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Human Performance Laboratory, Departments of Exercise and Sport Science and Physiology, East Carolina University, Greenville, North Carolina 27858
Address all correspondence and requests for reprints to: Dr. R. C. Hickner, 379 Ward Sports Medicine Building, East Carolina University, Greenville, North Carolina 27858. E-mail: hicknerr{at}mail.ecu.edu.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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Numerous laboratories have reported an association between obesity, particularly central adiposity, and high cortisol concentrations in adults. One impetus for the study of salivary or plasma cortisol has been the hypothesis that elevated fatty acid release can result in hepatic insulin resistance and increase the risk of the metabolic syndrome (9, 10). Obese adults often have a greater and more prolonged cortisol response to food than lean (LN) individuals, particularly after lunch (11, 12, 13, 14). During exercise, cortisol concentrations remain higher in OB than in LN subjects (15). It has been traditionally thought that cortisol aids in the release and mobilization of fatty acids from muscle and adipose tissue, indicating that the hormone increases lipolysis. More recently, it has been demonstrated that cortisol may actually suppress, rather than stimulate, lipolysis in fat cells (16). When fasting glycerol release (rate of lipolysis) is expressed per unit fat mass, a similar or lower glycerol release is apparent in OB compared with LN adults (17, 18). A reduced suppression of lipolysis by antilipolytic agents has been shown in OB adults, although this poor suppression of lipolysis with obesity is not always seen in sc fat (17, 19, 20). Furthermore, stimulation of lipolysis during exercise has been shown to be normal in OB adults (21, 22). The extent to which lipolytic responses are different in OB and LN children is yet to be determined.
Due to the lack of information on adipose tissue lipolysis and blood flow as well as the effect of cortisol on lipolysis in children, the purpose of this study was to assess interstitial glycerol concentration, adipose tissue nutritive flow, and salivary cortisol responses to food intake and physical activity during an 8-h study of LN and OB children.
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Subjects and Methods |
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Participants included 21 children, aged 8–10 yr. The male (n = 9) and female (n = 12) children were divided into two groups, OB and LN, based on anthropometric measures. Children were Tanner stage I or II, as reported by the parents, who were presented with a Tanner stage rating scale (23, 24). Subjects were free from known disease and were not taking any medications. The guardians of the participants each completed an informed consent form and a medical history questionnaire; each child signed an assent form. Approval was obtained from the East Carolina University and Medical Center institutional review board before any data were collected.
Study design
Preliminary measures. Children initially visited the Human Performance Laboratory for preliminary measurements, during which time they completed an assent document that outlined procedures, risks, and compensation ($50 for completion of the 8-h microdialysis visit). To determine food of choice for the 8-h microdialysis visit, children also completed a food questionnaire.
After completion of the forms, body mass and height were measured for calculation of body mass index (BMI; kilograms per meter squared). Minimum waist (the narrowest point between the xiphoid process and the umbilicus) and maximum hip circumferences (above the gluteal fold) were also measured while the participants in a standing anatomical position with a Gulick II measuring tape (Country Technologies, Inc., Gays Mills, WI). Skinfolds of the triceps and calf were measured using Harpenden skinfold calipers (Harpenden, UK). Two-site skinfold values were used to calculate percent body fat: the SE of estimate for the body composition analysis was 3.8% fat (r = 0.88) (25). Based on BMI, subjects were categorized as OB or LN. Obesity was defined as having a BMI in the 95th percentile or greater for a specific age and gender (26). The LN group was defined as having a BMI for age less than the 75th percentile. Children also came to the Human Performance Laboratory on a separate day (a Saturday visit from 0700–1515 h) for microdialysis monitoring of interstitial glycerol concentration and nutritive blood flow as well as collection of salivary cortisol and fasting blood samples.
Collection and analysis of fasting blood sample. A fasting fingerstick blood sample was initially obtained from each subject on the microdialysis study day using a microlance and a 50-µl capillary tube for sample collection. The sample was transferred to a microcentrifuge vial and subjected to 3000 x g centrifuge for 5 min. The sample was stored at –20 C until analyzed for plasma glycerol concentration as previously described (27).
Collection and analysis of salivary cortisol. During the Saturday microdialysis visit, salivary cortisol samples were collected in the fasted state at 0900 and 1000 h (before breakfast), 1345 h (before a standardized lunch), and 30, 45, and 60 min after lunch (28). Additional samples were collected each hour to coincide with the hourly collection of dialysate samples. Fifteen minutes before sample collection, children were instructed to rinse their mouths with water. After that, fluid intake was prohibited to prevent dilution of the sample. Saliva samples were collected by spitting into 2-ml vials. The samples were stored at –20 C until analyzed for cortisol in triplicate. Cortisol samples were analyzed using a salivary cortisol enzyme immunoassay (Diagnostic Systems Laboratories, Inc., Webster, TX). The coefficient of variation (CV) for triplicate analyses of salivary cortisol was 1.5 ± 2.0% in the analyses of this study. The CV for the analysis of salivary cortisol samples collected from the same person on 2 separate days was 18.3 ± 3.1%.
Microdialysis. Microdialysis of sc abdominal adipose tissue was performed on the 8-h visit to monitor interstitial glycerol (29) and adipose tissue nutritive flow (7, 8, 30). One LM-3 probe (Bioanalytical Systems, West Lafayette, IN) with a dialysis membrane diameter of 0.2 mm and a length of 30 mm was inserted percutaneously into the abdominal sc fat after the skin was desensitized to pain using topical ethyl chloride spray. Interstitial glycerol concentration was determined in all 21 children using microdialysis at a perfusate flow rate of either 0.3 µl/min (n = 12) or 2.0 µl/min (n = 9). Perfusion at 2.0 µl/min also allowed for the determination of nutritive blood flow in those nine children. Microdialysis probe membrane recovery of glycerol at 0.3 µl/min was assumed to be nearly 100%, and dialysate glycerol was taken to be interstitial concentration. For probes perfused at 2.0 µl/min, recovery was calculated according to extrapolation to zero flow based on dialysate concentrations at 0.3 and 2.0 µl/min perfusate flow rate (31). Dialysate samples were collected at 1000 (start of breakfast), 1100, 1200, and 1300 (end of exercise), 1400 (immediately before lunch), and 1515 (end of study) h. The dialysate and perfusate samples were stored at –20 C until analyzed for glycerol, using a CMA 600 automated analyzer (CMA/Microdialysis, Stockholm, Sweden), and ethanol using a previously described enzymatic fluorometric method (7). The CVs of the ethanol and glycerol assays were 4.5 ± 0.9% and 6.0 ± 1.0%, respectively, for the duplicate determinations of each sample analyzed in this study.
Physical activity. The children participated in a 26-min exercise session at 1230 h during the Saturday microdialysis visit. Exercise, which was performed on a treadmill, consisted of a 3-min warm-up, 20-min of exercise at 140 beats/min heart rate, and a 3-min cool-down. A slightly higher treadmill speed was needed in the LN than OB children to elicit the average heart rate of 140 beats/min for each person during the 20-min portion of exercise.
Meal composition. Both groups of children received standard meals for breakfast and lunch. The weight of food given to each child was measured before and after each meal. All meals were consumed within 15 min. Breakfast, given at 1000 h, consisted of a bowl of cereal, 1 cup of 2% milk, and a piece of fruit. The range of calories, carbohydrate, protein, and fat consumed for this meal were 295–345 kcal, 43–61 g, 9–12 g, and 5–6.5 g, respectively. For lunch, children were given the choice of a prepackaged lunch (Lunchable; Oscar Mayer, Madison, WI). Lunch was served at 1400 h. The ranges of food components consumed were 450–550 kcal, 59–71 g carbohydrate, 10–21 g protein, and 14–20 g fat. The OB children ingested a similar number of total calories, but approximately 20% fewer calories when expressed per kilogram of lean body mass (OB, 8.6 ± 0.64 kcal/kg lean mass; LN, 10.4 ± 0.37 kcal/kg lean mass; P < 0.05).
Calculations
The ethanol outflow/inflow ratio (O:I ratio) was calculated using the concentration of ethanol in the perfusate fluid and the concentration of ethanol in each dialysate sample (ethanol concentration in the dialysate/ethanol concentration in the perfusate fluid).
Estimation of glycerol release was calculated using the following equation adapted from the Fick principle: (V – A) x Q. In this equation V is capillary venous plasma glycerol concentration (micromoles per liter), A is arterialized plasma glycerol concentration (micromoles per liter), and Q is plasma flow rate (milliliters per 100 g per minute). Venous plasma glycerol concentration was calculated using V = (I – A) x (1 – e–PS/Q) + A, where I is interstitial glycerol, and PS is the permeability surface area. The permeability surface area was set at 5 ml/100 g·min (18, 32). Blood flow (Q) was set at 1.6 ml and 3.3 ml/100 g·min in LN and OB children, respectively, in line with previous research and the 2-fold higher ethanol O:I ratios (minimum of 2-fold lower blood flow) measured in OB than in LN children in the present study (32).
Statistical analysis
Fasting values of salivary cortisol, plasma glycerol, O:I ratios, and macronutrient content of the meals were compared between OB and LN groups using nonpaired t tests. Simple linear regression was used to determine the relationship between salivary cortisol and interstitial glycerol responses to breakfast, lunch, and exercise. Repeated measures analyses of covariance, with caloric intake per kilogram of lean body mass as a covariate, were used when analyzing the concentrations of salivary cortisol, interstitial glycerol, and O:I ratios over time in the LN and OB groups. Significant interactions were followed by simple effects test with Bonferroni adjustment. Data are presented ±SD.
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Results |
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A total of 21 subjects (11 LN and 10 OB) participated in the study. Descriptive characteristics of the two groups are presented in Table 1
. Percent body fat and BMI were different between the OB and LN groups by definition.
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The mean fasting plasma glycerol concentration in the OB group was 59.0 ± 30.1 µmol/liter (0.543±.277 mg/dl) compared with a mean fasting glycerol concentration of 42.1 ± 17.6 µmol/liter (0.388 ± 0.016 mg/dl) in the LN group. When fasting glycerol release was calculated (from first dialysate sample; see Subjects and Methods), glycerol release in the OB group was 70% that in the LN group (P < 0.05; Fig. 1). Figure 2 illustrates group trends of interstitial glycerol concentrations throughout the day. The interstitial glycerol concentration was higher in the OB than in the LN group throughout the day (P < 0.05). There was a reduction in dialysate glycerol in the LN, but not in the OB, group in response to breakfast (P < 0.05). Dialysate glycerol increased (P < 0.05) similarly in both groups in response to exercise and decreased (P < 0.05) similarly in response to lunch. There was no correlation between salivary cortisol concentration and interstitial glycerol in response to breakfast or lunch in either group (r = 0.33 and r = –0.39, respectively; not significant).
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The OB group had a significantly higher ethanol O:I ratio than the LN group during fasting and throughout the day (Fig. 3), indicating lower rates of adipose tissue nutritive flow in the OB group. Fasting O:I ratios were 0.31 ± 0.07 and 0.65 ± 0.08 in the LN and OB groups, respectively. The fasting O:I ratio was positively correlated with abdominal skinfold thickness (r = 0.84; P < 0.05). There were no differences in response to treatment stimuli between LN and OB groups.
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There was no significant difference between groups in fasting salivary cortisol concentrations. The LN group had a mean fasting cortisol concentration of 290.9 ± 56.0 nmol/liter (10.54 ± 2.03 µg/dl) compared with 259.2 ± 54.4 nmol/liter (9.39 ± 1.97 µg/dl) for the OB group. Figure 4 illustrates the group trends in salivary cortisol throughout the day.
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Discussion |
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Lipolysis
The measurement of sc interstitial glycerol concentration, in combination with the measure of adipose tissue nutritive blood flow, was used as an index of lipolysis in the present study. Higher abdominal sc interstitial glycerol concentrations were measured in the OB than in the LN group throughout the day, as has been previously reported in studies of adults (20, 33); however, this was probably due to the lower blood flow in the OB children, resulting in poor clearance of glycerol from the interstitium. Adipose tissue nutritive blood flow as well as glycerol release have been found to be lower in OB than in the LN adults in the postabsorptive state (18, 20, 32). Similar to these findings in OB adults, estimated fasting glycerol release was lower in the OB than LN children in the present study. This reduced fasting glycerol release may contribute to the accumulation and/or maintenance of excess fat stores in OB children.
Interstitial glycerol response to food intake
The suppression of lipolysis in response to a meal is of importance with respect to the production and handling of blood lipids. The rise in plasma lipids after a meal has been associated with insulin resistance in adults (34). An increase in circulating fatty acids from adipose tissue during times of increased plasma insulin concentration (after a meal) could lead to excess deposition of nonoxidized fatty acids in skeletal muscle and liver, contributing to insulin resistance. This problem would be exacerbated in the OB state due to the large adipose mass.
In the present study, sc abdominal adipose tissue lipolysis decreased in the LN group in response to breakfast and lunch, as indicated by decreases in interstitial glycerol concentrations and the lack of change in nutritive blood flow. In contrast to the antilipolytic response to breakfast in the LN group, lipolysis was not suppressed in the OB group in response to breakfast. Suppression of lipolysis during the postprandial period in adults has been previously reported (19, 20). Cortisol, which has been recently shown to suppress intracellular lipolysis (16), was not related to the decrease in interstitial glycerol concentrations of either group after breakfast or lunch.
Both LN and OB groups exhibited similar robust decreases in interstitial glycerol concentrations in response to lunch, without significant alterations in nutritive flow. This is in contrast to the breakfast response, in that the LN group, but not the OB group, displayed a suppression of lipolysis in response to breakfast. Exercise may have normalized the antilipolytic response to food intake (lunch) in the OB group. The time of day may have been a contributing factor, although this is unlikely, because the differential response to breakfast and lunch was observed only in the OB group. Salivary cortisol concentrations increased in both groups in response to lunch, although there was no correlation between the increased salivary cortisol concentration and the reduction in interstitial glycerol concentration in response to lunch.
Interstitial glycerol response to exercise
In the current study interstitial glycerol concentrations increased in both groups during and after exercise without significant changes in nutritive blood flow, indicating an increase in lipolysis as expected (21, 22). The changes seen in interstitial glycerol in response to exercise did not seem to be related to blood flow. The adipose tissue nutritive flow response to exercise was not significantly different between groups. Also, adipose tissue nutritive flow did not change significantly from pre- to postexercise periods in either group.
Adipose tissue nutritive blood flow
OB adults have a reduced adipose tissue nutritive blood flow that may alter the supply of nutrients and hormones to, and limit the removal of metabolites from, adipose tissue (18, 20). In the present study adipose tissue nutritive blood flow was reduced in the OB children, as evident from the higher ethanol O:I ratio in OB than in LN children throughout the study day. Lower adipose tissue blood flow per unit fat mass with increased adiposity or skinfold thickness in adults has been shown to be a result of increased adipocyte size without a compensatory increase in the number of capillaries per cell (18, 20). This inverse relationship between blood flow and skinfold thickness was supported by the positive correlation between the ethanol O:I ratio and abdominal skinfold thickness in the present study (r = 0.84; P < 0.05). This is the first demonstration, to our knowledge, of reduced adipose tissue nutritive blood flow in OB children and may signal the early development of the vascular changes seen in OB adults.
Adipose tissue nutritive blood flow response to food intake and exercise
The ethanol O:I ratio was not altered by food intake or exercise in either group of children, suggesting that abdominal sc adipose tissue nutritive blood flow is not altered by food intake or exercise. These data should be interpreted with caution, however, because nutritive blood flow was determined only on a subset of the subjects (n = 9). Carbohydrate and glucose ingestion stimulates an increase in adipose tissue blood flow in LN adults, although there is a delayed or blunted increase in OB adults (18, 20, 32). During exercise, adipose tissue nutritive flow has been reported to increase slightly or not change from resting flow (22, 35).
Salivary cortisol
In the present study the salivary cortisol concentrations of both groups tended to follow previously reported diurnal variation (35, 36), indicating normally functioning HPA axes. Salivary cortisol concentrations in the LN and OB groups were highest in the morning, reached low points in the early afternoon (1200–1300 h), and rose in response to lunch.
Salivary cortisol response to food intake
The salivary cortisol increase in response to food intake was not present in response to breakfast in the LN group. This could be due to the high pulsatile frequency and amplitude of cortisol in the morning masking the cortisol response to breakfast (37). The response of salivary cortisol to lunch was similar in both groups and was similar to that previously termed a normal response (13, 38, 39). OB adults, however, have been shown to have a greater and more prolonged cortisol response to lunch than LN adults (13, 39). The OB children in this study did not have a greater or more prolonged response to lunch, possibly due to the exercise session that ended 1 h before lunch was served. It is unlikely that cortisol is a major regulator of lipolysis in response to food intake, because there was no relationship between salivary cortisol and the change in interstitial glycerol concentration.
Salivary cortisol response to exercise
There were differences in salivary response to exercise, in that there was a change in salivary cortisol only in the OB group in response to exercise. The lack of change in salivary cortisol concentration in the LN group may have been due to the low relative exercise intensity (estimated to be 
60% maximal oxygen consumption, because the cortisol response in adults is consistently measured only with exercise intensities above 65% maximal oxygen consumption (40). It is not known whether cortisol response is dependent upon exercise intensity in children. Salivary cortisol concentration was unexpectedly decreased 32% in the OB group from pre- to post-exercise: the cause of which is not readily apparent.
Limitations
There are several limitations in the study design that should be considered. 1) There is a lack of lipolytic measures in visceral adipose tissue. 2) A control group that did not exercise was not included in the study. 3) The temporal proximity and order of the breakfast, lunch, and dinner may have affected the results. These limitations should therefore be considered for proper interpretation of the current data.
With respect to the first limitation, the lack of data in visceral adipose tissue is important, in that it has been suggested by numerous investigators that visceral adiposity is the best predictor for development of insulin resistance and the metabolic syndrome (41, 42). Unfortunately, visceral adipose tissue is not readily accessible with the microdialysis probe, and there is no other method of measuring visceral adipose tissue lipolysis in ambulatory children. With respect to the second limitation mentioned above, the lack of a nonexercise control group limits the conclusion that exercise may have normalized the antilipolytic response to food intake (lunch) in the OB group. It is, however, unlikely that the time of day was the principle contributing factor in this response, because the differential responses to breakfast and lunch were observed only in the OB group. The third limitation regarding the temporal proximity of the meals and exercise session should be considered when addressing the unexpected decrease in cortisol in the OB group in response to exercise. It is possible that the preceding breakfast meal influenced the cortisol response to exercise, although there was approximately 2.5 h between breakfast and exercise. Serum cortisol has been shown to return to baseline within 2 h after eating (43).
Regarding the macronutient content of the meals, LN and OB children ingested a similar number of total calories, but the heavier/OB children did ingest 20% fewer calories per kilogram of body weight and per kilogram of lean mass than the LN children at both breakfast and lunch. In previous studies of cortisol, meals have typically been given as a standard total number of calories, as in this study. We know of no studies of cortisol or lipolysis designed to investigate the relative influence of absolute caloric content ingested compared with caloric content ingested per kilogram of lean body mass or other relative expressions. There have been reports that large differences in meal composition can result in differential cortisol responses (44). However, in past studies of LN and OB adults, there has been a poor suppression of lipolysis and usually an increased cortisol response in OB individuals regardless of whether the food consumed resulted in the same absolute or the same relative caloric load (45, 46). We have nonetheless statistically controlled for differences in caloric intake per kilogram of lean mass in the present study.
Summary
In this study ambulatory microdialysis was used for the first time in children. Data from this study demonstrate that, based on the sc abdominal adipose tissue glycerol responses to food intake, OB children had less of a suppression of lipolysis in response to breakfast than LN children. However, the suppression of lipolysis and the salivary cortisol response to lunch were similar in OB and LN children. Normalization of the suppression of sc abdominal adipose tissue lipolysis may have been due to the exercise session performed before lunch, although the lack of a control (no exercise) group does not allow for this determination in this study. Subcutaneous abdominal adipose tissue nutritive blood flow was also monitored in ambulatory children for the first time. It was determined that OB children had a reduced nutritive blood flow in this depot throughout the course of the study day. This and other novel uses of the relatively noninvasive and unobtrusive ambulatory microdialysis in children may provide insight into the regulation of lipolysis and nutritive flow in overweight children, thereby providing a basis for treatment of childhood obesity and concomitant cardiovascular disease risk.
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Acknowledgments |
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Footnotes |
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Abbreviations: BMI, Body mass index; CV, coefficient of variation; LN, lean; OB, obese.
Received July 9, 2003.
Accepted June 11, 2004.
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different from before exercise; 
, different from before lunch.
