A long-acting LEAP2 analog reduces hepatic steatosis and inflammation and causes marked weight loss in mice.

OBJECTIVE: The number of individuals affected by metabolic dysfunction associated fatty liver disease [1] is on the rise, yet hormonal contributors to the condition remain incompletely described and only a single FDA-approved treatment is available. Some studies suggest that the hormones ghrelin and LEAP2, which act as agonist and antagonist/inverse agonist, respectively, for the G protein coupled receptor GHSR, may influence the development of MAFLD. For instance, ghrelin increases hepatic fat whereas synthetic GHSR antagonists do the opposite. Also, hepatic steatosis is less prominent in standard chow-fed ghrelin-KO mice but more prominent in 42% high-fat diet-fed female LEAP2-KO mice. METHODS: Here, we sought to determine the therapeutic potential of a long-acting LEAP2 analog (LA-LEAP2) to treat MAFLD in mice. LEAP2-KO and wild-type littermate mice were fed a Gubra-Amylin-NASH (GAN) diet for 10 or 40 wks, with some randomized to an additional 28 or 10 days of GAN diet, respectively, while treated with LA-LEAP2 vs Vehicle. Various metabolic parameters were followed and biochemical and histological assessments of MAFLD were made. RESULTS: Among the most notable metabolic effects, daily LA-LEAP2 administration to both LEAP2-KO and wild-type littermates during the final 4 wks of a 14 wk-long GAN diet challenge markedly reduced liver weight, hepatic triglycerides, plasma ALT, hepatic microvesicular steatosis, hepatic lobular inflammation, NASH activity scores, and prevalence of higher-grade fibrosis. These changes were accompanied by prominent reductions in body weight, without effects on food intake, and reduced plasma total cholesterol. Daily LA-LEAP2 administration during the final 10 d of a 41.5 wk-long GAN diet challenge also reduced body weight, plasma ALT, and plasma total cholesterol in LEAP2-KO and wild-type littermates and prevalence of higher grade fibrosis in LEAP2-KO mice. CONCLUSIONS: Administration of LA-LEAP2 to mice fed a MAFLD-prone diet markedly improves several facets of MAFLD, including hepatic steatosis, hepatic lobular inflammation, higher-grade hepatic fibrosis, and transaminitis. These changes are accompanied by prominent reductions in body weight and lowered plasma total cholesterol. Taken together, these data suggest that LEAP2 analogs such as LA-LEAP2 hold promise for the treatment of MAFLD and obesity.

Impact of Ghrelin on Islet Size in Nonpregnant and Pregnant Female Mice.

Reducing ghrelin by ghrelin gene knockout (GKO), ghrelin-cell ablation, or high-fat diet feeding increases islet size and ß-cell mass in male mice. Here we determined if reducing ghrelin also enlarges islets in females and if pregnancy-associated changes in islet size are related to reduced ghrelin. Islet size and ß-cell mass were larger (P = .057 for ß-cell mass) in female GKO mice. Pregnancy was associated with reduced ghrelin and increased liver-expressed antimicrobial peptide-2 (LEAP2; a ghrelin receptor antagonist) in wild-type mice. Ghrelin deletion and pregnancy each increased islet size (by ∼19.9-30.2% and ∼34.9-46.4%, respectively), percentage of large islets (>25 µm2×103, by ∼21.8-42% and ∼21.2-41.2%, respectively), and ß-cell mass (by ∼15.7-23.8% and ∼65.2-76.8%, respectively). Neither islet cross-sectional area, ß-cell cross-sectional area, nor ß-cell mass correlated with plasma ghrelin, although all positively correlated with LEAP2 (P = .081 for islet cross-sectional area). In ad lib-fed mice, there was an effect of pregnancy, but not ghrelin deletion, to change (raise) plasma insulin without impacting blood glucose. Similarly, there was an effect of pregnancy, but not ghrelin deletion, to change (lower) blood glucose area under the curve during a glucose tolerance test. Thus, genetic deletion of ghrelin increases islet size and ß-cell cross-sectional area in female mice, similar to males. Yet, despite pregnancy-associated reductions in ghrelin, other factors appear to govern islet enlargement and changes to insulin sensitivity and glucose tolerance in the setting of pregnancy. In the case of islet size and ß-cell mass, one of those factors may be the pregnancy-associated increase in LEAP2.

Ghrelin enhances tubular magnesium absorption in the kidney.

Osteoporosis after bariatric surgery is an increasing health concern as the rate of bariatric surgery has risen. In animal studies mimicking bariatric procedures, bone disease, together with decreased serum levels of Ca2+, Mg2+ and the gastric hormone Ghrelin were described. Ghrelin regulates metabolism by binding to and activating the growth hormone secretagogue receptor (GHSR) which is also expressed in the kidney. As calcium and magnesium are key components of bone, we tested the hypothesis that Ghrelin-deficiency contributes to osteoporosis via reduced upregulation of the renal calcium channel TRPV5 and the heteromeric magnesium channel TRPM6/7. We expressed GHSR with TRPV5 or TRPM6/7 channel in HEK293 cells and treated them with purified Ghrelin. Whole-cell current density was analyzed by patch-clamp recording. Nephron-specific gene expression was performed by tubular microdissection followed by qPCR in wild-type (WT) mice, and immunofluorescent imaging of GHSR-eGFP mice. Tubular magnesium homeostasis was analyzed in GHSR-null and WT mice at baseline and after caloric restriction. After Ghrelin exposure, whole-cell current density did not change for TRPV5 but increased for TRPM6/7 in a dose-dependent fashion. Applying the Ghrelin-mimetic (D-Trp7, Ala8,D-Phe10)-α-MSH (6-11) amide without and with the GHSR antagonist (D-Lys3)-GHRP6, we confirmed the stimulatory role of Ghrelin towards TRPM6/7. As GHSR initiates downstream signaling via protein kinase A (PKA), we found that the PKA inhibitor H89 abrogated TRPM6/7 stimulation by Ghrelin. Similarly, transfected Gαs, but not the Gαs mutant Q227L, nor Gαi2, Gαq, or Gα13 upregulated TRPM6/7 current density. In microdissected TALs and DCTs similar levels of GHSR mRNA were detected. In contrast, TRPM6 mRNA was expressed in the DCT and also detected in the TAL at 25% expression compared to DCT. Immunofluorescent studies using reporter GHSR-eGFP mice showed a strong eGFP signal in the TAL but surprisingly displayed no eGFP signal in the DCT. In 3-, 6-, and 9-month-old GHSR-null and WT mice, baseline serum magnesium was not significantly different, but 24-h urinary magnesium excretion was elevated in 9-month-old GHSR-null mice. In calorically restricted GHSR-null mice, we detected excess urinary magnesium excretion and reduced serum magnesium levels compared to WT mice. The kidneys from calorically restricted WT mice showed upregulated gene expression of magnesiotropic genes Hnf1b, Cldn-16, Cldn-19, Fxyd-2b, and Parvalbumin compared to GHSR-null mice. Our in vitro studies show that Ghrelin stimulates TRPM6/7 via GHSR and Gαs-PKA signaling. The murine studies are consistent with Ghrelin-GHSR signaling inducing reduced urinary magnesium excretion, particularly in calorically restricted mice when Ghrelin levels are elevated. This effect may be mediated by Ghrelin-upregulation of TRPM6 in the TAL and/or upregulation of other magnesiotropic genes. We postulate that rising Ghrelin levels with hunger contribute to increased renal Mg2+ reabsorption to compensate for lack of enteral Mg2+ uptake.

The intersection between ghrelin, metabolism and circadian rhythms.

Despite the growing popular interest in sleep and diet, many gaps exist in our scientific understanding of the interaction between circadian rhythms and metabolism. In this Review, we explore a promising, bidirectional role for ghrelin in mediating this interaction. Ghrelin both influences and is influenced by central and peripheral circadian systems. Specifically, we focus on how ghrelin impacts outputs of circadian rhythm, including neuronal activity, circulating growth hormone levels, locomotor activity and eating behaviour. We also consider the effects of circadian rhythms on ghrelin expression and the consequences of disrupted circadian patterns, such as shift work and jet lag, on ghrelin secretion. Our Review is aimed at both the casual reader interested in gaining more insight into the scientific context surrounding the trending topics of sleep and metabolism, as well as experienced scientists in the fields of ghrelin and circadian biology seeking inspiration and a comprehensive overview of how these fields are related.

Associations of postprandial ghrelin, liver-expressed antimicrobial peptide 2 and leptin levels with body composition, disease progression and survival in patients with amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: Loss of appetite contributes to weight loss and faster disease progression in amyotrophic lateral sclerosis (ALS). Impairment of appetite control in ALS may include altered production or action of orexigenic (i.e., ghrelin) and anorexigenic (i.e., liver-expressed antimicrobial peptide 2 [LEAP2] and leptin) hormones. We aimed to determine if postprandial circulating ghrelin levels, LEAP2 levels, LEAP2:ghrelin molar ratio and leptin levels differ in ALS patients compared to non-neurodegenerative disease controls, and whether they are associated with disease progression and body composition. METHODS: In this prospective natural history study, we assessed postprandial plasma levels of ghrelin, LEAP2 and leptin in patients with ALS (cases; n = 46) and controls (controls; n = 43). For cases, measures were compared to changes in body weight, body composition and clinical outcomes. RESULTS: Postprandial ghrelin level was decreased by 52% in cases compared to controls (p = 0.013). LEAP2:ghrelin molar ratio was increased by 249% (p = 0.009), suggesting greater ghrelin resistance. Patients with lower LEAP2:ghrelin tended to have better functional capacity at assessment, as inferred by the ALS Functional Rating Scale-Revised (τ = -0.179, p = 0.086). Furthermore, ghrelin and LEAP2:ghrelin molar ratio correlated with diagnostic delay (ghrelin, τ = 0.223, p = 0.029; LEAP2:ghrelin, τ = -0.213, p = 0.037). Baseline ghrelin level, LEAP2 level, LEAP2:ghrelin ratio and leptin level were, however, not predictive of change in functional capacity during follow-up. Also, patients with higher postprandial ghrelin levels (hazard ratio [HR] 1.375, p = 0.048), and lower LEAP2:ghelin ratios (HR 0.828, p = 0.051) had an increased risk of earlier death. CONCLUSIONS: Reduced postprandial ghrelin levels, coupled with increased LEAP2:ghrelin molar ratios, suggests a loss of ghrelin action in patients with ALS. Given ghrelin's actions on appetite, metabolism and neuroprotection, reduced ghrelin and greater ghrelin resistance could contribute to impaired capacity to tolerate the physiological impact of disease. Comprehensive studies are needed to explain how ghrelin and LEAP2 contribute to body weight regulation and disease progression in ALS.

Crosstalk between Gut Sensory Ghrelin Signaling and Adipose Tissue Sympathetic Outflow Regulates Metabolic Homeostasis.

The stomach-derived orexigenic hormone ghrelin is a key regulator of energy homeostasis and metabolism in humans. The ghrelin receptor, growth hormone secretagogue receptor 1a (GHSR), is widely expressed in the brain and gastrointestinal vagal sensory neurons, and neuronal GHSR knockout results in a profoundly beneficial metabolic profile and protects against diet-induced obesity (DIO) and insulin resistance. Here we show that in addition to the well characterized vagal GHSR, GHSR is robustly expressed in gastrointestinal sensory neurons emanating from spinal dorsal root ganglia. Remarkably, sensory neuron GHSR deletion attenuates DIO through increased energy expenditure and sympathetic outflow to adipose tissue independent of food intake. In addition, neuronal viral tract tracing reveals prominent crosstalk between gut non-vagal sensory afferents and adipose sympathetic outflow. Hence, these findings demonstrate a novel gut sensory ghrelin signaling pathway critical for maintaining energy homeostasis.

Ghrelin deletion and conditional ghrelin cell ablation increase pancreatic islet size in mice.

Ghrelin exerts key effects on islet hormone secretion to regulate blood glucose levels. Here, we sought to determine whether ghrelin's effects on islets extend to the alteration of islet size and ß cell mass. We demonstrate that reducing ghrelin - by ghrelin gene knockout (GKO), conditional ghrelin cell ablation, or high-fat diet (HFD) feeding - was associated with increased mean islet size (up to 62%), percentage of large islets (up to 854%), and ß cell cross-sectional area (up to 51%). In GKO mice, these effects were more apparent in 10- to 12-week-old mice than in 4-week-old mice. Higher ß cell numbers from decreased ß cell apoptosis drove the increase in ß cell cross-sectional area. Conditional ghrelin cell ablation in adult mice increased the ß cell number per islet by 40% within 4 weeks. A negative correlation between islet size and plasma ghrelin in HFD-fed plus chow-fed WT mice, together with even larger islet sizes in HFD-fed GKO mice than in HFD-fed WT mice, suggests that reduced ghrelin was not solely responsible for diet-induced obesity-associated islet enlargement. Single-cell transcriptomics revealed changes in gene expression in several GKO islet cell types, including upregulation of Manf, Dnajc3, and Gnas expression in ß cells, which supports decreased ß cell apoptosis and/or increased ß cell proliferation. These effects of ghrelin reduction on islet morphology might prove useful when designing new therapies for diabetes.

Elevated Ghrelin Promotes Hippocampal Ghrelin Receptor Defects in Humanized Amyloid-ß Knockin Mice During Aging.

BACKGROUND: Emerging evidence has revealed that dysregulation of the hormone ghrelin and its receptor, growth hormone secretagogue receptor (GHSR), contributes to the pathogenesis of Alzheimer's disease (AD). Specifically, defective GHSR function and resultant hippocampal ghrelin resistance are linked to hippocampal synaptic injury in AD paradigms. Also, AD patients exhibit elevated ghrelin activation. However, the detailed molecular mechanisms of hippocampal GHSR dysfunction and the relevance of ghrelin elevation to hippocampal ghrelin resistance in AD-relevant pathological settings are not fully understood. OBJECTIVE: In the current study, we employed a recently established mouse line of AD risk [humanized amyloid beta knockin (hAß KI mice), also referred to as a mouse model of late-onset AD in previous literature] to further define the role of ghrelin system dysregulation in the development of AD. METHODS: We employed multidisciplinary techniques to determine the change of plasma ghrelin and the functional status of GHSR in hAß KI mice as well as primary neuron cultures. RESULTS: We observed concurrent plasma ghrelin elevation and hippocampal GHSR desensitization with disease progression. Further examination excluded the possibility that ghrelin elevation is a compensatory change in response to GHSR dysfunction. In contrast, further in vitro and in vivo results show that agonist-mediated overstimulation potentiates GHSR desensitization through enhanced GHSR internalization. CONCLUSIONS: These findings suggest that circulating ghrelin elevation is a pathological event underlying hippocampal GHSR dysfunction, culminating in hippocampal ghrelin resistance and resultant synaptic injury in late-onset AD-related settings.

Ghrelin-responsive mediobasal hypothalamic neurons mediate exercise-associated food intake and exercise endurance.

Previous studies have implicated the orexigenic hormone ghrelin as a mediator of exercise endurance and the feeding response postexercise. Specifically, plasma ghrelin levels nearly double in mice when they are subjected to an hour-long bout of high-intensity interval exercise (HIIE) using treadmills. Also, growth hormone secretagogue receptor-null (GHSR-null) mice exhibit decreased food intake following HIIE and diminished running distance (time until exhaustion) during a longer, stepwise exercise endurance protocol. To investigate whether ghrelin-responsive mediobasal hypothalamus (MBH) neurons mediate these effects, we stereotaxically delivered the inhibitory designer receptor exclusively activated by designer drugs virus AAV2-hSyn-DIO-hM4(Gi)-mCherry to the MBH of Ghsr-IRES-Cre mice, which express Cre recombinase directed by the Ghsr promoter. We found that chemogenetic inhibition of GHSR-expressing MBH neurons (upon delivery of clozapine-N-oxide) 1) suppressed food intake following HIIE, 2) reduced maximum running distance and raised blood glucose and blood lactate levels during an exercise endurance protocol, 3) reduced food intake following ghrelin administration, and 4) did not affect glucose tolerance. Further, HIIE increased MBH Ghsr expression. These results indicate that activation of ghrelin-responsive MBH neurons is required for the normal feeding response to HIIE and the usual amount of running exhibited during an exercise endurance protocol.

Ghrelin signalling in AgRP neurons links metabolic state to the sensory regulation of AgRP neural activity.

OBJECTIVE: The sensory detection of food and food cues suppresses Agouti related peptide (AgRP) neuronal activity prior to consumption with greatest suppression occurring in response to highly caloric food or interoceptive energy need. However, the interoceptive mechanisms priming an appropriate AgRP neural response to external sensory information of food availability remain unexplored. Since hunger increases plasma ghrelin, we hypothesized that ghrelin receptor (GHSR) signalling on AgRP neurons is a key interoceptive mechanism integrating energy need with external sensory cues predicting caloric availability. METHODS: We used in vivo photometry to measure the effects of ghrelin administration or fasting on AgRP neural activity with GCaMP6s and dopamine release in the nucleus accumbens with GRAB-DA in mice lacking ghrelin receptors in AgRP neurons. RESULTS: The deletion of GHSR on AgRP neurons prevented ghrelin-induced food intake, motivation and AgRP activity. The presentation of food (peanut butter pellet) or a wooden dowel suppressed AgRP activity in fasted WT but not mice lacking GHSRs in AgRP neurons. Similarly, peanut butter and a wooden dowel increased dopamine release in the nucleus accumbens after ip ghrelin injection in WT but not mice lacking GHSRs in AgRP neurons. No difference in dopamine release was observed in fasted mice. Finally, ip ghrelin administration did not directly increase dopamine neural activity in the ventral tegmental area. CONCLUSIONS: Our results suggest that AgRP GHSRs integrate an interoceptive state of energy need with external sensory information to produce an optimal change in AgRP neural activity. Thus, ghrelin signalling on AgRP neurons is more than just a feedback signal to increase AgRP activity during hunger.

Ghrelin's orexigenic action in the lateral hypothalamic area involves indirect recruitment of orexin neurons and arcuate nucleus activation.

OBJECTIVE: Ghrelin is a potent orexigenic hormone, and the lateral hypothalamic area (LHA) has been suggested as a putative target mediating ghrelin's effects on food intake. Here, we aimed to investigate the presence of neurons expressing ghrelin receptor (a.k.a. growth hormone secretagogue receptor, GHSR) in the mouse LHA (LHAGHSR neurons), its physiological implications and the neuronal circuit recruited by local ghrelin action. METHODS: We investigated the distribution of LHAGHSR neurons using different histologic strategies, including the use of a reporter mice expressing enhanced green fluorescent protein under the control of the GHSR promoter. Also, we investigated the physiological implications of local injections of ghrelin within the LHA, and the extent to which the orexigenic effect of intra-LHA-injected ghrelin involves the arcuate nucleus (ARH) and orexin neurons of the LHA (LHAorexin neurons) RESULTS: We found that: 1) LHAGHSR neurons are homogeneously distributed throughout the entire LHA; 2) intra-LHA injections of ghrelin transiently increase food intake and locomotor activity; 3) ghrelin's orexigenic effect in the LHA involves the indirect recruitment of LHAorexin neurons and the activation of ARH neurons; and 4) LHAGHSR neurons are not targeted by plasma ghrelin. CONCLUSIONS: We provide a compelling neuroanatomical and functional characterization of LHAGHSR neurons in male mice that indicates that LHAGHSR cells are part of a hypothalamic neuronal circuit that potently induces food intake.

Ghrelin does not impact the blunted counterregulatory response to recurrent hypoglycemia in mice.

Introduction: Recurrent episodes of insulin-induced hypoglycemia in patients with diabetes mellitus can result in hypoglycemia-associated autonomic failure (HAAF), which is characterized by a compromised response to hypoglycemia by counterregulatory hormones (counterregulatory response; CRR) and hypoglycemia unawareness. HAAF is a leading cause of morbidity in diabetes and often hinders optimal regulation of blood glucose levels. Yet, the molecular pathways underlying HAAF remain incompletely described. We previously reported that in mice, ghrelin is permissive for the usual CRR to insulin-induced hypoglycemia. Here, we tested the hypothesis that attenuated release of ghrelin both results from HAAF and contributes to HAAF. Methods: C57BL/6N mice, ghrelin-knockout (KO) + control mice, and GhIRKO (ghrelin cell-selective insulin receptor knockout) + control mice were randomized to one of three treatment groups: a "Euglycemia" group was injected with saline and remained euglycemic; a 1X hypoglycemia ("1X Hypo") group underwent a single episode of insulin-induced hypoglycemia; a recurrent hypoglycemia ("Recurrent Hypo") group underwent repeated episodes of insulin-induced hypoglycemia over five successive days. Results: Recurrent hypoglycemia exaggerated the reduction in blood glucose (by ~30%) and attenuated the elevations in plasma levels of the CRR hormones glucagon (by 64.5%) and epinephrine (by 52.9%) in C57BL/6N mice compared to a single hypoglycemic episode. Yet, plasma ghrelin was equivalently reduced in "1X Hypo" and "Recurrent Hypo" C57BL/6N mice. Ghrelin-KO mice exhibited neither exaggerated hypoglycemia in response to recurrent hypoglycemia, nor any additional attenuation in CRR hormone levels compared to wild-type littermates. Also, in response to recurrent hypoglycemia, GhIRKO mice exhibited nearly identical blood glucose and plasma CRR hormone levels as littermates with intact insulin receptor expression (floxed-IR mice), despite higher plasma ghrelin in GhIRKO mice. Conclusions: These data suggest that the usual reduction of plasma ghrelin due to insulin-induced hypoglycemia is unaltered by recurrent hypoglycemia and that ghrelin does not impact blood glucose or the blunted CRR hormone responses during recurrent hypoglycemia.

Effects of thermoneutrality on food intake, body weight, and body composition in a Prader-Willi syndrome mouse model.

OBJECTIVE: Prader-Willi syndrome (PWS) is a multisystem genetic disorder. Unfortunately, none of several mouse models carrying PWS mutations emulates the entirety of the human PWS phenotype, including hyperphagia plus obesity. METHODS: To determine whether housing at thermoneutrality (TN, 30 °C) permits the development of hyperphagia and obesity in the Snord116del PWS mouse model, the effects of housing three different ages of Snord116del and wild-type (WT) littermates at TN versus room temperature (RT, 22-24 °C) for 8 weeks were compared. RESULTS: Snord116del mice born and maintained at TN exhibited lower body weight curves, lower percentage fat mass, and lower food intake than WT mice at RT. In 4- to 6-month-old high-fat diet-fed female mice, TN raised the Snord116del body weight curve closer to that of RT-housed WT mice although the TN-housed Snord116del mice did not gain more adiposity or exhibit greater food intake. In 6- to 8-month-old high-fat diet-fed male mice, body weight, adiposity, and food intake of TN-housed Snord116del mice remained far below levels in RT-housed WT mice. TN elicited hypotonia in Snord116del adults and exacerbated mortality of Snord116del newborns. CONCLUSIONS: In none of three tested TN protocols were greater food intake, body weight, or adiposity induced in Snord116del mice compared with RT-housed WT mice.

Liver-expressed antimicrobial peptide 2 elevation contributes to age-associated cognitive decline.

Elderly individuals frequently report cognitive decline, while various studies indicate hippocampal functional declines with advancing age. Hippocampal function is influenced by ghrelin through hippocampus-expressed growth hormone secretagogue receptor (GHSR). Liver-expressed antimicrobial peptide 2 (LEAP2) is an endogenous GHSR antagonist that attenuates ghrelin signaling. Here, we measured plasma ghrelin and LEAP2 levels in a cohort of cognitively normal individuals older than 60 and found that LEAP2 increased with age while ghrelin (also referred to in literature as "acyl-ghrelin") marginally declined. In this cohort, plasma LEAP2/ghrelin molar ratios were inversely associated with Mini-Mental State Examination scores. Studies in mice showed an age-dependent inverse relationship between plasma LEAP2/ghrelin molar ratio and hippocampal lesions. In aged mice, restoration of the LEAP2/ghrelin balance to youth-associated levels with lentiviral shRNA Leap2 downregulation improved cognitive performance and mitigated various age-related hippocampal deficiencies such as CA1 region synaptic loss, declines in neurogenesis, and neuroinflammation. Our data collectively suggest that LEAP2/ghrelin molar ratio elevation may adversely affect hippocampal function and, consequently, cognitive performance; thus, it may serve as a biomarker of age-related cognitive decline. Moreover, targeting LEAP2 and ghrelin in a manner that lowers the plasma LEAP2/ghrelin molar ratio could benefit cognitive performance in elderly individuals for rejuvenation of memory.

Toward a consensus nomenclature for ghrelin, its non-acylated form, liver expressed antimicrobial peptide 2 and growth hormone secretagogue receptor.

The stomach-derived octanoylated peptide ghrelin was discovered in 1999 and recognized as an endogenous agonist of the growth hormone secretagogue receptor (GHSR). Subsequently, ghrelin has been shown to play key roles in controlling not only growth hormone secretion, but also a variety of other physiological functions including, but not limited to, food intake, reward-related behaviors, glucose homeostasis and gastrointestinal tract motility. Importantly, a non-acylated form of ghrelin, desacyl-ghrelin, can also be detected in biological samples. Desacyl-ghrelin, however, does not bind to GHSR at physiological levels, and its physiological role has remained less well-characterized than that of ghrelin. Ghrelin and desacyl-ghrelin are currently referred to in the literature using many different terms, highlighting the need for a consistent nomenclature. The variability of terms used to designate ghrelin can lead not only to confusion, but also to miscommunication, especially for those who are less familiar with the ghrelin literature. Thus, we conducted a survey among experts who have contributed to the ghrelin literature aiming to identify whether a consensus may be reached. Based on the results of this consensus, we propose using the terms "ghrelin" and "desacyl-ghrelin" to refer to the hormone itself and its non-acylated form, respectively. Based on the results of this consensus, we further propose using the terms "GHSR" for the receptor, and "LEAP2" for liver-expressed antimicrobial peptide 2, a recently recognized endogenous GHSR antagonist/inverse agonist.

Growth hormone secretagogue receptor signaling in the supramammillary nucleus targets nitric oxide-producing neurons and controls recognition memory in mice.

Ghrelin is a stomach-derived hormone that acts via the growth hormone secretagogue receptor (GHSR). Recent evidence suggests that some of ghrelin's actions may be mediated via the supramammillary nucleus (SuM). Not only does ghrelin bind to cells within the mouse SuM, but ghrelin also activates SuM cells and intra-SuM ghrelin administration induces feeding in rats. In the current study, we aimed to further characterize ghrelin action in the SuM. We first investigated a mouse model expressing enhanced green fluorescent protein (eGFP) under the promoter of GHSR (GHSR-eGFP mice). We found that the SuM of GHSR-eGFP mice contains a significant amount of eGFP cells, some of which express neuronal nitric oxide synthase. Centrally-, but not systemically-, injected ghrelin reached the SuM, where it induced c-Fos expression. Furthermore, a 5-day 40% calorie restriction protocol, but not a 2-day fast, increased c-Fos expression in non-eGFP+ cells of the SuM of GHSR-eGFP mice, whereas c-Fos induction by calorie restriction was not observed in GHSR-deficient mice. Exposure of satiated mice to a binge-like eating protocol also increased c-Fos expression in non-eGFP+ cells of the SuM of GHSR-eGFP mice in a GHSR-dependent manner. Finally, intra-SuM-injected ghrelin did not acutely affect food intake, locomotor activity, behavioral arousal or spatial memory but increased recognition memory. Thus, we provide a compelling neuroanatomical characterization of GHSR SuM neurons and its behavioral implications in mice.

A closer look at alcohol-induced changes in the ghrelin system: novel insights from preclinical and clinical data.

Ghrelin is a gastric-derived peptide hormone with demonstrated impact on alcohol intake and craving, but the reverse side of this bidirectional link, that is, the effects of alcohol on the ghrelin system, remains to be fully established. To further characterize this relationship, we examined (1) ghrelin levels via secondary analysis of human laboratory alcohol administration experiments with heavy-drinking participants; (2) expression of ghrelin, ghrelin receptor, and ghrelin-O-acyltransferase (GOAT) genes (GHRL, GHSR, and MBOAT4, respectively) in post-mortem brain tissue from individuals with alcohol use disorder (AUD) versus controls; (3) ghrelin levels in Ghsr knockout and wild-type rats following intraperitoneal (i.p.) alcohol administration; (4) effect of alcohol on ghrelin secretion from gastric mucosa cells ex vivo and GOAT enzymatic activity in vitro; and (5) ghrelin levels in rats following i.p. alcohol administration versus a calorically equivalent non-alcoholic sucrose solution. Acyl- and total-ghrelin levels decreased following acute alcohol administration in humans, but AUD was not associated with changes in central expression of ghrelin system genes in post-mortem tissue. In rats, alcohol decreased acyl-ghrelin, but not des-acyl-ghrelin, in both Ghsr knockout and wild-type rats. No dose-dependent effects of alcohol were observed on acyl-ghrelin secretion from gastric mucosa cells or on GOAT acylation activity. Lastly, alcohol and sucrose produced distinct effects on ghrelin in rats despite equivalent caloric value. Our findings suggest that alcohol acutely decreases peripheral ghrelin concentrations in vivo, but not in proportion to alcohol's caloric value or through direct interaction with ghrelin-secreting gastric mucosal cells, the ghrelin receptor, or the GOAT enzyme.

Ghrelin cell-expressed insulin receptors mediate meal- and obesity-induced declines in plasma ghrelin.

Mechanisms underlying postprandial and obesity-associated plasma ghrelin reductions are incompletely understood. Here, using ghrelin cell-selective insulin receptor-KO (GhIRKO) mice, we tested the impact of insulin, acting via ghrelin cell-expressed insulin receptors (IRs), to suppress ghrelin secretion. Insulin reduced ghrelin secretion from cultured gastric mucosal cells of control mice but not from those of GhIRKO mice. Acute insulin challenge and insulin infusion during both hyperinsulinemic-hypoglycemic clamps and hyperinsulinemic-euglycemic clamps lowered plasma ghrelin in control mice but not GhIRKO mice. Thus, ghrelin cell-expressed IRs are required for insulin-mediated reductions in plasma ghrelin. Furthermore, interventions that naturally raise insulin (glucose gavage, refeeding following fasting, and chronic high-fat diet) also lowered plasma ghrelin only in control mice - not GhIRKO mice. Thus, meal- and obesity-associated increases in insulin, acting via ghrelin cell-expressed IRs, represent a major, direct negative modulator of ghrelin secretion in vivo, as opposed to ingested or metabolized macronutrients. Refed GhIRKO mice exhibited reduced plasma insulin, highlighting ghrelin's actions to inhibit insulin release via a feedback loop. Moreover, GhIRKO mice required reduced glucose infusion rates during hyperinsulinemic-hypoglycemic clamps, suggesting that suppressed ghrelin release resulting from direct insulin action on ghrelin cells usually limits ghrelin's full potential to protect against insulin-induced hypoglycemia.

LEAP2 deletion in mice enhances ghrelin's actions as an orexigen and growth hormone secretagogue.

OBJECTIVE: The hormone liver-expressed antimicrobial peptide-2 (LEAP2) is a recently identified antagonist and an inverse agonist of the growth hormone secretagogue receptor (GHSR). GHSR's other well-known endogenous ligand, acyl-ghrelin, increases food intake, body weight, and GH secretion and is lowered in obesity but elevated upon fasting. In contrast, LEAP2 reduces acyl-ghrelin-induced food intake and GH secretion and is found elevated in obesity but lowered upon fasting. Thus, the plasma LEAP2/acyl-ghrelin molar ratio could be a key determinant modulating GHSR signaling in response to changes in body mass and feeding status. In particular, LEAP2 may serve to dampen acyl-ghrelin action in the setting of obesity, which is associated with ghrelin resistance. Here, we sought to determine the metabolic effects of genetic LEAP2 deletion. METHODS: We generated the first known LEAP2-KO mouse line. Food intake, GH secretion, and cellular activation (c-fos induction) in different brain regions following s.c. acyl-ghrelin administration in LEAP2-KO mice and wild-type littermates were determined. LEAP2-KO mice and wild-type littermates were submitted to a battery of tests (such as measurements of body weight, food intake, and body composition; indirect calorimetry, determination of locomotor activity, and meal patterning while housed in metabolic cages) over the course of 16 weeks of high-fat diet and/or standard chow feeding. Fat accumulation was assessed in hematoxylin & eosin-stained and oil red O-stained liver sections from these mice. RESULTS: LEAP2-KO mice were more sensitive to s.c. ghrelin. In particular, acyl-ghrelin acutely stimulated food intake at a dose of 0.5 mg/kg BW in standard chow-fed LEAP2-KO mice while a 2× higher dose was required by wild-type littermates. Also, acyl-ghrelin stimulated food intake at a dose of 1 mg/kg BW in high-fat diet-fed LEAP2-KO mice while not even a 10× higher dose was effective in wild-type littermates. Acyl-ghrelin induced a 90.9% higher plasma GH level and 77.2-119.7% higher numbers of c-fos-immunoreactive cells in the arcuate nucleus and olfactory bulb, respectively, in LEAP2-KO mice than in wild-type littermates. LEAP2 deletion raised body weight (by 15.0%), food intake (by 18.4%), lean mass (by 6.1%), hepatic fat (by 42.1%), and body length (by 1.7%) in females on long-term high-fat diet as compared to wild-type littermates. After only 4 weeks on the high-fat diet, female LEAP2-KO mice exhibited lower O2 consumption (by 13%), heat production (by 9.5%), and locomotor activity (by 49%) than by wild-type littermates during the first part of the dark period. These genotype-dependent differences were not observed in high-fat diet-exposed males or female and male mice exposed for long term to standard chow diet. CONCLUSIONS: LEAP2 deletion sensitizes lean and obese mice to the acute effects of administered acyl-ghrelin on food intake and GH secretion. LEAP2 deletion increases body weight in females chronically fed a high-fat diet as a result of lowered energy expenditure, reduced locomotor activity, and increased food intake. Furthermore, in female mice, LEAP2 deletion increases body length and exaggerates the hepatic fat accumulation normally associated with chronic high-fat diet feeding.