Secretory dynamics of leptin in adolescent girls with anorexia nervosa and healthy adolescents

Madhusmita Misra,1,2 Karen K. Miller,1 Kelly Kuo,1 Kathryn Griffin,1 Victoria Stewart,1 Emily Hunter,1 David B. Herzog,3 and Anne Klibanski1

1Neuroendocrine Unit, Massachusetts General Hospital and Harvard Medical School, 2Pediatric Endocrine Unit, Massachusetts General Hospital for Children and Harvard Medical School, 3Eating Disorders Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts

Submitted 1 February 2005 ; accepted in final form 30 March 2005


    ABSTRACT
 TOP
 ABSTRACT
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Leptin, an adipocytokine that suppresses appetite and may regulate neuroendocrine pathways, is low in undernourished states like anorexia nervosa (AN). Although leptin exhibits pulsatility, secretory characteristics have not been well described in adolescents and in AN, and the contribution of hypoleptinemia to increased growth hormone (GH) and cortisol in AN has not been explored. We hypothesized that hypoleptinemia in AN reflects decreased basal and pulsatile secretion and may predict increased GH and cortisol levels. Sampling for leptin, GH, cortisol, and ghrelin was performed every 30 min (from 2000 to 0800) in 23 AN and 21 controls 12–18 yr old, and data were analyzed using Cluster and deconvolution methods. Estradiol, thyroid hormones, and body composition were measured. AN girls had lower pulsatile and total leptin secretion than controls (P < 0.0001) subsequent to decreased burst mass (P < 0.0001) and basal secretion (P = 0.02). Nutritional markers predicted leptin characteristics. In a regression model including BMI, body fat, and ghrelin, leptin independently predicted GH burst interval and frequency. Valley leptin contributed to 56% of the variability in GH burst interval, and basal leptin and fasting ghrelin contributed to 42% of variability in burst frequency. Pulsatile leptin independently predicted urine free cortisol/creatinine (15% of variability). Valley leptin predicted cortisol half-life (22% of variability). Leptin predicted estradiol and thyroid hormone levels. In conclusion, hypoleptinemia in AN is subsequent to decreased basal and pulsatile secretion and nutritionally regulated. Leptin predicts GH and cortisol parameters and with ghrelin predicts GH burst frequency. Low leptin and high ghrelin may be dual stimuli for high GH concentrations in undernutrition.

growth hormone; cortisol; insulin resistance; estradiol; ghrelin; thyroid hormones


LEPTIN IS A CYTOKINE expressed by adipose tissue that suppresses appetite by interactions with its receptor in the brain (5, 32, 46). There is evidence that leptin is secreted in a pulsatile manner (20, 22, 44), but secretory dynamics of leptin are unclear, especially in adolescents. Low levels of leptin have been reported in adolescent girls with anorexia nervosa (AN) by our group (15, 28, 38) and by other investigators (17, 40). Differences in leptin secretory dynamics in this model of undernutrition compared with healthy adolescents, however, have not been examined.

Published data suggest that leptin may modulate neuroendocrine function and levels of nutritionally regulated hormones such as growth hormone (GH), cortisol, thyroid-stimulating hormone (TSH), and luteinizing hormone (LH) in healthy adults. Anorexia nervosa is a unique model in which to examine the effects of undernutrition and hypoleptinemia on neuroendocrine function and its regulation. We have previously demonstrated high levels of GH with low levels of insulin-like growth factor-I (IGF-I), and elevations in serum cortisol in girls with AN compared with healthy adolescents (27, 29), and it is unclear whether these alterations are related to low leptin levels.

In this study, we determined secretory dynamics of leptin by frequent sampling overnight in adolescent girls with AN and in healthy controls of comparable maturity by Cluster and deconvolution methods. We hypothesized that decreased leptin concentrations in AN are a consequence of decreased basal and pulsatile secretion, which are nutritionally regulated. We also determined the relationship between leptin secretory characteristics and those of GH and cortisol. The relationship between leptin and other nutritionally regulated hormones was explored.


    SUBJECTS AND METHODS
 TOP
 ABSTRACT
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Subject selection. We studied 23 adolescent girls meeting the Diagnostic and Statistical Manual of Mental Disorders (4th edition; DSM-IV) criteria for AN and 21 healthy adolescents of comparable maturity (chronological age: 16.2 ± 1.6 vs. 15.4 ± 1.8 yr, P not significant; bone age: 15.8 ± 1.5 vs. 15.7 ± 2.1 yr, P not significant). The mean duration since diagnosis of AN was 7.9 ± 10.5 mo. Clinical characteristics and hormonal data (but not leptin data from frequent sampling) have been previously reported (27, 29). We recruited subjects with AN through referrals from Eating Disorder Units and providers in the New England region. Healthy adolescents were recruited through advertisements within the Partners HealthCare system and mass mailings to primary care providers in New England. None of the healthy controls had a present or past history of eating disorders. Use of medications and presence of disorders (other than AN) that might affect GH, cortisol, or leptin levels were exclusion criteria for the study. This study was approved by the Industrial Review Board of Partners HealthCare, and informed consent was obtained from all subjects and their parents.

Experimental protocol. Subjects were admitted overnight to the Clinical Research Center of Massachusetts General Hospital following a screening visit to confirm eligibility. Height was measured on a single stadiometer in triplicate and the average of three readings recorded. Weight was measured on a single electronic scale. Bone age was performed to assess maturity and was read by a single observer, a pediatric endocrinologist, using the methods of Greulich and Pyle (14). Percent body fat and percent trunk fat were determined using dual energy X-ray absorptiometry (DEXA; Hologic 4500, Waltham, MA).

We performed frequent sampling for leptin, ghrelin, GH, and cortisol through a 20-gauge intravenous catheter in the antecubital region every half-hour for 12 h overnight (2000 to 0800). Our subjects had supper before 1900 and were fasting thereafter. Fasting blood samples were obtained for IGF-I, glucose, insulin, leptin, and estradiol at 0800 the following morning. We also obtained levels of FSH, LH, TSH, free thyroxine (T4), total T4, and total triiodothyronine (T3). Girls with AN were followed over a 1-yr period, and frequent sampling was repeated at weight recovery (defined as a 10% increase in BMI; n = 10).

Biochemical assessment. Levels of creatinine, glucose, LH, FSH, and TSH were determined by the hospital laboratory using standard methods. Radioimmunoassay (RIA) was used to measure leptin (Linco Diagnostics, St. Louis, MO; sensitivity 0.5 ng/ml, coefficient of variation 3.4–8.3%), serum cortisol (Diagnostic Products, Los Angeles, CA; limit of sensitivity 1.0 µg/dl, coefficient of variation 2.5–4.1%), insulin (Diagnostics Products, Los Angeles, CA; coefficient of variation of 4.7–7.7%), ghrelin (Phoenix Pharmaceuticals, Belmont, CA; sensitivity 2 pg/ml, coefficient of variation 10%), and estradiol (Diagnostic Systems Laboratories, Webster, TX; limit of detection 2.2 pg/ml, coefficient of variation 6.5–8.9%). The ghrelin assay used measures total ghrelin. We used an immunoradiometric assay (IRMA) to measure GH (Nichols Institute Diagnostics, San Juan Capistrano, CA; detection limit 0.05 ng/ml, coefficient of variation 2.4–9.4%) and IGF-I (Nichols Institute Diagnostics; detection limit 30 µg/l coefficient of variation of 3.1–4.6%). Urine free cortisol (UFC) over 24 h was measured by the hospital laboratory using the GammaCoat 125I RIA (Diasorin, Stillwater, MN; detection limit 1 µg/dl, coefficient of variation 7%). UFC was standardized for creatinine (UFC/Cr) by dividing the UFC by creatinine excretion over the 24-h period. Samples were sent to Quest Diagnostics for analysis of free T4 by equilibrium dialysis and for total T4 levels by standard methods. Total T3 was measured using an RIA (Diasorin, Stillwater, MN; sensitivity 9.0 ng/dl, coefficient of variation 3.1–7.9%). All samples were stored at –80°C until analysis, and all samples were run in duplicate.

Glucose levels can be converted to Systeme International (SI) units (mmol/l) by dividing by 18. Levels of GH, IGF-I, and leptin can be converted to SI units (µg/l) by multiplying by 1, and serum cortisol can be converted to SI units (nmol/l) by dividing by 0.0363. HOMA-IR (homeostasis model of assessment) was used as a measure of insulin resistance (36) and was calculated using the formula: [fasting glucose (mmol/l) x fasting insulin (µU/ml)]/22.5.

Analysis of leptin, ghrelin, GH, and cortisol concentrations obtained from frequent sampling overnight. Leptin, ghrelin, GH, and cortisol data from frequent sampling were analyzed by Cluster (1 x 2) and deconvolution methods as previously described (27, 29). The half-life for leptin applied to this model was based on half-life determinations by Klein et al. [24.9 ± 4.4 min (19)]. Cluster analysis provides information about concentration characteristics of a hormone (42), which depends on its half-life, basal secretion, secretory burst mass, and burst frequency. However, deconvolution methods are necessary to determine secretory dynamics of a hormone (41, 43). Total basal secretion was determined as the product of basal secretion rate and the number of minutes of sampling (i.e., 720). Total pulsatile secretion was determined as the product of mean burst mass and frequency of secretory bursts. Total basal secretion and pulsatile secretion were added to give total hormone secretion overnight. Data for ghrelin, GH, and cortisol, but not leptin, have been reported earlier (27, 29). In this study, we have used values of area under the curve (AUC), basal secretion, burst frequency, burst interval, burst mass, and total secretion for GH, cortisol, and ghrelin for determination of relationships with leptin concentration and secretion parameters.

We performed cross-correlations between leptin and ghrelin, leptin and GH, and leptin and cortisol by lagging the concentration time series of one hormone relative to the concentration time series of the other. A lag period is the time (min) that separates consecutive samples in the paired-hormone series of interest. Significant cross-correlation values for the two groups at any particular lag time were tested against the null hypothesis of random associations by using the one-sample Kolmogorov-Smirnov statistic (2, 20).

Approximate entropy (ApEn) measures the disorderliness of secretion, and the ApEn score increases as secretion becomes more disorderly. ApEn was determined using methods described by Pincus (33, 34). Cross-ApEn (X-ApEn) quantifies the degree of lag-independent pattern synchrony between timed series (20, 35) and is the bivariate analog of ApEn. X-ApEn measurements were obtained for leptin and GH, leptin and cortisol, and leptin and ghrelin.

Statistical analysis. Data are reported as means ± SD. Student's t-test was used for comparison of means for normally distributed data. We performed correlational analyses followed by stepwise regression to determine predictors of hormonal secretion and concentration characteristics. A P value of 0.15 was used for entry into the model and a P value of 0.10 to leave the stepwise regression model. The paired t-test was used to compare baseline and follow-up characteristics in girls with AN who recovered weight.


    RESULTS
 TOP
 ABSTRACT
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Clinical characteristics of our subjects have been previously reported and are summarized here. Girls with AN had lower BMI (16.7 ± 1.2 vs. 21.7 ± 3.6 kg/m2, P < 0.0001), percent fat mass (18.3 ± 3.9 vs. 29.4 ± 5.4%, P < 0.0001), and percent trunk fat (33.4 ± 3.9 vs. 38.6 ± 5.6%, P = 0.001) than healthy adolescents. Girls with AN also had lower levels of fasting glucose (78.9 ± 9.0 vs. 85.6 ± 5.2 mg/dl, P = 0.006), insulin (6.8 ± 2.6 vs. 14.5 ± 4.1 µIU/ml, P < 0.0001), and insulin resistance measured as HOMA-IR (1.4 ± 0.6 vs. 3.1 ± 0.9, P < 0.0001) than healthy controls. Fasting IGF-I levels were significantly lower in girls with AN (315 ± 141 vs. 531 ± 153 ng/ml, P < 0.0001). Girls with AN had higher total AUC for GH (3,879 ± 2,007 vs. 2,596 ± 1,112 ng·ml–1·12 h, P = 0.01), and higher basal GH secretion (14.1 ± 12.2 vs. 8.0 ± 4.3 ng·ml–1·12 h, P = 0.03), GH secretory burst frequency (11.8 ± 1.4 vs. 10.3 ± 1.7/12 h, P = 0.004), total GH secretion (238 ± 135 vs. 169 ± 71 ng·ml–1·12 h, P = 0.04), and lower burst interval (60.9 ± 9.7 vs. 68.4 ± 10.5 min, P = 0.005) than healthy adolescents. GH burst mass did not differ between the groups. Girls with AN also had significantly higher UFC/Cr (0.050 ± 0.028 vs. 0.036 ± 0.017, P = 0.04), cortisol AUC (6,112 ± 1,467 vs. 4,117 ± 802 µg·dl–1·12 h, P < 0.0001), cortisol burst frequency (7.0 ± 1.2 vs. 5.8 ± 1.3/12 h, P = 0.001), total cortisol secretion (89.6 ± 18.8 vs. 71.2 ± 17.6 µg·dl–1·12 h, P = 0.002), and lower burst interval (113.7 ± 25.7 vs. 131.5 ± 32.9 min, P = 0.05) than healthy adolescents. Cortisol basal secretion and secretory burst mass did not differ between the groups. Ghrelin AUC was higher in AN (464,115 ± 158,329 vs. 320,563 ± 110,594 pg/ml, P = 0.002), as was ghrelin burst mass (812 ± 309 vs. 618 ± 262 pg/ml, P = 0.04) and total ghrelin secretion (11,976 ± 3,781 vs. 9,139 ± 4,210 pg/ml, P = 0.03). Basal ghrelin secretion, burst frequency, and interval did not differ in the two groups. Estradiol levels were lower in AN (16.7 ± 6.6 vs. 21.9 ± 8.8 pg/ml, P = 0.03), FSH levels were higher (5.6 ± 2.0 vs. 4.3 ± 2.0 U/liter, P = 0.04), and LH levels did not differ (4.4 ± 4.1 vs. 6.9 ± 8.0 U/liter, P not significant). Girls with AN had lower total T3 (87.3 ± 20.2 vs. 153.5 ± 48.8 ng/dl, P < 0.0001), free T4 (1.37 ± 0.48 vs. 1.59 ± 0.26 ng/dl, P = 0.07), and total T4 levels (5.3 ± 1.3 vs. 6.1 ± 1.3 µg/dl, P = 0.05) compared with controls, although TSH levels did not differ.

Cluster and deconvolution analysis of leptin concentration. Mean leptin concentrations at each time point from frequent sampling in the two groups are shown in Fig. 1. On Cluster analysis (Table 1), girls with AN had significantly lower leptin AUC, and also lower mean, valley, and nadir leptin, than healthy adolescents. On deconvolution analysis (Table 1), basal leptin secretion, leptin burst mass, burst amplitude, and pulsatile and total leptin secretion were all markedly lower in girls with AN than in controls. Burst frequency and half-life, however, did not differ between the groups. This suggests that decreased leptin concentration in AN is a consequence of decreased basal leptin secretion and also decreased leptin secretory burst mass.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. Mean leptin concentration from frequent sampling from 2000 to 0800 the following morning in girls with anorexia nervosa (AN; gray) and healthy adolescents of comparable maturity (black). Girls with AN had significantly lower leptin concentrations at each time point compared with controls (P < 0.0001 for each time point). Growth hormone (GH), cortisol, and ghrelin data with figures have been previously reported (30).

 

View this table:
[in this window]
[in a new window]
 
Table 1. Comparison of concentration and secretion characteristics of leptin in adolescent girls with anorexia nervosa and healthy adolescents

 
With weight recovery (n = 10 girls with AN), a trend was observed toward an increase in leptin burst mass (23.1 vs. 10.3 ng/ml, P = 0.07), burst amplitude (0.91 vs. 0.41 ng/ml, P = 0.07), and pulsatile (336 vs. 143 ng·ml–1·12 h, P = 0.06) and total leptin secretion (341 vs 148 ng·ml–1·12 h, P = 0.06) on matched-pair analysis. BMI increased in this group from 16.7 kg/m2 at baseline to 19.5 kg/m2 (P = 0.0001) at weight recovery, and percent fat mass from 16.4 to 22.4 kg (P = 0.002). Leptin concentration and secretion characteristics in a healthy control, a girl with AN, and the same girl with AN after weight recovery are shown in Fig. 2.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 2. Deconvolution analysis (top) of leptin concentration in a healthy adolescent, a girl with AN, and the same girl with AN after weight recovery. Bottom: individual secretory bursts for each individual. Burst mass and basal leptin secretion were lower in the girl with AN than in the healthy adolescent and increased markedly with weight recovery.

 
Eight of the girls who had repeat sampling at weight recovery had also resumed menses. Girls with menses and weight recovery similarly showed a trend toward increased leptin burst mass (24.8 vs. 10.3 ng/ml, P = 0.09), pulsatile leptin (361.8 vs. 143.2 ng·ml–1·12 h, P = 0.09), and total leptin secretion (366.8 vs. 148.7 ng·ml–1·12 h, P = 0.09). Other leptin measurements increased but did not reach statistical significance.

Leptin, nutritional status, and insulin resistance. Table 2 demonstrates the strong positive correlations observed between leptin concentration and secretion parameters and measurements of nutritional status, including BMI, fat mass, and insulin resistance. In a multiple regression model including these three predictors of leptin, percent fat mass was found to be the sole significant predictor of basal leptin secretion, contributing to 15.3% of the variability. BMI and HOMA-IR predicted leptin burst mass (67.5 and 5.0%) and pulsatile secretion (67.1 and 5.0%), and percent body fat, BMI and HOMA-IR predicted total leptin secretion (74.3, 7.2, and 4.0%). Total leptin AUC was predicted by percent body fat and HOMA-IR (62.0 and 5.5%), whereas mean leptin was predicted by percent body fat, BMI, and HOMA-IR (79.1, 6.8, and 5.1%). BMI and HOMA-IR predicted nadir (62.8 and 7.0%) and valley leptin (61.7 and 10.9%).


View this table:
[in this window]
[in a new window]
 
Table 2. Relationship between nutritional markers and leptin characteristics

 
Relationship between leptin and GH and leptin and ghrelin. The relationship between leptin and GH parameters is shown in Table 3. Strong correlations were observed of basal leptin secretion and measurements of leptin concentration with GH secretory burst frequency and burst interval. In a multiple regression model including BMI, percent body fat, IGF-I, ghrelin AUC, and leptin characteristics, we noted that leptin measurements were independent and the most significant predictors of GH burst interval and burst frequency but did not predict basal GH secretion or other measurements of GH secretion. Ghrelin is a GH secretatgogue that also increases cortisol secretion and thus was included in this model.


View this table:
[in this window]
[in a new window]
 
Table 3. Relationship between leptin characteristics and GH secretory parameters

 
For GH burst interval, leptin characteristics including basal leptin secretion, total leptin secretion, AUC leptin, and mean, nadir, and valley leptin contributed 33.9, 30.4, 33.6, 43.1, and 56.2% of the variability, respectively, when entered into a stepwise regression model individually with BMI, percent body fat, IGF-I, and ghrelin AUC. For GH burst frequency, similarly, basal leptin secretion, total leptin secretion, leptin AUC, and mean, nadir, and valley leptin contributed to 30.4, 22.0, 22.3, 25.0, 22.0, and 38.6% of the variability, respectively, when individually entered into this model. A smaller portion of the variability was contributed by BMI or body fat. Figure 3, A and B, shows the correlations between valley mean leptin and GH burst interval, and valley mean leptin and GH burst frequency. When fasting ghrelin was entered into the model instead of ghrelin AUC with BMI, percent body fat, and a leptin parameter, basal leptin secretion, total leptin secretion, leptin AUC, mean leptin, and valley leptin contributed to 26.4, 15.7, 18.2, 21.2, and 29.7%, respectively, of the variability of GH burst frequency. Fasting ghrelin was also an independent predictor of GH burst frequency, accounting for an additional 16.2, 5.8, 6.9, and 4.4% of the variability when entered into the model with basal leptin, total leptin, leptin AUC, and mean leptin, respectively.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 3. Relationship between valley leptin and GH burst interval (3a) and GH burst frequency (3b). A strong positive correlation was observed between valley mean leptin levels and GH burst interval, such that girls with lower leptin levels had lower GH burst interval, and thus increased frequency of secretory bursts. Valley leptin and other leptin concentration measures were significant predictors of GH burst interval and GH burst frequency independent of BMI, percent body fat, IGF-I or ghrelin AUC.

 
Strong inverse correlations were observed between GH AUC and leptin burst mass (r = –0.41, P = 0.005), pulsatile leptin secretion (r = –0.41, P = 0.006), total leptin secretion (r = –0.47, P = 0.001), leptin AUC (r = –0.42, P = 0.005), mean leptin (r = –0.49, P = 0.0006), and valley mean leptin (r = –0.44, P = 0.02). Total AUC leptin, mean leptin, and valley leptin were independent predictors of GH AUC, contributing to 24.7, 25.6, and 23.0% of the variability when entered into a repression model one at a time.

Inverse correlations were also observed between ghrelin AUC and mean leptin (r = –0.36, P = 0.03), leptin burst mass (r = –0.34, 0.02), and total leptin secretion (r = –0.33, P = 0.04). Similar inverse correlations were observed between nadir, valley, and mean ghrelin and mean leptin, leptin burst mass, and total leptin secretion (data not shown). However, none of the leptin secretion and concentration characteristics was an independent predictor of ghrelin concentration. Leptin secretory and concentration characteristics did not correlate with ghrelin burst mass, burst interval, or basal, pulsatile, or total ghrelin secretion.

Cross-correlational and X-ApEn analyses between leptin and GH and leptin and ghrelin. The lag time between GH and leptin did not differ between girls with AN and controls (31.4 ± 136.5 vs. 63.0 ± 150.1 min, P not significant; GH leads leptin). X-ApEn between GH and leptin did not differ between the groups (0.90 ± 0.14 vs. 0.96 ± 0.11, P not significant). However, the normalized X-ApEn was higher in AN than in controls (1.26 ± 1.84 vs. 0.33 ± 0.78, P = 0.04), suggesting greater disorderliness in the relationship between GH and leptin secretion in AN.

No differences were observed for the lag times between leptin and ghrelin in girls with AN vs. controls (44.3 ± 144.4 vs. 28.6 ± 203.6 min, P not significant). X-ApEn between the two groups did not differ (0.91 ± 0.10 vs. 0.89 ± 0.11, P not significant); neither did normalized X-ApEn (1.25 ± 1.41 vs. 1.45 ± 1.35, P not significant).

Relationship between leptin and cortisol. Correlations between leptin and cortisol secretion characteristics and AUC are demonstrated in Table 4. Although correlations were observed between measurements of leptin and cortisol AUC as well as cortisol total secretion, leptin was not a significant predictor of serum cortisol characteristics when entered into a regression model including BMI, percent body fat, and ghrelin AUC. Unlike with GH, leptin did not predict cortisol burst interval or basal cortisol secretion (data not shown).


View this table:
[in this window]
[in a new window]
 
Table 4. Relationship between leptin characteristics and cortisol secretory parameters

 
UFC/Cr correlated inversely with leptin burst mass (–0.40, P = 0.008), pulsatile leptin (–0.42, P = 0.005), total leptin secretion (r = –0.32, P = 0.03), leptin AUC and mean leptin (r = –0.28, P = 0.07), nadir leptin (r = –0.41, P = 0.03), and fasting leptin (r = –0.27, P = 0.08). In a regression model including BMI, percent body fat and ghrelin AUC, leptin burst mass, pulsatile leptin secretion, total leptin secretion, and nadir leptin were significant and independent predictors of UFC/Cr, contributing to 13.0, 14.6, 7.8, and 13.7% of the variability, respectively.

Given that leptin is an independent predictor of UFC/Cr, but not of serum cortisol, we also examined the relationship between cortisol half-life and leptin characteristics. Cortisol half-life correlated inversely with basal leptin secretion (r = –0.29, P = 0.05), leptin AUC (r = –0.29, P = 0.05), mean leptin (r = –0.30, P = 0.04), and valley mean leptin (r = –0.40, P = 0.04). On regression modeling (with BMI, %body fat, ghrelin AUC, and leptin), leptin was observed to be an independent predictor of cortisol half-life. Basal leptin secretion, leptin AUC, and mean and valley leptin contributed to 8.5, 11.0, 11.1, and 21.5%, respectively, of the variability of cortisol half-life when entered into the model one at a time. Ghrelin AUC was another independent predictor of cortisol half-life, contributing to 6.5–15.3% of the variability depending on the leptin parameter entered into the model. Figure 4, A and B, demonstrates the correlations between UFC/Cr and pulsatile leptin and cortisol half-life and valley leptin.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 4. Relationship between pulsatile leptin and urine free cortisol standardized for creatinine (UFC/Cr) (A) and valley leptin and cortisol half-life (B). An inverse correlation was observed between pulsatile leptin and UFC/Cr and between valley leptin and cortisol half-life, and leptin parameters were independent predictors of UFC/Cr and cortisol half-life when entered into a regression model with body mass index (BMI), %body fat, and ghrelin area under the curve (AUC).

 
Cross-correlational and X-ApEn analyses between leptin and cortisol. The lag time between cortisol and leptin did not differ in girls with AN vs. controls (–173.5 ± 291.3 vs. –226.5 ± 146.8 min, P not significant), with leptin secretion preceding cortisol. X-ApEn did not differ between the groups for cortisol and leptin (0.89 ± 0.03 vs. 0.89 ± 0.04, P not significant), and neither did normalized X-ApEn (1.25 ± 2.35 vs. 0.97 ± 1.29, P not significant).

Relationship between leptin and other hormones. Table 5 shows the relationship between leptin characteristics and the thyroid hormones and estradiol. In a regression model similar to the one previously described (including BMI, %body fat, ghrelin AUC, and a leptin secretion or concentration characteristic), pulsatile leptin secretion was an independent predictor of estradiol levels, contributing to 10.7% of its variability (Fig. 5). Weak positive correlations were observed between leptin burst frequency and FSH levels (r = 0.29, P = 0.06) and nadir leptin concentration and LH (r = 0.32, P = 0.09).


View this table:
[in this window]
[in a new window]
 
Table 5. Relationship between leptin characteristics and estradiol and thyroid hormones

 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 5. Relationship between puslatile leptin and estradiol. Pulsatile leptin was a significant positive predictor of estradiol levels and independent of BMI, %body fat, and ghrelin AUC.

 
Leptin did not predict free T4 or TSH levels but did correlate strongly and positively with total T4 and T3. By use of the same regression model, leptin AUC was an independent and significant predictor of total T3 and T4, contributing to 29.8 and 13.6% of the variability. Mean and valley leptin predicted total T3 and T4 levels (mean leptin: 31.5 and 12.7% of the variability; valley leptin: 26.4 and 15.0% of the variability), and nadir leptin predicted 19.7% of the variability of total T4. Ghrelin AUC was another independent predictor of total T3 levels.

Relationship between fasting leptin and leptin concentration and secretion parameters. Fasting leptin correlated with basal leptin secretion (r = 0.44, P = 0.002). Much stronger correlations were observed between fasting leptin and leptin burst mass (r = 0.83, P < 0.0001), pulsatile leptin secretion (r = 0.84, P < 0.0001), and total leptin secretion (r = 0.94, P < 0.0001). Similarly, fasting leptin correlated strongly with leptin concentration characteristics, including leptin AUC (r = 0.91, P < 0.0001), mean leptin (r = 0.97, P < 0.0001), valley leptin (r = 0.94, P < 0.0001), and nadir leptin (r = 0.84, P < 0.0001).


    DISCUSSION
 TOP
 ABSTRACT
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We demonstrate that low leptin concentrations in AN are a consequence of decreased basal leptin secretion and decreased leptin secretory burst mass with a trend toward improvement in burst characteristics with weight recovery. Leptin is strongly predicted by measurements of nutritional status, including BMI and HOMA-IR and also percent body fat. Leptin concentration is a strong and independent predictor of GH secretory burst interval and GH secretory burst frequency. Pulsatile leptin secretion predicts UFC/Cr and estradiol levels, and leptin concentration predicts total T4 and T3 levels.

The lack of a more significant improvement in leptin parameters with weight recovery among girls with AN may be a consequence of small sample size (for weight-recovered subjects), incomplete weight recovery, or a time lag between weight recovery and improvement in hormonal characteristics. Although final BMI and percent fat mass in the weight-recovered group were lower than in controls, they were significantly higher than the BMI and percent fat mass at study initiation on matched-pair analysis, even with this sample size. Despite significant increases in BMI and percent fat mass, the increase in leptin characteristics was less marked. These data suggest that there may indeed be a lag between weight recovery and improvement in secretory characteristics. More significant improvements in leptin characteristics may be demonstrable with persistent weight recovery in a larger number of subjects and with achievement of BMIs that approach values in healthy adolescents.

Although associations have been reported between fasting leptin levels and GH, and recombinant human (rh)GH administration results in a reduction in leptin (7, 911, 13, 20, 23, 2527), the relationship between secretory characteristics of leptin and GH has not been explored. We (27) have previously demonstrated that increased GH concentrations in AN are a consequence of increased basal GH secretion and increased GH burst frequency (decreased burst interval). Low levels of IGF-I occur in AN despite high levels of GH, suggestive of a nutritionally acquired "resistance" to GH effects (27). Nutritional status (BMI, %body fat) inversely predicts GH concentration, and ghrelin secretion predicts basal GH secretion and GH burst frequency (30). Low levels of IGF-I have been hypothesized to contribute to increases in GH levels by negative feedback, although our data have demonstrated only weak inverse correlations between GH and IGF-I in AN, likely because of additional effects of high cortisol on the GH-IGF-I axis in AN (27). This study demonstrates that leptin secretion is an independent and strong predictor of GH burst interval and burst frequency, and fasting ghrelin similarly independently predicts GH burst frequency. A combination of increased ghrelin and low leptin secretion in AN may thus contribute to the increased basal GH secretion and increased secretory burst frequency observed in girls with AN. Of importance, these are correlational analyses, which provide information about predictors of a variable but cannot prove causality.

There is evidence that glucocorticoids may increase leptin mRNA expression (8), whereas leptin administration to cell cultures reduces ACTH-induced cortisol secretion (12). Cushing's disease has been associated with high leptin levels (44), and an inverse relationship between leptin and cortisol has been demonstrated in healthy men (22). However, administration of rh-leptin did not produce changes in cortisol secretion compared with placebo administration in one study in acutely starved young women (37) or in young women with hypothalamic amenorrhea (45). We (28, 29) have previously shown that cortisol is a strong predictor of levels of the soluble leptin receptor, but not of fasting leptin. Consistent with results of studies of leptin administration and our studies with fasting leptin, we demonstrate that leptin secretory characteristics are not independent predictors of cortisol secretion, although they do predict UFC/Cr. Leptin parameters independently predicted cortisol half-life, and decreased cortisol clearance from a greater cortisol half-life in a state of hypoleptinemia may account for the relationship between UFC/Cr and leptin. We again mention the correlational nature of these analyses, which cannot determine causation.

An increase in leptin levels associated with increases in fat mass has been suggested to be a trigger for pubertal onset and for the recovery of the hypothalamic-pituitary-gonadal axis in conditions of undernutrition (1, 6, 16, 21). Administration of rh-leptin increases LH pulse frequency in women with hypothalamic amenorrhea (45) and increases LH peak width but decreases LH pulsatility in acutely starved young women (37). Consistent with these data, pulsatile leptin secretion was an independent predictor of estradiol levels, and resumption of menses and weight recovery was associated with a trend toward increased leptin burst mass. Studies in a larger number of patients exhibiting recovery of menstrual function would be useful to determine how an improvement in leptin secretory characteristics predicts changes in FSH and LH secretory characteristics.

Many reports suggest that leptin may modify hypothalamic production of TSH. rh-Leptin prevents the decrease in TSH secretion observed in acute starvation (37) and causes increased production of free T4 and T3 (45). In addition, a synchronicity has been observed between leptin and TSH levels (24). Endogenous leptin levels in our study were independent predictors of total T3 and total T4 levels, consistent with data observed with leptin administration, but did not predict single TSH levels.

Ghrelin and leptin have opposing effects on appetite, and inverse relationships between ghrelin and leptin have been previously reported (40). We did observe inverse correlations between the two hormones; however, ghrelin parameters did not independently predict levels of leptin, and vice versa. Conversely, ghrelin is present in the circulation in acylated and desacylated forms. Acylated ghrelin has traditionally been considered the active form of ghrelin, with effects mediated via the type 1a ghrelin receptor (3, 4, 18, 31, 39). Recent reports, however, suggest that desacylated ghrelin may also be biologically active, with effects noted specifically on adipose tissue (39). In our study, we measured total ghrelin levels. More studies are necessary to determine whether independent effects of leptin may exist on acyl or desacyl ghrelin and vice versa.

In conclusion, low leptin levels that occur in undernutrition are regulated by markers of nutritional status, and may, in turn, play a role in the modulation of nutritionally regulated hormones. Our data suggest that leptin may regulate levels of GH and cortisol by increasing GH burst frequency and decreasing cortisol half-life. Leptin may also modulate levels of other nutritionally regulated hormones such as estradiol and thyroid hormones.


    GRANTS
 TOP
 ABSTRACT
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported, in part, by National Institutes of Health Grants M01 RR-01066, DK-062249, and K23 RR-018851


    ACKNOWLEDGMENTS
 
We thank Ellen Anderson and the Bionutrition team, as well as the skilled nursing staff, of the General Clinical Research Center, Massachusetts General Hospital, for their help in competing this study. In addition, we thank Jeffrey Breu of the Core Laboratory of the Massachusetts Institute of Technology for help in analyzing our leptin samples, and Rita Tsay and team for performing and analyzing the DEXA scans. Most of all, we deeply thank our subjects, without whose participation this study would not have been possible.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. Klibanski, BUL 457B, Neuroendocrine Unit, Massachusetts General Hospital, 55 Fruit St., Boston, MA 02114 (e-mail: aklibanski{at}partners.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ahima R, Dushay J, Flier S, Prabakaran D, and Flier J. Leptin accelerates the onset of puberty in normal female mice. Science 99: 391–395, 1997.[CrossRef]
  2. Box G and Jenkins G. Time Series Analysis: Forecasting and Control. Oakland, CA: Holden-Day, 1976, p. 532.
  3. Broglio F, Benso A, Gottero C, Prodam F, Gauna C, Filtri L, Arvat E, van der Lely A, Deghenghi R, and Ghigo E. Non-acylated ghrelin does not possess the pituitaric and pancreatic endocrine activity of acylated ghrelin in humans. J Endocrinol Invest 26: 192–196, 2003.[ISI][Medline]
  4. Broglio F, Gottero C, Prodam F, Gauna C, Muccioli G, Papotti M, Abribat T, van der Lely AJ, and Ghigo E. Non-acylated ghrelin counteracts the metabolic but not the neuroendocrine response to acylated ghrelin in humans. J Clin Endocrinol Metab 89: 3062–3065, 2004.[Abstract/Free Full Text]
  5. Campfield L, Smith F, Guisez Y, Devos R, and Burn P. Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science 269: 546–549, 1995.[ISI][Medline]
  6. Cheung C, Thornton J, Kuijper J, Weigle D, Clifton D, and Steiner R. Leptin is the metabolic gate for the onset of puberty in the female rat. Endocrinology 138: 855–858, 1997.[Abstract/Free Full Text]
  7. De Barretto ES A, Gill MS, De Freitas ME, Magalhaes MM, Souza AH, Aguiar-Oliveira MH, and Clayton PE. Serum leptin and body composition in children with familial GH deficiency (GHD) due to a mutation in the growth hormone-releasing hormone (GHRH) receptor. Clin Endocrinol (Oxf) 51: 559–564, 1999.[CrossRef][ISI][Medline]
  8. De Vos P, Saladin R, Auwerx J, and Staels B. Induction of ob gene expression by corticosteroids is accompanied by body weight loss and reduced food intake. J Biol Chem 270: 15958–15961, 1995.[Abstract/Free Full Text]
  9. Elimam A, Lindgren A, Norgren S, Kamel A, Skwirut C, Bang P, and Marcus C. Growth hormone treatment downregulates serum leptin levels in children independent of changes in body mass index. Horm Res 52: 66–72, 1999.[CrossRef][ISI][Medline]
  10. Elimam A, Norgren S, and Marcus C. Effects of growth hormone treatment on the leptin system and body composition in obese prepubertal boys. Acta Paediatr 90: 520–525, 2001.[ISI][Medline]
  11. Fors H, Matsuoka H, Bosaeus I, Rosberg S, Wikland KA, and Bjarnason R. Serum leptin levels correlate with growth hormone secretion and body fat in children. J Clin Endocrinol Metab 84: 3586–3590, 1999.[Abstract/Free Full Text]
  12. Glasow A, Haidan A, Hilbers U, Breidert M, Gillespie J, Scherbaum W, Chrousos G, and Bornstein S. Expression of Ob receptor in normal human adrenals: differential regulation of adrenocortical and adrenomedullary function by leptin. J Clin Endocrinol Metab 83: 4459–4466, 1998.[Abstract/Free Full Text]
  13. Gregoire Nyomba BL, Johnson M, Berard L, and Murphy LJ. Relationship between serum leptin and the insulin-like growth factor-I system in humans. Metabolism 48: 840–844, 1999.[CrossRef][ISI][Medline]
  14. Greulich W and Pyle S. Radiographic Atlas of Skeletal Development of the Hand and Wrist (2nd ed.). Stanford, CA: Stanford Univ. Press, 1959.
  15. Grinspoon S, Gulick T, Askari H, Landt M, Lee K, Anderson E, Ma Z, Vignati L, Bowsher R, Herzog D, and Klibanski A. Serum leptin levels in women with anorexia nervosa. J Clin Endocrinol Metab 81: 3861–3863, 1996.[Abstract]
  16. Hardie L, Trayhurn P, Abramovich D, and Fowler P. Circulating leptin in women: a longitudinal study in the menstrual cycle and during pregnancy. Clin Endocrinol (Oxf) 47: 101–106, 1997.[CrossRef][ISI][Medline]
  17. Heer M, Mika C, Grzella I, Heussen N, and Herpertz-Dahlmann B. Bone turnover during inpatient nutritional therapy and outpatient follow-up in patients with anorexia nervosa compared with that in healthy control subjects. Am J Clin Nutr 80: 774–781, 2004.[Abstract/Free Full Text]
  18. Hotta M, Ohwada R, Katakami H, Shibasaki T, Hizuka N, and Takano K. Plasma levels of intact and degraded ghrelin and their responses to glucose infusion in anorexia nervosa. J Clin Endocrinol Metab 89: 5707–5712, 2004.[Abstract/Free Full Text]
  19. Klein S, Coppack S, Mohamed-Ali V, and Landt M. Adipose tissue leptin production and plasma leptin kinetics in humans. Diabetes 45: 984–987, 1996.[Abstract]
  20. Koutkia P, Canavan B, Johnson ML, DePaoli A, and Grinspoon S. Characterization of leptin pulse dynamics and relationship to fat mass, growth hormone, cortisol, and insulin. Am J Physiol Endocrinol Metab 285: E372–E379, 2003.[Abstract/Free Full Text]
  21. Laughlin G and Yen S. Hypoleptinemia in women athletes: absence of a diurnal rhythm with amenorrhea. J Clin Endocrinol Metab 82: 318–321, 1997.[Abstract/Free Full Text]
  22. Licinio J, Mantzoros C, Negrao A, Cizza G, Wong M, Bongiorno P, Chrousos G, Karp B, Allen C, Flier J, and Gold P. Human leptin levels are pulsatile and inversely related to pituitary-adrenal function. Nat Med 3: 575–579, 1997.[CrossRef][ISI][Medline]
  23. Lissett CA, Clayton PE, and Shalet SM. The acute leptin response to GH. J Clin Endocrinol Metab 86: 4412–4415, 2001.[Abstract/Free Full Text]
  24. Mantzoros CS, Ozata M, Negrao AB, Suchard MA, Ziotopoulou M, Caglayan S, Elashoff RM, Cogswell RJ, Negro P, Liberty V, Wong ML, Veldhuis J, Ozdemir IC, Gold PW, Flier JS, and Licinio J. Synchronicity of frequently sampled thyrotropin (TSH) and leptin concentrations in healthy adults and leptin-deficient subjects: evidence for possible partial TSH regulation by leptin in humans. J Clin Endocrinol Metab 86: 3284–3291, 2001.[Abstract/Free Full Text]
  25. Marzullo P, Buckway C, Pratt KL, Colao A, Guevara-Aguirre J, and Rosenfeld RG. Leptin concentrations in GH deficiency: the effect of GH insensitivity. J Clin Endocrinol Metab 87: 540–545, 2002.[Abstract/Free Full Text]
  26. Matsuoka H, Fors H, Bosaeus I, Rosberg S, Albertsson-Wikland K, and Bjarnason R. Changes in body composition and leptin levels during growth hormone (GH) treatment in short children with various GH secretory capacities. Eur J Endocrinol 140: 35–42, 1999.[Abstract/Free Full Text]
  27. Misra M, Miller K, Bjornson J, Hackman A, Aggarwal A, Chung J, Ott M, Herzog D, Johnson M, and Klibanski A. Alterations in growth hormone secretory dynamics in adolescent girls with anorexia nervosa and effects on bone metabolism. J Clin Endocrinol Metab 88: 5615–5623, 2003.[Abstract/Free Full Text]
  28. Misra M, Miller KK, Almazan C, Ramaswamy K, Aggarwal A, Herzog DB, Neubauer G, Breu J, and Klibanski A. Hormonal and body composition predictors of soluble leptin receptor, leptin, and free leptin index in adolescent girls with anorexia nervosa and controls and relation to insulin sensitivity. J Clin Endocrinol Metab 89: 3486–3495, 2004.[Abstract/Free Full Text]
  29. Misra M, Miller KK, Almazan C, Ramaswamy K, Lapcharoensap W, Worley M, Neubauer G, Herzog DB, and Klibanski A. Alterations in cortisol secretory dynamics in adolescent girls with anorexia nervosa and effects on bone metabolism. J Clin Endocrinol Metab 89: 4972–4980, 2004.[Abstract/Free Full Text]
  30. Nakai Y, Hosoda H, Nin K, Ooya C, Hayashi H, Akamizu T, and Kangawa K. Plasma levels of active form of ghrelin during oral glucose tolerance test in patients with anorexia nervosa. Eur J Endocrinol 149: R1–R3, 2003.[Abstract/Free Full Text]
  31. Pelleymounter M, Cullen M, Baker M, Hecht R, Winters D, Boone T, and Collins F. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269: 540–543, 1995.[ISI][Medline]
  32. Pincus S. Approximate entropy as a measure of system complexity. Proc Natl Acad Sci USA 88: 2297–2301, 1991.[Abstract/Free Full Text]
  33. Pincus S. Greater signal regularity may indicate increased system isolation. Math Biosci 122: 161–181, 1994.[CrossRef][ISI][Medline]
  34. Pincus S, Mulligan T, Iranmanesh A, Gheorghiu S, Godschalk M, and Veldhuis J. Older males secrete luteinizing hormone and testosterone more irregularly, and jointly more asynchronously, than younger males. Proc Natl Acad Sci USA 93: 14100–14105, 1996.[Abstract/Free Full Text]
  35. Radziuk J. Insulin sensitivity and its measurement: structural commonalities among the methods. J Clin Endocrinol Metab 85: 4426–4433, 2000.[Abstract/Free Full Text]
  36. Schurgin S, Canavan B, Koutkia P, DePaoli AM, and Grinspoon S. Endocrine and metabolic effects of physiologic r-metHuLeptin administration during acute caloric deprivation in normal-weight women. J Clin Endocrinol Metab 89: 5402–5409, 2004.[Abstract/Free Full Text]
  37. Soyka L, Misra M, Frenchman A, Miller K, Grinspoon S, Schoenfeld D, and Klibanski A. Abnormal bone mineral accrual in adolescent girls with anroexia nervosa. J Clin Endocrinol Metab 87: 4177–4185, 2002.[Abstract/Free Full Text]
  38. Thompson NM, Gill DAS, Davies R, Loveridge N, Houston PA, Robinson ICAF, and Wells T. Ghrelin and des-octanoyl ghrelin promote adipogenesis directly in vivo by a mechanism independent of the type 1a growth hormone secretagogue receptor. Endocrinology 145: 234–242, 2004.[Abstract/Free Full Text]
  39. Tolle V, Kadem M, Bluet-Pajot MT, Frere D, Foulon C, Bossu C, Dardennes R, Mounier C, Zizzari P, Lang F, Epelbaum J, and Estour B. Balance in ghrelin and leptin plasma levels in anorexia nervosa patients and constitutionally thin women. J Clin Endocrinol Metab 88: 109–116, 2003.[Abstract/Free Full Text]
  40. Veldhuis J, Carlson M, and Johnson M. The pituitary gland secretes in bursts: appraising the nature of glandular secretory impulses by simultaneous multiple-parameter deconvolution of plasma hormone concentrations. Proc Natl Acad Sci USA 84: 7686–7690, 1987.[Abstract/Free Full Text]
  41. Veldhuis J and Johnson M. Cluster analysis: a simple, versatile, and robust algorithm for endocrine pulse detection. Am J Physiol Endocrinol Metab 250: E486–E493, 1986.[Abstract/Free Full Text]
  42. Veldhuis J and Johnson M. Deconvolution analysis of hormone data. Methods Enzymol 210: 539–575, 1992.[ISI][Medline]
  43. Veldman R, Frolich M, Pincus S, Veldhuis J, and Roelfsema F. Hyperleptinemia in women with Cushing's disease is driven by high-amplitude pulsatile, but orderly and eurhythmic, leptin secretion. Eur J Endocrinol 144: 21–27, 2001.[Abstract/Free Full Text]
  44. Welt CK, Chan JL, Bullen J, Murphy R, Smith P, DePaoli AM, Karalis A, and Mantzoros CS. Recombinant human leptin in women with hypothalamic amenorrhea. N Engl J Med 351: 987–997, 2004.[Abstract/Free Full Text]
  45. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, and Friedman J. Positional cloning of the mouse obese gene and its human homologue. Nature 372: 425–432, 1994.[CrossRef][ISI][Medline]