title: Estrogen inhibits NPY secretion through membrane-associated estrogen receptor (ER)-α in clonal, immortalized hypothalamic neurons.
abstract: OBJECTIVE
  Estrogen (E(2)) has an inhibitory effect on food intake by acting centrally in the hypothalamus, although it is not clear which hypothalamic neurons are involved in this process. Earlier studies from our lab and others have implicated neuropeptide Y (NPY) as an important central anorexigenic target of E(2). This study was designed to investigate whether E(2) can directly regulate NPY secretion and examine the cellular mechanisms and receptors responsible for this anorexigenic action of E(2).
  
  
  METHODS
  Clonal, murine, hypothalamic neuronal cell models, mHypoE-42 and mHypoA-2/12, were investigated for NPY secretory responses to 17β-estradiol (E(2)) in the presence or absence of pharmacological inhibitors directed against the phosphatidylinositol-3-kinase (PI3K), mitogen-activated protein kinase (MAPK) and AMP-activated kinase (AMPK) pathways or to estrogen receptor (ER) specific agonists/antagonists.
  
  
  METHODS
  The presence of hypothalamic markers and characterization of neuronal cell lines was completed with polymerase chain reaction. NPY levels were measured using an enzyme immunoassay (EIA). The expression of ER-α and caveolin-1 was analyzed using immunocytochemistry.
  
  
  RESULTS
  E(2) significantly decreased NPY secretion in both the mHypoE-42 and mHypoA-2/12 neurons. The E(2)-mediated repression of NPY secretion in the mHypoE-42 and mHypoA-2/12 neurons required ER-α, but not ER-β, as shown by studies using an ER-specific agonist/antagonists. Additionally, using immunocytochemistry we detected colocalization of ER-α and the membrane-associated signaling protein caveolin-1. Importantly, using E(2)-conjugated bovine serum albumin (E(2)-BSA) and ER antagonists, we were able to show that the E(2)-mediated decrease in NPY secretion occurred through membrane-bound ER-α. Finally, using a combination of pharmacological inhibitors, we found that inhibition of the PI3K or AMPK pathway blocked the E(2)-mediated decrease in NPY secretion.
  
  
  CONCLUSIONS
  These findings indicate that the central anorexigenic action of E(2) occurs at least partially through hypothalamic NPY-synthesizing neurons. This regulation of NPY secretion occurs through rapid signaling mechanisms through membrane bound ER-α.
authors.affiliation: Department of Physiology, Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada.
authors.forename: S S
authors.initials: SS
authors.lastname: Dhillon
authors.forename: D D
authors.initials: DD
authors.lastname: Belsham
mesh_terms: D000818:Animals; D001069:Appetite Regulation / Q000235:genetics / Q000502:physiology*; D015415:Biomarkers; D002417:Cattle; D051242:Caveolin 1 / Q000378:metabolism; D002460:Cell Line; D004958:Estradiol / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000235:genetics / Q000378:metabolism*; D007031:Hypothalamus / Q000187:drug effects* / Q000378:metabolism; D007150:Immunohistochemistry; D051379:Mice; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D009478:Neuropeptide Y / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*
chemical_list: D015415:Biomarkers; D051242:Caveolin 1; D047628:Estrogen Receptor alpha; D009478:Neuropeptide Y; D004958:Estradiol