| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
From the Clinical Research Centers |
Department of Pediatrics and the Yale Children’s Clinical Research Center, Yale University School of Medicine, New Haven, Connecticut 06510
Address correspondence and requests for reprints to: Sonia Caprio, M.D., Department of Pediatrics, 333 Cedar Street, Yale University School of Medicine, New Haven, Connecticut 06520. E-mail: Sonia.Caprio{at}Yale.edu
 |
Abstract |
|---|
 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Like other hormones, leptin is secreted in a diurnal and pulsatile fashion (5, 8, 9). Maximum circulating leptin levels are reached after midnight, whereas the nadir occurs usually around noon (9). Although the nocturnal rise in leptin resembles the rise in GH secretion and the increase in free fatty acids (FFAs), the temporal relationship between the diurnal changes in leptin, GH, and FFA levels have not been studied previously. Consequently, we have examined the diurnal variation in circulating leptin levels and compared them to 24-h profiles of insulin, GH, and fatty acids in lean and obese adolescents. Puberty represents the ideal developmental stage to examine these relationships because it is normally associated with dramatic increases in circulating GH and insulin levels, as well as changes in body composition. In addition, the effect of obesity on the diurnal variation in plasma leptin was examined in obese adolescents.
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
The study population consisted of 9 lean and 10 obese boys and 9
lean and 10 obese girls. All children were recruited from the Yale
Pediatric Weight Management Clinic. Their clinical characteristics are
indicated in Table 1
. Each subject
underwent a complete physical examination, and a detailed medical and
nutritional history was obtained. During the physical examination
height and weight were measured while the subjects were wearing only
their undergarments, and Tanner stage of pubic hair, breast, or genital
development were assessed. All obese and lean girls had reached
menarche; in obese and lean boys Tanner stage ranged from III–IV.
Although the obese adolescents were 3 years younger than the lean
adolescents, it should be noted that they had similar Tanner stage
development to the lean controls. All obese adolescents had a body mass
index greater than the 95th percentile specific for age and sex
(10). Furthermore, the percent total body fat was 2-fold
greater in obese than lean adolescent boys and girls. As expected, the
percent body fat was 40% (P < 0.01), greater in lean
girls than boys. None of the obese subjects were participating in any
weight reduction program, and their weight had been stable for the last
3 months before the study; all subjects had a normal glycosylated
hemoglobin (normal range, 4–6.3%). None of the subjects who were
taking any medications had any irregular life habits or sleep
complaints. The nature and purpose of the study were carefully
explained to both parents and children before written voluntary consent
to participate in the study was obtained. The study protocol was
approved by the Human Investigation Committee of the Yale University
School of Medicine.
|
Total body composition was determined by dual-energy x-ray absorptiometry using a Hologic scanner (Boston, MA), which is calibrated before scans according to the manufacturer’s recommendations. Subjects were scanned in light clothing lying flat on their backs and with arms by their side. Dual-energy x-ray absorptiometry scans were analyzed using pediatric software (version 1.5e).
Study design
All studies were carried out in the Children’s General Clinic Center at Yale University. Before the day of the study, subjects were asked to follow a 3-day isocaloric diet containing 50% carbohydrate, 35% fat, and 15% protein; to avoid participating in any strenuous physical activity; and to sleep between 2200 h and 0700 h. The subjects were then admitted to the research center at 0700 h, and an iv catheter was placed at 0730 h and was kept patent by an infusion of normosaline. Starting at 0800 h, blood was collected every hour for 24 h for determination of plasma glucose, insulin, leptin, GH, and FFAs. Breakfast, lunch, and dinner were served at 0800 h, 1300 h, and 1800 h, respectively, and all children slept between 2200 h and 0700 h. The caloric distribution was 20% for breakfast, 40% for lunch, and 40% for dinner. Each subject received a caloric diet composed of 50% carbohydrate, 35% fat, and 15% protein, and only water was allowed between meals. The diet consisted of typically consumed whole foods and contained a total of 2200 kcal per day. The subjects did not exercise and most times played either video games, watched television, or read books or magazines.
Biochemical analyses
Plasma glucose was measured by the glucose oxidase method with a glucose analyzer (Beckman Coulter, Inc., Brea, CA). Plasma insulin and GH were measured by a double antibody RIA. Plasma FFA was assayed by a colorimetric method (11). Plasma leptin levels were measured in duplicate using a double antibody RIA (Human Ria Kit; Linco Research, Inc., St. Charles, MO). The limit of sensitivity for the human assay is 0.5 ng/mL, the intra-assay coefficient of variation was 6.4% for a mean level of 14 ng/mL, and the interassay coefficient of variation was 7.6% for a mean level of 15.5 ng/mL. All samples were measured in duplicate.
Data analyses
The area under the curve (AUC) was used to calculate the average of the 24-h glucose, insulin, and leptin concentrations. To quantitatively describe the diurnal rhythm of each leptin profile, we used cosinor analysis (12). Using this analysis, the following chronobiological parameters were obtained: the peak and nadir and the times at which leptin levels were highest and lowest in each individual during the 24-h period. The amplitude was calculated as the difference between the peak and nadir of plasma leptin levels. The 24-h leptin excursion was calculated by dividing the amplitude by the 24-h mean leptin levels. In addition, night (2000 h to 0800 h) and day (0900 h to 1900 h) ratios were calculated.
In addition, with the aid of a Power PC Macintosh microcomputer, Chronolab 3.0 (E.T.S.I. Telecommunication, Pontevedra, Spain), a software package for analyzing biological time series analysis by least squares estimation, was used to validate the results obtained from SAS software (SAS Institute Inc., Cary, NC). Linear least squares rhythmometry was designed for the detection of periodic components in short and noisy series (as they are usually present in clinical situations). In particular, individual circadian rhythm parameters obtained first by single cosinor from each subject were used as imputations or first-order statistics for a population mean cosinor analysis. In this method, the parameter estimates were based on the means of estimates obtained from individuals in the sample, and their confidence intervals depend on the variability among individual parameter estimates. Thus, one obtains for the period under consideration an estimate of the following chronobiological parameters and their confidence intervals: 1) the rhythm adjusted mean or midline estimate statistic of rhythm (MESOR), defined as the average value of the rhythmic function fitted to the data; 2) amplitude, defined as half the extent of rhythmic change in a cycle approximated by the fitted cosine curve (difference between the maximum and the MESOR of the fitted curve); and 3) the acrophase, the lag between a defined reference time (usually midnight of the first day of measurement when the fitted period is 24 h) and time of peak value of the crest time in the cosine curve fitted to the data.
To test the difference between the leptin cosine curves fitted for the groups, we compared the curves using both SAS and Chronolab. Before performing any statistical tests, it was necessary to check to see that the cosine model fit the data accurately. Rhythm characteristics between groups according to gender and age could then be compared with a population-mean cosinor analysis using Chronolab software that is specifically designed to analyze repeated biological data, and Bingham’s test was developed for testing cosinor parameters. When the option for Bingham’s test is selected, Chronolab includes the P values for testing the difference in amplitude, MESOR, and acrophase in its output. The specifics of this test are described in detail elsewhere.
Statistical analysis
Unless otherwise specified, data are expressed as mean ± SEM or as means with 95% confidence interval. Comparisons between groups over time were performed with a two-way ANOVA using SAS version 6.12 oz Chronolab 3.0 software. Pearson’s correlation coefficients were used to study the relations between plasma leptin and percent body fat and with other variables. Multiple linear regression analyses with a stepwise forward procedure was used to determine predictors of the 24-h leptin concentrations. Significance was accepted at less than 0.05. Before the day of the study, subjects were asked to follow a three-day isocaloric diet containing 50% carbohydrate, 35% fat, and 15% protein; to avoid participating in any strenuous physical activity; and to sleep between 2200 h and 0700 h.
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
The temporal pattern of meal-induced glucose and insulin
elevations in lean and obese boys and girls is shown inFig. 1. Plasma glucose profiles were not
significantly different in obese and lean boys and girls. In contrast,
in obese girls and boys fasting and postmeal plasma insulin levels were
significantly greater than the levels in lean boys and girls.
Consequently, the AUC for insulin were significantly greater in the
obese than lean adolescents (Table 2;
P < 0.001).
|
|
).
Circulating plasma leptin showed a diurnal pattern with a nadir
occurring in the early afternoon and a peak between midnight and
0300 h. The cosine fit of the 24-h leptin profile had a similar
time course in lean and obese adolescents. The absolute amplitude was
higher in obese adolescent boys and girls compared with lean controls.
In contrast, the 24-h leptin excursion calculated as the percentage of
the amplitude/24 h mean leptin was significantly smaller in obese girls
than lean girls (57 ± 3% vs. 76 ± 7%,
P < 0.004; Table 3).
Moreover, the night (2000 h to 0800 h) and day (0800 h to
2000 h) ratios were also lower in the obese girls than lean girls
(P < 0.03).
|
). Both a cosinor model using the
actual leptin values and a model using the natural logarithm
transformation of the leptin values were fit. Neither model exhibited a
lack of fit, and neither model’s residuals seemed to stray far from
normal. Thus, each model satisfied the assumptions. Each model was run
and reached identical conclusions. When testing the curve for each group individually, all curves had an amplitude significantly greater than zero. The cosine fit of the 24-h leptin profile had a similar time course in all subjects; all tests indicated that there was not a difference in the acrophase across the groups. However, the MESOR was significantly different when testing lean vs. obese (P < 0.001), lean male vs. lean female (P < 0.002), lean male vs. obese male (P < 0.001), and lean female vs. lean obese female (P < 0.001). Bingham’s test indicated that there was no gender difference in the amplitudes of the curves across gender. However, there was a significant difference in the amplitudes between the lean female and obese female groups (P < 0.002). A similar significant difference was evident between the lean male and obese male groups (P < 0.006). In each case, the obese groups had a larger amplitude than the lean groups.
GH and FFA profile
The pattern of GH levels in lean females showed several daytime
significant changes from baseline and, as expected, a much greater
increment in GH secretion during the sleeping hours (Fig. 2). By 0300 h circulating GH levels
returned to basal. A similar pattern also occurred in lean boys,
although throughout the daytime GH levels increased significantly only
during the early afternoon, returning thereafter to basal levels until
2300 h when it rapidly increased by 3- to 4-fold
(P < 0.001). In contrast, in obese boys mean serum GH
levels were fairly stable during the day. Furthermore, the nocturnal GH
rise was markedly blunted in both obese girls and boys than lean girls
and boys (P < 0.001). Although obese children had
lower GH levels than lean controls, the difference reached statistical
significance only in girls (lean girls vs. obese girls,
P < 0.03).
|
). Likewise,
plasma FFA levels throughout the day were significantly higher in obese
boys (P < 0.03) and girls (P < 0.04)
than respective controls. In both lean and obese boys and girls plasma
FFA levels were suppressed after meals. The nocturnal rise in plasma
FFA was more pronounced in girls than boys and also occurred
significantly earlier in obese girls compared with lean girls.
|
)In a univariate analysis, a significant positive correlation was found between mean plasma insulin levels between 0800 h and 2000 h and peak leptin in all four groups combined (r = 0.63, P < 0.0001). Peak leptin was inversely correlated with the area under the nocturnal (2200 h to 0400 h) GH levels in all groups (r = -0.31, P < 0.0003), whereas it was positively correlated with nocturnal peak in FFA levels (r = 0.45, P < 0.004).
|
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
In agreement with previous reports (9, 14), we found markedly higher fasting and 24-h leptin levels in obese boys and girls compared with lean controls. The time course of the diurnal leptin variation in the obese adolescents was not different from that in lean controls. Interestingly, however, in obese girls leptin 24-h excursion and leptin to night-day ratio were lower than those found in lean girls. This blunted diurnal excursions was also reported by Saad et al. (9) in both obese women and men. The pathophysiological implications of the blunted diurnal variation in leptin levels in obesity is unclear but could play a role in the leptin resistance, thereby contributing to the development and maintenance of obesity. Interestingly, Lagendonk et al. (15) reported in obese women after 50% weight reduction significantly lower relative amplitude in leptin than lean women. Additional studies are needed to establish whether this alteration in the 24-h leptin profile is a preexisting defect or a merely a consequence of the obese state.
The diurnal variation as well as the pulsatility of leptin secretion is entrained to meal pattern (16). The signal regulating leptin levels, and more importantly its pulsatility, by widely dispersed adipocytes, have not been identified. It is conceivable that insulin levels may be essential in regulating leptin production, because leptin diurnal rhythm is entrained to meals (16). Interestingly, the nocturnal rise in leptin does not occur during fasting (17). Moreover, the leptin rhythmicity can be restored during fasting by infusing glucose, which reverses the fall in plasma insulin that normally occurs during prolonged fasting. Absence of the leptin diurnal rhythm has been reported in women athletes with marked decrease in fat mass and decreased insulin levels (18). Moreover, severe insulin deficiency leads to a rapid fall in leptin levels (19). All together, these studies suggest that energy intake affects leptin secretion via changes in insulin secretion. Although insulin has not been found to acutely increase circulating leptin levels (14), it certainly does after chronic infusion (17). In the present study, we found a significant positive correlation between the amount of insulin released throughout the feeding period of the day (AUC, 0800 h to 2000 h) with peak leptin levels in all four groups (r = 0.61, P < 0.001). Similar findings were also reported by Saad et al. (9). Thus, postprandial insulin secretion may be playing a key role in modulating the late rise in leptin levels. Although insulin levels were markedly higher in the obese children, the magnitude of the leptin excursion was not higher compared with lean controls. This may reflect the impairment in insulin action that is commonly seen in obesity.
The temporal pattern of plasma GH in lean adolescents consisted of a number of small peaks during the daytime, followed by a much greater nocturnal increase starting after the onset of sleep and culminating around mid-sleep. In contrast, in obese adolescents mean GH levels varied much less during the day and night. Notably, the nocturnal rise in GH levels paralleled that of leptin secretion. In light of the emerging data on the influence of leptin in the regulation of a number of pituitary hormones, it is intriguing to speculate that the nocturnal rise in leptin may be potentially a metabolic signal that influences GH secretion. A possible role of leptin on the regulation of the GH axis is suggested by the existence of leptin receptors in GHRH neurons in the rat (20) and in human fetal (21) but not adult pituitary (22). Studies in children indicate an inverse correlation between serum leptin concentrations and basal and GHRH-stimulated GH secretion (22, 23, 24). Roemmich et al. (22) recently found that GH secretion and leptin levels were inversely related in pubertal boys and girls (23). In addition to the presence of leptin receptors in different hypothalamic nuclei, several studies have reported effects of leptin on the hypothalamic pituitary axis. For example, leptin administration increased plasma LH, FSH, and testosterone in fasting normal mice (2) and decreased ACTH and corticosterone levels (25). Of note, Licinio et al. (4) recently reported that the nocturnal increase in leptin levels is associated temporally with a profound change in LH pulse parameters in humans. Thus, leptin seems to regulate the minute-to-minute oscillations in LH and estradiol, and the nocturnal rise in leptin may determine the change in LH profile in healthy women (4). Recently, Carro et al. (7) found that leptin antiserum administration to normal fed rats led to a decrease in plasma GH levels, suggesting that physiological leptin levels are needed to ensure normal GH secretion. It is noteworthy that GH hormone response to stimulation is disturbed in the homozygous leptin-deficient adult patients (26). Likewise, obese children and adults have blunted GH secretion, even though they are hyperleptinemic (27). Taken together, these studies are suggestive of leptin having a neuroendocrine role in anterior pituitary hormone secretion. Additional studies are needed to determine the coupling or synchronicity between leptin and GH secretion in lean and obese individuals.
In both childhood and adolescent obesity, the GH-insulin-like growth factor I (IGF-I) is dramatically altered (28). The profound hyperinsulinemia seen in obese children may underlie the decreased production in IGF-binding proteins 1 and 2, which, in turn, may cause a rise in free IGF-I, the biological active form (29). As a result, increased circulating free IGF-I levels could feedback to reduce GH release (29).
During the day there were distinct preprandial increases and postprandial decreases in concentrations in FFA in lean and obese adolescents and an increase during the night. Increased rates of nocturnal lipolysis in adipose tissue could be responsible for the elevated FFA levels at night (30). GH secretion may be particularly important in the regulation of nocturnal lipolysis, in view of its well described lipolytic effect (31). It is also possible, however, that the nighttime rise in leptin mediates the nocturnal rise in circulating FFA levels. Clearly, the relationships between leptin, GH, and lipolysis must be investigated further.
In summary, plasma leptin levels follow a diurnal pattern peaking after midnight and reaching its lowest level in the early afternoon during puberty. Although girls have higher leptin levels, there is no gender difference in the pattern of the 24-h leptin levels in boys and girls. Adolescent obese girls have a blunted relative diurnal excursion in leptin levels. This abnormal rhythmicity may, in part, explain the leptin resistance of the obese state. The nocturnal rise in leptin was paralleled by a nocturnal rise in GH and FFA levels. Additional studies are, however, needed to examine the coupling between leptin and GH diurnal changes in humans.
|
Footnotes |
|---|
Received January 11, 2000.
Revised August 14, 2000.
Accepted October 3, 2000.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  J. Z. Kasa-Vubu, W. Ye, K. T. Borer, A. Rosenthal, and T. Meckmongkol Twenty-Four Hour Growth Hormone and Leptin Secretion in Active Postpubertal Adolescent Girls: Impact of Fitness, Fatness, and Age at Menarche J. Clin. Endocrinol. Metab., October 1, 2006; 91(10): 3935 - 3940. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Wallace, M. Matsuzaki, J. Milne, and R. Aitken Late but Not Early Gestational Maternal Growth Hormone Treatment Increases Fetal Adiposity in Overnourished Adolescent Sheep Biol Reprod, August 1, 2006; 75(2): 231 - 239. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Shea, M. F. Hilton, C. Orlova, R. T. Ayers, and C. S. Mantzoros Independent Circadian and Sleep/Wake Regulation of Adipokines and Glucose in Humans J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 2537 - 2544. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. M. Grundy Obesity, Metabolic Syndrome, and Cardiovascular Disease J. Clin. Endocrinol. Metab., June 1, 2004; 89(6): 2595 - 2600. [Full Text] [PDF]
|
|
|
|
|
|
P. Marzullo, C. Buckway, K. L. Pratt, A. Colao, J. Guevara-Aguirre, and R. G. Rosenfeld Leptin Concentrations in GH Deficiency: The Effect of GH Insensitivity J. Clin. Endocrinol. Metab., February 1, 2002; 87(2): 540 - 545. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
) girls and in lean (
) and obese (
) boys.
Arrows indicate breakfast, lunch, and dinner.
