Introduction and Objective: Ghrelin regulates food intake, exercise endurance, and blood glucose; its plasma levels are increased by fasting and exercise. During fasting, the SNS stimulates ghrelin secretion via norepinephrine release onto ghrelin cells. A role for the SNS in mediating ghrelin secretion during exercise is unknown. Here, we sought to characterize the dynamic nature of ghrelin cell innervation by the SNS, the chemical identity of these SNS neurons, and the requirement of the SNS for fasting- and exercise-induced ghrelin secretion. Methods: SNS subtypes in the celiac ganglion (CG-SMG) were identified by snRNA seq. CNS projections to gastric ghrelin cells were mapped with Cre-dependent PRV-2017-EGFP tracing in Ghrelin-Cre mice. The anatomical organization of NPY axonal terminals in relation to ghrelin cells was determined by histochemistry. NPY-induced ghrelin secretion was determined in 1° gastric cell cultures. The impact of CG-SMG NPY neuronal activation on ghrelin secretion and various physiological parameters was determined with chemogenetics. Results: CG-SMG snRNA seq identified 4 SNS neuron subtypes including NPY+ neurons. A 24h fast increased NPY+ neuron contacts with ghrelin cells by 226%; this reversed by 16h of refeeding. NPY directly stimulated ghrelin secretion. Cre-dependent PRV-2017-EGFP traveled retrogradely from gastric ghrelin cells of Ghrelin-Cre mice to several brainstem and hypothalamic nuclei via the CG-SMG (90% were NPY+). Chemogenetic inhibition of CG-SMG NPY neurons reduced exercise and fasting increases in plasma ghrelin, led to greater blood glucose drops after a 24h fast (by 65%) and reduced post-HIIE food intake (by 45%) and exercise endurance (by 27%) compared to controls. These effects were rescued by ghrelin delivery. Conclusion: CG-SMG NPY neurons mediate SNS-driven ghrelin release during exercise and fasting, coordinating various gut-brain metabolic responses including changes to blood glucose, food intake, and exercise endurance. Disclosure O. Singh: None. S. Sheybani Deloui: None. S. Varshney: None. D. Gupta: None. K. Shankar: None. S. Kulkarni: None. M. Lyu: None. C. Lawrence: None. I.B. Hogue: None. S. Osborne-Lawrence: None. J.M. Zigman: Stock/Shareholder; Current; Novo Nordisk, Eli Lilly and Company, Dexcom, Inc. Funding American Diabetes Association (1-25-PDF-88), National Institutes of Health (R01DK142092-01A1); Sprouts Grant Program of the Peter O’Donnell Jr. Brain Institute at UT Southwestern Medical Center

The transcriptional landscape of the gastric mucosa in response to opposing nutritional states remains poorly defined. Here, we profiled gastric mucosal gene expression changes induced by diet-induced obesity (DIO) and calorie restriction (CR) in mice and investigated the physiological role of Decidual Protein Induced by Progesterone 1 (Depp1) using newly generated Depp1-knockout (KO) mice. RNA sequencing revealed that DIO elicits a predominantly proinflammatory transcriptional program in the gastric mucosa, whereas CR upregulates genes involved in peptide transport and extracellular matrix organization while downregulating immunity-related pathways. Among CR-induced genes, Depp1 exhibited the strongest positive correlation with expression of gene encoding the gastric hormone ghrelin. qRT-PCR confirmed enrichment of Depp1 in gastric ghrelin cells and demonstrated CR-induced upregulation of Depp1 in additional tissues, including liver, kidney, and pancreas; notably, hepatic induction by CR was absent in ghrelin-KO mice. Despite this association, Depp1-KO mice displayed normal metabolic responses to CR, including preserved glucose homeostasis. In contrast, following 16 weeks of ad libitum high-fat diet feeding, male Depp1-KO mice exhibited greater weight gain, hyperphagia, increased fat and lean mass, and impaired glucose tolerance compared with wild-type littermates. These phenotypes were accompanied by selective hepatic gene expression changes affecting Pgc1a, Pck1, and Igf1. Collectively, these findings identify Depp1 as a CR-induced, ghrelin-associated gene that influences hepatic transcriptional responses yet is dispensable for short-term adaptation to CR, while also implicating Depp1 as a protective factor against metabolic dysfunction during DIO through mechanisms that remain to be defined.

Food consumption impacts body weight differently depending on the time of day. Here, we investigated whether suprachiasmatic nucleus (SCN) neurons responsive to the hormone ghrelin temporally regulate eating and body weight in mice. The chemogenetic stimulation of GHSR (growth hormone secretagogue receptor)-expressing SCN neurons during the mid-rest phase-when mice are most sensitive to ghrelin's orexigenic effects-increased food intake. Repeated chemogenetic inhibition of these neurons during this same time of day reduced the corresponding, typically low amount of food intake; it also reduced the cumulative feed efficiency and body weight. These effects were not observed at other times of day. The chemogenetic stimulation of GHSR-expressing arcuate hypothalamic nucleus neurons increased food intake independently of time of day. GHSR-expressing SCN neurons represented subpopulations of six distinct SCN neuronal clusters, which were predominantly GABAergic and exhibited light-sensitive, time-of-day-dependent transcriptomic profiles. Thus, our study identifies GHSR-expressing SCN neurons as a neuronal population that regulates eating, feed efficiency, and body weight in a mid-rest phase-dependent manner.

The hormone ghrelin serves a protective role in cancer-related anorexia-cachexia syndrome (CACS)-a condition in which plasma levels of ghrelin rise, its administration lessens CACS severity, and experimentally reduced signaling by its receptor (GHSR) worsens fat loss and anorexia and accelerates death. Yet, actions for the related hormone liver-expressed antimicrobial peptide-2 (LEAP2), which is an endogenous GHSR antagonist, are unexplored in CACS. Here, we found that plasma LEAP2 and LEAP2/ghrelin ratio were lower in Lewis lung carcinoma (LLC) and RM-9 prostate cancer CACS mouse models. Ghrelin deletion exaggerated losses of tumor-free body weight and fat mass, reduced food intake, reduced soleus muscle weight, and/or lowered grip strength in LLC or RM-9 tumor-bearing mice. LEAP2 deletion lessened reductions in tumor-free body weight and fat mass and increased food intake in LLC or RM-9 tumor-bearing mice. In a 55-subject cohort of patients with CACS or weight-stable cancer, the plasma LEAP2/total ghrelin ratio was negatively correlated with 6-month weight change preceding blood collection. These data demonstrate that ghrelin deletion exacerbates CACS in the LLC and RM-9 tumor-bearing mouse models while contrastingly, LEAP2 deletion reduces measures of CACS in these tumor-bearing mouse models. Further, they suggest that lower plasma LEAP2/ghrelin ratio protects against worsened CACS.

Reducing ghrelin by ghrelin gene knockout (GKO), ghrelin-cell ablation, or high-fat diet feeding increases islet size and β-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 β-cell mass were larger (P = .057 for β-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 β-cell mass (by ∼15.7-23.8% and ∼65.2-76.8%, respectively). Neither islet cross-sectional area, β-cell cross-sectional area, nor β-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 β-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 β-cell mass, one of those factors may be the pregnancy-associated increase in LEAP2.

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 β 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 β 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 β cell numbers from decreased β cell apoptosis drove the increase in β cell cross-sectional area. Conditional ghrelin cell ablation in adult mice increased the β 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 β cells, which supports decreased β cell apoptosis and/or increased β cell proliferation. These effects of ghrelin reduction on islet morphology might prove useful when designing new therapies for diabetes.

ObjectiveThe 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.MethodsHere, 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.ResultsAmong 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.ConclusionsAdministration 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.

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.

Abstract Disclosure: K. Shankar: None. S. Varshney: None. D. Gupta: None. O. Singh: None. S.B. Ogden: None. S. Osborne-Lawrence: None. N.P. Metzger: None. C.P. Richard: None. J.M. Zigman: Grant Recipient; Self; Novo Nordisk. Objective: The hormones ghrelin and LEAP2 both are endogenous ligands for the growth hormone secretagogue receptor (GHSR). Whereas ghrelin activates GHSR, LEAP2 blocks ghrelin from activating GHSR and also inhibits GHSR constitutive activity. It is via GHSR that the glucoregulatory actions of ghrelin and LEAP2 are mediated. These glucoregulatory actions are highlighted by the findings of severe hypoglycemia in ghrelin-KO mice and LEAP2-overexpressing mice when they are submitted to an acute-on-chronic caloric restriction protocol. Also, recently, our group showed that ghrelin-KO mice require a much higher glucose infusion rate (GIR) and exhibit markedly reduced elevation of counterregulatory hormones when submitted to a hyperinsulinemic-hypoglycemic clamp. In the current study, we investigated the potential of LEAP2 to protect against insulin-induced-hypoglycemia and counteract the effects of ghrelin deletion during the hypoglycemic clamp. Methods: Eight-ten week-old male wild-type (WT), Ghrelin-KO, LEAP2-KO, and LEAP2/Ghrelin-doubleKO (lacking both ghrelin and LEAP2 ) littermates were used for this study (n=7-10 per genotype). Mice were implanted with a right jugular vein catheter. Five days later, hyperinsulinemic-hypoglycemic clamps were performed in conscious, unrestrained mice. A low dose of insulin was infused at 4 mU/kg/min i.v. over 2 hr. A 20% glucose solution was simultaneously infused at a variable rate to achieve hypoglycemia (35–45 mg/dL) during the final 30 min. Blood glucose was measured via tail nicks every 5 min. Results: We were able to achieve the target hypoglycemic range in all four genotypes using this protocol. As compared to WT mice, Ghrelin-KO mice required markedly elevated (∼110% higher) GIRs (10±4 mg/kg/min in WT vs. 22±2 mg/kg/min in Ghrelin-KO mice; p=0.03) by the end of the 2-hr clamp. The GIRs required by LEAP2-KO mice were equivalent to those of WT mice (7±2 mg/kg/min in LEAP2-KO; p=n.s.). Furthermore, although during the first 60 min of the clamp, LEAP2/Ghrelin-doubleKO mice exhibited slightly lower blood glucose levels than the other genotypes and during the middle 60 min of the clamp, they required higher GIRs than the other genotypes, by the end of the 2-hr clamp, their GIRs were similar to Ghrelin-KO mice (25±3 mg/Kg/min in Ghrelin/LEAP2-doubleKO mice, p=n.s.). Conclusions: These data suggest a complex effect of LEAP2 on blood glucose. Alone, LEAP2 deletion does not impact insulin sensitivity. When coupled with ghrelin deletion, LEAP2 deletion lowers fasting blood glucose and causes greater insulin sensitivity early on during the hyperinsulinemic-hypoglycemic clamp. By the end of the clamp, this effect of LEAP2 deletion when coupled to ghrelin deletion is gone. Altogether, these results suggest that LEAP2 deletion does not counteract the effects of ghrelin deletion during insulin-induced hypoglycemia. Presentation: Friday, June 16, 2023

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.

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

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.

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.

Abstract Objective: Ghrelin regulates eating, body weight, and blood glucose. Upon binding to its receptor (growth hormone secretagogue receptor; GHSR), administered ghrelin increases food intake, body weight, and blood glucose. In contrast, blocking ghrelin lowers body weight and food intake. Also, mice that lack ghrelin or GHSR develop life-threatening hypoglycemia when submitted to a prolonged caloric restriction protocol providing only 40% of usual daily calories. Although GHSR was first identified in the pituitary, ghrelin was first defined by its ability to stimulate GH secretion via GHSRs, GH replacement prevents hypoglycemia in ghrelin-KO mice undergoing prolonged caloric restriction, and GH is known to modulate body composition, relatively little attention has been devoted to the role of GH-secreting pituitary somatotrophs (“GH cells”) in ghrelin action. The objective here was to determine the requirement for GHSR-expressing GH cells in mediating ghrelin’s metabolic actions. Methods: Mice with GH cell-selective GHSR deletion were generated by crossing novel GH-IRES-Cre mice to novel floxed-GHSR mice. GH cell-selective GHSR knockout mice and three control littermate groups were studied. Plasma GH, food intake, and blood glucose were measured after ip or sc ghrelin administration. Blood glucose and plasma GH were measured over the course of a 15-d calorie restriction protocol providing only 40% of usual daily calories. Results: In mice with GH cell-selective GHSR deletion, ghrelin-induced GH secretion and food intake were attenuated (by 84.1% at 15 min and by 35.3% at 45 min, respectively) as compared to controls; ghrelin-induced blood glucose elevation was unchanged. Mice with GH cell-selective GHSR deletion exhibited an attenuated GH rise (by 76.8%) over the 15-d calorie restriction period, yet they nonetheless resisted life-threatening hypoglycemia which is observed in similarly-treated ghrelin-KO mice, GHSR-null mice, and mice with hepatocyte-selective GH receptor deletion. Conclusions: These results suggest that GH cell-expressed GHSRs are required for ghrelin’s acute orexigenic and GH secretory actions but are dispensable for ghrelin’s glucoregulatory actions, at least in the settings assessed here. Although GH cell-expressed GHSRs are required for the progressive GH elevations associated with prolonged calorie restriction, they are not required for ghrelin’s overall protective effects to block prolonged calorie restriction-associated hypoglycemia.

Background: The hormone ghrelin stimulates food intake, promotes adiposity, increases body weight, and elevates blood glucose. Conse-quently, alterations in plasma ghrelin levels and the functioning of other components of the broader ghrelin system have been proposed as potential contributors to obesity and diabetes. Furthermore, targeting the ghrelin system has been proposed as a novel therapeutic strategy for obesity and diabetes. Scope of review: The current review focuses on the potential for targeting ghrelin and other proteins comprising the ghrelin system as a treatment for obesity and diabetes. The main components of the ghrelin system are introduced. Data supporting a role for the endogenous ghrelin system in the development of obesity and diabetes along with data that seemingly refute such a role are outlined. An argument for further research into the development of ghrelin system-targeted therapeutic agents is delineated. Also, an evidence-based discussion of potential factors and contexts that might influence the efficacy of this class of therapeutics is provided. Major conclusions: It would not be a "leap to" conclusions to suggest that agents which target the ghrelin system -including those that lower acyl-ghrelin levels, raise LEAP2 levels, block GHSR activity, and/or raise desacyl-ghrelin signaling -could represent efficacious novel treatments for obesity and diabetes. (c) 2020 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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 2x 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 10x 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. (c) 2021 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).