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Localization of leptin receptor immunoreactivity in the lean and obese Zucker rat brain
Brain Research 785 Ž1998. 80–90
Research report
Localization of leptin receptor immunoreactivity in the lean and obese Zucker rat brain Deborah O. Yarnell a , David S. Knight b
b, )
, Kathryn Hamilton b , Orien Tulp a , Patrick Tso
c
a Department of Bioscience and Biotechnology, Drexel UniÕersity, Philadelphia, PA 19104, USA Department of Cellular Biology and Anatomy, LSUMC, 1501 King’s Highway, ShreÕeport, LA 71130, USA c Department of Pathology, UniÕersity of Cincinnati, 231 Bethesda AÕe., Cincinnati, OH 45267, USA
Accepted 11 November 1997
Abstract Leptin, a product of the obese Žob. gene, is secreted by adipocytes and appears to act as a hormone to regulate food intake, metabolism and body weight. Subcutaneous administration of leptin causes reductions in food intake and body and fat-depot weights in both lean and genetically obese Žobrob. mice, and leptin infusion into the lateral cerebral ventricles decreases feeding with short latency, suggesting a central site of action. A gene defect in the Zucker obese rat causes an amino acid substitution in the leptin receptor and reduced leptin binding at the cell surface. An antiserum to a portion of the mouse leptin receptor ŽAA 877–894. located within the intracellular domain was used to label Zucker lean ŽFar?. and obese Žfarfa. rat brain sections. At optimal dilution Ž1:8000., only cells in the basal forebrain, preoptic area, hypothalamus and brainstem were moderately or intensely labeled. The most intensely-labeled nuclei, the anterior commissural, magnocellular paraventricular, supraoptic, circularis in the anterior hypothalamus and fornical in the lateral hypothalamus contain large neurons that synthesize and secrete vasopressin or oxytocin and their respective neurophysins. Diminished leptin transport into the central nervous system or defective signal transduction in Zucker obese rats may sufficiently compromise leptin regulation of the HPA axis, NPY-immunoreactive neurons or other hypothalamic elements to cause obesity. q 1998 Elsevier Science B.V. Keywords: Zucker; Vasopressin; Obesity; Oxytocin; Leptin; Neuropeptide Y
1. Introduction Leptin, a product of the obese Žob. gene, is secreted by adipocytes, and circulating leptin concentrations correlate positively with body mass index and percent body fat w14x. Leptin appears to act as a hormone to decrease food intake, body weight, and secretion of insulin and corticosterone w19x. Leptin levels are elevated in Zucker obese Žfarfa. rats and are increased by feeding, insulin and glucocorticoids in Zucker lean rats but not in the obese rats w21x. Levin et al. w30x demonstrated that subcutaneous leptin infusion causes reductions in food intake, body weight and fat-depot weight in both lean and genetically obese Žobrob. mice, and leptin infusion into the lateral cerebral ventricles decreases feeding with short latency, suggesting a central site of action w8x.
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Corresponding author. Fax: q1-318-675-5889.
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 3 8 8 - 7
Lynn et al. w33x used autoradiography to study the binding of w 125 Ix leptin to frozen sections of obrob and dbrdb mouse brains, and found dense specific binding only in choroid plexus in the third and lateral ventricles. Malik and Young w36x also found leptin binding sites only in the choroid plexus and leptomeninges of C57BLr6J mice. On the other hand, Mercer et al. w38x used a probe that recognized all known splice variants to demonstrate expression of the leptin receptor gene in mouse hypothalamus and other brain regions. There are at least six splice variants of the leptin receptor gene ŽOb-R. w27x. Splice variant Ob-Rb encodes a receptor with a long intracellular domain that may mediate signal transduction, whereas other variants that encode receptors with short intracellular domains may transport leptin across the blood–brain or other barriers w57x. Mutations in the Ob-R gene are believed to underlie the abnormalities in energy balance observed in some rodent models of obesity, and Chua et al. w12x showed by genetic mapping and genomic analysis that mouse diabetes Ždb., rat fatty Žfa. and Ob-R are the same
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gene. Chen et al. w10x identified a mutation in the dbrdb mouse leptin receptor gene that results in a 106 bp insertion and a truncated intracellular domain, suggesting that the long intracellular domain of Ob-Rb is critical for signal transduction. Chua et al. w11x identified a point mutation in the fa gene that causes an amino acid substitution at position 269 Žglutamine™ proline., a region of the extracellular domain. The mutant leptin receptor has a 10-fold reduced leptin binding at the cell surface with no decrease in affinity for leptin. Neither of these mutations was detected by Considine et al. w13x in humans but a mutation resulting in a substitution at position 223 Žglutamine™ arginine. was detected. Increased plasma leptin concentrations in animal models and in obese humans may indicate reduced sensitivity to leptin resulting from such mutations w32,34x. Determining the location of Ob-R in the brain is critical to elucidating the role it plays in energy balance. Reduced leptin binding in the Zucker obese rat may affect receptor density or distribution in the central nervous system. The present immunohistochemical study was designed to determine the specific brain structures that express the leptin receptor in Zucker lean ŽFarFa or Farfa. and obese Žfarfa. rats. An antiserum ŽM-18. to a portion of the mouse leptin receptor ŽAA 877–894. located within the intracellular domain was used to label Zucker rat brain sections. The fatty Žfa. mutation noted above produces hyperphagia, diminished thermogenesis by brown fat and profound obesity. The defect may exert an effect in the hypothalamus where concentrations or turnover of both orexigenic and anorexigenic amines and peptides are abnormal w62x. The obese rats have elevated basal plasma insulin, adrenocorticotropic hormone ŽACTH. and corticosterone ŽCS. concentrations, and pituitary and adrenal cortical hypertrophy, all of which may be induced by hypothalamic dysfunction w25x.
2. Materials and methods 2.1. Animals Six lean and four obese female Zucker rats from Charles River ŽWilmington, MS. were housed individually in plastic shoe box cages lined with pine shavings. Each rat was fed a pellet diet ŽHarlan Teklad, Rodent Diet a8640. and tap water ad libitum for 12–16 weeks prior to sacrifice, at which time the rats were 16–24 weeks old. 2.2. Antiserum An antiserum to a unique segment of the mouse leptin receptor and the corresponding control peptide were purchased from Santa Cruz Biotechnology ŽSanta Cruz, CA.. The epitope ŽAA 877–894. used to produce the goat
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polyclonal antiserum ŽM-18. lies in the intracellular domain of the leptin receptor. 2.3. Tissue fixation and sectioning The rats were anesthetized with sodium pentobarbital Ž45–90 mgrkg. then perfused transcardially with 150 ml of 0.1 M Sorensen’s phosphate buffer ŽpH 7.3–7.4. containing 0.5% procaine amide Ž p-aminobenzoic acid diethylaminoethylester. and 5000 U of heparin followed by 250 ml of 4% formaldehyde in cold Sorensen’s buffer. After perfusion, each brain was removed from the skull and fixed overnight in cold 4% formaldehyde, rinsed in the buffer, and immersed in 20% then 30% sucrose for 24 h each at 48C. After cryoprotection, each brain was quickly frozen at y558C and sectioned with a Reichert Frigocut cryostat. From each brain, six sets of 40-m m thick sections were collected and stored in Sorensen’s buffer at 48C. 2.4. Labeling and staining Sections were pretreated for 30 min in 0.2% H 2 O 2 in 50% methanol in Sorensen’s buffer, rinsed three times in the same buffer and presoaked for 1 h in the buffer containing 0.3% Triton X-100, 0.45% NaCl, 0.25% gelatin, and 4% normal horse serum prior to incubation in the primary antiserum. Serial dilutions of the M-18 antiserum Ž1:400 to 1:8000. in Sorensen’s buffer containing 0.3% Triton X-100, 0.45% NaCl and 0.1% sodium azide were used to determine an optimum dilution and differentiate specific labeling and background. Brain sections were incubated in primary antiserum for 16 h at room temperature, and control sections were incubated under the same conditions in primary antiserum preabsorbed overnight with 2 = 10y6 M control peptide and centrifuged for 30 min at 15,600 = g. All sections were incubated in biotinylated horse anti goat IgG and avidin biotinylated horseradish peroxidase ŽABC. complex ŽVector Labs, Burlingame, CA. for 1 h each with agitation and intervening buffer rinses. Horseradish peroxidase was visualized by reaction with 0.05% diaminobenzidine tetrahydrochloride in 0.1 M Tris buffer ŽpH 7.6. containing 0.003% H 2 O 2 . The sections were rinsed in buffer, mounted in serial order on slides, dehydrated in graded alcohols, counterstained with eosin, cleared in xylene and coverslipped with Permount. The Rat Brain in Stereotaxic Coordinates by Paxinos and Watson w44x was the source of most rat brain nomenclature. Nomenclature of magnocellular elements was derived from the study by Rhodes et al. w47x. Specific hypothalamic nuclei in labeled, counterstained sections were identified by comparison with sets of Zucker rat brain sections stained with acid thionin or with sections illustrated in The Rat Brain in Stereotaxic Coordinates w44x. Sections were photographed with an Olympus BH2 microscope and Kodak T-Max 100 film.
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3. Results
Incubation of brain sections in the M-18 antiserum at a dilution of 1:8000 produced moderate to intense labeling of specific cell populations in the basal forebrain, preoptic area, hypothalamus and brainstem. In each labeled cell, the DAB reaction product appeared to be deposited as a reticulum within the cytoplasm ŽFig. 1.. As shown in Fig. 2, all labeling was abolished by preabsorption of the M-18 antiserum diluted 1:8000 or 1:4000 with 2 = 10y6 M control peptide. Cells in the anterior commissural nucleus ŽACN. and a few cells scattered in the medial preoptic area ŽMPOA. were intensely labeled. In the hypothalamus, there were intensely labeled cells in the magnocellular subdivision of the paraventricular nucleus ŽPVN., the supraoptic nucleus ŽSON., and the nucleus circularis ŽNC. which consists of clusters of cells centrally located within the anterior nucleus. There was no apparent difference in labeling intensity or in the distribution of labeled cell bodies between Zucker lean and obese animals. This is illustrated by similar density of the DAB reaction product in the SON of lean ŽFig. 3. and obese ŽFig. 4. Zucker rats. The most intense labeling was seen in magnocellular elements in preoptic and hypothalamic areas. Labeled cells that comprise the ACN ŽFig. 5. lie in the preoptic area rostral to the PVN, ventral to the fornix and adjacent to the bed nucleus of the stria terminalis. Numerous cells in the magnocellular subdivision of the PVN ŽFig. 6. were labeled, whereas fewer cells in medial and posterior parvocellular subdivisions ŽFig. 6. were labeled. Cells throughout the SON ŽFigs. 3 and 4., including the retrochiasmatic subdivision ŽFig. 7. were intensely labeled. In the central region of each anterior hypothalamic nucleus, there were two or three small clusters of labeled cells that comprise the NC. A row of labeled cells accompanied some blood vessels which entered the hypothalamus lateral to the optic chiasm and penetrated the SON and anterior nucleus. Some of the clusters of cells that comprise the NC in the anterior hypothalamus appeared to lie at the distal extent of such rows of cells. Intense labeling was also seen in the dorsal hypothalamus ŽDA. and in the fornical nucleus ŽFN. and in other cells of the lateral hypothalamus ŽLH.. There were few labeled cells in the DA, whereas such cells in the LH ŽFig. 7. were numerous, and most were widely dis-
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persed in caudal regions of the lateral hypothalamus. In the lateral hypothalamus, the FN was the only nucleus that contained compact clusters of labeled cells. There were moderately-labeled cells in the nucleus of the horizontal limb of the diagonal band ŽHDB. and associated magnocellular preoptic nucleus ŽMCPO. and in the suprachiasmatic ŽSCN., ventromedial ŽVM. and arcuate ŽARC. hypothalamic nuclei. Labeled cells in the SCN ŽFig. 8. and ARC nucleus ŽFig. 9. were uniformly small and evenly distributed within the nucleus. Cells in the dorsomedial subdivision of the VM ŽFig. 9. were more intensely labeled than those in other subdivisions of the nucleus. Incubation of brainstem sections in M-18 antiserum at a dilution of 1:8000 selectively labeled cell populations in the periaqueductal gray ŽPAG., locus ceruleus ŽLC., dorsal motor nucleus of the vagus nerve ŽDMN-X., nucleus of the tractus solitarius ŽNTS. and inferior olivary nucleus ŽION.. Such cells were lightly or moderately immunolabeled compared with the more intensely labeled cells in the PVN or SON of the hypothalamus. Moderately labeled cells in the PAG ŽFig. 10. were sparse and most were located at the level of the superior colliculi near the Edinger–Westphal nuclei. Compact clusters of cells in the LC, DMN-X and ION were labeled with moderate intensity. Labeling intensity in the DMN-X ŽFig. 11., ION and LC ŽFig. 12. was well below that seen in the PVN. Cells of the NTS ŽFig. 11. had only a light, fine deposition of the DAB reaction product. Cell populations that appeared to be specifically labeled at the optimal antiserum concentration Ž1:8000 dilution. were intensely labeled at greater concentrations Ž1:400 to 1:4000 dilution. but at these concentrations, additional cell populations in the cerebral cortex, basal ganglia, hippocampus, thalamus, hypothalamus, and brainstem were lightly labeled as well. Cells lightly immunolabeled at 1:4000 included pyramidal cells of the cerebral cortex and hippocampus, sparsely distributed cells in the caudate putamen and substantia innominata, and cells in several thalamic nuclei Žanterodorsal, habenular, paraventricular, mediodorsal, rhomboid and reuniens.. Hypothalamic nuclei that contained lightly immunoreactive cells at this dilution included the periventricular, dorsomedial and ventral premammillary. There was no labeling of choroid plexus or leptomeninges at any antiserum concentration.
Fig. 1. A section of a Zucker lean hypothalamus showing cells of the supraoptic nucleus that are immunoreactive for amino acid segment 877–894 of the leptin receptor and illustrating cytoplasmic deposition of the DAB reaction product. Bar s 60 m m. Fig. 2. A section of a Zucker lean hypothalamus showing absence of labeling in a supraoptic nucleus ŽS. incubated with M-18 antiserum that had been preabsorbed with 2 = 10y6 M control antigen ŽAA 877–894.. Penetrating artery ŽA., Optic Chiasm ŽOC.. Bar s 60 m m. Fig. 3. The cells of the supraoptic nucleus in a Zucker lean rat brain section labeled with the M-18 antiserum. Optic chiasm ŽOC.. Bar s 120 m m. Fig. 4. The cells of the supraoptic nucleus in a Zucker obese rat brain section labeled with the M-18 antiserum. Optic chiasm ŽOC.. Bar s 120 m m.
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4. Discussion At optimal dilution of a polyclonal antiserum ŽM-18. raised against a unique segment ŽAA 877–894. of the leptin receptor, only cells in the basal forebrain, preoptic area, hypothalamus and brainstem were moderately or intensely labeled. The failure to detect obvious differences in labeling intensity or distribution of cell bodies between the Zucker lean and obese animals suggests that receptor distribution is not substantially altered by the elevated circulating leptin or diminished leptin receptor density in the obese animal w11x. Leptin receptors with a short intracellular domain which are found in choroid plexus and leptomeninges do not appear to bind the M-18 antibodies. The PVN, SON, NC, FN, SCN, DA, LH, VM and ARC nuclei of the hypothalamus and cells in the ACN and MPOA were moderately or intensely labeled with minimal background. Cells in the PAG, LC, DMN-X and ION of the brainstem were moderately labeled, and cells in the NTS were slightly immunoreactive. Mercer et al. w38x, using a probe that recognized all known splice variants, reported expression of the leptin receptor gene in the mouse hypothalamus and other brain regions. Such mRNA was visualized in cerebral cortex, hippocampus, thalamus and hypothalamus, and in choroid plexus and leptomeninges. In the same study, a probe specific for Ob-Rb, the variant with a long intracellular domain that is highly expressed in the hypothalamus, revealed strong expression in hypothalamic nuclei which have been implicated in regulation of energy balance ŽPVN, VM and ARC., suggesting that these nuclei are sites of leptin action. Baskin et al. w4x studied the distribution of leptin receptor mRNA in mice and found no differences in obrob, dbrdb and lean wild type mice each of which strongly expressed the receptor in the choroid plexus and the ARC, VM and dorsomedial hypothalamic nuclei with weaker expression in the PVN and hippocampus. In the present study, the most intensely-labeled nuclei, the ACN, magnocellular PVN, SON, NC in the anterior hypothalamus and FN in the lateral hypothalamus contain large neurons that synthesize and secrete arginine vasopressin ŽAVP. or oxytocin ŽOXT. and their respective neurophysins w47x. Although co-localization of leptin re-
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ceptors with AVP or OXT has not been demonstrated, it seems likely on the basis of receptor distribution that one or both types of cell may be involved in the response to leptin binding. Each peptide, administered intraperitoneally or intracerebroventricularly, has been reported to reduce food intake w2,26x. Cholecystokinin octapeptide and Dfenfluramine each enhances synthesis and secretion of AVP and OXT which may mediate, in part, the effects of these well-known anorexigenic agents w37,39x. The PVN is believed to play a prominent role in regulation of energy balance, and studies of farfa rats have implicated abnormally low activity of neurons that synthesize corticotropin releasing hormone ŽCRH., most of which are located in parvocellular subdivisions of the PVN w42x. Nevertheless, vasopressinergic neurons, some of which co-express CRH, may also be dysregulated in this and other rodent models of obesity w50x. CRH and AVP act in a synergistic manner to increase circulating ACTH, and neurons that express both peptides may function to rapidly modulate ACTH release w17,60x. AVP may also directly increase secretion of CRH w6x. Compared with Zucker lean rats, Zucker obese rats whose leptin receptor density is diminished have been reported to secrete 73% and 35% less CRH and AVP, respectively into the hypophyseal portal blood w45x. Adrenalectomy which prevents the development of obesity in the farfa rat increases synthesis of CRH and also enhances expression of AVP in hypothalamic neurons that synthesize CRH w61x. Slieker et al. w54x reported that leptin reduces adipose ob mRNA in vivo but has no effect on isolated rat adipocytes, and suggested negative feedback by leptin mediated by the hypothalamic–pituitary–adrenal ŽHPA. axis. Since leptin is known to decrease circulating corticosterone, one might expect decreased leptin binding in the hypothalamus to produce an increase rather than the decrease in CRH secretion reported in the Zucker obese rat. However, Schwartz et al. w52x found that leptin, administered during a 40-h fast, increased levels of CRH mRNA in the PVN of Long–Evans rats, and Raber et al. w46x showed that leptin increases CRH but not AVP release from hypothalamic slices, so that decreased binding in Zucker obese rats may decrease CRH secretion. If leptin increases secretion of CRH but not AVP, leptin resistance could decrease the ratio of CRH to AVP secreted. Such a change could potentially decrease
Fig. 5. A section through the preoptic region of a Zucker obese rat showing cells of the anterior commissural nucleus Žarrow. that are labeled by the M-18 antiserum. Third ventricle ŽIII.. Bar s 230 m m. Fig. 6. A section of a Zucker obese hypothalamus showing cells immunoreactive for amino acid segment 877–894 of the leptin receptor. Labeled cells are located in parvocellular and magnocellular subdivisions of the paraventricular hypothalamic nucleus. Bar s 120 m m. Fig. 7. A section through a caudal region of a Zucker lean hypothalamus illustrating intense labeling by the M-18 antiserum in the retrochiasmatic supraoptic nucleus Žarrow. and in other widely-dispersed cells in the lateral hypothalamus. Fornix ŽF.. Bar s 230 m m. Fig. 8. A section of a Zucker obese hypothalamus showing small, evenly-distributed cells of the suprachiasmatic nucleus labeled by the M-18 antiserum. Suprachiasmatic nucleus ŽSN., Optic chiasm ŽOC.. Bar s 120 m m.
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the ratio of CRH to ACTH secreted and alter energy balance in favor of weight gain. The anterior pituitary expresses leptin receptor mRNA as well w66x, and leptin has been reported to release ACTH from pituitary slices
w46x. It has been suggested that saturation of the leptin transport system may cause a loss of balance between the effects of leptin on the hypothalamus and pituitary in which CRH release is decreased whereas ACTH is in-
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creased w46x. Feedback inhibition by elevated corticosterone would tend to further decrease CRH secretion. The fact that circulating ACTH and corticosterone are elevated in the Zucker obese rat whereas CRH and AVP secretion appears to be diminished, may be explained by such a loss of balance. Other potential mediators of increased circulating ACTH and corticosterone are increased hypothalamic neuropeptide Y ŽNPY. or decreased neurotensin ŽNT. secretion, each of which is characteristic of Zucker rat and other models of obesity w5,16,59x. These peptides may directly influence anterior pituitary corticotrophs and modulate release of ACTH w23,35x. Diminished secretion of CRH would tend to increase synthesis of hypothalamic NPY as well as decrease sympathetic nervous system activity and thermogenesis w22x. Both parvocellular and magnocellular subdivisions of the PVN and other nuclei intensely immunoreactive for amino acid segment 877–894 of the leptin receptor ŽACN, SCN, SON. are principal sites of projection of NPY-immunoreactive neurons located in the ARC nucleus and in the LC. Cell populations in the ARC nucleus and in the LC were also moderately labeled by the M-18 antiserum in the present study. Hypothalamic NPY increases synthesis and secretion of both CRH and AVP w20,28,55,64x, effects apparently blocked or overridden by other mechanisms in the Zucker obese rat in which synthesis and secretion of hypothalamic NPY are enhanced. Schwartz et al. w52x showed that intraperitoneal injections of leptin lowered NPY mRNA selectively in the ARC nuclei of leptin-deficient obrob mice by 42.3%. Diminished leptin binding in the Zucker obese ARC nucleus may therefore facilitate increased synthesis and secretion of hypothalamic NPY. Such a defect is suggested by Glaum et al. w18x who studied the effects of leptin on synaptic transmission in Zucker lean and obese hypothalamic slices and found that it produced rapid modulatory effects in the ARC nucleus of lean rats but had no effect in obese rats. An effect in the ARC nucleus is also suggested by the failure of leptin to affect weight gain in monosodium glutamate-lesioned rats w15x. Some oxytocin-immunoreactive neurons in the anterior part of the magnocellular subdivision of the PVN co-express CRH and may play a role in energy balance w50x but few studies have implicated OXT in regulation of energy balance. Nevertheless, hypothalamic effects on thermogen-
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esis may be mediated, in part, by oxytocin-immunoreactive neurons that project from the PVN to the intermediolateral cell column w49x, whereas effects on insulin secretion may involve similar projections to the DMN-X w7x. CRH-induced anorexia may be mediated, in part, by OXT, as OXT antagonists have been shown to prevent the anorectic effects of central injections of CRH w43x. Loss of an anorexigenic effect of OXT may play a role in the Prader–Willi syndrome, a condition characterized by extreme obesity in which there is a 50% reduction in the number of OXT neurons in the PVN w56x. Nuclei with cells immunoreactive for amino acid segment 877–894 of the leptin receptor Žparvocellular and magnocellular PVN, SON, VM, ARC and NTS. have been shown to increase Fos expression in response to the potent orexigenic peptide NPY in combination with food intake w31x. Specific sites of leptin action are indicated by induction of Fos in response to altered leptin secretion. Mistry et al. w40x reported that the number of Fos-immunoreactive nuclei in the PVN of leptin-deficient obrob mice was three times that in lean control mice, and that food deprivation for 24 h increased the number of immunoreactive nuclei in lean mice but did not further increase the number in obese mice. Willing et al. w63x found increased Fos in the PVN, VM nucleus and LH after central leptin injection, whereas Woods and Stock w65x found that the PVN of obrob mice was the only neural structure strongly immunolabeled for Fos 3 h after peripheral leptin treatment, with slight labeling in the VM and ARC nuclei and zona incerta. In that study, increased immunoreactivity for Fos appears to lie in dorsal and medial parvocellular subnuclei of the PVN, in which neurons that synthesize CRH are located. Collectively, such studies suggest that there are leptin receptors in several nuclei which play a role in appetitive behavior, and that leptin receptor activity can influence transcription in the parvocellular PVN. Although the present study demonstrates more intense antibody binding in the magnocellular PVN, neurons that synthesize CRH or other parvocellular elements may be affected by leptin binding in closely-related nuclei. Other effects of leptin may involve two areas of the hypothalamus known to influence feeding behavior, the LH and VM nucleus, each of which contained cells intensely or moderately labeled by the M-18 antiserum respectively. Mizuno et al. w41x reported that transfer of the
Fig. 9. A section through the ventromedial ŽVM. and arcuate ŽARC. hypothalamic nuclei of a Zucker lean rat illustrating moderate immunoreactivity for amino acid segment 877–894 in cells of these two nuclei. Bar s 230 m m. Fig. 10. Cells Žarrow. labeled by the M-18 antiserum in the periaqueductal gray near the Edinger–Westphal nuclei of a Zucker lean rat. Bar s 230 m m. Fig. 11. A section through the medulla oblongata of a Zucker lean rat illustrating cells in the nucleus of the tractus solitarius ŽNTS. and dorsal motor nucleus of the vagus nerve ŽDMNX. labeled by the M-18 antiserum. Bar s 120 m m. Fig. 12. A section through the pons of a Zucker lean rat illustrating cells in the locus ceruleus ŽLC. labeled by the M-18 antiserum. Bar s 120 m m.
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ob gene into the VM of the Sprague–Dawley rat inhibits body weight gain and food intake. Immunoreactivity in the HDB and associated MCPO nuclei may suggest an interaction with olfactory systems, as at least two distinct neuronal populations in the HDB project to the olfactory bulb and may interact with inputs from the ACN which was intensely labeled by the M-18 antiserum in the present study. Shipley and Ennis w53x suggested that such interaction may help to regulate transmission of olfactory information between the two hemispheres. Brainstem immunoreactivity for amino acid segment 877–894 of the leptin receptor was visualized in the LC whose noradrenergic neurons co-express NPY and project rostrally to widespread regions of the telencephalon and diencephalon including the hypothalamus. Noradrenergic innervation of both parvocellular and magnocellular subdivisions of the PVN and of the SON is especially dense compared with other hypothalamic areas w29x. Clusters of labeled cells in the PAG may also project rostrally but the identity of such cells was not determined in this study. Leptin binding in the DMN-X could affect gastrointestinal motility and secretion, including secretion of insulin which is decreased by leptin and elevated in several models of obesity. A direct effect of insulin on adipocytes is suggested by studies of Kolaczynski et al. w24x who demonstrated a long-term effect of insulin to increase leptin production both in vivo and in vitro, and Saladin et al. w48x who showed that a single insulin injection increased ob mRNA in fasted rats to levels of fed controls. A synergistic interaction between leptin and cholecystokinin to activate gastric vagal afferents has also been reported w58x. Such afferents have central connections to the NTS, some of whose cells were lightly labeled by the M-18 antiserum in the present study. Several brainstem sites immunoreactive for amino acid segment 877–894 of the leptin receptor also express receptors for AVP or OXT ŽPAG, LC, DMNX, NTS, ION. w3x. Hypothalamic nuclei implicated in regulation of energy balance and other nuclei in the basal forebrain, preoptic area and brainstem of Zucker lean and obese rats were immunoreactive for amino acid segment 877–894 of the leptin receptor. Leptin may therefore interact with multiple central pathways to regulate food intake, metabolism and body weight. Less intense labeling of other cell populations in widespread areas of the brain may reflect cross reactivity with other receptor proteins or may indicate multiple regulatory roles for leptin including regulation of gonadal and thyroid axes in response to fasting w1x. Although CSF leptin levels show a positive correlation with adiposity and are elevated in obese individuals, CSF:plasma leptin ratios are lower in obese than in lean individuals w9x. Therefore, diminished leptin transport into the central nervous system, or defective signal transduction in obese individuals may sufficiently compromise leptin regulation of the HPA axis, NPY-immunoreactive neurons or other hypothalamic elements to cause obesity w13,51x.
Acknowledgements This work was supported by NIH grant DK 32288.
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Research report
Localization of leptin receptor immunoreactivity in the lean and obese Zucker rat brain Deborah O. Yarnell a , David S. Knight b
b, )
, Kathryn Hamilton b , Orien Tulp a , Patrick Tso
c
a Department of Bioscience and Biotechnology, Drexel UniÕersity, Philadelphia, PA 19104, USA Department of Cellular Biology and Anatomy, LSUMC, 1501 King’s Highway, ShreÕeport, LA 71130, USA c Department of Pathology, UniÕersity of Cincinnati, 231 Bethesda AÕe., Cincinnati, OH 45267, USA
Accepted 11 November 1997
Abstract Leptin, a product of the obese Žob. gene, is secreted by adipocytes and appears to act as a hormone to regulate food intake, metabolism and body weight. Subcutaneous administration of leptin causes reductions in food intake and body and fat-depot weights in both lean and genetically obese Žobrob. mice, and leptin infusion into the lateral cerebral ventricles decreases feeding with short latency, suggesting a central site of action. A gene defect in the Zucker obese rat causes an amino acid substitution in the leptin receptor and reduced leptin binding at the cell surface. An antiserum to a portion of the mouse leptin receptor ŽAA 877–894. located within the intracellular domain was used to label Zucker lean ŽFar?. and obese Žfarfa. rat brain sections. At optimal dilution Ž1:8000., only cells in the basal forebrain, preoptic area, hypothalamus and brainstem were moderately or intensely labeled. The most intensely-labeled nuclei, the anterior commissural, magnocellular paraventricular, supraoptic, circularis in the anterior hypothalamus and fornical in the lateral hypothalamus contain large neurons that synthesize and secrete vasopressin or oxytocin and their respective neurophysins. Diminished leptin transport into the central nervous system or defective signal transduction in Zucker obese rats may sufficiently compromise leptin regulation of the HPA axis, NPY-immunoreactive neurons or other hypothalamic elements to cause obesity. q 1998 Elsevier Science B.V. Keywords: Zucker; Vasopressin; Obesity; Oxytocin; Leptin; Neuropeptide Y
1. Introduction Leptin, a product of the obese Žob. gene, is secreted by adipocytes, and circulating leptin concentrations correlate positively with body mass index and percent body fat w14x. Leptin appears to act as a hormone to decrease food intake, body weight, and secretion of insulin and corticosterone w19x. Leptin levels are elevated in Zucker obese Žfarfa. rats and are increased by feeding, insulin and glucocorticoids in Zucker lean rats but not in the obese rats w21x. Levin et al. w30x demonstrated that subcutaneous leptin infusion causes reductions in food intake, body weight and fat-depot weight in both lean and genetically obese Žobrob. mice, and leptin infusion into the lateral cerebral ventricles decreases feeding with short latency, suggesting a central site of action w8x.
)
Corresponding author. Fax: q1-318-675-5889.
0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 3 8 8 - 7
Lynn et al. w33x used autoradiography to study the binding of w 125 Ix leptin to frozen sections of obrob and dbrdb mouse brains, and found dense specific binding only in choroid plexus in the third and lateral ventricles. Malik and Young w36x also found leptin binding sites only in the choroid plexus and leptomeninges of C57BLr6J mice. On the other hand, Mercer et al. w38x used a probe that recognized all known splice variants to demonstrate expression of the leptin receptor gene in mouse hypothalamus and other brain regions. There are at least six splice variants of the leptin receptor gene ŽOb-R. w27x. Splice variant Ob-Rb encodes a receptor with a long intracellular domain that may mediate signal transduction, whereas other variants that encode receptors with short intracellular domains may transport leptin across the blood–brain or other barriers w57x. Mutations in the Ob-R gene are believed to underlie the abnormalities in energy balance observed in some rodent models of obesity, and Chua et al. w12x showed by genetic mapping and genomic analysis that mouse diabetes Ždb., rat fatty Žfa. and Ob-R are the same
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gene. Chen et al. w10x identified a mutation in the dbrdb mouse leptin receptor gene that results in a 106 bp insertion and a truncated intracellular domain, suggesting that the long intracellular domain of Ob-Rb is critical for signal transduction. Chua et al. w11x identified a point mutation in the fa gene that causes an amino acid substitution at position 269 Žglutamine™ proline., a region of the extracellular domain. The mutant leptin receptor has a 10-fold reduced leptin binding at the cell surface with no decrease in affinity for leptin. Neither of these mutations was detected by Considine et al. w13x in humans but a mutation resulting in a substitution at position 223 Žglutamine™ arginine. was detected. Increased plasma leptin concentrations in animal models and in obese humans may indicate reduced sensitivity to leptin resulting from such mutations w32,34x. Determining the location of Ob-R in the brain is critical to elucidating the role it plays in energy balance. Reduced leptin binding in the Zucker obese rat may affect receptor density or distribution in the central nervous system. The present immunohistochemical study was designed to determine the specific brain structures that express the leptin receptor in Zucker lean ŽFarFa or Farfa. and obese Žfarfa. rats. An antiserum ŽM-18. to a portion of the mouse leptin receptor ŽAA 877–894. located within the intracellular domain was used to label Zucker rat brain sections. The fatty Žfa. mutation noted above produces hyperphagia, diminished thermogenesis by brown fat and profound obesity. The defect may exert an effect in the hypothalamus where concentrations or turnover of both orexigenic and anorexigenic amines and peptides are abnormal w62x. The obese rats have elevated basal plasma insulin, adrenocorticotropic hormone ŽACTH. and corticosterone ŽCS. concentrations, and pituitary and adrenal cortical hypertrophy, all of which may be induced by hypothalamic dysfunction w25x.
2. Materials and methods 2.1. Animals Six lean and four obese female Zucker rats from Charles River ŽWilmington, MS. were housed individually in plastic shoe box cages lined with pine shavings. Each rat was fed a pellet diet ŽHarlan Teklad, Rodent Diet a8640. and tap water ad libitum for 12–16 weeks prior to sacrifice, at which time the rats were 16–24 weeks old. 2.2. Antiserum An antiserum to a unique segment of the mouse leptin receptor and the corresponding control peptide were purchased from Santa Cruz Biotechnology ŽSanta Cruz, CA.. The epitope ŽAA 877–894. used to produce the goat
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polyclonal antiserum ŽM-18. lies in the intracellular domain of the leptin receptor. 2.3. Tissue fixation and sectioning The rats were anesthetized with sodium pentobarbital Ž45–90 mgrkg. then perfused transcardially with 150 ml of 0.1 M Sorensen’s phosphate buffer ŽpH 7.3–7.4. containing 0.5% procaine amide Ž p-aminobenzoic acid diethylaminoethylester. and 5000 U of heparin followed by 250 ml of 4% formaldehyde in cold Sorensen’s buffer. After perfusion, each brain was removed from the skull and fixed overnight in cold 4% formaldehyde, rinsed in the buffer, and immersed in 20% then 30% sucrose for 24 h each at 48C. After cryoprotection, each brain was quickly frozen at y558C and sectioned with a Reichert Frigocut cryostat. From each brain, six sets of 40-m m thick sections were collected and stored in Sorensen’s buffer at 48C. 2.4. Labeling and staining Sections were pretreated for 30 min in 0.2% H 2 O 2 in 50% methanol in Sorensen’s buffer, rinsed three times in the same buffer and presoaked for 1 h in the buffer containing 0.3% Triton X-100, 0.45% NaCl, 0.25% gelatin, and 4% normal horse serum prior to incubation in the primary antiserum. Serial dilutions of the M-18 antiserum Ž1:400 to 1:8000. in Sorensen’s buffer containing 0.3% Triton X-100, 0.45% NaCl and 0.1% sodium azide were used to determine an optimum dilution and differentiate specific labeling and background. Brain sections were incubated in primary antiserum for 16 h at room temperature, and control sections were incubated under the same conditions in primary antiserum preabsorbed overnight with 2 = 10y6 M control peptide and centrifuged for 30 min at 15,600 = g. All sections were incubated in biotinylated horse anti goat IgG and avidin biotinylated horseradish peroxidase ŽABC. complex ŽVector Labs, Burlingame, CA. for 1 h each with agitation and intervening buffer rinses. Horseradish peroxidase was visualized by reaction with 0.05% diaminobenzidine tetrahydrochloride in 0.1 M Tris buffer ŽpH 7.6. containing 0.003% H 2 O 2 . The sections were rinsed in buffer, mounted in serial order on slides, dehydrated in graded alcohols, counterstained with eosin, cleared in xylene and coverslipped with Permount. The Rat Brain in Stereotaxic Coordinates by Paxinos and Watson w44x was the source of most rat brain nomenclature. Nomenclature of magnocellular elements was derived from the study by Rhodes et al. w47x. Specific hypothalamic nuclei in labeled, counterstained sections were identified by comparison with sets of Zucker rat brain sections stained with acid thionin or with sections illustrated in The Rat Brain in Stereotaxic Coordinates w44x. Sections were photographed with an Olympus BH2 microscope and Kodak T-Max 100 film.
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3. Results
Incubation of brain sections in the M-18 antiserum at a dilution of 1:8000 produced moderate to intense labeling of specific cell populations in the basal forebrain, preoptic area, hypothalamus and brainstem. In each labeled cell, the DAB reaction product appeared to be deposited as a reticulum within the cytoplasm ŽFig. 1.. As shown in Fig. 2, all labeling was abolished by preabsorption of the M-18 antiserum diluted 1:8000 or 1:4000 with 2 = 10y6 M control peptide. Cells in the anterior commissural nucleus ŽACN. and a few cells scattered in the medial preoptic area ŽMPOA. were intensely labeled. In the hypothalamus, there were intensely labeled cells in the magnocellular subdivision of the paraventricular nucleus ŽPVN., the supraoptic nucleus ŽSON., and the nucleus circularis ŽNC. which consists of clusters of cells centrally located within the anterior nucleus. There was no apparent difference in labeling intensity or in the distribution of labeled cell bodies between Zucker lean and obese animals. This is illustrated by similar density of the DAB reaction product in the SON of lean ŽFig. 3. and obese ŽFig. 4. Zucker rats. The most intense labeling was seen in magnocellular elements in preoptic and hypothalamic areas. Labeled cells that comprise the ACN ŽFig. 5. lie in the preoptic area rostral to the PVN, ventral to the fornix and adjacent to the bed nucleus of the stria terminalis. Numerous cells in the magnocellular subdivision of the PVN ŽFig. 6. were labeled, whereas fewer cells in medial and posterior parvocellular subdivisions ŽFig. 6. were labeled. Cells throughout the SON ŽFigs. 3 and 4., including the retrochiasmatic subdivision ŽFig. 7. were intensely labeled. In the central region of each anterior hypothalamic nucleus, there were two or three small clusters of labeled cells that comprise the NC. A row of labeled cells accompanied some blood vessels which entered the hypothalamus lateral to the optic chiasm and penetrated the SON and anterior nucleus. Some of the clusters of cells that comprise the NC in the anterior hypothalamus appeared to lie at the distal extent of such rows of cells. Intense labeling was also seen in the dorsal hypothalamus ŽDA. and in the fornical nucleus ŽFN. and in other cells of the lateral hypothalamus ŽLH.. There were few labeled cells in the DA, whereas such cells in the LH ŽFig. 7. were numerous, and most were widely dis-
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persed in caudal regions of the lateral hypothalamus. In the lateral hypothalamus, the FN was the only nucleus that contained compact clusters of labeled cells. There were moderately-labeled cells in the nucleus of the horizontal limb of the diagonal band ŽHDB. and associated magnocellular preoptic nucleus ŽMCPO. and in the suprachiasmatic ŽSCN., ventromedial ŽVM. and arcuate ŽARC. hypothalamic nuclei. Labeled cells in the SCN ŽFig. 8. and ARC nucleus ŽFig. 9. were uniformly small and evenly distributed within the nucleus. Cells in the dorsomedial subdivision of the VM ŽFig. 9. were more intensely labeled than those in other subdivisions of the nucleus. Incubation of brainstem sections in M-18 antiserum at a dilution of 1:8000 selectively labeled cell populations in the periaqueductal gray ŽPAG., locus ceruleus ŽLC., dorsal motor nucleus of the vagus nerve ŽDMN-X., nucleus of the tractus solitarius ŽNTS. and inferior olivary nucleus ŽION.. Such cells were lightly or moderately immunolabeled compared with the more intensely labeled cells in the PVN or SON of the hypothalamus. Moderately labeled cells in the PAG ŽFig. 10. were sparse and most were located at the level of the superior colliculi near the Edinger–Westphal nuclei. Compact clusters of cells in the LC, DMN-X and ION were labeled with moderate intensity. Labeling intensity in the DMN-X ŽFig. 11., ION and LC ŽFig. 12. was well below that seen in the PVN. Cells of the NTS ŽFig. 11. had only a light, fine deposition of the DAB reaction product. Cell populations that appeared to be specifically labeled at the optimal antiserum concentration Ž1:8000 dilution. were intensely labeled at greater concentrations Ž1:400 to 1:4000 dilution. but at these concentrations, additional cell populations in the cerebral cortex, basal ganglia, hippocampus, thalamus, hypothalamus, and brainstem were lightly labeled as well. Cells lightly immunolabeled at 1:4000 included pyramidal cells of the cerebral cortex and hippocampus, sparsely distributed cells in the caudate putamen and substantia innominata, and cells in several thalamic nuclei Žanterodorsal, habenular, paraventricular, mediodorsal, rhomboid and reuniens.. Hypothalamic nuclei that contained lightly immunoreactive cells at this dilution included the periventricular, dorsomedial and ventral premammillary. There was no labeling of choroid plexus or leptomeninges at any antiserum concentration.
Fig. 1. A section of a Zucker lean hypothalamus showing cells of the supraoptic nucleus that are immunoreactive for amino acid segment 877–894 of the leptin receptor and illustrating cytoplasmic deposition of the DAB reaction product. Bar s 60 m m. Fig. 2. A section of a Zucker lean hypothalamus showing absence of labeling in a supraoptic nucleus ŽS. incubated with M-18 antiserum that had been preabsorbed with 2 = 10y6 M control antigen ŽAA 877–894.. Penetrating artery ŽA., Optic Chiasm ŽOC.. Bar s 60 m m. Fig. 3. The cells of the supraoptic nucleus in a Zucker lean rat brain section labeled with the M-18 antiserum. Optic chiasm ŽOC.. Bar s 120 m m. Fig. 4. The cells of the supraoptic nucleus in a Zucker obese rat brain section labeled with the M-18 antiserum. Optic chiasm ŽOC.. Bar s 120 m m.
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4. Discussion At optimal dilution of a polyclonal antiserum ŽM-18. raised against a unique segment ŽAA 877–894. of the leptin receptor, only cells in the basal forebrain, preoptic area, hypothalamus and brainstem were moderately or intensely labeled. The failure to detect obvious differences in labeling intensity or distribution of cell bodies between the Zucker lean and obese animals suggests that receptor distribution is not substantially altered by the elevated circulating leptin or diminished leptin receptor density in the obese animal w11x. Leptin receptors with a short intracellular domain which are found in choroid plexus and leptomeninges do not appear to bind the M-18 antibodies. The PVN, SON, NC, FN, SCN, DA, LH, VM and ARC nuclei of the hypothalamus and cells in the ACN and MPOA were moderately or intensely labeled with minimal background. Cells in the PAG, LC, DMN-X and ION of the brainstem were moderately labeled, and cells in the NTS were slightly immunoreactive. Mercer et al. w38x, using a probe that recognized all known splice variants, reported expression of the leptin receptor gene in the mouse hypothalamus and other brain regions. Such mRNA was visualized in cerebral cortex, hippocampus, thalamus and hypothalamus, and in choroid plexus and leptomeninges. In the same study, a probe specific for Ob-Rb, the variant with a long intracellular domain that is highly expressed in the hypothalamus, revealed strong expression in hypothalamic nuclei which have been implicated in regulation of energy balance ŽPVN, VM and ARC., suggesting that these nuclei are sites of leptin action. Baskin et al. w4x studied the distribution of leptin receptor mRNA in mice and found no differences in obrob, dbrdb and lean wild type mice each of which strongly expressed the receptor in the choroid plexus and the ARC, VM and dorsomedial hypothalamic nuclei with weaker expression in the PVN and hippocampus. In the present study, the most intensely-labeled nuclei, the ACN, magnocellular PVN, SON, NC in the anterior hypothalamus and FN in the lateral hypothalamus contain large neurons that synthesize and secrete arginine vasopressin ŽAVP. or oxytocin ŽOXT. and their respective neurophysins w47x. Although co-localization of leptin re-
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ceptors with AVP or OXT has not been demonstrated, it seems likely on the basis of receptor distribution that one or both types of cell may be involved in the response to leptin binding. Each peptide, administered intraperitoneally or intracerebroventricularly, has been reported to reduce food intake w2,26x. Cholecystokinin octapeptide and Dfenfluramine each enhances synthesis and secretion of AVP and OXT which may mediate, in part, the effects of these well-known anorexigenic agents w37,39x. The PVN is believed to play a prominent role in regulation of energy balance, and studies of farfa rats have implicated abnormally low activity of neurons that synthesize corticotropin releasing hormone ŽCRH., most of which are located in parvocellular subdivisions of the PVN w42x. Nevertheless, vasopressinergic neurons, some of which co-express CRH, may also be dysregulated in this and other rodent models of obesity w50x. CRH and AVP act in a synergistic manner to increase circulating ACTH, and neurons that express both peptides may function to rapidly modulate ACTH release w17,60x. AVP may also directly increase secretion of CRH w6x. Compared with Zucker lean rats, Zucker obese rats whose leptin receptor density is diminished have been reported to secrete 73% and 35% less CRH and AVP, respectively into the hypophyseal portal blood w45x. Adrenalectomy which prevents the development of obesity in the farfa rat increases synthesis of CRH and also enhances expression of AVP in hypothalamic neurons that synthesize CRH w61x. Slieker et al. w54x reported that leptin reduces adipose ob mRNA in vivo but has no effect on isolated rat adipocytes, and suggested negative feedback by leptin mediated by the hypothalamic–pituitary–adrenal ŽHPA. axis. Since leptin is known to decrease circulating corticosterone, one might expect decreased leptin binding in the hypothalamus to produce an increase rather than the decrease in CRH secretion reported in the Zucker obese rat. However, Schwartz et al. w52x found that leptin, administered during a 40-h fast, increased levels of CRH mRNA in the PVN of Long–Evans rats, and Raber et al. w46x showed that leptin increases CRH but not AVP release from hypothalamic slices, so that decreased binding in Zucker obese rats may decrease CRH secretion. If leptin increases secretion of CRH but not AVP, leptin resistance could decrease the ratio of CRH to AVP secreted. Such a change could potentially decrease
Fig. 5. A section through the preoptic region of a Zucker obese rat showing cells of the anterior commissural nucleus Žarrow. that are labeled by the M-18 antiserum. Third ventricle ŽIII.. Bar s 230 m m. Fig. 6. A section of a Zucker obese hypothalamus showing cells immunoreactive for amino acid segment 877–894 of the leptin receptor. Labeled cells are located in parvocellular and magnocellular subdivisions of the paraventricular hypothalamic nucleus. Bar s 120 m m. Fig. 7. A section through a caudal region of a Zucker lean hypothalamus illustrating intense labeling by the M-18 antiserum in the retrochiasmatic supraoptic nucleus Žarrow. and in other widely-dispersed cells in the lateral hypothalamus. Fornix ŽF.. Bar s 230 m m. Fig. 8. A section of a Zucker obese hypothalamus showing small, evenly-distributed cells of the suprachiasmatic nucleus labeled by the M-18 antiserum. Suprachiasmatic nucleus ŽSN., Optic chiasm ŽOC.. Bar s 120 m m.
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the ratio of CRH to ACTH secreted and alter energy balance in favor of weight gain. The anterior pituitary expresses leptin receptor mRNA as well w66x, and leptin has been reported to release ACTH from pituitary slices
w46x. It has been suggested that saturation of the leptin transport system may cause a loss of balance between the effects of leptin on the hypothalamus and pituitary in which CRH release is decreased whereas ACTH is in-
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creased w46x. Feedback inhibition by elevated corticosterone would tend to further decrease CRH secretion. The fact that circulating ACTH and corticosterone are elevated in the Zucker obese rat whereas CRH and AVP secretion appears to be diminished, may be explained by such a loss of balance. Other potential mediators of increased circulating ACTH and corticosterone are increased hypothalamic neuropeptide Y ŽNPY. or decreased neurotensin ŽNT. secretion, each of which is characteristic of Zucker rat and other models of obesity w5,16,59x. These peptides may directly influence anterior pituitary corticotrophs and modulate release of ACTH w23,35x. Diminished secretion of CRH would tend to increase synthesis of hypothalamic NPY as well as decrease sympathetic nervous system activity and thermogenesis w22x. Both parvocellular and magnocellular subdivisions of the PVN and other nuclei intensely immunoreactive for amino acid segment 877–894 of the leptin receptor ŽACN, SCN, SON. are principal sites of projection of NPY-immunoreactive neurons located in the ARC nucleus and in the LC. Cell populations in the ARC nucleus and in the LC were also moderately labeled by the M-18 antiserum in the present study. Hypothalamic NPY increases synthesis and secretion of both CRH and AVP w20,28,55,64x, effects apparently blocked or overridden by other mechanisms in the Zucker obese rat in which synthesis and secretion of hypothalamic NPY are enhanced. Schwartz et al. w52x showed that intraperitoneal injections of leptin lowered NPY mRNA selectively in the ARC nuclei of leptin-deficient obrob mice by 42.3%. Diminished leptin binding in the Zucker obese ARC nucleus may therefore facilitate increased synthesis and secretion of hypothalamic NPY. Such a defect is suggested by Glaum et al. w18x who studied the effects of leptin on synaptic transmission in Zucker lean and obese hypothalamic slices and found that it produced rapid modulatory effects in the ARC nucleus of lean rats but had no effect in obese rats. An effect in the ARC nucleus is also suggested by the failure of leptin to affect weight gain in monosodium glutamate-lesioned rats w15x. Some oxytocin-immunoreactive neurons in the anterior part of the magnocellular subdivision of the PVN co-express CRH and may play a role in energy balance w50x but few studies have implicated OXT in regulation of energy balance. Nevertheless, hypothalamic effects on thermogen-
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esis may be mediated, in part, by oxytocin-immunoreactive neurons that project from the PVN to the intermediolateral cell column w49x, whereas effects on insulin secretion may involve similar projections to the DMN-X w7x. CRH-induced anorexia may be mediated, in part, by OXT, as OXT antagonists have been shown to prevent the anorectic effects of central injections of CRH w43x. Loss of an anorexigenic effect of OXT may play a role in the Prader–Willi syndrome, a condition characterized by extreme obesity in which there is a 50% reduction in the number of OXT neurons in the PVN w56x. Nuclei with cells immunoreactive for amino acid segment 877–894 of the leptin receptor Žparvocellular and magnocellular PVN, SON, VM, ARC and NTS. have been shown to increase Fos expression in response to the potent orexigenic peptide NPY in combination with food intake w31x. Specific sites of leptin action are indicated by induction of Fos in response to altered leptin secretion. Mistry et al. w40x reported that the number of Fos-immunoreactive nuclei in the PVN of leptin-deficient obrob mice was three times that in lean control mice, and that food deprivation for 24 h increased the number of immunoreactive nuclei in lean mice but did not further increase the number in obese mice. Willing et al. w63x found increased Fos in the PVN, VM nucleus and LH after central leptin injection, whereas Woods and Stock w65x found that the PVN of obrob mice was the only neural structure strongly immunolabeled for Fos 3 h after peripheral leptin treatment, with slight labeling in the VM and ARC nuclei and zona incerta. In that study, increased immunoreactivity for Fos appears to lie in dorsal and medial parvocellular subnuclei of the PVN, in which neurons that synthesize CRH are located. Collectively, such studies suggest that there are leptin receptors in several nuclei which play a role in appetitive behavior, and that leptin receptor activity can influence transcription in the parvocellular PVN. Although the present study demonstrates more intense antibody binding in the magnocellular PVN, neurons that synthesize CRH or other parvocellular elements may be affected by leptin binding in closely-related nuclei. Other effects of leptin may involve two areas of the hypothalamus known to influence feeding behavior, the LH and VM nucleus, each of which contained cells intensely or moderately labeled by the M-18 antiserum respectively. Mizuno et al. w41x reported that transfer of the
Fig. 9. A section through the ventromedial ŽVM. and arcuate ŽARC. hypothalamic nuclei of a Zucker lean rat illustrating moderate immunoreactivity for amino acid segment 877–894 in cells of these two nuclei. Bar s 230 m m. Fig. 10. Cells Žarrow. labeled by the M-18 antiserum in the periaqueductal gray near the Edinger–Westphal nuclei of a Zucker lean rat. Bar s 230 m m. Fig. 11. A section through the medulla oblongata of a Zucker lean rat illustrating cells in the nucleus of the tractus solitarius ŽNTS. and dorsal motor nucleus of the vagus nerve ŽDMNX. labeled by the M-18 antiserum. Bar s 120 m m. Fig. 12. A section through the pons of a Zucker lean rat illustrating cells in the locus ceruleus ŽLC. labeled by the M-18 antiserum. Bar s 120 m m.
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ob gene into the VM of the Sprague–Dawley rat inhibits body weight gain and food intake. Immunoreactivity in the HDB and associated MCPO nuclei may suggest an interaction with olfactory systems, as at least two distinct neuronal populations in the HDB project to the olfactory bulb and may interact with inputs from the ACN which was intensely labeled by the M-18 antiserum in the present study. Shipley and Ennis w53x suggested that such interaction may help to regulate transmission of olfactory information between the two hemispheres. Brainstem immunoreactivity for amino acid segment 877–894 of the leptin receptor was visualized in the LC whose noradrenergic neurons co-express NPY and project rostrally to widespread regions of the telencephalon and diencephalon including the hypothalamus. Noradrenergic innervation of both parvocellular and magnocellular subdivisions of the PVN and of the SON is especially dense compared with other hypothalamic areas w29x. Clusters of labeled cells in the PAG may also project rostrally but the identity of such cells was not determined in this study. Leptin binding in the DMN-X could affect gastrointestinal motility and secretion, including secretion of insulin which is decreased by leptin and elevated in several models of obesity. A direct effect of insulin on adipocytes is suggested by studies of Kolaczynski et al. w24x who demonstrated a long-term effect of insulin to increase leptin production both in vivo and in vitro, and Saladin et al. w48x who showed that a single insulin injection increased ob mRNA in fasted rats to levels of fed controls. A synergistic interaction between leptin and cholecystokinin to activate gastric vagal afferents has also been reported w58x. Such afferents have central connections to the NTS, some of whose cells were lightly labeled by the M-18 antiserum in the present study. Several brainstem sites immunoreactive for amino acid segment 877–894 of the leptin receptor also express receptors for AVP or OXT ŽPAG, LC, DMNX, NTS, ION. w3x. Hypothalamic nuclei implicated in regulation of energy balance and other nuclei in the basal forebrain, preoptic area and brainstem of Zucker lean and obese rats were immunoreactive for amino acid segment 877–894 of the leptin receptor. Leptin may therefore interact with multiple central pathways to regulate food intake, metabolism and body weight. Less intense labeling of other cell populations in widespread areas of the brain may reflect cross reactivity with other receptor proteins or may indicate multiple regulatory roles for leptin including regulation of gonadal and thyroid axes in response to fasting w1x. Although CSF leptin levels show a positive correlation with adiposity and are elevated in obese individuals, CSF:plasma leptin ratios are lower in obese than in lean individuals w9x. Therefore, diminished leptin transport into the central nervous system, or defective signal transduction in obese individuals may sufficiently compromise leptin regulation of the HPA axis, NPY-immunoreactive neurons or other hypothalamic elements to cause obesity w13,51x.
Acknowledgements This work was supported by NIH grant DK 32288.
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