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The Journal of Clinical Endocrinology & Metabolism 91(10):39353940


Copyright 2006 by The Endocrine Society
doi: 10.1210/jc.2005-2841

Twenty-Four Hour Growth Hormone and Leptin


Secretion in Active Postpubertal Adolescent Girls:
Impact of Fitness, Fatness, and Age at Menarche
J. Z. Kasa-Vubu, W. Ye, K. T. Borer, A. Rosenthal, and T. Meckmongkol
Departments of Pediatrics (J.Z.K.-V., A.R., T.M.), Biostatistics (W.Y.), and Movement Science (K.T.B.), University of
Michigan, Ann Arbor, Michigan 48109
Context: GH is strongly related to body composition, physical activity, and pubertal progression. Adolescent girls decrease physical activity during puberty, whereas their weight increases. Because leptin
is a good index of energy balance in active young women, we hypothesized that leptin is related to GH secretion in this population while
taking into account fitness, fatness, and age at menarche.
Methods: We measured body composition and maximal oxygen consumption (VO2max) in 37 postpubertal adolescent girls aged 16 21
yr. GH was sampled every 10 min and leptin hourly for 24 h. We first
analyzed 6-h time blocks by repeated measures for GH and leptin,
with body mass index (BMI), percent body fat, and VO2max as covariates for the entire group and a lean subgroup. The deconvolution
method was used to characterize GH pulsatility from individual time
points.

HE ATTAINMENT OF puberty enhances the susceptibility to metabolic derangements related to energy balance and excess weight (1, 2). Puberty is characterized by
dramatic changes in the hormonal environment with evidence of a significant age-related decrease in insulin sensitivity and insulin action that merit further study. Concomitant to the insulin resistance of puberty, GH secretion rises
dramatically, and it has been suggested that this increase
mediates in part the changes in insulin dynamics (3). GH
secretion in youth is very sensitive to changes in energy
balance, and fasting can swiftly alter 24-h leptin and GH
secretion in obese prepubertal girls (4). We sought to investigate further relationships among weight, adiposity, and
GH in postpubertal girls.
The parallelism between GH and leptin, a marker of fat
mass (5), has been described in various populations of obese
and nonobese subjects (3, 4). Several recent studies also highlighted the link between GH and metabolic indices related to
weight control such as visceral abdominal fat, insulin (6),
leptin, and ghrelin (7) in adults and elderly subjects. Cardiovascular fitness is also linked to exercise and weight
control.
There is a rapidly declining rate of physical activity in
adolescent girls (8) as well as rising rates of obesity in women
First Published Online July 25, 2006
Abbreviations: BMI, Body mass index; CV, coefficient of variation;
VO2max, maximal oxygen consumption.
JCEM is published monthly by The Endocrine Society (http://www.
endo-society.org), the foremost professional society serving the endocrine community.

Results: GH varied through the day (P 0.0001), with the highest


concentrations overnight. BMI, percent body fat, and VO2max were
related to GH concentrations in the entire group, whereas leptin
predicted GH in the entire group as well as the lean subgroup of girls.
Higher leptin was related to lower GH concentrations (P 0.011),
regardless of time. A log leptin level increase by 1 unit decreased GH
by 27%. Pulsatility characteristics showed a 1-yr increase of age at
menarche increasing total GH input by 20% (P 0.0035) independently from BMI.
Conclusion: In postpubertal adolescent girls, leptin is related to GH
concentration across the lean to overweight BMI spectrum. GH pulsatile secretion was greater in girls with later age at menarche.
(J Clin Endocrinol Metab 91: 39353940, 2006)

(9). In contrast to this, regular, intense exercise is associated


with endocrine disruptions in adolescent girls. A state of
negative energy balance as found with athletic training has
been linked to a relative decrease in leptin in young women
(10). Whereas athleticism in lean girls has been extensively
studied (1113), sedentary or moderately active adolescent
girls have been the focus of considerably fewer studies.
Leptin is an adipocyte-derived hormone that is related to
body mass index (BMI) but is also negatively impacted by
fitness and can be used as an index of energy balance (10).
We thus hypothesized that leptin is related to GH secretion
in healthy postpubertal adolescent girls across the weight
and activity spectra.
Subjects and Methods
Subjects
The protocol was approved by the Institutional Review Board of the
University of Michigan Health System. Thirty-seven healthy adolescents
aged 16 21 yr with a BMI between the 10th and 95th percentile for age
(based on growth charts issued by the National Institute for Health
Statistics) were studied a minimum of 2 yr after the age at menarche as
part of a study on exercise and the menstrual cycle. Because the range
of menarche in the United States is 1116 yr (14), onset of menarche at
16 yr was selected as the upper limit for inclusion in the study. Young
women were recruited from local university campuses and high schools
and were enrolled after signing an institutional review board-approved
consent form. An additional signature was required from one parent or
legal guardian for all participants under 18 yr of age. All participants
were healthy and taking no medications. Individuals who had used any
hormonal method of birth control or reported a significant weight loss
(more than 10%) within the last year were excluded. A 3-d prospective
food diary was collected and analyzed. All girls were asked to estimate,
if any, how many hours of exercise they practiced every week (reported

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J Clin Endocrinol Metab, October 2006, 91(10):39353940

exercise) and describe the type of exercise. Self-described sedentary girls


who reported no planned physical activity were not excluded. All participants attended classes on a college or high school campus, and all
walked from class to class. In addition, we characterized fitness with the
measure of maximal aerobic capacity (VO2max) as determined by a
treadmill test following the Bruce protocol (15). Relative body fat was
obtained by dual-energy x-ray absorptiometry using a total body scanner (model DPX-L; Lunar Radiation Corp., Madison, WI) (16). As a
screen for potential causes of pituitary dysfunction, TSH and prolactin
were assessed in all adolescents. Pregnancy was excluded by serum
measurement of human chorionic gonadotropin.
All participants with an established menstrual cycle were instructed
to contact the principal investigator at the start of the menstrual cycle
and were admitted to the General Clinical Research Center of the University of Michigan within the first 10 d of the cycle. The follicular phase
of the cycle was further confirmed by a serum progesterone measured
on the night preceding the study and before proceeding with sample
collection. Girls with irregular menses were admitted at random, with
the provision that the prestudy progesterone had to be at a follicular
phase level, to proceed with data collection. This strategy allowed to
consistently target the follicular phase of the cycle (average serum progesterone of 0.4 sd 0.2 ng/ml). The admission to the General Clinical
Research Center was also timed on the night before the study to allow
for acclimatization. By 0700 h, an iv catheter was inserted into the
forearm, and blood was sampled 24 h for GH every 10 min as well as
leptin every hour.

Assays
Plasma samples were stored at 70 C until assayed. Plasma GH
concentrations were measured using a chemiluminescent enzyme immunometric assay for use with the Immulite automated analyzer (Diagnostic Products Corp., Los Angeles, CA). Assay sensitivity was 0.01
ng/ml. The intra- and interassay coefficients of variation (CVs) were 2.0
and 4.5%, respectively. Leptin was measured using reagents from Linco
Research (St. Charles, MO) with intra- and interassay CVs of 4.5 and
6.6%. To further characterize the GH axis (17), we measured IGF-I using
a chemiluminescent enzyme immunometric assay for use with the Immulite automated analyzer (Diagnostic Products). The assay sensitivity
was 20 ng/ml; intra- and interassay CVs were 2.5 and 10.7%,
respectively.

Analysis
The analysis applied two different approaches to first describe more
general patterns throughout the day, focused on large time blocks, and
then further detailing pulsatile characteristics of GH derived from frequent individual samples. We took into account fitness as measured by
maximal oxygen consumption, fatness as measured by total body percent body fat, and age at menarche.
Time-block means analysis. As a first step, we delineated 6-h time blocks
to see whether there were relationships that could be related to integrated windows of time reflecting different times of the day (overnight,
early morning, midday, and evening). Because of the nighttime prominence of GH in youth, 2300 h was arbitrarily chosen as the beginning
of the first time block or time 1. The 145 time points of GH and 25 hourly
leptin measurements were analyzed as four 6-h blocks defined as follows: time 1 2300 0450 h, time 2 0500 1050 h; time 3 1100 1650
h; time 4 1700 2250 h. The mean concentration of both hormones for
each time interval was then calculated and referred to as block mean.
Repeated-measures ANOVA was used to determine the effects of time
as well as the effect of body composition, VO2 max, and age at menarche
on GH and leptin. A Tukey adjustment was used for post hoc analysis
of different time periods. Each time block mean was log (base e) transformed before analysis.
Pulsatility analysis. As a second step, we analyzed the pulsatile characteristics of GH over 24 h. Deconvolution (18) was fitted to each series to
detect and characterize the pulsatility in GH. Total input was defined as
the sum of area under all the recognized GH pulses. Half-life, mean area
under the curve, total input, mean height, and number of pulses were
the summary measures of interest. Linear regression was used to study

Kasa-Vubu et al. Body Fat and GH Secretion in Adolescent Girls

the relationship among VO2max, BMI, and percent body fat on GH. All
analyses were adjusted for age at menarche. Half-life, mean area under
the curve, and total input were log (base e) transformed before analysis,
and the number of pulses was square root transformed before analysis.

Results

Subject characteristics are shown in Table 1. The average


age at menarche was concordant with national norms (14).
The average BMI corresponds to the 50 75th percentile for
late adolescent girls and ranged from a minimum at the fifth
to a maximum at the 95th percentile for age (19). IGF-I levels
were comparable with age-specific norms (20). Fitness range
was also comparable with published norms for adolescent
girls (21). The range of reported exercise ranged from sedentary (or no planned exercise) to daily planned physical
activity and averaged almost 4 h/wk at the time of enrollment. Types of reported exercise were all recreational and
varied; they included use of gymnasium, in-line skating,
snowboarding, diving, biking, resistance training, jogging,
skiing, exercise videotapes, and figure skating.
Leptin, BMI, percent body fat, and fitness

We examined relationships between leptin as an index of


energy balance and weight, adiposity, and fitness. Regardless of time of collection, a 1% increase in body fat related to
a 7% increase of leptin (P 0.0001), whereas a 1-unit increase
in BMI increased leptin by 15% (P 0.0001), and a 1-unit
increase in VO2max decreased leptin by 6% (P 0.0001).
When both percent body fat and VO2max were studied together, percent body fat was still significantly related to
leptin (P 0.0001), but the significance of VO2max disappeared (P 0.69). However, BMI and VO2max both retained
significance within the same model for leptin (P 0.0032 and
0.0154, respectively). Thus, the relationship between
VO2max and leptin is not mediated by BMI changes but
appears dependent on differences in percent body fat.
GH Time block analysis

There was marked diurnal variation for GH during the 6-h


time block, with the highest concentrations reached overnight between 2300 and 450 h as outlined in Table 2.
The relationships among BMI, percent body fat, VO2max,
and GH did not depend on the time of the day. However,
when the analysis was restricted to participants with a BMI
between 18 and 25 kg/m2, these relationships with GH lost
significance (P value for BMI 0.74, P value for percent body
fat 0.11, P value for VO2max 0.12).
TABLE 1. Subjects characteristics (n 37)
Mean

Age (yr)
BMI (kg/m2)
Body fat (%)
Age at menarche (yr)
Years from menarche
Average caloric intake (kcal/d)
Average dietary fat intake (%)
VO2max (ml/kgmin)
Reported exercise (h/wk)
IGF-I (ng/ml)

SE

18.9 1.5
23.1 0.5
30.0 1.4
12.7 0.2
6.1 0.3
1896.2 72.4
30.0 1.3
41.1 1.1
3.8 0.4
316.0 16.0

Range

16.0 21.0
18.231.8
17.0 49.7
10.0 16.0
2.0 11.0
1027.73027.0
13.0 51.8
25.752.2
0.0 7.0
110.0 573.0

Kasa-Vubu et al. Body Fat and GH Secretion in Adolescent Girls

TABLE 2. Summary for mean GH (ng/ml) for four time-block


periods
Time

Mean

1
2
3
4

3.9 0.4
1.5 0.2
1.9 0.2
2.0 0.2

SE

Minimum

Maximum

0.5
0.1
0.2
0.2

11.5
6.6
5.7
7.2

Time blocks in hours: time 1 2300 0450 h; time 2 0500 1050


h; time 3 1100 1650 h; time 4 1700 2250 h.

Univariate relationships between GH and BMI and percent body fat and VO2max for the entire group are shown in
Fig. 1. VO2max, BMI, and percent body fat were all highly
related. The Pearson coefficients of correlation were 0.87 for
BMI and percent body fat, 0.58 for BMI and VO2max, and
0.75 for percent body fat and VO2max. As a result, none of
the three variables emerged as a predominant predictor of
GH when analyzed in a multiple regression model.
Leptin and GH

Leptin showed a marked diurnal variation with a peak


between 2300 and 0450 h and a nadir in the afternoon followed by an evening rise (Table 3). Overall, higher leptin
concentrations were related to lower GH concentrations (P
0.011) and a log leptin level increase by 1 unit related to a
mean GH decrease of 27%. This relationship was maintained
regardless of the time block of observation. This relationship
was also maintained when the analysis was restricted to the
leanest girls with a BMI between 18 and 25 kg/m2 (P value
for leptin 0.015). Within the lean subgroup, a log leptin
level increase by 1 unit related to a mean GH decrease of 26%.
The correlation of leptin with weight and fitness indices was
strong, and there was no independent effect of either variable
on mean GH concentration.
GH pulsatility analysis

We studied five pulsatility parameters of GH: half-life,


mean area under the curve, total input, input, and mean peak
amplitude. Representative profiles are shown in Fig. 2. Halflife, number of pulses, and mean amplitude of GH were not
significantly related to VO2max, BMI, or percent body fat
(results not shown). The total input or pulse-related GH was
linked to age at menarche (P 0.0035). For every additional
year at menarche GH total input increased by 20%. The total
input of GH was not related to VO2max (P 0.16) or BMI
(P 0.11); however, there was a negative trend with percent
body fat that did not reach statistical significance (P 0.066).
TABLE 3. Summary for mean leptin (ng/ml) for four time-block
periods
Time

Mean

1
2
3
4

15.9 2.0
12.3 1.7
11.7 1.6
13.6 1.6

SE

Minimum

Maximum

3.1
2.7
2.3
2.7

46.1
41.9
41.6
37.3

Time 1 2300 0450 h; time 2 0500 1050 h; time 3 1100 1650


h; time 4 1700 2250 h.

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We did not find a significant effect of leptin on the five


pulsatility parameters.
Discussion

GH concentrations were higher in those girls who had


higher VO2 max and girls who were leaner. There was a high
correlation between fitness and body composition as well.
During and immediately after puberty, adolescent girls can
experience substantial changes in levels of physical activity
and body composition. By using VO2max as a surrogate
measure of fitness (22), we observed that its effect on GH
secretion pattern was not distinguishable from that of percent body fat. To characterize adiposity further, we measured
leptin concentrations (4, 23), and we found, as expected, that
percent body fat was the most critical determinant of leptin.
Our focus was a population spanning a wide range of fitness
from sedentary to very active and an equally wide range of
BMI, from lean to obese. Our findings are in agreement with
previous work in which athleticism in women represents a
hypoleptinemic state, even when relative adiposity is accounted for (10, 24). The wide range of leptin at all time
blocks was likely a reflection of the wide range of fat mass
as well as the range of athletic activities in our participants.
One additional possibility, which was not within the scope
of our study, is that there may be a range of set points for
leptin.
Although leptin was closely related to the other surrogate
measures of energy balance, namely BMI, VO2max, and percent body fat, it was associated to GH mean concentration in
the subgroup of lean girls with a BMI between 18 and 25
kg/m2. Our study further illustrates the limitations of BMI
as a weight measure and/or a surrogate measure of adiposity
in young subjects who can represent a wide range of physical
fitness and body composition. The importance of fitness is
likely to be more evident in youth because baseline activity
tends to be greater than in older adults. Analyzed together,
both BMI and VO2max remained significantly related to leptin. We propose that in obesity-related studies in adolescent
and young women, the assessment of BMI should be complemented by a measure of adiposity as well as a measure of
fitness or endurance to exercise.
Although all girls were studied a minimum of 2 yr after
menarche, there was higher GH secretion with menarche at
an older age. Because GH decreases after puberty (25) and
declines further with aging (26), one possible explanation is
that the girls with a later age at menarche were still immature
with stronger GH pulses, as can be seen in younger individuals. Our study highlights that the postpubertal maturation of neuroendocrine function continues for at least longer
than 23 yr. Because exercise habits are acquired over a long
period of time (27), one can speculate that girls with a later
age at menarche were likely to be more active as they entered
puberty, thus delaying their menarche. This would in part
explain why higher VO2max was related to significantly
higher GH secretion. Because adolescents with delayed puberty may also have a more prolonged course until they
achieve maturation, it is also possible that a slower tempo of
puberty would influence the results in girls with a later age
at menarche.

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Kasa-Vubu et al. Body Fat and GH Secretion in Adolescent Girls

FIG. 1. Time-block analysis relationships between mean GH and BMI and


percent body fat and VO2max.

In recent years, there have been reports suggesting


that GH is involved in pubertal maturation (28), facilitates folliculogenesis (29), and enhances the effectiveness of assisted reproductive techniques in some women
(30). Attempts to discriminate between women in which
GH was effective at facilitating ovulation from those who
were not GH responsive have relied on the response to
GH-provocative tests as predictors (31, 32). Salat-Baroux et
al. (33) reported that basal GH levels were higher in

women who were poor responders to assisted ovulation


techniques than those who had a good response. Due to
our participants young age and the cross-sectional nature
of this study, fertility potential could not be assessed here.
It is possible, however, that the concurrent finding of
higher GH secretion and decreased fertility merely represents the impact of acquired fitness during puberty
by delaying puberty and fertility and enhancing GH
secretion.

Kasa-Vubu et al. Body Fat and GH Secretion in Adolescent Girls

J Clin Endocrinol Metab, October 2006, 91(10):39353940

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FIG. 2. Selected individual GH profiles of girls with menarche before (left) or after (right) 13 yr.

Acknowledgments
We are most grateful to the adolescent girls who participated in this
study and the skilled nurses of the General Clinical Research Center of
the University of Michigan.
Received December 30, 2005. Accepted July 18, 2006.
Address all correspondence and requests for reprints to: Josephine Z.
Kasa-Vubu, M.D., M.S., Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan 48019-0718.
This work was supported by the Genentech Foundation for Growth
(to J.Z.K.-V.), National Institutes of Health Building Interdisciplinary
Research Careers In Womens Health Center Grant 1K12HD01438-01 (to
J.Z.K.-V.), 1K23DK065995 award (to J.Z.K.-V.), and General Clinical
Research Center Grant M01-RR-0042.
The authors have nothing to declare.

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JCEM is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the
endocrine community.