Introduction
Human ghrelin, a 28-amino acid peptide, is predominantly synthesized by the stomach and is the only identified endogenous ligand for the growth hormone secretagogue receptor 1a (GHS-R1a) [
1]. GHS-R1a is expressed in various tissues in different concentrations and has been found in pituitary, hypothalamus, heart, blood vessels, lung, pancreas, intestine, kidney, adipose tissue, B- and T-cells and neutrophils [
2‐
4]. This wide distribution of GHS-R1a suggests multiple roles of ghrelin with regard to cerebral, renal and pulmonary function, hemodynamics, gut barrier and immune system. Nevertheless, about two third of the circulating ghrelin is derived from the stomach and nearly all of the remaining one third from the small intestine [
5,
6]. Rat ghrelin is very similar to human ghrelin and differs only by two amino acids [
7]. Therefore animal models have been widely used to investigate potential functions of ghrelin. Ghrelin stimulates growth hormone secretion in rats and humans, regulates food intake and energy homeostasis and has vasodilatatory effects as a physiological antagonist of endothelin-1 [
8‐
10]. Moreover, protective effects of ghrelin in animal sepsis models have been reported. Specifically, ghrelin was found to mediate improvement of tissue perfusion in severe sepsis [
11], down-regulation of proinflammatory cytokines in sepsis through activation of the vagus nerve [
12], stabilization of gut barrier function in sepsis [
13], attenuation of sepsis-induced acute lung injury [
14] and protection against endotoxemia-induced acute kidney failure [
15]. On the basis of these findings GHS-R1a has been regarded as a possible drug target in critical care medicine with ghrelin or a ghrelin mimetic as a new therapeutic option [
16].
Nevertheless, the findings on the responses of ghrelin to endotoxin in animal models and in healthy humans are partially contradictory and inconsistent. Beyond that, there are no data concerning the mechanisms of regulation in critically ill patients from large cohorts. Before testing the possible therapeutic effects of ghrelin in humans, clinical studies on profiles of endogenous ghrelin regulation in the critically ill have been demanded [
17]. Up to now, there has been only a small pilot study with 16 ICU patients, reporting low initial, but, during ICU treatment, increasing ghrelin serum concentrations [
18]. The present study was conducted with a large cohort of well characterized critically ill patients to provide information on ghrelin serum concentrations in different circumstances of critical disease, to identify possible pathogenic functions of ghrelin by correlations with a wide number of markers of inflammation, organ dysfunction and metabolism and to examine potential protective effects of ghrelin in critically ill patients and human sepsis from a clinical point of view.
Materials and methods
Study design and patient characteristics
The present study was approved by the local ethics committee. Before inclusion, written informed consent was obtained from the patient, his or her spouse or the appointed legal guardian. We studied 170 patients (111 male, 59 female with a median age of 62 years; range 18 to 86 years) (Table
1) [
19]. Patients were included consecutively upon admission to the Medical ICU of the RWTH University Hospital Aachen due to critical illness. Patients were excluded from this study, if they were expected to have a short-term (<72 h) intensive care treatment, for example, due to post-interventional observation or acute intoxication. All patient data, clinical information and blood samples were collected prospectively.
Table 1
Baseline patient characteristics and ghrelin serum concentrations
Number | 170 | 122 | 48 |
Sex (male/female) n | 111/59 | 81/41 | 30/18 |
Age median (range) (years) | 63 (18 to 86) | 64 (20 to 86) | 60 (18 to 79) |
APACHE-II score median (range) | 14 (0 to 31) | 14 (0 to 31) | 15 (0 to 31) |
SAPS2 score median (range) | 44 (0 to 80) | 45 (0 to 79) | 41 (13 to 80) |
ICU days median (range) | 8.5 (1 to 137) | 10 (1 to 137) | 6 (1 to 45) |
Hospital days median (range) | 27 (2 to 151) | 30 (2 to 151) | 14 (2 to 85) |
Death during ICU n(%) | 54 (32%) | 42 (34%) | 12 (25%) |
Death during follow-up n(%) | 88 (52%) | 64 (53%) | 24 (50%) |
30-day mortality [%] | 32% | 32% | 31% |
60-day mortality [%] | 39% | 40% | 35% |
90-day mortality [%] | 42% | 44% | 35% |
180-day mortality [%] | 45% | 48% | 38% |
1-year mortality [%] | 49% | 51% | 44% |
Mechanical ventilation n(%) | 113 (67%) | 82 (67%) | 31 (65%) |
Ventilation time median (range) [h] | 66 (1 to 2,966) | 127.5 (1 to 2,966) | 31 (1 to 755) |
pre-existing diabetes n(%) | 56 (33%) | 39 (32%) | 17 (35%) |
BMI median (range) (m²/kg) | 25.8 (14.0 to 59.5) | 26.0 (14.0 to 59.5) | 25.1 (17.5 to 53.3) |
Serum IGF-1 median (range) (μg/L) | 54 (25 to 295) | 54 (25 to 295) | 49 (25 to 165) |
Serum growth hormone median (range) (μg/L) | 1.5 (0.1 to 128.0) | 1.3 (0.1 to 128.0) | 2.0 (0.1 to 22.3) |
Serum ghrelin median (range) (pmol/L) | 18.4 (5.0 to 129.5) | 18.4 (5 to 113.8) | 18.4 (5 to 129.5) |
Blood samples of 60 healthy non-diabetic blood donors (33 male, 27 female, with a median age of 46 years; range 31 to 58 years) with normal values for blood counts, C-reactive protein and liver enzymes have been examined as a control group.
Characteristics of sepsis and non-sepsis patients
A total of 122 of the 170 critically ill patients (72%) enrolled in this study, fulfilled the criteria of bacterial sepsis, according to the American College of Chest Physicians and the Society of Critical Care Medicine Consensus Conference Committee for severe sepsis and septic shock [
20]. In the majority of sepsis patients the identified origin of infection was pneumonia (Table
2). Non-sepsis patients were admitted to the ICU due to cardiopulmonary disorders (myocardial infarction, pulmonary embolism, and cardiac pulmonary edema), decompensated liver cirrhosis or other critical conditions and did not differ in age or sex from sepsis patients. As expected, significantly higher levels of laboratory indicators of inflammation (that is, C-reactive protein, procalcitonin, white blood cell count) were found in sepsis patients than in non-sepsis patients (Table
1, and data not shown). Both groups did not differ in acute physiology and chronic health evaluation (APACHE II) score, vasopressor demand, or laboratory parameters indicating liver or renal dysfunction (data not shown). ICU-mortality of all critical care patients was 32%, and 52% of the total initial cohort died during the overall follow-up of 900 days (Table
1).
Table 2
Disease etiology of the study population
Etiology of sepsis critical illness
| | |
Site of infection n (%) | | |
Pulmonary | 72 (59%) | |
Abdominal | 22 (18%) | |
Other | 28 (23%) | |
Etiology of non-sepsis critical illness
| | |
n (%) | | |
Decompensated liver cirrhosis | | 17 (35%) |
Cardiopulmonary diseases | | 18 (38%) |
Other | | 13 (27%) |
Comparative variables
The patients in the sepsis and non-sepsis groups were compared by age, sex, body mass index (BMI), pre-existing diabetes mellitus and severity of disease using the APACHE II score upon admittance to the ICU. Careful recording of intensive care treatment, such as volume therapy, vasopressor infusions, demand of ventilation and ventilation hours, antibiotic and antimycotic therapy, renal replacement therapy and nutrition, has been performed. Additionally, a large number of laboratory parameters that were routinely assessed during intensive care treatment have been analyzed.
Quantification of ghrelin, IGF-1 and growth hormone serum concentrations
Peripheral venous blood samples were obtained at admission before therapeutic intervention, immediately placed on ice, centrifuged and stored at 80°C. All patients had been fasting for at least three hours before admission to the ICU. All measurements were performed in a blinded fashion. Ghrelin serum concentrations were measured using an enzyme-linked immunosorbent assay (ELISA) according to manufacturer's instructions (Millipore, Schwalbach, Germany). Furthermore, growth hormone (Immulite 2000 hGH, Siemens, Erlangen, Germany) and IGF-1 (Immulite 2500 IGF-1, Siemens, Erlangen, Germany) were measured by chemiluminescent immunometric assay in the routine clinical laboratory.
Statistical analysis
Due to the skewed distribution of most of the parameters in critically ill patients, data are given as median and range. Differences between two groups were assessed by Mann-Whitney-
U-test and multiple comparisons between more than two groups have been conducted by Kruskal-Wallis-ANOVA and Mann-Whitney-
U-test for post hoc analysis. Box plot graphics were employed to illustrate comparisons between subgroups. They display a statistical summary of the median, quartiles, range and extreme values. The whiskers extend from the minimum to the maximum value excluding outside and far out values which are displayed as separate points. An outside value (indicated by an open circle) is defined as a value that is smaller than the lower quartile minus 1.5-times interquartile range, or larger than the upper quartile plus 1.5-times the interquartile range. A far out value is defined as a value that is smaller than the lower quartile minus three times the interquartile range, or larger than the upper quartile plus three times the interquartile range. All values, including
outliers, have been included for statistical analyses [
19]. Correlations between variables have been analysed using the Spearman correlation tests, where values of
P <0.05 were considered statistically significant. The prognostic value of the variables was tested by univariate and multivariate analysis in the Cox regression model. Kaplan Meier curves were plotted to display the impact on survival. All statistical analyses were performed with SPSS version 12.0 (SPSS, Chicago, IL, USA).
Discussion
Contradictory findings on the responses of ghrelin to endotoxin in animal models have been reported, and data are not fully consistent [
21,
22]. Current data either demonstrated increased or decreased ghrelin concentrations after administration of endotoxin. In a rat model of cecal ligation and puncture (CLP) induced sepsis, significantly decreased ghrelin serum concentrations at early (5 h after CLP) and late (20 h after CLP) stages of sepsis were reported. In contrast, elevated ghrelin serum concentrations and a strong association of ghrelin to markers of inflammation and hepatic and renal function were observed in dogs after endotoxinaemia [
22]. As a conclusion of this study, elevated ghrelin levels have been considered as an “adaptive protective response to endotoxin”. These findings are supported by a previous study in rats where serum ghrelin levels were found to be significantly increased upon endotoxin shock [
26]. Furthermore, this study demonstrated a therapeutic effect of ghrelin infusion by the means of a significantly decreased mortality rate and ameliorated hypotension due to septic shock.
Little is known about the function of ghrelin in sepsis in humans. A study on healthy volunteers identified ghrelin as one of the first hormones increasing in the physiological response to endotoxinaemia [
17]. There are no data on the profiles of circulating ghrelin levels in critically ill patients treated at a medical ICU. In a small study of 25 surgical patients with postoperative intraabdominal sepsis elevated ghrelin levels have been reported [
27]. In contrast, a study of 16 surgical and non-surgical ICU patients showed significantly reduced serum ghrelin levels [
18]. Before promoting ghrelin as a new therapeutic target in intensive care medicine it is (in our opinion) essential to elucidate the regulation of ghrelin in human critical illness, sepsis and septic shock from a clinical point of view. In the present study we can demonstrate for the first time in a large, well characterized cohort of patients from a medical ICU that ghrelin levels are significantly elevated in all critically ill patients as compared to healthy controls, albeit with a considerable overlap between both groups (Figure
1a). Ghrelin serum concentrations did not differ between sepsis and non-sepsis patients, which might indicate that high serum ghrelin levels rather reflect the impact of critical disease than being directly influenced by inflammatory cytokines in sepsis or septic shock (Figure
1b). According to this we could not demonstrate any correlation of ghrelin with
classical markers of inflammation as white blood cell count, C-reactive protein, procalcitonin, TNF-α or IL-6.
Ghrelin stimulates physiologically growth hormone (GH) secretion independent of hypothalamic GH-releasing hormone and causes weight gain and obesity by increasing food intake and diminishing lipid utilisation in non-critically ill individuals [
28,
29]. Before a meal ghrelin serum levels rise and show an abrupt decline at the beginning of food intake with trough levels within one hour after eating [
30]. Ghrelin serum levels are decreased in obese patients and elevated in patients with anorexia nervosa [
31,
32]. In our study population of critically ill patients we could not establish a significant correlation between ghrelin and the body mass index as a parameter reflecting the nutritional status upon admission to ICU. This observation strongly indicates that ghrelin regulation is not primarily driven by the current nutritional status, but by mechanisms related to the stress of critical disease.
In fact, the mechanisms of ghrelin release are not satisfyingly understood at present. The most important factor is food intake, but possibly blood glucose and insulin may participate in regulation [
24]. However, we found a close inverse correlation of ghrelin with serum glucose and insulin only in the subgroup of non-sepsis patients (Figure
3b, c), but not in sepsis patients. It is therefore very likely that additional, so far not apparent factors in the complex and multifactorial metabolic disturbance in critically ill patients impact serum ghrelin. Similarly, the growth hormone and IGF-1 axis, both targets of physiological ghrelin effects, have been reported to be heavily deranged in ICU patients [
33]. However, direct therapeutic intervention by administration of growth hormone in critically ill patients resulted in increased mortality [
34]. This underlines that the changes of metabolism in critical illness and sepsis are complex, multifactorial, and future studies are warranted to unravel these interactions.
Furthermore, we could identify high ghrelin levels as a prognostic marker for survival at the ICU in sepsis patients (Figure
4). Assuming that high ghrelin levels have protective effects in sepsis, as demonstrated by ghrelin administration in several animal studies [
11,
13‐
15], our findings support the concept to view ghrelin upregulation as beneficial in severe sepsis and septic shock in humans. Several mechanisms may concertedly mediate the benefical effect of circulating ghrelin. Specifically, intravenous Ghrelin administration in healthy humans or animal studies has been found to reduce peripheral vascular resistance and increase cardiac output without a significant change in heart rate, resulting in improved tissue perfusion [
11,
35]. Ghrelin also exerted protective effects in an experimental model of acute, endotoxin-induced kidney failure [
15]. Furthermore, ghrelin mediated protective effects on pulmonary function though inhibition of NF-κB in an animal model of acute lung injury [
14]. Regarding the necessity of mechanical ventilation as a surrogate parameter of pulmonary function in patients, we could demonstrate significantly higher ghrelin serum concentrations in spontaneous breathing critically ill patients as compared with mechanically ventilated patients (Figure
4b). That might advert to pulmonary protective effects of high ghrelin serum concentrations in critically ill patients, keeping in mind that ghrelin regulation is most likely multifactorial and not fully understood.
Conclusions
We could demonstrate, for the first time, high ghrelin levels in critically ill patients as compared to healthy controls, independent of the presence of sepsis or inflammatory markers. Moreover, high ghrelin levels were a positive predictor of ICU-survival in sepsis patients, matching previous results from animal models. Nevertheless, the regulation of cytokines, adipokines and hormones with metabolic functions in critical illness is complex, and both, future experimental and clinical studies are needed to identify and evaluate ghrelin as a potential new therapeutic agent in critical care medicine.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
AK, FT and CT designed the study, analyzed data and wrote the manuscript. ES, AH and SV collected data and assisted in patient recruitment.