Effects of age, malnutrition and refeeding on the expression and secretion of ghrelin

Methods Four groups were prospectively enrolled: 11 undernourished elderly (80 ± 6 y, BMI: 17.4 ± 1.9 [Mean ± SD]), nine well-nourished elderly (76 ± 9 y, 23.5 ± 2.0), 10 undernourished young (26 ± 6 y, 15.1 ± 1.9) and 10 well-nourished young (34 ± 8 y, 22.2 ± 2.7). Fasting and postprandial plasma ghrelin and other hormones (every 30 min) were measured at baseline and after a 21-day enteral nutrition in malnourished patients. Gastric ghrelin mRNA levels were measured by RT-PCR at baseline in all subjects. Results Ghrelin was significantly higher in undernourished (2151 ± 871 ng/L) than in well-nourished (943 ± 389 ng/L) adults, whereas there were no differences between undernourished (1544 ± 758 ng/L) and well-nourished (1154 ± 541 ng/L) elderly. Refeeding did not influence ghrelin levels. Gastric ghrelin mRNA levels were similar in all groups. Conclusions There is an absence of malnutrition-induced increase of plasma ghrelin levels in elderly subjects. This feature, post-transcriptional, may be important in the lack of adaptation of elderly subjects to malnutrition. Keywords Elderly Malnutrition Anorexia Ghrelin Enteral nutrition Non-standardized abbreviations LMI lean mass index FMI fat mass index BMC bone mineral content HOMA-IR homeostatic assessment model for insulin resistance Introduction Protein energy malnutrition is frequent in elderly subjects, a major determinant of morbidity, mortality, disability and health costs in this growing population. It is a multifactorial process, but age-related decrease in food intake may be one of the main risk factors for malnutrition in old age. 1,2 Roberts et al. have clearly demonstrated that after a period of underfeeding elderly people do not increase their food intake over their maintenance needs, contrarily to what young subjects do, and therefore fail to regain the weight lost or regain it at a slower rate. 3 A vicious circle can appear where on top of age-induced anorexia, disease adds another bout of anorexia decreasing energy intake further. 4 Food intake is regulated by numerous factors of both central (e.g. cocaine and amphetamine regulated transcript, neuropeptide Y) or peripheral origin (e.g. insulin, leptin, ghrelin) 5 which interact to determine a feeding response. Ghrelin is an orexigenic peptide produced primarily by the stomach. 6 Plasma levels of ghrelin have been reported to rise before and to fall after a meal. 7,8 Ghrelin causes hunger in humans and increases food intake in animals, 9,10 and it increases body weight when administered chronically to rodents. 9 Moreover, ghrelin is implicated in long-term body-weight regulation, in part because its levels display adaptive responses to body-weight alterations, at least in young people. Although many studies have demonstrated a compensatory increase of ghrelin levels in response to weight loss resulting from various conditions in younger adults, 11–14 few studies have been performed to investigate the effects of ageing on ghrelin regulation. It has been reported that ghrelin levels in healthy older individuals were lower than those in healthy younger people. 15 Other investigators found that ghrelin levels in undernourished older women were higher than those of well-nourished older and younger women, but no significant difference in ghrelin was noted between well-nourished older and younger women. 16 In both studies, only overnight fasting ghrelin levels were measured. However, the profile of secretion after meal initiation is not known in older individuals. The aim of the present study was to assess the respective effects of age, malnutrition and refeeding on ghrelin expression and secretion (both in the fasting and postprandial states). Materials and methods Patients Forty subjects were recruited into four groups, depending on their age (adults/elderly) and their nutritional status (well-nourished, malnourished). Undernourished subjects were eligible to receive cyclic enteral nutrition (CyEN) for 3 weeks. Undernourished subjects were included in the study if they had a body mass index below 18.5 (before the age of 70) or below 21 (after the age of 70) and/or a weight loss greater than 10% in three or less months. The cut-off for body mass index in the elderly was taken from French 17 and international recommendations and from the literature. 18 All patients suffered from starvation, either due to anorexia nervosa (8 adults), depression (2 adults and 7 elderly), or anorexia three to five weeks following an acute condition such as surgery (2 elderly) or infection (2 elderly). All subjects were ambulatory and encouraged to eat normally during the day. Subjects with an ongoing infection, a wasting disease such as chronic renal, respiratory, liver, or cardiac failure, cancer or diabetes mellitus, or with C-reactive protein concentrations higher than 10 mg/L were excluded. We also excluded subjects who were taking steroids, antibiotics or other drugs that may interfere with gastrointestinal motility, visceral sensitivity, oral intake or metabolism, and those who had undergone gastrectomy or vagotomy. Controls were selected in the out-patient clinic among volunteers without any identifiable disease and with a normal nutritional status and were subsequently paid. Prospective food records ascertained stability of their food intake. The acceptance rate for recruitment was over 75% and no comparison was performed between participants and non-participants. All subjects gave their written informed consent, and this study was performed according to the ethical rules for human experimentation according to the Declaration of Helsinki. The local Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale approved the study protocol. Experimental design The measurements were conducted over a two-day period. On the first day, all subjects underwent a body composition assessment as well as an upper gastrointestinal endoscopy. On the morning of the second day, all subjects underwent a metabolic assessment after a 12-h overnight fast. At 7 a.m., resting energy expenditure was measured by indirect calorimetry. At 8 a.m., subjects were given ad libitum breakfast (26 ± 12% of their daily resting energy expenditure) over a 20-min period. Venous blood samples were taken every 30 min starting 1 h and ending 4 h after snack consumption, for the determination of ghrelin, leptin, growth hormone, glucose, free fatty acids and insulin. Hunger and satiety were measured at the same time. In the 14 malnourished subjects (7 young and 7 elderly) who completed a 21-day CyEN period, all measurements were repeated at the end of the 21-days. Energy intake In all subjects, the daily amount of food eaten and the macronutrient distribution of energy were quantified. For subjects undergoing CyEN, oral and enteral energy and protein intake were measured by the same dietician. Food of known composition was selected for breakfast, lunch and dinner and weighed before and after serving. The difference between the serving weight and leftover weight represented the consumption. Snack consumption was also taken into consideration and carefully recorded by the nursing staff. Blood samples After centrifugation at 3000 rounds per minute for 15 min at 10 °C in a refrigerated centrifuge, plasma were separated and stored at −80 °C until assay. Plasma ghrelin was measured in duplicate using a commercial competitive radioimmunoassay (Linco Research Inc., Missouri, USA) 13,19 that uses 125 I-labeled bioactive ghrelin as a tracer and a rabbit polyclonal antibody against full-length octanoylated human ghrelin. The assay detects total ghrelin, including Ser3-octanoyl and Ser3-des-octanoyl ghrelins, and has no cross-reactivity with ghrelin (1–5), ghrelin (1–14), secretin, vasoactive intestinal peptide, galanin, growth hormone (GH)-releasing factor, neuropeptide Y, orexin A and B, prolactin-releasing peptide (kit data sheet), or leptin. 19 The limit of detection of this assay was 7.3 ng/L. Leptin was measured using a commercial radioimmunoassay method (Diagnostic System Laboratory, Inc. Texas, USA). Plasma concentrations of GH, insulin and leptin were determined by using a standard in-house laboratory methods. Glucose and free fatty acid concentrations were determined by an automated method (Hitachi 917, Boehringer). The homeostatic model assessment (HOMA-IR) was calculated to assess insulin resistance. 20 RNA isolation During endoscopy, three biopsy specimens were obtained from the middle portion of the corpus along the greater curvature. Samples were snap-frozen in an ethanol-dry ice mixture and then stored at −80 °C until quantitative analysis of ghrelin mRNA was performed. Biopsies samples were submerged in RNA later reagent (Ambion) to stabilize and to protect cellular RNA. Biopsies were then dissociated using a Polytron (PT3100, Kinematica AG). Total RNA was extracted with TRIzol reagent (Invitrogen). After treatment with DNAse I (Ambion), the RNA was quantified by spectrophotometry and its integrity was checked by agarose gel electrophoresis and ethidium bromide staining. Semi quantitative Real Time Polymerase Chain Reaction (RT-PCR) was performed as follows: 1 μg of each DNAse I-treated total RNA was reverse-transcribed by the Reverse Transcription System (PROMEGA) in a total volume of 20 μL. 1/10th of the RT reaction mix was used for quantitative PCR analysis with an ABI PRISM 7000 Sequence Detector System (Applied Biosystems). Real-time reaction mixtures contained TaqMan Universal Master Mix (Applied Biosystems) and TaqMan probe mixture containing primers (20× Assays-on-Demand, Applied Biosystems). Probe for detection of ghrelin sequence was GAAGTCCGGTTCAACGCCCCCTTG. Probe for detection of the housekeeping gene, cyclophilin A, sequence was CTGCACTGCCAAGACTGAGTGGTTG. Cyclophilin A was analyzed in parallel to ensure that equivalent amounts of template were amplified. Satiety and hunger evaluation Visual analogue scales were used to measure subjective satiety and hunger. Satiety was defined as the sensation of fullness after eating so that a person does not feel the need to eat for some time afterward. Hunger was defined as the subjective driving force for the search for, choice of, and ingestion of food. 21 Subjects were instructed to make a single vertical mark on a horizontal 10-cm bar to indicate their current feelings, ranging between “not hungry at all” and “really hungry” and between “empty” and “full”. Baseline evaluations were collected 1 h and 30 min before, immediately after and every 30 min during 4 h after breakfast. Body composition Body composition was quantified in subjects by dual energy X-ray absorptiometry (DEXA) scan. Subjects were weighed at the same time of day, wearing only underwear and after emptying their bladder. A digital electronic scale (SECA, Birmingham, UK) was used for the measurement of body weight and calibrated before each measurement was taken. Height was measured using a wall-mounted stadiometer. Whole body and regional body composition of malnourished and well-nourished subjects were estimated from a whole body scan obtained by DEXA using a QDR-4500 W scanner (Hologic, Bedford, MA; software version 11.2) as described previously. 22 Subjects were scanned in light, metal-free clothing while lying still, flat on their backs with their arms by their sides. Percentage body fat, total fat mass, fat-free mass, and whole body bone mineral content were determined. Indirect calorimetry All subjects were studied under similar conditions. Resting energy expenditure was measured by indirect calorimetry in the morning, after a 12-h overnight fasting. Subjects were studied while in a semi recumbent position, with a ventilated hood, open-circuit indirect calorimeter (Deltatrac, Datex Instruments, Helsinki, Finland). After equilibrium was reached (10 min), respiratory exchanges were monitored continuously during 20 min; data were collected every minute and averaged over 20 min. The system was checked weekly by burning ethanol under standard conditions and calibrated daily with two standard gases. Cyclic enteral nutrition CyEN was performed as previously described 23 via a nasogastric feeding tube in malnourished subjects who accepted it. Infusion began between 7 p.m. and 8 p.m. and lasted 12–14 h. A commercially available polymeric diet without lactose or gluten at the concentration of 5.56 kJ/mL (Sondalis HP, Nestlé Clinical Nutrition, Noisiel, France) was used, providing 20% protein (50% from casein and 50% from soy protein), 45% carbohydrates (maltodextrin) and 35% fat (corn oil, medium chain triglyceride oil: 47%). The flow rate was maintained constant and below 3 mL/min with a pump. During the day, subjects were encouraged to eat normally and walk inside or outside their room, alone or with the help of a physiotherapist. Statistical analysis Results are expressed as means ± standard deviation. With no relevant data found in the literature on which differences could be expected in ghrelin profile according to age and nutritional status, the number of participants was set to 10 per group (in the end two groups had 10 subjects, one 11 and one 9). Comparisons between the four groups were performed using one-way analysis of variance. The F test for factorial design was used to compare the four groups of subjects. When significant F values emerged, data were submitted to pair wise comparisons using the Fisher-protected least significant difference test for repeated measurements analysis of variance or the t test with Bonferroni correction for factorial design analysis of variance. Non-parametric analysis of the measurements with large standard deviations gave the same results as the parametric methods used. The area under the 0–300 min curve was calculated by the trapezoidal method 13 for variables with repetitive measures. In malnourished subjects, values at days 0 and 21 were compared using non-parametric Wilcoxon tests. Differences were considered statistically significant at P < 0.05. SPSS 11.0 for Macintosh was used for statistical analysis. Results The anthropometric and biological characteristics of the study population are summarized in Table 1 . Malnutrition in undernourished subjects had been present for at least one month because of a reduced food intake, whether primary or following acute stress. Indirect calorimetry confirmed the absence of hypermetabolism. In the fasting state, plasma total ghrelin concentrations were higher in young malnourished subjects compared to well-nourished subjects; the difference was absent in elderly subjects. In healthy elderly subjects, leptin concentrations were higher than in young subjects. In the postprandial state, the area under the plasma ghrelin curve was significantly ( P < 0.01) higher in undernourished young subjects (11,474 ± 4762 ng.h/L) compared to well-nourished young (4640 ± 1707 ng.h/L) and to all elderly subjects ( Fig. 1 ). However, no difference was observed between well-nourished and malnourished elderly subjects (7177 ± 2760 and 5496 ± 2242 ng.h/L, respectively). The same differences were observed with acylated ghrelin. No difference between groups was observed for metabolic consequences of the meal, with the exception of higher glucose areas under the curve in elderly compared to young subjects. However, there was no difference in insulin resistance between groups. There was no significant difference in ghrelin mRNA expression levels in the fundus between undernourished and well-nourished elderly, undernourished and well-nourished young subjects (2.7 ± 2.4 vs 3.8 ± 7.3 vs 2.4 ± 2.1 vs 2.6 ± 2.6 in arbitrary units, respectively). The fasting and postprandial hunger and satiety curves are shown in Figs. 2 and 3 . Young well-nourished subjects had a snack-based cycle of hunger and satiety: hunger increased before eating and satiety appeared few minutes after the meal. In young malnourished subjects, postprandial hunger was significantly higher than in well-nourished subjects. There was no difference between hunger areas under the curve between well-nourished and undernourished elderly. No differences were found between groups as regards the satiety areas under the curve. Seven elderly and seven young malnourished subjects were assessed again after 21-days of CyEN. During the 21-day period, they received an average 188.3 ± 41.8 kJ/kg body weight/d. The energy intake per kg of body weight did not differ between age groups. Table 2 shows changes in weight, body composition and hormonal parameters. There was a significant effect of refeeding on total weight (with a significant increase in lean and fat compartments only in young subjects) and leptin. No difference was observed for appetite, satiety and other biological parameters. Discussion In the recent years, several hormones and neuropeptides that influence appetite control have been discovered. However, animal data cannot be transferred easily to humans, and most studies that tried to find a causal relationship between hyperphagia/anorexia and the excess/deficiency in one given marker came up with disappointing results. For example, leptin has failed to validate a single mediator theory in obese patients. 24 Ghrelin is a prime candidate for situations of anorexia, as it is involved in the hypothalamic regulation of energy homeostasis and increases food intake via stimulation of neuropeptide Y/Agouti-related protein neurons in the hypothalamus. 25 It is indeed the only orexigenic gastrointestinal peptide. 26 Our results show that there is an adaptative increase of ghrelin in young malnourished patients, but that this phenomenon does not occur in elderly malnourished patients. This may explain the absence of compensatory hyperphagia in elderly subjects after weight loss, reported by Roberts et al. 3 among others, and frequently seen in clinical practice; this reduced oral intake, after a period of CyEN, predicts the relapse of malnutrition. 23 This feature, along with the preferential loss of fat-free mass during weight loss, 27 defines the lack of adaptation of the elderly to malnutrition, that may be responsible for the increased morbidity of malnutrition in this age group. Most young malnourished patients among our cohort suffered from anorexia nervosa. This explains why mostly female patients were included in this group, thus creating a potential gender bias in the analysis. 28 Authors have reported higher fasting ghrelin levels in young malnourished patients with anorexia nervosa compared to young healthy controls with a higher BMI. 15,29 We believe that the difference was explained by malnutrition in the latter and not by the pathophysiology of anorexia nervosa. Indeed, hunger is present in patients with anorexia nervosa, and the fact that these patients do not eat despite an orexigenic profile indicates that psychological determinism is predominant in this disease. 29 Lower fasting ghrelin levels have been described in healthy elderly subjects compared to young subjects of similar BMI. 15 However, a more recent study confirmed our findings of similar ghrelin levels between young and elderly healthy controls. 30 Acylated ghrelin is an endogenous ligand for the growth hormone secretagogue receptor GHS-R1a and stimulates both feeding and growth hormone release. In contrast, desacylated ghrelin which does not have the acyl side chain, has no affinity for the GHS-R1a 6 and may play a lesser – if not inverse 31 – role in energy regulation. In our study, acylated ghrelin levels followed those of total ghrelin, and differences between groups were similar for both; we therefore were unable to show a distinct effect of acylated ghrelin in these settings. Ghrelin secretion is known to be influenced by sleep duration. 32 We may have missed sleep-induced changes in our subjects by not monitoring their sleep; however, these changes seem rather minor. 33 Similar mRNA gastric ghrelin levels (even though isolation of the gastric mucosa from other layers may have provided different results) suggest that observed differences are post-transcriptional. These may implicate interactions with other peptides such as leptin, through an imbalance of their respective dynamics. 30,34 The other biological findings in our study were not surprising: hyperglycemia in elderly subjects, reflecting age-induced carbohydrate intolerance, lower bone mineral contents in undernourished elderly, two-fold difference in leptin values between well-nourished young and elderly that matched the two-fold difference in body fat; this latter finding is a well-known feature of ageing. 4 Postprandial insulin has been recently described to increase fullness and decrease hunger in healthy volunteers 35 ; it cannot be implicated in the differences in satiety and hunger observed between groups in our study, as areas under the curve were similar. The postprandial persistence of elevated satiety sensations in both elderly groups may also be a feature of the anorexia of ageing. Effects of refeeding on body compartments have been described before, thus making the non-significance of the increase in lean mass in the elderly not surprising. 36 The dramatic increase in leptin levels observed in the elderly cannot be explained by the modest (furthermore inverse) changes in fat mass in this group. 37 Whatever its cause, it may also participate in the resistance to refeeding by inhibiting spontaneous food intake in patients receiving CyEN. The small number of subjects in this part of the study may be responsible for non-significant differences in ghrelin levels between days 0 and 21 (type 2 error). Synthesis of purified ghrelin has led way to intervention studies. Ghrelin infusion increases short-term food intake in healthy volunteers by 28%. 10 The same team has recently showed that a similar infusion was able to increase energy intake by 31% in cancer patients 38 ; ghrelin levels are reportedly elevated in patients with cancer cachexia, 39 which indicates that exogenous ghrelin may be able to overcome any resistance to ghrelin in this setting. It may therefore be a logical step to study the effects of ghrelin administration in elderly malnourished patients, even if long acting agonists will need to be soon developed in order for a new drug to be used in a clinical setting. In conclusion, the absence of increase in ghrelin levels in the malnourished elderly may be an important feature of the lack of adaptation to malnutrition in this age group. Conflict of interest statement No author had a conflict of interest. Acknowledgements This research was supported by a joint action of Institut National de la Santé Et de la Recherche Médicale and Institut National de la Recherche Agronomique (ATC 2002). The sponsors were not involved in the study design, the collection, analysis and interpretation of data, the writing of the manuscript or in the decision to submit the manuscript for publication. SMS was involved in the study design and concept, analysis and interpretation of data, statistical analysis, and preparation of manuscript. JG, PF, EVO and XH were involved in the study design and concept, analysis and interpretation of data, and critical revision of manuscript. RAJ and CC were involved in acquisition, analysis and interpretation of data, and preparation of manuscript. KA, PF and FS were involved in acquisition, analysis and interpretation of data, and critical revision of manuscript. IMS was involved in analysis and interpretation of data, and critical revision of manuscript. 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