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 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.

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 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.

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.

High Coexpression of the Ghrelin and LEAP2 Receptor GHSR With Pancreatic Polypeptide in Mouse and Human Islets.

Islets represent an important site of direct action of the hormone ghrelin, with expression of the ghrelin receptor (growth hormone secretagogue receptor; GHSR) having been localized variably to alpha cells, beta cells, and/or somatostatin (SST)-secreting delta cells. To our knowledge, GHSR expression by pancreatic polypeptide (PP)-expressing gamma cells has not been specifically investigated. Here, histochemical analyses of Ghsr-IRES-Cre × Cre-dependent ROSA26-yellow fluorescent protein (YFP) reporter mice showed 85% of GHSR-expressing islet cells coexpress PP, 50% coexpress SST, and 47% coexpress PP + SST. Analysis of single-cell transcriptomic data from mouse pancreas revealed 95% of Ghsr-expressing cells coexpress Ppy, 100% coexpress Sst, and 95% coexpress Ppy + Sst. This expression was restricted to gamma-cell and delta-cell clusters. Analysis of several single-cell human pancreatic transcriptome data sets revealed 59% of GHSR-expressing cells coexpress PPY, 95% coexpress SST, and 57% coexpress PPY + SST. This expression was prominent in delta-cell and beta-cell clusters, also occurring in other clusters including gamma cells and alpha cells. GHSR expression levels were upregulated by type 2 diabetes mellitus in beta cells. In mice, plasma PP positively correlated with fat mass and with plasma levels of the endogenous GHSR antagonist/inverse agonist LEAP2. Plasma PP also elevated on LEAP2 and synthetic GHSR antagonist administration. These data suggest that in addition to delta cells, beta cells, and alpha cells, PP-expressing pancreatic cells likely represent important direct targets for LEAP2 and/or ghrelin both in mice and humans.

Disrupting the ghrelin-growth hormone axis limits ghrelin's orexigenic but not glucoregulatory actions.

OBJECTIVE: Acyl-ghrelin regulates eating, body weight, blood glucose, and GH secretion upon binding to its receptor GHSR (growth hormone secretagogue receptor; ghrelin receptor). GHSR is distributed in several brain regions and some peripheral cell-types including pituitary somatotrophs. The objective of the current study was to determine the functional significance of acyl-ghrelin's action on GHSR-expressing somatotrophs in mediating GH secretion and several of acyl-ghrelin's metabolic actions. METHODS: GH-IRES-Cre mice and loxP-flanked (floxed) GHSR mice were newly developed and then crossed to one another to generate mice that lacked GHSR selectively from somatotrophs. Following validation of mice with somatotroph-selective GHSR deletion, metabolic responses of these mice and control littermates were assessed following both acute and chronic acyl-ghrelin administration, a 24-h fast, and a prolonged 60% chronic caloric restriction protocol modeling starvation. RESULTS: In mice with somatotroph-selective GHSR deletion, a single peripheral injection of acyl-ghrelin failed to induce GH secretion or increase food intake, unlike wild-type and other littermate control groups. However, the usual acute blood glucose increase in response to the acyl-ghrelin bolus was preserved. Similarly, chronic s.c. acyl-ghrelin administration to mice with somatotroph-selective GHSR deletion failed to increase plasma GH, food intake, or body weight. Physiologically elevating plasma acyl-ghrelin via a 24-h fast also failed to raise plasma GH and resulted in a limited hyperphagic response upon food reintroduction in mice with somatotroph-selective GHSR deletion, although those mice nonetheless did not exhibit an exaggerated reduction in blood glucose. Physiologically elevating plasma acyl-ghrelin via a 15-day caloric restriction protocol which provided only 40% of usual daily calories failed to raise plasma GH in mice with somatotroph-selective GHSR deletion, although those mice did not exhibit life-threatening hypoglycemia. CONCLUSIONS: These results reveal that direct engagement of GHSR-expressing somatotrophs is required for a peripheral ghrelin bolus to acutely stimulate GH secretion and the actions of chronic acyl-ghrelin delivery and physiological plasma acyl-ghrelin elevations to increase plasma GH. These results also suggest that actions of acyl-ghrelin to increase food intake and body weight are reliant on direct activation of GHSRs expressed on somatotrophs. Furthermore, these results suggest that the glucoregulatory actions of acyl-ghrelin - in particular, its actions to raise blood glucose when acutely administered, prevent small blood glucose drops following a 24-h fast, and avert life-threatening hypoglycemia during an acute-on-chronic caloric restriction protocol - do not depend on GHSR expression by somatotrophs.

"A LEAP 2 conclusions? Targeting the ghrelin system to treat obesity and diabetes".

BACKGROUND: The hormone ghrelin stimulates food intake, promotes adiposity, increases body weight, and elevates blood glucose. Consequently, 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.

Ghrelin Protects Against Insulin-Induced Hypoglycemia in a Mouse Model of Type 1 Diabetes Mellitus.

Insulin-induced hypoglycemia is a major limiting factor in maintaining optimal blood glucose in patients with type 1 diabetes and advanced type 2 diabetes. Luckily, a counterregulatory response (1) system exists to help minimize and reverse hypoglycemia, although more studies are needed to better characterize its components. Recently, we showed that the hormone ghrelin is permissive for the normal CRR to insulin-induced hypoglycemia when assessed in mice without diabetes. Here, we tested the hypothesis that ghrelin also is protective against insulin-induced hypoglycemia in the streptozotocin (2) mouse model of type 1 diabetes. STZ-treated ghrelin-knockout (KO) (3) mice as well as STZ-treated wild-type (WT) littermates were subjected to a low-dose hyperinsulinemic-hypoglycemic clamp procedure. The STZ-treated ghrelin-KO mice required a much higher glucose infusion rate than the STZ-treated WT mice. Also, the STZ-treated ghrelin-KO mice exhibited attenuated plasma epinephrine and norepinephrine responses to the insulin-induced hypoglycemia. Taken together, our data suggest that without ghrelin, STZ-treated mice modeling type 1 diabetes are unable to mount the usual CRR to insulin-induced hypoglycemia.

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.

Ghrelin's Relationship to Blood Glucose.

Much effort has been directed at studying the orexigenic actions of administered ghrelin and the potential effects of the endogenous ghrelin system on food intake, food reward, body weight, adiposity, and energy expenditure. Although endogenous ghrelin's actions on some of these processes remain ambiguous, its glucoregulatory actions have emerged as well-recognized features during extreme metabolic conditions. The blood glucose-raising actions of ghrelin are beneficial during starvation-like conditions, defending against life-threatening falls in blood glucose, but they are seemingly detrimental in obese states and in certain monogenic forms of diabetes, contributing to hyperglycemia. Also of interest, blood glucose negatively regulates ghrelin secretion. This article reviews the literature suggesting the existence of a blood glucose-ghrelin axis and highlights the factors that mediate the glucoregulatory actions of ghrelin, especially during metabolic extremes such as starvation and diabetes.

Role of brown adipose tissue in modulating adipose tissue inflammation and insulin resistance in high-fat diet fed mice.

Obesity results in the chronic activation of innate immune system and subsequently sets in diabetes. Aim of the study was to investigate the immunometabolic role of brown adipose tissue (BAT) in the obesity. We performed both BAT transplantation as well as extirpation experiments in the mouse model of high-fat diet (HFD)-induced obesity. We carried out immune cell profiling in the stromal vascular fraction (SVF) isolated from epididymal white adipose tissue (eWAT). BAT transplantation reversed HFD-induced increase in body weight gain and insulin resistance without altering diet intake. Importantly, BAT transplantation attenuated the obesity-associated adipose tissue inflammation in terms of decreased pro-inflammatory M1-macrophages, cytotoxic CD8a T-cells and restored anti-inflammatory regulatory T-cells (Tregs) in the eWAT. BAT transplantation also improved endogenous BAT activity by elevating protein expression of browning markers (UCP-1, PRDM16 and PGC1α) in it. In addition, BAT transplantation promoted the eWAT expression of various genes involved in fatty acid oxidation (such as Elvol3 and Tfam,). In contrast, extirpation of the interscapular BAT exacerbated HFD-induced obesity, insulin resistance and adipose tissue inflammation (by increasing M1 macrophages, CD8a T-cell and decreasing Tregs in eWAT). Taken together, our results suggested an important role of BAT in combating obesity-associated metabolic complications. These results open a novel therapeutic option to target obesity and related metabolic disorders like type 2 diabetes.

ß1-adrenergic receptors mediate plasma acyl-ghrelin elevation and depressive-like behavior induced by chronic psychosocial stress.

The ghrelin system is a key component of the mood and metabolic responses to chronic psychosocial stress. For example, circulating acyl-ghrelin rises in several rodent and human stress models, administered acyl-ghrelin induces antidepressant-like behavioral responses in mice, and mice with deleted ghrelin receptors (GHSRs) exhibit exaggerated depressive-like behaviors, changed eating behaviors, and altered metabolism in response to chronic stress. However, the mechanisms mediating stress-induced rises in ghrelin are unknown and ghrelin's antidepressant-like efficacy in the setting of chronic stress is incompletely characterized. Here, we used a pharmacological approach in combination with a 10-day chronic social defeat stress (CSDS) model in male mice to investigate whether the sympathoadrenal system is involved in the ghrelin response to stress. We also examined the antidepressant-like efficacy of administered ghrelin and the synthetic GHSR agonist GHRP-2 during and/or after CSDS. We found that administration of the ß1-adrenergic receptor (ß1AR) blocker atenolol during CSDS blunts the elevation of plasma acyl-ghrelin and exaggerates depressive-like behavior. Neither acute injection of acyl-ghrelin directly following CSDS nor its chronic administration during or after CSDS nor chronic delivery of GHRP-2 during and after CSDS improved stress-induced depressive-like behavior. Thus, ß1ARs drive the acyl-ghrelin response to CSDS, but supplementing the natural increases in acyl-ghrelin with exogenous acyl-ghrelin or GHSR agonist does not further enhance the antidepressant-like actions of the endogenous ghrelin system in the setting of CSDS.

Temporal immmunometabolic profiling of adipose tissue in HFD-induced obesity: manifestations of mast cells in fibrosis and senescence.

BACKGROUND/OBJECTIVES: Chronic low-grade inflammation/meta-inflammation in adipose tissue leads to obesity-associated metabolic complications. Despite growing understanding, the roles of immune cell subsets, their interrelationship, and chronological events leading to progression of obesity-associated insulin resistance (IR) remains unclear. METHODS: We carried out temporal immunometabolic profiling of adipose tissue from C57BL/6 mice fed a high-fat diet (HFD) for 4, 8, 12, 16, and 20 weeks. We used clodronate sodium liposomes (CLODs) to deplete macrophages and disodium cromoglycate sodium liposomes (DSCGs) to stabilize mast cells. RESULTS: In the temporal HFD settings, mice showed progressive glucose intolerance, insulin resistance, and adipose tissue senescence. Histochemistry analysis of epididymal white adipose tissue (eWAT) using picro-sirius red and Masson's trichrome staining showed extensive collagen deposition in the 16th and 20th weeks. Flow cytometry analysis of the stromal vascular fraction (SVF) from eWAT revealed T-cell subsets as early-phase components and pro-inflammatory macrophages, as well as mast cells as the later phase components during obesity progression. In our therapeutic strategies, macrophage depletion by CLOD and mast stabilization by DSCG attenuated obesity, adipose tissue fibrosis, and improved whole-body glucose homeostasis. In addition, mast cell stabilization also attenuated senescence (p53 and X-gal staining) in eWAT, signifying the role of mast cells over macrophages during obesity. CONCLUSION: New-generation mast cell stabilizers can be exploited for the treatment of obesity-associated metabolic complications.

Ghrelin Receptor Agonist Rescues Excess Neonatal Mortality in a Prader-Willi Syndrome Mouse Model.

In the current study, we sought to determine the significance of the ghrelin system in Prader-Willi Syndrome (PWS). PWS is characterized by hypotonia and difficulty feeding in neonates and hyperphagia and obesity beginning later in childhood. Other features include low GH, neonatal hypoglycemia, hypogonadism, and accelerated mortality. Although the hyperphagia and obesity in PWS have been attributed to elevated levels of the orexigenic hormone ghrelin, this link has never been firmly established, nor have ghrelin's potentially protective actions to increase GH secretion, blood glucose, and survival been investigated in a PWS context. In the current study, we show that placing Snord116del mice modeling PWS on ghrelin-deficient or ghrelin receptor [GH secretagogue receptor (GHSR)]-deficient backgrounds does not impact their characteristically reduced body weight, lower plasma IGF-1, delayed sexual maturation, or increased mortality in the period prior to weaning. However, blood glucose was further reduced in male Snord116del pups on a ghrelin-deficient background, and percentage body weight gain and percentage fat mass were further reduced in male Snord116del pups on a GHSR-deficient background. Strikingly, 2 weeks of daily administration of the GHSR agonist HM01 to Snord116del neonates markedly improved survival, resulting in a nearly complete rescue of the excess mortality owing to loss of the paternal Snord116 gene. These data support further exploration of the therapeutic potential of GHSR agonist administration in limiting PWS mortality, especially during the period characterized by failure to thrive.

miR-876-3p regulates glucose homeostasis and insulin sensitivity by targeting adiponectin

miRNA has been known to regulate diverse cellular and molecular functions. In the earlier study, we have reported that adipocytes differentiated from human mesenchymal stem cells (hMSC) on 72-h chronic insulin (CI) treatment exhibit insulin resistance (IR). Present study has further explored above model to investigate the role of early expressed miRNAs within human adipocytes to modulate differential adipokine expression as observed during IR. Our results highlight that miR-876-3p regulate glucose homeostasis and its dysregulation leads to IR. We found that miR-876-3p level is a critical determinant of adiponectin expression by virtue of its target within adiponectin 3'UTR. Regulatory effect of miR-876-3p impacts crosstalk between adiponectin and insulin signaling. Rosiglitazone treatment in CI-induced IR adipocytes drastically reduced miR-876-3p expression and increased adiponectin level. In line with this, lentiviral-mediated inhibition of miR-876-3p expression ameliorated CI and high-fat diet (HFD)-induced IR in adipocytes differentiated from hMSC and C57BL/6 mice, respectively. Our findings thus suggest that modulating miR-876-3p expression could provide novel opportunities for therapeutic intervention of obesity-associated metabolic syndrome.