The use of ghrelin and ghrelin receptor agonists as a treatment for animal models of disease: Efficacy and mechanism

Abstract

Ghrelin is a stomach-derived hormone that acts at the ghrelin receptor (formerly called the Growth Hormone Secretagogue (GHS)-1a receptor) in multiple tissues throughout the body, exhibiting pleotropic effects potentially beneficial as a treatment in human disease states. Given its properties including increasing appetite, decreasing systemic inflammation, decreasing vascular resistance, increasing cardiac output, and increasing growth hormone and IGF-1 levels, ghrelin has been tested as a treatment in animal models of multiple disease states that produce the deficits in these processes. Thus, the efficacy of ghrelin has been testing in diseases involving anorexia, negative energy balance, cardiovascular compromise, systemic inflammation and gastroparesis. These diseases include cancer cachexia, chronic heart failure, chronic renal failure, chemotherapy, arthritis, gastroparesis and inflammatory bowel disease. Across this wide variety of diseases treatment with ghrelin and ghrelin agonists have produced benefits, though given ghrelin’s widespread effects, the exact mechanisms behind ghrelin’s action in these settings is frequently difficult to determine. Further investigation using animal models may help to determine mechanisms that are most operative in these disease states and narrow treatment parameters helpful for human application.

Introduction

The regulation of energy consumption, storage and expenditure is tightly regulated in normal physiology but can be perturbed in maladaptive ways in the setting of multiple disease states [1]. Many diseases including acute inflammatory processes as well as chronic conditions require the mobilization of energy stores to maintain a higher rate of metabolism that is beneficial for processes targeted at resolving the pathological process (such as mediating an inflammatory response to infection) or maintaining physiologic processes in the face of restrictions (such as increase heart rate in the setting of myocardial failure)[2,3]. In addition to these increases in energy utilization, however, many diseases also involve a component of a pathological decrease in appetite. This decrease in appetite may prove beneficial in short-term disease processes, for example to decrease efforts of foraging for food while energy reserves are needed for fighting an acute infection. However, in the setting of chronic disease, this decrease in appetite only exacerbates a negative energy balance and consumption of lean and fat mass [4,5]. Despite the current culture in our society of excess energy consumption and resultant obesity, it has proven difficult to pharmacologically increase appetite in the setting of inflammatory and other chronic diseases [6].

One potential treatment for diseases involving anorexia and loss of body mass is the orexigenic hormone ghrelin. Ghrelin is an endogenous hormone released primarily from the stomach that acts on the ghrelin receptor (previously known as the growth hormone secretagogue 1-a (GHS-1a) receptor) on multiple cell types including in the hypothalamus, heart, blood vessels and leukocytes [7]. In doing so, ghrelin produces a variety of effects of potential benefit in the treatment of many chronic disease processes. This has stirred hope that ghrelin may prove to be an effective treatment for energy imbalance in these underlying diseases [8].

Not surprisingly, animal models of disease have been utilized in guiding progress toward investigating the efficacy of ghrelin as a treatment and potential mechanisms by which ghrelin mediates changes. In this review we will consider basic science data and limited clinical data regarding cachexia—the most common of these disease processes—and the effects of ghrelin treatment in these settings. For each of the underlying diseases we will consider the animal models used for investigation, unique aspects of ghrelin treatment for the underlying disease state and how current knowledge can help in guiding future clinical application.

Ghrelin

Ghrelin was originally discovered because of longstanding observations that opiates could stimulate release of growth hormone at what was eventually discovered to be the GHS-1a receptor [9]. Individual oligo peptides were assembled to separate these growth hormone stimulatory actions from the analgesic actions of opiates, resulting in the manufacture of small molecule agonists at the GHS-1a receptor that preceded the discovery of ghrelin itself. These small molecule agonists include hexarelin and multiple growth hormone releasing peptides that have been used in animal models of disease.

Ghrelin itself was discovered using reverse-cloning technology and was first reported in 1999 as a 28 amino acid peptide hormone with a unique O-n-octanoylation at serine 3 [7]. This fatty acid addition was later found to placed by ghrelin O-n-acyltransferase (GOAT) and is required for activity at the ghrelin receptor [10]. Acyl ghrelin has a half life of approximately 30 minutes in the serum [11]. Degradation of acyl ghrelin including removal of the fatty acid moiety yields desacyl ghrelin which is the predominant form of ghrelin in the serum [12]. While there are no known receptors for desacyl ghrelin, there is a growing body of literature suggesting that desacyl ghrelin has activity also, as discussed in further sections.

Regulation

Ghrelin is released predominantly by the endocrine cells of the stomach in response to the amount of time since the last meal, though the availability of specific fatty acids in the diet also appears to play a role [10,13]. Ghrelin release is decreased in the presence of leptin [14] and is thus lower in the setting of obesity [15]. In the setting of acute inflammation (such as via injection of IL-1β or lipopolysaccharide) ghrelin secretion is decreased [14,16], consistent with observations regarding decreased food seeking behavior in these settings. The integration of these regulatory inputs—and in particular the daily curve of ghrelin peaking before meals—has contributed to a view of ghrelin as a meal-initiating signal [13].

Appetite effects

Although ghrelin was discovered because of its ability to directly stimulate growth hormone secretion at ghrelin/GHS-1a receptors, it became apparent that a more striking property of ghrelin was to stimulate appetite [17]. Ghrelin receptors are expressed in the hypothalamus [18], including in the central melanocortin system, a key appetite regulating system that has been implicated in the anorexia associated with chronic disease [19]. Given the potential importance of the melanocortin system in mediating some of ghrelin’s actions, we will consider it briefly here.

The melanocortin system consists of two major classes of neurons that are found in the arcuate nucleus of the hypothalamus [20]. One of these groups of neurons expresses pro-opiomelanocortin (POMC) and is anorexigenic in nature. The POMC peptide is cleaved into α-melanocyte stimulating hormone (α-MSH), which is released into synapses of downstream neurons and acts on melanocortin-4 receptor (MC4-R) to decrease feeding behavior. These POMC neurons thus provide a tonic restraint on food intake that can be modulated to be increased (e.g. in the setting of cachexia [19]) or decreased (e.g. in response to specific hunger signals).

The second class of neurons in the central melanocortin system expresses two orexigenic neuropeptides. Agouti related peptide (AgRP) is a natural antagonist to MC4-R). Thus, its release serves to decrease tonic anorexigenic signal POMC neurons and thus increase appetite. The other orexigenic neuropeptide is neuropeptide Y (NPY), which acts on second order neurons to stimulate appetite. Both POMC and AgRP neurons respond to systemic cues such as inflammatory cytokines to increase (in the case of a-MSH) or decrease (for AgRP) their output, playing a central role in anorectic symptoms of cachexia [21,22]. Administration of antagonists of the MC4-R (analogues of AgRP) has been another studied intervention as a treatment of cachexia [19,23,24].

When injected into the third ventricle of the hypothalamus of healthy animals, ghrelin stimulates c-fos activation of AgRP an increase in expression of AgRP and NPY transcript and protein, co-incident with increased food intake [17,25,26].

Other effects

In the time since the identification of ghrelin—and in the case of growth hormone releasing peptides, before its identification—it has become apparent that ghrelin’s actions are not limited to appetite stimulation and growth hormone secretion. As mentioned, ghrelin receptors are expressed in multiple tissues including leukocytes, vascular smooth muscle, and renal tissue [18]. In considering ghrelin’s effects in the disease models covered below, we will consider potential mechanisms to account for improvements in disease endpoints. In many cases these considerations are complicated by multiple concurrent effects, making it difficult to ascertain the exact mechanism of improvement in disease state. A recurring theme is that while animal models are an excellent means of deciphering between these mechanisms, in most cases the exact mechanism has not been determined, leaving room to speculate regarding mechanisms and leaving ample room for further exploration.

Cachexia: Underlying Physiology

In considering disease states associated with anorexia for which ghrelin treatment has been tested, many of the most concerning cases culminate in cachexia, a syndrome that is associated with multiple chronic diseases including cancer, chronic kidney disease, chronic heart disease and AIDS [1]. Cachexia consists of concurrent symptoms of increased resting energy expenditure, loss of appetite, and ultimately a wasting of lean and fat body mass [27]. The onset of cachexia associated with underlying diseases constitutes an ominous sign in that mortality is greatly increased in the presence of cachexia. The term cachexia itself is derived from the Greek “kakos” (bad) and “hexis” (condition) and indeed the process was recognized in antiquity by Hippocrates, who observed, “…the shoulders, clavicles, chest and thighs melt away. This illness is fatal…” [28]. Despite this long-standing nature of our recognition of this disorder, successful treatment has remained elusive, underscoring the need for further research regarding the underlying pathophysiology and treatments.

The similarity of cachexia symptoms between underlying diseases suggests that while diverse in their primary cause these conditions share common features driving the processes of cachexia. As we shall see, one common characteristic of these diseases that likely contributes to cachexia itself is systemic inflammation. Inflammatory cytokines are known to act at multiple levels, including direct effects on skeletal and smooth muscle [29,30] and the enteric nervous system [31] as well as centrally in the brainstem [32,33] and in hypothalamic centers regulating appetite and energy expenditure such as the central melanocortin system [21,22]. The central melanocortin system (a target of ghrelin action) has been shown to be of particularly high importance in this disease process in that antagonism of the system, either among animals with genetic deficiencies in the melanocortin 4 receptor (MC4R) or using agents to inhibit action at this receptor, ameliorate the appetite and metabolic manifestations of this system [20].

Further importance of appetite in the setting of cachexia have been shown by the strong tie between cancer-related anorexia and quality of life among patients [34]. This decrease in appetite—potentially as a sign of cachexia-associated disease state—can be an ominous prognostic factor in diseases associated with cachexia in one survey patients with terminal cancer and nausea or emesis had a 68% decrease in survival [35].

Because of these features of cachexia related to anorexia and energy imbalance, ghrelin has been hypothesized as a potential treatment for multiple models of cachexia, having been the subject of extensive investigation in animal models [36]. In the following sections we will consider three of these categories of cachexia-associated disease: cachexia associated with cancer, chronic heart failure and chronic kidney disease. We will pay particular consideration to the physiologic regulation of endogenous ghrelin in the setting of disease-associated cachexia, as well as the efficacy of treatment with ghrelin and other ghrelin receptor agonists in the setting of cachexia. We will also consider how discoveries from animal models will continue to influence the potential for human application.

Cancer Cachexia

Cancer cachexia remains a disease process with a high degree of morbidity, affecting 30-80% patients suffering from a wide variety of cancers and particularly from gastrointestinal cancers [37,38]. Ominously, cachexia is implicated in up to 20% of cancer deaths [37,39]. In many cases cancer cachexia is associated with an increase in metabolic rate, further taxing energy demands [40,41]. Perhaps most importantly, no treatment thus far has made significant clinical improvement in human disease [6].

Ghrelin has shown promise in application as a potential treatment for this disorder, and many of the available data have come from investigation using animal models [42]. In this section—and in our discussion of each of the other major cachexia sub-types, we will consider a) types of animal models of the underlying disease used to evaluate physiology and pharmacology; b) what is known regarding the pathophysiology linking the underlying disease process to cachexia; c) the efficacy of ghrelin and ghrelin receptor agonists as a treatment and the doses and route used; and d) the potential mechanisms by which ghrelin acts in these settings.

Animal Models of Cancer Cachexia

Animal models used to investigate the physiology and pharmacology of ghrelin and other ghrelin receptor agonists have predominantly utilized tumor cell lines that are implanted into a host animal. Examples of these cell lines include human melanoma cells [43], Lewis Lung Carcinoma (LLC)[19,24,44–48], colorectal tumors [49–51], syngenic sarcomas [19,52–56], and other types of stable, cachectogenic neoplasms. Following subcutaneous implantation into the host, these cell lines are allowed to grow to a particular burden at which point the animal develops symptoms of cachexia. The implanted cell lines differ from most human cancers in that they do not metastasize significantly but they are similar in many other ways to cachexia-causing neoplasms in humans. This includes stimulation of host-tumor interactions and in some cases release of cachexia-inducing factors [57,58]. Many of these implanted tumor cells express or induce inflammatory cytokines or prostaglandins [59–62]. Most importantly, these implanted tumor models result in the key features of cachexia, including anorexia, weight loss, loss of lean and fat mass and increased energy expenditure [19,24]. These models also result in an increase in mortality, though study of this as an endpoint has been limited in most investigations into treatment efficacy.

Pathophysiology of Cancer Cachexia

While causal associations between various cancer conditions and cachexia are well established in animal models and human cases of cachexia, the exact mechanisms are less well-established than may be expected [38]. This lack of clear cause is likely because of the diversity in both the underlying cancer types that can result in cachexia and in the pathophysiologic processes that accompany malignancy and the body’s response. Major contributors to the process appear to be systemic inflammation and action of tumor-produced mediators.

The inflammation that is seen in the setting of cancer has been implicated in cachexia in that up to 50% of humans with cachexia exhibit increases in acute phase proteins such as C-reactive protein and systemic inflammatory cytokines [63]. This inflammation has its source in both tumor-based production of cytokines as well as the host response to the tumor [64,65]. Animals with high degrees of implanted tumors have been found to have elevated levels of inflammatory cytokines compared to non-tumor-bearing animals. Chen et al demonstrated that mice implanted with colon-26 adenocarcimoma had levels of IL-6 and TNF-α that were 100-fold and 5-fold above those of healthy controls [66]. Similarly, our group in the laboratory of Dan Marks showed that rats bearing syngenic sarcoma had levels of IL-1β and IL-6 were increased by 2-3 fold above sham-operated animals [52]. The presence of cytokines along with tumor appear to be necessary in that tumor-bearing animals with a genetic deletion of the gene for IL-6 did not have as striking of anorexia as those who expressed IL-6, while animals receiving IL-6 without tumor did not exhibit signs of cachexia [60]. The suggestion of systemic inflammation as an etiology of cancer cachexia is significant in that inflammation has been shown to act on central appetite-regulating centers such as the central melanocortin system, targets where ghrelin receptor agonists are known to act [21,22,24]. As discussed later, these inflammatory effects are also significant in that ghrelin exhibits anti-inflammatory properties.

Another means by which cancer may result in cachexia is via tumor-based production of molecules that result in cachexia. One such molecule is proteolysis-inducing factor, which is produced by the implantable cell line MAC16 and acts on a receptor on muscle cells [57,58]. It should be acknowledged that the diversity of tumor types leaves open a wide variety of different molecules that may be operative in the production of cachexia. Currently additional specific factors related to cachexia production (besides cytokines themselves) are not known for other implantable tumor lines.

Treatment of cancer with chemotherapeutics can also have profound effects on appetite and weight through additional mechanisms [67]. These effects—and ghrelin’s role in their treatment will be considered in the section of “Other Conditions Associated with Cachexia and Anorexia.”

Regulation of Ghrelin in Cancer Cachexia

Ghrelin levels in the setting of animal models of cancer cachexia have been investigated in animal models, with data restricted to total ghrelin levels. In a model of implanted eicosanoid tumor-related cachexia Wang et al reported a 40% increase in serum levels of total ghrelin in tumor-bearing mice compared to controls [68]. In addition, tumor-bearing animals exhibit a 54% increase in ghrelin receptor expression in the hypothalamus, suggesting an elevated level of responsiveness to ghrelin. The increases in ghrelin levels likely demonstrates a physiologic response to changes in weight and food intake and, which were decreased by 15% and 20%, respectively [68]. It should be noted, however that a wide variety of tumors themselves express ghrelin, including in animal models, though it is uncertain how much this expression contributes to overall ghrelin levels [69,70]. Certainly an additional factor in ghrelin regulation in the setting of cancer cachexia is leptin. Hanada et al measured ghrelin content in the stomach, reporting an increase of 36% among mice bearing human melanoma tumors, accompanying a 96% decrease in serum levels of leptin [43].

In addition to these data regarding total ghrelin levels in animals, studies of ghrelin levels in humans with cancer have confirmed that these elevations are due to increases in both acyl and desacyl levels, which are both elevated 25-50% above normal in the setting of malignancies of lung, breast, prostate and colon [71–73].

These elevated ghrelin levels in the setting of implantable tumor models appear to respond to treatment with ghrelin as expected in terms of serum ghrelin levels and its production in the stomach. Wang et al demonstrated a 40% increase in serum ghrelin levels during treatment in tumor-bearing mice, demonstrate the efficacy of treatment [68]. Again, these represent total ghrelin levels; it is not known from these animal models whether acyl ghrelin levels increase even further. In one study of human application levels of acyl ghrelin rose 26-fold during treatment [74]. In addition to these changes in serum levels of ghrelin, apparent stomach production of ghrelin responds to ghrelin treatment as may be anticipated from negative feedback mechanisms. Hanada et al demonstrated a 15% decrease in stomach content of ghrelin during treatment [43]. Taken together, these data suggest that treatment with ghrelin results in further increases in serum levels of total ghrelin while endogenous production appears to decrease—potentially in response to treatment-induced changes in weight and leptin. Nevertheless, changes in serum ghrelin levels may also be tumor dependent in that Pourtau et al found serum levels of ghrelin that were approximately 50% lower in rats bearing a Moris hepatoma 7777 tumor [75].

Efficacy of Ghrelin Treatment in Cancer Cachexia

The optimism for using ghrelin as a treatment for cancer cachexia rests predominantly on demonstrations of the efficacy of treatment with ghrelin and other ghrelin receptor agonists in animal models. We will consider multiple endpoints used in evaluating this efficacy, including a) food intake, b) weight gain, c) lean body mass gain, d) fat mass gain. Untested—but in many ways most important from the standpoint of human disease—is the evaluation of ghrelin’s effect on survival in the setting of cancer cachexia.

Food intake

An increase in food intake evaluates one mechanism by which ghrelin may exert its anti-cachectic effect in cancer cachexia. However, given the central placement of eating in our culture, food intake can also be seen as a marker for quality of life [34].

Administration of ghrelin in animal models of cachexia has resulted in increases in food intake in some—but not all—reported studies. Using a human melanoma model of cachexia in mice, Hanada found that mice receiving injections with ghrelin had a 26% increase in food intake over 6 days of ghrelin injections compared to vehicle [43]. Wang et al used a methyl-cholantrene-induced sarcoma model to demonstrate in which mice receiving ghrelin had a 10% increase over tumor-bearing controls [68]. Our experiments in the laboratory of Dan Marks demonstrated a 40% increase in food intake in rats receiving ghrelin via continuous release of ghrelin (500 nmol/kg/d) over another tumor-bearing group that received vehicle [52]. In these experiments, a separate tumor-bearing group receiving a synthetic small molecule ghrelin receptor agonist, BIM-28131 (whose name has since been changed to RM-131), exhibited a similar 45% increase in food intake.

Weight gain

Weight gain has been the most-used endpoint in evaluating efficacy of potential treatment agents for animal models of cancer cachexia. This is likely because of the striking centrality of weight loss as a marker of human disease. In addition, assessment of weight integrates multiple potential effects of ghrelin as a treatment, including changes in food intake, catabolism and basal metabolic rate. The effects of ghrelin and ghrelin receptor agonists on animal models of cancer cachexia have reported consistent improvements in weight retention (or weight gain) in the tumor models reported. These findings have occurred in the setting of significant weight loss in tumor-bearing animals treated with vehicle.

Hanada et al reported a minor increase in weight among ghrelin-treated mice (1.4%), as compared to a 4% weight loss in tumor-bearing mice treated with control (Table 1)[43]. Wang et al reported a final weight of ghrelin-treated animals (carcass weight, following tumor removal) that was 10% greater than the control-treated mice [68]. Our group found a 13% increase in weight in tumor-bearing rats treated with ghrelin and a 9.5% increase in rats treated with BIM-28131, compared to a 10.3% weight loss in tumor bearing rats treated with control [52]. Certainly, the variability in these results between experiments is likely due to differences in model, dose, route and length of treatment. Overall, higher doses (Wang et al) and continuous infusion (DeBoer et al) appeared to be more effective than intermediate doses and twice-daily injections.

Table 1.

Summary of animal models of ghrelin and ghrelin receptor agonists for the treatment of cancer cachexia.

Publication [reference]	Underlying Disease Model (species)	GHS-1a Agent	Dose, Route, Duration of treatment	Effects
Hanada et al., 2003 [42]	SEKI human melanoma cells (mice)	ghrelin	800 nmol/kg/d as twice daily IP injections × 6 days	Weight: ghrelin +0.2%; vehicle -0.7%* Food intake (cumulative): ghrelin 26% more than vehicle*
Wang et al., 2006 [68]	methyl-cholanthrene-induced sarcoma (mice)	ghrelin	800 nmols/kg/d as twice daily IP injections × 5 days	Weight: ghrelin +10% more than vehicle* Food intake (cumulative): ghrelin +20% more than vehicle
DeBoer et al., 2007 [52]	methyl-cholanthrene-induced sarcoma (mice)	ghrelin, BIM-28131	500 nmol/kg/d as continuous mini-pump infusion × 5 days	Weight: ghrelin +13%***; BIM-28131 +9.5%***; vehicle -10.3% Food intake (cumulative): ghrelin 37% more than vehicle***; BIM-28131 43% more than vehicle*** Lean body mass: ghrelin -1.0%; BIM-28131 -2.7%; vehicle -12.6

Significance: NS=not significant (p>0.05);

^*p<0.05;

^**p<0.01;

^***p<0.001.

Change in Lean Mass

A higher degree of lean and fat mass stores may be protective in the setting of cancer cachexia and losses from each of these compartments can be predictive of risk of mortality [76]. In animal models of cancer cachexia, lean mass losses in particular are frequently reported as a marker of disease [44,45,77]. Change in lean mass in the setting of ghrelin treatment has only been reported in our group’s data, revealing that ghrelin largely ameliorated losses of lean mass compared to tumor-bearing controls (-1% for ghrelin, -2.7% BIM 28131 and -12.6% vehicle). These modest losses of lean mass were in comparison to an 11% gain in lean mass among non-tumor-bearing controls [52].

Change in Fat Mass

A commonly-noted effect of ghrelin administration in non-diseased states is an increase in fat mass. Given the importance of fat mass for survival in cachexia, this has been regarded as a potential benefit of ghrelin treatment in settings of cancer cachexia. Multiple models of cancer cachexia have evaluated these effects, with mixed results regarding the maintenance of fat mass. Wang et al demonstrated a higher total body fat content in tumor-bearing animals treated with ghrelin that was14% higher than tumor-bearing controls. It should be noted that this fat content was still 15% less than non-tumor-bearing controls [68]. In our experiments we found that ghrelin-treated rats had a similar loss of fat mass as tumor-bearing controls (-50%) while the BIM 28131-treated rats lost only 30% of their fat mass, which was similar to that of the non-tumor-bearing controls [52]. It is interesting to note that these sets of experiments differed 3-fold in the dose of ghrelin (1600 nmol/kg/day vs. 500 nmol/kg/d), which may account for the differences in fat mass retention. Hanada et al attempted to measure weight of white adipose tissue in the region of the kidneys and uterus at the end of treatment [43]. Whereas non-tumor-bearing animals had measureable levels of white adipose tissue, the tumor-bearing animals did not have visible white adipose tissue to measure. This may have resulted from the severity of their model.

Tumor growth

A concern regarding the use of ghrelin in cancer cachexia has been its ability to stimulate release of growth hormone and subsequently IGF-1 levels, each of which have been implicated in potential stimulation of tumor growth [78]. Thus far animal models of cancer cachexia have failed to demonstrate such effects. The studies reported here by Hanada, Wang, and DeBoer all reported no difference in tumor mass following use of ghrelin or ghrelin receptor agonists for as long as 6 days of treatment [43,52,68]. Nevertheless, given theoretical concerns, tumor size after treatment remains an important endpoint to measure in all animal model trials.

Survival time

While increases in survival are of major interest in clinical use of anti-cachexia medications, the effects of treatment on survival time has been difficult to examine in animal models of cachexia because of ethical concerns regarding the suffering of studied animals [42]. Effects of treatment on survival time have not yet been examined among mice receiving ghrelin, though other experimenters using treatment with growth hormone, insulin and indomethacin have revealed an increase in survival time [66].

Ghrelin’s Mechanism of Action in Cancer Cachexia

While evaluation of ghrelin’s efficacy in animal models of cancer cachexia helped to pave the way for trials in human disease, an equally-important role of animal models has been in the assessment of mechanisms of ghrelin’s actions in these settings. Not surprisingly, these mechanistic considerations are complex, given ghrelin’s widespread effects on multiple physiologic processes.

Action at appetite-regulating centers

Ghrelin’s ability to stimulate appetite via action at the ghrelin receptor in hypothalamic centers is a hallmark of its role in normal physiology and is clearly a major reason that ghrelin receptor agonists were a logical choice for testing in the treatment of cancer cachexia. As mentioned previously, multiple studies have revealed an increase in food intake in the setting of ghrelin treatment for animal models of cachexia. In addition, multiple studies have evaluated for additional evidence of direct stimulation of appetite-regulating centers such as the central melanocortin system, discussed in the section above on ghrelin’s appetite effects. During cancer cachexia, levels of AgRP have been noted to be decreased, potentially contributing to decreased appetite [75].

In the laboratory of Dan Marks, we used our model of cachexia in rats to evaluate effects of ghrelin administration on gene expression in these neurons in the setting of cancer cachexia. Using real-time RT-PCR on hypothalamic extracts, we noted that compared to tumor-bearing controls, tumor-bearing rats treated with ghrelin had an increase in expression of AgRP (+75%) and NPY (+30%) [52]. We did not note a change in expression of POMC in either the hypothalamus or the brainstem, another locus of POMC neurons regulating appetite. POMC expression in these tissues was already suppressed 50% between tumor-bearing and non-tumor-bearing groups and ghrelin treatment failed to suppress this expression further. Clearly it should be noted that these data are regarding expression levels of these neurons and not protein content or activity. Nevertheless, our conclusion from these results was that in the setting of cancer cachexia, ghrelin treatment results in a stimulus of orexigenic pathways (AgRP and NPY). Anorexigenic pathways already appeared to be significantly suppressed, potentially as a physiologic response to the low leptin levels or other conditions associated with cachexia. Regarding the effects of ghrelin on such appetite-stimulating centers, it should be noted that these data still do not prove a direct effect of ghrelin on these neurons during cancer cachexia, since indirect effects such as a decrease in inflammation may cause indirect orexigenic effects on these centers as well.

In addition to its effects on appetite regulation, the central melanocortin system also has effects on resting energy expenditure [24], with a decrease in energy expenditure during melanocortin inhibition. This potentially provides an additional means of achieving the improvements in weight gain seen in this setting. Changes in energy expenditure have not been measured during ghrelin treatment of cancer cachexia, providing an opportunity for further investigation.

Growth hormone stimulation

The property of ghrelin that originally lead to its discovered was its ability to stimulate release of growth hormone from the anterior pituitary. Treatment with ghrelin receptor agonists in non-cancer models have been shown to result in increases in IGF-1 levels. Given potential effects of IGF-1 on muscle mass, the growth hormone-IGF-1 axis represents another potential mechanism of ghrelin’s effects in the setting of cancer.

In our rat model of cancer cachexia treated with ghrelin, we did not note significant differences in basal levels of GH, though there appeared to be a trend toward increased levels of GH among animals treated with ghrelin and BIM-28131 [52]. We also noted a clear suppression of IGF-1 in tumor-bearing rats (a ≥50% reduction vs. non-tumor-bearing rats). The suppression of IGF-1 in this model was not affected by treatment with ghrelin or BIM-28131, suggesting that growth hormone resistance seen in severe cancer cachexia was not overcome with ghrelin treatment. Since that time human application has suggested an increase in IGF-1 levels following treatment with another ghrelin receptor agonist, RC-1291. Thus it remains unclear how significant a role GH stimulation plays in ghrelin’s effects in cancer cachexia, though in some settings IGF-1 can be up-regulated during ghrelin receptor agonist treatment.

Anti-inflammatory effect

As mentioned previously, systemic inflammation is thought to play a key role in the propagation of cancer cachexia and ghrelin has exhibited potential anti-inflammatory effects, at least in part through ghrelin receptors on leukocytes. As mentioned previously, anti-inflammatory effects may constitute a means by which ghrelin could affect orexigenic activity. The group of Wang et al tested for levels of prostaglandin E2 (PGE2, a marker of inflammatory response) among mice in their model of cancer cachexia, including during treatment with ghrelin. While tumor-bearing mice had high levels of PGE2 compared to non-tumor-bearing animals, treatment with ghrelin did not produce significantly lower levels of PGE2 [68]. Our group measured multiple systemic inflammatory cytokines in the setting of ghrelin treatment. Tumor-bearing animals exhibited a trend toward higher levels of cytokines than seen in non-tumor-bearing controls. However, we did not see a difference between tumor-bearing animals that were treated with ghrelin vs. placebo [52]. Thus, at this point no clear data exists to support a significant anti-inflammatory component to ghrelin’s action in cancer cachexia.

Other effects

As mentioned previously, ghrelin has been found to have cardiovascular effects in non-diseased animals. While these effects may play a role in other disease states associated with cachexia, they have not been studied in the setting of cancer cachexia. This thus remains fertile ground for further research.

Future Directions and How Animal Data Informs Human Investigation

Basic science approaches to studying ghrelin’s role in cancer cachexia are likely to continue to be a vital piece study to complement and inform human investigations. This is particularly true regarding efficacy and specific mechanisms of ghrelin’s action in these settings. Using genetic knock out strains, animal models could be used to determine the extent of ghrelin’s effects that are due to any of the above postulated mechanisms. Mice that have had knock out of ghrelin receptors in central appetite-regulating centers could be tested to see if ghrelin treatment maintained any efficacy in the absence of direct appetite stimulation. As mentioned, there are multiple other etiologies that have not yet been explored in the setting of cancer cachexia. Effects on cardiovascular function, particularly cardiac output, are mechanisms that may contribute ghrelin’s efficacy in the setting of cancer cachexia. More extensive evaluations of anti-inflammatory effects may also be enlightening. Animal models provide important opportunities to explore these relationships.

An important endpoint in considering clinical use is survival. While survival has been tested in animal models of cancer cachexia following treatment with growth hormone, insulin and indomethacin [66], these evaluations have not been performed in the setting of ghrelin treatment. Given improvements in weight retention, it may be hypothesized that ghrelin would result in increased survival, but this should be rigorously (and humanely) tested. Improved survival would greatly increase clinical interest in pursuing ghrelin receptor agonist molecules as a treatment option.

Animal models could be also be used to explore potential harmful effects of ghrelin in these settings. Although data have been reassuring thus far regarding the effects of ghrelin treatment on tumor growth, animal models could be used to provide further reassurance. For example, growth-hormone deficient mice could be employed and compared to wild-type mice regarding tumor growth and other aspects of treatment, including weight gain and survival.

There is some possibility that resistance or tolerance to treatment may develop over time in the treatment of cancer cachexia. This could occur through compensation of anorexigenic pathways or down-regulation of ghrelin receptors, for example. If resistance to ghrelin (or potentially other anti-cachectic treatments) occurs, animal models may also play an instrumental role in investigating combinations of therapies on a rotating basis. If down-regulation of receptors occurs, a staggered use of multiple different orexigenic compounds (such as melanocortin antagonists) may prove effective in either alternate means of stimulating key orexigenic pathways or stimulating parallel pathways that have not yet exhibited resistance to treatment. Again, animal models would prove useful in these tests.

Cachexia Due to Chronic Heart Failure

Worldwide, incidence of cardiovascular disease continues to rise in both developed and developing countries, placing a growing number of people at risk for chronic heart disease [79]. In the U.S., 5 million individuals have chronic heart failure, with an incidence of 3% among elderly individuals over 75 years old ([80]. Cachexia associated with chronic heart failure is thus likely to rise also, as up to 15% of individuals with chronic heart failure are found to have cachexia, exhibiting losses of lean mass, losses of fat mass and loss of appetite [81,82]. Weight loss approaches 18% of lean body mass and 37% of fat mass in patients with cachexia due to chronic heart failure as compared to those without cachexia. Additionally, the mortality rises by over 3-fold in the presence of significant weight loss, underscoring the need of interventions in this setting. Thus, it is not surprising that animal models of cardiac cachexia have been a well-studied setting to test ghrelin’s efficacy as a treatment of cachexia (Table 2), with some promising results but also with continued questions.

Table 2.

Summary of animal models testing ghrelin and ghrelin receptor agonists for the treatment of cachexia.

Publication [reference]	CHF Model (species)	Treatment Agent	Dose, Route, Duration	Effects Weight gain Lean mass LV ejection fraction
Tivesten et al., 2000 [109]	LAD ligation (rats)	hexarelin	114 nmol/kg/d × 2 wks	Body weight change: hexarelin -5% vs. saline (NS) Cardiac output: hexarelin +18% vs. saline *
Nagaya et al., 2001 [100]	LAD ligation (rats)	ghrelin	67 nmol/kg/d as twice daily SQ injections × 3 wks	Body weight change: ghrelin +10% vs. saline+3%* Cardiac output: ghrelin +22% vs. saline *
Xu et al., 2005 [108]	Aortic banding (rats)	GHRP-1,-2, -6 & hexarelin	225 nmol/kg/d as twice daily SQ injections × 3 wks	Body weight change: GHRP-1 +47%*; GHRP-2 +47%*; GHRP-6 +46%*; hexarelin +47%*; saline +41% LV ejection fraction: GHRP-1 86%*; GHRP-2 79%*; GHRP-6 77%*; hexarelin 80%*; saline 59%
Akashi et al., 2009 [107]	LAD ligation (rats)	ghrelin	50 or 500 nmol/kg/d by osmotic mini-pump × 4 wks	Body weight change: ghrelin +18-25% vs. saline *** Lean mass change: ghrelin +16-25% vs. saline ** Cardiac output: ghrelin +3% vs. pre-treatment; saline -11% vs. pre-treatment (NS)

Abbreviations: CHF, chronic heart failure; LAD left anterior descending coronary artery; Significance: NS=not significant (p>0.05);

^*p<0.05;

^**p<0.01;

^***p<0.001.

Pathophysiology of cachexia due to chronic heart failure

As is the case for most disease states associated with cachexia, cardiac cachexia is multi-factorial. Hypothesized causes have included metabolic dysfunction and malabsorption of nutrients [83]. One contributing cause has been postulated to be systemic inflammation in the setting of cardiac failure [84,85]. This has been seen in humans with chronic heart failure in which TNF-α and IL-6 levels can be elevated over 2-fold above controls [86–88] as well as in animal models of cardiac failure [89,90].

The cytokines seen in chronic heart failure may be due to leaking of bacterial endotoxin introduced though a bowel wall that is edematous and permeable because of chronically poor perfusion [80,91]. Other sources of inflammation may be release from the failing myocardium [92,93] or from other underperfused organs such as the liver [94]. The sum of these inflammatory processes may be activation of muscle catabolism (including activation of the ubiquitin proteosome pathway)[84] and the central melanocortin system [95,96], each of which is discussed in more depth elsewhere in this article.

Animal Models of cachexia due to chronic heart failure

Multiple animal models have been employed in studying ghrelin’s effects in the setting of chronic heart failure. These models are designed to mimic either infracted myocardium and/or restricted cardiac output. The myocardial infarction model involves surgical ligation of the anterior left descending artery. This can be performed in mice [97,98] and rats [99,100] using similar techniques. The procedure is performed via a central sternotomy to expose the heart and a suture being placed just inferior to the left atrium to occlude the left anterior descending (LAD) coronary artery. This can be a difficult procedure since this suture is placed in a relatively blind fashion (i.e., the coronary itself is not visible). Thus this model can result in a variable amount of myocardial infarction and a potentially high early mortality rate. Sham animals ideally receive identical needle placement without constricting the artery.

Following ligation, animals experience myocardial infarction and exhibit worsening systolic dysfunction. Over the ensuing weeks, they develop an indolent course of worsening appetite, decreased weight gain, lean and fat body wasting, and increased basal VO2 utilization. The effects of the procedure on weight gain range from frank weight loss (when performed on fully-growth male rats >12 mo old) to a decrease in weight gain (when performed on younger rats <8 months old) [101].

An alternative model of cardiac cachexia utilizes restriction of cardiac output via aortic banding, which can also be performed in mice and rats. This model involves use of a left thoracic incision and constricture of the left ascending aorta using titanium clips. Over the ensuing 2-4 weeks, the animal develops congestive heart failure, with a decrease in appetite and a decrease in weight gain [97,98]. Also, chronic rapid pacing using a catheterization-implanted pacemaker set to a heart rate approximately 75% above normal is another means of inducing heart failure utilized for studies of ghrelin receptor agonists [102].

Additional models of ghrelin’s effects in the setting of cardiovascular compromise utilize acute models of cardiac damage. These include a more severe model of myocardial infarction in which effects were measured 2 hours after infarction [103]. Finally, ex-vivo models are also employed assessing ghrelin effects on relaxation of aortic tissue following exposure to norepinephrine [104].

Regulation of ghrelin in cachexia due to chronic heart failure

Similar to cancer cachexia, acyl and total ghrelin levels are increased in cardiac cachexia. This has been convincingly demonstrated in human studies revealing that compared to healthy controls patients with chronic heart failure exhibit total ghrelin levels that are increased by approximately 40% [105,106], and these levels return to near-normal levels following heart transplant [106]. Interestingly, among patients with chronic heart failure, those with cachexia have ghrelin levels that are 50% higher than those without cachexia, suggesting a high degree of ghrelin resistance in this setting [105]. To our knowledge ghrelin levels have not been published in animal models of cardiac cachexia, suggesting the potential for research pursuits on the topic.

Efficacy of Ghrelin Treatment in Cardiac Cachexia

Efficacy of ghrelin in the treatment of animal models of cachexia requires the use of additional endpoints not typically followed in cancer cachexia. In cardiac cachexia, some traditional measures such as weight gain may not be as straightforward to follow, given that edema resulting from chronic heart failure may cause increases in body mass without improving muscle mass or fat mass. Some studies have used diuretics such as furosemide in infarcted animals for this reason [107]. In addition to food intake and weight gain, assessments related to cardiac function, including cardiac output, blood pressure, and left ventricular function are key endpoints in the setting of chronic heart failure. These endpoints have thus far not been evaluated in models of cancer cachexia or cachexia due to chronic kidney failure.

Food intake

Akashi et al used an LAD ligation model in rats treated with 50 or 500 nmol/kg/d of human ghrelin and reported food intake in two time periods: days 28-41 after LAD ligation and days 42-56 [107]. While receiving ghrelin from days 28-41 after infarction, rats on 50nmol/kg/d ate 2.3% more than saline-treated rats while those on 500 nmol/kg/d ate 15% more. Then, between days 42-56, the 50 nmol/kg/d group ate 9% more than saline-treated rats while those receiving 500 nmol/kg/d ate 12% more. Notably, during this first time period LAD-ligated rats treated with saline had only a 2% decrease in food intake relative to sham-treated controls (which is to say that ghrelin-treated rats with infarctions ate more than the sham-treated controls). During the second time period, saline-treated rats with infarctions had a 10% decrease vs. sham-treated animals, underscoring the worsening anorexia over the course of chronic heart failure [107].

Weight gain

Studies of ghrelin application in cachexia due to chronic heart failure have reported differing degrees of changes in weight gain during treatment. Nagaya et al used an LAD ligation model of MI and administered 67 nmol/kg/d of ghrelin for 3 weeks, reporting an 18% increase in weight gain (an additional 21 g or 8% body weight) in ghrelin-treated rats with CHF compared to CHF controls [100]. Similarly, in their LAD ligation model, Akashi et al reported that rats receiving 50 nmol/kg/d had a 41% improvement in weight gain over saline-treated controls (an additional 8.1 g or 2.4% body weight) while those receiving 500 nmol/kg/d had a 160% increase in weight gain vs. saline animals (an additional 40 g or 11.8% of body weight). The differences between these studies—which used similar models of chronic heart failure—on measured weight gain may stem from differences in length of treatment (3 weeks for Nagaya vs. 4 weeks for Akashi), dose of ghrelin or route of delivery (twice daily injections for Nagaya vs. continuous pump for Akashi). Also, Akashi treated all animals with furosemide, while it is not clear whether Nagaya did [100,107].

Using a model of aortic banding to produce chronic heart failure, Xu et al administered multiple ghrelin receptor agonists for 3 weeks and reported a 50% increase in weight gain (an additional 12 g or 3% body weight) with ghrelin receptor agonist treatments (including hexarelin) at 225 nmol/kg/d compared to banded controls [108]. In this case, the saline-treated animals with aortic banding gained 30% less than sham-treated controls. Thus, over the time period of treatment, the animals treated with GHS-1a agonists gained more weight than the sham-treated animals.

Finally, Tivesten et al used a LAD ligation model of heart failure and tested two doses of hexarelin (114 and 11.4 nmol/kg/d) for two weeks. They did not note any change in weight gain at these doses [109]. Interestingly, they did note a 50% increase in weight gain among a group of rats treated with growth hormone.

These data on weight are significant in that overall higher body weight—presumably including a combination of lean mass and fat mass—is protective among patients who have developed chronic heart failure, a concept known as the “obesity paradox,” though it is unclear whether additional lean and fat mass are beneficial or merely a marker of decreased cachexia severity [110]. The ability of ghrelin treatment to increase weight retention may benefit by providing improved energy reserve in the case of worsening disease.

Lean and fat mass

Retention of lean mass has been reported as an important prognostic factor in cardiac cachexia [81]. In their model, Akashi et al reported that treatment with 50 nmol/kg/d of ghrelin produced no additional increase in lean mass above saline-treated animals following LAD ligation. Rats treated with 500 nmol/kg/d, however, exhibited a 92% increase in lean mass above the changes seen in saline-treated rats (an additional 7.7 g lean mass or 3% body weight). The saline-treated animals had gained a similar amount of lean mass as had the sham-treated controls (+7.8% vs. +8.1%), and thus the high-dose ghrelin-treated rats gained more lean mass than non-infarcted controls.

Gain in fat mass has been a widely-reported finding in the setting of ghrelin treatment in non-cachexia models. Akashi found that saline-treated animals following ligation gained only a trace amount of fat mass (+1%) compared to sham-treated controls (+15%), while ligated rats treated with 50 nmol/kg/d ghrelin gained 21% and those treated with 500 nmol/kg/d gained 57% during treatment [107]. The retention of lean and fat mass may be beneficial from a perspective of the prognosis of chronic heart failure, as discussed in the section on ghrelin’s potential mechanisms of benefit.

Cardiac output

As mentioned previously, an additional important endpoint in studies of the effects of ghrelin in the setting of cardiac cachexia. These effects were first evaluated by Nagaya et al who assessed multiple cardiovascular parameters in their model and reported a 22% increase in cardiac output in ghrelin-treated rats following LAD ligation relative to saline-treated controls [100]. Relative to the saline-treated controls, the ghrelin-treated group also exhibited decreases in left ventricular end-diastolic pressure (31% lower) and left ventricular wall stress (lower by 42% in diastole, 22% in systole). Both ligated groups (ghrelin- and saline-treated) showed similar decreases in blood pressure relative to the sham surgery controls.

Akashi et al used a similar approach, utilizing echocardiographic data in the setting of ghrelin treatment following LAD ligation, but reported a lack of significant differences between groups [107]. It should be noted, however, that ligated rats that received saline had a 12% decrease in cardiac output between days 27 and 55 after ligation, while the group treated with 100 nmol/kg/d of ghrelin had a 3% increase over this same time frame. Relative to the saline-treated rats, ghrelin-treated rats following ligation had diastolic stress measurements that were 59% lower in diastole and 17% lower in systole. Again, these failed to reach statistical significance, as did measurements of blood pressure between groups. Akashi et al treated their animals for 4 weeks, which is longer than any of the other applications of ghrelin administration. This raises the possibility that the effects of ghrelin on cardiac function diminish over time, though more extensive investigation is needed [107].

In their model of LAD ligation, Tivesten et al found that even at a relatively low dose (114 nmol/kg/d), hexarelin increased stroke volume by 25%–even more so than they had noted with treatment with growth hormone. They noted these effects despite using lower doses than other groups and despite noting no effects on weight gain [109].

King et al used a chronic rapid pacing model of chronic heart failure in pigs and demonstrated that a ghrelin receptor agonist (CP-424,391, Pfizer Central Research) improved left ventricular pump function, including an increase in fractional shortening and a decrease in left ventricular peak wall stress [102].

Human trials have demonstrated increased left ventricular ejection fraction (by 4%) and an increase in peak oxygen consumption during exercise. There was no difference in blood pressure.

Other markers of cardiovascular disease

As a means of assessing the effects of ghrelin receptor agonist on other markers of chronic heart failure, Xu et al measured multiple serum markers of disease status. Treatment with any of the 4 ghrelin receptor agonists resulted in approximately 20% decreases in measures of norepinephrine, renin activity, angiotensin II, aldosterone and atrial natriuretic peptide [108]. Echocardiographic measures were not performed to see if these improvements all coincided with improvements in cardiac function.

Ghrelin’s Mechanism of Action in Cardiac Cachexia

Given the variety of effects that ghrelin exerts in the setting of chronic heart failure, the exact mechanisms of improvements related to ghrelin’s action in these experiments are not certain. This is particularly true because isolation of the exact effects would be difficult in a process as complex as cachexia. Ghrelin receptor agonists have long been noted to bind cardiac tissue [111,112] and GHSR transcript has been detected in cardiac tissue [7,113]. Furthermore, as discussed below cardiac effects of ghrelin receptor agonists have been well documented, potentially accounting for the increases in cardiac output and other effects seen during ghrelin treatment of cardiac cachexia. Nevertheless, the potential also exists that improvements in cardiac parameters in the setting of cachexia may also be related to decreases in inflammation or to a diminution of central processes related to cachexia. Conversely, improvements in weight gain could be due to improved cardiovascular function (and fewer processes contributing to cachexia) or to direct stimulation on appetite centers.

The degree of stimulation of ghrelin on appetite-regulating centers in cachexia due to chronic heart failure is unclear. Whereas changes in gene expression of transcripts related to appetite regulation have been demonstrated in the setting of cachexia and renal failure, these changes have not been demonstrated in cachexia from heart failure. As alluded, the increase in food intake seen by some in during ghrelin treatment in heart failure may be inferred as resulting from stimulation of these centers but may have also resulted from a decrease in cachexia-mediating processes. Similarly, potential effects that ghrelin may have on resting energy expenditure in the setting of chronic heart failure have not been tested and could account for changes in weight gain seen in this setting.

The effects of ghrelin treatment on levels of inflammatory cytokines, which have been tested in other settings of cachexia have also not been tested in the setting of chronic heart failure. Nevertheless, the potential remains that anti-inflammatory effects contribute to the improvements in weight gain and cardiovascular function that have been seen during ghrelin treatment of chronic heart failure.

Cardiac effects

Not surprisingly, then, the focus on mechanism of ghrelin receptor agonists have rested predominantly on cardiac effects, be it via changes from direct or indirect mechanisms. There are multiple individual effects of ghrelin that may play a role in the observed increases in cardiac output.

Cardiac myocyte preservation

Ghrelin’s effect on apoptosis of cardiac myocytes has received significant attention. This is particularly important given the degree of apoptosis in the setting of chronic heart failure [114]. Multiple investigators have demonstrated these effects on cultured cardiac myocytes and have noted effects in the presence of both ghrelin and desacyl ghrelin that appear to be due to mediated apart from ghrelin receptor. Ghrelin and desacyl-ghrelin block apoptosis and stimulate proliferation and differentiation of cultured cardiac myocytes [115,116]. Similarly, Lear et al evaluated effects of ghrelin treatment on cardiomyocyte cultures and reported an increase in medium chain uptake and insulin-induced glucose-transporter-4 in response to des-ghrelin but not acyl-ghrelin that appear to be mediated by non-GHS-1a receptors [117]. Others have used chemotherapy models of cardiac damage to demonstrate ghrelin’s efficacy in protecting from apoptosis. Filigheddu et al used rat model of doxorubicin exposure on cultured cardiomyocytes and found that hexarelin protected against apoptosis [118]. The extent of these effects in vivo and in the setting of cardiac cachexia is not known, but the consistency of the effects through multiple experiments suggests this may be an important property of ghrelin in the treatment of cardiac cachexia.

A decrease in apoptosis has been noted during in vivo experiments as well. Xu et al used terminal deoxynucleotide transferase mediated dUTP nick end labeling (TUNEL) to examine cardiac tissue from rats in their model of aortic banding, finding that whereas saline-treated rats had 3.1% apoptotic cells, rats treated with various ghrelin receptor agonists had significantly lower amounts of apoptosis at 0.8-1.4% [108]. At least some of this myocyte preservation activity appeared to be due to increases in expression of the anti-apoptotic protein BCL-2 in cardiac tissue and a decrease in expression of the pro-apoptotic protein Bax. Still, it remains unclear how much this decrease in apoptosis contributes to the improved cardiac function seen with ghrelin treatment.

Ischemia protection

A significant amount of data supports the effect of ghrelin on protection of cardiac function following ischemia/reperfusion. Treatment with ghrelin receptor agonists prior to inducing short term ischemia followed by reperfusion lead to improvements in left ventricular contractility [119–121]. These improvements appear to be independent of growth hormone/IGF-1 axis, given that they are seen even in growth hormone deficient mice [119,122]. However, given that these are short-term ischemic models, it is difficult to know how significantly these effects contribute to the mechanism of ghrelin’s action in chronic heart failure.

Inotropic effects

As mentioned previously, the data are mixed regarding the inotropic effects of ghrelin receptor agonsts in chronic heart failure. Nagaya et al found both inotropic effects and weight gain during ghrelin treatment [100]; Tivesten et al found an increase in cardiac output without weight gain during hexarhelin-treatment; and Akashi et al finding weight gain but no cardiac effects. As these experiments involved variations on common models, as well as different ghrelin receptor agonists and different doses, it is difficult to account for the exact source of the variable findings. It is important to note that inotropic effects have been seen in human application as well [123], including during short-term administration [124,125]. That these effects were noted prior to any effect on blood pressure suggests a direct effect [126]. Overall, the presence of body mass improvements in the absence of inotropic effects supports the notion that the body mass effects are not likely due solely to inotropic effects, or at least not in some of the experimental conditions that have been used.

Blood pressure

Isgaard et al found a modest decrease in blood pressure in hypophysectomized rats during ghrelin treatment [126], while Tivesten et al found a decrease in total peripheral resistance during hexarelin treatment of chronic heart failure [109]. Human studies also revealed a non-significant decrease in blood pressure during ghrelin treatment in the setting of heart disease [123].

The mechanism of decreased blood pressure seen during ghrelin administration may be due to vasodilatory effects. Shimzu noted an increase in acetylcholine-induced vasorelaxation from aortic ring explants in ghrelin-treated animals [104] while Rossoni and colleagues noted similar effects using hexarelin [120]. Nevertheless, it remains unclear how much the decrease in blood pressure during ghrelin treatment represents a further mechanism whereby ghrelin treatment improved cardiac output in the setting, warranting further investigation.

Sympathetic tone

Ghrelin has been shown to attenuate cardiac sympathetic tone within the first week after MI [103,127], as well as following in the setting of sepsis [128]. Intracerebroventricular injection of ghrelin in rabbits was shown to decrease renal sympathetic nerve activity, as well as decreases in arterial pressure and heart rate [129]. These sympathetic effects were also seen by the same researchers during microinjection into the nucleus of the solitary tract [130], suggesting a role for ghrelin action in brainstem mediated outflow. Not only is these decreased sympathetic activity seen in humans but it is absent in individuals who have undergone a vagotomy, confirming the central nature of the effects [131].

Stimulation of growth hormone and IGF-1

Growth hormone itself has been heavily studied as a treatment in the setting of chronic heart failure [132]. Growth hormone and IGF-1 related actions have thus remained important features in following the effect of GHS-1a agonists on cardiac cachexia.

In their experiments using multiple ghrelin receptor agonists in their LAD model of chronic heart failure, Xu et al reported increases in serum levels of both growth hormone (by 20%) and IGF-1 (by 40%), accompanying cardiovascular benefits during long-term ghrelin receptor agonist administration [108]. Similarly Nagaya et al reported increased serum levels of growth hormone (by 60%) and IGF-1 (by 34%) during ghrelin treatment, accompanied by improvements in cardiovascular function in an LAD model [100]. Using a model of chronic rapid pacing to mimic chronic heart failure, King et al tested the effects of a GHS1a agonist and found an increase in IGF-1 of 92% over the chronically-paced controls (and 75% above the sham-treated controls)[102]. These findings were also accompanied by improved cardiovascular function. Overall, these experiments thus leave open the potential for the GH/IGF-1 axis to play a role in these effects.

However, other investigators have reported a separation in ghrelin’s effects. Tivesten et al reported significant cardiovascular effects of hexarelin treatment in the absence of changes in IGF-1, suggesting against growth hormone related effects as a primary cause of ghrelin’s cardiovascular actions [109]. De Gennaro Colonna et al evaluated the effect of hexarelin on ischemia/reperfusion injury in mice that were growth hormone deficient and found that pre-treatment with hexarelin increased endothelium-dependent relaxing function following ischemia in the absence of growth hormone [119]. Thus, at a minimum some of ghrelin’s effects on cardiac function do not appear to be mediated apart from the growth hormone/IGF-1 axis. This is further supported by the effects of desacyl ghrelin, which does not act through the ghrelin receptor and does not stimulate growth hormone secretion.

How Animal Data Informs Human Investigation

Of all of the underlying diseases associated with cachexia, cardiac cachexia is the field of research in which ghrelin’s action was studied in humans earliest. Nagaya et al tested ghrelin administration (2 ug/kg IV twice daily over a 3 week period) to subjects with chronic heart failure, demonstrating a 15% increase in left ventricular ejection fraction and improvements in muscle strength and lean body mass [123]. These experiments followed the extensive animal model data that laid the ground work. Nevertheless, additional data regarding GHS-1a agonist treatment is necessary and again animal models should provide more detailed information regarding mechanism and longer-term application. The longest experiments thus far have been four weeks by Akashi et al. It is notable that Akashi et al did not find significant cardiovascular effects of ghrelin treatment, raising the possibility for tolerance to ghrelin over time [107]. Thus, more experiments are needed employing long-term use, as well as assessing for differences in survival during treatment. Overall, animal data continue to support the potential for using ghrelin in the treatment of cardiac cachexia and these additional data would be helpful in guiding potential further human application.

Chronic Kidney Disease

Chronic kidney disease (which includes both chronic renal insufficiency and chronic renal failure) produces uremia and is associated with an increase in protein energy metabolism, muscle wasting and loss of appetite. This condition affects up to 64% of individuals on dialysis [133], and given the growing number of patients on dialysis worldwide (an estimated 350,000 people in the U.S. alone), it also represents a growing cause of cachexia and a field in which ghrelin application has been investigated [134].

Animal Models of Chronic Kidney Disease

Animal models of CKD focus on worsening uremia in animals as a means of affecting changes in food intake and body composition. In testing for efficacy of compounds for the treatment of cachexia, surgical models have been employed in both mice and rats, gradually restricting the amount of functional kidney tissue and producing uremia.

The 5/6 nephrectomy model is a two-staged procedure resulting in only 1/6 of a functional kidney and subsequent uremia. In the first stage of the procedure, one of the animal’s kidneys is isolated with care to maintain an intact vascular supply. The upper 1/3 and the lower 1/3 of the kidney is then excised, and cautery is used to control bleeding and induce scarring. Attention is given, however, to preserving the middle 1/3 of the kidney and the renal artery and vein. Once bleeding has stabilized, this 1/3 remaining kidney is returned to the retroperitoneum and the wound sutured. The animal is then allowed to recover for 7-10 days before a second surgery is performed in which the contralateral kidney is completely resected following ligation of the renal vessels. In both cases, sham-operated animals have the kidney isolated and the renal capsule incised prior to replacement of the intact kidney and wound closure.

Following removal of the second kidney, the animal becomes rapidly uremic, with creatinine and blood urea nitrogen levels that increase by 100-200% over the ensuing 14 days [135]. It is important to measure these levels at experiment’s end to document renal insufficiency. It is further important to assess for acidosis, which may produce symptoms of cachexia, including muscle wasting [136,137]. The 5/6 nephrectomy model is not typically associated with the production of acidosis when followed for 21-day periods following second surgery [23,138,139].

Following the second stage surgery, appetite may decrease over a two-week period [135], though cumulative food intake may not differ from sham-operated animals [23,138,139]. Because of alterations in energy expenditure, poor weight gain is seen even without change in food intake, with a 60-85 percent decrease in weight gain vs. sham-operated animals [23,138,139]. Though animals do not loose overall weight over the course of the experiments, they exhibit changes in body mass compared to sham-operated controls [23,135,138,139]. These changes include a lack of lean body mass accrual and a sharp drop in fat mass—compared to sham controls that show gains in both components. In this way, these models are reasonable representations of human disease, as described above.

Other investigators have used models of acute renal injury to test the effects of ghrelin. This is frequently induced via inducing ischemia to the kidney via a complete clamping of the renal vessels followed by removal of the clamp and witness of the return of blood flow. Renal function is followed thereafter. While this model is primarily used to assess risks during renal transplantation and other surgeries, we will consider some data here related to ghrelin use in this setting, given potential importance of ghrelin physiology in this setting.

Pathophysiology of Cachexia in Chronic Kidney Disease

The appetite and metabolic changes associated with CKD have been attributed to a wide variety of causes including uremia, acidosis and high levels of leptin (due to impaired renal clearance).[140–142] As is the case with cancer cachexia, the cachexia of CRF is strongly linked to increased inflammatory markers, though the cause of the increased inflammation in CKD is not entirely clear.[143] Inflammatory markers such as c-reactive protein (CRP) are highly associated with clinical features of protein energy imbalance, increased metabolic rate and poor prognosis [144–146]. Both elevated CRP and malnutrition were likely to coincide with heart disease, and each is an independent risk factor for mortality.

A key feature of muscle wasting in chronic kidney disease is from activation of the ubiquitin proteosome system [147]. In this process, ubiquitin moieties are added to proteins that are then targeted for digestion by proteosomes into individual amino acids. This is mediated at least in part by acidosis during chronic kidney disease [136], though in animal models, activation has also been noted in the absence of acidosis [135] and can be activated by administration of cytokines [148].

Regulation of Ghrelin in Chronic Kidney Disease

Similar to the case of cachexia in the setting of chronic heart failure, to our knowledge data regarding changes in ghrelin levels in chronic kidney disease has not been tested in animal models and comes from testing in human with renal failure. Multiple studies have shown that while levels of desacyl [149,150] and total ghrelin are increased in chronic kidney disease compared to controls [149,151–153], while studies differ on the levels of acyl ghrelin, with some studies suggesting higher levels in chronic kidney disease [153,154] while others show no difference [149,155]. These levels of desacyl ghrelin normalize following dialysis [152,153] or transplant [151], with improvements being greater in patients on peritoneal dialysis as opposed to hemodialysis. These improvements likely underscore the importance of renal clearance of desacyl ghrelin. Elevated levels of ghrelin have been suggested as an ominous sign in patients with chronic kidney failure [156].

The role of obesity in these settings is unclear. One recent study demonstrated that while acyl ghrelin levels were higher in the setting of hemodialysis relative to healthy controls, these levels were particularly high in subjects with a BMI above 23 kg/m2 as compared to subjects with chronic kidney disease with a BMI below this level [157]. The opposite was true regarding levels of des-acyl ghrelin, which was higher in lean subjects. Even more intriguing findings in this study regarded levels of obestatin, a 23-amino acid peptide that is co-expressed with ghrelin and appears to have potential anorexic properties [158]. Leaner individuals on hemodialysis had higher levels of obestatin, though it remains unclear the cause of these differential levels—as well as any role they may have played in weight changes in the individual subjects.

Ghrelin’s Efficacy in Chronic Kidney Disease

Given the complexity of chronic kidney disease, it is difficult to know the most important measure in determining ghrelin’s efficacy of treatment in chronic kidney disease. Individuals with chronic kidney disease experience anorexia as in other diseases associated with cachexia. But chronic kidney disease also involves wasting of protein that sets it apart from other diseases associated with cachexia. As mentioned previously, this is associated with increases in ubiquitination of protein in muscles [159]. This raises the potential for testing additional outcome points not tested in other conditions. However, similar to cachexia associated with other diseases, chronic kidney disease also involves decreases in lean and fat mass, making traditional endpoints of weight gain and body composition changes important to follow. Because of its role in quality of life, data on food intake remain important. Finally, as in other animal models of cachexia, there are no data regarding what may be the most important outcome of all, namely survival.

To our knowledge, only one long-term trial of ghrelin treatment has been undertaken in an animal model of chronic kidney disease, which was a 5/6 nephrectomy model of renal failure in rats performed in the laboratory of Dan Marks [135]. We administered ghrelin or two other ghrelin receptor agonists via osmotic mini-pumps that delivered 150 nmol/kg/d over a 14-day period of worsening uremia. We followed multiple endpoints that are potentially pertinent to cachexia, as detailed below.

Food intake

In using food intake as an endpoint, both ghrelin and the other ghrelin receptor agonists resulted in an increase in food intake above the saline-treated nephrectomy group, with 14-day food intake amounts that were increased by approximately 15%. Again all of the groups remained significantly lower than the sham surgery group [135].

Effects on appetite and food intake have also been noted in a human study of ghrelin treatment in subjects on dialysis with objective signs of malnutrition were given ghrelin (12 mcg/kg/d SC) or placebo over a one week period in a blinded, cross-over manner with a one week wash-out period in between [160]. During the time receiving ghrelin subjects reported a higher perceived appetite and ate more than during the week on placebo. Given the connection between food intake and quality of life, increase in food intake may represent an important outcome.

Weight Gain and Body Composition

In terms of weight gain in this 5/6 nephrectomy model, one of the ghrelin receptor agonists—BIM 28125—resulted in a 100% increase in weight gain as compared to the saline-treated group after nephrectomy, while ghrelin and the other ghrelin receptor agonist—BIM 28131—resulted in non-significant increases of 85-95% in weight gain compared to saline. Each of the groups gained significantly less than the sham-treated group [135].

When change in total mass was measured via DEXA, all of ghrelin and GHS-1a-treated groups had a larger change in total mass as compared to the saline-treated controls. This was also true regarding changes in lean mass for the ghrelin-treated animals compared to the saline-treated nephrectomized group [135]. Interestingly, ghrelin treatment did not result in a preservation of fat mass, which was unexpected given known effects of ghrelin on fat mass in other settings [161], effects that appear to be independent of orexigenic effects [162].

Ubiquitination

Muscle catabolism of protein involves among other reactions, ubiquitination of actinomycin filaments, leading to degradation [147]. In our experiments, administering ghrelin following 5/6 nephrectomy, we evaluated for actinomycin cleavage in calf muscles, expressed as a ratio of the intact actin to the 14 kD degradation product [135]. We noted that while the saline-treated animals exhibited a 30% increase in actinomycin cleavage over sham-treated animals, animals treated with ghrelin had a complete amelioration of this cleavage, suggesting protection from ubiquitin-mediated muscle catabolism. Because of the importance of increased protein metabolism in chronic kidney disease [136], this serves as an important endpoint in considering ghrelin’s effects.

Renal function

Renal function is a natural outcome of interest in this field in cases where some residual renal function remains. In the 5/6 nephrectomy model, treatment with ghrelin and other GHS-1a agonists did not result in significant decreases in blood urea nitrogen or creatinine, with levels that were only 10-15% lower than the saline-treated control and still 4-fold higher than the sham surgery group.

The effect of ghrelin treatment on renal function has also been tested in an acute model of renal injury. Takeda et al used a model of acute renal injury, induced by bilateral renal artery clamp placement for a 45 minute period and assessed renal function in part by following blood urea nitrogen levels [163]. They reported that treatment with ghrelin prior to the production of renal injury resulted in an improvement in renal function, with blood urea nitrogen levels that were 40% lower than control-treated animals. The significance of these findings to chronic kidney disease are quite unclear, however, and are not likely to apply in cases were no residual renal function remains.

Ghrelin’s Mechanism of Action in Chronic Kidney Disease

As with cachexia associated with cancer and heart failure, the beneficial effects of ghrelin treatment in chronic kidney disease is potentially mediated by multiple concurrent actions, including stimulation of appetite-regulating centers, anti-inflammatory effects and direct kidney effects. There remains a great deal of opportunity for distinguishing between these effects using animal models.

Appetite regulation

As mentioned in the section on important endpoints of treatment, increases in food intake are observed during ghrelin treatment of chronic kidney disease. In our model of chronic kidney disease, we hypothesized that effects on the central melanocortin system may be one mechanism of action in ghrelin’s effect and used real time RT-PCR to assess for changes in transcript of key mediators of melanocortin output. Treatment with ghrelin resulted in an increase in mRNA levels of the orexigenic peptide AgRP in the hypothalamus, while treatment with the ghrelin receptor agonist BIM 28131 resulted in a decrease in expression of prohormone convertase-2 (PC-2), a key regulator of POMC processing [164]. If these expression changes resulted in changes in translation and activity of these gene products they would be expected to result in an increase in orexigenic output (in the case of AgRP) and a decrease in anorexigenic output (in the case of PC-2). Interestingly, all of the nephrectomized groups showed a decrease in expression of POMC in the brainstem, though this was most striking in the BIM 28131-treated group [135]. This may indicate that though the melanocortin system exhibits expected responses to loss of body weight, including a decrease in some markers of anorexigenic output, that treatment with ghrelin can further affect output of this system—potentially suggesting that the system is not saturated.

We also noted a decrease in expression levels of the IL-1 receptor in the hypothalamus [135]. It is not clear whether this was due to direct effects of ghrelin in the hypothalamus or due to the decrease in overall inflammation (discussed below). Given the importance of inflammation for output of the central melanocortin system, this decrease may have affected melanocortin output and thus appetite. Overall, these changes in transcription suggest a role for ghrelin in appetite regulation in the setting of chronic kidney disease. Changes in resting energy expenditure, also regulated in part by hypothalamic centers, may also play a role in ghrelin’s effect on weight gain in chronic kidney disease but have not been studied.

Anti-inflammatory effects

Chronic kidney disease is the only animal model in which ghrelin treatment has been shown to decrease systemic inflammation. The potential for anti-inflammatory effects had been previously raised in experiments in which ghrelin was administered to peripheral mononucleocytes [165] and mice with an animal model of arthritis, as described below [166,167]. In the laboratory of Dan Marks, we assessed for levels inflammatory cytokines over the course of treatment, demonstrating a decrease in pro-inflammatory cytokines overall. This decrease in inflammation involved sizable decreases in individual cytokines including IL-6, TNF-α, IL-6 and granulocyte macrophage colony simulating factor [135]. In addition, treatment with one of the ghrelin receptor agonists resulted in an increase in the anti-inflammatory cytokine IL-10. It should be noted that these effects may have contributed to decreased activation of the central melanocortin system, as well as other peripheral effects on muscle and cardiac tissue, further complicating interpretation of inter-related mechanisms.

These changes may require long-term treatment, as the one-week ghrelin treatment among dialysis patients discussed previously failed to produce significant changes in inflammatory mediators [160].

Cardiac effects

As mentioned in the section regarding ghrelin treatment of chronic heart failure, ghrelin exhibits a large number of cardiovascular effects that may contribute to improved clinical endpoints in that setting. While the majority of these effects, including cardiac output and decreased sympathetic activity, have not been tested in the setting of renal cachexia, these effects may also prove beneficial in the setting of chronic kidney failure. Patients with renal failure frequently develop heart disease also, which could in turn contribute to worsening clinical course. These details deserve further attention. As mentioned in the section below regarding renal effects of ghrelin, there are vascular smooth muscle effects of ghrelin that have been evaluated in the kidney that may play a role in ghrelin treatment of chronic kidney disease in some settings as well.

Growth hormone-IGF-1 axis

The growth hormone-IGF-1 axis is important in the setting of chronic kidney disease as demonstrated by what appears to be a state of growth hormone resistance in this setting [168]. Testing the potential benefit of growth hormone in this setting, two randomized controlled trials have administered recombinant human growth hormone or placebo over a 6 month period to adults on hemodialysis. Growth hormone treatment resulted in 3-6 kg gains in lean body mass accompanied by a 3-6 kg decreases in fat mass [168,169]. This raises the potential that ghrelin could act in chronic kidney disease via increases in growth hormone levels, potentially overcoming the level of growth hormone resistance similarly to treatment with growth hormone alone.

We measured random growth hormone levels in the context of ghrelin treatment in the 5/6 nephrectomy model, finding an increase in growth hormone levels in ghrelin-treated animals above that seen in the sham-surgery group [135]. Still, there was no difference between the ghrelin and ghrelin receptor agonist treated groups and the saline-treated nephrectomy group. Similarly, there were no differences in IGF-1 levels between groups. Similarly, there was no difference in IGF-1 levels in the 1-week trial of ghrelin among dialysis patients. Thus, while the theoretical potential for action through the growth-hormone-IGF-1 axis remains, there is at this point no data supporting this hypothesis.

Muscle catabolism

The decrease in actinomycin cleavage during ghrelin cleavage raises further questions regarding ghrelin’s mechanism in preservation of lean mass in the setting of chronic kidney disease. While ghrelin receptor agonists have been demonstrated to bind to skeletal muscle [116], there also remains the possibility of effects via innervation or via overall improvements in nutrition or decreases in inflammation. Thus whether decreased ubiquitination represents a direct mechanism of ghrelin’s action remains a further opportunity for investigation.

Mitochondrial changes

Mitochondrial dysfunction in skeletal muscle has been noted in the setting of chronic kidney disease and may contribute to insulin resistance seen in chronic kidney disease [170–172]. In turn, these processes may be contribute to increased mortality in chronic kidney disease [173]. Similarly to the findings of ubiquitination, during ghrelin treatment of chronic kidney disease in the 5/6 nephrectomy model, mitochondrial enzyme activity was noted to improve, co-inciding with lower muscle triglyceride content and higher levels of phospohorylated AKT, a key intermediate of insulin action [174]. The mechanism of these apparent improvements of mitochondrial activity and insulin action in the setting of ghrelin treatment are unclear regarding whether these are due to direct effects at the level of the myocyte or due to improvements in the overall clinical course. This also requires further investigation.

Renal function and renal vasculature

As mentioned previously, direct or indirect effects on kidney function could represent another means of improvement seen in chronic kidney disease. Ghrelin receptors are expressed in the distal tubules of the kidney, providing a potential source of effect on renal function in individuals with functioning kidneys [175]. Moreover, infusion of ghrelin into the renal interstitium of uninephrectomized rats results in an increase in Na reabsorption compared to dextrose infusion, an effect which is blocked by co-infusion of a ghrelin receptor antagonist, further suggesting the potential for renal effect during treatment [176].

In their model of acute injury, Takeda et al delivered ghrelin as every 6-hour injections starting 2 days prior to renal artery occlusion [163]. In addition to improving levels of blood urea nitrogen, this treatment resulted in an increase in acetylcholine-induced endothelium-dependent relaxation of the renal vessels, reminiscent of ghrelin’s relaxation properties seen in cardiac settings [126]. A similar set of experiments by Rajan et al expanded on these findings by demonstrating improvements when ghrelin was only given after reperfusion (i.e., without the extended pre-treatment), finding that over the ensuing 24 hours creatinine levels rose by 75% over sham-surgery controls compared to a 275% increase in saline-treated mice after ischemia-reperfusion [177]. Still, others have failed to note the same protection [178]. While ghrelin’s vasodilatory effects may play a role in the beneficial effects of ghrelin treatment in these settings, it is unclear whether such effects on renal function after short-term ischemic injury have bearing on ghrelin’s effects as a treatment in chronic kidney disease. The lack of significant effects of ghrelin treatment on creatinine and blood urea nitrogen during chronic kidney disease suggests against an effect on overall renal function during long-term treatment.

Future Directions and How Animal Data Informs Human Investigation

Human trials of ghrelin’s effect in the setting of chronic kidney disease have paralleled many of the endpoints and mechanisms followed in animal models. The human trials have confirmed the appetite-stimulating effects of ghrelin over the course of a week’s treatment, as well as investigating short-term effects of ghrelin on decreasing systolic and diastolic blood pressure. Anti-inflammatory effects were not observed at the doses and time-course tested. Given benefits observed in animal models, longer-term treatment studies in humans may be needed. Additionally, human studies will be needed to test the effect of longer-acting ghrelin receptor agonists in chronic kidney disease, as has been performed in animal models. Effects such as the decrease in blood pressure seen in the first hour after sub-cutaneous injection with ghrelin itself may be sustained for longer periods following injection of an agonist with a longer serum half-life. Ghrelin itself has a half life of approximately 30 minutes in humans, while small molecule GHS-1a agonists have as much as a 10-fold longer half-life [135].

As in the prior disease states associated with cachexia, further experiments in animal models of chronic kidney disease are needed to confirm the past findings and to isolate variables to better differentiate mechanisms behind these systemic effects. In particular, the extent of ghrelin’s cardiovascular effects on blood pressure and cardiac output in the setting of chronic kidney disease is unknown and represents a potential means by which ghrelin could improve clinical status among patients on dialysis.

Other animal models of disease

Chemotherapy

Individuals receiving chemotherapy exhibit striking decreases in appetite, weight loss and decreases in intestinal motility [67]. Perhaps most striking during chemotherapy treatment is the high incidence of nausea, vomiting and food avoidance caused by both central effects and intestinal effects [179]. In this way, chemotherapy shares multiple symptoms with cachexia, though it likely has a different set of underlying etiologies. Nevertheless, given ghrelin’s action on both hunger and gastrointestinal motility, ghrelin has been tested in animal models this setting.

Further underscoring differences in underlying pathophysiology as contrasted to cancer cachexia, during chemotherapy, ghrelin levels in the serum and ghrelin production in the hypothalamus have been shown to be decreased in humans in conjunction with cisplatin treatment [180,181]. These lower levels raise potential for less ghrelin resistance in this setting compared to the cachexia syndromes discussed above.

Specific chemotherapy agents

One common chemotherapy is cisplatin, which results a pronounced anorexia [67]. In animal models of cisplatin, rats consume 70-80% less chow in the days following cisplatin injection than seen in controls [182]. Liu et al reported that treatment with ghrelin (166 nmol/kg/d) resulted in a rapid increase in food intake over the ensuing hour, representing a 16-fold increase over vehicle-treated rats receiving cisplatin. Despite this rapid increase, ghrelin-treated rats ate a similar amount to the vehicle-treated rats over the first 24-hours but then ate twice as much as the vehicle-treated rats over the second day of treatment, suggesting a potential sustained effect. There was also a non-significant improvement in body weight preservation among the ghrelin-treated group.

Garcia et al also evaluated ghrelin’s effect on cisplatin-induced dyspepsia and reported a doubling of food intake over the 3 days following cisplatin treatment in rats receiving ghrelin (530 nmol/kg/d) compared to saline-treated rats [183]. The ghrelin-treated group also exhibited a more modest increase in food intake over the next 10 days of treatment as well. Ghrelin treatment partially protected against the decrease in IGF-1 levels seen in all groups treated with cisplatin.

Ghrelin effects have also been tested in the setting of doxorubicin chemotherapy. Using a rat model of doxorubicin exposure one set of researchers demonstrated a protective effect of hexarelin on myocardial ischemia-reperfusion [118] while another set of investigators reported a decrease in gastrointestinal epithelial damage during doxorubicin treatment [184].

Finally, the ghrelin receptor agonist GHRP-2 has also been tested in an intriguing mouse model of both implanted tumor (colon-26 cancer cells) and chemotherapy with 5-fluoruracil (5-FU)[185]. In this setting, treatment with GHRP-2 resulted in an early increase in food intake and body weight as compared to the tumor-bearing mice receiving 5-FU alone, though this difference appeared to dissipate over time. Interestingly, the mice treated with GHRP-2 and 5-FU had a non-significant trend toward longer median survival time (18 d 5-FU/GHRP-2 vs. 15.5 d for 5-FU alone)[185].

Mechanism

The mechanism of ghrelin’s effect on food intake and weight gain in the setting of chemotherapy is not known, though clearly central stimulation of appetite centers and improved gastric emptying may play roles. It is interesting to note that despite a trend toward gastrointestinal discomfort in some randomized, controlled studies of ghrelin administration [186] ghrelin has been given to good effect as a treatment in states of vomiting [187] and gastroparesis [188–190], suggesting that gastrointestinal properties may contribute to the improved food intake in the setting of chemotherapy.

Aging

Elderly individuals frequently exhibit a decrease in appetite, as well as a loss of lean body mass commonly referred to as sarcopenia [191,192]. Animal models of aging have replicated these findings, exhibiting a 10-15% declines in food intake and body weight after they reach 2 years of age [193]. In one set of experiments, ghrelin treatment (10 nmol/kg IV) resulted in short-term increases in food intake and growth hormone levels [193]. Another set of researchers, Yukawa et al, investigated the effect of chronic ghrelin administration (1 mg/kg/d), demonstrating consistent maintenance of body weight compared to a 2.5% loss of body weight in saline-treated animals [194]. They then used similarly aged animals that had undergone a surgery to cut down the external jugular vein and administered ghrelin (1 mg/kg/d SC) or saline, finding a 0.2% weight gain in ghrelin-treated rats compared to a 3% weight loss in saline-treated rats. These results using animal models of aging parallel a randomized, placebo-controlled trial in elderly adults treated with MK-677, an oral ghrelin receptor agonist. Given over a 2-year period, the treatment resulted in increased weight gain and increase in lean mass in MK-677-treated individuals, paralleling increases in growth hormone and IGF-1 [195]. These benefits have raised the question of whether such treatment should be offered in elderly individuals with anorexia [196].

Thermal injury

Thermal injury can result in a sharp increase in basal metabolism that is at least in part related to high levels of inflammatory cytokines [197] and catabolic hormones [198] and low levels of anabolic hormones, including growth hormone [199,200]. These processes combine to produce concerning losses of lean mass during tenuous recovery [200]. Ghrelin production in this setting is greatly decreased through unclear mechanisms. Ghrelin treatment in an animal model of burn injury (open flame burn) resulted in increases in short-term food intake and a doubling of growth hormone levels [199]. Other effects noted include a decrease in gastoparesis [201] and a decrease in skeletal muscle protein catabolism [202]. These processes may be related to a decrease in inflammatory cytokines [203] and a decrease in expression of ubiquitin enzymes [202].

Arthritis

Animal models of arthritis provided the first evidence for anti-inflammatory effects of ghrelin treatment. In one of these animal models Grandao et al injected rats with Freund’s adjuvant, resulting in arthritis with increases in paw swelling as well as decreased weight gain [167]. Associated with this arthritis, serum ghrelin levels increased along with decreases in leptin levels. What may be most surprising is that the amount of paw swelling and the severity of arthritis score were lower in the setting of growth-hormone releasing peptide 2, co-incident with decreases in serum IL-6. The investigators then tested peripheral monocytes by exposing them to LPS, revealing a decrease in expression of IL-6 when these moncytes were co-incubated in the presence of ghrelin or GHRP-2.

Granado et al. continued their investigation by evaluating changes in muscle fibers seen in their arthritis model [166]. Treatment with GHRP-2 decreased expression of TNF-α in these muscles in the setting of arthritis, as well as decreasing expression of genes for ubiquitin degradation ligases MuRF1 and MAFbx.

The adjuvant model of arthritis may exhibit differences from human experiments regarding ghrelin regulation. In studies of humans with arthritis, ghrelin levels are lower than in controls [204–206], which may reflect differences in the extent of weight loss between the animal model and human disease. Nevertheless, ghrelin levels increase following anti-TNF-α therapy [204,207]—notable results since the animal models demonstrated a decrease in inflammation during ghrelin receptor agonist treatment. This further underscores overlapping relationships between ghrelin and inflammation and emphasizes the need for further mechanism-based experiments.

Gastroparesis

Deficiencies in stomach and intestinal motility present clinical abnormalities for which ghrelin application has a high likelihood to offer benefit [208]. Gastroparesis is a common sequela of diabetes and is also present following surgery and chronic gastritis, and ghrelin’s pro-motility properties have been shown in animal models to improve gastric emptying.

Pointras et al used a post-operative model of illeus and demonstrated that the ghrelin receptor agonist RC-1139 increased gastric emptying rate up to 42% in a dose-dependent fashion [209]. Qiu et al assessed the effect of ghrelin and GHRP-6 in mouse [210] and guinea pig [211] models of diabetic gastroparesis, reporting increased rates of gastric emptying during administration of both compounds. Frasier et al used another ghrelin agonist TZP-101 to demonstrate an increased rate of gastric emptying in non-diseased animals [188].

Interestingly, gastroparesis represents a disease state in which human trials have exceeded animal models, with trials of multiple agents with significant improvement in clinical endpoints [187,190,212–216], suggesting that gastroperesis may represent the disease state for which ghrelin will first be approved for clinical use. If this occurs, it will be interesting to observe whether the increase in ghrelin receptor agonist availability will enable wider-spread testing of ghrelin’s effects in both animal models and human diseases.

Inflammatory Bowel Disease

Inflammatory bowel disease is an immune-mediated process resulted in denuded intestinal mucosa, frequently leading to abdominal pain and bloody diarrhea [217]. While not a cause of cachexia per se, IBD frequently results in a decrease in body weight similar to classical forms of cachexia. Other manifestations include decreased bone mineral density [218] and—when IBD occurs prior to puberty—suppression of growth and delay of puberty [219–222].

Ghrelin has been demonstrated to have beneficial effects on colitis in animal models of IBD [223]. In mice that had colitis induced with tri-nitrobenzene sulfate (TNBS) [224,225] and dextran sodium sulfate (DSS) [224] ghrelin treatment resulted in improved weight gain and an amelioration of intestinal inflammation. The effect of ghrelin on DSS colitis was explored by another group as well, who reported worsening in the setting of ghrelin treatment [226]. DSS colitis is a chemical model and past experiments have demonstrated worsened course in the setting of inflammatory blockade [227]. TNBS colitis, in which ghrelin has thus far exhibited consistent improvements, is immune mediated process and as such is a better model of human disease [228].

It is interesting to note that enteral nutrition is an effective treatment option in treatment of Crohn’s disease [229,230]. Thus, the increase in food intake during ghrelin treatment, mimicking enteral feeding, is a potential mechanism by which ghrelin may cause improvements in the course of colitis. Ghrelin’s effects on motility and its anti-inflammatory effect may also contribute to the findings [208]. Overall, however, the mechanisms behind these changes are unclear and IBD represents another model system in which much more data is needed regarding a potential role for ghrelin in treatment.

Unanswered Questions and Future Uses

As a hormone with widespread receptors and actions, ghrelin offers significant potential benefits to a variety of human diseases. As demonstrated in animal models, ghrelin’s actions related to energy balance, cardiovascular effects, anti-inflammatory effects and motility all represent potential avenues whereby ghrelin may confer improvements in human disease. Animal models—for example using genetic manipulations in which the ghrelin receptor is expressed more narrowly in tissues of interest—will prove particularly useful in demonstrating the specific mechanisms among these effects are most responsible for normalization in endpoints in disease states. These experiments may demonstrate synergy in different effects of ghrelin (e.g. cardiac effects and anti-inflammatory effects) that could be taken advantage with addition of yet other agents. Determining mechanism may also identify parameters most helpful for monitoring response to therapy.

Nevertheless, the widespread effects of ghrelin also raise the potential for unforeseen side effects. Thus far, the safety profile of ghrelin has remained favorable [36]. However, future studies using animal models (and later human application) will be needed to investigate for side effects during therapy (e.g. for hypotension, diarrhea, etc.). For some of these issues will be necessary to test in human subjects for issues such as gastrointestinal discomfort—which was suggested by one human trial [186] and which may be more difficult to score among animal models.

Thus far ghrelin has shown promise as a treatment tool, and while the field has accumulated a significant amount of knowledge regarding ghrelin’s actions there remain questions in each of the potential treatment settings that beg for further investigation, with hope that these efforts will enable optimizing use of ghrelin-related compounds to improve outcomes of human disease.

Figure 1. Potential mechanisms of ghrelin action in experimental models of disease.

Ghrelin acts on the GHSR-1a in widespread tissues, causing several effects that might contribute to efficacy in features pertaining to multiple disease states.

Table 3.

Summary of animal models of ghrelin and ghrelin receptor agonists for the treatment of cachexia due to chronic kidney disease.

Publication [reference]	Underlying Disease Model (species)	Treatment Agent	Dose, Route, Duration of treatment	Effects
DeBoer et al., 2008 [135]	5/6 nephrectomy (rats)	ghrelin, BIM-28125, BIM-28131	150 nmol/kg/d as continuous mini-pump infusion for 14 d	Total mass: ghrelin +22%***; BIM-28125 +27%***; BIM-28131 +21%***; vehicle +1% Food intake (cumulative): ghrelin 19% more than vehicle*; BIM-28125 19% more than vehicle*; BIM-28131 20% more than vehicle* Lean body mass: ghrelin +26%***; BIM-28125 +29%***; BIM-28131 +24%***; vehicle +2.8%

Significance: NS=not significant (p>0.05);

^*p<0.05;

^**p<0.01;

^***p<0.001.

Table 4.

Summary of animal models of ghrelin and ghrelin receptor agonists for multiple models of disease.

Publication [reference]	Underlying Disease Model (species)	GHS-1a Agent	Dose, Route, Duration of treatment	Effects
Chemotherapy
Liu et al., 2006 [182]	Cisplatin	Ghrelin	166 nmol/kg/d IP × 2 days	Food intake: 1st 24 hrs: similar food intake for cisplatin/ghrelin and cis/saline; 2nd 24 hrs: 100% increase food intake cis/ghrelin vs. cis/saline
Garcia et al., 2008 [183]	Cisplatin (rats)	Ghrelin	530 nmol/kg/d SC × 3 days	Food intake: 1st 3 days: cis/ghrelin 116% increase vs. cis/saline; next 10 days: 5.4% increase in cis/ghrelin vs. cis/saline Body weight change: +22.5% cis/ghrelin vs. +14.8% cis/saline**
Perboni et al., 2008 [185]	5-fluorouricil (mice)	GHRP-2	10 ug/mouse SC daily x up to 18 days	Food intake: 1st 24 hrs: 5-FU/GHRP-2 43% above 5-FU alone (NS); Body weight change: similar weight loss between 5FU/GHRP-2 and 5FU (NS)
Aging
Yukawa et al., 2008 [194]	>2 y.o. rats + surgery	Ghrelin	1 mg/kg/d SC × 14 or 17 days	Food intake: no cumulative differences between groups Body weight change: elderly rats: ghrelin -0.2% vs. saline -2.5%* over 17 days; elderly rats after minor surgery +0.2% ghrelin vs. -3% saline* over 14 days
Thermal injury
Balasubramaniam et al., 2006 [199]	Open-flame burn (rats)	Ghrelin	30 nmol/rat, IP on days 3, 5,7,9 after burn	Food intake: 2 hrs after injection, 11 d average: ghrelin 1.55 g vs. saline 0.45 g *; overnight food intake ghrelin 16.5 g vs. saline 12.4 g
Arthritis
Granado et al., 2005 [167]	Adjuvant injection (rats)	GHRP-2	100 ug/kg SC × 7 days	Body weight change: GHRP-2 +14 g vs. saline -+9.5 g* Paw volume: GHRP-2 3.05 mL vs. saline 3.85 mL**
Gastroparesis
Pointras et al., 2005 [209]	Post-operative ileus (mice)	RC-1139	Dose response, up to 10 mg/kg × 1 inj	Gastric residue: decrease vs. control from 25% (1 mg/kg RC-1139) to 42% (10 mg/kg)
Qui et al., 2008 [210]	Diabetes gastroparesis (mice)	Ghrelin, GHRP-6	Dose response, up to 0.2 mg/kg IP × 1 inj	Gastric emptying rate: increase vs. control from 23% (0.05 mg/kg) to 45% (0.2 mg/kg)
Inflammatory Bowel Disease
Gonzalez-Rey et al., 2006 [224]	TNBS and DSS colitis (mice)	Ghrelin	2 nmol/mouse IP × 1 12 hrs after TNBS induction	Body weight change: ghrelin +10% vs. saline -14% * Colitis score (day 10): ghrelin +10% above control vs. saline +400% above control*
Konturek et al., 2009 [225]	TNBS colitis (rats)	Ghrelin	20 ug/kg IP daily × 11 days	Colonic lesion area: ghrelin 20.5 mm2 vs. saline 39.5 mm2
deSmet et al., 2009 [226]	DSS colitis (mice)	Ghrelin	100 nmol/kg IP twice daily × 10 days	Food intake: ghrelin-treated mice ate 24% less than saline-treated mice* Body weight change: ghrelin -12% vs. saline -6%*

Significance: NS=not significant (p>0.05);

^*p<0.05;

^**p<0.01;

^***p<0.001.