Ghrelin Knockout Mice Research Articles

Germline and conditional ghrelin knockout increases islet size.

Conflicting studies in recent years report that genetic or pharmacological increases or decreases in ghrelin either increase or have no effect on islet size. In this issue of the JCI, Gupta, Burstein, and colleagues applied a rigorous approach to determine the effects of reducing ghrelin on islet size in germline and conditional ghrelin-knockout mice as well as across varying ages and weight. Both germline and conditional ghrelin-knockout mice associated with increased islet size, which was further exacerbated by older age and diet-induced obesity. These findings suggest that modulation of ghrelin may open a therapeutic window to prevent or treat diabetes.

THU309 Ghrelin Deficiency Does Not Improve Glucose Tolerance And Insulin Secretion In Female Mice

Abstract Disclosure: D. Gupta: None. K. Shankar: None. S. Varshney: None. O. Singh: None. S. Paul: None. S.B. Ogden: None. S. Osborne-Lawrence: None. A.W. Burstein: None. N.P. Metzger: None. C.P. Richard: None. J.M. Zigman: None. Background: Ghrelin is a peptide hormone secreted primarily from specialized enteroendocrine cells of the stomach. Ghrelin’s ability to regulate blood glucose has emerged as one of its well-recognized actions. For instance, administered ghrelin increases blood glucose in rodents and humans, while ghrelin-knockout (GKO) mice and ghrelin receptor-knockout (GHSR-KO) mice exhibit reduced blood glucose upon fasting, improved glucose tolerance when on regular chow or when on obesogenic diets, and frank hypoglycemia when chronically food restricted. Upon obesogenic diet or glucose challenge, plasma insulin is higher in GKO and GHSR-KO mice, suggesting that ghrelin affects blood glucose at least in part by inhibiting insulin secretion. Notably, ghrelin’s glucoregulatory actions, including its effects to alter insulin secretion, have almost exclusively been studied in males. Because sex significantly impacts the pathogenesis of metabolic disorders and type 2 diabetes, here we studied ghrelin’s effects on glucose tolerance and insulin secretion in female mice and in the setting of pregnancy. Methods: Separate cohorts of 12-13 week-old female GKO and wildtype (WT) littermates with 11.5 gestational days induced by timed pregnancy, and aged matched GKO and WT non-pregnant female mice were fasted for 6 h after which oral glucose tolerance test (OGTT) was performed. Briefly, mice were administered D-glucose (2 g/kg BW) by oral gavage at t = 0 min. Blood glucose was measured from nicked tails at t = 0, 15, 30, 60, 90 and 120 min and insulin was assayed in blood samples collected at t = 0, 15 and 30 min of glucose administration. Results: In OGTT, blood glucose increased significantly and similarly 15 min after oral glucose gavage in GKO and WT mice with or without pregnancy. Blood glucose AUC was 74.2% and 72.8% decreased with pregnancy in WT and GKO mice, respectively. No difference in plasma insulin was observed 15 min after glucose gavage in GKO mice vs WT mice with or without pregnancy. AUC of plasma insulin was significantly increased with pregnancy by 112.6% and by 123.8% in WT mice and GKO mice, respectively. Conclusions: With pregnancy, plasma insulin elevates (presumably as a means to maintain or improve glucose tolerance). Ghrelin deficiency during pregnancy is dispensable for this elevation in insulin and improvement in glucose tolerance. Ghrelin’s response to glucose tolerance and insulin secretion was blunted in female mice suggesting ghrelin’s actions on glucose tolerance and insulin secretion exhibit sexual diversity. Disclosure: The authors have no competing interest. Funding. This work was supported through research grants from the NIH (R01 DK103884 and R01 DK119341 to J.M.Z) Presentation: Thursday, June 15, 2023

Deletion of ghrelin alters tryptophan metabolism and exacerbates experimental ulcerative colitis in aged mice.

A major component of aging is chronic, low-grade inflammation, attributable in part by impaired gut barrier function. We previously reported that deletion of ghrelin, a peptidergic hormone released mainly from the gut, exacerbates experimental muscle atrophy in aged mice. In addition, ghrelin has been shown to ameliorate colitis in experimental models of inflammatory bowel disease (IBD), although the role of endogenous ghrelin in host-microbe interactions is less clear. Here, we showed that 22-month-old global ghrelin knockout (Ghrl-/-) mice exhibited significantly increased depressive-like behaviors, while anxiety levels and working memory were similar to littermate wild-type (WT) mice. Furthermore, old Ghrl-/- mice showed significantly increased intestinal permeability to fluorescein isothiocyanate (FITC)-dextran, significantly higher colonic interleukin (IL-1β) levels, and trends for higher colonic IL-6 and tumor necrosis factor-α (TNF-α) compared to WT mice. Interestingly, young Ghrl-/- and WT mice showed comparable depressive-like behavior and gut permeability, suggesting age-dependent exacerbation in gut barrier dysfunction in Ghrl-/- mice. While fecal short-chain fatty acids levels were comparable between old Ghrl-/- and WT mice, serum metabolome revealed alterations in metabolic cascades including tryptophan metabolism. Specifically, tryptophan and its microbial derivatives indole-3-acetic acid and indole-3-lactic acid were significantly reduced in old Ghrl-/-mice. Furthermore, in an experimental model of dextran sulfate sodium (DSS)-induced colitis, Ghrl-/- mice showed exacerbated disease symptoms, and higher levels of chemoattractant and pro-inflammatory cytokines in the colon. Overall, these data demonstrated that ghrelin deficiency is associated with gut barrier dysfunction, alterations in microbially derived tryptophan metabolites, and increased susceptibility to colitis. These data suggested that endogenous ghrelin contributes to maintaining a healthy host-microbe environment, ultimately impacting on brain function.

1449-P: Reducing Ghrelin Levels as a Novel Method to Increase Pancreatic Beta-Cell Mass

Objective: Exogenous insulin remains the leading therapy for type 1 diabetes mellitus, T1D. However, current insulin formulations and delivery methods do not match the precision of functioning beta-cells, and even intensive monitoring of blood glucose and use of insulin pumps are not enough to halt frequent hypoglycemia, hyperglycemia and morbidity. Thus, regenerating beta-cells has become a major target of diabetes research to treat T1D. While the hormone ghrelin is well known to increase blood glucose, at least in part by inhibiting insulin secretion, ghrelin’s action on beta-cell mass and proliferation has heretofore not been explored in detail. Here, we sought to test the hypothesis that reducing ghrelin levels increases beta-cell mass. Methods: Using histologic methods, we compared islet anatomy in ghrelin-knockout (ghrelin-KO) mice vs. wildtype littermates at three different ages: newborns (P0-P2) , juveniles (4-week-old) , and adults (10-12-week-old) . We also compared islet anatomy in adult wildtype mice, 4 weeks following conditional ghrelin-cell ablation vs. wildtype mice with intact ghrelin-cells. n= 4-8 mice per group and 33-433 islets per mouse. Results: Ghrelin gene deletion or ghrelin-cell ablation increased mean islet area and mean islet insulin-immunoreactive (insulin-IR) area by 52-82 % and by 45-51 %, respectively, in juveniles and adults. Increased islet size was driven by an increased % of large islets. Increased insulin-IR area was associated with increased beta-cell number per islet. An increased % of large islets was also observed in newborn ghrelin-KO mice. Conclusions: In mice, ghrelin plays an important role in controlling beta-cell mass. Reducing ghrelin levels for even short periods can increase islet size by increasing beta-cell numbers. Disclosure D.Gupta: None. C.P.Richard: None. J.M.Zigman: Research Support; Novo Nordisk Research Center Indianapolis, Novo Nordisk Research Center Indianapolis, Stock/Shareholder; Medtronic, Medtronic. A.W.Burstein: None. K.Shankar: None. S.Varshney: None. O.Singh: None. S.Paul: None. S.B.Ogden: None. S.Osborne-lawrence: None. N.Metzger: None.

Acyl-ghrelin Is Permissive for the Normal Counterregulatory Response to Insulin-Induced Hypoglycemia.

Insulin-induced hypoglycemia leads to far-ranging negative consequences in patients with diabetes. Components of the counterregulatory response (CRR) system that help minimize and reverse hypoglycemia and coordination between those components are well studied but not yet fully characterized. Here, we tested the hypothesis that acyl-ghrelin, a hormone that defends against hypoglycemia in a preclinical starvation model, is permissive for the normal CRR to insulin-induced hypoglycemia. Ghrelin knockout (KO) mice and wild-type (WT) littermates underwent an insulin bolus-induced hypoglycemia test and a low-dose hyperinsulinemic-hypoglycemic clamp procedure. Clamps also were performed in ghrelin-KO mice and C57BL/6N mice administered the growth hormone secretagogue receptor agonist HM01 or vehicle. Results show that hypoglycemia, as induced by an insulin bolus, was more pronounced and prolonged in ghrelin-KO mice, supporting previous studies suggesting increased insulin sensitivity upon ghrelin deletion. Furthermore, during hyperinsulinemic-hypoglycemic clamps, ghrelin-KO mice required a 10-fold higher glucose infusion rate (GIR) and exhibited less robust corticosterone and growth hormone responses. Conversely, HM01 administration, which reduced the GIR required by ghrelin-KO mice during the clamps, increased plasma corticosterone and growth hormone. Thus, our data suggest that endogenously produced acyl-ghrelin not only influences insulin sensitivity but also is permissive for the normal CRR to insulin-induced hypoglycemia.

Voluntary exercise is motivated by ghrelin, possibly related to the central reward circuit.

We previously reported that voluntary exercise contributed to the amelioration of abnormal feeding behavior with a concomitant restoration of ghrelin production in a rat model of obesity, suggesting a possible relationship between exercise and appetite-regulating hormones. Ghrelin is known to be involved in the brain reward circuits via dopamine neurons related to motivational properties. We investigated the relevance of ghrelin as an initiator of voluntary exercise as well as feeding behavior. The plasma ghrelin concentration fluctuates throughout the day with its peak at the beginning of the dark period in the wild-type (WT) mice with voluntary exercise. Although predominant increases in wheel running activity were observed accordant to the peak of plasma ghrelin concentration in the WT mice, those were severely attenuated in the ghrelin-knockout (GKO) mice under either ad libitum or time-restricted feeding. A single injection of ghrelin receptor agonist brought about and reproduced a marked enhancement of wheel running activity, in contrast to no effect by the continuous administration of the same drug. Brain dopamine levels (DAs) were enhanced after food consumption in the WT mice under voluntary exercise. Although the acceleration of DAs were apparently blunted in the GKO mice, they were dramatically revived after the administration of ghrelin receptor agonist, suggesting the relevance of ghrelin in the reward circuit under voluntary exercise. These findings emphasize that the surge of ghrelin plays a crucial role in the formation of motivation for the initiation of voluntary exercise possibly related to the central dopamine system.

Ghrelin knockout mice display defective skeletal muscle regeneration and impaired satellite cell self-renewal.

Muscle regeneration depends on satellite cells (SCs), quiescent precursors that, in consequence of injury or pathological states such as muscular dystrophies, activate, proliferate, and differentiate to repair the damaged tissue. A subset of SCs undergoes self-renewal, thus preserving the SC pool and its regenerative potential. The peptides produced by the ghrelin gene, i.e., acylated ghrelin (AG), unacylated ghrelin (UnAG), and obestatin (Ob), affect skeletal muscle biology in several ways, not always with overlapping effects. In particular, UnAG and Ob promote SC self-renewal and myoblast differentiation, thus fostering muscle regeneration. To delineate the endogenous contribution of preproghrelin in muscle regeneration, we evaluated the repair process in Ghrl-/- mice upon CTX-induced injury. Although muscles from Ghrl-/- mice do not visibly differ from WT muscles in term of weight, structure, and SCs content, muscle regeneration after CTX-induced injury is impaired in Ghrl-/- mice, indicating that ghrelin-derived peptides actively participate in muscle repair. Remarkably, the lack of ghrelin gene impacts SC self-renewal during regeneration. Although we cannot discern the specific Ghrl-derived peptide responsible for such activities, these data indicate that Ghrl contributes to a proper muscle regeneration.

Ghrelin deletion protects against age‐associated hepatic steatosis by downregulating the C/EBPα‐p300/DGAT1 pathway

SummaryNonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. NAFLD usually begins as low‐grade hepatic steatosis which further progresses in an age‐dependent manner to nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma in some patients. Ghrelin is a hormone known to promote adiposity in rodents and humans, but its potential role in hepatic steatosis is unknown. We hypothesized that genetic ghrelin deletion will protect against the development of age‐related hepatic steatosis. To examine this hypothesis, we utilized ghrelin knockout (KO) mice. Although no different in young animals (3 months old), we found that at 20 months of age, ghrelin KO mice have significantly reduced hepatic steatosis compared to aged‐matched wild‐type (WT) mice. Examination of molecular pathways by which deletion of ghrelin reduces steatosis showed that the increase in expression of diacylglycerol O‐acyltransferase‐1 (DGAT1), one of the key enzymes of triglyceride (TG) synthesis, seen with age in WT mice, is not present in KO mice. This was due to the lack of activation of CCAAT/enhancer binding protein‐alpha (C/EBPα) protein and subsequent reduction of C/EBPα‐p300 complexes. These complexes were abundant in livers of old WT mice and were bound to and activated the DGAT1 promoter. However, the C/EBPα‐p300 complexes were not detected on the DGAT1 promoter in livers of old KO mice resulting in lower levels of the enzyme. In conclusion, these studies demonstrate the mechanism by which ghrelin deletion prevents age‐associated hepatic steatosis and suggest that targeting this pathway may offer therapeutic benefit for NAFLD.

Suppression of Ghrelin Exacerbates HFCS-Induced Adiposity and Insulin Resistance.

High fructose corn syrup (HFCS) is widely used as sweetener in processed foods and soft drinks in the United States, largely substituting sucrose (SUC). The orexigenic hormone ghrelin promotes obesity and insulin resistance; ghrelin responds differently to HFCS and SUC ingestion. Here we investigated the roles of ghrelin in HFCS- and SUC-induced adiposity and insulin resistance. To mimic soft drinks, 10-week-old male wild-type (WT) and ghrelin knockout (Ghrelin−/−) mice were subjected to ad lib. regular chow diet supplemented with either water (RD), 8% HFCS (HFCS), or 10% sucrose (SUC). We found that SUC-feeding induced more robust increases in body weight and body fat than HFCS-feeding. Comparing to SUC-fed mice, HFCS-fed mice showed lower body weight but higher circulating glucose and insulin levels. Interestingly, we also found that ghrelin deletion exacerbates HFCS-induced adiposity and inflammation in adipose tissues, as well as whole-body insulin resistance. Our findings suggest that HFCS and SUC have differential effects on lipid metabolism: while sucrose promotes obesogenesis, HFCS primarily enhances inflammation and insulin resistance, and ghrelin confers protective effects for these metabolic dysfunctions.

Acyl ghrelin improves cognition, synaptic plasticity deficits and neuroinflammation following amyloid β (Aβ1-40) administration in mice.

Ghrelin is a metabolic hormone that has neuroprotective actions in a number of neurological conditions, including Parkinson's disease (PD), stroke and traumatic brain injury. Acyl ghrelin treatment in vivo and in vitro also shows protective capacity in Alzheimer's disease (AD). In the present study, we used ghrelin knockout (KO) and their wild-type littermates to test whether or not endogenous ghrelin is protective in a mouse model of AD, in which human amyloid β peptide 1-40 (Aβ1-40 ) was injected into the lateral ventricles i.c.v. Recognition memory, using the novel object recognition task, was significantly impaired in ghrelin KO mice and after i.c.v. Aβ1-40 treatment. These deficits could be prevented by acyl ghrelin injections for 7days. Spatial orientation, as assessed by the Y-maze task, was also significantly impaired in ghrelin KO mice and after i.c.v. Aβ1-40 treatment. These deficits could be prevented by acyl ghrelin injections for 7days. Ghrelin KO mice had deficits in olfactory discrimination; however, neither i.c.v. Aβ1-40 treatment, nor acyl ghrelin injections affected olfactory discrimination. We used stereology to show that ghrelin KO and Aβ1-40 increased the total number of glial fibrillary acidic protein expressing astrocytes and ionised calcium-binding adapter expressing microglial in the rostral hippocampus. Finally, Aβ1-40 blocked long-term potentiation induced by high-frequency stimulation and this effect could be acutely blocked with co-administration of acyl ghrelin. Collectively, our studies demonstrate that ghrelin deletion affects memory performance and also that acyl ghrelin treatment may delay the onset of early events of AD. This supports the idea that acyl ghrelin treatment may be therapeutically beneficial with respect to restricting disease progression in AD.

Caloric Restriction Protects against Lactacystin-Induced Degeneration of Dopamine Neurons Independent of the Ghrelin Receptor

Parkinson’s disease (PD) is a neurodegenerative disorder, characterized by a loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNc). Caloric restriction (CR) has been shown to exert ghrelin-dependent neuroprotective effects in the 1-methyl-4-phenyl-1,2,3,6-tetrathydropyridine (MPTP)-based animal model for PD. We here investigated whether CR is neuroprotective in the lactacystin (LAC) mouse model for PD, in which proteasome disruption leads to the destruction of the DA neurons of the SNc, and whether this effect is mediated via the ghrelin receptor. Adult male ghrelin receptor wildtype (WT) and knockout (KO) mice were maintained on an ad libitum (AL) diet or on a 30% CR regimen. After 3 weeks, LAC was injected unilaterally into the SNc, and the degree of DA neuron degeneration was evaluated 1 week later. In AL mice, LAC injection significanty reduced the number of DA neurons and striatal DA concentrations. CR protected against DA neuron degeneration following LAC injection. However, no differences were observed between ghrelin receptor WT and KO mice. These results indicate that CR can protect the nigral DA neurons from toxicity related to proteasome disruption; however, the ghrelin receptor is not involved in this effect.

Ghrelin is required for dietary restriction-induced enhancement of hippocampal neurogenesis: lessons from ghrelin knockout mice.

Neurogenesis occurs in the adult hippocampus and is enhanced by dietary restriction (DR), and neurogenesis enhancement is paralleled by circulating ghrelin level enhancement. We have previously reported that ghrelin modulates adult neurogenesis in the hippocampus. In order to investigate the possible role of ghrelin in DR-induced hippocampal neurogenesis in adult mice, ghrelin knockout (GKO) mice and wild-type (WT) mice were maintained for 3 months on DR or ad libitum (AL) diets. Protein levels of ghrelin in the stomach and the hippocampus were increased by DR in WT mice. One day after BrdU administration, the number of BrdU-labeled cells in the hippocampal dentate gyrus was decreased in GKO mice maintained on the AL diet. DR failed to alter the proliferation of progenitor cells in both WT and GKO mice. Four weeks after BrdU injection, the number of surviving cells in the dentate gyrus was decreased in AL-fed GKO mice. DR increased survival of newborn cells in WT mice, but not in GKO mice. Levels of brain-derived neurotrophic factor protein in the hippocampus were similar between WT and GKO mice, and were increased by DR both in WT and GKO mice. These results suggest that elevated levels of ghrelin during DR may have an important role in the enhancement of neurogenesis induced by DR.

Ghrelin stimulates proliferation, migration and differentiation of neural progenitors from the subventricular zone in the adult mice

Ghrelin has been shown to regulate neurogenesis in the hippocampus. The aim of this study was to investigate the possible influence of ghrelin on cell proliferation and neuroblast formation in the subventricular zone (SVZ) and rostral migratory system (RMS) and generation of interneurons in the olfactory bulb (OB). We found that ghrelin receptors were expressed in the SVZ–RMS–OB system. Ghrelin knockout (GKO) mice have fewer proliferating neural progenitor cells and neuroblasts in the SVZ, while ghrelin administration attenuated these changes. We also found that not only the number of BrdU-labeled cells but also the fraction of migratory neuroblasts in the RMS was decreased in the GKO mice compared with controls. Treatment of GKO mice with ghrelin restored these numbers to the wild-type control values. Far fewer BrdU/NeuN double-labeled cells were found in the OB of GKO mice than in wild-type mice 4weeks after labeling, which were increased by ghrelin replacement. GKO mice showed less numbers of BrdU/calbindin, BrdU/calretinin and BrdU/tyrosine hydroxylase double-labeled cells in the periglomerular layer of the OB. However, these numbers were increased to wild-type values after ghrelin administration. Finally, in the GH-deficient spontaneous dwarf rats, ghrelin increased the number of progenitor cells and neuroblasts in the SVZ, without significant effect on the differentiation in the OB. These findings suggest that ghrelin is involved in the regulation of proliferation of progenitor cells in the SVZ, the number of migratory neuroblasts in the SVZ, and the differentiation of interneurons in the OB.

Hexarelin Treatment in Male Ghrelin Knockout Mice after Myocardial Infarction

Both ghrelin and the synthetic analog hexarelin are reported to possess cardioprotective actions that are mainly exerted through different receptors. However, their effects on acute myocardial infarction have not been compared in vivo. This study aimed to clarify whether hexarelin treatment can compensate for ghrelin deficiency in ghrelin-knockout mice and to compare the effects of hexarelin (400 nmol/kg/d, sc) and equimolar ghrelin treatment after myocardial infarction. Myocardial infarction was produced by left coronary artery ligation in male ghrelin-knockout mice, which then received ghrelin, hexarelin, or vehicle treatment for 2 weeks. The mortality within 2 weeks was significantly lower in the hexarelin group (6.7%) and ghrelin group (14.3%) than in the vehicle group (50%) (P < .05). A comparison of cardiac function 2 weeks after infarction showed that in the ghrelin and hexarelin treatment groups, cardiac output was greater, whereas systolic function, represented by ejection fraction, and diastolic function, represented by dP/dt min (peak rate of pressure decline), were significantly superior compared with the vehicle group (P < .05). Hexarelin treatment was more effective than ghrelin treatment, as indicated by the ejection fraction, dP/dt max (peak rate of pressure rise), and dP/dt min. Telemetry recording and heart rate variability analysis demonstrated that sympathetic nervous activity was clearly suppressed in the hexarelin and ghrelin groups relative to the vehicle group. Our data demonstrated that hexarelin treatment can result in better heart function than ghrelin treatment 2 weeks after myocardial infarction in ghrelin-knockout mice, although both hormones have similar effects on heart rate variability and mortality.

Excessive Sympathoactivation and Deteriorated Heart Function After Myocardial Infarction in Male Ghrelin Knockout Mice

We have previously demonstrated the protective role of endogenous ghrelin against malignant arrhythmias in the very acute phase of myocardial infarction (MI). However, the role of endogenous ghrelin in the chronic phase is unknown. Therefore, the aim of the current study was to focus on the effects of endogenous ghrelin on cardiac function and sympathetic activation after acute MI. In 46 ghrelin-knockout (KO) and 41 wild-type (WT) male mice, MI was produced by left coronary artery ligation. The mortality due to heart failure within 2 weeks was 0% in WT and 10.9% in KO (P < 0.05). At the end of this period, lung weight/tibial length, atrial natriuretic peptide and brain natriuretic peptide transcripts, end-systolic and end-diastolic volumes were all significantly greater in KO mice, whereas systolic function, represented by ejection fraction (16.4 ± 4.7% vs 25.3 ± 5.1%), end-systolic elastance, and preload-recruitable stroke work, was significantly inferior to that in WT mice (P < 0.05). Telemetry recording and heart rate variability analysis showed that KO mice had stronger sympathetic activation after MI than did WT mice. Metoprolol treatment and ghrelin treatment in KO mice prevented excessive sympathetic activation, decreased plasma epinephrine and norepinephrine levels, and improved heart function and survival rate after MI. Our data demonstrate that endogenous ghrelin plays a crucial role in protecting heart function and reducing mortality after myocardial infarction, and that these effects seem to be partly the result of sympathetic inhibition.

Ghrelin knockout mice show decreased voluntary alcohol consumption and reduced ethanol-induced conditioned place preference

Recent work suggests that stomach-derived hormone ghrelin receptor (GHS-R1A) antagonism may reduce motivational aspects of ethanol intake. In the current study we hypothesized that the endogenous GHS-R1A agonist ghrelin modulates alcohol reward mechanisms. For this purpose ethanol-induced conditioned place preference (CPP), ethanol-induced locomotor stimulation and voluntary ethanol consumption in a two-bottle choice drinking paradigm were examined under conditions where ghrelin and its receptor were blocked, either using ghrelin knockout (KO) mice or the specific ghrelin receptor (GHS-R1A) antagonist “JMV2959”. We showed that ghrelin KO mice displayed lower ethanol-induced CPP than their wild-type (WT) littermates. Consistently, when injected during CPP-acquisition, JMV2959 reduced CPP-expression in C57BL/6 mice. In addition, ethanol-induced locomotor stimulation was lower in ghrelin KO mice. Moreover, GHS-R1A blockade, using JMV2959, reduced alcohol-stimulated locomotion only in WT but not in ghrelin KO mice. When alcohol consumption and preference were assessed using the two-bottle choice test, both genetic deletion of ghrelin and pharmacological antagonism of the GHS-R1A (JMV2959) reduced voluntary alcohol consumption and preference. Finally, JMV2959-induced reduction of alcohol intake was only observed in WT but not in ghrelin KO mice. Taken together, these results suggest that ghrelin neurotransmission is necessary for the stimulatory effect of ethanol to occur, whereas lack of ghrelin leads to changes that reduce the voluntary intake as well as conditioned reward by ethanol. Our findings reveal a major, novel role for ghrelin in mediating ethanol behavior, and add to growing evidence that ghrelin is a key mediator of the effects of multiple abused drugs.

Ghrelin regulates hypothalamic prolyl carboxypeptidase expression in mice

Hypothalamic Prolyl carboxypeptidase (PRCP) plays a role in the regulation of energy metabolism by inactivating hypothalamic α-melanocyte stimulating hormone (α-MSH) levels and thus affecting melanocortin signaling. Alpha-MSH production is highly regulated both at transcriptional and posttranslational levels. Here we show that fasting induces a hypothalamic-specific up-regulation of Prcp mRNA and protein levels. Since fasting is characterized by elevated circulating ghrelin levels, we tested the effect of peripheral and central administration of ghrelin, and found that ghrelin increases hypothalamic Prcp mRNA expression. No changes in Prcp mRNA levels were detected in ghrelin knockout mice compared to their controls. Finally, ghrelin effect on PRCP expression was ghrelin receptor-mediated. Altogether our data show that ghrelin is a key regulator of hypothalamic PRCP expression, and up-regulation of PRCP by ghrelin may be an additional mechanism to decrease melanocortin signaling.

Calorie-Restricted Weight Loss Reverses High-Fat Diet-Induced Ghrelin Resistance, Which Contributes to Rebound Weight Gain in a Ghrelin-Dependent Manner

Twelve weeks of high-fat diet feeding causes ghrelin resistance in arcuate neuropeptide Y (NPY)/agouti-related protein (AgRP) neurons. In the current study, we investigated whether diet-induced weight loss could restore NPY/AgRP neuronal responsiveness to ghrelin and whether ghrelin mediates rebound weight gain after calorie-restricted (CR) weight loss. Diet-induced obese (DIO) mice were allocated to one of two dietary interventions until they reached the weight of age-matched lean controls. DIO mice received chow diet ad libitum or chow diet with 40% CR. Chow-fed and high-fat-fed mice served as controls. Both dietary interventions normalized body weight, glucose tolerance, and plasma insulin. We show that diet-induced weight loss with CR increases total plasma ghrelin, restores ghrelin sensitivity, and increases hypothalamic NPY and AgRP mRNA expression. We propose that long-term DIO creates a higher body weight set-point and that weight loss induced by CR, as seen in the high-fat CR group, provokes the brain to protect the new higher set-point. This adaptation to weight loss likely contributes to rebound weight gain by increasing peripheral ghrelin concentrations and restoring the function of ghrelin-responsive neuronal populations in the hypothalamic arcuate nucleus. Indeed, we also show that DIO ghrelin-knockout mice exhibit reduced body weight regain after CR weight loss compared with ghrelin wild-type mice, suggesting ghrelin mediates rebound weight gain after CR weight loss.

Ghrelin directly stimulates adult hippocampal neurogenesis: implications for learning and memory

Adult hippocampal neurogenesis is important in mediating hippocampal-dependent learning and memory. Exogenous ghrelin is known to stimulate progenitor cell proliferation in the dentate gyrus of adult hippocampus. The aim of this study was to investigate the role of endogenous ghrelin in regulating the in vivo proliferation and differentiation of the newly generating cells in the adult hippocampus using ghrelin knockout (GKO) mice. Targeted deletion of ghrelin gene resulted in reduced numbers of progenitor cells in the subgranular zone (SGZ) of the hippocampus, while ghrelin treatment restored progenitor cell numbers to those of wild-type controls. We also found that not only the number of bromodeoxyuridine (BrdU)-positive cells but also the fraction of immature neurons and newly generated neurons were decreased in the GKO mice, which were increased by ghrelin replacement. Additionally, in the GKO mice, we observed impairment of memory performance in Y-maze task and novel object recognition test. However, these functional deficiencies were attenuated by ghrelin administration. These results suggest that ghrelin directly induces proliferation and differentiation of adult neural progenitor cells in the SGZ. Our data suggest ghrelin may be a plausible therapeutic potential to enhance learning and memory processes.

Profound Hypoglycemia in Starved, Ghrelin-deficient Mice Is Caused by Decreased Gluconeogenesis and Reversed by Lactate or Fatty Acids

When mice are subjected to 7-day calorie restriction (40% of normal food intake), body fat disappears, but blood glucose is maintained as long as the animals produce ghrelin, an octanoylated peptide that stimulates growth hormone secretion. Mice can be rendered ghrelin-deficient by knock-out of the gene encoding either ghrelin O-acyltransferase, which attaches the required octanoate, or ghrelin itself. Calorie-restricted, fat-depleted ghrelin O-acyltransferase or ghrelin knock-out mice fail to show the normal increase in growth hormone and become profoundly hypoglycemic when fasted for 18-23 h. Glucose production in Goat(-/-) mice was reduced by 60% when compared with similarly treated WT mice. Plasma lactate and pyruvate were also low. Injection of lactate, pyruvate, alanine, or a fatty acid restored blood glucose in Goat(-/-) mice. Thus, when body fat is reduced by calorie restriction, ghrelin stimulates growth hormone secretion, which allows maintenance of glucose production, even when food intake is eliminated. In humans with anorexia nervosa or kwashiorkor, ghrelin and growth hormone are known to be elevated, just as they are in fat-depleted mice. We suggest that these two hormones prolong survival in starved humans as they do in mice.