• Anaies Nazarians-Armavil, Jennifer A Chalmers, Claire B Lee, Wenqing Ye, and Denise D Belsham.
• Departments of Physiology, Obstetrics and Gynaecology Medicine, University of Toronto, Medical Sciences Building 3344, 1 Kings College Circle, Toronto, Ontario, Canada M5S 1A8 Division of Cellular and Molecular Biology, Toronto Genera Hospital Research Institute, University Health Network, Toronto, Ontario, Canada M5S 1A8.
• J. Endocrinol. 2014 Jan 1; 220 (1): 13-24.

AbstractPOMC neurons play a central role in the maintenance of whole-body energy homeostasis. This balance requires proper regulation of POMC neurons by metabolic hormones, such as insulin. However, the heterogeneous cellular population of the intact hypothalamus presents challenges for examining the molecular mechanisms underlying the potent anorexigenic effects of POMC neurons, and there is currently a complete lack of mature POMC neuronal cell models for study. To this end, we have generated novel, immortalized, adult-derived POMC-expressing/α-MSH-secreting cell models, mHypoA-POMC/GFP lines 1-4, representing the fluorescence-activated cell-sorted POMC population from primary POMC-eGFP mouse hypothalamus. The presence of Pomc mRNA in these cell lines was confirmed, and α-MSH was detected via immunofluorescence. α-MSH secretion in the mHypoA-POMC/GFP-1 was found to increase in response to 10  ng/ml ciliary neurotrophic factor (CNTF) or 10  nM insulin as determined by enzyme immunoassay. Further experiments using the mHypoA-POMC/GFP-1 cell line revealed that 10  ng/ml CNTF increases Pomc mRNA at 1 and 2  h after treatment, whereas insulin elicited an increase in Pomc mRNA level and decreases in insulin receptor (Insr (Ir)) mRNA level at 4  h. Furthermore, the activation of IR-mediated downstream second messengers was examined by western blot analysis, following the induction of cellular insulin resistance, which resulted in a loss of insulin-mediated regulation of Pomc and Ir mRNAs. The development of these immortalized neurons will be invaluable for the elucidation of the cellular and molecular mechanisms that underlie POMC neuronal function under normal and perturbed physiological conditions.

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