Humans, Clinical410

Malkova D et al. Ghrelin and Appetite Following Exercise … Horm Metab Res 2008 ; 40: 410 – 415

received 07.05.2007 accepted 15.10.2007

Bibliography DOI 10.1055/s-2008-1058100 Published online: March 14, 2008 Horm Metab Res 2008 ; 40: 410 – 415 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043

Correspondence Prof. M. A. Nimmo School of Sport and Exercise Sciences Loughborough University Ashby Road Loughborough Leicestershire LE11 3TU United Kingdom Tel.: + 44 (0)1509 / 22 63 11 Fax: + 44 (0)1509 / 22 63 01 m.a.nimmo@lboro.ac.uk

Key words � � ghrelin � � cytokines � � insulin � � leptin � � appetite

Effect of Moderate-intensity Exercise Session on Preprandial and Postprandial Responses of Circulating Ghrelin and Appetite

suppression after feeding suggest that ghrelin participates in the determination of food intake from meal to meal [13, 14] . Accordingly, it has been shown that intravenous administration of ghrelin enhances appetite and increases food intake in humans [6, 15] . Circulating concentrations of ghrelin have been reported to be unchanged after a single exercise session of running or cycling at moderate inten-sity [16 – 20] and after rigorous treadmill running [18, 21] . Long- or medium-term training studies also have shown no effect of exercise on total ghrelin independent of its impact on body weight [22, 23] , with one of these studies reporting an increase in acylated ghrelin after aerobic training for fi ve consecutive days [23] . In contrast there are reports of a signifi cant decrease in total ghre-lin concentration after concentric muscle actions [24] , single circuit resistance exercise [25] , and a standardised maximal exercise test [26] . In addi-tion, a recent study reported that running exer-cise reduces plasma concentrations of acylated

Introduction & Ghrelin is a 28-amino-acid peptide primarily secreted by endocrine cells in the gastrointesti-nal tract [1 – 3] and is found in the circulation of healthy humans in both acylated and desacyl forms [4] . Initially, ghrelin was known as a potent stimulus for growth hormone secretion [2] , but it was soon suggested that ghrelin is also a potent orexigenic substance [5, 6] that crosses the blood-brain barrier and stimulates food intake by acting on several classical body-weight regulatory cen-tres [7] . Accumulating evidence suggests that ghrelin secretion is upregulated under conditions of nega-tive energy balance and downregulated in the setting of positive energy balance [8 – 12] , which suggests an important role for this peptide dur-ing long-term energy imbalance. At the same time, studies reporting a preprandial rise in humans, whether being fed on a known schedule or initiating meals voluntarily without time- or food-related cues, together with a signifi cant

Authors D. Malkova 1 , R. McLaughlin 1 , 3 , E. Manthou 1 , A. M. Wallace 2 , M. A. Nimmo 4

Affi liations 1 Human Nutrition Section, Division of Developmental Medicine, Medical School, University of Glasgow, United Kingdom 2 Department of Clinical Biochemistry, Medical School, University of Glasgow, United Kingdom 3 Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom 4 School of Sport and Exercise Sciences, Loughborough University, United Kingdom

Abstract & Responses of plasma total ghrelin and appetite were investigated during preprandial and post-prandial stages of recovery from a moderate-intensity cycling session. Healthy recreationally active men underwent one exercise and one control trial. In the exercise trial, subjects exer-cised for approximately 60 minutes, while in the control trial they rested quietly for the same duration. After the intervention, subjects rested for 120 minutes and then consumed a test meal. Measurements were obtained immediately and 120 minutes after the intervention and then dur-ing 180 minutes of the postprandial period. The post-intervention concentration of total ghrelin was lower (p < 0.05) in the exercise than in the

control trial. The modulating effect of exercise was related to the reduction in the postprandial rather than preprandial concentration. Post-intervention scores of appetite were not differ-ent between the two trials, but when preprandial and postprandial responses were considered separately, postprandial hunger and desire to eat was higher (p < 0.05) in the exercise trial. In sum-mary, during recovery from moderate-intensity exercise, total ghrelin does not respond in a compensatory manner to disturbances in energy balance. Thus, an exercise-induced increase in appetite during the later stages of recovery coinciding with the postprandial state cannot be explained by changes in the plasma concentra-tion of total ghrelin.

Humans, Clinical 411

Malkova D et al. Ghrelin and Appetite Following Exercise … Horm Metab Res 2008 ; 40: 410 – 415

ghrelin and that this suppression lasts for as long as eight hours after exercise [27] . Given the present worldwide increase in the prevalence of obes-ity and the knowledge that physical inactivity is a signifi cant contributing factor [28] , an understanding of how ghrelin and exercise interplay to impact on appetite and thus energy balance is of great importance. Unfortunately, only a few of the exercise and ghrelin studies considered appetite measurements [19, 23, 27] . Findings of these studies suggest that during the early hours of post-exercise recovery, changes in fasting appetite may be predicted by changes in acylated ghrelin [23, 27] but not total ghrelin [19] and that during the later stages of post-exer-cise recovery that coincide with postprandial state, the appetite regulatory role of acylated ghrelin is diminished [27] . The main aim of this study was to investigate the impact of a single moderate-intensity exercise session with duration to expend 2.1 MJ on subjective measures of appetite and concen-trations of plasma ghrelin in preprandial and postprandial states of a recovery period lasting for fi ve hours. Furthermore, given the reported role of insulin, leptin, and interleukin-6 (IL-6) in the regulation of circulating ghrelin and appetite [29 – 32] , we also investigated changes in these hormones.

Materials and Methods & Subjects Eleven healthy, recreationally active men with the following characteristics (mean ± SD): age 23.8 ± 5.8 years; body mass index 23.3 ± 2.9 kg / m 2 ; body fat level 13.7 ± 3.4 % ; V ̇ O 2 peak 41.8 ± 7.2 ml / kg / min, participated in the study, after giving their written informed consent to the study protocol, which was approved by the Ethics Committees of Strathclyde and Glasgow Universities. None of the subjects was a smoker, was on any special diet, or was taking self-medicated or prescribed medication.

Study design Prior to the fi rst main trial, subjects attended the laboratory for lactate threshold (LT) and peak oxygen uptake ( V ̇ O 2 peak) determination. They subsequently underwent two trials, one control and one exercise, in a balanced design separated by at least three days. In the exercise trial, the intervention involved cycling at 90 % of LT for a duration to expend 2.1 MJ (E), while in the control trial subjects rested quietly for the same dura-tion. Fasting blood samples were collected before (baseline) and immediately and 120 minutes after the intervention. Subjects then consumed a meal and stayed in the laboratory for postprandial blood collections. Subjects kept weighed food records for two days preceding the fi rst trial and were requested to replicate their diet exactly two days before the second trial. The subjects were also asked to refrain from alcohol, caffeine, and exercise for the two days before each trial.

Preliminary exercise tests: determination of lactate threshold and V O 2 peak The participants were required to cycle on a friction-braked cycle ergometer (Monark 824E, Monark Exercise AB, Sweden). The initial load was set at 60 W and increased by 15 W at the end of each three-minute stage. The participants kept a constant pedal cadence of 60 rpm. During the last minute of each three-

minute stage, heart rate was determined (Favor, Polar Electro, Kempele, Finland), expired gas was collected and analysed for V O 2 and V CO 2 via an online gas analysis system (Oxycon-gamma, Mijnhardt B.V, Holland), and a fi ngertip capillary blood sample was taken in order to determine lactate concentration by using Lactate Pro (Arkray, Japan). When blood lactate concentration displayed a visually increased breakpoint, a further two stages were undertaken. On cessation of blood sampling, the 15-W increments continued every minute until volitional exhaustion and determination of V ̇ O 2 peak. LT was confi rmed using linear regression analysis according to the intersection of the two best-fi tting lines.

Main trials On the experimental days subjects attended the laboratory after a 12-hour fast, where a cannula (Vefl on 18G, Becton Dickinson Ltd, Helsingborg, Sweden) was inserted into an antecubital vein and a three-way stopcock (Connecta Plus, Becton Dickinson, Helsinborg, Sweden) was connected to the cannula. After 10 minutes, a baseline blood sample was collected. Subjects then either exercised on a friction-braked cycle ergometer (Monark 824E, Monark Exercise AB, Sweden) at 90 % LT for the duration of 57 ± 3 minutes to expend 2.1 MJ (exercise trial) or rested quietly for the same duration (control trial). Calculations of the duration of exercise sessions to expend ~ 2.1 MJ were done by using indi-rect calorimetry equations [33] and individual relationships between V O 2 and V CO 2 and work load obtained during prelimi-nary exercise tests. Blood samples were collected immediately and 120 minutes after the intervention. Subjects then consumed a meal and stayed in the laboratory for postprandial blood col-lection at 30, 120 and 180 minutes. The meal provided was iso-caloric to the individual ’ s habitual lunch and provided 3.9 ± 0.69 MJ. It consisted of a chocolate bar (Mars, Slough, UK) and a meal replacement (Complan, H. J. Heinz Co. Ltd., Hayes, UK) mixed with semi- skimmed milk. The amount of Complan powder to be provided and the volume of milk to be added were calculated by taking into consideration the fact that the energy content of the chocolate bar was ~ 1176 kJ and that 1 g of Comp-lan powder mixed with 3.5 ml of semi-skimmed milk resulted in an energy content of 25 kJ / g.

Estimation of energy intake in habitual lunch For the determination of energy intake in their habitual lunch, subjects were asked to record their lunch for three consecutive days. An experienced nutritionist inspected their records to ensure that they were completed and that suffi cient detail had been recorded. The dietary records were analysed using a com-puterised version of the food composition tables (Diet 5, Robert Gordon University, Aberdeen, UK) [34] .

Anthropometry Height and body mass measurements were made using stand-ard procedures. Skinfold thickness was measured using callipers (Holtain, UK) at four sites (triceps, subscapular, biceps, suprail-iac), and their sum was used to estimate percentage body fat using the equations of Durnin and Womersley [35] .

Blood preparation and plasma analysis For IL-6, insulin, and leptin analyses, venous blood was collected in pre-chilled EDTA vacutainers, and for ghrelin analysis blood was placed into pre-chilled vacutainers containing EDTA and aprotinin (0.6 TIU / ml of blood) to inhibit the activity of pro-

Humans, Clinical412

Malkova D et al. Ghrelin and Appetite Following Exercise … Horm Metab Res 2008 ; 40: 410 – 415

teinases. Plasma was separated by centrifugation and stored at − 70 ° C until analysis. Commercially available enzyme immu-noassays were used for the analysis of plasma ghrelin (Phoenix Pharmaceuticals, California, USA), IL-6 (R & D Systems, Minneap-olis, USA), and insulin (Mercodia, Uppsala, Sweden). Leptin con-centration was analysed by radioimmunoassay. All samples for each subject were analysed in the same run and performed in duplicate. The within-assay coeffi cients of variation were < 4 % for IL-6, leptin, and insulin and < 10 % for ghrelin over the mea-sured concentration range.

Measurement of the drive to eat The visual analogue scales (VAS) were used to evaluate the drive to eat, with subjects reporting ratings for hunger, desire to eat, prospective food consumption, satiety, and fullness [36] . The present VAS were horizontal lines (100 mm long), unbroken and unmarked except for word anchors at either end that describe extremes of the drive to eat. The subjects were instructed to mark the scale with a vertical line at a point that most accurately refl ected the intensity of their appetite feeling at that moment in time.

Statistical analysis Results are shown as means ± SE. Baseline data were compared by paired t -test, and then a two-way ANOVA with repeated measures was used on post-intervention responses to determine the effect of trial and time. Two-way ANOVA with repeated measures was also used to compare differences in the preprandial and postpran-dial period separately. Associations between variables were assessed by Pearson ’ s product-moment correlations. This was applied to baseline data and to the time-averaged concentrations of the post-intervention period (defi ned as the trapezium rule-derived areas under the concentration vs. time curve, divided by the duration of the observation in the corresponding period). The level of signifi cance was set at p < 0.05. Statistical analyses were performed using Statistica (version 6, Stat Soft Inc., Tulsa, Okla-homa) and SPSS (version 11).

Results & Responses of plasma ghrelin, leptin, insulin, and IL-6 Baseline concentrations of plasma ghrelin, IL-6, insulin, and lep-tin were not different between the control and exercise trials ( Table 1 ). The post-intervention concentration of plasma ghrelin was signifi cantly lower (p < 0.05) and the concentration of IL-6 higher (p < 0.05) in the exercise than in the control trial ( � � Fig. 1A, B ). When preprandial and postprandial responses of ghrelin and IL-6 were considered separately, the difference in concentration of IL-6 between trials was signifi cant (p < 0.05) only in the preprandial state, while the concentration of ghrelin differed signifi cantly (p < 0.05) only in the postprandial state ( � � Fig. 1A, B ). Post-intervention concentration of plasma leptin tended to be lower (p = 0.06) in the exercise than in the control trial ( � � Fig. 1C ), and responses of insulin were not different between the control and exercise trials ( � � Fig. 1D ). Over time, the plasma concentration of ghrelin, IL-6, insulin, and leptin did not differ in the preprandial state ( � � Fig. 1A – D ). Nor

Table 1 Baseline values of plasma ghrelin, IL-6, leptin, insulin, and appetite measures in the control and exercise trials (values are mean ± SE)

Parameter Control Exercise

ghrelin (pg / ml) 676 ± 58 645 ± 52 IL-6 (pg / ml) 1.3 ± 0.3 1.4 ± 0.4 leptin (ng / ml) 4.4 ± 1.1 3.3 ± 6.9 insulin ( � U / ml) 6.1 ± 0.9 5.2 ± 0.5 hunger (mm) 38 ± 9 39 ± 9 desire to eat (mm) 41 ± 10 51 ± 7 PFC (mm) 67 ± 7 69 ± 6 fullness (mm) 28 ± 8 30 ± 5

PFC = Prospective food consumption

A C

DB

Fig. 1 Plasma concentration of ghrelin ( A ), IL-6 ( B ), leptin ( C ) and insulin ( D ) during preprandial and postprandial periods in the exercise and control trials. Meal consumption is indicated by arrows. Values are means ± SE for 11 men. Signifi cant difference (p < 0.05) between trials over * the post-intervention, a the postprandial, and b the preprandial periods.

Humans, Clinical 413

Malkova D et al. Ghrelin and Appetite Following Exercise … Horm Metab Res 2008 ; 40: 410 – 415

were changes in plasma concentration of IL-6 ( � � Fig. 1B ) and leptin ( � � Fig. 1C ) signifi cant over time in the postprandial state. After the meal consumption, the concentration of ghrelin fell signifi cantly (p < 0.05) in both trials, but the duration of post-prandial ghrelin suppression was longer in the control trial ( � � Fig. 1A ). Changes in postprandial insulin concentration with time were similar in both trials ( � � Fig. 1D ).

Responses of appetite measures Baseline scores of hunger, desire to eat, prospective food con-sumption, and fullness were not different between the control and exercise trials ( Table 1 ). Post-intervention responses of appetite measures also were not different between the control and exercise trials. However, when preprandial and postprandial responses of appetite measures were considered separately, postprandial responses of hunger and desire to eat were signifi -cantly higher (p < 0.05) in the exercise trial compared with the control trial ( � � Fig. 2A, B ). As expected, a signifi cant effect of the meal was observed (p < 0.01), indicating a decrease in hunger, desire to eat, and prospective food consumption and an increase in fullness following the meal.

Correlations of ghrelin with insulin, IL-6, leptin, and appetite scores At baseline, the concentration of ghrelin correlated negatively with the concentration of insulin (p < 0.05) in both trials ( Table 2 ). In the post-intervention period, a signifi cant negative correla-tion was found between time-averaged concentrations of ghre-lin and insulin (p < 0.05) in the control trial and time-averaged concentrations of ghrelin and IL-6 (p < 0.05) in the exercise trial ( Table 2 ). In the post-intervention period, time-averaged con-centration of ghrelin also tended to correlate negatively with time-averaged concentration of leptin in both the control (p = 0.07) and exercise (p = 0.07) trials ( Table 2 ). No signifi cant associations of plasma ghrelin with appetite scores were noted.

Discussion & This study shows that when estimated for a duration of fi ve hours, the plasma concentration of total ghrelin in healthy adult males was lower after a single exercise session than after resting conditions. Notable was the fi nding that the difference in ghrelin concentration seen between exercise and control conditions was related to the difference in the response to the meal given two hours after the completion of the intervention. Despite an exercise-induced reduction in the concentration of postprandial ghrelin, hunger and the desire to eat in the postprandial state were higher in the exercise trial than in the control trial. These fi ndings contrast with the regulatory role of ghrelin suggested by intravenous administration studies in which ghrelin modi-fi ed appetite sensations in a dose-dependent manner [6, 15] and implies that during later hours of post-exercise recovery coin-ciding with the postprandial state, appetite regulation may be mediated by the action of factors other than ghrelin. Our fi nding that plasma total ghrelin concentrations during a recovery period lasting for fi ve hours were signifi cantly lower in the exercise than the control trial is similar to the recent report of Broom et al. [27] , which showed that concentrations of acylated ghrelin measured for eight hours after running were lower than those during the same period after control condi-tions. Separate consideration of preprandial and postprandial data, however, revealed that the responses of total and acylated ghrelin in these two studies were similar in the postprandial state but not during the preprandial state, which coincided with the fi rst two hours of the post-exercise recovery. In our study suppression of total ghrelin was seen only during the postpran-

A

B

Fig. 2 Hunger ( A ) and desire to eat ( B ) during the preprandial and postprandial periods in the exercise and control trials. Meal consumption is indicated by arrows. Values are means ± SE for 11 men. a Signifi cant difference (p < 0.05) between trials over the postprandial period.

Table 2 Pearson product moment correlations of ghrelin with IL-6, insulin, and leptin for baseline and time-averaged concentrations of the post-intervention period in the control and exercise trials

Parameter Ghrelin

Control Exercise

insulin baseline − 0.69 * − 0.61 * post-intervention − 0.65 * 0.33 IL-6 baseline 0.14 − 0.22 post-intervention − 0.21 − 0.64 * leptin baseline − 0.41 − 0.32 post-intervention − 0.60 − 0.57

* p < 0.05 signifi cant correlation between relevant variables

Humans, Clinical414

Malkova D et al. Ghrelin and Appetite Following Exercise … Horm Metab Res 2008 ; 40: 410 – 415

dial period, while in the study of Broom et al. [27] exercise-induced suppression of acylated ghrelin was evident in both the preprandial and postprandial periods. This fi nding suggests that mechanisms by which total and acylated ghrelin are modifi ed either immediately or up two hours after exercise may be differ-ent. On the other hand, a recent study in which total ghrelin measurements during two hours of post-exercise recovery were more frequent than in our study provided evidence of an acute decrease in total ghrelin after a standardised maximal exercise test [26] . As in the case of our study, the above study [26] had no measurements of acylated ghrelin, and the study of Broom et al. [27] had no measurements on total ghrelin; therefore, the extent of variations between responses of total and acylated ghrelin to exercise remains unclear. Although acute suppression of appetite could be expected dur-ing and for a short duration after moderate-intensity exercise [19, 27, 37, 38] , during the fi rst two hours of the post-exercise recovery in the current study, appetite scores did not differ between the control and exercise trials. Because the suppression of hunger is brief and in some studies returned to control values within 30 minutes [19] , it could be argued that our ability to detect the suppression was limited by the low frequency of appetite measurements. At the same time, it should be noted that exercise-induced acute suppression of appetite is not a uni-versal fi nding and that immediate effects of exercise on appetite also were not found in other studies [39 – 41] . Therefore, differ-ences in exercise intensity, duration, and exercise-induced energy expenditure also may be responsible for these confl icting results. Indeed, in this study the exercise intensity and the energy expended over the whole exercise session were lower than in some of the studies in which post-exercise suppression of appetite was reported [27, 37, 38] . Although in our study appetite measures were not different between the two trials during the fi rst two hours of the post-intervention observation, postprandial hunger and desire to eat measured in response to a test meal consumed two hours after an exercise session were higher in the exercise trial. A tendency toward exercise-induced increases in appetite during the later hours of post-exercise recovery coinciding with postprandial state also was found by Broom et al. [27] and during a meal-tol-erance test conducted after the last exercise session of a fi ve-day exercise training program [23] . Thus, it seems that exercise-induced energy defi cits may be maintained during early but not later stages of the post-exercise recovery. This study is one of very few studies in which post-exercise measurements of appetite and ghrelin were conducted under the same experimental conditions, and thus it investigates how ghrelin and exercise interplay to impact on appetite [19, 23, 27] . We found that in the preprandial state neither appetite nor ghrelin was infl uenced by the exercise session. Thus, our pre-prandial data cannot be compared with previous evidence sug-gesting that during the early hours of post-exercise recovery, changes in appetite may be predicted by changes in acylated ghrelin [23, 27] but not total ghrelin [19] . Our postprandial fi nd-ings suggest that the changes in appetite seen during the later stages of the post-exercise recovery coinciding with postpran-dial state cannot be explained by changes in total ghrelin. We appreciate that total ghrelin includes both acylated and non-acylated ghrelin [4] and that the nonacylated form of ghrelin may be unimportant in appetite regulation [23] , and we admit that the lack of acylated ghrelin measurements limits the inter-pretation of our results. At the same time, we note that the post-

prandial fi ndings of this study are very similar to the postprandial fi ndings of the recent study [27] in which acylated ghrelin and appetite responses to exercise and consecutive meal were mea-sured for a duration of eight hours. As expected [42] , the post-intervention concentration of IL-6 was higher in the exercise trial than in the control trial. How-ever, despite the suggestion that IL-6 may be one of the signals generated by the exercising body that feeds back to the brain to regulate central neuropeptide systems involved in the regula-tion of energy homeostasis [32] , post-intervention appetite scores were not different between the two trials, and postpran-dial hunger and desire to eat were higher in the exercise trial. Thus, the expected appetite-suppressive role of IL-6 during post-exercise recovery has not been confi rmed by this study. Further studies are required to determine whether exercise-induced increases in IL-6 have a role in appetite regulation after exercise sessions with different intensities, durations, and energy expen-ditures. In the current study changes in plasma ghrelin occur in the absence of any change in plasma insulin. This is consistent with the fi ndings of Weickert [43] but contrary to the suggestions implicating insulin in the regulation of ghrelin secretion [29, 30] . The analysis of correlation data between insulin and ghrelin, however, points to the speculation that, under the conditions of this experiment, the regulatory role of insulin was diminished only in the exercise trial. This may be due to the release of exer-cise-related hormones and metabolites, including increased secretion of IL-6. We found that the exercise-induced reduction in the post-intervention concentration of plasma ghrelin coin-cided with a signifi cant increase in the concentration of IL-6 and that time-averaged concentrations of ghrelin correlated nega-tively with concentrations of IL-6 during the post-exercise period. At the same time, we appreciate that regardless of the report allocating a role for IL-6 in the modifi cation of ghrelin secretion under conditions of increased fasting [31] , the correla-tions between ghrelin and IL-6 did not show a causal relation. In this study, the increased suppression of ghrelin at the post-prandial nadir seen in the exercise trial was followed by acceler-ated ghrelin rebound. Thus, future human studies aiming to understand how exercise and ghrelin interplay to impact appe-tite and thus energy balance may benefi t from consideration of the changes over an extended time scale. Indeed, in a recent study conducted on horses, plasma ghrelin concentrations were lower 30 minutes prior to and 15 minutes after a test meal given approximately three hours after the completion of exercise but were higher 12 hours post-exercise compared with control [44] . In summary, despite an exercise-induced reduction in post-intervention and postprandial concentrations of plasma ghrelin, the postprandial response of hunger and desire to eat was higher after an exercise session than after resting conditions. Thus, an exercise-induced increase in appetite during the later stages of recovery coinciding with postprandial state cannot be explained by changes in the plasma concentration of total ghrelin.

Acknowledgments & The authors would like to thank the Synergy Fund of Strathclyde and Glasgow Universities.

Humans, Clinical 415

Malkova D et al. Ghrelin and Appetite Following Exercise … Horm Metab Res 2008 ; 40: 410 – 415

References 1 Date Y , Kojima M , Hosoda H , Sawaguchi A , Mondal MS , Suganuma T ,

Matsukura S , Kangawa K , Nakazato M . Ghrelin, a novel growth hor-mone-releasing acylated peptide, is synthesized in a distinct endo-crine cell type in the gastrointestinal tracts of rats and humans . Endocrinology 2000 ; 141 : 4255 – 4261

2 Kojima M , Hosoda H , Date Y , Nakazato M , Matsuo H , Kangawa K . Ghre-lin is a growth-hormone-releasing acylated peptide from stomach . Nature 1999 ; 402 : 656 – 660

3 Kojima M , Kangawa K . Ghrelin: structure and function . Physiol Rev 2005 ; 85 : 495 – 522

4 Akamizu T , Shinomiya T , Irako T , Fukunaga M , Nakai Y , Nakai Y , Kangawa K . Separate measurement of plasma levels of acylated and desacyl ghrelin in healthy subjects using a new direct ELISA assay . J Clin Endocrinol Metab 2005 ; 90 : 6 – 9

5 Nakazato M , Murakami N , Date Y , Kojima M , Matsuo H , Kangawa K , Matsukura S . A role for ghrelin in the central regulation of feeding . Nature 2001 ; 409 : 194 – 198

6 Wren AM , Seal LJ , Cohen MA , Brynes AE , Frost GS , Murphy KG , Dhillo WS , Ghatei MA , Bloom SR . Ghrelin enhances appetite and increases food intake in humans . J Clin Endocrinol Metab 2001 ; 86 : 5992 – 5995

7 Cummings DE , Overduin J . Gastrointestinal regulation of food intake . J Clin Invest 2007 ; 117 : 13 – 23

8 Tschop M , Weyer C , Tataranni PA , Devanarayan V , Ravussin E , Heiman ML . Circulating ghrelin levels are decreased in human obesity . Diabetes 2001 ; 50 : 707 – 709

9 Nagaya N , Uematsu M , Kojima M , Date Y , Nakazato M , Okumura H , Hosoda H , Shimizu W , Yamagishi M , Oya H , Koh H , Yutani C , Kangawa K . Elevated circulating level of ghrelin in cachexia associated with chronic heart failure: relationships between ghrelin and ana-bolic/catabolic factors . Circulation 2001 ; 104 : 2034 – 2038

10 Shiiya T , Nakazato M , Mizuta M , Date Y , Mondal MS , Tanaka M , Nozoe S , Hosoda H , Kangawa K , Matsukura S . Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion . J Clin Endocrinol Metab 2002 ; 87 : 240 – 244

11 Tolle V , Kadem M , Bluet-Pajot MT , Frere D , Foulon C , Bossu C , Dardennes R , Mounier C , Zizzari P , Lang F , Epelbaum J , Estour B . Balance in ghrelin and leptin plasma levels in anorexia nervosa patients and constitutionally thin women . J Clin Endocrinol Metab 2003 ; 88 : 109 – 116

12 Monti V , Carlson JJ , Hunt SC , Adams TD . Relationship of ghrelin and leptin hormones with body mass index and waist circumference in a random sample of adults . J Am Diet Assoc 2006 ; 106 : 822 – 828

13 Cummings DE , Purnell JQ , Frayo RS , Schmidova K , Wisse BE , Weigle DS . A preprandial rise in plasma ghrelin levels suggests a role in meal initiation in humans . Diabetes 2001 ; 50 : 1714 – 1719

14 Cummings DE , Frayo RS , Marmonier C , Aubert R , Chapelot D . Plasma ghrelin levels and hunger scores in humans initiating meals voluntar-ily without time- and food-related cues . Am J Physiol 2004 ; 287 : 297 – 304

15 Akamizu T , Takaya K , Irako T , Hosoda H , Teramukai S , Matsuyama A , Tada H , Miura K , Shimizu A , Fukushima M , Yokode M , Tanaka K , Kangawa K . Pharmacokinetics, safety, and endocrine and appetite effects of ghrelin administration in young healthy subjects . Eur J Endo-crinol 2004 ; 150 : 447 – 455

16 Dall R , Kanaley J , Hansen TK , Moller N , Christiansen JS , Hosoda H , Kangawa K , Jorgensen JO . Plasma ghrelin levels during exercise in healthy subjects and in growth hormone-defi cient patients . Eur J Endocrinol 2002 ; 147 : 65 – 70

17 Kallio J , Pesonen U , Karvonen MK , Kojima M , Hosoda H , Kangawa K , Koulu M . Enhanced exercise-induced GH secretion in subjects with Pro7 substitution in the prepro-NPY . J Clin Endocrinol Metab 2001 ; 86 : 5348 – 5352

18 Schmidt A , Maier C , Schaller G , Nowotny P , Bayerle-Eder M , Buranyi B , Luger A , Wolzt M . Acute exercise has no effect on ghrelin plasma con-centrations . Horm Metab Res 2004 ; 36 : 174 – 177

19 Martins C , Morgan LM , Bloom SR , Robertson MD . Effects of exercise on gut peptides, energy intake and appetite . J Endocrinol 2007 ; 193 : 251 – 258

20 Pomerants T , Tillmann V , Karelson K , J ü rim ä e J , J ü rim ä e T . Ghrelin response to acute aerobic exercise in boys at different stages of puberty . Horm Metab Res 2006 ; 38 : 752 – 757

21 Kraemer RR , Durand RJ , Acevedo EO , Johnson LG , Kraemer GR , Hebert EP , Castracane VD . Rigorous running increases growth hor-mone and insulin-like growth factor-I without altering ghrelin . Exp Biol Med 2004 ; 229 : 240 – 246

22 Foster-Schubert KE , MacTiernan A , Frayo RS , Schwartz RS , Rajan KB , Yasui Y , Tworoger SS , Cummings DE . Human plasma ghrelin levels increase during a one-year exercise program . J Clin Endocrinol Metab 2005 ; 90 : 820 – 825

23 Mackelvie KJ , Meneilly GS , Elahi D , Wong AC , Barr SI , Chanoine JP . Regulation of appetite in lean and obese adolescents following exer-cise: role of acylated and desacyl ghrelin . J Clin Endocrinol Metab 2007 ; 92 : 648 – 654

24 Kraemer RR , Durand RJ , Hollander DB , Tryniecki JL , Hebert EP , Castracane VD . Ghrelin and other glucoregulatory hormone responses to eccentric and concentric muscle contractions . Endocrine 2004 ; 24 : 93 – 98

25 Ghanbari-Niaki A . Ghrelin and glucoregulatory hormone responses to a single circuit resistance exercise in male college students . Clin Bio-chem 2006 ; 10 : 966 – 970

26 Vestergaard ET , Dall R , Lange KH , Kjaer M , Christiansen JS , Jorgensen JO . The ghrelin response to exercise before and after growth hormone administration . J Clin Endocrinol Metab 2007 ; 92 : 297 – 303

27 Broom DR , Stensel DJ , Bishop NC , Burns SF , Miyashita M . Exercise-induced suppression of acylated ghrelin in humans . J Appl Physiol 2007 ; 102 : 2165 – 2171

28 Hill JO . Understanding and addressing the epidemic of obesity an energy balance perspective . Endocrine Rev 2006 ; 27 : 750 – 761

29 Toshinai K , Mondal MS , Nakazato M , Date Y , Murakami N , Kojima M , Kangawa K , Matsukura S . Upregulation of ghrelin expression in the stomach upon fasting, insulin-induced hypoglycemia, and leptin administration . Biochem Biophys Res Comm 2001 ; 281 : 1220 – 1225

30 Flanagan DE , Evans ML , Monsod TP , Rife F , Heptulla RA , Tamborlane WV , Sherwin RS . The infl uence of insulin on circulating ghrelin . Am J Physiol 2003 ; 284 : 313 – 316

31 Zoladz JA , Konturek SJ , Duda K , Majerczak J , Sliwowski Z , Grandys M , Bielanski W . Effect of moderate incremental exercise, performed in fed and fasted state on cardio-respiratory variables and leptin and ghrelin concentrations in young healthy men . J Physiol Pharmacol 2005 ; 56 : 63 – 85

32 Patterson CM , Levin BE . Role of exercise in the central regulation of energy homeostasis and in the prevention of obesity . Neuroendo-crinology 2007 Mar 19 , [Epub ahead of print]

33 Frayn KN . Calculation of substrate oxidation rates in vivo from gaseous exchange . J Appl Physiol 1983 ; 55 : 628 – 634

34 Holland B , Welch AA , Unwin ID , Buss DH , Paul AA , Southgate DAT . McCance and Widdowson’s The Composition of Foods . Cambridge: Royal Society of Chemistry and Ministry of Agriculture, Fisheries and Food 1991

35 Durnin JVGA , Womersley J . Body fat assessed from total body density and its estimation from skinfold thickness: measurements on 481 men and women aged from 16 to 72 years . Br J Nutr 1974 ; 32 : 77 – 97

36 Flint A , Raben A , Blundell JE , Astrup A . Reproducibility, power and valid-ity of visual analogue scales in assessment of appetite sensations in single test meal studies . Int J Obesity 2000 ; 24 : 38 – 48

37 King NA , Burley VJ , Blundell JE . Exercise-induced suppression of appe-tite: effects on food intake and implications for energy balance . Eur J Clin Nutr 1994 ; 48 : 715 – 724

38 King NA , Blundell JE . High-fat foods overcome the energy expenditure induced by high-intensity cycling or running . Eur J Clin Nutr 1995 ; 49 : 114 – 123

39 King NA , Snell L , Smith RD , Blundell JE . Effects of short-term exercise on appetite responses in unrestrained females . Eur J Clin Nutr 1996 ; 50 : 663 – 667

40 Hubert P . Uncoupling the effects of energy expenditure and energy intake: appetite response to short-term energy defi cit induced by meal omission and physical activity . Appetite 1998 ; 31 : 9 – 19

41 Imbeault P , Saint-Pierre S , Almeras N , Tremblay A . Acute effects of exer-cise on energy intake and feeding behaviour . Br J Nutr 1997 ; 77 : 511 – 521

42 Pedersen BK , Steensberg A , Schjerling P . Exercise and interleukin-6 . Curr Opin Hematol 2001 ; 8 : 137 – 141

43 Weickert MO , Spranger J , Holst JJ , Otto B , Koebnick C , Mohlig M , Pfeiffer AF . Wheat-fi bre-induced changes of postprandial peptide YY and ghrelin responses are not associated with acute alterations of satiety . Br J Nutr 2006 ; 96 : 795 – 798

44 Gordon ME , MacKeever KH , Betros CL , Manso Filho HC . Exercise-induced alterations in plasma concentrations of ghrelin, adiponectin, leptin, glucose, insulin, and cortisol in horses . Vet J 2007 ; 173 : 532 – 540