Ghrelin is a 28-amino acid peptide predominantly produced in the stomach and secreted into the circulation. Ghrelin is found in plasma and tissues in two major forms of n-octanoyl-modified at its N-terminal third serine residue and des-acyl ghrelin. The n-octanoyl group of ghrelin is essential for its growth hormone (GH)-releasing activity and appetite regulation mediated through growth hormone secretagogue receptor (GHS-R). We demonstrated that both ghrelin and des-acyl ghrelin evoke vasodilatation at remarkably low concentrations compared to acetylcholine (ACh) in phenylephrine (PE)-constricted perfused rat mesenteric vascular bed (MVB). This was abolished in endothelium-denuded preparations and in endothelium-intact preparations exposed to either a calcium-activated potassium channel (KCa) blocker or a depolarizing stimulus. While KATP channel blockade, nitric oxide synthase and cyclooxygenase inhibition had no effect, the responses were abolished in the presence of combinations of apamin and charybdotoxin, apamin and TRAM-34, and ouabain and Ba2+. The GHS-R antagonist, [D-Lys3]-GHRP-6, per se evoked vasodilatation. Inclusion of L-756867, a peptide antagonist of classical GHS-R, failed to evoke any vasodilator response or to affect vasodilatation evoked by ghrelin. Both non-peptide agonists of GHS-R, L-166446 and L-163255, demonstrated concentration-dependent decreases in perfusion pressure. All short peptides encompassing the first 20, 16, 10, 6, 4, and 3 residues of des-acyl ghrelin were able to evoke vasodilator responses to the same extent as des-acyl ghrelin. However, vasodilatation to single amino acids, L-serine and glycine, were significantly attenuated. Streptozotocin (STZ)-induced diabetes increased plasma ghrelin concentration. Diabetes for 4-weeks did not cause any significant reduction in ghrelin-evoked vasodilatation, whereas 8-weeks diabetes significantly reduced ghrelin-evoked vasodilatation. In contrast to ghrelin, there was a duration-dependent fall in vasodilator response to ACh from 4- to 8-weeks diabetes. These data suggest that the vasodilatation evoked by ghrelin is mediated by endothelium-dependent hyperpolarization (EDHF) by mechanism(s) that are independent of classical GHS-R activation. In addition, EDHF-dependent ghrelin-evoked vasodilator responses may not be affected, at least in the early stages of STZ diabetes, whereas the responses to ACh, predominantly mediated through nitric oxide, are progressively diminished right from the early stages of endothelial dysfunction in STZ diabetic rats.

Ghrelin is a 28-amino acid peptide predominantly produced in the stomach and secreted into the circulation. Ghrelin is found in plasma and tissues in two major forms of n-octanoyl-modified at its N-terminal third serine residue and des-acyl ghrelin. The n-octanoyl group of ghrelin is essential for its growth hormone (GH)-releasing activity and appetite regulation mediated through growth hormone secretagogue receptor (GHS-R). We demonstrated that both ghrelin and des-acyl ghrelin evoke vasodilatation at remarkably low concentrations compared to acetylcholine (ACh) in phenylephrine (PE)-constricted perfused rat mesenteric vascular bed (MVB). This was abolished in endothelium-denuded preparations and in endothelium-intact preparations exposed to either a calcium-activated potassium channel (KCa) blocker or a depolarizing stimulus. While KATP channel blockade, nitric oxide synthase and cyclooxygenase inhibition had no effect, the responses were abolished in the presence of combinations of apamin and charybdotoxin, apamin and TRAM-34, and ouabain and Ba2+. The GHS-R antagonist, [D-Lys3]-GHRP-6, per se evoked vasodilatation. Inclusion of L-756867, a peptide antagonist of classical GHS-R, failed to evoke any vasodilator response or to affect vasodilatation evoked by ghrelin. Both non-peptide agonists of GHS-R, L-166446 and L-163255, demonstrated concentration-dependent decreases in perfusion pressure. All short peptides encompassing the first 20, 16, 10, 6, 4, and 3 residues of des-acyl ghrelin were able to evoke vasodilator responses to the same extent as des-acyl ghrelin. However, vasodilatation to single amino acids, L-serine and glycine, were significantly attenuated. Streptozotocin (STZ)-induced diabetes increased plasma ghrelin concentration. Diabetes for 4-weeks did not cause any significant reduction in ghrelin-evoked vasodilatation, whereas 8-weeks diabetes significantly reduced ghrelin-evoked vasodilatation. In contrast to ghrelin, there was a duration-dependent fall in vasodilator response to ACh from 4- to 8-weeks diabetes. These data suggest that the vasodilatation evoked by ghrelin is mediated by endothelium-dependent hyperpolarization (EDHF) by mechanism(s) that are independent of classical GHS-R activation. In addition, EDHF-dependent ghrelin-evoked vasodilator responses may not be affected, at least in the early stages of STZ diabetes, whereas the responses to ACh, predominantly mediated through nitric oxide, are progressively diminished right from the early stages of endothelial dysfunction in STZ diabetic rats.

München, Techn. Universiẗat, Diss., 2007.

Thesis (M.S.)--North Carolina State University (Dept. of Animal Science). / "Caroline Elizabeth Brown" -- p. iii. Includes vita. Includes bibliographical references (p. 50-54).

Ghrelin is an appetite-stimulating hormone produced mainly in the stomach and duodenum. Poor ghrelin control is often caused by obesity-related hyperinsulinemia, which fails to suppress ghrelin and results in excess appetite and higher body fat storage that perpetuates even greater fat accumulation. High intensity exercise has been shown to acutely decrease plasma acylated ghrelin concentrations in healthy weight individuals. However, the evidence for how exercise affects ghrelin in obese individuals is currently lacking. PURPOSE: To compare the effects of high intensity interval exercise on acute plasma acylated ghrelin levels in obese and non-obese males. METHODS: Eighteen subjects with a mean age of 29.8 yr. (± 7.6) were assessed for body fat percent (BF%), acylated ghrelin and hunger. Subjects included 9 non- obese men (BF% mean= 13.7 ± 3.6) and 9 obese men (BF% mean = 31.7 ± 4.7) who agreed to participate in this study. Using a crossover design, participants were randomly assigned to an exercise or control condition, with each subject acting as their own control. The exercise trial consisted of participants cycling at high intensity intervals for 20 minutes (not including the 5 minute warmup and cool down) at a rate of 65% to 85% of their heart rate reserve on a cycle ergometer followed by 60 minutes of rest. The control trial consisted of 90 minutes of rest. Blood samples (3-4ml) were collected at baseline, 0.5, 1, and 1.5 hours post-intervention. Acylated ghrelin concentrations were determined from plasma. Hunger was assessed using a 10-point Likert-type scale while blood samples were being drawn. Group means for plasma ghrelin concentrations between groups were analyzed using an independent t-test. The effect of exercise on ghrelin was analyzed using paired t-test. The relationship between perceived hunger and ghrelin was assessed using Pearson correlations. RESULTS: Baseline plasma ghrelin levels were significantly higher in the non-obese group when compared to the obese group (t = 3.43, p = .036). While exercise was effective in reducing plasma acylated ghrelin levels in the non-obese group (t = 2.34, p = .047), no significant changes were found in acylated ghrelin in the obese group between baseline or any time point following the exercise intervention. CONCLUSIONS: The low resting levels of plasma ghrelin concentrations exhibited by the obese subjects, when compared to non-obese subjects, may result in long fasting periods that lead to hypoglycemia and a hyperinsulinemic response at the next eating opportunity. Furthermore, the lack of reduction in ghrelin following exercise may result in an overconsumption of energy. Both the sustained ghrelin with associated excess energy intake and the hyperinsulinemia may result in sustained or increased fat storage in obese individuals.

Proghrelin produces three proghrelin derived peptides (PGDP) known for their roles in glucose homeostasis: acylated ghrelin (AG), unacylated ghrelin (UAG), and obestatin (Ob). Only the receptor for AG, growth hormone secretagogue receptor 1a (GHS R1a), is known, however there is evidence for additional receptors for AG, UAG and Ob. Our aim was to determine the actions of PGDP on two beta cell lines, MIN6 and INS-1, which we have shown to contain and lack GHS R1a respectively. PGDP increased proliferation in INS-1 but not MIN6 cells, measured by BrdU incorporation. AG decreased apopotosis in both cell lines, measured by decreased levels of activated caspase 3. Insulin secretion was investigated in INS-1 cells, where PGDP modulated insulin release in a glucose dependent manner. Our results indicate that PGDP modulate beta cell mass in the presence and absence of GHS R1a, and present a detailed anaylsis of insulin secretion in INS-1 cells.

The incidence of obesity, and the associated morbidities and mortality are increasing. Strategies to manage this disease hinge on the balance of caloric intake and energy expenditure. This regulation depends largely on endocrine input from the periphery. The recently discovered stomach derived hormone, ghrelin, has emerged as a key player in the regulation of appetite and energy storage. Ghrelin achieves these functions through binding the ghrelin receptor in appetite regulating neurons and in peripheral metabolic organs including the pancreas and adipose tissue. Since ghrelin acts on energy regulating metabolic organs, its secretion from the stomach is tightly coupled to energy availability. Ghrelin levels increase during periods of fasting and decrease after a meal is consumed. Under chronic energy surplus (such as obesity) ghrelin levels decrease while in chronic energy deficit (anorexia nervosa, weight loss) ghrelin levels increase. While major advances have been made in understanding both the function of ghrelin and its dysregulation in disease, little is known about the cellular regulation of ghrelin secretion. This is due to the lack of cellular models of ghrelin secretion. In this thesis, I describe the development of a novel ghrelin secreting primary rat stomach cell culture. Using this system I elucidated the roles and mechanisms of neurotransmitters, hormones (insulin and glucagon), nutrients (glucose) and anti-diabetics (metformin) in the regulation of ghrelin secretion. These findings have clearly demonstrated the ability of ghrelin cells to sense energy availability and provide important insights for ghrelin altering therapies. To evaluate both the function of ghrelin and the feasibility of reducing circulating ghrelin, I developed a novel in vivo ghrelin-reducing strategy. In vivo expression of a decoy protein based on the ligand binding domains of the ghrelin receptor was expressed in mice. Mice treated with this plasmid construct had reduced circulating levels of ghrelin. Interestingly, reduced circulating ghrelin was protective from high fat diet-induced obesity and resulted in improved glucose metabolism. This work demonstrates both the importance of ghrelin in peripheral energy storage and the feasibility of this novel ghrelin reducing approach for the treatment of obesity and insulin resistance.

No description available.

Access to abstract permanently restricted to Ball State community only. / Access to thesis permanently restricted to Ball State community only.

Ghrelin is a 28 amino acid peptide that interacts with ghrelin receptors (GHS-Rs) to modulate brain reinforcement circuits. Systemic ghrelin infusions augment cocaine (COC) stimulated locomotion and conditioned place preference (CPP) in rats, whereas genetic or pharmacological ablation of GHS-Rs has been shown to attenuate the acute locomotor-enhancing effects of nicotine (NIC) and COC, and to blunt the CPP induced by food, alcohol, amphetamine and COC in mice. The stimulant NIC can induce CPP and like COC, repeated administration of NIC induces locomotor sensitization in rats. In experiment 1, we examined the effects of GHS-R antagonism with JMV 2959 on COC-induced locomotion and found that JMV 2959 suppresses COC-induced locomotor sensitization. In experiment 2, we examined the effects of GHS-R antagonism with JMV 2959 on NIC-induced locomotion and found that JMV 2959 suppresses NIC-induced locomotor sensitization. In experiment 3, we examined the effects of GHS-R knockout on COC-induced locomotion and found that animals sustaining GHS-R knockout display a suppression of COC-induced locomotor sensitization. In experiment 4, we examined the effects of GHS-R knockout on COC-induced locomotion and found that animals sustaining GHS-R knockout display a suppression of COC-induced locomotor sensitization. In experiment 5, we examined the effects of JMV 2959 on NIC-enhanced intracranial self-stimulation (ICSS) responding and found that JMV 2959 alone had no effect, but when combined with NIC,JMV 2959 pretreatment reversed the enhancement of responding produced by NIC. In experiment 6, we examined the effects of GHS-R knockout on ICSS responding and found that animals sustaining GHS-R knockout were unable to acquire ICSS at current intensity levels that would support responding by WT animals. It was not until the intensity was ramped up four fold that these knockout rats were able to acquire responding. These results show that antagonism of GHS-Rs diminishes the reinforcing effects of NIC and COC. This provides evidence that antagonists of GHS-Rs could be useful in the treatment of drug addiction, particularly that involving nicotine.