Ghrelin and glucose homeostasis

Peptides 32 (2011) 2309–2318

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Review

Ghrelin and glucose homeostasis P.J.D. Delhanty ∗ , A.J. van der Lely Department of Internal Medicine, Erasmus MC, 3000 CA Rotterdam, The Netherlands

a r t i c l e

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Article history: Received 11 January 2011 Received in revised form 1 March 2011 Accepted 1 March 2011 Available online 9 March 2011 Keywords: Ghrelin Obesity Metabolism Diabetes

a b s t r a c t 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. © 2011 Elsevier Inc. All rights reserved.

Contents 1. 2. 3.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidence of a role for ghrelin in glucose homeostasis in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanisms of action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Role of GH secretagogue activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Insulin regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. In vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. In vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Insulin sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Central glucose sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Hepatic glucose production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. GOAT expression/activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Interaction with UAG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.1. AG/UAG ratio and insulin sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7.2. Effect of UAG on fat metabolism and energy balance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction

∗ Corresponding author at: Room Ee532, Department of Internal Medicine, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands. Tel.: +31 10 704 35 77; fax: +31 10 703 54 30. E-mail address: [email protected] (P.J.D. Delhanty). 0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.03.001

In humans and rodents ghrelin is a 28 amino acid peptide hormone that is expressed at the highest levels in the stomach and pancreas, but also to a lesser extent in the hypothalamus [75,78,126]. It is derived from a 117 residue preprohormone by removal of a secretory signal peptide at its N-terminus, and prohormone convertase (PC)1/3 cleavage of the resultant 94 amino acid

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prohormone after Arg28 at its C-terminus [97,142]. A second peptide, obestatin, may also be derived by proteolytic cleavage of the prohormone, and its biological function is under investigation [50]. Although the length of the ghrelin peptide in other species varies, its third amino acid residue (Ser/Thr) is invariably O-acylated, predominantly by an octanoyl or, to a lesser extent, decanoyl moiety (acylated ghrelin, AG) [62,76]. Acylation is catalyzed by the recently characterized enzyme ghrelin O-acyl transferase (GOAT) whose expression occurs, like ghrelin, chiefly in the stomach and pancreas, but at very low to undetectable levels in the brain [56,137]. Acylation by GOAT is required for the activity of ghrelin at its cognate receptor, the growth hormone secretagogue receptor (GHSR) [11,48,86]. AG displays strong growth hormone (GH) releasing activity mediated by the growth hormone secretagogue receptor type 1a (GHSR) [75,116]. Initially identified as the receptor for a family of synthetic peptidyl and nonpeptidyl GH secretagogues (GHS) [63], the GHSR is mainly concentrated in the hypothalamo-pituitary unit coinciding with its function in regulating GH, prolactin and ACTH secretion [14,53]. Other brain regions in which the GHSR is expressed include the dentate gyrus, CA2 and CA3 regions of the hippocampus, the thalamus and several nuclei within the brain stem [54]. The GHSR is also expressed in the pancreas and stomach, as well as to a much lesser extent in other peripheral tissues [49]. Because of this pattern of receptor distribution the ghrelin system has been shown to display a wide variety of activities, particularly stimulation of appetite and food intake, but also modulation of gastric motility and acid secretion, pancreatic exocrine and endocrine function, immune function, cardiovascular function, biological rhythmicity, memory, learning, sleep and behavior [14,126]. Unacylated ghrelin (UAG) is the predominant form of the circulating peptide. In contrast to AG, at physiological concentrations UAG is unable to bind the GHSR or induce GH secretion and, partly because of this, UAG was initially considered to be devoid of physiological activity. Today, accumulating evidence indicates that although UAG has no direct effect on GH secretion and other pituitary functions, it is involved in some of the non-endocrine activities of the ghrelin system, particularly those at the metabolic level, either agonizing or antagonizing AG effects. This antagonistic effect does not appear to occur at the level of the GHSR [48], suggesting independent activity of UAG via a separate signaling mechanism.

2. Evidence of a role for ghrelin in glucose homeostasis in vivo The earliest studies with both non-peptidyl and peptidyl GHSs indicated that these compounds might affect glucose homeostasis. In human studies relatively subtle effects were observed. In lean subjects treated for four days with the spiroindoline, MK0677, fasting insulin was found to be significantly increased, but fasting glucose levels were unaffected [22]. However, postprandial insulin and glucose levels were elevated during treatment. In a longer-term study of 8 weeks of treatment in obese subjects, fasting insulin and glucose were not significantly modulated, but oral glucose tolerance tests indicated impaired glucose tolerance after 2 and 8 weeks [117]. More profound effects of these compounds were discovered in animal studies. Treatment of obese ZDF rats with the GH releasing peptide (GHRP) G7039 was shown to lead to worsening hyperglycemia after 7 days of treatment, and to exacerbate their hyperinsulinemic state [25]. In contrast, peptidyl GHSs showed no effects on glucose homeostasis in normal human subjects (e.g. [13,87,89]), although prolonged treatment with hexarelin (20 weeks) caused significantly increased HbA1c levels [101].

Fig. 1. Growth hormone related activities of AG in modulating glucose metabolism. AG and GH interact to regulate glucose homeostasis. Ghrelin stimulates GH release by the pituitary, with IGF-I regulating this activity via a negative feedback mechanism. GH negatively regulates AG stimulated hepatic glucose production and peripheral glucose uptake, via modulation of liver and pancreatic function and peripheral insulin sensitivity. During prolonged starvation fat and glycogen stores become depleted, and GH increases due to reduced IGF-I negative feedback and increased AG. Low insulin levels also sustain increased AG and GH, which facilitates maintenance of levels of glycemia that prolong survival. The role of GH in regulating AG, including its acylation by GOAT, remains to be worked out, although GOAT has been found to be affected by nutritional status. Adapted from Nass et al. 2010 PNAS 107:8501–8502.

Overall these studies suggested possible effects of GHSs on insulin sensitivity. However, many of the effects of these compounds on glucose homeostasis and intermediate metabolism were consistent with a GH-driven response [61], indicating an indirect mechanism of action (see Fig. 1). It was not until about the time ghrelin was discovered that it was found that GHSs could indeed modulate glucose metabolism via a GH-independent pathway (see Fig. 2). In a study on human subjects designed to examine the roles of GH and its receptor in the control of metabolic status during fasting, it was found that GHRP6 induced diabetogenic effects indicative of an insulin resistant state [89]. Injection of a bolus of GHRP6 caused acute, significant, increases in glucose and insulin, and suppression of free fatty acids. This effect only occurred in subjects whose GH receptors had been blocked with pegvisomant and were in the non-fasting state. This clearly demonstrated two important points: that under normal circumstances the diabetogenic activity of this peptidyl GHS was masked by the action of GH, and that its activity at peripheral target tissues, such as liver, pancreas and adipose tissue, was independent of GH. It was hypothesized that the mechanism of diabetogenic activity of GHRP6 was closely regulated at peripheral sites by GHR activation, and that the mechanism for GHRP6 activity was through tissue-specific modulation of insulin sensitivity, causing relative insulin resistance in liver and muscle and increased insulin sensitivity in adipose tissue. The first indication that ghrelin, like the synthetic GHS analogs, may have an influence on glucose metabolism came from a study in mice in which a bolus subcutaneous injection, during both the dark and light periods, caused a sustained increase in respiratory quotient (RQ) as assessed by indirect calorimetry [123]. This indicated that ghrelin causes a switch from lipid to carbohydrate as fuel for the animals’ energy requirements, consistent with its induction of accretion of fat and increased carbohydrate utilization [123].

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Fig. 2. Direct activities of AG and UAG in regulating glucose homeostasis. Some of the effects of AG (and GHSs) are revealed by the absence of GH, suggesting modulation of ghrelin activity/signaling by GH. Additionally, AG and UAG have activity both in vitro and in vivo in the absence of GH, and the primary effects in relation to glucose homeostasis are summarised in the diagram. The stomach generates the majority of circulating AG and UAG, although there is local production, particularly in the pancreas, but also in the brain. The effects of AG generally oppose those of UAG in the pancreas, liver and peripheral tissues. In addition to direct action, AG acts via central pathways to modulate pancreatic, and perhaps liver, function. Peripherally and centrally administered UAG can activate hypothalamic neurons, but the downstream pathway that modulates glucose metabolism remains to be elucidated.

The discovery of ghrelin led to a profusion of pharmacological investigations in humans aimed at examining its GH secretagogue activity, as well as its potential effects on glucose metabolism (see [105]), although so far these have been limited to relatively shortterm studies. The first such study demonstrated that injection of a bolus of AG caused a rapid hyperglycemic effect, which preceded a more transient suppression of insulin levels [13]. The hyperglycemia was sustained for almost 3 h, while insulin returned to normal levels within 2 h. This finding suggested that in humans ghrelin caused an acute suppression of glucose disposal, perhaps through reduced insulin sensitivity. However, the subsequent suppression of insulin in the face of persistent hyperglycemia suggested that AG was having direct modulatory effects on hepatic glycogenolysis and/or peripheral glucose uptake, and insulin secretion. Soon after, these findings were substantiated in another study in which it was also demonstrated that AG acutely stimulated pancreatic polypeptide (PP) and, particularly, somatostatin (SS), which could explain the suppression of insulin levels [2]. However, the simultaneous rise in PP, SS and glucose indicates that these peptide hormones do not have a direct glucoregulatory role under these circumstances, and again suggested direct effects of AG in inducing liver glucose output and/or suppressing peripheral glucose disposal. In longer-term infusion studies AG also raised glucose, but appeared to block glucose induced insulin secretion via a direct mechanism, which recovered after infusion was stopped [130]. During the post-infusion period insulin responded to the hyperglycemia and normalized glucose levels. Estimation of insulin sensitivity during the study period, however, suggested that in fact AG infusion caused peripheral insulin resistance that was sustained during the post-infusion phase, perhaps partly attributable to the induction of raised GH and free fatty acid levels. In rodents, prolonged treatment with ghrelin has also been found to disturb glucose homeostasis. In tundra voles (Microtus

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oeconomus) treatment with AG for 4 days caused elevated insulin levels and hyperglycemia, probably caused by release of glucose from peripheral glycogen stores [92]. In mice on a high fat diet AG treatment for 5 days caused a trend to raised glucose levels and significant hyperinsulinemia [4]; whereas a study in rats treated for 4 days showed increased glucose levels in the absence of significant hyperinsulinemia [6]. In the rats increased hepatic glucose-6-phosphatase gene expression suggested that the AG induced hyperglycemia was a consequence of increased gluconeogenesis. The above studies, although central to our understanding of mechanisms of AG action, all assess the effects of pharmacological levels of AG. However, strong links between AG and glucose homeostasis have been confirmed in clinical investigations assessing diurnal variations in ghrelin, glucose and insulin. An early study by Purnell et al. [100], for example, showed inverse correlation between total ghrelin (AG + UAG) levels and insulin during the day, and a direct relationship with insulin sensitivity. Furthermore, total ghrelin varied with feeding status during the waking period, rising before a meal, and declining directly following. In a similar, very recent study, Spiegel et al. [110] show in more detail changes in both total ghrelin levels and, importantly, AG levels in relation to changes in glucose and insulin levels during the day. In neither study was total ghrelin correlated with glucose. However, Spiegel et al. observed that AG levels show a remarkably stronger correlation with 24 h glucose levels than insulin levels, and a similar inverse relationship between total ghrelin levels and insulin to that described by Purnell et al. 3. Mechanisms of action The processes that are key to glucose homeostasis are regulation of insulin secretion by the pancreas, and sensitivity to insulin in peripheral tissues, particularly the liver, muscle and adipose tissue. The liver and gut are the main sources for circulating glucose, and the muscle, adipose tissue and brain are the major sites of glucose utilization. The mechanism by which AG modulates glucose homeostasis must impinge on insulin function at these sites. The evidence suggests that AG is a critical component of a system of insulin counter-regulatory factors, including GH (Figs. 1 and 2). 3.1. Role of GH secretagogue activity GH is counter-regulatory of insulin action on lipid and carbohydrate metabolism [131]. Because GHSs and AG seem to have similar effects to GH, initially it was thought they acted via a GHdependent mechanism. However, unlike GH, AG has obesogenic activity in the long-term causing accumulation of fat, independent of its effects on food intake and the presence or absence of GH [123], and much experimental evidence in vivo now strongly suggest a GH-independent component to the activity of AG. The first study demonstrating diabetogenic activity for AG in humans, also indicated that the effect may not be via a GH-related mechanism, since in the same study the peptidyl GHS hexarelin caused an induction of GH but no hyperglycemic effect [13]. The study in humans by Muller et al. [89] showed that GH in fact masks the activity of GHRP6. This GH-independent activity was substantiated in GHdeficient humans by Gauna et al. [46], in which AG was found to have rapid diabetogenic effects, causing increased postprandial circulating glucose and free fatty acid levels, and suppressed insulin. To dissect in greater detail the direct peripheral effects of ghrelin from indirect GH-dependent mechanisms, Vestergaard et al. performed a study where AG was infused during an SS induced pancreatic clamp [128]. It was demonstrated that ghrelin significantly suppressed glucose disposal rate under both basal

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and insulin-stimulated conditions, and induced peripheral insulin resistance, but had no effect on hepatic glucose production. In this report the SS infusion blocked AG induced GH, and suggested that most of the effects of AG on glucose metabolism were GHindependent. A more conclusive study by this group was performed in hypopituitary subjects in whom AG infusion induced hyperglycemia under basal conditions [129]. It was also shown that ghrelin suppressed insulin-stimulated glucose disposal and stimulated free fatty acid release. Further confirmation has been obtained in rodents where AG was found to have similar, rapid, hyperglycemic effects in wild type and GH-deficient mice [36]. Although AG has an important direct (or GH-independent) mechanism of action, studies in humans and recent findings in rodents indicate that an important function of ghrelin is to integrate the physiological responses to fasting (increased GH and suppressed insulin secretion, in particular) so as to ensure maintenance of glucose levels [125], as originally hypothesized by Cummings et al. [29]. Although Ghsr deficiency in mice causes no overt effect on glucose homeostasis under normal circumstances or when fed high fat diets, Sun et al. [115] showed that calorie restriction (50% of normal food intake) causes a significant suppression of blood glucose compared with calorie restricted wild type animals. Furthermore, the hypoglycemia produced during calorie restriction had a more rapid onset in Ghrl and Ghsr deficient mice than in controls. This discovery led the authors to suggest that the ghrelin system plays an important role in glucose homeostasis under conditions of negative energy balance. The discovery of GOAT enabled the development of a murine model of acylated ghrelin-deficiency by deletion of the Goat gene (Ghrl knockout causes deficiency of both acylated and unacylated forms of ghrelin). Initial studies with Goat-deficient mice showed moderate suppression of body and fat mass, together with increased energy expenditure, but no effect on glucoregulation [74]. However, when Goat-deficient mice were placed on a severe calorie restricted (40% of normal) diet they become moribund within 7 days [141]. It was found that during this period of calorie restriction wild type, AG-sufficient, mice were able to maintain circulating glucose levels, albeit at ∼50% of fed levels, while Goat-deficient animals exhibited steadily worsening hypoglycemia, in the absence of hyperinsulinemia. Zhao et al. [141] showed that inability to control glucose levels during calorie restriction in Goat-deficient mice is due to their relative GH deficiency. Although calorie restriction markedly increased GH in the circulation of the animals, it only reached ∼50% of wild type levels in Goat-deficient mice. Under these circumstances, the hypoglycemia could be corrected by infusing either AG or GH. This study may shed light on the observation that anorectic humans have elevated AG and GH levels, which is likely a physiological response to maintain blood glucose levels in the range required for survival, and particularly to prevent neuroglycopenia [90,126]. It appears, then, that AG may only become important in its role as a GH secretagogue under conditions of starvation when GH levels are greatly elevated. This explains why, under normally fed conditions, Ghrl and Ghsr deficient mice are not dwarves [20,54–56]. 3.2. Insulin regulation 3.2.1. In vitro Local regulation of insulin secretion by ghrelin was suggested by studies demonstrating expression of both ghrelin and the GHSR mRNAs in the pancreas [30,36,49,52,54,59,67,69,99,109,132,134]. The majority of studies now show that AG retards glucose stimulated insulin secretion by the pancreas and pancreatic islets [26,37–40,103], concurring with in vivo studies. However, some studies suggest that under certain conditions AG can amplify glucose-stimulated insulin secretion in the pancreas [30,81], and in insulinoma cell lines [43,51]. In addition to showing that AG

suppressed insulin release by islets, Dezaki et al. [36] showed that GHSR blockade amplified glucose-stimulated insulin release together with an increase in [Ca2+ ]i . This effect was abolished in the absence of exogenous Ca2+ . Conversely, the attenuation of [Ca2+ ]i together with delay of ATP-dependent outward K+ currents (with resultant decrease in electrical activity) in isolated ␤-cells upon AG treatment indicated a causal link with suppression of insulin secretion [39]. However, the concentration of AG required (10 nM) to suppress insulin release was higher than circulating levels. A subsequent study by Dezaki et al. [38] showed that AG levels in the pancreatic vein were in the insulin-suppressive range determined in vitro. These effects were not glucagon driven, since AG did not affect glucose-stimulated release of this peptide [36]. Isolated pancreatic preparations responded in the same way as islets, and showed suppression of both first and second phase glucosestimulated insulin release [38]. Intriguingly, AG-regulated [Ca2+ ]i and Kv driven K+ currents were suppressed both in the presence of dibutyryl cAMP and by pertussis toxin, an inhibitor of G␣i mediated GPCR signaling. This is surprising, since GHSR signaling is mediated primarily by G␣q/11 , and the phospholipase C/[Ca2+ ]i pathway [63]. Reportedly, however, G␣i (in this case subtype G␣i2 ) is expressed in ␤-cells, whereas G␣11 is expressed primarily in non-␤-cells in the pancreas [39]. G␣ subunits show high sequence identity and it is perhaps not so surprising that promiscuity in G-protein usage by GPCRs has been described [60], and GHSR/dopamine receptor heterodimers signal via an alternative G␣i/o pathway [68]. More work remains to elucidate AG-regulated intra-cellular signaling pathways in ␤-cells in relation to glucose-stimulated insulin secretion. 3.2.2. In vivo 3.2.2.1. Gain of function models. Investigations in rodents demonstrate that AG treatment can cause a rapid suppression of glucose stimulated insulin secretion into the circulation presumably by direct inhibition at the pancreas [36,103], although this finding is not universal [45]. Iwakura et al. [67] have generated transgenic mice with the preproghrelin gene driven either by the insulin II (RIP-G Tg) or the glucagon (RGP-G Tg) promoters. These mice overexpress ghrelin in the pancreatic islets, although ghrelin immunoreactivity was also determined in the brain of RIP-G Tg mice. No effects of diet were observed on plasma glucose or insulin levels. However, the insulin response to glucose loading was suppressed in RIP-G Tg mice, suggesting local and/or central effects of ghrelin overexpression on pancreatic insulin secretion, although the authors discuss the possibility that the concomitant increase in circulating UAG indirectly caused the suppression of insulin secretion by improving insulin sensitivity [67]. Bewick et al. [12] observed that glucose intolerance in AG transgenic mice is probably a result of suppressed glucose-stimulated insulin release since insulin sensitivity was unaltered. Similar findings were obtained in mice that overexpress Trp3 -ghrelin, a partial agonist of the GHSR [136]. At one year these mice have significantly impaired glucose tolerance, but are also more insulin resistant than their wild type counterparts. The difference in phenotype between the mice from these last two groups may be related to differences in the sites of overexpression of the transgenes. 3.2.2.2. Loss of function models. Blockade of AG and the GHSR, as well as Ghrl deficiency have been shown to improve the insulin response of rodents to glucose challenge [36,38,113]. Sun et al. have investigated the influence of Ghrl-deficiency on ␤-cell function [113]. Under normal conditions, levels of fasting blood glucose and insulin were indistinguishable from wild type mice. However, when challenged with a glucose tolerance test, the Ghrl-deficient mice showed relatively increased insulin output concomitant with faster glucose clearance suggesting improved pancreatic ␤-cell

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function [38,113]. Ghrl-deficiency has also been shown to counteract some of the effects of a high fat diet on glucose metabolism. Ghrl knockout mice on a high fat diet have a similar response to a glucose tolerance test as their normally fed counterparts, probably via compensation in pancreatic function since their insulin response is enhanced [38]. Similar to studies in Ghrl-deficient mice, it has been found that blocking AG activity using the GHSR antagonist [d-Lys3 ]GHRP6 improves glycemic control not only in normal mice, but also in the obese, diabetic, leptin-deficient ob/ob mouse [4,38]. However, it was unclear in these experiments if this was through direct inhibitory effects on AG signaling, or indirectly through the suppression of food intake that was observed in these mice (or, alternatively, through non-specific effects of [d-Lys3 ]-GHRP6 on serotonin receptor function [35]). To resolve this issue, Sun et al. developed combined Ghrl- and leptin-deficient mice, by crossing Ghrl knockouts with ob/ob mice [113]. Deletion of Ghrl in these mice did not affect the hyperphagic, obese phenotypes of the ob/ob mice, but it significantly reduced their hyperglycemia. This was driven by an increase in their circulating insulin levels, caused by pancreatic compensation, as shown by increased C-peptide levels [113]. Moreover, Ghrl-deficient ob/ob mice have significantly improved glucose tolerance and glucose stimulated first-phase insulin release, indicating an improvement in their diabetic phenotype. The mechanism by which pancreatic function in these mice was improved was shown to be linked, in part, to a marked reduction in expression of Ucp2 (uncoupling protein 2). Ucp2 deletion on an ob/ob background causes a similar improvement in the diabetic, rather than hyperphagic/obese, phenotype and it has been shown that UCP2 is an important component of pathways regulating insulin secretion [139]. A recent study using islets suggests that the effect of AG on Ucp2 expression in the pancreas is direct and glucose-dependent [24]. 3.2.2.3. Human studies. Initial studies in humans showed a lack of effect of AG on glucose stimulated insulin secretion, but suppression of arginine-stimulated release [15]. A later study showed an AG-induced increase in insulin following a meal [46], although AGinduced post-prandial hyperglycemia caused by worsened insulin sensitivity may also have contributed to this response. However, it has been found that a bolus injection of AG suppresses insulin in women with obesity, and obesity together with polycystic ovarian syndrome, but not in healthy subjects [42,55,118]. A recent study by Tong et al. [121] finds that infusion of AG at higher overall doses can suppress glucose-stimulated insulin secretion and deteriorated glucose tolerance in normal, healthy, humans. The doses of AG used (0.3, 0.9 and 1.5 nmol/kg/h) markedly increased steady state ghrelin levels to between 4- and 23-fold above average basal levels. However, the authors argue that the supraphysiological levels may be representative of concentrations present in the pancreas due to local production, as suggested by Dezaki et al. [38]. In further support of a physiological role for AG in regulating insulin secretion in humans, Spiegel et al. [110] find a direct correlation between AG and glucose levels during a 24 h frequent-sampling study. Remarkably, this relationship was found to be statistically stronger than that between glucose and insulin. Finally, ghrelin gene variants have been associated with impaired first-phase insulin secretion, although the precise mechanism was not elucidated [79]. 3.2.2.4. Central regulation. The pancreas is innervated by parasympathetic nerve fibers originating from the dorsal vagal complex (DVC) that modulate pancreatic secretion via cholinergic and neuropeptidic synapses [77]. AG and GHSs have also been shown to activate gluco-sensory neurons in the brainstem. The DVC contains AG sensitive neurons that mediate an orexigenic effect and pancreatic enzyme secretion [41,82,106,143]. The finding that AG

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can activate neurons in this region indicated an important role in efferent vagal modulation of pancreatic function, beyond the original effects described on feeding and gastric acid secretion [31,41]. Additionally, potential local activity of AG in the hypothalamus and brainstem suggest a gluco-sensory mode of action (see below, and [114]). Together with the finding that AG regulates glucose-induced insulin secretion from isolated islets, and glucose-stimulated insulin release in vivo, it appears that AG has both central and local roles in regulation of pancreatic function. Further studies are necessary to delineate the mechanisms by which endogenous AG may regulate pancreatic islet function. 3.3. Insulin sensitivity Studies in GH-deficient human subjects by Gauna et al. [46] show that injections of AG cause an acute increase in glycemia, followed by an eventual increase in insulin, and both glycemia and insulin levels are raised compared to placebo controls following a meal, also indicative of worsened insulin sensitivity. This was concomitant with an increase in circulating free fatty acids relative to placebo controls, which may contribute to the insulin resistant state. In normal healthy humans Vestergaard et al. have also shown that infusion of AG causes prompt induction of peripheral insulin resistance, especially in muscle, concomitant with increases in levels of free fatty acids [127,129]. Perhaps counter to the acute inhibitory effects of AG on peripheral insulin sensitivity, in vitro studies have suggested that AG has either a stimulatory effect, or no effect, on insulin stimulated glucose uptake in adipocytes and myocytes, respectively [72,80,96]. Correspondingly, hyperinsulinemic euglycemic clamp studies in mice suggested that AG improves peripheral insulin sensitivity, and AG treatment in rats was shown to enhance Akt signaling in skeletal muscle [7,58]. Moreover, several studies in humans indicate a negative correlation between ghrelin levels and the incidence of type 2 diabetes and insulin resistance [16,64,70,98], but it is unclear if low ghrelin levels in this case are a risk factor or a compensatory response. Ghrelin gene variants have been associated with altered insulin sensitivity, although reports tend to be conflicting, perhaps dependent on population size and other characteristics [98,112,138]. Long-term deficiency of AG does appear to affect insulin sensitivity. Ghrl- and Ghsr-deficient mice are more insulin sensitive than their wild type counterparts, indicating that this is an important mechanism by which AG modulates glucose metabolism [84,113]. This was further confirmed in hyperinsulinemic euglycemic clamp studies, where hepatic glucose production rate was suppressed during a low-dose insulin clamp in Ghrl-deficient compared with wild type mice [113]. These mice also exhibited significantly increased glucose disposal rate, and required a markedly increased glucose infusion rate [113]. Although gain of function models of AG over-expression have been difficult to assess, since tissue-specific expression is necessary to ensure correct processing of the peptide (particularly acylation), a transgenic mouse over-expressing the partial GHSR agonist [Trp3 ]-ghrelin has been developed (see above, [136]). When these mice reach a relatively old age (1 year) they develop impaired insulin sensitivity compared with their wild type counterparts. These mice were also insulin intolerant, as has been observed in two other transgenic AG over-expressing models [12,102], although here the mechanism involved impaired pancreatic function rather than insulin resistance. 3.4. Central glucose sensing The maintenance of glucose homeostasis must include the ability of the central nervous system, as well as peripheral tissues,

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to sense changes in glucose levels. The preprandial and fasting induced increase in circulating AG [110,126,141] and hypothalamic GHSR gene expression [73], suggest involvement of a central mechanism whereby the ghrelin system can sense declining glucose levels [114]. In the hypothalamus the activity of agouti-related peptide (AgRP) and neuropeptide Y (NPY) expressing neurons in the arcuate nucleus (ARC) is modulated by glucose [20,21]. Glucose responding neurons are also present in the ventromedial hypothalamic nucleus (VMH), the lateral hypothalamic area (LHA, orexin expressing neurons) and the parvocellular area of the paraventricular nucleus (PVN). Intriguingly, the GHSR is expressed in, and ghrelin gene expression is localized adjacent to, these regions [28,143], and all of these hypothalamic sites are important targets for the orexigenic and energy homeostatic effects of AG through NPY, AgRP, POMC and CRH expressing neurons [28,69], and glucose sensing neurons within these regions respond to ghrelin [23]. The nucleus of the solitary tract (NTS), within the DVC, relays GH-regulatory signals to the hypothalamus via noradrenergic projections [5,66], although the effects of GHSs at this site were shown to be independent of noradrenaline and GH [5]. The stimulation of neurons in the NTS by insulin-induced hypoglycemia triggers an orexigenic response that can be blocked by AG antibodies, indicating a role at this site for AG in sensing hypoglycemia [108]. Subsequently, Wang et al. [133] showed that the NTS contains AG regulated gluco-sensory neurons, and that the primary effect of AG on glucose-inhibited and glucose-excited neurons in the NTS is inhibitory. However, it remains unclear how these responses to AG might lead to its insulin counter-regulatory effects [133]. Overall, although clearly an important player in central control of energy homeostasis, the integrative role that AG may play in sensing glucose at the central level in mammals remains to be fully determined. 3.5. Hepatic glucose production The liver can generate glucose by utilizing two main processes, glycogenolysis and gluconeogenesis, which are down-regulated directly by insulin, mediated by the IRS-Akt signaling pathway. Early clinical experiments suggested that GHSs and AG could rapidly assimilate glucose into the circulation, possibly through induction of hepatic glucose production. Gauna et al. showed that AG induces glucose production by primary porcine hepatocytes in vitro, indicating a direct mechanism of action [44]. Barazzoni et al. have shown that AG treatment in rats suppressed hepatic Akt phosphorylation which was associated with a concomitant reduction in phosphorylation of glycogen synthase kinase 3 and increase in Ppargc1a gene expression [7]. Since PGC1␣ can stimulate gluconeogenesis in the liver, this could be a mechanism by which AG causes its hyperglycemic effect in the longer-term [7]. In confirmatory studies, hyperinsulinemic euglycemic studies in mice by Heijboer et al. [58] show that AG treatment prevents suppression of endogenous glucose production by insulin, and conversely, knockout of Ghrl in ob/ob mice markedly suppresses hepatic glucose production [113]. Hepatic glucose production is also influenced by the hypothalamus via efferent vagal input [19], a possible site of influence of AG. 3.6. GOAT expression/activity AG is relatively unstable, being rapidly deacylated to UAG by esterases in the circulation [10,33,107,127]. Therefore it follows that there is also a high turnover rate, with levels of AG dependent upon acylation rate by GOAT, and availability of substrate for octanoylation [74,93]. However, the regulation of Goat gene expression does not appear to fit well with alterations in circu-

lating levels of AG, for example during fasting (see [104]). GOAT deficiency in mice has very little impact, either under normal feeding conditions or high fat diet, on glucose levels, very similar to the phenotype of Ghrl-deficient mice [74,115]. Rather, Goat-deficiency has a beneficial effect on glucose stimulated insulin release and glucose tolerance, as assessed by glucose loading (glucose tolerance test), an effect that is accentuated by a high fat diet [141]. Recently it has been shown that GOAT protein and mRNA expression, and transcriptional activity are suppressed by insulin in insulinoma and pancreatic islet cells both in vitro and in vivo [1], consistent with the suppression of AG production in these cells. This effect was shown to be mediated by activation of the mTOR signaling pathway, concurring with an earlier report showing the involvement of this pathway in regulation of Goat gene expression in the stomach [135]. Clearly more work is required to clarify the mechanisms that regulate Goat gene expression in relation to its enzymatic activity, as well as a physiological role in regulating ghrelin function. On the other hand, pharmacologic blockade of GOAT has clear effects on glucose homeostasis through reduction of AG’s activity in the pancreas [9]. Suppression of GOAT activity using a specific inhibitor of ghrelin acylation (GO-CoA-Tat) amplifies glucosestimulated insulin secretion by pancreatic islets in vitro, reinforcing the concept that AG can blunt the insulin response of the pancreas by a paracrine mechanism [9,38]. The effect of GOAT inhibition in vivo was also investigated by Barnett et al. [9]. Treated mice showed a significant increase in response to a glucose challenge, as well as improved glucose tolerance. This was repeated in Ghrl knockout mice, in which GO-CoA-Tat had no effect during a glucose tolerance test, supporting the hypothesis that the inhibitor specifically blocked AG’s regulatory effects on insulin secretion. The mechanism behind the effect of blockade of ghrelin acylation is probably linked to a concurrent marked reduction in Ucp2 expression [9]. In summary, although natural regulation of GOAT and its impact on the function of ghrelin still needs further elucidation, it appears that GOAT is a promising target for pharmacologic intervention to control glucose homeostasis. 3.7. Interaction with UAG Initial clinical studies showed that intravenous injection of AG into healthy subjects induced a prompt and prolonged (>2 h) increase in glycemia and a decrease in circulating insulin levels [13]. This contrasted with only a transient increase in GH, suggesting that the effect on glucose and insulin was not GH-mediated. Subsequently, the effects on glucose and insulin of a single intravenous administration of UAG was investigated [17] and compared to administration of AG alone as well as to co-administration of AG and UAG. UAG was found neither to modulate the neuroendocrine effects of AG nor to have independent effects on pituitary hormones. As expected, glucose levels increased and insulin levels decreased following administration of AG. No change was noted following administration of UAG alone on either parameter, but the co-administration of UAG antagonized the AG-related rise in glucose. The beneficial effects of combined administration of UAG and AG on glucose levels has been confirmed in GH deficient patients [46]. Overall, these data indicated that both AG and UAG play a role in the regulation of glucose metabolism in humans, and that coadministration of AG and UAG improves insulin sensitivity. Data also suggest that these effects are independent of the GH-releasing activity of AG and are likely mediated by a receptor distinct from the GHSR. Based on this background, further pharmacological work has been undertaken including investigation of continuous administration in humans and animal models of diabetes. In humans, UAG has been continuously infused for up to 16 h into healthy subjects [18] and type 2 diabetes patients. Results

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from these studies indicate that UAG treatment decreased fasting (healthy and type 2 diabetes subjects) and post-prandial glucose levels (type 2 diabetes subjects) while no change was observed in insulin levels, suggesting improvement in insulin sensitivity. In streptozotocin-treated rats, both AG and UAG were able to reverse changes in plasma glucose and insulin and significantly increase the number of pancreatic islets but significant restoration of islet area was observed with UAG only [52]. In obese and insulin resistant (ob/ob) mice, constant infusion of UAG for 4 weeks significantly reduced glucose levels over this period (our unpublished data). Recent in vitro work has shown that AG and UAG interact in promotion of proliferation, inhibition of apoptosis and stimulation of insulin secretion by pancreatic ␤-cells [43,51]. These effects are mediated by the cAMP/PKA pathway. Recently, it has also been demonstrated using rat pancreatic islets that UAG prevents the suppression by AG of glucose-induced insulin secretion [91]. Based on earlier findings, Gauna et al. tested the effects of UAG on glucose-induced insulin secretion in vivo [45]. AG and UAG were administered to non-diabetic rats. Measurements included portal and systemic glucose and insulin levels following an intravenous glucose tolerance test (IVGTT). Results demonstrate that UAG dosedependently enhanced the first-phase insulin response to IVGTT in the portal circulation. The stimulatory effect was observed to a lesser extent in the systemic circulation [45]. AG and UAG have also been shown to modulate hepatocyte function in which glucose output was shown to be dose-dependently stimulated by AG and inhibited by UAG [44], independent of the influence of insulin. In line with the clinical data described above, this study showed that UAG counteracted the stimulatory effect of AG and glucagon on glucose release. Extending these findings, the in vivo effects of AG and UAG on hepatic insulin sensitivity have been examined by hyperinsulinemic–euglycemic clamp and determination of hepatic insulin resistance [58]. It was shown that in vivo (under hyperinsulinemic conditions) AG and UAG desensitize the liver to insulin, whereas coadministration of AG and UAG neutralized these effects and led to normalized hepatic insulin sensitivity. However, in a double-blind cross-over study in humans, injections of a combination of AG and UAG had no immediate effect on adiponectin levels, despite significant suppression of insulin [71]. Although rodent studies suggest that adiponectin has a direct insulin-sensitizing function in the liver, the relationship between adiponectin and insulin sensitivity in humans appears to be more complex [27]. More work is required to elucidate this relationship, and therefore, the significance of the lack of effect on combine AG/UAG treatment on adiponectin levels in humans remains unclear. Together, these findings reinforce the concept that AG and UAG interact in their regulation of hepatic glucose metabolism. 3.7.1. AG/UAG ratio and insulin sensitivity The studies described above indicate that UAG can have direct effects on glucose, lipid and energy homeostasis, but that these effects may also be interdependent with those of AG. For example, we found that UAG directly counter-regulates AG-induced glucose output by hepatocytes, and UAG reverses the diabetogenic effects of AG in GHD humans. UAG’s counter-regulatory effect on AG has also been shown to occur centrally [65,122]. Until recently the physiological relevance of these “AG-dependent” effects of UAG was not entirely clear since analytical methods could not clearly distinguish AG, UAG and total ghrelin levels in the circulation, and it was assumed that UAG and AG were co-regulated. However, improved methods for measuring AG and UAG have begun to demonstrate that UAG is regulated differently from AG. For example, 60 h of fasting in humans appears to reduce the AG/UAG ratio through suppression of AG levels [83], and suppress the diurnal rhythm of AG and UAG production into the circulation. Physiological changes in

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AG/UAG ratio have also been observed in a frequent sampling study of humans during a 24 h period, using assays that discriminate AG from total ghrelin [110]. In this study it was found that although total ghrelin levels peak at mealtimes, levels of AG show multiple peaks and troughs between meals, with consequent changes in AG/total ghrelin ratio during the day. The AG/total ghrelin ratio was significantly lower during sleep than during the waking period. However, it is unclear if these changes relate to diurnal changes in insulin sensitivity and insulin secretion, which decrease during the night, deserving further investigation [124]. In obesity basal total ghrelin levels are usually decreased and increase back toward normal values after weight loss, but little is known about the regulation of AG and UAG in obesity and insulin resistant conditions. Obese mice and humans have been reported to present lower UAG levels than normal weight subjects whereas AG levels are similar, indicating that obesity might be correlated with a relative UAG deficiency, and that UAG levels are regulated by the body [85,94,95]. It has been observed that insulin-resistant obese subjects have an elevated AG/UAG ratio when compared to insulin-sensitive obese subjects [8,111]. The mechanism by which the AG/UAG ratio is regulated is still not clear, but may be multi-factorial. For example, we have found that the AG/UAG ratio is greater in the portal than systemic circulation, driven by hepatic clearance of AG [47]. In a study of longer-term fasting (61.5 h) in humans the AG/UAG ratio was found to decrease without a change in total ghrelin levels, suggesting a change in rate of acylation, perhaps linked with suppression of substrate (octanoate/octanoyl-CoA) or GOAT activity [83]. In pregnancy the percentage of AG is suppressed, likely due to a decrease in acylation [119]. This was perhaps related to changes in activity of GOAT, since cholinesterase levels were similarly suppressed and octanoate levels were inversely related to AG. 3.7.2. Effect of UAG on fat metabolism and energy balance Dysregulated lipid metabolism is an important contributor to the pathogenesis of insulin resistance and markedly affects glucose homeostasis. Several studies in rodents have shown that AG stimulates body weight gain by increasing adiposity (e.g. [32,123]). AG and UAG have effects at peripheral sites in adipose tissue, promoting bone marrow adipogenesis [120] and inhibiting lipolysis in rat adipocytes [88]. Recent studies performed in mice have shown that, in contrast to AG, centrally or intraperitoneally administered UAG induces a negative energy balance by decreasing food intake and delaying gastric emptying [3]. Consistent with these results, peripherally injected UAG blocks the orexigenic effects of AG in rats [65] and transgenic mice that overexpress UAG in fat had improved insulin sensitivity and reduced fat mass [140]. In humans, our data also suggest effects on lipid metabolism; the co-administration of AG and UAG reduces plasma FFA in GHD patients [46] and the continuous infusion of UAG [18] decreases FFA in healthy and diabetic subjects, respectively. We have been able to extend these data further by utilizing Ghsr knockout mice [34]. UAG was found to significantly modulate both lipid and glucose metabolic pathways in adipose tissue, liver and muscle. In adipose tissue, lipogenic (fatty acid and cholesterol synthetic pathways), adipogenic, and glycolytic pathway genes were suppressed, whereas pathways that could improve insulin sensitivity (e.g. Akt/PKB) were increased. These findings are supported by mouse models in which fat mass is suppressed concurrent with increased UAG levels (Goat-deficient [74] and FABP4-Ghrl transgenic [140]). 4. Summary The gut hormone ghrelin plays an important physiological role in modulating GH secretion, insulin secretion and glucose

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metabolism. AG appears to act on glucose metabolic pathways via pathways that are GH-dependent as well as direct. For example, under severe food restriction the maintenance of GH by AG seems critical for the preservation of glucose levels in the range required for survival. On the other hand, AG has direct effects on pancreatic islet function. Therefore, AG is part of a mechanism that integrates the physiological response to fasting. However, pharmacologic studies indicate the important obesogenic/diabetogenic properties of AG. 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. In the western world, of course, this function for AG has become maladaptive, since food is continually available. These findings indicate the potential of AG 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 [16,64,70,98]. 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 [57]. Pharmacological suppression of the AG/UAG ratio by treatment with UAG may also be a viable alternative approach which appears to improve insulin sensitivity [125]. 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 [9].

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