Analytical characterization of full, intermediate, and empty AAV capsids.

Manufacturing of recombinant adeno-associated virus (AAV) vectors produces three types of capsids: full, intermediate, and empty. While there are different opinions about the impact of intermediate and empty capsids on safety and efficacy of AAV products, they are generally considered impurities because they are not the intended fully intact vector product. The presence of these impurities could impact product efficacy due to potential competition with fully packaged AAVs for cellular transduction, as well as have potential implications to patient safety due to increased capsid load during dosing. To determine the impact of intermediate capsids on potency, an AAV preparation was separated into fractions enriched for full, intermediate, or empty capsids. Using a matrix of in vitro (infectivity, gene expression, biological activity) and in vivo potency assays to determine potency as a function of capsid content, our results indicate that while intermediate capsids contribute to the vector genome titer of the product and are equally as infectious as full capsids, they do not contribute to the potency of the AAV product. This study confirms the criticality of reducing and controlling the level of intermediate capsids to ensure a more efficacious AAV product.

Acute Impacts of Ionizing Radiation Exposure on the Gastrointestinal Tract and Gut Microbiome in Mice.

Radiation therapy for abdominopelvic malignancies often results in damage to the gastrointestinal tract (GIT) and permanent changes in bowel function. An overlooked component of the pathophysiology of radiation-induced bowel injury is the role of the gut microbiome. The goal of this research was to identify the impacts of acute radiation exposure on the GIT and gut microbiome. C57BL/6 mice exposed to whole-body X-rays (0.1-3 Gy) were assessed for histological and microbiome changes 48 h post-radiation exposure. Within the ileum, a dose of 3 Gy significantly decreased crypt depth as well as the number of goblet cells, but increased overall goblet cell size. Overall, radiation altered the microbial distribution within each of the main phyla in a dose- and tissue-dependent manner. Within the Firmicutes phylum, high dose irradiation resulted in significant alterations in bacteria from the class Bacilli within the small bowels, and from the class Clostridia in the large bowels. The 3 Gy radiation also significantly increased the abundance of bacterial families from the Bacteroidetes phylum in the colon and feces. Overall, we identified various alterations in microbiome composition following acute radiation exposure, which could potentially lead to novel biomarkers for tracking patient toxicities or could be used as targets for mitigation strategies against radiation damage.

Muscle cooling modulates tissue oxidative and biochemical responses but not energy metabolism during exercise.

PURPOSE: This study investigated whether muscle cooling and its associated effects on skeletal muscle oxidative responses, blood gases, and hormonal concentrations influenced energy metabolism during cycling. METHODS: Twelve healthy participants (Males: seven; Females: five) performed two steady-state exercise sessions at 70% of ventilatory threshold on a cycle ergometer. Participants completed one session with pre-exercise leg cooling until muscle temperature (Tm) decreased by 6 °C (LCO), and a separate session without cooling (CON). They exercised until Tm returned to baseline and for an additional 30 min. Cardiovascular, respiratory, metabolic, hemodynamic variables, and skeletal muscle tissue oxidative responses were assessed continuously. Venous blood samples were collected to assess blood gases, and hormones. RESULTS: Heart rate, stroke volume, and cardiac output all increased across time but were not different between conditions. V̇O2 was greater in LCO when muscle temperature was restored until the end of exercise (p < 0.05). Cycling in the LCO condition induced lower oxygen availability, tissue oxygenation, blood pH, sO2%, and pO2 (p < 0.05). Insulin concentrations were also higher in LCO vs. CON (p < 0.05). Importantly, stoichiometric equations from respiratory gases indicated no differences in fat and CHO oxidation between conditions. CONCLUSION: The present study demonstrated that despite muscle cooling and the associated oxidative and biochemical changes, energy metabolism remained unaltered during cycling. Whether lower local and systemic oxygen availability is counteracted via a cold-induced activation of lipid metabolism pathways needs to be further investigated.

The Role of the Bacterial Muramyl Dipeptide in the Regulation of GLP-1 and Glycemia.

The host's intestinal microbiota contributes to endocrine and metabolic responses, but a dysbiosis in this environment can lead to obesity and insulin resistance. Recent work has demonstrated a role for microbial metabolites in the regulation of gut hormones, including the metabolic hormone, glucagon-like peptide-1 (GLP-1). Muramyl dipeptide (MDP) is a bacterial cell wall component which has been shown to improve insulin sensitivity and glucose tolerance in diet-induced obese mice by acting through the nucleotide oligomerization domain 2 (NOD2) receptor. The purpose of this study was to understand the effects of MDP on GLP-1 secretion and glucose regulation. We hypothesized that MDP enhances glucose tolerance by inducing intestinal GLP-1 secretion through NOD2 activation. First, we observed a significant increase in GLP-1 secretion when murine and human L-cells were treated with a fatty acid MDP derivative (L18-MDP). Importantly, we demonstrated the expression of the NOD2 receptor in mouse intestine and in L-cells. In mice, two intraperitoneal injections of MDP (5 mg/kg body weight) caused a significant increase in fasting total GLP-1 in chow-fed mice, however this did not lead to an improvement in oral glucose tolerance. When mice were exposed to a high-fat diet, they eventually lost this MDP-induced GLP-1 release. Finally, we demonstrated in L-cells that hyperglycemic conditions reduce the mRNA expression of NOD2 and GLP-1. Together these findings suggest MDP may play a role in enhancing GLP-1 during normal glycemic conditions but loses its ability to do so in hyperglycemia.

A Protective Role for Glucagon-like Peptide-2 in Heat-stable Enterotoxin b (STb)-Induced L-Cell Toxicity.

Enterotoxigenic Escherichia coli (ETEC)-derived purified heat-stable enterotoxin b (STb) is responsible for secretory diarrhea in livestock and humans. STb disrupts intestinal fluid homeostasis, epithelial barrier function, and promotes cell death. Glucagon-like peptide-2 (GLP-2) is a potent intestinotrophic hormone secreted by enteroendocrine L cells. GLP-2 enhances crypt cell proliferation, epithelial barrier function, and inhibits enterocyte apoptosis. Whether STb can affect GLP-2 producing L cells remains to be elucidated. First, secreted-His-labeled STb from transformed E coli was collected and purified. When incubated with L-cell models (GLUTag, NCI-H716, and secretin tumor cell line [STC-1]), fluorescent immunocytochemistry revealed STb was internalized and was differentially localized in the cytoplasm and nucleus. Cell viability experiments with neutral red and resazurin revealed that STb was toxic in all but the GLUTag cells. STb stimulated 2-hour GLP-2 secretion in all cell models. Interestingly, GLUTag cells produced the highest amount of GLP-2 when treated with STb, demonstrating an inverse relationship in GLP-2 secretion and cell toxicity. To demonstrate a protective role for GLP-2, GLUTag-conditioned media (rich in GLP-2) blocked STb toxicity in STC-1 cells. Confirming a protective role of GLP-2, teduglutide was able to improve cell viability in cells treated with H2O2. In conclusion, STb interacts with the L cell, stimulates secretion, and may induce toxicity if GLP-2 is not produced at high levels. GLP-2 or receptor agonists have the ability to improve cell viability in response to toxins. These results suggest that GLP-2 secretion can play a protective role during STb intoxication. This work supports future investigation into the use of GLP-2 therapies in enterotoxigenic-related diseases.

High-intensity interval exercise in the cold regulates acute and postprandial metabolism.

High-intensity interval exercise (HIIE) has been shown to be more effective than moderate-intensity exercise for increasing acute lipid oxidation and lowering blood lipids during exercise and postprandially. Exercise in cold environments is also known to enhance lipid oxidation; however, the immediate and long-term effects of HIIE exercise in cold are unknown. The purpose of this study was to examine the effects cold stress during HIIE on acute exercise metabolism and postprandial metabolism. Eleven recreationally active individuals (age: 23 ± 3 yr, weight: 80 ± 9.7 kg, V̇O2peak: 39.2 ± 5.73 mL·kg-1·min-1) performed evening HIIE sessions (10 × 60 s cycling, 90% V̇O2peak interspersed with 90 s active recovery, 30% V̇O2peak) in thermoneutral (HIIE-TN, control; 21°C) and cold environment (HIIE-CO; 0°C), following a balanced crossover design. The following morning, participants consumed a high-fat meal. Indirect calorimetry was used to assess substrate oxidation, and venous blood samples were obtained to assess changes in noncellular metabolites. During acute exercise, lipid oxidation was higher in HIIE-CO (P = 0.002) without differences in V̇O2 and energy expenditure (P ≥ 0.162) between conditions. Postprandial V̇O2, lipid and CHO oxidation, plasma insulin, and triglyceride concentrations were not different between conditions (P > 0.05). Postprandial blood LDL-C levels were higher in HIIE-CO 2 h after the meal (P = 0.003). Postprandial glucose area under curve was 49% higher in HIIE-CO versus HIIE-TN (P = 0.034). Under matched energy expenditure conditions, HIIE demonstrated higher lipid oxidation rates during exercise in the cold; but only marginally influenced postprandial lipid metabolism the following morning. In conclusion, HIIE in the cold seemed to be less favorable for postprandial lipid and glycemic responses.NEW & NOTEWORTHY This is the first known study to investigate the effects of cold ambient temperatures on acute metabolism during high-intensity interval exercise, as well as postprandial metabolism the next day. We observed that high-intensity interval exercise in a cold environment does change acute metabolism compared to a thermoneutral environment; however, the addition of a cold stimulus was less favorable for postprandial metabolic responses the following day.

The Protective Role of Hydrogen Sulfide Against Obesity-Associated Cellular Stress in Blood Glucose Regulation.

Circulating palmitic acid (PA) is increased in obesity and causes metabolic stress, leading to diabetes. This includes the impairment of the glucoregulatory hormone glucagon-like peptide-1 (GLP-1) secreted from intestinal L-cells. Recently, the anti-inflammatory gasotransmitter hydrogen sulfide (H2S) has been implicated in the enhancement of GLP-1 secretion. We hypothesized that H2S can reduce the oxidative stress caused by palmitate and play a protective role in L-cell function. This study was conducted on both human and mouse L-cells and a mouse model of Western diet (WD)-induced obesity. PA-induced L-cell stress was assessed using DCF-DA. H2S was delivered using the donor GYY4137. C57BL/6 mice were fed either chow diet or PA-enriched WD for 20 weeks with ongoing measurements of glycemia and GLP-1 secretion. In both L-cell models, we demonstrated that PA caused an increase in reactive oxygen species (ROS). This ROS induction was partially blocked by the H2S administration. In mice, the WD elevated body weight in both sexes and elevated fasting blood glucose and lipid peroxidation in males. Additionally, a single GYY4137 injection improved oral glucose tolerance in WD-fed male mice and also enhanced glucose-stimulated GLP-1 release. To conclude, H2S reduces oxidative stress in GLP-1 cells and can improve glucose clearance in mice.

Impact of the Escherichia coli Heat-Stable Enterotoxin b (STb) on Gut Health and Function.

Enterotoxigenic Escherichia coli (ETEC) produces the heat-stable enterotoxin b (STb), which is responsible for secretory diarrhea in humans and animals. This toxin is secreted within the intestinal lumen of animals and humans following ETEC colonization, becoming active on enterocytes and altering fluid homeostasis. Several studies have outlined the nature of this toxin and its effects on gut health and the integrity of the intestinal epithelium. This review summarizes the mechanisms of how STb alters the gastrointestinal tract. These include the manipulation of mucosal tight junction protein integrity, the formation of enterocyte cellular pores and toxin internalization and the stimulation of programmed cell death. We conclude with insights into the potential link between STb intoxication and altered gut hormone regulation, and downstream physiology.

Expression of PCSK1 (PC1/3), PCSK2 (PC2) and PCSK3 (furin) in mouse small intestine.

The family of serine proteases known as the proprotein convertases subtilisin/kexin type (PCSK) is responsible for the cleavage and maturation of many precursor hormones. Over its three successive regions, the duodenum, the jejunum and the ileum, the small intestine (SI) expresses over 40 peptide hormones necessary for normal intestinal physiology. Most of these hormones derive from proteolytic cleavage of their cognate inactive polypeptide precursors. Members of the PCSK family of proteases have been implicated in this process, although details of enzyme-substrate interactions are largely lacking. As a first step towards elucidating these interactions, we have analyzed by immunohistochemistry the regional distribution of PCSK1, PCSK2 and PCSK3 in mouse SI as well as their cellular co-localization with substance P (SP), cholecystokinin (CCK), glucose-dependent insulinotropic polypeptide (GIP) and somatostatin (SS), 4 peptide hormones known to result from PCSK-mediated processing. Results indicate that PCSK1 is found in all three regions of the SI while PCSK2 and PCSK3 are primarily expressed in the upper two, the duodenum and the jejunum. In these proximal regions, PCSK1 was detectable in 100% of SP-positive (+) cells, 85% of CCK+ cells and 50% of GIP+ cells; PCSK2 was detectable in 40% of SS+ cells and 35% of SP+ cells; PCSK3 was detectable in 75% of GIP+ cells and 60% of SP+ cells. These histological data suggest that the 3 PCSKs may play differential and overlapping roles in prohormone processing in the three regions of the SI.

Hydrogen sulfide suppresses ghrelin secretion in vitro and delays postprandial ghrelin secretion while reducing appetite in mice.

Ghrelin is a stomach-derived hormone that regulates several metabolic functions including growth hormone release, appetite, adiposity, and gastric motility. Nutrients, the autonomic nervous system, and other metabolic hormones have all been implicated in the regulation of ghrelin secretion. Despite this, ongoing efforts to develop modulators of ghrelin secretion in human diseases are still underway. Hydrogen sulfide (H2 S) is a gaseous signaling molecule that is produced both endogenously in many tissues and by the gut microbiome. H2 S has established roles in cardiovascular and immune health, however, more recently H2 S has been implicated in the regulation of metabolic hormone secretion. We hypothesized that H2 S is able to directly regulate ghrelin secretion and in turn, regulate appetite. We first demonstrated that GYY4137 (an H2 S donor molecule) directly suppresses ghrelin secretion in rat primary gastric culture, in part through the activation of the protein kinase B (AKT) pathway. We then demonstrated the colocalization of ghrelin-positive gastric cells with the H2 S producing enzyme cystathionine-γ-lyase (CSE). While GYY4137 suppressed ghrelin secretion, inhibition of CSE caused a stimulation in ghrelin secretion in primary gastric culture. In mice, GYY4137 treatment prolonged the postprandial drop of circulating ghrelin and caused reduced food consumption up to 4 h after treatment. These results demonstrate for the first time a role for H2 S in the regulation of ghrelin and appetite. Modulating H2 S levels may be a novel approach to regulate ghrelin secretion in the treatment of metabolic diseases.

Enhanced access to anthropogenic food waste is related to hyperglycemia in raccoons (Procyon lotor).

Urban landscapes have well-known effects on wildlife populations. Many species of urban wildlife feed on anthropogenic food wastes, and little is known regarding the sub-lethal physiological consequences of this novel diet. We use samples from three populations of raccoons to test the hypothesis that access to anthropogenic food waste will lead to elevated body mass, blood glucose and serum leptin. Each population varied in their presumed access to food waste. We found that raccoons from the site with the highest presumed access to food waste were significantly heavier and had significantly higher levels of glycated serum protein (GSP, a marker of elevated blood glucose). In addition, GSP concentration was positively related to body mass. No significant differences in serum leptin were detected, nor was serum leptin related to body mass. Urban diets may have significant physiological consequences for urban wildlife related to glucose metabolism. Further research will be needed to determine the evolutionary consequences of the novel urban diet, and whether adaptation is occurring.

A role for methanogens and methane in the regulation of GLP-1.

INTRODUCTION: The gastrointestinal (GI) microbiome has emerged as a potential regulator of metabolism. However, the precise mechanisms of how microorganisms may influence physiology remain largely unknown. Interestingly, GI microorganisms, including methanogens, are localized within the same regions as the glucagon-like peptide-1 (GLP-1) secreting L cells. GLP-1 plays key roles appetite and glucose regulation. Furthermore, both methane and GLP-1 levels are altered in obese humans with metabolic disease. We predict that high-fat diet-induced obesity alters the abundance of GI methanogens and that methane may play a role in the GLP-1 secretory response from the L cell. METHODS: To demonstrate this, GLP-1 secretion response and faecal methanogens were examined in mice given a high-fat diet for 14 weeks. In addition, the direct effect of methane on GLP-1 secretion was assessed in two L-cell models (NCI-H716 and GLUTag). RESULTS: High-fat diet caused a significant increase in both GLP-1 secretion and faecal methanogen content. There was a direct correlation between GLP-1 secretion response and faecal methanogen levels. In L cells, methane stimulated GLP-1 secretion and enhanced intracellular cAMP content. CONCLUSION: These results indicate that alterations in the methanogen communities occurring in obesity may play a vital role in directly enhancing GLP-1 secretion, and that methane can directly stimulate the secretion of GLP-1.

Hydrogen Sulfide and Sulfate Prebiotic Stimulates the Secretion of GLP-1 and Improves Glycemia in Male Mice.

Recently, the gastrointestinal microbiome, and its metabolites, has emerged as a potential regulator of host metabolism. However, to date little is known on the precise mechanisms of how this regulation occurs. Hydrogen sulfide (H2S) is abundantly produced in the colon by sulfate-reducing bacteria (SRB). H2S is a bioactive gas that plays regulatory roles in many systems, including metabolic hormone regulation. This gas metabolite is produced in close proximity to the glucagonlike peptide-1 (GLP-1)-secreting cells in the gut epithelium. GLP-1 is a peptide hormone that plays pivotal roles in both glucose homeostasis and appetite regulation. We hypothesized that H2S can directly regulate GLP-1 secretion. We demonstrated that H2S donors (NaHS and GYY4137) directly stimulate GLP-1 secretion in murine L-cells (GLUTag) and that this occurs through p38 mitogen-activated protein kinase without affecting cell viability. We then increased SRB in mice by supplementing the diet with a prebiotic chondroitin sulfate for 4 weeks. Mice treated with chondroitin sulfate had elevated Desulfovibrio piger levels in the feces and increased colonic and fecal H2S concentration. These animals also had enhanced GLP-1 and insulin secretion, improved oral glucose tolerance, and reduced food consumption. These results indicate that H2S plays a stimulatory role in GLP-1 secretion and that sulfate prebiotics can enhance GLP-1 release and its downstream metabolic actions.

Implications of Hydrogen Sulfide in Glucose Regulation: How H2S Can Alter Glucose Homeostasis through Metabolic Hormones.

Diabetes and its comorbidities continue to be a major health problem worldwide. Understanding the precise mechanisms that control glucose homeostasis and their dysregulation during diabetes are a major research focus. Hydrogen sulfide (H2S) has emerged as an important regulator of glucose homeostasis. This is achieved through its production and action in several metabolic and hormone producing organs including the pancreas, liver, and adipose. Of importance, H2S production and signaling in these tissues are altered during both type 1 and type 2 diabetes mellitus. This review first examines how H2S is produced both endogenously and by gastrointestinal microbes, with a particular focus on the altered production that occurs during obesity and diabetes. Next, the action of H2S on the metabolic organs with key roles in glucose homeostasis, with a particular focus on insulin, is described. Recent work has also suggested that the effects of H2S on glucose homeostasis goes beyond its role in insulin secretion. Several studies have demonstrated important roles for H2S in hepatic glucose output and adipose glucose uptake. The mechanism of H2S action on these metabolic organs is described. In the final part of this review, future directions examining the roles of H2S in other metabolic and glucoregulatory hormone secreting tissues are proposed.

Ghrelin Is a Novel Regulator of GLP-1 Secretion.

GLP-1 is a gastrointestinal L-cell hormone that enhances glucose-stimulated insulin secretion. Hence, strategies that prevent GLP-1 degradation or activate the GLP-1 receptor are used to treat patients with type 2 diabetes. GLP-1 secretion occurs after a meal and is partly regulated by other circulating hormones. Ghrelin is a stomach-derived hormone that plays a key role in whole-body energy metabolism. Because ghrelin levels peak immediately before mealtimes, we hypothesized that ghrelin plays a role in priming the intestinal L-cell for nutrient-induced GLP-1 release. The intraperitoneal injection of ghrelin into mice 15 min before the administration of oral glucose enhanced glucose-stimulated GLP-1 release and improved glucose tolerance, whereas the ghrelin receptor antagonist D-Lys GHRP-6 reduced plasma levels of GLP-1 and insulin and diminished oral glucose tolerance. The ghrelin-mediated improvement in glucose tolerance was lost in mice coinjected with a GLP-1 receptor antagonist as well as in Glp1r(-/-) mice lacking the GLP-1 receptor. The impaired oral glucose tolerance in diet-induced obese mice was also improved by ghrelin preadministration. Importantly, ghrelin directly stimulated GLP-1 release from L-cell lines (murine GLUTag, human NCI-H716) through an extracellular signal-related kinase 1/2-dependent pathway. These studies demonstrate a novel role for ghrelin in enhancing the GLP-1 secretory response to ingested nutrients.

Chronic Exposure to TNFα Impairs Secretion of Glucagon-Like Peptide-1.

Obesity is associated with systemic inflammation and elevated levels of TNFα, leading to impaired glucose tolerance. In humans, obesity is also associated with reduced nutrient-stimulated secretion of the intestinal incretin hormone, glucagon-like peptide-1 (GLP-1). We hypothesized that TNFα plays a direct role in the impairment of GLP-1 secretion from the enteroendocrine L-cell and that blocking TNFα can restore both GLP-1 secretion and glucose homeostasis. Expression of the TNFα receptor subytpe-1 was detected in the human NCI-H716 and murine GLUTag L-cell models and in mouse ileal sections. Although TNFα acutely increased GLP-1 release from NCI-H716 cells (P < .05-.001), preincubation with TNFα for 24 hours reduced proglucagon mRNA (P < .05) and GLP-1 cellular (P < .05) levels without affecting cell viability. Furthermore, both NCI-H716 and GLUTag cells pretreated with TNFα for 24 hours no longer responded to known GLP-1 secretagogues, an effect that was reversed by coincubation with the Nuclear Factor Kappa B inhibitor, 5-aminosalicylic acid, in the NCI-H716 cells. Mice given a high-fat diet (HFD) for 12 weeks developed impaired glucose tolerance, hyperinsulinemia, and increased TNFα mRNA expression in fat and ileal tissue. Hyperglycemia and hyperinsulinemia were reduced in HFD mice treated with the anti-TNFα biological, etanercept, for 2 weeks. In primary intestinal cultures from these animals, HFD control mice had impaired GLP-1 secretion, and this was not observed in the HFD etanercept-derived cultures (P < .05). In conclusion, chronic exposure to TNFα directly impairs GLP-1 secretion at the level of the intestinal L-cell, an effect that is reversed by anti-TNFα therapy in association with improved glucose tolerance.

Glucagon stimulates ghrelin secretion through the activation of MAPK and EPAC and potentiates the effect of norepinephrine.

Ghrelin is a stomach-derived orexigenic hormone whose levels in circulation are altered by energy availability. Like ghrelin, the glucotropic hormone glucagon increases in the fasting state and serves to normalize energy levels. We hypothesized that glucagon can directly stimulate stomach ghrelin production. To verify this hypothesis, we used a primary culture of dispersed rat stomach cells. We first demonstrated that stomach ghrelin cells express the glucagon receptor (GluR). Glucagon (1-100 nM) significantly stimulated ghrelin secretion and proghrelin mRNA expression, and co-incubation with a GluR inhibitor prevented glucagon's action. The MAP kinase inhibitor (PD98058) reduced the glucagon-stimulated ghrelin secretion and proghrelin mRNA expression. Furthermore, glucagon treatment increased the phosphorylation of ERK1/2. Glucagon also increased intracellular cAMP levels, and inhibition of adenylate cyclase reduced glucagon's effect on ghrelin secretion. Surprisingly, inhibiting protein kinase A (PKA) (using H89 and phosphorothioate [Rp]-cAMP) did not prevent glucagon-stimulated ghrelin secretion. Instead, inhibiting the exchange protein activated by cAMP (EPAC) with Brefeldin-A was able to significantly reduce glucagon-stimulated ghrelin secretion. Furthermore, the EPAC agonist (8-pCPT) significantly stimulated ghrelin secretion. Depleting endoplasmic reticulum calcium stores or blocking voltage-dependant calcium channels prevented glucagon stimulated ghrelin secretion. Finally, co-incubation with the sympathetic neurotransmitter norepinephrine potentiated the glucagon stimulation of ghrelin secretion. Our findings are the first to show a direct link between glucagon and stomach ghrelin production and secretion and highlight the role of MAPK, the PKA-independent EPAC pathway, and the synergy between norepinephrine and glucagon in ghrelin release.

Insulin and norepinephrine regulate ghrelin secretion from a rat primary stomach cell culture.

Ghrelin is a peptide hormone primarily produced in the previously unidentified X/A endocrine cells of the stomach. Extensive studies have focused on the effects of ghrelin on growth hormone release and appetite regulation. However, the mechanisms regulating ghrelin secretion are less understood. In the present study, we developed a primary culture of newborn rat stomach cells to investigate the mechanisms regulating ghrelin synthesis and secretion. We demonstrated that this cell preparation secretes ghrelin in a regulated manner through the increase of cAMP, intracellular calcium, and activation of protein kinase C. Norepinephrine (NE) (0.1-10 µm) stimulated ghrelin secretion through the ß1-adrenergic receptor via increased cAMP and protein kinase A activity, whereas acetylcholine had no effect. Because circulating ghrelin levels were previously shown to be inversely correlated with insulin levels, we investigated the effect of insulin on ghrelin secretion. We first demonstrated that ghrelin cells express the insulin receptor α- and ß-subunits. Next, we determined that insulin (1-10 nm) inhibited both basal and NE-stimulated ghrelin secretion, caused an increase in phosphorylated serine-threonine kinase (AKT) and a reduction in intracellular cAMP, but did not alter proghrelin mRNA levels. The inhibitory effect of insulin was blocked by inhibiting phospho-inositol-3 kinase and AKT but not MAPK. Higher dose insulin (100 nm) did not suppress ghrelin secretion, which prompted the investigation of cellular insulin resistance by pretreating the cells with 100 nm insulin for 24 h. This caused a reduction in insulin receptor expression and prevented the insulin-mediated AKT activation and the suppression of ghrelin secretion with no impact on NE-stimulated ghrelin secretion. Our findings highlight the role of the sympathetic nervous system, insulin, and insulin resistance in the regulation of ghrelin secretion.