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Accepted Preprint first posted on 8 April 2014 as Manuscript EJE-14-0183

Regulation of glucose metabolism by the ghrelin system: multiple players and multiple actions

Kristy M. Heppner1 and Jenny Tong2 1

Division of Diabetes, Obesity & Metabolism, Oregon National Primate Research Center,

Oregon Health and Science University, Beaverton, Oregon

2

Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of

Cincinnati, Cincinnati, OH

Corresponding author: Jenny Tong, MD, MPH 260 Stetson Street, Suite 4200 Cincinnati, OH 45219-0547 Email: [email protected]

Telephone: 513-558-4446 Fax: 513-558-8581 Word Count: 4248

Copyright © 2014 European Society of Endocrinology.

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Abstract Ghrelin is a 28-amino acid peptide secreted mainly from the X/A-like cells of the stomach. Ghrelin is found in circulation in both a des-acyl (dAG) and acyl form (AG). Acylation is catalyzed by the enzyme ghrelin O-acyltransferase (GOAT). AG acts on the growth hormone secretagogue receptor (GHSR) in the central nervous system (CNS) to promote feeding and adiposity, and also acts on GHSR in the pancreas to inhibit glucose-stimulated insulin secretion. These well-described actions of AG have made it a popular target for obesity and type 2 diabetes (T2DM) pharmacotherapies. However, despite the lack of a cognate receptor, dAG appears to have gluco-regulatory action, which adds an additional layer of complexity to ghrelin’s regulation of glucose metabolism. This review discusses the current literature on the glucoregulatory action of the ghrelin system (dAG, AG, GHSR and GOAT) with specific emphasis aimed towards distinguishing AG versus dAG action.

Key Words: ghrelin, des-acyl ghrelin, GHSR, GOAT, glucose metabolism

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Introduction Ghrelin is a 28-amino acid peptide that was discovered as the endogenous ligand for the growth hormone secretagogue receptor-1a (GHSR) 1. Ghrelin possesses a unique post-translational modification where an n-octanoic acid is esterified to the serine3 residue of the peptide molecule. The presence of a fatty acid (FA) side-chain attached to the ghrelin peptide is required for the full agonism of GHSR 1. Acylation of ghrelin occurs before secretion and is catalyzed by the enzyme, ghrelin O-acyltransferase (GOAT) 2, 3. Ghrelin was initially noted for its growth hormone (GH) releasing property and was later found to regulate energy metabolism. Ghrelin has potent orexigenic and adipogenic effects that are mediated through GHSR located in the central nervous system (CNS) 4, 5. These finding highlighted ghrelin as a key component of the gut-brain axis in regulating energy metabolism. In addition to being expressed in areas that regulate energy homeostasis, ghrelin and its receptor are both expressed in pancreatic islet cells, suggesting that ghrelin may have paracrine or autocrine action in the pancreas 6. Therefore, much effort was dedicated to exploring the role of ghrelin in regulating glucose homeostasis. The biological role of des-acyl ghrelin (dAG) has been questioned due to the lack of a known cognate receptor. However, increasing evidence suggests that the both dAG and acyl ghrelin (AG) have gluco-regulatory action in different species 7-9. The recent discovery of the ghrelin acylating enzyme, GOAT, has allowed for a more thorough dissection of AG versus dAG action. This review focuses on the regulation of glucose metabolism mediated by the ghrelin system, which consists of dAG, AG, GHSR and GOAT.

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In vitro action of AG on pancreatic hormone secretion Both ghrelin and GHSR1a are expressed in human, rat, and mouse islets from early gestation to adulthood 10-14. Ghrelin is expressed in both α- and β-cells 15, 16 as well as in a novel, developmentally-regulated endocrine islet cell type that shares lineage with glucagon-secreting cells 17, 18. While the functional role of ghrelin in the embryonic islet is not fully known, the ghrelin-expressing epsilon lineage has a significant impact on the final cell type composition of mature islets 19. It has been proposed that the presence of higher number of ghrelin expressing epsilon cells in the embryonic islet may modulate other islet hormone secretion (i.e. insulin) to prevent hypoglycemia in the fetal period 17. Moreover, the presence of GOAT expression in the pancreas suggests that ghrelin acylation can occur within pancreatic cells 2, 20. Numerous reports demonstrate that AG acts on isolated islets and pancreatic β-cells to inhibit glucose-stimulated insulin secretion (GSIS) 11, 18, 21, 22. However, this effect seems to be dependent on dose and experimental condition and a stimulatory effect of AG on insulin secretion has also been reported 13, 16, 21, 23

. Application of a GHSR antagonist to isolated rat islets or perfused pancreas enhances

glucose-induced insulin release 24 suggesting that the constitutive activity of the receptor 25-27 alone may regulate islet hormone secretion. However, other groups have reported that a GHSR antagonist itself has no effect on GSIS in isolated rat islets, but does act to block the inhibitory action of exogenous AG on GSIS 28.

The inhibitory action of ghrelin on insulin secretion initially seemed paradoxical because GHSR couples to a Gαq/11 signaling pathway as first described in pituitary cells 29. Activation of the Gαq/11 pathway stimulates rather than inhibits insulin secretion 30, 31. In the pancreas, AG attenuates cAMP and [Ca2+]i signaling in β-cells through a non-canonical pathway

11

. Further

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investigation demonstrated that AG acts through a Gαi2 signaling pathway leading to a reduction in cAMP accumulation and subsequent inhibition of insulin secretion 32. The underlying molecular mechanisms that explain why AG promotes GHSR coupling to a Gαi/o as apposed to a Gαq/11 signal transduction pathway in the β-cell have recently been explored by Park et al. 33. These investigators showed that GHSR heterodimerizes with the somatostatin receptor 5 (SST5) and this heteromer formation promotes GHSR coupling to Gαi/o. Enhanced GSIS is observed when AG is applied to INS-SJ cells that overexpress GHSR but lack SST5 expression. This data supports the requirement of SST5 for GHSR to couple to a Gαi/o pathway leading to AG inhibition of insulin secretion. The GHSR:SST5 heteromer formation is influenced by the relative abundance of ghrelin and SST 33: a high [ghrelin]/[SST] ratio promotes ghrelin suppression of cAMP accumulation, whereas lowering the [ghrelin]/[SST] ratio attenuates ghrelin inhibition of cAMP accumulation. Moreover, lowering the [ghrelin]/[SST] enhances ghrelin-induced [Ca2+]i mobilization. These data indicate that conditions that alter circulating ghrelin levels (such as fasting and feeding) can modulate the effect of ghrelin on insulin secretion (either inhibitory or stimulatory) though the interaction of ghrelin and SST signaling. Collectively, the in vitro data in the literature indicate that AG action on insulin secretion is influenced by: the cell type used, presence of other G-protein coupled receptors (GPCRs), as well as the concentration of ghrelin present. Therefore, differences in these factors may account for discrepancies in the literature that demonstrate that AG can have both stimulatory and inhibitory actions on GSIS.

In addition to regulating insulin secretion, AG acts on isolated islet cells and cultured α-cells to regulate glucagon secretion 34, 35. AG increases glucagon secretion from both isolated islets and

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from cultured α-cells 16, 34, 36. In contrast, islets isolated from Ghsr-/- mice show no changes in glucagon secretion in response to AG, indicating a GHSR-mediated action 34. This effect is mediated, in part, through AG induced rise in intracellular calcium as well as AG induced ERKphosphorylation in α-cells 34. Taken together, these data demonstrate that AG acts directly on pancreatic islets to regulate insulin and glucagon release.

In vitro action of dAG on pancreatic hormone secretion The des-acyl form of ghrelin was initially characterized as an inactive by-product of ghrelin secretion and degradation because it does not act as a full GHSR agonist 1. Despite this initial characterization, several studies investigated GHSR-independent actions of dAG on islet cell function. In INS-1E rat insulinoma cells, dAG promotes insulin secretion in the presence or absence of a GHSR antagonist 37. Furthermore, dAG promotes insulin secretion from HIT-T15 cells that lack the expression of GHSR further supporting a GHSR-independent mechanism of action 13. However, other groups have found no effect of dAG on GSIS from isolated rat islets even at high doses (1000nM) 28. There is also evidence that dAG alone does not regulate islet cell function, but rather, acts to antagonize the inhibitory action of AG on insulin secretion 21. The receptor that mediates these actions of dAG is still unknown, and although it is largely accepted that dAG does not bind and activate GHSR 1, studies in CHO-K1 cells transfected with human GHSR demonstrate that dAG can act as a full agonist of GHSR in the high nanmolar to micromolar range 38. Similarly, high nanomolar concentrations of dAG activates GHSR in HEK-293 cells transfected with the ghrelin receptor 9. Collectively, dAG action on pancreatic islet cell function is not fully understood, but most studies support a GHSR-independent action. Few studies demonstrate that dAG activates GHSR in cell types other than pancreatic islets.

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Further investigation of both GHSR-dependent and independent actions of dAG are necessary to fully understand the action of dAG on islet cell function.

Gluco-regulatory action of AG in rodents Pharmacological treatment as well as genetic manipulation of ghrelin in rodents indicate that AG regulates GSIS. Although some data suggest that systemic administration of AG enhances insulin secretion in rats 39, most rodent data support that acute systemic administration of AG has an inhibitory effect on insulin secretion. AG administered intravenously (iv) to rats or mice attenuates GSIS 40, 41. This effect is likely to be independent of AG-induced GH secretion as GH-deficient mice also demonstrated decreased GSIS and impaired glucose tolerance after AG administration 11. Pharmacological inhibition of AG synthesis with a GOAT inhibitor 42 or antagonizing GHSR 28 significantly increases GSIS and improves glucose tolerance. Together these data indicate that antagonizing AG or AG signaling enhances glucose-stimulated insulin release and improves glucose tolerance in rodents.

Consistent with pharmacological studies, genetic ablation of either ghrelin or GOAT leads to improved glucose tolerance and increased GSIS in 16-h fasted mice fed a standard chow diet 43, 44

. However, other groups have found no differences in glucose tolerance in Goat-/- or Ghrelin-

/- mice when a shorter fasting paradigm (6 h) was implemented 45, 46. The different fasting durations used in these studies may account for the differences in results. Fasting increases circulating ghrelin levels 47, 48 and it has been recently demonstrated that the [ghrelin]/[SST] ratio determines whether ghrelin will be stimulatory or inhibitory on insulin secretion 33. Under fasting conditions, the a high [ghrelin]/[SST] promotes GHSR:SST5 heteromer formation and

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GHSR then couples to Gai/o to inhibit insulin secretion. However, when the [ghrelin]/[SST] ratio is low as seen in fed conditions, the GHSR:SST5 heteromer destabilizes and GHSR no longer couples to Gai/o to inhibit insulin secretion. This could explain why the glucose tolerant phenotype in Goat-/- and Ghrelin-/- mice is only apparent under prolonged fasting conditions. However, further investigation is required to test this hypothesis.

Mice lacking Ghsr have similar glucose tolerance but decreased GSIS compared to wild-type (WT) mice suggesting that GHSR ablation leads to improved insulin sensitivity 49, 50. This is supported by studies that implement hyperinsulinemic euglycemic clamps in Ghsr-/- mice. When compared to WT controls, Ghsr-/- mice have an increased glucose infusion rate (GIR), increased glucose disposal (Rd), and decreased endogenous glucose production (EGP) indicating that peripheral and hepatic insulin sensitivity are both enhanced in the absence of GHSR signaling 50, 51. Similar to Ghsr-/- mice, improved hepatic and peripheral insulin sensitivity measured by hyperinsulinemic euglycemic clamps has also been reported in ghrelin deficient mice 43. Subjecting WT and Goat deficient mice to an ip insulin tolerance test led to a similar decrease in blood glucose levels in both groups suggesting similar insulin sensitivity 52. However, a more sensitive measure such as a hyperinsulinemic euglycemic clamp will be needed to more accurately determine whether Goat-/- mice have altered insulin sensitivity compared to WT animals. Together, pharmacological inhibition or genetic ablation of AG or AG signaling improves glucose metabolism by enhancing insulin secretion and insulin sensitivity.

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Gluco-regulatory action of dAG in rodents Although the receptor mediated actions of dAG have not been established, dAG has been shown to regulate glucose metabolism in vivo. During an ivGTT, dAG increased GSIS in rats 53. This effect was blocked by co-administration of AG 53. Chronic peripheral infusion of dAG does not alter glucose metabolism in mice on a standard chow diet 9, 54. However, chronic peripheral dAG treatment prevented high fat diet (HFD)-induced glucose intolerance and insulin resistance 54. Transgenic mice that express the preproghrelin gene under control of the mouse FABP4 promoter have elevated levels of dAG and display improved glucose tolerance and insulin sensitivity 55. These findings suggest that dAG analogs might be a viable option for the treatment of type 2 diabetes mellitus (T2DM). However, a better understanding of dAG physiology, pharmacology and receptor mediated actions are required before a safe and effective therapy can be developed.

Gluco-regulatory action of AG in humans The effect of AG on fasting plasma insulin and glucose levels in humans is still a matter of debate. Some groups have found no changes 56-58, whereas others have found that AG increased fasting blood glucose and decreased plasma insulin following ghrelin administration 59-61. Infusion of supra-physiological doses of AG to healthy humans decreased insulin secretion during an ivGTT which led to an impairment in glucose tolerance 56. Moreover, infusion of physiological concentration of AG also attenuated GSIS without altering insulin sensitivity 57. These finding highlight a pharmacological and physiological role of AG in regulating GSIS in humans.

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Under hyperinsulinemic euglycemic clamp conditions, ghrelin has also been reported to regulate insulin action in peripheral tissues. AG infusion decreases glucose infusion rate (GIR) and glucose disposal, while having no effect on endogenous glucose production (EGP) in healthy individuals 62, GH deficient patients 63 and those who underwent gastrectomy 64. These data are consistent with an acute effect of ghrelin to induce peripheral insulin resistance while sparing insulin sensitivity in the liver through a GH independent mechanism. AG infusion into the femoral artery of healthy men increased free fatty acid (FFA) levels suggesting that ghrelin can act directly on muscle to increase lipolysis which in turn leads to the development of insulin resistance 65. This concept was challenged by the finding that AG infusion directly into the muscle resulted in decreased interstitial blood glucose concentrations, which indicates improved muscle insulin sensitivity 66. Furthermore, no alteration in classical insulin signaling pathways was evident on muscle biopsy samples taken during ghrelin infusion in another study 63. Further studies are needed to understand the tissue specific actions of AG in regulating insulin sensitivity. As a whole, data in the literature demonstrate that acute administration of physiological and pharmacological doses of AG inhibit GSIS, whereas supraphysiological doses are required to decrease peripheral insulin sensitivity and glucose tolerance in humans.

The long-term effects of AG on glucose metabolism are not well defined. In a healthy elderly population, an oral ghrelin mimetic taken daily for 1 year caused an increase in fasting blood glucose levels and a decline in insulin sensitivity as estimated by the Quicki Index 67. Although ghrelin mimetics are currently being developed for the treatment of cancer cachexia, heart failure, and conditions related to GH deficiency, a better understanding of the long-term effects

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of these ghrelin-related pharmaceutical agents on glucose metabolism is necessary to avoid unwanted side-effects.

Gluco-regulatory action of dAG in humans The effects of dAG on glucose metabolism have not been extensively studied in humans (summarized in Table 1) and the findings have been inconsistent. During iv infusion in humans, AG is deacylated to dAG and therefore it is important to understand the possible actions of dAG. It is also important to note that dAG has a slower rate of clearance as compared to AG 68. An acute iv bolus of dAG in healthy subjects did not alter fasting GH, insulin or glucose levels 59, whereas co-administration of dAG with AG acted to abolish AG’s inhibitory action on fasting insulin in a similar study population 61. Similarly, dAG given for 4 days as an iv bolus once daily to obese non-diabetic patients had no effect on fasting or postprandial serum insulin, glucose or FFA levels 69. We recently showed that a 210-minute continuous infusion of AG or co-infusion of AG and dAG to healthy subjects yielded a decreased acute insulin response to glucose (AIRg) and iv glucose tolerance, whereas dAG infusion alone did not alter these parameters 70. However, the lack of effect of dAG on glucose metabolism is not universally observed. An overnight infusion of dAG in healthy humans decreased overall glucose and FFA levels but did not have a significant impact on overall insulin concentration during the 16-hr infusion period as compared to saline 71. In obese patients with T2DM, the same duration of dAG infusion decreases post-prandial glucose levels as well as fasting and postprandial AG levels without altering postprandial insulin. Insulin sensitivity, estimated by the M-index during a hyperinsulinemic euglycemic clamp, was improved as compared to saline infusion 72. Taken as a whole, the literature on dAG action in humans is very inconsistent and may be a result of

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different doses, subject populations, and length of infusion. Furthermore, the method employed to stabilize AG (and, therefore, dAG) in blood samples varies from study to study and this may also account for the discrepancies between studies. The long-term effects of dAG and how it interacts with AG to regulate glucose metabolism still warrants further investigation.

Ghrelin regulation of glucose homeostasis during calorie restriction Endogenous ghrelin levels rise during fasting or calorie restriction (CR) 4, 47, 48. Furthermore, exogenous ghrelin stimulates the secretion of all four counter-regulatory hormones (GH, cortisol, epinephrine and glucagon) and therefore has been implicated in maintaining blood glucose in states of negative energy balance. Ghrelin-/- and Ghsr-/- mice placed on a 50% calorie restricted diet have significantly lower blood glucose levels as compared to WT controls 73. Placing animals on a 60% calorie restricted diet depletes fat mass stores which then leads to severe hypoglycemia in both Ghrelin-/- and Goat-/- mice 44, 74. The hypoglycemia in these animals is attribute to the lack of AG induced GH secretion as AG or GH treatment rescues the hypoglycemic phenotype in calorie restricted Goat-/- mice 44. However, the hypoglycemic and relative GH deficient phenotype was not universally observed 75, 76. Adult-onset isolated GH deficient (AOiGHD) mice placed on a 60% calorie restricted diet lose significantly less fat mass, but maintain similar blood glucose levels as controls 75 indicating that GH may regulate adiposity during calorie restriction, but is not essential to maintain glycemia. Furthermore, mice lacking Ghrelin, Ghsr or Goat expression maintain similar blood glucose levels as WT controls when placed on a 60% calorie restricted diet even when fat mass stores were completely depleted 76. The reason for the discrepancies among these data is unclear, but differences in the age of the mice could be one of the contributing factors. Zhao et al. 44 and Li et al. 74 used young 8-week-

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old male mice that are still in the growing phase, whereas Yi et al. 76 and Gahete et al. 75 used mature (7-13 months) adult mice. In rodents, pancreatic ghrelin expression is highest prior to birth and slowly declines after birth 16 while gastric ghrelin gene expression increases rapidly after birth and then slowly declines with age 77. These data may suggest that ghrelin in the pancreas plays a more predominant role in regulating glucose metabolism at early stages of life. The role of ghrelin in glucose counter-regulation during acute or chronic CR needs to be more clearly defined.

CNS regulation of glucose metabolism A large body of literature provides clear evidence that the CNS is involved in regulating peripheral glucose metabolism 78, 79. Islet cell function is influenced by the CNS through the autonomic nervous system 80. Both parasympathetic and sympathetic neurons synapse on β- and α-cells 81 to activate or inhibit insulin secretion 82. GHSR is found in parasympathetic preganglionic neurons 83 and the brainstem, where ghrelin activates pathways controlling sympathetic and parasympathetic nerve activity 84-86. These data raise the possibility that in addition to direct effects, AG may suppress insulin secretion indirectly via neural signaling. For example, AG inhibited GSIS when infused into the portal but not the femoral vein, and hepatic vagotomy or intra-portal atropine diminished this inhibitory effect 41, 87. In humans, AG administration increased plasma epinephrine 88 and decreased heart rate variability 89, suggesting that AG mediates a sympathetic response that could affect islet secretion. An intact vagus nerve is required for many of the physiological effects of AG in mice and men 10, 89, 90, but no studies have directly tested the hypothesis that AG controls insulin via the autonomic nervous system. Intracerebroventricular (ICV) administration of AG to rodents has shown a stimulatory rather

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than an inhibitory action on plasma insulin levels. Chronic ICV AG administration to rats increases fasting insulin levels that is independent of the hyperphagia induced by AG 91. Similarly, chronic ICV administration of AG increases GSIS in mice 9. However, the central mechanisms that mediate the stimulatory action of AG on GSIS have not been identified. Furthermore, it is unclear as to why AG has an inhibitory action on GSIS in the periphery, whereas it appears to be stimulatory when administered centrally.

Limited data is available on the central effects of dAG on glucose homeostasis. One study demonstrated that ICV administration of dAG to mice increases GSIS through a GHSRdependent mechanism 9. Whether this is a pharmacological effect or also a physiological action of dAG requires further investigation.

Ghrelin interaction with Glucagon-like peptide (GLP-1) The post-prandial release of GLP-1 stimulates GSIS by enhancing cAMP signaling and increasing [Ca2+]i in pancreatic β-cells 92. AG inhibits insulin secretion by suppressing glucosestimulated [Ca2+]i in β-cells 11, and therefore, it was hypothesized that AG can counteract the stimulatory action of GLP-1 on insulin secretion 93. Indeed, the effect of GLP-1 on [Ca2+]i levels in isolated β-cells was attenuated by AG administration 93. Isolated rat pancreatic islets treated with both GLP-1 and a GHSR antagonist had increased insulin release as compared to treatment with GLP-1 alone 93. The clinical relevance of these findings was tested in a study of PraderWilli syndrome (PWS), a congenital disease that is associated with hyperphagia, T2DM and elevated ghrelin levels. A PWS patient treated with Liraglutide, a GLP-1 analog, for 12 months 94

lost 5.7 kg of weight, had improved glycemic control, and lower plasma ghrelin levels. The

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metabolic benefits seen with Liraglutide treatment may be in part due to GLP-1 suppression of ghrelin secretion but the mechanisms involved require further clarification. However, a recent study using GLP-1 infusion at 1 pmol/kg/min failed to observe any changes in ghrelin levels in either obese T2DM or healthy lean subjects 95. Conversely, ghrelin administration has been reported to accelerate gastric emptying and increase GLP-1 secretion following a test meal in healthy individuals 96. Collectively, these data indicate that GLP-1 and ghrelin interact with each other at the level of the pancreas to regulate islet cell function. Further studies are needed to address the interaction of ghrelin and GLP-1 in other tissues that regulate glucose metabolism such as the brain.

Metabolic benefits of bariatric surgery: is ghrelin a key player? Bariatric surgery is the most effective way to reduce body weight and improve glucose metabolism in obese individuals 97. Studies comparing the effects of the two most common bariatric procedures, Roux-en-Y gastric by-pass (RYGB) and vertical sleeve gastrectomy (VSG), in obese T2DM patients demonstrate that body weight reduction and remission of T2DM are comparable in patients who undergo one vs. the other operation 98. Furthermore, patients receiving either RYGB or VSG had similar improvements in oral glucose tolerance 6 months post-surgery 99 as well as improved insulin sensitivity as measured by Homeostasis Model Assessment Index (HOMA) 100. Studies in rodents and humans have demonstrated that both surgical procedures result in the development of improved glycemic control, which interestingly, occurs prior to a significant reduction in body weight and adiposity suggesting that other factors besides weight loss are involved 101-104. RYGB and VSG produce similar metabolic benefits through very different rearrangement of the gastrointestinal anatomy (reviewed in 105). A

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common characteristic of the two types of surgeries is that they both prevent nutrients from contacting ghrelin-producing cells in the stomach. A number of studies indicate that AG has detrimental effects on glucose metabolism, and therefore, many groups aimed to determine whether circulating ghrelin levels are lower following bariatric surgery. Indeed, ghrelin levels are lower in individuals that underwent RYGB as compared to normal weight controls as well as individuals that experienced diet-induced weight loss 106. However, these results have been inconsistent and some groups have found no changes in fasting or post-prandial ghrelin levels after RYGB, whereas other groups found decreased fasting and postprandial ghrelin levels (reviewed in 107). Ghrelin measurements following VSG have been more consistent, and most groups find reduced fasting and post-prandial ghrelin levels in VSG operated patients 108-111. Most studies find that VSG causes a 20-30% reduction in fasting ghrelin levels 108-110, 112 while some show a near 60% reduction from pre-surgical levels 6 months following surgery 111. The relevance that these changes in circulating ghrelin have to the metabolic benefits of bariatric surgery has recently been challenged by a study in which DIO Ghrelin-/- mice were given VSG. Interestingly, VSG-operated Ghrelin-/- mice achieved similar improvement in oral glucose tolerance as compared to their VSG-operated WT controls 113. In addition to improvements in glucose metabolism, a reduction in body weight and adiposity is also seen in the VSG Ghrelin-/animals indicating that ghrelin is not the critical hormone that determines the metabolic benefits of VSG 113. However, these genetically modified animals may undergo compensation by other metabolic signaling systems during development, which is a factor that cannot be ignored using this gene deletion approach. Therefore, the relevance of post-operative ghrelin changes for the overall metabolic benefits of bariatric surgery remains to be elucidated.

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Summary The gastrointestinal hormone ghrelin has received much attention for its ability to regulate glucose metabolism. The two major ghrelin isoforms found in circulation, AG and dAG, appear to have distinct actions. From in vitro studies to clinical studies in humans, the majority of reports demonstrate that AG has an inhibitory effect on GSIS and tissue glucose uptake when administered peripherally. Conversely, blocking AG synthesis improves glucose tolerance and enhances insulin secretion in rodents 42. Therefore, pharmaceutical agents that aim to antagonize AG or AG signaling could be potential therapeutics for treating T2DM. A summary of AG action in pancreatic islets is provided in Figure 1. The effects of dAG on β-cell function and insulin action are less clear. Some studies suggest that dAG has no effect, whereas others indicate that dAG can stimulate insulin secretion and improve glucose tolerance, and still others show that dAG acts to antagonize AG action. Before dAG analogs can be used for therapeutic purposes, it is essential to clearly define the physiological function of dAG and to uncover possible dAG receptor-mediated actions. The rise and fall of AG and dAG correspond to the duration of fasting 47. It is likely that the ghrelin action on the pancreas and/or the CNS is linked to the metabolic states of the species in order to maintain glucose homeostasis.

Collectively, the major effects of ghrelin are linked as a protective mechanism against starvation: orexigenic actions to promote food intake, stimulation of GH secretion to promote lipolysis and restrict peripheral glucose uptake, and restraint of insulin secretion to prevent hypoglycemia. Much research is still needed to learn about the contribution of each of the components of the ghrelin system (dAG, AG, GOAT, GHSR) to the regulation of these functions. Lastly, identifying the tissue specific actions of each of these components through the use of advanced

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genetic technology and pharmacology will help to pinpoint the underlying mechanisms involved in ghrelin system’s regulation of glucose and energy homeostasis.

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Figure Legend Figure 1: Acyl ghrelin (AG) regulation of islet cell function. Similar to the schematic depicted by Park et al. 33, high AG concentrations activates GHSR on alpha-cells to promote glucagon secretion and activates GHSR on beta-cells to inhibit insulin secretion. GHSR signaling in betacells may be regulated by heterodimerization with other GPCRs. These combined actions will lead to an overall increase in circulating glucose. High AG concentrations occur during CR, weight loss, in PWS and after pharmacological administration of AG analogs. When AG levels are low, AG does not act to regulate blood glucose through modulation of islet cell function. Low AG concentrations occur in obese and insulin resistant individuals, following pharmacological inhibition of GOAT, and following gastrectomy or gastric sleeve surgery. Acyl ghrelin (AG); ghrelin O-acyltransferase (GOAT); calorie restriction (CR); growth hormone secretagogue receptor (GHSR); somatostatin receptor subtype 5 (SST5); G-protein coupled receptor (GPCR)

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Table 1: Effects of dAG on glucose homeostasis in humans Reference

Tong et al. (2014)69

Ozcan et al. (2013)71

Benso et al. (2012)70

Sample size

17 healthy subjects

8 overweight T2DM patients

8 healthy subjects

Dose of dAG

210 min continuous infusion at (4µg/kg/hr)

Measurements

Results

Fasting glucose and insulin

 fasting insulin

FSIGT: AIRg, insulin sensitivity index (SI), disposition index (DI), and kg

 fasting glucose

 AIRg or SI

 DI

 kg

Overnight (16h) Glucose and infusion (3 or insulin response to a 10µg/kg/hr) standardized meal. Insulin sensitivity (MIndex calculated from H-E clamp)

↓ postprandial glucose

Overnight (16h) Meal-related (dinner and infusion breakfast) and (1µg/kg/hr) inter-meal glucose, insulin and FFA

↓ overall glucose AUC

 postprandial insulin ↑ insulin sensitivity during H-E clamp

 overall insulin AUC

↑ 1 hr postmeal insulin AUC

↓ overall FFA AUC

Kiewiet et al. (2009) 68

8 morbidly obese nondiabetic patients

Once daily IV bolus (200 µg/day) for 4 consecutive days

Fasting and postprandial insulin, glucose and FFA

 fasting or postprandial insulin  fasting or postprandial glucose  fasting or postprandial

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FFA

Gauna et al. (2004)113

8 adult-onset GH-deficient patients

IV bolus (1µg/kg)

Fasting and postprandial glucose, insulin, and FFA

↑ fasting and postprandial glucose

 fasting or postprandial insulin

 fasting or postprandial FFA

Broglio et al. (2004)60

Broglio et al. (2003)58

6 healthy subjects

7 healthy subjects

IV bolus (1µg/kg)

IV bolus (1µg/kg)

Fasting plasma glucose and insulin

 fasting glucose

Fasting plasma glucose and insulin

fasting glucose

 fasting insulin

fasting insulin

Table 1 summarizes the current literature of dAG action on glucose metabolism in humans. ↑ increase; ↓ decrease;  no change; type 2 diabetes mellitus (T2DM); des-acyl ghrelin (dAG); hyperinsulinemic euglycemic clamp (H-E); intravenous (IV); frequently sampled IV glucose tolerance test (FSIGT); IV glucose tolerance (kg); Acute insulin response to glucose (AIRg); free fatty acid (FFA); area under the curve (AUC).

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Low [AG]

High [AG]

(obesity/insulin resistance, GOAT inhibition, gastrectomy/gastric sleeve surgery)

(CR, weight loss, PWS, pharmacological AG administration)

GHSR AG

GHSR

glucagon

Insulin

AG

SST5

α-cell

Pancreatic Islets

Glucose

GHSR

β-cell

Other GPCRs?

Mechanisms in endocrinology: regulation of glucose metabolism by the ghrelin system: multiple players and multiple actions.

Ghrelin is a 28-amino acid peptide secreted mainly from the X/A-like cells of the stomach. Ghrelin is found in circulation in both des-acyl (dAG) and ...
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