275 Leptin stimulates pituitary prolactin release through an extracellular signal-regulated kinase-dependent pathway Christian K Tipsmark, Christina N Strom, Sean T Bailey and Russell J Borski Department of Zoology, North Carolina State University, Raleigh, North Carolina 27695-7617, USA (Correspondence should be addressed to C K Tipsmark who is now at Institute of Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark; Email: [email protected]) Abstract Leptin was initially identified as a regulator of appetite and leptin are mediated by the activation of extracellular signal- weight control centers in the hypothalamus, but appears to be regulated kinase (ERK1/2) but nothing is known about the involved in a number of physiological processes. This study cellular mechanisms by which leptin might regulate PRL was carried out to examine the possible role of leptin in secretion in vertebrates. We therefore tested whether regulating prolactin (PRL) release using the teleost pituitary ERK1/2 might be involved in the leptin PRL response and model system. This advantageous system allows isolation of a found that the ERK inhibitor, PD98059, hindered leptin- nearly pure population of lactotropes in their natural, in situ induced PRL release. We further analyzed leptin response by aggregated state. The rostral pars distalis were dissected from quantifying tyrosine and threonine phosphorylation of tilapia pituitaries and exposed to varying concentrations of ERK1/2 using western blots. One hour incubation with leptin (0, 1, 10, 100 nM) for 1 h. Release of PRL was leptin induced a concentration-dependent increase in stimulated by leptin in a potent and concentration-dependent phosphorylated, and thus active, ERK1/2. Our data show manner. A time-course experiment showed that the strongest that leptin is a powerful stimulator of in vitro PRL release and response in PRL release with leptin occurs within the first that its actions occur in part through stimulation of ERK1/2. hour (approximately sixfold), and stimulation was sustained Journal of Endocrinology (2008) 196, 275–281 after 16 h (approximately twofold). Many of the actions of Introduction Leptin is the 16 kDa product of the ob gene, and the hormone is primarily expressed and secreted by white adipose Among the pituitary hormones, prolactin (PRL) is the most tissue in mammals (Zhang et al. 1994). In teleosts, putative versatile in the spectrum and number of functions it regulates. leptin has been detected with mammalian antibodies and is PRL modulates virtually every aspect of vertebrate physiology, particularly abundant in the bloodstream and liver (Johnson including osmoregulation, growth, metabolism, development, et al. 2000). Recently, a teleost ortholog of mammalian leptin reproduction, parental behavior, and immune function was found in three fish species (Kurokawa et al. 2005) and (Freeman et al. 2000). The central biological importance of shown to be expressed in various tissues including liver and PRL highlights the need for defining those factors and adipose (Kurokawa et al. 2005, Huising et al. 2006a). Leptin intracellular mechanisms that govern its secretion from the receptors are also present in the genome of teleosts examined pituitary gland. Among its numerous functions, PRL plays a thus far (Huising et al. 2006b). critical role in freshwater osmoregulation (Manzon 2002)as Since its discovery, the biology of leptin has been extensively well as in the modulation of energy metabolism in teleost fishes studied and in mammals it seems important as a humoral (Sheridan 1986, Leena et al. 2001, Sangio-Alvarellos et al. ‘lipostat’, by increasing energy expenditure and decreasing 2006). PRL is regulated by a complex arrayof hormonal factors appetite (Sahu 2004). Accordingly, food intake is suppressed in and neurotransmitters, including the osmoregulatory and response to treatment with exogenous leptin in mammals metabolic hormones, insulin-like growth factor-I (IGF-I), (Campfield et al.1995, Sahu 2004)andbirds(Denbow et al. and cortisol (Nishioka et al. 1988, Fruchtman et al. 2000, 2000, Lohmus et al.2003). In fish some ambiguity exist as to Hyde et al. 2004). Gut stomach-derived ghrelin also modulates leptin’s role, since treatment with mammalian hormone was teleost PRL release (Riley et al. 2002, Kaiya et al. 2003a) and in reported to have either anorexigenic actions (sunfish, Johnson mammals this hormone may act in cooperation with leptin to et al.2000; goldfish, Volkoff et al.2003), or no effect at all (catfish, transfer information to the brain about the caloric state of the Silverstein & Plisetskaya 2000; coho salmon, Baker et al.2000; animal (Popovic & Duntas 2005). sunfish, Londraville & Duvall 2002). Food deprivation in Journal of Endocrinology (2008) 196, 275–281 DOI: 10.1677/JOE-07-0540 0022–0795/08/0196–275 q 2008 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/27/2021 04:23:32PM via free access 276 C K TIPSMARK and others . Leptin stimulates prolactin release via ERK mammals induces a drop in leptin expression (Bertile et al.2003) Falcon 96-well plate (Becton Dickinson, Oxnard, CA, USA) but a similar treatment elicits little effect in carp (Huising et al. containing 100 ml Krebs bicarbonate Ringers. Ringers solution 2006a), suggesting possible evolutionary differences in leptin contained glucose, glutamine and Eagle’s minimal essential regulation among endotherms and ectotherms. medium (355–360 mOsm, pH 7.2; Hyde et al.2004). Tissues In mammals it appears that leptin works on hypothalamic were incubated at 27 8C in a humidified chamber (95% O2/5% neurons to induce luteinizing hormone release, and when energy CO2). The chamber was continuously agitated on a gyratory stores are sufficient, promotes the initiation of sexual maturation platform at 60 r.p.m. To allow PRL release to stabilize to (Smith et al. 2002, Harvey & Ashford 2003). Leptin treatment baseline levels, the tissues were pre-incubated for 2 h, after also stimulates circulating PRL levels in rats (Gonzalez et al. 1999, which, medium was removed and replaced with treatment Watanobe 2002). Melanocortin-4 receptor antagonists decrease medium. Samples were taken after 1, 4, and 16 h, and medium the magnitude of leptin-induced PRL surges in vivo indicating and tissue were collected. Tissues were sonicated in RIA buffer interaction with this receptor and that hypothalamic control of (0.01 M sodium phosphate, 1% BSA, 0.01% NaN3,and0.1% the rat pituitary plays a significant role in the response (Watanobe Triton X-100; pH 7.3) and stored at K20 8C. For measures of et al. 1999). In addition to leptin’s effect via the hypothalamus, ERK1/2, tissues were directly placed into reducing sample leptin appears to act directly at the level of the rat pituitary to buffer for subsequent western blot analyses. stimulate gonadotropin and PRL release from anterior pituitary cells (Yu et al.1997). Leptin also stimulates PRL secretion from PRL measurements bovine pituitary explants (Accorsi et al. 2007), but has no effect on PRL release from primary cultures of porcine anterior pituitary The tilapia pituitary releases two PRLs (PRL177 and PRL188) cells (Nonaka et al. 2006). Whether leptin stimulates PRL release and both are regulated in a similar fashion by all secretagogue in nonmammalian vertebrates is unknown. In isolated pituitary examined to date (see Hyde et al. 2004). Thus, in the present cells from teleosts, recombinant mammalian leptin induces study only the release of the more abundant PRL188 was release of gonadotropins (European sea bass, Peyon et al. 2001; measured in a concentration–response and time-course rainbow trout, Weil et al. 2003)andsomatolactin(Peyon et al. experiment. Tilapia PRL188 was quantified using a homolo- 2003). We examined whether leptin might modulate PRL gous RIA as previously described (Ayson et al. 1993, secretion from the teleost pituitary, which contains a naturally, Tipsmark et al. 2005) and hormone release is expressed as a highly enriched population of lactotropes within the rostral pars percentage of the total amount of hormone in the incubations distalis (RPD, Nishioka et al. 1988,seeFruchtman et al. 2000, (tissueCmedia). In a separate experiment, the effect of a Kasper et al. 2006). The pituitary RPD,containing 95–99% PRL specific ERK1/2 inhibitor, PD98059, on leptin-induced cells, can be easily isolated and allows the study of a nearly pure PRL release was examined. In this experiment, PRL was population of PRL cells in their naturally aggregated in situ state quantified by colorimetric gel detection according to our using completely defined, hormone-free culture medium. We previously described and validated procedures where both characterized the concentration-dependent response and time- tilapia PRLs, due to their differences in sizes, could be course of leptin’s possible effect on PRL release from the RPD. measured simultaneously (Borski et al. 1991, Hyde et al. Extracellular signal-regulated protein kinases (ERK1/2) have 2004). In short, tissue and media samples were run on SDS- been reported to be essential signal transducers of leptin responses PAGE and the gels stained with Coomassie brilliant blue. in several tissues (Harvey & Ashford 2003) and it is involved in After destaining, both PRL177 and PRL188 bands were IGF-I induced PRL release from the teleost lactotrope quantified using an Odyssey scanner (LI-COR, Lincoln, NE, (Fruchtman et al. 2000, 2001). Whether