ARTICLE |
Correspondence to: Denis G. Baskin, Div. of Endocrinology and Metabolism, Research Service, Mail Stop 151, Veterans Affairs Puget Sound Health Care System, 1660 S. Columbian Way, Seattle, WA 98108. E-mail: baskindg@u.washington.edu
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reduced leptin (Ob protein) signaling is proposed to be a stimulus for the activation of neuropeptide Y (NPY) gene activity and increased expression of mRNA for the long form of the leptin receptor (Ob-Rb) in the hypothalamic arcuate nucleus. To determine if Ob-Rb protein is expressed in arcuate nucleus NPY neurons, we developed an affinity-purified polyclonal antibody against amino acids 9561102 of human Ob-Rb. This antibody specifically recognizes the cytoplasmic tail of Ob-Rb and does not react with shorter leptin-receptor variants. Western immunoblots of Ob-Rb-transfected COS cells showed a single 150-kD band, and immunofluorescence revealed intense perinuclear staining in the cytoplasm. A 150-kD band was also present in Western immunoblots of hypothalamus. Immunocytochemical staining of brain slices revealed immunoreactive Ob-Rb protein concentrated in many neuronal cell bodies in the same regions of the forebrain that also express Ob-Rb mRNA. In the hypothalamus, Ob-Rb-positive cell bodies were abundant in the arcuate nucleus and ventromedial nucleus, with lesser numbers in the dorsomedial nucleus and paraventricular nucleus. Immunostaining was also detected in cell bodies of pyramidal cell neurons of the pyriform cortex and cerebral cortex, in neurons of the thalamus, and on the surface of ependymal cells lining the third ventricle. The choroid plexus, which expresses the short Ob-Ra form, was negative. Combined immunocytochemistry for Ob-Rb protein and fluorescence in situ hybridization for NPY mRNA identified arcuate nucleus neurons containing both NPY mRNA and Ob-Rb protein. The present finding of Ob-Rb protein in neurons that express NPY mRNA supports the hypothesis that arcuate nucleus NPY neurons are direct targets of leptin and play an important role in regulation of food intake and body weight. (J Histochem Cytochem 47:353362, 1999)
Key Words: obesity, food intake, body weight, neuropeptide Y, arcuate nucleus, hypothalamus
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A MAJOR HYPOTHESIS in the field of obesity research is that leptin (Ob protein), a protein hormone secreted by fat cells, promotes loss of body weight by acting in the brain to decrease food intake and increase sympathetic nervous system activity (
Much attention is currently focused on the effects of leptin on the expression of arcuate nucleus neuropeptide Y (NPY), a potent hypothalamic stimulator of food intake. We have hypothesized that reduced leptin signaling in the hypothalamus is a stimulus for the activation of NPY gene activity in arcuate nucleus neurons (
A role for leptin in regulating the activity of arcuate nucleus NPY gene expression is supported by evidence that mRNA encoding the long form of the leptin receptor (Ob-Rb) (
Finding Ob-Rb receptor protein in arcuate nucleus NPY neurons would strengthen the hypothesis that the mechanism of leptin's effects on energy balance involves NPY neurons in the hypothalamus. Ob-Rb mRNA has been detected in arcuate nucleus NPY neurons by in situ hybridization (
To determine if the Ob-Rb receptor protein is expressed in arcuate nucleus neurons (NPY neurons in particular) we developed an affinity-purified antibody that specifically recognizes the cytoplasmic tail of Ob-Rb and does not recognize amino acid sequences found in shorter leptin receptor splice variants. The specificity of this Ob-Rb IgG was validated by analysis of transfected COS cells that express Ob-Rb and Ob-Ra. We used this Ob-Rb-specific IgG for immunocytochemical staining of rat brain, and combined Ob-Rb immunostaining with in situ hybridization for NPY mRNA.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reagents
Chemicals were obtained from Sigma (St Louis, MO), unless otherwise noted. Labeled antisera, second antibodies, and normal sera were obtained from Jackson ImmunoResearch (West Grove, PA), except where indicated.
Animals
Male Wistar rats (280300 g) (Simonsen Labs; Gilroy, CA) were housed individually and maintained on a 12:12-hr day: night cycle (07000900 hr) with ad lib access to standard rodent chow and water before the study. For collection of brains, animals were sacrificed in the morning by decapitation under CO2 inhalation. The Animal Research Committees of the Veterans Affairs Puget Sound Health Care System and the University of Washington approved all procedures.
Antiserum
To prepare an antibody that is specific for Ob-Rb, a cDNA encoding amino acids 9561102 of human Ob-Rb was fused to glutathione S-transferase (GST) (pGEX4T-1; Pharmacia, Uppsala, Sweden) and expressed in E. coli MC1061. The GST/Ob-Rb fusion protein was injected into rabbits (Pocono Animal Farms; Pocono, PA) to make Ob-Rb antiserum. The IgG was purified from the antiserum (
COS Cell Studies
To verify the specificity of the anti-Ob-Rb IgG for Ob-Rb protein, extracts of COS cells transfected with cDNAs encoding Ob-Rb or Ob-Ra (gift of L. Tartaglia) and grown for 48 hr (
Western Immunoblotting of Tissue
To characterize the reactivity of the anti-Ob-Rb IgG with rat brain proteins, hypothalamus and liver were homogenized at 4C in a lysis buffer (50 mM Hepes, pH 7.5, with 137 mM NaCl, 10 mM Na3VO4, 100 mM NaF, 100 mM tetrasodium pyrophosphate, 10 mM EDTA, 0.1% Triton X-100, 2 mM PMSF, and 100 µg/ml leupeptin and aprotinin, and centrifuged at 15,000 x g. Supernatants (with equivalent protein concentrations) were immunoprecipitated overnight at 4C with anti-Ob-Rb IgG (0.5 µg/ml) and mixed with Gammabind GSepharose (Pharmacia) for 90 min at 4C, followed by SDS-PAGE in a 7.5% gel. Proteins were transferred to PVDF membranes and blocked overnight in 5% dried milk, then immersed for 3 hr in anti-Ob-Rb IgG (0.5 µg/ml) followed by goat anti-rabbit IgGalkaline phosphatase and detected by chemiluminescence (Trophix). Controls for the specificity of anti-Ob-Rb in Western immunoblots of brain and liver included absorption of the anti-Ob-Rb IgG with the GST/Ob-Rb fusion protein and substitution of anti-GST IgG for the affinity-purified Ob-Rb IgG.
Immunostaining of Ob-Rb in Brain
Brains (n = 12) were fixed by vascular perfusion in 4% paraformaldehyde, sectioned coronally by a cryostat at 10 µm, thaw-mounted on slides, and stored at -80C until use. Sections for immunocytochemical staining were selected from the region between stereotaxic levels -2.5 and -3.5 mm posterior to the bregma (
Combined Immunocytochemistry and In Situ Hybridization
Sections of paraformaldehyde-perfused rat brains were obtained from the specimens prepared for immunocytochemical staining of Ob-Rb protein, as described above, and placed in freshly prepared 0.1 M TEA, pH 8.0. The sections were acetylated for 10 min in 350 ml TEA containing 875 µl acetic anhydride and were rinsed in 2 x SSC. Sections were dehydrated in ethanol and delipidated with chloroform, followed by 100% and 95% ethanol (2 min each) and PBS at 4C. Sections were immunostained with affinity-purified anti-Ob-Rb IgG and donkey anti-rabbit IgG-Cy3 (with the addition of 0.25% Tween in the buffer), as described above, followed by three 5-min rinses in PBS without Tween. The sections were then subjected to the fluorescence in situ hybridization (FISH) protocol to detect NPY mRNA (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Characterization of the Anti-Ob-Rb IgG in COS Cells
Immunoblots of Ob-Rb-transfected COS cells showed a single broad band of approximately 150 kD when incubated in anti-Ob-Rb IgG. This band was also present when Ob-Rb protein was concentrated from Ob-Rb-transfected COS cell extracts by immunoprecipitation with anti-Ob-Rb IgG or with leptin immobilized on Nugel beads. The 150-kD band was absent in mock-transfected COS cells and in COS cells transfected with Ob-Ra (Figure 1A). Immunofluorescence with anti-Ob-Rb IgG revealed intense perinuclear staining in Ob-Rb-transfected COS cells (Figure 1B). In controls, immunofluorescent staining was absent when anti-GST IgG was substituted for anti-Ob-Rb IgG and when Ob-Ra-transfected COS cells or mock-transfected cells were used (Figure 1C). Ob-Rb immunostaining was also observed in COS cells transfected with mouse Ob-Rb cDNA (not shown).
|
Immunocytochemical Staining of Ob-Rb in Brain
Brain sections that were incubated in with anti-Ob-Rb IgG showed many positively stained neuronal cell bodies throughout the arcuate nucleus (Figure 2A and Figure 2D). A high percentage of arcuate nucleus neuronal cell bodies contained Ob-Rb-like immunoreactive protein, although the precise numbers were not determined quantitatively. Staining was absent when anti-GST IgG was substituted for the anti-Ob-Rb IgG (Figure 2B) and when the anti-Ob-Rb IgG was absorbed with the GST/Ob-Rb fusion protein (Figure 2E). In individual neuronal cell bodies the immunoreactive Ob-Rb-like protein was concentrated intracellularly in a perinuclear distribution (Figure 2C and Figure 2F). No neuronal cell bodies showed a staining pattern suggestive of surface labeling, although a surface location of the immunoreactivity could not be precluded. This staining pattern was consistently found in all rats and was detected by both peroxidase and fluorescence immunocytochemical staining methods in all regions of the forebrain that were examined.
|
Positive staining of Ob-Rb-like immunoreactive protein was also observed in other hypothalamic regions. In the paraventricular nucleus, Ob-Rb-like immunoreactive protein was detected as small foci of bright fluorescence that appeared to be located in parvocellular neuronal cell bodies (Figure 3A). The intensity and abundance of Ob-Rb-like immunoreactive protein were relatively weak in the paraventricular nucleus compared to the arcuate nucleus. In contrast, the ventromedial nucleus showed many positively stained neuronal cell bodies with intense intracellular immunoreactivity to the anti-Ob-Rb IgG (Figure 3C). Positive immunostaining was seen in neuronal cell bodies in the supraoptic nucleus and dorsomedial nucleus (not shown). The surface of the ependymal cells lining the third ventricle in the region adjacent to the ventromedial nucleus was intensely stained with the anti-Ob-Rb IgG (Figure 2D). The ependymal immunoreactivity was absent in the region of the median eminence and arcuate nucleus. Throughout the hypothalamus, staining was at background levels in cells and neuropil when anti-GST IgG was substituted for the anti-Ob-Rb IgG and when the anti-Ob-Rb IgG was absorbed with the GST/Ob-Rb fusion protein (Figure 3B and Figure 3D).
|
Neurons in other forebrain regions also showed positive immunostaining for the Ob-Rb splice-variant protein. Immunostaining was markedly strong in cell bodies of pyramidal cell neurons in Layer III of the pyriform cortex (Figure 4A), in pyramidal cell bodies scattered throughout the cerebral cortex (Figure 4B), and throughout the thalamus, particularly in the anteromedial thalamic nucleus (Figure 4C). As in the hypothalamus, Ob-Rb-like immunoreactive protein was concentrated in the cytoplasm of the cell bodies. Cells showing surface labeling only were not detected. Staining of dendrites and terminals was not detected. The choroid plexus was negative for Ob-Rb-like immunoreactivity, whereas the ependymal lining of the ventricles showed bright positive immunocytochemical staining (Figure 4D). Immunostaining in nonhypothalamic regions was absent in all controls.
|
Co-localization of Ob-Rb Protein and NPY mRNA
To determine if Ob-Rb splice-variant protein is expressed by NPY neurons in the arcuate nucleus, we performed immunocytochemistry for Ob-Rb and FISH for NPY mRNAs in the same brain sections (Figure 5). Although the results were not quantified, visual examination suggested that about 75% of arcuate nucleus neuronal cell bodies that showed FISH for NPY mRNA also showed positive immunocytochemical staining for Ob-Rb-like immunoreactive proteins. Ob-Rb immunostaining was present in many arcuate nucleus neurons that did not express NPY mRNAs. Likewise, some neurons with NPY mRNA did not show immunocytochemical staining for Ob-Rb. Neurons with Ob-Rb-like immunoreactive protein were found in all regions of the arcuate nucleus.
|
Western Immunoblotting of Hypothalamus and Liver
Western immunoblots of hypothalamic extracts that were immunoprecipitated with anti-Ob-Rb IgG showed a prominent band representing a protein of about 150 kD, similar to the single band observed in COS cells transfected with Ob-Rb (Figure 6A). Additional bands representing smaller proteins (approximately 78, 100, and 120 kD) were also present in hypothalamic extracts. The 100-kD protein was sometimes present as a doublet. All of these bands were present in immunoprecipitates of boiled hypothalamus and in extracts prepared from both fresh and frozen hypothalamus. A variety of protease inhibitors and various buffer conditions were used in an attempt to eliminate the smaller molecular weight bands, but they were persistently present. However, all of these bands were absent when the blots were incubated in anti-Ob-Rb IgG that was absorbed with the Ob-Rb/GST fusion protein and when affinity-purified anti-GST IgG was substituted for the anti-Ob-Rb IgG. (Figure 6B). A smaller nonspecific band of about 66 kD was also present in controls. However, it probably did not contribute to the immunostaining because all immunostaining was abolished by these controls. As an additional control, Western immunoblots were performed on rat liver, which shows low expression of Ob-Rb. In immunoblots prepared from rat liver immunoprecipitated with anti-Ob-Rb IgG, the 150-kD protein band was very weak compared to hypothalamus (Figure 6C vs 6D).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The absence of direct evidence that Ob-Rb protein is present in the hypothalamus and expressed specifically by NPY neurons has been a significant gap in the data needed to validate the hypothesis that leptin acts in the arcuate nucleus to regulate the activity of NPY neurons. There is no doubt, on the basis of in situ hybridization analyses, that mRNA encoding the Ob-Rb form of the leptin receptor is abundant in the arcuate nucleus (
Previous immunocytochemical studies of Ob-Rb receptor protein in the arcuate nucleus used antisera that recognized amino acid sequences common to both Ob-Ra and Ob-Rb (
The present findings suggest that the relative levels of Ob-Rb receptor protein are roughly proportional to the levels of mRNA as shown by in situ hybridization in the hypothalamus and elsewhere in the forebrain. Although precise quantitation was not performed in the present analysis, Ob-Rb immunocytochemical staining was observed to be most abundant, both in terms of relative numbers of stained cells and in the intensity of the staining within cells, in the arcuate nucleus, where Ob-Rb mRNA levels are also highest (
Our finding of Ob-Rb protein by immunocytochemical staining in the parvocellular neuron cell bodies of the paraventricular nucleus is significant because leptin potently activates c-fos gene expression in this nucleus (
In neuronal cell bodies of the hypothalamus and elsewhere in the forebrain, most of the Ob-Rb-like immunoreactive protein was found in the cytoplasm. The distribution of immunoreactive protein was largely perinuclear, in a pattern consistent with localization in an intracellular vesicular compartment and in the Golgi apparatus. Although these neurons have an abundance of Ob-Rb in the cytoplasm, the degree to which Ob-Rb is expressed at the surface, where the receptor can interact with leptin, is unknown and cannot be determined from these results. The functional meaning of this distribution requires further investigation, but a similar intracellular distribution of immunoreactive Ob-Rb protein has been reported for endothelial cells (-opioid receptors in dorsal root ganglion neurons (
The observations from the immunocytochemical staining and Western immunoblotting analyses of transfected COS cells support the conclusion that the anti-Ob-Rb IgG recognizes Ob-Rb. The antibody detected a single band in the Western immunoblots from COS cells that express Ob-Rb but not from COS cells that express Ob-Ra. Furthermore, only the Ob-Rb-transfected COS cells showed positive immunocytochemical staining with the anti-Ob-Rb IgG. The mobility of the Ob-Rb band in the immunoblots of Ob-Rb-COS cells suggested a molecular size of about 150 kD, which is larger than the predicted size (approximately 130 kD) based on the amino acid composition of Ob-Rb but is consistent with previous reports (
An increasing body of evidence indicates that the mechanism of leptin's effects in the hypothalamus involves the altered expression of hypothalamic neuropeptides (
![]() |
Acknowledgments |
---|
Supported by the Merit Review and Career Scientist Programs of the Department of Veterans Affairs and by NIH grants DK-17047, DK-12829, DK-17844, DK-17847, HD-09171, and NS-32273.
We thank P. Bailon, W.J. Fung, and L. Tartaglia for valuable reagents.
Received for publication August 20, 1998; accepted October 27, 1998.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahima RS, Prabakaran D, Mantzoros C, Qu DQ, Lowell B, MaratosFlier E, Flier JS (1996) Role of leptin in the neuroendocrine response to fasting. Nature 382:250-252[Medline]
Banks WA, Kastin AJ, Huang WT, Jaspan JB, Maness LM (1996) Leptin enters the brain by a saturable system independent of insulin. Peptides 17:305-311[Medline]
Baskin DG, Schwartz MW, Sipols AJ, D'Alessio DA, Goldstein BJ, White MF (1994) Insulin receptor substrate-1 (IRS-1) expression in rat brain. Endocrinology 134:1952-1955[Abstract]
Baskin DG, Seeley RJ, Kuijper JL, Lok S, Weigle DS, Erickson JC, Palmiter RD, Schwartz MW (1998) Increased expression of mRNA for the long form of the leptin receptor in the hypothalamus is associated with leptin hypersensitivity and fasting. Diabetes 47:538-544[Abstract]
Baskin DG, Sipols AJ, Schwartz MW, White MF (1993) Immunocytochemical detection of insulin receptor substrate-1 (IRS-1) in rat brain: colocalization with phosphotyrosine. Regul Pept 48:257-266[Medline]
Bjorbaek C, Uotani S, da Silva B, Flier JS (1997) Divergent signaling capacities of the long and short isoforms of the leptin receptor. J Biol Chem 272:32686-32695
Campfield LA, Smith FJ, Guisez Y, Devos R, Burn P (1995) Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science 269:546-549[Medline]
Collins S, Kuhn CM, Petro AE, Swick AG, Chrunyk BA, Surwit RS (1996) Role of leptin in fat regulation. Nature 380:677[Medline]
Corp ES, Conze DB, Smith F, Campfield LA (1996) Regional localization of specific [I-125]leptin binding sites in rat forebrain. Brain Res 789:40-47
Couce ME, Burguera B, Parisi JE, Jensen MD, Lloyd RV (1997) Localization of leptin receptor in the human brain. Neuroendocrinology 66:145-150[Medline]
Cusin I, RohnerJeanrenaud F, Stricker Krongrad A, Jeanrenaud B (1996) The weight-reducing effect of an intracerebroventricular bolus injection of leptin in genetically obese fa/fa ratsreduced sensitivity compared with lean animals. Diabetes 45:1446-1451[Abstract]
Devos R, Richards JG, Campfield LA, Tartaglia LA, Guisez Y, Van der Heyden J, Travernier J, Plaetinck G, Burn P (1996) OB protein binds specifically to the choroid plexus of mice and rats. Proc Natl Acad Sci USA 93:5668-5673
Elmquist JK, Ahima RS, MaratosFlier E, Flier JS, Safer CB (1997) Leptin activates neurons in ventrobasal hypothalamus and brainstem. Endocrinology 138:839-842
Fei H, Okano HJ, Li C, Lee G, Zhao C, Darnell R, Friedman JM (1997) Anatomic localization of alternatively spliced leptin receptors (Ob-R) in mouse brain and other tissues. Proc Natl Acad Sci USA 94:7001-7005
Flier JS, MaratosFlier E (1998) Obesity and the hypothalamus: novel peptides for new pathways. Cell 92:437-440[Medline]
Ghilardi N, Skoda RC (1997) The leptin receptor activates janus kinase 2 and signals for proliferation in a factor-dependent cell line. Mol Endocrinol 11:393-399
Guan XM, Hess JF, Yu H, Hey PJ, Van der Ploeg LHT (1997) Differential expression of mRNA for leptin receptor isoforms in the rat brain. Mol Cell Endocrinol 133:1-7[Medline]
Hahn TM, Breininger JF, Baskin DG, Schwartz MW (1998) Colocalization of agouti-related protein and neuropeptide Y in arcuate nucleus neurons activated by fasting. Nature Neurosci 1:271-272[Medline]
Håkansson ML, Hulting AL, Meister B (1996) Expression of leptin receptor mRNA in the hypothalamic arcuate nucleusrelationship with NPY neurones. Neuroreport 7:3087-3092[Medline]
Hakimi J, Seals C, Kondas JA, Pettine L, Danho W, Kochan J (1990) The alpha subunit of the human IgE receptor (FcERI) is sufficient for high affinity IgE binding. J Biol Chem 265:22079-22081
Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, Lallone RL, Burley SK, Friedman JM (1995) Weight-reducing effects of the plasma protein encoded by the obese gene. Science 269:543-546[Medline]
Hoggard N, Hunter L, Duncan JS, Williams LM, Trayhurn P, Mercer JG (1997) Leptin and leptin receptor mRNA and protein expression in the murine fetus and placenta. Proc Natl Acad Sci USA 94:11073-11078
Huang XF, Koutcherov I, Lin S, Wang HQ, Storlien L (1996) Localization of leptin receptor mRNA expression in mouse brain. Neuroreport 7:2635-2638[Medline]
Jacob RJ, Dziura J, Medwick MB, Leone P, Caprio S, During M, Shulman GI, Sherwin RS (1997) The effect of leptin is enhanced by macroinjection into the ventromedial hypothalamus. Diabetes 46:150-152[Abstract]
Légrádi G, Emerson CH, Ahima RS, Flier JS, Lechan RM (1997) Leptin prevents fasting-induced suppression of prothyrotropin-releasing hormone messenger ribonucleic acid in neurons of the hypo-thalamic paraventricular nucleus. Endocrinology 138:2569-2576
Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah LT, Morgan PJ, Trayhurn P (1996a) Coexpression of leptin receptor and preproneuropeptide Y mRNA in arcuate nucleus of mouse hypothalamus. J Neuroendocrinol 8:733-735[Medline]
Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah LT, Tray-hurn P (1996b) Localization of leptin receptor mRNA and the long form splice variant (Ob-Rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization. FEBS Lett 387:113-116[Medline]
Mizuno TM, Kleopoulos SP, Bergen HT, Roberts JL, Priest CA, Mobbs CV (1998) Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting in ob/ob and db/db mice, but is stimulated by leptin. Diabetes 47:294-297[Abstract]
Osborne MA, Dalton S, Kochan JP (1995) The yeast trihybrid systemgenetic detection of trans-phosphorylated ITAM-SH2-interactions. Biotechnology 13:1474-1478[Medline]
Paxinos G, Watson C (1986) The Rat Brain in Stereotaxic Coordinates. 4th Ed. New York, Academic Press
Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, Collins F (1995) Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269:540-543[Medline]
Quintela M, Señaris R, Heiman ML, Casanueva FF, Dieguez C (1997) Leptin inhibits in vitro hypothalamic somatostatin secretion and somatostatin mRNA levels. Endocrinology 138:5641-5644
RohnerJeanrenaud F, Cusin I, Sainsbury A, Zakrzewska KE, Jeanrenaud B (1996) The loop system between neuropeptide Y and leptin in normal and obese rodents. Horm Metab Res 28:642-648[Medline]
Sahu A (1998) Evidence suggesting that galanin (GAL), melanin-concentrating hormone (MCH), neurotensin (NT), proopiomelanocortin (POMC) and neuropeptide Y (NPY) are targets of leptin signaling in the hypothalamus. Endocrinology 139:795-798
Satoh N, Ogawa Y, Katsuura G, Hayase M, Tsuji T, Imagawa K, Yoshimasa Y, Nishi S, Hosoda K, Nakao K (1997) The arcuate nucleus as a primary site of satiety effect of leptin in rats. Neurosci Lett 224:149-152[Medline]
Schwartz MW, Baskin DG, Bukowski TR, Kuijper JL, Foster D, Lasser G, Prunkard DE, Porte D, Woods SC, Seeley RJ, Weigle DS (1996a) Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice. Diabetes 45:531-535[Abstract]
Schwartz MW, Erickson JC, Baskin DG, Palmiter RD (1998) Effect of fasting and leptin deficiency on hypothalamic neuropeptide Y gene transcription in vivo revealed by expression of a lacZ reporter gene. Endocrinology 139:2629-2635
Schwartz MW, Peskind E, Raskind M, Boyko EJ, Porte DJ (1996b) Cerebrospinal fluid leptin levels: relationship to plasma levels and to adiposity in humans. Nature Med 2:589-593[Medline]
Schwartz MW, Seeley RJ (1997) Neuroendocrine responses to starvation and weight loss. N Engl J Med 336:1802-1811
Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG (1996c) Identification of targets of leptin action in rat hypothalamus. J Clin Invest 98:1101-1106
Schwartz MW, Seeley RJ, Woods SC, Weigle DS, Campfield LA, Burn P, Baskin DG (1997) Leptin increases hypothalamic pro-opiomelanocortin mRNA expression in the rostral arcuate nucleus. Diabetes 46:2119-2123[Abstract]
Seeley RJ, Van Dijk G, Campfield LA, Smith FJ, Burn P, Nelligan JA, Bell SM, Baskin DG, Woods SC, Schwartz MW (1996) Intraventricular leptin reduces food intake and body weight of lean rats but not obese Zucker rats. Horm Metab Res 28:664-668[Medline]
Shioda S, Funahashi H, Nakajo S, Yada T, Maruta O, Nakai Y (1998) Immunohistochemical localization of leptin receptor in the rat brain. Neurosci Lett 243:41-44[Medline]
SierraHonigmann MR, Nath AK, Murakami C, GarciaCardena G, Papapetropoulos A, Sessa WC, Madge LA, Schechner JS, Schwabb MB, Polverini PJ, FloresRiveros JR (1998) Biological action of leptin as an angiogenic factor. Science 281:1683-1686
Stephens TW, Basinski M, Bristow PK, BueValleskey JM, Burgett SG, Craft L, Hale J, Hoffmann J, Hsiung HM, Kriauciunas A, MacKellar W, Rosteck PR, Jr, Schoner B, Smith D, Tinsley FC, Zhang XY, Heiman M (1995) The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature 377:530-532[Medline]
Tartaglia LA (1997) The leptin receptor. J Biol Chem 272:6093-6096
Thornton JE, Cheung CC, Clifton DK, Steiner RA (1997) Regulation of hypothalamic proopiomelanocortin mRNA by leptin in ob/ob mice. Endocrinology 138:5063-5066
Van Dijk G, Thiele TE, Donahey JCK, Campfield LA, Smith FJ, Burn P, Bernstein IL, Woods SC, Seeley RJ (1996) Central infusions of leptin and GLP-1-(7-36) amide differentially stimulate c-FLI in the rat brain. Am J Physiol 271:R1096-1100
Weigle DS, Bukowski TR, Foster DC, Holderman S, Kramer JM, Lasser G, Lofton Day CE, Prunkard DE, Raymond C, Kuijper JL (1995) Recombinant ob protein reduces feeding and body weight in the ob/ob mouse. J Clin Invest 96:2065-2070[Medline]
White DW, Kuropatwinski KK, Devos R, Baumann H, Tartaglia LA (1997) Leptin receptor (OB-R) signalingcytoplasmic domain mutational analysis and evidence for receptor homo-oligomerization. J Biol Chem 272:4065-4071
Yarnell DO, Knight DS, Hamilton K, Tulp O, Tso P (1998) Localization of leptin receptor immunoreactivity in the lean and obese Zucker rat brain. Brain Res 785:80-90[Medline]
Zhang X, Bao L, Arvidsson U, Elde R, Hökfelt T (1998) Localization and regulation of the delta-opioid receptor in dorsal root ganglia and spinal cord of the rat and monkey: evidence for association with the membrane of large dense-core vesicles. Neuroscience 82:1225-1242[Medline]