Introduction

The coordinated regulation of food intake, energy expenditure and adiposity is dependent on a complex signalling network involving both peripheral signals and central nervous system. Growing evidence has shown that the gut plays a key role in this homeostatic process and may, therefore, be considered as the body’s largest endocrine organ [1]. The gastrointestinal tract is, in fact, able to release more than 20 different hormones, mostly in relation to the quality and quantity of nutrients in the tract. These have so far been characterized as able to produce a large and widespread set of effects, including, at local level, the regulation of gut motility, the modulation of glucose homeostasis and of peripheral insulin sensitivity as well as the stimulation of hunger or satiety feelings at central level [2]. Among these peptides, increasing interest is growing for ghrelin, the only gut hormone known to stimulate appetite [1, 2]. Since 1996, 9716 papers are currently (October 2018) recorded in Pubmed, as detected by performing a general search using the keyword “ghrelin”. This narrative review aims at presenting and discussing the most significant advancements in the understanding of the complex biology of this hormone and of its effects on the modulation of energy balance and metabolism.

Ghrelin: cell biology

Ghrelin is a gastric hormone, first identified in 1996 by Kojima et al. in rat stomach [3]. In the previous years, several small synthetic molecules had been discovered for their ability to induce growth hormone (GH) release by acting at hypothamical level independently from GH releasing hormone pathways [4] and were, therefore, named growth hormone secretagogues (GHS) [5, 6]. However, while a specific receptor for GHS (GHSR) had been identified in 1996 [7], its endogenous ligand was unknown until Kojima et al. finally identified a novel hormone that was able to simulate GH secretion through GHSR [3] and named it “ghrelin” after the Proto-Indo-European word root (“ghre”) meaning “grow” [8]. Almost contemporarily, Tomasetto and others identified the same hormone for its regulatory role in gastrointestinal motility and named it motilin-related peptide [9].

Ghrelin is mainly secreted by endocrine cells (P/D1 in humans and X/A-like in rats) located in the gastric fundus, and gastrectomy reduces ghrelin plasma concentrations by 65% [10], but its expression has also been described in duodenum, jejuna, ileum, colon and at lower concentrations in the pancreas, adipose tissue, kidneys, testes, placenta, hypophysis and nucleus arcuatus in the hypothalamus, an important region for appetite regulation [11,12,13,14,15,16,17,18,19,20].

Transcriptional regulation and polymorphisms

The gene coding for the ghrelin peptide, GHRL, is highly conserved in mammals [8] and spans 5 kb on chromosome 3p 25–36 in humans. The sequence includes four exons encoding a precursor 117 aa protein, preproghrelin [21], while two further exons with regulatory function were discovered later [22]. Preproghrelin undergoes splicing and editing ultimately resulting in the bioactive peptides obestatin and ghrelin (Fig. 1).

Fig. 1
figure 1

Adapted from Liu et al. and Sato et al. [23, 24]

Diagram showing ghrelin gene splicing products and ghrelin’s post translational modifications.

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Interestingly, obestatin, a 23 amino acid peptide discovered by Zhang et al. [25], is also involved in the complex-regulation of the gut–brain network, with initial reports showing its ability to counteract ghrelin’s effects [25], potentially suggesting GHRL as an important effector in the maintenance of energy homeostasis. However, while pleiotropic metabolic effects have been reported, other groups did not confirm the inhibitory effect on food intake, making of obestatin a controversial peptide whose effects are currently largely undefined [26,27,28,29,30].

Genomic variation of the ghrelin gene has been associated with obesity development in humans. Two polymorphisms have been reported in humans: Leu72Met and Arg51Gln [31,32,33]. Individuals presenting Leu72Met allele are reportedly protected against fat accumulation and associated metabolic comorbidities [34]. The Arg51Gln polymorphism changes the processing site of ghrelin within its precursor protein, preventing normal ghrelin editing. Importantly, its prevalence was shown to be 6.3% in obese subject, while it was not detectable among non obese individuals, showing a clear link with obesity development [33].

Post-translational modifications

It is important to note that the hormone described by Kojima et al. as an endogenous ligand for GHSR was an acylated peptide. The ghrelin peptide, in fact, undergoes post-translational modifications, the main being acylation on S3 by the membrane bound enzyme ghrelin-O-acyl-transferase (GOAT) [35] (Fig. 1). This enzyme, highly conserved across species, has a tissue expression profile similar to ghrelin, with highest expression in stomach, pancreas and intestine and ghrelin acylation is completely prevented in GOAT knock-out mice [36,37,38]. While fatty acids derived from acetic (C2) to tetradecanoic acid (C14:0) are all possible ligands, octanoic acid (C8:0) is the principal fatty acid involved in this reaction, with decanoic (C10:0) and likely decenoic (C10:1) acids also being reported as optimal ligands [37, 39,40,41]. No differential effects between these identified acylated forms on receptor binding and GH secretion activity in vitro has been found [40].

Ghrelin phosphorylation has also been reported, with induced protein structural changes which affect both acylation and membrane binding in vitro [42], but its potential importance in vivo is currently unclear.

Ghrelin secretion and circulating forms

Ghrelin release from the stomach has been reported to involve sympathetic nerves [43] and recent evidence shows that ghrelin secretion in gastric endocrine cells is mediated by a series of G-protein coupled receptors (GPCRs), allowing for its release to be integrated in a network of modulatory signals [44].

Both acylated (AG) and unacylated (UnAG) ghrelin forms are detectable in human and animal plasma. Interestingly, most circulating ghrelin is unacylated, whereas the acylated hormone is generally considered to only account for approximately 10% [35, 45,46,47] of total ghrelin, with possible variations depending also on the detection technique used. Mizutani et al. have shown that, while unacylated ghrelin is localized in both gastric open-type cells and closed-type round cells, the acylated form is present only in the latter [48, 49]. Both cell types are able to release hormone forms, with enhanced unacylated but not acylated ghrelin secretion at lower gastric pH [49], suggesting a potentially different physiological role for the two forms.

Circulating ghrelin is subject to de-acylation and cleavage, with a half-life of respectively 240 min in humans and 30 min in rats, depending on the mediation of different enzymatic systems across species [50].

Ghrelin acylation influences its transport across compartments, in particular across the blood–brain barrier (BBB). While octanoylated ghrelin crosses the mouse BBB mainly from brain to blood, passage for the unacylated peptide was observed only in the opposite direction [51]. Interestingly, later studies showed that whole body energy balance impacts on ghrelin transport at BBB level, with obese mice showing reduced permeability compared to lean animals. Moreover, triglyceride co-administration increased ghrelin transport [52], suggesting a role for nutrients in modulating ghrelin action at central level.

Ghrelin release modulation and feedback regulation

Regulation of ghrelin secretion is still partly unknown but it is well established that ghrelin mRNA and plasma concentrations are increased during fasting [53, 54], and in humans circulating ghrelin is characterized by a peak just before meals, suggesting a potential role in meal initiation [55, 56]. On the contrary, ghrelin expression and plasma concentrations are decreased by food intake [56, 57] and in relation to food composition, with maximum inhibitory effect observed after carbohydrate ingestion, compared to proteins and lipids [58, 59].

In the long term, plasma ghrelin levels are known to be related to body weight and composition, with lower levels in obese patients and higher concentration in anorexia and in negative energy balance conditions including cachexia [8, 60, 61]. Importantly, among selected obese individuals, lower ghrelin levels were specifically related to a decrease in UnAG with no change in AG levels compared to non-obese [62]. Interestingly, the same study also showed that AG levels in obese individuals which did not meet the diagnostic criteria for metabolic syndrome diagnosis were comparable to both lean and obese subjects with the metabolic syndrome [62] and in obese patients undergoing bariatric surgery, the obesity-associated altered AG/UnAG ratio was found to be maintained even at 12 months after surgery despite weight loss [63]. This evidence suggests that the modulation of ghrelin acylation and its kinetics may be a potentially interesting target for further research not just in the treatment of obesity and its related complications, but also in the mechanisms underlying the pathogenesis of obesity per se.

At molecular level, ingested fatty acids are directly used for AG acylation and GOAT activation is reported to be modulated by ingestion and availability of medium chain fatty acids and triglycerides [8, 64,65,66]. GOAT is also potently inhibited by octanoylated ghrelin end-products, suggesting the existence of a negative feedback regulation in AG synthesis [37]. Moreover, recent evidence shows that GOAT expression levels decrease during prolonged fasting, leading to an increase in UnAG rather than AG in that condition. Interestingly, GOAT-null mice, while not expressing AG, present a marked increase in UnAG levels in association with lower body weight and fat mass, and opposite effects are observed in transgenic mice overexpressing GOAT [37, 64]. This body of evidence strongly supports the hypothesis that the GOAT-ghrelin system acts as a nutrient sensor providing information on the presence of nutrients, potentially leading to the optimization of nutrient partitioning and growth signals [64, 65, 67].

Ghrelin receptor

Acylated ghrelin’s receptor is a G-protein coupled receptor produced in two isoforms by alternative splicing of an mRNA transcript of a single gene, located on chromosome 3 q26-27 [8]. AG binding sequence has been identified in the first four residues at the N-terminus of ghrelin, which include the octanoylation site in Ser3 [68], and its interaction with ghrelin receptor (GHSR) leads to a Gq-mediated activation of phospholipase C and subsequent production of inositol 3 phosphate and diacylglycerol. In turn, this leads to Ca2+ release from the sarcoplasmic reticulum and ultimately to GH secretion.

GHSR1a is highly expressed in the pituitary and hypothalamus but also at lower levels in other brain areas including hippocampus, ventral tegmental area, nucleus tractus solitarius and substantia nigra. Interestingly, also numerous peripheral tissues express GHSR including intestine, pancreas, heart, lung, kidney and adipose tissue [69,70,71,72,73]. Evidence by several authors is consistent in failing to detect GHSR1a expression in both skeletal muscle and liver [69, 74,75,76,77,78]. Interestingly the expression of both ghrelin and GHS-R1b has been reported also in tissues not expressing the active receptor form, including liver [69, 77], suggesting that ghrelin may anyway produce tissue-specific effects by activating different pathways [69, 79].

Supported by observations showing differential biological activities between ghrelin forms, as well as by evidence from activation experiments of GHS-R1 variants in different cell types, some authors have proposed the existence of a novel class of receptors specifically binding UnAG [80, 81]. However, no receptor for UnAG has been currently identified [67, 80].

Ghrelin effects on energy metabolism, body mass and composition

Since its discovery, ghrelin has been progressively characterized as a hormone involved in energy balance homeostasis as well as in GH secretion, as its functions span from central regulation of feeding to the modulation of whole body and tissue-specific metabolism [67]. With regard to ghrelin acylation, AG has long been considered the active form of the hormone for its interaction with GHSR and for its impact on GH secretion and on appetite stimulation, while UnAG was regarded as a precursor/degraded form without specific biological activities. As a consequence, until recent years most studies were focused on AG, or did not differentiate the two forms [82].

Food intake and energy balance

Ghrelin has a modulatory role in the regulation of energy homeostasis, including appetite stimulation [83, 84]. Both peripheral and central treatment with AG increase food intake and body weight in experimental models [85,86,87]. In agreement with the described low permeability of BBB to AG in the blood-to-brain direction [51], one study reports that ghrelin signalling from the stomach to the central nervous system (CNS) is principally mediated by afferent vagal nerve, and ghrelin-induced stimulation of appetite and GH secretion are prevented by blocking vagal fibres [20]. However, this point remains controversial since other studies show that vagal afferents are not necessary for AG effects on appetite stimulation and ghrelin analogues are effective also after gastrectomy and related vagotomy [88,89,90]. Importantly, effects of AG in appetite stimulation are preserved in GH-deficient rats, showing its independence from GH release [85].

At CNS level AG-induced effect on appetite stimulation is mediated by hypothalamic neuropeptide Y (NPY) secretion but also by interaction with other known appetite regulators at this level, including AgRP, orexin, endocannabinoids and leptin [8, 85, 91, 92]. NPY-producing cells largely express ghrelin receptors, and ghrelin i.v. administration in mice largely stimulates hypothalamic activity in the same neurons [93, 94].

Nucelus caudatus and mesolimbic centres are also involved in long term energy homeostasis regulation by ghrelin [55] and effects on appetite possibly involve hedonic appetite regulation pathways [95]. In agreement, using functional magnetic resonance imaging, Davis et al. have shown that ghrelin administration increases activity in food-reward brain regions in humans [96].

UnAG effects on food intake poorly understood and likely marginal. While some authors report that in rodent models peripheral UnAG treatment decreases food intake in association with slower gastric transit [97], others do not confirm this effect but describe an inhibitory effect of UnAG on AG-induced increase in food intake when both forms are administered simultaneously [98]. This effect appears to be independent of GHSR1a modulation and at least in part mediated by UnAG-induced release of nesfatin-1, an inhibitor of NPY. Central administration of UnAG, on the contrary, is reportedly also orexigenic [99], indicating that further investigation on UnAG effects and receptor interaction is needed.

Whole body glucose homeostasis

Ghrelin also causes several direct effects on systemic and tissue metabolism, independently of food intake. At whole body level, ghrelin has an important impact on glucose homeostasis, as an important player in the pathophysiology of obesity-related metabolic complications [100]. Not long after ghrelin’s discovery, Broglio et al. reported that AG increases blood glucose levels and reduces insulin secretion [101]. Later studies showed that AG reduces glucose-stimulated insulin secretion, rather than fasting insulin levels [102]. Consistently, GHSR null mice have lower fasting glycaemia compared to control [103]. Underlying mechanisms were later investigated, showing that AG inhibits insulin release at pancreatic level by acting on voltage-dependent K channels (Kv) in β-cells. In fact, AG interaction with GHSR activates Kv channels through the receptor coupled G-protein αi, thus preventing Ca2+ signalling and limiting insulin exocytosis [104].

Ghrelin also modulates insulin sensitivity. In humans, total circulating ghrelin levels are positively associated with insulin sensitivity both in the general population [105] and in insulin-resistant diseases, including chronic renal disease [106] and obesity [107]. In addition, epidemiological data clearly shows that total plasma ghrelin levels are also inversely associated to the risk of developing type 2 diabetes and to several cardiovascular risk factors [105, 108].

With specific regard to the unacylated form, in 2004 Broglio et al. reported that UnAG coadministration with AG in humans counteracted the decrease in insulin levels induced by AG alone [109]. Later evidence showed that, at variance with AG, UnAG potently rises insulin release in glucose-stimulated conditions in rats [110], suggesting a potential independent role for UnAG in regulating glucose and lipid metabolism. Studies performed in a cohort of 45 metabolic syndrome patients clearly showed different associations of ghrelin forms with insulin resistance. AG ghrelin levels were, in fact, positively correlated with insulin resistance (HOMA index), while UnAG levels were markedly inversely correlated with the same parameter [62].

Importantly, in a population cohort from the Mo.Ma epidemiological study [111], UnAG was independently positively associated with insulin sensitivity, and lower UnAG plasma levels predicted 5-year insulin resistance [112]. Although it has been reported that UnAG may show a positive modulatory effect on insulin release in vitro [113, 114], in vivo studies collectively strongly suggest that UnAG metabolic effects are mainly related to the modulation of insulin action at tissue level. In excellent agreement, other studies show that acute administration of UnAG does not impact basal or stimulated insulin secretion in β-cells in humans [115].

However, further investigation on the molecular mechanisms involved in ghrelin forms’ metabolic actions is required, as several studies show that ghrelin signalling transduction is interlinked with other pathways, and may be modulated by different expression of receptor forms and by interactions among tissues [116,117,118,119].

Ghrelin and liver metabolism

Ghrelin reportedly modulates hepatic gluconeogenesis, and therefore, glucose release from the liver. Moreover, AG and UnAG have differential effects on glucose release in cultured hepatocytes, with AG stimulating gluconeogenesis and UnAG suppressing it [120]. It should be pointed out that the underlying mechanisms need further investigation as the same effects were not replied with the GHSR1a agonist hexarelin, in agreement with reports of no expression of GHSR in hepatocytes [120] and with the fact that hexarelin administration in humans does not increase plasma glucose levels [120].

However, consistently with in vitro experiments, the expression of PGC1α, a gluconeogenesis inducer, is increased in the liver of AG-treated rats [121], and mice studies with radiolabeled glucose showed that AG partially antagonizes insulin-induced suppression of gluconeogenesis [116]. Moreover, AG reduces insulin signalling in rodents, and this effect is not associated with changes in mitochondrial function [117, 121]. The same authors showed that sustained AG treatment also causes modulation of liver lipid metabolism by inducing a pro-lipogenic gene expression pattern, increasing tissue triglyceride content and reducing the activation of the stimulator of fatty acid oxidation AMP-activated protein kinase (AMPK) [117].

Both antioxidant and anti-inflammatory effects of AG have also been reported in the liver. In in vivo experiments of liver injury in rodent models, as well as in in vitro experiments on primary human stellate cells exposed to chemical damage, AG blunted liver pro-oxidant and pro-inflammatory changes and this result was associated with reduced fibrosis [76, 122].

AG was also reported to improve liver redox state in association with improved inflammation markers in high fat diet-fed rats [123, 124]. However, studies in high fat fed rats show that the beneficial impact of AG on liver redox state and inflammation is not paralleled, expect in one study [123], by improved hepatic insulin signalling, but rather by decreased activating phosphorylation at AKT and GSK-3β levels [121, 124, 125]. This finding, which is also in agreement with in vitro studies in hepatoma cells [125], is consistent with reports showing that in rodent high-fat feeding models, liver AKT activation may directly contribute to hepatic lipogenesis, oxidative stress and inflammation [126].

Fewer reports on UnAG effects on liver metabolism are available. Recent evidence has shown that 4-day UnAG administration does not modify liver redox state, mitochondrial function, inflammation and insulin signalling in young healthy rats, and that these findings are tissue-specific [127]. These results have also been confirmed in transgenic mice with UnAG expression upregulation [128]. However, recent evidence both in vivo and in vitro suggests that in tissue metabolic stress conditions, such as during ischemia/reperfusion, UnAG may improve liver mitochondrial function and protect against apoptosis [129].

Ghrelin and adipose tissue

Appetite stimulating effects of ghrelin were very soon associated with increased body weight, and particularly with fat mass [87]. Further studies in animal models have shown that ghrelin-induced effects are mainly observed in retroperitoneal fat mass and only to a lesser extent in subcutaneous adipose tissue [130]. In a model of daily peripheral ghrelin administration these effects were found to be independent of appetite-induced increased food intake but instead related to reduced fat utilization [87], and in vitro experiments confirmed that ghrelin inhibits lipolysis in adipocytes [131]. Also, AG administration did not impact on food intake in high fat diet feeding but increased adipose tissue mass and favoured the expression of lipogenesis markers [132]. Consistently, ghrelin- or GHRS-null mice were protected from high fat diet induced obesity [133, 134].

Ghrelin promotes adipocyte differentiation [135] and ghrelin’s proadipogenic effect are at least in part mediated by peroxisome proliferator-activated receptor γ (PPARγ2), a transcription factor which favours triglyceride synthesis and downregulates lipolysis [136].

Interestingly, in white adipose tissue, ghrelin enhances the expression of the uncoupling protein 2 (UCP-2), a protein involved in the regulation of mitochondrial reactive oxygen species (ROS) generation, and in UCP-2 null mice, ghrelin enhances its lipogenic effects, suggesting a possible feedback regulation mechanisms involving mitochondrial function [137].

UnAG impact on adipose tissue has been less investigated. While in vitro reports suggest that it may induce at least in part superimposable effects to those produced by AG on adipogenesis upregulation and lipolysis inhibition [131, 138], in vivo studies in rodents show that UnAG peripheral administration may reduce fat mass [139]. Although further studies are needed on the potential role of UnAG on adipose tissue regulation, reported evidence suggests that UnAG is an active hormone with modulatory functions in the complex context of lipid homeostasis.

Ghrelin and skeletal muscle

Skeletal muscle metabolism is characterized by a cluster of interlinked metabolic functional pathways, including mitochondrial function, redox state regulation, inflammation and insulin signalling and action [84, 140,141,142,143,144]. Increased muscle ROS production and inflammation are linked at the level of IκB/NF-κB activation, and may cause insulin resistance by inhibition of insulin signalling downstream of insulin receptor [145,146,147,148]. Interestingly, ghrelin has been reported to be an important modulator of these factors at several levels.

Mitochondrial function

Mitochondrial respiration may be modulated by several mechanisms including UCPs, which selectively reduce mitochondrial ROS generation by inducing mild uncoupling [149,150,151]. In skeletal muscle, both UCP2 mRNA and protein levels are increased after 4-day AG treatment at non orexigenic doses in healthy rats, and this finding is importantly associated with enhanced mitochondrial enzyme activities [117]. Moreover, the same AG treatment improved altered mitochondrial oxidative capacity and transcription of mitochondrial regulatory genes in both uremic rats and in mice with chronic heart failure [152, 153], and was associated with preserved muscle triglyceride accumulation in high fat diet-fed rodents [121].

On the contrary, recent evidence has shown that UnAG treatment is associated with reduced ATP synthesis in healthy rats and that this finding is also present in obese mice with UnAG overexpression [127].

Redox state

In addition, a role for ghrelin in blunting oxidative stress is supported by several studies at whole body level and in several tissues. In obese patients ghrelin levels negatively correlate with systemic oxidative stress marker 8-epi-prostaglandin F2α (8-epi-PGF2α) [154] and in normobaric hypoxia, ghrelin administration attenuates hypoxia-induced increase in plasma levels of malondialdehyde (MDA), another marker of oxidative stress [155]. Moreover, evidence supporting ghrelin as a negative modulator for oxidative stress has been reported also tissues. In experimental models of ischemic or alendronate-induced gastric injury, intravenous ghrelin treatment lowered tissue damage in association with lower ROS production [156, 157], and reperfusion with ghrelin in a rat model of cardiac cachexia decreased myocardial lipid peroxidation [158].

Underlying mechanisms may involve a negative modulation in ROS generation. While AG effect on ROS production by inducing mild uncoupling in mitochondria has already been described, some observations suggest that AG may also act by increasing antioxidant mechanisms [159,160,161].

Few data are available on ghrelin effects on muscle oxidative stress. AG potential role in skeletal muscle redox state modulation has been investigated in a rodent model of diet-induced obesity. In 1-month high fat diet-fed rats, sustained 4-days AG treatment did not modify obesity-induced increase in muscle glutathione peroxidase (GPx) or glutathione oxidation status [162].

On the contrary, some interesting in vitro evidence shows that UnAG, differently from AG, reduces mitochondrial ROS generation in neonatal ventricular myocytes [163]. Moreover, Togliatto et al. have shown that increased skeletal muscle ROS imbalance in a mouse model of limb ischemia was counteracted by UnAG but not AG treatment via an increase in SOD-2 expression [164]. The same authors also observed similar protection of skeletal muscle from ROS in a mouse model of glucose intolerance with peripheral artery disease [165]. A potential role for UnAg in ROS modulation in skeletal muscle was further confirmed and defined by recent evidence, which showed both in vivo and in vitro that UnAG lowers mitochondrial ROS generation, thus improving overall redox state and that this finding is unrelated to changes in antioxidant systems [127, 166]. Improved redox state was also observed in the gastrocnemius muscle of diet-induced obese mice overexpressing UnAG, with levels comparable to wild-type control [127].

Inflammation

AG has been reported to lower inflammation in different experimental settings [167]. GHSR is expressed in both human T lymphocytes and monocytes, and in these cells ghrelin inhibits the expression of the pro-inflammatory cytokines IL-1β, IL-6 e TNF-α [168]. Accordingly, ghrelin levels are increased in septic dogs [169] and ghrelin is among the first increasing hormones responding to endotoxic shock in humans [170], further supporting its potential anti-inflammatory role. Moreover, AG-induced reduction of pro-inflammatory cytokines is paralleled by increased levels of the anti-inflammatory cytokine IL-10 in several cell types [171, 172]. This consistent evidence on the systemic and tissue anti-inflammatory effects of ghrelin has suggested its potential use as a therapeutic agent in clinical settings characterized by high inflammation. Clinical trials have so far shown that ghrelin treatment suppresses airway neutrophil-dominant inflammation in patients with chronic respiratory infection [173] and that postoperative ghrelin administration in patients with oesophageal cancer inhibited inflammatory mediators and ameliorated their clinical course [174].

The role of ghrelin in skeletal muscle inflammation is largely to be investigated. However, sustained administration of AG markedly lowered tissue NF-κB nuclear translocation and tissue TNFα expression in a rodent model of diet-induced obesity, independently from changes in redox state [162]. Interestingly, AG effects in lowering inflammation are also associated with improved redox state in different models [156, 157], strongly suggesting an interplay between ROS production or scavenging and inflammation modulation. Since in skeletal muscle high TNFα levels may reduce mitochondrial function [175], AG might improve mitochondrial function with a mechanism at least in part involving the reduction of TNF-α levels.

UnAG effects on muscle inflammation have only been recently investigated, with reports showing that hormone-induced improvements in muscle redox state are paralleled by the development of an anti-inflammatory cytokine pattern at tissue level both in vivo and in vitro [127, 166].

Tissue insulin signalling and action

Available data globally suggests that AG enhances mitochondrial function, with reports showing also associated improvements in redox state and inflammation. In several models and experimental settings, it has been shown that these effects, alone or combined, are associated with increased tissue insulin sensitivity and action [146,147,148, 153], indicating that AG may potentially improve insulin sensitivity at least partly through these pathways.

In several tissues AG has in fact been reported to activate protein kinase B (AKT), a main mediator of insulin signalling pathway, in association with beneficial effects in different experimental settings [176,177,178,179,180]. Acute AG infusion in humans [181, 182] or experimental models [116] has provided conflicting results in term of systemic or muscle insulin sensitivity changes, with reports of enhanced [116, 121, 168], unchanged [182] or reduced [181] insulin action.

Fewer reports are available on UnAG effects on insulin signalling. Interestingly, one study by Lear et al. showed that in HL-1 cardiac cells and in primary cultures of neonatal rat cardiomyocytes, while both ghrelin forms do not activate AKT, UnAG but not AG increases insulin-induced GLUT4 activation [183]. Recent evidence shows that in young adult rats as well as in vitro, UnAG increases activating phosphorylation at AKT level and downstream, with activation of GSK-3β, and of the protein anabolic mediators PRAS40 and P70S6K [127, 144, 166]. Importantly, UnAG treatment was able to counteract muscular mass wasting in a rodent model of chronic kidney disease [166].

Autophagy

Autophagy is an intracellular selective auto-degradation process that contributes to amino acid recycling for essential proteins synthesis, but may also eliminate dysfunctional mitochondria, thus removing inefficient energy consumption and also excess ROS generation [184, 185].

In 2012 Słupecka et al. showed that enteral AG administration was able to favour small intestine mucosa renewal in new-born piglets in association with enhanced autophagy [186], demonstrating for the first time a link between ghrelin and autophagy. These findings were followed by other studies in different tissues, experimental models and conditions, and mostly confirmed ghrelin as an autophagy inducer.

Interestingly, experiments in an in vitro model of cardiac hypoxic injury showed that AG stimulated autophagy with parallel reduction of ROS generation [187]. In skeletal muscle cells, both AG and UG reportedly enhanced autophagy markers, while blunting apoptosis. Moreover, in a mouse model of gene-induced insulin resistance, UnAG was also able to improve muscle insulin signalling and GLUT4 activation in association with increased autophagy [188]. Recent evidence both in vivo and in vitro further shows that UnAG-induced beneficial effects on skeletal muscle metabolism, with lower mitochondrial ROS production, lower inflammation and enhanced insulin signalling and action in rat muscle are at least in part be mediated through upregulation of autophagy [127, 166] (Fig. 2). In vitro experiments with prolonged hormone incubation of C2C12 myotubes confirmed UnAG-relate findings, while only highest AG doses selectively induced a moderate increase of GSK-3βS9 phosphorylation but failed to reduce ROS production and to enhance downstream insulin signalling [127], suggesting that AG may be a weaker autophagy inducer than UnAG.

Fig. 2
figure 2

Proposed interactions between UnAG and clustered metabolic alterations in skeletal muscle: chronic UnAG over-exposure lowers mitochondrial production of reactive oxygen species (ROS), inflammation and insulin signalling activation levels. Our findings further indicate UnAG to directly lower mitochondrial ROS generation through autophagy stimulation

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Ghrelin in obesity-induced insulin resistance

Despite the fact that ghrelin was at first characterized for its effects on GH secretion, polymorphism studies in ghrl gene have also established a link between ghrelin, obesity and obesity-correlated comorbidities development [33, 34]. Moreover, effects of ghrelin on appetite stimulation and therefore on body weight and fat mass increase were soon identified [67]. Also, patients with Prader–Willy Syndrome (PWS), a complex genetic disease characterized by hypomentia, hormonal impairments and early obesity development, present high levels of circulating AG but not UnAG both in fasting and in postprandial conditions [189,190,191,192]. These findings soon led to the hypothesis that ghrelin agonists could be potentially used as anti-obesity drugs. Some studies further supported this hypothesis showing that engineered mice lacking GHSR expression, as well as mice with induced expression of an inactive form of GHSR, did not develop obesity under high fat diet treatment [133, 134, 193]. However, enthusiasm for treating obesity by counteracting ghrelin-mediated effects on appetite was replaced by scepticisms, as data from other studies showed that decreased ghrelin action does not always result in hypophagia and loss of body mass [194, 195], and that the ablation of ghrelin cells in adult mice does not decrease response to HFD [196].

However, observations suggesting that ghrelin could regulate glucose homeostasis and energy metabolism also independently of food intake, led to further investigations of its potential role in obesity and obesity-related co-morbidities, given their complex metabolic pathophysiology [197,198,199]. The first evidence linking ghrelin with human obesity and insulin resistance was reported soon after the discovery of the hormone. In 2001, Tschöp et al. clearly showed that plasma ghrelin levels were decreased in obese humans [61] and a few years later McLaughlin et al. showed that among obese individuals, ghrelin levels were lower in insulin-resistant subjects [107]. This finding was further investigated in studies in selected patients with the metabolic syndrome which demonstrated that obesity-associated reduction in total plasma ghrelin levels was related to a decrease of the unacylated form, while absolute AG levels were not modified compared to non-obese [62]. The same study also showed that in metabolic syndrome patients, among markers [200], while AG levels were positively associated with insulin resistance, waist circumference and BMI, UnAG levels strongly negatively correlated with HOMA [62]. More recently, data analysis from a large cohort has confirmed these findings both in the general population and more specifically in overweight subjects. In addition, UnAG levels were also found to be independently negatively associated with the development of insulin resistance after 5 years [201].

At tissue level, ghrelin has shown to produce a pattern of tissue-specific effects in obese models. In rats fed with high fat diet (HFD) (60% of calories from fat) diet for 4 weeks, 4 day sustained AG administration improved liver oxidative stress and inflammation [124] and similar results were obtained also by other authors after 8 weeks HFD and AG treatment throughout the whole period [123]. These findings were not paralleled, except in one study [123], by improved hepatic insulin signalling, but rather associated with decreased activating phosphorylation at AKT and GSK-3β levels [121, 124, 125]. Decreased hepatic insulin sensitivity in obese rats is also reportedly associated with a decrease in liver expression of insulin receptor substrate 1 (IRS-1), an important mediator and modulator of insulin signalling transmission from insulin receptor to AKT [202]. Interestingly, this finding was reversed by UnAG administration [203]. Collectively, these observations are consistent with reports showing that liver AKT activation may directly contribute to hepatic lipogenesis, oxidative stress and inflammation in rodent models of obesity [126]. Mechanisms involved in AG modulation of liver metabolism in diet-induced obesity are unknown, but a recent report has shown that in fat-induced obese rodents, and in in vitro hepatocytes incubated with saturated fatty acids, AG treatment reduced lipotoxicity via autophagy induction [204].

With regard to adipose tissue, Perez-Tilve et al. have shown that chronic central AG administration in HFD-fed rats, while not increasing food intake, was however associated with enhanced lipogenesis and increased body fat mass, indicating that AG modulation of adiposity is independent of orexigenic effects [132].

In skeletal muscle, sustained 4-day AG treatment was associated with the prevention of obese-related tissue triglyceride content after 4 weeks of HFD [121, 205]. Moreover, although in a similar study by the same group no effect on obesity-induced increase in muscle oxidized glutathione was observed, sustained administration of AG markedly lowered tissue NF-κB nuclear translocation and tissue TNFα expression [162]. This finding was not however associated with significant increases in muscle insulin signalling activation at AKT and GSK-3β levels compared to non-treated obese rats [162].

On the contrary, recent evidence shows for UnAG more clear results. The beneficial effects of UnAG on muscle mitochondrial function, redox state, inflammation and insulin signalling observed in lean rats were in fact confirmed in transgenic mice, in which UnAG overexpression was able to normalize obesity-induced insulin resistance [127].

Conclusions

Collectively, the literature shows that ghrelin forms may play a major role in energy homeostasis and in the regulation of energy metabolism with complex interactions at several levels. While AG is a main appetite modulator at CNS level that also induces a complex set of tissue-specific metabolic effects at tissue level, UnAG is emerging as a novel independent hormone, which is directly able to reduce skeletal muscle ROS generation also by increasing autophagy, with associated improved tissue inflammation and insulin activity. Moreover, reports of beneficial effects induced by ghrelin forms in different models of pathological conditions, including obesity, suggest that further research is strongly needed to investigate the potential use of ghrelin forms in clinical practice. Further study of the molecular mechanisms involved might also lead to the discovery of important new therapeutic targets.