title: Evidence suggesting phosphodiesterase-3B regulation of NPY/AgRP gene expression in mHypoE-46 hypothalamic neurons.
abstract: Hypothalamic neurons expressing neuropeptide Y (NPY) and agouti related-protein (AgRP) are critical regulators of feeding behavior and body weight, and transduce the action of many peripheral signals including leptin and insulin. However, intracellular signaling molecules involved in regulating NPY/AgRP neuronal activity are incompletely understood. Since phosphodiesterase-3B (PDE3B) mediates the hypothalamic action of leptin and insulin on feeding, and is expressed in NPY/AgRP neurons, PDE3B could play a significant role in regulating NPY/AgRP neuronal activity. To investigate the direct regulation of NPY/AgRP neuronal activity by PDE3B, we examined the effects of gain-of-function or reduced function of PDE3B on NPY/AgRP gene expression in a clonal hypothalamic neuronal cell line, mHypoE-46, which endogenously express NPY, AgRP and PDE3B. Overexpression of PDE3B in mHypoE-46 cells with transfection of pcDNA-3.1-PDE3B expression plasmid significantly decreased NPY and AgRP mRNA levels and p-CREB levels as compared to the control plasmid. For the PDE3B knockdown study, mHypoE-46 cells transfected with lentiviral PDE3BshRNAmir plasmid or non-silencing lentiviral shRNAmir control plasmid were selected with puromycin, and stably transfected cells were grown in culture for 48h. Results showed that PDE3BshRNAmir mediated knockdown of PDE3B mRNA and protein levels (∼60-70%) caused an increase in both NPY and AgRP gene expression and in p-CREB levels. Together, these results demonstrate a reciprocal change in NPY and AgRP gene expression following overexpression and knockdown of PDE3B, and suggest a significant role for PDE3B in the regulation of NPY/AgRP gene expression in mHypoE-46 hypothalamic neurons.
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: Prashanth
authors.initials: P
authors.lastname: Anamthathmakula
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: Maitrayee
authors.initials: M
authors.lastname: Sahu
authors.affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. Electronic address: asahu@pitt.edu.
authors.forename: Abhiram
authors.initials: A
authors.lastname: Sahu
mesh_terms: D054369:Agouti-Related Protein / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3 / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D007031:Hypothalamus / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D009478:Neuropeptide Y / Q000378:metabolism*
chemical_list: D054369:Agouti-Related Protein; D009478:Neuropeptide Y; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3
keywords: AgRP; Hypothalamic neurons; NPY; PDE3B; p-CREB