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Original Studies |
Departments of Pediatrics and Physiology, University of Turku (M.B.), FIN-20520 Turku, Finland; Endocrinology Section, Medicine Service, Salem Veterans Affairs Medical Center (A.I.), Salem, Virginia 24513; and Division of Endocrinology and Metabolism and the NSF Center for Biological Timing, University of Virginia Health Sciences Center (W.S.E., J.D.V.), Charlottesville, Virginia 22908
Address correspondence and requests for reprints to: J. D. Veldhuis, Division of Endocrinology and Metabolism, Department of Internal Medicine, Box 202 McKim Hall, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908. E-mail: JDV{at}Virginia.edu
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Abstract |
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In summary, short-term (2.5-day) fasting profoundly suppresses 24-h serum leptin concentrations and pulsatile leptin release in the sex steroid-sufficient midluteal phase of healthy women via mechanisms that selectively attenuate leptin pulse area and incremental amplitude. In contrast, the pulse-generating, nyctohemeral phase-determining, and entropy-control mechanisms that govern 24-h leptin release are not altered by acute nutrient restriction at this menstrual phase. Leptin-GH (but not leptin-LH) showed nutrient-dependent positive (fed) and negative (fasting) cross-correlations. Whether similar neuroendocrine mechanisms supervise altered leptin signaling during short-term nutrient restriction in men, children, or postmenopausal women is not known.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The hormonal product of the OB gene, leptin, is a signal of nutritional
status and also a regulator of reproduction in the rodent. In adult
animals, leptin administration corrects the sterility defect in
homozygous obese (OB/OB) female mice (9) and reverses hypogonadism in
mice starved for 2 days (10). Licinio et al. (11, 12) and
others (13, 14) have described significant ultradian and diurnal
fluctuatious in leptin concentrations in humans. Riad-Gabriel et
al. (15) further observed that leptin concentrations rise by
50% in the late follicular and luteal phases of the menstrual
cycle. In addition, a gender difference exists wherein the 24-h plasma
leptin concentration profile is more than 2-fold higher in women
studied in the mid-to-late follicular phase of the menstrual cycle than
in young healthy normal-weight men (12).
To our knowledge, there are no studies of pulsatile leptin secretion in fasting young women evaluated in the midluteal phase of the normal menstrual cycle. This neuroendocrine interval after ovulation is of reproductive significance because it serves critically to prepare the endometrium for blastocyst implantation. Short-term fasting is sufficient to investigate the nutritional regulation of leptin secretion because leptin concentrations decline steadily during the first 12 h of fasting, reaching a nadir after 36 h in lean and obese subjects (16). Indeed, in one study, serum leptin concentrations fell to 65% and 70% of basal (fed) values after, respectively, 10 and 20 h of fasting in lean and obese humans and then plateaued during the remaining 52-h fasting period (17).
In the present study, we investigated the effects of short-term (2.5-day) fasting on frequently sampled 24-h leptin release in healthy young women in the midluteal phase of the menstrual cycle. We used model-free Cluster analysis to quantify the pulsatile mode of leptin release (18). In complementary assessments of the nonpulsatile features of leptin secretion, we applied cosinor analysis to quantitate the nyctohemeral rhythmicity of leptin and used the approximate entropy (ApEn) statistic to estimate the pattern orderliness of the leptin release process (19, 20, 21).
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Subjects and Methods |
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Eight young healthy women within ±25% of normal body weight (body mass indices 21 ± 1.3 kg/m2) and age 21–28 yr were studied after provision of written informed consent approved by the Human Investigation Committee of the University of Virginia. Each woman was a nonsmoker, was not taking birth control pills or other medications, had not undertaken transmeridian travel of three or more time zones in at least 2 weeks, and had an unremarkable clinical history and physical examination. Volunteers had normal adult sexual maturation, regular (28 ± 4 days) menstrual cycles, normal screening biochemical tests of renal, hepatic, metabolic, and hematologic function, and normal fasting serum concentrations of total and free T4, TSH, GH, PRL, estradiol, immunoreactive LH and FSH, and insulin-like growth factor-I (IGF-I). The secretion of GH and LH in this paradigm was described earlier (present study).
Subjects were admitted to the General Clinical Research Center of the University of Virginia during the midluteal phase of their menstrual cycles (see below) on the night before blood sampling in the fed state, and again before the 2.5-day fasting session. Ovulation was documented during each study cycle by the development of a normal preovulatory follicle (mean diameter, 20 mm; range, 16–26 mm), followed by its disappearance, as characterized using daily or alternate-day transvaginal ovarian ultrasonography. The fed and fasting admissions were assigned on days 5–8 after ovulation and in randomized order, at least 1 month apart.
In both the fed and fasted states, blood sampling was carried out at 10-min intervals for 24 h beginning at 0800 h at least 1 h after initial venipuncture. In the fasting admission, blood sampling was performed during hours 32–56 (i.e. beginning 11/3 days into the fast). Blood was withdrawn through an iv catheter placed in a forearm vein, and samples were allowed to clot at room temperature. The subsequent sera were frozen at -20 C for later assays. Subjects remained in bed or a chair during sampling, except for bathroom privileges. In the fed state, three isocaloric meals were given per day (at 0800, 1200, and 1800 h). During the 2.5-day fast, the volunteers received caffeine- and calorie-free liquids, potassium chloride (40 meq) and water-soluble vitamins only, slept in the Clinical Research Center, and had urinary ketones monitored daily to corroborate compliance with the fast.
Assays
Serum leptin concentrations were measured by RIA, as described previously (12). The assay sensitivity is 0.2 µg/L, and the median inter- and intraassay coefficients of variation were less than 9% and 6%, respectively. All 145 samples in each admission were assayed together. Serum concentrations of GH, LH, FSH, estradiol, progesterone, PRL, IGF-I, cortisol, and insulin were reported earlier from 24-h pools of serum (present study), as determined by RIA, chemiluminescence, or immunoradiometric assay (3, 7, 19, 22, 23, 24).
Cluster analysis
Cluster, a largely model-free computerized peak-detection algorithm, was used to identify statistically significant pulses in relation to dose-dependent measurement error in serum leptin concentration vs. time series (25). Samples obtained at 10-min intervals over the 24-h period were used to assess mean 24-h leptin levels, leptin pulse frequency (number of significant peaks/24 h), interpulse interval (time separating consecutive peak maxima), peak duration in minutes, height (maximal hormone concentration in a peak), amplitude (as percentage of increase over preceding baseline; 100% corresponds to preceding nadir), area (peak mass), and increment (increase above nadir), along with interpulse valley mean and nadir concentrations. The variance model used in Cluster analysis was the between-replicate SD expressed as a power function of dose. Test cluster sizes were 2 x 2 in the moving nadir and peak with t = 2.0 as the significance level for both test upstrokes and downstrokes in the data (18). Cluster analysis was validated to quantitate the pulsatile properties of leptin release in earlier studies (11, 12, 26).
Nyctohemeral (24-h) rhythmicity
Diurnal rhythms of serum leptin concentrations were appraised using cosinor analysis, as described previously (19, 27).
ApEn
ApEn was used as a scale- and model-independent statistic to quantify the serial orderliness or regularity of leptin release over 24 h. Normalized ApEn parameters of m = 1 and r = 20% of the intraseries SD were used, as described previously (20, 21). For this parameter set, ApEn is, hence, designated ApEn (1, 20%). ApEn quantifies the regularity of subordinate (nonpulsatile) patterns in the data and, as such, yields information complementary to either cosinor or peak analyses. Cross-ApEn evaluates relative orderliness between two series (21). Higher absolute ApEn values denote greater disorderliness or irregularity of neurohormone release, as observed in acromegaly (28), Cushing’s disease (29, 30), aldosteronoma (31), the aging LH (21), GH (32), and insulin (33) axes, and for the GH axis in women compared with men (20).
Statistical analyses
Due to non-Gaussian distributions, differences between fed and fasting measures of pulsatile or rhythmic leptin release were assessed after logarithmic transformation using a paired two-tailed Student’s t test. Mean (24-h) concentration values were compared via paired two-tailed Student’s t tests. Identical inferences were obtained with the (nonparametric) Kolmogorov-Smirnov statistic using untransformed data. Results are presented as the mean ± SEM (and median). Statistical significance was accepted for P less than 0.05. Linear regression and cross-correlation were used to test for significant linear and colinear (synchronous) relationships (34).
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Results |
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The 2.5-day fast resulted in a 4.6-fold decrease in mean 24-h
serum leptin concentrations [15.2 ± 2.3 (median, 15.5)
vs. 3.4 ± 0.56 µg/L (median, 3.5), P
= 0.0007, Fig. 1
]. None of the 145 serum
leptin concentrations in any of the eight women was undetectable.
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Figure 2 illustrates the 24-h
profiles of serum leptin concentrations, which were pulsatile by visual
inspection in both the fed and fasting states. Cluster was used to
resolve leptin pulses in women studied in the midluteal phase of the
menstrual cycle. The quantitative attributes of
(Cluster-estimated) leptin release are summarized in Table 1. The number of significant leptin
pulses was not changed in response to a 2.5-day fast; viz.,
14 ± 1.1 (median, 14) in the fasting state and 14 ± 1.1
(median, 14) leptin peaks per 24 h during the fed study
(P = NS). The mean leptin interpeak interval averaged
89 ± 5.4 (median, 91) in the fed and 92 ± 5.3 (median, 96)
min in the fasting session (P = NS; Table 1). On the
other hand, the calculated incremented area of leptin peaks (integrated
peak above flanking nadirs) decreased significantly from 155 ± 21
(median, 154) in the fed session to 37 ± 7.0 (median, 32)
µg/Lxmin in the fasting environment (P = 0.0004; Fig. 3A). There was a parallel diminution in
leptin peak amplitude (maximal height of pulse) in midluteal phase
women during fasting [i.e. fed 18.3 ± 2.8 (median,
18.5) vs. fasting 4.6 ± 0.68 (median, 4.1) µg/L,
P = 0.0008 (Fig. 3B)], as well as leptin pulse
increment (algebraic increase above preceding nadir) from 4.4 ±
0.7 (median, 4.5) in the fed to 1.0 ± 0.1 (median, 1.0) µg/L in
the fasting state (P = 0.0011; Fig. 3C). The fractional
leptin peak amplitude (percentage of increase above nadir) remained
unchanged during fasting (P = NS, Table 1), reflecting
the equivalent decline in interpeak valley (and nadir) values.
Specifically, the 2.5-day fast resulted in a 74% and 75% decrease in
the interpulse valley mean [from 14.5 ± 2.3 (median, 14.4) to
3.7 ± 0.57 (median, 3.3) µg/L; P = 0.0009; Fig. 3D] and in the interpulse valley nadir [from 13.1 ± 2.0
(median, 12.9) vs. 3.3 ± 0.5 (median, 3.0) µg/L;
P = 0.0009].
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The 24-h variation(s) in serum leptin concentrations are
summarized in Table 2. Maximal
nyctohemeral serum leptin concentrations occurred between 2310 and 0036
clocktime (95% confidence intervals) in the fed state and between 1642
and 2234 in the fasting state (P = NS). Fasting-induced
decreases in the mesor (average value about which the diurnal rhythm
oscillates); namely, fed 14.6 ± 2.2 vs. fasting
3.7 ± 0.52 µg/L (P = 0.0007 compared with
values in the fed state), and the amplitude (one-half the absolute
difference between the nadir and peak value); viz., fed
3.2 ± 0.85 vs. fasting 0.8 ± 0.3 µg/L
(P = 0.043). Individual mesor data are given in
Fig. 4.
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ApEn averaged 1.406 ± 0.083 (median, 1.393) during the fed
admission and 1.052 ± 0.224 (median, 1.172) during the fasting
period (P = NS; Table 1
). Cross-ApEn values were not
significant for fed vs. fasting leptin and GH, or leptin and
LH joint synchrony.
Correlations of leptin with other hormones
Serum mean (24-h) leptin concentrations correlated negatively with FSH in the fed (r = -0.765, P = 0.027), but not fasting (r = 0.438, P = NS), study. No other correlations were evident in either the fed or fasting states for leptin vs. LH, FSH, PRL, cortisol, IGF-I, insulin, estradiol, or progesterone in pooled sera. Cross-correlation was significantly positive for leptin-GH in the fed state and negative for leptin-GH during fasting with maxima at -40 and +10-min lags, respectively.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Several authors have reported menstrual cycle differences in leptin release with higher values in the late follicular and especially luteal phases (15, 35, 36). Comparing the present data with those of Licinio et al. (namely, luteal vs. follicular phases) (12) would also support this distinction. A second difference is the lower leptin pulse frequency in the luteal phase, compared with peak frequency reported in the late follicular phase of fed young women (11). Possible mechanistic explanations for differences in leptin release across the menstrual cycle include variations in caloric intake, given the sensitivity of leptin to short-term fasting (present study and Refs. 16, 17) and acute overfeeding (37). Alternatively, hypothalamic modulatory mechanisms may be important. Full-length leptin receptors are present in the hypothalamus (38, 39), and leptin administration can inhibit the expression of NPY (37), stimulate TRH release (38), inhibit somatostatin secretion (40), modulate synaptic transmission (39), and enhance gonadotropin secretion possibly by joint hypothalamo-pituitary actions (10, 40, 41). Leptin receptors and defects are also demonstrable in reproductive organs, including the ovary (42, 43). Indeed, we cannot exclude a contribution to plasma leptin elevations in the midluteal phase from nonadipose sites, since both granulosa and cumulus cells express leptin messenger RNA and protein (42).
Leptin concentration-dependent pulse parameters, such as incremental and maximal peak heights and interpulse (nadir) leptin levels, explained earlier gender differences (i.e. lower leptin values in men) (12). Analogously, here short-term fasting suppressed these (frequency-independent) measures. This finding agrees with a pilot study of one obese and one normal-weight woman (11). Nonetheless, the exact mechanisms driving pulsatile leptin release and its amplitude-modulation by fasting remain incompletely defined (44).
We earlier observed that short-term fasting in the mid-luteal phase of the menstrual cycle suppresses 24-h serum insulin and IGF-I concentrations, augments cortisol and GH secretion, and fails to alter serum LH, FSH, estradiol, or progesterone concentrations (present study). Indeed, cortisol and leptin production tend to vary inversely (11). Here, we assessed the synchrony between moment-to-moment leptin and LH, and leptin and GH, release in the fed and fasting state. Nutritionally replete vs. deplete women showed, respectively, positive and negative cross-correlations between leptin and GH (but no synchrony of leptin and LH) release in the midluteal phase. In the fasted male rat, leptin infusions stimulate otherwise suppressed GH release by inhibiting hypothalamic somatostatin expression. Here, fasting women exhibited a negative leptin-GH association, and fed women a positive leptin-GH relationship, across the 24-h sampling session. A negative correlation between leptin and GH has also been recognized in sarcopenic postmenopausal women (45) and middle-aged men and women (46). The putative hypothalamic mechanisms that underlie these bihormonal relationships are not yet known in the human, but in the rat may involve neuropeptide Y (47). The present short-term fasting paradigm may provide one context, in which to explore relevant mechanisms further.
By sampling both leptin and LH every 7 min for 24 h in fed mid-to-late follicular phase women, Licinio et al. (26) recently observed an inverse correlation between sample-by-sample LH and leptin release. Here, in group analyses, we found no leptin-LH correlations but that mean daily serum leptin concentrations correlated negatively with (pooled 24-h) serum FSH concentrations in fed, but not fasting, midluteal phase women. How fasting might disrupt the latter negative relationship between leptin and FSH release in midluteal phase women is not known.
Short-term fasting significantly decreased nyctohemeral rhythmicity of leptin release in the young midluteal phase women. This finding is concordant with that of Boden et al. (48), who did not observe any nocturnal rise in plasma leptin concentrations in fasting subjects. The latter study evaluated five normal-weight (2 male, 3 female volunteers) and five obese subjects (2 males, 3 females) with less frequent blood sampling (at 2-h intervals). Our use of 10-min blood sampling in eight women assessed at a single menstrual-cycle-stage further establishes the stability of the leptin acrophase (timing of maximal nyctohemeral serum leptin concentration) in the face of fasting. This observation suggests preservation of the phase-determining mechanisms of leptin production in fasting; e.g. circadian and/or sleep-entrained leptin secretion (36). Although sleep stages were not monitored here, our determination of the mean timing of the (fed) diurnal leptin peak at 2130 clocktime agrees with two earlier estimates (11, 13). Sinha et al. suggested that the nocturnal rise in leptin secretion in humans may be related to appetite suppression during sleep (13). The 24-h rhythm in leptin release can also be modulated by the sleep-wake activity cycle, since Schoeller et al. (49) observed a 12-h phase-shift after day-night reversal in a simulated jet-lag study, whereas the rhythm in the circulating cortisol concentration, which maintains a true circadian dependence, persisted.
Fasting did not affect the orderliness or regularity of leptin release patterns, as analyzed by the approximate entropy statistic. To our knowledge, by way of possible comparison, there are no studies of the orderliness of leptin release in fasting young women evaluated at other stages of the menstrual cycle. However, Licinio et al. (25) demonstrated significant synchrony in the fluctuations of leptin and LH (or estradiol) as determined by cross-approximate entropy in healthy young midfollicular-phase women, and suggested notion of temporal coupling between activities of the leptin and the reproductive axes.
In conclusion, fasting suppresses pulsatile leptin release profoundly (by 4.6-fold) in the sex-steroid sufficient midluteal phase of the menstrual cycle in healthy women. This adaptive response is achieved via mechanisms that selectively attenuate leptin pulse area and its incremental amplitude. The frequency-generating, phase-determining, and entropy-control properties of leptin regulation are not altered by acute nutrient restriction. Whether similar dynamic mechanisms subserve altered leptin signaling in food-deprived men, children, or postmenopausal women is not known.
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Acknowledgments |
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Footnotes |
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Received March 25, 1999.
Revised July 2, 1999.
Accepted October 7, 1999.
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