Delhanty, P.J.D.

In vitro and in vivo stability and pharmacokinetic profile of unacylated ghrelin (UAG) analogues (Article)

2012-11-20T00:00:00Z

Ghrelin, an endocrine hormone predominantly produced by the stomach, exists in acylated and unacylated forms in the circulation. Unacylated ghrelin (UAG), the more abundant form in blood, possesses similar, independent or opposite physiological actions as acylated ghrelin (AG). AZP502, a linear 8-amino acid peptide from the central region of UAG (UAG6-13), and its full (AZP531) and partially (AZP533) cyclised derivatives, exhibit the same pharmacological profile as UAG both in vitro and in vivo, independently of AG receptor binding. We investigated the stability of these three fragments in vitro in human blood samples and in vivo after subcutaneous and intravenous injection in rats and dogs using liquid chromatography-mass spectrometry. In both species, AZP502 is rapidly degraded generating two major metabolites. Partial cyclisation of AZP502 and acylation at its N-terminus (AZP533 peptide) improves its stability in human plasma in vitro. Full cyclisation of AZP502 (AZP531 peptide) also completely protects the peptide from peptidase degradation in vitro in human blood samples. Moreover this cyclisation strongly improves the stability and the bioavailability of this peptide in vivo in both dogs and rats (mean bioavailability of 10-15% and 85-95% for AZP502 and AZP531 respectively). Taken together these results support the rationale for developing AZP531 as a long-acting UAG analogue for subcutaneous injection for the treatment of type 2 diabetes mellitus and other metabolic disorders.

Ghrelin: a new treatment for non-alcoholic fatty liver disease? (Article)

2012-09-20T00:00:00Z

Ghrelin and glucose homeostasis (Article)

2011-11-01T00:00:00Z

Ghrelin plays an important physiological role in modulating GH secretion, insulin secretion and glucose metabolism. Ghrelin has direct effects on pancreatic islet function. Also, ghrelin is part of a mechanism that integrates the physiological response to fasting. However, pharmacologic studies indicate the important obesogenic/diabetogenic properties of ghrelin. This is very likely of physiological relevance, deriving from a requirement to protect against seasonal periods of food scarcity by building energy reserves, predominantly in the form of fat. Available data indicate the potential of ghrelin blockade as a means to prevent its diabetogenic effects. Several studies indicate a negative correlation between ghrelin levels and the incidence of type 2 diabetes and insulin resistance. However, it is unclear if low ghrelin levels are a risk factor or a compensatory response. Direct antagonism of the receptor does not always have the desired effects, however, since it can cause increased body weight gain. Pharmacological suppression of the ghrelin/des-acyl ghrelin ratio by treatment with des-acyl ghrelin may also be a viable alternative approach which appears to improve insulin sensitivity. A promising recently developed approach appears to be through the blockade of GOAT activity, although the longer term effects of this treatment remain to be investigated.

Preoperative fasting induced protection against renal ischemia/reperfusion injury is independent of ghrelin in mice (Article)

2010-12-01T00:00:00Z

One of the factors negatively influencing the outcome after kidney transplantation is ischemia-reperfusion (I/R) injury. Preoperative fasting is able to confer protection against I/R injury. We hypothesized that the protection imposed by preoperative fasting is mediated by increased levels of acylated ghrelin. Male C57BL/6 mice, 10 to 12 weeks old, were fasted for 1, 2, or 3 days, after which, acylated ghrelin levels were determined. Ad libitum fed mice were injected with acylated ghrelin or phosphate-buffered saline before renal I/R injury. Furthermore, mice were fasted for 3 days during which they were injected with a growth hormone secretagogue receptor antagonist, to block the effects of ghrelin, or a vehiculum. Bilateral renal I/R injury was induced by clamping the artery and vein of the left and right kidney simultaneously for 37 minutes. Kidney function was assessed by means of serum urea values determined at 24 and 48 hours after reperfusion. Fasting significantly increased acylated ghrelin serum levels. Ghrelin suppletion in ad libitum fed animals or ghrelin receptor blockade in fasted animals did not affect renal function after I/R injury. Our data suggest that the increased levels of acylated ghrelin induced by fasting do not mediate its protection against renal I/R injury.

Unsaturated fatty acids prevent desensitization of the human growth hormone secretagogue receptor by blocking its internalization (Article)

2010-09-01T00:00:00Z

The composition of the plasma membrane affects the responsiveness of cells to metabolically important hormones such as insulin and vasoactive intestinal peptide. Ghrelin is a metabolically regulated hormone that activates the G protein-coupled receptor GH secretagogue receptor type 1a (GHSR) not only in the pituitary gland but also in peripheral tissues such as the pancreas, stomach, and T cells in the circulation. We have investigated the effects of lipids and altered plasma membrane composition on GHSR activation. Oligounsaturated fatty acids (OFAs) disrupt the structure of membranes and make them more fluid. Prolonged (96 h), but not acute, treatment of the GHSR cells with the 18C OFAs oleic and linoleic acid caused a significant increase in sensitivity of the receptor to ghrelin (EC50reduced by a factor of 2.4 and 2.9 at 60 and 120 μM OFAs, respectively). OFAs were found to block the inhibitory effects of ghrelin pretreatment on subsequent ghrelin responsiveness, suggesting that OFAs suppress desensitization of GHSR. Radioligand displacement studies did not show a significant shift in receptor binding after incubation with OFAs. However, it was found that OFA treatment suppressed GHSR internalization, likely explaining OFA-induced refractoriness to ligand-induced desensitization. The involvement of lipid rafts in this process was indicated by the altered responsiveness of GHSR under conditions that alter membrane cholesterol. In conclusion, our findings demonstrate the importance of membrane composition for GHSR activation and desensitization and indicate at least part of the mechanism through which OFAs and cholesterol could affect ghrelin's activity in vivo. Copyright

Unacylated ghrelin rapidly modulates lipogenic and insulin signaling pathway gene expression in metabolically active tissues of GHSR deleted mice (Article)

2010-08-23T00:00:00Z

Background: There is increasing evidence that unacylated ghrelin (UAG) improves insulin sensitivity and glucose homeostasis; however, the mechanism for this activity is not fully understood since a UAG receptor has not been discovered. Methodology/Principal Findings: To assess potential mechanisms of UAG action in vivo, we examined rapid effects of UAG on genome-wide expression patterns in fat, muscle and liver of growth hormone secretagogue receptor (GHSR)-ablated mice using microarrays. Expression data were analyzed using Ingenuity Pathways Analysis and Gene Set Enrichment Analysis. Regulation of subsets of these genes was verified by quantitative PCR in an independent experiment. UAG acutely regulated clusters of genes involved in glucose and lipid metabolism in all three tissues, consistent with enhancement of insulin sensitivity. Conclusions/Significance: Fat, muscle and liver are central to the control of lipid and glucose homeostasis. UAG rapidly modulates the expression of metabolically important genes in these tissues in GHSR-deleted mice indicating a direct, GHSRindependent, action of UAG to improve insulin sensitivity and metabolic profile.

Effects of acute administration of acylated and unacylated ghrelin on glucose and insulin concentrations in morbidly obese subjects without overt diabetes (Article)

2009-11-27T00:00:00Z

Objective: To investigate the effects of unacylated ghrelin (UAG) and co-administration of acylated ghrelin (AG) and UAG in morbid obesity, a condition characterized by insulin resistance and low GH levels. Design and method: Eight morbidly obese non-diabetic subjects were treated with either UAG 200 μg, UAG 100 μg in combination with AG 100 μg (Comb) or placebo in three episodes of 4 consecutive days in a double-blind randomized crossover design. Study medication was administered as daily single i.v. bolus injections at 0900 h after an overnight fast. At 1000 h, a standardized meal was served. Glucose, insulin, GH, free fatty acids (FFA) and ghrelin were measured up to 4 h after administration. Results: Insulin concentrations significantly decreased after acute administration of Comb only, reaching a minimum at 20 min: 58.2 ± 3.9% of baseline versus 88.7 ± 7.2 and 92.7± 2.6% after administration of placebo and UAG respectively (P<0.01). After 1 h, insulin concentration had returned to baseline. Glucose concentrations did not change after Comb. However, UAG administration alone did not change glucose, insulin, FFA or GH levels. Conclusion: Co-administration of AG and UAG as a single i.v. bolus injection causes a significant decrease in insulin concentration in non-diabetic subjects suffering from morbid obesity. Since glucose concentration did not change in the first hour after Comb administration, our data suggest a strong improvement in insulin sensitivity. These findings warrant studies in which UAG with or without AG is administered for a longer period of time. Administration of a single bolus injection of UAG did not influence glucose and insulin metabolism.

Ghrelin and Bone (Article)

2007-11-06T00:00:00Z

A consequence of gastrectomy is loss of bone mass. Several mechanisms have been proposed, such as malabsorption of vitamins and minerals. Additionally, a peptide hormone produced in the stomach has been shown to mediate a calcitropic effect on bone. The identity of this peptide has not been elucidated, but ghrelin, produced by A-like cells in the fundus of the stomach, could be a good candidate. Ghrelin stimulates growth hormone (GH) secretion both in vivo and in vitro, and could by this means have a positive effect on bone. There is also evidence for direct effects of ghrelin on bone. We discuss here the role that ghrelin may play in bone metabolism, based on the most recent literature.

Intravenous glucose administration in fasting rats has differential effects on acylated and unacylated ghrelin in the portal and systemic circulation: A comparison between portal and peripheral concentrations in anesthetized rats (Article)

2007-11-01T00:00:00Z

Ghrelin is produced by the gastrointestinal tract, and its systemic concentrations are mainly regulated by nutritional factors. Our aim was to investigate: 1) endogenous portal and systemic acylated and unacylated ghrelin levels (AG and UAG, respectively); 2) whether an iv glucose tolerance test (IVGTT) modifies AG and UAG; and 3) whether the liver passage plays a role in regulating systemic AG and UAG. To elucidate this, we evaluated the effects of IVGTT or saline injection on endogenous portal and systemic concentrations of glucose, insulin, AG, and UAG in anesthetized fasting rats. Hepatic extraction of insulin, AG, and UAG and the ratio of AG to UAG were also measured. IVGTT suppressed both portal (P < 0.03) and peripheral (P < 0.05) UAG, whereas it only blunted prehepatic, but not peripheral, AG. During fasting, hepatic clearance of UAG was 11%, and it was decreased to 8% by IVGTT. AG was cleared by the liver by 38% but unaffected by glucose. The AG to UAG ratio was higher in the portal than the systemic circulation, both in the saline (P < 0.004) and IVGTT (P < 0.0005) rats. In conclusion, this study shows that: 1) the ratio of AG to UAG is very low in the portal vein and decreases further in the systemic circulation; 2) IVGTT in anesthetized fasting rats inhibits UAG, whereas it only blunts prehepatic, but not systemic, AG; and 3) hepatic clearance of AG is much higher than that of UAG. Thus, our results suggest that peripheral AG metabolic regulation and action are mainly confined within the gastrointestinal tract. Copyright

Unacylated ghrelin acts as a potent insulin secretagogue in glucose-stimulated conditions (Article)

2007-09-01T00:00:00Z

Acylated and unacylated ghrelin (AG and UAG) are gut hormones that exert pleiotropic actions, including regulation of insulin secretion and glucose metabolism. In this study, we investigated whether AG and UAG differentially regulate portal and systemic insulin levels after a glucose load. We studied the effects of the administration of AG (30 nmol/kg), UAG (3 and 30 nmol/kg), the ghrelin receptor antagonist [D-Lys3]GHRP-6 (1 μmol/kg), or various combinations of these compounds on portal and systemic levels of glucose and insulin after an intravenous glucose tolerance test (IVGTT, D-glucose 1 g/kg) in anesthetized fasted Wistar rats. UAG administration potently and dose-dependently enhanced the rise of insulin concentration induced by IVGTT in the portal and, to a lesser extent, the systemic circulation. This UAG-induced effect was completely blocked by the coadministration of exogenous AG at equimolar concentrations. Similarly to UAG, [D-Lys3]GHRP-6, alone or in combination with AG and UAG, strongly enhanced the portal insulin response to IVGTT, whereas exogenous AG alone did not exert any further effect. Our data demonstrate that, in glucose-stimulated conditions, exogenous UAG acts as a potent insulin secretagogue, whereas endogenous AG exerts a maximal tonic inhibition on glucose-induced insulin release. Copyright

Unacylated ghrelin is not a functional antagonist but a full agonist of the type 1a growth hormone secretagogue receptor (GHS-R) (Article)

2007-08-15T00:00:00Z

Recent findings demonstrate that the effects of ghrelin can be abrogated by co-administered unacylated ghrelin (UAG). Since the general consensus is that UAG does not interact with the type 1a growth hormone secretagogue receptor (GHS-R), a possible mechanism of action for this antagonistic effect is via another receptor. However, functional antagonism of the GHS-R by UAG has not been explored extensively. In this study we used human GHS-R and aequorin expressing CHO-K1 cells to measure [Ca2+]ifollowing treatment with UAG. UAG at up to 10-5M did not antagonize ghrelin induced [Ca2+]i. However, UAG was found to be a full agonist of the GHS-R with an EC50of between 1.6 and 2 μM using this in vitro system. Correspondingly, UAG displaced radio-labeled ghrelin from the GHS-R with an IC50of 13 μM. In addition, GHS-R antagonists were found to block UAG induced [Ca2+]iwith approximately similar potency to their effect on ghrelin activation of the GHS-R, suggesting a similar mode of action. These findings demonstrate in a defined system that UAG does not antagonize activation of the GHS-R by ghrelin. But our findings also emphasize the importance of assessing the concentration of UAG used in both in vitro and in vivo experimental systems that are aimed at examining GHS-R independent effects. Where local concentrations of UAG may reach the high nanomolar to micromolar range, assignment of GHS-R independent effects should be made with caution.

Ghrelin and its unacylated isoform stimulate the growth of adrenocortical tumor cells via an anti-apoptotic pathway (Article)

2007-07-01T00:00:00Z

Ghrelin is expressed in normal human adrenocortical cells and induces their proliferation through growth hormone secretagogue receptor 1a (GHS-R1a). Consequently, it was of interest to us to determine whether acylated ghrelin and its predominant serum isoform, unacylated ghrelin, also act as factors for adrenocortical carcinoma cell growth. To examine a potential ghrelin-regulated system in adrenocortical tumors, we measured proliferative effects of acylated and unacylated ghrelin in the adrenocortical carcinoma cell lines SW-13 and NCI-H295R. We also examined the expression of ghrelin, GHSR1a, and corticotrophin-releasing factor receptor 2 (CRF-R2). Acylated and unacylated ghrelin in the nanomolar range dose-dependently induced adrenocortical cell growth up to 200% of untreated controls, as measured by thymidine uptake and WST1 assay. The proliferative effects of acylated and unacylated ghrelin in SW-13 cells was blocked by [D-Lys3]growth hormone-releasing peptide 6 (GHRP6), but a CRF-R2 antagonist had no effect on unacylated ghrelin growth stimulation. Cell cycle analysis suggests that acylated and unacylated ghrelin suppress the sub-G0/apoptotic fraction by up to 50%. Measurement of DNA fragmentation and caspase-3 and -7 activity in SW-13 cells confirmed that acylated and unacylated ghrelin suppress apoptotic rate. SW-13 cells express preproghrelin mRNA and secrete ghrelin, and [D-Lys3]GHRP6 suppresses their basal proliferation rate, strongly suggesting that ghrelin could act as an auto/paracrine growth factor. Acylated and unacylated ghrelin are potential auto/paracrine factors acting through an antiapoptotic pathway to stimulate adrenocortical tumor cell growth. Unacylated ghrelin-stimulated growth is suppressed by an antagonist of GHS-R1a, suggesting either that unacylated ghrelin is acylated before its action or that ghrelin, unacylated ghrelin, and [D-Lys3]GHRP-6 bind to a novel receptor in these cells. Copyright

Ghrelin stimulates, whereas des-octanoyl ghrelin inhibits, glucose output by primary hepatocytes. (Article)

2005-02-01T00:00:00Z

Ghrelin exerts various metabolic activities, including regulation of glucose levels in humans. To verify whether the glucose response to ghrelin reflects a modulation of an insulin-independent hepatic phenomenon, we studied glucose output by primary porcine hepatocytes in suspension culture, after incubation with acylated ghrelin (AG), unacylated ghrelin (UAG), and hexarelin (HEX). AG induced glucose output dose dependently after 20 min of incubation (P < 0.001), whereas HEX, a GH secretagogue receptor type 1a (GHS-R1a) agonist, had no effect. UAG inhibited glucose release also dose dependently and after 20 min (P < 0.001). Moreover, UAG completely reversed AG-induced glucose output (P < 0.01). Using real-time PCR, GHS-R1a gene expression was undetectable in all the hepatocyte preparations studied. The lack of efficacy of HEX, the efficacy of UAG, and the absence of GHS-R1a expression indicate the involvement of a yet uncharacterized ghrelin receptor type. In conclusion, glucose output by primary hepatocytes is time- and dose-dependently stimulated by AG and inhibited by UAG. Moreover, UAG counteracts the stimulatory effect of AG on glucose release. These actions might be mediated by a different receptor than GHS-R1a, and apparently, we must consider AG and UAG as separate hormones that can modify each other's actions on glucose handling, at least in the liver.

Administration of acylated ghrelin reduces insulin sensitivity, whereas the combination of acylated plus unacylated ghrelin strongly improves insulin sensitivity. (Article)

2004-10-01T00:00:00Z

We investigated the metabolic actions of ghrelin in humans by examining the effects of acute administration of acylated ghrelin, unacylated ghrelin, and the combination in eight adult-onset GH-deficient patients. We followed glucose, insulin, and free fatty acid concentrations before and after lunch and with or without the presence of GH in the circulation.We found that acylated ghrelin, which is rapidly cleared from the circulation, induced a rapid rise in glucose and insulin levels. Unacylated ghrelin, however, prevented the acylated ghrelin-induced rise in insulin and glucose when it was coadministered with acylated ghrelin. Surprisingly, the injection of acylated ghrelin induced an acute increase in unacylated ghrelin and therefore total ghrelin levels. Finally, acylated ghrelin decreased insulin sensitivity up to the end of a period of 6 h after administration. This decrease in insulin sensitivity was prevented by coinjection of unacylated ghrelin. This combined administration of acylated and unacylated ghrelin even significantly improved insulin sensitivity, compared with placebo, for at least 6 h, which warrants studies to investigate the long-term efficacy of this combination in the treatment of disorders with disturbed insulin sensitivity.