ghrelin mRNA in the brain and intestine using a real-time PCR method. The level of ghrelin mRNA in the intestine, but not in the brain, obtained from fish fasted for 7 days was significantly higher than that in fish that had been fed normally. These results suggest that, in the goldfish, acyl ghrelin, but not des-acyl ghrelin, stimulates locomotor activity and enhances food intake via central and peripheral pathways.