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Obesity: Original Article |
Noll Physiological Research Center and the Department of Kinesiology, Pennsylvania State University, University Park, Pennsylvania 16802
Address all correspondence and requests for reprints to: Nancy I. Williams, 108 Noll Laboratory, Pennsylvania State University, University Park, Pennsylvania 16802. E-mail: niw1{at}psu.edu.
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Abstract |
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Introduction |
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Previous short-term studies have shown that ghrelin levels increase in response to an energy deficit created by decreased food intake (2, 3). Therefore, it would be reasonable to predict that a deficit created by exercise in combination with a reduction in food intake would also lead to an increase in ghrelin. However, a possible independent effect of the physical stress of exercise, apart from an impact on energy homeostasis, on circulating ghrelin has not been explored. Thus, a secondary purpose of this study was to test whether chronic exercise leads to changes in circulating ghrelin in the absence of an overt energy deficit. To this end, we assessed ghrelin levels in individuals who performed exercise training but maintained body weight.
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Subjects and Methods |
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This study was part of a larger prospective study originally designed to assess changes in reproductive function in response to a controlled feeding and exercise intervention. Inclusion in the study was based on the following criteria: 1) no history of serious medical conditions; 2) no current evidence of disordered eating or history of an eating disorder; 3) age, 18–30 yr; 4) weight, 105–160 lb (48–73 kg); 4) body fat, 15–30%; 5) BMI, 18–25; 6) nonsmoking; 7) no medication use that would alter metabolic hormone levels; 8) no significant weight loss/gain ±2.3 kg (±5 lb) in the last year; 9) less than 1 h of purposeful aerobic exercise per week; 10) not taking hormonal contraceptives for the past 6 months; 11) documentation of at least two ovulatory menstrual cycles ranging in length from 25–35 d; and 12) suitable candidate for a controlled feeding and exercise study. Each subject was informed of the purpose, procedures, and potential risks of participation in the study before signing an informed consent approved by the University Biomedical Institutional Review Board. Thirty-eight subjects met the initial screening criteria and began the study; a total of 22 finished the study. Ghrelin was measured in blood samples from a subset of subjects from the larger study who remained weight stable or lost weight.
Subject groupings
All subjects were studied for a baseline period equivalent to one menstrual cycle, followed by three menstrual cycles during which the controlled diet and exercise intervention occurred. Before the baseline period, subjects were randomly assigned to one of four groups that were defined by a particular prescription for the quantity of calories provided as food and the quantity of calories expended as exercise. The groups consisted of a control group who performed no exercise and were provided enough calories to maintain initial body weight and three groups who performed exercise but were provided varying quantities of calories as food. The three exercising groups consisted of an energy balance group that exercised at a high level and were provided extra calories to match those expended through exercise and two exercising groups that were provided fewer calories than that required to maintain initial body weight. By design, one of the latter groups was in a moderate energy deficit, and the other group was in a high energy deficit. Energy intake was quantified daily, and exercise energy expenditure was quantified during each workout. For the current study on ghrelin, subjects were regrouped after completion of the intervention into the following three groups: 1) control group (no exercise, weight maintenance diet), 2) weight-stable exercisers (exercised but body weight did not change significantly, and 3) weight-loss exercisers (exercised and lost a significant amount of weight). To determine the magnitude of weight change deemed to represent significant weight loss, the normal variability in body weight in this group of subjects was estimated by examining the maximum weight change observed during the study in the control group, which was ±1.5 kg. Thus, a change in body weight of 1.5 kg or more became the minimum criteria for inclusion into the weight-loss exercising group, whereas those with weight gain/loss less than ±1.5 kg were included in the weight-stable exercising group.
Dietary intake during the intervention
All meals were made and provided at the metabolic kitchen in the General Clinical Research Center during the three intervention months, and each subject was required to eat at least two of the three weekday meals at this facility. Food items were measured to the nearest gram to achieve the prescribed calorie level. The diet was comprised of 55% carbohydrates, 30% fat, and 15% protein. The dietary protocol used an 8-d meal rotation. The calorie level required to maintain weight for each subject was calculated based on measurement of resting metabolic rate in combination with estimations of 24-h energy expenditure as assessed with a triaxial accelerometer worn on the hip for 7 d (RT3 Accelerometer, Stayhealthy, Inc., Monrovia, CA). This amount of calories was then fed to the subjects for 7 d during the baseline period, and minor adjustments were made to this level of calories if body weight fluctuated during this time. To meet the target level of calorie intake during the intervention, caloric intake was either increased or reduced from the weight maintenance level depending on the experimental group to which the subject was initially assigned.
Subjects were instructed to eat all the food provided to them and only the food provided to them by the study. Any uneaten food was reweighed and recorded for later subtraction from the prescribed intake total. Eating food not provided by the study was highly discouraged, but if this occurred extra food was recorded on a log sheet and calories and macronutrient composition were calculated using Nutritionist Pro (First Data Bank, Indianapolis, IN). Daily and weekly averages of 24-h calorie intake were closely monitored throughout the study. During the intervention, body weight and 24-h energy expenditure were repeatedly monitored and minor adjustments in caloric intake levels and exercise energy expenditure were made.
Exercise training protocol during the intervention
Both the weight-loss and weight-stable exercising groups performed aerobic exercise five times per week at 70–80% of maximum heart rate as determined from tests of maximal aerobic capacity (maximal capacity for oxygen consumption, VO2max). Exercise duration was the number of minutes required to achieve the prescribed exercise expenditure calorie level. All training was supervised by personal trainers in a research training room. The total amount of calories expended during each exercise session was measured using the OwnCal feature on the Polar S610 heart rate monitor (Polar Electro Oy, Kempele, Finland). Exercise calories per week were closely monitored at the beginning of each week to determine whether target exercise calories were met. Throughout the study the heart rate monitors were continually reinitialized with the most recent values of weight, maximum heart rate, maximal aerobic capacity, and age.
Body composition, resting metabolic rate, and cardiorespiratory fitness assessment
All of the following measurements were completed during the pre-, mid-, and postintervention periods in all subjects. Percentage of body fat, fat mass, and fat-free mass were determined by hydrostatic weighing after correcting for residual lung volume (21). Several trials were repeated until three tests yielded a difference of less than 0.5%. Body density was used to calculate body composition using the Brozek equation (21). Body weight was measured to the nearest 0.01 kg on the same day as body composition and twice per week throughout the intervention. All body weight measurements were obtained with subjects wearing shorts and a tee shirt, without shoes. Resting metabolic rate was measured between 0600 and 1000 h after an overnight fast. Upon arriving at the laboratory, the subject would lie in a supine position on a bed for 20–30 min to acclimate to room temperature and undergo familiarization with the equipment and procedures. A ventilated hood was then placed over the subject’s head for 30 min. Expired air was analyzed each minute for carbon dioxide and oxygen concentration using a carbon dioxide analyzer (URAS 4, Hartmann & Braun, Frankfurt, Germany) and a paramagnetic oxygen analyzer (Magnos 4G, Hartmann & Braun). The values for minutes in which steady state was achieved were averaged and the resting metabolic rate in kilocalories per minute was determined using the Weir equation (22). Measurement of maximal aerobic capacity (VO2max) was performed during treadmill running [Modified Astrand Protocol (23)] using indirect calorimetry (Sensormedics Metabolic Cart Model no. 229, Sensormedics Corp., Yorba Linda, CA).
Ghrelin
Fasting morning blood samples were collected and circulating ghrelin was measured using an RIA for total ghrelin (Linco Research, St. Charles, MO). Twenty-two subjects completed the study and underwent pre- and poststudy blood sampling. In 17 of 22 subjects, a blood sample at the midpoint of the study was also obtained. The intraassay and interassay coefficients of variation for the high control were 2.7 and 16.7%, respectively; the intraassay and interassay coefficients of variation for the low control were 1.2 and 14.7%, respectively. The sensitivity of the assay is 30 pmol/liter. All samples from a given subject were analyzed in duplicate and in the same assay.
Statistical analyses
Baseline measurements as well as changes between pre- and postintervention time points in key variables were examined using a one-way ANOVA in 22 subjects. When main effects were detected, post hoc analyses were performed using the least-significant difference procedures. An ANOVA with repeated measures was performed on ghrelin at pre-, mid-, and postintervention in those subjects who had data for all three time points. When main effects were detected, post hoc analyses were performed using t tests employing the Bonferoni correction factor. To determine the time course of changes in ghrelin and changes in key energy balance parameters in the weight-loss exercisers, paired t tests were performed on pre-to-mid and mid-to-post time points. Pearson correlation coefficient analyses were used to examine the relationship between changes in circulating ghrelin and other variables. In all analyses, P < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS software (version 11.0; SPSS Inc., Chicago, IL). Data are reported as mean ± SD.
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Results |
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. No significant differences between groups existed in initial body weight or fat-free mass. However, initial body fat, fat mass, and resting metabolic rate were significantly higher in the weight loss exercisers compared with the controls and weight-stable exercisers. Baseline calorie intake was significantly lower in the control group compared with both exercising groups. Body weight, body mass index, percentage of body fat, and fat mass did not change significantly in the control group and weight-stable exercise group, but all decreased significantly in the weight-loss exercise group. Body weight changes ranged from –2.10 to –4.33 kg in the weight-loss group, +1.5 to –1.25 kg in the weight-stable exercising group, and +1.35 to –1.25 kg in the control group. The change in VO2max was significantly greater in the exercising groups (21.6 ± 11.1%) compared with controls (11.5 ± 9.6%).
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No significant differences were observed in circulating ghrelin between groups before the intervention. There were no correlations between baseline ghrelin and initial body weight, BMI, percentage of body fat, fat mass, fat-free mass, resting metabolic rate, or baseline calorie intake (data not shown). In response to the intervention, ANOVA using pre-, mid-, and post-time points revealed significant changes in ghrelin concentrations over time and a significant time by experimental group interaction. Post hoc testing revealed that ghrelin concentrations increased significantly from pre- to postintervention (770 ± 296 to 1322 ± 664 pmol/liter) in the weight-loss exercisers but did not change significantly in the controls (403 ± 102 to 466 ± 161 pmol/liter) or weight-stable exercisers (744 ± 339 to 646 ± 263 pmol/liter) (Fig. 1). Changes in ghrelin were negatively correlated with changes in body weight in the exercising subjects (r = –0.607; P < 0.05) (Fig. 2). Changes in ghrelin in the exercising subjects were also negatively correlated with changes in fat-free mass (P = 0.064), fat mass (P = 0.078), and calorie intake (P = 0.077), but these correlations did not reach statistical significance. When the time course of the changes in ghrelin and other key energy balance parameters were examined in the weight-loss exercising group, body weight, fat-mass, percentage of body fat, BMI, and resting metabolic rate decreased before a change in ghrelin. Significant changes in these variables occurred from the pre- to mid time points (P < 0.05), whereas ghrelin exhibited significant increases from the mid- to post time points, but not from the pre- to mid time points (P < 0.05) (Fig. 3).
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Discussion |
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Prior cross-sectional and prospective studies have been performed in individuals of differing metabolic status, i.e. obesity (9, 11, 12, 14), and anorexia nervosa (10, 13). These studies show that circulating levels of ghrelin are inversely related to the energy status of the individual in these extreme conditions, i.e. levels are low in obesity and high in anorexia. We failed to observe a correlation between baseline ghrelin concentrations and baseline body weight, BMI, percentage of body fat, fat mass, or fat-free mass. The latter finding suggests that ghrelin levels reflect energy status and body composition only in subjects who have experienced significant alterations in energy homeostasis as opposed to the relatively homogenous energy and body composition status represented by our subjects. The stimulatory effect on food intake attributed to ghrelin (6, 8) suggests a potential role for ghrelin in returning the body to a prior set point for body weight after weight loss. However, cross-sectional studies on individuals of differing energy and body composition status cannot confirm that ghrelin levels have changed over time because energy balance has changed with the development of states such as anorexia or obesity. In these situations, one might hypothesize that ghrelin would be reduced in response to a positive energy balance to promote a reduction in food intake, and increased in response to a negative energy balance to promote an increase in food intake. Of course, the success of reestablishing a theoretical set point for body weight is most likely dependent on a multitude of factors, and the failure of such a mechanism in states like anorexia or obesity may involve cognitive factors as well. Only one prospective study, performed in overweight men, has examined the effects of an exercise intervention program that resulted in weight loss on circulating ghrelin levels. Although there was a significant decrease in body weight, no significant changes in circulating ghrelin were found (15). Our findings differ such that in our normal-weight, healthy young women, ghrelin does appear to act as part of the normal physiological process involved in the regulation of energy balance. Although the design of our study did not allow self-imposed adjustments in caloric intake as weight changed, future studies might test whether an increase in circulating ghrelin in response to weight loss would be associated with an increase in volitional food intake and the restoration of original body weight.
Although ghrelin appears responsive to an energy deficit that results in weight loss, it is unclear what factors specifically modulate its secretion. Clearly, there are a multitude of factors that are changing together when energy homeostasis is altered. Other studies have demonstrated changes in ghrelin in response to insulin and leptin administration (3, 24). Although the possibility of endocrine modulators of ghrelin secretion is not addressed here, we found the strongest relationship between the change in body weight and the change in ghrelin in our exercising subjects. However, other factors such as changes in fat-free mass, fat mass, calorie intake, and resting metabolic rate exhibited a trend toward significance in relation to the change in ghrelin in response to the intervention. Of these variables, we also observed that resting metabolic rate, fat mass, and body weight significantly decreased before ghrelin during the intervention. This suggests that factors related to changes in body weight and perhaps other components of body composition stimulate a compensatory increase in ghrelin in response to weight loss. Although a clear link between ghrelin and food intake has been established in studies in which ghrelin has been administered (6), we were not able to detect a significant correlation between baseline calorie intake and baseline ghrelin concentrations, nor did we find a correlation between the change in calorie intake during the intervention and the change in ghrelin. This may indicate a large interindividual variability in the relationship between ghrelin and calorie intake in individuals of normal body weight who are in energy homeostasis. Additionally, the existence of many other physiological and behavioral factors that modulate food intake must be considered.
Although acute physical exercise has been shown to produce acute decreases in hunger and food intake, studies examining the impact of chronic exercise on food intake are equivocal (25, 26, 27), and none have examined the effects of chronic exercise on ghrelin. Additionally, physical exercise had been shown stimulate various neuroendocrine systems independent of its impact on energy balance (28), but an independent effect on ghrelin has not been explored. In the current study, we showed that exercise training, in the absence of weight loss, had no impact on circulating ghrelin, even though the subjects expended from 517 to 664 kcal per exercise bout. Had we found that exercise training itself produced an increase in circulating ghrelin, this result might have discouraged the incorporation of exercise into weight-loss programs, therefore offsetting other potential exercise-related health outcomes. Alternatively, had we shown that exercise training in the absence of weight loss decreased ghrelin, this result might lead to the recommendation that only exercise be used to achieve weight loss in free-living individuals. The absence of changes in ghrelin in our weight-stable group demonstrates that exercise training itself has little impact on at least one powerful modulator of food intake. This finding also reinforces the concept that the increase in ghrelin in the weight-loss group was in response to the overall energy deficit created by the combination of reduced food intake and exercise, and not due to the endocrine and or metabolic effects of physical exercise itself.
In summary, our findings suggest that ghrelin may be part of a mechanism to reestablish a body weight set point that has been disturbed by diet and exercise in normal-weight young women. Changes in ghrelin appear to be most sensitive to changes in body weight created by an overall energy deficit, independent of specific effects of reduced food intake or physical exercise. Additional analyses comprehensively examining the time course of changes in ghrelin with respect to changes in critical energy balance parameters are necessary to begin to continue to address the mechanism whereby ghrelin contributes to the maintenance of energy homeostasis. Additionally, no studies have clearly established the specific physiological significance of baseline vs. premeal rises in ghrelin. Whether changes in the diurnal rhythm of ghrelin occur with the imposition of a chronic energy deficit also has not been examined.
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Footnotes |
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Abbreviations: BMI, Body mass index; VO2max, maximal capacity for oxygen consumption.
Received August 25, 2003.
Accepted January 28, 2004.
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, Mid- vs. post time points.