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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 7 3486-3495
Copyright © 2004 by The Endocrine Society

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

Madhusmita Misra, Karen K. Miller, Cecilia Almazan, Kavitha Ramaswamy, Avichal Aggarwal, David B. Herzog, Gregory Neubauer, Jeffrey Breu and Anne Klibanski

Neuroendocrine Unit (M.M., K.K.M., C.A., K.R., A.A., A.K.), and Eating Disorders Unit (D.B.H.), Massachusetts General Hospital and Harvard Medical School; Pediatric Endocrine Unit (M.M.), Massachusetts General Hospital for Children and Harvard Medical School; Core Laboratory (G.N.), General Clinical Research Center, Massachusetts General Hospital, Boston, Massachusetts 02114; and Core Laboratory (J.B.), General Clinical Research Center, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Address all correspondence and requests for reprints to: Anne Klibanski, M.D., BUL 457B, Neuroendocrine Unit, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114. E-mail: aklibanski{at}partners.org.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Anorexia nervosa (AN) is associated with very low levels of leptin, a cytokine secreted by adipose tissue and known to suppress appetite. Leptin may play a permissive role in onset of puberty and in resumption of gonadal function in conditions of undernutrition. The soluble leptin receptor (sOB-R) is the main leptin binding protein, and the ratio of serum leptin to sOB-R provides a measure of the free leptin index (FLI), which may be a more accurate determinant of leptin function. Determinants of sOB-R and FLI have not been examined in an adolescent population. We examined levels of sOB-R, leptin, and FLI, and body composition and hormonal determinants of these variables in 23 adolescent girls with AN and 21 healthy adolescent girls of comparable maturity prospectively over 1 yr. Measures of insulin resistance and adiponectin were also examined. We determined changes in levels of sOB-R, leptin, and FLI with weight recovery (defined as an increase in body mass index of ≥10%, n = 11), and with resumption of menstrual cycles (n = 13).

Girls with AN had significantly higher levels of sOB-R (P = 0.0008) and significantly lower levels of leptin and FLI (P < 0.0001 for both) than healthy controls, and levels of FLI were reduced more than levels of leptin in girls with AN compared with controls. An inverse correlation was noted between levels of leptin and sOB-R for the group as a whole (r = –0.64, P < 0.0001) but not in girls with AN considered alone. The most important predictor of levels of sOB-R was cortisol in the group as a whole (r = 0.61, P < 0.0001) and in girls with AN considered alone (r = 0.66, P = 0.0008). Other independent predictors of sOB-R levels for the entire group were percent body fat (r = –0.44, P = 0.003) and levels of IGF-I (r = –0.37, P = 0.01). The most important predictors of leptin and FLI were body mass index and percent body fat. An inverse relationship was noted between measures of insulin resistance and sOB-R levels, whereas a positive association was noted between these measures and leptin and FLI. Adiponectin values did not differ in girls with AN compared with healthy controls and did not correlate with sOB-R, leptin, or FLI. Weight recovery resulted in significant decreases in levels of the sOB-R (24.7 ± 1.7 to 17.6 ± 1.2 U/ml, P = 0.004), and increases in levels of leptin (4.4 ± 1.0 to 13.7 ± 2.9 µg/liter, P = 0.02). Resumption of menstrual function, but not weight recovery alone, was associated with significant increases in FLI (0.19 ± 0.04 to 0.50 ± 0.09 µg/U x 10–3, P = 0.02).

We demonstrate an increase in levels of sOB-R and a decrease in the FLI in adolescent girls with AN, and also demonstrate that cortisol is the most important predictor of levels of sOB-R in this condition. Levels of leptin and FLI, conversely, are primarily predicted by body composition. Weight recovery is associated with a decrease in sOB-R and an increase in leptin. Resumption of menses is associated with significant increases in the FLI, suggesting that free leptin may be an important determinant of menstrual recovery.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
LEPTIN, A CYTOKINE expressed by adipose tissue, regulates food intake by interactions with the leptin receptor in the brain (1, 2, 3). Leptin-deficient and leptin receptor-deficient mice (1, 4) and humans (5, 6) are obese, and leptin administration to leptin-deficient mice results in a decrease in food intake (2). Most obese individuals, however, have very high leptin levels (7, 8, 9), and anorexia nervosa (AN), a condition of self-imposed starvation and hypogonadotropic hypogonadism, is associated with low levels of leptin (10, 11, 12, 13, 14). Leptin levels correlate with fat mass in both overweight and underweight groups (10, 15, 16, 17), and changes in leptin appear to be a consequence of changes in fat mass.

In addition to effects on appetite, a role for leptin in onset of puberty has been postulated. A permissive level of body fat and leptin appears necessary for onset of puberty and for recovery of the gonadal axis in conditions of nutritional deprivation (18, 19, 20, 21, 22). A 50% increase in leptin levels occurs just before pubertal onset (23), and leptin administration prevents a fasting-associated decrease in testosterone in adult men (24). However, studies have not demonstrated a cutoff value for leptin above which the gonadal axis becomes functional in subjects recovering from AN (25, 26). An important limitation of prior studies is that levels of total, rather than free, leptin were examined.

The leptin receptor belongs to the type 1 cytokine receptor family (27, 28), and various mRNA splice variants of this receptor have been identified (28). A soluble form of the leptin receptor lacking the cytoplasmic domain is generated by proteolytic cleavage of the membrane-bound receptor and represents the main leptin binding activity in human blood (29). Levels of the soluble leptin receptor (sOB-R) thus provide an indication of free leptin; the free leptin index (FLI) being defined as the ratio of leptin to sOB-R (30). In AN, sOB-R levels are high (31, 32), whereas these levels are low in obesity (9, 33, 34).

Body composition and hormonal determinants of leptin have been examined in several studies, and a role for fat mass (10, 15, 16, 17) and GH (14, 16, 35, 36, 37, 38, 39, 40, 41, 42), insulin (16, 43, 44), cortisol (16, 43, 45, 46, 47, 48), and possibly ghrelin (13) has been reported. Little is known regarding hormonal and body composition determinants of sOB-R and FLI in healthy adolescents and in AN. Studies that do exist have been primarily conducted in healthy adult men and women (30, 49). Body mass index (BMI) predicts sOB-R levels in children (32, 49), but regional body composition determinants have not been examined. Leptin production is higher in sc than omental fat (17), suggesting that regional fat distribution may regulate sOB-R and FLI. Moreover, data do not exist regarding the relationship between levels of sOB-R and FLI and measures of insulin resistance in adolescents. Adiponectin, another product of adipocytes, decreases insulin resistance (50), and high levels occur in adults with AN (51, 52). Almost identical 24-h variations in sOB-R and adiponectin that are out of phase with leptin have been noted in adult men (53). The relationship of adiponectin to sOB-R, leptin, and FLI in this young adolescent group has not been examined.

AN in adolescence is associated with specific changes in regional body composition (54) and serves as a model of decreased resistance to insulin. Adolescents with AN have changes in the GH-IGF-I axis (14, 55, 56) and in levels of ghrelin (57), cortisol (14), and estradiol (15, 25). This condition of undernutrition with important changes in body composition and in levels of various hormones thus serves as a unique model in which to examine predictors of levels of sOB-R and FLI.

In this study, we have examined levels of sOB-R, leptin, and FLI in adolescent girls with AN and in healthy controls of comparable maturity, who were followed prospectively for 1 yr. We hypothesized that sOB-R levels and FLI would be predicted by specific changes in body composition such as a decrease in fat mass, with trunk fat being a more important predictor than extremity fat, based on data from previous studies in which we demonstrated marked reductions in trunk fat in girls with AN (54). We also hypothesized that the GH-IGF-I axis and levels of ghrelin and cortisol would predict sOB-R levels and FLI, and that sOB-R levels and FLI, in turn, would predict the extent of insulin resistance. In addition, we expected to observe a greater increase in FLI than in levels of total leptin with weight recovery and recovery of menses in AN.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects

We studied 23 adolescent girls with a Diagnostic and Statistical Manual of Mental Disorders-IV diagnosis of AN and 21 healthy controls of comparable maturity (similar bone age and pubertal stage) at the General Clinical Research Center (GCRC) of Massachusetts General Hospital. The girls were Caucasian and ranged in age between 12.2 and 18.8 yr. The mean duration of illness for girls with AN was 7.9 ± 2.2 months (1–36 months). The BMI of healthy controls ranged between 16.8 and 28.0 kg/m2, and controls had no history of eating disorders. Subjects were, for the most part, postmenarchal except for four girls with AN and three controls. Subjects with AN were recruited through referrals from eating disorder specialists and primary care providers in Massachusetts, New Hampshire, and Maine. Healthy controls were recruited through mass mailings to primary care providers in the Greater Boston area. Subjects with AN were all receiving outpatient multidisciplinary treatment at enrollment. The study was approved by the Institutional Review Board of the Partners HealthCare system. We obtained informed consent and assent from all subjects and parents.

Experimental protocol

All subjects were screened to rule out conditions other than AN that might cause hypogonadism by measuring prolactin, FSH, and LH. No subject had taken hormonal medications within 3 months of study initiation. Eligible subjects were admitted to the GCRC for a baseline visit, which included frequent sampling for GH (every 30 min, from 2000 h on the night of admission to 0800 h the following morning). A bone age and dual-energy x-ray absorptiometry (DXA) evaluation (for body composition) were obtained for all subjects. Height, weight, and BMI were measured. Blood was sent to the hospital laboratory for real-time analysis of fasting glucose levels. Fasting serum was collected for analysis of sOB-R, leptin, IGF-I, estradiol, ghrelin, insulin, and adiponectin. Subjects submitted a 24-h urine sample for urinary free cortisol (UFC) and creatinine (cr), and the ratio of UFC/cr was calculated (58). Healthy postmenarchal subjects were studied in the early follicular phase of their menstrual cycles.

Subjects were studied at 3, 6, and 12 months after the baseline with an outpatient visit. Subjects with AN who recovered weight during the study course were admitted for a repeat overnight stay at the GCRC. Weight recovery was defined as a 10% increase in BMI from baseline. Nineteen girls with AN and 20 healthy controls completed the yearlong follow-up. Eleven subjects with AN had an increase of 10% or more in BMI over the 1-yr period of follow-up, and 13 girls with AN resumed menses during this period. Height, weight, and BMI were measured at these visits. Serum was obtained as at baseline. Bone age, DXA for body composition, and 24-h urine for cortisol and cr were repeated at 6 and 12 months and at weight recovery.

Anthropometric measurements

A single stadiometer at the GCRC was used for measurement of height at each visit, and an average of three readings was recorded. Subjects were weighed on an electronic scale in a hospital gown. BMI was calculated using the formula: weight in kilograms/height in meters2, and compared with standard charts (59). Bone age was determined using the methods of Greulich and Pyle (60). Pubertal Tanner staging was performed for all subjects. The reading of bone age and Tanner staging was performed by a single investigator, a pediatric endocrinologist. Because breast tissue can be quite atrophic in girls with AN, Tanner staging of pubic hair rather than of breasts is reported.

Biochemical assessment

Glucose levels, UFC, and cr measurements were carried out by the hospital laboratory using published methods (61). All other assays were performed at the core laboratory of the GCRC. Levels of the soluble leptin receptor were measured using an ELISA (Alexis Biochemicals, San Diego, CA), with a limit of sensitivity of 0.4 U/ml and intraassay coefficient of variation of 6.0–6.3%. Leptin was measured with a RIA (Linco Diagnostics, Inc., St. Charles, MO; sensitivity, 0.5 µg/liter; coefficient of variation, 4.6–5.7%). FLI was calculated as the ratio of leptin to sOB-R (leptin/sOB-R) (30).

GH was measured by an immunoradiometric assay (Nichols Institute Diagnostics, Inc., San Juan Capistrano, CA) with a detection limit of 0.05 µg/liter and intraassay coefficient of variation of 2.4–9.4%. An immunoradiometric assay was also used to determine IGF-I levels (Nichols Institute Diagnostics, Inc.), with a detection limit of 30 µg/liter and coefficient of variation of 3.1–4.6%. RIA was used to measure serum estradiol (Diagnostic Systems Laboratories, Inc., Webster, TX; limit of detection, 2.2 pg/ml; coefficient of variation, 6.5–8.9%), ghrelin (Phoenix Pharmaceuticals, Belmont, CA; sensitivity, 2 pg/ml; coefficient of variation, 9.9–10.5%), insulin (Diagnostics Products, Los Angeles, CA; coefficient of variation of 4.7–7.7%), and adiponectin (Linco Diagnostics, Inc.; lowest detectable concentration, 1 µg/liter; coefficient of variation, 6.4–8.4%). The ratio of fasting glucose to fasting insulin (62) and homeostasis model of assessment insulin resistance (HOMA-IR) were used as measures of insulin resistance. HOMA-IR was calculated using the formula: [fasting glucose (mmol/liter) x fasting insulin (mU/ml)]/22.5 (63).

Analysis of GH concentration

We examined GH data from frequent sampling by Cluster analysis. Data from these analyses have been previously reported (14). In this paper, we have used nadir GH concentration and total area under the curve from Cluster analysis (64) for determination of relationships with levels of sOB-R, leptin, and FLI.

Body composition

Body composition was determined by DXA (QDR 4500; Hologic Inc., Waltham, MA). Percent trunk fat was calculated as the ratio of trunk fat to total fat x 100. Percent extremity fat was calculated as the ratio of total extremity fat (right and left arm fat and right and left leg fat) to total fat x 100. Trunk to extremity fat ratio was determined by dividing percent trunk fat by percent extremity fat (54).

Statistical analysis

All data are described as mean ± SEM. The data were analyzed using the JMP (Cary, NC) program (version 4). Student t test was used to calculate differences between means. Univariate and multiple regression analyses were employed to determine predictors of sOB-R, leptin, and FLI. The paired t test was used to compare baseline and follow-up characteristics.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Clinical characteristics

Adolescent girls with AN did not differ from healthy adolescents in age, bone age, or Tanner stage (Table 1Go). Some baseline characteristics, but not regional body composition, sOB-R, FLI, insulin resistance, and adiponectin data, of 22 girls with AN and 20 controls have been previously reported (14, 57). Girls with AN had lower BMIs, fat mass, percent trunk fat, and trunk to extremity fat ratio than controls. Estradiol and IGF-I levels were lower in AN; whereas GH, ghrelin, and UFC/cr were higher in AN.


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  		TABLE 1. Clinical characteristics

 
sOB-R, leptin, and FLI

Levels of sOB-R were significantly higher in girls with AN (27.7 ± 3.01 vs. 15.9 ± 0.9 U/ml, P = 0.0008), whereas leptin levels were lower in girls with AN than in controls (4.3 ± 0.6 vs. 15.8 ± 1.3 µg/liter, P < 0.0001) (Fig. 1Go). FLI was markedly lower in the AN group (0.18 ± 0.03 vs. 1.10 ± 0.13 µg/U x 10–3, P < 0.0001). Levels of the sOB-R were increased by 74%, leptin levels decreased by 72%, and FLI decreased by 84% in AN compared with healthy adolescents. When levels of sOB-R, leptin, and FLI were standardized for weight, BMI, and fat mass, the differences between AN and controls were even more marked (Table 2Go). An inverse relationship was noted between leptin and the sOB-R (r = –0.48, P = 0.0008) for the group as a whole (Fig. 2Go). Weaker correlations were noted within individual groups (r = –0.39, P = 0.08 for controls; and r = –0.43, P = 0.05 for AN).



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  		FIG. 1. Levels of sOB-R, leptin, and FLI in girls with AN (black bars) and healthy adolescents of comparable maturity (white bars). Girls with AN had significantly higher levels of the sOB-R and significantly lower levels of leptin than healthy controls (A). FLI was significantly lower in girls with AN than in healthy controls (B).

 

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  		TABLE 2. Comparison of levels of sOB-R, leptin, and FLI corrected for weight, BMI, and fat mass in girls with AN and controls

 


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  		FIG. 2. Correlation between levels of the sOB-R and levels of leptin in girls with AN (gray triangles) and healthy controls (black diamonds). The correlation coefficient was 0.64 for the entire group (P < 0.0001).

 
Predictors of levels of sOB-R, leptin, and FLI

Body composition. Table 3Go demonstrates the correlations between body composition variables and levels of sOB-R, leptin, and FLI for the group taken together (AN and controls), in AN alone, and in healthy controls.


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  		TABLE 3. Correlation between levels of sOB-R, leptin, and FLI with body composition parameters in all subjects, girls with AN, and healthy controls

 
sOB-R.
When all subjects were considered together, an inverse correlation was observed between sOB-R and BMI as well as fat mass. For AN alone, body composition did not predict sOB-R levels; whereas for controls, BMI was a weak predictor of sOB-R. For the group as a whole, using stepwise regression in which BMI, fat mass, and percent fat mass were entered into the model, percent body fat was the only body composition determinant of sOB-R, contributing to 20% of the variability. For the groups considered separately, on stepwise regression, body composition did not predict sOB-R. Lean body mass and regional body composition did not predict sOB-R levels in any group (data not shown).

Leptin and FLI.
When subjects were considered together and also when girls with AN and healthy controls were considered separately, BMI and fat mass were important predictors of serum leptin and FLI. Percent trunk fat and trunk/extremity fat predicted levels of leptin (r = 0.53, P = 0.0004; r = 0.40, P = 0.009) and FLI (r = 0.48, P = 0.001; r = 0.36, P = 0.02) in the group as a whole but not for the groups considered separately. Lean body mass did not predict leptin levels. For leptin and FLI, on stepwise regression, BMI contributed to 78% and 71% of the variability, respectively, whereas percent body fat contributed to another 4% and 7% of the variability. In girls with AN alone and controls alone, fat mass was the sole predictor of leptin, using multiple regression analysis, contributing to 67% and 48% of the variability, respectively. Fat mass was also the only determinant of FLI, contributing to 68% of the variability in AN and 43% of the variability in controls.

GH-IGF-I axis, ghrelin, and cortisol. Table 4Go demonstrates correlations between hormonal factors and levels of sOB-R, leptin, and FLI for the group taken together, for AN, and for healthy controls.


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  		TABLE 4. Correlation between levels of sOB-R, leptin, and FLI and levels of IGF-I, GH, and cortisol in all subjects, girls with AN, and controls

 
sOB-R.
In the group as a whole, levels of sOB-R correlated inversely with IGF-I, but no correlation was noted with GH concentration. A weak positive correlation was observed between sOB-R and fasting ghrelin (r = 0.28, P = 0.07). sOB-R levels correlated strongly and positively UFC/cr. Using stepwise regression analysis including GH concentration, levels of IGF-I, UFC/cr, and body composition variables for the group as a whole, levels of sOB-R were predicted by UFC/cr, percent body fat, and IGF-I (contributing to 38%, 9%, and 5% of the variability).

In girls with AN, a strong positive correlation was noted between sOB-R and UFC/cr; whereas in healthy controls, a positive correlation was noted between sOB-R and GH concentration. In AN, using stepwise regression including body composition and hormonal variables, sOB-R levels were predicted by UFC/cr (44% of the variability). Ghrelin did not correlate with sOB-R levels in the groups considered separately.

Leptin and FLI.
In the group as a whole, strong inverse correlations were observed between serum leptin and GH concentration, whereas a direct relationship was noted between leptin and IGF-I. Similar relationships were noted between FLI and GH concentration, and FLI and IGF-I. A weak inverse correlation was observed between leptin and FLI and fasting ghrelin (r = –0.28, P = 0.07 for leptin; r = –0.26, P = 0.09 for FLI). UFC/cr correlated weakly and inversely with leptin and the FLI. Using stepwise regression including body composition and hormonal variables, BMI and percent body fat were the only significant predictors of leptin (contributing to 77.5% and 4% of the variability) and of FLI (contributing to 71% and 7% of the variability).

In girls with AN, leptin and FLI were predicted by IGF-I alone; whereas in healthy controls, an inverse correlation was observed between leptin and GH, and FLI and GH concentration. On stepwise regression including body composition and hormonal variables, fat mass was the only significant predictor of leptin and FLI both in girls with AN (65% and 60% of variability) and in controls (60% and 61% of the variability).

Estradiol. For AN and controls considered together, leptin levels correlated positively with estradiol (r = 0.30, P = 0.05), whereas a trend toward a positive correlation was noted between FLI and estradiol (r = 0.28, P = 0.07). No correlation was noted between sOB-R and estradiol. In girls with AN and healthy controls considered separately, leptin and FLI did not correlate with estradiol.

Measures of insulin resistance. Table 1Go shows levels of fasting glucose, leptin and adiponectin, and insulin/glucose ratio and HOMA-IR in girls with AN and controls. Fasting glucose and insulin values were lower in girls with AN than in controls. Girls with AN had significantly lower insulin resistance, as measured by the ratio of fasting insulin to fasting glucose and HOMA-IR, than healthy adolescents. Adiponectin levels did not differ between the groups. An inverse relationship was noted between sOB-R levels and measures of insulin resistance when girls with AN and controls were considered together (Table 5Go).


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  		TABLE 5. Correlations between levels of soluble leptin receptor, leptin, FLI, and measures of insulin resistance and adiponectin in girls with AN and controls

 
Effects of weight recovery on levels of sOB-R, leptin, and FLI

Table 6Go shows paired analysis from baseline and at 10% weight recovery in 11 girls with AN who recovered weight during the study period, baseline and 12 month follow-up data in eight girls with AN who did not recover weight, and from 20 healthy controls followed over the same period. Weight recovery was associated with a significant decrease in levels of sOB-R (P = 0.004) and an increase in levels of leptin (P = 0.02). Estradiol values (P = 0.02), BMI (P < 0.0001), and IGF-I levels (P = 0.02) increased with weight recovery. No change in fasting ghrelin levels was observed. In contrast, no significant differences were noted in BMI, sOB-R, leptin, FLI, IGF-I, estradiol, and ghrelin in girls with AN who completed the study but did not recover weight. Similarly, paired analysis in 20 healthy controls who completed the study showed no differences in these variables over 12 months except for an increase in estradiol (P = 0.003), likely from expected advancement in pubertal status, and a small increase in sOB-R (P = 0.03).


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  		TABLE 6. Paired analysis at baseline and over 1 yr of follow-up in girls with AN who recovered weight, did not recover weight, and in healthy controls followed over the same period

 
Levels of sOB-R, leptin, and FLI by recovery of menstrual function

Thirteen girls with AN resumed menses over the study period (Table 7Go). Girls resuming menses were noted to have significant increases in BMI (P = 0.0009), leptin (P = 0.02), FLI (P = 0.005), estradiol (P = 0.01), and IGF-I (P = 0.03) and a trend toward lower sOB-R levels at the time of recovery of menses compared with baseline levels. Ghrelin values did not differ from baseline levels. In contrast, seven girls with AN who did not recover menses over the study period did not demonstrate such increases in BMI, leptin, FLI, estradiol, or IGF-I. No cut-off for FLI could be identified above which recovery of menstrual function occurred. Figure 3AGo shows FLI at baseline and at resumption of menses in the 13 girls who did resume menses over the study period. Three girls with AN who resumed menses had no change in FLI over time. All others had an increase in FLI over time. Figure 3BGo shows initial and final FLI in the seven girls with AN who did not resume menses over the study period. Two of these seven girls had an increase in FLI, but the others had minimal changes in FLI over time.


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  		TABLE 7. Paired analysis at baseline and over 1 yr of follow-up in girls with AN who resumed menses or did not resume menses over the study period

 


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  		FIG. 3. FLI in girls with AN (A) who resumed menses over the study period and (B) did not resume menses over the study period. FLI increased (n = 10) or remained the same (n = 3) in girls with AN who resumed menses over the study period. FLI increased in two subjects who did not resume menses over the study period, but remained about the same or decreased in the others.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
We demonstrate increased levels of sOB-R and decreased FLI in girls with AN compared with healthy adolescents, and show that BMI and fat mass are the most important predictors of leptin and FLI. Regional fat distribution does not independently predict sOB-R, leptin, or FLI. To our knowledge, this is the first demonstration that cortisol is an independent and important predictor of sOB-R levels in adolescents, especially in a state of nutritional deprivation. Measures of insulin resistance relate closely to levels of sOB-R and FLI. Recovery of menses is associated with increases in leptin and FLI.

Our data demonstrate increased levels of sOB-R levels in adolescents with AN, similar to adult women (31). The one study that examined sOB-R levels in adolescents with AN reported median levels of sOB-R in 13 girls but did not compare them to healthy adolescents (32). We demonstrate markedly elevated values of the sOB-R in AN, with values 74% higher than in controls. The FLI in our study was decreased to a greater extent than was total leptin in girls with AN (84% decrease in FLI vs. 72% decrease in total leptin). However, correlations of body composition parameters with FLI were not stronger than correlations of these parameters with total leptin. We noted an inverse correlation between sOB-R and leptin in the group as a whole, and this has been reported in healthy adults and adolescents (32, 34, 49). In AN alone, however, this correlation was weak, possibly from insufficient power. It has been suggested that leptin may regulate levels of the sOB-R, its own binding protein (34). The weak correlation between leptin and sOB-R in AN, a condition associated with very low levels of leptin, suggests that other variables, such as cortisol, may determine levels of both leptin and the sOB-R.

Few studies have examined body composition and hormonal determinants of sOB-R and FLI in adolescents. An inverse relationship between BMI and sOB-R has been reported in children and young adults (32, 49); however, correlations with fat mass and regional fat distribution were not determined. We report significant contributions of both BMI and fat mass to levels of FLI and leptin on stepwise regression in the group as a whole, with these two variables contributing to 82% and 78% of the variability of leptin and FLI; however, only fat mass remained a significant predictor of sOB-R on stepwise regression, and contributed to only 20% of the variability. Regional fat distribution did not predict sOB-R levels. In the groups considered separately, body composition parameters did not predict sOB-R, but fat mass did predict FLI and leptin. Thus, body composition, a strong predictor of FLI and leptin, is a weak predictor of sOB-R.

Of the hormones examined, in the group as a whole, cortisol and IGF-I correlated with sOB-R levels, whereas IGF-I, GH, and cortisol correlated with FLI and leptin. Two studies (in healthy adults and children) that examined the relationship between sOB-R and IGF-I reported contradictory findings. Kratzsch et al. (32) observed an inverse correlation between sOB-R and IGF-I levels similar to our study. Chan et al. (34) found no correlation of sOB-R or leptin with IGF-I levels. In our study, IGF-I was an independent predictor of sOB-R levels on multiple regression for the group as a whole.

The relationship between GH concentration and sOB-R levels or the FLI has not been previously examined. Measures of GH concentration did not predict levels of sOB-R but correlated inversely with FLI and leptin in the group as a whole, and also in controls but not in AN. We have previously reported that girls with AN have a nutritionally acquired resistance to GH with low levels of IGF-I and high levels of GH (14), and a correlation between GH and bone formation markers in healthy adolescents that is lost in AN. The lack of any correlation between GH concentration and FLI or leptin in AN may well represent another aspect of this resistance. However, patients with true GH resistance, as occurs in Laron’s dwarfs, have high levels of leptin (35, 41). Administration of recombinant human GH to GH-deficient children results in a decrease in leptin, presumably through lipolytic effects of GH (42). On stepwise regression analysis including body composition variables and GH levels in controls in our study, fat mass remained the only significant predictor of leptin and FLI, suggesting that associations between GH and leptin may merely reflect associated alterations in fat mass. However, Elimam et al. (36) reported a decrease in leptin levels with recombinant human GH therapy for GH deficiency before changes were noted in fat mass, suggesting a more direct regulation of leptin by GH. Ghrelin, a GH secretagogue that also up-regulates appetite, has been shown to correlate inversely with leptin (13). We noted only weak correlations of ghrelin with sOB-R, leptin, and FLI.

Few studies have examined the relationship between sOB-R levels and cortisol, although available literature suggests that the adrenal axis and leptin secretion are interrelated. Glucocorticoid administration results in a dose-dependent increase in leptin mRNA expression (65), and a decrease occurs in ACTH-induced cortisol secretion after addition of leptin to adrenal cell cultures (66). A synchronicity between leptin and cortisol pulse dynamics in healthy men has been reported, with changes in leptin preceding changes in cortisol, suggesting a relationship between the two hormones independent of changes in fat mass (16). In our study, on multiple regression analysis, cortisol was not an independent predictor of leptin or FLI. Conversely, we observed strong positive correlations between sOB-R and cortisol levels in the group as a whole and in AN. In fact, cortisol was the most significant predictor of sOB-R levels on multiple regression analysis in these groups. Consistent with these data, Chan et al. (34) have reported a positive correlation between cortisol and sOB-R levels in healthy adults. The very strong correlation noted between sOB-R and cortisol levels in AN suggests that cortisol is an important predictor of sOB-R levels in conditions of underweight.

Measures of insulin resistance were significantly lower in girls with AN than in controls. An inverse correlation was noted between levels of the sOB-R and measures of insulin resistance, whereas a positive correlation was noted between these measures and levels of leptin and the FLI, suggesting that leptin or FLI may regulate the amount of insulin secreted, similar to adult data (34). However, correlational analysis does not imply causation, and the underlying condition of undernutrition may well be responsible for decreases in both leptin and insulin. In fact, leptin administration did not prevent fasting-related decrease in insulin levels in normal men (24).

Similar to our study, Kratzsch et al. (32) reported an increase in levels of sOB-R (median values) in girls with AN with weight recovery. However, changes in levels of the sOB-R in girls with AN not recovering weight and in healthy controls were not reported. We unequivocally show that whereas weight recovery in AN is associated with a decrease in sOB-R levels and an increase in leptin (less marked changes in FLI), these changes do not occur in non-weight-recovered AN and in healthy adolescents. Conversely, the study of adult women with AN reported no change in sOB-R levels with refeeding (31).

A change in levels of the sOB-R and FLI with resumption of menses in AN has not been previously reported. We demonstrate a trend toward a decrease in sOB-R levels and significant increases in leptin, FLI, and estradiol at the time of resumption of menses. These changes do not occur in AN not recovering menses. Our data are consistent with findings of Chan et al. (24), who reported that leptin administration prevented fasting-related decreases in testosterone levels in healthy men, suggesting that leptin is necessary for intact functioning of the hypothalamo-gonadal axis. The fact that the FLI is significantly higher at the time of resumption of menses, but not at weight recovery alone, suggests that the FLI, rather than total leptin, may be an important determinant of menses recovery. However, we found no cutoff for FLI values above which girls with AN resumed menses. Also, two girls with AN had increases in FLI but did not resume menses. Both had a 2.1-kg/m2 increase in BMI from baseline, and it is likely that these patients were at the point of resuming menses, although this did not occur during the study. Although an increase in FLI in girls resuming menses was associated with an increase in estradiol, we observed only weak correlations between estradiol and leptin levels in our study, and we did not observe any correlations between sOB-R and estradiol. In contrast, in healthy adults, significant correlations have been demonstrated between sOB-R and both estradiol and testosterone (34). One reason for this lack of correlation may be the very low levels of estradiol in our AN group, and the fact that healthy controls were examined in the early follicular phase of their cycles, when estradiol values are lowest.

We thus demonstrate high levels of sOB-R in adolescent girls with AN with a correspondingly low FLI compared with healthy girls of comparable maturity. We show that cortisol is an important determinant of sOB-R levels, whereas BMI and fat mass predict leptin and FLI. Recovery of menses is associated with significant increases in FLI and estradiol. We could not demonstrate a value for FLI above which menses resumed in AN, thus further studies are necessary to determine predictors of return of menstrual function in this group. In addition, it will be important for future studies to establish the mechanisms whereby cortisol regulates sOB-R levels.


   Acknowledgments
 
We thank the skilled nursing staff of the General Clinical Research Center (GCRC), and Ellen Anderson and her Bionutrition staff for the care provided to our subjects at the GCRC. We also thank our study coordinators and study assistants, Jennifer Bjornson, Catherine Chamay-Weber, and Joyce Chung, for the respective roles they played in the completion of this study. Not the least, we thank our study volunteers, without whose participation this study would not have been possible.


   Footnotes
 
This work was supported, in part, by National Institutes of Health Grants M01-RR-01066, DK 52625–05, and DK 062249.

Abbreviations: AN, Anorexia nervosa; BMI, body mass index; cr, creatinine; DXA, dual-energy x-ray absorptiometry; FLI, free leptin index; HOMA-IR, homeostasis model of assessment insulin resistance; sOB-R, soluble leptin receptor; UFC, urinary free cortisol.

Received December 31, 2003.

Accepted March 29, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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