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Central Nervous System Effects of Leptin
Central Nervous System Effects of Leptin Clint Buchanan, Virendra Mahesh, Pedro Zamorano and Darrell Brann
Evidence exists demonstrating the importance of leptin in the control of energy homeostasis, feeding behavior and reproductive function. Leptin receptors are localized in several regions of the brain implicated in regulation of energy balance and reproductive function, including the arcuate nucleus/median eminence, paraventricular nucleus, and ventromedial nucleus. Administration of exogenous leptin has been shown to alter function of the hypothalamic–pituitary–adrenal axis and stimulate gonadotropin release through hypothalamic and pituitary actions. Results from in situ hybridization studies demonstrate the ability of leptin to modulate the expression of key neuropeptides (neuropeptide Y, corticotropin-releasing hormone) implicated in the regulation of energy homeostasis. This suggests that leptin is an important component in the neuroendocrine transmission line that regulates appetite, energy balance and reproduction.
Obesity is a risk factor associated with a myriad of health problems including hypertension, cardiovascular disease, infertility and non-insulin-dependent diabetes mellitus (NIDDM). This nutritional disorder, resulting from disequilibrium between energy expenditure and ingestive behavior, is a cause of significant morbidity, and is becoming more common in the developed world (Bouchard and Perusse 1993). For this reason, the etiology of obesity has been investigated intensely, in order to provide information necessary for the development of therapeutic approaches for its treatment. Results from cross-circulation (parabiosis) experiments involving obese ob/ob and db/db mice provided initial evidence suggesting the existence of a circulating satiety factor responsible for a reduction in food intake and subsequent weight loss. A single point C. Buchanan, V. Mahesh, P. Zamorano and D. Brann are at the Department of Physiology and Endocrinology, Medical College of Georgia, Augusta, GA 30912, USA.
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mutation in the leptin receptor gene, as observed in C57BL/Ks db/db mice, results in alternative splicing of the receptor coding region and subsequent production of mutant leptin receptor (Chen et al. 1996, Lee et al. 1996). When the circulatory systems of ob/ob mice were connected indirectly to those of db/db mice, ob/ob mice lost weight and reduced their food intake, whereas db/db mice did not lose weight or reduce their food intake. This suggested that ob/ob mice were deficient in the circulating factor but retained the ability to respond to it with subsequent reduction in body weight. In contrast, db/db mice were unable to respond to adequate circulating levels of the factor. It was concluded that the ob gene encoded the satiety factor, while the db gene encoded the receptor for this ligand (Coleman 1973 and 1978, Chen et al. 1996). Recently, this hypothesis received important confirmation with the cloning of the ob gene and the subsequent demonstration that the gene product acts as a blood-borne
hormone responsible for weight maintenance (Zhang et al. 1994, Pellymounter et al. 1995). Leptin, the product of the adipose-specific ob gene, is a 167-amino acid secreted peptide that exerts its effects on central neural networks through regulation of energy homeostasis and feeding behavior. The ob/ob mouse has provided a useful model for investigating the physiological role of leptin in the body. A single base mutation of the leptin gene at codon 105, as observed in the C57BL/6J ob/ob mouse, involves the replacement of arginine by a premature stop codon and the subsequent production of an inactive form of leptin (Weigle et al. 1995). The genetically obese ob/ob mouse exhibits hyperinsulinemia, infertility, hyperglycemia and impaired thyroid function (Bray and York 1979). Interestingly, treatment of obese ob/ob mice with recombinant leptin lowers body weight and percent body fat. This treatment also corrects the hyperinsulinemia, infertility and hyperglycemia of ob/b mice. However, diet restriction alone was ineffective in restoring fertility in ob/ob mice, which suggests that leptin may be essential for normal reproductive function (Campfield et al. 1995, Chehab et al. 1996). Many reviews have discussed leptin actions in feeding behavior; therefore, this review will focus on the central nervous system (CNS) effects of leptin. •
Leptin Receptors and Signaling Pathways
Description of Leptin Receptors The leptin receptor (OB-R) is a single membrane-spanning protein homologous to members of the class I cytokine receptor family. Isoforms of the leptin receptor, which differ in the length of their cytoplasmic domains, include OB-Ra, OB-Rb, OB-Rc and OB-Rd. OB-Ra and OB-Rb, the short and long isoform, respectively, are generated by alternative splicing of a common transcript, and are highly conserved between mice and humans. OB-Rd is generated when a short exon is spliced between the OB-Rb splice donor (Lys-889) and splice acceptor (Pro-890) sites, while OB-Rc is synthesized when
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splicing does not occur after the splice donor (Lys-889) site. Recent evidence suggests the existence of a soluble leptin receptor OB-Re, but the physiological role of this isoform remains unclear (Tartaglia et al. 1995, Chen et al. 1996, Ghilardi et al. 1996, Fei et al. 1997). Results from experiments investigating signal transduction pathways of OB-Ra and OB-Rb isoforms suggest that OB-Rb may play an important role in mediating the biological effects of leptin. In these studies, COS cells and a factor-dependent hematopoietic cell line BaF3 were transfected with constructs encoding OB-Rb or OB-Ra isoforms. COS cells expressing OB-Rb responded to leptin exposure with activation of components in the signal transducers and activators of transcription (STAT) pathway, including STAT5, STAT6 and STAT3, while cells expressing OB-Ra were inactive in STAT signaling upon leptin treatment. BaF3 cells transfected with OB-Rb responded to leptin with increased proliferation and activation of Janus kinase 2 (Jak2), a cytoplasmic tyrosine kinase associated with the intracellular domain of receptors in the cytokine receptor superfamily. However, cells expressing OB-Ra, the short isoform, were inactive in proliferation and janus kinase activation (Ihle 1995, Ghilardi and Skoda 1997). Leptin treatment has also been shown to induce dose-dependent hypothalamic STAT3 activation in female C57BL/6J ob/ob and C57BL/6J +/? wild-type mice, but not C57BLKS/J db/db mice, which lack OB-Rb (Vaisse et al. 1996). This suggests that the OB-Rb isoform may be necessary for hypothalamic STAT3 activation. However, STAT activation was not observed in other tissues that express OB-Rb. This could be due to the presence of truncated isoforms acting as dominant negatives, thereby reducing the ratio of OB-Rb (long isoform) to OB-Ra, OB-Rc and OB-Rd (short isoforms). Interestingly, the ratio of OB-Rb to truncated isoforms has also been reported to be greatest in the hypothalamus (Ghilardi et al. 1996). Taken as a whole, these results suggest that STAT activation may be an important TEM Vol. 9, No. 4, 1998
component in hypothalamic OB-Rb signal transduction. Localization of Leptin Receptors in the Central Nervous System Results from in situ hybridization studies demonstrate that the leptin receptor gene is expressed strongly in several regions of the mouse brain (Mercer et al. 1996a, Fei et al. 1997). Utilizing a probe recognizing all OB-R isoforms, leptin receptor gene expression was detected in the choroid plexus, leptomeninges and parts of the hypothalamus implicated in the regulation of energy balance, including the arcuate nucleus (ARC), paraventricular nucleus (PVN) and ventromedial nucleus (VMH). A similar hybridization pattern was observed in adjacent sections utilizing a probe specific for the Ob-Rb isoform; however, there was minimal hybridization to the leptomeninges and choroid plexus. Quantification of the signal intensity indicated that the OB-R gene expressed in the hypothalamus consisted mainly of OB-Rb. Reverse transcription polymerase chain reaction (RT-PCR) analysis and immunohistochemical studies from our laboratory have demonstrated OB-R gene expression and localization in neuroendocrine tissues of the rat, including the anterior pituitary, choroid plexus and arcuate nucleus/median eminence (ARC/ME) of the hypothalamus (Zamorano et al. 1997). OB-R gene expression was also demonstrated in the immortalized gonadotropin-releasing hormone (GnRH)-producing GT1-7 and NLT neuronal cell lines. The localization of OB-R in the ARC, a site involved in control of both reproductive function and feeding behavior, indicates that leptin could potentially act at the ARC to modulate these two functions. Results from immunohistochemical studies using an antibody recognizing all OB-R isoforms demonstrated that OB-R is present in regions of the human brain, including the choroid plexus, nucleus basalis of Meynert, inferior olivary nuclei, ependymal lining of the lateral ventricle and cerebellar Purkinje cells. Immunoreactivity was also localized in
several hypothalamic nuclei, including suprachiasmatic, ARC, PVN, mamillary, dorsomedial (DMH), supraoptic and posterior nuclei (Couce et al. 1997). Studies demonstrating OB-R gene expression and localization in the choroid plexus of the mouse, rat and human provide further evidence to support the idea that leptin binds to the choroid plexus and is subsequently transported across the blood–brain barrier into the cerebrospinal fluid, thereby allowing it to travel to specific brain sites (Lynn et al. 1996). •
Leptin Effects on Hypothalamic and Pituitary Function
GnRH and Gonadotropin Secretion The secretion of the hypothalamic decapeptide GnRH is considered a primary neural signal involved in induction of the preovulatory luteinizing hormone (LH) surge. Upon receiving stimulatory signals, hypothalamic GnRH neurons secrete GnRH into the hypophyseal portal veins of the median eminence, allowing it to travel to the anterior pituitary and, after a cascade of events, initiate the LH surge (Levine and Ramirez 1982, Moenter et al. 1991). Recently, McCann and colleagues reported that leptin may stimulate gonadotropin release through pituitary and hypothalamic actions (Yu et al. 1997). Results from tissue culture studies demonstrated that hemi-anterior pituitaries of adult male rats respond to leptin exposure with a significant increase in LH release, supporting previous reports demonstrating the ability of leptin to increase significantly serum LH levels in fasted mice (Ahima et al. 1996). Yu et al. (1997) also reported that leptin can significantly enhance GnRH secretion in vitro from hypothalamic ARC/ME explants, suggesting that leptin also acts centrally at the level of the hypothalamus to influence reproductive function. A hypothalamic site of action of leptin is supported further by the findings that leptin injected into the third ventricle stimulates LH release (Yu et al. 1997) while, conversely, administration of leptin antiserum into the third ventricle of female rats induces a 147
marked decrease in LH pulsatility and disrupts cyclicity (Carro et al. 1997). In addition, leptin has also been shown to stimulate the reproductive endocrine system in ob/ob mice (Barash et al. 1996, Chehab et al. 1996, Cioffi et al. 1996). Leptin treatment induced a significant increase in plasma levels of LH in female ob/ob mice, while ob/ob male mice exhibited a significant increase in plasma levels of follicle stimulating hormone (FSH). Both sexes also responded to leptin treatment with increased uterine or seminal vesicle weight.
on feeding behavior and energy balance may involve, at least in part, antagonism of NPY neuronal systems (Erickson et al. 1996). Recent studies suggest that melanocortin receptor (MC4) signaling may also play a role in mediating leptin’s effects on feeding behavior. Along these lines, pretreatment of male rats with SHU9119, an MC4 receptor antagonist, abolished the ability of centrally administered leptin to induce cFos-like immunoreactivity in the PVN and to decrease feeding and body weight (Seeley et al. 1997).
Neuropeptide Y and Corticotropinreleasing Hormone Results from in situ hybridization studies have suggested that leptin can alter expression of hypothalamic neuropeptides implicated in the regulation of energy homeostasis. Leptin treatment has been reported to lower neuropeptide Y (NPY) mRNA levels in the ARC of ob/ob mice (Mercer et al. 1997). Similar results were observed in the male rat; however, leptin also induced a significant increase in corticotropin-releasing hormone (CRH) mRNA levels in the PVN (Schwartz et al. 1996). NPY is considered one of the most potent inducers of feeding behavior, and CRH is thought to act at the PVN to increase energy expenditure and inhibit ingestive behavior; thus, the ability of leptin to inhibit NPY and stimulate CRH gene expression may partially account for the weightreducing effect of leptin (Clark et al. 1984, Rothwell 1989). Interestingly, leptin receptor mRNA is reportedly coexpressed in NPY neurons of the ARC, suggesting that the effect of leptin on NPY gene expression may be through a direct mechanism (Mercer et al. 1996b). In support of a partial role of NPY in leptin effects on energy homeostasis, double mutant NPY−/− ob/ob mice were less obese than ob/ob mice, owing to reduced feeding behavior and increased energy expenditure. The absence of NPY also improved reproductive function in ob/ob mice, and double mutants were less severely affected by diabetes. These results suggest that leptin’s regulatory actions
Regulation of the Hypothalamic–Pituitary–Adrenal Axis The obesity and chronic hyperadrenocorticism exhibited by ob/ob mice can be attenuated by adrenalectomy; therefore, hypothalamic–pituitary–adrenal (HPA) axis abnormality might play a role in mediating the expression of the obesity syndrome in ob/ob mice (Saito and Bray 1984, Bray et al. 1989). Studies investigating HPA axis function demonstrated increased synthesis and secretion of pituitary adrenocorticotropic hormone (ACTH), along with an enhanced adrenal response to ACTH in ob/ob mice (McGinnis et al. 1992). ob/ob mice also exhibit chronically elevated serum corticosterone (CS) levels, suggesting that glucocorticoid negative feedback may also be impaired at the level of the pituitary or hypothalamus. Leptin has also been reported to influence changes in the HPA axis induced by starvation of male rats (Ahima et al. 1996). Starvation-induced increases in plasma ACTH and CS levels were reduced by leptin and, interestingly, fed rats exhibited an inverse correlation between serum CS and leptin levels. Similar results have demonstrated that fluctuations in the 24-h patterns of circulating human serum leptin levels are the inverse of cortisol and ACTH levels (Licinio et al. 1997). This provides additional evidence implicating leptin as a potential modulator of HPA axis function, although further studies are needed to elucidate the mechanisms underlying the HPA axis hyperactivity observed in the leptin-deficient state.
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Puberty Previous evidence suggests a connection between body weight, adipose tissue mass and time of puberty onset (Kennedy and Mitra 1963, Frisch 1980). In addition, recent work has suggested that leptin may play a role in the process of sexual maturation and acquisition of reproductive competence. Administration of exogenous leptin resulted in an earlier onset of vaginal opening, estrus and cycling in female mice, indicating that leptin may be a factor in determining the timing of puberty (Ahima et al. 1997, Chehab et al. 1997). Although these studies did not address the site of action of leptin in the induction of puberty, the previously reported ability of leptin to facilitate GnRH release suggests that the action of leptin could involve hypothalamic effects (Yu et al. 1997). However, it should be pointed out that other studies failed to find a facilitatory effect of leptin on the timing of puberty in female rats, questioning whether leptin is the primary signal that initiates the onset of puberty (Cheung et al. 1997). In humans, a significant rise in serum leptin levels was observed before the onset of puberty in boys; however, a cause and effect relationship has not been established (Mantzoros et al. 1997). Clearly, more work is needed to determine the precise role and importance of leptin in the achievement of puberty. •
Conclusions
The evidence presented in this review demonstrates the central nervous system effects of leptin in the regulation of energy homeostasis, feeding behavior and reproductive function. Leptin receptor gene expression and localization have been demonstrated in regions of the brain involved in the control of ingestive behavior and energy balance. In addition, leptin has been shown to alter the expression of hypothalamic neuropeptides implicated in the regulation of these functions. The ability of leptin to modulate HPA axis function along with gonadotropin and GnRH secretion also demonstrates its ability to affect reproductive function. The precise role of leptin in the TEM Vol. 9, No. 4, 1998
induction of puberty remains unclear, but evidence suggests that it may be a factor determining the timing of puberty. Evidence presented here suggests that leptin plays an important role in regulation of appetite, energy balance and reproduction. Future studies identifying potential effectors of leptin action may be useful in the development of therapeutic approaches for use in the treatment of obesity and reproductive dysfunction. References Ahima RS, Prabakaran D, Mantzoros C, et al.: 1996. Role of leptin in the neuroendocrine response to fasting. Nature 382:250–252. Ahima RS, Dushay J, Flier SN, Prabakaran D, Flier JS: 1997. Leptin accelerates the onset of puberty in normal female mice. J Clin Invest 99:391–395. Barash IA, Cheung CC, Weigle DS, et al.: 1996. Leptin is a metabolic signal to the reproductive system. Endocrinology 137:3144–3147. Bouchard C, Perusse L: 1993. Genetics of obesity. Annu Rev Nutr 13:337–354. Bray GA, York DA: 1979. Hypothalamic and genetic obesity in experimental animals: an autonomic and endocrine hypothesis. Physiol Rev 59:719–809. Bray GA, York DA, Fisher JS: 1989. Experimental obesity: a homeostatic failure due to defective nutrient stimulation of the sympathetic nervous system. Vitam Horm 45:1–125. 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. Carro E, Pinilla L, Seoane LM, et al.: 1997. Influence of endogenous leptin tone on the estrous cycle and luteinizing hormone pulsatility in female rats. Neuroendocrinology 66:375–377. Chehab F, Lim M, Lu R: 1996. Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat Genet 12:318–320. Chehab FF, Mounzih K, Lu R, Lim ME: 1997. Early onset of reproductive function in normal female mice treated with leptin. Science 275:88–90. Chen H, Charlat O, Tartaglia LA, et al.: 1996. Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell 84:491–495.
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Cheung CC, Thornton JE, Kuijper JL, Weigle DS, Clifton DK, Steiner RA: 1997. Leptin is a metabolic gate for the onset of puberty in the female rat. Endocrinology 138:855–858. Cioffi J, Shafer A, Zupanic T, et al.: 1996. Novel B219/OB receptor isoforms: possible role of leptin in hematopoiesis and reproduction. Nat Med 2:585–588. Clark JT, Kalra PS, Crowley WR, Karla SP: 1984. Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology 115:427–429. Coleman DL: 1973. Effects of parabiosis of obese with diabetes and normal mice. Diabetologia 9:294–298. Coleman DL: 1978. Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice. Diabetologia 14:141–148. Couce ME, Burguera B, Parisi JE, Jenson MD, Lloyd RV: 1997. Localization of leptin receptor in the human brain. Neuroendocrinology 66:145–150. Erickson JC, Hollopeter G, Palmiter RD: 1996. Attenuation of the obesity syndrome of ob/ob mice by the loss of neuropeptide Y. Science 274:1704–1707. Fei H, Okano HJ, Li C, et al.: 1997. Anatomic localization of alternately spliced leptin receptors (OB-R) in mouse brain and other tissues. Neurobiology 94:7001–7005. Frisch RE: 1980. Pubertal adipose tissue: is it necessary for normal sexual maturation? Evidence from the rat and human female. Fed Proc 39:2395–2400. 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. Ghilardi N, Ziegler S, Wiestner A, Stoffel R, Heim MH, Skoda RC: 1996. Defective STAT signaling by the leptin receptor in diabetic mice. Proc Natl Acad Sci USA 93:6231–6235. Ihle JN: 1995. Cytokine receptor signaling. Nature 377:591–594. Kennedy GC, Mitra J: 1963. Body weight and food intake as initiating factors for puberty in the rat. J Physiol (London) 166:408–418. Lee GH, Proenca R, Montez JM, et al.: 1996. Abnormal splicing of the leptin receptor in diabetic mice. Nature 379:632–635. Levine JE, Ramirez VD: 1982. Luteinizing hormone releasing hormone release during the rat estrous cycle and after ovariectomy as estimated with push pull cannulae. Endocrinology 111:1439–1448. Licinio J, Mantzoros C, Negrao AB, et al.: 1997. Human leptin levels are pulsatile and
inversely related to pituitary-adrenal function. Nat Med 3:575–579. Lynn RB, Cao GY, Considine RV, Hyde TM, Caro JF: 1996. Autoradiographic localization of leptin binding in the choroid plexus of ob/ob and db/db mice. Biochem Biophys Res Commun 219:884–889. Mantzoros CS, Flier JS, Rogol AD: 1997. A longitudinal assessment of hormonal and physical alterations during normal puberty in boys. V. Rising leptin levels may signal the onset of puberty. J Clin Endocrinol Med 82:1066–1070. McGinnis R, Walker J, Margules D, Aird F, Redel E: 1992. Dysregulation of the hypothalamus–pituitary–adrenal axis in male and female, genetically obese (ob/ob) mice. J Neuroendocrinol 4:765–771. Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah LT, Trayhurn P: 1996a. 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. Mercer JG, Hoggard N, Williams LM, et al.: 1996b. Coexpression of leptin receptor and proneuropeptide Y mRNA in the arcuate nucleus of mouse hypothalamus. Neuroendocrinology 8:733–735. Mercer JG, Moar KM, Rayner DV, Trayhurn P, Hoggard N: 1997. Regulation of leptin receptor and NPY gene expression in hypothalamus of leptin-treated obese (ob/ob) and cold exposed lean mice. FEBS Lett 402:185–188. Moenter SM, Caraty A, Locatelli A, Karsch FJ: 1991. Pattern of gonadotropin releasing hormone (GnRH) secretion leading up to ovulation in the ewe: existence of a preovulatory GnRH surge. Endocrinology 111:1175–1182. Pellymounter MA, Cullen MJ, Baker MB, et al.: 1995. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269:540–543. Rothwell N: 1989. Central effects of CRF on metabolism and energy balance. Neurosci Biobehav Rev 14:263–271. Saito M, Bray GA: 1984. Adrenalectomy and food restriction in the genetically obese (ob/ob) mouse. Am J Physiol 248:E20–E25. Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG: 1996. Identification of targets of leptin action in rat hypothalamus. J Clin Invest 98:1101–1106. Seeley RJ, Yagaloff KA, Fisher SL, et al.: 1997. Melanocortin receptors in leptin effects. Nature 390:349.
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Tartaglia LA, Dembski M, Weng X, et al.: 1995. Identification and expression cloning of a leptin receptor, OB-R. Cell 83:1263–1271.
Weigle DS, Bukowski TR, Foster DC, et al.: 1995. Recombinant ob protein reduces feeding and body weight in the ob/ob mouse. J Clin Invest 96:2065–2070.
Vaisse C, Halass JL, Horvath CM, Darnell JE, Stoffel M, Friedman JM: 1996. Leptin activation of STAT3 in the hypothalamus of wild-type and ob/ob mice but not db/db mice. Nat Genet 14:95–97.
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Zamorano PL, Mahesh VB, De Sevilla L, et al.: 1997. Expression and localization of the leptin receptor in endocrine and neuroendocrine tissues of the rat. Neuroendocrinology 65:223–228. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM: 1994. Positional cloning of the mouse obese gene and its human homologue. Nature 372:425–432.
•
The Androgen Receptor as Mediator of Gene Expression and Signal Transduction Pathways Andrew C.B. Cato and Heike Peterziel
The current model of action of androgens involves activation of a cytoplasmic receptor that migrates into the nucleus to regulate the expression of specific genes, either positively or negatively. While positive regulation requires direct binding of the receptor to DNA, negative regulation occurs mainly through protein–protein interactions of the receptor and other transcription factors. More recent findings have shown that the receptor also mediates non-conventional responses attributed hitherto only to activated growth factor receptors. These actions proceed principally through activation of cytoplasmic kinases and they suggest that in addition to its genomic functions, the androgen receptor also regulates non-genomic processes.
Androgens play an important role in the differentiation and development of the male sexual organs (Wilson et al. 1981). In target cells, they bind to the androgen receptor (AR), a ligandbinding transcription factor, to make it competent for the regulation of expression of various genes. The AR has also been implicated in a number of physiological disorders, including partial and complete androgen insensitivity syndromes (Patterson et al. 1994), bulbar and spinal muscular atrophy (Warner et al. 1992) and neoplastic transformation of the prostate (Cunha et al. A.C.B. Cato and H. Peterziel are at the Forschungszentrum Karlsruhe, Institut für Genetik, Postfach 3640, D-76021 Karlsruhe, Germany.
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1987). In some of these disorders, the contribution of the AR to the altered phenotype is not fully understood. This is especially the case in prostate carcinomas, where attempts to block androgen action for therapeutic purposes have been only partially successful (Gittes 1991). Therefore, there is a need for a thorough analysis of the mode of action of the AR, with a view to identifying novel regulatory pathways that can provide a better understanding of the function of this receptor. In this article, we review the action of the AR, incorporating its classic function with some more recently discovered regulatory activities. When put together, these findings provide a new model of AR action that combines both genomic and non-genomic processes.
Transactivation
The classic mode of action of the AR is the positive and negative regulation of genes in a ligand-specific manner (Fig. 1). In positive regulation of gene expression, the AR binds first as a homodimer to discrete nucleotide sequences on the promoter of inducible genes to enhance their activity. The sequence to which the AR binds has been shown in previous studies to be the motif 5⬘-GGTACAnnnTGTTCT-3⬘ (Beato 1989) in the mouse mammary tumor virus (MMTV) DNA, that is also recognized by the progesterone, mineralocorticoid and glucocorticoid receptors (PR, MR and GR) (Cato et al. 1987, Ham et al. 1988) (Table 1). Recently, a more specific recognition site of the AR has been identified (Claessens et al. 1996). A sequence 5⬘GGTTCTTGGAGTACT-3⬘ from the probasin gene promoter mediates only the androgen response in transfection experiments when linked to an indicator gene (Claessens et al. 1996) (Table 1). Mutational analyses showed that this specificity comes from the left half 5⬘-GGTTCT-3⬘ of the binding sequence, which excludes other receptors from binding. Preferential binding of the AR in a head-to-tail fashion and activation of an indicator gene containing binding sites of the direct repeat (DR-1)-type sequence, have also been described (Zhou et al. 1997) (Table 1). Furthermore, the AR has been shown to bind, together with the GR, as a heterodimer in a head-tohead fashion (Chen et al. 1997). In this case, the transactivation function of the AR is repressed by the GR. Binding of the AR to discrete sequences on the promoter of inducible genes allows the receptor to interact with the basal transcription machinery.
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Evidence exists demonstrating the importance of leptin in the control of energy homeostasis, feeding behavior and reproductive function. Leptin receptors are localized in several regions of the brain implicated in regulation of energy balance and reproductive function, including the arcuate nucleus/median eminence, paraventricular nucleus, and ventromedial nucleus. Administration of exogenous leptin has been shown to alter function of the hypothalamic–pituitary–adrenal axis and stimulate gonadotropin release through hypothalamic and pituitary actions. Results from in situ hybridization studies demonstrate the ability of leptin to modulate the expression of key neuropeptides (neuropeptide Y, corticotropin-releasing hormone) implicated in the regulation of energy homeostasis. This suggests that leptin is an important component in the neuroendocrine transmission line that regulates appetite, energy balance and reproduction.
Obesity is a risk factor associated with a myriad of health problems including hypertension, cardiovascular disease, infertility and non-insulin-dependent diabetes mellitus (NIDDM). This nutritional disorder, resulting from disequilibrium between energy expenditure and ingestive behavior, is a cause of significant morbidity, and is becoming more common in the developed world (Bouchard and Perusse 1993). For this reason, the etiology of obesity has been investigated intensely, in order to provide information necessary for the development of therapeutic approaches for its treatment. Results from cross-circulation (parabiosis) experiments involving obese ob/ob and db/db mice provided initial evidence suggesting the existence of a circulating satiety factor responsible for a reduction in food intake and subsequent weight loss. A single point C. Buchanan, V. Mahesh, P. Zamorano and D. Brann are at the Department of Physiology and Endocrinology, Medical College of Georgia, Augusta, GA 30912, USA.
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mutation in the leptin receptor gene, as observed in C57BL/Ks db/db mice, results in alternative splicing of the receptor coding region and subsequent production of mutant leptin receptor (Chen et al. 1996, Lee et al. 1996). When the circulatory systems of ob/ob mice were connected indirectly to those of db/db mice, ob/ob mice lost weight and reduced their food intake, whereas db/db mice did not lose weight or reduce their food intake. This suggested that ob/ob mice were deficient in the circulating factor but retained the ability to respond to it with subsequent reduction in body weight. In contrast, db/db mice were unable to respond to adequate circulating levels of the factor. It was concluded that the ob gene encoded the satiety factor, while the db gene encoded the receptor for this ligand (Coleman 1973 and 1978, Chen et al. 1996). Recently, this hypothesis received important confirmation with the cloning of the ob gene and the subsequent demonstration that the gene product acts as a blood-borne
hormone responsible for weight maintenance (Zhang et al. 1994, Pellymounter et al. 1995). Leptin, the product of the adipose-specific ob gene, is a 167-amino acid secreted peptide that exerts its effects on central neural networks through regulation of energy homeostasis and feeding behavior. The ob/ob mouse has provided a useful model for investigating the physiological role of leptin in the body. A single base mutation of the leptin gene at codon 105, as observed in the C57BL/6J ob/ob mouse, involves the replacement of arginine by a premature stop codon and the subsequent production of an inactive form of leptin (Weigle et al. 1995). The genetically obese ob/ob mouse exhibits hyperinsulinemia, infertility, hyperglycemia and impaired thyroid function (Bray and York 1979). Interestingly, treatment of obese ob/ob mice with recombinant leptin lowers body weight and percent body fat. This treatment also corrects the hyperinsulinemia, infertility and hyperglycemia of ob/b mice. However, diet restriction alone was ineffective in restoring fertility in ob/ob mice, which suggests that leptin may be essential for normal reproductive function (Campfield et al. 1995, Chehab et al. 1996). Many reviews have discussed leptin actions in feeding behavior; therefore, this review will focus on the central nervous system (CNS) effects of leptin. •
Leptin Receptors and Signaling Pathways
Description of Leptin Receptors The leptin receptor (OB-R) is a single membrane-spanning protein homologous to members of the class I cytokine receptor family. Isoforms of the leptin receptor, which differ in the length of their cytoplasmic domains, include OB-Ra, OB-Rb, OB-Rc and OB-Rd. OB-Ra and OB-Rb, the short and long isoform, respectively, are generated by alternative splicing of a common transcript, and are highly conserved between mice and humans. OB-Rd is generated when a short exon is spliced between the OB-Rb splice donor (Lys-889) and splice acceptor (Pro-890) sites, while OB-Rc is synthesized when
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splicing does not occur after the splice donor (Lys-889) site. Recent evidence suggests the existence of a soluble leptin receptor OB-Re, but the physiological role of this isoform remains unclear (Tartaglia et al. 1995, Chen et al. 1996, Ghilardi et al. 1996, Fei et al. 1997). Results from experiments investigating signal transduction pathways of OB-Ra and OB-Rb isoforms suggest that OB-Rb may play an important role in mediating the biological effects of leptin. In these studies, COS cells and a factor-dependent hematopoietic cell line BaF3 were transfected with constructs encoding OB-Rb or OB-Ra isoforms. COS cells expressing OB-Rb responded to leptin exposure with activation of components in the signal transducers and activators of transcription (STAT) pathway, including STAT5, STAT6 and STAT3, while cells expressing OB-Ra were inactive in STAT signaling upon leptin treatment. BaF3 cells transfected with OB-Rb responded to leptin with increased proliferation and activation of Janus kinase 2 (Jak2), a cytoplasmic tyrosine kinase associated with the intracellular domain of receptors in the cytokine receptor superfamily. However, cells expressing OB-Ra, the short isoform, were inactive in proliferation and janus kinase activation (Ihle 1995, Ghilardi and Skoda 1997). Leptin treatment has also been shown to induce dose-dependent hypothalamic STAT3 activation in female C57BL/6J ob/ob and C57BL/6J +/? wild-type mice, but not C57BLKS/J db/db mice, which lack OB-Rb (Vaisse et al. 1996). This suggests that the OB-Rb isoform may be necessary for hypothalamic STAT3 activation. However, STAT activation was not observed in other tissues that express OB-Rb. This could be due to the presence of truncated isoforms acting as dominant negatives, thereby reducing the ratio of OB-Rb (long isoform) to OB-Ra, OB-Rc and OB-Rd (short isoforms). Interestingly, the ratio of OB-Rb to truncated isoforms has also been reported to be greatest in the hypothalamus (Ghilardi et al. 1996). Taken as a whole, these results suggest that STAT activation may be an important TEM Vol. 9, No. 4, 1998
component in hypothalamic OB-Rb signal transduction. Localization of Leptin Receptors in the Central Nervous System Results from in situ hybridization studies demonstrate that the leptin receptor gene is expressed strongly in several regions of the mouse brain (Mercer et al. 1996a, Fei et al. 1997). Utilizing a probe recognizing all OB-R isoforms, leptin receptor gene expression was detected in the choroid plexus, leptomeninges and parts of the hypothalamus implicated in the regulation of energy balance, including the arcuate nucleus (ARC), paraventricular nucleus (PVN) and ventromedial nucleus (VMH). A similar hybridization pattern was observed in adjacent sections utilizing a probe specific for the Ob-Rb isoform; however, there was minimal hybridization to the leptomeninges and choroid plexus. Quantification of the signal intensity indicated that the OB-R gene expressed in the hypothalamus consisted mainly of OB-Rb. Reverse transcription polymerase chain reaction (RT-PCR) analysis and immunohistochemical studies from our laboratory have demonstrated OB-R gene expression and localization in neuroendocrine tissues of the rat, including the anterior pituitary, choroid plexus and arcuate nucleus/median eminence (ARC/ME) of the hypothalamus (Zamorano et al. 1997). OB-R gene expression was also demonstrated in the immortalized gonadotropin-releasing hormone (GnRH)-producing GT1-7 and NLT neuronal cell lines. The localization of OB-R in the ARC, a site involved in control of both reproductive function and feeding behavior, indicates that leptin could potentially act at the ARC to modulate these two functions. Results from immunohistochemical studies using an antibody recognizing all OB-R isoforms demonstrated that OB-R is present in regions of the human brain, including the choroid plexus, nucleus basalis of Meynert, inferior olivary nuclei, ependymal lining of the lateral ventricle and cerebellar Purkinje cells. Immunoreactivity was also localized in
several hypothalamic nuclei, including suprachiasmatic, ARC, PVN, mamillary, dorsomedial (DMH), supraoptic and posterior nuclei (Couce et al. 1997). Studies demonstrating OB-R gene expression and localization in the choroid plexus of the mouse, rat and human provide further evidence to support the idea that leptin binds to the choroid plexus and is subsequently transported across the blood–brain barrier into the cerebrospinal fluid, thereby allowing it to travel to specific brain sites (Lynn et al. 1996). •
Leptin Effects on Hypothalamic and Pituitary Function
GnRH and Gonadotropin Secretion The secretion of the hypothalamic decapeptide GnRH is considered a primary neural signal involved in induction of the preovulatory luteinizing hormone (LH) surge. Upon receiving stimulatory signals, hypothalamic GnRH neurons secrete GnRH into the hypophyseal portal veins of the median eminence, allowing it to travel to the anterior pituitary and, after a cascade of events, initiate the LH surge (Levine and Ramirez 1982, Moenter et al. 1991). Recently, McCann and colleagues reported that leptin may stimulate gonadotropin release through pituitary and hypothalamic actions (Yu et al. 1997). Results from tissue culture studies demonstrated that hemi-anterior pituitaries of adult male rats respond to leptin exposure with a significant increase in LH release, supporting previous reports demonstrating the ability of leptin to increase significantly serum LH levels in fasted mice (Ahima et al. 1996). Yu et al. (1997) also reported that leptin can significantly enhance GnRH secretion in vitro from hypothalamic ARC/ME explants, suggesting that leptin also acts centrally at the level of the hypothalamus to influence reproductive function. A hypothalamic site of action of leptin is supported further by the findings that leptin injected into the third ventricle stimulates LH release (Yu et al. 1997) while, conversely, administration of leptin antiserum into the third ventricle of female rats induces a 147
marked decrease in LH pulsatility and disrupts cyclicity (Carro et al. 1997). In addition, leptin has also been shown to stimulate the reproductive endocrine system in ob/ob mice (Barash et al. 1996, Chehab et al. 1996, Cioffi et al. 1996). Leptin treatment induced a significant increase in plasma levels of LH in female ob/ob mice, while ob/ob male mice exhibited a significant increase in plasma levels of follicle stimulating hormone (FSH). Both sexes also responded to leptin treatment with increased uterine or seminal vesicle weight.
on feeding behavior and energy balance may involve, at least in part, antagonism of NPY neuronal systems (Erickson et al. 1996). Recent studies suggest that melanocortin receptor (MC4) signaling may also play a role in mediating leptin’s effects on feeding behavior. Along these lines, pretreatment of male rats with SHU9119, an MC4 receptor antagonist, abolished the ability of centrally administered leptin to induce cFos-like immunoreactivity in the PVN and to decrease feeding and body weight (Seeley et al. 1997).
Neuropeptide Y and Corticotropinreleasing Hormone Results from in situ hybridization studies have suggested that leptin can alter expression of hypothalamic neuropeptides implicated in the regulation of energy homeostasis. Leptin treatment has been reported to lower neuropeptide Y (NPY) mRNA levels in the ARC of ob/ob mice (Mercer et al. 1997). Similar results were observed in the male rat; however, leptin also induced a significant increase in corticotropin-releasing hormone (CRH) mRNA levels in the PVN (Schwartz et al. 1996). NPY is considered one of the most potent inducers of feeding behavior, and CRH is thought to act at the PVN to increase energy expenditure and inhibit ingestive behavior; thus, the ability of leptin to inhibit NPY and stimulate CRH gene expression may partially account for the weightreducing effect of leptin (Clark et al. 1984, Rothwell 1989). Interestingly, leptin receptor mRNA is reportedly coexpressed in NPY neurons of the ARC, suggesting that the effect of leptin on NPY gene expression may be through a direct mechanism (Mercer et al. 1996b). In support of a partial role of NPY in leptin effects on energy homeostasis, double mutant NPY−/− ob/ob mice were less obese than ob/ob mice, owing to reduced feeding behavior and increased energy expenditure. The absence of NPY also improved reproductive function in ob/ob mice, and double mutants were less severely affected by diabetes. These results suggest that leptin’s regulatory actions
Regulation of the Hypothalamic–Pituitary–Adrenal Axis The obesity and chronic hyperadrenocorticism exhibited by ob/ob mice can be attenuated by adrenalectomy; therefore, hypothalamic–pituitary–adrenal (HPA) axis abnormality might play a role in mediating the expression of the obesity syndrome in ob/ob mice (Saito and Bray 1984, Bray et al. 1989). Studies investigating HPA axis function demonstrated increased synthesis and secretion of pituitary adrenocorticotropic hormone (ACTH), along with an enhanced adrenal response to ACTH in ob/ob mice (McGinnis et al. 1992). ob/ob mice also exhibit chronically elevated serum corticosterone (CS) levels, suggesting that glucocorticoid negative feedback may also be impaired at the level of the pituitary or hypothalamus. Leptin has also been reported to influence changes in the HPA axis induced by starvation of male rats (Ahima et al. 1996). Starvation-induced increases in plasma ACTH and CS levels were reduced by leptin and, interestingly, fed rats exhibited an inverse correlation between serum CS and leptin levels. Similar results have demonstrated that fluctuations in the 24-h patterns of circulating human serum leptin levels are the inverse of cortisol and ACTH levels (Licinio et al. 1997). This provides additional evidence implicating leptin as a potential modulator of HPA axis function, although further studies are needed to elucidate the mechanisms underlying the HPA axis hyperactivity observed in the leptin-deficient state.
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Puberty Previous evidence suggests a connection between body weight, adipose tissue mass and time of puberty onset (Kennedy and Mitra 1963, Frisch 1980). In addition, recent work has suggested that leptin may play a role in the process of sexual maturation and acquisition of reproductive competence. Administration of exogenous leptin resulted in an earlier onset of vaginal opening, estrus and cycling in female mice, indicating that leptin may be a factor in determining the timing of puberty (Ahima et al. 1997, Chehab et al. 1997). Although these studies did not address the site of action of leptin in the induction of puberty, the previously reported ability of leptin to facilitate GnRH release suggests that the action of leptin could involve hypothalamic effects (Yu et al. 1997). However, it should be pointed out that other studies failed to find a facilitatory effect of leptin on the timing of puberty in female rats, questioning whether leptin is the primary signal that initiates the onset of puberty (Cheung et al. 1997). In humans, a significant rise in serum leptin levels was observed before the onset of puberty in boys; however, a cause and effect relationship has not been established (Mantzoros et al. 1997). Clearly, more work is needed to determine the precise role and importance of leptin in the achievement of puberty. •
Conclusions
The evidence presented in this review demonstrates the central nervous system effects of leptin in the regulation of energy homeostasis, feeding behavior and reproductive function. Leptin receptor gene expression and localization have been demonstrated in regions of the brain involved in the control of ingestive behavior and energy balance. In addition, leptin has been shown to alter the expression of hypothalamic neuropeptides implicated in the regulation of these functions. The ability of leptin to modulate HPA axis function along with gonadotropin and GnRH secretion also demonstrates its ability to affect reproductive function. The precise role of leptin in the TEM Vol. 9, No. 4, 1998
induction of puberty remains unclear, but evidence suggests that it may be a factor determining the timing of puberty. Evidence presented here suggests that leptin plays an important role in regulation of appetite, energy balance and reproduction. Future studies identifying potential effectors of leptin action may be useful in the development of therapeutic approaches for use in the treatment of obesity and reproductive dysfunction. References Ahima RS, Prabakaran D, Mantzoros C, et al.: 1996. Role of leptin in the neuroendocrine response to fasting. Nature 382:250–252. Ahima RS, Dushay J, Flier SN, Prabakaran D, Flier JS: 1997. Leptin accelerates the onset of puberty in normal female mice. J Clin Invest 99:391–395. Barash IA, Cheung CC, Weigle DS, et al.: 1996. Leptin is a metabolic signal to the reproductive system. Endocrinology 137:3144–3147. Bouchard C, Perusse L: 1993. Genetics of obesity. Annu Rev Nutr 13:337–354. Bray GA, York DA: 1979. Hypothalamic and genetic obesity in experimental animals: an autonomic and endocrine hypothesis. Physiol Rev 59:719–809. Bray GA, York DA, Fisher JS: 1989. Experimental obesity: a homeostatic failure due to defective nutrient stimulation of the sympathetic nervous system. Vitam Horm 45:1–125. 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. Carro E, Pinilla L, Seoane LM, et al.: 1997. Influence of endogenous leptin tone on the estrous cycle and luteinizing hormone pulsatility in female rats. Neuroendocrinology 66:375–377. Chehab F, Lim M, Lu R: 1996. Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat Genet 12:318–320. Chehab FF, Mounzih K, Lu R, Lim ME: 1997. Early onset of reproductive function in normal female mice treated with leptin. Science 275:88–90. Chen H, Charlat O, Tartaglia LA, et al.: 1996. Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell 84:491–495.
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Cheung CC, Thornton JE, Kuijper JL, Weigle DS, Clifton DK, Steiner RA: 1997. Leptin is a metabolic gate for the onset of puberty in the female rat. Endocrinology 138:855–858. Cioffi J, Shafer A, Zupanic T, et al.: 1996. Novel B219/OB receptor isoforms: possible role of leptin in hematopoiesis and reproduction. Nat Med 2:585–588. Clark JT, Kalra PS, Crowley WR, Karla SP: 1984. Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology 115:427–429. Coleman DL: 1973. Effects of parabiosis of obese with diabetes and normal mice. Diabetologia 9:294–298. Coleman DL: 1978. Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice. Diabetologia 14:141–148. Couce ME, Burguera B, Parisi JE, Jenson MD, Lloyd RV: 1997. Localization of leptin receptor in the human brain. Neuroendocrinology 66:145–150. Erickson JC, Hollopeter G, Palmiter RD: 1996. Attenuation of the obesity syndrome of ob/ob mice by the loss of neuropeptide Y. Science 274:1704–1707. Fei H, Okano HJ, Li C, et al.: 1997. Anatomic localization of alternately spliced leptin receptors (OB-R) in mouse brain and other tissues. Neurobiology 94:7001–7005. Frisch RE: 1980. Pubertal adipose tissue: is it necessary for normal sexual maturation? Evidence from the rat and human female. Fed Proc 39:2395–2400. 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. Ghilardi N, Ziegler S, Wiestner A, Stoffel R, Heim MH, Skoda RC: 1996. Defective STAT signaling by the leptin receptor in diabetic mice. Proc Natl Acad Sci USA 93:6231–6235. Ihle JN: 1995. Cytokine receptor signaling. Nature 377:591–594. Kennedy GC, Mitra J: 1963. Body weight and food intake as initiating factors for puberty in the rat. J Physiol (London) 166:408–418. Lee GH, Proenca R, Montez JM, et al.: 1996. Abnormal splicing of the leptin receptor in diabetic mice. Nature 379:632–635. Levine JE, Ramirez VD: 1982. Luteinizing hormone releasing hormone release during the rat estrous cycle and after ovariectomy as estimated with push pull cannulae. Endocrinology 111:1439–1448. Licinio J, Mantzoros C, Negrao AB, et al.: 1997. Human leptin levels are pulsatile and
inversely related to pituitary-adrenal function. Nat Med 3:575–579. Lynn RB, Cao GY, Considine RV, Hyde TM, Caro JF: 1996. Autoradiographic localization of leptin binding in the choroid plexus of ob/ob and db/db mice. Biochem Biophys Res Commun 219:884–889. Mantzoros CS, Flier JS, Rogol AD: 1997. A longitudinal assessment of hormonal and physical alterations during normal puberty in boys. V. Rising leptin levels may signal the onset of puberty. J Clin Endocrinol Med 82:1066–1070. McGinnis R, Walker J, Margules D, Aird F, Redel E: 1992. Dysregulation of the hypothalamus–pituitary–adrenal axis in male and female, genetically obese (ob/ob) mice. J Neuroendocrinol 4:765–771. Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah LT, Trayhurn P: 1996a. 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. Mercer JG, Hoggard N, Williams LM, et al.: 1996b. Coexpression of leptin receptor and proneuropeptide Y mRNA in the arcuate nucleus of mouse hypothalamus. Neuroendocrinology 8:733–735. Mercer JG, Moar KM, Rayner DV, Trayhurn P, Hoggard N: 1997. Regulation of leptin receptor and NPY gene expression in hypothalamus of leptin-treated obese (ob/ob) and cold exposed lean mice. FEBS Lett 402:185–188. Moenter SM, Caraty A, Locatelli A, Karsch FJ: 1991. Pattern of gonadotropin releasing hormone (GnRH) secretion leading up to ovulation in the ewe: existence of a preovulatory GnRH surge. Endocrinology 111:1175–1182. Pellymounter MA, Cullen MJ, Baker MB, et al.: 1995. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269:540–543. Rothwell N: 1989. Central effects of CRF on metabolism and energy balance. Neurosci Biobehav Rev 14:263–271. Saito M, Bray GA: 1984. Adrenalectomy and food restriction in the genetically obese (ob/ob) mouse. Am J Physiol 248:E20–E25. Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG: 1996. Identification of targets of leptin action in rat hypothalamus. J Clin Invest 98:1101–1106. Seeley RJ, Yagaloff KA, Fisher SL, et al.: 1997. Melanocortin receptors in leptin effects. Nature 390:349.
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•
The Androgen Receptor as Mediator of Gene Expression and Signal Transduction Pathways Andrew C.B. Cato and Heike Peterziel
The current model of action of androgens involves activation of a cytoplasmic receptor that migrates into the nucleus to regulate the expression of specific genes, either positively or negatively. While positive regulation requires direct binding of the receptor to DNA, negative regulation occurs mainly through protein–protein interactions of the receptor and other transcription factors. More recent findings have shown that the receptor also mediates non-conventional responses attributed hitherto only to activated growth factor receptors. These actions proceed principally through activation of cytoplasmic kinases and they suggest that in addition to its genomic functions, the androgen receptor also regulates non-genomic processes.
Androgens play an important role in the differentiation and development of the male sexual organs (Wilson et al. 1981). In target cells, they bind to the androgen receptor (AR), a ligandbinding transcription factor, to make it competent for the regulation of expression of various genes. The AR has also been implicated in a number of physiological disorders, including partial and complete androgen insensitivity syndromes (Patterson et al. 1994), bulbar and spinal muscular atrophy (Warner et al. 1992) and neoplastic transformation of the prostate (Cunha et al. A.C.B. Cato and H. Peterziel are at the Forschungszentrum Karlsruhe, Institut für Genetik, Postfach 3640, D-76021 Karlsruhe, Germany.
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1987). In some of these disorders, the contribution of the AR to the altered phenotype is not fully understood. This is especially the case in prostate carcinomas, where attempts to block androgen action for therapeutic purposes have been only partially successful (Gittes 1991). Therefore, there is a need for a thorough analysis of the mode of action of the AR, with a view to identifying novel regulatory pathways that can provide a better understanding of the function of this receptor. In this article, we review the action of the AR, incorporating its classic function with some more recently discovered regulatory activities. When put together, these findings provide a new model of AR action that combines both genomic and non-genomic processes.
Transactivation
The classic mode of action of the AR is the positive and negative regulation of genes in a ligand-specific manner (Fig. 1). In positive regulation of gene expression, the AR binds first as a homodimer to discrete nucleotide sequences on the promoter of inducible genes to enhance their activity. The sequence to which the AR binds has been shown in previous studies to be the motif 5⬘-GGTACAnnnTGTTCT-3⬘ (Beato 1989) in the mouse mammary tumor virus (MMTV) DNA, that is also recognized by the progesterone, mineralocorticoid and glucocorticoid receptors (PR, MR and GR) (Cato et al. 1987, Ham et al. 1988) (Table 1). Recently, a more specific recognition site of the AR has been identified (Claessens et al. 1996). A sequence 5⬘GGTTCTTGGAGTACT-3⬘ from the probasin gene promoter mediates only the androgen response in transfection experiments when linked to an indicator gene (Claessens et al. 1996) (Table 1). Mutational analyses showed that this specificity comes from the left half 5⬘-GGTTCT-3⬘ of the binding sequence, which excludes other receptors from binding. Preferential binding of the AR in a head-to-tail fashion and activation of an indicator gene containing binding sites of the direct repeat (DR-1)-type sequence, have also been described (Zhou et al. 1997) (Table 1). Furthermore, the AR has been shown to bind, together with the GR, as a heterodimer in a head-tohead fashion (Chen et al. 1997). In this case, the transactivation function of the AR is repressed by the GR. Binding of the AR to discrete sequences on the promoter of inducible genes allows the receptor to interact with the basal transcription machinery.
© 1998, Elsevier Science Ltd, 1043-2760/98/$19.00. PII: S1043-2760(98)00039-3
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