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Department of Pediatrics, Division of Pediatric Endocrinology (J.Z.K.-V., P.O., T.M., C.M.F.), and Department of Internal Medicine, Division of Endocrinology and Metabolism (A.B.), University of Michigan Medical School, Ann Arbor, Michigan 48019; and Department of Biostatistics, University of Michigan School of Public Health (N.E.C.), Ann Arbor, Michigan 48109
Address all correspondence and requests for reprints to: Dr. Josephine Z. Kasa-Vubu, Department of Pediatrics, Division of Pediatric Endocrinology, University of Michigan Medical Center, 11th Floor, 300 North Ingalls, Ann Arbor, Michigan 48019-0718.
Abstract
We studied nutrition and GH in eight obese girls, aged 6–11 yr. Blood was sampled every 15 min for 24 h. A 48-h diet providing 25% of assumed caloric needs was imposed, with repeat sampling during the last 24 h. Six nonfasting lean girls were also studied, and their mean GH was 3 times that of the obese girls in the fed state (P = 0.024). Dieting increased mean GH by 60% (P = 0.0028). There was no difference in pulse number for either group, but total secretion for lean girls was 3.9 times greater than that in obese girls during the fed state. With dieting, obese girls increased their total GH secretion by 60% (P = 0.010), but maintained lower total secretion, approximately 40% that of lean girls (P = 0.014). Mean leptin in obese girls in the fed state was 6.2 times greater than mean leptin in lean girls (P = 0.0001), with higher concentrations at night (P < 0.05) and lowering of total mean leptin while dieting. We conclude that in early pubertal obese girls, short-term caloric restriction partially reverses the low GH state that is characteristic of obesity. The change is concomitant with a decrease in leptin and a lessening of circadian differences.
IN PREPUBERTAL children, growth and stature are related to circulating GH concentrations. Obese children present the paradoxical combination of tall stature associated with decreased GH concentrations. GH release is induced by GHRH and is inhibited by hypothalamic somatostatin, which antagonizes GHRH-mediated GH release (1). In addition to the central role played by these hypothalamic peptides, GH secretion appears to be regulated by peripheral peptides. Circulating IGF-I is synthesized primarily by the liver and is a peripheral index of GH secretion (2). It is believed that low GH concentrations, characteristic of obesity, are related to relatively high IGF-I concentrations that feed back at the pituitary and/or hypothalamic level to suppress GH secretion. The iv infusion of recombinant IGF-I suppresses GH secretion in young men with greater potency than in young women. This evidence of sexual dimorphism in the regulation of GH secretory patterns suggests that there may be other unidentified modulators of the GH axis (3).
The pathophysiology of GH secretion in obese children has been inferred from studies performed in adults. However, inferences from adults may be misleading regarding hypothalamic feedback mechanisms. Brain maturation and the effects of sex steroids result in marked differences in gonadotropin secretion in both children and adults. The changes in GH concentration before, during, and after puberty suggest that inferences from adult GH physiology and pathophysiology may be problematic. We therefore tested the hypothesis that fasting would increase GH secretion in obese children.
Recent studies on the role of nutrition in GH physiology have identified leptin, an adipocyte-derived peptide, as a potential indicator of growth. Human models of congenital leptin deficiency suggest that this protein may be a fat messenger generated from peripheral stores to report the status of energy balance to the brain (4, 5). There is evidence that leptin has a role in regulating the GH axis during the prepubertal transition (6). Recent data suggest that a functional and reversible defect in GH secretion in food-deprived rats is closely linked to a decrease in leptin concentration (7). Furthermore, leptin appears to be a significant determinant of GH-binding protein (GHBP), which suggests that there may be a physiological pathway involving GH receptor expression and leptin (8).
In this study we have examined the physiological relationships among nutrition, GH secretion, and leptin concentrations in eight early pubertal girls, 6–11 yr of age. Overweight early pubertal girls should have greater leptin and IGF-I concentrations compared with normal lean girls. However, despite rapid growth and accelerated pubertal maturation, obese girls represent a relatively low GH concentration state. We measured GH concentration at short intervals for an extended time period to characterize the secretion patterns before and during a 48-h modified fast. Secretory profiles of GH during fed and fasting states were compared with those in normal lean girls in a nonfasting state. Leptin concentrations, whether tied to GH secretion or not, are useful in assessing fasting state; hence, we measured leptin concentrations in our subjects.
Subjects and Methods
Subjects
Eight obese girls in early to midpuberty, characterized by pubertal staging as well as bone age, were enrolled in the study. Six additional lean girls with bone ages within the same ranges as the obese subjects were studied as controls. All subjects had normal thyroid function.
Protocol
Studies were conducted in the General Clinical Research Center of the University of Michigan. The institutional review board committee of the University of Michigan approved the study protocol (Fig. 1
). Informed written consent was obtained from a parent. The girls were admitted to the General Clinical Research Center during the evening before the study. At 0600 h the following day, an iv line was inserted in the forearm to allow for blood withdrawal from subjects during a 24-h period. GH was sampled every 15 min. Equal aliquots from each sample were pooled to yield 3-h interval measurements for leptin. Obese girls were then placed on a modified fast, during which 25% of their estimated lean weight’s caloric needs were provided, for the following 48 h. The General Clinical Research Center registered dietitian directly supervised the diet plan. During the last 24 h of caloric restriction, a second 24-h blood sampling every 15 min for GH and leptin was collected. During this study trained nursing personnel monitored the quality of sleep. Lights were turned off at 2200 h and were turned back on at 0600 h the following day. Meals were provided at 0800, 1200, and 1700 h, with afternoon and bedtime snacks.
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Blood samples were centrifuged, and plasma was stored at -20 C until assayed. All assays were run in duplicate. Using reagents from Linco Research, Inc. (St. Charles, MO), we performed leptin assays with intra- and interassay coefficients of variation of 4.5% and 5.0%, respectively. The assay sensitivity was 0.5 ng/ml. Plasma GH was measured by a chemiluminometric assay (Nichols Institute Diagnostics, San Juan Capistrano, CA). The assay sensitivity was 0.1 µg/liter (1 mU/liter = 0.4 µg/liter), and the mean coefficient of variation was 6.5%. All samples from a given subject were run in the same assay. Esoterix Laboratories (Las Calabas, CA), a commercial laboratory, measured IGF-I and IGF-binding protein-1 (IGFBP-1). Leptin concentrations were measured in all lean girls and in seven of the eight obese subjects. GH, IGF-I, and IGFBP-1 were measured in all subjects.
Statistical analysis
Mean analysis. To determine whether GH concentrations differ between groups and over time, the 96 time points of observation were divided into 4 6-h intervals as follows: time 1 (T1), 2300–0445 h; T2, 0500–1045 h; T3, 1100–1645 h; and T4, 1700–2245 h. The mean concentration was then calculated for each of the four intervals and referred to as a block mean. For both hormones (GH and leptin), repeated measures ANOVA was used to determine the effect of time and group on mean hormone concentrations. A Tukey adjustment was used when comparing the means of different time periods. IGF and IGFBP-1 differences were compared using a two-sample t test assuming unequal variances or a paired t test where appropriate. Each block mean or observation was log (base-e) transformed before analysis.
Pulsatility analysis. Deconvolution was fitted to each series to detect and characterize the pulsatility in GH. Half-life, total input, and number of pulses were the summary measures of interest. Each measure was analyzed using a paired t test for comparison between fed and fasting states in the obese girls and a two-sample t test assuming unequal variances for comparing obese girls vs. lean girls. The half-life and total input were log (base-e) transformed before analysis, and the number of pulses was square root transformed before analysis.
Results
Subjects’ characteristics are shown in Table 1. Body mass index (BMI) (9) was consistently elevated (P = 0.0001) in all obese girls.
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shows the mean GH concentration over time for the lean girls and the obese girls during the fed and fasting states. The mean GH concentration was significantly higher during the night (T1) compared with the day (T2–4) for all groups. Depending on the group comparison, the mean GH for T1 was approximately 2.5–2.8 times the mean GH for T3 and T4 and approximately 2.5–5.1 times the mean GH for T2 (P < 0.05 for all comparisons). The lean girls’ mean GH concentration was approximately 2.9 times [95% confidence interval (CI), 1.2–7.3; P = 0.024] that of the obese girls during the fed state. Caloric restriction significantly increased the obese girls’ mean GH concentration (P = 0.0028), such that mean GH after caloric restriction was approximately 1.6 times (95% CI, 1.2–2.0) their mean GH during the fed state. Mean GH in the obese girls during caloric restriction was no longer significantly lower than that in the lean girls (P = 0.10).
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). During the fed state, total pulsatile secretion in the lean girls was approximately 3.9 times (95% CI, 1.7–9.1; P = 0.022) that of the obese girls. Caloric restriction in the obese girls increased total pulsatile secretion by 60% (95% CI, 20–110%; P = 0.010) compared with that in their fed state, but the total pulsatile input was still only 40% (95% CI, 20–80%; P = 0.014) that in the lean girls. There was no difference in pulse number or half-life for any of the groups.
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, GH profiles and secretion rate, deduced from deconvolution analysis, are represented for two lean (A and B) and two obese (C1–2, and D1–2) girls in both fed and fasting states.
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shows the mean leptin concentrations over time for all three groups. The mean leptin concentration in obese girls during the fed state was approximately 6.2 (95% CI, 3.4–11.2; P = 0.0001) times that in lean girls. Caloric restriction in obese girls decreased the mean leptin concentration by approximately 30% (95% CI, 17–38%; P = 0.0017), but mean leptin in calorie-restricted girls was still approximately 4.4 times that in lean girls (95% CI, 2.3–8.7; P = 0.0010). During the fed state, obese and lean girls had a significant circadian pattern for leptin. In general, leptin levels were lower in the morning, increased throughout the day, reached maximal concentrations in the late night (2300 h), and began to decrease in the early morning hours. See Table 3 for statistical comparisons. The time pattern in the obese girls marginally depended on their eating state (P = 0.055). Obese girls during the fed state had a significant time trend (described above), whereas obese girls during their calorie-restricted state did not have a significant time trend. A difference in time trend was not detected when comparing calorie-restricted obese girls with lean girls because of the small sample size in the two-sample design, but there was still an overall significant time trend (P = 0.0015) that was consistent with the circadian pattern found when comparing the obese girls during the fed state and the lean girls.
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).
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Obesity is a disorder with significant morbidity in children. The incidence of this disease is rising to epidemic proportions in Northern America, and an estimated 25% of children are now considered overweight. The onset of obesity often becomes more noticeable as the child approaches pubertal age; statistically, a child who is obese at age 12 yr has a 75% chance of being obese as an adult (10). GH concentrations are relatively low in obese children despite increased linear growth and increased IGF-I. Decreased GH in overweight individuals has been reported across ages from childhood (11), and throughout the pubertal transition (12). The lower GH concentrations observed in obese individuals are refractory to interventions that increase GH secretion in lean individuals, such as exercise (13). In obese children, multiple abnormalities of the GH and IGF-I axes stand in sharp contrast to their tall stature. This paradox is poorly understood (14).
The low GH concentrations characteristic of obesity have been attributed to diminished pituitary secretion and increased plasma clearance of the hormone (15). The secretion of bioactive GH nonmeasurable by standard assays has been explored in a number of physiological states (16). The reduction of GH secretion in obese children can be acutely overcome by the administration of a synthetic GH-releasing peptide alone or in combination with GHRH (17). Pharmacological blockade of somatostatin with agents such as pyridostigmine (18), galanin (19), and atenolol (20) will acutely reverse the obesity-induced low GH state. These reports suggest that an increased somatostatin tone is likely to play a role in the decreased GH concentrations associated with obesity. A fasting-induced reduction of somatostatin could explain the results of our study. However, an alternate possibility would be that low GH concentrations in obese individuals are the direct result of a decrease in GHRH. Further studies using a GHRH antagonist during fasting would help elucidate whether GHRH plays a key role in obesity physiology.
Lasting changes in energy balance resulting from decreased caloric intake and increased energy expenditure can reverse the metabolic effects of obesity. Indeed, significant weight loss is known to restore the GH concentration in previously obese subjects (21). In our study we have observed that a supervised short-term modified fast induces a rapid increase in GH concentrations in obese early pubertal girls. These findings indicate that in obese girls changes in GH concentrations may be evident before weight loss becomes significant.
It has been suggested that hyperinsulinemia associated with obesity inhibits GH secretion while maintaining IGF-I levels and decreasing IGFBP-1 (14). Low GH concentrations in obese girls could be explained by high insulin levels, which may help maintain elevated IGF-I, as seen in our subjects. As a result of the negative feedback of IGF-I, GH secretion is blunted. One hypothesis is that obesity-induced hyperinsulinemia decreases IGFBP-1, which would further increase IGF-I bioavailability (11). These changes could mediate accelerated growth velocity in obese children despite suppressed GH release. Taking into account the large numbers of IGFBPs and possible interactions among themselves, free circulating IGF-I could change significantly with fasting. Our study does not show any significant changes during short-term caloric restriction with either total IGF-I or IGFBP-1. Larger studies as well as longer periods of monitored caloric restriction may be necessary to induce a significant decrease in free IGF-I and/or a concomitant increase in IGFBP-1. Although IGF-I and its binding proteins are likely to play a major role in mid- to long-term changes in energy balance, the changes in GH shown in this study suggest that other agents are implicated in this acute response to caloric restriction.
Recently, leptin, the adipocyte-derived hormone, has emerged as a potential regulator of the GH axis. In contrast to the suppression of GH seen in obesity, leptin increases with fat mass (4). Multiple studies have shown a close relationship between BMI and circulating leptin concentrations. Although this relationship is present in both males and females, puberty is associated with greater circulating leptin concentrations in females, even after correction for differences in BMI (5). This protein appears to play an important role in the regulation of energy balance by controlling the appetite set-point (4, 5) and may also affect the metabolic rate. During the modified fast, the decrease in leptin occurs in parallel to the increase in GH in obese early pubertal girls. Previously described as a mere reflection of fatness, leptin may actually play the role of a metabolic signal transmitting the state of peripheral energy balance to the hypothalamus (5). In rats, the administration of leptin antiserum leads to a decrease in spontaneous GH secretion. The administration of leptin to normal fed rats will not affect GH secretion, whereas the administration of leptin to fasting animals will reverse the inhibitory effect of fasting on GH secretion (22). In humans, changes in leptin have been linked to another nutritional factor, GHBP. GHPB is elevated in obese subjects and is linked to nutritional status, as it is low in underweight subjects and rises with increased BMI (23). In the study by Llopis et al. (23), there was a significant correlation between leptin and GHBP, the soluble fraction of the GH receptor. Llopis et al. (23) speculate that in obesity the increased leptin concentration may induce GHBP/GH receptor expression, which would explain blunted GH concentrations in contrast to high IGF-I levels. The abrupt decrease in leptin in our study with a concomitant increase in GH would suggest that leptin might have a role in the fasting-mediated changes in GH concentrations. The rapidly suppressive effects of a modified fast on leptin concentrations suggest that it is not merely an index of total body fat; rather, our data indicate that leptin may function as an adaptive mechanism in response to food deprivation. Whether the administration of exogenous leptin can prevent the GH changes induced by this modified fast is unknown. The administration of leptin induces fat loss in all mammalian species tested, but trials on its safety and efficacy for the treatment of obesity in humans are still underway (24).
Although GH showed substantial day-night differences in these same girls, our data indicate that leptin could fulfill functions that differ depending on the nutritional state (23). In a short-term modified fast, changes in leptin concentration before significant weight loss can be documented, suggesting a physiological role for leptin in relation to acute or long-standing differences in energy balance. Via leptin, peripheral adipose stores play an active role in the modulation of the GH axis in both the fed and fasting states. Our study shows that the effect of fasting on leptin and GH concentrations are also evident after a modified fast providing 25% of caloric requirements.
The recent discovery of ghrelin as a probable regulator of pituitary GH secretion may improve our understanding of the effect of obesity on GH secretion. Subcutaneous injection of ghrelin induces adiposity in rodents without an associated change in appetite, whereas intracerebroventricular administration generates a dose-dependent increase in food intake and body weight (25). A recent report showed that intracerebroventricular administration of ghrelin also stimulates GH secretion and food intake (26). This novel hypothalamic peptide, also synthesized in the stomach is currently hypothesized to act as an integrator of food intake, GH secretion, and energy balance (27). Further studies are needed to determine whether ghrelin plays a role in the changes in GH concentrations associated with caloric restriction, as seen in our obese subjects.
Spontaneous GH secretion is enhanced by fasting in adults and elderly individuals regardless of the state of wakefulness and with no day-night difference (28). This study shows that short-term modified fasting can partially reverse the suppressive effect of obesity on GH secretion. These changes are evident before any demonstrable effect of fasting on IGF-I or IGFBP-1 concentrations. Our data suggest that the adipocyte-derived peptide, leptin, may play a role in the regulation of GH secretion in overweight individuals. The age- and gender-related differences in the nutritional dependency of the GH axis need further study.
Acknowledgments
We thank Alice Rolfes-Curl and Roberta DeMott-Friberg for their expert technical assistance.
Footnotes
This work was supported by the Genentech Foundation for Growth, NIH Grant RO1 DK-384449, and General Clinical Research Center Grant M01-RR-0042.
Abbreviations: BMI, Body mass index; CI, confidence interval; GHBP, GH-binding protein; IGFBP-1, IGF-binding protein-1; T1, time 1.
Received February 6, 2001.
Accepted October 29, 2001.
References
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  M. Misra, M. A. Bredella, P. Tsai, N. Mendes, K. K. Miller, and A. Klibanski Lower growth hormone and higher cortisol are associated with greater visceral adiposity, intramyocellular lipids, and insulin resistance in overweight girls Am J Physiol Endocrinol Metab, August 1, 2008; 295(2): E385 - E392. [Abstract] [Full Text] [PDF]
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 B. Martin, M. Pearson, L. Kebejian, E. Golden, A. Keselman, M. Bender, O. Carlson, J. Egan, B. Ladenheim, J.-L. Cadet, et al. Sex-Dependent Metabolic, Neuroendocrine, and Cognitive Responses to Dietary Energy Restriction and Excess Endocrinology, September 1, 2007; 148(9): 4318 - 4333. [Abstract] [Full Text] [PDF]
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 J. Z. Kasa-Vubu, A. Rosenthal, E. G. Murdock, and K. B. Welch Impact of Fatness, Fitness, and Ethnicity on the Relationship of Nocturnal Ghrelin to 24-Hour Luteinizing Hormone Concentrations in Adolescent Girls J. Clin. Endocrinol. Metab., August 1, 2007; 92(8): 3246 - 3252. [Abstract] [Full Text] [PDF]
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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]
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