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The Journal of Clinical Endocrinology & Metabolism Vol. 83, No. 11 4140-4147
Copyright © 1998 by The Endocrine Society

Original Studies

Sex Differences in Circulating Human Leptin Pulse Amplitude: Clinical Implications1

Julio Licinio, André B. Negrão, Christos Mantzoros2, Virginia Kaklamani, Ma-Li Wong, Peter B. Bongiorno, Paula P. Negro, Abeda Mulla, Johannes D. Veldhuis, Laura Cearnal, Jeffrey S. Flier and Philip W. Gold

Clinical Neuroendocrinology Branch, National Institute of Mental Health (J.L., A.B.N., M.-L.W., P.B.B., P.P.N., A.M., P.W.G.), and the Warren Grant Magnuson Clinical Center (L.C.), National Institutes of Health, Bethesda, Maryland 20892; the Division of Endocrinology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School (C.M., V.K., J.S.F.), Boston, Massachusetts 02215; the Department of Internal Medicine, and National Science Foundation Center for Biological Timing, University of Virginia Health Sciences Center (J.V.D.), Charlottesville, Virginia 22908

Address all correspondence and requests for reprints to: Julio Licinio, M.D., Clinical Neuroendocrinology Branch, National Institute of Mental Health, National Institutes of Health, Building 10/2D46, 10 Center Drive, MSC 1284, Bethesda, Maryland 20892-1284. E-mail: licinio{at}nih.gov


   Abstract
Top
 Abstract
Introduction
 Experimental Subjects
 Materials and Methods
 Results
 Discussion
 References
 
Leptin, a product of fat cells, provides a signal of nutritional status to the central nervous system. Leptin concentrations have ultradian and diurnal fluctuations. We conducted this study to assess sex differences in the levels of organization of frequently sampled leptin concentrations in healthy, normal weight women and men. Leptin levels were sampled every 7 min for 24 h in 14 healthy, normal weight individuals (6 women and 8 men). The 14 leptin time series containing a total of 2898 leptin measurements were assessed by 1) algorithms that characterize statistically significant pulsatility, 2) Spectral (Fourier) analysis, 3) analysis of time intervals and variability, and 4) approximate entropy. We found that frequently sampled plasma leptin concentrations have a 24-h profile that is numerically more than twice as high in women as in men, and leptin pulse amplitude is likewise more than twice as high in women. However, healthy men and women have nearly identical concentration-independent and frequency-related 24-h and ultradian patterns. Leptin concentrations have nonrandom fluctuations over 24 h, independent of their absolute value and underlying 24-h periodicity, that are similar in men and women. Ultradian periodicities detected by Fourier time series have similar values in men and women. The strongest distinction between the sexes in the level of organization of leptin concentration is not at the level of pulse organization or oscillation frequency, but, rather, in the mass or amount of leptin released (or removed) per unit time, indicating that women might be more resistant to the effects of leptin than men. Because leptin is clinically relevant to the regulation of body weight, future studies should examine whether the relative leptin resistance exhibited by women might contribute to their increased susceptibility to disorders whose pathophysiology involves dysregulation of food intake and body weight.


   Introduction
Top
 Abstract
 Introduction
Experimental Subjects
 Materials and Methods
 Results
 Discussion
 References
 
THE OBESE (ob) gene is expressed in white fat cells, encoding a peptide hormone, leptin, that communicates a signal of nutritional status to the brain and peripheral organs (1, 2). Leptin has a crystal structure with a four-helix bundle similar to that of the long chain helical cytokine family (3). One leptin receptor gene has been cloned, encoding five or more leptin receptor splice variants (2, 4, 5, 6). After it is secreted, leptin undergoes rapid uptake from the blood into the brain (7) and acts in key hypothalamic nuclei, such as arcuate, paraventricular, dorsomedial, ventromedial, and ventral premammillary, that contain leptin receptors (8, 9, 10, 11, 12, 13). Leptin administration to rodents reduces body weight by suppressing food intake and increasing motor activity, energy metabolism, oxygen consumption, and body temperature (14, 15, 16). Several studies have indicated that insulin and glucose contribute to regulate insulin secretion (17, 18). Leptin is essential for the regulation of body weight in humans; a genetically determined congenital deficiency of leptin or its receptor results in severe obesity (19, 20, 21). Moreover, low plasma leptin concentrations predicted weight gain in Pima Indians (22). Body fat gain has been shown to be inversely proportional to nocturnal leptin excursion in humans (23).

Sex differences in fasting basal plasma levels of leptin have been identified across a broad spectrum of age, body mass indexes (BMIs), and body fat composition in both rodents and men (24, 25, 26, 27, 28, 29, 30). Circulating plasma levels of leptin exhibit diurnal (24-h) fluctuations as well as short term (ultradian) pulsatility (31, 32, 33). We have recently characterized leptin pulsatility in healthy men and have determined that leptin pulses are of short duration and can therefore be best assessed by the use of very rapid (every 7 min) plasma sampling (33). Previous studies have not assessed sex differences in 24-h leptin concentrations, nyctohemeral patterns and pulsatility, or in ultradian oscillations of this hormone. Moreover, certain hormones show strong sex contrast in the orderliness or regularity of the release process over time (34), as assessed by approximate entropy (ApEn), a statistic that distinguishes random variation from orderly structure within the data in a model-free and scale-invariant fashion. Sex-related differences in ApEn of leptin time series have not been previously determined. To address the question of whether there are sex differences at various levels of organization of leptin concentrations throughout the 24-h period, we conducted very rapid sampling of plasma leptin for 24 h in healthy, normal weight men and women and analyzed sex differences in 24-h profiles, nyctohemeral patterns, pulsatility, ultradian variations, and ApEn.


   Experimental Subjects
Top
 Abstract
 Introduction
 Experimental Subjects
Materials and Methods
 Results
 Discussion
 References
 
Using a clinical protocol approved by the NIMH institutional review board, we conducted a 24-h blood collection study in 14 Caucasian subjects (6 women and 8 men) who gave informed consent. Partial results that did not include male/female comparisons were previously reported (33, 35). Subjects had an average age of 27.1 ± 1.9 yr (25.3 ± 1.6 and 28.4 ± 3.1 yr for women and men, respectively) and an average BMI (calculated as the weight in kilograms divided by the square of the height in meters) of 22.7 ± 0.7 kg/m2 (women, 22.3 ± 0.8 kg/m2; men, 23.0 ± 1.1 kg/m2). We screened all subjects for any personal history of mental illness, medical illness, obesity, smoking, or substance abuse. All subjects gave medical histories and underwent physical examination, electrocardiogram, and screening laboratory examinations, including tests of hematological, thyroid, liver, and renal function and measurements of serum calcium, magnesium, and phosphate. For the 30-day period preceding each study as well as during the study, no subject was taking prescribed or over the counter medications, hormones, or dietary supplements. Women were studied on days 8–11 of the menstrual cycle.


   Materials and Methods
Top
 Abstract
 Introduction
 Experimental Subjects
 Materials and Methods
Results
 Discussion
 References
 
Clinical protocol

All subjects were acclimatized to a research bed in the NIH Clinical Center for 48 h before the study. Blood collection started at 0800 h via an indwelling catheter that was inserted during the previous evening; samples were collected every 7 min for 1442 min, for a total of 207 samples/subject. Subjects had 4 standardized meals/day: breakfast at 0830 h, lunch at 1230 h, dinner at 1730 h, and an evening snack at 2100 h. Each subject received a total amount of calories per day that was calculated to keep each individual at their admission body weight, with 20% of calories served at breakfast, 35% at lunch, 35% at dinner, and 10% at the evening snack. Subjects were exposed to light from 0700–2300 h and were studied in bed in the dark from 2300–0700 h, during which time they slept. Sleep was monitored by the NightCap apparatus (Healthdyne Technologies, Marietta, GA) (36, 37). Subjects were allowed to walk from their bed to the bathroom and to an adjacent hospital day room, but were not allowed to exercise, so as to maintain their levels of physical activity at a comparable baseline.

Hormone assays

Total human leptin was measured as previously described (33). Briefly, 50 µL test plasma were incubated for 24 h at 4 C with phosphate-buffered saline containing 0.1% Triton X-100 and a 1:2000 dilution of antileptin antiserum. Twenty-four hours later, 125I-labeled leptin (~15,000 cpm) was added to a tube, and the reagents were incubated for an additional 24 h. Antibody-bound leptin was precipitated by addition of 500 µL precipitating reagent. Tubes were centrifuged for 45 min at 2500 rpm, after which supernatants were decanted, and pellets were counted in a {gamma}-counter. Recovery experiments showed that recovery of added human leptin (±SD) was always between 100–105 ± 2–5%. For this leptin assay, the limit of detection was 0.2 ng/mL. The within-assay coefficient of variation (CV) was 4.4% for low levels (2.9 ng/mL) and 5.7% for high levels (14.1 ng/mL). The interassay CV was 6.9% and 9% for low and high levels, respectively. This assay has a sensitivity of 0.2 ng/mL.

To determine an index of experimental variation, leptin was measured from 1 pool of blood 207 times, providing a series of replicates with the same length as the experimental series obtained from female and male subjects.

Pulse analysis

We used Cluster (38), a computerized pulse analysis algorithm, to identify statistically significant pulses in relation to measurement error in each individual leptin time series. We used a CV of 10% in the settings of the program that was just above 9%, which was the highest interassay CV in our assay. Consequently, the program only detected statistically significant pulses after taking into account both the limit of detection of the assay and a CV of 10%. As previously described, this procedure constrains the false positive rate to less than 5% (33). Measurement error for the 207 samples in each series was modeled as a power function of sample concentration, and significantly increased or decreased hormone concentrations were judged by pooled t statistics, which were applied to moving test nadirs and peak clusters that began with the onset of the experimental series and traversed all points. We identified the following properties of pulsatile leptin concentrations: pulse frequency (number of significant peaks per 24 h), mean interpeak interval (time separating consecutive peak maxima), mean pulse duration in minutes, mean pulse height (maximal leptin concentration in a peak), pulse height as percent increase over preceding baseline (100% corresponds to preceding baseline), and interpulse valley mean (a valley has been defined as a region embracing nadirs without intervening peaks).

Less intensive datasets

Surrogate datasets were created listing leptin levels in women and men every 7, 14, 21, 28, 35, 42, 49, and 56 min to permit an assessment of the effect of sampling interval affected on leptin pulsatility. Cluster analysis was applied to each subordinate dataset.

Assessment of 24-h leptin time series

Twenty-four-hour leptin levels were assessed both as raw values (nanograms per mL) as well as percent variability. Variability at each time point t, as previously described (33), is defined as a percentage of 24-h averages, using the formula: variability at time t = (leptin level at time t/24-h average level) x 100.

Spectral analysis

The temporal nature of leptin concentrations in women and men was assessed using the Spectral (Fourier) analysis methods described by Jenkins and Watts (39) (Statistica software program, StatSoft, Inc., Tulsa, OK). Leptin profiles from women and men were detrended, and then spectral estimations were obtained using a Tukey window with a width of 33 data points (4 h) to define 24-h rhythmicity. To identify further ultradian oscillations (namely rhythms with periods less than 24 h in duration), we fit unsmoothed, linearly detrended time series to significant underlying sine and cosine periodicities whenever such periodicities significantly reduced the fitted variance (P < 0.05) and also exhibited a significant nonzero amplitude (P < 0.05 vs. noise, defined by spectral power at that same period and its 95% confidence intervals after 1000 random shufflings of the data series).

ApEn

ApEn is a model-independent regularity statistic developed to quantify the orderliness of sequential measures (40), such as hormonal time series. Larger ApEn values correspond to greater randomness (irregularity). Technically, ApEn measures the logarithmic likelihood that runs of patterns that are similar remain so for the next incremental comparison. The basic derivation and calculation of ApEn were previously presented (34, 41). Two input parameters, m (window length) and r (tolerance), must be specified to compute ApEn. For this study, we calculated ApEn values for each leptin profile with window length m = 1 and tolerance parameter r = 20% of the average SD of the individual subject’s leptin time series. Thus, this calculated ApEn is denoted as ApEn (1, 20%). Previous theoretical analyses and clinical applications have demonstrated that these input values produce good statistical validity of ApEn for time series of 50 or more data points (34, 41, 42, 43). Mathematically stated, the ApEn application with m = 1 is said to estimate the rate of entropy for a first order (m = 1) approximating Markov chain to the underlying true process.

In choosing the r input parameter (tolerance) in ApEn as a fixed percentage of each dataset’s SD, we normalize ApEn for each profile. This so-called normalized ApEn is both translation and scale invariant; adding to or multiplying each data value in the hormone profile by a constant would produce an identical ApEn value (34, 41). This point is important when different absolute hormone levels are expected, as they are here.

ApEn is stable to small changes in noise characteristics and infrequent, albeit significant, outliers (34, 41). This statistic evaluates a variety of dominant and subordinate patterns in the data; for example, ApEn can detect and quantify changes in the underlying regularity of hormone release that are not necessarily reflected in changes in peak frequency or amplitude (34, 41). ApEn identifies consistency of point by point variations in the data, rather than macroscopic patterns or diurnal trends. Indeed, the latter are removed by first differencing of the data. Additionally, ApEn provides a barometer of feedback changes in many coupled systems (34, 41) .

ApEn is a family of statistics that individually provide a relative, not absolute, measure of process regularity. ApEn thus can show significant variation in absolute value with changing m or r input parameters, N (data series length), and/or noise characteristics (experimental variability) (42). As ApEn will generally increase with increasing N and noise (and, hence, increasing intraassay CVs), it is important to compare datasets with similar N and assay CV values, as we do here. Technical and statistical properties of ApEn, including so-called mesh interplay, relative consistency of (m, r) pair choices, asymptotic normality under general assumptions, statistical bias, and error estimation for general processes were discussed previously (34, 41). To compare observed ApEn measures with those expected on the basis of purely chance patterns occurring within the leptin profiles, each leptin time series was shuffled 1000 times to produce randomly assigned sample sequences. The resultant so-called random ApEn values were assumed to reflect assay and sampling noise without biological rhythmicity. Hence, the ratio of random to observed ApEn, given the calculated SD of this ratio for 1000 simulations in each series, monitors the extent of significantly nonrandom structure or patterning in the data.

Statistical analysis

Results are expressed as the mean ± SEM. Sex differences in mean leptin levels and pulse parameters were compared with the use of Student’s t tests for independent samples. Differences in pulse numbers detected by Cluster in each surrogate dataset and differences in mean leptin levels in men and women during the 24-h period divided into 4-h intervals were assessed by one-factor ANOVA with post-hoc correction (Scheffe’s test). Analysis of covariance and single linear (Pearson) correlations were used to assess the effects of age, BMI, and pulse height on mean 24-h individual circulating leptin levels. Significance was assumed for P < 0.05.


   Results
Top
 Abstract
 Introduction
 Experimental Subjects
 Materials and Methods
 Results
Discussion
 References
 
Comparison of 24-h leptin profiles in men and women

Our groups of women and men had demographic characteristics that are listed in Table 1Go. There were no statistically significant differences in age or BMI between women and men in this study. The age of the female subjects was 25.3 ± 1.6 yr, and that of the males was 28.4 ± 3.1 yr (average, 27.1 ± 1.9 yr); the BMI of the females was 22.3 ± 0.8 kg/m2, and that of the males was 23.0 ± 1.1 kg/m2 (average, 22.7 ± 0.7 kg/m2). The 24-h profiles of circulating leptin in women and men are shown in Fig. 1Go. Women and men had 24-h leptin levels with both ultradian and 24-h variability. Raw leptin levels were consistently higher in women than in men throughout the 24-h period; however, when the values were normalized and expressed as variability over 24-h averages, the 24-h leptin profiles of women and men showed no significant difference. All subjects showed evidence of 24-h (diurnal) periodicity in 24-h leptin levels as assessed by two procedures. First, spectral analysis of the smoothed 24-h profiles of women and men showed one dominant peak per series, indicative of a 24-h cycle (Fig. 2Go). Unsmoothed data disclosed highly significant ultradian rhythms of leptin concentrations in seven of the eight men and all six of the women (Fig. 3Go). Periodicities in the two genders were similar and approximated 4–10 h in women and 4–12 h in men. Ultradian rhythms had amplitudes greater than randomly shuffled leptin time series at P < 0.05. Additionally, when leptin levels were normalized, expressed as percent variability, and averaged in women and men by 4-h intervals starting at 0800 h, we showed a clear and statistically significant 24-h pattern in both women and men, with no significant differences in leptin variability between women and men at each 4-h interval (Fig. 4Go). Both women and men had a nadir of leptin levels during the 0800–1200 h period and highest levels during the 0000–0400 h period. ANOVA showed that leptin values during the 0800–1200 h period in women and men were lower than those in any other periods (P < 0.0001).


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  		Table 1. Twenty-four-hour pulsatility parameters in healthy men and women

 


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  		Figure 1. Frequently sampled 24-h profiles of total plasma leptin concentrations in men and women. The top panel shows absolute levels, which are higher in women throughout the 24-h period. The bottom panel shows leptin levels normalized and expressed as variability, defined as a percentage of the individual 24-h averages, using the formula: variability at time t = (hormone level at time t/24 h individual average level) x 100.

 


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  		Figure 2. Spectral analysis of detrended and smoothed data series showing 24-h periodicity in leptin levels.

 


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  		Figure 3. Individual significant periodicities by nonlinear Fourier time series analysis of 24-h leptin profiles in men and women. The periodicities of significant rhythms are given for the two genders. Each ultradian rhythm achieved statistical significance at P < 0.05 vs. the amplitude of random periodicity within the data as estimated by 1000 shufflings of each time series after linear detrending.

 


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  		Figure 4. Leptin levels were averaged for six 4-h time periods (0800–1200, 1200–1600, 1600–2000, 2000–0000, 0000–0400, and 0400–0800 h) and expressed as a percentage of the 24-h average. Values are shown as the mean ± SEM and were analyzed by ANOVA with post-hoc correction (Fisher’s protected test). All subjects had leptin levels that were higher at all other time periods than at 0800–1200 h.

 
ApEn and Fourier time series analysis

The mean circulating 24-h leptin concentration for all subjects was 5.80 ± 1.11 (mean ± SEM) ng/mL, with a mean level of 8.49 ± 1.44 ng/mL for women and 3.79 ± 1.24 ng/mL for men. All subjects, except 1 man, had a 24-h pattern of total circulating leptin levels that exhibited statistically significant order or patterning, as quantified by ApEn compared to randomly shuffled leptin time series (1000 shuffled realizations of 7-min leptin data over 24 h; Fig. 5Go). There were no sex differences in ApEn (Fig. 5Go). In contrast, by applying an identical analysis to the 207-replicate pooled series, we detected no significant structure or orderliness by ApEn analysis, as the random and observed ApEn values were statistically identical. In addition, unsmoothed Fourier time series analysis showed no rhythms with periods within the 1–1440 min periodicity in the replicate pool data. Thus, there is no consistent orderliness, regularity, or structure, or rhythmicity of either ultradian or 24-h period within the replicate pool.



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  		Figure 5. Individual ratios of random ApEn estimates to observed ApEn estimates in six women and eight men. ApEn is a quantitative measure of the disorderliness of the hormone release profile over time. For the data depicted here, the ApEn value was calculated for each series from the original measurements and then recalculated 1000 times after random shuffling of the data series to estimate a random ApEn. The latter denotes the apparent regularity of the leptin measurements after random reordering of the series and thus reflects noise unrelated to biological structure. According to the ratio of random to observed ApEn, the extent that it exceeds 1.0 defines significantly nonrandom order or regularity within the serial leptin concentration measurements. Each data point is as the mean ± SD of 1000 random shufflings. Note that where error bars are not visible, they are smaller than the depicted signal.

 
Sex differences in leptin pulsatility

All series had significant pulsatility as assessed by Cluster analysis (Table 1Go). Concentration-independent pulse parameters were very similar in men and women, with no significant differences between the sexes. Pulse frequency per 24 h was 30 ± 1 in women and 30 ± 2 in men (average, 30 ± 1), interpeak interval was 47.4 ± 1.6 min in women and 46.2 ± 2.2 min in men (average, 46.7 ± 1.4 min), pulse duration was 35.9 ± 1.5 min in women and 34.6 ± 2.0 min in men (average, 35.2 ± 1.3 min), and pulse height (percent increase) was 127% in women and 134% in men (average, 131 ± 4%). In contrast, the concentration-dependent pulse parameters, which are pulse height and valley concentration, that define pulse amplitude, were significantly higher in women than in men. Maximal pulse height was 9.9 ± 1.7 ng/mL in women and 4.6 ± 1.6 ng/mL in men (average, 6.9 ± 1.4 ng/mL), and valley concentration was 8.1 ± 1.4 ng/mL in women and 3.5 ± 1.1 ng/mL in men (average, 5.5 ± 1.0 ng/mL). Incremental pulse heights were 1.8 ± 0.4 ng/mL in women and 1.2 ± 0.6 ng/mL in men (average, 1.4 ± 0.4 ng/mL).

Analysis of covariance as well as Pearson correlations showed no effect of age or BMI on mean 24-h circulating leptin levels in this group of healthy men and women. We found a highly significant correlation between mean 24-h circulating leptin levels and pulse height (r2 = 0.991; P < 10-9) and between mean 24-h circulating leptin levels and valley concentration (r2 = 0.995; P < 10-9; Fig. 6Go). This indicates that in healthy women and men, mean 24-h leptin levels have a linear relation to the average pulse amplitude, which is defined by pulse height (Fig. 6AGo) and valley concentration (Fig. 6BGo). There was no significant correlation between mean 24-h leptin levels and pulse number (Fig. 6CGo), interpeak interval, pulse duration, or pulse height expressed as percent increase.



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  		Figure 6. Leptin mean 24-h values are highly correlated to pulse amplitude, as defined by pulse height (r2 = 0.995; P < 10-9; A) and valley concentration (r2 = 0.991; P < 10-9; B), but not to frequency-related parameters, such as pulse number (r2 = 0.006; P < 0.8, nonsignificant; C).

 
Surrogate datasets

We created daughter data series from each individual original 24-h profile, corresponding to sampling at the following intervals: 7, 14, 21, 28, 35, 42, 49, and 56 min. Applying Cluster analysis to each surrogate dataset, we show that leptin pulsatility in women and men can be best characterized by frequent sampling. The average pulse number per 24 h at 7 min of sampling was 30; at 14 min, it was 11.5; at 21 min, it was 8; at 28 min, it was 6; at 35 min, it was 3; at 42 min, it was 4; at 49 min, it was 1.5; and at 56 min, it was 0. The increase in the sample interval from 7–14 min resulted in a significant loss of 62% in the number of pulses per 24-h period (P < 0.0001). The decreases in pulse sample interval from 14–21 min, from 21–28 min, and from 42–49 min also resulted in statistically significant changes in pulse number per 24 h (Fig. 7Go).



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  		Figure 7. Assessment of leptin pulse numbers in datasets of women and men corresponding to sampling every 7, 14, 21, 28, 35, 42, 49, and 56 min, assessed by Cluster. The asterisk indicates a significant decrease in pulse number over that during the preceding time interval (determined by ANOVA with post-hoc Scheffe’s test).

 

   Discussion
Top
 Abstract
 Introduction
 Experimental Subjects
 Materials and Methods
 Results
 Discussion
References
 
In this study we show that frequently sampled plasma leptin concentrations have a 24-h profile that is numerically more than twice as high in women as in men; however, a careful analysis of 24-h and ultradian periodicity shows that healthy men and women have nearly identical concentration-independent and frequency-related 24-h and ultradian patterns. We observe a 24-h cycle in leptin levels in both women and men and nearly identical concentration-independent and frequency-related pulse parameters in women and men: those include pulse number, interpeak interval, and pulse duration. We conclude that in healthy individuals, mean 24-h leptin concentrations are a direct function of pulse amplitude, defined by pulse height and valley concentration, and that significantly higher pulse amplitude contributes to the observed higher plasma leptin concentrations exhibited by women in this study compared to those in men. We found that the absolute peak leptin concentrations for men and women are 4.6 and 9.9 ng/mL, respectively (2 times higher in women). However, it should be noted that the total pulse heights are occurring over baseline (or valley) concentrations of 3.5 and 8.1 ng/mL, respectively. Thus, the actual pulse heights correspond to incremental pulse heights of 1.2 and 1.8 ng/mL in men and women, respectively (50% higher in women). Therefore, there is a 2-fold higher baseline leptin production rate in women vs. men, and in addition, 50% higher pulses occur in women. We conclude that a higher baseline production rate of leptin in women causes the majority of the sex differences in plasma leptin concentrations, with the increased pulse amplitude observed in women contributing to a lesser degree. Because normal weight women have a higher percentage of body fat than men (25), it is possible that the 50% increases in incremental pulse amplitude that we have observed in this study might be explained by sex differences in the amount of adipose tissue, which may occur in the context of similar BMI values. However, an animal study that carefully measured amount of leptin per g body fat in animal carcasses found that females have more leptin per g body fat compared to males (24).

Additionally, by shuffling the 7-min leptin time series for each subject 1000 times and then reexploring the presence or absence of significant oscillations or apparent orderly structure in the data (respectively, Fourier time series, and ApEn analyses), we could achieve a significant model-free definition of nonrandom leptin release profiles by way of ApEn and identify ultradian oscillations of leptin in the 24-h data by way of Fourier time series analysis. Importantly, ApEn is both a model-independent and scale-invariant statistic that strongly distinguishes varying degrees of randomness and deterministic processes confounded by randomness (34, 41, 42). The model-free nature of ApEn is useful in analyzing leptin, because the kinetics of its removal from plasma remain unknown, and the waveform (profile of secretion rate) of leptin release episodes over time has not been determined directly. Our ApEn estimates strongly indicate that 13 of the 14 individuals studied here released leptin in a nonrandom manner over 24 h, independently of their absolute values and underlying 24-h periodicities. Of interest, ApEn was similar in men and women, in contrast to a strong gender distinction that exists for regularity/orderliness of GH release in men and women (34). Moreover, ultradian periodicities detected by Fourier time series had similar values in men and women, thus indicating that the strongest gender distinction is not at the level of pulse organization or oscillation frequency, but, rather, in the mass or amount of leptin released and/or removed per unit time in the two sexes.

Although we did not determine circadian free running vs. sleep-activity entrainable characteristics of leptin release, there were strong 24-h rhythms in serum leptin concentrations. To establish a circadian nature for such variations, one would need to find a free running rhythm of approximately 24 h in the absence of external cues and masking events, show that the rhythm is temperature compensated and entrainable, and demonstrate distinct phase responses to relevant zeitgebers as being phase related to core temperature, sleep data, etc. To date and to our knowledge, such estimates of truly circadian leptin release are not available in humans or experimental animals.

Because the leptin gene is expressed in fat cells, leptin levels vary as a function of body adiposity. However, female sex is associated with higher leptin levels, independent of adiposity. The studies conducted to date have used far fewer measures of leptin dynamics and are limited by infrequent sampling. Several studies, however, have indicated that for the same level of adiposity, leptin levels are higher in females than in males in both rodents and humans. It has been demonstrated that leptin levels reflect total body lipid content, as assessed by carcass analysis, in normal and transgenic mice across a broad range of body lipid content; importantly, at any body fat content, female mice have higher leptin levels than males (24). Similarly, in humans, leptin levels are a direct function of adiposity, with considerable individual variation. Four large studies with a combined number of 654 individuals have recently reported that women have higher fasting leptin levels than men independent of BMI, adiposity, and body fat composition (26, 27, 28, 44). Likewise, studies in 1691 children have shown sex dimorphism of fasting plasma leptin concentrations independent of body fat distribution. In 112 lean and obese children with an age range of 9.6 ± 0.5 to 14 ± 0 yr, Lahlou and colleagues (29) observed a strong correlation between leptin levels and BMI, but for any level of normal or elevated BMI, leptin concentrations were higher in girls than in boys. Likewise, the study by Hassink et al. of 77 normal weight and obese children (mean age, 11.3 yr) (45), the study by Blum et al. of 713 children (312 boys and 401 girls; age range, 5.8–19.9 yr) (46), and the study by Garcia-Mayor et al. of 789 children (343 girls and 446 boys; age range, 5–15 yr) (47) showed that compared with boys, girls have increased leptin concentrations independent of adiposity.

Interestingly, in newborns, leptin levels measured in cord blood are 63–90% higher in girls than in boys despite similar leptin concentrations in the plasma of mothers who gave birth to boys (18.1 ± 1.7 µg/L) or to girls (19.5 ± 2.3 µg/L) (30, 48): such sex differences in fetal leptin levels are less likely to be due either to body fat content or distribution or to reproductive hormone status and may reflect genetic differences between females and males. However, it must be noted that an independent study could not replicate the observed sex dimorphism in leptin levels in newborns (49). In all of these studies, both females and males exhibited considerable variation in leptin levels as a function of adiposity or BMI. This finding in light of the narrow BMI range of our subjects explains why in our study we could not detect a direct relation between leptin concentrations and normal BMI.

Recently, Saad et al. conducted a 24-h study with sampling every 20 min for measurements of glucose, insulin, and leptin in lean and obese males and females (50). In that study, plasma leptin profiles were higher in obese than in lean subjects and higher in women than in men regardless of fat mass. Leptin showed diurnal rhythmicity, with peaks between 2200–0300 h (median, 0120 h) and nadirs between 0800–1740 h (median, 10:33 h). The relative diurnal amplitude also was higher in men than in women, controlling for adiposity. The main difference between that study and the present one is that we sampled 3 times as often during the 24-h period, examining only leptin. As shown in Fig. 7Go, based on analysis of surrogate datasets, we estimated that sampling at intervals longer than 7 min would substantially decrease the ability to detect leptin pulsatility. Thus, in our study, with sampling at 7-min intervals we showed 30 ± 1 pulses/24 h, whereas Saad et al. showed only 3.6 ± 0.3 pulses/24 h due to sampling that was one third as frequent as that in our study. In their study, pulse frequency correlated negatively with fat mass and insulinemia, possibly indicating that obesity is associated not only with higher leptin levels but also with blunted diurnal excursions and dampened pulsatility. However, it must be noted that such an association might be an artifact of infrequent sampling. Thus, future studies employing intensive sampling methods should be undertaken to confirm the association among insulinemia, obesity, and patterns of leptin pulsatility.

We show that within a normal range of BMI, women, as previously reported (24, 25, 26, 27, 28, 29, 30, 44, 46, 47, 48, 50), have higher leptin levels than men, and we find that this difference is due solely to higher leptin pulse amplitude in women. All other concentration-independent and frequency-related pulsatility parameters as well as diurnal variation and ApEn were the same in women and men. Elevations in circulating levels of leptin have been reported to result from accelerated secretion rates of the peptide from adipose tissue due to increased leptin gene expression (51). Those findings would lead us to suggest that individual bursts of leptin may contain more leptin molecules in women than in men. Assuming similar plasma leptin half-lives in women and men, we infer that there may be sex dimorphism in the production of leptin; to maintain normal body weight women appear to require a higher leptin output per pulse. This would indicate that women may be more resistant to the actions of leptin than men. A careful review of all existing data on sex dimorphism in leptin concentrations indicates that genetic factors may be of importance, independent of body fat composition and hormonal milieu (27, 29, 52). Thus, the sex differences in circulating plasma leptin pulse amplitude, resulting in higher 24-h leptin concentrations in women than in men without alterations in oscillator mechanisms and the orderliness (ApEn) of the leptin secretion process could be attributed to the combined effects of genetics, body fat distribution, and sex steroid levels.

Additional factors contribute to regulate human plasma leptin concentrations over a 24-h period. First, the nocturnal rise of plasma leptin concentrations does not occur and leptin levels decrease progressively if the subjects are fasted; the decrease in leptin during fasting can be prevented by glucose infusion, which reverses the decreases in plasma glucose and insulin that occur during fasting (53). Schoeller et al. (54) have shown in a simulated jet-lag study that the nocturnal rise in plasma leptin concentrations is shifted forward by 12 h after day-night reversal, whereas the rhythm of circulating cortisol, which has a true circadian pattern, was unchanged over this short period of time. These investigators also showed that a 6.5-h forward shift in the time meals were consumed shifted the leptin peak forward by an equivalent amount of time, indicating that leptin rhythmicity is related to meal timing rather than the light-dark cycle. Additionally, food intake might affect leptin secretion through increases in blood glucose and insulin secretion. In this regard, Laughlin and Yen have shown that the nocturnal rise in leptin is proportional to the insulin response to meals (17), and Mueller et al. have shown that adipocyte glucose metabolism is involved in insulin-mediated leptin production (18).

Anorexia nervosa, bulimia nervosa, major depression, binge eating disorder, and obesity are disorders characterized by abnormalities in the regulation of food intake and body weight. Those disorders are more prevalent in women. The female to male ratio is 10:1 for anorexia nervosa (55), 6:1 for bulimia nervosa (56), 5:2 for major depression (57), 3:2 for binge eating disorder (58), and 11.3:10 for obesity (59). Our results show that women and men have the same 24-h diurnal periodicity in leptin levels, with the same concentration-independent and frequency-related leptin pulse parameters, including ApEn; however, women have higher leptin pulse amplitude than men, resulting in circulating plasma leptin levels more than twice those in men throughout the 24-h period. Our data may indicate that women require twice as high circulating leptin levels to maintain normal body weight and are therefore relatively resistant to the effects of leptin compared to men. Leptin is an important regulator of body weight in humans (19). The hypothesis that the relative leptin resistance exhibited by women might contribute to their increased susceptibility to disorders whose pathophysiology involves dysregulation of food intake and body weight should be explored in further studies.


   Acknowledgments
 
We are very grateful to Anna Esposito, Jonathan Gold, Pooja Khatri, Ramesh Kumar, Rachel Lisman, Zeba Qaadri, Amer Al-Shekhlee, and Fuyiu A. Yip for their outstanding technical assistance. This study could not have been conducted without the expertise of the 4W nursing staff, NIH Clinical Center.


   Footnotes
 
1 This work was supported by grants from the NIDDK, NIH (to J.S.F.); the NIA, NIH (1R01–147991; to J.D.V.); and National Alliance for Research on Schizophrenia and Depression (to M.-L.W.). Back

2 Supported by the Division of Endocrinology, Beth Israel Deaconess Medical Center, and the Harvard-MIT Clinical Investigators Training Program in collaboration with Pfizer, Inc.. Back

Received February 10, 1998.

Revised June 10, 1998.

Revised July 30, 1998.

Accepted August 10, 1998.


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

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