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Original Article |
Department of Cell Biology & Physiology (M.L.B.-G., A.S., T.M.P.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; and Department of Physical Therapy (C.R.P.), School of Health Sciences, Duquesne University, Pittsburgh, Pennsylvania 15282
Address all correspondence and requests for reprints to: Dr. Tony M. Plant, Department of Cell Biology & Physiology, University of Pittsburgh School of Medicine, S-828A Scaife Hall, Pittsburgh, Pennsylvania 15261. E-mail: plant1{at}pitt.edu.
Abstract
The purpose of this study was to examine the hypothesis that the pubertal reaugmentation of pulsatile GnRH release in male primates is triggered by a rise in circulating leptin concentrations. Agonadal juvenile male rhesus monkeys (n = 7) were implanted with indwelling venous catheters and housed in specialized cages that allow continuous access to the venous circulation. GnRH release was monitored indirectly using LH secretion from the in situ pituitary sensitized to the LH releasing action of GnRH as a bioassay for the hypothalamic peptide. Infusion of recombinant human leptin (5 µg/kg body weight ·h for 16 d resulted in a marked square wave increment in circulating leptin concentration from approximately 2–20 ng/ml but did not elicit precocious GnRH release. GH secretion, however, was stimulated confirming that the heterologous leptin preparation was bioactive in the monkey. Parenthetically, recombinant human leptin was found to be immunogenic in the monkey and circulating antileptin IgG was demonstrable 22–35 d after the initial exposure to the human protein. These findings further support the view that circulating leptin is unlikely to provide the signal that triggers the onset of puberty in male primates.
IN HIGHER PRIMATES, INCLUDING man, the pubertal activation of the pituitary-gonadal axis is triggered by a reaugmentation in hypothalamic GnRH secretion as the juvenile phase of development wanes (1). The cue that times this fundamental developmental event in the primate brain has been of interest for many years but remains enigmatic. A view posited by some is that this hypothalamic activation is timed by an increase in circulating leptin concentrations (2, 3). This notion, as it relates to primates, has been based, in part, on the report in boys of an increase in plasma leptin concentrations preceding the pubertal rise in nocturnal testosterone secretion (4). Although analogous studies by this laboratory have failed to demonstrate such a correlation in the male rhesus monkey (5), we have been haunted by the idea that leptin may provide a signal for the onset of primate puberty for the following reasons. First, in the male rhesus monkey, the pubertal reaugmentation in GnRH pulsatility is temporally correlated with a decline in both expression of the gene encoding neuropeptide Y (NPY) and the content of this peptide in the mediobasal hypothalamus (6). Second, injection of NPY into either the lateral or third cerebroventricle of the postpubertal monkey inhibits GnRH and LH release (7, 8, 9). Third, studies of rodents have demonstrated that leptin inhibits NPY gene expression, NPY secretion, and NPY action (10, 11, 12, 13, 14, 15). Lastly, NPY neurons in the mediobasal hypothalamus of the monkey (16), as in other species (17, 18, 19, 20), express leptin receptors.
For the foregoing reasons, we sought to test the hypothesis that circulating leptin may provide the cue to the brain for initiating the pubertal increase in GnRH release in male primates by using a direct approach. That was to determine whether chronic iv administration of recombinant (r) human (hu) leptin to juvenile male monkeys results in a precocious activation of the pubertal mode of GnRH release, as measured indirectly by monitoring LH secretion. Because leptin has been shown to exert a direct effect on the testis of rat (21, 22), agonadal monkeys were used in the present study. Here it is to be noted that, in the rhesus monkey, the pubertal reaugmentation of pulsatile GnRH secretion occurs in the absence of the testis (1) and, therefore, the agonadal paradigm is an appropriate model to address the present question.
As the suppressive effect of leptin on food intake is well known (23, 24, 25, 26), attempts were made in initial experiments to measure food consumption to establish the bioactivity of the heterologous hormone. In addition, stimulatory effects of leptin on GH secretion have been demonstrated in various mammals (27, 28, 29, 30, 31), and therefore plasma GH concentrations were also monitored in the present study.
Materials and Methods
Animals
Seven male rhesus monkeys (Macaca mulatta) ranging in age from 16–20 months and weighing between 3 and 3.5 kg were studied. All monkeys were born at the Primate Research Laboratory of the Center for Research in Reproductive Physiology at the University of Pittsburgh School of Medicine. The monkeys were maintained under a 12-h light, 12-h dark cycle with lights on at 0700 h, and each morning fed approximately 30 pellets of a commercial monkey chow (Lab Diet 5045; PMI International Inc., Brentwood, MO), together with seeds and fruit. Additional fruit was given in the afternoon, and water was provided ad libitum. Before initiating the experiments, the monkeys were first adapted to remote-infusion cages and to wearing a jacket and tether to permit continuous access to the venous circulation at the time of experimentation (32). Food intake was monitored daily. Initially, attempts were made to precisely quantitate food consumption recording the number of pellets fed and the number remaining immediately before the next feed. This proved to be unreliable, however, as pellets were discarded by the monkeys to be recovered the following morning as fragments in the bottom of the cage mixed with stool and urine soaked sawdust. The difficulty in analyzing food intake by rhesus monkeys has been previously noted (33). Therefore, attempts to precisely monitor food intake were abandoned, and other parameters of leptin action were considered. Body weight (BW) was generally obtained on a weekly basis. All experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee.
Surgery
Surgical procedures were generally conducted after sedation with ketamine hydrochloride (20 mg/kg BW iv; Phoenix Pharmaceuticals, Inc., St. Joseph, MO) followed by inhalation anesthesia (1.5–2.5% isoflurane in oxygen), unless stated otherwise. During surgery, plasma pO2, heart rate, respiratory rate, and body temperature were monitored.
Chronic-indwelling venous catheters, which were made from medical grade SILASTIC brand tubing (Dow Corning, Midland, MI) (0.132'' x 0.183'', inner diameter x outer diameter; Tri-Animal Health, Youngwood, PA), were implanted into the internal jugular vein and into the femoral vein as previously described (32), between 14 and 19 months of age. The catheters were tunneled sc to the back and exteriorized through a small fistula in the subscapular region. The monkey was then refitted with its jacket and the catheters fed through the stainless steel flexible tether and connected to a three channel fluid swivel device (Alice King Chatham Medical Arts, Hawthorne, CA). The monkey, fitted with its jacket and tether, was then returned to its cage and the swivel located in position on the cage top. Additional tubing was attached to the ports of the swivel and led through the wall into an adjacent laboratory for remote blood sampling and infusion.
Bilateral castration was usually performed at the time that iv catheters were implanted, as described previously (32). However, in two monkeys this was conducted 3 wk after catheterization, and in one monkey, this procedure was conducted at 4 months of age. In the latter case, surgery was conducted under anesthesia with ketamine hydrochloride (20 mg/kg BW).
Postoperative care
After placement of iv catheters and after castration of monkeys bearing an iv line, the animals were returned to their remote sampling cages, and body temperature was maintained with an infrared lamp until consciousness was regained. Animals were treated prophylactically with penicillin (300,000 U im; Phoenix Pharmaceuticals, Inc.) for 1 d, and given cephalosporin (100 mg, iv; Cefazolin; Apothecon, Princeton, NJ) and meperidine hydrochloride (0.6 mg, iv; Demerol; Abbott Laboratories, Chicago, IL) twice daily for 4 d. Monkeys were infused iv with normal saline for the first 24 h postoperatively, and after this period lines were kept patent with heparinized saline (4 U/ml).
The monkey that was castrated at 4 months of age was given an im injection of penicillin (300,000 U) and returned to its mother when it had regained consciousness.
Hormones
GnRH. GnRH was obtained from the Contraceptive Development Branch, National Institutes of Health (Bethesda, MD). A stock solution (1 mg/ml) was prepared as previously described (34). The GnRH stock was diluted with normal saline to give a working concentration of 0.3 µg/ml, which was kept at -20 C until needed.
Leptin.
Lyophilized rhu leptin was obtained from the National Hormone and Peptide Program (Lot AFP 496C; Los Angeles, CA) or R&D Systems (398-LP; Minneapolis, MN). The leptin infusates were custom prepared for each animal to deliver 5 µg/kg BW·/h. In brief, leptin was first dissolved in 0.01 M Na2CO3 to make a stock solution containing approximately 10 µg/µl. For a given monkey, an appropriate amount of the leptin stock solution was then further diluted with saline to achieve a leptin concentration of 15–19 µg/ml. Additionally, the infusates contained the animals’ own serum (
1%) and a broad spectrum antibiotic (Cefazolin, 0.1 µg/ml). The vehicle infusates were also custom made in a similar manner. Leptin and vehicle infusates were filtered through a 0.22-µm filter before administration.
Experimental preparation
The agonadal prepubertal monkey, in which pituitary responsivity to GnRH had been previously heightened with an intermittent iv infusion of the synthetic decapeptide, was employed. Before the start of the experiments, each monkey received an intermittent iv infusion of exogenous GnRH (0.1 µg/min for 3 min once every hour) for approximately 3 wk to sensitize the pituitary to GnRH, as described earlier (32). In the majority of animals, pulsatile GnRH treatment was initiated following castration. In two of the monkeys, however, this was conducted before orchidectomy. The pituitary was considered to be adequately sensitized to GnRH either when circulating LH values exceeded 2 ng/ml in the castrate males, or when testosterone exceeded 4 ng/ml in the two intact monkeys. Circulating LH and testosterone concentrations in monkeys aged between 14 and 19 months are generally less than 0.15 ng/ml and less than 0.5 ng/ml, respectively. The iv administration of synthetic GnRH was discontinued 5–7 d before initiating the leptin or vehicle infusion to allow circulating LH levels to decline to low levels while maintaining pituitary responsivity to GnRH. To restore pituitary responsivity between leptin and vehicle infusions, the GnRH priming was reestablished for periods of 9–11 d.
Experimental protocol
A continuous iv infusion of rhu leptin (5 µg/kg BW·h), or vehicle, administered at a rate of 1 ml/h was initiated on d 1 and maintained for 16–22 d. A leptin exposure of 3 wk was selected in the first place because the pubertal reaugmentation of pulsatile GnRH release in the agonadal male monkey occurs over this time frame (32). Three monkeys received leptin followed by vehicle, whereas in the other four animals the order was reversed. Because the pubertal increase in LH release in the rhesus monkey is first observed at night (35), nocturnal blood samples were collected every 20 min for 3 h between 2100–2400 h before initiation of the infusions, and on d 1, 2, 4, 8, and 16 thereafter. Red blood cells were returned to the animals at the end of the collection period. In monkeys that received infusions for 22 d, GnRH priming was reinitiated on d 17, 18, and 19, to maintain pituitary responsivity for the duration of the leptin or vehicle infusion, and a final series of nocturnal samples was collected on d 22. The response of the pituitary to an iv bolus of GnRH (0.3 µg) was tested in all animals on d 9 and after termination of the leptin or vehicle infusion. In those animals infused for 22 d, GnRH responsivity was also tested on d 17. For this purpose, a blood sample was withdrawn immediately before injection of GnRH and again at 10–15 min after the injection. Responsivity was defined as the difference between these two values. Following termination of the infusions, blood samples were collected every hour for 6 h and thereafter, in some animals, less frequently for up to 14 d to monitor the post-infusion changes in circulating leptin concentrations. A minimum of 12 d was allowed to elapse between the end of a leptin or vehicle infusion and the initiation of the next infusion.
Assays
LH. Circulating LH levels were assessed by a double antibody RIA using recombinant cynomolgus monkey LH (AFP 6936A) for standard and radioiodinated trace, and a polyclonal rabbit antiserum (AFP 342994) raised against the recombinant LH as first antibody. The mean assay sensitivity (n = 14) was 0.11 ng/ml, and the average ED50 was 1.1 ng/ml. For three volumes of a standard pool of monkey serum (25, 50, 100 µl), the intraassay coefficients of variation (CV) were 6.5%, 4.6%, and 3.9% at 71%, 47%, and 25% binding, respectively. The corresponding interassay CV were 12%, 8.3%, and 9.5%.
Leptin. Leptin was assayed in 100-µl plasma aliquots with a commercially available primate leptin RIA kit (Linco Research, Inc., St. Charles, MO), which employs a primary antibody raised against hu leptin in rabbits, iodinated hu leptin as trace, and hu leptin as standard. The assay, which displays 100% cross-reactivity with primate leptin, has been previously validated for the measurement of monkey leptin in plasma (5). The mean assay sensitivity (n = 8) was 0.24 ng/ml. The average ED50 was 4.5 ng/ml. Using two quality control pools provided with the kit (3.63 ng/ml, and 21 ng/ml), the intraassay CV was 13.1% and 4.3% at 55.5% and 17.4% binding, respectively. The corresponding interassay CV were 9.5% and 9.8%.
GH.
Plasma GH was assessed with a commercially available double antibody huGH kit (Diagnostic Products Corp., Los Angeles, CA). This assay employs an antiserum directed against huGH, iodinated huGH as trace, and huGH as standard. Goat antirabbit 
-globulin-polyethylene glycol was supplied as the precipitating reagent. The mean assay sensitivity (n = 9) was 0.77 ng/ml. The average ED50 was 7.4 ng/ml. Using two serum pool standards (5.1 ng/ml and 10 ng/ml), the mean intraassay CV was 3.74% and 2.59% at 63.2% and 39.2% binding, respectively. The corresponding interassay CV were 4.9 and 4.7%, respectively.
Leptin binding activity. Leptin binding activity in monkey plasma was examined by gel filtration. For this purpose, plasma (100 µl), together with iodinated hu leptin (12,000–15,000 cpm; Linco RIA kit), was diluted 1:2 in 0.1% gel PBS. A 200-µl aliquot of the diluate was incubated overnight at 4 C, and then transferred to an 8 ml G-100 Sephadex column and eluted with 0.01 M PBS. Eluate was collected in approximately 0.5-ml fractions and counted for 2 min in a gamma counter. The elution profiles of labeled leptin incubated with plasma were compared with that obtained when labeled leptin was incubated with the rabbit anti-hu leptin antibody supplied with the Linco RIA kit.
Anti-leptin IgG. The presence of antileptin IgGs in monkey plasma was determined using protein A precipitation. For this purpose, monkey plasma (5 µl) was diluted 1:10 with 0.01 M PBS and incubated overnight at 4 C with 50 µl of iodinated hu leptin (6,000–10,000 cpm) supplied with the Linco RIA kit. After incubation, 40 µl of protein A (100 µg/µl; Sigma-Aldrich, St. Louis, MO) in 0.01 M PBS was added to the solution and gently shaken for 2–3 h at 4 C. The solution was centrifuged at 10,000 rpm for 10 min, the supernatant discarded, and the radioactivity in the pellets containing the IgG-iodinated leptin complex was counted in a gamma counter over 2 min. Aliquots of a pool of monkey serum generated for other purposes were used as a nonimmune control in all assays.
Numerical analysis
An average for circulating LH concentrations during each window of sequential sampling (10 samples) was calculated for each monkey for each time point, and these averages were used to obtain mean LH values for each time point for all animals. It is to be noted that, in one monkey (the oldest animal), a pulsatile mode of LH secretion was evident during vehicle infusion, indicating that removal of the brake that holds the GnRH pulse generator in check during prepubertal development had already been initiated. Hormonal data from this monkey were therefore excluded from numerical analysis. Differences in mean plasma LH concentrations were analyzed by a two-way ANOVA with repeated measures with treatment (vehicle vs. leptin) as the first factor and time as the second factor. Changes in plasma GH concentration were examined in an identical fashion. The significance of differences in mean pituitary responsivity to GnRH was tested using the t test. It should be noted that the response to GnRH on d 17 in two animals was excluded from analyses because of a technical error. In all tests, a probability of P < 0.05 was considered as significant.
Results
Before describing our findings on the effects of leptin administration on GnRH release in the prepubertal male rhesus monkey, it is first necessary to present an unexpected set of results that led us to the conclusion that rhu leptin is antigenic in this species. This property of rhu leptin emerged when, on completion of the 22-d infusion in the first four monkeys, the declines in circulating leptin were being described. As anticipated, when the leptin infusions were stopped, plasma levels of this hormone initially fell precipitously toward preinfusion concentrations (approximately 2 ng/ml), but within 1–5 d this decline was arrested and circulating levels of immunoactive leptin began to rebound. Elevated levels of immunoactive leptin ranging from approximately 5–30 ng/ml persisted in these four animals for the remainder of the study. Consequently, we discovered post hoc that immunoactive leptin concentrations were spuriously elevated during the infusion of vehicle in the first two animals that received leptin followed by vehicle. At the time the foregoing data were emerging, the fifth monkey was receiving a 22-d infusion of leptin.
The presence of a circulating leptin binding protein in one of the first four monkeys studied was confirmed using gel filtration. As shown in Fig. 1
, when plasma collected 11 wk after terminating leptin treatment was incubated with iodinated leptin and then chromatographed using a Sephadex G 100 column, a peak in radioactivity was eluted in fractions corresponding to those in which antibody-bound-iodinated-leptin-complexes also eluted. Subsequent experiments with protein A demonstrated that the leptin binding protein was due to IgG, and outlined the time course of the immune response (Fig. 2). Leptin binding IgG activity during the first 2 wk of the leptin infusions was indistinguishable from control values obtained with preimmune plasma. An increase in protein A precipitated IgG bound labeled leptin complex was present by d 23 in two animals, and by d 36 in the remaining two animals. In the fifth animal that received a 22-d leptin infusion, leptin binding IgG was also detected by d 22. Because of the consistent immune response observed in the first five monkeys following a 22-d exposure to rhu leptin, the duration of the infusions in the last two animals was reduced to 16 d. During these abbreviated leptin infusions, antileptin IgG levels, as revealed by protein A precipitation, were indistinguishable from those in preimmune plasma. Leptin binding IgG, however, was found in the circulation of these animals on d 24, 8 d after terminating the leptin infusion (Fig. 2). For the foregoing reasons, only results for the first 16 d of leptin and vehicle administration were subjected to numerical analysis.
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, and a composite of the results for six monkeys are shown in Fig. 4. Following initiation of a continuous iv infusion of rhu leptin (5 µg/kg BW·h) to each of six monkeys, circulating concentrations of this hormone increased from 1.6 ± 0.37 ng/ml (mean ± SE) to reach, within 12 h, a plateau of approximately 15–17 ng/ml. In spite of the marked and sustained elevation in circulating leptin levels, GnRH secretion, as reflected by plasma LH concentrations, was not stimulated. Mean plasma LH levels during d 1, 2, 4, 8, and 16 of leptin administration were similar to those at corresponding times during infusion of vehicle (P > 0.05 for all times). Moreover, mean LH levels at all times during the leptin infusion were lower than that immediately before the start of leptin treatment. A similar situation was seen during the vehicle infusion. Administration of a GnRH bolus on d 9 and 16 of the leptin and vehicle infusions elicited a robust discharge of LH. The magnitudes of the pituitary responses to GnRH during leptin infusion (2.46 ± 0.52 and 1.11 ± 0.27; d 9 and 16, respectively) was indistinguishable from that during vehicle infusion (2.32 ± 0.21 and 0.64 ± 0.13; d 9 and 16, respectively).
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). Two-factor ANOVA with repeated measures revealed a significant main effect of treatment [f 1,5 = 11.18; P = 0.02] and time [f 5,25 = 2.80; P = 0.04] and a significant interaction of treatment and time [f 5,25 = 3.97; P = 0.01]. Briefly, post hoc analysis using one factor repeated measures ANOVA to analyze treatment (vehicle vs. leptin) effects, revealed that mean plasma GH levels after leptin infusions were significantly higher than those after vehicle infusions on d 4 and 16 of the infusion (P < 0.02 and P < 0.01, respectively). Although the pulsatile mode of GH secretion was not formally analyzed, the increase in mean GH concentrations during leptin infusion appeared to reflect an increase in GH pulse amplitude.
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Discussion
Human and rhesus monkey leptin both contain 167 amino acids, and there is a 91% homology between the species (36, 37). Therefore, the finding that the continuous administration of rhu leptin to the monkey elicited a very rapid immune response resulting in the appearance of antileptin IgGs in the circulation 22–34 d after the initial exposure to leptin was not anticipated. Male rhesus monkeys have been previously treated with rhu leptin for up to 45 h (16, 26, 38), but in these earlier studies the need to screen for antileptin antibodies never arose. Interestingly, an antibody response was observed after 1–24 months administration (sc) of rhu leptin to human subjects (39, 40). The appearance of circulating leptin antibodies in these subjects, however, did not appear to influence the biological effect of leptin, as reflected by the ability of the leptin replacement to reduce BW (39, 40). Additionally, the antibodies in the patient described by Farooqi et al. (40) were reported to be nonneutralizing.
Whether the immune response to rhu leptin in the present study also generated nonneutralizing antibodies in the monkey was not studied. This may well have been the case, however, because in those animals that received leptin for 22 d, circulating GH concentrations did not appear to decline during the last week of treatment. In any event, the leptin induced increase in GH secretion demonstrated that rhu leptin was indeed biologically active in the rhesus monkey. That rhu leptin is biologically active in the macaque has been previously reported by Finn et al. (16), who administered a very high dose (134 µg/kg BW·h) of this hormone by iv infusion to fasted adult male rhesus monkeys for 45 h and observed that the fasting induced suppression of LH and FSH secretion was blocked by the leptin treatment. In another study, a suppression of feeding and a reduction in BW was observed after a 1-µg bolus injection of rhu leptin into the third cerebroventricle of the rhesus monkey (26).
While pulsatile patterns of GH secretion were not formally analyzed in the present study, the increase in GH secretion appeared to result from an increase in the amplitude of the secretory discharges of GH. Peripheral administration of leptin to fasted sheep (30) and fasted rats (41), and central administration of the hormone in rat, sheep, and swine, have been shown to increase spontaneous or GHRH induced GH secretion (27, 28, 29, 31, 42). In man, homozygous mutations in the leptin receptor gene are associated with impaired GH secretion (43), and circulating leptin levels have been shown to be positively correlated with GH secretion in children (44).
While the antigenicity of rhu leptin in the monkey led us to reduce the duration of leptin exposure in the last two animals studied from 22 to 16 d, and to restrict the formal analysis of the results to those obtained before the earliest time an immunogenic response was manifest (d 22), it seems reasonable to proceed on the assumption that the present experimental design was sufficiently robust to identify a triggering role of leptin should this, in fact, exist. In this regard, it should be noted that a sustained, adult-like hypophysiotropic drive may be immediately elicited from the GnRH neuronal network of the prepubertal monkey by intermittent stimulation with the glutamate receptor agonist, N-methyl-D-aspartate (45). The latter finding indicates that the molecular machinery necessary for GnRH release is extant in the juvenile hypothalamus and that it may be immediately activated by an appropriate stimulus. Moreover, in the absence of the testis, the spontaneous pubertal reaugmentation of pulsatile GnRH release occurs explosively (32). Lastly, in the rat, the inhibitory effects of leptin on hypothalamic NPY gene expression occur within hours (10, 12), and, in the monkey, as in the rat, physiological and behavioral responses to central leptin administration occur within 24 h (26),
Thus, the present finding that, in the agonadal juvenile monkey, a sustained 16-d period of hyperleptinemia did not elicit precocious GnRH release, as reflected indirectly by the pattern of LH secretion from the in situ pituitary sensitized to stimulation by GnRH, provides new evidence for the view that a rise in circulating leptin does not serve as the trigger that times the pubertal reaugmentation of pulsatile GnRH release in male primates. The failure to observe evidence of precocious GnRH secretion in our experimental paradigm cannot be accounted for by a loss in the responsiveness of the pituitary to GnRH during the leptin infusion because a bolus injection of 300 ng synthetic GnRH on d 16 elicited a robust LH response. This dose of GnRH has been previously shown to produce a peak circulating GnRH concentration of 200 pg/ml and to provide a physiological stimulus to the gonadotrophs of the male monkey (34). In this regard, it is interesting to note that the response of the pituitary to exogenous GnRH on d 16 of the leptin infusion was indistinguishable to that on the same day of vehicle administration. Thus, it would appear that leptin may have less of an impact on the gonadotroph of the primate pituitary than that reported for the rat (46).
The failure of hyperleptinemia to elicit a precocious pubertal reaugmentation of GnRH in the male monkey is entirely consistent with the unremarkable time course of circulating concentrations of leptin in this species at the time that puberty is initiated spontaneously (5, 47, 48). As in boys, elevated nocturnal testicular testosterone secretion driven by hypothalamic GnRH release is an endocrine herald of the onset of puberty in the rhesus monkey (1), but there is no evidence that a rise in circulating leptin concentrations precedes this critical developmental event in macaques. The relationship between circulating leptin concentrations and the pubertal reaugmentation of LH or pulsatile GnRH release has also been examined in the agonadal male monkey (5, 49). While the authors of one of these studies have argued that a rise in circulating leptin precedes puberty (49), a reanalysis of the same data by one of us failed to provide any optimism for such a view (50).
Thus, in the case of the male monkey, the weight of evidence argues against a role for circulating leptin in triggering the pubertal reaugmentation of pulsatile GnRH release and, therefore, the onset of puberty. This conclusion forces a reexamination of the earlier clinical studies that examined the relationship between circulating leptin concentrations and pubertal development in boys. The first of these, in which eight boys were studied longitudinally, reported that a rise in circulating leptin concentrations preceded the nocturnal activation of testosterone secretion (4). As previously recognized (51), however, in the latter study the values reported for plasma leptin before the elevations in testosterone secretion were extremely variable, and the seemingly tantalizing increments of circulating levels of the adipocyte hormone were actually rather trivial in many of the subjects. A similar variability in leptin levels was reported in another study of boys (52). In a comprehensive cross-sectional study of several hundred boys, mean leptin concentrations were found to rise to a peak at 9–10 yr of age, which preceded the pubertal increase in morning testosterone secretion (53). Although additional cross-sectional and longitudinal studies have also examined changes in circulating leptin levels during peripubertal development in boys, the binning of data with respect to Tanner stage does not address the question of whether circulating leptin concentrations increase immediately before the onset of puberty (54, 55, 56, 57). Similarly, while circulating levels of leptin have been described in children with central precocious puberty (58), understandably, measurements of the hormone before manifestation of this pathophysiology were not reported.
The conclusion that circulating leptin does not provide the trigger that times the initiation of puberty does not contradict the notion that, in a permissive sense, the adipose hormone may be obligatory for pubertal development. The issue of whether leptin is required for puberty to unfold in human and nonhuman primates, however, is unclear because the data pertaining to this issue are limited, contradictory and largely of a correlative nature. A 21-yr-old male subject with a prepubertal phenotype has been described with a homozygous missense mutation in the gene encoding leptin (59, 60), and primary amenorrhea at 13.5 and 19 yr of age was noted in two sisters, respectively, with a mutation of the leptin receptor (43). Interestingly, an obese, leptin-deficient 9-yr-old girl, who received leptin replacement for 12 months, was observed to manifest a pattern of pulsatile gonadotropin secretion consistent with early puberty at 10 yr of age (40). On the other hand, puberty with a normal tempo has been reported in two severely leptin-deficient women with lipotrophic diabetes (61).
A related question of whether a permissive action of leptin is required postpubertally for maintaining activity in the neuroendocrine axis governing testicular function in men and other adult male primates is also unclear. Certainly, reduced food intake and weight loss appear to inhibit GnRH release in adult male monkeys and men (62, 63, 64, 65). Although it has been proposed that a decline in circulating leptin may represent the signal relaying the compromised metabolic status to the reproductive axis (see Ref. 66), studies of the male monkey addressing this issue have yielded contradictory results. While Finn et al. (16) were able to prevent fasting induced inhibition of LH secretion in young adult rhesus monkeys by the concomitant administration of a high dose of rhu leptin, Lado-Abeal et al. (38, 67), also studying the rhesus monkey and using either rhu leptin or recombinant monkey leptin for replacement, were unable to confirm the earlier observation. Additionally, in adult male rhesus monkeys fasted for 48 h, the restoration of circulating leptin to prefast levels did not terminate the fasting induced arrest of LH secretion (68). Thus, the role of leptin in maintaining activity in the neuroendocrine axis governing testicular function in the adult remains to be fully defined.
Acknowledgments
We acknowledge the help of Drs. Anthony J. Zeleznik and Suresh Ramaswamy (Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine) with evaluating leptin binding activity in plasma and collecting samples, respectively. We are also grateful to Dr. A. J. Daniels, GlaxoSmithKline, for the gift of the NPY receptor ligand, 1229; to Dr. A. F. Parlow and the National Hormone and Peptide Program for rhu leptin and for the reagents used in the LH RIA; and to Dr. G. Bialy, Contraceptive Development Program, for the synthetic GnRH.
Footnotes
We acknowledge the support of the Primate and Assay Cores of the Center for Research in Reproductive Physiology, University of Pittsburgh School of Medicine. A preliminary report of this work, which was supported by NIH Grants HD-13254 and HD-08610 (to T.M.P.), was presented at the 31st Annual Meeting of the Society for Neuroscience, November 2001, San Diego, California.
Abbreviations: BW, Body weight; CV, coefficient(s) of variation; hu, human; NPY, neuropeptide Y; r, recombinant.
Received May 21, 2002.
Accepted July 30, 2002.
References
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 M. E. Wilson, J. Fisher, K. Chikazawa, R. Yoda, A. Legendre, D. Mook, and K. G. Gould Leptin Administration Increases Nocturnal Concentrations of Luteinizing Hormone and Growth Hormone in Juvenile Female Rhesus Monkeys J. Clin. Endocrinol. Metab., October 1, 2003; 88(10): 4874 - 4883. [Abstract] [Full Text] [PDF]
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), with plasma obtained from a monkey previously treated for 22 d with rhu leptin (
) or with preimmune plasma (
). The elution of large amounts of iodinated leptin in high molecular weight fractions (nos. 7–12) after incubation with both the antileptin antibody and plasma from the leptin-treated monkey indicate the presence of a leptin binding protein in the plasma of the leptin-treated monkey.
