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Program in Nutritional Metabolism (S.S., B.C., P.K., S.G.), Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114; and Amgen Inc. (A.M.D.), Thousand Oaks, California 91319
Address all correspondence and requests for reprints to: Steven Grinspoon, M.D., Program in Nutritional Metabolism, Massachusetts General Hospital, 55 Fruit Street, LON 207, Boston, Massachusetts 02114. E-mail: sgrinspoon{at}partners.org.
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Study design
Baseline testing. Before baseline testing, calorie, protein and fat intake were determined from 4-d outpatient food records (version 8A/2.6; Minnesota Nutrition Data Systems, Minneapolis, MN). Subjects returned for baseline testing after an overnight fast, and weight, resting energy expenditure (REE), and body composition were determined. REE was measured by indirect calorimetry with the VMAX 29 metabolic cart using standardized techniques (Sensor Medics Corp., Yorba Linda, CA). All subjects were fasting and tested in a resting position (lying on back with the head of the bed raised 30 degrees). Light and room temperature were adjusted for comfort. The subjects were instructed to lie quietly, not talk, and not fall asleep. The testing period was approximately 15 min. Total body fat was determined using dual-energy x-ray absorptiometry (Hologic 4500; Hologic Inc., Waltham, MA) at baseline and at the end of the 4-d fast.
Fasting blood was drawn for T4, T3, IGF-I, IGF binding protein-1 (IGFBP-1), estradiol, progesterone, glucose, insulin, and free fatty acids. Subjects were then maintained for 24 h on an isocaloric diet, with similar proportions of protein and carbohydrate to the prebaseline diet. Frequent sampling of blood was performed every 10 min for LH and every hour for TSH, GH, and cortisol over 24 h.
Randomization. Subjects were randomly assigned to receive either recombinant methionyl human leptin (Amgen, Inc., Thousand Oaks, CA) 0.05 mg/kg sc daily or identical placebo in divided doses (0800, 1400, 2000, and 0200 h). Study drug was stored at the Massachusetts General Hospital research pharmacy at 2–8 C before reconstitution and administration by the nursing staff of the Massachusetts General Hospital General Clinical Research Center. r-metHuLeptin and placebo were supplied in a single-use vial and reconstituted with sterile water immediately before sc injection. The Massachusetts General Hospital Pharmacy performed the randomization. All investigators, study staff, and subjects were blinded to drug assignment.
Fasting protocol. Study drug continued over 4 d during which time subjects underwent a complete fast, except for water and a daily multivitamin, and had daily blood drawing at 0800 h for leptin and electrolytes. On the final day of fasting, subjects again underwent frequent sampling every 10 min for LH and every hour for TSH, GH, and cortisol, while the study drug continued. On the final morning of the protocol, while still fasting, repeat hormone evaluations for T4, T3, IGF-I, IGFBP-1, estradiol, progesterone, glucose, insulin, and free fatty acids were performed, and weight, REE, and body composition again determined.
A visual analog scale (VAS) evaluating hunger, thirst, nausea, and fatigue was administered by a bionutritionist to each subject in the morning, after awakening, just before the end of the 4-d fasting period. Subjects were asked to rate their level of hunger on a 10-cm scale, with 0 cm being not at all hungry and 10 cm being extremely hungry. Subjects were asked to mark the VAS at the point on the scale that they consider to be appropriate to their hunger sensation at the time of the testing (9). Similarly, thirst, nausea, and fatigue were also evaluated (data not shown). After all testing was complete, the fast was broken, and the subjects were allowed to eat breakfast ad libitum.
LH pulse analysis
Subjects underwent frequent LH sampling every 10 min for 24 h at baseline and on d 4 of the 4-d fast. To assess LH pulsatility we used CLUSTER, a largely model-free computerized pulse analysis algorithm to identify statistically significant pulses in relation to dose-dependent measurement error in each hormone time series (10). In performing the analysis, we specified individual test cluster sizes for the nadir of 2 and a peak width of 1 (2 x 1), a minimum and maximum intraseries coefficient of variation, a t statistic to identify significant increase, and a t statistic to define a significant decrease (11). A coefficient of variation (CV) of 4.0%, the intraassay CV for our LH assay, was used in the settings of the program. Day and nighttime 10-min samples were analyzed separately because LH CLUSTER analysis patterns are known to be distinct in the overnight period, compared with the daytime, in normal women. Data are presented from overnight (from 2000–0750 h) LH CLUSTER analysis, determined from LH samples collected every 10 min (Table 1
). Daytime (from 0800–1950 h) and 24-h LH CLUSTER were analyzed as well (data not shown).
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All samples from the same patient were run in duplicate in the same assay. The intraassay CVs were developed using pooled sera covering the range of the assay. Serum leptin levels were determined using an RIA kit [Linco Research Inc. (St. Charles, MO) intraassay CV of 3.4%, interassay CV of 3.0–6.2%, sensitivity of 0.5 ng/ml]. LH levels were assessed by a single measurement at each time point during the overnight frequent sampling in each subject by RIA [Nichols Institute (San Juan Capistrano, CA), intraassay CV, 1.0–1.6%, interassay CV, 2.2–7.1%]. Serum estradiol was measured by RIA [Diagnostic Systems Laboratories, Inc. (Webster, TX), intraassay CV of 6.5–8.9%]. Serum progesterone was measured by RIA kit [Diagnostic Products Corp. (Los Angeles, CA), intraassay CV of 3.9–8.8%, sensitivity of 0.2 ng/ml]. Serum TSH levels were run using a two-site RIA [DiaSorin, Inc. (Stillwater, MN), intraassay CV average of 3.1–3.3%, sensitivity of 0.01 µIU/ml based on serial dilutions]. Reverse T3 was measured by RIA kit (Adaltis Italia S.p.A., Casalecchio di Reno, Italy). Serum GH levels were run using a two-site RIA (Nichols Institute Diagnostics, intraassay CV average of 2.8–4.2%, interassay CV of 3.5–7.2%). Serum IGF-I was measured by RIA (Diagnostics Systems Laboratories, intraassay CV of 3.9–7%). IGFBP-1 was measured by two-site RIA (Diagnostic Systems Laboratories, intraassay CV of 4.2%, sensitivity of 0.33 ng/ml). Serum cortisol levels were run using a two-site RIA (DiaSorin, intraassay CV average of 5.1–6.1%, interassay CV of 8.8–9.8%, calculated sensitivity of 0.21 µg/dl). Serum insulin levels were run using RIA (Diagnostic Products, intraassay CV of 3.1–9.3%). Serum free fatty acids were assessed using RIA [Wako Chemicals USA, Inc. (Richmond, VA), intraassay CV of 1.1–2.7%].
Statistical analysis
Baseline comparisons were made by Wilcoxon test between the groups. Change from baseline between the groups was compared by the Wilcoxon test. For leptin, morning levels were compared between treatment groups on each day of the study using the Wilcoxon test, and change from baseline was compared within each treatment group using a nonparametric analysis. For LH, treatment effect was determined by analysis of covariance (ANCOVA). The measurements obtained during the final day of the study was the outcome variable; treatment assignment was the main effect; and baseline measurement of the variable, cycle day, weight, and 24-h serum cortisol were used as covariates. Regression equations were determined within each treatment group for the relationship of hunger and leptin levels (Spearman’s 
) and change in progesterone and estradiol (Pearson’s coefficient). All values are expressed as mean values ± SEM. Outlier analysis for LH pulse dynamics was performed using the criteria of Dixon and Massey (12), and data were analyzed in 17 subjects. Statistical analyses were performed using JMP software (SAS Institute, Inc., Cary, NC). Statistical significance was defined as a two-tailed 
-value of P 
0.05.
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Results |
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The baseline clinical characteristics of the study subjects are shown in Tables 1
and 2. Baseline clinical characteristics including age, weight, body mass index, endocrine and metabolic parameters including REE, and total fat mass by dual-energy x-ray absorptiometry scan were not significantly different between the two groups at baseline. Ethnicity was similar in both groups (67 vs. 63% Caucasian, P = 0.73, leptin vs. placebo treated, respectively). Baseline leptin levels were similar in both groups (14.9 ± 2.5 vs. 11.4 ± 1.9 ng/ml, leptin vs. placebo, P = 0.29). Last menstrual period was recorded for each patient and menstrual cycle day at baseline was not different between the groups (d 3.0 ± 0.8 vs. 3.9 ± 0.8, leptin vs. placebo, P = 0.32).
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Daily leptin levels were lower after baseline in the placebo compared with the leptin-treated group (Fig. 1, P < 0.05 for each time point after the baseline). Leptin levels were significantly decreased from baseline with fasting in the placebo-treated group. In contrast, leptin levels in the leptin-treated group tended to decrease but did not change significantly from baseline with fasting and remained in the low physiologic range (Fig. 1).
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Both treatment groups demonstrated significant reductions in weight (P < 0.01 for change from baseline within each group), but these differences were not significant between the groups (Table 2). Fat mass decreased significantly only in the leptin group and decreased significantly more in the leptin than the placebo group (P = 0.03 for comparison of change between groups, Table 2).
Hormonal and metabolic parameters
LH, estradiol, and progesterone.
Overnight LH pulse parameters were not significantly different between the groups at baseline (Table 1). LH area (38.9 ± 21.5 vs. 1.2 ± 11.1 µIU/ml·min, P = 0.05, Table 1, Fig. 2A) and LH peak width increased (15.8 ± 7.1 vs. –2.3 ± 6.7 min, P = 0.06, Table 1, Fig. 2B) more in the leptin vs. the placebo group. The number of LH peaks (–2.0 ± 0.9 vs. 1.0 ± 0.8 peaks/12 h, P = 0.03, Table 1, Fig. 2C) and valleys (–2.0 ± 0.8 vs. 0.4 ± 0.8 valleys/12 h, P = 0.02, Table 1) decreased more in the leptin vs. placebo group. Approximate entropy, a measure of pattern regularity, was significantly lower at study end in the leptin vs. placebo-treated subjects, indicating a greater regularity of LH pulsatility in the leptin-treated group (0.53 ± 0.06 vs. 0.77 ± 0.09, P = 0.02). Daytime LH CLUSTER analysis was performed, and no significant differences were observed between the leptin- and placebo-treated groups (data not shown). Estradiol concentrations were similar at baseline, increased in both groups over the course of the study, and did not differ significantly between the groups after the leptin administration. Progesterone levels at baseline and at the end of the fast remained nonovulatory in all the subjects studied (Table 1). The change in progesterone levels was highly correlated with the change in estradiol levels during the study in the leptin-treated group (r = 0.91, P = 0.0007) but not in the placebo-treated group (r = 0.04, P = 0.93).
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Twenty-four-hour mean TSH decreased with fasting in the placebo-treated group (P < 0.01 for change from baseline) but not in the leptin-treated group, and the change from baseline was significantly different between the treatment groups (P = 0.03; Table 2 and Fig. 3). T3 levels dropped significantly in both groups, but the change was not significant between the groups. Conversely, reverse T3 levels increased significantly in both groups, but the change was not significant between groups (Table 2). T4 levels did not decrease significantly with fasting and did not change significantly between the groups (Table 2).
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Twenty-four-hour mean GH and IGFBP-1 increased, whereas IGF-I decreased significantly in both groups with fasting (P < 0.01 from baseline), but no significant differences were seen between the groups in these parameters (Table 2).
Cortisol
Twenty-four-hour mean cortisol levels increased significantly with fasting in both groups (P < 0.01 for change from baseline), but the changes between the groups were not significant (Table 2).
Metabolic parameters
Fasting glucose decreased, whereas fatty acid levels increased significantly with fasting within both groups (P < 0.01 for change from baseline, Table 2). Changes between groups were not significant for these parameters. Whereas insulin levels dropped in both groups, only the leptin-treated group demonstrated a trend toward significance (P = 0.06) in change from baseline, and there were no differences in the changes between the treatment groups (Table 2).
Hunger
Hunger was not related to leptin at the end of the study among the subjects receiving placebo (r = –0.18, P = 0.70, Fig. 4A), all of whom demonstrated very low leptin levels of approximately 3 ng/ml. In contrast, a significant inverse linear relationship (r = –0.76, P = 0.03, Fig. 4B) was seen between hunger score and end of study leptin level among the subjects receiving leptin. Diminished hunger sensation was seen among those subjects achieving the highest leptin levels.
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Discussion |
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Leptin may be an important modulator of reproductive function. Leptin levels are low in association with reduced body fat (4) and increase with weight gain in anorexia nervosa (13). Among patients with hypothalamic amenorrhea, leptin levels are associated with decreased body fat (14). Leptin administration prevented the acute starvation-induced delay in ovulation in female mice (5) and was recently shown to prevent the fasting-induced decline in testosterone and gonadotropin pulsatility in healthy men (8). In the study of Chan et al. (8), the higher of the two doses studied (0.08 mg/kg·d) reversed the fasting-induced changes in gonadotropin pulsatility and testosterone in men but was associated with supraphysiologic leptin levels to approximately twice the level seen before fasting. The lower of the two doses (ranging from 0.001 to 0.008 mg/kg·d based on baseline leptin level) did not significantly blunt the fall in leptin level with fasting and was not shown to reverse the fasting-induced changes in gonadotropin pulsatility.
In contrast, we studied healthy, normal-weight, fasting women and demonstrate that acute administration of low dose physiologic leptin increases nighttime LH pulse area and pulse width, compared with placebo, during acute caloric deprivation. Nighttime LH pulse frequency declined but pulse regularity, as measured by approximate entropy, was increased in the leptin compared with the placebo-treated group. Estradiol increased significantly within both groups over 4 d in the follicular phase but did not increase more with the relatively low doses of leptin given in this study. Gonadotropin changes in the placebo-treated group are consistent with data from Olson et al. (15) among healthy women, in whom the normal rise in LH area between cycle d 6 and 11 was reduced with fasting with minimal effect on LH pulsatility and estradiol.
Physiological leptin may preserve a more normal relationship between estradiol and progesterone, which is disrupted with fasting. As anticipated from the study design in the early follicular phase, progesterone levels remained preovulatory in all subjects. However, a tight relationship between change in estradiol and progesterone was noted in the leptin-treated but not in the placebo-treated group. Our data suggest a permissive effect of low-dose physiologic leptin on the normal pattern of nighttime gonadotropin change across 4 d of the follicular phase, e.g. LH area increased more in response to leptin than placebo. Licinio et al. (16) demonstrated a significant relationship between the release pattern of leptin and LH in healthy women and proposed that the nocturnal rise in leptin determines the nocturnal LH profile in the late follicular phase. In this study we demonstrate that administration of very low doses of r-metHuLeptin to maintain leptin within the physiological range permits the normal preprogrammed changes to occur in gonadotropin dynamics across the follicular phase during acute caloric deprivation. Our study does not address whether such changes would be seen in response to physiologic leptin administration in chronic undernutrition.
Administration of low-dose physiologic leptin prevented the fasting-associated decline in TSH. In animal studies, leptin increases transcriptional regulation of the TRH gene (17) and abrogates the reduction in T4 associated with acute fasting (5). Chan et al. (8) demonstrated that leptin administration prevented fasting-induced changes in TSH pulsatility with minimal effects on T4 or T3 in healthy fasting men. In our study of healthy, fasting women, TSH decreased significantly in the placebo-treated but not the leptin-treated group. Similar to Chan et al., we showed no effect of leptin on changes in T4 and T3 with acute fasting, but such changes may take longer to manifest than 3–4 d due to the half-life of T3 and particularly T4. In addition, reverse T3 increased significantly with fasting in both treatment groups, with no differences seen between the groups. These data suggest that in humans, the reduction in TSH associated with acute caloric deprivation may, in part, be related to decreased leptin. Because changes in euthyroid sick syndrome involve both decreased TSH production and changes in deiodinase function, our model of acute fasting may best address acute changes in pituitary secretion of TSH, rather than downstream effects on T4, T3, and reverse T3.
Leptin administration did not affect 24-h mean cortisol levels during fasting. Ahima et al. (5) demonstrated increased ACTH in response to 48 h of fasting in mice, which was abrogated by recombinant leptin infusion. Leptin administration during starvation may decrease neuropeptide Y and prevent the anticipated hypothalamus-pituitary-adrenal (HPA) activation during acute starvation. However, leptin effects on the HPA axis were not seen during fasting in healthy men (8). Similarly, cortisol increased with acute fasting but was not affected by leptin in our study. These data argue against an effect of physiologic leptin on cortisol during acute caloric deprivation in healthy women, but further studies are needed to assess the chronic effects of leptin on the HPA axis.
We found that leptin administration did not affect the GH-IGF-I axis. Our data are concordant with the data of Chan et al. (8) demonstrating that leptin administration does not prevent the fasting-induced changes in GH pulsatility or decline in IGF-I levels in healthy men. Taken together, these data argue against an effect of leptin on the GH-IGF-I axis during acute caloric deprivation.
The effects of leptin on body composition and regulation of fat mass have been demonstrated in a limited number of human models. Farooqi et al. (18) demonstrated significant effects of r-metHuLeptin to decrease fat mass and improve gonadal function in subjects with congenital leptin deficiency. In contrast, relatively large doses of leptin (0.3 mg/kg·d) are needed to achieve significant weight loss in generalized obesity, possibly the result of leptin resistance in this population (19). In a study of 10% weight-reduced subjects (two men and two women), leptin administration over 5 wk resulted in modest reductions in weight and fat mass at lower doses of 0.08 mg/kg fat mass per day in men and 0.14 mg/kg fat mass per day in women (20). In the current study, we show that short-term leptin administration (0.05 mg/kg·d, in divided doses) results in a significant reduction in total fat mass, compared with placebo administration, even after 4 d in women undergoing a short-term fast. REE did not increase in the leptin-treated patients, nor did REE decrease in the placebo group. Further studies of leptin effects on energy expenditure in humans are necessary. The strong effects of leptin on fat in short-term fasting may result from increased sensitivity to leptin in undernutrition in contrast to obesity.
We assessed hunger and demonstrate a significant inverse relationship between leptin concentrations and hunger score at the end of the fast among those subjects receiving leptin. Subjects achieving the highest leptin levels demonstrated the least hunger after 4 d of fasting. In contrast, among those subjects receiving placebo, in whom leptin levels were very low, hunger was not related to the leptin concentration. These data suggest that there is a physiologic threshold below which leptin is not related to hunger in acute caloric deprivation. At very low leptin levels, the degree of hunger resulting from acute caloric deprivation may be a function of other factors. In contrast, in the leptin-sensitive state of caloric deprivation, achievement of higher leptin levels with acute leptin administration, even within the physiologic range, is associated with reduced hunger. Subjects at normal weight, or in particular who have experienced chronic or acute undernutrition, may be more sensitive to the anorexigenic effects of leptin. In contrast, if leptin administration decreased hunger, this might promote weight loss in obese patients. However, the absence of a significant leptin effect on fat in obesity (19) suggests that a similar tight relationship between achieved leptin level and hunger may not hold in obesity. Our study provides novel data on the role of physiologic leptin to regulate hunger in humans.
In summary, we have examined the effects of physiologic leptin administration on endocrine and metabolic function during acute caloric deprivation in normal-weight, healthy women. In this model, leptin has significant effects to alter nighttime gonadotropin pulsatility and may be permissive to the normal organization of LH pulsatility through the follicular phase. Furthermore, leptin prevents the fasting-induced fall in TSH levels. Importantly, leptin reduces fat mass and is associated with minimized hunger in this model, suggesting potent effects of physiologic low-dose leptin in the highly leptin-sensitive state of acute caloric deprivation. These data stand in contrast to the less potent effects of leptin reported in human models of obesity and leptin resistance. Leptin is an important physiologic regulator of endocrine and metabolic function during undernutrition in women and functions as a critical signal of energy availability and nutritional status to the hypothalamus and pituitary in this setting.
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Acknowledgments |
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Footnotes |
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Abbreviations: ANCOVA, Analysis of covariance; CV, coefficient of variation; HPA, hypothalamus-pituitary-adrenal; IGFBP-1, IGF binding protein-1; REE, resting energy expenditure; VAS, visual analog scale.
Received June 14, 2004.
Accepted August 2, 2004.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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