Ghrelin Receptor Knockout Mice Research Articles

Effects of disrupted ghrelin receptor function on fear processing, anxiety and saccharin preference in mice

BackgroundObesity is a risk factor for stress-related mental disorders such as post-traumatic stress disorder. The underlying mechanism through which obesity affects mental health remains poorly understood but dysregulation of the ghrelin system may be involved. Stress increases plasma ghrelin levels, which stimulates food intake as a potential stress-coping mechanism. However, diet-induced obesity induces ghrelin resistance which in turn may have deleterious effects on stress-coping. In our study, we explored whether disruption of ghrelin receptor function though high-fat diet or genetic ablation affects fear processing, anxiety-like behavior and saccharin preference in mice. MethodsAdult male C57BL6/J mice were placed on a standard diet or high-fat diet for a total period of 8 weeks. We first established that high-fat diet exposure for 4 weeks elicits ghrelin resistance, evidenced by a blunted hyperphagic response following administration of a ghrelin receptor agonist. We then carried out an experiment in which we subjected mice to auditory fear conditioning after 4 weeks of diet exposure and evaluated effects on fear extinction, anxiety-like behavior and saccharin preference. To explore whether fear conditioning as such may influence the effect of diet exposure, we also subjected mice to auditory fear conditioning prior to diet onset and 4 weeks later we investigated auditory fear extinction, anxiety-like behavior and saccharin preference. In a final experiment, we further assessed lack of ghrelin receptor function by investigating auditory fear processing, anxiety-like behavior and saccharin preference in ghrelin receptor knockout mice and their wild-type littermates. ResultsHigh-fat diet exposure had no significant effect on auditory fear conditioning and its subsequent extinction or on anxiety-like behavior but significantly lowered saccharin preference. Similarly, ghrelin receptor knockout mice did not differ significantly from their wild-type littermates for auditory fear processing or anxiety-like behavior but showed significantly lower saccharin preference compared to wild-type littermates. ConclusionTaken together, our data suggest that disruption of ghrelin receptor function per se does not affect fear or anxiety-like behavior but may decrease saccharin preference in mice.

Anticonvulsant effect of a ghrelin receptor agonist in 6Hz corneally kindled mice.

Ghrelin has anticonvulsant and neuroprotective effects in models of chemoconvulsant-induced seizures and status epilepticus. In this study we investigated whether deletion of the ghrelin receptor could alter the kindling process in the 6 Hz corneal kindling model and whether ghrelin receptor ligands possess anticonvulsant effects in fully kindled mice. Ghrelin receptor wild-type and knockout mice were electrically stimulated at a subconvulsive current twice daily via corneal electrodes until they reached the fully kindled state. Mice lacking the ghrelin receptor showed similar seizure severity during kindling acquisition as well as in the maintenance phase when compared to their wild-type littermates. Subsequently we proceeded by investigating possible anticonvulsant effects of the ghrelin receptor ligands in the acute 6 Hz seizure model and the fully 6 Hz kindled mice. The ghrelin receptor agonist JMV-1843 decreased the seizure severity score both in acutely 6 Hz stimulated mice and in fully kindled ghrelin receptor wild-type mice, but not in fully kindled ghrelin receptor knockout mice. No effect on seizure severity was observed following the ghrelin receptor antagonist JMV-2959 in both models. This finding indicates that JMV-1843 exerts an anticonvulsant effect in kindled mice via the ghrelin receptor.

Ghrelin receptor modulates T helper cells during intestinal inflammation.

The orexigenic peptide ghrelin has anti-inflammatory properties in colitis, however, the mechanism of action and the immune cells targeted remain still to be elucidated. Here, we assessed the possible effect of ghrelin on T helper (Th) cells in a T cell transfer model of chronic colitis. Disease was induced in the recombination activating gene 1 knockout mice (Rag1(-/-) ) by adoptive transfer of naïve Th cells from ghrelin receptor knockout mice (GRLN-R(-/-) ) or littermate wild-type (WT) mice. The course and severity of colitis was assessed by monitoring body weight, diarrhea score, histological analysis, gene expression, and flow cytometry analysis. The possible effects of ghrelin on Th cell proliferation, polarization, and apoptosis was examined invitro. Our data showed that Rag1(-/-) mice injected with GRLN-R(-/-) Th cells displayed increased severity of colitis compared to mice injected with WT Th cells. In addition, Rag1(-/-) mice injected with GRLN-R(-/-) Th cells had significantly higher intestinal inflammation and increased accumulation of Th1 and Th17 cells in the colon. In vitro, ghrelin directly affected proliferation of Th cells and induced apoptosis whereas it did not influence Th cell polarization. Our observations suggest that ghrelin modulates Th effector cells in the gut controlling proliferation and inducing apoptosis. Our findings further support the use of ghrelin as a novel therapeutic option to treat intestinal inflammatory diseases.

Des-acyl ghrelin attenuates pilocarpine-induced limbic seizures via the ghrelin receptor and not the orexin pathway

Des-acyl ghrelin, widely accepted to work independently of the ghrelin receptor, is increasingly being implicated in a number of biological functions. The involvement of des-acyl ghrelin in epilepsy has only been recently reported. In this study, apart from unravelling the effect of des-acyl ghrelin on seizure thresholds and seizure severity in two models of pilocarpine-induced seizures, we mainly attempted to unravel its anticonvulsant mechanism of action. Since it was found that des-acyl ghrelin administration affected food intake via the orexin pathway, we first determined whether this pathway was responsible for des-acyl ghrelin's seizure-attenuating properties using the dual orexin receptor antagonist almorexant. We noted that, while des-acyl ghrelin showed dose-dependent anticonvulsant effects against focal pilocarpine-evoked seizures in rats, almorexant did not affect seizure severity and did not reverse des-acyl ghrelin's anticonvulsant effect. Subsequently, to investigate whether the ghrelin receptor was implicated in des-acyl ghrelin's anticonvulsant properties, we tested this peptide in ghrelin receptor deficient mice and wild type mice, all infused with pilocarpine intravenously. Unexpectedly, we found that des-acyl ghrelin significantly elevated seizure thresholds in C57Bl/6 and wild type mice but not in ghrelin receptor knock-out mice. Taken together, our results indicate the involvement of the ghrelin receptor in the anticonvulsant effects of des-acyl ghrelin on pilocarpine-induced seizures. We also show for the first time that dual antagonism of hippocampal orexin receptors does not affect seizure severity.

Cortistatin‐14 Mediates its Anticonvulsant Effects Via sst2 and sst3 but Not Ghrelin Receptors

Cortistatin (CST)-14, a neuropeptide that is structurally and functionally related to somatostatin-14 (SRIF) binds all five somatostatin receptor subtypes (sst1-sst5). Using in vivo microdialysis and telemetry-based electroencephalographic recordings, we provide the first experimental evidence for anticonvulsive effects of CST-14 in a pilocarpine-induced seizure model in rats and mice and for the involvement of sst2 and sst3 receptors in these anticonvulsant actions of CST-14. Both receptor subtypes are required for the anticonvulsant effects of CST-14 given that co-perfusion of a selective sst2 antagonist (cyanamid15486) or a selective sst3 antagonist (SST3-ODN-8) reversed anticonvulsant effect of CST-14, and this, independently of each other. Next, as the ghrelin receptor has been proposed as a target for the biological effects of CST-14, we used ghrelin receptor knockout mice and their wild type littermates to study the involvement of this receptor in the anticonvulsive actions of CST-14. Our results show a significant decrease in seizure duration in both genotypes when CST-14 treated mice were compared with corresponding control animals receiving only pilocarpine. In addition, this CST-14-induced decrease was comparable in both genotypes. We here thus provide the first evidence that ghrelin receptors are not involved in mediating anticonvulsant actions of CST-14 in vivo.

Gastric motility in ghrelin receptor knockout mice

The aim of this study was to investigate the effects and possible mechanisms of ghrelin receptor (GHS-R) deficiency on gastric motility in GHS-R deficient (Ghsr-/-) mice. Ghsr-/- and control (Ghsr+/+) mice were genotyped by PCR. The percentage of gastric emptying (GE%) was calculated following the intraperitoneal adminis-tration of ghrelin. Invitro, the contractile response of smooth muscle strips to ghrelin and electrical field stimulation (EFS) and the intraluminal pressure change of isolated stomach to carbachol were observed in an organ bath. The staining of nerve cells in the gastric muscle layer was performed by immunofluorescence. Delayed gastric emptying was observed in the Ghsr-/- mice; ghrelin enhanced the GE% in the Ghsr+/+ mice but had no effect on the GE% in the Ghsr-/- mice. Invitro, the response of the strips to ghrelin and EFS and the intraluminal pressure change to cabarchol was reduced in the Ghsr-/- mice. GHS-Rs were predominantly expressed on nerve cells in gastric muscle layers. The number of nerve cells was observed to be decreased in the Ghsr-/- mice. The delayed gastric emptying may relate to the loss of GHS-Rs and the reduction in the number of nerve cells in the gastric muscle layers of the GHS-R-deficient mice.

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.

Bitter taste receptors and α-gustducin regulate the secretion of ghrelin with functional effects on food intake and gastric emptying

Ghrelin is a hunger hormone with gastroprokinetic properties but the factors controlling ghrelin secretion from the stomach are unknown. Bitter taste receptors (T2R) and the gustatory G proteins, α-gustducin (gust) and α-transducin, are expressed in the gut and are involved in the chemosensation of nutrients. This study aimed to investigate whether T2R-agonists affect (i) ghrelin release via α-gustducin and (ii) food intake and gastric emptying via the release of ghrelin. The mouse stomach contains two ghrelin cell populations: cells containing octanoyl and desoctanoyl ghrelin, which were colocalized with α-gustducin and α-transducin, and cells staining for desoctanoyl ghrelin. Gavage of T2R-agonists increased plasma octanoyl ghrelin levels in WT mice but the effect was partially blunted in gust(-/-) mice. Intragastric administration of T2R-agonists increased food intake during the first 30 min in WT but not in gust(-/-) and ghrelin receptor knockout mice. This increase was accompanied by an increase in the mRNA expression of agouti-related peptide in the hypothalamus of WT but not of gust(-/-) mice. The temporary increase in food intake was followed by a prolonged decrease (next 4 h), which correlated with an inhibition of gastric emptying. The delay in emptying, which was partially counteracted by ghrelin, was not mediated by cholecystokinin and GLP-1 but involved a direct inhibitory effect of T2R-agonists on gastric contractility. This study is unique in providing functional evidence that activation of bitter taste receptors stimulates ghrelin secretion. Modulation of endogenous ghrelin levels by tastants may provide novel therapeutic applications for the treatment of weight -and gastrointestinal motility disorders.

Ghrelin receptor deficiency does not affect diet-induced atherosclerosis in low-density lipoprotein receptor-null mice

Objective: Ghrelin, a stomach-derived, secreted peptide, and its receptor (growth hormone secretagogue receptor, GHSR) are known to modulate food intake and energy homeostasis. The ghrelin system is also expressed broadly in cardiovascular tissues. Since ghrelin has been associated with anti-inflammatory and anti-atherogenic properties, but is also well known to promote obesity and impair glucose metabolism, we investigated whether ghrelin has any impact on the development of atherosclerosis. The hypothesis that endogenous ghrelin signaling may be involved in atherosclerosis has not been tested previously. Methods and Results: We crossed ghrelin receptor knockout mice (GHSr−/−) into a low-density lipoprotein receptor-null (Ldlr−/−) mouse line. In this model, atherosclerotic lesions were promoted by feeding a high-fat, high-cholesterol Western-type diet for 13 months, following a standard protocol. Body composition and glucose homeostasis were similar between Ldlr−/− and Ldlr/GHSR−/−ko mice throughout the study. Absence or presence of GHSr did not alter the apolipoprotein profile changes in response to diet exposure on an LDLRko background. Atherosclerotic plaque volume in the aortic arch and thoracic aorta were also not affected differentially in mice without ghrelin signaling due to GHSR gene disruption as compared to control LDLRko littermates. In light of the associations reported for ghrelin with cardiovascular disease in humans, the lack of a phenotype in these loss-of-function studies in mice suggests no direct role for endogenous ghrelin in either the inhibition or the promotion of diet-induced atherosclerosis. Conclusion: These data indicate that, surprisingly, the complex and multifaceted actions of endogenous ghrelin receptor mediated signaling on the cardiovascular system have minimal direct impact on atherosclerotic plaque progression as based on a loss-of-function mouse model of the disease.

Impact of food restriction and cocaine on locomotion in ghrelin- and ghrelin-receptor knockout mice

Food restriction (FR) augments the behavioral and reinforcing effects of psychomotor stimulants such as cocaine or amphetamine; effects that may be related to the capacity of FR to increase plasma levels of ghrelin (GHR), a 28-amino acid orexigenenic peptide linked to activation of brain dopamine systems. The present study used wild-type (WT) mice or mutant mice sustaining knockout of either GHR [GHR((-/-)) ] or of the growth hormone secretagogue receptor [GHS-R((-/-)) ] and subjected to FR or not to evaluate the role of GHR and GHS-R in cocaine-stimulated locomotion. WT, GHR((-/-)) , and GHS-R((-/-)) mice were either restricted to 60% of baseline caloric intake or allowed to free-feed (FF). Mice were treated with 0, 1.25, 2.5 and 5.0 mg/kg cocaine on separate test days (in random dose order) and forward locomotion was recorded on each drug day for 45 minutes after drug dosing. Food (and water) was available immediately after (but not during) each activity test. For FF mice, there was no interaction between cocaine and GHR status on locomotion. FR-WT mice treated with saline exhibited significant increases in anticipatory locomotion (relative to FF-WT mice), whereas FR-GHS-R((-/-)) mice did not. Cocaine significantly increased locomotion in FR-GHR((-/-)) and FR-GHS-R((-/-)) mice to the levels noted in FR-WT mice. These results suggest that GHS-R activity, but not GHR activity, is required for FR to augment food-associated anticipatory locomotion, but do not support the contention that GHR pathways are required for the capacity of FR to augment the acute effect of cocaine on locomotion.

Ghrelin Receptor Antagonism Decreases Alcohol Consumption and Activation of Perioculomotor Urocortin‐Containing Neurons

The current therapies for alcohol abuse disorders are not effective in all patients, and continued development of pharmacotherapies is needed. One approach that has generated recent interest is the antagonism of ghrelin receptors. Ghrelin is a gut-derived peptide important in energy homeostasis and regulation of hunger. Recent studies have implicated ghrelin in alcoholism, showing altered plasma ghrelin levels in alcoholic patients as well as reduced intakes of alcohol in ghrelin receptor knockout mice and in mice treated with ghrelin receptor antagonists. The aim of this study was to determine the neuroanatomical locus/loci of the effect of ghrelin receptor antagonism on alcohol consumption using the ghrelin receptor antagonist, D-Lys3-GHRP-6. In Experiment 1, male C57BL/6J mice were injected with saline 3 hours into the dark cycle and allowed access to 15% (v/v) ethanol or water for 2 hours in a 2-bottle choice experiment. On test day, the mice were injected with either saline or 400 nmol of the ghrelin receptor antagonist, D-Lys3-GHRP-6, and allowed to drink 15% ethanol or water for 4 hours. The preference for alcohol and alcohol intake were determined. In Experiment 2, the same procedure was followed as in Experiment 1 but mice were only allowed access to a single bottle of 20% ethanol (v/v), and alcohol intake was determined. Blood ethanol levels were analyzed, and immunohistochemistry for c-Fos was carried out to investigate changes in neural activity. To further elucidate the mechanism by which D-Lys3-GHRP-6 affects alcohol intake, in Experiment 3, the effect of D-Lys3-GHRP-6 on the neural activation induced by intraperitoneal ethanol was investigated. For the c-Fos studies, brain regions containing ghrelin receptors were analyzed, i.e. the perioculomotor urocortin population of neurons (pIIIu), the ventral tegmental area (VTA), and the arcuate nucleus (Arc). In Experiment 4, to test if blood ethanol concentrations were affected by D-Lys3-GHRP-6, blood samples were taken at 2 time-points after D-Lys3-GHRP-6 pretreatment and systemic ethanol administration. In Experiment 1, D-Lys3-GHRP-6 reduced preference to alcohol and in a follow-up experiment (Experiment 2) also dramatically reduced alcohol intake when compared to saline-treated mice. The resulting blood ethanol concentrations were lower in mice treated with the ghrelin receptor antagonist. Immunohistochemistry for c-Fos showed fewer immunopositive cells in the pIIIu of the antagonist-treated mice but no difference was seen in the VTA or Arc. In Experiment 3, D-Lys3-GHRP-6 reduced the induction of c-Fos by intraperitoneal ethanol in the pIIIu but had no effect in the VTA. In the Arc, there was a significant increase in the number of c-Fos immunopositive cells after D-Lys3-GHRP-6 administration, but the antagonist had no effect on ethanol-induced expression of c-Fos. D-Lys3-GHRP-6-pretreatment also did not affect the blood ethanol concentrations observed after a systemic injection of ethanol when compared to saline-pretreated mice (Experiment 4). These findings indicate that the action of ghrelin on the regulation of alcohol consumption may occur via the pIIIu.

Improved insulin sensitivity and metabolic flexibility in ghrelin receptor knockout mice

Stimulation of the ghrelin receptor (GhrR) by ghrelin results in a variety of metabolic changes including increased food intake, fat storage and insulin resistance. Loss of ghrelin signaling is protective against diet-induced obesity, suggesting that ghrelin plays a significant homeostatic role in conditions of metabolic stress. We examined glycemic control in GhrR −/− mice fed a high-fat diet, and used indirect calorimetry to assess fuel substrate usage and energy expenditure. GhrR −/− mice fed a high-fat diet had several measures of greater insulin sensitivity, including: lower fasted blood glucose and plasma insulin, lower %Hb A1c, lower insulin levels during glucose tolerance tests, and improved performance in hyperinsulinemic-euglycemic and hyperglycemic clamp studies. GhrR −/− mice fed a high-fat diet did not develop hepatic steatosis and had lower total cholesterol, relative to controls. Furthermore, GhrR −/− mice demonstrated a lower intestinal triglyceride secretion rate of dietary lipid. GhrR −/− mice have higher respiratory quotients (RQ), indicating a preference for carbohydrate as fuel. The range of RQ values was wider in GhrR −/− mice, indicating greater metabolic flexibility and insulin sensitivity in these animals. We therefore propose that loss of ghrelin signaling promotes insulin sensitivity and metabolic flexibility, and protects against several fatty diet-induced features of metabolic syndrome due to convergent changes in the intake, absorption and utilization of energy.

Characterization of adult ghrelin and ghrelin receptor knockout mice under positive and negative energy balance.

Ghrelin and the ghrelin receptor (GH secretagogue receptor, GHS-R), are believed to have important roles in energy homeostasis. We describe results from the first studies to be conducted in congenic (N10) adult ghrelin(-/-) and Ghsr(-/-) mice under conditions of both positive (high-fat diet) and negative (caloric restriction) energy balance. In contrast to results from young N2 mutant mice, changes in body weight and energy expenditure are not clearly distinguishable across genotypes. Although respiratory quotient was lower in mice fed a high-fat diet, no differences were evident between littermate wild-type and null genotypes. With normal chow, a modest decrease trend in respiratory quotient was detected in ghrelin(-/-) mice but not in Ghsr(-/-) mice. Under caloric restriction, the weight loss of ghrelin(-/-) and Ghsr(-/-) mice was identical to wild-type littermates, but blood glucose levels were significantly lower. We conclude that adult congenic ghrelin(-/-) and Ghsr(-/-) mice are not resistant to diet-induced obesity but under conditions of negative energy balance show impairment in maintaining glucose homeostasis. These results support our hypothesis that the primary metabolic function of ghrelin in adult mice is to modulate glucose sensing and insulin sensitivity, rather than directly regulate energy intake and energy expenditure.

Genetically engineered mice: a new paradigm to study gastric physiology

To summarize key aspects from recent research as well as review articles on the topic of genetic mouse models, particularly in knockout mice, that have considerably contributed to understanding the pathways and mechanisms underlying gastric physiology. A series of knockout mouse models has proven to be invaluable in elucidating the mechanism and validating the current model of acid secretion. The interaction between the gastrin-histamine and cholecystokinin-somatostatin pathways was identified using the genetic approach as being critical in regulating acid secretion. Curiously, neither ghrelin nor ghrelin receptor knockout mice displayed the expected lean phenotype. Importantly, the study of obestatin in GPR39 knockout mice could be misleading, as zinc rather than obestatin is the endogenous ligand for GPR39. The physiological roles of ghrelin and obestatin have yet to be confirmed using knockout mouse models. The knockout mouse continues to serve as an excellent model to dissect the complexity of the mechanism of gastric acid secretion and to study the physiological importance of gastric ghrelin.