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Leptin Does Not Mediate Short-Term Fasting-Induced Changes in Growth Hormone Pulsatility but Increases IGF-I in Leptin Deficiency States

Division of Endocrinology, Diabetes, and Metabolism (J.L.C., C.J.W., P.R., J.B., T.K., I.K., C.S.M.), Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215; and Division of Endocrinology and Metabolism (M.L.J., M.O.T.), University of Virginia Health System, Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Christos S. Mantzoros, M.D., D.Sc., Division of Endocrinology, Diabetes, and Metabolism, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, ST 816, Boston, Massachusetts 02215. E-mail: cmantzor{at}bidmc.harvard.edu.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: States of acute and chronic energy deficit are characterized by increased GH secretion and decreased IGF-I levels.

Objective: The objective of the study was to determine whether changes in levels of leptin, a key mediator of the adaptation to starvation, regulate the GH-IGF system during energy deficit.

Design, Setting, Patients, and Intervention: We studied 14 healthy normal-weight men and women during three conditions: baseline fed and 72-h fasting (to induce hypoleptinemia) with administration of placebo or recombinant methionyl human leptin (r-metHuLeptin) (to reverse the fasting associated hypoleptinemia). We also studied eight normal-weight women with exercise-induced chronic energy deficit and hypothalamic amenorrhea at baseline and during 2–3 months of r-metHuLeptin treatment.

Main Outcome Measures: GH pulsatility, IGF levels, IGF and GH binding protein (GHBP) levels were measured.

Results: During short-term energy deficit, measures of GH pulsatility and disorderliness and levels of IGF binding protein (IGFBP)-1 increased, whereas leptin, insulin, IGF-I (total and free), IGFBP-4, IGFBP-6, and GHBP decreased; r-metHuLeptin administration blunted the starvation-associated decrease of IGF-I. In chronic energy deficit, total and free IGF-I, IGFBP-6, and GHBP levels were lower, compared with euleptinemic controls; r-metHuLeptin administration had no major effect on GH pulsatility after 2 wk but increased total IGF-I levels and tended to increase free IGF-I and IGFBP-3 after 1 month.

Conclusions: The GH/IGF system changes associated with energy deficit are largely independent of leptin deficiency. During acute energy deficit, r-metHuLeptin administration in replacement doses blunts the starvation-induced decrease of IGF-I, but during chronic energy deficit, r-metHuLeptin administration increases IGF-I and tends to increase free IGF-I and IGFBP-3.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
States of acute starvation are associated with increased GH pulse frequency, higher peaks, and elevated interpulse levels in humans (1, 2). Despite the increase in GH secretion, levels of IGF-I are decreased (3). Similarly, states of chronic energy deficit such as anorexia nervosa or hypothalamic amenorrhea are associated with decreased IGF levels and increased GH secretion (4, 5, 6, 7), although the pattern of GH secretion may differ (7). However, the exact signal(s) mediating the development of the increased GH and decreased IGF-I are not well understood.

The adipocyte-secreted hormone leptin plays a critical role in signaling energy availability and mediating the neuroendocrine adaptation to starvation in rodents (8) and humans (9, 10). Thus, leptin may be a potential candidate linking energy deficiency with changes in the GH-IGF system. Some evidence for this derives from humans with congenital leptin deficiency who have abnormalities in the GH-IGF axis and decreased GH response to insulin-induced hypoglycemia (11). Leptin replacement for 18 months in three such individuals resulted in weight loss and increased IGF binding protein (IGFBP)-1 and IGFBP-2 but not IGFBP-3; however, it is possible that this may have been due to weight loss rather than leptin administration per se or any other potential confounding factors, given the lack of a control group (12). We previously found no effect of recombinant methionyl human leptin (r-metHuLeptin) administration on the fasting-associated increase in GH pulse frequency but partial restoration of decreased total IGF-I levels in normal-weight men during short-term starvation (9) and no effect on decreased IGF-I levels in women during similar conditions (10). In a model of more chronic relative leptin deficiency [women with hypothalamic amenorrhea (HA)], we found that r-metHuLeptin administration for 2–3 months resulted in an increase in total IGF-I and IGFBP-3 levels (13).

Despite the above initial observations, the exact mechanisms mediating the fasting-induced changes in the GH-IGF axis in healthy humans, including whether leptin plays a role, have not yet been fully characterized. More specifically, important questions remain unanswered regarding whether leptin regulates: 1) other components of the GH-IGF axis such as IGFBPs and IGF-II in short- or long-term energy deficit as well as GH binding protein (GHBP), which may reflect functional GH receptor status (14), and 2) GH pulsatility in women (who have higher baseline leptin levels, differential regulation of the GH axis, and possible differences in regulation of IGF-I) in states of short-term energy deficit as well as more chronic energy deficit. These questions have relevance not only for advancing our understanding of the pathophysiology of GH resistance in short- and long-term energy deficiency states (e.g. malnutrition, anorexia nervosa, HA), but also might have therapeutic implications if the GH hypersecretion and/or low IGF-I levels associated with energy deficit were related, wholly or in part, to leptin deficiency.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Human subjects

The study protocols were approved by the Institutional Review Board of the Beth Israel Deaconess Medical Center (BIDMC), and subjects gave written informed consent to participate. Clinical-quality r-metHuLeptin was supplied by Amgen, Inc. (Thousand Oaks, CA) and administered under an investigational new drug application submitted to the Food and Drug Administration by one of the authors (C.S.M.).

Short-term energy deficit study

Eight men (aged 23.0 ± 3.6 yr, mean ± SD) and six women (aged 24.3 ± 3.8 yr) with body mass index (BMI) less than 25 kg/m² were studied during three separate admissions in the BIDMC General Clinical Research Center (GCRC) as part of a larger study to evaluate the role of leptin in the neuroendocrine (9, 10) and immune (10) response to fasting: baseline assessment during an isocaloric weight-maintaining diet; 72 h fasting with administration of placebo; and 72 h fasting with administration of replacement-dose r-metHuLeptin to normalize the fasting-induced decline in leptin levels. For women, the treatment order for the two fasting studies was random (10), and for men, the fasting with r-metHuLeptin administration study was performed after the fasting with placebo study (9). Women had regular menstrual cycles and were not on oral contraceptives for at least 6 months before the study. Subjects were admitted to the GCRC the night before the first study day. During each study in the fed or fasting state, blood samples were obtained at 0800 h on d 1 (men and women) and at 0800 h on d 3 (men) or d 4 (women) for measurement of hormone and binding protein levels. Starting at 0800 h on d 3, blood samples for GH were drawn every 15 min for 24 h through an indwelling peripheral iv line. Body composition (fat mass) was measured by dual-energy x-ray absorptiometry in men (9) and bioelectric impedance analysis (10). For women, the frequent-sampling day was matched to a cycle day within 2 d of the cycle day of the other two frequent sampling studies and for all admissions within cycle d 6–11. During the baseline-fed condition, subjects received a standardized isocaloric diet. During both fasting studies, subjects received only water and calorie-free liquids for 3 d (to which they had free access) and NaCl (500 mg), KCl (40 mEq), and a standard multivitamin with minerals daily. r-metHuLeptin was administered at a dose of 0.04 (men) or 0.08 (women) mg/kg·d on the first day and 0.1 (men) or 0.2 (women) mg/kg·d on the second and third days. The total daily leptin dose for each day was divided into four equal doses given every 6 h by sc injection. Placebo (a buffer solution) was administered at the same volume as the corresponding r-metHuLeptin dose and according to the same schedule.

Chronic energy deficit study

Eight women (aged 24.8 ± 5.4 yr, BMI 20.5 ± 2.0 kg/m², mean ± SD) with chronic energy deficit, relative leptin deficiency (baseline leptin level < 4 ng/ml), and HA for at least 6 months related to strenuous exercise or low weight were studied to evaluate the effects of r-metHuLeptin on neuroendocrine parameters (13). Subjects self-administered r-metHuLeptin (0.08 mg/kg·d) sc twice daily for 2–3 months, with 40% of the daily dose at 0800 h and 60% at 2000 h to mimic the normal diurnal variation of leptin levels. One subject participated in the study for only 1 month for reasons unrelated to the study and is included only in the analysis of GH pulsatility for which data were complete. Blood samples for measurement of hormone and binding protein levels were obtained 1 month before as well as just before the initiation of r-metHuLeptin (with these two values averaged for the baseline level, except for IGF-II measured 1 month prior only), after 1, 2, and 3 months of treatment and after a 1-month washout period after completion of treatment (n = 7, except third month because two subjects completed the study at 2 months by design). Just before initiation of r-metHuLeptin and after 2 wk of r-metHuLeptin treatment, subjects underwent a 12-h frequent sampling in the BIDMC GCRC, during which blood was sampled every 10 min (1900 to 0700 h).

Hormone measurements

Hormone levels were run in duplicate using standard immunoassays and within the same run for a given subject as follows: leptin [RIA; Millipore, Billerica, MA; GH immunoradiometric assay (IRMA), Diagnostics Systems Laboratory, Webster, TX; free IGF-I (IRMA; Diagnostics Systems Laboratory), IGF-II (IRMA; Diagnostics Systems Laboratory), IGFBP-1 (IRMA; Diagnostics Systems Laboratory), IGFBP-II (RIA; Diagnostics Systems Laboratory), IGFBP-4 (ELISA; Diagnostics Systems Laboratory), IGFBP-6 (RIA; Diagnostics Systems Laboratory), GHBP (ELISA; Diagnostics Systems Laboratory)]. Total IGF-I, free IGF-I, IGFBP-1, IGFBP-2, and IGFBP-3 were measured in all subjects as previously described (9, 10, 13).

GH pulsatility analysis and approximate entropy (ApEn) calculation

Multiparameter deconvolution analysis (Deconv) allows for simultaneous determination of quantitative properties of underlying secretory bursts and endogenous GH hormone half-life (15). To quantify irregularity in GH concentration time series, ApEn is used (16).

Statistical methods

Data are presented as mean ± SD. For the short-term study, we also report percentage change in hormone and binding protein levels from initial to final day of each condition. We used both paired t tests and nonparametric Wilcoxon rank sum tests to assess changes in hormone and binding protein levels for each condition. To determine whether changes in study variables (including pulsatility parameters) varied between conditions, we conducted a comparison of mean final to initial day differences using one-way ANOVA and Kruskal-Wallis tests, with pairwise t tests and Wilcoxon rank sum tests for post hoc analysis and a Bonferroni-corrected P = 0.017 to adjust for multiple comparisons. For the long-term study, we used repeated measures mixed-effects models to assess changes in variables over the course of the study, with adjustment for estradiol and/or body weight. Nonnormally distributed variables were logarithmically transformed for analysis. Post hoc analyses for comparison of on-treatment values with baseline and follow-up values were also conducted using mixed-effects models, with a Bonferroni-corrected P = 0.017 to account for multiple comparisons. Differences in pulsatility parameters between baseline and post r-metHuLeptin were determined using paired t tests and nonparametric Wilcoxon rank sum tests. We report results only from nonparametric testing because findings were similar for parametric and nonparametric methods. All P values are two sided. The short-term energy deficit study had 80 and 90% power to detect differences of 0.81 and 0.94, respectively, per unit of SD at the = 0.05 level. The chronic energy deficit study had 80 and 90% power to detect differences of 1.9 and 2.21, respectively, per unit of SD at the = 0.05 level.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Effect of short-term energy deficit with and without leptin replacement on the GH-IGF axis

In the baseline-fed condition, total and free IGF-I, IGF-II, IGFBP-1, IGFBP-4, and IGFBP-6 levels remained stable in healthy normal-weight subjects (men and women) (Table 1). IGFBP-2 and GHBP decreased by approximately 15–18%, whereas body weight increased slightly by 0.9 kg (Table 1).

Effect of chronic energy deficit and leptin replacement on the GH-IGF axis

We then evaluated the effect of leptin replacement on the GH-IGF axis using a model of chronic energy deficiency, women with HA and relative leptin deficiency. At baseline, leptin levels were significantly lower in HA women, compared with eumenorrheic controls of similar body weight (3.4 ± 1.8 vs. 13.9 ± 2.7 ng/ml, P < 0.05) (Table 3). Administration of r-metHuLeptin increased serum leptin to levels similar to that of controls after 1 month of treatment and higher than controls but within the physiological range for normal-weight women after 2 months and to high physiological levels in the third month (Table 3).

View larger version (16K): [in this window] [in a new window]   FIG. 1. Twelve-hour GH pulsatility profile (1900 to 0700 h) in normal-weight eumenorrheic controls (n = 6) (based on sampling every 15 min) and in subjects with exercise-induced energy deficit, hypoleptinemia, and hypothalamic amenorrhea (n = 8) at baseline and after 2 wk of r-metHuLeptin administration (based on sampling every 10 min).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Prior studies have demonstrated increased GH secretion in response to short-term starvation due to more frequent GH secretory bursts (1, 2) and greater mass per burst from larger amplitude pulses (1, 2, 17), both of which contribute to the increase in mean GH levels and AUC. Our findings herein demonstrate not only that 72 h fasting increases GH pulsatility with increased secretion events and decreased interpulse interval but also increases total GH production, mean concentration, AUC, and ApEn (i.e. less regularity). This extends findings from our prior study in men (9) and demonstrates a similar response in women. A recent study using a highly sensitive chemiluminescent assay found no change in GH pulse frequency with short-term fasting (nonsignificant increase of 10%); however, subjects fasted for only 33 h, in contrast to 72 h herein (18). Although the GH assay we used is less sensitive (0.01 ng/ml) than highly sensitive chemiluminescent assays (0.003 ng/ml) (18), it is more sensitive than other IRMAs (0.2 ng/ml). Importantly, this study clearly demonstrates that the fasting-induced increase in GH pulsatility is not mediated by leptin deficiency in healthy men and women because increased GH pulsatility is observed during both fasting alone and fasting with normalized leptin levels.

Short-term starvation also decreases IGF-I levels. It appears that at least approximately 2.5–3 d of complete fasting (17, 19, 20) are required to have an effect because 36 h fasting did not result in major alterations of IGF-I levels in healthy subjects (21). Because normalizing leptin levels during fasting did not substantially normalize the decreased IGF-I, this study cannot test the hypothesis that the decrease in IGF-I plays an important role in increased GH secretion (22). IGFBP-1 consistently increases in response to short-term starvation (19, 20, 21), likely due to loss of the suppressive effect of insulin on IGFBP-1, but this occurs independently of leptin based on our findings herein. Prior studies have shown no effect of fasting on IGFBP-3 (19, 21), whereas we found that IGFBP-3 decreased modestly by approximately 8% during fasting but with no effect of leptin replacement. We found that fasting decreased IGFBP-4 and IGFBP-6 by 33 and 25%, respectively, but these changes are also leptin independent. Prior studies on IGF-II have been conflicting, showing no effect of 5 d fasting on IGF-II (23) or a 27% decrease with 72 h fasting (24). We found a trend toward decreased IGF-II with fasting and reversal with r-metHuLeptin, but the absolute magnitude of the change was small and did not reach statistical significance.

States of chronic energy deficit are also generally associated with increased GH but characterized by a different pattern of secretion. In women with anorexia nervosa, a prototypical model of severe chronic energy deficit, GH secretion has been reported to be increased (6, 7) due to a 20-fold increase in nonpulsatile secretion as well as a 4-fold increase in the pulsatile component (7). HA, whether due to excessive exercise or functional, is also characterized by energy deficiency (25), hypoleptinemia (26, 27), and a more disorderly pattern of GH secretion with an unstable baseline and less discrete pulses (28), consistent with our findings. Women with functional HA had increased GH secretion only at night (5, 28), whereas amenorrheic athletes (who may have greater energy deficit) had increased 24-h mean GH levels (4). Amenorrheic athletes also had higher GH interpulse levels and pulse frequency, compared with eumenorrheic athletes (4), indicating a specific effect of their hypometabolic state distinct from exercise alone. We did not find increased mean overnight GH levels in our subjects (having exercise induced amenorrhea), perhaps related to differences in the severity of the underlying energy deficit. Importantly, normalization of leptin levels with r-metHuLeptin did not substantially alter GH pulsatility after 2 wk, although there was a trend for mean GH concentration and AUC to increase at 2 wk. This stands in contrast to the marked improvement in LH pulsatility over the same time frame in the same subjects (13). Whether the trend toward increased GH secretion would continue with continued r-metHuLeptin treatment and explain the increased IGF-I levels seen after the first month of r-metHuLeptin treatment requires further investigation through more long-term studies evaluating GH pulsatility in the context of leptin replacement. In addition, although estrogen administration can increase GH secretion, which may be due to decreased IGF-I (29), estradiol levels did not change significantly until after 2 wk of r-metHuLeptin in our study.

IGF-I levels, a sensitive indicator of nutritional status, are decreased in states of chronic energy deficit, including protein-energy malnutrition (30) and anorexia nervosa (6, 31, 32). However, whereas some studies have found no difference in IGF-I or IGFBP-3 levels but a lower IGF-I to IGFBP-1 ratio in women with functional HA (5) and exercise-induced HA (4), others demonstrate lower IGF-I levels in HA women, compared with controls (25), similar to our findings. Similarly, data are also conflicting on whether weight recovery in women with anorexia nervosa normalizes the low IGF-I levels (32) or not (31).

IGFBP-1 and/or IGFBP-2 are increased in protein energy malnutrition (30) and anorexia nervosa (31, 32), whereas IGFBP-3 levels are decreased (30, 31, 32). These abnormalities improve or normalize after weight recovery or refeeding in most studies (30, 31, 32), except IGFBP-3 in one study (31). The effect of HA on free IGF-I and IGF binding proteins has not been as well characterized. In this study, we found that IGFBP-1 and IGFBP-2 tended to be higher and IGFBP-3 tended to be lower in HA women at baseline, compared with controls, although not statistically significant due to high interindividual variability. We report novel findings that IGFBP-4 levels were higher, whereas free IGF-I, IGFBP-6, and GHBP levels were lower and IGF-II levels were not different in HA subjects. r-metHuLeptin administration for 2–3 months was associated with a significant increase in total IGF-I levels, independent of changes in weight or estradiol levels, which may have been due to the tendency for IGFBP-3, the main IGF-I binding protein, to increase. Free IGF-I also tended to increase and became significant after adjustment for estradiol and weight changes.

Although a few individuals with mutations in the leptin receptor gene were reported to have abnormal GH stimulation testing, low IGF-I and IGFBP-3 levels, and growth delay in early childhood (33), a recent larger study of eight individuals with leptin receptor mutations found normal linear growth and IGF-I levels but lack of a pubertal growth spurt leading to reduced final heights (34). r-metHuLeptin administration to three adults with congenital leptin deficiency led to an increase in IGFBP-1 and IGFBP-2 but not IGF-I, IGFBP3, IGFBP-6, or IGF-II (12); however, leptin replacement also led to dramatic weight loss and improved insulin sensitivity, which may have contributed to the increase in IGFBP-1. Leptin treatment in another model of leptin deficiency (congenital or acquired severe lipodystrophy) increased IGF-I levels by approximately 70% after 1 yr (IGFBPs were not reported) (35, 36), but again improvement in insulin sensitivity may have been a confounder. Reasons for differences in these study findings may be related to differing durations of leptin deficiency and/or leptin replacement as well as the small number of subjects and lack of placebo control in prior studies.

The mechanism by which IGF-I levels are decreased in starvation despite the increased GH secretion is not completely understood but may relate to down-regulation of hepatic GH receptors, postreceptor defects, direct effect of starvation on IGF-I gene expression, changes in other hormone levels (such as insulin, T₃), and/or changes in IGFBPs (3). GHBP, the extracellular cleaved component of the GH receptor, is thought to reflect the status of GH receptor levels in the liver (14). In observational studies, GHBP levels are highly correlated with leptin levels (standardized for gender) even after adjustment for BMI (37), suggesting that leptin may be a potential factor regulating GHBP. We observed no differential decrease in GHBP levels with fasting similar to a prior study finding no effect of fasting for 5 d on GHBP levels in men (38). However, states of chronic energy deficit, including anorexia nervosa (31, 32) and HA [both functional (5) and exercise-induced (4)], are consistently associated with lower GHBP levels, and recovery of weight in women with anorexia nervosa normalized GHBP levels (31, 32). We found decreased GHBP levels in HA women at baseline, compared with controls, but no effect of normalizing leptin levels for up to 3 months. If GHBP indeed reflects hepatic GH receptor levels, this suggests an effect of leptin to regulate IGF-I levels independent of changes in hepatic GH receptor expression and is consistent with data indicating that GH and IGF-I may become dissociated during energy deficit (31).

In summary, we demonstrate herein using an interventional controlled study design that the increased GH associated with short-term starvation in humans is not due to changes in leptin. In more chronic energy deficit, r-metHuLeptin administration for 2 wk has minimal effects on GH secretion but after the first month increases IGF-I and tends to increase free IGF-I and IGFBP-3. Studies using more long-term administration of r-metHuLeptin are needed to clarify the chronic effects of leptin in regulating GH pulsatility, IGF-I, and IGFBPs and GHBPs in humans.

	Acknowledgments

 
We thank the BIDMC GCRC nurses for assistance with collecting the samples, the GCRC nutritionists for assistance with the isocaloric diet and fasting studies, Paula Veldhuis for assistance with the pulsatility analysis, and Lauren Kuhn for assistance with the manuscript.

	Footnotes

 
This work was supported by National Institutes of Health Grants MO1-RR01032 and R01–58785, an Amgen grant, a Center for Nutritional Research Charitable Trust grant, and Beth Israel Deaconess Medical Center discretionary grant (to C.S.M.); and National Institutes of Health Grant K23 RR018860 (to J.L.C.).

Author Disclosure Summary: J.L.C., C.J.W., P.R., J.B., T.K, I.K., and M.L.J. have nothing to declare. M.O.T. consults for Novo Nordisk, Asubio, and Tercica and received lecture fees from Genentec. C.S.M. received an honorarium for two lectures from Amylin.

First Published Online April 29, 2008

Abbreviations: ApEn, Approximate entropy; AUC, area under the curve; BIDMC, Beth Israel Deaconess Medical Center; BMI, body mass index; GCRC, General Clinical Research Center; GHBP, GH binding protein; HA, hypothalamic amenorrhea; IGFBP, IGF-binding protein; IRMA, immunoradiometric assay; r-metHuLeptin, recombinant methionyl human leptin.

Received January 9, 2008.

Accepted April 21, 2008.

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