ARTICLE |
Correspondence to: Sergio Vidal, Dept. of Anatomy, Laboratory of Histology, University of Santiago de Compostela, Campus Universitario de Lugo, 27002 Lugo, Spain. E-mail: svidal@lugo.usc.es
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Summary |
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Leptin is a key mediator in the maintenance of neuroendocrine homeostasis. Recently, leptin and leptin receptor expression were demonstrated in non-tumorous and adenomatous human pituitaries. This study was performed to determine the subcellular localization of leptin in human adenohypophyses (n = 3) and in various types of pituitary adenoma (n = 16). Immunoelectron microscopy showed leptin immunolabeling in most hormone-producing cells of the human non-tumorous adenohypophysis, but not in stellate cells. Labeling was noted over secretory granules. Immunocytochemistry using double labeling revealed leptin expression in GH-, ACTH-, TSH-, and FSH/LH-containing cells but not in PRL cells. The percentage of immunopositive cells and the intensity of immunostaining varied considerably among the various cell types. Immunoelectron microscopy with double gold labeling showed co-localization of leptin and adenohypophysial hormones in the same secretory granules. Among pituitary tumors, leptin immunolabeling was evident only in corticotroph adenomas. Compared to non-tumorous corticotrophs, leptin immunoexpression was less abundant in corticotroph adenomas. The presence of leptin and adenohypophysial hormones in the same secretory granules suggests that leptin is secreted concomitantly with various adenohypophysial hormones and that its release is under the control of hypothalamic stimulating and inhibiting hormones. (J Histochem Cytochem 48:11471152, 2000)
Key Words: adenohypophysial hormones, immunoelectron microscopy, human, leptin, pituitary tumors
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Introduction |
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Leptin, a circulating hormone secreted by adipocytes, influences body weight homeostasis through effects on food intake and energy expenditure (
On the basis of previous studies, the anterior pituitary may play a role in the regulatory effects of leptin. For example, it has been reported that leptin receptor is expressed in rodent anterior pituitary and that its gene expression is regulated by GH and/or GHRH (
The present study is the first to reveal the subcellular localization of leptin in non-tumorous and adenomatous human pituitary cells, using immunoelectron microscopy.
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Materials and Methods |
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Nineteen transsphenoidally resected pituitary specimens were examined. Three represented normal (non-tumorous) adenohypophysis removed in association with an adenoma. On the basis of immunohistochemical and ultrastructural studies, the samples included four GH (two sparsely and two densely granulated), three PRL, three TSH, three FSH/LH, and three oncocytic null-cell adenomas.
All specimens were fixed in 2.5% glutaraldehyde in Sorensen's phosphate buffer (pH 7.4). After a thorough washing in Sorensen's buffer, the samples were dehydrated in a graded ethanol series, embedded in an EponAraldite mixture, and investigated by transmission electron microscopy. Portions of the tissues were postfixed in 1% osmium tetroxide, whereas other portions remained unosmified.
The postembedding double-immunogold labeling technique of
To ensure specificity, three control procedures were used successively with each immunolabeling procedure. (a) The specific primary antibody was replaced with the antibody diluent (0.2 M PBS admixed with 0.2% coldwater fish gelatin). (b) The specific primary antibody was substituted by normal serum rabbit (not immune serum). (c) The specific polyclonal antiserum was preabsorbed with homologous and heterologous antigens. Absorption tests were carried out as described previously (
After staining, immunoreactive cells were counted to express the percentage of leptin-immunopositive cells in each pituitary sample. In such cells, the percentage of leptin-labeled secretory granules was also determined.
Data were tested for statistical significance using the SPSS statistical computer program (SPSS; Chicago, IL). All data were evaluated by one-way analysis of variance and the Student's t-test as a multiple comparison method. Differences of p<0.05 were considered statistically significant.
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Results |
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Immunoelectron microscopy demonstrated leptin immunolabeling in most hormone-producing glandular cells of the human non-tumorous adenohypophyses. No positive reaction was found in the stellate cells. Leptin staining was detected only in unosmified tissue and was abolished by absorption of the specific primary antibody with 50 µg/ml purified leptin (Eli Lilly; Indianapolis, IN). Positive cells showed specific immunolabeling over secretory granules (Fig 1 Fig 2 Fig 3 Fig 4). Leptin-positive secretory granules were fewer in number than immunonegative granules.
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Because immunopositive normal adenohypophysial cells varied in ultrastructural appearance, the double immunogold method was applied to specifically identify those cells expressing leptin. The double immunocytochemical method demonstrated that, with the exception of lactotrophs, all forms of hormone-producing adenohypophysial cells showed leptin immunolabeling (Fig 1 Fig 2 Fig 3 Fig 4). The percentage of immunopositive cells varied considerably within each of the various cell types. Studies of the co-localization of leptin and pituitary hormones showed that corticotrophs were most frequently leptin labeled (7080% of ACTH- immunopositive cells). Significantly lower frequencies of leptin immunopositivity were observed among other pituitary cell types, including somatotrophs (1015%), thyrotrophs (2025%), and gonadotrophs (2530%). Immunocytochemistry also showed that intensity of staining varied significantly in each pituitary cell type. Lastly, whereas 2025% of secretory granules in corticotrophs were labeled for leptin, only 510% were positive in somatotrophs. The frequency of leptin-labeled granules in both in gonadotrophs and thyrotrophs was 1520%.
The double immunogold method showed co-localization of leptin and specific pituitary hormone in the same secretory granules. A few secretory granules that were usually smaller in size contained only leptin (Fig 2).
Analysis of 16 pituitary adenomas by immunoelectron microscopy demonstrated leptin immunolabeling only in corticotroph adenomas (Fig 5). Positive immunostaining was not identified in somatotroph, lactotroph, thyrotroph, gonadotroph, and oncocytic null- cell adenomas. Furthermore, leptin immunoreactivity was significantly less in corticotroph adenomas than in non-tumorous corticotrophs; only approximately 3040% of adenomatous coticotrophs were leptin-immunopositive.
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Discussion |
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All endocrine cells possess secretory pathways for the transportation of their products to the plasma membrane. In pituitary cells and in various other endocrine cells that store proteins, the secretory pathway includes their accumulation in secretory granules. Previous studies have shown that secretory granules of adenohypophysial cells store not only pituitary hormones but other proteins as well. These include vascular endothelial growth factor (
Our immunoelectron microscopic findings indicate that in the non-tumorous adenohypophysis leptin is expressed in all secretory cells except lactotrophs. Similar observations were previously reported using double-label immunohistochemistry and in situ hybridization at the light microscopic level (
The present study revealed stronger leptin immunoreactivity in normal adenohypophysial cells than in adenomas. This is in agreement with the findings of
Although the significance of leptin expression in the adenohypophysis is unknown, various lines of evidence suggest that leptin plays an important role in regulating adenohypophysial activity. Prior studies have demonstrated that leptin acts via specific receptors expressed by most human and rodent adenohypophysial cells. In addition, it was found that leptin stimulates GH, LH/FSH, and PRL release (
Further studies are needed to establish the mechanism controling leptin expression at the pituitary level and to examine the possible association between decreased leptin expression and secretory activity as well as growth of pituitary adenomas.
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Acknowledgments |
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Supported in part by the Research Council of St. Michael's Hospital, by Mr and Mrs Stephen Jarislowsky, and by the Lloyd CarrHarris foundation. Dr. Sergio Vidal was supported by a research grant from the University of Santiago de Compostela, Spain.
We are indebted to Ms Sandy Briggs and Ms Elizabeth McDermott for technical assistance and to the staff of St. Michael's Health Sciences Library for their contribution to this study.
Received for publication February 19, 2000; accepted April 26, 2000.
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Literature Cited |
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Barash IA, Cheung CC, Weigle DS, Ren H, Kabigting EB, Kuijper JL, Clifton DK, Steiner RA (1996) Leptin is a metabolic signal to the reproductive system. Endocrinology 137:3144-3147[Abstract]
Baskin DG, Schwartz MW, Seeley RJ, Woods SC, Porte D, Jr, Breininger JF, Jonak Z, Schaefer J, Krouse M, Burghardt C, Campfield LA, Burn P, Kochan JP (1999) Leptin receptor long-form splice-variant protein expression in neuron cell bodies of the brain and co-localization with neuropeptide Y mRNA in the arcuate nucleus. J Histochem Cytochem 47:353-362
Bendayan M (1982) Double immunocytochemical labeling applying the protein Agold technique. J Histochem Cytochem 30:81-85[Abstract]
Cai A, Hyde JF (1998) Upregulation of leptin receptor gene expression in the anterior pituitary of human growth hormone-releasing hormone transgenic mice. Endocrinology 139:420-423
Cai A, Hyde JF (1999) The human growth hormone-releasing hormone transgenic mouse as a model of modest obesity: differential changes in leptin receptor (OBR) gene expression in the anterior pituitary and hypothalamus after fasting and OBR localization in somatotrophs. Endocrinology 140:3609-3614
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]
Carro E, Senaris R, Considine RV, Casanueva FF, Dieguez C (1997) Regulation of in vivo growth hormone secretion by leptin. Endocrinology 138:2203-2206
Casanueva FF, Dieguez C (1999) Neuroendocrine regulation and actions of leptin. Front Neuroendocrinol 20:317-363[Medline]
Dannies PS (1999) Protein hormone storage in secretory granules: mechanisms for concentration and sorting. Endocrine Rev 20:3-21
Farnworth PG (1995) Gonadotrophin secretion revisited. How many ways can FSH leave a gonadotroph? J Endocrinol 145:387-395[Abstract]
Farquhar MG, Reid JJ, Daniell LW (1978) Intracellular transport and packaging of prolactin: a quantitative electron microscope autoradiographic study of mammotrophs dissociated from rat pituitaries. Endocrinology 102:296-311[Abstract]
Fei H, Okano HJ, Li C, Lee GH, 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 (1997) Leptin expression and action: new experimental paradigms. Proc Natl Acad Sci USA 94:4242-4245
Gainsford T, Willson TA, Metcalf D, Handman E, McFarlane C, Ng A, Nicola NA, Alexander WS, Hilton DJ (1996) Leptin can induce proliferation, differentiation, and functional activation of hemopoietic cells. Proc Natl Acad Sci USA 93:14564-14568
Guan XM, Hess JF, Yu H, Hey PJ, van der Ploeg LH (1997) Differential expression of mRNA for leptin receptor isoforms in the rat brain. Mol Cell Endocrinol 133:1-7[Medline]
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]
Hardie LJ, Guilhot N, Trayhurn P (1996) Regulation of leptin production in cultured mature white adipocytes. Horm Metab Res 28:685-689[Medline]
Huang Q, Rivest R, Richard D (1998) Effects of leptin on corticotropin-releasing factor (CRF) synthesis and CRF neuron activation in the paraventricular hypothalamic nucleus of obese (ob/ob) mice. Endocrinology 139:1524-1532
Jin L, Burguera BG, Couce ME, Scheithauer BW, Lamsan J, Eberhardt NL, Kulig E, Lloyd RV (1999) Leptin and leptin receptor expression in normal and neoplastic human pituitary: evidence of a regulatory role for leptin on pituitary cell proliferation. J Clin Endocrinol Metab 84:2903-2911
Jin L, Zhang S, Burguera BG, Couce ME, Osamura RY, Kulig E, Lloyd RV (2000) Leptin and leptin receptor expression in rat and mouse pituitary cells. Endocrinology 141:333-339
Karlsson C, Lindell K, Svensson E, Bergh C, Lind P, Billig H, Carlsson LM, Carlsson B (1997) Expression of functional leptin receptors in the human ovary. J Clin Endocrinol Metab 82:4144-4148
Kovacs K, Lloyd R, Horvath E, Asa SL, Stefaneanu L, Killinger DW, Smyth HS (1989) Silent somatotroph adenomas of the human pituitary. A morphologic study of three cases including immunocytochemistry, electron microscopy, in vitro examination, and in situ hybridization. Am J Pathol 134:345-353[Abstract]
Kovacs K, Stefaneanu L, Horvath E, Lloyd RV, Lancranjan I, Buchfelder M, Fahlbusch R (1991) Effect of dopamine agonist medication on prolactin producing pituitary adenomas. A morphological study including immunocytochemistry, electron microscopy and in situ hybridization. Virchows Arch 418:439-446. [A]
Levin N, Nelson C, Gurney A, Vandlen R, de Sauvage F (1996) Decreased food intake does not completely account for adiposity reduction after ob protein infusion. Proc Natl Acad Sci USA 93:1726-1730
Mayer G, Bendayan M (1999) Immunogold signal amplification: application of the CARD approach to electron microscopy. J Histochem Cytochem 47:421-430
Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah LT, Trayhurn P (1996) 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]
Morash B, Li A, Murphy PR, Wilkinson M, Ur E (1999) Leptin gene expression in the brain and pituitary gland. Endocrinology 140:5995-5998
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]
Shimabukuro M, Koyama K, Chen G, Wang MY, Trieu F, Lee Y, Newgard CB, Unger RH (1997) Direct antidiabetic effect of leptin through triglyceride depletion of tissues. Proc Natl Acad Sci USA 94:4637-4641
Shimada O, Suzuki S, TosakaShimada H, Ishikawa H (1998) Detection of deoxyribonuclease I in a hormone-secretory pathway of pituitary cells in humans and rats. Cell Struct Funct 23:49-56[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 (1995) The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature 377:530-532[Medline]
Tannenbaum GS, Gurd W, Lapointe M (1998) Leptin is a potent stimulator of spontaneous pulsatile growth hormone (GH) secretion and the GH response to GH-releasing hormone. Endocrinology 139:3871-3875
Thomas SG, Clarke IJ (1997) The positive feedback action of estrogen mobilizes LH-containing, but not FSH-containing secretory granules in ovine gonadotropes. Endocrinology 138:1347-1350
Vidal S, Kovacs K, Cohen SM, Stefaneanu L, Lloyd RV, Scheithauer BW (1999) Localization of vascular endothelial growth factor in nontumorous human pituitaries. Endocrine Pathol 10:109-122
Vidal S, Oliveira MC, Kovacs K, Scheithauer BW, Lloyd RV (2000) Immunolocalization of vascular endothelial growth factor (VEGF) in the GH3 cell line. Cell Tissue Res 300:83-88[Medline]
Vidal S, Roman A, Moya L (1997) Description of two types of mammosomatotropes in mink (Mustela vison) adenohypophysis: changes in the population of mammosomatotropes under different physiological conditions. Acta Anat (Basel) 159:209-217[Medline]
VilaPorcile E, Corvol P (1998) Angiotensinogen, prorenin, and renin are co-localized in the secretory granules of all glandular cells of the rat anterior pituitary: an immunoultrastructural study. J Histochem Cytochem 46:301-311
Yu WH, Kimura M, Walczewska A, Karanth S, McCann SM (1997) Role of leptin in hypothalamic-pituitary function. Proc Natl Acad Sci USA 94:1023-1028
Zamorano PL, Mahesh VB, De SL, Chorich LP, Bhat GK, Brann DW (1997) Expression and localization of the leptin receptor in endocrine and neuroendocrine tissues of the rat. Neuroendocrinology 65:223-228[Medline]