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Endocrinology Vol. 141, No. 11 3965-3975
Copyright © 2000 by The Endocrine Society

ARTICLES

Leptin Regulates Pulsatile Luteinizing Hormone and Growth Hormone Secretion in the Sheep1

Shoji Nagatani, Yanhua Zeng, Duane H. Keisler, Douglas L. Foster and Craig A. Jaffe

Reproductive Sciences Program (S.N., D.L.F.), Departments of Medicine (Y.Z., C.A.J.), Obstetrics and Gynecology (D.L.F.), and Biology (D.L.F.), University of Michigan, Ann Arbor, Michigan 48109; Ann Arbor Veterans Affairs Medical Center (C.A.J.), Ann Arbor, Michigan 48105; and Department of Animal Sciences, University of Missouri (D.K.), Columbia, Missouri 65211

Address all correspondence and requests for reprints to: Dr. Craig A. Jaffe, Division of Endocrinology and Metabolism, 3920 Taubman Center, Box 0354, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0354. E-mail: cjaffe{at}umich.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Administration of leptin during reduced nutrition improves reproductive activity in several monogastric species and reverses GH suppression in rodents. Whether leptin is a nutritional signal regulating neuroendocrine control of pituitary function in ruminant species is unclear. The present study examined the control of pulsatile LH and GH secretion in sheep. We determined whether exogenous leptin could prevent either the suppression of pulsatile LH secretion or the enhancement of GH secretion that occur during fasting. Recombinant human met-leptin (rhmet-leptin; 50 µg/kg BW; n = 8) or vehicle (n = 7) was administered sc every 8 h during a 78-h fast to estrogen-treated, castrated yearling males. LH and GH were measured in blood samples collected every 15 min for 6 h before fasting and during the last 6 h of fasting. Leptin was measured both by a universal leptin assay and by an assay specific for ovine leptin. During the fast, endogenous plasma leptin fell from 1.49 ± 0.16 to 1.03 ± 0.13 ng/ml. The average concentration of rhmet-leptin 8 h after leptin administration was 18.0 ng/ml. During fasting, plasma insulin, glucose, and insulin-like growth factor I levels declined, and nonesterified fatty acid concentrations increased similarly in vehicle-treated and leptin-treated animals. In vehicle-treated animals, LH pulse frequency declined markedly during fasting (5.6 ± 0.5 vs. 1.1 ± 0.5 pulses/6 h; fed vs. fasting; P < 0.0001). Leptin treatment prevented the fall in LH pulse frequency (5.0 ± 0.4 vs. 4.9 ± 0.4 pulses/6 h; P = 0.6). Neither fasting nor leptin administration altered GH pulse frequency. Fasting produced a modest increase in mean concentrations of circulating GH in control animals (2.4 ± 0.5 vs. 3.4 ± 0.6 ng/ml; P = 0.04), whereas there was a much greater increase in GH during leptin treatment (2.7 ± 0.6 vs. 8.6 ± 1.6 ng/ml; P = 0.0001). GH pulse amplitudes were also increased by fasting in control (P = 0.04) and leptin-treated sheep (P = 0.007). The finding that exogenous rhmet-leptin regulates LH and GH secretion in sheep indicates that this fat-derived hormone conveys information about nutrition to mechanisms controlling neuroendocrine function in ruminants.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
FOOD AVAILABILITY is an important environmental factor regulating the reproductive (1) and somatotropic (2) axes. Reduced nutrition results in the suppression of pulsatile LH release and the cessation of gonadal activity in several monogastric species including rats (3), monkeys (4), and sheep rendered monogastric by a milk diet (5) as well as in ruminant sheep (6, 7, 8, 9, 10, 11). The reduced LH pulse frequency reflects the slow frequency of GnRH release that occurs when food availability is limited (12). Subsequent refeeding restores pulsatile secretion of LH (4, 5, 13). The effect of nutritional restriction on GH secretion is less consistent across species (2, 14). Food deprivation suppresses GH secretion in rodents, but stimulates it in humans and sheep. Because the circulating leptin concentration increases directly with fat mass (15), this product of adipose tissue could be an important metabolic signal mediating nutritional regulation of LH and GH secretion. Indeed, leptin treatment of fasting animals was reported to maintain estrous cyclicity in mice (16) and hamsters (17) and to prevent the fasting-associated decrease in LH pulse frequency in rats (18) and monkeys (19). Similarly, the fasting-induced decrease in GH secretion was prevented by leptin treatment in rats (20, 21, 22, 23, 24).

In ruminants, however, the effect of leptin on LH and GH secretion is less clear. In well fed sheep, intracerebroventricular (icv) infusion of leptin was reported to depress appetite, but had no apparent influence on the neuroendocrine control of pituitary function (25). In a preliminary communication, Morrison and colleagues reported that GH secretion in sheep was increased, but LH secretion was unaltered by feed restriction (26). In that study, central leptin treatment further augmented GH release, but did not affect the secretion of LH.

The present investigation reevaluated whether leptin plays a role in pituitary regulation during food deprivation in sheep. Our approach was to establish a model in which acute food deprivation altered pulsatile LH and GH secretion in sheep. We then determined whether systemic leptin administration prevented these fasting-induced changes in neuroendocrine function.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
General
All studies were approved by the university committee on use and care of animals at the University of Michigan. In Exp 1, we used postpubertal Suffolk cross-bred yearling male sheep (40 weeks of age, 55–60 kg BW, born November). Six months later they were again studied in Exp 2. The sheep had been gonadectomized at 2 weeks of age. Three months before Exp 1 they began treatment with a small sc implant containing crystalline estradiol, which resulted in circulating estradiol concentrations of less than 1 pg/ml (Foster, D. L., unpublished). The capsule consisted of SILASTIC brand tubing (od, 0.46 cm; id, 0.34 cm; Dow Corning Corp., Midland, MI) with a 10-mm packed column of crystalline 17ß-estradiol (Sigma, St. Louis, MO), which was sealed with SILASTIC brand adhesive type A (Dow Corning Corp.). A stainless steel jacket was placed over the capsule such that only 3 mm of the tube length was exposed in addition to the end. Estradiol implants were preincubated in water overnight before insertion to prevent a postimplantation peak in steroid release (27). We chose this size of implant because larger (28), but not this size (Foster, D. L., unpublished), implants can completely block LH secretion in fed animals. Before each study the sheep were maintained outdoors on fresh pasture and hay at the Reproductive Sciences Program Sheep Research Facility at the University of Michigan. The sheep were fed ad libitum before the start of the experiments (0800 h on day 0) and then were fasted for 78 h. Access to water was unrestricted. During the experiments, the animals were freely moving within 3 x 3-m2 pens, with two sheep per pen.

Exp 1: establishment of a fasting-mediated, hypogonadotropic model
To develop an animal model in which acute fasting inhibits pulsatile LH secretion, we compared the effect of estrogen on the secretion of LH during food deprivation. In rats, estrogen has been found either to be required for (29, 30) or to potentiate (18) fasting-mediated inhibition of LH. In sheep, the role of estrogen is unclear, and several studies in castrated male sheep failed to show an affect of food restriction on LH secretion (26, 31). In contrast to these sheep data, Beckett and colleagues found an estradiol-dependent suppression of LH in chronically undernourished wethers that did not occur in better nourished animals (32).

During the initial development of a model, we studied LH secretion in agonadal, yearling female sheep (40 weeks of age, 55–60 kg BW, November). LH secretory profiles in the fed animals were obtained by sampling every 15 min for 4 h beginning at 0800 h on day 0. The second frequent blood collection using the same protocol began at 0800 h on day 3 during the final 4 h of the 78-h fast. We determined that in these females, a 78-h fast produced no significant change in mean circulating LH concentrations (13.3 ± 1.2 vs. 14.9 ± 2.0 ng/ml; fed vs. fasting), LH pulse frequency (6.8 ± 0.5 vs. 7.5 ± 0.4 pulses/6 h), or LH amplitude (3.7 ± 0.6 vs. 5.5 ± 1.9 ng/ml).

We then studied six similarly aged males treated with low dose estrogen using the same fasting protocol. In contrast to our results in estrogen-deficient females, there was unequivocal LH suppression in the estrogen-treated males. Because of the clear effect of fasting on LH secretion in the presence of steroids, this identical experimental design was used in the same six males, in addition to nine others, for Exp 2.

Exp 2: effects of leptin on hormone secretion
Castrated males bearing estradiol implants (n = 15) were fed ad libitum for 14 days before the start of the protocol. At 0800 h on day 0, jugular blood was collected at 15-min intervals for 6 h to obtain estimates of the frequency and amplitude of LH and GH pulses before fasting. The animals were then stratified by weight into two groups, leptin treated (n = 8) and control (n = 7), and begun on a 78-h fast as in Exp 1. The leptin-treated animals received recombinant human met-leptin (rhmet-leptin; Amgen, Inc., Thousand Oaks, CA; 50 µg/kg, sc, every 8 h) from 2400 h on day 0 until 2400 h on day 2 for a total of sevem doses. This dose was based on the hypothesis that the low systemic GH level found in obese humans (33) was the result of high circulating concentrations of leptin and on published data for peripheral leptin concentrations in obese men and women (34, 35, 36). Control animals received the same volume of PBS sc every 8 h. In a preliminary experiment (n = 2), we determined that 1.5 h after a sc leptin injection of 30 µg/kg, the peak leptin concentrations was 34 ng/ml, and that the exogenous leptin had an estimated circulating half-life of 4 h. Peripheral (jugular) blood samples were obtained every 8 h, just before each leptin injection. Beginning at 0800 h on day 3, a second frequent blood sampling (15-min intervals for 6 h) was performed to assess the influence of exogenous leptin on LH and GH secretion.

Upon completion of blood sample collections, the effect of leptin on feeding was assessed. The penned sheep were allowed free access to preweighed pelleted food, with two sheep per pen. The sum of weight of food consumed by the two sheep during the next 45 min was measured.

Hormone and metabolite assays
Plasma samples were stored at -20 C until assayed. Plasma LH concentrations were determined in duplicate by a double antibody RIA and were expressed in terms of NIH LH-S12 as previously described (37). Assay sensitivity, defined as 2 SD from the zero standard, averaged 0.4 ng/ml for 50 µl plasma. The mean intra- and interassay coefficients of variation were 8% and 14%, respectively, at a bound/free ratio (B/Bo) of 75%. Plasma GH was measured in duplicate in a double antibody RIA with National Hormone and Pituitary Program reagents using GH standard GH-2 as previously described (38). The mean GH intraassay coefficient of variation (CV) was 5% at a B/Bo of 30% and 4% at a B/Bo of 70%. Assay sensitivity was 0.4 ng/ml using 100 µl plasma. Cortisol was measured in pooled plasma and basal samples by RIA (Diagnostic Products, Los Angeles, CA). The pools were made by combining equal aliquots of hourly samples obtained during the frequent sampling periods. The cortisol assay sensitivity was 3.8 ng/ml, and the intraassay CV was 3%. All LH, GH, and cortisol determinations for an individual sheep were run in the same assay. Plasma insulin was measured in the pooled plasma by RIA (ICN Pharmaceuticals, Inc., Costa Mesa, CA). The sensitivity of the insulin RIA was 0.21 IU/ml, and the intra- and interassay CVs were 5% and 17%, respectively.

Plasma leptin was measured in the pooled plasma and basal tubes by two different methods. The first method used a recently developed RIA for ovine leptin (39). The oleptin standard curve and data from a serial dilution of a sheep plasma sample are shown in Fig. 1Go. The detection limit of the assay, using 100 µl plasma/tube, was 0.06 ng/ml. The cross-reactivity between the antioleptin antibody and rhmet-leptin was less than 0.5% compared with the oleptin standard. Sheep plasma spiked with rhmet-leptin did not dilute with the zero standard in a parallel fashion to the oleptin standard. All samples were run in a single assay, and the intraassay CV was 8% at a B/Bo of 85%. For the second method, leptin was measured in 100-µl aliquots of the basal and pooled plasma samples using a multispecies leptin RIA kit (Linco Research, Inc., St. Charles, MO). The assay sensitivity for the multispecies RIA averaged 0.29 ng/ml. The intra- and interassay coefficients of variation were 3% and 13%, respectively, at a B/Bo of 70%. In contrast to the specific oleptin RIA, there was 100% cross-reactivity of rhmet-leptin with the assay standard, and fed endogenous leptin immunoreactivity was near the detection limit of the multispecies RIA. Thus, this assay was used to quantify the exogenous hormone. Plasma insulin-like growth factor I (IGF-I) and nonesterified fatty acids (NEFA) were measured in the fed and fasting pools and in the basal sample collected after 24 h of fasting. Plasma IGF-I was measured by immunoradiometric assay after extraction (Diagnostics Systems Laboratories, Inc., Webster, TX), and NEFA were measured by a calorimetric method (Wako Chemicals, Inc., Richmond, VA). Glucose was measured in single 5-µl aliquot of the pooled plasma using an enzymatic method (Glucose Trinder kit, Sigma).



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  		Figure 1. Standard curve for ovine leptin RIA. •, The percent binding for recombinant oleptin standard. A sheep plasma sample that was serially diluted with the zero standard ({circ}) gave a percent binding curve that was parallel with the ovine standard.

 
Statistical analysis
LH and GH pulses were quantified by Cluster analysis (40). The parameters for LH pulse detection were previously described (41). For GH pulse detection, a 1 x 1 matrix with a 1.5 cut-off was used.

The effects of fasting and treatment on hormonal parameters, metabolites, feed intake, and weight were determined by repeated measures ANOVA using treatment group and time (duration of fasting) as the between- and within-group factors, respectively. Pooled samples from the two frequent sampling time periods as well as intermediate time points were included in the analyses. When the primary analysis demonstrated a significant (P < 0.05) treatment x time interaction, subsequent repeated measures ANOVAs were performed on the data from control and leptin-treated animals individually. Contrasts comparing fasting values to the fed baseline or comparing between-treatment groups were performed as appropriate when significant main effects or interactions were identified by ANOVA. Data were log transformed before analysis when appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exp 1: model development
Figure 2Go provides patterns of LH secretion for estrogen-treated males during the fed and fasted states. All animals exhibited rapid LH pulse frequency during the fed period, but during fasting few LH pulses occurred (3.5 ± 0.6 vs. 0.8 ± 0.3 pulses/4 h; fed vs. fasting, P = 0.02). There was a trend to lower mean LH concentrations during fasting (19.1 ± 3.0 vs. 11.8 ± 2.8 ng/ml; P = 0.11). LH pulse amplitudes were similar between the two studies (24.5 ± 5.6 vs. 40.9 ± 14.0 ng/ml; P = 0.26).



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  		Figure 2. Plasma LH profiles from estradiol-treated castrated male sheep fed (left panel) and after a 72-h fast (right panel). During fasting, there was a clear decrease in LH pulse frequency.

 
Exp 2: influence of leptin on LH and GH secretion
Figure 3Go shows the effect of fasting on ovine plasma leptin concentrations. Plasma leptin measurements using the specific ovine leptin RIA are presented for samples collected before leptin administration or from control sheep (no leptin treatment). As rhmet-leptin partially cross-reacted in the ovine leptin assay, only the fed baseline and 16 h fasting data, which were obtained before rhmet-leptin administration, were included in the initial ANOVA. A repeated measures ANOVA using these two time points demonstrated a strong time effect (P < 0.001), but no treatment x time interaction (P = 0.9), indicating a parallel fall in oleptin during fasting in the two treatment groups. Equivalency in baseline oleptin concentrations in the two groups was further supported by a two-sample t test (1.49 ± 0.16 vs. 1.79 ± 0.25 ng/ml; control vs. leptin, P = 0.35). During fasting, plasma leptin fell approximately 30% within the first 16 h (1.06 ± 0.09 ng/ml; P = 0.0005 vs. 0800 h on day 0). A repeated measures ANOVA using the fasting time points demonstrated that ovine leptin concentrations in control animals remained stable throughout the remainder of the experiment.



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  		Figure 3. Effect of fasting on plasma oleptin concentration. Plasma leptin was measured in a RIA specific for oleptin. There was partial cross-reactivity of the oleptin assay with rhmet-leptin; therefore, only leptin concentrations before the first dose of rhmet-leptin at 16 h are presented. *, P < 0.05, fed vs. fasting.

 
Circulating oleptin concentrations before leptin administration were near the limit of detection of the multispecies RIA. Leptin immunoreactivity measured by this assay remained low in the fasted control animals. In leptin-treated animals, plasma leptin concentrations in samples obtained 8 h after sc leptin administration averaged 18 ng/ml in the multispecies assay. By ANOVA, leptin concentrations measured by the multispecies RIA did not vary over the time of treatment.

Figure 4Go presents the effect of fasting on glucose and insulin in vehicle or leptin-treated sheep, and Fig. 5Go shows the effects of fasting on NEFA and IGF-I. There were no treatment effects (P > 0.3), and there were highly significant time effects (P < 0.0001) for each of these metabolic parameters. There were no treatment group x time interactions for plasma glucose (P = 0.9), insulin (P = 0.6), NEFA (P = 0.6), or IGF-I (P = 0.7). Therefore, there was no effect of treatment group on either baseline or fasting measurements. Plasma glucose reached a nadir 32 h into the fast. Plasma insulin concentrations mirrored the decline in glucose, falling from 23.9 ± 1.2 µU/ml at baseline to 8.47 ± 0.33 µU/ml at 72 h (P < 0.0001). NEFA increased from 0.20 ± 0.01 mEq/liter at baseline to 0.88 ± 0.05 mEq/liter at 24 h (P < 0.0001) and was even higher at 72 h (1.56 ± 0.08 mEq/liter; P < 0.0001). There was a small decline in plasma IGF-I after 24 h of fasting (208 ± 14 vs. 180 ± 20 ng/ml; P = 0.01), and after 72 h of fasting, plasma IGF-I had decreased further (70 ± 13 ng/ml; P < 0.0001 vs. fed and 24 h).



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  		Figure 4. Effects of fasting and leptin on plasma insulin and glucose. By repeated measures ANOVA, there was no treatment effect. *, P < 0.05; **, P < 0.0001 (fed vs. fasting).

 


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  		Figure 5. Effects of fasting and leptin on plasma NEFA and IGF-I. By repeated measures ANOVA, there was no treatment effect. *, P < 0.05; **, P < 0.0001 (fed vs. fasting).

 
Figure 6Go illustrates plasma cortisol concentrations in the vehicle- and leptin-treated sheep during the fed and fasting periods. There was no treatment effect (P = 0.4). There was, however, a time effect (P = 0.01) and a treatment x time interaction (P = 0.005), indicating that the changes in cortisol between the two groups were not parallel. The treatment x time interaction was driven by significant differences in plasma cortisol late in the fast, with higher plasma cortisol (P < 0.05) after 64 h of fasting in the control group. Repeated measures ANOVA performed on the control data found a time effect (P = 0.02), and post-hoc contrasts demonstrated that plasma cortisol increased over the baseline fed value late in the fast. A similar analysis of cortisol concentrations during leptin administration also demonstrated a time effect (P = 0.01); however, plasma cortisol levels at all time points, except for 32 h, were similar to the fed baseline level.



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  		Figure 6. Effects of fasting and leptin on plasma cortisol. *, P < 0.05; **, P < 0.01 (fed vs. fasting). #, P < 0.05; ##, P < 0.01 (vehicle vs. leptin).

 
Parameters of pulsatile LH secretion in control and leptin-treated sheep are presented in Table 1Go. Plasma LH concentrations in vehicle- and leptin-treated sheep during the fed and fasting periods are shown in Fig. 7Go (mean ± SE) and Fig. 8Go (individual LH profiles). Repeated measures ANOVA for LH concentration demonstrated a treatment effect (P = 0.03), a strong time effect (P < 0.0001), and a strong treatment group x time interaction (P < 0.0001) for LH concentration. During the fed period, mean concentrations of LH were similar for the two groups (P = 0.5). As shown in Fig. 7Go, within 32 h of fasting, plasma LH in the control animals fell below the mean baseline concentration (P = 0.0005). In contrast, fasting LH concentrations in the leptin group were unchanged or increased slightly above the fed control level.


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  		Table 1. Effect of leptin on fasting-induced changes in LH pulse parameters in gonadectomized sheep treated with estrogen

 


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  		Figure 7. Effects of fasting and leptin treatment on plasma LH. *, P < 0.05; **, P < 0.01 (fed vs. fasting). #, P < 0.05; ##, P < 0.005 (vehicle vs. leptin).

 


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  		Figure 8. Individual fed (0–6 h) and fasting (72–78 h) LH profiles for sheep. Those on the left were treated with vehicle; those on the right were treated with rhmet-leptin (50 µg/kg) every 8 h sc. Jugular blood samples were collected every 15 min.

 
There were also treatment (P = 0.02) and time (P < 0.0001) effects on LH pulse frequency as well as a strong treatment x time interaction (P < 0.0001). LH pulse frequencies in the two treatment groups were the same during the fed baseline study (P = 0.40). In the control animals, there was a marked decrease in LH pulse frequency during fasting (P < 0.0001). In contrast, the LH pulse frequency in leptin-treated sheep during fasting remained similar to that measured during the fed period (P = 0.6).

ANOVA for LH pulse amplitude showed a very significant time effect (P < 0.001), but no treatment effect and no interaction. LH pulse amplitudes were similar in the two groups during the fed period (P = 0.54). There was a marginal increase in LH pulse amplitude in the vehicle-treated sheep during fasting (P = 0.04). This increase was a result of infrequent, large amplitude LH pulses in the fasted control sheep (Fig. 8Go). Moreover, three control animals had no LH pulses during the fasting period. Therefore, definite conclusions relative to the LH pulse amplitude data in the control animals cannot be made. LH pulse amplitudes in the leptin-treated sheep were marginally above amplitudes during the fed period (P = 0.04).

Parameters characterizing pulsatile GH secretion in control and leptin-treated sheep are presented in Table 2Go. Individual GH profiles for each sheep are shown in Fig. 9Go. By repeated measures ANOVA, there was a marginally insignificant effect of treatment group on mean GH (P = 0.06), but a very strong time effect (P < 0.0001) and treatment x time interaction (P = 0.001). The mean GH concentration (P = 0.8), mean GH pulse amplitudes (P = 0.7), and pulse frequency (P = 0.9) during the fed baseline were similar in the two groups. The equivalency of the baseline values and the strong interaction between treatment and time indicated that the effects of fasting on mean GH and GH pulse amplitude were different between the vehicle- and leptin-treated animals. In the control animals, there was a modest increase in both mean GH concentration (P = 0.03) and GH pulse amplitude (P = 0.04) during fasting. In contrast, in the leptin-treated animals there was a more than 3-fold increase in both mean GH concentration (P = 0.0001) and pulse amplitude (P = 0.007) over the fed baseline measurements. GH pulse frequency did not change with either fasting or leptin treatment.


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  		Table 2. Effect of leptin on fasting-induced changes in GH pulse parameters in gonadectomized sheep treated with estrogen

 


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  		Figure 9. Individual fed and fasting GH profiles for sheep treated with vehicle (left panels) or rhmet-leptin (right panels).

 
Treatment with leptin was well tolerated. The mean weights of the control and leptin-treated animals during the fed period were similar (P = 0.44) as was the amount of weight lost during the fast (21% vs. 20% of starting weight; P = 0.46). As shown in Fig. 10Go, there was a weak trend for less food intake in the leptin-treated sheep (P = 0.14). Of interest, three of eight sheep treated with leptin attempted to mount other males around the time of the last leptin injection, and two of eight exhibited mounting behavior during the second frequent blood collection. None of control animals demonstrated this type of sexual behavior.



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  		Figure 10. Effect of fasting and leptin on feed intake (upper panel) and body weight (lower panel). There was a trend to decreased feed intake after the 78-h fast in leptin-treated animals (P = 0.14). Both groups lost weight (*, P < 0.0001) during the fast, and the decreases in weight were similar in the two groups (P = 0.46).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We sought to establish a convenient model to study the effects of leptin on the neuroendocrine regulation of pituitary function in ruminants. We used a short-term fast in the presence of a small amount of estrogen, one that does not inhibit pituitary function in the fed state but becomes effective in this respect during fasting. Using the castrated male in which estradiol was replaced with a small sc capsule, we determined that leptin administration prevented the fasting-induced suppression of pulsatile LH secretion and stimulated GH secretion. These findings support the hypothesis that in sheep, leptin is an important nutritional signal that modulates reproductive activity through the regulation of LH secretion. These findings stand in contrast to recent reports in which leptin was not found to influence LH secretion (25, 26). As discussed below, this discrepancy may be a function of the model system used to assess leptin action. The importance of leptin in the normal physiological regulation of GH is less apparent from our study.

Whether food deprivation alters pulsatile LH secretion in sheep has been unclear. Long-term feed restriction suppresses LH pulse frequency (6, 7, 8, 9, 10, 11, 32). However, virtually no reports have documented that acute fasting inhibits sheep reproductive neuroendocrine function. In our preliminary acute experiments with gonadectomized females without estradiol replacement, we also found no effect of fasting on LH secretion. In contrast, fasting clearly suppressed LH in gonadectomized males administered physiological doses of estradiol. Whether this difference in LH response to fasting reflects a sex difference rather than a difference in steroid status cannot be determined from our study because the appropriate comparisons were not made.

These results, however, are consistent with other data demonstrating interactions between estrogen and nutritional status in the control of LH secretion. Several studies suggest that estrogen could alter the set-point for gonadotropin suppression during caloric restriction. Indeed, a role for estrogen in the modulation of LH secretion during caloric restriction in rats (42) and sheep (32) has been previously proposed. We recently demonstrated a decrease in LH secretion during fasting in ovariectomized rats and that sex steroid replacement enhanced this gonadotropin suppression (18). We further determined that this modulatory effect of estrogen in rats involves sex steroid feedback to brain areas exhibiting fasting-mediated increases in estrogen receptors (30, 43). Similar observations have been made in sheep. In orchidectomized sheep without gonadal steroid replacement, feed restriction did not suppress pulsatile LH secretion (32). Systemic estrogen infusion inhibited LH secretion in these feed-restricted animals, but not in more liberally fed ones (32). Of interest, feed restriction in lambs reduced LH secretion and up-regulated the number of estrogen receptor-positive cells in the preoptic area (44), which is a site for estrogen negative feedback on LH secretion in sheep (45). Therefore, enhancement of estrogen’s negative feedback on GnRH secretion is a likely mechanism for fasting-induced LH suppression in sheep and other species.

The estrogen dependency for suppression of pulsatile LH secretion in acutely fasted sheep might be an important mechanism that increases survival. During periods of starvation, fertility is not desirable, and metabolic signals to the brain would tend to suppress LH secretion. When estrogen is absent, the brain would sense that the reproductive axis is "off." In contrast, the presence of systemic estrogen would inform the central mechanisms controlling the hypothalamic-pituitary-gonadal axis that the axis is "on." As fertility during periods of food scarcity would be detrimental to survival, neuroendocrine mechanisms that actively turn off LH secretion would be beneficial to survival. This teleological argument fits well with observations from this and other (30, 32, 42, 43) studies demonstrating that caloric restriction enhances estrogen negative feedback.

If leptin serves as a metabolic hormone that connects the level of nutrition with reproductive hormone secretion, it probably has a permissive role. Although it was reported that icv leptin administration increased LH pulse amplitude in nonfasted, well fed rats (46), most studies have found an effect of leptin on gonadotropin secretion only in fasting animals. For example, we recently demonstrated that leptin administration to fed rats did not affect LH pulses (47). Similarly, an icv leptin infusion did not further increase pulsatile LH secretion in fed sheep (25). Our finding that exogenous leptin prevents fasting-induced suppression of pulsatile LH secretion in sheep is consistent with previous reports using ovariectomized, fasted rats (18) and gonad-intact, fasted male monkeys (19). Overall, these data indicate that the effect of leptin on pulsatile LH secretion is only manifest in hypogonadotropic animals with low plasma leptin concentrations. Artificially increasing leptin concentrations further would fail to increase LH secretion in the nonhypogonadotropic individual. This agrees with the ideas that high levels of leptin provide a signal about energy balance and that once an appropriate energy balance is achieved, greater concentrations of circulating leptin do not provide any additional information to the reproductive neuroendocrine axis.

The neuroendocrine mechanism(s) through which leptin regulates GnRH neuronal activity is uncertain. One potential mechanism is activation of the hypothalamic-pituitary-adrenal (HPA) axis by the stress of fasting (48). Arguments supporting this possibility include an association between hypogonadotropism and elevated plasma corticosterone in the fasting rat (16) and restoration of the fasting-induced LH suppression by icv injection of a CRH antagonist (49). In addition, leptin treatment of fasting ob/ob mice ameliorates both hypercortisolemia (50) and infertility (51, 52, 53). Similarly, leptin treatment of wild-type mice prevents fasting-induced increases in corticosterone and suppression of estrous cyclicity (16). Other findings do not support the view that HPA activation by fasting mediates hypogonadotropism. Foot shock stress and fasting both inhibit LH secretion in the CRH knockout mouse (54).

Our data suggest that if activation of the HPA axis is involved, then it is unlikely that an increase in glucocorticoid per se is what inhibits the reproductive axis. In our leptin-treated animals, plasma cortisol concentrations remained at fed levels. In the control animals, cortisol only increased consistently above the fed state levels late in the experiment (64–72 h of fasting), whereas there was clear suppression of LH by 32 h of fasting. These data do not, however, rule out the possibility that activation of the HPA axis is important in stress-mediated hypogonadotropism. In the case of the knockout mouse, an alternative pathway to the HPA axis might develop in response to stress. In the case of our results, central activation of the HPA axis could mediate gonadotropin suppression without an increase in cortisol secretion. Studies in vivo suggest that leptin increases paraventricular nuclei CRH messenger RNA (55) and hypothalamic CRH content (56). Leptin has been reported to either increase (57) or decrease (58) CRH release from hypothalamic tissue in culture. In addition, leptin inhibits glucocorticoid release from human (59) and bovine (60) primary adrenal cultures. The degree to which central activation of the HPA axis produces the fasting-induced suppression of GnRH secretion is yet unknown.

We have also investigated the effects of leptin on the GH-IGF-I axis. In rats, fasting potently inhibits GH secretion, presumably through increasing hypothalamic somatostatin secretion (61). Recent data have shown that arcuate nucleus neuropeptide Y (NPY) neurons regulate periventricular somatostatin (62) and that NPY inhibits GH in rats (63). Fasting suppresses GH in the rat by increasing NPY, and the fasting-mediated changes in NPY, somatostatin, and GH can be reversed by treatment with exogenous leptin (20, 21, 22, 23, 24, 64, 65).

Whether these data apply to species other than rats is uncertain. In contrast to the suppression of GH that occurs in rats, mean GH increased 3-fold in humans within 24 h of the initiation of fasting (66). In sheep, the effects of nutritional deprivation on GH secretion are less well defined. During chronic feed restriction, mean GH concentrations in lambs (9) or adult ewes (7) increased. Acute fasting of sheep was reported to either increase (67) or have no effect (68) on GH secretion. In our study, 72 h of fasting modestly increased mean GH, but did not affect GH pulse frequency. Consistent with observations in rats, food restriction increased hypothalamic NPY in sheep (31). Although it was originally reported that icv NPY had no effect on GH in sheep (69), a more recent study observed that NPY might stimulate GH in this species (70).

Based on the concordant changes in NPY and the discordant changes in GH during fasting in rats and sheep, we had hypothesized that leptin infusion would prevent an increase in GH secretion in fasting sheep. The dose used, 50 µg/kg every 8 h, resulted in circulating leptin concentrations of a magnitude similar to that measured in obese humans (34, 35, 36). Contrary to our hypothesis, GH was potently stimulated by leptin administration to fasting sheep. Of interest, a single 10- to 100-µg icv injection of porcine leptin acutely stimulated GH release in pigs (71). In a preliminary experiment we similarly observed that icv infusion of rhmet-leptin (2.5 µg/kg·day) for 3 days to fasting sheep also stimulated GH secretion (Jaffe, C., unpublished data). In contrast, a continuous icv infusion of recombinant human leptin at the dose of 480 µg/day had no effect on GH in fed sheep (25). The contradictory results from fasting and underfed animals again underscore the importance of careful delineation of metabolic status when studying the effects of leptin on the neuroendocrine axes.

Whether our results were influenced by the dose of leptin used is uncertain. As noted above, a relatively large range of icv leptin doses stimulated GH secretion in sheep and swine. Limited in vitro data, however, suggest that the leptin dose, in addition to the nutritional status and the species of the animal, might be an issue. Although leptin did not influence GH secretion from primary rat pituitary cell cultures (65), high leptin concentrations increased GH release from pig pituitaries in culture, whereas lower concentrations suppressed GHRH-stimulated GH release leptin (71). Sheep pituitary cells express leptin receptor messenger RNA (72), so it is possible that leptin has a direct pituitary effect in this species. Human leptin had no acute effect on GH release in primary cultures of sheep pituitaries, but in concentrations comparable to those used in the pig study, more chronic leptin exposure inhibited the GH response to GHRH (73). This suppression of GH response to GHRH could conceivably account for the low spontaneous and GHRH-stimulated GH levels in obese humans. Further studies are needed to accurately define the interactions between GH secretion and leptin, nutritional status, and IGF-I.

As opposed to the effects of leptin on gonadotropin and GH secretion, we did not find a clear effect of leptin on food intake. This is contrary to the recent report that icv leptin infusion suppresses food intake (25). There are several potential explanations for this difference. We had anticipated that the effect of leptin on feed intake would be large, so that few animals would be required to see a significant effect on feeding. We therefore penned two animals together to avoid isolation stress. It is possible that our experimental design, in which we measured the combined feed intake of two sheep, did not give us adequate power to see a difference. It is also possible that a higher dose of leptin might have resulted in decreased feed intake. However, the fact that we obtained unequivocal effects on both LH and GH secretion suggests that the dose used (150 µg/kg·day) did have a significant central effect. Alternatively, rhmet-leptin, which is similar but not identical to ovine leptin, might be less anorexigenic than the ovine peptide in sheep. It is also conceivable that either sheep have relative leptin resistance with regard to satiety or that leptin does not play a role in feed intake in this species. The previous report that icv leptin suppressed food intake (25) could have been due to a toxic effect of administering the peptide icv. Finally, it is most likely that the model used for study accounts for the observed differences in feeding during leptin treatment. Henry et al. (25) demonstrated decreased feed intake in chronically feed-restrained sheep. Our studies were performed with sheep fasted for 78 h. This more extreme acute nutritional deprivation might have stimulated pathways that overruled any anorexigenic input from the exogenous leptin.


   Acknowledgments
 
We thank Amgen, Inc., for providing rhmet-leptin. We thank Dr. Morton Brown of the Michigan Diabetes Research and Training Center for his assistance with statistical analyses; Mr. Douglas D. Doop, Ms. Juanita Pelt, and Mr. Christopher Reavill for their technical advice and assistance; Drs. Gordon D. Niswender, Colorado State University (Fort Collins, CO), and Leo E. Reichert, Jr., Albany Medical College (Albany, NY), for providing reagents used in the LH RIA. We are grateful to the staff of the Core Facilities of the Center for the Study of Reproduction, Mr. Gary R. McCalla of the Sheep Research Core Facility for careful animal care, the staff of the Assays and Reagents Core Facility for standardization of hormone assay reagents, and the staff of the Administrative Core Facility for administrative assistance.


   Footnotes
 
1 This work was supported by a V.A. Merit Award (to C.A.J.), NIH Grants HD-18258 and HD-18394 (to D.L.F.), and Michigan Diabetes Research and Training Center Grant 2P60-DK-20572-21. A preliminary report of this work was presented at the 82nd Annual Meeting of The Endocrine Society. Back

Received February 25, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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