Research ReportLeptin-dependent STAT3 phosphorylation in postnatal mouse hypothalamus
Introduction
Leptin, the product of the ob (obese) gene, is a circulating peptide produced primarily by adipocytes in proportion to the body's fat energy stores (Zhang et al., 1994). In adult animals leptin is an important regulator of the energy balance, mainly through its actions in the brain in relation to food intake and energy expenditure (Friedman, 2002, Coll et al., 2007).
Leptin receptors (Ob-R) are the product of several alternatively spliced forms of the db (diabetes) gene (Chen et al., 1996, Lee et al., 1996). They are found in several tissues and organs, including a number of brain areas (Tartaglia et al., 1995, Fei et al., 1997). One splice variant, Ob-Rb, encoding a transmembrane protein with a longer cytoplasmic domain, is often referred to as the “long” or “functional form” of the receptor. Ob-Rb is highly expressed in the hypothalamus (Lee et al., 1996, Mercer et al., 1996, Schwartz et al., 1996, Fei et al., 1997, Elmquist et al., 1998). Mutations in the protein result in an obese phenotype (db/db mouse and Zucker fa/fa rat), demonstrating that leptin signalling in the hypothalamus via this receptor is required for normal energy homeostasis in adult rodents (Chen et al., 1996, Lee et al., 1996). Ob-Rb belongs to the family of cytokine receptors that lack the intrinsic catalytic kinase domain and function through cytoplasmic kinases. The cytoplasmic chain of Ob-Rb has a consensus amino acid sequence that is involved in the activation of Janus kinase-signal transducer and activator of transcription (JAK-STAT) tyrosine kinases and specifically activates STAT3 proteins (Baumann et al., 1996, Vaisse et al., 1996). Phosphorylated STAT3 proteins dimerize and translocate to the cell nucleus of leptin-sensitive neurons, where they bind to DNA, activate gene transcription and regulate neuroendocrine, autonomic and behavioural responses (Myers, 2004). Given the absence of specific Ob-Rb antibodies, detection of nuclear phospho-STAT3 immunoreactivity (P-STAT3-IR) after a single leptin injection has been proposed as a reliable neuroanatomical tool for the functional mapping of central leptin actions and the characterization of leptin-responsive, Ob-Rb-bearing neurons (Hubschle et al., 2001).
While it is generally accepted that leptin has an adipostatic action in adult animals, inhibiting food intake and boosting energy expenditure through the central activation of the JAK2-STAT3 signalling pathway (Sweeney, 2002, Myers, 2004), its physiological role in the rodent postnatal period, i.e. between birth and weaning, is unclear. Circulating leptin is detectable in pups as early as the first few days after birth, its level progressively rising to a peak around postnatal day 10 (P10), and then slowly declining to the concentration found in adults (Devaskar et al., 1997, Ahima et al., 1998, Yura et al., 2005). Although the neonatal mouse brain expresses all receptor isoforms (Proulx et al., 2002), administration of intraperitoneal (Mistry et al., 1999, Proulx et al., 2002) or intracerebroventricular (Mistry et al., 1999) leptin fails to reduce food intake and body weight during lactation and until after weaning. In addition, leptin-deficient rodent models (ob/ob mouse, db/db mouse and fa/fa rat) do not show hyperphagia or increased fat accumulation and body weight before the fourth week of postnatal life (Boissonneault et al., 1978, McLaughlin and Baile, 1981, Mistry et al., 1999). Leptin does not therefore appear to affect food intake and body weight in suckling mice, whereas it may have a neurotrophic role in postnatal hypothalamus (Bouret et al., 2004a), cortical neurons (Valerio et al., 2006) and hippocampus (Walker et al., 2007).
The present study was designed to determine whether leptin modulates STAT3 signalling in the postnatal hypothalamus. To do this, mice in the first three weeks of life received intraperitoneal (i.p.) mouse recombinant leptin (3 mg/kg of body weight); the pattern of leptin-induced P-STAT3 activation was evaluated by immunohistochemistry and the degree of STAT3 activation was quantified by Western blotting at specific developmental ages. The phenotype of leptin-sensitive cells was characterized by detecting both P-STAT3 and specific mature and immature neuronal and glial markers in hypothalamic slices by double-labelling immunofluorescence histochemistry and confocal microscopic analysis. Finally, a distinctive group of subependymal cells found to be responsive to leptin from P5 to P13 was studied with leptin-induced c-Fos pre-embedding immunoelectron microscopy.
Section snippets
P-STAT3-IR in normal and db/db\ adult mouse hypothalamus
In normal adult mice P-STAT3 was detected by immunohistochemistry in cell nuclei of the hypothalamic arcuate nucleus (ARC; Fig. 1A). Leptin induced a marked increase in P-STAT3-IR in the ARC; specific nuclear staining was also detected in several cells in hypothalamic areas such as medial preoptic area (MPO), anterior hypothalamic area (AHA), retrochiasmatic area (RCh), dorsomedial (DMH) and, to a lesser extent, ventromedial (VMH) hypothalamic nuclei, lateral hypothalamic area (LHA; Fig. 1B)
Discussion
Leptin is a pleiotropic hormone produced and secreted by adipocytes that acts on a distributed network of CNS neurons expressing Ob-Rb, the signalling form of its receptor. In adult mammals the metabolic, neuroendocrine and autonomic effects of leptin are mainly dependent on the activation of the JAK2-STAT3 intracellular pathway in hypothalamic neurons, even though non-hypothalamic centres, such as those located in the brain stem and ventral tegmental area, and other signalling pathways may
Animals and tissue processing
CD1 Swiss and C57BL/6J db/+ and db/db mice purchased from Charles River Laboratories (Calco, Italy) were housed in plastic cages in constant environmental conditions (12 h light/dark cycle at 22 °C) and ad libitum access to food and water. Litters of female mice (n = 6 per mother) were suckled ad libitum. Handling was limited to cage cleaning. All efforts were made to minimize animal suffering and to reduce the number of animals used. Experiments were carried out in accordance with EC Council
Acknowledgments
The work was financed by grants from Marche Polytechnic University (Contributi Ricerca Scientifica) and the Italian Ministry of University (PRIN 2005 to AG and FIRB Internazionalizzazione RBIN047PZY 2005 to SC).
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