Phosphodiesterase-3B Regulates The Activity of NPY/AgRP-Expressing Hypothalamic Neurons

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: MON 649-675-Central Regulation of Appetite & Feeding/GI Regulatory Peptides
Bench to Bedside
Monday, June 17, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board MON-666
Prashanth Anamthathmakula*, Maitrayee Sahu and Abhiram Sahu
University of Pittsburgh School of Medicine, Pittsburgh, PA
Hypothalamic neurons that express neuropeptide Y (NPY) and agouti-related protein (AgRP) are critical regulators of feeding behavior and body weight. NPY/AgRP neurons transduce the action of many peripheral signals including leptin and insulin. Both leptin and insulin inhibit NPY/AgRP neuronal activity and the knockdown of NPY/AgRP in the arcuate nucleus of adult animals causes starvation and body weight loss. However, intracellular signaling molecules involved in regulating NPY/AgRP neuronal activity are incompletely understood. We have previously shown that leptin and insulin stimulates PDE3B activity in the hypothalamus (1, 2), PDE3B mediates hypothalamic action of leptin on feeding (1), and NPY neurons express PDE3B (3). Thus, PDE3B could play a significant role in regulation of NPY/AgRP neuronal activity. To investigate the direct regulation of NPY/AgRP neuronal activity by PDE3B, we used a clonal hypothalamic neuronal cell line (mHypoE-46) expressing NPY/AgRP.  We first confirmed PDE3B expression in this cell line and then examined the effects of gain-of-function or loss-of-function of PDE3B on NPY/AgRP gene expression.  For the gain-of-function study, mHypoE-46 cells were transfected with 1 or 2µg of pcDNA-3.1-PDE3B expression plasmid or control pcDNA-3.1-GFP plasmid, and 24 to 48 hr later, cells were harvested to determine mRNA levels by qPCR and protein by western blot. We observed that PDE3B was over-expressed in a dose and time dependent manner. NPY and AgRP mRNA levels were decreased by ~55% at 24 hr or by 32-55% at 48 hr following overexpression of either dose of PDE3B-expressing plasmid as compared to the control plasmid (p < 0.05). For the loss-of-function study, mHypoE-46 cells were transfected with lentiviral PDE3BshRNAmir plasmid or non-silencing lentiviral shRNAmir control plasmid (Open Biosystems) and selected with puromycin. Selected cells were grown in culture for 48 hr, and processed for PDE3B mRNA and protein levels as well as NPY and AgRP mRNA measurement. Results showed that PDE3BshRNAmir significantly knockdown both PDE3B mRNA and protein levels (~60%; p < 0.05) as compared to control shRNAmir treatment. Importantly, decrease in PDE3B expression was associated with a 2-fold increase (p < 0.05) in both NPY and AgRP gene expression. Together, these results demonstrate a reciprocal change in NPY and AgRP gene expression following overexpression and knockdown of PDE3B in mHypoE-46 neuronal cells, and suggest that PDE3B plays a significant role in regulation of NPY/AgRP neuronal activity. Since obesity is associated with increased NPY/AgRP neuronal activity, targeting the PDE3B pathway in NPY/AgRP neurons may be considered as one of the approaches to treat obesity and related disorders.

(1) Zhao AZ et al. Nature Neuroscience 2002; 5: 727-728. (2) Sahu A et al. Program No.389.24. 2012 Neuroscience Meeting Planner, New Orleans, Society for Neuroscience. 2012. Online. (3) Sahu M et al. Neuroscience Letters 2011; 505:93-97.

Nothing to Disclose: PA, MS, AS

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Sources of Research Support: NIH Grant DK78068 awarded to AS