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Prince Henry’s Institute of Medical Research (B.A.H., A.R., I.J.C.), Clayton, Victoria 3168; the Department of Immunology and Pathology, Monash University Medical School (J.W.G.), Prahran, Victoria 3181; the Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital (W.S.A.), Parkville, Victoria 3050; the Department of Physiology, Monash University (A.J.T., B.J.C.), Clayton, Victoria 3168; Victorian Institute of Animal Science (F.D.), Werribee, Victoria 3030; and Swinburne University of Technology (A.M.), Hawthorn, Victoria 3122, Australia
Address all correspondence and requests for reprints to: Dr. Iain J. Clarke, Prince Henry’s Institute of Medical Research, P.O. Box 5152, Clayton, Victoria 3168, Australia. E-mail: iain.clarke{at}med.monash.edu.au
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Abstract |
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Introduction |
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One question that is central to the issue of how leptin may affect endocrine function is whether there is a direct effect on neuroendocrine cells in the hypothalamus or whether there is an indirect effect via neuronal systems that possess the leptin receptor. For example, neuropeptide Y (NPY) cells express the leptin receptor and could act to regulate appetite (2, 17) and neuroendocrine function (18, 19, 20). Thus, centrally administered leptin could act on appetite regulatory systems within the hypothalamus, which could, in turn, influence the function of neuroendocrine cells. There are various precedents for this, one being that melanin-concentrating hormone regulates appetite (21), but also appears to regulate the secretion of GnRH (22) and CRF (18, 23). Another possibility is that leptin could directly affect neuroendocrine cells. Zamorano et al. (24) used RT-PCR to demonstrate the existence of the leptin receptor in GT1–7 cells. Others (Moenter, S., personal communication), however, found that leptin cannot affect the secretion of GnRH from GT cells unless the immortalized neurons are transfected with the receptor.
We investigated the effect of centrally administered leptin on the secretion of pituitary hormones in the sheep. It was hypothesized that a dose of leptin that acted to inhibit food intake would be capable of influencing endocrine function.
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Materials and Methods |
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Animals, surgery, infusions, and blood sampling
Corriedale ewes with a mean body weight of 44.6 ± 1.2 kg
were used during the breeding season. The animals were ovariectomized
at least 1 month before surgery and were fitted with guide tubes into
the third cerebral ventricle (3V) as previously described by
Barker-Gibb et al. (19). The animals were tamed and
familiarized with the experimental facilities and were housed in
individual pens for the duration of the experiment. On the day before
commencement of infusions and blood sampling, one external jugular vein
was cannulated, and the cannula was kept patent with heparinized (50
U/ml) normal saline. The animals were fed 1.5 kg Lucerne chaff/day, and
refusals were weighed to monitor food intake from 5 days before the
start of the infusion until the end of the experiment.
For the infusion of leptin or vehicle, we used Graseby MS16A infusion pumps (Graseby Medical Ltd., Gold Coast, Australia) strapped onto the backs of the animals. Polyethylene tubing was connected to a 2.5-ml plastic syringe and to a 19-gauge stainless steel tubing assembly that was introduced into the 3V at least 2 mm beyond the end of the guide tube. The patency of the system was verified by checking that cerebrospinal fluid flowed out of the infusion cannulas. Groups of five sheep were randomly assigned to two groups to receive either vehicle or leptin, infused into the 3V at a rate of 110 µl/h (20 µg/h). The infusion syringes were filled once a day, using a side port in the infusion line.
Blood samples (8 ml) were taken from the jugular venous cannulas that were extended with a manometer line (Portex Ltd., Kent, UK) and closed with a three-way tap. Samples were collected into heparinized tubes and centrifuged at 4 C to obtain plasma, which was stored at -20 C until assayed. The animals were sampled at 10-min intervals for 6 h (commencing at 0900 h) on days -1 and 2 relative to the start of the infusion and again on day 3 for 3 h. After this, the animals were injected (iv) with an overdose of pentobarbitone (Lethabarb, May and Baker Pty. Ltd., Australia) and decapitated. The hypothalamus was dissected, frozen on dry ice (within 2 min of decapitation), and stored at -80 C for in situ hybridization.
Recombinant human leptin
Leptin complementary DNA was isolated from human adipose tissue
by the PCR and cloned into the bacterial expression vector pCAL-n
(Stratagene, La Jolla, CA), and its authenticity was
verified by DNA sequencing. Recombinant leptin was produced by
transformation of BL-21(LysS) Escherichia coli, followed by
growth in trypton-phosphate broth and induction with isopropyl
ß-D-galactoside. Bacteria were lysed by freezing and
thawing followed by sonication, and DNA was digested with
deoxyribonuclease I. Inclusion bodies were washed twice in 10
mM Tris-HCl, pH 8, containing 0.1% Triton X-100, followed
by one wash in the same buffer lacking detergent. Inclusion bodies were
then solubilized in 9 M urea and 5 mM
dithiothreitol at room temperature, and the insoluble material was
removed by centrifugation. Leptin was refolded by slow dropwise
dilution into 10 vol 10 mM HCl with rapid stirring and was
finally purified by reverse phase HPLC using C4-silica and a linear
gradient of 0.1% trifluoroacetic acid in water to 70%
acetonitrile-0.1% trifluoroacetic acid, followed by lyophilization. A
single major peak that eluted late in the gradient was the only species
that had biological activity, as assessed by induction of proliferation
in the factor-dependent cell line BAF-3 that had been transfected with
the human leptin receptor (25). Biological activity per unit mass was
comparable to that purified by nondenaturing affinity chromatography
from supernatant of the same gene expressed in COS cells, compatible
with the idea that refolding was essentially complete. The recombinant
leptin had a molecular mass of 16,000 daltons, as determined by
SDS-PAGE. Figure 1
shows the bioactivity
of this material assessed in the in vitro cell proliferation
bioassay. The material was stored in 0.1 M HCl at 4 C
before infusion and infused in the same vehicle at a concentration of
0.18 µg/µl. A high dose of leptin was used to ensure a significant
reduction in food intake; this was based on the results of preliminary
studies.
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FSH. Every tenth sample was assayed (100 µl) in duplicate using the method of Bremner et al. (27) with the ovine standard NIAMMD oFSH-RP-1. The sensitivity of two assays was 1.3 ng/ml; the intraassay CV was less than 10% between 0.95–21.8 ng/ml, and the interassay CV was 6.1%.
PRL. Samples were assayed in duplicate at a volume of 10 µl following the method of Clarke et al. (28) and with the ovine standard Sigma Chemical Co. (lot 114F-0558, St. Louis, MO). The sensitivity of nine assays was 0.15 ng/ml; the intraassay CV was < 10% over the range 1.5–6.8 ng/ml, and the interassay CV was 20%.
GH. Samples were assayed in duplicate at a volume of 200 µl using the method of Thomas et al. (29) with NIDDK oGH-I-4 as the standard. The sensitivity of 8 assays was 0.5 ng/ml; the intraassay CV was less than 10% over the range of 1.9–27.1 ng/ml, and the interassay CV was 20%.
Cortisol. All samples were assayed in duplicate at 100 µl, using the RIA outlined by Bocking et al. (30). One hundred microliters from every sample were placed into a daily pool for each individual sheep. These pools were used for cortisol, glucose, lactate, nonesterified fatty acid, and insulin. For four cortisol assays, the sensitivity was 0.2 ng/ml; the intraassay CV was 9.9%, and the interassay CV was 13.3% at 20.7 ng/ml and 10% at 14.8 ng/ml.
Insulin. Samples were assayed using a kit (Linco Research, Inc., St. Charles, MO) with human insulin as a standard and validated for ovine insulin in our laboratory. All analyses were performed in a single assay, and the intraassay CV was 2.5%.
Nonesterified fatty acids (NEFA). Plasma NEFA were analyzed using an enzymatic kit assay (31). All analyses were performed in a single assay, and the intraassay CV was 4.1%.
Glucose and lactate. Blood glucose and lactate concentrations were measured in 25-µl samples of plasma using a YSI2300 STAT glucose/L-lactate analyzer (Yellow Springs Instrument Co., Yellow Springs, OH). The measurable range for blood glucose was between 0–30 mM and was 0–16 mM for lactate.
In situ hybridization
Hypothalamic sections (16 µm) were cut on a freezing microtome
and were thaw-mounted onto warm slides. In situ
hybridization was performed using a 35S-labeled 48-mer
oligonucleotide sequence complementary to 159–206 of the coding region
of human NPY, as previously described (32). Hybridization was carried
out at 42 C in a humid chamber; after posthybridization treatment,
sections were taped to an x-ray cassette. Slides were exposed to
Kodak X-omatic AR film (Kodak, Australasia, Coburg,
Australia) for 12 days at room temperature before being
developed. Carbon14 standards (Americal Radiolabeled
Chemicals, Inc., St. Louis, MO) were used to calibrate the index of
labeling and therefore quantify the total amount of hybridization. The
smallest two standards were plotted against a known index of staining
(disintegrations per min/mm2), and the background was
defined as zero, providing a standard curve for the quantification of
labeling density. Slides were then dipped in Ilford K5 photographic
emulsion (Ilford Australia, Mount Waverly, Australia), exposed at 4 C
for 7 nights, and developed using Ilford phenisol x-ray developer, stop
bath, and Hypam fixer. Sections were counterstained with 1% cresyl
violet and coverslipped using DPX (BDH Laboratory Supplies, Melbourne,
Australia). Analysis at the cellular level was carried out on
the emulsion-dipped slides. Five labeled cells per section were
analyzed by counting silver grains under x400 magnification, and the
number of cells expressing NPY messenger RNA (mRNA) was counted at x20
magnification. Densitometry and silver grain counts were performed
using the microcomputer imaging device M1 system from the Imaging Research, Inc. (Brock University, St. Catherines, Canada).
Data analysis
Pulse analysis was performed for patterns of LH and GH
secretion, and mean daily levels were used to study effects on the
other hormones and metabolic parameters. LH pulses were defined as
previously described (33). The mean GH concentration, interpulse
interval, and pulse amplitude were determined using the TURBOPULSAR
program (34). This program enables the selection of both high narrow
peaks and smaller broad peaks based on the G parameters. The G
parameters used were G(1) = 4.4, G(2) = 2.0, G(3) = 1.0, G(4) = 0.75,
and G(5) = 0.5. The within-assay CV and assay sensitivity were also
taken into consideration, as defined by Fletcher and Clarke (34) in the
following quadratic equation: y = 24.4 (log
x)2 - 42.7 log x + 24.1.
Statistical analysis
All data were checked for homogeneity of variance; the average
plasma concentrations were subjected to square root transformation, and
the data for plasma glucose and lactate were transformed to log values.
Nontransformed data are presented as the mean ± SEM.
Repeated measures ANOVA was used to analyze the in vivo
data, and paired comparisons were made between groups on each day of
the experiment. Least significant differences were used to test for
significant differences between means. Data on the expression of NPY
mRNA in the arcuate nucleus were analyzed by single factor ANOVA. Data
for the number of silver grains per cell were subjected to square root
transformation.
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Results |
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)
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and 4)
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)
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and 7).
There was no significant difference in the number of cells labeled for
NPY mRNA in the arcuate nucleus between the two groups, but a
significant (P < 0.01) decrease was observed in the
number of silver grains per cell in the leptin-treated animals (Figs. 6 and 7).
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Discussion |
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It is now clear that leptin is a satiety factor in a wide range of species, including rodents (1, 2) and pigs (8). In addition, a range of studies in the rodent (5, 7, 8, 12, 35) and pig (8) has shown that leptin may regulate neuroendocrine function. The hypophysiotropic peptides of the hypothalamus regulate pituitary hormone secretion, and it is most likely that the effects of centrally administered leptin on endocrine function would involve some alteration in the secretion of these peptides. In rodents, either centrally (7) or peripherally (5, 12, 36) administered leptin will alter endocrine function. Whether this is due to direct action on the hypophysiotropic cells of the hypothalamus or on the endocrine cells of the pituitary gland is not yet clear. Leptin receptors have been identified in the hypothalamus (17, 24, 37, 38), but there has as yet been no report of these receptors on neuroendocrine cells. Likewise, receptors have been demonstrated at the level of the pituitary gland, but the cell types on which they are found have not been defined. As the hypothalamic receptors can be localized to neurons that contain neuropeptide Y (17) and POMC (39), it would seem most likely that any effects on neuroendocrine function would be relayed through such cells. These neuropeptides affect both endocrine function (20) and appetite (40); it might be supposed that leptin would alter both. This is not necessarily the case in the sheep and raises doubts about extrapolating the data that have been obtained in rodents to nonrodent species. It should be noted, however, that the types of cells that express leptin receptor in the ovine brain remains unknown, and species differences may well exist in this regard.
Bronson (41) has indicated that alterations in food intake in rodents have a profound impact on reproductive function in a short time frame (<1 day), because these animals do not generally have large fat reserves. This contrasts with other species in which fat reserves are considerable. For example, the ideal body fat composition of the human female is 22% (42). Likewise, it has been shown that peripheral (5) and central (7) administration of leptin to starved mice/rats has a profound short term effect on the secretion of pituitary hormones. There have, however, been only limited studies carried out on other species. In pigs, an icv injection of 10 µg (and higher doses) leptin increased the secretion of GH. Our present data, in contrast, show no effect on GH levels during a 3-day infusion of a appetite-reducing dose of leptin.
The ruminant animal is capable of maintaining glycemia during periods of moderate negative energy balance (43). In the present study, icv leptin infusion and the associated reduction in food intake moved the sheep into negative energy balance, as indicated by plasma metabolites. For example, the increase in plasma NEFA concentrations that we observed is a clear indication of the mobilization of considerable amounts of fat reserves to spare glucose utilization (44, 45). During a period of extended undernutrition, plasma lactate normally decreases as glycogen stores are depleted (46), but over a period of semiacute undernutrition, as in the present study, plasma lactate concentrations would increase with mobilization of muscle glycogen and an increase in glucose carbon recycling. Accordingly, the sheep in the present study were able to maintain blood glucose levels by using mobilized NEFA as an alternative energy source and increasing gluconeogenesis from mobilized lactate, glycerol, and amino acids. It has been shown previously that restricted food intake does not alter plasma insulin levels in the sheep (43), and the present results are in accord with these earlier findings.
The situation is somewhat different from that obtained with food deprivation over 1–2 days in the primate (47), in which plasma glucose and insulin levels fall. Insulin levels probably play a significant role in the production of leptin in nonruminant species (48). Thus, to put the present results in context, we emphasize that the ruminant is a special case. Using the sheep, we have demonstrated the ability of leptin to reduce food intake over a period of 3 days without an effect on the secretion of pituitary hormones. This strongly suggests that when blood glucose and insulin levels are maintained, leptin does not have a direct effect on the neuroendocrine system.
The animals of this study were ovariectomized, and this may have a significant bearing on the results, especially with respect to gonadotropin secretion. The secretion of GnRH is not restrained by gonadal hormone feedback in the ovariectomized animal, and the ability of leptin to increase the secretion of GnRH or the gonadotropins (the former was not measured in this study) might be minimal. Yu et al. (6) reported small effects of leptin on the secretion of GnRH from hypothalamic fragments in vitro, but there are, as yet, no studies on the effects of leptin on GnRH secretion in vivo. Although it has been reported that immortalized GnRH neurons contain leptin receptors (24), these cells do not respond to leptin with an increase in GnRH secretion (Moenter, S., personal communication). To warrant the measurement of GnRH secretion in vivo, an effect on gonadotropin secretion would first have to be demonstrated. It is possible that leptin acts directly on the pituitary gland to influence the secretion of the gonadotropins, PRL, and other pituitary hormones, but iv infusion of the same amount of leptin (20 µg) over the same time course (3 days) had no effect (Clarke, I. J., unpublished data). A recent publication (49) showed that leptin prevents the reduction in pulsatile LH secretion that is seen in fasted female rats that received either vehicle or estrogen. As leptin treatment restored the frequency of LH pulses, this suggested action at the central level to influence GnRH secretion. Leptin did not overcome the negative feedback effect of estrogen. It remains possible that an interaction exists between leptin and steroid effects on GnRH secretion in the sheep, and current studies are investigating this.
We (21) have previously shown that low dietary intake, which decreased body weight in ewes, elevated plasma GH levels, whereas overweight sheep have reduced GH levels (29, 33). If leptin is to be regarded as the metabolic indicator that signals the amount of fat stores, then it might be expected to reduce plasma GH levels in sheep. It was somewhat surprising, therefore, that leptin did not alter the plasma levels of GH in the present study. Carro et al. (7) showed that leptin could restore plasma levels of GH to normal if administered to starved rats, in which GH secretion is reduced, but an effect was not seen in normally fed animals. Recent in vitro studies (50) have shown that leptin reduces the responsiveness of ovine pituitary somatotropes to GH-releasing hormone, but stimulates GH secretion. In pigs, icv injection of 10 µg leptin (and higher doses) (8) reduced food intake and increased plasma GH levels. Whether this reflects a difference between monogastric species and ruminants is not clear at this stage. It would seem likely that these doses in rodents are probably supraphysiological. Continuous infusion of smaller doses, rather than large bolus doses, of leptin may be expected to have different effects.
As mentioned above, a single icv injection of leptin caused an increase in GH secretion in pigs (8). This is not consistent with the idea that leptin signals an increase in adiposity that is associated with reduced plasma GH levels in most species. On the other hand, it is consistent with the well documented effect of fasting, which increases plasma GH levels (51). Thus, it might be the case that the effect of leptin in the pig is an indirect effect of reduced food intake rather than a direct action of the satiety factor. It should also be noted that these results were obtained with a single injection of leptin. We have been unable to show an effect of a 24-h infusion of 8.3 µg/h leptin on food intake or hormone levels in sheep (Henry, B., and I. J. Clarke, unpublished data). This suggests that a longer infusion time might be required to obtain any meaningful effect in this species. It was not possible to do this in the current study with the dose that we used, because food intake was severely reduced in some animals, and continuation would not have been ethically acceptable. Using lower doses of leptin would enable a longer period of treatment and could yield different results.
In addition to the effects on the reproductive axis and GH levels in rodents, leptin has been shown to influence the function of the hypothalamo-pituitary-adrenal (5, 36) and the hypothalamo-pituitary-thyroid (5, 35) axis. The importance of leptin in maintaining endocrine function is evident in ob/ob mice, which suffer a number of anomalies, including diabetes, hypercorticoidism, and hypothyroidism (52). Consistent with the lack of an effect of icv leptin infusion on plasma gonadotropin and GH levels, we have also shown that this treatment does not affect plasma PRL and cortisol levels. The latter gives some representation of the hypothalamo-pituitary-adrenal axis. Thus, we conclude that satiety-inducing doses of leptin do not affect the neuroendocrine systems, although we have not studied the hypothalamo-pituitary-thyroid axis.
Leptin treatment decreased the expression of NPY mRNA in the arcuate nucleus, which is consistent with previous studies in the rodent species (5, 53). The decrease was due to a reduction in the expression of NPY and not to a decrease in the number of NPY-labeled cells. The leptin receptor is expressed in NPY-containing cells in the rodent (17), and this neuropeptide plays a central role in the control of appetite. The obesity syndrome in ob/ob mice is attenuated in NPY gene knockout mouse. Double mutant mice (ob/ob NPY-/-) are less obese, have lowered food intake and increased energy expenditure, and do not show the same incidence of diabetes, sterility, and somatotropic defects that is seen in ob/ob (54). In the sheep, there is evidence that NPY regulates the GnRH-LH axis (19) and CRF-arginine vasopressin secretion (18). In the present study, however, we observed a decrease in the expression of NPY, but this was without effect on the secretion of pituitary hormones or cortisol. One possible explanation could be that leptin acts on a subset of NPY cells that regulates appetite, but not on those NPY cells that regulate hypophysiotropic neurons. Other cells (e.g. POMC) in the arcuate nucleus (39, 55, 56) or in other parts of the brain (57, 58) may also mediate the effect of leptin. In conclusion, we have shown that leptin can inhibit food intake in sheep, but, in contrast to the results obtained in other species, the satiety-inducing dose of leptin was unable to alter plasma levels of LH, FSH, GH, PRL, and cortisol. A catabolic state was achieved with leptin treatment, but this did not alter the plasma glucose concentration. These results suggest that the metabolic factors that regulate food intake may be different from those that regulate neuroendocrine function.
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Acknowledgments |
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Footnotes |
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Received July 9, 1998.
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