Ghrelin: a journey from GH secretagogue to regulator of metabolism

Introduction

In the last 40 years, the human lifestyle has been suffering radical changes, where the availability and accessibility to food as well as food advertising and marketing contribute to create an obesogenic environment. A ‘side effect’ of our modern lifestyle is that the incidence of obesity has grown to pandemic proportions. According to the National Health and Nutrition Examination Study (NHANES), 33.8% of adults are obese in the United States (1). Moreover, the prevalence of obesity worldwide has more than doubled over the past three decades (2). Obesity is associated with a range of metabolic diseases, cardiovascular diseases and the development and progression of several cancers (3,4). To prevent the development of obesity it is crucial to understand the mechanisms that regulate food intake and energy expenditure.

Feeding and energy metabolism is essential for species survival. The complex homeostatic mechanism regulating body weight involves interactions between peripheral organs, such as white adipose tissue (WAT), the gastrointestinal system (GIS) and the central nervous system (CNS), through signals, that inform brain centers of the nutritional and metabolic status of the animal (5).

A primary site for controlling energy balance is the hypothalamus; a region that is intimately associated with the regulation of basic functions such as reproduction, temperature, hormonal balances and biological rhythms. Hypothalamic nuclei and areas that are associated with regulation of energy balance include the arcuate nucleus of the hypothalamus (ARH), ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), paraventricular hypothalamus (PVH) and lateral hypothalamus (LH) area (6). These hypothalamic areas form interconnected neuronal circuits that respond to changes in energy status by altering the expression of specific molecules, especially neuropeptides, resulting in changes in energy intake and expenditure. These neurons also project to other regions of the brain, like the brainstem and spinal cord. Apart from the hypothalamus, the brainstem in particular, plays an important role in the regulation of feeding behavior.

A large number of peripheral signals, particularly peptides, have been identified in the last three decades. Such peptides have been shown to regulate food intake and energy expenditure. Most of the peptides (hormones) that regulate appetite are anorexigenic “satiety” factors (5). However, there is an exception; Ghrelin, a hormone secreted mainly by the gut, displays an exclusive characteristic; and so far, it is a unique known orexigenic hormone, which confers a predominant role on feeding control (7).

In this review, we will provide an overview of the current knowledge in the literature regarding the integration between the brain and gastrointestinal signals to regulate energy homeostasis. We will also highlight the role of ghrelin in maintaining energy and glucose homeostasis.

Signals to the brain from GIS

To maintain energy homeostasis, the brain must closely monitor the peripheral energy state within the body. Since the brain does not store energy, it is dependent on a continuous supply of nutrients from the general circulation to survive. The brain receives signals via vagal afferents or through the circulation. The enteric nervous system that interconnects with the autonomic nervous system transmits information of mechanical (distension, contraction), chemical (presence of nutrients in the gut lumen) and neurohumoral stimuli (gut hormones, neurotransmitters and neuromodulators) to the CNS through vagal and sympathetic nerves (8). The signal peptides released locally from enteroendocrine cells of the GIS activate receptors of the vagal afferent fibers, informing the brainstem of luminal composition (9,10).

The hindbrain (caudal brainstem) contains neurons and circuits that involve autonomic control of ingestion, digestion, and absorption of food (8) independently of the forebrain (11). The nucleus of the solitary (NTS) tract is one of the major processors of vagal afferent signals that conveys messages to higher neural centers involved in appetite control, such as the ARH, PVH and DMH (12). The integration of all these afferent signals related to food presence in the gut in turn regulates the meal size of individuals (8,10,11). Therefore, vagal afferent nerves are the major conduit by which nutrients signal to the brain and influence motility and secretion, as well as hunger and satiety (8-10).

Hormones secreted from WAT and the GIS can influence several specific brain regions and neurons via the circulation. Among these regions, the NTS—area postrema (AP) complex in the hindbrain and the ARH in the forebrain are two of the major targets of those hormones.

The gastrointestinal tract secretes several satiety signals, such as Cholecystokinin (CCK), Bombesin, Glucagon-like peptide-1 and 2 (GLP-1 and GLP2), Amylin, Peptide YY (PYY), Oxyntomodulin, somatostatin and enterostatin in response to gut nutrient content and most of them play an important role in the control of energy homeostasis (13,14).

The circulating signals are often categorized as long-acting adiposity signals, and short-acting gastrointestinal factors (Figure 1). Long-acting signals characteristically reflect the levels of energy stores and regulate body weight and the amount of energy stored as fat over time. The two adiposity signals that are best known are insulin and leptin. The functional ability of insulin and leptin as adiposity signals to the brain have been reviewed several times and are beyond the scope of the current review [see (15,16) for review]. Several hormones are a representation of short-acting signals that regulate appetite and the majority of them decrease food intake.

Figure 1 Model depicting the short and long term signals to the brain centers involves in body weight regulations. Hypothalamic areas form interconnected neuronal circuits that respond to changes in energy status. It includes the arcuate (ARH) paraventricular nuclei (PVH), lateral hypothalamus (LH), Dorsomedial hypothalamus and ventromedial hypothalamus. In the brainstem, the area postrema, the nucleus of the solitary tract and the dorsal motor nucleus of the vagus nerve, which together compose the dorsal vagal complex (DVC) also plays a vital role in the regulation of feeding. Insulin secreted by the pancreas and leptin secreted by white adipose tissues (WAT) are long-acting signals (blue arrows). They reflect the levels of energy stores and regulate body weight, and act in hypothalamic centers and DVC. The majority of the short-acting signals that decrease food intake are generated in the gastrointestinal (GI) tract during meals. They provide information about mechanical and chemical properties of the food (as indicated by CCK release). The signals are conveyed via sensory axons in the vagus and sympathetic (SNS) nerves into the DVC of the brainstem. Short-term signals can also reach to the hypothalamus and DVC through systemic circulation. In the diagram, this is represented by the yellow (satiety signals -Glucagon-like peptide-1 and Peptide YY) and red arrow (orexigenic peptide ghrelin).

Neural circuits within the hypothalamus and the brainstem can regulate both food intake as well as integrate short and long-term signals of energy balance (17-19). Both types of signalling are able to act together. For example, leptin enhances the satiation effect of GLP-1 at vagal afferent fibers and the hindbrain (20). Similarly, the activation of GLP-1R-expressing neurons of the NTS, which project to hypothalamic areas involved in appetite regulation, can modulate the activity of those areas (12,14).

Ghrelin acts in the brain to increase food intake via activation of its ghrelin receptors (GHS-R). These receptors are expressed in several caudal brainstem nuclei, including area postrema, NTS and the dorsal motor nucleus of the vagus nerve, forming the dorsal vagal complex (DVC) (21). Stimulation of GHS-R in the caudal brainstem leads to a hyperphagic response (22). A direct action in the same area (on the dorsal vagal complex) that increases food intake was demonstrated using microinjections of ghrelin (23). Moreover, fourth-ventricle ghrelin delivery increases Fos in the NTS but not in the hypothalamic areas involved in food regulation such as ARH and LH. This suggests the existence of hypothalamic circuits within the forebrain and the hindbrain that respond independently to ghrelin (24).

In the hypothalamus, the ARH is the most studied neural circuit that regulates energy balance. ARH and PVH are two critical brain centers that transmit the orexigenic action of ghrelin. Several lines of evidence have demonstrated that ghrelin induces feeding by stimulating an orexigenic population of neurons in the ARH; the neuropeptide Y and agouti-related peptide neurons (NPY/AgRP neurons) (25-33). More than 90% of NPY/AgRP neurons express the GHS-R1a in the ARH (34). The GHS-R1a is also expressed on growth hormone releasing neurons (34,35) and tyrosine hydroxylase neurons (21,36) in the ARH. The GHS-R1a-expressing neurons of the PVH are also activated by ghrelin to increase food intake and to promote the intake of carbohydrate rich diets and increase adiposity (37).

The ghrelin axis: peptides, receptors and enzymes

Ghrelin synthesis and structure

Ghrelin is a peptide that was discovered as an endogenous ligand for the growth hormone secretagogue receptor (GHSR-1a) in 1999 (38) through which it stimulates GH release from the anterior pituitary.

Ghrelin is predominantly synthesized in the stomach and secreted into the circulation. The X/A-like cells of gastric oxyntic glands of the stomach are the most abundant source of circulating ghrelin. The small intestine also synthesizes ghrelin to a lesser extent, with the amount of ghrelin produced diminishing with an increased distance to the pylorus (39). Additionally, it has been demonstrated that ghrelin is expressed in many tissues such as the lung, heart, pancreas, kidney, testis, pituitary, and hypothalamus (40).

Ghrelin has a unique structure with 28 amino acids and a n-octanoyl ester at its third serine residue, which is essential for its potent biological activity at the GHS-R1a. Several steps involving different enzymes detail the process from gene synthesis to the final structure. The ghrelin gene is transcribed as a pre pro-ghrelin mRNA isoform, which leads to the translation of a 117 amino-acid peptide. This prohormone is cleaved into pro-ghrelin, a 94 amino acid signalling peptide. Pro-ghrelin then requires posttranslational acylation with n-octanoic acid or n-decanoic acid at the third serine residue for its biological activity at the GHS-R1a. Ghrelin O-Acyltransferase (GOAT) is the enzyme responsible for pro-ghrelin acylation (41) and it is also found predominantly in the stomach and digestive tract (41,42). In the stomach and duodenum, GOAT co-localizes with ghrelin expressing cells (43), where it readily acylates newly synthesized pro-ghrelin. GOAT can also acylate pro-ghrelin with other fatty acid substrates besides octanoate (44). Intake of diet enriched with either medium chain fatty acids or medium-chain triglycerides could lead to a modification in the proportions of octanoyl or decanoyl ghrelin stored in the same granules in gastric cells, suggesting that GOAT is likely to use the most available substrate to perform ghrelin acylation (45). Pro-ghrelin is further processed by prohormone convertase (PC 1/3) to produce the 28 amino-acid mature ghrelin peptide (46,47). Other forms of ghrelin (truncated peptides) have also been described; more of them arise from alternatively spliced variants that may act on autocrine/paracrine pathways (48). It has been suggested that these truncated peptides could play multiple roles in several diseases, particularly in breast and prostate cancer (46).

Circulating ghrelin

Ghrelin exists as two forms in the plasma, acylated ghrelin and des-acylated ghrelin. In humans, approximately 80-90% of plasma ghrelin is des-acyl ghrelin (49) however the mechanisms that control the rate at which acyl-ghrelin converts into des-acylated ghrelin is unknown. Recently a ghrelin deacetylation enzyme; acyl-protein thioesterase-1 (APT1) has been described, and it can des-acylate ghrelin in the plasma (50). The high des-acyl to acyl ghrelin ratio in the circulation can be explained by the shorter half-life of ghrelin compared to des-acyl ghrelin (51,52). The proportion of acyl to des-acyl ghrelin seems to be physiologically important since des-acyl ghrelin is unable to activate GHS-R1a. While most des-acyl ghrelin circulates as a free peptide, the vast majority of acyl-ghrelin is bounded to larger molecules such as lipoproteins (53).

Ghrelin levels exhibit a circadian rhythm and closely follow feeding schedules; peripheral ghrelin levels rise sharply before main meals in a scheduled meal-fed sheep and decrease once the animal has been fed (54). Under fasting conditions, plasma ghrelin peaks match the previous pattern of daily meals in humans (55).

Ghrelin levels show a marked gender distribution, with higher levels in women as compared to men and the levels decline with age, BMI, hypertension and others markers of metabolic syndrome (56).

A large number of studies that evaluate ghrelin levels do not distinguish between total ghrelin, acyl ghrelin, and des-acyl ghrelin levels. Appropriate sample collection and storage strategies are necessary to limit the acyl ghrelin degradation in order to elucidate the physiological and pathophysiological roles of ghrelin (see Delporte’s review) (57).

Ghrelin receptors

Two isoforms of Ghrelin receptors have been identified; GHS-R1a is a 366 amino acid protein belonging to the G-protein coupled receptor (GPCR) superfamily and is characterized by seven transmembrane receptor domains (58), whereas GHS-R1b is a truncated form that lacks part of transmembrane domains 6 and 7. GHS-R1a is the functional ghrelin receptor required to elicit growth hormone release or food intake in response to exogenous administered ghrelin. On the contrary, GHS-R1b is regarded as a non-functional receptor due to its inability to bind to acyl or des-acyl ghrelin (58).

Recent evidence has unravelled some of the important regulatory functions of GHS-R1b. For example, GHS-R1b forms heterodimers with GHS-R1a to decrease the constitutive activity of GHS-R1a (59,60). Both isoforms of the GHSR are widely expressed. GHS-R1a expression was first identified in the pituitary and hypothalamus, where it is highly expressed (58). GHS-R1a expression has also been demonstrated in a number of brain regions and in a wide range of peripheral tissues, including the stomach, intestine, pancreas, spleen, thyroid gland, adrenal gland, kidney, heart, lung, liver, lymphocytes and adipose tissue (58-61).

Functional activity of ghrelin

Ghrelin acylation is required to bind GHS-R1a and exert its biological activity. Interestingly, acylated ghrelin is also able to produce some endocrine effects in tissues where there is no expression of GHS-R1a (9), suggesting the presence of an unknown alternate ghrelin receptor.

Even though des-acyl ghrelin is unable to bind to GHS-R1a, it is not functionally inactive. Extensive studies have demonstrated that des-acyl ghrelin can modulate cell proliferation, apoptosis, metabolism and glucose homeostasis (9,57,62-65), indicating the presence of an alternative receptor. Nevertheless, further studies are required to unveil these pathways to understand the potential biological importance of des-acyl ghrelin, the most abundant isoform in circulation.

Hypothalamic circuits that are involved in ghrelin-mediated feeding.

It is believed that neural circuits within the ARH regulate the effects of ghrelin on feeding. Two neuronal populations in the ARH are considered ‘first-order’ sensory neurons in the control of food intake. One population expresses the appetite-suppressing peptides, α-MSH [derived from the proopiomelanocortin (POMC) precursor] and cocaine- and amphetamine-regulated transcript (CART). The other co-expresses two appetite-stimulating peptides, neuropeptide Y (NPY) and agouti-related peptide (AgRP) (66). Both NPY/AgRP and POMC neurons project to the PVH and other hypothalamic nuclei (67).

Importantly, these neurons receive different metabolic signals such as glucose, hormones and fatty acids, and subsequently project them to second-order hypothalamic nuclei to regulate food intake. In addition, using novel human diphtheria toxin targeted therapy, the conditional deletion of NPY/AgRP neurons results in a rapid decrease in food consumption and body weight (68,69). Various methods including electrophysiology (25,26), or c-fos immunoreactivity (25,27,28), peptide secretion (29), or gene expression (30-33) have all revealed that ghrelin robustly stimulates NPY and AgRP to mediate feeding. To further bolster the role of ghrelin in energy homeostasis, selective ablation of AgRP in adult mice abrogates the orexigenic effects of ghrelin entirely (70) and double NPY/AgRP knockout mice fail to increase food intake in response to ghrelin (30). Collectively, these studies suggest that the orexigenic effects of ghrelin are mainly exerted through the ARH.

Upon the stimulation of NPY/AgRP neuronal activity with Ghrelin, POMC neurons are concurrently suppressed through the inhibitory γ-aminobutyric acid (GABA)-eric inputs from activated NPY/AgRP neurons (26). The inhibitory tone on POMC neurons is reversed when the vesicular GABA transporter was genetically ablated in AgRP neurons, causing subsequent anorexia following activation of the melanocortin system (71). Ghrelin increases GABA-mediated inhibitory inputs from NPY/AgRP neurons and alters POMC neuronal synaptic plasticity by increasing the number of inhibitory perikaryal synapses to POMC neurons, thus lowering POMC neuronal activation and results in a decrease in energy consumption (25).

Recent evidence has shed some light on a unique intracellular signaling modality that unravels how ghrelin activates NPY neurons to initiate changes in feeding behavior. However in this review, the molecular mechanisms are only discussed in brief. Of note, both intraperitoneal or intracerebroventricular delivery of ghrelin increases AMP-activated protein kinase (AMPK) phosphorylation and activity in the hypothalamus and consequently leads to increased calorie intake (25,72,73). AMPK plays an important role in energy homeostasis as central compound C abolishes AMPK activity and suppresses ghrelin-mediated food intake (25,74). In a nutshell, ghrelin binds to the GHSR and initiates a signal transduction cascade that begins with Ca²⁺ influx in identified NPY neurons (75-77). Ca²⁺ subsequently interacts with calmodulin (CaM) to activate CaM-dependent protein kinase kinases (CaMKK), which in turn leads to AMPK phosphorylation and increases NPY messenger RNA and protein expression (78,79).

The downstream intracellular actions after ghrelin-induced AMPK activation involve phosphorylation of acetyl CoA carboxylase (ACC), which causes the suppression of malonyl CoA and disinhibition of carnitine palmitoyl transferase 1 (CPT1) (25,74). The notion that CPT1 mediates ghrelin-induced food intake is strengthened by data showing that inhibition of CPT1 prevented ghrelin’s ability to increase NPY and AgRP mRNA expression in the hypothalamus (25). The downstream mechanism is unraveled when ghrelin stimulates palpitate-driven uncoupled respiration in isolated hypothalamic mitochondria in an UCP2-dependent manner. In essence, upon binding to GHS-R1a, ghrelin initiates AMPK-CPT1-UCP2 axis and mitochondrial respiration that necessitates mitochondrial biogenesis in NPY/AgRP neurons, depolarization of NPY/AgRP neurons and ghrelin-mediated synaptic plasticity of POMC neurons. The activation of GHS-R1a also triggers the opening of calcium channels, causing an intracellular influx of Ca²⁺ through the adenylate cyclase-protein kinase A (PKA) pathway (75). Altogether, both molecular mechanisms converge to increase mRNA expression of NPY and ultimately resulting in increased food intake.

Ghrelin and obesity

In line with the notion that ghrelin stimulates food intake, ghrelin levels are reported to be elevated in fasted mice and rats, and decreased in obesity (80,81). It is believed that the increased ghrelin levels during the fasting/starvation state serve to restore neutral energy balance and combat hunger (82). However in diet induced obesity (DIO), peripheral ghrelin does not stimulate food intake (83), ghrelin transport across the blood brain barrier is impeded (84), NPY/AgRP feeding circuits are perturbed (85) and basal hypothalamic AMPK activity is suppressed (86). In addition, DIO mice display ghrelin resistance in NPY/AgRP neurons at the level of the ARH (29,87) but not in other hypothalamic nuclei such as the PVH (6). An example of a similar condition is when mice are fed on a high-density diet; they develop leptin resistance in the ARH neurons while leptin sensing is preserved in second order neuronal region like the VMH (6,88). To date, it is still unclear how this phenomenon can impact obesity. However, studies have suggested that ghrelin resistance could be a factor in the decline of cognitive functions, as ghrelin signalling in the brain is essential for memory, learning and neuroprotection (89-92).

Interestingly, central ghrelin signaling in the brain can selectively modulate body weight without affecting food intake, because RNA interference of GHSR expression in the PVH significantly reduces body weight and blood ghrelin levels independent of food intake (93). Moreover, chronic central ghrelin infusion in pair-fed animals significantly increases respiratory quotient (RQ), which is indicative of increased fat deposition (94). The evident increase in mRNA of lipogenic enzymes lipoprotein lipase (LPL), acetyl-CoA carboxylase α (ACC), fatty acid synthase (FAS), and stearoyl-CoA desaturase-1 (SCD1) in pair-fed animals further substantiates that the effects on WAT is independent of changes in food intake (94). Importantly, there are studies in the literature showing that ghrelin administration in NPY-deficient mice increases body weight (95), overexpression of NPY in PVH induces obesity (96) and NPY deficiency decreases body weight when mice are fed on a high-density diet (97). Therefore, in terms of energy homeostasis, the actions of ghrelin in the hypothalamus are likely to be mediated by either ghrelin acting on NPY/AgRP neurons in the ARH to increase food consumption and body weight or the direct actions of ghrelin on GHSR-expressing neurons in the PVH to increase adiposity and body weight.

Ghrelin and lipogenesis

Besides the actions of ghrelin in the brain, it also has direct effects on lipogenesis in peripheral adipose tissue. Recent in vitro studies have provided compelling evidence that both ghrelin isoforms (acyl and des-acyl ghrelin) trigger the activation of adipogenic factors such as PPAR gamma and C/EBPα to induce cell proliferation, and adipocyte differentiation in 3T3-L1 preadipocytes (98-101). In human visceral adipocytes, both ghrelin isoforms stimulate lipid accumulation (102) and in cultured rat adipocytes, ghrelin directly increases leptin production (103). In vivo studies have revealed that ghrelin regulates adipogenesis by binding directly to the GHSR, which is expressed in adipose tissue (102,104,105). Ghrelin increases triglyceride content in adipose tissues (106) and Davies et al. (104) demonstrated that acyl-ghrelin increases abdominal adipose tissue in a depot-specific manner. In contrast, the GHSR-independent effect is proposed to be centrally mediated when in vivo studies with ghrelin delivered iv, ip or via minipumps altered adipogenesis (9,94). Even though it was mentioned previously that both ghrelin isoforms could stimulate adipogenesis in cultured adipocytes, studies have emerged to discriminate the differential effects of acyl and des-acyl ghrelin in whole animals. Des-acyl ghrelin is shown to increase adiposity (9), or have no effect on adiposity (104) or decrease adiposity and improve insulin sensitivity (107). More recent studies support the idea that des-acyl ghrelin maintains insulin sensitivity and prevents adipogenesis (65). There is no doubt that ghrelin stimulates weight gain by increasing food intake and adiposity. However, there are many equivocal issues that need to be addressed in the future. For example, does ghrelin primarily influence adiposity through central or peripheral mechanisms? How do acyl ghrelin, des-acyl and the ratio of acyl/des acyl ghrelin affect adipogenesis and insulin sensitivity? Understanding these critical issues will help design specific therapies to combat obesity and diabetes.

As there is a wealth of literature showing that exogenous ghrelin administration increases food intake and adiposity, it is noteworthy that studies from knockout models suggest that ghrelin signaling has only modest effects on body weight and food intake. There are reports showing that GHSR-/- mice have significantly reduced body weight on a regular chow diet (108,109), while others did not observe any change in body weight on a regular diet, possibly due to the employment of different GHSR-/- mouse lines (110,111).

Due to the equivocal nature of these knockout studies, it remains possible that animals could develop compensatory mechanisms to counteract ghrelin deletion. Thus, to understand the true physiological role of ghrelin in regulation of food intake and body weight, it will be useful to generate temporal ghrelin knockout mouse models (i.e., ghrelin ablation in adult mice).

Ghrelin regulates glucose homeostasis

Knockout models illustrate that ghrelin plays a major role in glucose homeostasis. Tschöp and colleagues were the first to document a possible interaction between ghrelin and glucose by demonstrating that a single subcutaneous ghrelin injection reduces serum ghrelin levels in rats (80).

Although the functionality of knockout models in evaluating ghrelin-mediated energy homeostasis is limited, they are beneficial to dissociate ghrelin’s actions in regulating glucose homeostasis. In fact, ghrelin gene deletion prevents the development of glucose intolerance when mice were fed on a high fat diet despite having similar body weight and food intake between ghrelin KO mice and wild type littermates (112). Furthermore, disruption of the ghrelin gene in leptin deficient (ob/ob) mice ameliorates glucose intolerance and augmented insulin secretion (113). Despite the fact that Pfluger and colleagues observed no difference in glucose disposal between chow fed mice after deleting both ghrelin and its receptors (111), we believe that it is beyond one’s imagination to improve a normal condition further and that may elucidate the clearer effects of ghrelin on glucose homeostasis seen in mice on an obesogenic HFD.

It is valuable to note that ghrelin potently stimulates GH secretion, and GH increases plasma glucose concentrations, free fatty acid uptake and suppresses glucose transport in skeletal muscles (38). It was proposed that the effect of ghrelin on glucose metabolism is driven by increased GH secretion. However, ghrelin infusion in humans given GHR antagonist (pegvisomant) increased plasma glucose concentration and decreased insulin sensitivity, proving the theory that ghrelin regulates blood glucose through a GH-independent mechanism (114,115).

Possible mechanistic action of ghrelin

Ghrelin is primarily produced in the stomach, as well as in the hypothalamus and other peripheral tissues albeit at low levels. On that note, it remains feasible that paracrine, endocrine, and neural pathways are all possible alternatives to explain ghrelin’s effects on glucose. In order to achieve normal glucose balance, it requires the activation of glucose sensing mechanisms in the hypothalamus and peripheral tissues. We will discuss some of them in the section below. For more in depth information, refer to the reviews of Sangiao-Arvarellos et al. (116) and Heppner et al. (117).

Ghrelin activates glucose sensing neurons in the brain

Emerging studies have identified glucosensing neurons within the hypothalamus and brainstem that are involved in orchestrating the activity of the autonomic nervous system, hormone secretion, and changes in fuel metabolism, which include glucose production and glucose uptake and utilization (118). It is plausible that hormonal signals such as ghrelin could converge and act directly on melanocortin circuits to modulate peripheral glucose balance, because POMC neuronal activity is triggered by glucose (119) whereas, NPY neuronal activity seems to be suppressed by glucose (120,121). It was also proposed that ghrelin is synthesized by a group of neurons close to the third ventricle between the dorsal, ventral, paraventricular and ARH (26). These ghrelin expressing neurons project to key hypothalamic circuits that synthesizes neuropeptides such as NPY, AgRP, POMC and CRH, clearing the uncertainty that hypothalamic ghrelin contributes to whole body glucose regulation. Importantly, the activation of NPY neurons in the hypothalamus affects peripheral glucose regulation by increasing hepatic glucose production and gluconeogenic enzymes such as Glucose-6-phophatase, consequently inducing hepatic insulin resistance (122).

Peripheral targets of ghrelin to modulate glucose homeostasis

Peripheral glucose responsive tissues such as the pancreas, liver, skeletal muscle and WAT are vital to ensure lipid and glucose balance in our body. Here, we will outline some of the pertinent studies about the interaction between ghrelin and these tissues to achieve glucose balance.

Pancreas

Ghrelin and its receptor are expressed in the islets (α-, β- and ε cells) of rodents and human pancreas (123,124). β-cells sense glucose through its metabolism and the resulting increase in ATP and the activation of ATP-sensitive K⁺ channels and Ca²⁺ influx stimulates insulin secretion. UCP2 is a key component in glucose sensing in pancreatic β cells because UCP2 mediates mitochondrial proton leak, decreasing ATP production and consequently decreases insulin secretion. The importance of UCP2 as a negative regulator of insulin secretion was demonstrated by the fact that UCP2-deficient mice display higher islet ATP levels and increased glucose-stimulated insulin (125,126). Moreover, the generation of ob/ob mice lacking UCP2 restores first-phase insulin secretion (acute insulin response to glucose) and ameliorates the hyperglycemia condition (125,126).

According to the in vitro study in rat pancreatic islets, ghrelin inhibits glucose-stimulated insulin secretion in a dose dependent manner (124). Interestingly, the ablation of ghrelin reduces expression of UCP2 mRNA in the pancreas, which contributes toward enhanced glucose-induced insulin secretion (113). Hence, ghrelin may play a part in glucose homeostasis by chronically regulating pancreatic UCP2 expression and subsequently glucose stimulated insulin secretion.

Whilst animal models demonstrate an important effect of ghrelin on insulin secretion, it is analogous to studies in humans as Tong et al. recently assessed the effect of continuous infusion of ghrelin on dynamic insulin secretion and glucose metabolism. By and large, they showed that exogenous ghrelin markedly reduced the first-phase insulin response to intravenous glucose in healthy humans (7). All together, these studies strongly suggest an inhibitory effect of ghrelin on insulin secretion.

Liver

Phosphorylation of AKT is associated with the suppression of hepatic gluconeogenesis, as both in vitro and in vivo studies in rats demonstrate that ghrelin reduces insulin-mediated AKT signalling in the liver (127,128), resulting in potential concomitant blood glucose increments (127,128). On the contrary, ghrelin infusion studies done in humans did not affect hepatic glucose production despite inducing peripheral resistance (115). It is important to take into account the differential effects of the nutritional status on ghrelin regulation of glucose metabolism, as neutralization of ghrelin with a specific Spiegelmer compound during the fasting-refeeding cycle decreases hepatic glucose and lipid metabolism in rats (129).

Skeletal muscle

There are reports showing that ghrelin administration in rats increased insulin sensitivity in skeletal muscle by enhancing AKT-dependent insulin signalling selectively in oxidative muscle (128). However, studies in human subjects showed otherwise. It is worth mentioning that high doses of ghrelin may cause undesired secretions of many pituitary hormones, such as GH, Prolactin and ACTH that may exert influence on systemic glucose homeostasis (130). To circumvent these interferences, Vestergaard and colleagues compared a population of human subjects with hypopituitarism to healthy individuals and showed that ghrelin infusion during a hyperinsulinemic-euglycemic clamp acutely reduces insulin sensitivity in skeletal muscle together with stimulation of lipolysis, indicating the direct effects of ghrelin on skeletal muscle is independent of GH (115).

In summary, most of the literature support the idea that ghrelin acts to impair insulin sensitivity, particularly in the muscle; nevertheless the underlying mechanism remains to be elucidated.

White adipose tissue

In terms of adipocytes, there has been quite a disagreement in the literature regarding the impact of ghrelin on insulin sensitivity of white adipose tissue. Some studies showed that ghrelin acts directly on isolated epididymal adipocytes to enhance insulin-stimulated glucose uptake (131) and adipocyte differentiation (9). On the other hand, others showed that ghrelin increases lipolysis in WAT (106,115). This inconsistency may potentially be explained by the use of different modes of ghrelin exposure and analysis of adipocytes from different parts of the body. Consistent with this, there are reports demonstrating fat-depot specific sensitivity to ghrelin, which utilizes different signal transduction pathways through GHS-R1a (104). For the understanding of the physiological relevance of ghrelin on glucose uptake in adipocytes, additional in vitro and in vivo studies are required.

In brief, the above studies have highlighted the main effects of ghrelin on peripheral tissues; (I) increases glucose production in the liver, (II) attenuates glucose-stimulated insulin release from the pancreas, (III) impairs insulin sensitivity in skeletal muscle and lastly (IV) differential effects of ghrelin on specific fat-depots. Under non-physiological conditions, all these undesired events may converge into the development of diabetes (113). Even so, it is imperative we consider this action of ghrelin in a physiological context as metabolic status has been implicated in the regulation of ghrelin’s influence in the brain and circulating ghrelin levels (87). Furthermore, ghrelin levels are elevated in calorie restricted mice, rats and humans and that may aid in the future development of anti-obesity and anti-diabetic therapies to combat the metabolic dysregulation(29,81,92,132-135).

Ghrelin and cancer

Concurrent with the discovery of ghrelin, GHSR1a and ghrelin expression was uncovered in the pituitary and neuroendocrine tumors (136), suggesting a role in pituitary pathogenesis (137). However, due to the important effects of ghrelin in the stimulation of food intake and adiposity; attention was shifted to its effect on regulating of body weight.

Recently, ghrelin has been “re-discovered’ in relation to its role in the dynamics of cellular proliferation. Accumulating literatures has implicated ghrelin in a number of processes in relation to cancer, including cell proliferation, apoptosis, cell invasion and migration, and angiogenesis, presumably through an autocrine/paracrine mechanism (46).

To date, it is widely known that ghrelin and its receptor are expressed in a range of peripheral tumour types. It is thought that ghrelin and its receptor may function as autocrine/paracrine growth factors, which underlies the development of a number of cancers. However, it is imperative to be mindful of the complex relationship between ghrelin and cancer. Some studies have shown that colorectal carcinoma cells secrete excessive ghrelin in vitro and it promotes cell proliferation in an autocrine/paracrine manner (138,139), similar to the results observed in human pancreatic and hepatoma cell lines (140,141). On the contrary, others studies have showed that ghrelin induces both, proliferative and anti-proliferative effects, which are often dependent of the cell type and dose of ghrelin administration (46).

Throughout the literatures, there are scant reports that examined the relationship between serum ghrelin levels and gastrointestinal cancer. However, to emphasize on the role of serum ghrelin in gastric cancers, a prospective epidemiological study has demonstrated that individuals with lower baseline concentration of ghrelin have a significant increase risks of both gastric and esophagogastric adenocarcinomas (142). This finding is recently validated by Sadjadi et al. (143); suggesting atrophic changes of ghrelin-producing cells.

In light of the limited information on the role of ghrelin in the aetiology of gastrointestinal cancers, further studies are required to define the interaction between ghrelin and carcinogenesis in different organs.

Conclusions

To recapitulate our knowledge based on the above studies, ghrelin is produced in the stomach and its unique structure requires acylation for its biological activity. Earlier studies have identified ghrelin as an endogenous agonist for the growth hormone secretagogue receptor involved in growth hormone secretion. As the research of ghrelin progresses, there is increasing evidence that hypothalamic circuits are implicated in ghrelin-mediated feeding. Presently ghrelin appears to have a greater importance for the regulation of blood glucose during starvation.

Since the initial discovery of ghrelin, the range of physiological and pathophysiological functions attributed to this hormone has grown rapidly. Ghrelin not only functions as a regulator for inflammation and the cardiovascular system, it also governs a number of processes in relation to cancer, including cell proliferation, apoptosis, cell invasion and migration, and angiogenesis. Whether or not ghrelin plays a role in the progression of cancer remains to be determined.

Acknowledgements

This work was supported by Monash University, and NHMRC grant. We thank Zachary Chow for his kind help in editing this manuscript.

Disclosure: The authors declare no conflict of interest.

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